Research Report 2014

Research Report 2014 Table of Contents

Inhalt

II Table of Contents Inhalt VIII Introduction by the Scientific Director Vorwort des Wissenschaftlichen Vorstands XII Editorial Remarks Redaktionelle Hinweise 1 Cardiovascular and Metabolic Disease Coordinator: Norbert Hübner

4 Mathematical Cell Physiology Martin Falcke 6 Neuromuscular and Cardiovascular Cell Biology Michael Gotthardt 8 Angiogenesis and Cardiovascular Pathology Ferdinand le Noble 10 Molecular Muscle Physiology Ingo Morano 14 Electrochemical Signaling in Development and Disease Daniela Panáková 16 Anchored Signalling Walter Rosenthal, Enno Klußmann 20 Experimental and Translational Kidney Research Kai Schmidt-Ott 22 Zebrafish Cardiovascular Developmental Genetics Salim Seyfried 24 Molecular and Cellular Basis of Embryonic Development Francesca M. Spagnoli 26 Molecular Cardiovascular Research Thomas Willnow 30 Mathematical Modelling of Cellular Processes Jana Wolf 32 Cardiovascular Hormones Michael Bader 36 Genetics and Genomics of Cardiovascular Diseases Norbert Hübner 40 Mobile DNA Zsuzsanna Izsvak 42 Molecular Genetics of Chronic Inflammation and Allergic Disease Young-Ae Lee 44 Hypertension Genetics, Regulators, and Environment Friedrich C. Luft 48 Ultrahigh Field Magnetic Resonance (UHF-MR) Thoralf Niendorf 52 Cell Signalling and Mass Spectrometry Matthias Selbach 54 Cardiovascular Molecular Genetics Ludwig Thierfelder

II MDC Research Report 2014 TABLE OF CONTENTS

Table of Contents

Inhalt

58 Molecular Epidemiology Tobias Pischon 62 microRNA and Molecular Mechanisms of Metabolic Diseases Matthew N. Poy 64 Genetics of Metabolic and Reproductive Disorders Mathias Treier 69 Cancer Research Coordinator: Claus Scheidereit

72 Computational Biology and Data Mining Miguel Andrade 74 Signal Transduction in Development and Cancer Walter Birchmeier 78 Molecular Immunology and Gene Therapy Thomas Blankenstein 82 Clinical and Molecular Oncology Peter Daniel 84 Structure and Function of Membrane-associated GTPases Oliver Daumke 88 Haematoloy, Oncology and Tumorimmunology Bernd Dörken 92 Experimental Pharmacology Iduna Fichtner 96 Macromolecular Structure and Interaction Udo Heinemann 100 Cell Differentiation and Tumorigenesis Achim Leutz 104 Molecular Tumor Genetics and Immunogenetics Martin Lipp 108 Molecular Immunotherapy Antonio Pezzutto 110 Immune Regulation and Cancer Klaus Rajewsky 114 Spatio-temporal Control of Rho GTPase Signaling Oliver Rocks 116 Signal Transduction in Tumor Cells Claus Scheidereit 120 Cancer Genetics and Cellular Stress Responses Clemens A. Schmitt 122 Stem Cell and Macrophage Biology Michael Sieweke 124 Intracellular Proteolysis Thomas Sommer 128 Molecular Cell Biology and Gene Therapy Wolfgang Uckert

MDC Research Report 2014 III

TABLE OF CONTENTS

131 Diseases of the Nervous System Coordinator: Carmen Birchmeier

134 Developmental Biology / Signal Transduction Carmen Birchmeier 138 Physiology and Pathology of Ion Transport Thomas J. Jentsch 142 Developmental Neurobiology Fritz G. Rathjen 146 Molecular Physiology of Somatic Sensation Gary Lewin 150 RNA Editing and Hyperexcitability Disorders Jochen Meier 152 Signaling and Transport Processes Björn Christian Schroeder 154 Neural Circuits and Behaviour James Poulet 156 Cellular Neurosciences Helmut Kettenmann 160 Proteomics and Molecular Mechanisms of Neurodegenerative Diseases Erich Wanker 164 Molecular Pathways in Cortical Development Annette Hammes 166 Cellular Sensing of the Physical Environment Kathryn Poole 169 Berlin Institute for Medical Systems Biology Coordinator: Nikolaus Rajewsky

172 Non-coding RNAs and Mechanisms of Cytoplasmic Gene Regulation Marina Chekulaeva 174 Novel Sequencing Technology, Functional and Medical Genomics Wei Chen 176 Metabolic Regulation Stefan Kempa 178 RNA Biology and Posttranscriptional Regulation Markus Landthaler 180 Signaling Dynamics in Single Cells Alexander Loewer 182 Evolutionary Modeling and Inference in Computational Systems Biology Benedikt Obermayer 184 Computational Regulatory Genomics Uwe Ohler 188 Epigenetic Regulation and Chromatin Architecture Ana Pombo

IV MDC Research Report 2014 TABLE OF CONTENTS

192 Systems Biology of Gene Regulatory Elements Nikolaus Rajewsky 196 Gene Regulation and Cell Fate Decision in C. elegans Baris Tursun 198 Systems Biology of Neural Tissue Differentiation Robert P. Zinzen 201 The Experimental and Clinical Research Center (ECRC) Coordinator: Friedrich C. Luft

206 Muscle Research Unit and Outpatient Clinic for Muscle Disorders of the Charité Simone Spuler 208 Nephrology and Inflammatory Vascular Diseases Ralph Kettritz 210 Cardiac MRI Jeanette Schulz-Menger 212 Registry Diabetic Nephropathy / Blood Vessel Function and Target-Organ Damage Maik Gollasch 214 Cardiovascular Genetics Silke Rickert-Sperling 216 Endocrinology, Diabetes and Nutritional Medicine Joachim Spranger 218 Hypertension-induced Target Organ Damage Dominik N. Müller, Ralf Dechend 220 Translational Oncology of Solid Tumors Ulrike Stein 223 Technology Platforms

226 Advanced Light Microscopy Anje Sporbert 228 BIMSB Bioinformatics Altuna Akalin 230 BIMSB Genomics Wei Chen 232 BIMSB Proteomics & Metabolomics Stefan Kempa 234 Electron Microscopy Bettina Purfürst 236 Interactomics Erich Wanker 238 Magnetic Resonance (Berlin Ultrahigh Field Facility, B.U.F.F.) Thoralf Niendorf

MDC Research Report 2014 V

TABLE OF CONTENTS

240 Mass Spectrometry Gunnar Dittmar 242 Pathophysiology Arnd Heuser 244 Preparative Flow Cytometry Hans-Peter Rahn 246 Transgenics Ralf Kühn 249 Technology Transfer

252 Enabling Technologies for Drug Discovery in Alzheimer’s and Further Protein Misfolding Diseases Annett Böddrich 252 Development of inhibitors of intracellular protein-protein interactions for the treatment of heart failure Adeeb Eldahshan 253 Exploitation of novel transduction targets for pain relief Christiane Wetzel and Gary R. Lewin 253 Development of novel compounds that inhibit growth of prostate cancer cells David Miles Carter 254 Alternative Splicing as a Therapeutic Target for Cardiac Disease Michael Radke 257 Academics Akademische Aktivitäten

258 International PhD Program Internationales Doktorandenprogramm 261 MDC Postdoctoral Program

262 MDC International

264 Appointments Berufungen 268 Awards Preise 269 European Research Council (ERC) Grants

270 Third-party Funding 2012-2013 Drittmittelprojekte 272 Conferences and Scientific Meetings Kongresse und wissenschaftliche Tagungen 274 Seminars 2012-2013

VI MDC Research Report 2014 TABLE OF CONTENTS

281 Annex Anhang

282 Helmholtz Association Helmholtz-Gemeinschaft 286 Berlin Institute of Health (BIH) Berliner Institut für Gesundheitsforschung 290 German Centre for Cardiovascular Research Deutsches Zentrum für Herz-Kreislauf-Forschung 291 EU-Life

292 Biotechnology Park with Innovation and Founding Center Biotechnologiepark mit Innovations- und Gründerzentrum 296 Health Care Industry Cluster Cluster Gesundheitswirtschaft 298 Press Department Pressestelle 300 Communications Department Kommunikationsabteilung 302 Organization Chart Organigramm 304 Organizational Structure Organisationsstruktur 310 Publications

312 Facts and Figures Kennzahlen 316 Index

327 Campus Map

MDC Research Report 2014 VII

Introduction by the Scientific Director

Vorwort des Wissenschaftlichen Vorstands

his research report looks back to two ieser Forschungsbericht blickt auf zwei eventful years at the Max Delbrück Cen- ereignisreiche Jahre am Max-Delbrück- Tter. We have not only launched the Berlin DCentrum zurück. So haben wir nicht nur Institute of Health, but also have co-founded das Berliner Institut für Gesundheitsforschung/ the German Centre for Cardiovascular Research Berlin Institute of Health auf den Weg gebracht, (DZHK). In 2012, we celebrated our 20-year sondern auch das Deutsche Zentrum für Herz- anniversary and we have set the course for the Kreislauf-Forschung mitgegründet. Wir haben

site in the heart of Berlin. wir haben die Weichen dafür gestellt, dass das MDC to open for the first time a second major MDC2012 erstmalsunser 20-jähriges einen zweiten Bestehen großen gefeiert Standort und im Herzen Berlins eröffnen wird. changes in many respects. In the coming two years,With alllaws this, will the come MDC into looks effect ahead that will to majorregu- Das MDC steht damit in vielerlei Hinsicht vor late the establishment of the Berlin Institute großen Veränderungen. In den kommenden of Health as a new public body and which thus beiden Jahren werden Gesetze auf den Weg will regulate also the future of our center. The gebracht, die die Gründung des Berliner Insti- MDC and the Charité – Universitätsmedizin tuts für Gesundheitsforschung als neue Körper- Berlin will both become member institutions schaft des öffentlichen Rechts und damit auch of the Berlin Institute of Health (BIH). This die Zukunft unseres Zentrums regeln. Das MDC way, we will institutionalize our partnership und die Charité – Universitätsmedizin Berlin with the Charité that we have been maintain- - ing since our founding in 1992. At the same lin Institute of Health (BIH) werden. Auf diese time, both parts of the new institute will main- Wwerdeneise institutionalisieren jeweils Gliedkörperschaften wir die seit desunserer Ber tain their respective identities and their very Gründung 1992 bestehende Partnerschaft mit own range of tasks. In the case of the MDC, der Charité. Beide Teile des neuen Instituts these tasks are biomedical basic research and disease-oriented research that is embedded ihre ureigenen Aufgaben behalten. Im Falle into the strategic research of the Helmholtz deswerden MDC dabei ist dasihre die jeweils biomedizinische eigene Identität Grund und- Association, Germany’s largest research orga- lagenforschung und die krankheitsorientierte nization. Forschung, die eingebettet ist in die strategische Forschung der Helmholtz-Gemeinschaft, The driving force in conceiving the BIH and in Deutschlands größter Forschungsorganisation. developing the funding programs was science with its desire to close the gap between basic Treibende Kraft bei der Konzeption des BIH research and clinical application research. The und der Ausarbeitung der Förderprogramme approach that was used here focuses on sys- war die Wissenschaft mit ihrem Wunsch, die tems medicine: away from the traditional no- Lücke zwischen Grundlagenforschung und kli- tion of “one disease – one mechanism”. Rather, nischer Forschung zu schließen. Der Ansatz ist we know today that a disease can be caused dabei systemmedizinisch: Er geht weg von der by various mechanisms and their interactions, traditionellen Vorstellung „eine Erkrankung – ein Mechanismus“. Vielmehr wissen wir heu- involved in causing several different diseases te, dass eine Erkrankung durch verschiedene (“onejust as disease a given – molecularmany mechanisms” mechanism and can “one be Mechanismen und deren Wechselwirkungen mechanism – many diseases”). The Charité verursacht werden kann, ebenso wie anderer- and the MDC perceive themselves to be pre- seits ein molekularer Mechanismus an unter- schiedlichen Erkrankungen beteiligt ist („eine closely cooperating with each other from the Erkrankung – viele Mechanismen“ und „ein destinedvery start. for In this 1992, project translation, as they that have is, been the Mechanismus – viele Erkrankungen“). Charité fastest possible transfer of basic research in- und MDC sehen sich als prädestiniert für dieses sights into clinical application, formed part of

Vorhaben an, kooperieren sie doch seit jeher VIII MDC Research Report 2014 INTRODUCTION

Prof. Dr. Walter Photo: Steffen Weigelt/MDC Steffen Photo: Rosenthal

the founding charter of the MDC. Already in eng miteinander. Die Translation, also die mög- 2007, against this background, we established lichst rasche Übertragung der Erkenntnisse aus together with the Charité the Experimental der Grundlagenforschung in die Klinik, gehör- and Clinical Research Center (ECRC), which te 1992 zum Gründungsauftrag des MDC. Vor has been headed by Fred Luft since that time. diesem Hintergrund haben wir mit der Chari- The BIH will build on the experiences we have té bereits 2007 das Experimental and Clinical gathered with the ECRC and it will add further Research Center (ECRC) gegründet, dem Fred value to the site of Berlin-Buch. Luft seither vorsteht. Das BIH wird auf den

I would like to use this opportunity to express gesammelt haben, und es wird den Standort my gratitude to policy-makers on both a fed- Berlin-BuchErfahrungen weiter aufbauen, aufwerten. die wir mit dem ECRC eral government and federal state level who have placed such great trust in us and whose Ich möchte an dieser Stelle Dank sagen an die initiative for establishing the new institute Politik in Bund und Land, die uns mit einem großen Vertrauensvorschuss ausgestattet hat and the MDC. I would also like to thank the und deren Initiative zur Gründung des neuen matched the scientific plans of the Charité Instituts zu den wissenschaftlichen Plänen von Charité und MDC passte. Ebenfalls bedanken thanksHelmholtz go to Association, entrepreneur which Johanna finances Quandt, the will ich mich bei der Helmholtz-Gemeinschaft, whoBIH during will provide the first the two new years. institute And with my special a total - of 40 million Euro in the context of her private ziert. Und mein ganz besonderer Dank gilt der excellence initiative. Thanks to this initiative, Untdie ernehmerindas BIH in den Johanna ersten Quandt, beiden Jahrendie mit finan ihrer which is administrated by the Charité Foun- privaten Exzellenzinitiative insgesamt 40 Milli- dation, we have managed to win over Nobel onen Euro für das neue Institut zur Verfügung Prize laureate Thomas Südhof to come to Ber- stellen wird. Dank dieser Initiative, die von der Stiftung Charité administriert wird, ist es uns (BIH Visiting Fellow). gelungen, den Nobelpreisträger Thomas Süd- lin for a BIH project as a first visiting scientist hof als ersten Gastwissenschaftler (BIH Visit- The German Centre for Cardiovascular Re- search (DZHK) started operations in April holen. 2012. It is part of an initiative by the German ing Fellow) für ein BIH-Projekt nach Berlin zu Federal Ministry of Education and Research, Das Deutsche Zentrum für Herz-Kreislauf-For- which has initiated health research centers schung DZHK nahm im April 2012 seine Arbeit and provides funding for these. The MDC is auf. Es ist eingebettet in eine Initiative des Bun- involved in the DZHK by way of a seat on the desministeriums für Bildung und Forschung,

MDC Research Report 2014 IX

INTRODUCTION

das Zentren der Gesundheitsforschung initiiert and the administration of funding. hat und diese fördert. Das MDC beteiligt sich board as well as by way of scientific projects am DZHK sowohl über einen Sitz im Vorstand In the past two years, we have also paved the - way for the MDC’s Berlin Institute for Medical wie mit der Administration der Fördermittel. Systems Biology (BIMSB) to be allocated a site als auch mit wissenschaftlichen Projekten so in Berlin-Mitte. It is part of the future concept Wir haben in den letzten beiden Jahren auch of the Humboldt Universität (HU) and thus den Weg dafür geebnet, dass das Berlin Insti- has contributed to the HU winning its status tute for Medical Systems Biology (BIMSB) des of “University of Excellence”. Together with us MDC einen Standort in Berlin-Mitte erhalten and the Charité, the HU has established the In- wird. Es ist Bestandteil des Zukunftskonzeptes tegrative Research Institute (IRI) for the Life der Humboldt-Universität (HU) und hat damit Sciences. The aim of the IRI for the Life Sci- zum Gewinn des Exzellenzstatus der HU beige- ences is to further develop the HU’s Campus tragen. Gemeinsam mit uns und der Charité hat die HU das Integrative Forschungsinstitut (IRI) in the heart of Berlin; the BIMSB is expected für Lebenswissenschaften gegründet. Ziel des toNor moved to into become a new an building influential on the science site as park of IRI für Lebenswissenschaften ist es, den Cam- 2018. pus Nord der HU zu einem maßgebenden Wis- senschaftspark im Herzen Berlins weiterzuent- Moreover, in 2013 we established the Euro- wickeln; das BIMSB wird dort voraussichtlich pean EU-Life alliance with twelve partnering ab 2018 in einen Neubau ziehen. institutions in the life sciences. It is intended to facilitate the exchange of information and Wir haben überdies im Jahr 2013 mit zwölf career moves within Europe and to provide Partnerinstitutionen aus den Lebenswissen- the life sciences with more political impact in schaften das europäische Konsortium EU-Life dealing with European Union committees and gegründet. Es soll dem Informationsaustausch bodies. dienen, Karrierewege innerhalb Europas er- leichtern und den Lebenswissenschaften ge- The basis for all these successes and develop- genüber den Gremien und Organen der Eu- ments in the world of science policy is the ex- ropäischen Union mehr politisches Gewicht cellent work carried out by our scientists. In verleihen. 2012, more than 30 international reviewers assessed the Max Delbrück Center. Our center Basis all dieser wissenschaftspolitischen Er- received the best possible mark of “outstand- folge und Entwicklungen ist die exzellente ing”. This success of our working groups is Arbeit unserer Wissenschaftlerinnen und Wis- senschaftler. Im Jahr 2012 haben mehr als 30 and in individual awards. For instance, Niko- internationale Gutachter das Max-Delbrück- reflected also in the number of publications Centrum evaluiert. Dabei schnitt unser Zent- 2012 and, since the publication of the last rum mit der Bestnote „Outstanding“ ab. Die- lausresearch Rajewsky report, was we awarded have attracted the Leibniz two Prize new ser Erfolg unserer Arbeitsgruppen bildet sich ERC grants. This makes the total number of auch ab in der Anzahl der Publikationen und in individuellen Preisen. So erhielt Nikolaus with several million Euro by the European Re- suchsearch projects Council at ERC the tenMDC (originally that each eleven;are funded the Veröffentlichung des letzten Research Reports ERC grant winner Jan Siemens followed a call habenRajewsky wir denzwei Leibniz-Preis neue ERC Grants 2012, eingeworben. und seit der to Heidelberg in 2013). Other well-respected awards went to James Poulet (Paul Ehrlich and Ludwig Darmstaedter Prize for Young ForschungsratDamit liegt die ERCGesamtzahl gefördert dieser werden, Projekte, am MDC die beimit zehn je mehreren (ursprünglich Millionen waren vom es Europäischenelf; der ERC- Carreras Award 2013, Medal of Honor of the Grant-Gewinner Jan Siemens nahm 2013 einen SignalResearchers, Transduction 2012) and Society Klaus – RajewskySTS) as well (José as Ruf nach Heidelberg an). Weitere ehrenvolle to many other researchers at the MDC. You Preise gingen an James Poulet (Paul-Ehrlich- can read more about these awards in the an- und Ludwig Darmstaedter-Nachwuchspreis, nex of this research report.

2012) und Klaus Rajewsky (José Carreras

X MDC Research Report 2014 INTRODUCTION

As a publicly funded research center, we are Award 2013, Ehrenmedaille der Gesellschaft aware of our duty to ensure the sustainability für Signaltransduktion) sowie an viele weite- of our work. This is why we place great impor- re Forscherinnen und Forscher des MDC. Sie - können im Anhang dieses Forschungsberichts cluding also school students. Currently, some mehr zu den Preisen lesen. 350tance y oungon training women young and menscientific are workingtalents inat the MDC to complete their doctoral theses. Als öffentlich gefördertes Forschungszentrum This takes place in the context of a structured - training provision within the Helmholtz Grad- beiten. Daher legen wir großen Wert auf die uate School “Molecular Cell Biology” and two Asehenusbildung wir uns des in wissenschaftlichen der Pflicht, nachhaltig Nachwuch zu ar- other international exchange programs at the ses und auch von Schülerinnen und Schülern. MDC. The training provision is structured into - zeit am MDC, um eine Doktorarbeit zu machen. metabolic diseases, cancer and immunology, RDiesund geschieht 350 junge inner Frauenhalb und einer Männer strukturierten sind der neurobiology,six fields of application: developmental cardiovascular biology, medical and Ausbildung durch die Helmholtz-Graduier- systems biology and bioinformatics as well tenschule „Molekulare Zellbiologie“ und zwei as structural biology. In training our doctoral weitere internationale Austauschprogramme candidates, we cooperate with universities am MDC. Die Ausbildung ist untergliedert in and research institutions in Europe, Israel and sechs Bereiche: Herz-Kreislauf- und Stoffwech- the USA. selerkrankungen, Krebs und Immunologie, Neurobiologie, Entwicklungsbiologie, Medizi- - nische Systembiologie und Bioinformatik so- tion starts much earlier: by means of a na- wie Strukturbiologie. Wir kooperieren bei der tionwideYet our work much-noticed in the field ofteacher providing education educa Doktorandenausbildung mit Universitäten und program (“Labor trifft Lehrer” – “Laboratory Forschungseinrichtungen in Europa, Israel und Meets Teacher”), we carry the working meth- den USA.

molecular biology into schools. This is sup- Unsere Bildungsarbeit setzt aber noch früher portedods and also results by the of Gläsernesour research Labor, in thewhich field was of an: Mit einem bundesweit viel beachteten Leh- initiated by the MDC and which will celebrate rerbildungsprogramm („Labor trifft Lehrer“) its 15th anniversary in 2014. With our activi- bringen wir die Arbeitsweise und die Ergebnis- ties we intend to contribute to a broader unse unserer molekularbiologischen Forschung in derstanding of science in society, for our work die Schulen. Dazu trägt auch das Gläserne Labor is made possible by the tax payer’s money. bei, das vom MDC initiiert wurde und im Jahr And today’s school students are the ones who 2014 15 Jahre alt wird. Wir wollen mit unse- will continue our work as the researchers of ren Aktivitäten zu einem breiteren Verständnis tomorrow, thus further strengthening our von Wissenschaft in der Gesellschaft beitragen, knowledge-based society. denn es sind die Steuerzahler, die unsere Arbeit ermöglichen. Und es sind die Schülerinnen und Schüler von heute, die morgen als Forscherin- Wishing you informative reading, nen und Forscher unsere Arbeit fortführen und so die Wissensgesellschaft stärken werden.

Ich wünsche Ihnen eine informative Lektüre

Prof. Dr. Walter Rosenthal Prof. Dr. Walter Rosenthal Scientific Director of the MDC Wissenschaftlicher Vorstand des MDC

MDC Research Report 2014 XI

Editorial Remarks

Redaktionelle Hinweise

his research report showcases the ieser Forschungsbericht gibt ei- work of the Max Delbrück Center for nen Überblick über die Arbeit des TMolecular Medicine (MDC) with a fo- DMax-Delbrück-Centrums für Mole- cus on the years 2012 and 2013. The MDC kulare Medizin (MDC) der Jahre 2012 und conducts biomedical basic research on the 2013. Das MDC betreibt biomedizinische Campus Berlin-Buch within the framework Grundlagenforschung auf dem Campus in of the Helmholtz Association, Germany’s Berlin-Buch und als Mitglied der Helm- largest research organization. The Max holtz-Gemeinschaft, Deutschlands größter Delbrück Center’s aim is to transfer the Forschungsorganisation. Ziel der Arbeit am MDC ist es, die Ergebnisse der Grundlagen- applications with a focus on the diagnosis forschung möglichst rasch in die klinische andfindings therapy as soonof diseases as possible as well into as on clinical pre- Anwendung zu überführen, sei es in der vention. The MDC cooperates closely with Diagnose, der Therapie oder der Präven- the Charité – Universitätsmedizin Berlin, tion von Krankheiten. Das MDC arbeitet dabei eng mit der Charité - Universitäts- school of the Free University and the Hum- medizin Berlin zusammen; letztere ist die boldti.e. the Universityjoint university in Berlin. hospital Both, and medicalCharité gemeinsame medizinische Fakultät und and MDC, are part of the Berlin Institute of Klinik der Freien Universität Berlin und Health (BIH) that was founded in 2013. der Humboldt-Universität zu Berlin. Beide, MDC und Charité, sind Teil des Ber liner Research at the Max Delbrück Center is di- Instituts für Gesundheitsforschung/Ber- vided into four main areas: cardiovascular lin Institute of Health, das 2013 gegründet and metabolic diseases, cancer, diseases of wurde. the nervous system and systems biology. Die Forschung am MDC ist in vier Bereiche book which in its main parts contains por- gegliedert: Herz-Kreislauf- und Stoffwech- traitsYou will of findindividual this division research reflected groups. in As this a selerkrankungen; Krebs; Erkrankungen des Nervensystems sowie medizinische Experimental and Clinical Research Cen- Systembiologie. Diese Gliederung spiegelt terfifth (ECRC) section which we have we includedrun together groups with of the sich im vorliegenden Bericht, der in seinem Hauptteil Porträts aller Arbeitsgruppen groups in this report. enthält. In einem fünften Bereich stellen Charité. All in all, you will find 70 research sich die Gruppen des Experimental and Cli- We also provide excellent support to our nical Research Centers (ECRC) vor, das das scientists by high-throughput technol- MDC gemeinsam mit der Charité betreibt. - cytometry, confocal and two-photon mi- pen in diesem Bericht. croscopy,ogy platforms electron for mass microscopy spectrometry, and magflow- Insgesamt finden Sie 70 Forschungsgrup netic resonance tomography, along with Das MDC bietet seinen Forscherinnen und bioinformatics services and advanced data Forschern exzellente Arbeitsbedingungen. Dazu gehört die Ausstattung mit Hoch- these technology platforms in this report, durchsatz-Technologieplattformen für Mas- too.modeling. You will find brief descriptions of konfokaler und 2-Photonen-Mikroskopie, Even though our work is focussed on ba- Elektronenmikroskopiesenspektrometrie, Durchflusszytometrie, und Magnetreso- sic biomedical research, we encourage our nanztomographie. Hinzu kommen Bioin- formatik und mathematische Modellierung. business-models if possible. To facilitate Kurze Beschreibungen der MDC-Technolo- technologyscientists to transfer transfer we theirhave findingsa program into at the MDC called Pre-GoBio which provides diesem Bericht. gieplattformen befinden sich ebenfalls in

XII MDC Research Report 2014 EDITORIAL REMARKS Photo: Guido Rottmann/MDC Photo:

MDC Campus in Berlin-Buch

initial support for scientists who then can Zwar konzentriert sich die Arbeit am MDC apply for other programs within the Helm- auf Grundlagenforschung, aber wir ermu- holtz Association or outside. We have dedi- tigen unsere Wissenschaftler dazu, ihre cated a section of this research report to Ergebnisse in die Wirtschaft zu übertra- gen. Für diesen Technologietransfer haben wir das MDC-Programm „PreGoBio“, das Atour the technology end of 2013, transfer we projects.hosted more than eine anfängliche Unterstützung für For- 350 PhD students in different programs. scherinnen und Forscher gewährt, die sich The part of the research report called Aca- dann um andere Förderungen innerhalb demics deals with our international net- der Helmholtz-Gemeinschaft oder darüber working programs, the Graduate School at hinaus bewerben können. Einige dieser the MDC, and offers overviews of awards that our scientists received and of the con- diesem Bericht vor. ferences in the past two years. Also includ- Technologietransferprojekte stellen wir in Ende 2013 gab es mehr als 350 Studie- that are realised through third-party fund- rende am MDC, die einen PhD-Abschluss ing.ed is a selection of our research projects anstreben. Der Berichtsteil mit dem Titel „Academics“ enthält Beschreibungen unserer internationalen Programme innerhalb information, our outreach activities and und außerhalb der Graduiertenschule so- In the Annex you will find, amongst other- wie einen Überblick über Preise und Aus- ships of our institute, e.g. the Berlin Insti- zeichnungen unserer Forscherinnen und tutethe various of Health, affiliations the German or strategic Centre partnerfor Car- Forscher. Außerdem gibt es Listen unse- diovascular Research “Deutsches Zentrum rer Konferenzen und Seminare sowie eine für Herz-Kreislauf-Forschung (DZHK)”. EU-Life, and the Helmholtz Association. We Drittmitteln gefördert wurden. Auswahl von Forschungsprojekten, die mit

also provide selected facts and figures that Aktivitäten unserer Pressestelle und der we hope you’ll find useful. Kommunikationsabteilung,Im Anhang finden Sie dann unterBeschreibun anderem- If you are missing any information in this gen unserer Zugehörigkeiten und strategi- report, please let us know. Our team will schen Partnerschaften, etwa Informationen be happy to provide this information and zur Helmholtz-Gemeinschaft, zum Berliner we will see whether we can include ad- Institut für Gesundheitsforschung/Berlin ditional sections in future reports. For any Institute of Health, dem Deutschen Zent- comments write to: rum für Herz-Kreislauf-Forschung (DZHK) [email protected] und zu EU-Life. Ausgewählte Zahlen und Statistiken haben wir überdies für Sie zu- sammengestellt.

Wenn Sie Informationen in diesem Bericht vermissen, geben Sie uns bitte Bescheid. Unser Team wird sie gerne für Sie bereit- stellen und wir werden prüfen, ob wir in künftigen Berichten weitere Kapitel anfügen. Ihre Fragen und Anmerkungen richten Sie bitte an: [email protected]

MDC Research Report 2014 XIII

EDITORIAL REMARKS

XII MDC Research Report 2014 Cardiovascular and Metabolic Disease

Coordinator: Norbert Hübner

MDC Research Report 2014

Cardiovascular and Metabolic Disease

Norbert Hübner

ardiovascular and metabolic disease duction to energy homeostasis. Together are the most common cause of death with the Hübner lab he studied the splicing Cworldwide. Most cases of cardiovas- regulator of titin, the RNA-binding protein cular diseases are likely to be the result RBM20. This work emphasized the role of of contributions from multiple genes, and post-transcriptional regulation in the eti- possibly complex epigenetic mechanisms ology of myocardial pathology. RBM20 inthat have yet to be discovered. Getting an teracts not only with titin but potentially experimental handle on these topics is a in a network of 30 genes previously linked to cardiomyophathy, ion-homeostasis, and increasing number of animal models – sarcomere biology. particularlymajor challenge strains for of the rats next and decade.mice – are An available, yet novel models will be needed Metabolic disturbances are emerging as

tools. Multifactorial diseases pose special but also neurodegenerative and oncological to convert findings into relevant clinical diseases.major risk However, factors not the only molecular of cardiovascular mechanisms that link the control of metabolism smallproblems number including of cases, finding and patients understand with a- to other biological systems remain poorly ingparticular complex profile, interactions extrapolating within from cells. a very All understood. The group of Thomas Willnow of these challenges will require new types is studying a novel group of cell surface of modeling and a thorough integration of receptors, the VPS10P domain receptors, bioinformatics, clinical and experimental which represent a unique class of signaling approaches. receptors with dual functions in the control of glucose and lipoprotein homeostasis in The molecular analysis of cardiovascular metabolic tissues and in the regulation of and metabolic functions at the cellular level neuronal viability in the brain. is a prerequisite for a mechanistic understanding of disease origins. It also provides Organogenesis involves multiple sequential a basis for the rational implementation of novel therapies to target cardiovascular differentiation, and morphogenesis, which ultimatelyevents such result as fate in the specification, development growth, of a program Fokus on key molecules and path- functional mature organ. This step-wise and metabolic disorders. Projects in the process relies on a coordinated and highly cardiovascular and metabolic systems. complex interplay of signaling events and ways involved in major processes of the transcriptional networks. Importantly, ab- For example, the group of Michael Got- errant developmental processes or a loss thardt studies the role of titin in the regu- of their coordination are common causes lation of contractility in cardiac, skeletal, of congenital defects of the cardiovascula- and smooth muscle. Specialized knock-in ture, liver, or pancreas. Learning to treat diabetes and various types of cardiovascular lesions will require a deeper knowledge themouse functional models areroles used of inthe conjunction many protein with - domainsbiomechanics of titin and that proteomics link mechanotrans to define- creatic progenitor cells. Deciphering the of the genetic profile of myocardial or pan

2 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

portant regulatory functions for circRNAs and provides evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to- tail splicing of exons, suggesting a previously unrecognized regulatory potential of coding sequences.

The group of Matthew Poy is interested in Whole mount immunofluorescence staining with Prox1 microRNA regulation in metabolic states. antibody shows the liver (lv) and both dorsal (dp) and They found that miR-184 is silenced in the ventral (vp) pancreatic buds in a E9.5 mouse embryo. pancreatic islets of insulin-resistant mouse adapted from Rodriguez-Seguel et al., Spagnoli, 2013 Their studies identify the targeting of Ago2 bymodels miR-184 and type as an 2 diabeticessential human component subjects. of the compensatory response to regulate ß molecular signatures of “stem cell-ness” of cell proliferation according to insulin sen- particular cell types of the heart, liver, and sitivity. They show that loss of Ago2 during pancreas, will allow us to isolate such cells for therapeutic purposes and possibly to relieved the regulation of miR-375-targeted instruct stem cells or progenitor cells to genes.insulin resistance blocked β cell growth and activate appropriate differentiation programs. Francesca Spagnoli is analyzing the Understanding diseases at the molecular transcriptome of hepatic and pancreatic and cellular level is an important step in progenitor cells through massive parallel elucidating mechanisms that ultimately sequencing isolated in vivo from mouse have their effects at the level of organs and embryos before and after the initiation organisms. But ultimately, the best model of organogenesis to reveal key regulatory of human disease remains man. Following genes that are differentially regulated in the the idea of translational medicine the MDC two populations and are possibly involved cooperates with the Charité through the in the pancreas- versus liver-fate decision. Experimental and Clinical Research Center The group of Salim Seyfried studies lateral (ECRC) encompassing novel imaging technology for whole-animals and man at the heart. They show that cardiac left-right Ultrahigh-Field Magnetic Imaging Center asymmetrydevelopmental is regulated processes by in Bmp, the zebrafisha growth (BUFF) run by Thoralf Niendorf pursuing factor of the TGF signaling pathway, and Nodal, and that Bmp signaling impairs the imaging. Our translational activities will be motility of cardiac progenitor cells. strengthendprojects including in the future cardiac through and metabolic the new Berlin Institute of Health (BIH) that was re- Genetic variability among individuals plays cently founded by MDC and Charité. In ad- an important role in the development and dition our program plays an eminent role in progression of cardiovascular disease. Ge- the German Center for Cardiovascular Re- netic and genomic studies in human popu- search and the National Cohort for proband and patient oriented research in the predic- are investigated by Young-Ae Lee to under- tion and prevention of cardiovascular and lations with chronic inflammatory disease metabolic diseases. that is often shared by other forms of com- monstand human the etiology cardiovascular of chronic and inflammation metabolic The challenge remains to understand the diseases. In a collaborative systems biology cardiovascular and metabolic disease risk, and Ferdinand LeNoble dicovered circular toregulatory link molecular genetic networksnetworks that with influence physi- RNAsapproach, systematically the groups ofand Nikolaus detected Rajewsky thou- ological data, and to identify key regulatory sands of well-expressed, stable circRNAs, nodes that can be targeted therapeutically often showing tissue/developmental-stage- to reduce the burden of cardiovascular and - metabolic diseases in patients.

specific expression. Analysis revealed im MDC Research Report 2014 3

CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Martin Falcke

Mathematical Cell Physiology

The group develops mathematical models and the waiting time distributions can be for cell biology from single molecules, via directly measured. We have successfully applied these methods to IP -induced Ca2+ protein complexes to cell level. Modelling 3 spiking and published the results (1, 2). serves to test and formulate biological hypotheses in a quantitative way as well We develop mathematical models of cell as to predict the behavior of complex morphodynamics in another line of research. Our model focusses in its current pathways and networks. Current proj- state on cells plated on a 2d substrate. Cell ects comprise basic methods of stochas- morphodynamics can then be recorded as tic modelling of cellular processes, cell the motion of the cell contour in a top down morphodynamics, motility and protrusion view on the cell. Two basic types of dynamics can be distinguished: dynamic genera- generation, IP -induced Ca2+-spiking and 3 tion and retraction of protrusions on the modelling of excitation contraction coupling time scale of minutes (3) and the motion of in cardiac myocytes. the leading edge of existing protrusions on the time scale of tens of seconds (4). We are interested in the mechanisms causing these types of morphodynamics. stochastic modelling addresses both of the problemsThe project of state developing space explosion basic methods and veri of- - tions for the existence of stable protrusions the exponential growth of the number of likeAs a lamellipodia. first step, we They investigated form in mesenchy the condi- (microscopic)fiability of models. states State of a spacestochastic explosion system is mal cell motion and then guide the cell with the number of stochastic elements. while moving on a 2d substrate, which may Since each state requires a differential also be a membrane, tissue or tumor sur- equation for its probability dynamics, that face in an organism during cell motion in entails also the explosion of the mathemati- the context of development, metastasis or cal effort to model these systems. Many wound healing. Lamellipodia maintain their equations entail large numbers of param- shape by the support of a network of actin eters, the values of which are usually not - the direction of motion. Cross-linking mol- ify these models. On top of these technical filaments preferentially aligned around known in vivo and hence it is difficult to ver- - edecules by branching turn the ensemble off existing of filaments ones. Filament into a experimentally.difficulties, the statesThe concept defined we in thisdeveloped math polymerizationstable network. generatesNew filaments leading are edge generat pro- isematical based onsense observable can usually states not instead. be identified It de- trusion. Polymerization is terminated by

like ‘closed’ or ‘open’ than based on mi- tip with a force dependent rate. We predict- croscopicfines states molecule rather by states. measurable Each functional function edproteins this force capping dependence the polymerizing which has filament by now state is a group of microscopic states. That entails mathematically a description by waiting time distribution functions instead leadingbeen experimentally edge membrane confirmed. may protect Binding them of transition rates and a Master equation of filament tips via linker molecules to the- as system of integro-differential equations instead of differential equations. But the from capping. Additionally, severing of fila advantages outweigh these technical dif- Wements found decreases (5) that the no filament stable lamellipodium number. exists for low branching rates, because the

ficulties: The number of states stays small 4 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Comparison of simulated leading edge motion (A,B) (3) with experimental results (C,D) during repetitive pro trusion generation of epithelial cells, Burnette et al., Nat. Cell Biol (2011). The black lines in B,D show leading edge velocity. The red line in B shows the network retrograde flow velocity, the light blue line the motion of the point where network stabilization by cross-linking is complete.

- by branching. That generation is self-am- plifying since the rate of branching is pro- cappingcreation ofand new severing. filaments The by branchingstable protru can- - sionnot compensate vanishes for for small filament cross-linking extinction rates, by portional to the number of existing fila - toments. severing In these and cells,capping, filaments bend thenand growform since the filaments grow long. That entails arcs.very long,That becometerminates very the floppy, existence susceptible of an large severing rates and renders the fila individual protrusion. Our results agree increasingments floppy, capping which rate. increases the capping very well with measurements including the rate. The filament density decreases with highly non-trivial existence of sharp peaks A larger external force decreases the capping rate via its force dependence. Further- velocities. We could also reproduce the ex- - perimentallyin the leading observed edge and increase retrograde in period flow ternal force, which decreases the severing and amplitude of protrusion generation rate.more, Hence, filaments applying shorten an externalto adapt forceto the may ex due to myosin inhibition (3). This excellent cause protrusion formation. agreement between measurements and the mathematical model very much supports This prominent role of external force moti- the idea of self-organized repetitive protru- vated us to investigate how cell velocity de- sion generation. pends on it. We explained the mechanism of the dynamic force-velocity relation recently (report 2012). Cells experience lasting forces in tissue and therefore, the sta- Selected Publications tionary force-velocity relation is relevant 1. K. Thurley, A. Skupin, R. Thul, M. Falcke, Fundamental properties of Ca2+ signals. there. This relation has not been measured, 1820, 1185 (2012). yet. We predict it to be distinctly different 2. BiochimicaG. Moenke, M.et BiophysicaFalcke, K. Thurley, Acta (BBA) Hierarchic - General stochastic Subjects modelling applied to intra- from the dynamic one, with larger velocity cellular Ca2+ signals. PLoS ONE 7, e51178 (2012). values, a single maximum for certain pa- 3. J. Zimmermann, M. Falcke, Formation of Transient Lamellipodia. PLoS one 9, rameter ranges and a linear decrease of the e87638 (2014). velocity towards the stall force (5). 4. A. Gholami, M. Enculescu, M. Falcke, Membrane waves driven by forces from actin 14, 115002 (2012). We were also interested in the mechanism 5. filaments.J. Zimmermann, NEW JM. PHYS Falcke, On the existence and strength of stable membrane and conditions for repetitive protrusion protrusions. New Journal of Physics 15, 015021 (2013). generation. In particular, we aimed at contributing to the discussion on the control of this process: Is the generation of each protrusion initiated by a signaling event upstream from nucleation promoting fac- Group Leader PhD tors or is it self-organized by the actin po- Dr. Martin Falcke Gregor Moenke lymerization machinery downstream from Janine Vierheller it. Our results demonstrate the possibility Scientist Juliane Zimmermann, of the latter (3). In that model, protrusions Janine Vierheller till end 2012

are generated due to filament generation MDC Research Report 2014 5

CARDIOVASCULAR AND METABOLIC DISEASE Photo: Uta Wrackmeyer/MDC Uta Photo:

Michael Gotthardt

Neuromuscular and Cardiovascular Cell Biology

Our long-term goal is to establish how changes that regulate cardiac alternative splicing and in biomechanics are translated into molecu- to identify various regulatory, metabolic, and lar signals and vice versa. Specifically, we are structuraltheir target proteins profiles. that This determine approach cardiovashas led us- interested in understanding how increased cular physiology and adaptation. In addition filling of the cardiac ventricle leads to improved to improving our knowledge of splicing as a contraction (Frank-Starling mechanism of the basis for cardiac adaptation, we translate our - heart), how alternative splicing relates to dia- tic options for patients with cardiovascular stolic heart failure, and how exercise or immo- diseasefindings towardsemphasizing developing diastolic novel dysfunction therapeu bilization change skeletal muscle growth. We with titin as a main contributor to the elastic focus on titin, the largest protein in the human splice active compounds, which are currently body and the multifunctional coxsackie-adeno- testedproperties in animal of the models heart. of We human have disease identified as a virus receptor (CAR). To lay the groundwork for prerequisite for future clinical studies. the in vivo analysis of titin’s multiple signaling, Titin based mechanotransduction elastic, and adaptor domains, we have gener- Michael Radke, Thirupugal Govindarajan, ated various titin deficient mice (knock-in and Christopher Polack, Douaa Megahed conditional knockout animals) and established a Titin is a unique molecule that contains elas- tissue culture system to study titin’s muscle and tic spring elements and a kinase domain, as well as multiple phosphorylation sites. There- non-muscle functions. We utilize a combina- fore, it has been frequently speculated that tition of cell-biological, biochemical, and genetic tin could act as a stretch sensor in muscle. So tools to establish titin as a stretch sensor con- far it has remained unknown how the stretch verting mechanical into biochemical signals. signal is processed, i.e. how the mechanical stimulus stretch is converted into a biochemi- Using a comparable loss of function approach cal signal. we have created a conditional knockout of the To investigate the stretch signaling pathway, coxsackie-adenovirus receptor to demonstrate we apply mechanical strain in vivo (plaster cast for skeletal muscle; aortic banding for that CAR is crucial for embryonic development the heart) and in tissue culture (cultivation and determines the electrical properties of the of primary cells on elastic membranes). The heart. resulting changes in the proteome of our titin

Cardiac alternative splicing are used to map the mechanotransduction Michael Radke, Thirupugal Govindarajan, pathway.kinase and spring element deficient animals Martin Liss, Vita Dauksaite, Florian Hinze, Piotr Grabokwski Sarcomeric and non-sarcomeric functions of titin - Thirupugal Govindarajan, Franziska Ru- tialAlternative requirements splicing of isembryonic a major mechanism and postnatal to dolph, Claudia Fink, Joanna Kaldrack physiology.adjust the cardiacWe use a proteome naturally occurring to the differen splice Overlapping titin molecules form a continu- - ogy, and in silico analysis to identify factors with the multiple binding sites for sarco- deficient rat strain, high throughput technol ous filament along the muscle fiber. Together

6 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Schematic diagram of the mechanical and electrical connection between cardiomyocytes. Titin (black) forms a continuous filament system along the muscle fiber. Its isoform expression is regulated by RBM20 to adapt the elastic properties of the myofilament. The proper transition of myofilaments between cells and their electrical coupling is regulated by the coxsackie and andenovirus receptor (CAR), which determines the localization of connexins and the attachment of filaments to the plasma membrane. Both titin and CAR have been linked to diverse cardiac diseases such as cardiomyopathy, diastolic dysfunction, and arrhythmia.

meric proteins, this makes titin a suitable blueprint for sarcomere assembly. The use Patents / Patent applications of transgenic techniques does not only allow Polynucleotides for use in medicine. Max-Delbrück-Centrum für Molekulare Medizin us to address the function of titin’s individual Berlin-Buch Dec, 29 2010: WO 2010/149332 domains in sarcomere assembly, but also to Inducible site-directed mutagenesis through conditional gene rescue. Max-Delbrück- Centrum für Molekulare Medizin Berlin-Buch Oct, 21 2004: WO 2004/090131 follow sarcomere assembly and disassembly LDL Receptor Signaling Pathways - US Patent Number 6,428,967. The University of - Texas, 2001 standing the structural and biomechanical Selected Publications functionsusing fluorescently of titin will tagged help elucidate proteins. theUnder Marsman R.F.; Bezzina C.R.; Freiberg F.; Verkerk A.O.; Adriaens M.E.; Podliesna S.; pathomechanisms of various cardiovascular Chen C.; Purfuerst B.; Spallek B.; Koopmann T.T.; Baczko I.; Dos Remedios C.G.; George diseases and ultimately aid the development A.L.; Bishopric N.H.; Lodder E.M.; de Bakker J.M.; Fischer R.; Coronel R.; Wilde A.A.; of suitable therapeutic strategies. cardiac conduction and arrhythmia vulnerability in the setting of myocardial ischemia. Recently titin has been proposed to perform GotthardtJournal of M.;the RemmeAmerican C.A. College Coxsackie of Cardiology and adenovirus 18;63(6):549-59 receptor (CAR) (2014) is a modifier of non-muscle functions following its localization to mitotic chromosomes and cleavage H.; Lincoln S.E.; Parrish A.M.; Dauksaite V.; Vakeel P.; Klaassen S.; Gerull B.; Thierfelder GuoL.; Regitz-Zagrosek W.; Schafer S.; Greaser V.; Hacker M.L.; T.A.; Radke Saupe M.H.; K.W.; Liss Dec M.; G.W.; Govindarajan Ellinor P.T.; T.; MacRae Maatz H.;C.A.; Schulz furrows as well as the D-titin related chromo- Spallek B.; Fischer R.; Perrot A.; Ozcelik C.; Saar K.; Hubner N.; Gotthardt M. RBM20, a some abnormalities and aneupoidity found gene for hereditary cardiomyopathy, regulates titin splicing. Nature Medicine 18 (5): in Drosophila melanogaster. Our preliminary 766-773 (2012) data indicate that titin is present in virtually da Silva Lopes K.; Pietas A.; Radke M.H.; Gotthardt M. Titin visualization in real time reveals an unexpected level of mobility within and between sarcomeres. Journal of every cell-type tested and our knockout of Cell Biology 193 (4): 785-798 (2011) titin’s M-band region displays a defect in im- Shi Y.; Chen C.; Lisewski U.; Wrackmeyer U.; Radke M.; Westermann D.; Sauter M.; plantation. Currently we are extending our Tschoepe C.; Poller W.; Klingel K.; Gotthardt M. Cardiac deletion of the coxsackievirus- adenovirus receptor abolishes coxsackievirus b3 infection and prevents myocarditis analysis to the titin dependent regulation of in vivo. Journal of the American College of Cardiology 53 (14): 1219-1226 (2009) the cell-cycle. Lisewski U.; Shi Y.; Wrackmeyer U.; Fischer R.; Chen C.; Schirdewan A.; Juettner

regulates cardiac conduction and cell-cell communication. Journal of Experimental Functional analysis of the Cox- R.;Medicine Rathjen 205 F.; Poller(10): 2369-2379 W.; Radke M.H.; (2008) Gotthardt M. The tight junction protein CAR sackie-Adenovirus Receptor Uta Wrackmeyer, Joanna Kaldrack, Meghna Thakkar, Anne Merks CAR was cloned as a receptor used by adeno- and coxsackievirus to enter cells but Group Leader Douaa Megahed its physiological role has remained obscure. Prof. Dr. Christopher Polack We have generated both tissue culture and Michael Gotthardt Franziska Rudolph animal models to study CAR’s function in Meghna Hiren Thakkar - PostDoc Anne Merks* myopathy, and basic cellular processes Dr. Vita Dauksaite suchcardiac as remodeling,endocytosis inflammatoryand cell-cell contact cardio Technical Assistants formation. Our data suggest a critical role of Dr. Michael Radke Janine Fröhlich CAR in the conduction of electrical signals Dr. UtaThirupugal Wrackmeyer Govindarajan Beate Goldbrich-Hannig from the atria to the cardiac ventricle. The Carmen Judis PhD Mathias Pippow has enabled us to completely block the en- Claudia Fink tryinducible of coxsackievirus heart-specific into knockout cardiomyocytes of CAR Piotr Grabowski Secretariat - Florian Hinze Sylvia Olbrich diomyopathy. Developing inhibitors of CAR Joanna Kaldrack function,and prevent we aimall signs to improve of inflammatory cardiac remod car- Martin Liss eling in myocardial disease. * jointly with Daniela Panáková

MDC Research Report 2014 7

CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Ferdinand le Noble

Angiogenesis and Cardiovascular Pathology

Vascular network remodeling and the addressed tip cell formation and angiogenic sprout guidance using a genetic approach formation of new blood vessels – angiogen- and in vivo imaging techniques in mouse, esis and arteriogenesis – play an important role in the pathophysiology of ischemic like 4 (Dll4) - Notch signalling plays a criti- cardiovascular disease and cancer. My lab caland rolezebrafish. in the We differentiation demonstrated of that endothe Delta-- lial cells into tip cells in response to VEGF aims at generating novel genetic insights in the regulation of vascular development VEGF receptor-1, Flt1, in tip cell formation that can translate into therapeutic strate- gradients. Recently, we defined the role of Flt1 produced by sprouting vessels acts, in gies. We focus on (1) differentiation and ausing Notch-dependent zebrafish. We manner, showed as that a negative soluble guidance of angiogenic vessels; (2) im- regulator of tip cell formation and sprout printing of arterial-venous identity in blood guidance. Soluble Flt1 mediated sprout vessels by neural guidance genes and guidance is a process that involved interaction with the nervous system. This interac- hemodynamic factors; (3) native collateral tion determines the distribution of soluble formation and vascular adaptation including Flt1 surrounding the sprout, and controls arteriogenesis and angiogenesis in arterial the direction of sprout expansion. We are occlusive diseases (stroke, heart infarct). currently investigation the role of Flt1 at the neuro-vascular interface in the context We address vascular development using of vessel guidance and neurogenesis. an integrative molecular and physiological approach in (transgenic) zebrafish, and Arteriogenesis and Ischemic Disease mouse models. Our signalling pathways of interest include Dll4-Notch, VEGF-recep- Arteries form highly branched networks tor-1, (post) transcriptional regulators of that ramify throughout the body. Most ar- these pathways, and mechanosensing of teries branch into progressively smaller diameter arterioles that in turn branch into hemodynamic factors, blood flow. even smaller sized capillaries, which supply oxygen and nutriments to tissues and organs. However, some arterioles directly connect with other arterioles, usually from Tip Cell biology and Vessel a neighboring arterial tree, and form so- Guidance called native collateral arteries. The native

Recent studies of vascular network devel- conduit for blood supply to tissues after oc- clusioncollateral of one circulation of the feed represents arteries. Increases a major novel aspects of angiogenesis crucial to - generateopment ina functional, the embryo and identified stable branched several tory pathways upon occlusion trigger the vascular network. These aspects include: outwardin shear stressremodeling and activation of these ofpre-existing inflamma the differentiation and guidance of endo- bypasses. Despite the clinical importance, thelial tip cells in angiogenic vessels, and - the formation of functional branches. We phogenesis and native collateral network factors influencing arterial branching mor

8 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

size remain poorly characterized. In the endothelial miR-30a opposes Dll4 expres- mouse brain, native arteriolar collateral sion and accordingly promotes angiogenic networks appear as distal end-to-end arte- cell behavior and arteriolar branching, in a riolar anastomoses. We recently found that the density of these collaterals is highest miR-30a on branching could be traced to an at time of birth, after which collateral den- effectNotch ondependent tip-cell differentiation. manner. The influence We are cur of- sity decreases with an increase of age. The rently evaluating whether miR-30 mimetic Dll4-Notch signaling pathway is essential may be used to augment angiogenesis in for embryonic vascular development and ischemic settings. While miRNAs can directly regulate expression of most mRNAs and vessel sprouting. Using genetic loss in a diverse range of biological functions, andmediates gain of specification function approaches of arterial in mice identity we surprisingly little is know about how and if found that Dll4-Notch signaling restricts mRNAs can escape regulation by a miRNA. pial collateral artery formation by modulat- A recently discovered mechanism for miR- ing arterial branching morphogenesis during embryogenesis and adult Dll4+/- mice is based on target sites acting as decoys or showed increased pial arterial collateral NA removal in a sequence specific manner numbers. The Dll4-Notch pathway thus acts - as a genetic determinant of native collateral lymiRNA exploring sponges. their impact We recently on tissue identified morpho a- genesisset of sponges and function. in zebrafish and are current be evaluated in experimental stroke models,number. and humanThe impact stroke of cohorts these findings in collabo will- ration with the Center for Stroke Research Berlin (CSB). - ing blood contribute to vascular branch- Selected Publications ingHemodynamic morphogenesis forces during exerted embryogenesis by flow and trigger collateral arteriogenesis in the Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, Gregersen LH, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C, Le Noble adult. Using special in vivo imaging tech- F - niques and systematic analysis of blood tory potency. Nature 495, 333-8. , and Rajewsky N. (2013). Circular RNAs are a large class of animal RNAs with regula Cristofaro B, Shi Y, Faria M, Suchting S, Leroyer AS, Trindade A, Duarte A, Zovein AC, networks and collateral arteries we were Iruela-Arispe ML, Nih LR, Kubis N, Henrion D, Loufrani L, Todiras M, Schleifenbaum J, ableflow characteristicsto identify a unique in developing biophysical vascular sig- Gollasch M, Zhuang ZW, Simons M, Eichmann A, and Le Noble F. (2013). Dll4-Notch nal essential for triggering arterial identity signaling determines the formation of native arterial collateral networks and arterial function in mouse ischemia models. Development 140, 1720-9. gene expression, and outward remodeling. - Krueger, J., Liu, D., Scholz, K., Zimmer, A., Shi, Y., Klein, C., Siekmann, A., Schulte-Merker, S., Cudmore, M., Ahmed, A., and Le Noble, F. (2011). Flt1 acts as a negative regulator - - Expression profiling of flow activated col ment 138, 2111-2120. lateral networks identified strong regula of tip cell formation and branching morphogenesis in the zebrafish embryo. Develop Klein, C., Mikutta, J., Krueger, J., Scholz, K., Brinkmann, J., Liu, D., Veerkamp, J., Siegel, intion mice of Gja5 resulted and members in reduced of the native bradykinin collat- D., Abdelilah-Seyfried, S., and Le Noble, F. (2011). Neuron navigator 3a regulates liver signaling pathway. Genetic ablation of Gja5 that bradykinin receptors act as positive organogenesisBuschmann, I., duringPries, A., zebrafish Styp-Rekowska, embryogenesis. B., Hillmeister, Development P., Loufrani, 138, 1935-1945.L., Henrion, D., Shi, modulatorseral number of and arteriogenesis arteriogenesis. in mice We findand Y., Duelsner, A., Hoefer, I., Gatzke, N., Wang, H., Lehmann, K., Ulm, L., Ritter, Z., Hauff, rats involving a contribution of peripheral Le Noble, F. (2010). Pulsatile shear and - immune cells, thereby linking hemodynam- P.,esis. Hlushchuk, Development R., Djonov, 137, 2187-2196. V., van Veen, T., and Gja5 modulate arterial identity and remodeling events during flow-driven arteriogen

icPost-transcriptional conditions with inflammatory regulators: pathways. miRNAs and sponges Group Leader Technical Assistants Since the Dll4-Notch signaling pathway is Prof. Dr. Ferdinand le Noble evolutionarily highly conserved, we hypoth- Janine Mikutta esized the existence of microRNA families Scientist Katja Meier Dr. Janna Krüger vessel branching. The miR-30 family mem- SecretariatAnja Zimmer berscapable are ofhighly targeting conserved Dll4 between and influencing human, PhD Regina Philipp Giulia Caglio matching with the 3’UTR of Dll4 mRNA in Anna Klaus Other thesemouse, species. and zebrafish Using loss with and perfect gain basepair of func- Alina Klems Raphael Wild

tion approaches in zebrafish, we found that MDC Research Report 2014 9

CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Ingo Morano

Molecular Muscle Physiology

Contraction of all muscle types is elicited by N- and C-terminal domains of es- increasing myoplasmic Ca2+ and interaction sential myosin light chains (ELC) of Type II myosins with thin (actin) filaments modulate cardiac functions Janine Lossie, Clemens Köhnke, of the sarcomere. In striated muscle, Ca2+ Petra Domaing bind to troponin C, which turn the thin filament “on”, allowing myosin actin interaction We modelled the missing 46 N-terminal and force generation. We are studying the amino acid of the ELC to the contemporary actin-myosin-S1 complex (Figure 1). The functional roles of subunits of key proteins N-terminus of ELC showed a rod-like 91 Å of Ca2+ handling and force generation, i.e. structure being long enough to bridge the the L-type Ca2+ channel and type II myosins gap between the ELC core of myosin-S1 and the appropriate binding site of the ELC on in muscle. Any change of these proteins by mutation, differential gene expression, could be inhibited by a peptide-competition alternative splicing of the transcripts, post- approachthe actin with filament. synthetic ELC/actin peptides interaction compris- translational modification, or sex modulate ing the most N-terminal amino acids 1-15, which bind to the appropriate ELC binding muscle function. Understanding muscle site of actin. Recordings of cardiac func- contraction regulation at the molecular and tion during peptide competition could then functional levels provides the opportunity direct into a possible physiological role of to develop new therapeutic strategies for ELC/actin interaction. To test the hypothesis that expression of N-terminal ELC pep- the treatment of cardiovascular and skeletal tides could modulate intrinsic contractility muscle dysfunction. of the whole heart, we generated transgenic rats (TGR) that overexpressed minigenes encoding the N-terminal 15 amino acids of human atrial or ventricular ELC isoforms in cardiomyocytes. Systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human ELC peptides.

We investigated structural properties and protein-protein interactions of a recombinant

- tatedhead-rod (M149V, fragment E143K, of ratA57G, cardiac E56G, β-myosin R154H) heavy chain (MYH) and normal or five mu

10 MDC Research Report 2014 Figure 1. 3D-model of the actomyosin complex. (A) Gauss–Connolly surfaces are used to visualize the molecular complex. Actin units are coloured orange, brown, and yellow. The myosin S1 head (green), the regulatory light chain (red), the shortened essential light chain (white), the 46 N-terminal residues of A1 (blue), and clusters of acidic residues on actin (pink) are shown. (B) More detailed view on the potential interaction of N-terminal APKK of A1 with acidic residues on actin. Ionic interactions between lysine residues (K3 and K4) of APKK and acidic residues (E361 and E364) on actin were assumed.

vealed that the isometric force generation A57G, and R154H mutated hVLC-1/MYH - hVLC-1 forms. Affinity of M149V, E143K,- sure development of perfused hearts from pared with the normal hVLC-1/MYH complex TgME56Gof skinned (8.3mN/mm2fibers and left ventricularand 66.2mmHg, pres interaction.complexes wereIn particular significantly the E56G lower mutation com - pared with TgMhVLC-1 (13.9mN/mm2 and Disturbed myosin binding of mutated hVLC-1 respectively) were significantly lower com- mayinduced provide an about a pathomechanism 30fold lower MYH for affinity.the de- ings provide valuable insights into the basal velopment of hypertrophic cardiomyopathy. function80.0mmHg, of hVLC-1 respectively). in the heartThese and novel the find development of familial hypertrophic cardio- We therefore hypothesized that hVLC-1 myopathy relatedhVLC-1 mutations. with weak myosin binding decreases myosin stiffness and deteriorates myosin and cardiac muscle functions. To test this hy- The functional role of the ahnak pothesis, we generated transgenic mouse protein family in skeletal muscle strains expressing hVLC-1 (TgMhVLC-1) fibers or hVLC-1E56G (TgME56G) in the heart. Hannelore Haase, Frank Suhr Steffen Lutter Echocardiographic analysis revealed no changes in cardiac dimension of TgME56G. In the heart, ahnak1 is located at the sar- Protein expression evaluated by SDS-PAGE colemma and T-tubuli of cardiomyocytes and Western-blot analysis showed that the indirectly associated with the voltage- de- portion of hVLC-1 or hVLC-1E56G expres- pendent L-type Ca2+ channel (L-VDCC) via sion was 39.1% and 40.7%, respectively, of L-VDCC current. AHNAK functions and and in vitro motility assays showed that subcellularits β2-subunit localization (ß2) where in skeletal it stimulates mus- stiffnesstotal VLC-1 and in actin TgM sliding myofibrils. velocity Laser of myo trap- cle are unclear. Co-localization of ahnak1 sin prepared from TgME56G (1.67pN and and ahnak2 with vinculin clearly demonstrates that both proteins are components decreased compared with myosin from of the costameric network. In contrast, TgMhVLC-12.3µm/s, respectively) (1.25pN and were 1.7µm/s, significantly respec- no ahnak expression was detected in the tively). Furthermore, investigation with T-tubule system. A laser wounding assay

and isolated perfused hearts of TgM re- ahnak1 is not involved in membrane re- chemically skinned cardiac muscle fibers with ahnak1-deficient fibers suggests that

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CARDIOVASCULAR AND METABOLIC DISEASE

Figure 2. ERα-overexpression in cardiomyocytes accelerates both angiogenesis and lymphangiogenesis predominantly in the peri-infarct areas in both sexes. A-B: Representative images of H&E-stained paraffin sections of LV tissues from WT- and ERα-OE mice after myocardial infection (MI). ERα-OE mice hearts showed an increased occurrence of vascular-like structures in the peri-infarct area. 20x magnification, scale bar 100µm. C-F: Representative immunofluorescence photographs of LV tissues 2 weeks after sham and MI operation from WT- (C & E) and ERα-OE (D & F) mice with CD31 and LYVE-1 staining (scale bar 100µm). For a better illustration of CD31 and LYVE-1 signals, higher magnification of the LV areas with strongest signals, i.e. in the peri-infarct area, have been shown. CD31-positive vessels (green), LYVE-1 positive vessels (red), nuclei (DAPI, blue).

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pair. Using atomic force microscopy (AFM), - - - tectsERα andfemale subjected mouse them cardiomyocytes to myocardial from in we observed a significantly higher trans thefarction. sequelae Our ofstudy ischemia indicates through that inductionERα pro proteinsverse stiffness in skeletal of ahnak1-/- muscle. fibers.Interestingly, These of neovascularization (Figure 2) in a para- humanfindings muscle suggest biopsy novel specimens functions ofobtained ahnak from patients with limb girdle muscular together may contribute to the attenuation dystrophy showed that ahnak1 lost its sar- ofcrine cardiac fashion remodelling. and impaired fibrosis, which colemmal localization and appeared in the extracellular space in both muscular dystrophies.

Identification of adipocyte-derived cardiodepressant factor and their receptors Christiane Woischwill, Karin Karczewski, in collaboration with Valeria Lamounier- Zepter, Dresden

The causal relationship between obesity and heart failure is broadly acknowledged; however, the pathophysiological mechanisms involved remain unclear. We observed that adipose tissue exerts highly potent activity with an acute depressant effect directly on cardiomyocytes, which may well contribute to increased heart failure risk in Selected Publications overweight patients. Through mass spectrometry and immunoblotting, we have Lättig-Tünnemann G, Prinz M, Hoffmann D, Behlke J, Palm-Apergi C, Morano I, Herce HD, Cardoso MC. (2011). Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides. Nat. Commun 2:453. FABP4. FABP4 acutely depressed shorten- Lamounier-Zepter V, Look C, Alvarez J, Christ T, Ravens U, Schunck W-H, Ehrhart-Bornstein ingidentified amplitude this as cardiodepressant well as intracellular factor sys as- M, Bornstein SR, Morano I (2009). Adipocyte fatty acid-binding protein suppresses cardio- tolic peak Ca2+ in a dose-dependent manner myocyte contraction: a new link between obesity and heart disease. Circ. Res 105:326-34. in isolated rat cardiomyocytes. Aydt EM, Wolff G, Morano I (2007). Molecular modelling of the myosin-S1(A1) isoform. J Struct Biol 159:158-63. Haase H, Dobbernack G, Tünnemann G, Karczewski P, Cardoso C, Petzhold D, Schlegel WP, Lutter S, Pierschalek P, Behlke J, Morano I. (2006). Minigenes encoding N-terminal domains Role of estrogen receptor alpha in of human cardiac myosin light chain-1 improve heart function of transgenic rats. FASEB J sexually dimorphic cardioprotec- 20:865-73. tion against myocardial injury Mahmoodzadeh S, Leber J, Zhang X, Jaisser F, Messaoudi S, Morano I, Furth PA, Dworatzek Shokoufeh Mahmoodzadeh, Petra Sakel, in col- - laboration with Vera Regitz-Zagrosek, Berlin E,cardial Regitz-Zagrosek infarction. J V.Cell (2014). Sci Ther Cardiomyocyte-specific 2014, 5:1 (in press). estrogen receptor alpha increases angiogenesis, lymphangiogenesis and reduces fibrosis in the female mouse heart post myo Heart failure is associated with changes in myocardial architecture leading to cardiac dysfunction. Females have an improved heart failure prognosis and better survival. Group Leader Animal studies showed that E2 protects the Prof. Dr. Ingo Morano Janine Lossie - Romie Siegert gest cardioprotective effects of the female Scientist Ines Pankonien heart from ischemic injury. These data sug Dr. Hannelore Haase However, the mechanisms how E2 regulates Dr. Clemens Koehncke Technical Assistants myocardialsex hormone, functions estrogen have (17β-estradiol, not yet been E2). ful- Dr. Shokoufeh Mahmoodzadeh Petra Domaing ly elucidated. E2 exerts its effects genomi- Dr. Frank Suhr Karin Karczewski cally and non-genomically by activating at Dr. Christiane Woischwill Steffen Lutter Petra Sakel - Graduate and Undergraduate cytesleast in two the E2 setting receptors, of myocardial ERα and ischemia, ERβ. To Philipp Baumert Secretariat weinvestigate recently thegenerated role of transgenic ERα in cardiomyo mice with Oriane Larcheveque Manuela Kaada

cardiomyocyte-specific overexpression of MDC Research Report 2014 13

CARDIOVASCULAR AND METABOLIC DISEASE Photo: Peter Himsel/MDC Peter Photo:

Daniela Panáková

Electrochemical Signaling in Development and Disease

Physiological cues that include electrical and ionic impulses or metabolic interme- PKG. Next, Wnt signaling could interfere diates have been long implicated in the lation via specific kinases like PKA, PKC or- orchestration of organ development during brane microdomains, and thus modulate Cawith2+ current LTCC localization through LTCC. to specific Alternatively, mem early embryonic patterning. While much is Wnt signaling might interfere with LTCC known about genetically encoded developmental pathways, the mechanisms by molecular mechanism by which Wnt11 reg- which the ions and small molecules interact ulateschannel LTCC trafficking. at a subcellular In order resolution, to define thewe with traditional morphogenetic signals are embryonic hearts. poorly understood. We use state-of-the-art use cell-based models as well as zebrafish technologies to explore the role of elec- Characterization of the trochemical and other physiologic stimuli components of the Wnt non- canonical transduction pathway during zebrafish embryogenesis. We focus involved in the L-type Ca2+ channel on elucidating the interactions between attenuation calcium signaling and Wnt11 non-canonical signaling, a major developmental pathway biological effects of Wnt signaling are me- regulating tissue morphogenesis, during The specificity and a wide range of diverse cardiovascular development. Wnts to their corresponding receptors, Friz- zleds.diated It via has binding been shown and affinitythat Wnt11 of distinct interacts with Fzd7 genetically as well as physi- Molecular bases of interactions cally. A crucial step in Wnt signaling is the between Wnt non-canonical path- translocation of Dishevelled (Dsh) from the way and L-type Ca2+ channel cytoplasm to the plasma membrane, relay- ing the Wnt signal to various downstream We have demonstrated that Wnt11 pat- effectors. Dsh has been implicated so far in terns intercellular electrical coupling in the all known aspects of Wnt signaling, canoni- myocardial epithelium through effects on cal and non-canonical. The N-terminal DIX transmembrane Ca2+ conductance medi- domain regulates Wnt canonical signaling, ated via the L-type calcium channel (LTCC). while the central PDZ and C-terminal DEP The cardiac LTCC belongs to a family of volt- domains of Dsh are responsible for the spe- age-gated Ca2+ channels that are essentially 2+ ions into cell polarity (PCP) pathway. In addition, the the cytoplasm. Even though channel open- DEPcific domain activation has of been the shown non-canonical, to have the planar abil- ingthe ismajor primarily point regulated of entry byfor membrane Ca po- ity to activate Wnt/Ca2+ signaling. It has been tential, many other molecular mechanisms recently reported that a polybasic stretch have been implicated in the modulation of within the DEP domain of Dsh is required for LTCC function. Channel phosphorylation is Dsh recruitment by Fzd to activate PCP sig- the most commonly explored way of LTCC activation and Wnt signaling might be a the Wnt transduction machinery in the pro- plausible pathway to test for LTCC modu- cessnaling. of attenuationWe aim to define of L-type the Carequirement2+ channel byof

14 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Wnt11 regulates Ca2+ transient amplitudes in cardio- Cellular rearrangements drive cardiac chambers formation: myocytes: a,b, Colour maps of Ca2+ transient amplitudes from a,b,c, Vertices analysis in wildtype hearts stained with the membrane wildtype hearts (a) and Wnt11 morphants (b). Colour code de- marker, zn8 at 26 hpf (a), at 54 hpf (b) and at 72 hpf (c). Blue dots picts Ca2+ transient amplitudes in fluorescent ratio units (F340/ mark 3-way vertices; red dots mark n-way vertices (n≥4). d, Graph F380). Square indicate automated ROIs for measurements showing the reduction of n-way vertices over time, between 26 hpf averaged in c. c, Averaged Ca2+ transients from ROIs in a,b. and 72 hpf. hpf=hours of post fertilization, scale bar=10μm.

measuring the changes in intracellular Ca2+ Selected Publications

loss-of-function experiments. Panáková D., Galindo C.L., Zhong L., Shin J.T., Coy S.M., Kelly A.E., Roden D.M., Lim C.C., and MacRae C.A. (2014). Differen- fluxes in the series of Fzd and Dsh gain- and Becker,tial activation J.R., Chatterjee of natriuretic S., Robinson peptide T.Y., receptors Bennett modulate J.S., cardiomyocyte proliferation during development. Development 141, 335-45. Wnt non-canonical signaling patterns cardiac form Wang, J.H., Panáková, D., Kikuchi, K., Holdway, J.E., Gemberling, M., Burris, J.S., Singh, S.P., Dickson, A.L., Lin, Y.F., Sabeh, M.K., Werdich, A.A., Yelon, D., Macrae, C.A., and Poss,

The profound morphogenetic changes that genetic cardiomyocyte depletion. Development 138, 3421-3430.

K.D. (2011). The regenerative capacity of zebrafish reverses cardiac failure caused by lead to the looped and chambered verte- Becker, J.R., Deo, R.C., Werdich, A.A., Panáková, D., Coy, S., and MacRae, C.A. (2011). Hu- brate heart occur concomitantly with the man cardiomyopathy mutations induce myocyte hyperplasia and activate hypertrophic - Panáková, D.*, Werdich, A.A.*, and MacRae, C.A. (2010). Wnt11 patterns a myocar- lular communication between cardiomyo- pathways during cardiogenesis in zebrafish. Disease Models & Mechanisms 4, 400-410. dial electrical gradient through regulation of the L-type Ca(2+) channel. Nature 466, cytes.establishment However, and the refinementmolecular and of intercelcellular 874-878. mechanisms that underlie the patterning Panáková, D. *, Sprong H. *, Marois E., Thiele C., and Eaton, S. (2005). Lipoprotein of functional myocardial syncytium dur- particles are required for Hedgehog and Wingless signalling. Nature 435, 58-65. ing early embryonic development are not * contributed equally thoroughly explored. We investigate how

from the linear heart tube. Although cardiac musclethe two-chambered cells are of mesodermal zebrafish heart origin, forms detailed studies demonstrated that embryonic Group Leader Graduate and Undergraduate Dr. Daniela Panáková Ines Uhlenhut characteristics. Thus, we hypothesize that thezebrafish mechanisms myocardium that drive retains epithelial its epithelial tissue Scientist Research Assistants remodeling might play an important role Dr. Tareck Rharass Alexander Meyer during cardiac chamber formation. We were able to show that cardiomyocyte rearrange PhD Secretariat within the myocardial epithelium to form Marie Swinarski Petra Haink two chambers. We are currently investigat- Laura Bartolini ing how Wnt non-canonical pathway guides Anne Merks* Michael Gotthardt rearrangements during this process. the junctional remodelling and the cellular Kitti Csályi * jointly with

MDC Research Report 2014 15

CARDIOVASCULAR AND METABOLIC DISEASE Photo: Steffen Weigelt/MDC Steffen Photo: Photo: David Ausserhofer/MDC Photo:

Walter Enno Rosenthal Klußmann

Anchored Signalling

Vasopressin-mediated water water reabsorption from primary urine and reabsorption (Fig. 1). Antidiuretic hormone (Arginin-vasopres- through this fine-tunes water homeostasis - Diabetes insipidus is associated with a mas- stasis. Its binding to vasopressin V2 recep- sive loss of hypotonic urine and polydipsia torssin, AVP)on the fine surface tunes of bodyrenal collecting water homeo duct (massive thirst). Loss of function muta- principal cells leads to a raise of the level tions in the vasopressin V2 receptor or of the second messenger cAMP, which ac- AQP2 genes cause congenital nephrogenic tivates protein kinase A (PKA). PKA phos- diabetes insipidus (NDI), lithium treatment phorylates the water channel aquaporin-2 of depression leads to acquired nephro- (AQP2) and thereby induces its redistri- genic diabetes insipidus; Elevated levels bution from intracellular storage vesicles of AVP with excessive water retention can into the plasma membrane. The membrane be found in heart failure, liver cirrhosis or insertion of AQP2 increases the water per- the syndrome of inappropriate antidiuretic meability of the collecting duct, facilitates hormone secretion (SIADH).

Fig. 1. Model of vasopressin (AVP)- mediated water reabsorption in renal collecting duct principal cells.

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Enno Klußmann

Fig. 2. In silico model of the interaction of the D/D domain of RIIα subunits of PKA with the AKAP18δ- derived peptide AKAP18δ-L314E.

Small molecules as tools for mechanistic insight into the functioning the elucidation of the molecular AVP-mediatedA major focus water of our reabsorption group is through to gain mechanisms underlying the control of AQP2 interacting partners that participate in the controlidentification of AQP2. of The novel redistribution proteins and of AQP2 their We pursued a small molecule screening as resembles cAMP-induced exocytic processes. Thus the elucidation of molecular mech- of novel proteins involved in controlling anisms underlying the control of AQP2 will AQP2.an unbiased In cooperation approach forwith the the identification screening not only help to understand this but also unit at the Leibniz Institute for Molecular other physiologically highly relevant cAMP- Pharmacology (FMP), we screened a library dependent exocytic processes such as re- of 17,700 small molecules for inhibitors of the cAMP-induced redistribution of AQP2 proton secretion from gastric parietal cells. utilizing mouse collecting duct-derived nin secretion from juxtaglomerular cells or- MCD4 cells as model system and automat- teins and their interacting partners partici- patingWe have in thepreviously control ofidentified AQP2. These several include pro approach yielded 17 inhibitors; amongst A-kinase anchoring proteins (AKAPs), the themed immunofluorescence 4-acetyldiphyllin (4AD), microscopy. a selective This GTP-binding proteins RhoA and Rab11, blocker of vacuolar H+-ATPase. 4AD in- the cytoskeletal components F-actin and creased pH levels of intracellular vesicles myosin and the motor proteins myosin Vb and caused an accumulation of AQP2 in the and dynein, which mediate intracellular Golgi. The compound prevented the cAMP- dependent and PKA-catalysed phosphory- - lation of AQP2 at serine 256, the key trigger tiontrafficking of these of and AQP2-bearing several other vesicles proteins along has for its redistribution into the plasma mem- shedthe cytoskeleton. some light on Althoughmechanisms the controlling identifica brane. 4AD did not affect cAMP levels, PKA AQP2, the precise molecular mechanisms activity or cAMP-induced phosphorylations underlying the regulation of AVP-mediated at other sites of AQP2. Thus our phenotyp- water reabsorption are largely unclear. ic cell-based screening approach showed functional vacuolar-H+-ATPase is required - for the cAMP-induced phosphorylation of velopment of novel strategies for pharma- AQP2 at S256 and its redistribution to the cologicalA second majorinterference focus of with the group AVP-mediated is the de plasma membrane but is not required for phosphorylation of AQP2 at other residues. ways for the treatment of cardiovascular - diseaseswater reabsorption characterised in orderby excessive to find AVP- new ecule inhibitors of the redistribution of dependent water reabsorption, in particu- AQP2.We have In the identified future, we additional aim to identify small their mol lar heart failure. - tion of the targets is expected to reveal fur- targets and modes of action. The identifica

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CARDIOVASCULAR AND METABOLIC DISEASE

PKA consists of a dimer of regulatory type Fig. 3. Model of a terpyridine helix mimetic interaction with the D/D RI or type RII and two catalytic (C) subunits. domain of RIIα subunits. The red rings indicate carboxyl groups of Binding of cAMP to the R subunits induces the first pyridine that are likely to interact with Q4’ and Q14 of the release of the C subunits that then phos- two protomers of the D/D dimer. The black ellipses indicate the ortho phorylate their substrates in close proxim- methyl group of the second pyridine, the condensed cyclopentyl ring of ity. The N-terminal dimerization/docking the third pyridine and the following two benzyl rings of the terpyridine (D/D) domains of the R subunits (amino molecule, which mimic the hydrophobic phase of the AKAP18δ-L314E acids 1-44) mediate dimerization and form helix and interact with the hydrophobic bottom of the binding pocket. a hydrophobic pocket that directly interacts with a conserved 14–25 amino acids long

Synthetic peptides, derived from the PKA- bindingα-helix ofdomain AKAPs of (RII-bindingAKAPs disrupt domain). AKAP- ther insight into the mechanisms control- PKA interactions by competitive binding to the D/D domain of R subunits. We derived molecules may serve as starting points for such PKA disruptor peptides from the PKA theling developmentthe trafficking of ofdrug AQP2. candidates Moreover, for the treatment of cardiovascular diseases characterised by excessive AVP-mediated water binding domain of AKAP18δ, which we had reabsorption, such as heart failure. previously identified. One of the peptides,- AKAP18δ-L314E binds PKA RII subunits units with KD = 0.4 nM. Pharmacological interference with with subnanomolar affinity, e.g. to RIIα sub AKAP-dependent protein-protein We observed that inhibition of AKAP- interactions – searching novel PKA interactions in primary inner med- strategies for the treatment of ullary collecting duct (IMCD) cells with heart failure membrane-permeable versions of peptide

Cells respond to a certain stimulus in a insertion of AQP2 into the plasma membrane,AKAP18δ-L314E presumably prevents because the theAVP-induced peptides displace PKA from its close proximity to tetheringhighly specific of components manner. of A signalling fundamental cas- AQP2 and thereby prevent the PKA phos- principle underlying this specificity is the phorylation of AQP2 at S256. Other groups to their sites of action. AKAPs are a family as well as ours showed that disruption of cades to defined cellular compartments, i.e. AKAP-PKA interactions in cardiac myocytes this task. Their unifying property is their and whole rat hearts increases contractil- abilityof around to directly50 scaffolding bind PKA. proteins However, that fulfilthey ity. Since inhibition of AVP-mediated water all bind additional signalling proteins such retention and improvement of cardiac con- as other kinases, phosphatases or phospho- tractility should relieve symptoms of heart diesterases via direct protein-protein inter- failure, inhibition of AKAP-PKA interactions actions. Thereby, they form signalling hubs in renal principal cells and cardiac myo- to coordinate cellular signalling spatially cytes may pave the way to a new concept and temporally. for the treatment of heart failure.

18 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Terpyridine helix mimetics In the future, we aim to develop our small targeting AKAP-PKA interactions molecules and helix mimetics towards drug candidates for the treatment of heart Although PKA anchoring disruptor peptides failure. are invaluable molecular tools for studying functions of AKAP-PKA interactions, the generally poor oral availability and stability of peptides limits their use in animal studies and their potential as drugs. Small molecules and non-peptide helix mimetics are considered as Patents / Patent applications small molecule disruptors of AKAP-PKA in- Klußmann, E., Tröger, J., Rosenthal, W. AKAP-Lbc-Hemmstoffe zur Therapie teractionsalternatives by to high-throughput peptides. We identified screening several and der kardialen Hypertrophie. EP12174673.9. are currently validating and optimising these molecules. Selected Publications Non-peptide helix mimetics including terpy- Abdul Azeez KR, Oder A, Moutty MC, Masada N, Beerbaum M, Schlegel B, Niquet S, - SchäferSchmieder G, Milić P, Krause J, Eldahshan G, von Kries A, Götz JP, CooperF, Zühlke DM, K, SchillingerKnapp S, Rademann C, Kreuchwig J, Rosenthal A, Elkins W, JM, ing side chains of amino acids from a rod-like Klussmann E. Highly Functionalized Terpyridines as Competitive Inhibitors of AKAP- axis.ridine Such scaffolds mimetics mimic are α-helixesdesigned to by combine project PKA Interactions. Angew. Chem. Int. Ed. 52:12187-12191, 2013 Bogum J, Faust D, Zühlke K, Eichhorst J, Moutty MC, Furkert J, Eldahshan A, Neuen- with the stability and membrane permeabil- schwander M, von Kries JP, Wiesner B, Trimpert C, Deen PM, Valenti G, Rosenthal W, itythe of specificity/target small molecules. selectivity Based on our of peptides peptide king. J. Am. Soc. Nephrol. 24:744-58, 2013 Klussmann E. Small-molecule screening identifies modulators of aquaporin-2 traffic helix mimetics, taking advantage of the favor- Schmieder, P., Götz, F., Göttert, H., Joshi, M., Reif, B., Haase, H., Morano, I., Kass, R., ableAKAP18δ-L314E solubility, conformational we developed pyridinesorientation, as Christian,Hampel, K., F., Kashin, Szaszák, D., M., Genieser, Drewianka, H.-G., S., Herberg, Friedl, S., F. Lorenz,W., Willoughby, D., Furkert, D., Cooper, J., Vargas, D. C.,M. F., and biological activity of helix mimetics of Baillie, G. S., Houslay, M. D., Kries, J. P., Zimmermann, B., Rosenthal, W. and Klussmann, E. Small molecule AKAP/PKA interaction disruptors that activate PKA increase cardiac this class. Starting point was an interaction contractility. J. Biol. Chem. 286, 9079–9096, 2011. model in which hydrophobic residues (L301, L304, L308) of the peptide interact with the Nedvetsky, P.I., Tabor, V., Tamma, G., Beulshausen, S., Mutig, K., Boltzen, M., Petrucci, O., Vossenkämper, A., Wiesner, B., Bachmann, S., Rosenthal, W., Klussmann, E. Recipro- hydrophobic bottom of the D/D domain of cal regulation of aquaporin-2 abundance and degradation by protein kinase A and p38-MAP kinase. J. Am. Soc. Nephrol. 21:1645-56, 2010 Recommended for reading by chains of the peptide interact with hydrophil- Faculty of 1000 icRIIα residues subunits such and as whereQ4 at the the peripheral hydrophilic rim side of Hundsrucker, C., Skroblin, P., Christian, F., Zenn, M., Popara, V., Joshi, M., Herberg, F. the pocket formed by the D/D dimer (Fig. 2). W., Reif, B., Rosenthal, W. and Klussmann, E. The domain of unknown function (DUF) 727 of glycogen synthase kinase interaction protein (GSKIP) facilitates anchoring of Fig. 3 shows one of the mimetics docked into protein kinase A. J. Biol. Chem. 285, 5507–5521, 2010. - - philicthe D/D residues domain at of the RIIα rim subunits. of the hydrophobic The carbox pocketyl groups and of mimic, the first for pyridine example, address the side hydro chain of E300 of the peptide that is likely to interact Group Leader with Q4. The ortho methyl group of the sec- Prof. Dr. Walter Rosenthal Joao Miguel Parente Fernandes ond pyridine, the condensed cyclopentyl ring Dirk Hampf of the third pyridine and the following two Co-Head Ekaterina Perets benzyl rings of the molecule mimic the hydro- PD Dr. Enno Klußmann Katharina Schrade

and interact with the hydrophobic core (Fig. 2 Scientists Technical Assistants andphobic 3) of phase the binding of the pocket. AKAP18δ-L314E These new helix Dr. Frank Goetz (externally Beate Eisermann mimetics interfere with AKAP-PKA interac- funded) Andrea Geelhaar tions in vitro and in cultured cells. Therefore, Dr. Micha Schroeter Baerbel Pohl Dr. Kerstin Zühlke biologically active and non-peptidic com- PhD poundsthe terpyridine that block molecules the D/D represent domain ofthe regu first- Veronika Deak Secretariat latory subunits of PKA and thereby interfere Alessandro Dema Jasmin Bonkowski with AKAP-PKA interactions. Doerte Faust Shirley-Ann Jennifer Felsenberg

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: Christiane Trabert Christiane Photo:

Kai Schmidt-Ott

Experimental and Translational Kidney Research

The kidney is a central organ of cardiovas- ogy, in which branching morphogenesis and cular homeostasis. It excretes toxins into the tubulogenesis occur in parallel. The sequence of events can be closely monitored in develop- urine, regulates volume status and solute ing mice or in organ cultures, in which key in concentrations in the body, and produces vivo aspects of nephrogenesis are recapitulat- hormones. Chronic kidney disease consti- ed. Exogenous or genetic perturbations of kid- tutes an important risk factor for many other ney development result in congenital kidney diseases. Furthermore, adult kidney epithelia diseases, including cardiovascular diseases preserve the ability to reactivate molecular and cancer. The kidney is composed of struc- pathways from earlier developmental stages tural units called nephrons, which consist in certain disease states, including tumors, kid- of several different types of renal epithelial a detailed understanding of these molecular cells that facilitate directional transport. Our mechanismsney fibrosis, will and help acute to tubular improve injury. the molecu Hence,- group studies the molecular mechanisms of lar diagnosis of kidney diseases and provide epithelial morphogenesis during embryonic novel targets for therapy. development and the maintenance of epithe- Transcription factors in epithelial lial integrity later in life. We use experimental development and homeostasis model systems, including cell and organ culture techniques and animal models, and carry We study the role of transcription factors in cellular differentiation and morphogenesis, and out clinical studies to carry our findings from in regeneration and tumorigenesis. We focus bench to bedside. on transcription factos from the TCF/Lef family and from the grainyhead family, which we From kidney development to kidney and others found to govern critical aspects of disease epithelial morphogenesis and differentiation,

During embryonic development, formation of For instance, two members of the grainyhead - family,and in healingcalled Tcfcp2l1of injured and epithelial Grhl2, structures.are highly teric bud, an epithelial tubule extending from expressed by nephron epithelia during devel- the definitive kidney is initiated, when the ure opment and in adulthood. In ongoing studies, - we are analyzing the roles of these transcrip- the posterior Wolffian duct, interacts with an- tion factors in development and disease. The dergoesadjacent branchingprogenitor morphogenesis cell population, to the give meta rise tonephric the ureter, mesenchyme. renal pelvis The and Wolffian collecting duct duct un with the groups of Jonathan Barasch (Colum- system, while the metanephric mesenchyme biaprojects University), benefit fromFrank our Costantini close collaborations (Columbia converts into epithelial cells that subsequently University, New York), Carmen Birchmeier get patterned along the proximal-distal axis to (MDC), Thomas Willnow (MDC), and Michael form the different cell types of the nephron. As Bader (MDC). these cells differentiate, they obtain epithelial characteristics, including the establishment Systems biology of kidney diseases of apico-basal polarity and the formation of - - bryonic kidney development constitutes a tions of kidney epithelia in congenital and classicalepithelial-specific model system junctions. in developmental Mammalian biol em- neoplasticKidney injury kidney and diseases phenotpyical are associated transforma with

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Developing kidneys are analyzed by whole- mount in situ hybridization (A) or immunofluorescent staining (B). (C) shows an example of an experiment to detect genome-wide binding of a transcription factor to DNA as determined by chromatin immunoprecipitation followed by next generation sequencing. Binding intensity (green) is shown relative to the genomic position. Genes (blue) are shown on the bottom.

widespread alterations of the genome and its expression. We are combining next generation sequencing based techniques and bioinformatics to map alterations of the genome, epigenome, and transcription factor binding sites in normal and diseased kidney cells. For this purpose we are collaborating with the Depart- ment of Urology at Charité (Kurt Miller), with Selected Patents the group of Walter Birchmeier (MDC), and Compositions for modulating growth of embryonic and adult kidney tissue and uses with systems biology groups at the Berlin Insti- for treating kidney damage. US-Patent, Application Number: 11807250, Filed on: tute of Medical Systems Biology (BIMSB). We 2007-05-25;, Inventors: Kai M. Schmidt-Ott, Jonathan Barasch, Jun Yang (via Columbia University) believe that a detailed elucidation of the ge- - nome-wide alterations in diseased renal cells meldung, EP 2397559 A1, US 61/355,723, Filed on: 2010-06-17, Inventors: Kai M. will yield an unprecedented opportunity to un- BiomarSchmidt-Ottkers ,for Anne determination Wuebken (via of temporalMDC/Ascenion) phase of acute kidney injury, PCT-An derstand in detail the molecular mechanisms of renal disease. Selected Publications Paragas N, Qiu A, Zhang Q, Samstein B, Deng SX, Schmidt-Ott KM, Viltard M, Yu W, For-

Molecular diagnosis of renal injury DW, D’Agati V, Lin CS, Barasch J. The Ngal reporter mouse detects the response of the ster CS, Gong G, Liu Y, KulkarniNature R, Mori Medicine. K, Kalandadze 2011;17(2):216-22. A, Ratner AJ, Devarajan P, Landry

Re-expression of embryonic marker molecules Nickolas TL*, Schmidt-Ott KM*, Canetta P, Forster C, Singer E, Sise M, Elger A, Maarouf kidney to injury in real time. is a common feature in disease states and is be- O, Sola-Del Valle DA, O’Rourke M, Sherman E, Lee P, Geara A, Imus P, Guddati A, Polland lieved to participate in compensation and regeneration. Neutrophil gelatinase-associated A,Emergency Rahman W,Department Elitok S, Malik Using N, Urinary Giglio J, Biomarkers El-Sayegh S, of Devarajan Nephron P,Damage Hebbar - S,A SaggiMulti -SJ, lipocalin (NGAL) is a protein in the developing Hahncenter B, Prospective Kettritz R, LuftCohort FC, Study. Barasch Journal J. Diagnostic of the Americanand Prognostic College Stratification of Cardiology. in the 2012, 59:235-44. * Co-First und Co-Corresponding authors. - Singer E, Elger A, Elitok S, Kettritz R, Nickolas TL, Barasch J, Luft FC, Schmidt-Ott KM. tion in embryonic renal epithelial progenitors. Urinary neutrophil gelatinase-associated lipocalin distinguishes pre-renal from intrin- kidney that is sufficient to induce differentia sic renal failure and predicts outcomes. Kidney International. 2011;80(4):405-14. NGAL is also markedly reactivated in tubular Werth, M., Walentin, K., Aue, A., Schonheit, J., Wuebken, A., Pode-Shakked, N., Viliano- is closely correlated with the temporal onset vitch, L., Erdmann, B., Dekel, B., Bader, M., Barasch, J., Rosenbauser, F., Luft, F.C., and Schmidt-Ott, K.M. 2010. The transcription factor grainyhead-like 2 (Grhl2) regulates injury of the kidney and its urinary excretion Development. 2010;137(22):3835-45.. with Jonathan Barasch (Columbia University, the molecular composition of the epithelial apical junctional complex. Newand severity York), Klemensof tubular Budde injury. (DepartmentIn collaboration of Schmidt-Ott KM, Masckauchan TN, Chen X, Hirsh BJ, Sarkar A, Yang J, Paragas N, Wal- Nephrology, Charité), Friedrich C. Luft (ECRC), a differentiation-associated transcriptional program in renal epithelial progenitors. Ralph Kettritz (ECRC), we are conducting basic Development.lace VA, Dufort D,2007;134(17):3177-90. Pavlidis P, Jagla B, Kitajewski J, Barasch J.?-catenin/TCF/Lef controls and clinical studies to understand the biology and clinical applications of NGAL in kidney

algorithms that utilize NGAL measurements to injury. We are aiming to optimize diagnostic Group Leader Prof. Dr. med. Kai Schmidt-Ott aimpredict to renalidentify injury additional and to differentiatenovel and phase- renal injury from related clinical entities. We also- Postdoctoral Scientists pare them to the available diagnostic methods Dr. rer. nat. Katharina Walentin MD Thesis Students inspecific clinical biomarkers medicine. of kidney injury and com Dr. med. Christian Hinze Carolin Elfriede Markus Dr. med. Eugenia Singer Ricarda Merle Viol Acknowledgements PhD students Jutta Sybille Swolinsky Our research is generously supported by the Annekatrin Aue Antje Elger Deutsche Forschungsgemeinschaft and by Zeliha Yesim Yurtdas Technical Assistants the Stiftung Urologische Forschung Berlin. Janett Ruffert For additional information please visit: Emilia Vigolo Gabriel Kirchgraber http://www.mdc-berlin.de/schmidt-ott Tatjana Luganskaja

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Salim Seyfried

Zebrafish Cardiovascular Developmental Genetics

Vertebrate organs are derived from epithelial ary approach included detailed expression or endothelial sheets of cells that undergo analyses combined with functional studies, high-resolution live imaging, and mathemati- complex morphogenetic transformations. The Seyfried lab studies the early zebrafish that Nodal, via modulating the extracellular heart, a relatively simple organ compared cal modeling of cardiac jogging. We found with its mammalian counterpart, to better matrix(Veerkamp within et theal., 2013).left cardiac The mechanismfield, dampens by understand the signaling events that instruct whichthe efficiency Nodal modulates of BMP signaling Bmp signaling on the leftac- the assembly of the early heart tube. Initially tivity involves the Nodal target Hyaluronan this organ consists of only the outer myo- synthase 2 (Has2), which has an asymmetric cardial and inner endocardial cell layers. The and Spaw-dependent expression on the left side of the cardiac cone and is required for ze- group would like to understand: What are the signals that regulate the morphogenesis involved in the production of hyaluronic acid, of myocardium and endocardium? To what brafish cardiac laterality. Has2 is an enzyme matrix. Since high expression levels of has2 extent do these two tissues communicate ona major the left macromolecule side of the cardiac of the cone extracellular correlate during cardiac jogging and looping morpho- with dampened Bmp signaling activity, we genesis? What determines the differentiation tested whether clonal misexpression of has2 of endocardium into its different morpho- within myocardial cells also reduces Bmp signaling activity. Indeed, clonal myocardial logical derivatives such as cushion cells? In expression of has2 resulted in strong damp- collaboration with clinical researchers, the ening of Bmp activity. Comparative microar- group uses developmental genetics combined with cell biological and pharmacologi- cardiac effector genes of Bmp signaling that includedray expression cell adhesion, analyses polarity identified and relevant epithe- cal approaches to develop animal models for lial development factors, which were all in human cardiovascular diseases. The group’s strong support of a role of Bmp signaling for long-term interest is to understand how the suggested that minor left-right differences cellular mechanisms controlling zebrafish cardiac progenitor cell adhesion. Our findings cardiogenesis shape the human heart and its determine bi-phasic states: cardiac progeni- associated blood vessels. torin Bmp cells activitywithin thewithin right the cardiac cardiac cone field being may slightly more adhesive, and cells on the left side exhibiting a slightly more motile char- Control of cardiac laterality by com- acter. Thus, cardiac left-right asymmetry binatorial TGF-ß network signaling may be explained by Nodal modulating an Justus Veerkamp, Florian Priller, Zoltan anti-motogenic Bmp activity. In clonal misex- Cseresnyes, Cécile Otten, Marc Renz pression studies, in which BMP activity was In an extension of previous studies on the cell biology of heart tube formation, we per- we observed an inversion of cardiac lateral- formed a comprehensive analysis of complex ity.clonally Moreover, reduced reducing within BMP the right activity cardiac to below field, TGF-ß network signaling during early cardiac normal levels on the left side even enhanced morphogenesis. Our highly-interdisciplin- -

cardiac jogging towards the left. One implica 22 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Figure 1: Effect of Nodal on L/R asymmetric BMP activity within the heart. (A,A’) Establishment of Bmp signaling asymmetry within the heart(outlined with white dotted line) between 16-21 hpf. The reporter line Tg(BRE-AAVmlp:dmKO2)mw40 drives expression of destabilized monomeric Kusabira-Orange 2 (dmKO2) which indicates Smad-1/5/8-mediated activity. (B) The L/R asymmetry of nuclear pSmad-1/5/8 intensities in WT, as indicated by the quotient of L- versus R-sided pSmad-1/5/8 intensities, is abolished in spaw morphant embryos at 19 hpf (***, p<0.0005). (C) Reversal of Bmp activity and cardiac laterality upon misexpression of spaw within the right cardiac field (asterisks, GFP false-colored in white). Signal- ing intensities are indicated by color range

tion of these clonal studies was that changes in individual cell motility rates impact the tissue displacement of larger coherent groups

that left-right differences in cell motility are of cells. Indeed, in silico modeling confirmed- tion of cardiac tissue. Based on these experiments,sufficient we to propose induce robust that aspects directional of cardiac migra laterality may be explained by a random cell motility gradient mechanism.

Establishment of a neuroepithelial - barrier by Claudin5a is essential for tions for small molecules (molecular mass of zebrafish brain ventricular lumen lanthanumof hindbrain nitrate: neuroepithelial 0.5 kDa). However, cell tight since junc expansion brain ventricular lumen expansion was not Jingjing Zhang, Cécile Otten (in collabora- entirely blocked by loss of Claudin-5a, oth- tion with Ingolf Blasig, Leibniz Institute for er Claudins may potentially contribute to Molecular Pharmacology, Berlin) the tightening of the neuroepithelial barrier as well. neuroepithelial-ventricular barrier is tightly coupledFormation with and neuroepithelial physiology of thematuration. zebrafish In all vertebrates, the embryonic neuroepi- Selected Publications thelium is initially organized as a columnar Veerkamp J, Rudolph F, Cseresnyes Z, Priller F, Otten C, Renz M, Schaefer L, Abdelilah- epithelial sheet which undergoes neurula- Seyfried S. (2013). Unilateral dampening of Bmp activity by nodal generates cardiac left-right asymmetry. Dev Cell. 24:660-7. tion to form the neural tube. Previous studies

the process of brain morphogenesis, which Wieffersorting regulatesM, Cibrián Wnt Uhalte signaling. E, Posor Curr Y, Otten Biol. 23:2185-90.C, Branz K, Schütz I, Mössinger J, Schu P, Abdelilah-Seyfried S, Krauß M, Haucke V. (2013). PI4K2β/AP-1-based TGN-endosomal involvesin zebrafish the expansion had already of brain demonstrated ventricles, that re- Tay HG, Schulze SK, Compagnon J, Foley FC, Heisenberg CP, Yost HJ, Abdelilah-Seyfried quires coherence among neuroepithelial cells S, Amack JD. (2013). Lethal giant larvae 2 regulates development of the ciliated organ Kupffer’s vesicle. Development. 140:1550-9. - - ling TF, Herzog W, Abdelilah-Seyfried S, Hammerschmidt M, Bakkers J. (2012). Bmp suevia apical polarity junctional defects complexes.or with Cadherin-based Furthermore, designaling Pater E, e xertsCiampricotti opposite M, effects Priller on F, cardiacVeerkamp differentiation. J, Strate I, Smith Circ K,Res. Lagendijk 110:578-87. AK, Schil cell-cellzebrafish adhesion mutants defects with neuroepithelialfail to expand brain tis Zhang, J, Piontek, J, Wolburg, H, Piehl, C, Liss, M, Otten, C, Christ, A, Willnow, TE, Blasig, ventricular lumens. In functional knock-down IE, Abdelilah-Seyfried, S.(2010). Establishment of a neuroepithelial barrier by Clau- USA, 107:1425-30. regulates the permeability properties of neu- din5a is essential for zebrafish brain ventricular lumen expansion. Proc. Natl. Acad. Sci studies, we showed that zebrafish Claudin-5a

moleculeroepithelial lanthanum cell tight nitratejunctions into as brain assessed ventri by- Group Leader Aylar Tafazzoli clesinjection between of the 17-20 small hours electron-dense post fertilization. tracer Dr. Salim Seyfried This correlated with a failure of brain ventri- Graduate and Undergraduate - Scientist Kristina Haehn din-5a. Strikingly, this effect was not caused Dr. Nana Bit-Avragim Carolin Scheffner bycles a togeneral expand loss in ofembryos neuroepithelial deficient tissue for Clau po- Dr. Veronica Lombardo larity since the apical localization of the tight Dr. Cecile Otten Technical Assistants Robby Fechner PhD Sabine Kraft ionjunctions pump were associated not affected. marker These protein analyses ZO-1 Ann-Christin Dietrich Jana Richter demonstratedand the normal that distribution the early ofneuroepithelial the Atp1 α1 Stefan Donat layer lining the brain ventricles represents an Marc Andreas Renz Secretariat embryonic cerebral-ventricular barrier and Marta Rocha Lourenco Iris Apostel-Krause that Claudin-5a determines the permeability

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David AUsserhofer/MDC Photo:

Francesca M. Spagnoli

Molecular and Cellular Basis of Embryonic Development

Understanding how distinct cell types arise research aims at investigating the origin of from multipotent progenitor cells is a major the pancreas and liver divergence at the cellular and molecular level. We used RNASeq to quest in stem cell biology. The endoderm - germ layer gives rise to a number of vital or- creas progenitor cells isolated by FACS from gans, including the lungs, liver, pancreas and thedefine mouse the molecular embryo at identity the time of ofliver the and lineage pan intestine. This remarkable diversity derives divergence (E8.5-E10.5). By integrating the temporal and spatial gene expression pro- from a homogenous population of multipotent cells. Our goal is to elucidate how liver signatures in hepatic and pancreatic progeni- and pancreas arise from common endoderm tors.files, weDeciphering defined mutually the molecular exclusive signaturesignaling of particular progenitor cell types holds the progenitors in the embryo and acquire spe- promise of isolating such cells for therapeu- cialized shape to form functional organs. This tic purposes and, possibly, of instructing fascinating question in developmental biology stem cells to activate appropriate differen- has direct clinical relevance. In the long run, tiation programs. For instance, our results have provided us with guidelines not only on our investigations will have direct implications how to generate hepatocytes and pancreatic in developing novel strategies to generate pancreas progenitors and β-cells either from to formulate strategies to promote liver-to- programming of stem cells or from re-pro- pancreasβ-cells from lineage human reprogramming. ES and iPS cells Notably,but also we have uncovered a unique non-canonical gramming of adult hepatic cells, closing the Wnt signaling signature in the emergence of gap between studies of basic processes in pancreas versus liver from endoderm pro- model systems and clinical research. genitors, which appears conserved across phylogenetically distant species. In addition - Comprehensive analysis of the meoproteins, being differentially expressed hepatic and pancreatic lineage betweento the Wnt-signature, pancreatic and we identifiedhepatic progenitor TALE ho divergence cells; and epigenetic modulators, as potential regulators of cell identity. The pancreas consists of two organs in one: the exocrine compartment, which produces Lineage reprogramming approaches digestive enzymes, and the endocrine com- to generate functional pancreatic partment, which houses the insulin-secreting cells - - Our previous studies provided novel insights sultsβ-cells in anddiabetes, is critical a still for incurable blood glucose metabolic ho into cellular plasticity and the basis for for- disorder.meostasis. Currently, Loss or dysfunction much effort of is β-cells targeted re mulating reprogramming strategies between toward generating renewable sources of liver and pancreatic cells. Targeted functional - studies of developmental regulators of the tes, and these efforts rely on the knowledge liver and pancreas fate decision and their ofβ-cells pancreas for cell development. replacement therapyThe pancreas of diabe is lineage reprogramming activity are ongoing closely related to the liver in terms of embry- in my group. We focus on both extrinsic and onic origin and gene regulatory network. Our intrinsic regulators of this cell fate decision.

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Figure 1. (A) Schematic of hepatic and pancreatic cell lineage specification (1) and fate conversion (2). (B-D) Characterization of Tg(Prox1-EGFP) mouse embryos. Prox1 is the earliest specific marker in common between hepatic and pancreatic endoderm from gastrulation onwards. Geneti- cally labeled Prox1-EGFP+ hepatic and pancreatic progenitors were purified by FACS at distinct developmental stages (E8.5-E10.5 interval) and analyzed by RNA-Seq. In insets, endoderm regions analyzed by RNA-Seq. Abbreviations: BP, bipotent progenitor; DE, definitive endoderm; fg, foregut; mfg, mid-foregut; hf, head fold; lv, liver; pa, pancreas; dp, dorsal pancreas; vp, ventral pancreas. (adapted from Rodriguez-Seguel et al. 2013).

How and when extrinsic factors, such as tor cells and, subsequent, organ hypoplasia. BMPs, control the emergence of liver versus The Stard13 mutant phenotype analysis has raised many interesting questions about cel- To address this question, we have developed lular mechanisms controlling the formation geneticpancreas approaches from the to same perturb embryonic the signaling region? in of the tips and progenitor allocation. Novel vivo in a controlled temporal and spatial fash- lines of research in my lab are aimed at inves- ion. These experiments will elucidate: if BMP tigating 1) the epithelial dynamics of branch- exerts a role in vivo in hepatic versus pancre- ing morphogenesis and 2) temporal and atic fate decision in mammals; and different spatial allocation of pancreatic tip progeni- potential functions of BMP in the endoderm tor cells. In the long run, we expect that a full according to the embryonic stages. Moreover, comprehension of the 3D organization of the pancreatic tissue will have practical implica- TGIF2 as an intrinsic regulator of the pan- tions in any attempt of cell-based therapy of creaswe have versus identified liver fate the decision TALE homeoprotein in the endo- diabetes based on ex vivo expansion of pancreatic progenitors and generation of insulin convert differentiated liver cells to a pancre- aticderm progenitor and whose state. expression This study is uncovers sufficient the to lineage reprogramming. reprogramming activity of TGIF2 and opens producing β-cells either from stem cells or to new investigation into the mechanistic aspects of the liver-to-pancreas lineage conver- Selected Publications sion. Wang Y, Chen W, Andrade-Navarro MA, Spagnoli FM. “Mutually exclusive signaling sig- Rodríguez-Seguel E, Mah N, Naumann N, Pongrac IM, Cerdá-Esteban N, Fontaine J-F, Control of morphogenesis natures define the hepatic and pancreatic progenitor cell lineages divergence” Genes - and tissue-architecture in the & Development 2013; Vol. 27: 1932–1946. developing pancreas ferentiation and Reprogramming” Developmental Dynamics 2014; Vol. 243: 76-87. Cerdá-Esteban N, Spagnoli FM. “A Glimpse Into Hox and TALE Regulation of Cell Dif Petzold K, Naumann H, Spagnoli FM. “Rho signaling restriction by the RhoGAP Stard13 integrates growth and morphogenesis in the pancreas” Development 2013; Vol. 140: During organogenesis, tissue-architecture 126-135. Gong M, Simaite D, Kühnen P, Heldmann M, Spagnoli FM, Blankenstein O, Hübner N, Hussain K, Raile K. “Two novel GATA6 mutations cause childhood-onset diabetes mel- anddefines subsequent particular differentiation. local microenvironments, For example, litus, pancreas malformation and congenital heart disease” Horm. Res. Paediatr. 2013; thewhich distal in turn tips impactof the branchingprogenitor pancreas specification epi- Vol. 79: 250-6. Petzold K, Spagnoli FM. “A System for Ex vivo Culturing of Embryonic Pancreas” J. Vis. thelium contain fast proliferating progeni- Exp. 2012; Vol. 66: e3979. - ing “niche”. How branching morphogenesis occurstors and, and possibly, is coordinated define awith special proliferation support and differentiation in the pancreas is largely Group Leader Julia Kofent unknown. We have developed mouse models Dr. Francesca M. Spagnoli Igor Pongrac* and time-lapse imaging of ex vivo embryonic Juan Zhang pancreas cultures to study morphogenetic, Scientist proliferation and differentiation events as Dr. Valeriya Arkhipova Graduate and Undergraduate they occur in the developing embryo. Using Dr. Anne Sophie Escot Lisa Conrad these approaches, we showed that Rho and Dr. Elisa Rodriguez-Seguel* Olivia Hertel its negative regulator, the RhoGAP protein Stard13, control branching morphogenesis PhD Technical Assistants of the pancreatic epithelium. Conditional Nuria Cerda-Esteban Heike Naumann ablation of Stard13 expression in the mouse Corinna Cozzitorto pancreatic epithelium disrupts morphogen- Secretariat esis and tip domain organization, resulting *part of the period reported Iris Apostel-Krause in hampered proliferation of tip progeni-

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Thomas Willnow

Molecular Cardiovascular Research

We study orphan endocytic receptors to totype of this gene family is the LDL recep- uncover their roles in normal physiological tor that mediates cellular uptake of cholesterol-rich lipoproteins. Since other family processes and in diseases. Recently, we members also bind lipoproteins, roles for demonstrated that LRP2, a member of the these receptors in lipid metabolism had LDL receptor gene family, acts as a recep- been anticipated. Surprisingly, studies un- tor for sonic hedgehog, and is required for covered many more functions performed by these receptors, changing our perception signaling of this morphogen in the mam- of lipoprotein receptors from mere cargo malian forebrain. Lack of LRP2 activity, in transporters to key regulators of numerous mouse models and in patients, results in physiological processes. In particular, the forebrain malformations and in holopros- transduction in embryonic development is encephaly. We also characterized SORLA significance of these receptors for signal and sortilin, members of a novel class of a glimpse at their contributions to develop- neuronal endocytic receptors. We showed mentalnoteworthy. processes, Yet, studies with many so far details provide still just to be uncovered. that both receptors control distinct steps in the catabolism of the Aβ peptide, the caus- Whereas studies on the LDL receptor gene ative agent in Alzheimer’s disease (AD). We family yielded exciting insights into the sig- found that SORLA acts as an intracellular sorting receptor for Aβ that directs nascent receptorsnificance ofcalled endocytosis VPS10P for domain cell signaling, recep- peptides to lysosomal degradation, while torsidentification or Sortilins of anotherdirected groupour attention of orphan to sortilin clears Aβ from the brain interstitial intracellular protein transport. The founding member of this gene family is SORLA, fluid (ISF) when bound to apolipoprotein a receptor that shares structural elements E. Both receptors effectively reduce the of the LDL receptor gene family (Fig. 1). In amount of neurotoxic Aβ in the brain and constitute promising drug targets for the VPS10P, a sorting protein in yeast. Subse- addition, it includes a domain identified in- treatment of AD. lian proteins that share the VPS10P domain uncoveredquent identification the existence of four of othera new mamma class of receptors that act in endocytosis and/or Introduction intracellular protein transport. Their many

Receptor-mediated endocytosis is the main mechanism for selective transport functions remain yet to be identified. LRP2 is an auxiliary SHH re- progress has been made in elucidating the ceptor required for forebrain ofvarious macr stepsomolecules of endocytosis into cells. at the Significant cellular development level. However, the physiological relevance Annabel Christ, Anna Christa of many endocytic pathways for organ (dys) function remains elusive. LRP2 is a receptor expressed in the neural tube, the embryonic tissue that gives rise The main class of endocytic receptors is the to the central nervous system. Early on, LDL receptor gene family (Fig. 1). The pro- we recognized the importance of LRP2 for

26 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Fig. 1: The structural organization of members of the LDL receptor and VPS10 domain receptor (Sortilins) gene families is shown.

- tor patched 1, a step essential to induce cient for the receptor suffer from holopros- forebrain patterning (Fig. 2) encephaly,brain development a defect asin micethe separation genetically of defi the - forebrain hemispheres and the most com- phogen signaling and revealed the mo- mon forebrain anomaly in humans. The sig- lecularOur data mechanism identified a novelunderlying concept forebrain in mor anomalies in patients with Donnai-Barrow syndrome. Our ongoing studies focus on defectsnificance as ofthe this cause receptor of Donnai-Barrow for humans syn was- exploring the relevance of this novel signal- drome,confirmed a monogenic by others, disorder demonstrating characterized LRP2 ing pathway for other clinically relevant as- by forebrain anomalies. pects of SHH signaling as in retinal degeneration or cancer. Recently, we succeeded in elucidating the mechanism of LRP2 action in brain development (Christ et al., Developmental Cell SORLA controls production of Aβ 2012; Willnow et al., Development 2012). Vanessa Schmidt, Tilman Burgert, Safak - Caglayan mary defect in Lrp2-/- embryos to a failure to establishSpecifically, signaling Annabel of Christthe morphogen traced the sonic pri hedgehog (SHH) in the rostral diencepha- proteolytic processing of the amyloid pre- - cursorAβ is aprotein neurotoxic (APP). peptide Excessive produced production by brain organizer. At neurulation, SHH origi- natinglon ventral from midline the prechordal (RDVM), plate a major moves fore to dysfunction and memory impairment ob- the overlying RDVM to induce its own ex- servedof Aβ is in held patients responsible with AD. for theAccordingly, synaptic pression and to establish its signaling do- - main (Fig. 2). Annabel demonstrated that sent promising targets for prevention of in Lrp2 mutants, SHH fails to localize to the thismechanisms devastating that disease. reduce Aβ levels repre RDVM, suggesting a defect in morphogen sequestration. Using cell lines, whole em- Previously, we documented the ability of bryo cultures, and neural explants, Annabel SORLA to act as an intracellular sorting - timately demonstrating that LRP2 acts as of this precursor into intracellular com- auxiliaryand Anna SHH further receptor refined that this sequesters pathway, this ul receptor for APP that prevents trafficking-

signaling of SHH through its cognate recep- lowpartments SORLA wherelevels seen processing in some into individuals Aβ oc morphogen in its target field and enables curs. These findings suggested that the

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CARDIOVASCULAR AND METABOLIC DISEASE

Fig. 2: In the developing forebrain, active SHH (SHH-N) is produced in the prechordal plate by SHH precursor cleavage and moves by unknown mechanisms to the apical surface of the rostral diencephalon ventral midline (RDVM), a forebrain organizer region. In the RDVM, SHH-N is sequestered by LRP2 to trigger patched 1 (Ptch1)-dependent induction of target gene transcription (through cellular mediators Smo and GLI).

with AD may be the underlying cause of - interacts with the cytosolic adaptor PACS1 thatand hisshuttles collaborators SORLA/APP identified complexes that SORLA from productionenhanced Aβ rates, production. Vanessa To Schmidt confirm and the her in endosomes to the Golgi, reducing the ex- colleaguesverse correlation combined of SORLAquantitative levels biochem with Aβ- tent of APP processing in endosomal com- ical studies with mathematical modeling to partments. Mutating the binding site for establish a kinetic model of amyloidogenic PACS1 in the cytoplasmic domain of SORLA resulted in aberrant retention of this recep- SORLA on this process. Their studies dem- tor (and its target APP) in endosomes and, onstratedprocessing thatand tosecretases evaluate the(the influence proteases by cleaving APP) represent allosteric enzymes (Burgert et al., MCB 2013). that require cooperativity by APP oligomer- consequently, in enhanced Aβ production - To provide proof that overexpression of ity enables swift adaptive changes in secre- in vivo, Safak Ca- taseization activity for efficient with processing.even small Cooperativalterations glayan generated mice that overexpress in APP concentration. They also showed thisSORLA receptor. reduces In Aβ line levels with our hypothesis, that SORLA prevents APP oligomerization increasing levels of SORLA in the brain eliminating the preferred form of the substrate and causing secretases to switch to a animals. Exploring the underlying mechanism,profoundly Safak reducedand his colleagues Aβ burden made in these the (Schmidt et al., EMBO J 2012). surprising observation that SORLA not only less efficient non-allosteric mode of action acts as sorting receptor for APP, but also Given the importance of SORLA-dependent - lysosomes for degradation, reducing the man Burgert set out to elucidate the molec- amountmoves newly of neurotoxic synthesized peptides Aβ molecules released to ulartrafficking mechanisms of APP controlling for Aβ production, SORLA sort Til- by neurons (Caglayan et al., Science Trans ing in neurons. Using mouse models that Med, in press). Safak also documented that -

express trafficking mutants of SORLA, he the ability of SORLA to sort Aβ is lost in a re 28 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

ceptor mutant expressed in a familial form (as for APP processing), or alters extracel- of AD. His studies substantiated SORLA as a potential drug target in AD and the loss data have shown that receptors of the LDL of receptor function as a possible cause of andlular VPS10P levels of domain metabolites receptor (as gene for Aβ). families Our early-onset AD. play key roles in these disease processes. - ular details how these receptors affect traf- Sortilin mediates clearance of Aβ Our future work aims at refining the molec in the brain molecules and how receptor dysfunctions Anne-Sophie Carlo contributeficking and tofunctional the most expression devastating of defects target in brain development and function occur- In the brain, astrocytes secrete lipoproteins ring in humans. containing apolipoprotein E (apoE) that are cleared from the brain ISF by apoE receptors expressed in neurons. These uptake pathways provide neurons with essential cholesterol (transported in lipoprotein particles) to sustain membrane integrity and synaptogenesis. Intriguingly, APOE is also the most important genetic risk factor for sporadic AD. Among other functions, APOE Selected Publications

the ISF, delivering them to cellular catabo- Caglayan, S., Takagi-Niidome, S., Liao, F., Carlo, A.-S., Schmidt, V., Burgert, T., Kitago, Y., lismis proposed via APOE to receptors. sequester The Aβ receptors molecules in in- Füchtbauer, E.-M., Füchtbauer, A., Holtzman, D.M., Takagi, J., and T.E. Willnow. Novel volved in this process remain controversial. role for SORLA/SORL1 as lysosomal sorting receptor for amyloid-beta peptide is impaired by familial Alzheimer disease mutation. Science Trans Med (in press) - Carlo, A.-S., Gustafsen, C., Mastrobuoni, G., Nielsen, M.S., Burgert, T., Hartl, D., Rohe, M., tilin as an apolipoprotein receptor in the liver,Because Anne-Sophie we had previously Carlo tested identified whether sor this protein may also be an apoE receptor Nykjaer, A., Herz, J., Heeren, J., Kempa, S., Petersen, C.M., and T.E. Willnow. (2013) The Pro-Neurotrophin Receptor Sortilin Is a Major Neuronal Apolipoprotein E Receptor in neurons. In line with her hypothesis, she for Catabolism of Amyloid-β Peptide in the Brain. J Neurosci 33: 358-70. and function. Trends Neurosci 35: 261-70. showed that sortilin binds APOE with high Nykjaer A., and T.E. Willnow. (2012). Sortilin, a receptor to regulate neuronal viability Schmidt, V., Baum, K., Lao, A., Rateitschak, K., Schmitz, Y., Teichmann, A., Wiesner, B., Pe- - resulted in the accumulation of APOE and affinity. Lack of sortilin expression in mice- disease.tersen, C.M., EMBO Nykjaer, J 31: 187-200. A., Wolf, J., Wolkenhauer, O., and T.E. Willnow. (2012). Quantita tive modeling of amyloidogenic processing and its influence by SORLA in Alzheimer’s Christ, A., Christa, A., Kur, E., Lioubinski, O., Bachmann, S., Willnow, T.E.*,and Hammes, A. (2012). LRP2 is an auxiliary SHH receptor required to condition the forebrain ven- animalsof Aβ in thedisplayed brain, asanomalies well as in in an brain aggravat lipid tral midline for inductive signals. Dev Cell 22: 268-278. *corresponding author ed amyloidogenic burden. Sortilin-deficient

APOEmetabolism uptake seen pathways in APOE-deficient (Carlo et al., J mice,Neu- indicating functional deficiency in cellular - Group Leader rosci 2013; Nykjaer and Willnow, Trends- Prof. Dr. Dr. h.c. wayNeurosci for 2012).APOE-containing Taken together, lipoproteins these find in Thomas Willnow Julia Klippert vivoings andidentified uncovered sortilin the asrelevance a neuronal of this path re- AygulKatja HerzogSubkhangulova ceptor for brain lipid homeostasis and AD. Scientists Dr. Anne-Sophie Carlo-Spiewok Technical Assistants Dr. Annabel Christ Kristin Kampf Outlook Dr. Sonya Dumanis Christine Kruse Dr. Vanessa Schmidt Melanie Liekweg Dysregulation of protein transport is emerging as a molecular mechanism of ma- PhD students Maria Schmeisser Tilman Burgert Tatjana Pantzlaff Among other functions, faulty transport im- Safak Caglayan Secretariat pactsjor importance cellular signal in many reception disease (as processes. for SHH), Anna Christa Verona Kuhle results in abnormal protein homeostasis

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Jana Wolf

Mathematical Modelling of Cellular Processes

Complex diseases are often characterised Modelling mammalian signal by an accumulation of multiple perturba- transduction pathways: IKK/ NF- κB signalling and Wnt/ β-catenin tions in rather large and complex cellular signalling networks. The consequences of these perturbations, such as mutations or In a collaborative approach with the group over-expression of proteins, can hardly of Claus Scheidereit at the MDC we aim for a systems level understanding of the IKK/ be analysed by pure reasoning. Here, mathematical modelling contributes to a consists of a canonical and a non-canonical deeper understanding of the regulatory branch.NF-κB signallingBoth have pathway. a distinct This timing pathway and distinct biological functions but are inter- systems and provides thus a better basis connected. On the one hand substrates and for the interpretation of high-throughput inducers of the non-canonical branch are data and identification of effective drug produced in the canonical branch, on the targets. other hand a control of the canonical part by the non-canonical branch was reported. Our group develops and analyses We are interested in the regulation of the mathematical models of signalling pathways long-time behaviour of the overall path- and gene-regulatory networks in normal way and its malfunction in diseases, e.g. and disease states. For our investigations Hodgkin-lymphoma. In particular, we want to dissect the contribution of canonical and we use tools such as simulations, non-canonical modules under these condi- bifurcation analyses and sensitivity tions. To that end we are investigating the analyses. These give insights into the kinetic properties, feedback regulations and interacting modules of both signalling dynamical properties of the systems and branches. help to identify most sensitive processes and critical regulations. Another important - aspect is the investigation of cell specific portant signal transduction pathway. Its de- regulationThe Wnt/ β-catenin is associated pathway with isvarious another types im differences in signalling and gene-regulatory of cancer. We use mathematical modelling networks since these are critically involved in the prediction of the efficiency and analyse the effect of transcriptional feed- of the canonical Wnt/ β-catenin pathway to possible side-effects of drugs. otherbacks pathways.(e.g. via Axin, β-TrCP/ HOS) and the cross-talk of Wnt/ β-catenin signalling to

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Robustness of cellular rhythms that a local addition of substrate led to the Many examples for rhythmic phenom- formationyeast cells. ofWe intercellular could for the glycolytic first time waves show ena can be found on the cellular level, e.g. in these cell layers. The theoretical analy- circadian rhythms, the cell cycle, calcium sis of this phenomenon showed that the or metabolic oscillations. These rhythms radial wave velocity arises from the sub- are involved in various functions and react strate gr adient. Overall, the results demonstrate that metabolic processes introduce - an additional level of local intercellular terminesspecifically the to balanceenvironmental between changes. sensitivity Our coordination. andproject robustness addresses of athe cellular question rhythm. of what To thisde end we compare the dynamical properties of various well-established mathemati-

models for calcium oscillations to be very sensitive,cal models those for differentfor circadian rhythms. rhythms We very find

the biological functions since for calcium Selected Publications oscillationsrobust. These a period-encoded findings correspond signal well trans to- duction was shown, whereas circadian Benary U.; Kofahl B.; Hecht A.; Wolf J. (2013), Modeling Wnt/beta-catenin target gene expression in APC and Wnt gradients under wild type and mutant conditions, Fron- oscillations generate an internal time in- tiers in Physiology 4: 21. formation. In a subsequent analysis, the impact of local kinetic parameters versus that Schmidt V.; Baum K.; Lao A.; Rateitschak K.; Schmitz Y.; Teichmann A.; Wiesner B.; of the system topology on the robustness is studied. Petersendisease, EMBO C.M.; NykjaerJournal 31A.; Wolf(1): 187-200. J.; Wolkenhauer O.; Willnow T.E. (2012), Quantitative modelling of amyloidogenic processing and its influence by SORLA in Alzheimer’s Schwanhaeusser B.; Busse D.; Li N.; Dittmar G.; Schuchhardt J.; Wolf* J.; Chen* W.; Sel- Metabolic synchronisation by 473 (7347): 337-342 (* corresponding authors). bach* M. (2011), Global quantification of mammalian gene expression control , Nature travelling waves in cell layers Schuetze J.; Mair T.; Hauser M.J.; Falcke M.; Wolf J. (2011), Metabolic synchronization by traveling waves in yeast cell layers, Biophysical Journal 100 (4): 809-813. The coordination of cellular dynamics is a Kofahl B.; Wolf J. (2010), Mathematical modelling of Wnt/beta-catenin signalling. prerequisite of the functionality of tissues Biochemical Society Transactions 38 (5): 1281-1285. and organs. Generally, this coordination may occur by signal transduction, neuronal control, or exchange of messenger molecules. The extent to which metabolic processes are involved is less understood. Group Leader Previously, a communicaton between cells Dr. Jana Wolf via the exchange of metabolites, called dynamic quorum sensing, was shown in Scientist stirred cell suspensions. However, it was Dr. Dorothea Busse Dinto Jose an open question whether the coupling of Dr. Bente Kofahl Janina Mothes

for the coordination of cellular behaviour. PhD Secretariat Wecells studied without this stirring question would by an be experimen sufficient- Katharina Baum Sabine Fröse tal-theoretical approach using a simple ex- Uwe Benary Petra Haink perimental system, that is, a layer of resting

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Michael Bader

Cardiovascular Hormones

The group focuses on the molecular biology function of these molecules. Moreover, a and function of hormone systems involved in novel peptide of the RAS, alamandine, with its receptor, MrgD, was discovered. To- cardiovascular regulation. The physiological gether with transgenic rats overexpressing and metabolic functions of these systems ACE2 or angiotensin(1-7), ACE2- and Mas- are evaluated by the production and analy- knockout mice characterized the ACE2/ sis of transgenic and gene-targeted animal angiotensin(1-7)/Mas system as a cardioprotective axis that counteracts the classi- models. Among the hormones studied, cal RAS effects in particular improving en- serotonin is of special interest, since it is not dothelial function. Moreover, these animals only involved in vascular homeostasis and showed that angiotensin(1-7) and Mas are important for insulin sensitivity and the other peripheral functions, but also serves as pathogenesis of metabolic syndrome. In potent and multifunctional neurotransmitter a newly established transgenic rat model in the brain. In addition, the group is inter- with inducible diabetes mellitus type 2 a ested in embryology and stem cell research, novel oral formulation of angiotensin(1-7) was shown to exert potent antidiabetic ac- in particular applying these fields to the rat. tions revealing the therapeutic potential of the protective RAS axis.

Renin-angiotensin system Kallikrein-kinin system and che- Natalia Alenina, Diogo Cavalcanti, Carlos mokines Fraga, Irina Lapidus, Valeria Nunes, Gabin Carlos Barros, Mike Becker, Johan Duchene, Sihn, Rafael Morais, Daisy Motta, Aline Lorena Gomez, Paola Guimaraes, Anne Järve, Oliveira, Luiza Rabelo, Martijn Rotteveel, An- Silke Mühlstedt, Katrin Nitz, Fatimunnisa thony Rousselle, Laura Souza, Mihail Todiras Qadri

The renin-angiotensin system (RAS) is of The kallikrein-kinin system (KKS) is an im- central importance in blood pressure regu- portant hormone system for cardiovascular lation and in the initiation of target organ regulation also mostly counteracting the damage. In particular, local angiotensin-II effects of the RAS. As models for the func- generating systems in tissues such as brain, tional analysis of the KKS in intact animals, heart, vessels, and kidney are involved in transgenic rats were generated expressing these processes. Therefore, transgenic rats different components of the system, such with local up- or downregulation of RAS as tissue kallikrein, the kinin B1 or the B2 components in these organs, e.g. by the local expression of antisense-RNA or of a in cardiovascular organs. These animals peptide-liberating protein, were produced supportedreceptor either the protective ubiquitously role or of specifically the KKS in and analyzed to clarify the local functions kidney and heart against ischemic, diabetic, of angiotensin II. Other genetically altered mouse and rat models for non-classical RAS the kinin B1 receptor were generated and components such as ACE2, the (pro)renin revealedand hypertrophic important injury. functions Knockout of this mice pro for- receptor, angiotensin(1-7) and its receptor Mas, have elucidated the physiological Moreover, the B1 receptor turned out to be tein in pain perception and inflammation.

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Figure 1: CXCL5 blockade increases macrophage foam cell formation in atherosclerosis Blockade of CXCL5 is associated with macrophage foam cell accumulation in atherosclerotic plaques in Apolipoprotein E deficient mice fed a Western diet and treated with either IgG isotype control (IgG) or anti-CXCL5 Ab (CXCL5 Ab) for 12 weeks. Staining for DAPI (nuclei), Mac2 (macrophages, upper panels), and adipophilin (fat containing vesicles, lower panels). Scale bars: 100 μm.

involved in arteriogenesis, sepsis, stroke, coding for tryptophan hydroxylase, the multiple sclerosis and high-fat diet induced rate-limiting enzyme in serotonin synthe- obesity. Mice lacking both kinin receptors and thereby being devoid of a functional the isoform responsible for the synthesis of KKS were also generated and shown to be serotoninsis, TPH1 andin the TPH2. gut, Mice showed deficient that in periph TPH1,- completely normal at baseline suggesting eral serotonin is involved in thrombosis, that the KKS is irrelevant for development pulmonary hypertension, remodelling of and the basic regulation of the cardiovascu- mammary glands, tumor angiogenesis, liver lar system but is involved in the pathogene- regeneration, and hepatitis, but not in bone sis of multiple diseases and, thus, a relevant metabolism as previously suggested. Mice drug target. for the synthesis of serotonin in the brain, Moreover, downstream mediators of kinins weredeficient surprisingly in TPH2, viable the isoform and fertile, responsible despite such as the chemokine CXCL5 and its re- a near complete lack of serotonin in the ceptor CXCR1 as well as other chemokines, brain, and showed growth retardation and such as CXCL12, are functionally analyzed altered autonomic control leading to im- in newly developed knockout mouse mod- pairment of sleep, respiration, cardiovas- els. Mice with a local deletion of CXCL12 in cular and metabolic parameters. Our recent cardiomyocytes presented an unexpected data revealed that exercise-induced neural deteriorating role of this chemokine for the cell proliferation in the dentate gyrus (adult heart function after cardiac damage. CXCL5 neurogenesis) is abolished in animals with life-long depletion in brain serotonin. In ad- macrophage foam cell formation in athero- dition, TPH2-deletion is correlated with in- sclerosis.was characterized as major determinant of creased aggression, maternal neglect, loss of aversive and anxiety-like behavior and depression-like phenotypes in mice. Since Serotonin system further behavior analysis of this model was Natalia Alenina, Annika Balke, Saleh Bas- hampered by poor ability of the mouse for hammakh, Daniel Beis, Michael Flinders, complex social interactions and learning Yasmine Graf, Maik Grohmann, Cornelia capacity, we now perform these studies Hainer, Friederike Klempin, Susann Matthes, Valentina Mosienko, Ashish Ranjan, Magda- lena Zaniewska in TPH2-deficient rats generated via zinc-- minantfinger-nuclease in the (ZFN)pathogenesis based gene-deletion. of psychi- Serotonin is a monoamine which functions atricSince disorders, brain serotonin such as is depression, a major deter we as an important neurotransmitter in the develop modulators of TPH activity as - novel therapeutic drugs together with the ripheral mediator produced by enterochro- high-throughput screening platform of the central nervous system and as a major pe Research Institute for Molecular Pharma- released by platelets in the circulation. We cology (FMP) and the Helmholtz Protein discoveredmaffin cells that of the vertebrates gut and transported have two genes and Sample Production Facility (PSPF).

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Figure 2: Serotonin is essential for exercise induced neurogenesis BrdU labeling in the dentate gyrus in wildtype (A’) and TPH2 deficient mice (A) after one week of running exercise. B, approximately 40% of newly generated cells (BrdU) express the transient immature neuronal marker Doublecortin (DCX) whereas in Tph2-deficient mice (C) increased numbers of proliferating microglia (iba1-positive) have been observed (immunohistochemistry and confocal imaging).

Figure 3: Novel modulators of TPH activity: Crystal structure of the TPH1 active site with bound specific inhibitor (brown, inhibitor shown as electron density map, feature-enhanced at 0.7 σ) super- imposed with a structure obtained with BH4 cosubstrate binding (blue, 1MLW.pdb).

Furthermore, we developed protocols for trophy and failure, the group generated and the in vitro differentiation of embryonic characterized animal models with altered stem (ES) and induced pluripotent stem androgen receptor expression in distinct (iPS) cells and transdifferentiation of cell types of the heart and vasculature. - Transgenic rats overexpressing the andro- ing neurons and performed gene expres- gen receptor in cardiomyocytes exhibited sionmouse analysis. fibroblasts To evaluate into serotonin-producif candidate genes marked alterations in cardiac rhythmicity. revealed by microarray are crucial for the development of serotonergic neurons in vivo, the morpholino-based knockdown of Importins Stefanie Hügel, Na Liu, Franziska Rother, Ariane Schabe, Phillip Schiele these genes is performed in zebrafish. Androgen receptor Importins are essential components of the Marko Bartusch, Albrecht Keil, Tina Kienitz, machinery that transports proteins into the Meike Latz, Silke Mühlstedt, Gabin Sihn nucleus of eukaryotic cells. In a collaborative approach with the University of Lübeck In a collaborative work with the Charité to study the physiological functions of al- studying gender effects in cardiac hyper- pha importins we have generated knockout

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Selected Publications phenotype was discovered in mice lack- Rousselle, A., Qadri, F., Leukel, L., Fontaine, J.-F., Rüstem, Y., Sihn, G., Bader, M., Ahlu- ingmice importin for five alpha7: paralogs. Both The sexes most of obvious these walia, A., Duchene, J. Cxcl5 limits macrophage foam cell formation in atherosclero- animals are infertile. The molecular basis of sis. J. Clin. Invest. 2013, 123, 1343-1347 the male infertility is currently being ana- Klempin, F., Beis, D., Mosienko, V., Kempermann, G., Bader, M., Alenina, N. Serotonin is required for exercise-induced adult hippocampal neurogenesis. J. Neurosci. lyzed. The female infertility is based on an 2013, 33, 8270-8275 essential function of importin alpha7 dur- Cui, Y., Niziolek, P.J., MacDonald, B.T., Zylstra, C.R., Alenina, N., Robinson, D.R., Zhong, ing zygotic genome activation of developing Z., Matthes, S., Jacobsen, C.M., Conlon, R.A., Brommage, R., Liu, Q., Mseeh, F., Powell, embryos. Furthermore, importin alpha7 is D.R., Yang, Q., Zambrowicz, B., Gerrits, H., Gossen, J.A., He, X., Bader, M., Williams, B.O., Warman, M.L., Robling, A.G. LRP5 functions in bone to regulate bone mass. Nat. Med. 2011, 17, 684-691 In parallel, proteomic analyses were per- Alenina, N., Kikic, D., Todiras, M., Mosienko, V., Qadri, F., Plehm, R., Boye, P., Vilia- pivotal for influenza virus infection of cells.- novich, L., Sohr, R., Tenner, K., Hörtnagl, H., Bader, M. 2009, Growth retardation and gos of single alpha importin paralogs. altered autonomic control in mice lacking brain serotonin. Proc. Natl. Acad. Sci. USA. 106, 10332-10337 formed identifying the specific import car Shmidt, T., Hampich, F., Ridders, M., Schultrich, S., Hans, V.H., Tenner, K., Vilianovich, L., Qadri, F., Alenina, N., Hartmann, E., Köhler, M., Bader, M. 2007, Normal brain Transgenic and stem cell technology development in importin alpha 5 deficient mice. Nat. Cell. Biol. 9, 1337-1338. Natalia Alenina, Ilya Chuykin, Venjamin Fishman, Alexander Krivokharchenko, Mi- Group Leader chael Lyskovikh, Markus Petermann, Elena Prof. Dr. Michael Bader Popova, Larissa Vilianovitch, Tianwu Qiao Scientist Daisy Motta* The group has also a strong emphasis in Dr. Natalia Alenina Katrin Nitz* Dr. Saleh Bashammakh Valeria Nunes* research. The rat is the preferred animal in Dr. Mike Becker* Aline Oliveira* physiologicalthe field of rat and embryology behavioural and studies. stem cellIn Dr. Carlos Barros* order to obtain rat pluripotent stem cells Dr. Ilya Chuykin* Anthony Rousselle two methodologies were applied in our Dr. Johan Duchene ArianeAshish Schabe*Ranjan group: isolation of ES cells from rat pre- Dr. Lorena Gomez* Laura Souza* implantation embryos and generation of Dr. Maik Grohmann* induced pluripotent stem (iPS) cells from Dr. Stefanie Hügel Graduates and Undergraduates Dr. Anne Järve* Annika Balke* carrying pluripotency genes. These cells Dr. Tina Kienitz* Marko Bartusch* arefibroblasts used to upon explore infection the signalling with lentiviruses cascades Dr. Friederike Klempin* Michael Flinders* underlying mechanisms of pluripotency in Dr. Alexander Krivokharchenko Yasmine Graf* the rat, to develop protocols in regenera- Dr. Irina Lapidous Albrecht Keil* tive therapy, and to establish homologous Dr. Na Liu Meike Latz* recombination and thereby allowing gene Dr. Valentina Mosienko Markus Petermann* targeting in the rat. Furthermore, trans- Dr. Silke Mühlstedt* genic rats have been produced carrying Dr. Elena Popova Phillip Schiele* constructs, which express small interfer- Dr. Fatimunnisa Qadri Martijn Rotteveel* Dr. Luiza Rabelo* Technical Assistants - Dr. Franziska Rother Adelheid Böttger* linence receptor, RNAs suited apolipoprotein to downregulate E, and thespecific LDL Dr. Gabin Sihn Susanne da Costa Goncalves receptor.genes. The first target genes were the insu Prof. Dr. Mihail Todiras Cathrin Gerhard Dr. Larissa Vilianovitch* Sabine Grüger Dr. Tianwu Qiao* Reika Langanki Dr. Magdalena Zaniewska* Lisa Mallis Keven Mallow* PhD Andrea Müller Daniel Beis Ralph Plehm Diogo Cavalcanti* Thorsten Riepenhausen Patents / Patent applications Veniamin Fishman* Madeleine Skorna-Nussbeck Method for diagnosing neuronal diseases and for the treatment of Carlos Fraga* Paola Guimaraes* Neuronally expressed tryptophan hydroxylase and its use, EP1521823 Cornelia Hainer* SecretariatTanja Schalow deficient primary hemostasis, US7049336 (B2) (23.05.2006) (B1) (23.05.2012) Michael Liskovykh* Iris Apostel-Krause Method for modulating, treating and/or preventing metabolic syn- Susann Matthes drome using MAS-G-protein-coupled receptor agonists, US8586054 (B2) (19.11.2013) Rafael Morais* * part of the period reported

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Norbert Hübner

Genetics and Genomics of Cardiovascular Diseases

Systematic perturbations of biological networks through complete knockout or over- genes for genetically complex cardio-meta- Over the past years our lab has identified expression of individual genes gave insights rat model, all of which have been translated for an understanding of the molecular basis tobolic advance and inflammatory understanding phenotypes of related human in the of human diseases. The current challenge is phenotypes. to understand systems perturbations due to more subtle changes induced by naturally a systems biology approach to understand occurring DNA variation or environmen- commonWe have initiatedcardiovascular the EURATRANS and metabolic project, distal conditions that are relevant to complex eases. We have assembled a multinational diseases in human populations. We are consortium of world-class investigators that use technologies to generate genomic, using novel large scale technologies and transcriptomic, and epigenomic datas- advanced computation in multidisciplinary ets. The EURATRANS consortium integrates approaches to expand the systems con- these datasets in relational and dynamic models that can be used for comparative cept and to accelerate discovery of gene analyses to understand human gene func- networks underlying complex cardiovas- tion. cular and metabolic disease phenotypes in the rat model, and to translate the findings Genetic analysis of disease to the molecular pathology of related hu- phenotypes in the rat. man disorders. Our particular interests are inflammatory and metabolic genetic risk The spontaneously hypertensive rat (SHR) factors that lead to cardio- and cerebrovas- is the most widely studied animal model of human hypertension and it displays a large cular target organ damage. We use primar- number of other physiological and patho- ily the rat as a model organism to identify physiological phenotypes, including stroke, the major gene pathways for human cardio- cardiac hypertrophy and failure, and features of the human metabolic syndrome. Starting vascular and metabolic disorders. from traditional genetic linkage studies and congenic approaches, we elected to study the SHR and SHR derived substrains as a model for hypertension associated heart failure and cardiac hypertrophy. Our initial studies iden-

gene expression – or expression quantitative traittified loci thousands (eQTLs) of – genetic across controlthe SHR points genome. for However, we realized that the power of genome-wide, microarray-based expression

36 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Similar Splicing Patterns in Human (blue) and direct RBM20 - RNA Interaction (red)

and pathophysiological phenotypes that may carried out extensive microarray expression be shared in common with similar disorders studiesprofiling in had7 tissues more from to offer.the BXH/HXB We therefore panel in humans. In key collaborations with T Ait- of recombinant inbred (RI) strains bred from man, S Cook and M Pravenec we explored the SHR and BN founders. Our collaborators and we have devised a systems approach for the genetic architecture of eQTLs across mul- dissecting genetic networks systematically tiplepower tissues, of the usingeQTL newapproach, methods, first andto define then, across the biological scale, from molecular to by correlating gene expression and linkage physiological, using segregating populations data, to identify genes for the individual risk of rats. RI strains have several advantages factors that lead to heart failure, namely Ogn over studies in other experimental crosses and EndoG for cardiac mass and CD36 for hy- including the ability to increase trait herita- pertension. Each of these genes was shown bility by phenotyping multiple genetically to have conserved function in human disease identical rats from each strain, and the abil- cohorts or tissues. In addition we were able ity to accumulate data from genetically stable to demonstrate that we not only could iden- strains over years or decades, obviating the tify genes that modify individual risk factors need for de novo genotyping. A particular that subsequently contribute to heart failure strength of the eQTL approach is the ability but that the clinically very complex endpoint to separate genetically determined differenc- - es in gene expression into local cis-regulated netic risk factors. In addition, we generate control, where sequence variants controlling large-scaleheart failure high is resolution directly influenced mapping data by ge in gene expression reside within or close to the Heterogeneous Stock, a rat resource colony differentially expressed gene, as compared derived from 8 ancestral strains and outbred to trans regulated control, where sequence for more than 40 generations. They serve as variation resides in another gene remote a valuable genetic resource for simultane- from the differentially expressed gene. By identifying several of robustly mapped cis- phenotypes in multiple disease areas, and and trans-acting expression QTLs in a model forous robustfine mapping integration, of susceptibility at high resolution, genes for of with large number of existing physiological detailed rat genetic and physical maps. We QTLs we generated a permanent resource further extend and complement the data- to test the hypothesis that genetic variation bases of gene expression and physiological in gene expression has a key role in the mo- phenotypes by generating transcriptome lecular evolution of complex physiological inventories, and develop new metabonomic,

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Multilevel datasets will be generated in rat models for human complex diseases. We generate genomic, transcriptomic, and epigenomic datasets. We are applying quantitative metabolomic and proteomic tools to provide in-depth coverage, at multiple phenotypic levels, of the gene, cell, organ, and organism. Multiple-QTL inference models and Bayesian approaches will identify casual regulators for the disease network that will be functionally validated by in vivo and in vitro experiments. Integrating these datasets in relational and dynamic models can be used for comparative analyses to understand human gene function.

proteomic, and epigenomic datasets in RI - and selected Heterogeneous Stock animals. ing proteins that contribute to pathologi- Mapping the genetic determinants of these calWe haveposttranscriptional identified mutations regulation in RNA of bind an new phenotypes to the genome using exist- entire network of target genes that lead to ing high-resolution maps will permit us to cardiomyopathy and heart failure. In col- identify cis and trans-regulatory control loci laboration with Michael Gotthardt (MDC) of these phenotypes. Using these multi-mo- we used genome-wide approaches in a rat dality QTL datasets we will identify the most important pathways leading to clinically rel- a loss-of-function mutation in RBM20 as evant phenotypes. thestrain underlying deficient cause in titin for splicing the pathological and found isoform expression of titin. Mutations in RBM20 in humans have previously been Integrated genomic approaches shown to cause dilated cardiomyopathy. for identification of gene net- - works. sembled the human disease phenotype. WithWe showed Cemil Özcelik that RBM20 (Charite) deficient we then rats iden re- Our advances in rat genomics are facilitating systems-genetics approaches. We have with an RBM20 missense mutation that led extended the use of integrated genome- totified RBM20 a dilative loss of cardiomyopathyfunction. Deep sequenc patient- wide approaches to identify gene networks ing of the human and rat cardiac transcrip- and the loci underlying their regulation.

tomes with RBM20 deficiency and controls 38 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

revealed an RBM20 dependent regulation Patents / Patent applications of alternative splicing events. In order to understand post-transcriptional RNA pro- 2010 Polynucleotides for use for Medicine cessing and particularly the function of 2008 Transposon mediated mutagenesis in spermatogonial stem cells splicing factors in the heart, we, together Selected Publications with Markus Landthaler and Nikolaus Ra- Atanur SS, Diaz AG, Maratou K, Sarkis A, Rotival M, Game L, Tschannen MR, Kaisaki PJ, Otto GW, Ma MC, Keane TM, Hummel O, Saar K, Chen W, Guryev V, Gopalakrish- RBM20 in common forms of heart failure. nan K, Garrett MR, Joe B, Citterio L, Bianchi G, McBride M, Dominiczak A, Adams Usingjewsky, PAR-CLIP are investigating we perform gene regulationhigh resolu of- DJ, Serikawa T, Flicek P, Cuppen E, Hubner N, Petretto E, Gauguier D, Kwitek A, tion mapping of the binding sites of RBM20 Underlie Disease Phenotypes in the Laboratory Rat. Cell 154(3):691-703, 2013. Jacob H, Aitman TJ. Genome Sequencing Reveals Loci under Artificial Selection that across the transcriptome as well as those Baud A, Hermsen R, Guryev V, Stridh P, Graham D, McBride MW, Foroud T, Calde- of other RNA binding proteins (RBP) inter- rari S, Diez M, Ockinger J, Beyeen AD, Gillett A, Abdelmagid N, Guerreiro-Cacais AO, Jagodic M, Tuncel J, Norin U, Beattie E, Huynh N, Miller WH, Koller DL, Alam I, Falak acting with RBM20. Additionally to titin we S, Osborne-Pellegrin M, Martinez-Membrives E, Canete T, Blazquez G, Vicens-Costa E, Mont-Cardona C, Diaz-Moran S, Tobena A, Hummel O, Zelenika D, Saar K, Patone 30 genes that is conserved between human G, Bauerfeind A, Bihoreau MT, Heinig M, Lee YA, Rintisch C, Schulz H, Wheeler DA, Worley KC, Muzny DM, Gibbs RA, Lathrop M, Lansu N, Toonen P, Ruzius FP, de andidentified rat and an enrichedRBM20 dependent for genes networkpreviously of linked to cardiomyophathy, ion-homeosta- T, Jones EY, Ekman D, Lopez-Aumatell R, Dominiczak AF, Johannesson M, Holmdahl BruijnR, Olsson E, Hauser T, Gauguier H, Adams D, Hubner DJ, Keane N, Fernandez-Teruel T, Atanur SS, Aitman A, Cuppen TJ, Flicek E, Mott P, Malinauskas R, Flint sis, and sarcomere biology. We will use the J. Combined sequence-based and genetic mapping analysis of complex traits in resulting binding site data in knowledge- outbred rats. Nat Genet 45(7):767-775, 2013. based approaches to extend our studies and to determine the impact of genomic Schulz H, Lincoln S E, Parrish A M, Dauksaite V, Vakeel P, Klaassen S, Gerull B, GuoThierfelder W*, Schafer L, Regitz-Zagrosek S*, Greaser M L,V, RadkeHacker M T H,A, LissSaupe M, KGovindarajan W, Dec G W, EllinorT, Maatz P H,T, - MacRae C A, Spallek B, Fischer R, Perrot A, Ozcelik C, Saar K, Hubner N, Gotthardt tions in humans. Our studies emphasize the M. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med importancevariability on of posttranscriptional posttranscriptional modifica regula- Heinig M, Petretto E, Wallace C, Bottolo L, Rotival M, Lu H, Li Y, Sarwar R, Langley tion in cardiac function and provide novel 18(5):766-773, 2012. *shared first author SR, Bauerfeind A, Hummel O, Lee YA, Paskas S, Rintisch C, Saar K, Cooper J, Buchan mechanistic insights into the pathogenesis R, Gray EE, Cyster JG, Braund P, Gracey J, Krishnan U, Moore JS, Nelson CP, Pol- of human heart failure. lard H, Attwood T, Crisp-Hihn A, Foad N, Jolley J, Lloyd-Jones H, Muir D, Murray E, O’Leary K, Rankin A, Sambrook J, Godfroy T, Brocheton J, Proust C, Schmitz G, Heimerl S, Lugauer I, Belz S, Gulde S, Linsel-Nitschke P, Sager H, Schroeder L, Lund- We are interested in epigenetic variation mark P, Syvannen AC, Neudert J, Scholz M, Deloukas P, Gray E, Gwilliams R, Niblett and gene expression in cardiovascular dis- D, Erdmann J, Hengstenberg C, Maouche S, Ouwehand WH, Rice CM, Samani NJ, Schunkert H, Goodall AH, Schulz H, Roider HG, Vingron M, Blankenberg S, Muenzel T, Zeller T, Szymczak S, Ziegler A, Tiret L, Smyth DJ, Pravenec M, Aitman TJ, Cam- main component of chromatin and undergo bien F, Clayton D, Todd JA, Hubner N*, Cook SA*. A trans-acting locus regulates an ease and inflammation. Histones are the- anti-viral expression network and type 1 diabetes risk. Nature467:460-464, 2010. cluding methylation. They play an impor- *Corresponding authors. tantseveral role posttranslational in genome organization, modifications stability in Joutel A, Monet-Lepretre M, Gosele C, Baron-Menguy C, Hammes A, Schmidt S, Lemaire-Carrette B, Domenga V, Schedl A, Lacombe P, Hubner N. Cerebrovascular and the control of gene expression. We will dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease. J Clin Invest. - 120(2):433-445, 2010. ternsaddress in the whether rat and there test whether are strain-specific those dif- differences in histone modification pat sequence variants. We will use likelihood basedferences model are selection determined procedures by allele to specific better

on differential gene expression. These stud- Group Leader PhD iespredict shall the lead influence to a better of genetic understanding variability of Prof. Dr. Norbert Hübner Eleonora Adami the interplay between epigenetic and ge- Christin Mieth netic variability. Scientist Sebastian Schäfer Deimante Simaite Dr. Samreen Falak Dr. KatharinaAnja Bauerfeind Grunz Technical Assistants Dr. Matthias Heinig Susanne Blachut Oliver Hummel Mathias Gerhard Dr. Henrike Maatz Anita Müller Dr. Carola Rintisch Dr. Giannino Patone Dr. Klaus Rohde Sabine Schmidt Dr. Franz Rüschendorf Gabriele Born Dr. Kathrin Saar Dr. Herbert Schulz Secretariat Dr. Nina Henriette Uhlenhaut Maren Stauch

MDC Research Report 2014 39

CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Zsuzsanna Izsvak

Domesticated, transposon-derived cellular genes Mobile DNA A. Szvetnik, T. Raskó Occasionally transposons are co-opted for cellular fuctions. We focus on two domesticated versions of the piggyBac transposon in Transposons (jumping genes) are segments the human genome. We are currently investi- of DNA that have the ability to move and rep- gating the cellular function of these genes by integrated genomic and transcriptomic ap- licate within genomes across the tree of life. proaches. Transposons offer a new model to study DNA recombination in higher organisms, as well as Transposon mutagenesis in stem host-parasite interaction. Our lab tries to un- cells I. Grabundzija, S. Bashir, A. Osiak derstand the mechanism of transposition and Transposons can be harnessed as vehicles for its regulation and to translate this knowledge introducing mutations into genes. Our goal is to derive transposon-based genetic tools for to establish tools based on SB as well as on the piggyBac and Tol2 transposon systems genome manipulation or gene therapy. to manipulate vertebrate genomes in organisms. One particular application relates to loss-of-function insertional mutagenesis with Quality controls in Sleeping Beauty the goal of generating knockout rats. The element (SB) transposition genes inactivated by transposon insertion are I. Bilic, H. Escobar tagged by the transposable element, which Our understanding of how eukaryotic recom- can be used for subsequent cloning of the binases are working is mostly based on as- mutated allele. With the goal of knocking out suming analogies to V(D)J recombination. Be- genes implicated in disease, we carried out a sides the basic chemical reaction, the different pilot screen in rat spermatogonial stem cells. elements have a variety of built-in regulatory mechanisms, often involving host factors, to Genetic background of hormone- - induced breast cancer tion. One main function of such regulation is J. Menyhért toprovide impose specificity quality control to the ontransposition transposition reac in The SB transposon is suitable for somatic mu- the form of regulatory checkpoints. The role tagenesis and emerged as a new tool in can- of these checkpoints is to avoid accumulation cer research. Transposon-based insertional of incorrect reaction products in genomes. oncogenes and tumor-suppressor genes. We Transposon-host interactions mutagenesis screens are able to identifiy both- A. Deveraj, V. Raghunathan, J. Rugor, M. Singh covery of novel driver mutations associated withare engaged neuroblastoma in a project (in mice) aiming and at estrogen- the dis - induced mammary cancer (in rats). posonsTransposons remain occupy silent due a significant to accumulated portion mu of- tationsour genomes in their but genomes. the vast Themajority transposition of trans Stress-induced transcriptional of the few, active copies is strongly regulated, activation of transposable elements but this control is sensitive to environmen- and gene expression tal stress. Our results show that transposons N. Fuchs, M. Singh, S. Narayanavari might exist in a latent form and are able to Recent comparative genomics studies have sense developmental and environmental shown that many constrained noncoding ele- changes and manipulate stress signaling. ments unique to mammals arose from mobile Thus, cellular mechanisms directly involved in development or stress-response have a force in the evolution of mammalian gene crucial role in establishing stable host-trans- regulation.elements, pointing We recently to TEs set as out a major to investigate creative poson co-existence. the cellular functions of hyper-conserved,

40 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

TE-derived repeats in the evolution of gene regulation.

Transposons as vectors for mammalian transgenesis A. Deveraj, S. Bashir DNA-based transposons are natural gene delivery vehicles, and molecular reconstruction of SB is a cornerstone in applying transposition-mediated gene delivery in vertebrates, including humans. Our recently developed 100-fold hyperactive SB system, SB100X (Molecule of the Year, 2009) opened new avenues for gene delivery. Recently, we demonstrated that the SB100X system supports

species. This seems to work with similar or efficient transgenesis in various mammalian- egies, but it is a non-viral technique and is muchimproved simpler efficacies and safer to retro/lentiviral to use. One of strat the A) Age-related macular degeneration (AMD) results from retinal pig- mented epithelium degeneration. (B) The ex vivo approach is to (i) remove transgenesis produces trangenics of supe- autologuos retinal cells, (ii) introduce the therapeutic PEDF gene with important findings is that transposon-based anti-angiogenic and/or neurotrophic activities into the pigment cells ex observed. vivo by nucleofection, and (iii) re-implant the cells. Stable integration and rior quality, in that no major mosaicism is secretion of the therapeutic PEDF (pigment epithelial derived factor) was Transposons as non-viral vectors demonstrated in >75% of the proliferating cells after several passages for gene therapy (followed up for 2 years). J. Wang, H. Escobar, H. Cai, A. Garcia-Perez, I. Bilic Using the SB100X system, we are currently Selected Publications performing preclinical studies for gene therapy of Wiskott-Aldrich syndrome, chronic granulomatous disease (CGD), Age–related Ivics,Transposon-mediated Z, Mátés, L, Tien Yau,germline TY, Bashir, transgenesis S, Zidek, in V, rodents. Landa, V, Nature Geurts, Prot. A, Hoffmann, (tripple bac-to- O. macular Degeneration (AMD) and dysfer- I,back), Hiripi, 773-827. L, Bosze, Z, Garrels, W, Kues, W. A, Pravenec, M, Rülicke, T, Izsvák, Z. 2014. linopathy. SB transposition occurs into chromosomes in a random manner, which is Wangin Eukaryotic Y, Wang Cells J, Devaraj, PLOS A,Genet. Singh, Mar M, 13;10(3):e1004103.Jimenez Orgaz A, Chen JX, Selbach M, Ivics Z, clearly undesired for human applications due Izsvák Z. 2014. Suicidal Autointegration of Sleeping Beauty and piggyBac Transposons to potential genotoxic effects. We succeeded Z., and Hamra, F.K. (2010) Generating Knockout Rats by Sleeping Beauty Transposon in targeting SB transposition into predeter- Izsvák,Mutagenesis Z., Fröhlich, in Spermatogonial J., Grabundzija, Stem I., Shirley,Cells. Nature J.R., Powell, Methods H.M., 7(6):443-5. Chapman, K.M., Ivics, mined chromosomal loci. We employed modular targeting fusion proteins, in which the Wang,Variability Y., Liska, in Rat F., Inbred Gosele, Strains. C., Šedová, Genome L., Křen, Research V., Křenová, 20(1):19-27. D., Ivics, Z., Hubner, N. and module responsible for target binding can be Izsvák, Z.(2010) A Novel Active Endogenous Retrovirus Family Contributes to Genome a natural DNA-binding protein or domain, or Schmitt, A, Becker, K, Matrai, J, Ma, L, Samara-Kuko, E, Gysemans, C, Pryputniewicz, D, Mátés, L, Chuah, MK, Belay, E, Jerchow, B, Manoj, N, Acosta-Sanchez, A, Grzela, DP, - tion of a novel hyperactive Sleeping Beauty transposase enables robust stable gene could be a powerful method for safe trans- trMiskey,ansfer C, in Fletcher, vertebrates. B, VandenDriessche, Nature enet. 41(6), T, Ivics, 753-761. Z., Izsvák, Z. (2009). Molecular evolu genean artificial integration protein. in human Targeted applications. transposition The - ing Beauty transposon, will be conducted fromfirst European 2015 to treat clinical AMD trials, (supported using the by Sleep FP7) and cancer patients by T cell-mediated immu- Group Leader Vaishnavi Raghunathan notherapy (supported by BIH), respectively. Dr. Zsuzsanna Izsvak Julia Rugor Manvendra Singh Genome-wide impact of epigenetic Scientist Chuanbo Sun reset triggered by induced pluripo- Dr. Nina Fuchs Jichang Wang tency I. Grabundzija, J. Wang, N. Fuchs, E. Nagy Dr. Judit Menyhert Graduate and Undergraduate Due to the superior safety features of the SB- Dr. EnikoIvana GrabundzijaEva Nagy Dora Fazekas mediated gene delivery, we have established Dr. Suneel Narayanavari an SB100X-mediated protocol of generating Dr. Tamas Rasko Technical Assistants induced pluripotent stem cells (iPSCs). The Malgorzata Anna Dalda aim is to assess the risk of genetic destabili- PhD Sandra Neuendorf Ana Jimenez Orgaz transcriptional or functional upregulation of Huiqiang Cai humanzation inflictedendogenous by the TEs mobilization on the genome and/or of HelenaIlija Bilic Escobar Secretariat pluripotent stem cell lines and their differen- Angelica Garcia-Perez Regina Philipp tiated derivatives.

MDC Research Report 2014 41

CARDIOVASCULAR AND METABOLIC DISEASE Photo: Steffen Jänicke Steffen Photo:

Young-Ae Lee

Molecular Genetics of Chronic Inflammation and Allergic Disease

The allergic disorders, atopic dermatitis, Our recent studies highlight the genetic asthma, and hay fever, are the most common overlap between risk loci for allergic dis- - chronic diseases in childhood. The preva- tory conditions that have previously been lence of allergic diseases has increased to thoughtease with to those be unrelated,for other chronic such as inflamma autoim- epidemic dimensions over the past decades. mune diseases. We performed a systematic In the industrialized countries, 25-30% of the population are affected.Our group is using dermatitisinvestigation (AD). of genetic A strong markers excess influencing of AD- genetic and genomic approaches in large associatedinflammatory markers traits as was risk observedfactors for among atopic study groups of affected patients and families the selected candidate SNPs, corroborating our hypothesis. Three novel loci were sig- to identify genes and genetic variants that predispose to atopic dermatitis, asthma, and functional amino acid-change in the inter- chronic inflammation. Enhanced understand- leukin-6nificantly receptor associated (IL6R with Asp358Ala). AD, including Inter a- ing of how the interaction between environ- estingly, investigation of two independent population-based birth cohorts showed mental factors, genetics, and the immune system results in inflammation will enable the to the persistent form of AD. This variant development of novel diagnostic and predic- determinesthat IL6R 358Ala the balance specifically between predisposes the clas- tive clinical tools, and of targets for innovative sical membrane-bound versus the soluble IL6R signaling pathways. Carriers of 358Ala therapies. had elevated serum levels of soluble IL6R with homozygote carriers showing a 2-fold increase. Moreover, we demonstrated that soluble IL6R levels were higher in AD pa- core paradigms of chronic human disease un- tients than in controls. Thus, the IL6R As- derlyingChronic inflammationnumerous conditions has become including one of theal- p358Ala is a new risk factor for persistent lergic and autoimmune disorders, cardiovas- AD which increases plasma levels of soluble cular, and neurological disease. Regardless of IL6R, suggesting that targeted blocking of - IL-6-transsignaling may be a novel thera- - peutic approach for persistent AD refrac- tal,the mechanismmicrobial or of self inflammation, antigens contribute inappropri to tory to conventional treatments, especially dysfunctionate inflammatory and destruction responses ofto target environmen organs. in carriers of this very common variant. Epidemiologic data provide strong evidence for a steady rise in the incidence of diverse The cross-disease approach was extended in the international Immunochip consortium. In industrialized countries over the past de- this study we investigated over 24,000 indi- cades,types ofindicating chronic inflammatorythat genetic variants disorders that in viduals to evaluate 188 genetic loci involved have evolved to arm our immune system - against multiple infectious/environmental novelin chronic susceptibility inflammatory loci disordersfor AD that as pointrisk fac to - genestors for involved atopic dermatitis.in the proliferation, We identified prolifera four- easeexposures in the nowhygienic influence environment immune of response modern tion, migration, and functional regulation of civilization.thresholds to favor chronic inflammatory dis immune cells.

42 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Genome-wide association studies (GWAS) Figure 1: Soluble have provided fundamental insight into the IL6 receptor plasma genetic determinants of complex allergic levels dependent on the functional IL6R than 80% of the presumed heritability re- variant (rs2228145) mainsdiseases. unexplained. Despite numerous We participate findings, in moresev- genotype and dis- eral international consortia enlarging study ease status populations and increasing the power to de- Box: 25th-75th percen- tect allergic disease variants (e.g. GABRIEL tiles; solid bar: median; consortium, EAGLE consortium). However, whiskers: 10th-90th these approaches must be paralleled by dif- percentiles; circles: ferent strategies to uncover the unexplained outliers. Number of heritability. The application of current and individuals per each emerging genome-sequencing technology, group is shown below new techniques for analysis of gene expres- each plot. A) soluble sion and structural genome variation, as well IL6R levels stratified by as statistics and genome informatics will con- rs2228145 genotypes. tribute to this aim on several levels. A and C are the non- risk and risk alleles, Clinical and molecular respectively; B) soluble subphenotyping. IL6R levels stratified To better understand the clinical diversity by atopic dermatitis status. we have recently established a research out- patientand temporal clinic (Hochschulambulanz) trajectory of allergic for disease pedi- atric allergy at the Experimental and Clinical Research Center (ECRC of Charité and MDC) for the prospective evaluation of allergic sensitization patterns, disease course, and physiological evaluation in children with allergic disease. Complementing the clinical pheno- Selected Publications type with precise molecular phenotypes may Ellinghaus D, Baurecht H, Esparza-Gordillo J, Rodriguez E, Matanovic A, Marenholz I, Hubner N, Schaarschmidt H, Novak N, Michel S, Maintz L, Werfel T, Meyer-Hoffert U, characterization of individual functional Hotze M, Prokisch H, Heim K, Herder C, Hirota T, Tamari M, Kubo M, Takahashi A, Na- kamura Y, Tsoi LC, Stuart P, Elder JT, Sun L, Zuo X, Yang S, Zhang X, Hoffmann P, Nothen pathways.significantly enhance the identification and MM, Folster-Holst R, Winkelmann J, Illig T, Boehm BO, Duerr RH, Buning C, Brand S, Glas J, McAleer MA, Fahy CM, Kabesch M, Brown S, McLean WH, Irvine AD, Schreiber S, Lee - Functional analysis ceptibility loci for atopic dermatitis. Nat Genet 2013;45:808-812. * shared senior author of disease genes. Esparza-GorYA *, Franke Adillo *, Weidinger J, Schaarschmidt S *. High-density H, Liang L, genotyping Cookson W, study Bauerfeind identifies A, four Lee-Kirsch new sus MA, Nemat K, Henderson J, Paternoster L, Harper JI, Mangold E, Nothen MM, Ruschen- of the susceptibility genes for allergic and dorf F, Kerscher T, Marenholz I, Matanovic A, Lau S, Keil T, Bauer CP, Kurek M, Ciecha- nowicz A, Macek M, Franke A, Kabesch M, Hubner N, Abecasis G, Weidinger S, Moffatt A major goal is to characterize the effects M, Lee YA. A functional IL-6 receptor (IL6R) variant is a risk factor for persistent better understand the earliest events in hu- atopic dermatitis. J Allergy Clin Immunol 2013;132:371-377. manchronic physiology inflammatory that lead diseases to this in type order of in to- Marenholz I, Esparza-Gordillo J, Lee YA. Shared genetic determinants between eczema and other immune-related diseases. Curr Opin Allergy Clin Immunol 2013;13:478-486. DNA re-sequencing to extract the full varia- Dizier MH, Margaritte-Jeannin P, Madore AM, Esparza-Gordillo J, Moffatt M, Corda E, Monier F, Guilloud-Bataille M, Franke A, Weidinger S, Annesi-Maesano I, Just J, Pin tionflammatory spectrum response. of regulatory We apply loci and systematic to pin- I, Kauffmann F, Cookson W, Lee YA, Laprise C, Lathrop M, Bouzigon E, Demenais F. point culprit variants in patients and con- The ANO3/MUC15 locus is associated with eczema in families ascertained through trols. Functional assays in model systems asthma. J Allergy Clin Immunol 2012;129:1547-1553. are performed to complement the molecular Scholtens S, Postma DS, Moffatt MF, Panasevich S, Granell R, Henderson AJ, Melen E, characterization of allergic disease risk vari- F, Kauffmann F, Siroux V, von ME, Ege MJ, Braun-Fahrlander C, Genuneit J, Brunekreef ants. Parent-of-origin effects. We direct an Nyberg F, Pershagen G, Jarvis D, Ramasamy A, Wjst M, Svanes C, Bouzigon E, Demenais Freidin MB, Bragina EI, Deev IA, Puzyrev VP, Daley D, Park J, Becker A, Chan-Yeung international systematic analysis of parent- B, Smit HA, Wijga AH, Kerkhof M, Curjuric I, Imboden M, Thun GA, Probst-Hensch N, of-origin effects for eczema and asthma in Franke L, Nolte IM, Vonk J, Kumar A, Farrall M, Cookson WO, Strachan DP, Koppelman four independent data sets in which genome- M,GH, Kozyrskyj Boezen HM. AL, Novel Pare P, childhood Marenholn asthma I, Lau genesS, Keil interact T, Lee YA, with Kabesch in utero M, and Wijmenga early-life C, tobacco smoke exposure. J Allergy Clin Immunol 2013. wide genotype data is available in complete nuclear families with allergic disease, and to - Group Leader dent families. Moreover, we aim to use ge- Prof. Dr. Young-Ae Lee Dr. Birgit Kalb nome-widereplicate the gene findings expression in over levels 1000 in indepen periph- Tamara Kerscher eral blood mononuclear cells of a subset of Scientist Dr. Ingo Marenholz 100 nuclear families with eczema as quanti- Jamina Eckhard tative trait loci and intermediate phenotypes Dr. Jorge Esparza-Gordillo Till-Yong Mohr in our parent-of-origin analysis. Anja Matanovic

MDC Research Report 2014 43

CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Friedrich C. Luft

Hypertension Genetics, Regulators, and Environment

Fig. 1. The CISTR-ACT locus (purple) and DA125942, a lncRNA transcribed from the CISTR-ACT locus, interact in cis with PTHLH and in trans with SOX9. Maass et al found that DA125942 organizes chromatin to coordinate transcription of multiple loci involved in cartilage differentiation. A chromosomal translocation found in BDE patients disrupts the ability of DA125942 to bind the PTHLH locus in cis, potentially causing premature chondrocyte differentiation at the expense of proliferation and bone elongation.

44 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Molecular genetics genetic mechanism that increases periph- Our goal is to reduce cardiovascular mor- eralthis syndrome.vascular resistance, We have identified independent a novel of tality; hypertension is the most important salt-reabsorptive processes. Moreover, we risk factor. We pursue genetic, mechanistic, show that the BDE phenotype is dependent upon the same mutation (unpublished). are investigating the molecular genetics ofand hypertension environmental and influences.brachydactyly First, type we E (BDE). The brachydactyly phenotype has Eicosanoids led us to elucidate cis- and trans-regulatory epigenetic phenomena. Second, our group Eicosanoids are signaling molecules made actively contributes to the elucidation of by oxygenation of arachidonic acid (AA) the cytochrome P450 (CYP) pathway of and other essential omega-6 and omega-3 eicosanoid formation that has been only polyunsaturated fatty acids (n-6 and n-3 recently discovered to play a pivotal role PUFAs). They exert complex control over in the pathogenesis of hypertension and - target organ damage. We follow therapeu- mation or immunity, and as second mes- tic strategies based on the mechanisms of sengersmany bodily of diverse systems, growth mainly factors in inflam and CYP-eicosanoid action for the prevention hormones throughout the cardiovascular - system. Our research interest has been mias. Third, we are studying environmental focused on the so-called third, and most of acute kidney injury and cardiac arrhyth- recently discovered branch of eicosanoid sequent sodium metabolism that we can formation that is catalyzed by cytochrome followinfluences with particularly sodium magnetic salt intake resonance and sub P450 (CYP) enzymes. CYP enzymes func- spectroscopy and imaging (Na-MRI) tech- tion as hydroxylases or epoxygenases niques. and convert n-6 and n-3 PUFAs to unique sets of biologically active hydroxy- and Earlier we showed that parathyroid hor- epoxy-metabolites, collectively termed mone-related peptide (PTHrP), encoded CYP-eicosanoids. Prominent examples in- by the PTHLH, is a BDE gene. We recently clude 20-hydroxyeicosatetraenoic acid used chromosome conformation capturing (20-HETE) and epoxyeicosatrienoic acids (EETs).We recently showed that enhanced 12q that interacts in cis with PTHLH over cardiac EET biosynthesis protects against aand 24.4-megabase identified a regulatordistance andon chromosomein trans with electrical remodeling, ventricular tachyar- the sex-determining region Y-box 9 (SOX9) - gene on chromosome 17q. The element ity during maladaptive cardiac hypertrophy also harbored a long noncoding RNA (ln- asrhythmia, described and in atrial (Fig. 2).fibrillation We collaborate susceptibil with cRNA). Silencing of the lncRNA, PTHLH, or John R. Falck (UT Southwestern, Dallas) in SOX9 revealed a feedback mechanism in- developing CYP-eicosanoid-derived phar- volving an expression-dependent network macological approaches to prevent these in humans. In the BDE patients, the human disease conditions. To follow this idea lncRNA was upregulated by the disrupted from basic research to clinical phase I and chromosomal association. Moreover, the phase II trials, the team founded OMEICOS lncRNA occupancy at the PTHLH locus was Therapeutics as a spin-off company of the MDC on the BioTech-Campus Berlin-Buch in trans-acting DNA and lncRNA regulatory (http://www.omeicos.com). feedbackreduced. Weelement identified that is a reciprocally novel in cis- regu and- lated by coding genes. We also provided a view of how enhancers encoding lncRNAs Environment may affect gene expression in normal development (Fig. 1.). However, our goal is Our group has worked together with Jens to elucidate the hypertension syndrome. Titze and his group (Erlangen/Nürnberg- - Vanderbilt University) to elucidate salt- nisms responsible for the phenotypes in We have recently identified genetic mecha related mechanisms. Briefly, we have MDC Research Report 2014 45

CARDIOVASCULAR AND METABOLIC DISEASE

Fig. 2. Thoracic aortic constriction (TAC) elicits ventricular-tachycardia (VT) susceptibility in wild-type (WT) mice but not in CYP2J2 (CYP) transgenic mice (A-B). Representative immunofluorescence staining of left ventricle from WT (C) and CYP2J2-TG (D) mice show normal Cx43 localization was in CYP2J2-TG mice, whereas WT mice featured TAC-induced redistribution of Cx43 to the cytoplasm and lateral borders of the cardiomyocytes (pink arrows). Nuclei were stained with DAPI (blue). Scale bar: 50 µm.

Fig. 3. Na-MRI scan of the lower leg im- mediately below the knee joint in a patient with an aldosterone- secreting tumor, before (left) and after (right) removal.

46 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

found that sodium balance follows infradian rhythms totally independent of salt intake. Total body sodium stores cycle independent of salt intake. Although high- salt intake is associated with higher blood pressure, the relationship is regulated by unknown mechanisms and not by pressure-natriuresis, as we currently understand it. We developed magnetic resonance imaging (MRI) based on sodium stimulation (Na-MRI). Na-MRI reveals hitherto fore unknown sources of sodium storage

“see” sodium in humans (Fig 3). The so- diumin skin is and stored skeletal in soft-tissues muscle.Briefly, and wecan can be removed from stores by removal of adrenal adenomas secreting aldosterone.

Perspectives Selected Publications

Maass PG, Rump A, Schulz H, Stricker S, Schulze L, Platzer K, Aydin A, Tinschert S, Goldring MB, Luft FC, Bähring S. A misplaced lncRNA causes brachydactyly in humans. J Clin Invest. 2012;122:3990-4002. directWe are increase optimistic. in peripheral We have vascular identified resis the- first Mendelian hypertension related to a Westphal C, Spallek B, Konkel A, Marko L, Qadri F, DeGraff LM, Schubert C, Bradbury JA, Regitz-Zagrosek V, Falck JR, Zeldin DC, Müller DN, Schunck WH, Fischer R. CYP2J2 based research group that had the courage overexpression protects against arrhythmia susceptibility in cardiac hypertrophy. totance. establish We are an the independent first (and only) company. clinically We PLoS One. 2013;8:e73490. pursue public health (global health) goals Wiig H, Schröder A, Neuhofer W, Jantsch J, Kopp C, Karlsen TV, Boschmann M, Goss J, based on genetic, cellular, animal-based, and Bry M, Rakova N, Dahlmann A, Brenner S, Tenstad O, Nurmi H, Mervaala E, Wagner direct translational (Na-MRI) techniques. cells control skin lymphatic electrolyte homeostasis and blood pressure. J Clin Invest. H,2013;123:2803-15. Beck FX, Müller DN, Kerjaschki D, Luft FC, Harrison DG, Alitalo K, Titze J. mmune

Rakova N, Jüttner K, Dahlmann A, Schröder A, Linz P, Kopp C, Rauh M, Goller U, Beck L, Agureev A, Vassilieva G, Lenkova L, Johannes B, Wabel P, Moissl U, Vienken J, Gerzer R, Eckardt KU, Müller DN, Kirsch K, Morukov B, Luft FC, Titze J. Long-term space

2013;17:125-31. flight simulation reveals infradian rhythmicity in human Na(+) balance. Cell Metab. Titze J, Dahlmann A, Lerchl K, Kopp C, Rakova N, Schröder A, Luft FC. Spooky sodium balance. Kidney Int. 2014;85:759-765.

Group Leader Prof. Dr. Friedrich C. Luft Eicosanoids Gene Lab Dr. Wolf-Hagen Schunck Dr. Sylvia Bähring Dr. Christina Westphal Dr. Atakan Aydin Maximilian Blum Dr. Philipp Maass Ramona Zummach Yvette Wefeld-Neuenfeld Christel Andrée Astrid Mühl Environment Eireen Bartels-Klein Natalia Rakova Irene Hollfinger

MDC Research Report 2014 47

CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Thoralf Niendorf

Ultrahigh Field Magnetic Resonance (UHF-MR)

Our research concentrates on the develop- Technology Focus: MR Methodology ment of MR methodology and MR technology & MR Technology for Ultrahigh Field MR (UHF-MR) with a focus on new ways of mapping and probing morphology, function, physiology and The sensitivity gain afforded by UHF-MR is metabolism together with explorations into the driving force behind our technological de- the benefits and challenges of ultrahigh field velopments. We pioneered multi-channel radiofrequency (RF) coil technology including (UHF) imaging. These efforts are designed the design, implementation and clinical eval- to spatially resolve and characterize (patho) uation of 4-, 6-, 8-, 12- and 16-channel coil physiological and biophysical processes to brain, ophthalmic, spine and shoulder MRI at harmonize basic research with clinical science 7.0configurations T (Figure 1)tailored. To stay for cardiovascularat the forefront (1), of while balancing technical developments with research we developed a modular 32 chan- clinical applications. To meet this goal small nel cardiac coil array (2). Our technology was animal (9.4 T) and whole-body human (7.0 T put to good clinical use for myocardial tissue characterization (3), for uncovering subtle & 3.0 T) MR scanners are put to good use at - B.U.F.F. Our research is built on collaborations sels, and to drive novel ways of cardiac imag- with partners from the Charité, the PTB, Sie- inganatomical including details susceptibility of the heart mapping. and major Going ves 1 mens Healthcare, Bruker Biospin and several beyond conventional proton ( H) MR we are other (inter)national institutions. We form a nuclei to gain a better insight into metabolic key/integral part of major initiatives including andexploring (nano)molecular MRI of sodium, processes. fluorine, 23 Na,and 19 otherF, 1H the Helmholtz Alliance for Imaging and Curing RF arrays designed for clinical or preclinical applications were developed to form basis Environmental Diseases (ICEMED), the popu- of (inter)national collaborations. The group lation MRI program of the German National also focuses on developing novel imaging Cohort (GNC), the DFG research group FOR techniques (4) and ancillary hardware. These 1368 on Hemodynamics of Acute Kidney efforts include our push towards RF based hyperthermia that makes use of the traits Injury, the imaging program of the German of the short wave length regime of UHF MR. Center for Cardiovascular Research (DZHK), Our group developed a hybrid applicator for the EU funded FP7 project INSERT on hybrid simultaneous targeted RF heating, tempera- SPECT/MR imaging, the BMBF initiative on so- ture mapping and diagnostic imaging at 7.0 T (Figure 2) (5). To master the physics of dium MRI (NAMRIS) and the German Ultrahigh Field Imaging (GUFI) network. Our initiatives even higher magnetic fields we devoted our resulted in 39 peer-reviewed publications dur- (EMF) simulations up to B0=30 T. Our activi- researchties culminated to numerical in our drive electromagnetic towards hybrid field ing 2012/13, 55 abstract contributions to the imaging (SPECT/MR, Doppler ultrasound/ 2012/13 annual meetings of the International MR, MR/PET) to exploit the opportunities of Society of Magnetic Resonance in Medicine combining anatomical, functional and meta- plus 12 (inter)national patent applications. bolic imaging. MR safety is also one of our focuses, to gain a better insight into RF heating

48 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Figure 1: MR technology focus Our research concentrates on the development of MR methodology and MR technology including pioneering novel multi-channel transmit/receive RF coil technology. Photographs of (a) 32-channel, (b) 6-channel and (c) 12-channel transceiver RF coil arrays designed, implemented and clinically evaluated by our group. This enabling RF technology was put to clinical use for (d) high spatial resolution blood oxygenation level dependent mapping of the heart (in collaboration with A. Meloni, CNR Pisa, Italy and J. Schulz-Menger, ECRC), (e) for hybrid anatomical and functional ophthalmic imaging (in collaboration with O. Stachs et.al., University Rostock and S. Langner et.al., University of Greifswald) and (f) for MR microscopy of the shoulder (in collaboration with T. Herold et.al. Helios Clinic Berlin-Buch).

Figure 2: Next generation MR technology focus Our group focuses on explorations into promising innovative MR applications that are not considered clinical mainstream yet. We developed ancillary hardware with the ultimate goal to enhance the capabilities for advanced diagnostics and therapy. These efforts include our push towards RF based hyperthermia that makes use of the traits of the short wave length regime of UHF MR. Our group developed a (a) hybrid applicator for simultaneous (b) diagnostic imaging, (c) targeted RF heating and temperature mapping at 7.0 T which was validated by (d) numerical temperature simulations (collaboration with P. Wust et.al. Charité and W. Hoffmann et. al., PTB).

of stents and other medical devices, which is rant an early diagnosis of pathologies (e.g. of paramount relevance before driving UHF- in hypertrophic cardiomyopathy), advance MR into the clinic. diagnostic imaging (e.g. of stroke, Figure 3), secure a differential diagnosis of pathologies Clinical Focus: Explorations into (e.g. multiple sclerosis and other neuroin- Cardiovascular and Neurovascular Figure 3) and drive Diseases using UHF-MRI emerging clinical applications (e.g. diagnosis offlammatory ocular masses conditions and other( diseases of the - eye, orbit and optical nerve (Figure 3). cular MR are in the spotlight of our group’s clinicalThe fields applications-oriented of cardiovascular andresearch. neurovas En Experimental Focus: Quantitative route to novel imaging-guided diagnostic and Mapping and (Nano)Molecular Prob- therapeutic approaches we strive towards ing the non-invasive assessment and early diagnosis of cardiac and neurovascular diseases The ultimate aim of the group is to harmonize through MR-biomarker based tissue charac- research carried out in the area of preclinical terization, quantitative parametric mapping, animal imaging with that of clinical imag- high spatial resolution and microstructural ing. The team helps monitor glioblastoma- imaging of the heart (Figure 3), brain and induced microglia attraction and tumor ex- other target anatomy. These efforts will war- pansion (collaboration with H. Kettenmann,

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Figure 3: Clinical applications focus Top: En route to novel imaging-guided diagnostic and therapeutic approaches we strive towards high spatial resolution and microstructural imaging of the heart. Short axis views of the heart derived from 2D CINE FLASH acquisitions of the heart at 7.0 T using a spatial resolution of left: (1.8 x 1.8 x 6) mm3, middle: (1.4 x 1.4 4) mm3 and right: (1.1 x 1.1 x 2.5) mm3. These results demonstrate that our novel multi-channel RF coil arrays tailored for cardiac MR at 7.0 T afford an order of magnitude spatial resolution enhancement versus standardized protocols used in today’s clinical practice. This resolution gain improves the delineation of subtle anatomical details and supports microstructural imaging of the heart. Bottom: Our explorations into neurovascular diseases left: advance diagnostic imaging of stroke (in collaboration with J. Sobesky and V. Madai et.al., Charité) , middle: secure a differential diagnosis of pathologies (e.g. multiple sclerosis with the lesion marked in blue and other neuroinflammatory conditions, in collaboration with F. Paul & J. Würfel et.al., Charité) and right: drive emerging clinical applications (e.g. diagnosis of ocular masses and other diseases of the eye, orbit and optical nerve, collaboration with O. Stachs, University of Rostock & S. Langner et.al., University of Greifswald).

Figure 4: Experimental focus Top: Key examples of our experimental research include a) non-invasive assessment of cardiac function of mice heart (collaboration with M. Bader, MDC), b) monitoring of brain tumour growth (collaboration with H. Kettenmann, MDC), c) MR microscopy of pre-symptomatic cerebellar lesions in an EAE model (collaboration with J. Millward, Charité) and d) tracking of 19F labeled cell migration in real time with greater sensitivity in vivo together withvisualization of brain inflammation. Bottom: Gaining a better understanding of the mechanisms behind acute kidney injury (AKI) is in the center of our experimental research radar screen. These efforts are designed to change the landscape of non-invasive renal diagnostic imaging by benchmarking parametric MR including f) blood oxygenation level sensitized MRI, g) arterial spin labeling perfusion MRI and h) water diffusion weighted MRI against established quantitative but invasive physiological methods using our integrated MR-PHYSIOL setup (collaboration with E. Seeliger et.al., Charité; D. Dragun et.al., Charité ; H-W. Schunck et.al., MDC; P. Persson et.al. Charité).

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development. The proposed integrated tracer discovery and metabolic imaging center pro- ofMDC) autoimmune and has made encephalomyelitis major progress (EAE). to study Our vides a unique platform to form a powerful brain inflammation during19 theF) andpathogenesis physico- alliance that bridges the gap between molec- chemical characteristics of nanoparticles is ular medicine/pharmacology and imaging- know-how on fluorine ( driven (pre)clinical research and provides processes and cell migration in real time with outcome data guiding personalized medicine. greaterbeing put sensitivity to good usein vivo. to follow Gaining inflammatory a better understanding of the mechanisms behind acute

of our experimental research radar screen. Patents / Patent applications Thesekidney efforts injury are(AKI) motivated is in the bycentral the gaps segment that EP 11156004.1, WO/EP 12710456.0, WOP 11708427.7, WO/US 14/001,506, US 13/644,947, DE 102012109419.5, US 13/853,825, EP 13161942.1, EP 13161948.8, AKI prevention and therapies still largely EP 13161952.0, EP 13163723.3, EP 13156151.6, DE 102013005612.8 rely on empirical clinical knowledge and that translational approaches are still scarce. We Selected Publications successfully combined MRI and quantitative 1. Niendorf T, Graessl A, Thalhammer C, Dieringer MA, Kraus O, Santoro D, Fuchs physiological techniques to monitor renal K, Hezel F, Waiczies S, Ittermann B, Winter L. Progress and promises of human hemodynamics in an innovative hybrid setup 2013;229:208-222. cardiac magnetic resonance at ultrahigh fields: a physics perspective. J Magn Reson (MR-PHYSIOL) with the goal to translate the 2. Graessl A, Renz W, Hezel F, Dieringer MA, Winter L, Oezerdem C, Rieger J, Kellman diagnostic capabilities of parametric MRI P, Santoro D, Lindel TD, Frauenrath T, Pfeiffer H, Niendorf T. Modular 32-channel from experimental research to improved clin- transceiver coil array for cardiac MRI at 7.0T. Magn Reson Med, doi: 101002/ mrm24903 2013. ical understanding of hemodynamics in AKI. 3. Meloni A, Hezel F, Positano V, Keilberg P, Pepe A, Lombardi M, Niendorf T. Detailing - Future Directions dial effective transverse relaxation rate at 1.5, 3.0, and 7.0 T. Magn Reson Med, doi: magnetic101002/mrm24856 field strength 2013. dependence and segmental artifact distribution of myocar Our strategic research will continue to be 4. Fuchs K, Hezel F, Klix S, Mekle R, Wuerfel J, Niendorf T. Simultaneous dual contrast weighting using double echo rapid acquisition with relaxation enhancement formed around MR physics and drive/sup- (RARE) imaging. Magn Reson Med, doi 101002/mrm25066 2013. port collaborations and translational explora- 5. Winter L, Ozerdem C, Hoffmann W, Santoro D, Muller A, Waiczies H, Seemann R, tions into cardio- and neurovascular diseases. Graessl A, Wust P, Niendorf T. Design and evaluation of a hybrid radiofrequency ap- We are dedicated to throw further weight plicator for magnetic resonance imaging and rf induced hyperthermia: electromag- behind the solution of the remaining unmet 2013;8(4):e61661. clinical needs. To meet this goal MR technol- netic field simulations up to 14.0 Tesla and proof-of-concept at 7.0 Tesla. PLoS One ogy remains to be developed and perfected. The development of next generation RF coil technology and imaging techniques will be tailored to demonstrate the clinical feasibil- Group Leader Eva Irene Oberacker ity and value of UHF-MR. On the MR physics Prof. Dr. Thoralf Niendorf Eva Peper side we strive to extend our activities and Joao dos Santos Periquito master MR electrodynamics at ultrahigh Scientists Michael Schwerter - Dr. Karen Arakelyan Tiago Fernando Ferreira da cal simulations and by seeking collaboration Dr. Min-Chi Ku Silva withfields institutions by progressing equipped further with our9.4 T, numeri 10.5 T Dr. Andreas Pohlmann or 11.7 T magnets, with the ultimate goal to Dr. Sonia Waiczies Technical Assistants - Babette Dieringer tem to B.U.F.F. We also want to extend our ex- PhD students plorationsattract an extremeinto very field promising whole innovativebody MR sys MR Katharina Maria Fuchs Sabrina Klix applications including feasibility studies on Andreas Graessl StefanieAntje Els Kox targeted drug delivery facilitated by RF heat- Fabian Hezel Blanca Lopez-Aranguren ing. Following the unanimously outstanding Marko Hoehne Blazquez and high praise (2013 POF evaluation) of our Till Huelnhagen Darius Georg Lysiak Tracer Discovery and Metabolic Imaging Oliver Kraus Karolin Voß Program (including MR-PET imaging)pro- Stefano Lepore posalwe will proceed and push to turn this Celal Özerdem Secretariat - Christof Thalhammer Rosita Knispel hind the CMD program of the MDC and per- Lukas Winter initiative into reality. It puts major weight be Other/Guests BIH. The establishment of this program will Graduate and Undergraduate Matthias Dieringer furtherfectly fits serve into to the close strategic the gap objectives between of in thevi- Students Dr. Petr Dusek tro discoveries and human (patho)physiology Martin Guenther Dr. Vince Madai and will change the landscape of molecular- Yiyi Ji Dr. Jason Millward oriented metabolic phenotyping. It provides Roman Leicht Dr. Florence Razoux strong synergies between the MDC’s and Conrad Martin Henning Reimann FMP’s present strength in target discovery, Maximilian Muhle Dr. Jens Würfel screening technologies and omics with tracer

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Matthias Selbach

Cell Signalling and Mass Spectrometry

Understanding how the genomic informa- corresponding proteins. SILAC can be used tion is interpreted to yield a specific pheno- to quantify differences in steady-state protein levels (Fig. 1, left). In addition, pulsed type is perhaps the most important ques- SILAC can be employed to measure differ- tion in the post-genomic era. Proteins are ences in protein synthesis (Fig. 1, middle). central players in this process: On the one Finally, dynamic SILAC can reveal protein hand, they are the final product of most turnover (Fig. 1, right). Combined with high throughput mass spectrometry, the differ- genes. On the other hand, proteins are also directly responsible for cellular phenotypes. of different aspects of proteome dynamics Proteins thus represent the central link be- onent a variants global scale. of SILAC enable quantification tween the genome and the phenotype. We Gene expression control are using quantitative mass spectrometry- based proteomics as our central technol- The four fundamental cellular processes in- ogy to investigate proteome dynamics on volved in gene expression are transcription, mRNA degradation, translation and protein a global scale. The lab is interested in two degradation. Each of these four steps is con- major questions. First, how is the genomic trolled by gene-regulatory events. So far, lit- information processed to yield a specific tle is known about how the combined effect proteome? Second, how do proteins that of all regulatory events shapes gene expression. The fundamental question of how ge- are expressed at a specific cellular condi- nomic information is processed to obtain a tion affect the phenotype? Answering the first question requires information about largely unknown. We are using metabolic pulsespecific labelling cellular approaches proteome is to therefore comprehen still- protein synthesis and degradation. System- sively quantify gene expression. Protein atic analysis of protein-protein interactions and posttranslational modifications can help SILAC. Similarly, newly synthesized RNA answering the second question. canturnover be labelled can be with quantified nucleoside using analogues. dynamic Mass spectrometry and next generation sequencing (in collaboration with the lab of Recently developed quantitative methods Wei Chen) then allows us to quantify the make it possible to obtain precise func- absolute abundance of mRNAs and pro- tional information and to monitor temporal teins and their half-lives in parallel. These changes in the proteome by mass spectrom- data can then be used to calculate synthesis etry. In one approach, named SILAC (for rates of mRNAs and proteins by mathemati- stable-isotope labelling with amino acids in cal modeling (collaboration with the group cell culture), cells are differentially labelled of Jana Wolf). Our results indicate that gene by cultivating them in the presence of ei- - ther normal or a heavy isotope–substituted inantly controlled at the level of translation amino acids. Due to their mass difference, (Schwanhausser,expression in mouse 2011). fibroblasts Consistently, is predom we pairs of chemically identical peptides of different stable-isotope composition can be during Schwann cell development in vivo distinguished in a mass spectrometer. The (collaborationfind that translation with the is lab actively of Carmen regulated Birch- ratio of intensities for such peptide pairs ac- meier, Sheean et al., 2014). Currently, we are investigating how the different levels of

curately reflects the abundance ratio for the 52 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Different variants of SILAC for the analysis of cellular proteome dynamics. Cells are metabolically labelled with light (L), medium-heavy (M) or heavy (H) stable isotope-encoded amino acids. Differentially labelled peptides can be distinguished by mass spectrometry. The different variants of SILAC allow global quantification of different aspects of proteome dynamics.

gene expression change upon perturbation. eas of biomedical research, especially in ge- In addition, we are using pulsed SILAC (pSI- netics and development. We have extended LAC) to directly quantify changes in protein the SILAC technology to Caenorhabditis ele- synthesis. For example, we are studying the gans (collaboration with the lab of Nikolaus impact of microRNAs and RNA-binding pro- Drosophila melanogaster teins on protein production. (Sury et al., 2010; Grun et al., 2014). Cur- Rajewsky)rently, we are and using these models to study Protein-protein interaction protein-protein interactions in vivo.

Proteins typically interact with other pro- Selected Publications Identifying interaction partners therefore Sheean, M.E., McShane, E., Cheret, C., ..., Selbach, M., and Birchmeier, C. (2014). Activa- providesteins to exertdirect a insights specific into cellular protein function. function of MAPK overrides the termination of myelin growth and replaces Nrg1/ErbB3 signals during Schwann cell development and myelination. Genes Dev 28, 290-303. tion and can reveal disease mechanisms. We are using quantitative mass spectrom- (2014). Conservation of mRNA and Protein Expression during Development of C. etry and to analyze protein-protein inter- Grun,elegans. D., CellKirchner, Rep. M., Thierfelder, N., Stoeckius, M., Selbach, M., and Rajewsky, N. actions (Paul et al., 2011). This approach Paul, F.E., Hosp, F., and Selbach, M. (2011). Analyzing protein-protein interactions by has two unique advantages. First, accurate quantitative mass spectrometry. Methods 54, 387-395. - Schwanhausser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W., and

Nature 473, 337-342. quantification allows us to distinguish spe Selbach, M. (2011). Global quantification of mammalian gene expression control. cific interaction partners from background- actionscontaminants change with in response very high to confidence. perturba- Sury, M.D., Chen, J.X., and Selbach, M. (2010). The SILAC fly allows for accurate protein tion.Second, We quantificationare using this reveals method how to interstudy quantification in vivo. Mol Cell Proteomics 9, 2173-2183. how disease-associated mutations and cell signaling events affect protein-protein interactions. We are also investigating how Group Leader Prof. Dr. Matthias Selbach Erik McShane virus proteins differ between strains with Katrina Meyer differentcellular interaction pathogenic partners potential. of influenza A Scientist Florian Paul Dr. Katrin Eichelbaum In vivo quantitative proteomics: Dr. Koshi Imami The SILAC zoo Dr. Marieluise Kirchner TechnicalDjordje Vasiljevic Assistants Dr. Henrik Zauber Martha Hergeselle Cell culture-based experiments cannot re- Christian Sommer capitulate all of the complex interactions PhD among different cell types and tissues that Boris Bogdanow Secretariat occur in vivo. Small animal models such as Jiaxuan Chen Sabine Fröse - Kamila Kutz Petra Haink tives that are extensively used in many ar- worms and fruit flies are attractive alterna

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Ludwig Thierfelder

Cardiovascular Molecular Genetics

Inherited cardiomyopathies, programming Arrhythmogenic right ventricular of the fetal heart and the cardiac role of cardiomyopathy Arnd Heuser nuclear receptors represent the major foci of our research group. Arrhythmogenic right Arrhythmogenic right ventricular cardio- ventricular cardiomyopathy, a difficult-to- myopathy (ARVC) is a heritable primary cardiac muscle disorder characterized by diagnose inherited heart muscle disorder, - originates from mutated desmosomal pro- brofatty tissue, predominantly in the right teins ultimately causing severe structural al- ventriclethe replacement (RV). The of cardiomyocytesresulting disruption with fiof terations, dangerous ventricular arrhythmias normal myocardial architecture can lead to RV dysfunction, life-threatening arrhyth- and heart failure. Correct fetal programming mias, and sudden cardiac death. Disease is known as a prerequisite for normal function in adulthood but little is known on how mainly in genes encoding desmosomal pro- this is achieved at the molecular level. A teins.causing mutations have been identified second focus of our research is, therefore, the molecular dissection of adequate pro- plakophilin2 (PKP2) and desmocollin2 gramming steps in the fetal heart. Inter- (DSC2)We were as the a cause first toof identifyautosomal mutations dominant in - fering with cardiac mitochondrial function cant proportion of ARVC cases (10-45%). during development is our tool to shed light PKP2ARVC. PKP2and DSC2 mutations are accountcomponents for a signifiof the on this issue. Whether or not liver X recep- - tor agonists are potential targets for the plex (Figure 1) known to be essential for maintainingdesmosomal tissue intercellular integrity junction and increas com- treatment of metabolic, inflammatory and ingly implicated in cell signaling. While cardiovascular diseases is the third topic of the involvement of multiple desmosomal our interest. protein in ARVC has led to speculations about the sensitivity of the myocardium to mechanical disruption, the true pathogenic mechanisms of ARVC causing mutations are largely unknown.

theDesmosomes intercalated and disc, adherence the area composita. junctions Maturebuild a specialdesmosomes “mixed” with type extended of junction area in composita structures in the heart form in a late, primarily postnatal process. Accord- ingly, we hypothesize that desmosomes

54 MDC Research Report 2014 CARDIOVASCULAR AND METABOLIC DISEASE

Figure 1: Structural organization of desmosomes. (A) Electron micrograph of desmosomes from murine heart. (B) The schematic drawing shows desmosomal cadherins (DSG, desmoglein; DSC, desmocollin); the armadillo-family members plakoglobin (PG) and the plakophilins (PKP); the desmosomal linker-protein desmoplakin (DSP); and the intermediate filament-binding desmin (DES).

play an essential role for the mechanical synthase (Hccs), an enzyme essential for integrity of cardiomyocytes in the adult normal function of the mitochondrial elec- heart. To test this hypothesis we investi- - veloping mouse heart. Loss of Hccs activity different desmosomal components in adult resultstron transport in cellular chain, energy specifically starvation in the caus de- mice.gate cardiac-specificAblation of the essential knockout desmosomal models of ing disturbed cardiomyocyte differentia- proteins Pkp2 and Dsp lead to substantial tion and ultimately cellular degeneration. structural changes at the intercalated disc. In contrast to the observed mid-gestational - lethality of hemizygous Hccs knockout (KO) naling properties of these proteins in the males, heterozygous females appeared nor- heart.Ongoing This work should defines shed the light adhesive on the and patho sig- mal during postnatal life with surprisingly genicity of ARVC, thereby facilitating the few clusters of defective myocardium, con- development of new diagnostic procedures sidering an expected mosaic of affected and and therapeutic strategies. normal cardiomyocytes as a result of random X chromosomal inactivation. However, analyses of heterozygous female (Hccs+/-) Embryonic heart regeneration, embryos revealed the expected 50:50 ratio fetal programming and cardiac organ size control mid-gestation with a progressive reduction Jörg Drenckhahn inof diseaseHccs deficient tissue to 10%normal prior cardiac to birth. cells We at

The prenatal heart possesses an impres- accounted for by increased proliferation of sive growth plasticity in response to both remainingcould show healthy that this cardiac significant cells. These change data is endogenous as well as environmental or reveal a previously unrecognised but im- maternal conditions. In this regard, our repressive regenerative capacity of the mid- - gestational heart that can compensate for onic murine heart has a remarkable regen- an effective loss of at least 50% of cardiac erativecent findings capacity. have We shown have thatinactivated the embry the tissue to enable formation of a functional X-linked gene encoding Holocytochrome c heart at birth.

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Yet despite this regeneration, hearts of however did not affect cardiac contractility. neonatal Hccs+/- females are hypoplastic at - birth evident as a reduction in heart weight, ability appears to be primarily important thinning of left ventricular (LV) walls and forIn conclusion,postnatal hypertrophic sufficient amino but not acid prenatal avail - proliferative growth of the heart. Current myocytes compared to littermate controls. Thea significantly reduction reducedin LV mass, number however, of cardio nor- the role of amino acid availability during malizes until adulthood and this could be theprojects transition intend from to thoroughly pre- to postnatal characterize car- attributed to an increase in cardiomyocyte diac growth pattern. size (hypertrophy) in Hccs+/- females (while proliferation rates are unaltered suggesting Unfavorable intrauterine growth condi- that cardiomyocyte number is not normal- tions have been shown to predispose the ized). These data suggest the activation of heart for cardiac disease later in life, a pro- compensatory cardiac growth mechanisms cess referred to as fetal programming. To in the postnatal heart after disturbed heart study the consequences of embryonic heart development. Investigation of the under- - lying molecular mechanisms revealed ed Hccs+/- females to chronic Angiotensin alterations of several metabolic regula- regenerationII infusion. Although for the adult Ang heartII did we not subject affect tors, such as mTOR and Ampk, as well as cardiac function when compared to con- activation of ER (endoplasmic reticulum) trols, it dramatically altered cardiomyocyte stress and amino acid metabolism. mTOR, growth kinetics as well as molecular stress an important regulator of translation and response resulting in hyperactivation of cell growth, is intensively studied in the JAK/STAT3 signaling in Hccs+/- hearts. This context of pathological as well as physiological cardiac hypertrophy in adulthood, cardiac development renders the response however, surprisingly little is known about ofclearly the postnatal confirmed heart that to disturbingpathological prenatal condi- its relevance for the prenatal heart. There- tions. Additionally, pharmacological inhibi- fore, we developed a rapamycin treatment tion of JAK/STAT3 during Ang II stress impairs the compensatory growth response in inhibit mTOR in fetuses during late gesta- Hccs+/- hearts resulting in contractile dys- tion.protocol Prenatal in pregnant rapamycin mice treatment to efficiently results function when compared to controls. Based in general growth restriction, evident as on these data we propose a new model link- a 12% reduction in body weight at birth. ing fetal programming to growth and organ Importantly, heart weight was reduced by size control in the adult and ageing heart. 33% (Figure 2), resulting in a reduced heart weight to body weight ratio in rapamycin versus vehicle treated neonates. Other or- Role of Nuclear Receptors in Car- gans were less affected, highlighting the diac Metabolism and Hypertrophy importance of mTOR for perinatal cardiac Florian Blaschke

study the role of mTOR during embryonic Cardiac hypertrophy leading to heart fail- andgrowth. fetal Current heart development projects intent using to genetic further - models of heart conditional inactivation of tality worldwide. The reason why cardiac mTOR components in mice. dilatationure is a major and causefailure of eventually morbidity occurand mor are unknown although alterations in energy To assess the importance of amino acid status and metabolism are proposed to play availability for cardiac growth and organ an important role. An understanding of the size control, we kept pregnant mice on a role of cardiac energy metabolism and hy- low protein diet throughout pregnancy. pertrophic gene expression in the pathol- Such amino acid restriction did not alter ogy of cardiomyopathy may consequently developmental patterning or growth of the lead to more effective novel therapeutic embryonic heart, but results in reduced strategies for the prevention and treatment heart size in adult mice if kept on a low of heart failure. We are currently elucidat- protein diet after birth. The latter is the re- ing the role of nuclear receptors in cardiac sult of reduced cardiomyocyte size, which metabolism and hypertrophy and charac-

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Figure 2: Prenatal mTOR inhibition results in impaired cardiac growth and reduced heart size at Selected Publications birth. Pregnant female mice were treated with either Heuser A, Plovie ER, Ellinor PT, Grossmann, KS, Shin JT, Wichter T, Basson CT, Lerman vehicle or the mTOR inhibitor rapamycin during late BB, Sasse-Klaassen S, Thierfelder L, MacRae CA, Gerull B. (2006). Mutant desmocol- gestation. Newborn hearts of the resulting offspring lin-2 causes arrhythmogenic right ventricular cardiomyopathy (ARVC). Am J Hum were embedded in paraffin, sectioned and stained Genet. Dec;79 (6):1081-8. with hematoxylin & eosin (H&E). Overview images Gerull B, Heuser A, Wichter T, Paul M, Basson CT, McDermott DA, Lerman BB, Markow- itz SM, Ellinor PT, Macrae CA, Peters S, Grossmann KS, Michely B, Sasse-Klaassen S, show a striking reduction in heart size in rapamycin Birchmeier W, Dietz R, Breithardt G, Schulze-Bahr E, Thierfelder L.(2004). Muta- compared to vehicle treated newborns. LV/RV = left tions in the desmosomal protein plakophilin-2 are common in arrhythmogenic right and right ventricle, LA/RA = left and right atrium, ventricular cardiomyopathy. [Corrigendum in: Nat Genet. 2005 Jan;37(1):106]. Nat. Genet. 36, 1162-1164. Ao = aorta (scale bar = 300 μm) Kirchner F, Schuetz A, Boldt LH, Martens K, Dittmar G, Haverkamp W, Thierfelder L, Heinemann U, Gerull B.(2012). Molecular insights into arrhythmogenic right ventricular cardiomyopathy caused by plakophilin-2 missense mutations. Circ Cardiovasc Genet. 5(4):400-11. Drenckhahn JD, Schwarz QP, Gray S, Laskowski A, Kiriazis H, Ming Z, Harvey RP, Du XJ, Thorburn DR, Cox TC (2008). Compensatory growth of healthy cardiac cells in the terize the molecular mechanism(s) utilized presence of diseased cells restores tissue homeostasis during heart development. to regulate gene expression involved in Developmental Cell 15 (4): 521-533. cardiomyocyte growth and energy metabo- Blaschke F, Takata Y, Caglayan E, Collins A, Tontonoz P, Hsueh WA, Tangirala RK. A lism. nuclear receptor corepressor-dependent pathway mediates suppression of cytokine- induced C-reactive protein gene expression by liver X receptor. Circ Res. 2006 Dec 8;99(12):e88-99. Epub 2006 Nov 16. Given their pleiotropic effects of nuclear

ligands in lipid and glucose homeostasis, cardiacreceptors energy and theirbalance activation and regulation by specific of Group Leader targeting nuclear receptor are emerging as Prof. Dr. Ludwig Thierfelder promisinginflammatory therapeutics gene expression, for the treatment new drugs of diabetes, obesity, atherosclerosis, resteno- Scientist Graduate and Undergraduate sis and heart failure. PD Dr. Florian Blaschke Charmaine Bridger Dr. Joerg-Detlef Drenckhahn Dr. Arnd Heuser Dominik Ewald Patrick Langner KarolineMaja Kinga Kokot Dziegelewska Dr. Daniel Romaker Kathleen Persicke Arsene Wabo PhD Maria Hennig Technical Assistants Ute Rimpler Robert Zinke Beatrice Leip Anja Conrad

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Tobias Pischon

Molecular Epidemiology

The Molecular Epidemiology Group studies metabolism (Aleksandrova, Cancer Prev Res 2011). Abdominal obesity is mostly ac- at the molecular level the relationship counted for by visceral fat, which is meta- between lifestyle, diet, genetic, metabolically active and secretes a variety of bolic, and environmental factors with risk cytokines and hormones. We investigated and outcome of chronic diseases in hu- the role of the adipocyte-derived hormones adiponectin and leptin (Aleksandrova, Curr man populations. The focus is on biomark- Nutr Rep. 2013). Adiponectin improves in- ers that have the potential for accurate sulin sensitivity and has antiangiogenic and precise assessment of exposure, properties. Leptin is a regulator of food intake and energy balance, and may also in- intermediary effects, and early disease duce tumor angiogenesis, reduce apoptosis, development. Our aims are to contribute and promote cell growth and migration. In to the understanding of disease etiology and pathogenesis in humans, to allow a between leptin concentrations and colon cancerEPIC we risk, found but noa strong significant inverse relationship relation- more precise prediction of diseases, and ship for the soluble leptin receptor (sOB- to improve the identification of high risk individuals as well as of the quantification of (Aleksandrova, the effect of interventions, thus envisioning CancerR), which Res was 2012). statistically In addition, significant we found even highafter concentrations adjustment for of leptin non-high molecular a reduction of chronic disease risk through weight (HMW) adiponectin associated with targeted prevention. lower colorectal cancer risk (Aleksandrova, Carcinogenesis 2012). Out of 9 biomarkers, the association between abdominal obesity and colon cancer risk was accounted mostly for by 3 markers, HDL-C, non-HMW Metabolic factors and cancer risk adiponectin, and sOB-R (Aleksandrova, Int J Cancer. 2014). We are currently investigat- Obesity is associated with metabolic dis- ing the association between fetuin-a (a liver eases, including cardiovascular diseases biomarker related to insulin resistance) and cancer (Aleksandrova, Front Biosci and risk of colorectal cancer (Nimptsch). (Elite Ed) 2013). Abdominal obesity contributes to chronic disease risk and mortal- The liver is central in human metabolism, ity beyond general obesity (Pischon, N Engl and obesity is closely associated with non- J Med 2008), and is associated with abnor- alcoholic steatosis, steatohepatitis, liver mal glucose metabolism, elevated blood cirrhosis, and liver cancer. We studied pro- pressure, dyslipidemia, and type 2 diabetes mellitus, which cluster within the Metabolic and metabolic biomarkers with risks of Syndrome. Within the EPIC study we found liverspectively and bilary the association tract cancers of (Aleksandrova, inflammatory that the Metabolic Syndrome is associated Hepatology 2014). After multivariable ad- with a higher risk of colon cancer and that this association is largely accounted for by of CRP, IL-6, C-peptide and non-HMW adi- abdominal obesity and abnormal glucose ponectinjustment, associated we found with higher higher concentrations risk of he-

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patocellular carcinoma, suggesting that ele- examined the association of red meat and and hyperinsulinemia are associated with whole-grainiting inflammation bread andconsumption oxidative stress.with bio We- highervated levels risk of of HCC,biomarkers independent of inflammation of obesity markers related to metabolic pathways in and established liver cancer risk factors. the EPIC-Potsdam study (Montonen, Eur J Nutr. 2013). Higher consumption of whole-

Metabolic factors and diabetes with lower levels of GGT, ALT and hs-CRP, risk whereasgrain bread higher was consumption significantly of associatedred meat

A number of biomarkers have been proposed for the estimation of type 2 diabetes potentialwas significantly confounding associated factors with related higher to (T2D) risk. We previously showed that out levels of GGT and CRP when adjusted for of 7 biomarkers the highest relative con- BMI and waist circumference attenuated tribution to risk of T2D was observed for thelifestyle association and diet. between Further red adjustment meat and CRP for adiponectin, followed by GGT and HDL-C, (P=0.19). These data suggest that high con- and less so for CRP (Montonen, Eur J Epi- sumption of whole-grain bread is related to demiol 2011). Design and advancement of lower levels of GGT, ALT and hs-CRP, where- high-throughput techniques determined as high consumption of red meat is associ- the emergence of metabolomics, which is ated with higher circulating levels of GGT the simultaneous study of numerous low- and CRP. molecular weight compounds. We prospec- tively investigated the association between 163 serum metabolites measured by tar- source of iron intake in humans and related geted metabolomics and risk of T2D in EP- toConsumption T2D risk. Impaired of red meatglucose is metabolism the major IC-Potsdam (Floegel, Diabetes 2013). Serum and diabetes mellitus are common clini- hexose; phenylalanine; and diacyl-phos- cal manifestations of iron overload in pa- phatidylcholines C32:1, C36:1, C38:3, and tients with haemochromatosis, a disorder C40:5 were independently associated with of abnormal iron absorption resulting in increased risk of T2D and serum glycine; the progressive accumulation of iron in sphingomyelin C16:1; acyl-alkyl-phospha- inner organs. We examined the associa- tidylcholines C34:3, C40:6, C42:5, C44:4, tion of body iron stores with risk of type 2 and C44:5; and lysophosphatidylcholine diabetes in EPIC-Potsdam (Montonen, Dia- C18:2 with decreased risk. Variance of the betologia 2012). - metabolites was largely explained by two ment, higher serum ferritin concentrations metabolite factors in opposing directions. were associated Afterwith multivariablea higher risk ofadjust type The data indicate that metabolic alterations, including sugar metabolites, amino observed for soluble transferrin receptor. acids, and choline-containing phospholip- These2 diabetes. results No support significant the associationhypothesis that was ids, are associated early on with a higher higher iron stores below the level of hae- risk of T2D. mochromatosis are associated with risk of type 2 diabetes.

Diet, nutrition, lifestyle Diet and physical activity are among

High consumption of red meat is associ- chronic diseases in Western civilizations. ated with increased risk of colorectal can- Yet,the mainit is less determinants clear what for determines risk of major diet cer, cardiovascular disease and type 2 dia- and physical activity. As part of the BMBF betes, whereas high intake of whole grains funded Knowledge Hub on the Determi- is related to reduced risk. Consumption of nants of Diet and Physical Activity Choice red meat may increase insulin resistance, (DEDIPAC), which is part of the European Joint Programme Initiative “A Healthy Diet intake of whole grains may provide protec- for a Healthy Life” we are investigating de- tionoxidative by improving stress and insulin inflammation, sensitivity, whereas lower- terminants of diet and physical activity. In ing insulin and glucose levels, and by inhib- addition, within the ActivE study, we are in-

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Serum metabolites and risk of type 2 diabetes. (a) metabolite factors identified by principal component analysis; (b) association of metabolite factors with risk of incident type 2 diabetes. Source: Floegel et al, Diabetes 2013

vestigating determinants of physical activity related energy expenditure. Further, we network of German research institutions, examine associations of adipose tissue gene includingprevention the of MDC, major has chronic started diseases, a large a - prospective cohort to be used as a common, tion, diet, and lifestyle. national resource for studies on risk factors expression profiles with metabolism, nutri in the German population. The National Genetic Variation in metabolic Cohortand etiologic will include mechanisms 200,000 of participants,major diseases to factors and risk of chronic be recruited through a network of 18 study diseases centers, organized in 8 clusters throughout Germany. At each center, a random sample of We previously observed a positive associa- the general population will be drawn within - strata of age and sex. All participants will be matory marker CRP and risk of colorectal invited to the study centers to take part in cancertion between (CRC) circulating in the EPIC levels study of the (Aleksan inflam- physical and medical examinations, collec- drova, Am J Epidemiol 2010). In a further tion of biomaterials, personal interviews, step, we applied the concept of Mendelian Randomization (MR) to investigate whether will be re-invited for a second examina- genetic variants associated with lifelong dif- and to fill in questionnaires. All participants ferences in CRP concentrations are associ- Participants will be re-contacted every 2-3 ated with risk of CRC. We observed that CRP tion five years after baseline recruitment.- genetic variability leading to raised CRP naires about changes in lifestyle and other concentrations was associated with higher characteristicsyears and asked and to about fill in the short occurrence question of risk of CRC (Nimptsch), supporting the hy- - pothesis that circulating CRP may play a tality follow-up and systematic record link- causal role in the etiology of this cancer. MR ageselected, with majorexisting diseases. disease In registries parallel, willa mor be studies will also be conducted to investigate performed periodically. Biomaterials will potential causality of the adipokines adipo- be stored in a centr alized biobank and in nectin, leptin, sOB-R and fetuin-a. decentralized storages. Within this initiative, the Molecular Epidemiology Group coordinates the Cluster Berlin-Brandenburg, The National Cohort which consists of the MDC, the Charité- University Medical Center Berlin, and the With the aim to develop new strategies German Institute of Human Nutrition Pots- for risk assessment, early detection, and dam-Rehbruecke (DIfE). The Cluster com-

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prises three study centers, which will re- Brandenburg study centers of the German cruit 30,000 participants into the National National Cohort. The measurements will Cohort. be the cornerstone for future analysis on exposure disease relationships.

Pretests for the National Cohort Selected Publications Aleksandrova K, Boeing H, Nothlings U, Jenab M, Fedirko V, Kaaks R, Lukanova A, preparation of the National Cohort was the Trichopoulou A, Trichopoulos D, Boffetta P, Trepo E, Westhpal S, Duarte-Salles T, St- implementationA major foundation of pretests for the to planning assess andthe Lagiou P, Bamia C, Benetou V, Agnoli C, Palli D, Panico S, Tumino R, Vineis P, Bueno-de- feasibility of methods for recruitment of epienMesquita M, Overvad B, Peeters K, PH,Tjonneland Gram IT, A, Lund Halkjaer E, Weiderpass J, Boutron-Ruault E, Quiros MC, JR, DossusAgudo A, L, SanchezRacine A, study participants and for assessment of MJ, Gavrila D, Barricarte A, Dorronsoro M, Ohlsson B, Lindkvist B, Johansson A, Sund study data. In addition, the pretests formed biomarkers and risk of liver and bilary tract cancer. Hepatology 2014. M, Khaw KT, Wareham N, Travis RC, Riboli E, Pischon T. Inflammatory and metabolic the basis to build up recruitment centers Aleksandrova K, Drogan D, Boeing H, Jenab M, Bas Bueno-de-Mesquita H, Jansen E, van

pretests, we have recruited 420 partici- DuijnhovenChapelon F, LukanovaFJ, Rinaldi A, S, TrichopoulouFedirko V, Romieu A, Trichopoulos I, Kaaks R, Riboli D, Vidalis E, Gunter P, Panico MJ, S,Romaguera Agnoli pantsand hire from qualified Berlin personnel.and Brandenburg. Within these The D,C, PalliWesthpal D, Tumino S, Overvad R, Vineis K, Tjonneland P, Buckland A, G, Halkjaer Sanchez-Cruz J, Boutron-Ruault JJ, Dorronsoro MC, M, Clavel- Diaz MJ, participants underwent a base set of de- Barricarte A, Ramon Quiros J, Peeters PH, May AM, Hallmans G, Palmqvist R, Crowe FL, Khaw KT, Wareham N, Pischon T. Adiposity, mediating biomarkers and risk of colon tailed interviews and physical examination cancer in the European Prospective investigation into cancer and nutrition study. Int J that were standardized across all study cen- Cancer 2014;134:612-21. ters. In addition, we have conducted feasi- Floegel A, Stefan N, Yu Z, Muhlenbruch K, Drogan D, Joost HG, Fritsche A, Haring HU, bility studies in collaboration with partner Hrabe de Angelis M, Peters A, Roden M, Prehn C, Wang-Sattler R, Illig T, Schulze MB,

institutions, to evaluate methods to assess risk of type 2 diabetes using a targeted metabolomic approach. Diabetes 2013;62:639- Adamski48. J, Boeing H, Pischon T. Identification of serum metabolites associated with and oral health. Further, we have developed - methodsphysical activity,for shipment physical and fitness, long-term nutrition, stor- hoven FJ, Rinaldi S, Fedirko V, Romieu I, Riboli E, Gunter MJ, Westphal S, Overvad Aleksandrova K, Boeing H, Jenab M, Bueno-de-Mesquita HB, Jansen E, van Duijn age of biomaterials. During the pretests, R, Lukanova A, Trichopoulou A, Lagiou P, Trichopoulos D, Mattiello A, Pala V, Palli blood samples have been collected from K,D, TjonnelandTumino R, Vineis A, Halkjaer P, Buckland J, Racine G, Sanchez A, Boutron-Ruault MJ, Amiano MC, P, Huerta Clavel-Chapelon JM, Barricarte F, Kaaks A, 1576 participants from the three study Menendez V, Peeters PH, Soderberg S, Palmqvist R, Allen NE, Crowe FL, Khaw KT, Wareham N, Pischon T. Leptin and soluble leptin receptor in risk of colorectal cancer centers of the Cluster Berlin-Brandenburg, in the European Prospective Investigation into Cancer and Nutrition cohort. Cancer and are being stored at the MDC. Further- Res 2012;72:5328-37. more a study protocols for the assessment Montonen J, Boeing H, Steffen A, Lehmann R, Fritsche A, Joost HG, Schulze MB, Pischon of body measures using a 3-dimensional T. Body iron stores and risk of type 2 diabetes: results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study. Diabetologia. 2012 body surface scanner has been developed Oct;55(10):2613-21. and evaluated.

MRI measurements within Group Leader the National Cohort Prof. Dr. Tobias Pischon, MPH

Magnetic resonance imaging (MRI) permits Scientists comprehensive imaging using highly stan- Dr. Jürgen Janke Ben Jacoby dardized protocols without ionizing radia- Sabine Mall Annelie Richter tion. In prospective settings, it offers the Dr. Katharina Nimptsch Manuela Stendal opportunity to study the relevance of pre- Dr. Astrid Steinbrecher Jana Treutler clinical morphological changes for chronic Anette Veauthier disease risk. In the German National Co- PhD students hort, whole-body 3T MRI measurements Stephanie Jeran Secretariat will be taken from 30.000 participants. Stefan Konigorski Marit Schneidereit At the MDC’s Berlin Ultrahigh Field Facil- Lina Jaeschke ity (B.U.F.F.), the Molecular Epidemiology MPH/MSE Students Group, the Experimental Ultrahigh-Field Technical Assistants Dr. Bettina Broecheler MR group, and the Cardiac MRI group Henning Damm Dr. Daniela Hoedke will conduct MRI measurements of 6.000 Julia Glöde Carolina Schwedhelm study participants from the three Berlin-

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Matthew N. Poy

microRNA and Molecular Mechanisms of Metabolic Diseases

Type II diabetes has finally become recog- understanding of how these small RNAs in- nized as a major challenge to global health. tegrate into the already complex landscape of regulating gene expression. Most nota- It is imperative to improve our understand- bly, based on a number of miRNA knockout ing of the molecular mechanisms behind mouse models showing subtle phenotypes this disorder and develop new drug thera- under steady state conditions, a hypothesis pies. The pathophysiology of diabetes is has gradually emerged suggesting the miR- NA pathway contributes to cellular stress undoubtedly complex, typically character- responses. Moreover, these observations ized by hyperglycemia resulting from vary- are further supported by our recent study ing states of insulin resistance and impaired focusing on the role of Argonaute2 (Ago2) b-cell function. Oftentimes, the failure to in the compensatory proliferation of the regulate circulating blood glucose levels is a consequence of the inability to produce yearspancreatic since β-cell our initial during study insulin which resistance. identi- sufficient amounts of insulin by the pan- These recent findings culminate over 10 among the highest expressed sequences in creatic -cells. In our group, we focus on β thisfied cell miR-375, type. a miRNA now established fundamental pathways regulating glucose metabolism and how altered pancreatic islet physiology contributes to metabolic disor- studies revealed this miRNA as a nega- tiveThe regulator very first of loss insulin and gain-of-functionrelease via the ders such as type 2 diabetes. direct targeting of the gene myotrophin. The role of miR-375 in insulin release was

mouse knockout (375KO). Furthermore, further confirmed in vivo using the total To date, numerous counter-regulatory several additional targets of miR-375 in- mechanisms have been characterized in the cludingthis model Elavl4/HuD, facilitated theCadm1, identification Gphn, and of cell illustrating the dynamic nature of the Rasd1. Moreover, 375KO mice exhibited intracellular environment. Among the more recently discovered is the microRNA (miR- and these effects were further exacerbated NA) pathway and as of this writing, 2578 afterhyperglycemia crossing the and knockout decreased onto β-cell the leptin- mass, mature human miRNAs have been anno- - tated in the miRBase database which now dicated that miR-375 plays an essential role catalogs 206 different plant and animal deficient ob/ob background. This result in- - duced by insulin-resistance in ob/ob mice. tion in C.elegans, a canonical pathway has Whilein the compensatorythe precise causes β-cell proliferationare not under in- species. Importantly, since their identifica- stood, insulin resistance is one of the most NAs are processed and incorporated into common metabolic stress conditions and Argonaute-containingnow been identified to complexes describe how to main miR- a consequence of chronic over-nutrition. tain their effect on gene expression. Many Moreover, our recent observations show- insights into the functional role of miRNAs ing loss of Ago2 also blocked proliferation have been recently made, improving our during insulin resistance further supports

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a role for the miRNA pathway in mediating such as miR-375 are expressed in several cellular stress responses. Importantly, we unique tissues such as the pituitary and showed that Ago2 is up-regulated in the adrenal gland, it is unclear how this miRNA pancreatic islets of both genetic and diet- may regulate growth and secretion in these induced models of insulin resistance and other neuroendocrine cell types. obesity as a result of the silencing of miR- 184. Furthermore, the inverse relationship - tifying components of the RNAi machinery - inIn summary,the nucleus in lightincluding of recent Ago2, findings Dicer, iden and between Ago2 and miR-184 was confirmed- GW182, it appears only a matter of time tionin islets in these of human mouse subjects models. further Of note, under miR- before new mechanistic insights into the 184lining was the shown relevance to target of studying the gene β-cell Slc25a22, func role of this pathway are discovered. While a mitochondrial glutamate transporter, indicating miRNAs may regulate many stages - remainsthe identification to be studied of direct in greater targets ofdetail, the ing to insulin secretion. themiRNAs role of and small their RNAs biological in the nucleus significance also of the canonical pathway in the β-cell lead remains to be described. The story of non- While miR-184 has been shown to potently coding RNAs and their functional role in the regulate both growth and secretion of the exciting time of rapidly evolving technolo- changes in insulin sensitivity as shown in gies,pancreatic the next β-cell 10 isyears far from are certain complete. to bringIn an theβ-cell, models it remains of obesity to may be determinedcontribute to how the clarity to this expanding narrative. direct regulation and function of all miR- NAs in this cell type. Meanwhile, it is un-

may also change in response to alterations withinclear whether its metabolic miRNA environment function in the such β-cell as levels of extracellular nutrients (glucose, amino acids or fatty acids), signaling hormones, neurotransmitters, or perturbations in cell-to-cell contact with neighboring endocrine, endothelial, mesenchymal, or neuronal cells. Moreover, key transcrip-

well as RNA-binding proteins that may spe- tional regulators of miRNAs in the β-cell as- Selected Publications - cifically bind miR-184 and target it for deg ty, SJ., Musahl, A., You, X., Chen, W., Herrera, P., O’Carroll, D., Eliasson, L., Zavolan, M., mayradation coordinately remain unidentified. target genes It to is promote unclear Tattikota, SG., Rathjen, T., Hausser, J., Pandey, AK., Wessels, HH., Esguerra, JLS., McAnul (2014) Cell Metabolism. 19(1):122-34. insulinhow the release additional as well abundant as facilitate β-cell compen miRNAs- and Poy, MN. Argonaute2 mediates compensatory expansion of the pancreatic β-cell.

- demand increases. While miR-184 was the Tattikota, SG., Sury, MD., Rathjen, T., Wessels, HH., Pandey, AK., You, X., Becker, C., Chen, satory expansion of the β-cell as metabolic W., Selbach, M., and Poy, MN. (2013) Argonaute2 regulates the pancreatic β-cell secre islets of insulin-resistant mice, additional tome. Molecular & Cellular Proteomics. 12, 1214–1225. miRNAsmost significantly have been regulatedshown to miRNAparticipate in the in

It will presumably be determined that nu- Group Leader Technical Assistants merousthe β-cell miRNAs growth act using in a human concerted islet fashion cells8. Dr. Matthew N. Poy Paulina Aranda Chale to regulate multiple targets that mediate Dr. Thies Kluessendorf cell growth and the continual recruitment Monika Schwarz of insulin-containing granules to the plas- PhD Rachel Schweiker ma membrane. Interestingly, it is still not Maria Bikou known whether the same miRNAs perform Secretariat an identical regulatory function in different Sudhir Gopal Tattikota Sylvia Olbrich cell types. As Ago2-associated sequences Thomas Rathjen

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CARDIOVASCULAR AND METABOLIC DISEASE Photo: David Ausserhofer/MDC Photo:

Mathias Treier

Genetics of Metabolic and Reproductive Disorders

Our research is focused on the genetic Stem cells, transcription factors regulation of metabolic and developmental and epigenetic regulation processes. Transcriptional regulators are at Stem/progenitor cell populations consti- the center of our investigations. In particu- tute the basic building units from which or- lar, we have started to look how metabolic gans and whole organisms are created. changes influence the epigenetic landscape modulating the transcriptional response to SALL4 as a key player that is required to meet the challenge during adaptation to maintainWe identified the pluripotencythe transcriptional state of regulator embry- novel environmental conditions. With a se- onic stem cells. SALL4 is highly expressed in the inner cell mass (ICM) of the blasto- ries of mouse models for human diseases cyst that gives rise to the embryo and the that we created over the last years, we are primitive endoderm. During the course of now in a position to dissect even complicated physiological questions at the organ- of the Notch pathway, as a Sall4 interaction our studies we identified Rbpjk, the effector- ismal level. partner. Subsequent purification of the Rb pjk protein complex confirmed the Sall4/- cupancyRbpjk interaction. of common F urthermore,DNA loci throughout ChIP-seq theanalysis genome. for Sall4 Furthermore, and Rbpjk we confirmed could show oc that Sall4 directly regulates transcriptional - ments providing a possible molecular ex- planationactivity of for a the Notch/Rbpkj observed neural response differen ele- tiation of ES cells upon Sall4 depletion.

At the moment, our interests are focused on how embryonic stem cell pluripotency and self-renewal is maintained at the molecular and biochemical level. Answers to these fundamental questions should enhance our

underlying embryonic stem cell pluripo- tencyunderstanding and self-renewal of epigenetic and provide modifications novel insights into the process of reprogramming of somatic genomes into a pluripotent or alternative cell fate.

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Murine embryonic stem cells stained with antibodies against DNMT3 and Nanog.

Sall4 staining of the inner cell mass (ICM) in a mouse blastocyst.

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Mouse brain cleared using the CLARITY technique. Shown is the expression of Histone2B-GFP nuclear reporter in hypothalamic BSX neurons. The whole intact tissue imaging technique allows an accurate representation of the 3D morphology of BSX neurons in the mouse hypothalamus.

With optogenetics it is possible to stimulate or inhibit the BSX neuronal network in its entirety or only in subpopulations. Subsequently, we can determine the impact on neuronal excitablity in response to dietary stressors such as high fat and carbohydrate-rich diets.

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Energy homeostasis and eating lations in order to understand its role in behavior the regulation of behavioral and metabolic pathways. In this respect, we will also chal- The ultimate goal for any living organism is to maintain energy homeostasis in its ability by dietary stressors including high fat and to survive. We are particularly interested in carbohydrate-richlenge the BSX-deficient diets to neuronal explore their network im- the neuronal circuits of the central nervous pact on neuronal excitability. system (CNS) that are involved in the (dys) regulation of energy homeostasis. With the In order to deconstruct the BSX hypothalamic network we will also employ CLAR- ITY (Clear, Lipid-exchanged, Anatomically brain-specificneuronal network homeobox within the protein hypothalamus BSX we Rigid, Imaging compatible, Tissue hYdrogel), thatidentified orchestrates an essential eating be playerhavior, marking sleep and a a technique that turns the brain transpar- higher cognitive functions. ent using the detergent SDS, which strips away lipids that normally block the pas- It was generally believed that increased body weight always goes hand in hand with acrylamide, which binds proteins, with reduced physical activity. In other nucleicsage of acidslight. andHere, other the brainbiomolecules. is first infused When words, reducing body weight would lead the acrylamide is heated, it polymerizes and to increased physical activity. However, we forms a tissue-wide mesh that secures the could show in ob/ob mice, which are de- molecules. By using this technique it is possible to visualize and trace, quantify and map levels were not restored when body weight - wasficient reduced for leptin, upon deletion that locomotor of the BSX activity gene. ergic neurons in an intact system in whole e.g. the axonal projections of the histamin

partThus, genetically our findings determined. demonstrated for the fluorochrome-expressing brains. first time that physical activity is at least in For us, the most fundamental question right Selected Publications now is to elucidate the molecular mechanisms that allow the BSX hypothalamic net- Coldren CD, Lai Z, Shragg P, Rossi E, Glidewell SC, Zuffardi O, Mattina T, Ivy DD, Curfs LM, Mattson SN, Riley EP, Treier M, Grossfeld PD (2009). Chromosomal work to coordinate the dialogue between microarray mapping suggests a role for BSX and Neurogranin in neurocognitive and cognitive and metabolic pathways. We are behavioral defects in the 11q terminal deletion disorder (Jacobsen syndrome). Neuro- in the process to characterize in detail the genetics. 2009 Apr;10(2):89-95 contributions of the different BSX neuronal Uhlenhaut N, Jakob S, Anlag K, Eisenberger T, Sekido R, Kress J, Treier AC, Klug- subpopulations throughout the hypothala- mann C, Klasen C, Holter N, Riethmacher D, Schütz G, Cooney A, Lovell-Badge R mus that contribute to this cross talk. & Treier M (2009). Somatic sex reprogramming of adult ovaries to testes by FOXL2 ablation. Cell 139:1130-1142. Experimentally, our studies will rely on the Attanasio M, Uhlenhaut NH, Sousa V, O’Toole JF, Otto E, Anlag K, Klugmann C, Treier AC, Helou J, Sayer JA, Seelow D, Nürnberg G, Becker C, Chudley AE, Nürn- generation of novel mouse models with con- berg P, Hildebrandt F & Treier M (2007) Loss of GLIS2 causes nephronophthisis in ditional inactivation of BSX itself or its identi- - humansSakkou M,and Wiedmer mice by increased P, Anlag K,apoptosis Hamm A,and Seuntjens fibrosis. Nature E, Ettwiller Genetics L,Tschop 39:1018-1024. M & ronal network. Subsequently, we will analyze Treier M thefied consequences targets in subpopulations of their altered of the expression BSX neu Hyperphagia and Locomotory Behavior. Cell Metabolism 5:450-463 with respect to feeding and sleep behavior. (2007). A Role for Brain-Specific Homeobox Factor Bsx in the Control of Elling U, Klasen C, Eisenberger T, Anlag K & Treier M (2006). Murine inner cell mass derived lineages depend on Sall4 function. PNAS 103(44), 16319-24. Taking advantage of two state-of-the-art methods, optogenetics and CLARITY initially developed by the Deisseroth lab in Stanford, California, we will further unravel the function of the BSX neuronal network in the brain Group Leader by correlating brain architecture to behav- Prof. Dr. Mathias Treier PhDs iors such as eating, movement and cognition. Mehrshad Aschmann Scientific Coordinator Jenny Rätzer Optogenetics is a technique that combines Dr. Nicole Huebener optics and genetics in order to control and Yu-Ting Tseng measure the activity of certain neurons in Scientists Fabio Rojas animal tissue. It involves inserting light- Christian Klasen Technical Assistants sensitive proteins from algae into neurons, Dr. Zuzanna Makowska Franziska Block allowing researchers to switch neuronal ac- Dr. Anna-Corina Treier tivity on and off with light. This will allow Dr. Petra Wiedmer Enya Longmuss us to stimulate or inhibit the BSX neuronal Dr. Xiushan Yin AnnikaAntje Brouwer-Lehmitz Winkler network in its entirety or only in subpopu-

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68 MDC Research Report 2014 Cancer Research

Coordinator: Claus Scheidereit

MDC Research Report 2014

Cancer Research

Claus Scheidereit

lmost half a million cancer cases are molecular understanding of mitochondria- diagnosed in Germany each year, based processes relevant for diseases, such Amaking advancements in diagnosis, as cancer. challenge in health research. Our mission is Selected scientific highlights totreatment understand and preventionthe principles of cancer of cancer a major development and progression and to provide Chronic myeloid leukemia (CML) often re- the basis for improved diagnosis and treat- sults from chromosomal translocations, ment. One aim is to discover and character- generating the oncogenic tyrosine kinase ize genes and cellular components that are BCR-ABL. The impact of other disease pro- important in the development and treat- moting pathways is poorly understood. The ment of cancer and that are promising as group of Achim Leutz reported that the novel targets for therapies. Another aim is to understand and therapeutically utilize interactions of the immune system with Wnt signal transducer β-catenin acts as an tumors. theamplifier deletion of a ofCML-specific the interferon gene signature.inducible transcriptionActivation of β-catenininfactor Irf8 combinationresults in highly with The MDC Cancer Research Program cov- aggressive leukemic stem cells and causes ers the three topics, (i) signaling pathways, the progression of CML into a fatal blast cell biology and cancer, (ii) structural and functional genomics and (iii) tumor im- targets in combinatorial therapies (Scheller munology and combines a spectrum of in- etcrisis, al., J. implying Exp. Med., Irf8 2013). and β-catenin as future terdisciplinary basic research laboratories and clinically oriented groups. A broad Estrogen-receptor (ER)-negative basal spectrum of experimental expertise is cov- breast cancers represent an aggressive ered, ranging from cell biology, biochem- subtype with poor prognosis. The group istry, structural, developmental and stem of Walter Birchmeier modeled cancer cell biology to immunology, experimental formation by combining activating muta- tumor models and clinical oncology. Cancer studies are conducted in close collabora- mouse mammary gland epithelial cells and tion with clinically oriented groups at the determinedtions of Wnt/β-catenin a Wnt-Met anddependent HGF/Met gene in Charité - Medical Faculty Berlin. signature, which predicts poor survival of human patients. The chemokine system In the reporting period, the international German-Israeli Research School “Frontiers driver of Wnt-Met tumors. Thus, targeting in Cell Signaling and Gene Regulation, Sign- CXCR4,CXCL12/CXCR4 Wnt and was Met identified might provide as a crucial an ef- Gene” (spokesman: Claus Scheidereit), funded by the Helmholtz Association, was (Holland et al., Cell Rep, 2013). ficient strategy for breast cancer treatment the Hebrew University of Jerusalem (HUJI), To model Burkitt lymphoma (BL), Klaus theestablished Technion as - aIsrael joint Instituteprogram of of Technolo the MDC,- Rajewsky and colleagues used targeted gy (TEC) in Haifa, the Humboldt University conditional mutagenesis, combining on- and the Charité. Oliver Daumke has been cogenic c-MYC overexpression and consti- awarded one of the prestigious European tutive PI3K signaling in germinal center Research Council (ERC) grants. He plans to B cells. The resulting mice reproduce the investigate the internal structure and dy- human disease in terms of histological ap- namics of mitochondria, to gain a precise pearance, gene expression signature, and

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“tertiary” mutations. Overexpression of c- Sieweke found that macrophage colony- MYC and constitutive PI3K signaling turned stimulating factor (M-CSF) promotes HSC out to be critical for the progression of the differentiation into myeloid cells. M-CSF human disease. The BL model allows the induces the expression of transcription fac- elucidation of disease progression, such as torproduced PU.1 in uponHSCs, infectionto promote or differentiation inflammation theidentification dissemination of new into drug the centraltargets nervousand the - system (Sander et al., Cancer Cell, 2012). matory and stress conditions directly in- into the myeloid cell lineage. Thus, inflam T cell surveillance protects against virus- (Mossadegh-Keller et al., Nature, 2013). associated tumor development, but occa- fluence hematopoiesis at the level of HSCs sionally fails, particularly in case of hepa- The ubiquitin proteasome system (UPS) titis B/C virus-associated hepatocellular maintains the integrity of cellular pro- carcinoma (HCC). To explore this phenom- cesses by controlling protein degradation, enon, the group of Thomas Blankenstein which is important for many cellular func- infected mouse hepatocytes with oncogenic tions. Degradation is triggered by the pro- SV40 large T antigen (TAg) expressing ad- tein ubiqutination and subsequent recog- enoviruses. Infection induced cytotoxic T nition of ubiquitin-binding domain (UBD) lymphocytes (CTLs), leading to clearance containing proteins. The group of Thomas of the cells. However, a few virus-infected Sommer explored the mechanistic details cells “sneaked through” and progressed to of ubiquitin binding to Cue1, a crucial regulator of the Endoplasmic Reticulum (ER)- associated protein degradation (ERAD) inhibitedHCC. T cells in theircan onlyfunction. poorly The infiltrate established HCC pathway (Bagola et al., Mol Cell, 2013). modeland the may few help infiltrating to better T cellsunderstand are locally human HCC (Willimsky et al., J. Clin. Invest., Activated oncogenes or chemotherapy can 2013). lead to cellular senescence, an irreversible exit from the cell cycle. In this state, tumor To monitor and to combat viral infections - and cancer, the immune system must be matory reactions, which can promote tumor formation.cells do not The proliferate group of but Clemens induce Schmitt inflam observed that therapy-induced senescent releasedarmed with by Tefficient cells and effector NK cells. mechanisms, Uta Höp- cancer cells display changes in energy me- kensuch, Armin as the Rehm cytokine and interferoncolleagues (IFN)-γ,showed tabolism (increased glucose and oxygen that the sorting receptor Sortilin controls consumption and ATP production), most - likely due to massive proteotoxic stress, which is a consequence of the senescence- exocytic trafficking of IFN-γ and of gran associated secretory phenotype (SASP). inzyme impaired A. Sortilin-deficient immune responses mice upon revealed infec- Both, inhibition of glucose production or tiondecreased and reduced release autoimmune of interferon-γ, responses resulting in blocking of autophagy caused cell death of the senescent tumor cells, implying a novel Immunity, 2012). two-step therapy approach, which com- inflammatory bowel disease (Herda et al., bines chemotherapy and metabolic therapy The control of lineage commitment of hae- (Dörr et al., Nature, 2013). matopoietic stem (HSCs) and progenitor cells is largely unknown. The Inserm/CNRS- and MDC-associated group of Michael

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Miguel Andrade

Computational Biology and Data Mining

Our group develops and applies computa- in-1 are enriched in coiled-coil regions. We tional methods for the study of gene and propose that while interactions of coiled-coil proteins with polyQ regions might increase protein function with emphasis on molecules aggregation, blocking this interaction, or in- related to human disease. Recently, our main teracting with other regions, might reduce fields of research have been the study of pro- aggregates and neurodegeneration. tein interaction networks, gene expression, and data and text mining of the biomedical Protein sequence analysis literature. David Fournier, Arvind Mer, Nancy Mah

Study of protein interaction We have updated a neural network method networks Martin Schaefer, Jean-Fred Fontaine and Armadillo, which form similar structures(ARD2) composed that identifies of alpha-helices. repeats likeWe evalu HEAT- We created HIPPIE, a database that inte- ated the phylogenetic distribution of these grates protein-protein interaction (PPI) data repeats, pointing to multiple likely events of from several databases scored according to independent emergence of these repeats in attached experimental information. We re- distant taxa and to their increased frequency cently showed that measured interactions in organisms of high cellular complexity such where the interacting proteins are expressed as eukarya in general, and cyanobacteria and in the same tissue, or cellular location, and planctomycetes within prokarya. involved in similar processes are more reli- In general, proteins perform their functions able (with Yoshihiro Kawaoka and Hiroaki Ki- in concrete sub-cellular locations (e.g., cy- tano). These properties are useful to focus on toplasmic, nuclear, extra-cellular). It was relevant PPI subnetworks; we implemented hypothesized that proteins adapt to their mechanisms to do this in HIPPIE and illus- physicochemical environment by mutations trated how to apply this to the study of bio- on the residues exposed to the media not logical cases. closely implicated in function and that there- Expanded polyglutamine (polyQ) stretches fore the composition of the exposed residues are observed in the proteins of patients with of a protein can be used for the prediction of different neurodegenerative diseases and protein sub-cellular location. We studied the are thought to trigger disease. We integrated predictive power of amino acid composition genomic, phylogenetic, protein interaction at variable ranges from buried to exposed network and functional information to add and found that both buried and exposed ami- evidence that polyQ tracts modulate protein no acids carry complementary information interactions (with Erich Wanker) (see Fig- about subcellular location. An optimized two ure). PolyQ expansion results in gain of ab- step predictor trained with vectors of amino normal interactions, leading to pathological acid composition in different ranges of expo- effects like protein aggregation. Furthermore, sure and using a Support Vector Machine fol- we characterized proteins that interact with lowed by a Neural Network (NYCE), reaches ataxin-1, which has a polyQ tract whose ab- an accuracy of 62% when predicting nuclear, normal expansion results in disease. Interac- nucleocytoplasmic, cytoplasmic or extracel- tors that enhance the toxicity of mutant atax- lular location of eukaryotic proteins.

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How polyQ modulates protein-protein interactions. Left: unbound state of a protein containing a polyQ region (red) in disordered state, N-terminally preceded by a coiled-coil region (blue), and C-terminally followed by a disordered polyP region (green). Right: upon interaction of the polyQ protein with a protein X (brown) through a coiled-coil interaction, the polyQ region adopts a coil conformation extending the preceding (blue) coiled coil, therefore increasing the strength of the interaction. The polyP region cannot adopt a coiled-coil conformation effectively providing a cap to the interaction-dependent conformational change induced by the polyQ.

duplications by their abnormally low or high general gene expression. We have implemented a method (CAFE) as an R package to do such analysis and visualize the results.

Data and text mining Jean-Fred Fontaine Transcript regulation and prediction Enrique Muro, Nancy Mah, Marie Gebhardt We develop web tools to assist researchers exploring the biomedical literature to under- Upstream open reading frames (uORFs) are stand the molecular basis of disease. Recently, (generally) short open reading frames that we developed the Alkemio web server to rank appear in the 5’UTRs of many transcripts. thousands of chemicals for their relevance to uORFs may interfere with the protein expres- any topic according to their associated bib- sion of a transcript but activatory effects have liography (in PubMed). We are also working also been described. In collaboration with on methods to mine the biomedical literature Achim Leutz, we are studying experimentally for relevant manuscripts. In this respect, we and computationally their properties and we recently examined how the references cited developed uORFdb, a database about uORFs by a manuscript improve document retrieval, in eukaryotic organisms. and also how to associate experts to particular We are also developing computational meth- topics using the lists of authors of manuscripts ods to predict ncRNAs and to evaluate experi- in PubMed. We provide the latter analysis as mental results identifying ncRNAs, including transcripts arising from pseudogenes and the given manuscript (peer2ref). characterization of miRNA function based on a tool to find peer reviewers appropriate for a the overlap of their targets to those of particular transcriptional repressors as derived from Selected Publications ChIP-seq experiments. Schaefer, M.H., E.E. Wanker and M.A. Andrade-Navarro. 2012. Evolution and function of CAG/polyglutamine repeats in protein-protein interaction. Nucleic Acids Research. 40, 4273-4287. Gebhardt, M.L., S. Reuter, R. Mrowka, and M.A. Andrade-Navarro. 2014. Similarity in Analysis of gene expression targets with REST points to neural and glioblastoma related miRNAs. Nucleic Acids Nancy Mah, Jean-Fred Fontaine, Marie Research. In press. Gebhardt, Sander Bollen Fournier, D., G.A. Palidwor, S. Shcherbinin, A. Szengel, M.H. Schaefer, C. Perez-Iratxeta and M.A. Andrade-Navarro. 2013. Functional and genomic analyses of alpha-solenoid Recently we have collaborated with several proteins. PLoS ONE. 8, e79894. Mer, A.S. and M.A. Andrade-Navarro. 2013. A novel approach for protein subcellular location prediction using amino acid exposure. BMC Bioinformatics. 14, 342. gene expression: to demonstrate that higher Bollen, S., M. Leddin, M.A. Andrade-Navarro and N. Mah. 2014. CAFE: an R package for expressiongroups on theof GPx3 interpretation correlates of with profiles higher of the detection of gross chromosomal abnormalities from gene expression microarray proliferation of Leukemia hematopoietic Stem data. Bioinformatics. In press. Cells (with Guy Sauvageau), to show that knock-down of transcription factor Irf8 transforms dendritic cell progenitors into neutrophils indicating that the DC progenitors have Group Leader David Fournier a default commitment (with Frank Rosenbau- Prof. Dr. Miguel Andrade Marie Gebhardt Arvind Mer recurrence based on the lower expression of Scientists immune-systemer), to define a signature related genes of colorectal (with Wolfgang cancer Dr. Enrique Manuel Muro Visitors Kemmner), and with Francesca Spagnoli in the Sanchez Sander Bollen study of the molecular mechanisms involved Dr. Jean-Fred Fontaine in fate decision for progenitors that originate Dr. Nancy Mah liver and pancreas. Khadija El Amrani We also develop methods for the study of PhD students AdministrativeDr. Adrián González Assistant López DNA microarray data, for example to detect Martin Schaefer Sylvia Olbrich chromosomal regions with gross deletions or

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Walter Birchmeier

Signal Transduction in Development and Cancer

Our research is primarily focused on two In the present report period, we have pub- signaling systems: Wnt/beta-catenin lished the following investigations: and receptor tyrosine kinase signaling. Components of both systems are frequently Wnt/beta-catenin and Bmp signals mutated or deregulated in a variety of control distinct sets of transcription factors in cardiac progenitor developmental disorders and cancers in cells humans. A. Klaus, M. Müller, H. Schulz, Y. Saga, JF. Martin, W. Birchmeier

In the previous years, the laboratory has studied adhesion and signaling of E-cadherin/Wnt/beta-catenin by biochemical transcriptionProgenitor cells factors of the for first their and differen second- means. We have shown that beta-catenin tiation.heart fieldsUsing dependconditional on mutagenesis cardiac-specific of binds to the transcription factors Lef1/ Tcf, and that this translocates beta-catenin of signaling events that controls the ex- to the nucleus. Beta-catenin is recruited to mouse embryos, we defined the hierarchy its degradation complex containing Axin2/ factors during differentiation of cardiac Conductin and APC. We have also investi- progenitorspression of at cardiac-specific embryonic day transcription 9.0. Wnt/ gated the role of scatter factor/hepatocyte beta-catenin and Bmp act downstream of growth factor (SF/HGF) and its receptor, the Notch/RBPJ at this developmental stage. Met tyrosine kinase, in the morphogenesis Mutation of Axin2, the negative regulator of epithelial cells. Signals of Met are trans- of canonical Wnt signaling, enhances Wnt mitted by the cytoplasmic multi-adapter Gab1 and the tyrosine phosphatase Shp2. arrest of cardiac differentiation caused by Conventional ablations of beta-catenin and lossand Bmp4of RBPJ. signals Using and FACS suffices enrichment to rescue of car the- Gab1 in mice result in gastrulation defects diac progenitors in RBPJ and RBPJ/Axin2 and embryonic organ failures, respectively. mutants, embryo cultures in the presence of the Bmp inhibitor Noggin, and by cross- In recent years, we have examined the role ing a Bmp4 mutation into the RBPJ/Axin2 of Wnt/beta-catenin and Met/Gab1/Shp2 mutant background, we showed that Wnt by conditional mutagenesis in mice. Be- ta-catenin regulates precursor and stem cells in the nervous system, the hair and theand cardiac Bmp4 signalingprogenitors: activate Nkx2-5, specific Isl1 and the heart; Gab1 and Shp2 control precur- Baf60cnonoverlapping are controlled cardiac-specific by Wnt/beta-catenin, genes in sor cells in liver, limbs, and kidney. Met and Gata4, SRF, and Mef2c are controlled regulates wound healing in the skin. Activa- by Bmp signaling. Our study contributes to tion of beta-catenin and HGF/Met in adult the understanding of the regulatory hierar- mouse tissues induces tumors and cancer chies of cardiac progenitor differentiation stem cells. -

and outflow tract development and has im 74 MDC Research Report 2014 CANCER RESEARCH

Notch signaling induces the formation of the right ventricle of the heart (RV, on the left), given the reduction seen in the RBBJ mouse mutants (orange arrow). Forced Wnt/beta- catenin signaling substantially rescues this phenotype, as seen in the RBPJ/Axin2 double mutants (green arrow). A heatmap shows (on the right) that the expression of many genes is rescued in the Notch/Wnt double mutants, like heart (orange), Wnt (green) and BMP genes (magenta). From Klaus et al., PNAS 109, 1092-1096 (2012).

plications for understanding and modeling Wnt/Rspondin/beta-catenin sig- heart development (published in Proc. Natl. nals control axonal sorting and Acad Sci. USA, 109, 10921-10926, 2012). lineage progression in Schwann cell development Tamara Grigoryan, Simone Stein, Jingjing Qi, Wnt/beta-catenin signalling in- Hagen Wende, Alistair N. Garratt, Klaus- duces MLL to create epigenetic Armin Nave, Carmen Birchmeier, and Walter changes in salivary gland tumors Birchmeier P. Wend, L. Fang, Q. Zhu, JH. Schipper, C. Loddenkemper, F. Kosel, V. Brinkmann, K. During late Schwann cell development, Eckert, S. Hindersin, JD. Holland, S. Lehr, M. immature Schwann cells segregate large Kahn, U. Ziebold, W. Birchmeier axons from bundles, a process called “axonal radial sorting”. Here we demonstrate We show that activation of Wnt/beta- that canonical Wnt signals play a critical catenin and attenuation of Bmp signals, by role in radial sorting and assign a role to combined gain- and loss-of-function muta- Wnt and Rspondin ligands in this process. tions of beta-catenin and Bmpr1a, respec- Mice carrying beta-catenin loss-of-function tively, results in rapidly growing, aggressive mutations show a delay in axonal sorting; squamous cell carcinomas (SCC) in the sali- conversely, gain-of-function mutations re- vary glands of mice. Tumors contain trans- plantable and hyperproliferative tumor are accompanied by abnormal process ex- propagating cells, which can be enriched tension,sult in accelerated differentiation, sorting. and Sorting aberrant deficits cell cycle exit of the Schwann cells. Using pri- Single mutations stimulate stem cells, but mary cultured Schwann cells, we analyze tumorsby fluorescence are not formed. activated We show cell that sorting. beta- the upstream effectors, Wnt and Rspondin catenin, CBP and Mll promote self-renewal ligands that initiate signaling, and down- and H3K4 tri-methylation in tumor propa- stream genetic programs that mediate the gating cells. Blocking beta-catenin-CBP in- Wnt response. Our analysis contributes to teraction with the small molecule ICG-001 a better understanding of the mechanisms and small-interfering RNAs against beta- of Schwann cell development and fate de- catenin, CBP or Mll abrogate hyperprolifer- cisions (published in Proc. Natl. Acad. Sci. ation and H3K4 tri-methylation, and induce USA, 110, 18174-18179, 2013). differentiation of cultured tumor propagating cells into acini-like structures. ICG-001 decreases H3K4me3 at promoters of stem Combined Wnt/beta-Catenin, HGF/ cell-associated genes in vitro and reduces Met, and CXCL12/CXCR4 Signals tumor growth in vivo. Remarkably, high Characterize Basal Breast Cancer Wnt/beta-catenin and low Bmp signalling and Predict Disease Outcome also characterize human salivary gland SCC Jane D. Holland, Balázs Györffy, Regina and head and neck SCC in general. Our work Vogel, Klaus Eckert, Giovanni Valenti, Liang Fang, Philipp Lohneis, Sefer Elezkurtaj, signals remodel chromatin and control in- Ulrike Ziebold, and Walter Birchmeier ductiondefines mechanismsand maintenance by which of tumorbeta-catenin propagating cells. Further, it supports new Prognosis for patients with estrogen-re- strategies for the therapy of solid tumors ceptor (ER)-negative basal breast cancer (published in EMBO J. 17, 1977-89, 2013). is poor, and chemotherapy is currently the

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Cells derived from mammary gland tumors of Wnt-Met compound mutant mice grown as 3D organoids. It is shown that treatment with a combination of Wnt and Met inhibitors induce the appearance of normal-like mammary gland acini (upper): hollow structures composed of a polarized outer layer of basal epithelial cells (red staining for keratin 5) encapsulating a luminal cell layer (green for keratin 8). Untreated organoids produce filled, transformed structures (lower). From Holland et al., Cell Reports 5, 1214-1227 (2013).

best therapeutic option. We have generated control different functions, selfrenewal and a compound-mutant mouse model com- differentiation. Molecular therapy targeting bining the activation of beta-catenin and HGF (Wnt-Met signaling), which produced delayed tumor development. The expres- rapidly growing basal mammary gland tu- sionWnt, ofMet, a Wnt-Met and CXCR4 322 in gene mice signature significantly was found to be predictive of poor survival of CXCL12/CXCR4 as a crucial driver of Wnt- human patients with ER-negative breast Metmors. tumors, We identified given that the compound-mutantchemokine system cancers. Thus, targeting CXCR4 and its upstream activators, Wnt and Met, might pro- tumor resistant. Wnt-Met activation rapidly expandedmice also deficienta population in the of CXCR4cancer-propagat gene were- treatment (published in Cell Reports 5, ing cells, in which the two signaling systems 1214-1227,vide an efficient 2013). strategy for breast cancer

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Shp2/Mapk signaling controls ise human kidney cancer stem cells (Annika goblet/paneth cell fate decisions Fendler, together with Klaus Jung and Jonas in the intestine Busch). We also examine the function of Julian Heuberger, Frauke Kosel, Jingjing Gab1 and Mapk signaling in hair cycle and Qi, Katja Grossmann, Klaus Rajewsky and stem cell quiescence (Özlem Akilli-Öztürk) Walter Birchmeier and of Shp2 in cancer stem cells and senescence of PyMT mammary gland tumors In the development of the mammalian in- (Linxiang Lan, together with EPO). We testine, Notch and Wnt/beta-catenin signals also study how Wnt/beta-catenin-target control stem cell maintenance and their dif- genes promote metastasis and migration of ferentiation into absorptive and secretory - cells. Mechanisms that regulate differentia- eration of EPO). tion of progenitors into the three secretory colorectal cancer cells (Jingjing Qi, in coop lineages, goblet, paneth or enteroendocrine cells, are not fully understood. Using conditional mutagenesis in mice, we observed that Shp2-mediated Mapk signaling determines the choice between paneth and gob- Selected Publications let cell fates, and also affects Lgr5+ stem cells. Ablation of the tyrosine phosphatase Shp2/Mapk signaling controls goblet/paneth cell fate decisions in the intestine. Proc Shp2 in the intestinal epithelium reduced Heuberger,Natl Acad Sci J., USA,Kosel, 111: F., Qi, 3472-3477. J., Grossmann, K.S., Rajewsky, K., and Birchmeier, W. 2014.

Mapk signaling and led to a reduction of - goblet cells while promoting paneth cell development. Conversely, conditional Mek1 Handolland, CXCL12/CXCR4 J.D., Győrffy, signals B., Vogel, characterize R., Eckert, basalK., Valenti, breast G., cancer Fang, andL., Lohneis, predicts P., disease Elez outcome.kurtaj, S., Ziebold,Cell Reports U., and 5: 1214-1227. Birchmeier, W. 2013. Combined Wnt/beta-catenin, Met activation rescued the Shp2 phenotype, promoted goblet cell and inhibited paneth Grigoryan T, Stein S, Qi J, Wende H, Garratt AN, Nave KA, Birchmeier C, and Birchmeier W. 2013. Wnt/Rspondin/beta-catenin signals control axonal sorting and lineage pro- cell generation. The Shp2 mutation also ex- gression in Schwann cell development. Proc Natl Acad Sci USA 110: 18174-18179. panded Lgr5+ stem cell niches, which could be restricted by activated Mek1 signaling. Wend P, Fang L, Zhu Q, Schipper JH, Loddenkemper C, Kosel F, Brinkmann V, Eckert K, Hindersin S, Holland JD et al. 2013. Wnt/beta-catenin signalling induces MLL to create Changes of Lgr5+ stem cell quantities were epigenetic changes in salivary gland tumours. EMBO J 32: 1977-1989. accompanied by alterations of paneth cells, indicating that Shp2/Mapk signaling might Klaus A, Muller M, Schulz H, Saga Y, Martin JF, and Birchmeier W. 2012. Wnt/beta- catenin and Bmp signals control distinct sets of transcription factors in cardiac affect stem cell niches directly or via paneth progenitor cells. Proc Natl Acad Sci USA 109: 10921-10926. cells. Remarkably, inhibition of Mapk signaling in intestinal organoids and cultured cells changed the relative abundance of Tcf4 isoforms and by this, promoted Wnt/ beta-catenin activity. The data thus show Group Leader that Shp2-mediated Mapk signaling con- Prof. Dr. Walter Birchmeier Giovanni Valenti trols the choice between goblet and paneth QionghuaJingjing Qi Zhu cell fates by regulating Wnt/beta-catenin Scientists activity (published in Proc. Natl. Acad. Sci. Dr. Özlem Akilli-Öztürk Undergraduate Students USA, 111, 3472-3477, 2014). Dr. Liang Fang Michael Gerloff* Dr. Annika Fendler Miriam Heuer* - Dr. Tamara Grigoryan cerned with the function of the E3 ubiquitin Dr. Stefanie Grosskopf* Technical Assistants ligaseNew projectsHakai by of conditional the laboratory gene areablation con Dr. Ritesh Gupta* Frauke Kosel and by proteomics approaches (Julian Heu- Dr. Julian Heuberger Marion Müller berger, together with Matthias Selbach). In Dr. Jane Holland Simone Stein cooperation with the Leibniz Institute of Dr. Alexandra Klaus Regina Vogel Molecular Pharmacology in Berlin-Buch, Dr. Linxiang Lan Christin Hesse* we develop further small molecule inhibitors of beta-catenin/Tcf4 (Liang Fang, to- PhD Students Secretariat gether with Edgar Specker and Jens von Irmgard Wiznerowicz Kries) and Shp2 (Judith Holz and Marc Naz- Hubert Pakula aré). In cooperation with the Department of Majid Momeny* (*part of the time reported) Urology of the Charité Berlin, we character- Zsofia Penzvalto*

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Thomas Blankenstein

Molecular Immunology and Gene Therapy

We focus on three areas in cancer Virus-induced hepatocellular immunology: carcinomas cause antigen-specific local tolerance. 1. Developing novel cancer models that better reflect human disease. T cell surveillance is often effective against 2. Employing the mechanisms of rejection virus-associated tumors because of their of established tumors by adoptively trans- high immunogenicity. It is not clear why surveillance occasionally fails, particularly ferred T cells with specific emphasis of the against hepatitis B virus- or hepatitis C tumor stroma as target. virus-associated hepatocellular carcinoma 3. Employing T cell therapy of cancer in the (HCC). We established a transgenic murine model of virus-induced HCC by hepatocyte- clinic. In the recent years we have developed a series of novel transgenic mouse the oncogenic SV40 large T antigen (Tag). models that allowed to analyze spontane- Adenovirusspecific adenovirus-induced infection induced activation cytotoxic of ous anti-tumor responses, to track tumor T lymphocytes (CTLs) targeted against the virus and Tag, leading to clearance of or T cells by non-invasive bioluminescence the infected cells. Despite the presence of imaging and to modulate oncogene expression in vivo. In addition transgenic mice virus-infected cells escaped immune clear- functional, antigen-specific T cells, a few are used that allow the isolation of TCRs to ance and progressed to HCC. These cells expressed Tag at levels similar to HCC iso- redirect T cells towards superior antitumor lated from neonatal Tag-tolerant mice, sug- activity. gesting that CTL not select for cells with low immunogenicity. Virus-infected mice - tration in early-stage HCC compared with thatrevealed in late-stage significantly HCC, greaterdemonstrating T cell infilpro- gressive local immune suppression through

cell death protein-1 (PD-1) and its ligand inefficient T cell infiltration. Programmed CD8+ T cells and HCC, respectively, which PD-L1 were expressed in all Tag-specific tolerance. Thus, we have developed a model ofcontributed virus-induced to local HCC tumor-antigen-specific that may allow for a better understanding of human HCC.

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Differently immunogenic cancers “passenger” mutation) by TE cells requires in mice induce immature myeloid cells that suppress CTL in vitro avoid selection of antigen-loss variants. but not in vivo following transfer. Becauseaction of “cancer-driving” IFNγ on tumor antigens stroma cells(CDAs) to

Tumors frequently induce immature my- expected to be dispensable in elimination ofare cancers rarely by counterselected, targeting a CDA. IFNγ Here, may initial be and unrelated CTL- responses and are regression of large, established tumors re- termedeloid cells myeloid-derived (iMC), which suppresssuppressor specific cells + (MDSC). Mainly analyzed by in vitro assays transferred CD8 TE cells targeting SV 40 quired neither IFNγ, FasL, nor perforin by in tumor transplantation models, little is large T as CDA. However, cytotoxic TE cells known about their function in autochtho- - nous tumor models in vivo. We analyzed - iMC in 3 SV40 large T (Tag)-driven con- genlacking by the IFNγ cancer or FasL cells. could Complete not prevent tumor re- ditional autochthonous cancer models lapse despite retention of the rejection anti

with different immune status: (1) Early tumor stroma. Therefore, TE cells lacking - jection required IFNγ-regulated Fas by the chastic Tag activation leading to sporadic antigenic cancer because the tumor stroma cancer,Tag-specific which CTL induces competence an aberrant and rareimmune sto escapesIFNγ or FasLdestruction cannot preventif its Fas progression expression ofis response and CTL tolerance; (2) Cre/LoxP down-regulated. recombinase-mediated hepatocellular carcinoma (HCC) development in neonatal Tag-tolerant mice; and (3) Tag-activation Depot formation of doxycycline through Cre recombinase-encoding viruses impairs Tet-regulated gene ex- in the liver and HCC development with sys- pression in vivo.

but not two latter models, tumors induced The tetracycline (Tet) system is widely CTLtemic hyporesponsiveness anti-Tag CTL immunity. to tumor-unrelat In the first- used for regulation of gene expression in ed antigens. Regardless of the model, tu- vitro and in vivo. We constructed C57BL/6 mors produced interleukin-6 and vascular transgenic mice (rtTA-CM2) with strong endothelial growth factor but not granulo- and ubiquitous reverse transactivator cyte macrophage–colony-stimulating fac- (rtTA2(S)-M2) gene expression. rtTA-CM2 tor (GM-CSF) and induced iMC (CD11b(+) mice were crossed to Tet-responsive report- Gr-1(int)) that suppressed CTL responses er mice (LC-1) conditionally expressing the in vitro. None of the iMC from the different tumor models suppressed CTL responses in of a Tet-responsive element, which allowed adoptive cell transfer experiments unless firefly luciferase (FLuc) gene under control- GM-CSF was provided in vivo. Together, iMC tor activity by bioluminescent imaging. Fol- expand independent of the type of antitu- lowingsensitive doxycycline quantification (dox) of theapplication, transactiva up mor response and are not immunosuppres- to 105-fold increase in BL signal was mea- sive in a cell-autonomous fashion. sured. rtTA activity was inducible in most analyzed organs. After dox withdrawal the

Fas expression by tumor stroma is not disappear completely, most likely due to required for cancer eradication. aBL dox signal depot decreased formation significantlyin vivo. The residual but did

The contribution of molecules such as per- a Tet-off controlled oncogene in a tumor - transplantationdox was sufficient experiment, to partly down-regulate resulting in gand (FasL) by transferred CD8+ effector T reduced tumor growth. rtTA-CM2 mice may forin, IFN-γ (IFNγ), and particularly Fas li (TE be a useful tool to analyze the function of - genes in various organs but also reveal that cient) cells attack to againstrejection large of large, tumors established carrying down-regulation of gene expression is not atumors surrogate is incompletely tumor antigen understood. (mimicking Effi a complete.

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B-cells and IL-4 promote methyl- development in magnitude comparable to cholanthrene-induced carcinogen- that previously attributed to differences esis but there is no evidence for a in immunocompetence. This suggested role of T/NKT-cells and their effec- that genetic drift between separate inbred tor molecules (Fas-ligand, TNF-α, colonies of mice and/or environmental perforin). factors (e.g., transport of the animals) influenced carcinogenesis. Therefore, litter- Mice deficient either in subtypes of im- mates were used as control in subsequent mune cells, cytokines or lytic pathways experiments. Although deficiency of T - cells, NKT cells, perforin, Fas-ligand, TNF- genesis by methylcholanthrene to evalu- - atehave whether been subjected these components to chemical of carcino the im- ferences in tumor development, the pres- mune system affect tumor development. enceα-receptor of B cells failed and to IL-4reveal enhanced significant tumor dif Inbred mice of the same genotype but development under similar experimental from different sources differed in tumor conditions.

IFNγ maintains MHC I and Fas expression on tumor stroma. a) Large established 16.113 tumors express SV 40 large as target antigen (Tas) and contain abundant stroma.

b) Rag-/-, Rag-/-/IFNγ-receptor-/- and Rag-/-/ Fas-/- mice with large 16.113 tumors were treated with SV40 large T antigen-immune CD8+ T cells (TE) and tumor growth was followed overtime. Schematic scheme of data published in Listopad et al., 2013. Note that expression of IFNγ-Receptor and Fas on the tumor stroma is necessary to prevent tumor recurrence.

c) In the absence of IFNγ, the tumor stroma es- capes by MHC I/Fas down-modulation, which allows immunogenic cancer to progress.

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Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion.

Introducing human genes into mice offers Selected Publications the opportunity to analyze their in vivo function or to obtain therapeutic mole- Willimsky, G., Schmidt, K., Loddenkemper, C., Gellermann, J. and Blankenstein, Th. cules. For proper gene regulation, or in case - ance. J. Clin. Invest. 123: 1032-1043. of multigene families, megabase (Mb)-sized (2013). Virus-induced hepatocellular carcinomas cause antigen-specific local toler DNA fragments often have to be used. Yeast Listopad, J., Kammertoens, T., Anders, K., Silkenstedt, B., Willimsky, G., Schmidt, K., Kühl, A., Loddenkemper, C. and Blankenstein, Th. (2013). Fas expression by tumor stroma is required for cancer eradication. Proc. Natl. Acad. Sci. USA 110: 2276-2281. transgenesis is irreplaceable for this purpose,artificial because chromosome alternative methods (YAC)-mediated such as Schmidt, K., Zilio, S., Schmollinger, J., Bronte, V., Blankenstein, Th. and Willimsky, G. (2013). Differently immunogenic cancers in mice induce immature myeloid cells that suppress CTL in vitro but not in vivo following transfer. Blood 121: 1740-1748. (BACs) cannot introduce DNA fragments largerthe use than of bacterial 500 kb artificialinto the chromosomes mouse germ Kammertoens, T. and Blankenstein, Th. Cancer Cell 23: 429-431. line. However, YAC libraries often contain (2013). It’s the peptide-MHC affinity, stupid. only partial gene loci. Time-consuming re- Li, L.-P. and Blankenstein, Th. (2013). Generation of transgenic mice with megabase- construction of YACs, genetic instability and fusion. Nat. Protoc. 8: 1567-1582. sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell above a certain size impede the generation ofthe humanized difficulty inmice. obtaining Here we intact describe YAC DNAhow to reconstruct YACs containing Mb-sized Group Leader (TRA) gene locus, thus facilitating the in- Prof. Dr. Katerina Thiede troductionhuman DNA, of suchlarge asDNA the fragments T cell receptor-α into the Thomas Blankenstein Lara Le Wuehrmann mouse germ line. Fusion of YAC-containing yeast and embryonic stem (ES) cells avoids Scientist Technical Assistants Dr. Kathleen Anders Stephanie Fuerl cells are then used to stably introduce the Mathias Friedrich* Angelika Gärtner functionalthe need for TRA YAC gene DNA purification.locus into the These mouse ES Dr. Ioannis Gavvovidis Monika Harm germ line. The protocol takes 1 year to com- Dr. Thomas Kammertoens Markus Hensel plete, from reconstruction of the entire TRA Dr. Joanna Listopad Marion Rösch gene locus from YACs containing partial but Christel Westen overlapping TRA regions to germline trans- Karin Schmidt* mission of the YAC. Dr. Ana TextorMilojkovic Secretariat Dr. Gerald Willimsky Sylvia Klahn

PhD Staff Other Kathrin Borgwald KatrinXiaojing Hönig Chen Christian Friese Meng-TungArunraj Dhamodaran Hsu Evelyn Hesse Dana Hoser Sabrina Horn Josephine Jung* (MD) Karin Karamatskos Matthias Obenaus* (MD) Anette Madel Lucia Poncette Kristin Retzlaff Michael Rothe Diana Tarak Christian Schön Maya Schreiber (MD) (*part of the time reported)

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Peter Daniel

Clinical and Molecular Oncology

Anticancer drug therapies rely on the induc- cence or death by apoptosis or autophagy tion of cell cycle arrest or cell death through and demise of the affected cell. The molecular basis of these events has been studied cellular stress programs. Deregulation of extensively during recent years and com- such stress pathways in cancer cells by prehensive models are now established for genetic or epigenetic inactivation results in large parts of these signaling events. Novel evasion of the tumor cells from cancer ther- disease-related resistance mechanisms and apy, insufficient responses to treatment with group in leukemias and solid tumors. In this selection of therapy-resistent subclones context,therapeutic we recentlytargets weredescribed, identified e.g. selective by our and, consequently, tumor relapse, metasta- loss of multiple cell death genes, including BH3-only proteins Nbk and Bim, in carcino- sis coinciding with poor disease prognosis. ma of the kidney. This is a unifying feature The aim of the group is to identify genetic of renal carcinoma and appears to be linked defects in cancer that result in such ag- to the impressive clinical resistance of this gressive disease and resistance to clinical tumor entity to anticancer therapy. Similar cancer therapy. Recent data from our group Links to epigenetic regulation and mTOR/ indicate that specific defects in cell signaling PI3events kinase were signaling identified were in addressed.acute leukemias. for apoptosis, autophagy or other forms of cell death such as mitotic catastrophe can Regulation of cell death by be overcome by rational selection of target- pro-apoptotic Bcl-2 family members ed anticancer drugs. Further projects are aimed to gain insights into novel aspects of Apoptosis is mediated through at least cell cycle and cell death regulation and their (1) the death receptors, (2) the mitochon- intricate interactions, e.g. in the context of dria,three andmajor (3) pathways the endoplasmic that are regulated reticulum by tumor suppression by p14ARF or targeted (ER). In most cells, these pathways are con- cancer therapies. trolled by the Bcl-2 family of proteins that can be divided into anti-apoptotic (Bcl-2,

Bcl-xL, Mcl-1) and pro-apoptotic members. Understanding resistance to Although the overall amino acid sequence anticancer therapy homology between the family members In cooperation with F. Essmann (Interfaculty is relatively low, they contain highly con- Institute for Biochemistry, Univ. Tübingen), served domains, referred to as Bcl-2 ho- C. Belka (Clinic and Polyclinic for Radia- mology domains (BH1 to BH4) that are tion Therapy and Radiation Oncology, LMU essential for homo- and hetero-complex München), and T. Blankenstein (MDC); formation as well as for their cell death in- supported by the German Consortium for ducing capacity. Structural and functional Translational Cancer Research (DKTK) analyses revealed that the pro-apoptotic homologs can be subdivided into the Bax Many anticancer therapies activate nuclear subfamily (Bax, Bak, Bok) and the growing stress responses to induce cell cycle arrest BH3-only subfamily. BH3-only proteins link and DNA repair. When repair fails, the same upstream signals from different cellular or stress responses trigger cellular senes- functional compartments to the mitochon-

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Therapeutic targeting of XIAP as a switch between Type I and Type II apoptosis signaling in therapy resistant malignant tumors The death ligand TRAIL (TNF-related apoptosis inducing ligand) initiates TRAIL-receptor-mediated recruitment and activation of the death protease zymogen pro-caspase-8. In type I cells, this is sufficient to induce caspase-3 processing and execution of apoptosis. Most tumor cells are, however, of a type II and rely upon a mitochondrial, Bcl-2 family regulated cell death pathway to achieve sufficient activation of executioner caspases. Mitochondrial release of SMAC/DIABLO, the second mitochondrial activator of caspases, into the cytosol thereby plays a key role in caspase activation by displacing the E3-Ubiquitin-Ligase XIAP from binding to activated caspase-3 thereby impeding proteasomal degradation. Inhibition of XIAP by small molecule SMAC mimetics, e.g. LBW242, or the SP1 inhibitor Mit A or XIAP siRNA facilitates caspase-3 activation, converts cells from a type II to a type I. This may circumvent the frequently found central apoptosis defects in malignant tumors that are caused by up-regulation of anti-apoptotic Bcl-2 members (e.g. Bcl-2, Bcl-xL, Mcl-1), loss of pro-apoptotic Bax and/or Bak, or BH3-only proteins. Proteasome inhibitors such as bortezomib (BZM) and MG132 restore caspase-3 activity by interfering with proteasomal degradation. Mit A: Mithramycin A.

drial apoptosis pathway. BH3-only proteins death by the Inhibitor of Apoptosis Pro- Puma, Noxa, Hrk, and Nbk (Bik) are induced teins (IAP) with XIAP playing a dominant by p53 and mediate cell death originating role in TRAIL resistance. These studies from the nucleus, e.g. upon DNA damage. also showed that interference with the E3- Nbk localizes to the ER and activates the ubiquitin ligase function of XIAP appears to pro-apoptotic multidomain protein Bax be an important mechanism of proteasome (but not the homologous Bak) indirectly, targeting drugs such as Bortezomib. through a ER-initiated death pathway that has been recently elucidated by our group. Bid links the death receptor pathway to the mitochondrial death machinery where it Selected Publications triggers activation of Bax and Bak follow- Gillissen B, Richter A, Richter A, Overkamp T, Essmann F, Hemmati PG, Preissner R, Belka C, Daniel PT. Targeted therapy of the XIAP/proteasome pathway overcomes ing limited proteolysis by caspase-8/-10 to TRAIL-resistance in carcinoma by switching apoptosis signaling to a Bax/Bak-independent ‘type I’ mode. Cell Death Dis. 2013, 23:e643 tBid. Zaltsman Y, Shachnai L, Yivgi-Ohana N, Schwarz M, Maryanovich M, Houtkooper RH, Aims of our work are (1) to gain structur- Vaz FM, De Leonardis F, Fiermonte G, Palmieri F, Gillissen B, Daniel PT, Jimenez E, al and functional insights into how Bcl-2 facilitator of tBID recruitment to mitochondria. Nat Cell Biol. 2010, 12:553-62.

subfamilies promote or inhibit cell death Walsh S, Koehler CM, Roy SS, Walter L, Hajnóczky G, Gross A. MTCH2/MIMP is a major signals and how these properties may be utilized for development of cell death- Andersgenetically K, Buscho unstablew C, tumors, Herrmann while A, oncogene Milojkovic inactivation A, Loddenkemper selects escapeC, Kammertoens variants. T, DanielCancer P,Cell, Yu H,2011, Charo 20: J, 755-67. Blankenstein T. Monospecific CD8+ effector T cells reject large promoting cancer therapies, (2) to address Gillissen B, Wendt J, Richter A, Richter A, Müer A, Overkamp T, Gebhardt N, Preiss- interactions with oncogene sigalling and ner R, Belka C, Dörken B, Daniel PT. Endogenous Bak inhibitors Mcl-1 and Bcl-xL: p53 dependent and independent sigalling ARF 188:851-62. by the p14 tumor suppressor and (3) to differential impact on TRAIL resistance in Bax-deficient carcinoma. J Cell Biol. 2010, understand pathway interactions with cell Gillissen B, Essmann F, Hemmati P, Richter A, Richter A, Öztop I, Chinnadurai G, Dörken B and Daniel PT. Mcl-1 mediates the Bax dependency of Nbk/Bik-induced death signalling as resistance mechanisms apoptosis. J Cell Biol 2007, 179: 701-15. to targeted cancer therapy, e.g. in mTOR/ PI3K survival signalling.

We recently showed how to resensitize tumor cells for drug- induced cell death Group Leader Ph.D. Students Prof. Dr. med. Peter Daniel the anti-apoptotic Bcl-2 homolog Mcl-1. Tim Overkamp Wein Bax-deficientalso found that carcinomas such central by defects targeting in MelanieAnja Müller Steinborn cell death resistance can be overcome even Postdocs Research Technicians targeting drugs that mimic the function of Dr. rer. nat. Bernd Gillissen themore endogenous efficiently bycell a newdeath class activator of apoptosis SMAC Dr. med. Martina Hampel that interferes with protection from cell Anja Richter Antje Richter

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Oliver Daumke

Structure and Function of Membrane-associated GTPases

GTPases of the dynamin and septin Structure and antiviral function of superfamilies are molecular machines MxA GTPases that assemble at the surface of cellu- The interferon-induced Myxovirus-resis- lar membranes. Some of these proteins tance (Mx) GTPases are central players of act as dynamic scaffolds that remodel innate immunity. They mediate resistance the underlying membrane in a GTPase- against a wide range of pathogens, includ- - dependent fashion; others orchestrate ative-strand viruses. As typical members the recruitment of interaction partner in a ofing the influenza dynamin viruses superfamily, and many they other can oligo neg- temporally and spatially defined manner. We merize in ring-like structures around tubular membranes. are interested to understand principles of assembly, function and regulation of these Using X-ray crystallography, we found that GTPases. To this end, structural studies are the virus binding domain, the stalk, of hu- combined with biochemical and cell-based man MxA has a four-helix bundle fold. In the crystals, the stalks assembled via three approaches. interfaces in a zigzag fashion. We showed that all three interfaces are required for the assembly of MxA at membrane surfaces and for its antiviral activity. Based on these results, we suggested a model how MxA assembles into ring-like oligomers and how these rings mediate the antiviral activity (Fig. 1a).

We also determined the structure of the

bundle signalling element (BSE), a three- helixfull-length bundle, MxA which molecule. bridges We GTPase identified (G) dothe- main and stalk. Based on functional experiments, we suggested a molecular pathway how nucleotide-driven rearrangements in the MxA oligomer are transmitted from the GTPase domain to the stalk.

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Oligomerization of dynamin and septin-like GTPases. a) Structural model of ring-like oligomers of the MxA GTPase. We suggested that these rings assemble around ribonucleoprotein complexes of negative-strand RNA viruses, thereby interfering with viral replication. b) Model of helical dynamin oligomers. Similarly to MxA, assembly proceeds via three conserved interfaces in the stalks. The dynamin helix oligomerizes around the neck of clathrin-coated vesicles leading to vesicle scission. c) Oligomeric ring model of the EHD2 ATPase. Oligomerization is solely mediated via the GTPase domains, but not the helical domains. This assembly mode suggests an alternative mechanism of EHD2 in membrane remodelling. d) Linear GTP-dependent oligomer of GIMAP2. GIMAP2 assembles via two interfaces in the GTPase domain into a linear scaffold. An amphipathic C-terminal a-helix might mediate assembly of interaction partners.

Structural insights into dynamin- BSE, a stalk and a lipid-binding pleckstrin mediated endocytosis homology (PH) domain. Interestingly, we found that the interaction site between the Dynamin is the founding member of the dy- stalk and the PH domain is often mutated namin superfamily. The multi-domain pro- in patients suffering from centronuclear tein oligomerizes around the neck of clath- myopathy, a congenital disease leading to rin-coated vesicles and induces membrane progressive muscle weakness. Supported scission in a GTPase-dependent fashion. We by biochemical and cell-based functional are interested to understand the molecular experiments, we proposed a molecular mechanisms how dynamin mediates mem- model for helical dynamin oligomers (Fig. brane scission. 1b). Furthermore, we suggested how the interplay between the dynamin domains con- Based on our MxA work, we determined tributes to the mechano-chemical coupling. the X-ray structure of nearly full-length dy- Finally, we introduced a dynamic model namin. Dynamin shows a four-domain ar- how GTP binding and hydrolysis induces chitecture comprising a GTPase domain, a scission of the vesicle neck.

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Crystals of GIMAP2, observed under polarized light, with a typical size of 0.2 x 0.1 x 0.05 mm3. In these crystals, the GIMAP2 molecules are arranged in a 3-dimensional lattice. Using X-ray diffraction, 3D information on the structure of GIMAP2 was obtained.

Dynamin-related GTPases in mito- mitochondrial membranes, e.g. mitofusin chondrial remodelling mediates fusion of the outer mitochondrial membrane whereas OPA1 catalyzes fusion A new focus of our group has been the of the inner mitochondrial membrane. structural and functional characterization of mitochondrial membrane remodelling We recently determined the crystal struc- events. Mitochondria are dynamic membra- ture of the mitochondrial membrane scis- nous structures which continuously under- sion protein DNM1L. Based on a range of go scission and fusion. These processes are biochemical and cell-based experiments, crucial for the maintenance of respiratory we deduced an alternative assembly mode and metabolic activity and for the preserva- of DNM1L at the outer mitochondrial mem- tion of mitochondrial DNA. Malfunctioning brane for the constriction of mitochondria. of this process has been linked to numerous These results may have implications for pathologies, such as neurodegenerative dis- mitochondria-based diseases in which DN- eases, cardiomyopathy and cancer. Three M1L function is compromised. dynamin-like proteins, dynamin-like-protein 1 (DNM1L), OPA1 (optical atrophy 1) and mitofusin are implicated in mitochon- Oligomerization and cellular func- drial dynamics. Unlike in endocytosis, mi- tion of Eps15 homology domain- tchondrial membrane fusion and division containing ATPases (EHDs) involves the double-membrane system of mitochondria. Consequently, mitochondrial EHDs are ubiquitously expressed dyna- dynamin-related GTPase act on distinct min-related ATPases which are built of an

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amino-terminal GTPase domain, followed Using co-localization experiments, we re- by a helical domain and a carboxy-terminal regulatory Eps15 homology domain. EHDs lipid droplets. In contrast to GIMAP2, GI- are found at the plasma membrane and at cently identified GIMAP7 at the surface of vesicular and tubular endocytic membrane stimulated the GTPase activity of GIMAP2. structures. They are implicated in several TheMAP7 structure efficiently of a hydrolyzedGTP-bound GTP.GIMAP7 It also di- - mer elucidated the mechanism of dimeriza- tably during the recycling of receptor pro- tion-induced GTPase activation. These data teinsmembrane to the plasma trafficking membrane. pathways, most no suggested that GIMAP7 acts as GTPase activating protein for GIMAP2. Furthermore, We previously showed that EHD2 oligo- we found GIMAPs differentially down-reg- merizes in ring-like structures around tu- ulated in a variety of different anaplastic bulated liposomes. By solving the crystal large cell lymphoma lines compared to their structure of an EHD2 dimer, we found that T-cell progenitors, implicating a function of stable dimerization of EHD2 is mediated certain GIMAPs as tumor suppressors.

lipid-binding site at the tip of the helical domains.via the GTPase More domains.recently, Wewe identifiedshowed that the also the N-terminus of EHD2 can insert into the membrane, thereby regulating membrane recruitment and/or oligomerization Selected Publications of EHD2. Based on functional experiments, Gao, S., von der Malsburg, A., Paeschke, S., Behlke, J., Haller, O., Kochs, G., and Daumke, we suggested a model for the EHD2 oligo- O. (2010). Structural basis of oligomerization in the stalk region of dynamin-like MxA. meric rings (Fig. 1c). These rings have a re- Nature 465: 502-506. markably different architecture from those Gao, S., von der, M.A., Dick, A., Faelber, K., Schroder, G.F., Haller, O., Kochs, G., and formed by MxA and dynamin. Eventually, Daumke, O. (2011). Structure of myxovirus resistance protein a reveals intra- and - intermolecular domain interactions required for the antiviral function. Immunity 35: 514-525. ponent of caveolae opening up a wide range ofwe cell-based identified approaches EHD2 as a novel to study integral the physi com- Faelber, K., Posor, Y., Gao, S., Held, M., Roske, Y., Schulze, D., Haucke, V., Noe, F., and ological function of this protein. Daumke, O. (2011). Crystal structure of nucleotide-free dynamin. Nature 477: 556- 560. Fröhlich C., Grabiger S., Schwefel D., Faelber K., Rosenbaum E., Mears J., Rocks O., Daumke O. (2013) Structural insights into oligomerization and mitochondrial remod- GIMAPs - cellular scaffolds elling of dynamin 1-like protein. EMBO J 32: 1280-92. controlling lymphocyte survival Schwefel D., Arasu B.S., Marino S., Lamprecht B., Köchert K, Rosenbaum E.,Eichhorst J., Wiesner B, Behlke J., Rocks O., Mathas S., Daumke O. (2013) Structural insights into GTPase of the Immunity Associated Proteins the mechanism of GTPase activation in the GIMAP family. Structure 21: 550-9 (GIMAPs) comprise a septin-related GTPase family in vertebrates. The seven human GI- MAP members are predominantly expressed in cells of the immune system. Some GIMAPs were proposed to regulate apoptosis by con- Group Leader Chris Fröhlich (PhD defense 2013) trolling the activity of Bcl2 proteins. Prof. Dr. Oliver Daumke Stephen Grunwald Manuel Hessenberger We determined four crystal structures of Scientist Kathrin Schulte a representative member, GIMAP2, in dif- Claudio Shah (PhD defense 2013) ferent nucleotide-loading states. In com- Dr. Stephen Marino bination with biochemical experiments, Dr. DanielKatja Fälber Olal Technical Assistants this work elucidated the molecular basis Dr. Eva Rosenbaum Susanne Scheu of GTP-dependent oligomerization via the Vivian Schulz GTPase domains (Fig. 1d). We also showed PhD Jeanette Schlegel that GIMAP2 in Jurkat cells localizes to the Arthur Alves de Melo Sabine Werner surface of lipid droplets which are cellular Arasu Balasubramaniyam storage and signalling compartments. In- Sivanandam Secretary terestingly, GIMAP2 bound GTP with high Birgit Cloos

Alexej Dick affinity but did not hydrolyze it. MDC Research Report 2014 87

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Bernd Dörken

Haematoloy, Oncology and Tumorimmunology

Biology and Targeted A detailed molecular understanding of the oncogenic process is fundamental for the Therapy of Lymphoma development of new therapeutic strategies for human lymphomas. Using cell and molecular biology techniques, high-throughput approaches as well as in vivo mouse models, we aim to gain insight into the network of oncogenic defects in transformed B and T cells. In particular, we are interested in the link between deregulated signaling and transcription factor activities, disruption of cellular differentiation and oncogenic transformation. Using classical Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) as model systems, we analyze the role of lineage infidelity, cellular reprogramming and activation of lineage-inappropriate survival signals in lymphomagenesis, as exempli- fied by our work on the transcription factor E2A, which is essential for the development of B and T lymphoid cells. Complementary to studying lymphoma cell autonomous processes, we focus on the elucidation of lymphoma-stroma interactions. We have defined growth and survival niches for B cell lymphoma cells in secondary lymphoid organs, and we are further dissecting stromal cell subpopulations to determine essential signals that cause B cell lymphomas to be addicted to their local microenvironment. Collectively, our approaches do not only address fundamental aspects of leukemia and lymphoma pathogenesis, but they are also aimed at providing therapeutic solutions.

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The role of deregulated transcrip- tors or multi-tyrosine kinase inhibitors like tion factor networks for differen- BAY 43-9006 resulted in blocked prolifera- tiation programs, proliferation and tion and enhanced cell death of lymphoma survival of malignant lymphoma cells. Further investigations regarding the cells molecular reprogramming mechanisms, M. Janz, S. Mathas, B. Dörken therapeutic implications and, in coopera-

Deregulated transcription factor (TF) ac- generation of transgenic mouse models are tivities are commonly observed in human tion with the group of Klaus Rajewsky, the lymphomas and leukemias, and the link between structural or functional alterations in ongoing projects of our group. TFs and malignant transformation has been Molecular defects in T cell-derived documented in various in vitro and in vivo lymphoproliferative disorders studies. Apart from the direct modulation S. Mathas, M. Janz, B. Dörken of cellular processes like cellular growth or cell death, alterations of the activity of - even single TFs might enforce malignant ed TF networks as outlined above, we are transformation by switching differentiation Apart from the identification of deregulat programs and consequently altering the and genomic defects of T cell-derived lym- cellular fate of the respective cells, as ex- phoproliferativeinterested in the identificationdisorders. In of particular,molecular emplarily demonstrated for the B lymphoid we focus on the pathogenesis of ALCL and TF Pax5. A number of lymphoid tumors dis- Sézary syndrome, a subtype of cutaneous T play a phenotype that is in accordance with cell lymphoma. In ALCL, we have developed such a reprogramming process, including a concept for the generation of the ALCL- HL, primary effusion lymphoma (PEL) and related chromosomal translocation t(2;5) ALCL. Among these, cHL constitutes the - delity and reprogramming: in striking con- involved(p23;q35). in Inthe this dedifferentiation project we have process already as trastmost to prominent their origin example from B forcells, lineage the malig infi- wellidentified as growth a number and survial of deregulated of these cells. genes It nant Hodgkin-/Reed-Sternberg (HRS) cells is the aim of our ongoing work to investi- of cHL have almost completely lost their B gate the mechanisms leading to the t(2;5)- translocation in ALCL and evaluate the have acquired the expression of genes char- consequences of breakpoint-proximal gene acteristiccell-specific for geneother expression hematopoietic program lineages. and deregulation for prognosis and develop- In our previous work, we could show that in ment of new treatment strategies for ALCL. particular the activity of TF E2A is severely disrupted in the malignant cells of HL and - ALCL. We could furthermore demonstrate ment of Dermatology of the Charité, Uni- that this disruption is directly linked to a versitätsmedizinIn a cooperation Berlin, project we with could the identify Depart reprogramming process that results in the the loss of TF E2A as a hallmark of Sézary up-regulation of genes usually suppressed syndrome (SeSy). Genomic loss of E2A in these lymphoma cells, including genes normally expressed in myeloid cells such as cases, and this loss is required for the un- the CSF1 receptor (CSF1R) gene. Remark- controlledis found in growth the majority of SeSy lymphoma of SeSy patient cells. ably, we observed that expression of CSF1R in HL cells is not mediated by the canonical genes in SeSy tumor cells supporting a role myeloid promoter, but by an upstream long ofIn E2A addition, as a tumor we have suppressor defined gene E2A-driven in hu- terminal repeat (LTR) that is aberrantly ac- man lymphoid cells. Interestingly, among tivated due to a loss of epigenetic control. E2A-regulated genes we identifed the on- Apart from these insights into the mecha- cogene MYC, which points to a direct link nisms of aberrant gene expression in lym- between the loss of differentiation pro- - grams and activation of oncogene activi- pact for the development of new treatment ties. In line with the known requirement to strategies.phoma cells, For our example, work inhibition has significant of CSF1R im counteract the MYC-driven induction of cell - death, we have shown that the tumor sup-

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The sorting receptor Sortilin has a SeSy patient samples and non-functional in dual function in effector molecule nearlypressor all gene investigated TP53 is lost lymphoma in the majority samples. of release from T and NK cells Armin Rehm and Bernd Dörken; in collabo- of further molecular and genomic defects ration with Uta E. Höpken (MDC), Gerald asOngoing well as projects their impact include on thethe identificationdevelopment Willimsky (Charité, Institut für Immunolo- of new treatment strategies for these T cell- gie), and Thomas Willnow (MDC) derived malignancies. One goal of our acitivities in tumor immunology is the development of an adoptive Tumor and microenviron- T cell therapy (ATT) that is highly effective ment: Cellular and molecular and has low/no side effects. Prior to trans- prerequisites for onset and lating ATT to a broader range of cancer progression of leukemia and types, several immunological, clinical and lymphoma technological obstacles must be overcome. Armin Rehm and Bernd Dörken; in collaboration with Uta E. Höpken (MDC) and Georg basic cell biological discoveries in T cells. Lenz (Charité-MKFZ) Hence,Our contribution we are addressing to this field the isquestion fueled byof how the secretory pathway in T cells regu- A hallmark of human Hodgkin and non- lates effector molecule release, e.g. perfo- Hodgkin lymphomas is their propensity to enter secondary lymphoid organs (SLO). Thus, two principal questions arise: what Inrin, recent granzymes work, and we IFN-γ. have shown that the - sorting receptor Sortilin controls constitu- phoma cells to SLOs and secondly, what se- lectivemechanisms growth regulate advantage trafficking might those of lymma- the regulated secretory pathway (Herda et lignant lymphoid cells encounter in their al.,tive Immunity,release of IFN-γ2012). and In granzyme murine Sortilin- A along niches. - cretion of granzyme A and cytotoxic killing Applying a Myc-driven B cell lymphoma wasdeficient enhanced CTLs and NKcorrelated cells, regulated with an sein- creased availability of the SNARE-molecule chemokine receptor CCR7 regulates ac- VAMP7. In contrast, loss of Sortilin reduced cessmodel of inlymphoma mice, we Bhave cells identified to the T cell that zone the - of SLOs (Rehm et al., Blood 2011). Lodging teria monocytogenes infections. From both within this microanatomical niche launched infectionthe release models of IFN-γ we upon infer adenovirusthat enhanced or Lis se- - cretion of granzyme A cannot compensate ticular cells and DCs, a process triggered by thea reciprocal interaction crosstalk of lymphotoxin-expressing with fibroblastic re because liver damage caused by prolonged - adenovirusfor the strong persistence reduction was in IFN-γstronger release and tor-activated stromal cells. Subsequently, titers for Listeria monocytogenes in liv- stromallymphoma cells B cells released and lymphotoxin chemokines β forrecep tumor cell recruitment and growth factors for their protection from apoptosis. apparatusers of Sortilin-deficient required the presence mice were of higher.Sorti- lin,Mechanistically, which acts as exit a sorting of IFN-γ receptor from the for Golgi the Targeting the stromal environment is an - appealing treatment option for lymphoma icles destined for endosomes. In contrast, because this benign compartment is sub- inclusionother cytokines of IFN-γ important into clathrin-coated for T cell vesdif- ferentiation and effector functions, such as and epigenetic changes compared with the - tumorject to lowcells selective themselves. pressure for mutations thermore, we tracked the transport route IL-2 and TNF-α, remained unaffected. Fur

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endosome sorting step, demonstrating that of IFN-γ further downstream of the early- cling endosomes and late endosomes. Our resultsIFN-γ is suggest secreted that independently Sortilin can contributefrom recy to the functional avidity of T cells through exocytic sorting of key effector molecules.

Selected Publications

Lamprecht*, B, Walter*, K, Kreher*, S, Kumar, R, Hummel, M, Lenze, D, Köchert, K, Bouhlel, MA, Richter, J, Soler, E, Stadhouders, R, Jöhrens, K, Wurster, KD, Callen, D,

Siebert, R, Dörken, B, Bonifer, C, Mathas, S. (2010). De-repression of an endogenous Harte,long terminal MF, Giefing, repeat M, activates Barlow, R, the Stein, CSF1R H, Anagnostopoulos,proto-oncogene in I, human Janz, M, lymphoma. Cockerill, P,Nat. Med. 16, 571-579. *) equal contribution.

Steininger, A, Möbs, M, Ullmann, R, Köchert, K, Kreher, S, Anagnostopoulos, I, Hummel, M, Richter, J, Beyer, M, Janz, M, Klemke, CD, Stein, H, Dörken, B, Sterry, W, Schröck, E, Mathas, S, Assaf, C. (2011). Genomic loss of the putative tumor suppressor gene E2A promotes lymphoma in human. J. Exp. Med. 208, 1585-1593.

Calado,the formation DP, Sasaki, and maintenanceY, Godinho, SA, of Pellerin, germinal A, centers. Köchert, Nat. K, Sleckman,Immunol. 13,BP, de1092-1100. Alborán, IM, Janz, M, Rodig, S, Rajewsky, K. (2012). The cell cycle regulator c-Myc is essential for Rehm, A., Mensen, A., Schradi, K., Gerlach, K., Winter, S., Büchner, G., Dörken, B., Lipp, M., Höpken, U.E., (2011). Cooperative function of CCR7 and lymphotoxin in the formation of a lymphoma-permissive niche within murine secondary lymphoid organs. Blood 118, 1020-1033.

Herda, S., Raczkowski, F., Mittrücker, H.-W., Willimsky, G., Gerlach, K., Kühl, A.A., Breiderhoff, T., Willnow, T.E., Dörken, B., Höpken, U.E.,Rehm, A. (2012). The sorting

granzyme A in T cells. Immunity 37, 854-866 receptor Sortilin exhibits a dual function in exocytic trafficking of interferon-g and

Group Leader Kathrin Wurster Prof. Dr. Bernd Dörken Katrin Ulrich*

Senior Scientists Undergraduate Dr. Armin Rehm Antonia Niedobitek* Dr. Stephan Mathas Henrike Sczakiel Dr. Martin Janz Markus Biedermann* Christof Bauer Postdocs Nikolai Schleussner

Dr. Stephan Kreher Technical Assistants Dr. Björn Lamprecht Kerstin Gerlach PhD Simone Lusatis Ute Nitschke Marleen Gloger Maria Kamprath StefanieMarcel Gätjen Herda Franziska Hummel Shuang Li Brigitte Wollert-Wulf Wiebke Winkler * part of the period reported

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CANCER RESEARCH Photo: David Ausserhofer/MDC Photo:

Iduna Fichtner

Experimental Pharmacology

In the recent years, our group has inten- Patient derived xenografts for sified in close collaboration with the Ex- the preclinical development of anticancer drugs perimental Pharmacology and Oncology GmbH the accomplishment of the biobank An extended panel of more than 400 PDX with patient-derived xenograft (PDX) mod- models has been collected and character- els (Figure 1). The collected samples have ized in close cooperation with the Charité and several clinics in Europe representing been used for the identification of biomark- the broad variety and heterogeneity of tu- ers with a special focus on gene muta- mour tissue. Among that are large cohorts tions. Further, the testing of novel targeted compounds has been continued within the (46), renal (22) cancer and leukemias (47).of specimen Each PDX of colonmodel (200),represents head an & neckindi- framework of large grant-financed con- vidual patient tumour (Figure 2) as cor- sortia. Alltogether, the PDX tumours have relation analyses at protein and gene level been regarded worldwide as the best avail- have revealed. The response rates of the PDX models clearly resemble the clinical able models for preclinical drug develop- situation both concerning standard of care ment because of their unlimited availability, treatments and novel targeted therapies. their close relation to the clinical situation Genomic analyses of 67 colorectal carci- and the preservation of heterogeneity and nomas proved that mutations in KRAS, BRAF and PIK3CA clearly could discrimi- complexity. nate between Cetuximab responder and In the field of nanoparticle research we nonresponder. It was shown in a case were focusing on the generation of ves- study of an individual patient that a com- icles for the treatment of brain tumours plete remission of the tumour after Ce- tuximab treatment was observed despite and metastases. The developed platform of the presence of a KRAS mutation. That technology proved the general feasibility to overcome the blood brain barrier and can are now searching for biomarkers of that unexpectedresponse was response reflected in in several the clinics. colorec We- be used for the optimization of treatment tal PDX models with similar features. of brain malignancies. Within our stem cell Extensive studies with PDX models of head research we were interested in the estab- - lishment of standardized procedures for vidual response to clinically used agents like& neck Docetaxel, cancer confirmed5-Fluorouracil, the veryCarbopla indi- the preclinical monitoring of engraftment tin and Cetuximab. Microarray studies and differentiation with the aim to integrate revealed a clear difference between Ce- the technology into risk assessment proce- tuximab responder and nonresponder in dures for the use of embryonic stem cells. relevant predictive biomarkers. Compara- genetive mutational profile enabling analyses the proved identification a stable of

mouse xenografted samples if the frequen- mutation profile between patient and

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Figure 1: Use of patient-derived xenografts (PDX) for the preclinical development of novel anticancer agents

cy of the mutation was higher than 20%. In our previous work regarding liposomal Within the studies of renal cancer we were drug transport of Mitoxantrone across the able to compare both biomarker expres- - sion and response data of several regions somes, targeted to the LRP receptor are suitableBBB we drug found transporters that fluid membrane for the treat lipo- the high intratumour heterogeneity making ment of experimental brain tumors. In aof successful the same tumour.treatment The of resultscancer confirmedvery chal- the recent period, LRP-targeted and non- lenging. targeted liposomes were prepared to en- capsulate oxaliplatin (OxP) and tested for tumour growth inhibition of human MT-3 Development of nanoparticles for breast cancer cells, transplanted both sub- the treatment of brain malignan- cutaneously (s.c.) and into the brain of nude cies mice in order to demonstrate the potential of these liposomes to treat both the pri- A systemic treatment of primary and sec- mary tumour and intracerebral metastases. ondary brain tumours still remains chal- It was demonstrated in a pharmacokinetic lenging because of the existence of the - blood-brain barrier (BBB), preventing ef- creased Cmax and AUC in plasma compared approachto the free that drug the liposomes(3.5-4.5fold). significantly Accumula in-

ficient drug passage. MDC Research Report 2014 93

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tion of OxP in the s.c. growing tumors was tiation. We simulated that approach by us- enhanced up to 3.4fold. In the brain, both ing both human and murine stem cells in non-targeted and targeted liposomes accu- preclinical studies aiming at the develop- mulated better than the free drug (increase ment of standardised analytical methods. in AUC in comparison to free drug: 3,7fold Live cell imaging by bioluminescence tech- or 2.9fold, respectively). In a therapeutic ex- nique was applied to follow murine em- periment, treatment with free OxP had no ef- bryonic stem cell engraftment after trans- fect, while the liposomal formulations clear- plantation via different application routes ly inhibited the growth of brain tumours (s.c., i.splen., i.hep., i.v.). Distribution and by 64% and 59% using non-targeted and organ engraftment were monitored in a targeted liposomes, respectively (Figure 3). time dependent manner. Diff erentiation The results further indicate that the lipo- of stem cells into germ layer progeni- somes developed in our group represent a tors was measured in the formed tera- platform technology for different drugs to tomas by histology, RT-PCR and immu- improve a systemic treatment of brain malignancies. embryotoxicitynofluorescence/immunhistochemistry. test. Agents with known embryotoxicThis technique potentialwas further (none,qualified weak,as an Stem cell engraftment and strong) were investigated in the validat- differentiation ed teratoma model and cytotoxic activity

A potential application of stem cells in pa- The established procedures were further tients requires validated methods for the usedcould to be investigate quantified cell in theengraftment expected way.and monitoring of engraftment and differen- distribution of human embryonic and

Figure 2: Identity of immunohistological staining of patient non-small cell lung carcinomas (upper row) and the corresponding PDX models (lower row). SQC squamous cell carcinoma, ADC adeno carcinoma

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Figure 3: Inhibition of intracerebral growing MT-3 tumours. Mice (n=6 per treatment arm) were treated with 6 mg/kg OxPt in free form (OxP), in non-targeted (nTL) and in targeted liposomes (TL) at day 3,7,12,17. At day 19 mice were sacrificed and the tumour area was measured.*significantly different to vehicle and OxP treated group (p<0.05).

mesenchymal stem cells in cooperation with Charité and BCRT. We further estab- Selected Publications lished standard operating procedures allowing the characterization and the live cell imaging for testing the tumour for- StachelscheidGerlach JC. (2013). H, Wulf-Goldenberg Teratoma formation A, Eckert of human K, Jensen embryonic J, Edsbagge stem J, cells Björquist in three- P, Rivero M,dimensional Strehl R, Jozefczuk perfusion J, culture Prigione bioreactors. A, Adjaye J, J UrbaniakTissue Eng T, RegenBussmann Med. P, 7, Zeilinger 729-741 K, mation and regenerative capacity of stem Wulf-Goldenberg, A., Keil M., Fichtner I, Eckert K. (2012). Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce dif- In addition to the research with embry- ferential organ engraftment. Tissue Cell. 44, 80-86 oniccells stem in experimentallycells we carried injuredout studies organs. with human hematopoietic stem cells. In vivo Kretschmar K, Stecker K, Mantke R, Pauli R, Pertschy J, Hertel K, Ridwelski K, Hellwig studies concerning the function of hema- PK,echańska Pross M, P,Radke Becker C, FichtnerM, Mayr T,I, HoffmannHinzmann J,B, Rosenthal Adams H-P, A. Klaman(2013). MutationI, Kretschmar status K-H, of topoietic stem cells are limited due to low KRAS, BRAF, PIK3CA and expression level of AREG and EREG identify responders to levels of engraftment and failure of the en- cetuximab in a large panel of patient derived colorectal carcinoma xenografts of all four UICC stages. Journal of Cancer Therapy 4, 678-693. grafted cells to differentiate into functional immune cells. Cord blood stem cells were Gries J, Schumacher D, Arand J, Lutsik P, Markelova MR, Fichtner I, Walter J, Sers C, transplanted into the liver of newborn and Titanium technology. Epigenetics 8, 765-771. Tierling S. (2013). Bi-PROF: Bisulfite profiling of target regions using 454 GS FLX on organ engraftment and differentiation Orthmann A, Zeisig R, Süss R, Lorenz D, Lemm M, Fichtner I. (2012). Treatment of adult immunodeficient mice and the effect- targeting on the therapeutic result. Pharm Res 29, 1949-1959. ment was found after transplantation into experimental brain metastasis with MTO-Liposomes: Impact of fluidity and LRP- thewas liver analysed. of newborn A significantly NOD/SCID high mice engraft compared to adult mice, with the highest level of human cells in bone marrow and spleen. These human cells showed CD19 B-cell, Group Leader Graduate students: CD34 and CD38 hematopoietic and CD33 Dr. Iduna Fichtner Marlen Keil myeloid cell differentiation, but lacked any Maria Rivera Markelova T-cell differentiation. On contrary, human Maria Stecklum stem cell transplantation into the liver of Scientists: Maxine Silvestrov adult NOD/SCID mice resulted only in mi- Dr. Michael Becker nor recruitment of human cells in mouse Dr. Klaus Eckert Technical assistants: liver and other organs. The results indicate Dr. Reiner Zeisig Margit Lemm the usefulness of the intrahepatic applica- Dr. Diana Behrens Carsta Werner tion route into the liver of newborn mice Dr. Konrad Klinghammer for the establishment of a human immune Dr. Annika Wulf-Goldenberg Secretariat: system with at least partial differentiation. Dr. Jana Rolff Sylvia Schulz

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Udo Heinemann

Macromolecular Structure and Interaction

Structural biology can reveal the molecular Structural basis of gene basis of cellular biology in exquisit detail and expression regulation is uniquely powerful in generating testable It is increasingly recognizied that gene ex- hypotheses regarding molecular function. pression control is not limited to the regu- We are combining the core technique of lation of transcription initiation. Instead, structural biology, macromolecular crystal- the maintenance of mRNA homeostasis by RNA-binding proteins and microRNAs lography, with biochemical and biophysical appears to be of equal importance. Tran- analyses to study the molecular basis of scriptome-wide cross-linking studies have two different cellular processes. Firstly, we revealed the presence of up to 1000 RNA- binding proteins in human cells. Many of are interested in macromolecular interac- these proteins contain recurrent domains tions that govern the regulation of gene that mediate RNA binding. We have a long- expression, both at the transcriptional and standing interest in one of these structural translational level. Secondly, we study the modules, the cold-shock domain (CSD) which is present in thousands of differ- molecular machineries supporting intracel- ent proteins across all kingdoms of life. In lular transport processes. Here, we focus bacteria, CSDs usually occur in isolation in on two aspects, endoplasmic reticulum- small proteins. They bind single-stranded associated protein degradation (ERAD) DNA and RNA with limited sequence selectivity to a conserved surface that presents and vesicular transport. In addition, we up seven nucleotide-binding subsites. are engaged in numerous collaborations with laboratories needing structural insight In eukaryotes, CSDs are often associated with natively unfolded polypeptide regions into biological problems of their choice. and rarely found together with other folded This collaborative work is facilitated by the domains in the same protein. Lin28 contains Helmholtz Protein Sample Production Facil- a CSD that is followed by a zinc-knuckle do- ity (PSPF). As part of the Joint Berlin MX main (ZKD) and is thus an exception to this rule. LIN28 is up-regulated in embryonic Laboratory we are engaged at the macro tissue and down-regulated in differentiated molecular crystallography facility at the cells. Up-regulation of LIN28 in adult tissue Berlin BESSY synchrotron to which we enjoy is correlated with carcinogenic transforma- priviledged access. tion. Lin28 acts in a negative feedback loop with the let-7 microRNA which is present in large amounts in differentiated cells. This loop is maintained by the Lin28-mediated inhibition of let-7 maturation through blocking the processing of pri-let-7 by Dro- sha and of pre-let-7 by Dicer as well as by recruiting terminal uridylate transferases that mark the miRNA precursor for degradation. We are interested in describing this

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Binding of a DNA single strand to the cold-shock domain of Xenopus tropicalis Lin28b. Center: The positively charged DNA- and RNA-binding surface of the cold-shock domain accommodates seven nucleotide residues which bind predominantly through stacking of nucleobases onto aromatic protein side chains. Sequence selectivity of binding is provided by several hydrogen bonds, some of which are water-mediated. Panels A-C: Detailed views of the subsites binding nucleosides T-1 and T-2 (A), T-4 and T-5 (B), and T-6 and T-7 (C). From Mayr et al. (2012).

remarkable regulatory network in structur- 2). In further work we could contribute al and biochemical terms. to a study of the Landthaler group of the MDC who showed that Lin28 binds a large To elucidate structural features of Lin28 number of other RNA sequences in addition binding to pre-let-7 miRNA, we produced to pre-let-7. With this laboratory we share various constructs of the protein ranging in a strong interest in other CSD-containing length from the full-length polypeptide to proteins such as the Y-box factors and in the isolated CSD or ZKD. Crystal structure further RNA-binding proteins, notably analysis revealed clear structural similarity RC3H1 whose crystal structure we could of the Lin28 CSD with bacterial cold shock recently elucidate. proteins. Binding of single-stranded oligo- nucleotides, in addition, followed the same pattern as in cold shock proteins (Figure Structural aspects of intracellular 1). An analysis of RNA sequence prefer- transport ences of Lin28 and the isolated Lin28 domains suggested that the CSD binds to the In the compartmentalized eukaryotic cell terminal loop of pre-let-7 whereas the every molecule has to be moved to its site of ZKD requires a GGAG motif next to the activity. For proteins with functions outside terminal loop for binding. Importantly, we the cytosol this necessitates their trans- could show that the CSD by itself, but not location across cellular mebranes. Mem- the isolated ZKD is capable of pre-let-7 brane-enclosed vesicles are responsible for binding and inhibition of Dicer cleavage. many steps in either endocytotic or exo- It was therefore concluded that the Lin28 CSD is required for remodeling the let-7 we study the cellular machinery respon- precursor so that the ZKD is able to as- cytoticsible for protein directing trafficking. vesicles and In thisestablishing context, sociate with its binding sequence (Figure their initial physical contact to target mem-

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Model of Lin28-mediated binding and inhibition of pre-let-7 maturation. Our structural and biochemical analyses suggest that Lin28 binds the terminal loop (preE) of the let-7 precursor in a two-step process. Initial binding of the Lin28 cold-shock domain (CSD) to the terminal loop leads to a structural remodeling of preE that renders a GGAG sequence competent for tight and specific binding to the Zn-knuckle domain (ZKD) of Lin28. When both Lin28 domains occupy their sites on pre-let-7, cleavage by Dicer is blocked and maturation is inhibited. In addition, Lin28 recruits terminal uridylate transferases (TUT4/Zcchc11 or PUP-2) to pre-let-7 which mark the miRNA precursor for degradation. From Mayr et al. (2012).

branes. Our work started with investigating binding sites were mapped on the surface structural aspects of the vesicular transport pf p115. The coiled-coil Golgi marker pro- complex TRAPP (transport protein par- tein GM130 bridges between p115 and the ticle) which facilitates the tethering of COP PDZ domains of the membrane-anchored II vesicles at the cis-Golgi membranes. The protein GRASP65, a process for which we structures of several TRAPP subunits and also provided structural insight. sub-complexes were studied and served to elucidate architectural and functional Proteins entering the exocytotic route are aspects of TRAPP. Recently, this work was co-translationally channeled to the lumen extended with the structure analysis of of the endoplasmic reticulum (ER) where the yeast TRAPP-associated protein Tca17. they assume their native fold and undergo Also, we have started to analyze structural features of the yeast HOPS (homotypic fu- incompetent luminal proteins are cleared sion and vacuole protein sorting) tether- frompost-translational the ER in a process modification. called ER-associat Folding-- ing complex which mediates the matura- ed degradation (ERAD) which is studied in tion of vacuoles from endocytic vesicles. detail by the Sommer group of the MDC. The Apart from multi-subunit tethering com- luminal lectin Yos9 of yeast is associated to plexes, Rab GTPases and proteins with ex- the HRD ubiquitin-ligase complex, a central tended coiled-coil structures are important functional module of ERAD. We determined molecules in vesicle tethering. Our struc- the crystal structure of the central domain tural and biochemical analyses of human of Yos9 (Figure 3) and showed that this p115 provided evidence for a tetrameric domain is responsible for the formation of arrangement of the protein supported by Yos9 dimers in solution. We could thus pro- interactions in both its globular head and vide evidence for a dimerization interface coiled-coil regions. Multiple Rab1 GTPase within the ER lumen that is likely to sup-

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Structure of the dimerization domain of the ERAD component Yos9, a lectin associated to the HRD ubi quitin ligase complex. The central dimerization domain (DD, amino acids 266-422) of the ER luminal protein Yos9 is dimeric in the crystal and in solution. The dimerization interface comprises residues anchored in β-strands β5, β6 and β7. Site-directed mutagenesis of interface residues abrogates dimerization of both full-length Yos9 and Yos9 DD. Identification of a Yos9 dimer supports the model of a dimeric HRD-ligase complex which is further stabilized by protein-protein contacts within the ER membrane and in the cytosol. From Hanna et al. (2012).

port a dimeric structure of the HRD ligase PSPF provides a platform through which - many groups from the MDC and beyond ization sites on the surface of HRD subunits have access to crystallization-ready protein locatedcomplex in in the conjunction membrane with bilayer similar or thedimer cy- samples. In cases of mutual interest, this tosol. The cellular ERAD pathways are well service can be extended to biophysical and conserved between yeast and mammals, structural characterizations of proteins and but the lectin Yos9 has two human homo- protein complexes. In recent years, MDC logs, OS-9 and XTP3B. We have recently laboratories from all research programs generated structural data for XTP3B which have made use of the PSPF services. shed further light on the function of these proteins in ERAD substrate recognition and recruitment. Selected Publications The cytosolic ERAD component Cdc48 of yeast is involved in substrate retro-trans- Structural and biochemical basis of Yos9 protein dimerization and possible contribu Hanna,tion to self-associationJ., Schütz, A., Zimmermann, of 3-hydroxy-3-methylglutaryl-coenzyme F., Behlke, J., Sommer, T. & Heinemann, A reductase U. (2012) degrada - location to the cytosol and delivery of tion ubiquitin-ligase complex. J. Biol. Chem. 287, 8633-8640. polyubiquitinated protein chains to the proteasome for degradation. Its ortholog, remodels pre-let‑7 microRNA. Nucleic Acids Res. 40, 7492-7506. the AAA+ ATPase p97 (or VCP for valosin- Mayr, F., Schütz, A., Döge, N. & Heinemann, U. (2012) The Lin28 cold shock domain containing protein) is among the most Sachs, R.*, Max, K.E.A.*, Heinemann, U.# # (2012) RNA single strands bind abundant proteins in human cells. In addi- 18, 65-76. & Balbach, J. tion to ERAD, p97 plays important roles in to a conserved surface of the major cold shock protein in crystals and solution. RNA homotypic membrane fusion, DNA repair regulation - A structural and functional perspective. Int. J. Mol. Sci. 14, 16532-16553. and as an inhibitor of apoptosis. p97 is a Mayr, F. & Heinemann, U. (2013) Mechanisms of Lin28-mediated miRNA and mRNA hexameric molecular machine whose activ- Seul, A.*, Müller, J.J.*, Andres, D., Stettner, E., Heinemann, U.# # (2014) Bac- teriophage P22 tailspike: Crystal structure of the complete protein and function of the ity is regulated by binding of adaptor pro- interdomain linker. Acta Crystallogr. D, in press. & Seckler, R. teins, many of which contain ubiquitin-X regulatory (UBX) domains. In close collaboration with the Wanker laboratory of the MDC we have determined crystal structures of complexes of p97 with the UBX adaptor Group Leader ASPL (alveolar soft-part sarcoma locus). Prof. Dr. Udo Heinemann Qianqian Ming These complexes show a very unexpected structure and stiochiometry which has far- Scientist reaching functional implications. Dr. Ulrich Gohlke TechnicalSaša Petrović Assistants Dr. Jürgen-Joachim Müller Tracy Alice Dornblut Andreas Knespel Protein Sample Production Facility Dr. Karine Sparta Silke Kurths supports collaborative research Dr. Anja Schütz Dr. Yvette Roske PhD Janett Tischer The Helmholtz Protein Sample Produc- Heide Behrmann Secretariat dedicated to the production of proteins AnkurSofia Banchenko Garg Birgit Cloos fortion structural Facility (PSPF) and biophysical led by Anja studies. Schütz The is

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Achim Leutz

Cell Differentiation and Tumorigenesis

Self-renewal of somatic stem cells and Structure & Function of C/EBP differentiation into functional cells are epi- C/EBP transcription factors regulate func- genetic events that are initiated by tran- tions in stem cells and differentiating scription factors downstream of signaling cells and are involved in tumor formation. cascades. Self-renewal vanishes with cell C/EBPs harbor a strong trans-differentia- differentiation but may recur in tumor stem tion and cell lineage instructive potential. Our research is focused on the dynamic cells. Understanding the molecular mecha- regulation of C/EBPs and how these factors nisms involved is central to the application balance self-renewal versus cell differentia- of stem cell based bio-medicine and to the tion. identification of the “Achilles’ heel” in tumorigenesis. We study transcription factors members and probably the result of gene of the CCAAT Enhancer Binding Protein quadruplicationC/EBPα,β,δ,ε, are in early highly vertebrate related evolu family- (C/EBP) family that are key players in cell tion. The C/EBPs display modular structures of conserved short amino acid regions fate decisions in many organs. Somatic that are intervened by low-complexity re- “stemcellness” is often regulated by the Wnt-β-catenin signaling pathway. We have N-termini harbor strong trans-activation domainsgions, suggesting (TAD) whereasstructural the flexibility. center These- explored the function of β-catenin in hema- quences represent regulatory domains topoiesis and leukemogenesis. (RD). The C-terminal basic DNA binding leucine zipper domains (bZip) determine homo- and hetero-dimerization with other transcription factors and interaction with cis-regulatory DNA sequence motifs.

Compound gene deletion studies in the - sential for tissue formation and homeosta- mouse revealed that C/EBPα and β are es Nevertheless, various C/EBP isoforms are generatedsis. Both, C/EBPα,βby alternative are single translation exon genes. initiation and by a multitude of post-transla-

downstreamtional amino ofacid signaling side chain events. modifications They determine(PTM). Boththe C/EBP-interactome types of modifications and play are key roles in the orchestration of diverse C/EBP functions.

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Translational regulation of C/EBPβ and osteoclast formation. C/EBPβ translation is regulated by an upstream open reading frame (uORF) that senses the activity of the translational machinery and directs initiation to alternative start sites. The resulting isoforms, termed LAP*, LAP, and LIP, have distinct gene regulatory and epigenetic potential. The ratio between the isoforms determines expression of the key regulatory MafB protein in osteoclasts and giant cells. MafB in turn regulates several factors involved in cell fusion and differentiation.

Translational regulation of C/EBP In contrast to the LIP mouse strain, mice - - of different N-terminal lengths (termed deficient for the C/EBPβ uORF initiation co LAP*,The C/EBPα LAP, and and LIP) C/EBPβ are generatedprotein isoforms by al- butdon (C/EBPβΔuORF)express the long fail isoforms to initiate LAP*/LAP. transla ternative translation initiation (Figure 1). tion of the truncated C/EBPβ LIP isoform,- - teoclastogenesis, liver regeneration, and bor small upstream open reading frames branchingC/EBPβΔuORF morphogenesis mice are deficientin the mammary for os (uORF)Both, C/EBPα that sense and the C/EBPβ activity mRNAsof the trans har- - lation initiation machinery and relay trans- active acute phase response and persistent lation starts to alternative in-frame initia- repressiongland. C/EBPβΔuORF of E2F-regulated mice display genes (Weth hyper- tion sites. High activity of the translation machinery favors generation of truncated proteins, which lack part of the N-terminus, isoformsmar et al, is 2010, important Genes in & regeneration, Dev., 24:15-20). cell whereas lower translation activity favors differentiation,In summary, switching and prevention between of cancer. C/EBPβ long C/EBP isoforms. Using targeted genetics we have generated knockin mice that can Post-translational C/EBP express either only the long (LAP*, LAP) or modifications provide an index of not switch between C/EBPβ isoforms but epigenetic functions mouse strains are susceptible to many diseases,the truncated including C/EBPβ cancer, isoform defects (LIP). in bone These homeostasis, liver regeneration, mammary gland development, and metabolism. For Several years ago, we found that C/EBPα- example, constitutive expression of trun- broblasts.and β may Accordingly, instruct expression C/EBPs ofentail myeloid the cated LIP isoform enhances the formation epigeneticgenes in many competence cell types, to orchestrate even in skin cell fi of multi-nucleated osteoclasts and induces fate. Considering the modular structural/ functional C/EBP features and seeking for osteoclast transcriptome revealed MafB as an explanation of cell fate determination, it bone diseases. Profiling of the LIP-mouse- came to our attention that several arginine teoclastogenesis (Smink et al. 2009, EMBO (R) and lysine (K) residues were targets J.a C/EBPβ28:1769-81). target These and as results an inhibitor suggest of that os interference with translational initiation and that occurrence of PTMs depends on may represent a potential target for treat- signalingfor post-translational events. Using modifications a systematic (PTM) mass ment of degenerative bone diseases. spectrometric analysis (in collaboration

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Connection between Wnt and interferon signaling in hematopoiesis and leukemogenesis. Wnt signaling induces expression of Irf8 that inhibits expansion of granulocytic precursor cells and limits Wnt pathway functions. In the absence of Irf8 a chronic myeloid leukemia phenotype develops. Loss of Irf8, together with excessive Wnt signaling, causes acute leukemia. The human Philadelphia chromosome BCR-ABL oncoprotein suppresses Irf8 and enhances Wnt signaling, suggesting both pathways as key events in the formation of leukemic stem cells.

genetic functions to direct differentiation into diverse innate immune cells. with Gunnar Dittmar, MDC), we identified- ylation.many novel Biochemical modifications and molecular on C/EBPα genetic and Observations of trans‐differentiation were analysisβ, including revealed K-acetylation that e.g. methylation and K-, R-meth of N- initially regarded merely as the result of arti- - with both, SWI/SNF and Mediator complex- respondence. However, incoherent cell dif- esterminal and that C/EBPβ activation R3 abrogated of ras-MAPK interaction signal- ferentiationficial manipulation, states werewith noreported physiological to emerge cor ing inhibited methylation of R3 by PRMT4/ in pre‐neoplastic tissue e.g. as epithelial CARM1 and of K39 by G9a. Moreover, mu- - tagenesis of R3 and K39 enhanced gene mia/lymphoma, echoing results from experi- activation. These data imply the existence mentalmetaplasia cell andreprogramming. ‘lineage infidelity’ Both, inmetapla leuke- of signal-dependent, intra-molecular cross- sia and biphenotypic leukaemia/lymphoma may represent examples of dysregulated cell - chinerytalk and (Leutzmodification et al, directed2011, Transcription, interactions differentiation and/or epigenetic reprogram- 2:3-8).between C/EBPβ and the epigenetic ma ming.differentiation Studies are that underway reflect a tohistory determine of trans- the C/EBP interaction partners involved in re- An assay that converts primary mouse B programming to elucidate the mechanism of lymphoid progenitors into myeloid cells trans-differentiation and to address the question whether reprogramming is involved in directs alternative cell fates. The results was employed to examine how C/EBPβ 2013, EMBO Mol Med, 5:1154-64) . - tumorigenic transformation (Regalo & Leutz, tiationshowed into that different various C/EBPβtypes of isoforms monocytes/ and We surmise the existence of a post-trans- macrophages,PTMs define the dendritic outcome cells, of trans-differen and granu- lational “Indexing Code” on C/EBPs that may direct distinct protein interactions. The functional consequences of individual locytes. For example, a region in C/EBPβ towas induce identified myeloid-lymphoid that, in conjunction conversion. with the require the knowledge of differentially in- bZip domain, is necessary and sufficient teractingC/EBP-PTMs partners are difficult for further to discern functional and chromatin remodeling complexes controls investigations. We therefore employ high- alternativeMoreover, the trans-differentiation ability of C/EBPβ into to recruit mac- end mass spectrometry and a systematic rophages and granulocytes (Stoilova et al., differential proteomic screening approach - to detect PTM-dependent interactors. Our ings revealed that PTMs and the structural screens revealed epigenetically important 2013, PLoS ONE, 8:e65169). These find proteins that differentially bind to distinct properties that integrate and rewire epi- C/EBP isoforms or C/EBP-PTMs. We ex- plasticity of C/EBPβ are adaptable modular

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ABL inhibitors. Thus, crosstalk between on C/EBPs orchestrate co-factor interac- Wnt and IFN signaling represents a BCR- tionsplore thein orderpossibility to direct that thethe modificationsgene regula- ABL-independent mechanism of disease tory and epigenetic machineries in a gene progression and establishes development of leukemia initiating cells. On the molecu- - dinarilycontext specifichigh numberfashion. Theof multiplicitycombinatorial of interactionsC/EBP modifications that mediate comprise functional an extraor and lar level, activated β-catenin enhances a evolutionary plasticity. We anticipate that causespre-existing progression Irf8-deficient of disease gene and signature, the shift unraveling the PTM-dependent C/EBP in- ofidentifying CML into blast β-catenin crisis. as Since an amplifierelimination that of teraction network might help to elucidate - complex functions in cell differentiation, etic stem cells, and since Irf8 antagonized regeneration, and tumorigenesis. BCR-ABL-inducedβ-catenin did not affect leukemia, normal targeting hematopoi of both pathways together with BCR-ABL inhibitor treatment may pave the way to Crosstalk between Wnt- more effective combinatorial therapeutic and interferon pathways in strategies in the treatment of CML. hematopoiesis & leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative stem cell disor- Selected Publications der resulting from the stable recurrent Kowenz-Leutz, E., Pless, O., Dittmar, G., Knoblich, M., and Leutz, A. (2010). Crosstalk Philadelphia chromosomal translocation between C/EBPbeta phosphorylation, arginine methylation, and SWI/SNF/Mediator implies an indexing transcription factor code. The EMBO J., 29, 1105-1115. in hematopoietic stem cells that gives rise to the oncogenic BCR-ABL fusion protein. Leutz, A., Pless, O., Lappe, M., Dittmar, G., and Kowenz-Leutz, E. (2011). Crosstalk Administration of BCR-ABL kinase inhibi- between phosphorylation and multi-site arginine/lysine methylation in C/EBPs. Transcription 2, 3-8.

Pless, O., Kowenz-Leutz, E., Dittmar, G., and Leutz, A. (2011). A differential proteome tors (e.g. Imatinib) can efficiently restrain achieve due to persistence of inhibitor re- interactions. Nature Protocols 6, 359-364. sistantCML, but leukemia complete initiating remission stem is difficult cells that to screening system for post-translational modification-dependent transcription factor may re-establish CML and cause relapse Scheller, M., Schonheit, J., Zimmermann, K., Leser, U., Rosenbauer, F., and Leutz, A. (2013). Cross talk between Wnt/beta-catenin and Irf8 in leukemia progression and of disease. CML eventually progresses to a drug resistance. The Journal of Experimental Medicine 210, 2239-2256. fatal blast-crisis phase that is characterized by accumulation of differentiation-arrested Wethmar, K., Begay, V., Smink, J.J., Zaragoza, K., Wiesenthal, V., Dorken, B., Calkhoven, C.F., and Leutz, A. (2010). C/EBPbetaDeltauORF mice: a genetic model for uORF-medi- and therapy resistant blast cells, highlighting the need to understand the emergence of leukemia initiating cells. ated translational control in mammals. Genes & Development 24, 15-20.

Using mouse genetics and a BCR-ABL-model of CML, we observed crosstalk between Group Leader Prof. Dr. Achim Leutz Johanna Schiller (INF)-inducible transcription factor Irf8 Julia Schulz (FigureWnt/β-catenin 2). In signalingnormal hematopoiesis, and the interferon ac- Scientist Sabine Schwarz - Dr. Susann Förster tion of Irf8, which in turn limits oncogenic Dr. Elisabeth Kowenz-Leutz Technical Assistants tivation of β-catenin results in up-regula Dr. Qingbin Liu Maria Hofstätter Dr. Jörg Schönheit Sarah Jaksch β-catenin functions. In BCR-ABL CML cells,- Dr. Klaus Wethmar Ilse-Maria Knoblich opIrf8 a isCML-like suppressed disease and and β-catenin self-renewal tends toof Romy Leu hematopoieticbe activated. Irf8-deficient stem cells and animals myeloprolif devel- PhD Victoria Malchin, part time Julia Wiebke Böhm Ruth Zarmstorff Combined deletion of Irf8 and constitutive Branko Cirovic eration becomes dependent on β-catenin.- Undine Hill Secretariat sion of CML into fatal blast crisis, elevated Günther Kahlert Sylvia Sibilak leukemicactivation potential of β-catenin and resultsresistance in progresto BCR-

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Martin Lipp

Molecular Tumor Genetics and Immunogenetics

The chemokine system controls lymphocyte recirculation in immune system homeosta- expressinggene expression the chemokine profiling that receptor B helper CXCR5, cell sis as well as the activation-dependent and whichactivity guides is largely these confined cells into to CD4B cell T follicles,cells co- tissue-selective trafficking of lymphocytes and costimulatory molecules of the CD28 and dendritic cells during immune respons- family such as the inducible costimulator es. Our main focus is the role of homeo- ICOS. We have demonstrated that a transcription factor, that has long been consid- static chemokine receptors like CXCR5 and CCR7 in lymphoid organ development, development of TFH cells. The role of this systemic immune responses, cancer, and eredfactor a in B T cell-specific cells has long factor, been promotes overlooked the chronic inflammatory diseases. We are also for this factor by multiple defects in many interested in the immune modulatory and stagesas its function of B cell development.is masked in miceTherefore, deficient the growth-inducing functions of chemokine re- most important outcome of our study was ceptors encoded by human herpesviruses. factor operated in a T cell intrinsic man- In our translational program we have devel- the finding that the identified transcription ner to control gene expression in TFH cells. oped efficient recombinant and bispecific antibodies targeting B cell Non-Hodgkin changes in the expression of costimulatory moleculesWithout this on T factor, cells weas well observed as a dramatic major lymphoma and multiple myeloma. FH reduction in the frequency of TFH cells.

Differentiation and transcriptional Follicular regulatory T cells (TFR) control of follicular B helper T control humoral autoimmunity via

cells (TFH) NFAT2-regulated CXCR5 expression T cell help to B cells is essential to the (collaboration: M. Vaeth/F. Berberich- development of B cells into antibody-se- Siebelt, Department of Molecular Pathology, creting plasma cells and of immunological Julius-Maximilians-Universität, Würzburg) memory. B cell help is mediated by a specialized subset of CD4 T cells, called follicu-

lar T helper (TFH) cells. Cognate interaction is precisely controlled during the germi- Maturation of high affinity B lymphocytes of antigen presenting B cells with TFH cells at nal center reaction. This is dependent on + + the edges of follicles in secondary lymphoid CD4 CXCR5 TFH cells and inhibited by organs enables the B cells to enter B cell fol- CD4+CXCR5+Foxp3+ follicular regulatory T

licles. TFH cells are also required during the cells (TFR). NFAT2 was found to be highly germinal center reaction, when class switch expressed and activated in follicular T cells. - Unexpectedly, ablation of NFAT2 in T cells cur, as they control multiple steps of B cell caused an augmented GC reaction upon im- recombination and affinity maturation oc munization. Consistently, however, TFR cells presenting plasma cells or long-lived mem- were reduced in the follicular T cell popula- orydifferentiation B cells. We intocould high-affinity show by large-scale antigen tion due to impaired homing to B cell fol-

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Healthy BALB/c mice exhibit a unique periarticular lymph node (paLN) near the knee joint (Left panel): Section of paraffin-embedded hind leg from BALB/c mouse in the early chronic phase of ACIA with hematoxylin eosin staining. Arrows: periarticular lymph node (paLN) close to the knee joint and popliteal lymph node (pLN) embedded in fat tissue. Black bars, 400 mm. (Middle panel): paLN and popliteal lymph node stained for B cells (B220, blue) and T cells (CD3, red). (Right panel): Ten days into the development of chronic arthritis, the paLN expands to a huge size (bottom right). [Nat. Commun. 2013;4:1644. DOI: 10.1038/ncomms2625]

licles. This was TFR-intrinsic since only in in the development of autoimmune gastri- these cells NFAT2 was essential to upregu- late CXCR5. The physiological relevance for both, Ccr7-/- -/-, succumbed early to humoral (auto-) immunity was corroborat- tis and chronic diarrhea. Mice deficient for- ed by exacerbated lupus-like disease in the Rorγt - acute destructive inflammation, character FR cells. andized autoantibody by massive recruitment production, ofand inflamma wasting presence of NFAT2-deficient T disease.tory leukocytes, Antibiotic-treatment pro-inflammatory of mice cytokine be- Transition from an autoimmune- fore disease onset reduced the overall gut prone state to fatal autoimmune disease (Uta E. Höpken; collaboration: M. Schumann, presencemicroflora of and commensal abrogated gut the bacteriadevelopment cru- B. Siegmund, A. Fischer, M.M. Heimesaat and ciallyof fatal contributes mucosal inflammation. to this condition Hence, which the S. Bereswill, Charité) eventually facilitates transformation of a non-life threatening autoimmune condi- Autoimmunity is associated with a strong genetic component, but onset and persis- -/- or the Ccr7-/- mouse strain, into a tence of clinically apparent autoimmune rapidlytion, as developing observed lethal in the autoimmune single-deficient dis- diseases often require an additional envi- easeRorγt in Ccr7-/- -/- mice. ronmental trigger. The balance between immunity and tolerance is regulated by Rorγt molecular factors including nuclear hor- Development of a novel chronic mone and homeostatic chemokine recep- murine model of rheumatoid arthritis and the chemokine receptor CCR7 are both tors.essentially The nuclear involved hormone in functional receptor lymphoid RORγt Rheumatoid arthritis (RA) is a common au- organogenesis and maintenance of lympho- toimmune disease with unknown etiology cyte homeostasis. Lack of one or the other that affects around 1% of the population. In impairs thymic T cell development and al-

revealed an immune-mediated condition, RA, chronic inflammation of the synovium whichters T cell caused homeostasis. a dosed CCR7-deficient immune response mice andof diarthrodial disability. Current joints leadsanimal to models irreversible of RA havejoint damage,fundamental which limitations results in chronicas they pain are

of pro-inflammatory cytokines resulting MDC Research Report 2014 105

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lacking the complexity of the human dis- access of lymphoma cells to the splenic T cell ease. Among the restrictions is the failure zone led to a survival advantage compared of most models to induce the formation were excluded from this zone. Within the the rare development of an auto-antigen niche,to CCR7-deficient lymphoma cells lymphoma stimulated cells a recipro which- drivenof RA-specific chronic course autoantibodies of the disease, like ACPA, and cal crosstalk with gp38+ the restriction to few susceptible strains. cells. This reciprocal cooperation program Therefore, we developed a novel murine was mediated by lymphomafibroblastic B cell-present reticular- arthritis model by combining classical antigen-induced arthritis (AIA) and the cells followed by alteration of stromal cellu- collagen-induced arthritis (CIA) model. The ed LT which acted on LTβR-bearing stromal rationale behind this set-up was to mimic - a chronic disease associated with autoanti- globulinlar composition. fusion protein Crosstalk impaired inhibition lymphoma by LTα body production as observed in human RA growth.deletion Ourand byaim employing is the dissection a LTβR -immuno of cellu- in commonly used mouse strains such as lar requirements and molecular pathways BALB/c and C57BL/6. In BALB/c mice, the which contribute to the transformation of novel antigen- and collagen-induced arthri- secondary lymphoid organs toward lympho- tis (ACIA) model leads to a profound syno- ma-permissive niches. vitis and strong cartilage and bone erosion.

and rapid formation of high serum levels A viral chemokine receptor ofMoreover, IgG against it is characterizedcyclic citrullinated by an peptidesefficient (vGPCR)-triggered animal model (CCP) and collagen II. Concomitant to the reveals epigenetic mechanisms observation of a more severe pathology, we in immune escape and tumor discovered a previously undescribed small progression periarticular lymph node in close proximity (collaboration: Wei Chen/G. Dittmar, MDC)

as the primary draining lymph node for the The human herpes virus 8-encoded G pro- synovialto the knee cavity. joint Our of BALB/c model mice,more whichclosely acts re- tein-coupled chemokine receptor (vGPCR) has been implicated in the pathogenesis of than classical models and is hence particu- Kaposi´s sarcoma (KS) particularly because larlyflects suitable the pathology for further of rheumatoid studies. arthritis of its high constitutive signaling activity. We have used retroviral transduction to generate vGPCR-transformed BALB/c-3T3 Cellular and molecular dissection of the lymphoma-stroma in B cell mice, but as expected fail to induce tumors lymphoma infibroblasts their immunocompetent that are tumorigenic counterparts. in nude (Uta E. Höpken, collaboration: A. Rehm/B. However, tumor fragments obtained from Dörken, MDC, Charité) nude mice grow progressively in BALB/c mice. Unexpectedly, vGPCR-expressing cells The crosstalk between lymphoid tumor cells established from grafted tumor fragments and their environment provides pivotal sig- gave rise to tumors in immunocompetent nals for the initiation and progression of mice. We have now compared gene expres- lymphoma. Histomorphological and immu- nophenotypical aspects of lymphoma re- tumors, cell lines established from these capitulate features characteristic of benign tumorssion profiles as well of as primary, the parenteral vGPCR-induced BALB/c- lymphoid neogenesis. We investigated the 3T3 and vGPCR-transformed BALB/c-3T3 cooperation between homeostatic chemo- cells. This approach, in combination with kine receptor-controlled dissemination and - lymphotoxin (LT)-promoted niche formation in a transgenic mouse model of Myc-driven ChIP-on-Chip histone modification analy- aggressive B cell lymphoma. We showed that quencingses, large-scale (RRBS), DNA and methylation mass spectrometry- profiling using Reduced-Representation Bisulfite Se- to lymph nodes and distinctive microana- cation of critical gene networks comprising tomicCCR7 regulatessites of the Eμ-Myc spleen. lymphoma CCR7-controlled homing signalingbased SILAC and proteomics immunomodulatory led to the identifi genes,

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- cells in vitro and in vivo, substantially pro- cations related to the successive passage of longs tumor-free survival under therapeu- thewhich vGPCR are expressing regulated by 3T3 epigenetic cells in nude modifi and tic conditions in a xenograft mouse model, immunocompetent mice. Hence, this novel and is therefore a promising option for the animal model will contribute to our under- effective treatment of MM and autoimmune standing of the role of the tumor microen- diseases. vironment for the induction of chromatin remodeling and epigenetic changes, which in turn trigger immune escape and progressive tumorigenesis. Patents / Patent applications

Andate antibody 01.11. 2012). that binds a plasma cell-specific antigen suitable for use in the treatment Immunotherapy with bispecific of plasma cell diseases such as multiple myeloma and autoimmune diseases (filing and recombinant antibodies Selected Publications targeting Non-Hodgkin lymphoma and multiple lymphoma Berberich-Siebelt F (2014) Follicular regulatory T cells control humoral autoimmunity Vaethvia NFAT2-regulated M, Müller G, Stauss CXCR5 D, Dietzexpression L, Klein-Hessling J Exp Med. 211:545-561. S, Serfling E, Lipp [Epub M, 2014 Berberich Mar 3] I, (collaboration: G. Moldenhauer, DKFZ, Sallusto F, Lenig D, Förster R, Lipp M, Lanzavecchia A. Pillars in immunology article: Heidelberg) two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature. 1999. 401:708-712 (2014) J Immunol. 192:840-844. for immunotherapy. We have developed a Bispecific antibodies are promising agents Baddack U, Hartmann S, Bang H, Grobe J, Loddenkemper C, Lipp M, Müller G (2013) A antibody binding the chemokine receptor BALB/c mice. Nat Commun. 4:1644. doi: 10.1038/ncomms2625. quadroma-based trifunctional bispecific- chronic model of arthritis supported by a strain-specific periarticular lymph node in Wichner K, Fischer A, Winter S, Tetzlaff S, Heimesaat MM, Bereswill S, Rehm A, Lipp M, ciently prevents tumor growth in a mouse B Höpken UE (2013) Transition from an autoimmune-prone state to fatal autoimmune CXCR5 and the T cell antigen CD3 that effi Autoimmun. 47:58-72. [Epub 2013 Sep 24] disease in CCR7 and RORγt double-deficient mice is dependent on gut microbiota. J effectorcell lymphoma T cells model.to CXCR5 In vitro, expressing the bispecific B cells Schumann M, Winter S, Wichner K, May C, Kühl AA, Batra A, Siegmund B, Zeitz M, andCXCR5::CD3 induced antibodya costimulation-independent efficiently recruited + + with altered ion transport in colonocytes in the absence of overt colitis. Mucosal Im- activation of CD8 and CD4 T cells. In vivo Schulzkemunol. 5:377-387. JD, Lipp M, [Epub Höpken 2012 UE Mar (2012) 7] CCR7 deficiency causes diarrhea associated imaging revealed that CXCR5::CD3 and its

tumor growth in a xenograft model of B cellparental lymphoma CXCR5 in antibody mice and efficiently prolonged prevent their Group Leader CXCR5 as a promising target for antibody- Dr. rer. nat. habil. Martin Lipp Vanessa Stache basedsurvival therapies significantly. in the Our treatment results of identify B cell Dennis Stauß malignancies. Senior Scientist Katharina Wichner PD Dr. Uta Höpken (collaboration: S. F. Marino, O. Daumke, MDC; Dr. Gerd Müller Technical Assistants J. Westermann, Charité) Andra Eienmann* Multiple myeloma (MM) is an aggressive Scientist Jenny Grobe* incurable plasma cell malignancy. The lack Dr. Uta Baddack Branka Kampfrath Dr. Kristina Heinig Kerstin Krüger MM cells led us to develop a human-mouse Susanne Scheu chimericof immunotherapies antibody directed specifically against targeting an ex- Heike Schwede tracellular domain of a receptor molecule, PhDDr. Hossein Panjideh Karolin Voß which is almost exclusively expressed on Franziska Brand - Claudia Krause Secretariat ity antibody blocks the binding of the na- Daniela Keyner plasma cells and MM cells. The high affin Felix Oden rationalized by the high resolution crystal FlorianNedjoua Scholz Mallem Other structuretive ligand of to thethis Fabreceptor. fragment This findingin complex was Edward G. Michaelis with the extracellular domain. Most impor- (* part of the period reported) Franziska Peter tantly, the antibody effectively depleted MM

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Antonio Pezzutto

Molecular Immunotherapy

Goal of our work is the implementation of Clinical vaccination studies using basic research into preclinical models and dendritic cells or modified tumor cells for the induction of tumor spe- clinical trials. Our strategies cover passive cific immune responses and active immunotherapy. A pilot clini- Jörg Westermann and J. Kopp, in cooperation cal study with a gene-modified tumor cell with Th. Blankenstein and W. Uckert vaccine developed in cooperation with Th. Blankenstein (MDC) and D. Schendel (GSF immunogenic tumor cell vaccine that express- Munich) for treatment of advanced meta- esIn renalco-stimulatory carcinoma molecules (RCC), a gene-modified,and secretes static renal cancer has been concluded Interleukin-7, generated in cooperation with Th. Blankenstein and W. Uckert (MDC) as well demonstrating feasibility and safety. We as D. Schendel (GSF, Munich) has been pro- would like to combine this vaccine with duced according to GMP rules in our labora- antibodies targeting the PD1/PD-L1 pathway tory at the Robert-Rössle Building. The pilot in patients who have residual disease after trial has been safely concluded. Evaluation of the immune response of vaccinated patients, therapy with tyrosine kinase inhibitors such and our recent data concerning the numbers as sorafenib and sunitinib. of regulatory T cells in RCC patients undergo- A multicenter clinical trial of Dendritic cell ing therapy with the multikinase inhibitors vaccination in chronic myeloid leukemia in sorafenib and sunitinib have led us to design - patients with persisting minimal residual dis- ies that increase T-cell stimulation by reduc- ease after imatinib treatment is due to start inga follow-up regulatory trial T-cells. in conjunction with antibod in early 2014. Preclinical models evaluate Following on a successfully concluded clinical the costimulatory role of cytokines and che- vaccination trial using in vitro-generated, bcr- mokines such as CCL19 and CCL21 in DNA abl positive dendritic cells (DC) in patients vaccination. This strategy is used to target with chronic myeloid leukemia (CML) we are Her-2, an antigen overexpressed in breast going to start a multicenter DC-vaccination clinical trial in CML patients with persisting cancer where we plan a clinical study using minimal residual disease upon treatment a gene-gun delivery system. T-cell receptor with tyrosine-kinase inhibitors in 2014 with (TCR) gene transfer is a promising strategy the aim of eradicating residual leukemia cells. for adoptive immunotherapy of Lymphomas and Leukemias. Epitopes of the B-cell anti- DNA Vaccination: preclinical models gens CD22 and CD79a/b are being evaluat- Jörg Westermann, Tam Nguyen-Hoai in coop- ed as targets in cooperation with Th. Blan- eration with U. Höpken and M. Lipp kenstein. We also evaluate the possibility DNA vaccination offers several advantages of targeting hematopoietic lineage specific over the use of peptides as vaccines (DNA antigens in the context of allogeneic stem covers several MHC-I and MHC-II epitopes, cell transplantation. directly targets the endogenous presentation pathway and contains immunostimulatory CpG sequences). Furthermore, DNA vaccines

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can be easily produced on large-scale for the not been thymically selected for the human use in clinical trials across HLA barriers. In CD22 or CD79a/b proteins in the murine sys- cooperation with the group of M. Lipp (Mo- - lecular Tumor Genetics) we have explored ly, we are investigating if CD22 and CD79a/b the possibility of recruiting immune cells by aretem suitableand are thereforetargets for of TCRhigh geneaffinity. therapy Current by using plasmid DNA coding for the chemo- analyzing their potential epitopes and test- - Coexpression of CCL19 or CCL21 with tumor antigenskines CCL19 results and CCL21in enhancement as possible adjuvants.of a Th1- ing specific TCRs for gene therapy. In the con polarized immune response with substantial oftext lymphocyte of a cooperative pathways project such we alsoas evaluateMYD88, improvement of the protective effect of the CARD11,mutation-specific EZH2, A20, epit opesCD79a of and key regulatorsCD79b as vaccine. Further improvement of the vaccine possible target for TCR recognition. Several potency has been achieved using a gene-gun for intradermal vaccine application (Coopera- also being evaluated as targets in the context tion with O.Hohn/S. Norley, the Robert-Koch ofhematopoiesis allogeneic stem lineage cell transplantation. specific antigens are Institute). These encouraging results have been extended to a preclinical model using the human breast-cancer associated antigen Her- Patents / Patent applications 2/neu as a target antigen, with the aim of de- PCT-Anmeldung PCT/EP2013/069569 “CD22-BINDING PEPTIDES” (O. Schmetzer veloping a vaccine for breast cancer patients. and A. Pezzutto) Selected Publications Scholz C, Pinto A, Linkesch W, Linden O, Viardot A, Keller U, Hess G, Lastoria S, Lerch Adoptive T-cell therapy for lympho- K, Frigeri F, Arcamone M, Stroux A, Frericks B, Pott C and Pezzutto A. 90Yttrium- ma and leukemia Ibritumomab-Tiuxetan as First Line Treatemnt for Follicular Lymphoma: 30 months of Simone Reuß, Sara Boiani & Neşe Çakmak follow-up Data from an International Multicetner Phase II Clinical Trial. (2013) J Clin in cooperation with Th. Blankenstein and W. Oncol 31: 308-313 Nguyen-Hoai T, Baldenhofer G, Ahmed MS, Pham-Duc M, Gries M, Lipp M, Dörken B, Uckert Pezzutto A, Westermann J. CCL19 (ELC) improves TH1-polarized immune responses and protective immunity in a murine Her2/neu DNS vaccination model. J Gene Med. We are focusing on the treatment of hema- 2012 14:128-137 tological malignancies, particularly of B cell Nguyen-Hoai, T., Baldenhofer, G., Sayed Ahmed, M.S., Pham-Duc, M., Vu, M.D., Lipp, M., Doerken, B., Pezzutto, A., and Westermann, J. (2012). CCL21 (SLC) improves tumor origin. Addressing possible therapies, we are protection by a DNA vaccine in a Her2/neu mouse tumor model. Cancer Gene Therapy working on the development of T cell receptor 19: 69-76. gene therapy. The retroviral transfer of TCR Westermann, J., Florcken, A., Willimsky, G., van Lessen, A., Kopp, J., Takvorian, A., genes into donor T lymphocytes equips them Johrens, K., Lukowsky, A., Schonemann, C., Sawitzki, B., Pohla, H., Frank, R., Dorken, B., Schendel, D.J., Blankenstein, T., and Pezzutto, A. tumor cells (RCC-26/IL-7/CD80) as a vaccine in patients with metastatic renal cell against antigens presented by the malignant cancer: a clinical phase-I study. Gene Therapy 18, 354-363.(2011). Allogeneic gene-modified cells.with aUpon new specificitytransfer into which patients, can be the directed gene- Buchner A, Pohla H, Willimsky G, Frankenberger B, Frank R, Baur-Melnyk A, Siebels - M, Stief CG, Hofstetter A, Kopp J, Pezzutto A, Blankenstein T, Oberneder R, Schendel tigen-expressing cells. As a target antigen ex- IL-2 in patients with metastatic renal cell carcinoma. Hum Gene Ther. 21: 285-297. pressedmodified by T cellsmost are B cell-caused able to attack hematological and kill an DJ. (2010). Phase I trial of allogeneic gene-modified tumor cell vaccine RCC-26/CD80/ disorders we have chosen the receptor CD22 which is responsible for B cell interaction and regulation of B cell signaling. Besides CD22, we are interested in a second B cell antigen Group Leader for targeted immunotherapy. CD79 is a het- Prof. Dr. Antonio Pezzutto erodimeric receptor which is associated with Graduate and Undergraduate the B cell receptor complex and regulates its Scientist Isabell Math (2013) signaling. It is expressed on almost all B cell Dr. Simone Reuss Daniel Pannwitz (2013) neoplasms. So far, these antigens (CD22 and Dr. Tam Hoai Nguyen Agnieszka Szmitkowska (2013) CD79a/b) have been targeted mainly by anti- PD Dr. med. Jörg Westermann Lauri Käsk (2013-2014) bodies but not by T cell therapy. In cooperation with Th. Blankenstein (MDC), we make PhD Students Technical Assistants use of a humanized mouse model expressing Sara Boiani Margarete Gries the human TCR gene repertoire to identify Nese Cakmak Nicole Hellmig

CD22 or CD79a/b-specific T cells which have MDC Research Report 2014 109

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Klaus Rajewsky

Immune Regulation and Cancer

Having moved from Harvard Medical Mechanisms controlling B cell School to the MDC towards the end of 2011, development and homeostasis Dinis Calado, Emmanuel Derudder, Robin our group has continued its exploration Graf, Verena Labi, Sandrine Sander of signals, transcriptional networks and post-transcriptional regulators like miR- While our previous work in this area had NAs controlling cell-autonomous and cell focused on survival and activation signals emanating from the B cell antigen receptor interaction-based mechanisms of nor- (BCR) and the BAFF receptor (BAFFR) and mal and malignant B cell development, using conditional targeted mutagenesis in signal in resting mature B cells downstream identified PI3K signaling as a key survival mice as our main experimental tool. Main signals downstream of BAFFR and canoni- achievements have been the elucidation of of the BCR, apart from “alternative” NF-κB the role of c-Myc in germinal center (GC) through the BCR and Toll-like receptors B cell differentiation and the generation cal NF-κB signaling upon B cell activation years has been to clarify the role of the proof faithful pre-clinical models of certain to-oncogene(TLRs) & CD40, c-Myc a main in the effort GC reaction.over the Thelast classes of human B cell lymphomas and GC reaction is a key process in T cell depen- Epstein-Barr-Virus (EBV) pathologies, in- dent antibody responses, where antigen-ac- cluding the immune surveillance of EBV-in- tivated B cells proliferate vigorously while undergoing programmed DNA breaks ac- fected normal and transformed cells. New, companying somatic hypermutation (SHM) highly efficient systems of targeted muta- of antibody variable (V) region genes and genesis in mammalian cells, in particular antibody class switch recombination (CSR). CRISPR/Cas9-mediated mutagenesis, are Earlier work had suggested that GC B cell presently revolutionizing the fields of mam- proliferation might be independent of c- malian somatic cell genetics and the gen- Myc, driven perhaps by a program con- eration of genetically modified experimental trolled by the transcription factor Bcl6. Us- ing conditional gain- and loss-of-function animals. Accordingly, a main recent activity alleles of c-Myc we showed that c-Myc is of the group has been the introduction of critical at two distinct stages of the GC re- this new approach into our experimental sponse, namely its initiation and when routine. somatic antibody mutants arising in the course of the reaction are selected for

antigen(Caladofurther rounds et of al. cell 2012). division This result & somatic pro- videsmutation an explanation on the basis for ofthe their frequent affinity (tran to- scription-dependent) c-Myc translocations in GC B cell–derived B cell lymphomas, by far the most frequent class of such tumors.

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Modeling Burkit lymphoma, a GC-B cell-derived human malignandy, in mice

GC B cells (= PNA expressing cells (A)) are important for the generation of antigen- specific immune responses; additionally, they are the cellular origin of most B cell derived lymphomas, like e.g. BL. We have generated a pre-clinical model of BL by conditional targeted mutagenesis allowing the expression of oncogenic factors specifically in GC B cells that resemble the human disease in several aspects including histology (“starry sky” pattern (B)) and expression of GC B cell specific genes (C) (Sander et al. 2012).

- Modeling human lymphomas and dressed the role of BCR signaling in B cell EBV-driven pathologies and im- subsetOther experiments differentiation, on this substantiating subject have ourad mune surveillance in mice

Burkitt lymphoma canprevious differentiate finding into that cells mature of the follicular so-called B Sandrine Sander, Daniela Bolgehn, Van B1cells, subset the major upon subsetexchange of peripheral of their BCR B cells, by a Trung Chu, Karl Koechert, Dinis Calado BCR typical for B1 cells. Present work addresses the mechanism of this “transdiffer- Burkitt lymphoma (BL) is a GC-derived hu- entiation” process, which proceeds through man B cell lymphoma, known to be driven an initial phase of rapid proliferation, and in 100% of the cases by deregulated c-Myc its relation to B1 cell development in ontog- expression. The latter is mostly mediated eny. by cMyc translocations into the immunoglobulin (Ig) loci of the cells. As these trans- We have also continued studies on the role locations involve the non-productively rear- of miRNAs in hematopoietic development, ranged Ig loci and thus keep BCR expression with a focus on B cell differentiation and by the cells intact, we speculated that the miRNA-142 and the miRNA cluster miR- pro-survival signal required to neutralize 17~92. This work relies heavily on attempts c-Myc’s pro-apoptotic properties would be to identify miRNA targets at a genome-wide the PI3K survival signal downstream of the level (collaborating with colleagues at the BIMSB), and conditional mutagenesis of Indeed, combining conditional constitu- miRNA seed matches in the 3’ UTRs of pre- tiveBCR, c-Myc which expression we had previously and PI3K identified.signaling sumed target genes. A surprising cell type in GC B cells by conditional targeted mutagenesis in mice resulted in a faithful pre- emerge from these experiments. The work clinical model of BL in terms of localization, alsospecificity relates of to miRNA the impact control of miRNAis beginning control to on the pathogenesis of lymphomas (Jin et gene expression signature. The tumors also al. 2013). sharedhistological recurrent & cell tertiary surface somatic phenotype mutations and

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Analysis of tumor associated mutations by CRISPR/Cas9

Mutations are selected based on their incidence in our various lymphoma mouse models as well as in published data sets of human lymphomas. The impact of selected candidates on tumor maintenance is tested initially in suitable (mouse and human) cell lines according to the indicated workflow of CRISPR/Cas9 mediated mutagenesis.

with their human counterparts (Sander et al. contexts. A case of special interest is Hodgkin - Lymphoma (HL), where the tumor cells have ited the same tendency to disseminate into undergone a clonal EBV infection in about the2012). central Significantly, nervous systemthe mouse as human tumors BL, exhib the 40% of the cases. most threatening clinical complication of the disease. Ongoing experiments address this is- Starting from our discovery many years ago sue and use CRISPR/Cas9-based mutagenesis that the tumor cells in HL, the so-called Hodg- to address the impact of recurrent somatic kin and Reed-Sternberg (HRS) cells, arise mutations on BL pathogenesis. from pro-apoptotic (“crippled”) GC B cells, we have over the years attempted to model EBV-driven pathologies and immune sur- HL and EBV infection in the mouse. With re- veillance; Hodgkin Lymphoma (collabo- spect to HL pathogenesis, this includes the ration with the Janz/Mathas/Dörken conditional activation of HL-associated tran- group) scriptional regulators and survival factors in Tomoharu Yasuda, Shuang Li, Thomas Som- mouse B cells, particularly at the GC stage of mermann, Timm Weber, Tristan Wirtz cooperation with the Janz/Mathas/Dörken group.differentiation, Our attempts a project to modelthat is EBVcarried infection out in population, with over 90% of people infected are based on the transgenic expression of key worldwide.EBV is a γ herpes The virus virus infects endemic and in transformsthe human EBV proteins in the B cell lineage of mice, a human B cells, but is contained by powerful T species from which EBV is absent. Focus- and NK cell immunosurveillance. In infected ing on the EBV proteins LMP1 and LMP2A, individuals EBV persists in a few B cells for known to play key roles in the transformation life. Reactivation under conditions of immune of human B cells, we showed in work which suppression like in post-transplantation or was already initiated in Boston that LMP-ex- AIDS patients can lead to a life-threatening lymphoproliferation and EBV-driven B cell immune surveillance in mice, and that LMP- lymphomas. These complications also occur drivenpressing lymphomas (mouse) B rapidlycell blasts develop induce when efficient im- upon primary EBV infection, in rare patients mune surveillance is compromised (Zhang, which are genetically unable to control EBV. Kracker, Yasuda, Casola et al. 2012). More In X-linked Lymphoproliferative Syndrome recent work has shown that we can faith- (XLP), T and NK cell defense against EBV is fully model primary EBV infection in mice by compromised by mutation of the X-linked conditional activation of LMP expression in gene encoding the signal-transducer SAP. a small fraction of mouse B cells at various EBV-driven lymphomas also occur in other stages of differentiation (Yasuda et al. 2013).

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A mouse model of primary EBV infection in mice

Lymphoma of the Activated B cell-type (ABC-

also functionally contribute to DLBCL pathogenesis,DLBCL), in the concert alternative with other NF-κB genetic pathway altera can- tions. We are also in the process of functionally elucidating the role of mutations in the TLR and BCR signaling pathways in the pathogenesis of DLBCL, through targeted conditional mutagenesis in mice. Finally, ongoing work in collaboration with Wiebke Winkler and Martin Janz addresses the generation of new mouse models of human multiple myeloma.

Mouse model for primary EBV infection. (A) Tamoxifen-induced expression of LMP1 in mouse B cells leads to expansion of splenic B cells in experimental Selected Publications (CD19creERT2;R26LMP1), but not control mice (CD19creERT2;R26YFP). (B) Proliferat- Yasuda T, Wirtz T, Zhang B, Wunderlich T, Schmidt-Supprian M, Sommermann T, ing lymphocytes six days after tamoxifen- Reconstructing EBV Infection in Mice. Cold Spring Harbor Symposia on Quantitative treatment of a CD19creERT2;R26LMP1 mouse. RajewskyBiology. 2013 K. Studying Nov 15. Epstein-Barr Virus Pathologies and Immune Surveillance by Ki-67 staining of proliferating cells in a spleen section. (C) T cell expansion after LMP1+ B cell Jin HY, Oda H, Lai M, Skalsky RL, Bethel K, Shepherd J, Kang SG, Liu W-H, Sabouri- expansion in spleen. (D) An effector/memory in lymphomagenesis by coordinating multiple oncogenic pathways. EMBO J. 2013 phenotype of T cells (CD44+/CD62L-) follow- GhomiAug;32(17):2377–91. M, Cullen BR, Rajewsky K, Xiao C. MicroRNA-17~92 plays a causative role ing expansion (Yasuda et al. 2013). Calado DP, Sasaki Y, Godinho SA, Pellerin A, Köchert K, Sleckman BP, de Alboran IM, - tion and maintenance of germinal centers. Nat Immunol. 2012 Nov;13(11):1092–100. Janz M, Rodig S, Rajewsky K. The cell-cycle regulator c-Myc is essential for the forma Sander S, Calado DP, Srinivasan L, Köchert K, Zhang B, Rosolowski M, Rodig SJ, Holz- In this model, an initial rapid expansion of - naling and MYC in Burkitt Lymphomagenesis. Cancer Cell. 2012 Aug 14;22(2):167–79. LMP-expressing B cells is followed by the mann K, Stilgenbauer S, Siebert R, Bullinger L, Rajewsky K. Synergy between PI3K Sig activation and expansion of T cells, with sub- Zhang B, Kracker S, Yasuda T, Casola S, Vanneman M, Hömig-Hölzel C, Wang Z, Derud- sequent elimination of the B cell blasts and der E, Li S, Chakraborty T, Cotter SE, Koyama S, Currie T, Freeman GJ, Kutok JL, Rodig establishment of T cell memory. Interference Epstein-Barr virus protein LMP1 in a mouse model. Cell. 2012 Feb 17;148(4):739–51. with T cell defense results in fatal lymphopro- SJ, Dranoff G, Rajewsky K. Immune surveillance and therapy of lymphomas driven by liferation as in human XLP. Ongoing work addresses the mechanisms controlling immune surveillance in this system, which apparently Group Leader Timm Peer Christopher Weber Prof. Dr. Klaus Rajewsky Tristan David Wirtz toreflects model a strategyhuman XLPof the and virus EBV-driven to establish lym a- phomagenesisprofitable coexistence in preclinical with its mouse (human) models, host, Scientists Technical Assistants and to elucidate cell-autonomous and cell Dr. Van Trung Chu Claudia Grosse interaction-based mechanisms driving EBV- Dr. Emmanuel Derudder associated pathologies. Dr. Verena Labi Kerstin Petsch Dr. Ulrike Sack (until 12/2013) JanineDr. Anja Woehlecke Koehler Other related work Dr. Marie Sandrine Sander Dinis Calado, Ulrike Sack, Kristin Schmidt, Van Dr. Thomas Sommermann Secretariat Trung Chu, Baochun Zhang; collaboration Dr. Tomoharu Yasuda Hannah Rose Dunham with Wiebke Winkler and Martin Janz Riikka Lauhkonen-Seitz (periods PhD of maternity leave) In work that has been submitted for publica- Daniela Bolgehn (until 12/2013) tions, we have shown that in addition to the Robin Graf Guest Scientists Kristin Schmidt Dr. Kaaweh Molawi in the pathogenesis of Diffuse Large B Cell well-known role of canonical NF-κB signaling

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Oliver Rocks

Spatio-temporal Control of Rho GTPase Signaling

Cells frequently change their shape and their attachment to the environment in order to move or to adopt specialized structures in tissues. These events are fundamental during embryonic development, immune surveillance or wound repair. The reorganization of the cytoskeleton and the adhesion machinery thereby is highly dynamic and requires precise coordination. Any deregulation can contribute to cancer metastasis and various developmental, cardiovascular and neuro- Schematic representation of the molecular switch function of logical disorders. Our lab is interested in the Rho GTPases and the function of their regulatory proteins. family of Rho GTPase proteins which have emerged as master regulators of the cyto- downstream of a signaling pathway. This skeleton. We combine advanced microscopy cycle is controlled by three classes of regula- with cell biological and genetic approaches tory proteins: the large families of activating to study the precision control that ensures RhoGEFs (Rho guanine nucleotide exchange factors) and inactivating RhoGAPs (Rho GT- proper Rho signaling dependent cellular re- Pase activating proteins), and by RhoGDI pro- sponses. Specificity of Rho GTPase function teins (Rho guanine nucleotide dissociation is achieved by the huge family of RhoGEF and inhibitors), which mediate the sequestration of the membrane anchored Rho GTPases into GAP regulatory proteins. We aim to identify the cytosol (Fig. 1). The Rho family comprises mechanisms by which these proteins couple about 20 genes, with the most prominent Rho activity to specific environmental cues members RhoA, Rac1 and Cdc42. Numer- and functional contexts in a cell. Insights into ous Rho-regulated signaling pathways have been outlined over the past years, many of this spatio-temporal regulation will provide a which drive central aspects of cellular mor- deeper understanding of the processes that phodynamics. The different members of the drive specific cell morphology changes, in Rho family thereby have distinct functions normal and disease settings. and can crosstalk with each other, resulting in a complex interplay of pathways. A precise spatio-temporal control of their activities Signaling Specificity - of Rho GTPases is essential to assure specificity in the bio Rho GTPases are molecular switches that logical response. The major challenge in the cycle between an ‘on’ and ‘off’ state. Only in GDIfield regulatory thus is to understand proteins. Notably, how this deregulated signaling the activated state they bind effector proteins Rhospecificity GTPase is signaling achieved in by pathological the RhoGEF/GAP/ settings and thereby relay incoming signals further is mostly caused by alterations at the level of

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Modular domain architecture of the human RhoGEF and RhoGAP proteins.

assembly is regulated, how they relay signals to the cytoskeleton or are interlinked with it and how they communicate with other cellular signaling pathways.

Temporal framework of Rho GTPase signaling

Very little is known also about the time control of Rho signaling. Most Rho family GTPases

associated to properly exert their signaling function.are lipid modifiedExactly how and on need membranes to be membrane the formation of Rho signaling complexes is initiated, maintained and terminated and whether a precise temporal control is important for proper signaling is unclear and is technical-

state-of-the-art imaging techniques to ana- the regulatory proteins and not by mutations lyzely difficult the dynamics to be studied of membrane in cells. Weinteraction employ of the Rho proteins themselves. of Rho GTPases and its modulation by regulatory and effector proteins. We thereby aim to elucidate the temporal framework in which RhoGEFs and RhoGAPs novel modes of signal modulation. The genome encodes over 145 different Rho signaling complexes operate and to find RhoGEFs and GAPs which contain a diverse spectrum of subcellular targeting and protein Selected Publications interaction domains, besides their catalytic Rocks, O., Gerauer, M., Vartak, N., Koch, S., Huang, ZP., Pechlivanis, M., Kuhlmann, domains (Fig. 2). This multidomain archi- J., Brunsveld, L., Chandra, A., Ellinger, B., Waldmann, H., Bastiaens, PI. (2010) The Palmitoylation Machinery Is a Spatially Organizing System for Peripheral Membrane protein complexes to target Rho activity to Proteins. Cell 141(3):458-471 distincttecture signaling enables themcontexts to such assemble as focal specific adhe- Dekker, FJ.*, Rocks, O.*, Vartak*, N., Menninger*, S., Hedberg, C., Balamurugan, R., Wet- - zel, S., Renner, S., Gerauer, M., Schölermann, B., Rusch, M., Kramer, JW., Rauh, D., Coates, - GW., Brunsveld, L., Bastiaens, PI., Waldmann, H. (2010) Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat Chem Biol 6(6):449-56 *equal GEFssions, andcell junctions,GAPs is only endocytic poorly vesiclescharacterized or mi contribution andtotic the spindles. cellular However, repertoire the of majority spatio-temporal of Rho Rocks, O. - control of Rho GTPases not fully explored. nals: one ship, three anchors, many harbors. Curr Op Cell Biol 18(4):351-7 Therefore, we have recently carried out a sys- , Peyker, A. & Bastiaens, PI. (2006) Spatio-temporal segregation of Ras sig tematic initial characterization of all mamma- Rocks, O., Peyker, A., Kahms, M., Verveer, PJ., Koerner, C., Lumbierres, M., Kuhlmann, lian RhoGEFs and GAPs, yielding a complete Localization and Activity of Palmitoylated Ras Isoforms. Science 307:1746-1752 cDNA library of all regulators and datasets J., Waldmann, H., Wittinghofer, A. & Bastiaens, PI. (2005) An Acylation Cycle Regulates of their interactome, subcellular localization, overexpression and knockdown phenotypes. We now exploit this unique resource and study novel RhoGEFs and GAPs with Group Leader implications in different signaling contexts Dr. Oliver Rocks such as cell adhesion, guided cell migration Graduate and Undergraduate or angiogenesis. To this end, we use live cell Scientist Laura Reckzeh microscopy and biosensors to obtain infor- Victoria Casado Sebastian Reusch mation on appropriate spatial and temporal Philipp Trnka microscales and employ three-dimensional PhD cell culture systems and model organisms Matti Baumann Technical Assistants Juliane Brümmer Marlies Grieben processes in the organismal context. We aim Rebecca Eccles forthat a closelybroader reflect understanding the underlying of the function biological of Paul Markus Müller Secretariat Carolin Riemer Petra Haink essential components they contain, how their the context-specific signaling scaffolds, which

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Claus Scheidereit

Signal Transduction in Tumor Cells

The major interest of our laboratory is to un- The molecular constituents of IKK derstand gene expression and signal trans- and NF-κB signaling cascades duction processes in tumorigenesis and in development. The inducible transcription factor nuclear factor-κB (NF-κB) provides andThe RelB, mammalian which form NF-κB hetero- family and comprises homodi- five related members, p50, p52, p65, c-Rel an excellent model system with broad mers and regulate hundreds of genes in the context of multiple important physiological physiological and medical significance. The and pathological processes. The inhibitory NF-κB system controls important steps in immunity and inflammation, as well as cell - IκB proteins, e.g. the cytoplasmic IκBα or death, proliferation or metabolism. We have subunits,the nuclear p50 Bcl-3 and and p52, IκB-ζ, are are generated fundamen by investigated various aspects of the regula- proteasomaltal regulators processing NF-κB activity. of their Two precursor NF-κB tion of NF-κB transcription factors, whose proteins, p105 and p100, respectively. activities are controlled by inhibitory IκB proteins and the IκB kinase (IKK) complex. (IKK) complex, composed of the two ki- Our major efforts are to decipher the mech- NF-κB activation requires the IκB kinase anisms that underlie gene regulation by IKKnases complex (IKKα andresponds IKKβ) to and a wide the regulatory variety of IKK and NF-κB in development and in the extra-subunit and IKKγ intracellular (also known stimuli as NEMO). to catalyze The pathogenesis of human diseases. as well as of other substrates. A character- isticphosphorylation feature of canonical of IκB and signaling NF-κB cascadesproteins

with K63-linked or linear polyubiquitin chains,is the modification which mediates of TRAF recruitment or RIP proteins of signaling components with ubiquitin binding - ing in IKK activation. Catalytically active domains, including IKKγ, finally result and liberation of prototypic p50-p65. The IKK complexes trigger degradation of IκBs

non-canonical NF-κB pathway is activated p52by a and specific nuclear set translocation of receptors andof p52-RelB induces complexes.C-terminal processingThe non-canonical of NF-κB2/p100 pathway to proceeds with much slower kinetics and

DNArequires double NF-κB-inducing strand breaks kinase and depends (NIK) and on IKKα. A third IKK pathway is activated by

nuclear shuttling and SUMO-modification of IKKγ, as well as on the kinase ATM. 116 MDC Research Report 2014 CANCER RESEARCH

Figure 1: DNA damage invokes two signaling axes, triggered by ATM and PARP-1, to activate NF-κB. PARP-1 is activated and sequesters PIASy, ATM and IKKγ, which shuttles between nucleus and cytoplasm, resulting in IKKγ SUMOylation. ATM is exported to the cytoplasm and binds to TRAF6 to induce Ubc13 dependent K63- linked polyUb synthesis. TRAF6 ubiquitination activates TAK1 and cIAP1-dependent IKKγ monoubiquitina- tion. IKKγ-monoUb requires both, PARP-1 and ATM signals and is essential for IKK activation. Other components acting downstream of TRAF6 and IKK, respectively, are the linear ubiquitin chain assembly complex (LUBAC) and the Hsp90/Cdc37 chaperone complex.

Mechanism of IKK and NF-kB ate DNA repair, cell cycle checkpoint con- activation by genotoxic stress trol and the activation of transcription fac- (Seda Çöl Arslan, Patrick Beaudette, Daniel Heinze, Michael Hinz, Marina Kolesnichenko, assumed that p53 induces cell-cycle arrest Nadine Mikuda, Giulietta Roel, Michael Stil- tors such as NF-κB and p53. It is generally- mann, Michael Willenbrock) vival and may counteract apoptotic action ofor genotoxicapoptosis, therapy.while NF-κB However, triggers it could cell sur be Metabolic and external DNA-damaging - agents, such as ionizing radiation or toxic chemicals, constantly endanger the genom- thedemonstrated induction thatof a NF-κB senescence-associated activation by che ic integrity of cells. To counter these threats, secretorymotherapy phenotype can also be(Jing beneficial et al. 2011). through We mammalian cells have developed a network could demonstrate that genotoxic stress of DNA damage response. DNA lesions are triggers two corresponding signaling axis recognized by sensor proteins, which initi-

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Figure 2: Molecular controls of early hair follicle development in the mouse (see Zhang et al., 2009, for details). At embryonic day E12.5, WNT is uniformly active in the upper dermis. First focal epidermal and dermal WNT activity was observed at E13.5, and is entirely dependent on epidermal WNT/β-catenin activity. The only focally expressed WNT at E13.5 is WNT10b, suggesting that it may be the HF placode inducing WNT. Early WNT10b activity initiates expression of the WNT inhibitor Dkk4, which may prevent WNT signaling in the direct surroundings of the developing placode. Between E13.5 and E14.5 Edar expression is focally up-regulated by WNT/β-catenin. This leads to activation of EDAA1/EDAR/NF-κB signaling, which induces expression of NF-κB target genes, such as Dkk4, Wnt10b and Wnt10a. Higher concentrations of DKK4 protein caused by joint NF-κB and β-catenin transcriptional activity will further refine hair follicle placode patterning. Apart from Dkk4, EDA-A1/EDAR/NF-κB signaling also maintains expression of Wnt10b, and perhaps also of WNT10a and Lef-1, leading to a positive feedback loop between WNT and EDA-A1 signaling.

- Mechanism of NF-κB precursor processing and selective functions initiatedanalogously by tothe the DNA canonical strand NF-κBbreak signalsensor of canonical and non-canonical poly(ADP-ribose)-polymerase-1ing cascades (Figure 1). The first (PARP-1), axis is NF-κB/IKK gene networks which assembles a transient nucleoplas- (Patrick Beaudette, Inbal Ipenberg, Eva mic complex and triggers PIASy mediated Kärgel, Kívia A. Pontes de Oliveira, Linda von SUMOylation and most likely ataxia telan- Hoff, Buket Yilmaz) giectasia mutated (ATM) mediated phos- Hodgkin’s lymphoma (HL), diffused large B-cell lymphoma (DLBCL) and many other shuttlesphorylation back of to nuclearthe nucleus IKKγ and (Stilmann integrates et - intoal., 2009). IKK complexes.It is assumed ATM that translocates modified IKKγ to mote, amongst others, their proliferation cytoplasm, binds to TRAF6 and triggers its andtumor survival. types relyIn HL on cells,IKK and canonical NF-κB asto prowell K63-linked polyubiquitination. Activated TRAF6 recruits cIAP1 and TAB2-TAK1, re- constitutively activated. We investigate at a - genomeas non-canonical and transcriptome IKK/NF-κB wide signaling level how is canonical (p50, p65), non-canonical (p52, sulting in TAK1 activation and IKKβ phos phorylation (Hinz et al., 2010). The final activation of the IKK complex requires IKKγ- tumor-biologicalIKKα, NIK) and functions accessory and components how gene ing”mono-ubiquitination (SRM) mass spectrometry of IKKγ at lysine(MS) (col285,- (Bcl-3) of the NF-κB system control distinct laborationidentified bywith “selected G. Dittmar). reaction In monitorongoing studies, we investigate characteristics of fullspecificity dissection is reached of the (collaborationIKK-dependent with onco N.- the genotoxic pathway in vivo by bio-im- genicHübner network and M. in Andrade). HL cells in The order final to aim predict is a aging, analyze how DNA damage induced novel therapeutic targets. We have deter- - mined the complete genomic binding maps tivation and search for novel components - inIKKγ the modifications signaling cascade. contribute Furthermore, to NF-κB acwe cies by ChIP sequencing and characterized investigate the cross talk between p53 and theirfor canonical differential and transcriptomic non-canonical impact,NF-κB spe using siRNA-mediated knockdown. In further

NF-κB in transcriptional regulation. 118 MDC Research Report 2014 CANCER RESEARCH

studies, we investigate the mechanisms and structures that underlie non-canonical Selected Publications - Stilmann, M., Hinz, M., Çöl Arslan, S., Zimmer, A., Schreiber, V.and Scheidereit, C. sion, using biochemical analyses, MS-based (2009) A nuclear poly(ADP-ribose) dependent signalosome confers DNA damage NF-κB signaling, e.g. p100 to p52 conver induced IkB kinase activation. Mol Cell 36, 365-378. mathematical modeling (collaboration with Zhang, Y., Tomann, P., Andl, T., Gallant, N., Huelsken, J., Jerchow, B., Birchmeier, W., Paus, Gunnarquantification Dittmar of and pathway Jana Wolf). components and R., Piccolo, S., Mikkola, M.L., Morrisey E.E., Overbeek, P.A:, Scheidereit, C., Millar, S.E., Dev. Cell 17, 49-61. and Schmidt-Ullrich, R*. (2009) Reciprocal requirements for Eda/Edar/NF-κB and Wnt/β-cateninHinz, M.,Stilmann, signaling M., Çöl pathways Arslan, S., in Khanna, hair follicle K.K., induction.Dittmar, G., and Scheidereit, C. Role of Eda-A1/Edar/NF-κB (2010) A cytoplasmic ATM-TRAF6-cIAP1 module links nuclear DNA damage signaling signaling in development and Mol Cell, 40, 63-74. organ regeneration toJing ubiquitin-mediated H, Kase J, Dörr JR, MilanovicNF-κB activation. M, Lenze D, Grau M, Beuster G, Ji S, Reimann M, Lenz (Karsten Krieger, Philip Tomann und Ruth P, Hummel M, Dörken B, Lenz G, Scheidereit C, Schmitt CA, Lee S. (2011) Opposing Schmidt-Ullrich) - tions. Genes Dev 25, 2137-2146. roles of NF-κB in anti-cancer treatment outcome unveiled by cross-species investiga

embryonic development and organ regen- Wolf A, Frick M, Dietze K, Madle H, Tzankov A, Hummel M, Dörken B, Scheidereit C, To elucidate the in vivo function of NF-κB in Nogai H, Wenzel SS, Hailfinger S, Grau M, Kaergel E, Seitz V, Wollert-Wulf B, Pfeifer M, eration, various loss-of-function, gain-of- gene network and survival of ABC DLBCL. Blood 122, 2242-2250. function and reporter mouse models were Janz M, Lenz P, Thome M, Lenz G. (2013) IκB-ζ controls the constitutive NF-κB target generated in our laboratory. We previously

induction of hair follicle and secondary lymphdemonstrated node development, a key role for and NF-κB in the in mor the- phogenesis of ectodermal organs such as teeth or mammary glands. Further studies mainly concentrated on hair follicle development. Essential signals that regulate early hair follicle development include WNT/

ectodysplasin A1 (EDA-A1) and its receptor β-catenin, as well as TNF family member the initial hair fate decision of epidermal EDAR. WNT/b-catenin signaling specifies Group Leader Inbal Ipenberg * hair bud formation. We have demonstrated Prof. Dr. Claus Scheidereit Karsten Krieger keratinocytes, while NF-κB is required for Nadine Mikuda EDA-A1/EDAR/NF-kB signaling which is re- Scientists Kivía A. Pontes de Oliveira quiredan interdependence for focal hair budof WNT/β-catenin formation and patand- Dr. Seda Çöl Arslan Linda von Hoff * terning, as well as hair bud down-growth Dr. Irene Coin * Michael Willenbrock (Zhang et al. 2009). To unravel further func- Dr. Michael Hinz - Dr. Eva Kärgel Technical Assistants Dr. Marina Kolesnichenko * Lydia Blankenstein targettions of gene NF-κB signatures in ectodermal during organogenehair follicle Dr. Giulietta Roel Sabine Jungmann sis, we have investigated NF-κB-dependent Dr. Ruth Schmidt-Ullrich Inge Krahn has a multifunctional role in hair follicle in- Dr. Philipp Tomann * Doris Lange duction,development. including Our ECMdata remodelingindicate that and NF-κB the regulation of stem cell markers. The func- Secretariat PhDDr. Zekiye Students Buket Yιlmaz Daniela Keyner regeneration in hair follicles and in the in- Patrick Beaudette testinetion of NF-κBis currently in stem under cell biology investigation. and organ Daniel Heinze (* part of the time reported)

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Clemens A. Schmitt

Cancer Genetics and Cellular Stress Responses

Our scientific interests focus on cellular stress Role of deregulated NF-κB and p53 responses (so called ‘failsafe mechanisms’) signaling networks in lymphoma development and therapy that may serve as oncogene-evoked anti-tu- Soyoung Lee, Maurice Reimann, Julia Kase and mor barriers. Ultimate stress responses such collaboration partners (Claus Scheidereit, Jana as apoptosis or cellular senescence – both ter- Wolf, Gunnar Dittmar and others) minal “cell-cycle exit” programs – do not only - counter tumorigenesis, but are induced as ef- mately linked, since they regulate each other fector mechanisms in response to anti-cancer NF-κBin comple andx p53ways. signaling They partly networks cooperate are inti in chemotherapy as well. Importantly, many of effector mechanisms (i.e. senescence), and partly mediate opposing functions (regarding the mutations now being increasingly unveiled vs. apoptosis [p53]). More- by large-scale sequencing efforts of primary - patient tumor biopsies throughout virtually survival [NF-κB] all cancer entities might impair these cellular asover, part NF -κBof the promotes “senescence-associated the secretion of pro-in secre- flammatory cytokines, which are often found stress responses. However, the actual func- a tumor entity in which both hyperactivating tion of a distinct mutant gene product – deter- tory phenotype (SASP)”. Lymphomasp53 reflectmuta- mined by its integration in a complex signaling tions are frequent. However, the dynamic NF- NF-κB mutations and inactivating network – often remains elusive. Moreover, - cations of distinct mutations or a deregulated additional layers of information – namely the κB/p53 interactions and the functional impli transcriptome, proteome and metabolome – Utilizing a mouse lymphoma model and ana- are needed for a better understanding of the lyzingNF-κB/p53 transcriptome network are and poorly clinical understood. data from lymphoma patients, we recently showed that malignant blueprint, and, ultimately, to make therapy-induced senescence presents with the concept of “personalized medicine” a clinical reality. To test the impact of molecular reapplied mouse model-derived genetic in- lesions in cellular stress response programs and depends on active NF-κB signaling, and clinically relevant patient subcohorts (“cross- on tumor development and treatment out- formationspecies investigations”). to human data setsWe tohave define now novel, ex- come under most physiological conditions in tended these analyses by modeling distinct vivo, we generate mouse models harboring lymphomas (and other tumor entities) with de- mutations in Eµ-myc transgenic mouse lymphomas.lymphoma Preliminary patient-derived data referring NF-κB andto tumor p53 fined genetic defects, and study their biology, biology and treatment responses indicate metabolism and long-term outcome in vitro common but also unique functional features of and in vivo.

p53distinct co-governed NF-κB pathway control as of well tumor as growth,p53 lesions. metabolism,Next, we aim cellular to functionally stress responses, dissect theand NF-κB/ cross-

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talk to the host immune system to identify novel vulnerabilities (as therapeutic “Achilles’ Heels”), potential biomarkers of activity and drug action, and seek to mathematically model

Senescence-associatedthe relationship between NF-κB proteotox and p53. - icity accounts for a hypermetabolic phenotype that can be exploited for senescence-selective synthetic Therapy-induced senescence as a cancer-exclusive condition that lethal therapies can be selectively targeted in a conceptually novel “next-generation Jan Dörr, Yong Yu, Soyoung Lee, Jan Lisec and synthetic lethal fashion”. A cancer-associated condition on one hand collaboration partners (Stefan Kempa et al.) and a pharmacological targeting strategy on the other hand, where only their coincidence is cytotoxic. Functional non-invasive positron emission tomography (PET)-based imaging using either glucose or thymidine as tracers prompted the discovery of an unexpected feature of cellular senescence: a glucose-avid but thymidine-negative hypermetabolic phenotype. Extensive Selected Publications Dörr, J.R., Y. Yu, M. Milanovic, G. Beuste, C. Zasada, J.H.M. Däbritz, J. Lisec, D. Lenze, A. boosted glycolysis and oxidative phosphory- Gerhardt, K. Schleicher, S. Kratzat, B. Purfürst, S. Walenta, W. Mueller-Klieser, M. Gräler, M. Hummel, U. Keller, A.K. Buck, B. Dörken, L. Willmitzer, M. Reimann, S. Kempa, S. Lee, lationbiochemical in senescence, and genetic and led analyzes to the confirmeddissection and C.A. Schmitt. 2013. Synthetic lethal metabolic targeting of cellular senescence in of the underlying mechanism. Senescent cells cancer therapy. Nature, 501:421-425. present with a massive secretory phenotype (SASP, see above) that overwhelms the capa Bywater,Ryckman, M.J., W.G. G. Rice, Poortinga, C. Schmitt, E. Sanij, S.W. N. Lowe, Hein, R.W. A. Peck, Johnstone, C. Cullinane, R.B. Pearson, M. Wall, G.A. L. Cluse, McArthur, D. city of proper protein biosynthesis. As a con- Drygin,and R.D. K. Hannan. Anderes, 2012. N. Huser, Inhibition C. Proffitt, of RNA J. Bliesath,Polymerase M. Haddach, I as a therapeutic M.K. Schwaebe, strategy D.M. to sequence of endoplasmic reticulum stress and Cancer Cell 22:51-65. an unfolded protein response, senescent cells Jing, H., J. Kase, J.R. Dörr, M. Milanovic, D. Lenze, M. Grau, G. Beuster, S. Ji, M. Reimann, promote cancer-specific activation of p53. cope with proteotoxicity by energy-consum- P. Lenz, M. Hummel, B. Dörken, G. Lenz, C. Scheidereit, C.A. Schmitt, and S. Lee. 2011. Opposing roles of NF-kB in anti-cancer treatment outcome unveiled by cross-species ing autophagy. In turn, energy depletion or investigations. Genes Dev. 25: 2137-2146. inhibition of autophagy results in selective cell Reimann, M., S. Lee, C. Loddenkemper, J.R. Dörr, V. Tabor, P. Aichele, H. Stein, B. Dörken, death out of senescence, thereby utilizing se- T. Jenuwein, and C.A. Schmitt. 2010. Tumor stroma-derived TGF-b limits Myc-driven nescence and these subsequent, conceptually lymphomagenesis via Suv39h1-dependent senescence. Cancer Cell 17: 262-272. novel treatment approaches in a “synthetically Braig, M., S. Lee, C. Loddenkemper, C. Rudolph, A.H.F.M. Peters, B. Schlegelberger, H. Stein, B. Dörken, T. Jenuwein and C.A. Schmitt. 2005. Oncogene-induced senescence as lethal-like” fashion to eliminate potentially an initial barrier in lymphoma development. Nature 436: 660-665. harmful senescent cancer but no normal cells.

Chemotherapy promotes cancer stemness via senescence-related reprogramming Group Leader Graduate students Maja Milanovic, Yong Yu, Henry Däbritz et al. Prof. Dr. Clemens Schmitt, M.D Damaris Anell Rendon Gregor Beuster Many key signaling components of the senes- Vice Group Leader Jan Dörr cence machinery also operate as critical regu- Dr. Soyoung Lee, Ph.D. Philip Schröder lators of stem cell functions. In cancer cells, Yong Yu altered stemness might have profound impli- Scientists cations for tumor aggressiveness and clinical Dr. Henry Däbritz, M.D. Technicians outcome. We investigated whether chemo- Dr. Julia Kase, M.D. Nadine Burbach therapy-induced senescence may change stem Sven Maßwig cell-related functionalities in malignant cells of Dr. Maurice Reimann, Ph.D. Sandra Wegener murine and human origin. Dr. Maja Milanovic, Ph.D.

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Michael Sieweke

Stem Cell and Macrophage Biology

Our research is located at the interface of immunology and stem cell research with the overarching question of how cell identity and cell numbers are controlled. Often these processes appear intrinsically linked. By defini- tion stem cells are immature and have not assumed specific effector functions but show an extended self-renewal capacity. As cells differentiate into specific functionally mature cells, self-renewal capacity is typically lost. Re- cent experiments have shown that cell identity is more malleable than previously thought and that the introduction of defined transcription Origin and self-renewal of tissue macrophages factor cocktails can reprogram mature dif- Katharine Sutliff © SCIENCE ferentiated cells into pluripotent stem (iPS) Sieweke MH, Allen JE, Beyond Stem Cells : self-renewal of differentiated macrophages cells or directly into other differentiated cells Science, 22;342(6161) :1242974 (2013) of other lineages. Essential for all projected stem cell applications, both for therapy and hematopoietic stem cells via blood monocyte drug screening, is the generation of large intermediates but can also be derived from embryonic progenitors and be maintained numbers of cells with defined identity. Rather long term in tissues without monocyte input. than manipulating cell identity we have taken This may involve stem-cell like self renewal the alternative and unique approach of iden- mechanisms (reviewed in Sieweke and Al- tifying mechanisms that could induce self-re- show that inactivation of two macrophage newal and thus increase cell numbers without len, Science 2013; figure 1). Indeed we could change of cellular identity. Understanding the term self-renewal of macrophages without difference of such mechanisms to tumorigenic lossspecific of cell transcription identity or tumorigenic factors enabled transfor long- mation (Aziz et al., Science 2009). Further- transformation would also be of critical impor- more macrophages have recently entered the tance for cancer research. limelight of immunology and beyond (Geiss- man et al., Science 2010), because they are involved in many physiological and pathologi- Learning from macrophages immune functions, such as regeneration, can- To address these questions we investigate the cercal processesand metabolic of major diseases. interest Macrophages beyond their are molecular and cellular mechanisms of self- present in essentially every tissue of the body, renewal and cell fate decision during the dif- where they can play trophic roles in tissue ferentiation from hematopoietic stem cells to homeostasis, metabolism and repair. Macro- macrophages. These cells appear particularly phages could thus be considered systemic tis- suited to identify self-renewal mechanisms sue guardians, whose function and manipula- independent of lineage conversion since tissue macrophages cannot only originate from disease of parenchymal cells. tion could have major impact on health and

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dent mechanism. It thus appears possible to amplify functional differentiated cells without malignant transformation or stem cell intermediates (Aziz et al, Science 2009). We currently investigate the mechanisms of self- renewal in differentiated cells and explore - phages and their utility as a research tool. the therapeutic potential of amplified macro Macrophages in tissue regeneration

- ten linked processes but require precise tim- ingInflammation and coordination and tissue of different regeneration cell types. are of In this context we investigate the cell fate decisions that give rise to different macrophage subtypes. We want to identify the transcrip- Single cell gene expression analysis of HSC tion factors controlling these choices and aim after 16h culture with M-CSF. The genes to understand how their manipulation affects analyzed are classified and color coded on tissue repair. top. Cells are clustered according to lineage identity identicated on the right. Patents / Patent applications In this framework we have developed re- Method for expanding monocytes

commitment of hematopoietic stem cells to Method for generating, maintaining and expanding monocytes, and/or macro- United States patent US 8,574,903 B2, filed 10.01.2008, delivered 05.11.2013 thesearch macrophage projects investigatinglineage, self-renewal the lineage and phages and/or dendritic cells in long term culture cellular identity of mature macrophages and European patent EP 07300717.1.-2405; filed 10.01.2007 the role of macrophage activation states in in- A method for inducing extended self-renewal of functionally differentiated United States patent PCT/EP2008/050221;filed 10.01.2008, delivered 08.04.2014 somatic cells

Lineageflammation commitment and tissue regeneration. of European patent EP 10734946.6-2401: filed 08.07.2009 Methods and compositions for use in preventing or treating myeloid cytopenia United States patent PCT/EP2010/059843:filed 08.07.2010 hematopoietic stem cells and related complications

MafB mutants and uses thereof monocyte differentiation that are initiated by European patent EP 13305464.3-1456: filed 09.04.2013 myeloidWe have lineage investigated commitment the very in first hematopoi steps of- SelectedEuropean patent Publications EP13306661.3: filed 03.12.2013 etic stem cells (HSC). Previously it had been Sieweke MH, Allen JE, Beyond stem cells: self-renewal of differentiated macro- widely assumed that cell fate choice in stem phages, Science, 2013, 22;342(6161): 1242974 cells is largely determined by stochastic mech- Mossadegh-Keller N, Sarrazin S, Kandalla PK, Espinosa L, Stanley RE, Nutt SL, Moore J, anisms. By the use of multiple methods of sin- Sieweke MH, M-CSF instructs myeloid lineage fate in single haematopoietic stem cells, Nature, 2013, 9;497(7448):239-43

could demonstrate that the cytokine M-CSF Geissmann F, Manz M, Jung S, Sieweke M, Merad M, Ley K, Development of monocytes, macrophages and dendritic cells, Science, 2010, 327 (5966):656-61 gle cell analysis (fig.2) in vitro and in vivo we- eloid fate (Mossadegh,Sarrazin et al., Nature Aziz A, Soucie E, Sarrazin S, Sieweke MH, MafB/c-Maf deficiency enables self-renewal of differentiated functional macrophages, Science, 2009, 326 (5954):867-71 2013).can instruct We also highly showed purified that HSC this to isadopt mediated a my Sarrazin S, Mossadegh-Keller N, Fukao T, Aziz A, Mourcin F, Kelly L, Vanhille L, Kastner by an integrated circuit of the transcription P, Chan S, Duprez E, Otto C. Sieweke MH, MafB restricts M-CSF-dependent myeloid factor MafB with M-CSF, where MafB acts as commitment divisions of hematopoietic stem cells, Cell, 2009, 138(2):300-13 a threshold setter for M-CSF driven asymmetric commitment divisions (Sarrazin et al., Cell - July 2012: Start of the franco-german research group with the Centre ing debate about the capacity of cytokines to d’Immunology de Marseille-Luminy, Marseille, France, as part of a new instruct2009). These cell fate findings and about resolved the acoordination long-stand Helmholtz-INSERM Franco-German research cooperation initiative of cell intrinsic and extrinsic regulators in Group Leader Research Assistants lineage choice. These studies have important Dr. Michael Sieweke Jérémy Favret implications for stem cell differentiation in Julien Maurizio general beyond the hematopoietic system. Senior Scientists Noushine Mossadegh Dr. Carole Berruyer-Pouyet Pierre Perrin Self-renewal of differentiated cells Dr. Sandrine Sarrazin PhD students Terminal differentiation is typically tightly Post-docs Stephanie Vargas Aguilar linked to cell cycle withdrawal. However, Dr. Rebecca Gentek Meryam Beniazza Dr. Prashanth Kandalla Laufey Geirsdottir enables self-renewal of differentiated func- Dr. Kaaweh Molawi Francesco Imperatore tionalwe discovered macrophages that by MafB/cMaf a cMyc/KLF4 deficiency depen-

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CANCER RESEARCH Photo: David Ausserhofer/MDC Photo:

Thomas Sommer

Intracellular Proteolysis

The Mechanisms of Protein Quality Control

In all cells, both newly synthesized and One of the best investigated PQC path- pre-existing proteins are constantly ways exist in the endoplasmic reticulum (ER). The ER is a cellular organelle through endangered by misfolding and aggregation. which a large number of proteins travel on The accumulation of such damaged - proteins perturbs cellular homeostasis and branes, exocytic and endocytic compart- their way to their final destination in mem provokes aging, pathological states, and ments, or the cell exterior. Since proteins enter the ER in an unfolded conformation, even cell death. To avert these dangers, - cells have developed protein quality control (PQC) strategies that counteract protein itthis hosts organelle an array is aof major molecular folding chaperones, compart whichment of assist eukaryotic in protein cells. foldingTo fulfill and this matu task,- damage in a compartment-specific man- ration. Still, protein biogenesis is an error- ner. PQC systems channel misfolded pro- prone process. A considerable fraction of all teins either into re-folding pathways or newly synthesized polypeptides fail to at- initiate their proteolysis by the Ubiquitin tain their native conformation due to mutations, transcriptional and translational er- Proteasome System (UPS). The work of this rors, folding defects, or imbalanced subunit group focuses on how specific branches synthesis. Mature proteins can be damaged of the UPS selectively dispose aberrant by environmental stress conditions, such proteins without affecting correctly folded as high-energy radiation, chemical insults, - polypeptides. nents of the UPS eliminate such misfolded proteinsor metabolic in a processby-products. termed Specific ER associated compo protein degradation (ERAD). The ERAD pathway cooperates with the Unfolded Protein Response (UPR) in maintaining homeostasis in the ER. The UPR measures the degree of misfolding in the ER and regulates folding and proteolytic capacities according to the load with aberrant proteins (Figure 1).

ERAD appears to be conserved from yeast to mammals. Thus, many basic principles and components have been discovered in the model organism Saccharomyces cere- visiae. One of the highly conserved components of the ER is the HRD ubiquitin ligase. This membrane-bound complex recognizes signals contained in misfolded proteins of the ER-lumen and ER-membrane. Proteins

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Protein homeostasis in the endoplasmic reticulum. Proteins translocate into the ER via the narrow protein translocation channel. Newly synthesized and unfolded proteins associate with molecular chaperones to fold. Only correctly folded proteins leave the ER for their final destination. Misfolded proteins are retained in the ER and either channeled again into folding pathways or subjected to protein dislocation initiated by UPS components (ERAD pathway). The UPR component Ire1 measures the content of misfolding in the ER, signals it to the nucleus, and regulates expression of a multitude of genes. This transcriptional program includes up-regulation of genes coding for molecular chaperones and UPS components of the ERAD branch.

committed for degradation are exported from the ER in a process termed protein does only rely on its ER-luminal domain dislocation. Subsequently, dislocated sub- wefied expressed components. the luminal Since the domain Hrd3 in function insect strate molecules are ubiquitylated and de- cells. We also expressed Yos9 in E. coli or graded by cytoplasmic 26S proteasomes (Figure 2). luciferaseinsect cells. but Expressed not the folded and purifiedenzyme. Hrd3Both Hrd3and Yos9and Yos9 bind deploy directly holdase denatured activity firefly as and characterized components of ligase they prevent the aggregation of a dena- complexesIn the last decade,using genetics, the group molecular has identified biol- tured protein. Our in vitro data imply that the HRD ligase directly recognizes misfold- However, this laboratory has now estab- ing of proteins. lishedogy, and powerful protein in purificationvitro approaches strategies. and - ponents to understand the biochemical A Ubiquitin-Binding Domain principlesquantitative of the methods ERAD usingpathway. purified com regulates ubiquitin chain synthesis. Katrin Bagola and Maximilian von Delbrück Hrd3 and Yos9 possess ‘Holdase’ activity and function as substrate Ubiquitin-binding domains (UBD) recog- receptors of the HRD ligase. Franziska Zimmermann, Sathish Lakshimpa- - thy and Maren Berger nize ubiquitin (ub) modifications. Some of- uitinthem chains.specifically They bind are mono-ubcrucial for while the othUPS In order to understand how substrates bind sinceers recognize they could specifically ‘read’ the linked ubiquitin poly-ubiq code. to the ER-luminal module of the HRD ligase (Hrd3, Yos9) we started to work with puri- has been observed for the UBD of yeast Thus far, no significant ubiquitin binding

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CANCER RESEARCH

The Hrd ubiquitin ligase as an example of a quality control element. The ER-luminal modul of the ligase consists of Hrd3 and Yos9 and associates with the Hsp70 type chaperone Kar2. Der1, Usa1, and the transmembrane (TM) domains of Hrd1 collectively build the membrane module of the ligase. Some of these TM regions are most likely involved in building a pore in the ER-membrane through which misfolded proteins are dislocated. The cytosolic module consisting of Hrd1 and Cue1/Ubc7 modifies dislocated proteins with K48 linked poly-ubiquitin chains. These chains and Ubx2 recruit the AAA-ATPase Cdc48 and its co-factors Ufd1 and Npl4 to the ligase complex. Cdc48 most likely provides energy for the dislocation event and hands-over the ubiquitylated proteins to the 26S proteasome for degradation.

Cue1, the CUE domain. Cue1 integrates the tions the tip of a growing ubiquitin chain in E2 enzyme Ubc7 into membrane-bound close proximity to the active site center of ubiquitin ligases at the Endoplasmic Re- ticulum (ER) and it furthermore activates elongation (Bagola et al, Mol Cell, 2013). it. Therefore, Cue1 is crucial for ER-associ- the E2 enzyme to promote efficient chain ated protein degradation (ERAD). We were able to provide evidence that the CUE do- The function of other main of Cue1 binds oligo-ubiquitin chains, Ubiquitin-conjugating enzymes. Katrin Bagola, Annika Weber and Lukas of K48-linked poly-ubiquitin chains in vi- Pluska which is pivotal for the efficient synthesis- tions that abolish ubiquitin binding by Cue1 In addition to Ubc7 and Cue1 other ubiqui- andtro. Inaffect addition turnover we of have ERAD identified substrates muta in vivo. Our data point strongly to a molecular as well. For example Ubc1 works together mechanism in which the CUE domain posi- withtin-conjugating Hrd1 in a enzymesparallel pathway function toin Cue1/ERAD

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and novel mechanisms how this E2 enzyme signal is generated (Man7GlcNAc2), which promotesUbc7. Preliminary ubiquitin results chain formation. indicate specific issidases recognized and mannosidases by the HRD ubiquitin until a ligase. specific Htm1p is a mannosidase in this trimming Ubc6 cooperates with the Doa10 ubiquitin pathway that interacts with the oxidore- ligase and also in this case we are investi- ductase Pdi1. Using an in vitro approach gating the molecular mechanisms how this as well as molecular biology approaches enzyme contributes to ubiquitin chain for- this Delbrück group investigates how the mation on substrates of this ligase complex heterodimer of Htm1 and Pdi1 recognizes at the ER. quality control substrates and trims their oligosaccharides to make them visible for downstream acting factors. A channel for ERAD in the ER-membrane. Martin Mehnert and Ernst Jarosch

Dislocation of selected misfolded proteins through the lipid bilayer is assumed to occur via a protein-conducting channel of Selected Publications unknown nature. We were able to show Horn, S.C., Hanna, J., Hirsch, C., Volkwein, C., Schütz, A., Heinemann, U., Sommer, T., and that the integral membrane protein Der1 Jarosch, E. (2009) Usa1 functions as a scaffold of the HRD ubiquitin ligase. Mol Cell oligomerises which relies on its interaction 36, 782-793.

with the scaffolding protein Usa1. Muta- Buchberger, A, Bukau, B, and Sommer, T. (2010) Protein quality control in the cytosol tions in the transmembrane domains of and the endoplasmic reticulum: brothers in arms. Mol Cell 40, 238-252. Der1 block the passage of soluble proteins across the ER-membrane. As determined M., Lashuel, H. A., Sommer, T., Brik, A., and Ciechanover, A. (2012) The size of the pro- Shabek,teasomal N., substrate Herman-Bachinsky, determines Y.,whether Buchsbaum, its degradation S., Lewinson, will O.,be Haj-Yahya,mediated by M., mono- Hejjaoui, or the ER-luminal exposed parts of Der1 are in polyubiquitylation. Mol Cell 48, 87-97. spatialby site-specific proximity in to vivo the substrate photocrosslinking, receptor Bagola, K., von Delbrück, M., Dittmar, G., Scheffner, M., Ziv, I., Glickman, M. H., Ciecha- Hrd3 whereas the membrane-embedded nover, A., and Sommer, T. (2013) Ubiquitin Binding by a CUE Domain Regulates Ubiquitin Chain Formation by ERAD E3 Ligases. Mol Cell 50, 528-539.

ligase Hrd1. Intriguingly, both regions also Mehnert, M., Sommer, T., and Jarosch, E. (2014) Der1 promotes movement of mal- formdomains crosslinks reside to adjacent client proteins. to the ubiquitin In sum- folded proteins through the endoplasmic reticulum membrane. Nature Cell Biol 16, mary our data imply that Der1 initiates 77-86 the export of aberrant polypeptides from the ER-lumen by threading such molecules Structure of the Group into the ER-membrane and routing them to Hrd1 for ubiquitylation (Mehnert et al., Na- Group Leader ture Cell Biol., 2014). Prof. Dr. Thomas Sommer Holger Brendebach Senior Scientist Maximilian von Delbrück Htm1p is a mannosidase that Dr. Ernst Jarosch Anett Köhler generates N-linked Man7GlcNAc2 Susanne Kressner glycans to accelerate the degrada- Delbrück Fellow Marcel Nowak tion of misfolded glycoproteins. Dr. Christian Hirsch Lukas Pluska Anett Köhler and Christian Hirsch Annika Weber PostDoc N-linked glycans are essential for the Dr. Katrin Bagola Technical Assistants breakdown of glycoproteins. The covalently Dr. Sathish Lakshmipathy Corinna Volkwein attached oligosaccharide structure is used Dr. Martin Mehnert Angelika Wittstruck as a signal indicating the folding status of the protein. Newly synthesized proteins PhD Secretariat Maren Berger Sylvia Klahn This glycan structure is trimmed by gluco- receive a Glc3Man9GlcNAc2 modification.

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Wolfgang Uckert

Molecular Cell Biology and Gene Therapy

Over the past decade, the genetic introduction of T cell receptor (TCR) genes antibodiesvirus-derived and envelopes which were whose blinded specificity for their was into T cells has been developed as a strat- redirected by CD4- or CD8-specific single-chain egy to provide defined antigen-specific T cell allow the targeted delivery of MHC class I- and immunity. TCR gene transfer aims to target classnative II-restricted receptors. SuchTCRs subset-specific into CD8+ or CD4 vectors+ T tumor or virus-infected cells by genetic modi- cells without the need for cell sorting. Further, fication of T cells. The potential value of this expression of immune-modulating transgenes, strategy - designated as TCR gene therapy – they can be employed for T cell subset-specific- was established in mouse models and phase nally, in vivo application of targeting vector which could enhance anti-tumor efficacy. Fi I clinical trials. However, most studies were particles would abrogate the need for ex vivo T cell transduction and expansion. of limited efficacy and accompanied by side effects and revealed i), the importance to Mario Bunse select the right candidate as target antigen heterodimer can form potentially self-reactive and ii), the need to improve the methodology mixedIn TCR-modified TCR dimers, T cells composed the transduced of endogenous TCRα/β for TCR gene therapy. We address questions and transferred TCR chains, which have been related to: generation of TCR-modified T cells demonstrated to cause lethal autoimmunity with new antigen specificity and high func- in a mouse model of TCR gene therapy. To prevent mixed TCR dimer formation, we de- tional avidity, safety aspects of TCR-modified veloped an RNAi-TCR replacement vector that T cells with respect to on- and off-target simultaneously silences the endogenous TCR effects, adoptive transfer of TCR-modified and expresses an RNAi-resistant, therapeutic T cells in preclinical models and the use of TCR. Importantly, lethal TCR gene transfer- induced autoimmunity caused by mixed TCR TCR-modified Treg in the treatment of auto- dimer formation was almost completely pre- immune diseases. vented when the cells were transduced using the RNAi-TCR replacement vector. Presently, Multi-functional vectors for TCR we are adapting this technology for the silenc- gene therapy ing of human endogenous TCR. Furthermore, we constructed a transposon vector analogous For clinical application TCR gene transfer to the viral RNAi-TCR replacement vector. into - Julian Clauß in collaboration with Z. Izsvak our grToup cells develops needs to highly be i) sophisticated efficient and gene spe (MDC, Berlin) cific,transfer ii) safe vectors and iii)based cost-effective. on retroviral To orthis trans end,- Currently, most clinical protocols use viral poson systems. vectors for gene transfer into T cells. How- ever, GMP manufacturing of DNA plasmids is Inan Edes in collaboration with C. Buchholz cheaper and faster compared to the produc- (PEI, Langen) tion of retroviral supernatants and plasmid In collaboration with the group of C. Buchholz vectors have no strict size limitation of the we have constructed lentiviral vectors, which expression cassette. The DNA-based Sleep- ing Beauty transposon system (SBTS) enables human and murine CD8+ or CD4+ T cell subsets. stable integration of the transgene cassette allowThese targetedvectors were gene pseudotyped transfer specifically with measles into into the genome of primary human T cells by

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nucleofection. Using the optimized hyperac- suppress self-reactivity. Activation of endogenous Treg or adoptive transfer of in vitro primary human T cells yielding high and long- expanded Treg has been applied to treat vari- termtive transposase transgene expression SB100Xo we with gene-modified integration ous autoimmune disorders. Several studies rates of approx. 45% (GFP) or 30% (TCR). Cell viability after nucleofection varied between 20% and 60%. Provision of the transposase hamethodve demonstrated to generate high that numbers therapeutic of antigen- efficacy as in vitro-transcribed RNA (ivtRNA) instead depends on their antigen-specificity. A novel of plasmid DNA resulted in increased gene the cells. specific Treg is the transfer of TCR genes into SBTS-mediated TCR gene transfer generated We have transduced the genes encoding TCR Ttransfer cells with efficacy tumor and cell-directed improved T reactivitycell viability. as directed against the CNS antigen myelin oli- godendrocyte glycoprotein (MOG) into Treg. - - ingmeasured cells. This by IFNγtechnology release will after be furtherco-cultivation devel- tion of effector T cells in vitro and adoptive opedof TCR-modified into a robust T non-viral cells and vector antigen-present platform to TCR-modified Treg could suppress prolifera - Treg – but not polyclonal Treg – could inhibit ized cancer immunotherapy. trdevelopmentansfer of very of low MOG numbers peptide-induced of TCR-modified ex- generate tumor-specific T cells for personal perimental autoimmune encephalitis (EAE) Pre-clinical in vivo models to test in mice. The therapeutic effect was dependent the efficacy of TCR gene therapy Matthias Leisegang and Peter Meyerhuber in CTLA-4 by the Treg as Tregs transduced with collaboration with Thomas Blankenstein (MDC, on high affinity of the TCR and expression of Berlin), Helga Bernhard (Darmstadt), Dolores 4 were not able to suppress EAE upon transfer. Schendel (Helmholtz Zentrum, Munich) low-affinity TCRs or a miRNA targeting CTLA-

We developed mouse models to evaluate the

therapy for established cancer. We generated therapeutic efficiency and safety of TCR gene mice (HHDxRag-/-) and a syngeneic tumor cell immune-deficient, human MHC-transgenic Selected Publications tumor antigens of interest. T cells engineered Reuss S, Sebestyen Z, Heinz N, Loew R, Baum C, Debets R, Uckert W (2014). TCR- line that can be modified to express human- engineered T cells: A model of inducible TCR expression to dissect the interrelation- ship between two TCRs. Eur J Immunol 44: 265-274. with human tyrosinase-specific TCR of dif Stärck L, Popp K, Pircher H, Uckert W (2014). Immunotherapy with TCR-redirected into mice bearing tyrosinase-positive tumors. T cells: comparison of TCR-transduced and TCR-engineered hematopoietic stem cell- Whileferent transfer affinities of were T cells adoptively engineered transferred with a derived T cells. J Immunol 192: 206-213. Sommermeyer D, Conrad H, Kronig H, Gelfort H, Bernhard H, Uckert W (2013). NY- ESO-1 antigen-reactive T cell receptors exhibit diverse therapeutic capability. Int J selectedhigh-affinity for escape tyrosinase-specific variants and TCRrelapse. resulted In a Cancer 132: 1360-1367. secondin complete mouse tumor model rejection, (NOD-SCIDxHHD) low TCR affinity T cells Zhou Q, Schneider IC, Edes I, Honegger A, Bach P, Schönfeld K, Schambach A, Wels WS, Kneissl S, Uckert W, Buchholz CJ (2012). T-cell receptor gene transfer exclusively to transduced with a panel of TCR recognizing human CD8(+) cells enhances tumor cell killing. Blood 120: 4334-4342. human epidermal growth factor receptor 2 Pouw N, Debets R, Kieback E, Uckert W, Song J-Y, Haanen JBAG, Schumacher TNM. Bendle(2010). .GM,Lethal Linnemann Graft-versus-host C, Hooijkaas disease AI, de in Wittemouse MA, models Jorritsma of T cell A, Bies receptor L, Kaiser gene ADM, therapy. Nature Med 16: 565-570. human(HER2) HER2-positivewith different tumorsaffinities and are ii) tested their on-for targeti) their toxicity efficacy on in HER2-expressing rejecting large establishednormal tissue. This latter is possible as humans and mice Structure of the Group share the same HER2-dervied epitope that is recognized by these TCRs. These in vivo mod- Group Leader els provide a versatile, pre-clinical test system Prof. Dr. Wolfgang Uckert Graduate and Undergraduate for TCR gene therapy of cancer and allows the Maria Lietz prediction whether or not TCR-engineered T Scientist cells eradicate tumors or select escape vari- Dr. Elisa Kieback Technical Assistants ants and whether the targeted antigen is safe. Dr. Matthias Leisegang Carolin Genehr Dr. Lilian Staerck Janina Hauchwitz TCR gene transfer into regulatory T Kordelia Hummel cells for the suppression of autoim- PhD Mareen Kamarys mune disease Mario Bunse Michaela Naschke Elisa Kieback in collaboration with Simon Fil- Julian Clauß Melanie Schwochow latreau (DRFZ, Berlin) Inan Edes Felix Lorenz Other The function of regulatory T cells (Treg) is to Peter Meyerhuber Erika von Buehler (SFB TR36) limit the extent of immune reactions and to

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130 MDC Research Report 2014 Diseases of the Nervous System

Coordinator: Carmen Birchmeier

MDC Research Report 2014

Diseases of the Nervous System

Carmen Birchmeier

he nervous system is fundamental to schools, among them the Helmholtz Inter- the body’s ability to maintain itself, to national Research School MolNeuro, the Tsense the environment, to move and Charité International Medical Neurosci- react to stimuli, and to generate and con- ences program, and MyoGrad. The curricu- trol behavior. Hence, disorders of the ner- lum of these Graduate Schools encompass- vous system that manifest themselves as neurological and psychiatric disease often they fund students to attend international conferences.es lecture series, journal clubs, courses, and impose a heavy burden on patients, families,severely caregivers impair and afflicted society individuals alike. Research and Noteworthy are also other honors received, in the Neuroscience Program of the MDC for instance James Poulet obtained the focuses on determining the molecular basis Paul Ehrlich und Ludwig Darmstaedter of normal nervous system function, and dis- Award for Junior Scientists and Helmut covering changes responsible for inherited Kettenmann was elected President of the or acquired defects are important priorities German Neuroscience Society, and Thomas Jentsch became member of the Committee of the Louis-Jeantet Foundation. Scientific inNeur theoscientists fight against of nervousthe MDC system are important disease. highlights from the Department participants in many Berlin-based cooperative networks (e.g. several Collaborative Re- Patients with high grade gliomas have a search Centers, Transregios and Research very poor prognosis because these tumors Units funded by the DFG). Seven of the elev- are rarely treatable. Rainer Glass and en principle investigators of the program Helmut Kettenmann found that endog- are associated with NeuroCure, the Berlin- enous neural precursor cells exert anti- based excellence cluster in neuroscience. tumour activity by activating the vanilloid NeuroCure was evaluated and will receive receptor TRPV1 on glioma cells, thus iden- funding for a second period (2012-2017). tifying TRPV1 agonists as potential glioma In addition, many of the groups received therapeutics. (Stock et al., Nat Med. 2012). grants from the European Union, in particu- In vertebrates, hearing and touch both rely lar Gary Lewin and Thomas Jentsch were on the detection of mechanical stimuli. Us- awarded ERC advanced grants. ing a classical twin study design, the group of Gary Lewin - The success of the Neuroscience Program tion between genes governing touch and - hearing acuity foundin healthy a significant humans. correla They searchers received offers for independent positionsis also reflected from outside in the factand thatleft youngthe MDC. re both sensory systems, USH2A (Frenzel et Alistair Garratt became Associate profes- al.,also PLoS identified Biol. 2012). one gene that contributes to sor (Charité) in 2012, Stefan Lechner became independent group leader (University The sense of touch relies on detection of of Heidelberg) in 2013, and Jan Siemens mechanical stimuli by specialized mecha- was appointed extraordinary professor nosensory neurons. The groups of Carmen (University of Heidelberg). On the other Birchmeier and Gary Lewin showed that the transcription factor c-Maf is crucial for the Program, Kate Poole (2013) and An- mechanosensation in mice and humans. netteside, two Hammes new principle (2014). investigators joined Pacinian corpuscles are specialized to detect high-frequency vibration and are at- Scientists from the Neuroscience Program rophied in c-Maf mutant mice. In line with participated in research and graduate this, sensitivity to high-frequency vibration

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is reduced in humans carrying a dominant ing direction instead of branching when mutation in c-MAF. (Wende et al., Science they enter the central nervous system (Ter- 2012). Avetisyan et al., J. Neuroscience 2014).

Mutations inactivating the potassium chan- The inhibitory neurotransmitter whose nel KCNQ4 lead to deafness in humans effects are mediated by glycine receptors and mice. The groups of Thomas Jentsch (GlyR). The group of Jochen Meier discov- and Gary Lewin have detected KCNQ4 ered a gain-of-function variant of GlyR in in the peripheral nerve endings of cuta- the brain of epileptic patients. They now ex- neous mechanoreceptors from mice and pressed this variant GlyR in mice in an neu- humans. Electrophysiological analyses of - mice demonstrated elevated mechanosen- tamatergic neurons resulted in recurrent sitivity in KCNQ4 mutant mice, similar to epileptiformron type-specific discharge, pattern: expressionwhereas expres in glu- the increased vibrotactile acuity observed sion in inhibitory interneurons resulted in in patients that lack a functional KCNQ4 gene (Heidenreich et al., Nat. Neuroscience pathophysiological mechanisms in epilepsy 2011). (Winkelmannincreased anxiety. et al., The J. Clin. work Invest., identifies 2014). the

Proteins with long polyglutamine sequenc- Cortical circuits spontaneously generate es have an enhanced propensity to self- coordinated activity even in the absence assemble into disease-causing insoluble of external inputs. James Poulet and his protein aggregates. The inherited neurode- collaborators in the group of Carl Peterson generative disease Spinocerebellar Ataxia investigated the impact of the thalamus Type 1 is caused by mutations in ataxin-1. on cortical activity. They demonstrate that The group of Erich Wanker the desynchronized cortical state is driven by an increase in thalamic action potential misfolding; some enhance and has others identified sup- press21 human misfolding proteins and that aggregation influence (Petrakis ataxin-1 stimulation of the thalamus (Poulet et al., et al., PLoS Genetics 2012). Natfiring, Neurosci. which 2012). is mimicked by optogenetic

High blood pressure is the leading risk fac- Sonic hedgehog (SHH) is a regulator of fore- tor for death worldwide. The group of Björn brain development that acts through the Schröder, in collaboration with Christian A. patched 1 receptor. The group of Annette - Hammes nel, TMEM16A, as an important calcium- LRP2, a member of the LDL receptor gene activatedHübner (Jena), chloride identified channel a newthat ioncontrols chan family, as anda component Thomas Willnow of SHH identifiedsignaling. peripheral vascular resistance. Their data The data reveal the molecular mechanism data suggest that TMEM16A is a promising underlying congenital forebrain anomalies target for hypertension treatment (Heinze (Christ et al., Dev. Cell 2012). et al., J. Clin. Invest., 2014). In sensory neurons, mechanotransduction Axonal branching is a prerequisite for for- is sensitive, fast and requires mechanosen- mation of complex neuronal circuits and sitive ion channels. Kate Poole developed a for parallel information processing. Fritz new method to directly monitor mechano- Rathjen and Hannes Schmidt foundthat Npr2 activity is needed for bifurcation of substrate interface (Poole et al, Nat. Comm. cranial sensory axons, with axons only 2014).transduction at defined regions of the cell- turning in either an ascending or descend-

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DISEASES OF THE NERVOUS SYSTEM Photo: Thomas Müller Photo:

Carmen Birchmeier

Developmental Biology / Signal Transduction

Our lab primarily focused on the functional analysis of genes important in embryonic development and adult stem cells, con- centrating on muscle, the nervous system and endocrine cells. For our work, we use mouse genetics and cell culture analyses of stem cells, and define molecular mecha- Fig. 1 Muscle stem cells require Notch signal- nisms using genome and proteome tech- ing to settle in their niche. nologies. (A) Schematic diagram showing the anatomical localization of the stem cells of the muscle that are also called satellite cells (SC, red) wedged between the basal lamina (BL) and the myofiber (MF) plasma membrane (PM). Satellite cells nuclei express Pax3/Pax7 and are shown in Muscle stem cells and their niche red, nuclei of the myofiber (myonuclei, MN) are Dominique Bröhl, Elena Vasyutina, Maciej T. shown in yellow. (B,C) Analysis of emerging satel- Czajkowski, Joscha Griger, Claudia Rassek, lite cells at E17.5 using antibodies against Pax3 Hagen Wende, in collaboration with Hans- (red) and laminin (green). Shown are sections Peter Rahn (Preparative Flow Cytometry of back muscle from control and Rbpj; MyoD Facility, MDC) and Bettina Purfürst (Elec- mutant mice. Arrows point towards satellite cells tron Microscopy Core Facility, MDC). located below the basal lamina, and arrowheads point towards the Pax3+ cells located in the Skeletal muscle growth and regeneration interstitial space of Rbpj/MyoD double mutants. rely on myogenic progenitor cells. Satel- lite cells are the progenitor/stem cells of the postnatal muscle and they reside between basal lamina and plasma membrane

called the stem cell niche. Previous data showof myofibers. that generation This anatomical and maintenance position of is satellite cells depends on Notch signals. For - However, rescued myogenic progenitors tional mediator of Notch signals, results in prematureinstance, elimination differentiation of Rbpj, of myogenic the transcrip pro- assume a satellite cell position (Fig. 1). In- genitors and the formation of tiny muscle stead,in the they Rbpj/MyoD locate to double the interstitial mutants space do not of groups that lack satellite cells (Vasyutina the muscle and contribute poorly to myo- et al., 2007). We recently found that this drastic effect of the loss of Notch signals is signaling stimulates emerging satellite cells rescued by the additional mutation of the fiber growth. Our work shows that Notch muscle differentiation factor MyoD. Thus, a to the production of the basal lamina that - eventuallyto adhere surrounds to myofibers both, and the to satellite contribute cell genesis is the repression of MyoD function. major function of Notch during fetal myo and the myofiber (Bröhl et al., 2012). 134 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

Fig. 2 c-Maf is required for the development of Pacinian corpuscles, the mechanosensory organs that detect high frequency vibration. (A,B) c-Maf mutation in mice causes a loss of Pacinian corpuscles and disrupts the morphologies of remaining corpuscles. Pacinian corpuscles were visualized using RT97 and S100 antibodies that detect neurofila- ment 200 present in axons and S100 present in glial cells that surround the axons and form the end organ of the Pacinian corpuscle. (C) Pedigree of a family with the R288P c-MAF mutation; members carrying the mutant allele (black symbols) suffer from early-onset cataracts. (D,E) Detection threshold of vibrotactile stimuli. (D) Example of a vibration stimulus and response and (E) vibration detection threshold in control and four carriers of the R288P c-MAF mutation. In healthy participants and unaffected family members, the detection threshold decreases with increasing frequencies. The detection threshold of affected family members is aberrant at high but not low frequencies. Scale bars, 100 μm. Error bars, SEM.

The transcription factor c-Maf con- gans are observed, and Pacinian corpuscles, trols touch receptor development Meissner corpuscles, and lanceolate endings and function (RAMs), as well as Merkel cell-neurite com- Hagen Wende, Cyril Cheret, Maria E. Sheean, Katja Reuter, in collaboration with Stefan G. Lechner and Gary R. Lewin (Molecular Physiol- inplexes the developing and Ruffini murine corpuscles nervous (SAMs) system, have we ogy, MDC), Patrick Carroll, Steeve Bourane, foundbeen defined. that the In transcription a screen for factors genes expressedc-Maf and Alexandre Pattyn (NSERM U.1051, Montpel- MafA are co-expressed in mechanosensory lier) and Francis L. Munier (Jules Gonin Eye neurons (RAMs). Hospital, Lausanne). We analyzed the role of c-Maf in mechanosen- The sense of touch relies on detection of me- sory neurons using mouse genetics. Pacinian chanical stimuli by specialized mechanosen- corpuscles are the primary detectors of high- sory neurons. The scarcity of molecular data frequency vibration. In c-Maf mutants, the quantity of Pacinian corpuscles was reduced by more than 50%, and remaining corpuscles ofhas their made diversity it difficult and function.to analyze Physiologically, development were small and irregularly shaped (Fig. 2A,B). of mechanoreceptors and to define the basis In humans, dominant c-MAF mutations are adapting, slowly adapting, and D-hair mecha- associated with cataracts, but the effects of noreceptorsmechanoreceptors (RAMs, are SAMs, classified and D-hairs). as rapidly In the mutation on mechanosensation had not addition, morphologically diverse end-or- been examined. Encouraged by our results in

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Fig. 3 Activation of MAPK overcomes the signals that end myelination. (A) Sustained activation of MAPK by expression of Mek1DD in Schwann cells; expression of Mek1DD was initiated by the use of the cre/loxP technology at the onset of myelination. Displayed are nerves from control and Mek1DD mice at different ages (P0–P90) that were analyzed by electron microscopy (representative axons of similar diameters are false-colored in light blue). In control mice, myelin growth ends around P15, but in Mek1DD animals myelin grows continuously. (B) Quantification of myelin thickness in control and Mek1DD mice shows that hypermyelination of small diameter axons is particularly pronounced.

mice, we tested touch sensitivity in humans ing strategy based on stable, non -radioac- that carry dominant c-MAF mutations, and tive isotope labeling with amino acids in cell observed that the mutation interferes with culture (SILAC) to measure the dynamics of normal vibration detection (Fig. 2C-E). Thus, myelin protein production in mice. This re- our analyses show that the transcription fac- vealed that protein synthesis slows down in tor c-Maf/c-MAF is crucial for mechanosen- the maturing postnatal peripheral nervous sory function in mice and humans (Wende et system. Remarkably, sustained activation of al., 2012). MAPK signaling by expression of the Mek1DD allele overcomes the signals that end myelination, resulting in continuous myelin growth Activation of MAPK overrides the (Fig. 3A,B). MAPK activation is accompanied termination of myelin growth and by minor changes in transcript levels and by replaces Nrg1/ErbB3 signals dur- a massive up-regulation of protein synthesis. ing Schwann cell development and Nrg1 and the ErbB2/3 receptors provide piv- myelination otal signals for Schwann cell development Maria E. Sheean, Cyril Cheret, Thomas Müller, and myelination that are mediated by the in collaboration with Erik McShane and Mat- tyrosine phosphatase Shp2. We found that thias Selbach (Cell Signaling/Mass Spectrom- activation of MAPK signaling by the Mek1DD etry, MDC), Jan Walcher and Gary R. Lewin allele releases Schwann cells from their natu- (Molecular Physiology, MDC), Annika Wulf- ral ErbB3/Shp2 dependence. Nrg1/ErbB sig- Goldenberg (Exp. Pharmacology and Oncol- nals control different aspects of Schwann cell ogy Berlin-Buch GmbH), Soraya Hoelper and development, i.e. precursor proliferation and Markus Krüger (MPI, Bad Nauheim), Alistair N. migration in early development, the onset of Garratt (Charité, Berlin), Dies Meijer (Erasmus myelination, myelin growth and myelin pro- University, Rotterdam), Klaus Rajewsky and tein production. Interestingly, activation of Walter Birchmeier (Cancer Program, MDC). MAPK signaling by the Mek1DD allele largely

Myelination depends on the synthesis of protein synthesis and myelination and thus large amounts of myelin transcripts and pro- rescues deficits in proliferation, migration, teins and is controlled by Nrg1/ErbB/Shp2 in Schwann cells during early development signaling. We developed a novel pulse label- andsuffices myelination to replace (Sheean Nrg1/ErbB/Shp2 et al., 2014). signals

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Our biochemical analysis indicates that

Bace1 processes the Ig-containing β1 Nrg1- cle(IgNrg1β1) contraction isoform. and are Muscle innervated spindles by pro are- prioceptivespecialized musclesensory fibers axons that (Fig. detect 4). Muscle mus spindles are critical for motor coordination.

signal strength in mice results in increas- We find that a graded reduction in IgNrg1- ration of muscle spindles. Furthermore, weingly show severe that deficits Bace1 in is formation required forand forma matu- tion and maturation of the muscle spindle. Finally, pharmacological inhibition and conditional mutagenesis in adult animals demonstrate that Bace1 and Nrg1 are essential to sustain muscle spindles and to maintain motor coordination. Our results assign to Bace1 a role in the control of coordinated movement through its regulation of muscle spindle physiology, and implicate IgNrg1-dependent processing as a molecular mechanism (Cheret et al., 2013).

Selected Publications RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells.Vasyutina E, Lenhard DC, Wende H, Erdmann B, Epstein JA, Birchmeier C. Proc Natl Acad Sci U S A. 2007;104(11):4443-8.

Colonization of the satellite cell niche by skeletal muscle progenitor cells depends

Purfürst B, Wende H, Birchmeier C. Dev Cell. 2012;23(3):469-81.

on Notch signals.Bröhl D, Vasyutina E, Czajkowski MT, Griger J, Rassek C, Rahn HP, The transcription factor c-Maf controls touch receptor development and function. Wende H, Lechner SG, Cheret C, Bourane S, Kolanczyk ME, Pattyn A, Reuter K, Munier FL, Carroll P, Lewin GR, Birchmeier C. Science. 2012; 335(6074):1373-6.

Fig. 4 Appearance of a muscle spindle, the propriocep- Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles. Cheret C, Willem M, Fricker FR, Wende H, Wulf-Goldenberg A, Tahirovic tive organ of the muscle. The spindle was visualized by S, Nave KA, Saftig P, Haass C, Garratt AN, Bennett DL, Birchmeier C. EMBO J. 2013; 32(14):2015-28. immunohistology using antibodies against NF200 (green) that stain the proprioceptive sensory axon that wraps the Activation of MAPK overrides the termination of myelin growth and replaces Nrg1/ muscle spindle and conveys information about muscle ErbB3 signals during Schwann cell development and myelination. Sheean ME, McShane E, Cheret C, Walcher J, Müller T, Wulf-Goldenberg A, Hoelper S, Garratt AN, contraction, antibodies against Egr3 (red) that stain the spindle fiber, and antibodies against collagen IV (blue) that C. Genes Dev. 2014; 28(3):290-303. visualize the capsule surrounding the spindle. Krüger M, Rajewsky K, Meijer D, Birchmeier W, Lewin GR, Selbach M, Birchmeier

Bace1 and Neuregulin-1 cooperate to control formation and mainte- Group Leader nance of muscle spindles Prof. Dr. Carmen Birchmeier PhD students Cyril Cheret, Hagen Wende, in collaboration Jie-Shin Chen with Michael Willem, Sabina Tahirovic and Senior Scientist Christian Haass (LMU and DZNE Munich), Dr. Thomas Müller Joscha Griger Florence R. Fricker and David L. Bennett UlrichMaciej KoestnerCzajkowski (University of Oxford), Annika Wulf-Golden- Scientists Claudia Rassek (until December berg (Experimental Pharmacology & Oncol- Dr. Dominique Broehl 2013) ogy Berlin-Buch GmbH), Klaus-Armin Nave Dr. Cyril Cheret (MPI Gottingen), Alistair N. Garratt (Charité Dr. Luis Rodrigo Hernandez Technical Assistants Berlin) and Paul Saftig (Universität Kiel). Miranda Bettina Brandt Dr. Shiqi Jia Sven Buchert Dr. Ines Lahmann Karin Gottschling - Dr. Maria Elzbieta Sheean (until Ivonne Schiffner The protease β-secretase 1 (Bace1) was May 2013) Mandy Terne identified through its critical role in pro Dr. Michael Strehle Alzheimer’sduction of amyloid-βdisease. Bace1 peptides is considered (Aβ), the a Dr. Hagen Wende (until March Animal care takers promisingmajor component target for of the amyloid treatment plaques of this in 2013) Claudia Päseler pathology, but processes additional sub- Dr. Kira Balueva Petra Stallerow strates, among them Neuregulin-1 (Nrg1).

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DISEASES OF THE NERVOUS SYSTEM Photo: Stefan Jentsch Stefan Photo:

Thomas J. Jentsch

Physiology and Pathology of Ion Transport

Ion transport across cellular membranes By means of KO mouse models for most is crucial for cellular homeostasis and has CLCs, in the past couple of years we iden- integrative functions such as transepithe- Ostm1), discovered that certain vesicular lial transport or signal transduction. We CLCstified are associated electrogenic β-subunits Cl-/H + (barttin-exchangers, and study ion transport at various levels: from performed structure-function analysis, and biophysical and structure-function analysis uncovered several new pathologies resulting from their dysfunction. Vesicular CLCs of transport proteins, their role in cellular were believed to be Cl- channels that fa- functions – such as cell volume regulation or endocytosis – to the role in the organism. proton pump currents, but surprisingly, cilitate vesicular acidification by shunting- + The physiological importance of ion trans- most (or all) vesicular CLCs are Cl /H - exchangers. In 2010 we reported on mice port proteins is often evident from patholo- in which ClC-5 and ClC-7 were converted gies resulting from their disruption in mice with single point mutations to uncoupled, or men. We have discovered several human pure Cl- channels. Surprisingly, these studies showed that these mice have largely the ‘channelopathies’ and have generated and same phenotype as the corresponding KO analyzed many mouse models. lines, suggesting an important role of these We focus on CLC chloride channels and transporters in vesicular Cl- accumulation. transporters, Anoctamin Ca2+-activated Cl- However, some of the phenotypes of the ‘uncoupled’ Clcn7unc/unc mice were less se- channels, KCNQ potassium channels, and vere than in Clcn7‑/‑ mice, which may indi- KCC potassium-chloride cotransporters. cate a rescue of the lacking Cl-/H+-exchange Their mutational inactivation led to patholo- by the Cl- conductance, or ion transport- gies ranging from epilepsy, deafness, and independent functions of the ClC-7 protein that is still present in Clcn7unc/un mice, but neurodegeneration to osteopetrosis and absent from Clcn7‑/‑ mice. To distinguish kidney stones. We are particularly inter- between these possibilities, we generated ested in the control of neuronal excitability, a mouse with a point mutation that totally abolishes ion transport, but expresses nor- sensory physiology and in the role of chlo- mal amounts of correctly localized ClC-7. ride and pH in endosomes and lysosomes. Analysis of these mice is in progress.

We also characterized functionally ClC-7 (1) CLC chloride channels and transporters and osteopetrotic cattle, and titrated the Sabrina Jabs, Lilia Leisle, Carmen Ludwig, amountmutants of identifiedosteoclast-expressed in human ClC-7 patients with Tobias Stauber, Stefanie Weinert respect to osteopetrosis in different transgenic mouse lines expressing ClC-7 in the The CLC gene family, discovered in our background of Clcn7‑/‑ mice. In biophysical laboratory in 1990, encodes plasma mem- studies we dissected further the slow volt- brane chloride channels and chloride age-dependent gating of ClC‑7/Ostm1. It transporters of intracellular membranes. corresponds to the common gating known

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Gating kinetics of ion flux through a ClC-7 monomer Myelin vacuolization in a Glialcam mouse model. are influenced by the adjacent subunit of the dimer. H&E stainings of sagittal paraffin section of cerebellum ClC-7 is a homodimeric lysosomal chloride transporter of a 52 weeks old GlialCAM-/- mouse. Novel mouse models important for lysosomal function and bone degradation. deficient for the cell adhesion molecule, GlialCAM, and Osteopetrosis and lysosomal storage disease are associ- the membrane protein MLC1, manifest myelin vacuoliza- ated with accelerating mutations in the ClC-7 C terminus tion in cerebellar white matter tract. These two proteins and the contacting intramembrane part. in addition with ClC-2, a chloride channel, underlie forms We discovered that altered gating kinetics of one subunit of leukoencephalopathy, a pathology of the white matter, affect the kinetics of the other subunit. Gating of ClC-7 seen in humans. GlialCAM, MLC1, and ClC-2 in vivo form a involves both CLC subunits and requires non-covalent protein complex at glial domains such as oligodendrocytes binding of cytoplasmic domains. to regulate chloride transport. The graph shows individual and averaged time constants For background information, see Jeworutzki E., López-

of activation (τact) of currents recorded from oocytes Hernández T., Capdevila-Nortes X., Sirisi S., Bengtsson L., injected with RNA encoding WT ClC-7, the ‘fast’ mutant Montolio M., Zifarelli G., Arnedo T., Müller C.S., Schulte R762Q , a 1:4 ratio of WT and the transport deficient U., Nunes V., Martínez A., Jentsch T.J., Gasull X., Pusch M., double mutant R762Q/td and a 1:4 co-injection of R762Q Estévez R. (2012). GlialCAM, a protein defective in a leu- with the E314A (td) mutant. Note that the transport-defi- kodystrophy, serves as ClC-2 Cl- channel auxiliary subunit. cient fast subunit accelerates gating in dimers with a WT Neuron 73: 951-961. subunit and that the transport-deficient WT subunit slows gating in dimers with a fast subunit, resulting in indistin- MLC1, and in GlialCAM, an Ig-like cell ad- guishable gating kinetics. hesion molecule that physically interacts Symbols above diagrams are as follows: WT subunits are with MLC1. In a collaboration with R. Es- shown in white, subunits with an accelerating point muta- tévez (Barcelona) and M. Pusch (Genova) tion are in gray, and subunits carrying a transport-abol- we showed that GlialCAM also interacts ishing point mutation (td; E314A in ClC-7) are symbolized with ClC-2 and drastically changes its cur- by a lack of the central ”hole.“ rent properties. Moreover, GlialCAM directs For details, see Ludwig C.F., Ullrich F., Leisle L., Stauber T., both MLC1 and ClC-2 to cell-cell contacts Jentsch T.J. (2013). Common gating of both CLC subunits of transfected cells. We now generated underlies voltage-dependent activation of the 2Cl–/H+- Glialcam‑/‑ and Glialcamdn/dn mice, the lat- exchanger ClC-7/Ostm1. J. Biol. Chem. 288: 28611-28619. ter carrying a mutation found in human leukodystrophy, and studied these mice in comparison with Mlc1‑/‑ and Clcn2‑/‑ mice. from CLC Cl- channels and affects both Immunohistochemstry indicated that Gli- pores in parallel, with a crucial role played alCAM is important for the localization of by the cytoplasmic CBS domains that can ClC-2 and MLC1 also in vivo, and that sur- bind non-covalently to the channel back- prisingly the lack of MLC1 also affected the bone when expressed for an independent localization of ClC-2 and GlialCAM in both cDNA. astrocytes and oligodendrocytes. ClC-2 currents were reduced in oligodendrocytes We had previously shown that the disrup- of all three mouse models, suggesting that tion of ClC-2 in mice leads to leukodystro- the lack of glial chloride contributes to the pathology of all three forms of leukodystro- mutations in a clinical subform of human phy. ClC-2 may be involved in extracellular leukodystrophy.phy, and recently In others humans, identified megalence CLCN2- ion homeostasis during neuronal activity. phalic leukoencephalopathy with subcorti- The observation that leukodystrophy was cal cysts (MLC) can be caused by mutations more severe in ClC-2/GlialCAM double KOs in the multimembrane spanning protein than in Clcn2‑/‑ mice, however, indicates

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Cell type-specific deletion of Kcc2 in the cerebellum. Progression of Kcc2 expression on Purkinje cells (PC) from Kcc2lox/lox (control) and PC-ΔKcc2 mice, av eraged over all regions in the vermis. PCs showing somatic membrane staining were counted as positive cells (average±standard deviation, n=2, 1, 4, and 2 mice for P17, P21, P25, and P30, respectively (for each genotype); ~200 cells counted per animal). Also in granule cells of the vermis, Kcc2 deletion was complete at P25. (a–h) Immunofluorescent labelling for Kcc2 (green), parvalbumin (red), and nuclei (blue). (e–h) on sagittal sections of cerebellar cortex from adult mice (scale bar: 40 μm). (a, e) Kcc2lox/lox mice (control). (b, f) GC-ΔKcc2 mice with specific Kcc2 deletion in granule cells (GC). (c, g) PC-ΔKcc2 mice with specific deletion of Kcc2 in Purkinje cells. (d, h) PC;GC-ΔKcc2 mice with Kcc2 deletion in both PCs and GCs. Kcc2 expression remains in cerebellar interneurons (c, d, arrows). Asterisks in (c, d) indicate PC somata. Taken from Seja P., Schonewille M., Spitzmaul G., Badura A., Klein I., Rudhard Y., Wisden W., Hübner C.A., De Zeeuw C.I., Jentsch T.J. (2012). Raising cytosolic Cl- in cerebellar granule cells affects their excitability and vestibulo-ocular learning. EMBO J. 31: 1217-1230.

that loss of GlialCAM has additional patho- Patch-clamp analysis showed that Kcc2 was genic effects. Conditional ClC-2 mice are in - progress to pinpoint the cell type in which in both types of neurons. Its deletion nearly loss of ClC-2 is pathogenic. aabolished major player GABA-induced in lowering hyperpolarization cytoplasmic Cl

affected excitability by membrane depolar- (2) K-Cl- cotransporters of Purkinje cells, but in granule cells it merely Kathrin Gödde, Guillermo Spitzmaul, from granule cells impaired consolidation of Patricia Seja long-termization. Granule-specific phase learning deletionin the vestibulo- of Kcc2

We have previously knocked-out all KCl-co- role of these cells. transporter isoforms (KCC1-4) in mice and ocular reflex, revealing a previously unknown - phenotypes. We are continuing our studies ies on Ancotamin Cl- channels and olfaction, have obtained specific and highly interesting- In another project that fits well to our stud cus is on KCCs expressed in neurons, where the olfactory bulb in order to investigate the KCC2on KCCs in withparticular conditional lowers KOs. the Our cytoplasmic major fo rolewe specifically of synaptic deleted inhibition Kcc2 to inodour mitral discrimi cells of- chloride concentration. Such a low concentra- nation. Electrophysiological and behavioural tion is necessary for the inhibitory action of analysis of these mice is in progress. GABA and glycine, which act on ligand-gated chloride channels. In the reporting period we have been investigating various mouse lines (3) KCNQ potassium channels Pawel Fidzinski, Matthias Heidenreich, sets of neurons. Guillermo Spitzmaul in which KCC2 has been inactivated in specific

of its rather simple architecture and the avail- (Kv7) potassium channels, KCNQ1- KCNQ5. We first focused on the cerebellum because KCNQ2-KCNQ5There are five differentmediate isoforms‘M-currents’ of KCNQ that regulate neuronal excitability. We had pre- ability of Cre lines that allow specific gene deletions in Purkinje cells and granule cells. 140 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

viously shown that KCNQ2 and KCNQ3 un- mice lacking both Ano1 and Ano2 in olfac- derlie a form of human epilepsy and that tory sensory neurons and will cross them dominant KCNQ4 mutations are a cause to mice lacking the upstream Trpc2 cation of human deafness and have published a channel to genetically and functionally dis- few years ago a mouse model for KCNQ4 sect the poorly understood signal trans- deafness. In the last reporting period we duction cascade in the VNO. In addition, showed that KCNQ4 also tunes rapidly we are working on other members of the adapting skin mechanoreceptor neurons Anoctamin family. - vestigating the role of other KCNQ isoforms into mechanosensation.specific frequencies. We are currently in Selected Publications

Novarino G., Weinert S., Rickheit G., Jentsch T.J. (2010). Endosomal chloride-proton KCNQ4 is not only expressed in (outer) exchange rather than chloride conductance is crucial for renal endocytosis. Science cochlear hair cells, but also in the vestibu- 328: 1398-1401.

lar organ where staining with KCNQ4 and Weinert S., Jabs S., Supanchart C., Schweizer M., Gimber N., Richter M., Rademann J., KCNQ5 suggested that both channels are Stauber T., Kornak U., Jentsch T.J. (2010). Lysosomal pathology and osteopetrosis differentially expressed at the basolateral upon loss of H+-driven lysosomal Cl- accumulation. Science 328:1401-1403. membrane of sensory hair cells. However, Jentsch T.J. (2011). Ca2+-activated Cl--currents are our investigation of the corresponding KO dispensable for olfaction. Nature Neurosci. 14: 763-769. and KI mouse models now show that they Billig G.M., Pál B., Fidzinski P., Heidenreich M., Lechner S.G., Vardanyan V., Wetzel C., Cremers C.W., De Leenheer E.M., are not expressed in hair cells, but in the Jentsch T.J.*, Lewin G.R.* (2012). KCNQ4 K+ channels tune mechanoreceptors for normal touch sensation in mouse and man. Nature NeurAránguezosci. 15:G., Moreno-Pelayo 138-145. M.A., showedadjacent moderate large postsynaptic changes in membrane the corre of- spondingcalyx synapses. mouse models, Vestibulo-ocular with KCNQ4 reflexes play- C.A., De Zeeuw C.I., Jentsch T.J. (2012). Raising cytosolic Cl- in cerebellar granule cells affectsSeja P., theirSchonewille excitability M., Spitzmaul and vestibulo-ocular G., Badura A., learning. Klein I., EMBO Rudhard J. 31: Y., 1217-1230.Wisden W., Hübner

ing the major role. CNS function of KCNQ5, the least known memberWe also made of this major family. efforts Our topreliminary delineate there- sults, which include results from immuno- Group Leader histochemistry, slice electrophysiology, in Prof. Dr. Dr. Thomas J. Jentsch Till Stuhlmann vivo registrations and behavioural studies FlorianPatricia UllrichSeja (*) (*) indicate that KCNQ5 plays a role in damp- Scientists Felizia Voß ening synaptic inhibition, whereas KCNQ2 Dr. Gwendolyn Billig (*) and 3 dampen excitation. Dr. Matthias Heidenreich (*) MD Student Momsen Reincke (*) Dr. Sabrina Jabs (4) Anoctamin (TMEM16) Ca2+-acti- Dr. Hermann-JosefMaja Hoegg-Beiler Kaiser (*) Technical Assistents vated Cl- channels Dr. Ian Orozco Carolin Backhaus (*) Jonas Münch, Sebastian Albrecht, Gwendolyn Dr. Rosa Planells-Cases (*) Anyess von Bock Billig Dr. Tobias Stauber Johanna Jedamzick (*) Dr. Janis Vogt (*) Nicole Krönke Dr. Stefanie Weinert Janet Liebold functions of members of the newly identi- We have started new projects2+-activated to define Cl- PhD Students Katrin Räbel Sebastian Albrecht MarioRuth Pareja Ringler Alcaraz (*) fiedthat AnoctaminAno2 is the familylong-sought of Ca Ca2+-activated Patrick Seidler Clchannels.- channel As of a olfactory first result, sensory we have neurons shown of Andreia Cruz e Silva Andrea Weidlich the main olfactory epithelium (MOE), but KathrinAnja Blessing Gödde (*) Silke Zillmann that it is dispensable for olfaction. We are Lilia Leisle (*) now turning our attention to the vomerona- Carmen Ludwig Animal Care Attendant sal organ (VNO) that is responsible for the Darius Lutter (*) Petra Göritz detection of pheromones and other cues Jonas Münch like MHC peptides. The signal transduc- Karina Oberheide Research Coordinator tion cascade in the VNO is different from Sebastian Schütze Dr. Norma Nitschke (*) the MOE and their sensory neurons express Kristin Schnuppe (*) (*) = part of the period both Ano1 and Ano2. We are generating

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Fritz G. Rathjen

Developmental Neurobiology

During embryonic development the guid- A cGMP signaling cascade composed of CNP, ance of axons and the precise recognition Npr2 and cGKI is implicated in the bifurcation of DRG axons of targets are crucial for establishing the correct neuronal circuits in the brain. For Many neurons within the brain display ex- the navigation to their target areas, growth tremely complex patterns of axonal arbo- cones of axons respond rapidly to guidance root ganglion neurons (DRG) into the spi- cues in their environment. Proper wiring of nalrization. cord display In contrast, a stereotyped projections and of simple dorsal the brain is orchestrated by multiple mo- pattern of axonal branching. The central lecular factors and cellular mechanisms including electrical activity. The research sites in a temporally controlled order: DRG axonsprojections enter of the DRG spinal neurons cord branch and bifurcate at three of Fritz G. Rathjen’s group focuses on two into two arms resembling a ‘T’, and then major topics of the “wiring” problem: the extend in rostral and caudal directions. branching of axons during outgrowth and Roughly two days later, the two daughter axons generate collaterals which grow in a modulation of electric activity by cell adhe- ventral direction to distinct laminae of the sion proteins which in turn might affect the spinal cord where they branch a third time. formation of neuronal circuits. Due to this simple pattern of branching the spinal cord represents an attractive system to identify factors implicated in axonal branching.

Previous studies by us using transgenic techniques showed that a cGMP-dependent signaling cascade composed of the ligand CNP, the receptor guanylyl cyclase Npr2 Axonal branching allows nerve and the kinase cGKI is essential for axonal cells to communicate with distinct branching at the dorsal entry zone of the neuronal targets spinal cord. In the absence of any one of these components, DRG axons do not bi- The branching of axons is a morphological furcate but are able to turn either rostrally hallmark of virtually all neurons, and al- or caudally (Fig. 1). In contrast, collateral lows an individual nerve cell to innervate formation is not affected by this signaling several distinct targets and to communicate system. with neurons in different parts of the brain. Axonal branching is therefore a key mechanism in the generation of neuronal circuits. The cGMP signaling pathway that triggers It is crucial for normal brain function and bifurcation of DRG axons is also an essential impairments of branching might result in regulator of the arborization of central af- neurological disorders. In the last decades ferents from cranial sensory ganglia axonal branching has been therefore the In the past years, we asked which neuro- vitro cultures, but our knowledge of the un- nal populations apart from DRG neurons derlyingsubject ofmolecular intense studiessignaling primarily mechanisms by in use this signaling system to regulate axonal is still fragmentary. branching. Using a transgenic technique to

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Figure 1: Loss of bifurcation of axons from DRG neurons in the absence of Npr2-mediated cGMP signaling. Individual DRG neurons were labeled by a transgenic technique in wild-type mice (A) or in Npr2-mutant mice (B). The arrow heads point to a bifurcating and a non-bifurcating axon in the wild-type and Npr2-mutant mouse, respectively. Rostral is to the left. DRG, dorsal root ganglion; RP, roof plate; SC, spinal cord; scale bar, 250 µm.

Figure 2: Bifurcation of axons from cranial sensory neurons depend on the receptor guanylyl cyclase Npr2. Individual neurons from cranial sensory ganglia were labeled by a transgenic technique in wildtype (A) or in Npr2-mutant mice (B). The arrow heads point to bifurcating and non-bifurcating axons in the wild-type and Npr2 mutant mouse. gV, trigeminal ganglion; gVII, geniculate ganglion; gVIII, vestibular and spiral ganglion; gIX, glos- sopharyngeal ganglion; gX, vagus ganglion; sup, superior; inf, inferior; Hb, hindbrain; scale bar, 200 µm.

label selectively a subset of Npr2-positive of established circuits. Cell adhesion mol- neurons, we demonstrated that the absence ecules (CAM) might be considered as candi- of Npr2 causes a complete loss of bifurca- dates to link electric activity of neurons to tion of all cranial sensory axons, which in- structural changes within neuronal circuits. stead establish only either ascending or de- CAMs establish initial cell-cell contacts and provide a platform for contact mediated in- branching of cranial and dorsal root ganglia tercellular signaling. Four structural groups isscending regulated projections in an analogous (Fig. 2). manner Thus, byaxonal this of CAMs are expressed in the developing cGMP signaling cascade. In contrast, collat- nervous system: cadherins/protocadherin; eral formation from the stem axons at the IgCAMs (immunoglobulin cell adhesion lateral margin of the hindbrain was not af- molecules), integrins and neurexins/neu- fected in the absence of cGMP – as also ob- roligins. Of these several IgCAMs are ex- served for DRG axons (Fig. 3). pressed at early stages and reveal a strong localization on axonal surfaces. We have

Link between electric activity and electric activity during development and cell adhesion molecules at devel- testedtherefore whether asked the whether absence IgCAMs of an IgCAMinfluence af- opmental stages of the brain fects calcium signalling. We observed that - In the mature brain electric activity is the tracellular calcium levels by regulating in- language for communication between ternalthe IgCAM calcium CAR stores. specifically Our studies modulates revealed in neurons. However, during embryonic and an unexpected link between adhesion pro- postnatal development electric activity of cesses, calcium release and electric activity. neurons is also implicated in shaping the structure of neuronal circuits of the brain. Further characterization indicated that As soon as functional circuits forms, spon- CAR is a typical cell adhesion molecule that taneous activity in form of calcium waves becomes correlated between neighbouring the immunoglobulin superfamily. CAR is primarilybelongs to expressed a specific in subgroup the developing of CAMs ner of-

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Figure 3: Schematic representation of the bifurcation defects observed in the absence of cGMP signaling in axons from dorsal root ganglia (DRG) in the spinal cord and cranial sensory ganglia (CSG) in the hindbrain. The localization of Npr2 and cGKI or CNP in the axons, spinal cord or hindbrain is colored. The left part illustrates the wild-type, the right the mutant situation. r1-r8, rhombomeres 1-8.

vous system and heart and is almost absent coordination. Analysis of the neuronal con- at mature stages. It reveals homophilic as well as heterophilic interactions. Our struc- recordings demonstrated impairments of tural, binding as well as adhesion studies presynapticnectivity of Purkinjematuration cells of byinhibitory patch-clamp syn- - ing conformational shifts for cis or trans revealed decreased evoked amplitudes, al- homophilicpredict a flexible interactions ectodomain on neurons. of CAR allow teredapses. paired-pulse GABAergic synapses facilitation on Purkinje and reduced cells depression after repetitive stimulation at early postnatal but not at mature stages. Impaired presynaptic function and elimination of synapses in the ab- Furthermore, the elimination of supernu- sence of CALEB cells was found to be impaired in the ab- CALEB, also termed neuroglycan C or sencemerary of climbing CALEB. fiberFor example, synapses at on postnatal Purkinje CSPG5, is a neural protein composed of an day 8 in wild-type mice 54 percent of Pur- N-terminal segment containing chondroitin - sulfate chains followed by an acidic stretch, ber synapses in contrast to mutants where an EGF-like domain, a transmembrane and thiskinje number cells have is decreasedthree or more to less climbing than 25fi a cytoplasmic segment. CALEB expression percent (Fig. 4). The basic properties of is restricted to the central nervous system and appears to be generated as a precur- remained unaffected. The alterations ob- sor protein that becomes converted to a servedthe climbing by patch-clamp fiber Purkinje recordings cell synapse corre- truncated transmembrane form with an exposed EGF domain and a secreted form. The conversion from a long form to a trun- dynamicallylated with a regulatedspecific pattern during and development timing of cated form is promoted by electric activity. fromexpression a high of chondroitinsulfate-containing CALEB in Purkinje cells. It is Our previous investigation on this protein form to a non-chondroitinsulfate-contain- showed an impaired synapse function in ing form. Thus, our results demonstrated the absence of CALEB at developing but not an involvement of CALEB in presynaptic mature stages. differentiation of cerebellar GABAergic synapses and revealed a role for CALEB in In past years we studied the role of CALEB in the developing cerebellum which was observations are restricted to early postna- motivated by our observation that CALEB- talsynapse stages elimination suggesting ina transientPurkinje cells.role for These CA- LEB in synapse maturation.

deficient mice displayed impaired motor 144 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

Figure 4: Elimination of climbing fiber- Purkinje cell synapses is impaired in the absence of CALEB. The scheme (A) illustrates the electrophysiological design to estimate the Selected Publications number of climbing fiber synapses on Purkinje cells. Example traces (B) and mean number cranial sensory neurons is disabled in the absence of Npr2-induced cGMP signaling. J. of steps (C), representing the mean number of Ter-Avetisyan,Neuroscience 34: G., Rathjen,737-747. F.G. and Schmidt, H. (2014) Bifurcation of axons from synapses from wild-type and CALEB -/- Pur- - kinje cells. D) The percentage of Purkinje cells paired presynaptic function and elimination of synapses at premature stages during with three and more climbing fiber synapses at Jüttner,postnatal R., de Montag,velopment D., Craveiro, of the cerebellum R.B., Babich, in theA., Vetter,absence P. andof CALEB Rathjen (CSPG5/neurogly F.G. (2013) Im - different developmental stages in the absence can C). Europ. J. Neuroscience 38(9):3270-80. of CALEB in comparison to wild-type Purkinje Patzke, C., Max, K., Behlke, J., Schreiber, J., Schmidt, H., Dorner, A., Kröger, S., Henning, cells is shown. CF - climbing fiber; PF - parallel (CAR) reveals complex homophilic and heterophilic binding activities on neural cells. fiber; rec – recording pipette; stim – stimula- M.,J. Neurosci. Otto, A., 30(8):Heinemann, 2897-2910 U. and Rathjen, F.G. (2010) The Coxsackie-Adenovirs receptor tion pipette.

C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. PNAS Schmidt,106(39):16847-16854 H., Stonkute, A., Jüttner, R., Kösling, D., Friebe, A. and Rathjen, F.G. (2009)

Group Leader Prof. Dr. Fritz Rathjen Graduate and Undergraduate Scientist Isabel Groß* Dr. René Jüttner Nina Hartmann* Patricia Preston* Marlon Kazmierczak* Dr. Hannes Schmidt* Dr. Gohar Ter-Avetisyan* Laura Schütz* Katja Nitze* PhD students Technical Assistants Alexandre Dumoulin* Madlen Driesner Florian Hetsch Mechthild Henning Hanna Langhorst Anne Köhn Claudia Matthaeus Vincent Ramillon* Assistent: Gohar Ter-Avetisyan* Birgit Cloos (part time) Philip Tröster Animal care taker: * Part of the time reported Karola Bach (part time)

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Gary Lewin

Molecular Physiology of Somatic Sensation

Somatic sensation includes all those sensa- Molecular Basis of tions that we consciously feel after stimula- Mechanotransduction Yinth Andrea Bernal-Sierra, Caglar Gök, tion of the body, e.g. touch, warmth, cool- Regina Herget, Liudmilla Lapatsina, Stefan G. ing, or even limb movement. We experience Lechner, Mirko Moroni, Kate Poole, Christiane these sensations as a direct result of the Wetzel activation of sensory neurons that are lo- Mechanotransduction is the process where cated in the dorsal root ganglia (DRG). In by receptor proteins present in the endings our group we are interested in the molecu- of sensory neurons are able to detect me- lar mechanisms that allow these neurons chanical stimulation of the tissue they innervate. We have used information from ge- to transduce these varied stimuli. Sensory netic experiments with the nematode worm neurons can, for example, detect changes C.elegans to identify possible vertebrate can- in temperature of the skin in non-noxious didate proteins that might detect mechanical (not painful) as well as the noxious range stimuli. Genetic screens for touch insensitive worms have turned up around 15 genes (painful heat, or cold). They can also de- whose function is necessary to confer touch tect gentle movement of the skin as well as sensitivity. These genes were named mec intense mechanical stimulation of the skin for mechanically insensitive and we have that is normally harmful. The nature of the focused on identifying a role mammalian orthologs of these genes in touch sensation. transduction molecules involved together The mec genes in C.elegans have been pro- with the developmental events that lead posed to work together in a mechanotrans- to specification of the appropriate sensory duction complex (Figure 1). An essential component of this complex is the membrane neuron sub-types are actively investigated protein MEC-2 that forms a hairpin in the in the lab. membrane and might regulate the activity of the mechanotransduction channels. We have cloned and characterized vertebrate homologues of mec genes and have created mouse mutant alleles to characterize the in vivo function of these genes. MEC-2 is a member of a large family of proteins that contain a stomatin-like domain. A member of this family called STOML3 (stomatin like protein-3) was cloned by our group, and we subsequently generated a mouse model with a null mutation of the STOML3 locus. In STOML3 mutant mice many mechanoreceptors (or touch receptors) in the skin do not work in the absence of the STOML3 protein. In order to analyze touch sensation in mice we also developed a novel behavioral assay

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teins. The structural and molecular basis of such regulation remained a mystery until recently. Together with the Daumke group we have used structural biology techniques and structure function studies to examine the molecular mechanisms of stomatin-domain protein function. By using X-ray crystallography techniques the structure of the mammalian stomatin pro- Model of the mechanotransduction complexes and how tein was solved we showed that the basic they may be inserted into the plasma membrane. building block of this protein is a banana- shaped dimer that can form the basis of higher order oligomeric structures in the membrane (Figure 2). The oligomerization of stomatin-domains appears to be neces- for touch driven behavior in rodents. This as- sary for their ability to modulate both ASIC say is based on the ability of mice to detect proteins as well as mechanosensitive ion channels like Piezo1 and Piezo2 (Brand et to have a textured quality. STOML3 mutant al 2012; Poole et al 2014). Ongoing work and react to gratings, which are fine enough in the lab seeks to examine the precise in detect such textured surfaces. vivo function of other poorly understood mice have severe deficits in their ability to stomatin-domain containing proteins such The central core of any mechanotransducer is as STOML1. The use of mouse models and an ion channel or channels(s), unfortunately advanced cellular imaging techniques as the molecular nature of this channel has not revealed an unexpected role for STOML1 in the metabolic disorders. neurons. There is some evidence that acid sensingyet been ion definitively channels (ASICs) identified which in belong sensory to The activity of mechanosensitive ion chan- the Deg/ENaC family of ion channels have an nels expressed by sensory neurons can be measured using high-resolution electro- stimuli by sensory neurons. Indeed there is physiology techniques. We have recently alsoinfluence evidence on the of genetic transduction interactions of mechanical between shown that such ion channels in the mem- stomatin–domain proteins and acid sensing branes of cultured DRG neurons can be ac- ion channels. Recently, two new mechano- tivated by stimuli in the nanometer range (Poole et al 2014). We have also gathered in mammals called Piezo1 and Piezo2. Us- considerable evidence that the mechano- ingsensitive a newly ion developed channels haveassay beenbased identified on elas- sensitive channels are actually opened via tomeric pili we could recently show that the a protein tether that attaches to laminin- sensitivity of Piezo ion channels to mechani- containing extracellular matrices. In order cal displacement is exquisitely tuned by the - STOML3 protein (see Poole et al 2014). This lular matrices on mechanotransduction work has been carried out in collaboration andto study to quantifythe influence the tinyof different forces extracelthat are with Dr Kate Poole who is now a Cecille-Vogt required to open mechanosensitive chan- fellow (see her pages in the research report). nels we have started to a use variety of new micro-fabrication techniques. For example, we have used micro-patterning of matrix Stomatin domain proteins, molecules in order force neurons in culture molecular membrane scaffolds with to adopt morphologies that better match multiple roles in physiology and the in vivo situation. We can also use such pathophysiology Caglar Gök, James Hall, Liudmilla Lapatsina, Kate Poole, Jane Reznick abilitypatterning or axon to branching test the localbehavior. influence We have of shownspecific that matrix sensory molecules neurons on can transduction be made to Stomatin-domain proteins can regulate grow to produce highly structured patterns both ion channels and transporter pro- in vivo (see Figure 1). Another application

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Two stomatin domains from the stomatin protein form a dimeric interaction that gives rise to a banana- shaped protein (left). A high resolution structure of the stomatin-domain was obtained together with the Daumke group at the MDC (right) (see Brand et al 2012).

of micro-engineering is to make neurons tion of the mechanotransduction channels. grow on three dimensional surfaces that al- We are currently working on further hearing low us to gauge the forces needed to open genes that may also affect cutaneous mecha- mechanosensitive ion channels in single nosensation. The same genes as we study in cells (Poole et al 2014). the mouse are also mutated in humans and it is possible that the perception of cutaneous touch stimuli is altered in such patients. Touch, hearing and the We have measured pyschometric functions development of mechanosensation in healthy and hearing impaired people in Henning Frenzel, Regina Herget, Stefan G. order to describe quantitatively differences Lechner, Rabih Moshourab and Jan Walcher in the perception of touch. We carried out a large twin study which showed that touch Hereditary deafness is a relatively common and also thermosensation are both herita- phenomenon and a large number of genes ble in humans. The results of these studies have also revealed that there appears to be lead to deafness in mouse and man. We are a strong link between the sense of touch and workinghave been with identified several deaf that mutant when mutatedmice to the sense of hearing (Frenzel et al 2012). We examine whether genes required for nor- could demonstrate that one gene, mutations mal mechanotransduction in the inner ear in which, cause severe hearing loss and may also be required for normal cutaneous blindness is also associated with poor touch sensation. Our data indicate that members performance in humans. This gene USH2A of the unconventional myosin protein family encodes a large extracellular protein that is have a common function in sensory neurons thought to form a structural link within the and in hair cells, mechanotransducing cells sterocillia of inner ear hair cells. The role of of the inner ear. In both cell types these pro- this protein in touch receptors remains to teins may function to regulate the adapta- be investigated (Frenzel et al. 2012).

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We have been interested in the development of mechanosensation for many years Selected Publications and it was remarkable how little was known in this area. We have recently shown, in a J, Lewin GR, Daumke O (2012) A stomatin dimer modulates the activity of acid-sens- very detailed study, that sensory neurons Branding ion J, channels. Smith ESJ, EMBO Schwefel J 31:3635–3646. D, Lapatsina L, Poole K, Omerbašić D, Kozlenkov A, Behlke acquire their competence to detect mechanical stimuli very early in embryonic Fforrenzel mechanosensory H, Bohlender traits J, Pinsker in humans. K, Wohlleben PLoS Biol B, Tank 10:e1001318. J, Lechner SG, Schiska D, Jaijo T, development. Interestingly, very distinct Rüschendorf F, Saar K, Jordan J, Millán JM, Gross M, Lewin GR (2012) A genetic basis developmental mechanisms are used to Lapatsina L, Jira JA, Smith ESJ, Poole K, Kozlenkov A, Bilbao D, Lewin GR, Heppenstall induce such competence in neurons that PA (2012) Regulation of ASIC channels by a stomatin/STOML3 complex located in a mobile vesicle pool in sensory neurons. Open Biol 2:120096. underlie touch sensation as opposed to no- ciception (Painful stimuli). For example, we Poole K, Herget, R, Lapatsina L, Ngo HD, Lewin GR (2014) Tuning Piezo ion channels have shown that NGF plays a critical role in 24;5:3520. doi: 10.1038/ncomms4520 the acquisition of transduction competence to detect molecular-scale movements relevant for fine touch. Nat Commun Mar by nociceptors. The development of touch molecular basis of acid insensitivity in the African naked mole-rat. Science 334:1557– receptors is poorly understood but now Smith1560. ESJ, Omerbašić D, Lechner SG, Anirudhan G, Lapatsina L, Lewin GR (2011) The in collaboration with Carmen Birchmeiers group it was found that one transcription factor c-Maf plays a decisive role in controlling the development of such receptors. In- triguingly, this factor is also necessary for the initiation of KCNQ4 expression in mechanoreceptors (Wende et al 2012).

The Naked Mole Rat an extremeo- phile mammal Group Leader Tetiana Kovalchuk Damir Omerbasic, Tania Kovalchuk, Jane Prof. Dr. Gary R. Lewin Damir Omerbasic Reznick, Valerie Begay-Müller Christina Picci Scientist Martha Servin Vences (with The naked mole rat is an unusual subter- Dr. Valerie Begay-Mueller* Kathryn Poole) ranean rodent in many respects. It is the Dr. Henning Frenzel* Jan Walcher only known poikilothermic mammal (ie. Dr. Liudmilla Lapatsina cold blooded), it lives in colonies with an Dr. Stefan G. Lechner* Graduate and Undergraduate insect-like social structure, and it is also Dr. Mirko Moroni Johanna Homfeld the longest-lived rodent species known Dr. med Rabih Moshourab* Steven Middleton (lifetimes in excess of 25 yrs). Although Dr. Kathryn Poole (now Cecille- Oriana Salazar Thula this animal has normal acute pain respons- Vogt fellow) es it displays no hypersensitivity (so called Dr. Jane Reznick Technical Assistants Dr. Jeffrey Stear Maria Braunschweig and chemical stimuli. The naked mole rat Dr. Christiane Wetzel (MDC Liana Kosizki completelyhyperalgesia) lacks to aa varietyneuronal of inflammatoryor behavioral Franziska Kressin response to acid. We have shown that dis- Dr Yinth Andrea Bernal Sierra Anke Scheer GoBio project leader) tinct gene variants encoding NaV1.7 ion Heike Thränhardt channels can account for the loss of acid PhD sensitivity in this species. However, our Tobias Albert Secretariat interest in naked mole-rat physiology goes Ole Eigenbrod Manuela Brandenburg beyond the study of sensory systems. We Caglar Gök are studying molecular changes in naked James Hall Staff mole rats that may account for their com- Julia Haseleu Raluca Fleischer plete lack of thermogenesis, and uniquely Regina Herget *for part of the time reported low metabolic rates.

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Jochen Meier

RNA Editing and Hyperexcitability Disorders

Excitatory and inhibitory transmission between sustains disease progression. In a recent study, neurons in the brain needs to be thoroughly plasticity by studying RNA editing of the neu- regulated and coordinated. Deregulation of rotransmitterwe identified areceptor maladaptive for glycine form of(GlyR). neuronal The this coordination ultimately results in nervous initial observation was that the expression of system disorders. A core aspect of my work an RNA-edited GlyR variant is increased in hip- concerns the study of the brain at the mo- pocampi from patients with temporal lobe epilepsy (TLE) (Nat Neurosci 8:736, 2005; J Cell lecular level, by investigating RNA editing and Mol Med 12:2848, 2008). We therefore gener- splicing. In particular, we analyze key compo- ated a new animal model that allows the tar- nents of the molecular machine responsible the RNA-edited GlyR variant. This model was for inhibition of electrical impulses, i.e. glycine thengeted used and neuronto study type-specific cognitive function, expression learn of- receptors (GlyR) and GABA type A receptors ing and memory, and emotional behavior (J (GABA(A)R) as well as gephyrin. We elucidate Clin Invest 124:696, 2014). The RNA-edited the function of these molecules in physiologi- terminals, and its presence increases the func- cal and pathophysiological processes on a tionalGlyR isweight specifically of such expressed synapses at in presynaptic the hippo- molecular, cellular and systemic level. campal network. Thereby, homeostatic control of synaptic transmission and neural network excitability is changed, and the mice display Disease mechanisms symptoms that are reminiscent of the pathology of TLE. Notably, targeted expression of the Epilepsy is a devastating neurodegenerative RNA-edited GlyR in excitatory glutamatergic disease that severely deteriorates life quality neurons provoked seizure-like activity and due to unpredictable occurrence of seizures cognitive dysfunction, and expression in in- and associated cognitive dysfunction. More- hibitory synapses of parvalbumin-positive in- over, epilepsy patients suffer from severe psy- terneurons resulted in anxiety. Thus, the same chiatric comorbidities including anxiety and molecule triggered distinct psychopathologi- depression. Most epilepsy syndromes have no discernable genetic component. This indicates maladaptive and disease-causing mechanism that epileptogenesis is governed by disease- calof neuronal symptoms plasticity of TLE. that This will study serve identified as a good a promoting molecular and cellular mecha- starting point for the development of an in- nisms of neuronal plasticity, which may vary dividualized treatment. We are expanding from patient to patient, resulting in diverse these experiments to target the expression of clinical pictures of cryptogenic/idiopathic epi- the RNA-edited GlyR variant to other neuro- lepsies. Therefore, new therapeutic strategies nal types and neurotransmitter systems. Fur- are needed to satisfy the variable demands of thermore, we are applying advanced imaging patients. techniques using molecular beacons to gain Prior to the development of effective individu- - alized therapies, it must be elucidated whether tion of RNA editing. a disease-associated mechanism represents Gephinsightsyrin into is a thepostsynaptic neuron type-specific scaffold protein regula re- either an adaptive form of plasticity that is quired for stabilization of GlyR and GABA(A) able to compensate for the disease-causing R at inhibitory synapses (Nat Neurosci 4:253, insult, or a maladaptive form of plasticity that 2001; Eur J Neurosci 37:544, 2013). Consider-

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DISEASE MECHANISMS: Deamination com- mutes cytidine to uridine. C-to-U RNA editing produces gain-of-function GlyR and is increased in patients with TLE. In a corresponding knock- in mouse model, expression of the GlyR RNA variant in glutamatergic principal neurons impaired cognitive function and memory formation. Altered gamma frequency network oscillatory activity due to spontaneous recurrent epileptiform network activity and resulting conflict of interest of neurons during processing of sensory context can explain the animal phenotype (J Clin Invest 124:696, 2014). THERAPEUTIC STRATEGIES: The new molecular tool ‘CIPRESS’ identifies stress-susceptible neurons (Brain 133:3778, 2010) and effectuates neuronal self-defense through expression of selected proteins (protein X). Using optogenetics and electrical stimulation of the trisynaptic hippocampal loop, it will also help identifying neuronal activity patterns that cause cellular stress and induce protein expression.

able functional heterogeneity of gephyrin aris- date proteins (or combinations) can suppress es from alternative mRNA splicing (Mol Cell seizure activity in vivo by using in utero elec- Neurosci 16:566, 2000; J Neurosci 24:1398, troporation of different expression constructs, or by lentiviral expression in slice culture of - the human epileptic hippocampus. We will ronal2004). cells We identified(J Biol Chem gephyrin 283:17370, splice variants 2008), also determine the characteristics of the net- indicatingthat are specifically that geph yrinexpressed RNA splicing in glial isor regu neu- work activity that causes cellular stress and candidate gene expression using optogenetics. isolated from hippocampi of TLE patients This work should allow the characterization of latedirregularly in a cell spliced type-specific gephyrin way. RNA Recently, variants. we disease causing mechanisms of plasticity, for These variants lack several exons and encode instance changes in the regulation of neuronal neuronal gephyrins with dominant-negative pH and chloride, energy metabolism, calcium- activities that de-stabilize postsynaptic recep- dependent proteolysis, and inhibitory actions tors and weaken transmission at inhibitory of GABA. synapses. We discovered that neuronal activity or calcium-dependent cellular stress induces Selected Publications the skipping of exons in the gephyrin-coding A.Winkelmann, N.Maggio, J.Eller, G.Caliskan, M.Semtner, U.Häussler, R.Jüttner, mRNA, leading to frameshift and prema- G.Rechavi, C.A.Haas, A.Kulik, T.Gloveli, U.Heinemann, J.C.Meier (2014). Changes in ture termination of protein synthesis (Brain T.Dugladze,neural network B.Smolinsky, homeostasis S.Kowalczyk, trigger neuropsychiatric E.Chronowska, G.Schwarz,symptoms. F.G.Rathjen,Journal of Clinical 133:3778, 2010). Thus, changes in RNA pro- Investigation 124:696-711. cessing of GlyR-coding mRNA and impaired T.Dugladze, N.Maziashvili, C.Börgers, S.Gurgenidze, U.Häussler, A.Winkelmann, C.A.Haas, J.C.Meier, I.Vida, N.Kopell, T.Gloveli (2013). GABA autoreceptor-mediated splicing of the gephyrin-coding mRNA may B Proceedings of the Na- cooperate in the vicious circle of pathogenic tional Academy of Sciences 110:15073-15078. cell-type specific reduction of inhibition in epileptic mice. mechanisms that impair neural network ho- B. Förstera, A.A.Belaidi, R.Jüttner, C.Bernert, M.Tsokos, T.-N.Lehmann, P.Horn, meostasis and sustain the disease progression. G.Schwarz, J.C.Meier (2010). Irregular RNA splicing curtails postsynaptic gephyrin in the cornu ammonis of patients with epilepsy. Brain 133: 3778-3794. Therapeutic strategies J.C. Meier*, C.Henneberger, I.Melnick, C.Racca, R.J.Harvey, U.Heinemann, V.Schmieden, R.Grantyn (2005). RNA editing produces P185L amino acid substitution in glycine receptor a3 resulting in high agonist potency. Nature Neuroscience 8:736-744. Several possibilities exist that can interrupt J. Meier, C.Vannier, A.Sergé, A.Triller, D.Choquet (2001). Fast and reversible trapping of the vicious circle of impaired plasticity and surface glycine receptors by gephyrin. Nature Neuroscience 4:253-260. disease progression. First, we are performing * Corresponding author

of the pathogenic GlyR variant produced by drug screening to identify specific antagonists be validated in the mouse model described RNA editing. The identified compounds will Group Leader PhD skipping in gephyrin-coding mRNA induces Prof. Dr. Jochen Meier Aline Winkelmann frameshiftabove. Second, in the based protein on thecoding finding sequence, that exon we developed a new molecular tool for neuro- Technical Assistants Carola Bernert neuron-eranet.eu/en/317.php). This system Scientist Andra Eisenmann nalpermits self-defense induction (cf. of ’CIPRESS’ protein project;expression www. in Anne Schäfer response to cellular stress that accompanies Dr. Marcus Semtner Sebastian Tausch epileptogenesis. We are asking which candi- Dr. Benjamin Förstera

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Björn Christian Schroeder

Signaling and Transport Processes

Our work focuses on signal and transport in vascular smooth muscle cells (VSMCs) of processes involving members of the recently the aorta and carotid arteries. CaCCs were small or even absent in medium sized vessels identified TMEM16 family of membrane pro- like mesenteric arteries or larger retinal arte- teins. Mutations in several TMEM16 genes rioles. In small vessels, which are responsible cause human inherited diseases including for about 80% of the total vascular resistance, muscular dystrophy, the rare bleeding dis- where VSMCs are gradually replaced by contractile pericytes, CaCCs were particularly order Scott syndrome, and variants of cer- large in wild-type but absent in Tmem16a ebellar ataxia and dystonia. So far two of mutant mice. Expression analysis for Tme- the TMEM16 proteins have been shown to m16a along the vascular tree showed a simi- mediate calcium activated chloride currents lar distribution (Figure 1). Out data suggest that Tmem16a plays a general role in arterio- (CaCCs), known to be important for various - functions including photo transduction, pain ing target for the treatment of hypertension. perception, and smooth muscle contraction. lar and capillary blood flow and is a promis It is still unknown whether other members of The role of Tmem16a in hearing this family are ion transport proteins and how they operate within cells and whole organ- Inner supporting cells in Kölliker’s organ, a isms. Using cultured cells and transgenic ani- developmentally transient structure within the organ of Corti, have been shown to initiate mals, we try to better understand the function calcium waves and suggested to be the origin of TMEM16 proteins under physiological and of the spontaneous activity. Cellular crenation pathophysiological conditions. events with unknown function occur during these waves.

Chloride channel Tmem16a is expressed in small, but Calcium activated chloride chan- not in large arterioles. Retinal whole-mount, double-im- nels in blood pressure regulation munostainings for the endothelial cell marker CD31 (left), which is expressed in arterial and capillary endothelial High blood pressure is the leading risk factor cells, and Tmem16a (middle) showing a large Tmem16a- for death worldwide. One of the hallmarks is negative primary arteriole (arrow head) and second and a rise of peripheral vascular resistance, which higher-order arterioles branching from it, and overlay largely depends on the tone of arterioles. (right). Arrows indicate strongly-labeled cells at branch- We generated a vascular smooth muscle cell ing points of second-order arterioles.

CaCCs are involved in the regulation of sys- temicspecific arterial Tmem16a blood knock pressure. out mouse Disruption to test of if the calcium activated chloride channel Tme- m16a in vascular cells resulted in decreased systemic blood pressure, as well as a decrease in angiotensin II-induced hypertension in mice. Analysis of calcium activated chloride currents (CaCCs) along the vascular tree of

wild-type mice identified substantial CaCCs 152 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

Tmem16a expression is necessary for the generation of volume changes in Kölliker‘s organ. Spontaneous epithelial cell volume changes in acutely isolated cochlea devoid of Tmem16a of P5-P7 animals are almost absent. Compared to wild-type, typical event sizes are very small (left) and rare (right).

Especially the negatively charged phospho- Because chloride channels have been impli- lipid phosphatidylserine is normally located cated in cellular volume regulation, we investigated whether Tmem16a is involved in this activation of platelets phosphatidylserine process. We found high Tmem16a expression in the inner but not the outer leaflet. During in Kölliker’s organ (Figure 2). Disruption it provides a binding side for interacting of Tmem16a under the control of the Pax2 coagulationis redistributed factors. to theIn outerScott leafletsyndrome, where a promoter resulted in the complete absence bleeding disorder caused by mutations in of crenation events, identifying Tmem16a the TMEM16F gene, this translocation is dis- as key mediator for these volume changes. turbed, resulting in impaired blood clotting, Moreover calcium waves in Kölliker’s organ and changed morphology of activated plate- were reduced to small calcium puffs in knock- lets. out mice. This might be explained by the absence of mechanical stress, which facilitates - ATP release of epithelial cells during activa- vestigate the role of Tmem16f in other cells, tion and is required for the formation of cal- especiallyWe generated of the Tmem16f immune deficient system. B-cellsmice to and in cium waves. The concept of chloride channels T-cells isolated from Tmem16f knock-out being important for the generation of calcium mice showed a strong diminished phosphati- waves is new and has never been proposed. dylserine exposure after stimulation (Figure It is likely that Tmem16a plays a similar role 3). In addition we found a reduced secretion for calcium waves in other organs like airway of cytokines, and microparticle shedding epithelial cells. mice, which suggests that Scott patients also havefrom anTmem16f altered deficientimmune response.cells from knockout Molecular functions of Tmem16f in the immune system We hope that our research will not only help to understand the function of various TMEM16 genes, but also to prevent diseases membrane have different lipid compositions. and conditions like hypertension, stroke or Inner and outer leaflet of the cellular plasma autoimmune disorders. Diminished phosphatidylserine exposure of Tmem16f-deficient T-cells after activation. T-cells isolated from spleen of wild-type and Selected Publications Tmem16f knockout mice were stimulated with - anti-CD3 + anti-CD28 and assayed for surface baum J, Vitzthum H, Gollasch M, Ehmke H, Schroeder BC, Hübner CA. (2014). Disrup- phosphatidylserine using an Annexin V binding tionHeinze of vascularC, Seniuk Ca2+-activated A, Sokolov MV, chloride Huebner currents AK, Klementowicz lowers blood AE, pressure. Szijártó IA,J Clin Schleifen Invest. 124, 675-86. assay at the time indicated. The increase in surface expression of phosphatidylserine found in wild-type cells is absent in mutant T-cells.

Group Leader Dr. Björn Christian Schroeder

Scientist Technical Assistants Dr. Maxim Sokolov Agnieszka Klementowicz

PhD Secretariat Hanad Farah Sylvia Olbrich Alena Maul

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James Poulet

Neural Circuits and Behaviour

We study sensory processing and percep- Synaptic mechanisms of tion in vivo to understand healthy brain somatosensory integration function. We work on a relevant sensory In vivo, cortical neurons integrate thou- system in a genetically tractable model sands of synaptic inputs to generate action animal - the mouse forepaw somatosen- potential outputs during different brain sory system. In awake, behaving animals, states. How brain states affect synaptic integration is debated as almost nothing is sensory processing involves the integration known about the properties of monosynap- of external and internally generated signals tic transmission in vivo. We developed tri- in the brain to generate a single coherent ple two-photon targeted whole-cell patch percept. We are especially interested in clamp recordings and optogenetic techniques to allow simultaneous recordings synaptic and cellular mechanisms underly- of synaptically connected neurons in vivo. ing “sensory integration” in neocortex - an This work provides new approaches to in- area of the brain that can integrate across vestigate active synapses in vivo and will sensory modalities and is under profound allow us to target molecular mechanisms underlying state-dependent synaptic inte- internal control by distinct brain states. To address these questions we use a combi- types in vivo. nation of techniques including two-photon gration between genetically identified cell microscopy, optogenetics, electrophysiol- Somatosensory processing by ogy (especially whole-cell patch clamp) and subtypes of cortical neuron behavioural training. Neighbouring layer 2/3 cortical excitatory neurons show considerable heterogeneity in their responses to sensory stimulation. One explanation is that different subtypes The mouse forepaw system of cortical pyramidal neurons exist with different sensory response properties in The mouse forepaw somatosensory sys- layer 2/3, but there are no molecular mark- tem has a similar structure and function to ers available to investigate this hypothesis. the human hand (Figure 1). Mice use their forepaws not only for locomotion but also investigate a subset of layer 2 excitatory neuronsIn this project in mouse we use somatosensory the fosGFP mouse cortex to sensing their immediate environment. The that are distinguished by expression of the for manipulating and grasping objects and activity-dependent fosGFP reporter gene more of a stump than a thumb. The gla- under basal conditions. We showed that brousmouse skin forepaw surface has is 5 innervated digits, albeit with the a first full fosGFP+ neurons have higher spontane- complement of primary sensory afferent - neurons that process light touch, proprio- midal neurons (Yassin et al., 2010). Recent ception, deep touch, temperature and pain. two-photonous firing rates targeted in vivo dual than whole-cell fosGFP- pyra re- Somatosensation is therefore a multimodal, cordings in combination with optogenetic integrative sense. (See Figure 1) and sensory stimulation have shown that

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Figure 1. The mouse forepaw system. A, a cartoon of the mouse forepaw system. Blue represents the sensory pathway from paw via spinal chord and thalamus to primary somatosensory cortex (S1), red highlights the motor pathway from motor cortex (M1) to spinal cord and forelimb that can drive paw movement. B, image of the glabrous skin of the right forepaw, mice have 5 digits (D1-D5) and 5 pads (P1-3, Thenar pad, TH, and the HypoThenar pad, HT).

fosGFP+ve neurons also have distinct sensory response properties and thalamocorti- cal wiring.

Somatosensory perception

- Selected Publications portant information for animals performing Barth A., and Poulet J.F.A. (2012) Experimental evidence for sparse cortical coding. tactileThe temperature tasks. Temperature of an object and provides touch input im Trends Neurosci. 35:345-55 are thought to closely interact in the brain Poulet J.F.A*., Fernandez, L*. Crochet S.* and Petersen C.C. (2012) Thalamic control of but the primary sensory afferents in the skin cortical brain states. Nature Neuroscience. 15:370-372 *equal contribution and central circuits they engage to enable the perception of surface temperature are Poulet J.F.A.*, Leibold C.* and Schmitz D.* (2011) Coherent phasic excitation during not clear. Here we have developed training Maierhippocampal N.*, Tejero-Cantero ripples. Neuron. A.*, Dorrn2011 72:137-52. A, Winterer *equal J., Beed contribution P, Morris G., Kempter R., routines for mice to report thermal and tactile cues delivered to the forepaw with high Yassin L, Benedetti B.L., Jouhanneau J-S., Wen J.A., Poulet J.F.A., Barth A.L., An embedded subnetwork of highly active neurons in the neocortex. (2010) Neuron 68: 1043- accuracy. We found that the forepaw prima- 50. ry somatosensory cortex, a region normally Poulet J.F.A. and Petersen C. C. (2008) Internal brain state regulates membrane poten- associated with tactile perception, mediates tial synchrony in barrel cortex of behaving mice. Nature, 454: 881-885. mild cooling processing and perception. In collaboration with the Lewin group at the

in the skin, that are normally thought of asMDC, pain we receptors, show that are sensory likely candidatesafferent fibres for Group Leader PhD forwarding mild cold information to the Dr. James Poulet - Leiron Ferrarese tablished a new genetically tractable model Scientist Wen-JieAnja Luise Zhao Dorrn systemcentral tonervous study thermalsystem. Thisperception project and has will es Dr. Luc Estebanez allow us to examine how multimodal sen- Dr. Jean-Sebastien Jouhanneau Diana Hoffmann (MD/PhD) sory inputs are integrated and perceived – a Technical Assistants fundamental yet poorly understood func- Dr. Birgit Voigt Janett König tion of neocortex. Dr. Nevena Milenkovic-Zujko

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Helmut Kettenmann

Cellular Neurosciences

Our goal is to understand the role of glial structures during development and rep- cells in physiology and pathology. We focus resent important elements for controlling the composition of the extracellular space on questions how glial cells communicate mediating signals between the brain endo- among each other. We study the expres- thelium and neurons. They form intimate sion of transmitter receptors in microglial contact with synapses and neuronal activi- cells and how activation of these receptors ty results in astrocyte responses. Microglial cells are immune-competent cells in the influences microglial functions. This is of brain and their functional role is best de- particular interest within the context of pa- - thology and we are currently studying this ing pathologic events. The present research fined as the first responsive elements dur question in stroke, glioma, Alzheimer’s dis- program is focused on four topics: (1) the ease and multiple sclerosis. A fourth line of the glial network and its impact on brain research addresses the question as to how functionextent of(2) gap the junctionfunctional coupling expression within of glioma cells interact with the intrinsic brain transmitter receptors in microglia (3) the cells, specifically microglia and astrocytes. and (4) the interaction of gliomas with mi- We are aiming to understand this interac- crogliaresponse and of astrocytes. microglial cells to brain injury tion on a molecular level, in particular with the hope of identifying therapeutic targets. Structural and functional analyses of panglial coupling networks and its impact on brain signaling Nadine Richter, Christiane Nolte (in collabo- Introduction ration with Christian Steinhäuser, University of Bonn) The central nervous system contains two

cells. The neurons are regarded as the ele- plays an important role for brain homeosta- mentsmajor cellmediating populations, the electrical neurons activity and glial in sis,Gap brain junction communication coupling among and brain glial energy cells the brain. As a consequence, neuroscience supply. Most previous studies have focused research of the past has focused on this cell on the network formed by astrocytes. We type. The functional role of glial cells is not now characterize communication between all types of glial cells, including communi- as cells providing only structural support to cation of astrocytes with oligodendrocytes, neurons,as obvious: a series while ofthey more were recent first studiesdescribed on with the newly described class of glial cells, glial cell function has attracted the atten- the NG2 cells, and with microglia. We ad- tion of the neuroscience community. It has dress the question whether these panglial become evident that glial cells are essential networks are heterogeneous with respect for the proper functioning of the brain. The to brain region, comparing different white and gray matter regions. We will character- tasks. Oligodendrocytes are the myelin- - formingdifferent cells types of of the glial central cells fulfilnervous distinct sys- lation by neurotransmitters. We will also tem and ensure a rapid signal conduction addressize junctional the question properties whether and theirthe coupled modu in the white matter. The role of astrocytes network has functional implications for energy supply to neurons. We aim to present

is less well defined; they provide guiding 156 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

Figure 1: The confocal image shows a biocytin filled panglial network in the cortex of a GFAP/eGFP mouse. In this mouse astrocytes express the enhanced green fluorescent protein under the GFAP (glial fibrillary acidic protein) promoter. A single astrocyte was injected with the gap junction permeable tracer biocytin via the whole-cell patch clamp technique. The biocytin is subsequently labeled with streptavidin Cy3 (red) while the oligodendrocytes (blue) in the network are immunolabeled with the marker Olig2. Apparently in this panglial network some astrocytes also express Olig2 (e.g. the green and blue fluorescent astocytes in the bottom right corner). Scale bar, 10µm.

a more comprehensive view of the panglial somatostatin, angiotensin II, vasopressin, network and its heterogeneity and function neurotensin, dopamine or nicotine. In cul- in the brain. (Funded by DFG). tured microglia from neonatal and adult mice, a similarly small population of cells responded to these neurotransmitters/- The functional expression of transmitter receptors in microglia Maria Pannell, Stefan Wendt, Laura Korvers, cultureshormones. for To 24 induceh. Several a pro-inflammatory of the respond- Marina Matyash, Vitali Matyash, Susanne A. ingphenotype, populations we applied increased, LPS orbut IFN-γ there to was the Wolf no uniform pattern when comparing adult

We have addressed the question whether microglia would also express receptors to substancewith neonatal increased microglia the or histamine, LPS with IFN-γsub- sense neuronal activity. We have previous- stancetreatment. P and IL-4 somatostatin as an anti-inflammatory sensitive popu- - lations only in microglia from adult, but mitters/neurohormones GABA, adrenaline, not in neonatal cells. We also found that dopamine,ly identified bradykinin, receptors serotonin,for the neurotrans endothe- the expression of different receptors was lin-1, substance P and histamine. We found not strongly correlated indicating that that activation of these receptors can mod- there are many different populations of ulate microglial functions such as migra- microglia with a distinct set of receptors. tion or cytokine release. Using freshly iso- A subpopulation (11%) of freshly isolated lated microglia from healthy adult mice, we adult microglia respond to the muscarinic found that only a small fraction (1 - 20 %) acetylcholine receptor agonist carbachol responded to the application of endothelin, with a Ca2+ increase, indicating the expres- histamine, substance P, serotonin, galanin, sion of functional receptors. The carbachol-

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Figure 2: Mouse brains injected with GL261 glioma cells were stained for the microglial marker Iba-1(green) and for metalloprotease-9 (red). In the tumor free area the level of metalloprotease-9 was low (left, outside of the dashed line) while within the tumor metalloprotease-9 is expressed mainly in Iba-1 positive cells (right).

sensitive population increased in microg- growth factors. We are studying microglial lia/brain macrophages isolated from the properties in different mouse models of tissue of mouse models for stroke (60%) disease including Alzheimer’s disease (with and Alzheimer’s disease (25%), but not Frank Heppner, Neuropathology, Charité) for glioma and multiple sclerosis. Microg- and stroke (in collaboration with Matthias lia cultured from adult and neonatal brain Endres, Neurology, Charité). In collabora- contained a small carbachol-sensitive sub- tion with Christine Winter (TU Dresden population (8% and 9%), which was in- and Charité) we study the impact of deep brain stimulation on microglial properties. around 60% after 12 hours. This increase We are currently investigating whether mi- wascreased sensitive by treatment to blockers with of interferon-γ protein syn to- thesis and correlated with an upregula- of the stimulated target. (Funded by DFG) tion of the M3 receptor subtype. Carbachol croglia are activated in the projection areas was a chemoattractant for microglia and decreased their phagocytic activity. Thus Microglia as therapeutic target in microglia are a heterogeneous population psychiatric disorders with respect to chemosensitivity, and the Daniele Mattei, Anaïs Djodari-Irani, Marina carbachol-sensitive population expands in Matyash, Susanne A. Wolf

Many studies discuss an immunological defined diseases. (Funded by DFG). component in the onset and progression of The response of microglial cells to psychiatric disorders. While the peripheral brain injury immune system has been intensively in- Petya Georgieva, Philipp Jordan, Anaïs vestigated, little is known about the role of Djodari-Irani, Susanne A. Wolf microglia, the intrinsic immune competent cells of the brain. We thus aimed to study Microglial cells are the pathologic sensors the microglia phenotype derived from ro- and represent the immune cells of the cen- dent models of schizophrenia and depres- tral nervous system. During any kind of sion including epigenetic factors. We used a disease or any pathological event such as maternal immune stimulation rodent mod- after trauma, stroke or in multiple sclero- el of schizophrenia in which polyinosinic- sis, the resting microglial cell transforms into an activated form characterized by an into pregnant rats to mimic an anti-viral ameboid morphology. Activated microglia polycytidilic acid (Poly I:C) was injected

phagocytose, and release a variety of factors factorsimmune correlating response. Webest identified with decreases microglia in likecan proliferate,cytokines, chemokines, migrate to the nitric site oxide of injury, and net-neurogenesisIL-1β and TNF-α increaseand impairment constituting in pre- the

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pulse inhibition of a startle response in the that underwent temporal lobe surgery. In Poly I:C model. Treatment with the antibi- the mouse model we also distinguish be- otic minocycline (3mg/kg/day) normalized tween blood-derived monocytes and mi- microglial cytokine production in the hip- croglia invaded from the brain parenchyma. pocampus and rescued neurogenesis and behavior. We could show that enhanced populations will indicate new pathways of microgliaGenetic profiling glioma ofinteraction. the different We microglial also de- the hippocampus was accompanied by a termine how bulk glioma cells and glioma decreasemicroglial in TNF-α the andpro-proliferative IL-1β production TNFR2 in stem cells differentially affect the microglial receptor expression on neuronal progeni- phenotype. We will try to identify poten- tor cells, which could be attenuated by mi- tial therapeutic targets that interfere with pathways of microglia glioma interactions with the ultimate goal to convert microglia nocycline. These findings strongly support from a pro- to an anti-tumorigenic pheno- therapythe idea into schizophrenic use anti-inflammatory patients. drugs to type. (Funded by DFG and NIH) target microglia activation as an adjunctive

Do microglial cells influence Selected Publications glioma cells? Krabbe G., Matyash V., Pannasch U., Mamer L., Boddeke HW., Kettenmann H. (2012) Feng Hu, Frank Szulzewsky, Felipe Sassi, Susanne A. Wolf (In collaboration with Mi- attenuates phagocytic activity. Brain Behav Immun. 26:419-428. Activation of serotonin receptors promotes microglial injury-induced motility but chael Synowitz, Neurosurgery, Charité, and Stock K*, Kumar J*, Synowitz* M, Petrosino S, Imperatore R, Smith ESJ, Wend P, Dolores Hambardzumyan, Cleveland Clinic, Purfürst B, Nuber UA, Gurok U, Matyash V, Wälzlein JH, Chirasani SR, Dittmar G, Ohio, USA) Cravatt BF, Momma S, Lewin GR, Ligresti A, De Petrocellis L, Cristino L, Di Marzo V, Kettenmann H*, Glass R* (2012) Neural precursor cells induce cell-death of high- grade astrocytomas via stimulation of TRPV1. Nat. Med., 18:1232 - 1239. * These authors contributed equally tumors and patients have a poor prognosis Tress O., Maglione M., May D., Pivneva T., Richter N., Seyfarth .J, Binder S., Zlomuzica A., sinceGliomas there comprise is essentially the majority no concept of cerebral for suc- - cessful treatment. Gliomas include astrocy- tional communication is essential for maintenance of myelin in the central nervous tomas, oligodendrogliomas, and the most Seifertsystem. G., J. Neurosci.,Theis M., Dere 32:7499-7518, E., Kettenmann * These H*, authorsand Willecke contributed K* (2012) equally Panglial gap junc malignant (and untreatable) brain tumor, Krabbe*, G., Halle*, A., Matyash, V., Rinnenthal, J. L., Eom, G. D., Bernhardt, U., Miller, K. the glioblastoma multiforme. A large frac- R., Prokop, S. Kettenmann#, H., Heppner# F. L. (2013) Functional impairment of microglia coincides with beta-amyloid deposition in mice with Alzheimer-like pathology. Plos tion of the cells within a glioma are brain One, 8:e60921. doi: 10.1371in press. #,* equal contribution macrophages/microglial cells which can amount up to 30%. We have found that Kettenmann H., Kirchhoff F. and Verkhratsky A. (2013) Microglia: New roles for the synaptic stripper. Neuron 77: 10 – 18. microglial cells strongly promote glioma growth and invasion. There is an interesting interplay between microglial and glioma cells. Glioma cells release the metalloprotease MMP2 which is important for degradation of extracellular matrix and promotes Group Leader invasion. This metalloprotease is, however, Prof. Dr. Helmut Kettenmann Graduate and Undergraduate released in an inactive, larger form and it Anne Hagen needs to be cleaved to acquire its activity. Scientists Philipp Jordan This cleavage is accomplished by the ecto- enzyme MT1-MMP. A factor released by the Dr. Masataka Ifuku Technical Assistants glioma cells activates TLR2 toll-like recep- Dr. MarinaAnais Djodari-Irani Matyash Irene Haupt tors in the surrounding microglial cells and Dr. Vitali Matyash Regina Piske triggers the expression of MT1-MMP. Thus, Dr. Christiane Nolte Nadine Scharek glioma cells exploit microglial cells to pro- Dr. Susanne Wolf Michaela Seeger-Zografakis mote their invasion. Thus interfering with TLR receptors or their intracellular path- PhD Secretariat ways might reduce the rapid expansion of Petya Georgieva Birgit Jarchow glioma cells and microglia have become a Feng Hu new target for glioma research Laura Maria Korvers Other Daniele Mattei We have established a procedure to purify Maria Pannell Meino Alexandra Gibson and culture human and mouse glioma-asso- Nadine Richter VeraTatjana Heinemann Butzek ciated and non-associated microglial cells. Frank Szulzewsky Judith Hoherz Corresponding control (non-glioma–asso- Felipe de Almeida Sassi Stefanie Korthals ciated microglial cells) human cells are ob- Stefan Wendt Victoria Rothe tained from patients with epileptic seizures

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Erich Wanker

Proteomics and Molecular Mechanisms of Neurodegenerative Diseases

1 The UBX protein ASPL rapidly Thus, our investigations provide biochemi- converts functional p97 hexamers cal and structural evidence that ASPL func- into non-functional heterotetramers controlling the cellular activity of p97/VCP. tions as a highly specific hexamer segregase, P97/VCP is a multifunctional ATPase that interacts with various adaptor proteins and 2 Identification of small molecule - modulators of light chain aggre- tio-temporal manner. However, the number, gates in AL amyloidosis sizeinfluences and biological their cellular properties function of p97/VCPin a spa containing protein complexes in mammali- In the context of protein misfolding disor- an cells are currently unknown. Recently, we ders, the study of a disease called amyloid - light chain amyloidosis (AL amyloidosis), ed proteins by automated Y2H interaction has only recently entered our lab. In AL screening;have identified one often these novel - the p97/VCP UBX containing associat amyloidosis the overproduction of immu- protein ASPL - was characterized in detail. noglobulin light chains in B cells leads to We found that ASPL directly associates with the formation of amyloid deposits, most VCP hexamers and rapidly converts these commonly in the heart and kidney, causing structures into highly stable VCP:ASPL dysfunction and failure. As the initial symp- heterotetramers. The crystal structure of toms are subtle, early disease diagnosis is p97/VCP bound to a C-terminal fragment of ASPL revealed that a unique helical lar- from AL amyloidosis have a short life ex- iat structure extending the UBX domain is pectancydifficult. after Consequently, diagnosis. patientsTo date, sufferingthe most crucial for the disruption of inter-protomer common therapy is to destroy the overpro- interactions and the disassembly of p97/ liferating monoclonal plasma cells by che- VCP hexamers. Site-directed mutagenesis motherapy. However, there are currently no of residues facilitating the VCP:ASPL inter- therapies that have a curative effect. There- action abrogated the disassociation of hex- fore, new therapeutic strategies for this se- amers. Overexpression of wild-type ASPL in vere disease are of high interest. The focus mammalian cells induced the activation of effector caspases, which was not observed molecule compounds, which can either ac- with a mutant ASPL variant that does not tivelyof our remove project theis the toxic identification amyloid deposits of small or disassemble p97/VCP hexamers in vitro. promote their degradation.

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Figure 1. Induced pluripotent stem (iPS) cells are differentiated in neural precursor cells (NPCs). (A) iPS cells derived from patients affected by Leigh syndrome (LS) and Huntington’s disease (HD) express pluripotency associated markers including TRA1-60. (B) Multipotent NPCs, positive for NPC markers such as SOX2 and NESTIN, are obtained from LS-iPS cells and HD-iPS cells. (C) Patient and control NPCs are coaxed into post-mitotic neurons expressing TUJ1 and other specific markers of basal ganglia.

3 Induced pluripotent stem (iPS) Huntington’s disease mechanisms and po- cell-based modelling of neurode- tentially identify novel therapeutic strate- generative disorders gies for these debilitating disorders.

The technology of induced pluripotent stem (iPS) cells may become a novel paradigm in 4 The protein interactome of the drug discovery for brain diseases by allow- chemical synapse ing live patient neurons to be employed in phenotype-based drug screening. We are Central to all neuronal functions are the exploring this approach by generating iPS trillions of chemical synapses within the cells from two genetic disorders affect- human brain that transmit chemical sig- ing the basal ganglia: Leigh syndrome and nals. Signal transmission is performed and Huntington’s disease. iPS cells are estab- regulated by complex protein networks in lished from patients using a footprint-free the pre- and postsynaptic termini. In the episomal plasmid-based reprogramming past, efforts in synaptic proteomics have protocol. Patient iPS cells are then differ- been focused on identifying the constitu- entiated into neural precursor cells (NPCs), ents of the synaptic sub-compartments. which are highly proliferative and retain Over 2000 gene products have been identi- their multipotent nature for several passages. Since current methods for NPC deri- previously been studied on a large scale. vation are complex and time-consuming, Tofied, gain but theirmore binary insight interactions into multi-protein have not we have developed a small molecule-based complexes formed at the synapse we have protocol that converts iPS cells into NPCs in adopted a high-throughput screening strat- a rapid and adherent fashion (Bukowiecki egy; approximately 1000 proteins are being et al, in preparation). Leigh syndrome-NPCs tested for pair wise interaction using the and Huntington’s disease-NPCs are cur- yeast-2-hybrid technique. rently being differentiated into basal ganglia neurons, whose identity and functional Following Y2H screening, selection of inter- properties are under evaluation (Fig. 1). Fi- acting protein pairs for validation will be nally, by combining standard functional and based upon a scoring system which takes biochemical assays with global -omics-driv- into account six factors: 1) known interac- en analyses on patient-derived neurons, we tions between orthologues, 2) the presence wish to unravel the Leigh syndrome and of domains known to interact in each of the

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proteins, 3) co-expression, 4) co-localisa- 6 Interaction network filtering tion, 5) participation in the same biological identifies CRMP-1 as a suppressor process and 6) distance in an interaction of mutant huntingtin misfolding network made up of all known interactions. and neurotoxicity - dated using the LUMIER co-immunoprecip- Assemblies of huntingtin fragments with itationHigh confidence method. interactions will be vali expanded polyglutamine tracts are a pathological hallmark of Huntington’s disease. The protein interaction network generated The molecular mechanisms of how these by this study will be a useful resource for structures cause neuronal dysfunction and studies on synapse dysfunction, which is toxicity are poorly understood. We utilized central to the etiology and progression of several neurological disorders including brain regions in health and disease for sys- neurodegeneration, autism and schizo- gene expression information from specific- phrenia. action data set. Our step-by-step computa- tionaltematic strategy filtering facilitated of a protein-protein the generation inter of a small, focused interaction network, which 5 Protein-protein interaction stud- directly or indirectly connects 13 potential- ies using a split-ubiquitin yeast- - 3-hybrid assay teins with the disease protein huntingtin. Thisly dysregulated, network enabled brain-region-specific the discovery of pro the - - velopment and biochemical investigation. tein CRMP-1, which targets aggregation- WeThis are project validating encompasses a new bothstrategy method for de- prone,heat shock N-terminal inducible huntingtin neuron-specific fragments pro tection of protein-protein interactions: the and suppresses their spontaneous self-as- split-ubiquitin yeast-3-hybrid assay. This sembly into proteotoxic structures in vari- assay involves co-expression of a consti- ous disease models. We propose that our tutively active kinase with two potentially interacting proteins within a yeast cell. The is a simple but highly valuable strategy for effect of the kinase on the protein interac- predictingcomputational potentially network disease-relevant filtering procedure pro- tion event is monitored through activation - toxicity of polyglutamine disease proteins. teins that influence misfolding and neuro Ourof the study reporter focuses genes on HIS3 phosphorylation and β-GAL. of Munc-18 (STXBP1), a synaptic protein that 7 Interactome mapping identifies plays a critical role in synaptic vesicle dock- the oncoprotein BMI1 as a stimu- ing at the pre-synaptic membrane prior lator of autophagosome formation to neurotransmitter release. It has been shown that phosphorylation of Munc-18 by Autophagy is an evolutionarily conserved catabolic process involved in the degrada- release the SNARE protein, syntaxin-1, al- tion and recycling of proteins and damaged lowingprotein the kinase SNARE C alpha protein (PKCα) complex causes to form. it to organelles by the lysosome. Currently, the The SNARE complex then mediates both molecular mechanisms that control au- vesicle docking and neurotransmitter re- tophagy induction are poorly understood. lease. To further understand the sequence We generated a binary yeast two hybrid of events that regulate this process we (Y2H) interaction network, connecting aim to identify proteins that interact with 1177 human proteins via 2407 high-quality interactions to the autophagy machinery.

Munc-18 in a manner regulated by PKCα.

162 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

This network facilitated the discovery of the nuclear oncoprotein BMI1 that inter- Selected Publications acts with ATG12, a critical mediator of au- Petrakis, S., Rasko, T., Russ, J., Friedrich, R.P., Stroedicke, M., Riechers, S.P., Muehlen- tophagosome formation. Strikingly, BMI1, berg, K., Moeller, A., Reinhardt, A., Vinayagam, A., Schaefer, M.H., Boutros, M., Tricoire, which shuttles from the nucleus to the cytoplasm under stress conditions and tar- modify misfolding and proteotoxicity of pathogenic ataxin-1. PLoS Genetics 8 (8): He1002897.., Andrade-Navarro, M.A., Wanker, E.E. (2012). Identification of human proteins that gets ATG12 through its RING domain, is a potent stimulator of autophagosome bio- Vinayagam, A., Stelzl, U., Foulle, R., Plassmann, S., Zenkner, M., Timm, J., Assmus, H.E., Andrade-Navarro, M.A., Wanker, E.E. (2011). A directed protein interaction network genesis in various cell model systems. Cy- for investigating intracellular signal transduction. Sci Signal. 4 (189): rs8. toplasic BMI1 protein levels are increased in mammalian cells that harbor misfolded, Bieschke, J., Herbst, M., Wiglenda, T., Friedrich, R.P., Boeddrich, A., Schiele, F., Kleckers, D., Lopez del Amo, J.M., Grüning, B.A., Wang, Q., Schmidt, M.R., Lurz, R., Anwyl, R., proteotoxic protein aggregates of hunting- Schnoegl, S., Fändrich, M., Frank, R.F., Reif, B., Günther, S., Walsh, D.M., Wanker, E.E. tin, a protein central to pathogenesis of the - neurodegenerative disorder Huntington’s (2011). Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amy disease (HD). Expression and subcellular loidBieschk fibrils.e, J., Nat Russ, Chem J., Friedrich, Biol. 8(1):93-101. R.P., Ehrnhoefer, D.E., Wobst, H., Neugebauer, K., Wanker, localization of BMI1 are altered in brains of reduces cellular toxicity. Proc Natl Acad Sci U S A. 107(17):7710-5. HD patients. We propose a model in which E.E. (2010). EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and nuclear proteins such as BMI1 directly reg- Stelzl, U., Worm, U., Lalowski, M., Haenig, C., Brembeck, F.H., Goehler, H., Stroedicke, M., ulate autophagy induction in mammalian Zenkner, M., Schoenherr, A., Koeppen, S., Timm, J., Mintzlaff, S., Abraham, C., Bock, N., Kietzmann, S., Goedde, A., Toksoez, E., Droege, A., Krobitsch, S., Korn, B., Birchmeier, cells and stimulate the degradation of mis- W., Lehrach, H., Wanker, E.E. (2005). A human protein-protein interaction network: a folded, proteotoxic proteins. These results resource for annotating the proteome. Cell 122 (6): 957-968 could be highly relevant to therapy development, both for cancer and neurodegenerative diseases.

Group Leader Prof. Dr. Erich Wanker

Scientists Engineers Dr. Annett Böddrich Lydia Brusendorf Dr. Alexander Buntru Gerlinde Grelle Christian Hänig Ronny Kalis Dr. Angeli Kishor Möller Kirstin Rau Dr. Alessandro Prigione Martina Zenkner Dr. Thomas Wiglenda Technical Assistants PhD students Daniela Kleckers Kathrin Andrich Susanne Köppen Anup Arumughan Alexandra Redel Raul Bukowiecki Nancy Schugardt Lisa Maria Diez Anne Hahmann (trainee) Franziska Hesse Christin Hesse (trainee) Sha Jin Franziska Wiedemann (trainee) Konrad Klockmeier Young-In Ko Secretariat Anne Steinhof Erika Pisch Philipp Trepte Staff Master students Sigrid Schnögl

(project manager) Ulrike Hübner Other CarmenAnja Briese Lorenz Dr. Sarah Stricker (guest, clinical Eileen Schormann training program ECRC)

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DISEASES OF THE NERVOUS SYSTEM Photo: David Ausserhofer/MDC Photo:

Annette Hammes

Molecular Pathways in Cortical Development

Receptor-mediated endocytosis provides the main mechanism whereby cells take up of SHH to recycling endosomes (Christ et al., trols internalization and cellular trafficking- metabolites and signaling molecules from the ure of the ventral neuroepithelium to respond extracellular space. One class of endocytic 2012).to the morphogen LRP2 deficiency SHH inand mice consequently leads to fail to receptors, termed low-density lipoprotein receptor-related proteins (LRPs), is particu- structures. Our data substantiate the emerg- impropering concept specification that auxiliary of receptors forebrain are midline criti- larly relevant for embryonic brain develop- cal modulators of morphogen delivery and ment. We investigate the role of LRPs and signal reception in target tissues, and identi- their interaction with morphogens and signaling molecules to regulate developmental in the ventral forebrain (Figure 2). fied an important role for LRP2 in SHH action events during early cortical development. In particular we are interested in how endocytic Endocytic receptor mediated receptor dysfunction leads to developmental control of folate uptake during neurulation disturbances of the CNS. Nora Mecklenburg, Esther Kur

Having established a role for LRP2 in SHH LRP2 is an auxiliary SHH receptor signaling in the ventral midline of the rostral required for cortical development neural tube, the function of the receptor in lat- Annabel Christ, Anna Christa in collaboration eral and dorsal neural tube domains, however, with Prof. Thomas Willnow remained unclear. Cranial neural tube defects

LRP2, the largest member of the LRP fam- the etiology of HPE, hinted at an additional ily, is a multifunctional endocytic receptor ex- role(NTDs) for inLRP2 LRP2 in deficientreceptor mice,mediated unrelated uptake to pressed on the apical surface of the entire neuroepithelium during neurulation (Figure 1). Loss of LRP2 activity in the developing CNS in embryosof a specific show ligand impaired in the closure developing of the rostral neural gene targeted mice causes holoprosencephaly neuraltube. Thirty-eight tube. Similar percent neural oftube LRP2 defects deficient have (HPE), a failure of the forebrain hemispheres previously been attributed to impaired folate

to separate along the midline. Patients with (vitamin B9) metabolism in mice and humans. autosomal recessive LRP2 gene defects suffer Neural tube defects are a group of congenital from Donnai-Barrow syndrome, a disorder malformations that occur when the neural associated with forebrain anomalies. In col- tube fails to close during embryonic develop- laboration with the group of Prof. Thomas ment. In human pregnancies, NTDs are the second most frequent malformations after mechanism underlying the HPE phenotype by congenital heart defects. identifyingWillnow we LRP2 previously as a novel clarified component the molecular of the sonic hedgehog (SHH) signaling machinery in We therefore asked whether LRP2 might be the ventral forebrain neuroepithelium. LRP2 required for delivery of folate to neuroepithe- acts as a SHH binding protein and co-receptor lial cells during neurulation. Uptake assays in to patched1 that sequesters SHH at the api- whole embryo cultures showed that LRP2 de- cal surface of the neuroepithelium and con- -

ficient neuroepithelial cells are unable to me 164 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

Figure 1: Expression of LRP2 in the developing mouse forebrain. The scheme depicts the structure of Figure 2: Model of LRP2 function in SHH signaling. SHH LRP2, a single-spanning transmembrane endocytic re- produced in the prechordal plate moves to the neuroepithe- ceptor of the LDL receptor gene family. Immunohisto- lium to be sequestered apically by a co-receptor complex of logical detection of LRP2 (green) on coronal sections LRP2 and patched 1 (Ptch1). SHH binding induces uptake of the rostral neural tube of the mouse embryo shows of receptor-ligand complexes and triggers Ptch1-dependent that the receptor is expressed uniformly on the apical activation of smoothened (Smo) resulting in target gene neuroepithelial surface. induction through GLI.

diate uptake of folate bound to folate receptor the folate receptor, to convert extracellular 1 (FOLR1). stimuli into intracellular signals. Shedding light on the mechanisms how LRP2 and other So far a putative co-receptor for FOLR1 had members of the LDL receptor family can function in a dual receptor mode to confer ligand endocytosis of soluble FOLR1 and the mem- notbrane yet anchored been identified. GPI-FOLR1, Our datarequires suggest interac that- the developing neural tube will contribute to tion with the endocytic receptor LRP2. This thespecificity emerging in a conceptcontext thatdependent LRPs aremanner critical in “dual receptor complex” hypothesis for the modulators of signal reception. uptake of folate is analogous to the uptake of

renal and intestinal vitamin B12 bound to intrinsic factor (IF) via cubilin, a peripherally attached glycoprotein. Internalization of the Selected Publications cubilin-ligand complex strongly relies on the Kur, E., Mecklenburg, N., Cabrera, R.M., Willnow T.E., and Annette Hammes. (2014) interaction with LRP2. LRP2 mediates folate uptake in the developing neural tube J Cell Science March 17

Christ, A., Christa, A., Kur, E., Lioubinski, O., Bachmann, S., Willnow, T.E., and Hammes, Our results demonstrate that LRP2 plays an A. (2012). LRP2 is an auxiliary SHH receptor required to condition the forebrain essential role for the uptake of folate in the de- ventral midline for inductive signals. Dev Cell 22: 268-278 veloping neural tube, a crucial step for proper Willnow, T.E., Christ, A., and A. Hammes (2012). Endocytic receptors control morphogen signalling. Development 139(23): 4311-4319. to impaired cellular uptake of folate during neurulationneural tube and closure. reduced LRP2 tissue deficiency folate concen leads- - shikawa, K., Bachmann, S., Christensen, E.I., Götz, M., Kempermann, G., Peterson, A.S., trations, which consequently contributes to GajerWillnoa,w, C.R., T.E. Emich, and A. H., Hammes. Lioubinski, (2010). O., Christ, Ependymal A., Beckervordersandforth-Bonk, cells regulate BMP signaling R., in Yothe NTDs in Lrp2-/- embryos. adult neurogenic niche through LRP2. J. Cell Science 123: 1922-30.

Hammes, A., Andreassen, T. K., Spoelgen, R., Raila, J., Huebner, N., Schulz, H., Metzger, J., - Perspectives ment of the reproductive organs in mice lacking megalin, an endocytic receptor for stSchweigert,eroid hormones. F. J., Luppa, Cell 122: P. B., 751-62. Nykjaerr, A. and T. E. Willnow. (2005) Impaired develop Future work aims at substantiating the relevance of LRP2 as part of the SHH receptor- some in developmental pathways of medical relevance including forebrain development Group Leader Graduate Students and HPE, retina formation and retinopathies, Dr. Annette Hammes Fabian Alexander Paul as well as adult neurogenesis. Scientists Technical assistance Further we expect to elucidate the complex Dr. Nora Mecklenburg Kristin Kampf interaction of LRP2 with other receptors, like

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DISEASES OF THE NERVOUS SYSTEM Photo: David Ausserhofer/MDC Photo:

Kathryn Poole

Cellular Sensing of the Physical Environment

The process by which cells can convert role of mechanically-gated channels in cell mi- mechanical and physical stimuli into a gration and chondrocyte mechanotransduction. biochemical response is known as mecha-

notranduction, a broad term that covers Mechanotransduction in sensory neurons is a number of processes involved in cellular sensing of the physical environment. These - influencedponents of bybasement the laminin membranes, network toan whichECM- include the fast channel-mediated trans- thebased cells compartment are bound. Laminins with w arehich major neurites com duction of mechanical stimuli into electrical from DRG cells are associated in vivo. Using responses (mechanoelectrical transduction) micro-contact printing to pattern surfaces to slower transduction events mediated by to polarize cells in terms of both neurite out- intracellular force-sensing proteins asso- growthwith specific and mechanotransdution. laminin mixtures we We were found able ciated with the cytoskeleton or integrins that by spiking an EHS-laminin substrate with at the cell surface. Mechanotransductive laminin-332 the EHS-laminin became inhibitory for both growth-cone bifurcation and mechanisms are not only fundamental to mechanotransduction (Fig 1). Not only did our senses of touch and hearing but also to these results elucidate two novel functions of cellular processes such as differentiation, laminin-332 they also demonstrate that there migration and tissue formation and the dis- is a contact-dependant effect on mechanotransduction that is a local, not a global phe- ruption of mechanotransduction pathways nomenon. This suggests that mechanically- can have a negative impact on health, impli- gated ion channels are primarily activated at cated in osteoarthritis, cancer development the cell-substrate interface. An atomic force microscopy analysis of the effect of adding and metastasis and mechanical dysfunction laminin-332 to EHS-laminin demonstrated of the heart, to name but a few. that increasing amounts of laminin-332 led to a decrease in stability of the EHS-laminin network by modulating its topography and rigid- ity1. This suggests that the physical features of We are interested in how individual cells de- the substrate are critical in supporting robust tect and respond to applied forces and to the mechanotransduction. We are currently test- physical features of their immediate environ- ing this hypothesis using a panel of laminin ment, namely the extracellular matrix (ECM). substrates that vary in either their composi- In the last years we have been using a number tion or their structure. of biophysical techniques to be able to better characterize the physical features of the ECM that cells can detect and how these signals are cell-matrix interactions on mechanotransduc- transduced across the membrane. Initially this Ourtion highlighted investigation a need of the for influence a new approach of local work was carried out in mammalian somato- to quantify mechanoelectrical transduction sensory neurons, i.e. those cells that mediate where the physical stimuli are applied at the our sense of touch. More recently we have relevant cellular compartment. In order to been extending this work to investigate the achieve this we have developed a highly sen-

166 MDC Research Report 2014 DISEASES OF THE NERVOUS SYSTEM

Figure 1. Laminin-directed polarization of DRG sensory neurons. Distinct mixes of laminin subtypes can be printed on a surface using microcontact printing (A- red, EHS laminin; green, EHS laminin: laminin 332, 15:1) AFM can be used to image structural properties of these substrates (B, EHS laminin; C, EHS laminin: laminin 332, 15:1). Neurite outgrowth is polarized on these substrates (D). Neurite segments from a single cell, over each distinct substrate, can be probed (E), resulting in either presence (F, red trace, EHS laminin) or absence of a response (F, green trace EHS laminin: laminin 332, 15:1)

Figure 2: Quantitative measurement of mechanoelectrical transduction. (A) A pillar array is cast from the elastomer PDMS (insert), coated with ECM molecules and cells, shown here are sensory neurons, can be cultured on top of the pillar array. (B) Individual pili underneath the cell can then be deflected using a nano-stimulator and mechanically-gated channel activity is monitored using whole-cell patch-clamp. (C) This method is particularly sensitive and quantitative, as the precise movement of the stimulating pili can be calculated and correlated to the resulting currents. (D) Stimulus-response curves of mechanotransduction in subsets of sensory neurons. Note that TypeII mechanoreceptors are extremely sensitive to mechanical stimuli.

sitive method where precise, molecular-scale mechanically-gated ion channels in chondrocyte mechanotr ansduction and in proliferative cell-matrix interface2 cell migration. stimuliis cast in are the applied elastomer to defined PDMS regionsand the of tops the ______of the pili are printed. Briefly,with ECM a pillar molecules array 1 Chiang, L-Y*., Poole, K*., et al (2011) Nat. Neurosci. upon which the cells of interest (in this case 14, 993-1000. 2 Poole, K. et al (2014) Nat. Comm. In press. somatosensory neurons) are cultured (Fig 2). Stimulus-response curves are generated by Selected Publications neurite or cell segment and measuring the cel- K. Poole§, R. Herget, L. Lapatsina, HD. Ngo, G.R. Lewin§. (2014) Nanoscale tuning of lulardeflecting response an individualusing whole-cell pilus underneath patch-clamp. a mechanosensitive ion channels by stomatin-domain proteins. Nat Comm (2014) Using this system, we have been able to pre- 5: doi:10.1038/ncomms4520 cisely measure the amplitude of mechano- J. Brand, E. St. J. Smith, D. Schwefel, L. Lapatsina, K. Poole, A. Kozlenkov, J. Behlke, G. R. Lewin and O. Daumke (2012) A stomatin dimer modulates the activity of acid-sensing electrical transduction currents in response ion channels. EMBO J. 31: 3635 - 3646 S.G. Lechner* ,S. Markworth*, K. Poole, E. St.J. Smith, L. Lapatsina, S. Frahm, M. Suzuki, I. Ibañez-Tallon, F.C. Luft, J. Jordan and G.R. Lewin (2011) The molecular and cellular tonlyo defined 13 nm, stimuli. or half theWe widthhave foundof a microtubule. that some identity of peripheral osmoreceptors. Neuron 69: 332-344 Ascells such, can these transduce cells can substrate detect molecular-scale deflections of L.Y. Chiang*, K. Poole*, B.E. Oliveira, N. Duarte, Y.A. Bernal-Sierra, L. Bruckner-Tuder- man, M. Koch, J. Hu and G. R. Lewin (2011) Laminin-332 coordinates mechanotrans- stimuli. We found that this sensitivity was de- duction and growth cone bifurcation in sensory neurons. Nat. Neurosci. 14: 993–1000 pendent on the presence of a membrane scaffolding protein (STOML3), which functions to tune the sensitivity of ion channels that are directly gated by mechanical stimuli2. These Group Leader data indicate that mechanically-gated channel Dr. Kathryn Poole thresholds can be modulated by a scaffolding (Cécile Vogt Programme) protein, suggesting a mechanism by which dif- PhD Students ferent cell types can tune their sensitivity to Post-doc Martha Rocio Servin Vences various mechanical stimuli. We are currently Jeffrey Stear (with GR Lewin) Regina Herget (with GR Lewin) using this technique to characterize the role of

MDC Research Report 2014 167

168 MDC Research Report 2014 Berlin Institute for Medical Systems Biology

Coordinator: Nikolaus Rajewsky

MDC Research Report 2014 169

Berlin Institute for Medical Systems Biology

Nikolaus Rajewsky

revalent and devastating diseases such ing in spring 2014. Additional Technology as cancer, cardiovascular disease, meta- Platforms will be established in the future. Pbolic diseases and neurodegenerative disorders are usually multifactorial diseases Scientific group leaders in BIMSB involving many genes. To extend our understanding of the interplay of functions involv- In addition to Junior group leaders Markus ing different genes in health and disease, an Landthaler and Alexander Löwer, BIMSB important goal of systems biology is to ana- was fortunate to recruit several outstanding lyze gene regulatory networks. To this end new Junior and Senior group leaders since we collect genome-wide quantitative data to 2012. They investigate important aspects of integrate them into predictive models. This gene regulatory mechanisms (for example challenge is at the heart of Medical Systems epigenetics) and model systems (e.g. Dro- Biology and is by nature highly interdisci- sophila) which were not represented on cam- plinary, combining molecular and cellular pus before: Marina Chekulaeva ‘Non coding biology, biochemistry, mathematics, phys- RNA’s and mechanims of cytoplasmic regula- ics and engineering. The past ten years have tion’, Baris Tursun, ‘Gene regulation and cell particularly highlighted that cells employ fate decision in C. elegans’, Uwe Ohler ‘Com- multiple sophisticated levels of gene regula- putational regulatory genomics’, Ana Pombo tion, which include organization of chromo- ‘Epigenetic regulation and chromatin archi- somes in the nucleus, epigenetic gene regula- tecture’, Robert Zinzen ‘Systems biology of tion, transcription, post-transcriptional gene neural tissue differentiation’. Benedikt Ober- - mayer tions. Importantly, all of these levels interact BIMSB as a Max Delbrück Fellow. andregulation regulate and each post-translational other and are involved modifica in ‘Computation and Theory’ joined disease development. At the MDC, the grand Publications - lin Institute for Medical Systems Biology” BIMSB scientists have published outstand- (BIMSB)challenge is and to Scientificintegrate Missionthese levels of the of “Bergene regulatory control into comprehensive and scope and collaborative nature within BIMSB predictive models of how gene regulatory ising highlighted and highly-cited for example papers. by Baltz The et scientific al. Mol changes are linked to phenotypes. collaborated with the Dieterich, Selbach and Scientific Technology Platforms BonneauCell 2012. (NYU) In this labs, project, to answer the Landthaler a fundamen lab- tal question: Can we comprehensively identi- High throughput technologies are key for fy the RNA binding proteins (RBPs) which are many research aspects at BIMSB, we have

Technology Platforms for ‘omics’-technolo- RNAactive in in a a human human cell cell? line. The Numerous answer is thatof these 800 gies.therefore The newly established recruited essential PIs have Scientificinstalled RBPsproteins came can as besurprises found specifically(see also Hentze, bound Cell to and integrated these technologies in sci- 2012). The Landthaler lab also developed a

other labs at the BIMSB/MDC. Wei Chen of RNAs in a human cell are actually bound directsentific projects,the Genomics often platform in collaboration which offers with bynew these method RBPs. which This quantifiesmethod allows which now parts to diverse applications, including genome and record transcriptome-wide “occupancy pro- transcriptome sequencing and assembly, and ChIP-Seq. Stefan Kempa develops cutting paper not only provides a much needed cata- edge methods to quantify the metabolome loguefiles” ofof the RNA:protein repertoire interactions. of RBPs, it also Thus, opens the as well as the proteome, with a special focus the door to study RNA:protein occupancy on aspects of the metabolism of tumors and stem cells. The Bioinformatics Platform was or stimuli. These approaches are expected to headed by Christoph Dieterich until August pinpointprofiles inparticularly response important to different RNA:protein conditions 2013 and will be led by Altuna Akalin start- interactions and to allow modeling of cross-

170 MDC Research Report 2014 BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY

(copyright MDC) (copyright Scheme of gene-regulatory processes .

Additional Activities

BIMSB has established an Exchange program to train the next generation of System Biologists. The program allows PhD students - tween BIMSB labs and partners at the Center forto work Genomics on collaborative and Systems research Biology project at New be York University. In addition to seminar series, guest seminars and lecture series, BIMSB talk between regulatory components. The organizes the highly successful annual Ber- lin Summer Meeting, the Berlin Summer the BIMSB-NYU exchange program and prof- School MRC-Clinical itedtwo firstfrom authors their experiences of this study at areNYU. members The paper of Sciences Centre (UK), and internal BIMSB retreats., jointIn 2012, retreats MDC/BIMSB with the PIs discussed MDC scientists collaborate as lead authors on in a two day retreat how to connect, develop, keyalso papers. exemplifies Further that examples often, several are Gruen BIMSB/ et al. and marry traditional Genetics and Systems - Biology. czak et al. Nature 2013 (Landthaler, Löwer, Cell Reports 2014 (Selbach, Rajewsky), Mem Grants, Awards & Peer reviews

2013leNoble, (Doerken, Rajewsky), Kempa, Oenal Schmitt),et al. EMBO Baltz J 2013 et In addition to the start-up funding of BIMSB, al.(Chen, Molecular Kempa, Cell Rajewsky), 2012 (Selbach, Doerr et al.Dieterich, Nature the labs have successfully competed for extra- - mural funding. Since 2012, BIMSB labs have sky, Landthaler, Dieterich). In total BIMSB acquired a total of 9,5 Mio € additional funds teamsLandthaler), published Anders 65 paperset al NAR in peer-reviewed 2012 (Rajew from the DFG, BMBF, EC, NIH, GIF, and others. - Some of the highlights are: the Gottfried Wil- pers had an impact factor (IF) higher than 5 helm Leibniz Prize Ger- andjournals 13 articles (2012-Feb. an IF 2014),higher ofthan which 10 (“High- 43 pa man Epigenome Consortium ‘DEEP’, and impact papers”). The 65 new papers have (N. Rajewsky), the - been cited 348 times (until Feb. 2014). tems biology and bioinformatics. In two peer- BMBF funding for national projects in sys Model of the new The new BIMSB building well as the overall concept were rated “out- BIMSB building in the standing”.reviews, scientific achievements of BIMSB as center of Berlin In 2013 an open architect competition took place, based on extensive preplanning. The contruction for the winning, highly innovative architectural plan is starting in 2014 (see Fig. 2). The new building houses up to 25 research groups, approximately half experimental and half computational. On 5100 qm

workspaces and communication areas, which arethe buildingall essential supports for high-techthe interdisciplinary labs, flexible BIMSB activities. BIMSB is a key element for the Integrative Research Institute for Life Sci- ences IRI, part of the successful “Exzellenzini- tiative” (funded by the DFG) of the Humboldt University and the Charité.

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BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY Photo: private Photo:

Marina Chekulaeva

Non-coding RNAs and Mechanisms of Cytoplasmic Gene Regulation

We are focusing on understanding the to translational repression, deadenylation molecular mechanisms that regulate mRNA reconciles literature data and provides a translation, localization and stability and newand foundation decay of target from which mRNAs. to explore This finding miR- role of non-coding RNAs in this process. Up NA function. What are the functions of the to 90% of human DNA is estimated to be repressive W-motifs outside of the GW182 transcribed into so called non-coding RNAs family, taking into account the pleiotropic role of the CCR4-NOT complex in RNA me- that are not translated into proteins. Many tabolism and its involvement in processes of them act as potent modifiers of gene ex- like cell growth, proliferation and tumori- pression. miRNAs are a class of such short non-coding RNAs. They regulate expression genegenesis? expression How is miRNAvia alternative function and regulated previ- of more than a half of eukaryotic genes, in time and space? Can miRNAs regulate thus, affecting multiple biological processes, are the roles of extracellular miRNAs in including cell proliferation, differentiation, intercellularously uninvestigated cross-talk mechanisms?and propagation What of apoptosis and senescence. Not surprisingly, combination of biochemical, computational miRNAs are involved in many human pa- approachesdiseases, such and as imaging cancer? to We address are using these a thologies, including cancer and neurological questions. disorders and hold great potential as drug targets, disease markers, as well as thera- What are the functions of the peutic agents. W-motifs/CCR4-NOT interaction platform in proteins outside of the miRNA repression complex?

One of our interests is to understand the W-motifs recruiting the CCR4-NOT complex mechanisms of miRNA function. In animals, represent a novel interaction platform em- miRNAs regulate gene expression post- ployed by miRNAs to regulate gene expres- transcriptionally: they act as guides, by sion. But W-motifs can be found not only in pairing with partially complementary sites GW182, but also in many other proteins. present in their target mRNAs, and thereby recruiting a complex of proteins that inhib- with W-motifs function as competitors of its mRNA function. The core components of GW182What are to their modulate functions? miRNA Do silencing these proteins in re- this complex are AGO and GW182 proteins. sponse to various stimuli or do they regulate We have shown that GW182 proteins con- gene expression in a miRNA-independent tain a novel and conserved type of protein way, by recruiting the CCR4-NOT complex motifs, termed W-motifs. These motifs act in additive manner to recruit the CCR4-NOT can regulate multiple steps of gene expres- deadenylation complex and thereby lead sion,to specific including targets? transcription, The fact that CCR4-NOTchromatin

172 MDC Research Report 2014 BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY

Scheme illustration the mechanisms of miRNA. GW182 proteins are recruited to mRNA via direct interaction with the miRNA–AGO complex. Through the W-motifs, GW182 recruits the CCR4–NOT complex that represses translation an leads to mRNA deadenylation

mRNA stability, argues that proteins with Selected Publications W-motifsmodification, have RNA the export, potential translation to regulate and Chekulaeva, M., Mathys, H., Zipprich, J., Attig, J., Colic, M., Parker, R., and Filipowicz, gene expression via multiple mechanisms. W. (2011). miRNA repression involves GW182-mediated recruitment of CCR4–NOT Using a combination of computational and through conserved W-containing motifs. Nature Structural and Molecular Biology biochemical approaches, we are studying 18(11): 1218-26. the roles of the W-motif/CCR4-NOT repres- Chekulaeva, M., Parker, R., and Filipowicz, W. (2010). The GW/WG repeats of Dro- sive module in differential gene expression. sophila GW182 function as effector motifs for miRNA-mediated repression. Nucleic Acids Research 38(19): 6673-83.

Chekulaeva, M. and Filipowicz, W. (2009). Mechanisms of miRNA-mediated post-tran- What are roles of miRNAs in mRNA scriptional regulation in animal cells. Current Opinion in Cell Biology 21(3): 452-60. localization and localized transla- Chekulaeva, M., Filipowicz, W., and Parker, R. (2009). Multiple independent domains of tion? dGW182 function in miRNA-mediated repression in Drosophila. RNA 15: 794-803. Chekulaeva, M., Hentze, M.W., and Ephrussi, A. (2006). Bruno acts as a dual repressor The role of miRNAs in translational control of oskar mRNA translation promoting mRNA oligomerization and formation of silenc- and mRNA decay has been well established. ing particles. Cell 124: 521-533. However, for a cell it is important not only how much of a given protein it produces, but also where in the cell this protein is produced. This is often achieved by localizing - pressing their translation prior to localiza- Start of the Group: January 2013 tion.mRNAs These to specific mechanisms sites ofunderly the cell establish and re- ment of body axis, cell migration, synaptic Group Leader Graduate and Undergraduate plasticity and other important processes. Dr. Marina Chekulaeva Cinthia Claudia Amaya Ramirez A number of human pathologies, including Karthika Annamalai neurological disorders such as the Fragile X PhD Michael Persicke syndrome, are associated with a failure to Marta Mauri - Alessandra Zappulo Technical Assistants lular compartments. To analyze the roles David van den Bruck Sarah Joana Herrmann oflocalize miRNAs certain in subcellular mRNAs to translation specific subcel and Larissa Ruhe localization of mRNAs, we are using spatial Secretariat transcriptomic, proteomic and bioinfor- lab) Ines Krock matic analyses. (joint with Nikolaus Rajewsky’s

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BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY Photo: David Ausserhofer/MDC Photo:

Wei Chen

Novel Sequencing Technology, Functional and Medical Genomics

“DNA makes RNA makes protein.” After addition to 239 known mouse pre-miRNAs, transcription, mRNAs undergo a series of miRGrep predicted 41 novel ones with high intertwining processes to be finally trans- miRNAs derived from most of these novel loci lated into functional proteins. The ‘post- showedconfidence. both Similar reduced as known abundance ones, the following mature transcriptional’ regulation provides cells an Dicer knockdown and the binding with Argo- extended option to fine-tune their proteomes. naute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis To meet the demands of complex organism sp.11 demonstrated that miRGrep could be development and the appropriate response widely used for miRNA discovery in metazo- to environmental stimuli, every step in these ans, especially in those without genome reference sequences. processes needs to be finely regulated and the deregulation could result in pathological Ultra-deep profiling of alternatively condition(s). To understand the underlying spliced Drosophila Dscam isoforms molecular mechanism, we have been de- by circularization assisted multi- segment sequencing (CAMSeq) veloping various genomic assays based on The Drosophila melanogaster gene Dscam novel sequencing technology to study post- (Down syndrome cell adhesion molecule) transcriptional regulation in the context of hu- can generate thousands of different ectodo- man diseases as well as during development mains via mutual exclusive splicing of three large exon clusters. The isoform diversity and evolution. plays a profound role in both neuronal wiring and pathogen recognition. However, the Global microRNA precursor profiling isoform expression pattern at the global level in mammalian cells remained unexplored. Here, we developed a MicroRNAs (miRNAs) constitute an impor- novel method, CAMSeq that allows for direct tant class of small regulatory RNAs that are derived from distinct hairpin precursors combinations from over hundreds of millions (pre-miRNAs). In contrast to mature miRNAs, ofquantification Dscam transcripts of the alternatively in one sequencing spliced exon run which have been characterized in numerous (Figure 1). With unprecedented sequencing genome-wide studies of different organisms, depth, we detected a total of 18496 isoforms, out of 19008 theoretically possible combina- is limited. Using massive parallel sequencing, tions. Importantly, we demonstrated that al- weresearch have performed on global global profiling characterization of pre-miRNAs of ternative splicing between different clusters both mouse mature and precursor miRNAs. is independent. Moreover, the isoforms were Our analysis revealed new aspects of miRNA/ expressed across a broad dynamic range, with - - cluding eight Ago2-cleaved pre-miRNAs, new - instancespre-miRNA of processing miRNA editing and modification,and exclusively in significantpreciated, suchbias in bias cell/tissue can dramatically and developmen reduce 5’ tailed mirtrons. Furthermore, based on thetal stage-specificability of neur onspatterns. to display Hitherto unique underap surface the sequences of both mature and precursor receptor codes. Therefore, the seemingly ex- miRNAs, we developed a miRNA discovery cessive diversity encoded in the Dscam locus pipeline, miRGrep, which does not rely on the might nevertheless be essential for a robust availability of genome reference sequences. In self and non-self discrimination in neurons.

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Figure 1. Outline of CAMSeq. In brief, first, using RT–PCR with the barcode-indexed primers targeting constitutive exon 3 and exon 10, Dscam mRNA was reverse-transcribed and amplified (barcodes are depicted in yellow and marked with ‘B’). After circularization of the 2 kb RT–PCR product and another round of PCR with the primers targeting constitutive exon 7 and exon 8, the amplification product of 1 kb in length was then sequenced. Here, using a modified sequencing procedure with four specific sequencing primers Figure 2. Mechanistic model for RBM10-mediated splicing targeting constitutive exon 3, regulation. RBM10 binding in the vicinity of splicing sites might exon 5, exon 8, and exon 10, repress the splicing of flanking introns and delay the splicing respectively (blue arrows), we obtained from every template DNA molecule choice, thereby facilitate the skipping of cassette exons flanked four sequencing reads derived from exons 4, 6, 9, and barcode, respectively. with relatively weaker splice sites.

Integrative analysis of alternative splicing regulated by RBM10 Selected Publications RBM10 encodes an RNA binding protein. Mu- Wang, Y., Gogol-Döring, A., Hu, H., Fröhler, S., Ma, Y., Jens, M., Maaskola, J., Murakawa, Y., Quedenau, C., Landthaler, M., Kalscheuer, V., Wieczorek, D., Wang, Y., Hu, Y*. and Chen, tations in RBM10 are known to cause multiple W*: Integrative analysis revealed the molecular mechanism underlying RBM10-medi- congenital anomaly syndrome in male hu- ated splicing regulation. EMBO Mol Med. 2013 Sep;5(9):1431-42 mans, the TARP syndrome. However, the mo- Sun W, You X, Gogol-Döring A, He H, Kise Y, Sohn M, Chen T, Klebes A, Schmucker D*, lecular function of RBM10 is unknown. Here circularization-assisted multi-segment sequencing. EMBO J. 2013 Jul 17;32(14):2029- we used PAR-CLIP to identify thousands of 38Chen W*: Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by - Li N, You X, Chen T, Mackowiak SD, Friedländer MR, Weigt M, Du H, Gogol-Döring A, cant RBM10–RNA interactions in the vicinity - bindingof splice sitessites. ofComputational RBM10 and observedanalyses of signifi bind- sors: insights into miRNA processing and novel miRNA discovery. Nucleic Acids Res. ing sites as well as loss-of-function and gain- 2013Chang Apr Z, Dieterich 1;41(6):3619-34 C, Hu Y, Chen W: Global profiling of miRNAs and the hairpin precur of-function experim ents provided evidence Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J*, Chen W*, Selbach for the function of RBM10 in regulating exon 19;473(7347):337-42 M* Global quantification of mammalian gene expression control Nature. 2011 May skipping and suggested an underlying mecha- Adamidi C, Wang Y, Gruen D, Mastrobuoni G, You X, Tolle D, Dodt M, Mackowiak nistic model (Figure 2), which could be subse- SD, Gogol-Doering A, Oenal P, Rybak A, Ross E, Alvarado AS, Kempa S*, Dieterich - quently validated by minigene experiments. tome by massive parallel sequencing and shotgun proteomics. Genome Res. 2011 Furthermore, we demonstrated the splicing Jul;21(7):1193-200C*, Rajewsky N*, Chen W*. De novo assembly and validation of planaria transcrip defects in a patient carrying an RBM10 muta- *shared corresponding author tion, which could be explained by disrupted function of RBM10 in splicing regulation. Overall, our study established RBM10 as an important regulator of alternative splicing, presented a mechanistic model for RBM10- mediated splicing regulation and provided a Group Leader Qingsong Gao molecular link to understanding a human con- Dr. Wei Chen Jingyi Hou genital disorder. Wei Sun Scientist Martina Weigt Systematic identification of genetic Dr. Sebastian Froehler Mei-Sheng Xiao factors underlying congenital disor- Dr. Andreas Gogol-Doering Xintian You ders (till 04/2013) To expedite the molecular elucidation of ge- Dr. Yuhui Hu Technical Assistants netic factors underlying rare diseases, we Dr. Yongbo Wang (till 09/2013) have established a high-throughput muta- Dr. Xi Wang Claudia Langnick tion screening approach by combining target MadlenMirjam FeldkampSohn region enrichment and next generation se- PhD Claudia Quedenau quencing. Together with the clinical partners Ana Babic from Charite, we have succeeded in the identi- Tao Chen Secretariat Hang Du Sabrina Deter in a variety of congenital disorders. fication of causative genetic defects implicated

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BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY Photo: David Ausserhofer/MDC Photo:

Stefan Kempa

Metabolic Regulation

The central carbon metabolism (CCM) is Decoding the blood metabolome the principal source of energy and building The blood summarizes all metabolic activi- blocks for all cellular processes. Although ties and cross talks between the individual the structure of this super-pathway is highly organs. Due to specialized functions of the conserved in higher organisms, the regu- organs, e.g. sink and source relationships, it lation is rather complex, and appears at patterns to the metabolic activity of organs. all levels of gene regulation - epigenetic, Themay liver be possible as a central to relate metabolic specific organ metabolic regu- (post-) transcriptional (post-) translational lates the supply of fuel sources in the ground and on an allosteric level. The structure of state and adapts the fuel supply during star- the regulatory and signaling network and the metabolome after sampling blood all ten the expression of alternative enzyme vari- minutesvation or during activity. a Inlong a case distance study run. we profiledThe re- ants determine the metabolic state and sults about the association of the known fuel flexibility. To gain insights in regulatory sources during that time course were visualized - we were able to follow a switch within properties at a mechanistic level we apply the metabolic program ‘online’. metabolomics and proteomics techniques allowing the quantitative and dynamic mea- Visualization of distribution of fuel sources during surement of the biochemical network in exercise in a human individual. Blood samples were collected and analyzed by GC-MS based metabolomics. high resolution. The circos-plot visualizes the three main energy sources The interest of our group is twofold; on the and the time profile during a long distance run. Samples one hand, we are interested in decoding were taken all 10 min. The different phases of the exer- the metabolic composition of the blood me- cise are fasted (short before exercise, R1-6 (round 1-6) PL post exercise. tabolome (in vivo) as it represents the overlay of the activity of all organs. On the other hand, we are studying metabolic regulatory processes at the cellular levels in molecular details (in vitro). The final goal is to merge these informations and to understand the consequences of a gene defect and its expression in metabolic signatures during the next years to come.

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Visualization of the metabolic changes within CCM after 15 min applic ation of two glycolytic inhibitors (3-bromo pyruvate, 2-deoxyglucose). The size of the circles encode the x-fold change of the concentration and the color visualizes the change of stable isotope incorporation resulting from a three 13 minutes pulse with C6 glucose.

Regulation of cellular metabolism Selected Publications

The intense crosstalk between regulatory Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. networks and metabolism emerges as a new Doerr, J.R. and Yu, Y. and Milanovic, M. and Beuster, G. and Zasada, C. and Daebritz, research focus. To analyze the dynamics of J.H.M. and Lisec, J. and Lenze, D. and Gerhardt, A. and Schleicher, K. and Kratzat, S. and Purfuerst, B. and Walenta, S. and Mueller-Klieser, W. and Graeler, M. and Hummel, M. cellular metabolism we have established and Keller, U. and Buck, A.K. and Doerken, B. and Willmitzer, L. and Reimann, M. and Kempa, S. and Lee, S. and Schmitt, C.A. Nature 501 : 421-425. 19 September 2013 techniques and developed pulse stable iso- Deregulated MYC expression induces dependence upon AMPK-related kinase 5. topeand modifiedresolved GC-MSmetabolomics based metabolomics(pSRIM). Us- Liu, L. and Ulbrich, J. and Mueller, J. and Wuestefeld, T. and Aeberhard, L. and Kress, T.R. and Muthalagu, N. and Rycak, L. and Rudalska, R. and Moll, R. and Kempa, S*. and ing this technique, we studied reprogram- Zender, L. and Eilers, M. and Murphy, D.J. Nature 483 (7391): 608-612. 29 March 2012 ming of metabolism and synthetic lethal (corr. for metabolomics/proteomics) interactions in cancer cells (Liu et al. 2012, Proteome dynamics and early salt stress response of the photosynthetic organism Dorr et al. 2013). Chlamydomonas reinhardtii. Mastrobuoni, G. and Irgang, S. and Pietzke, M. and Wenzel, M. and Assmus, H.E. and Schulze, W.X. and Kempa, S. BMC Genomics 13 (1): 215. 31 May 2012 Metabolic reprogramming is a key step in De novo assembly and validation of planaria transcriptome by massive parallel oncogenic transformation including the sequencing and shotgun proteomics. activation of energy- and anabolic-metabo- Adamidi, C. and Wang, Y. and Gruen, D. and Mastrobuoni, G. and You, X. and Tolle, D. and Dodt, M. and Mackowiak, S.D. and Gogol-Doering, A. and Oenal, P. and Rybak, A. lism. Thus, differentiated cells reenter the cell cycle and proliferate. Central to this is Chen, W. Genome Research 21 (7): 1193-1200. July 2011 (*co-corresponding author) and Ross, E. and Alvarado, A.S. and Kempa, S*. and Dieterich, C. and Rajewsky, N. and a strong up-regulation of glucose and glu- Muscle-type creatine kinase interacts with central domains of the M-band proteins tamine consuming pathways. Ongoing from myomesin and M-protein. Hornemann, T. and Kempa, S. and Himmel, M. and Hayess, K. and Fuerst, D.O. and Wal- observations made already one century limann, T. Journal of Molecular Biology 332 (4): 877-887. 26 September 2003 ago, the aerobic glycolysis also termed as ‘Warburg effect’, raised the questions for metabolic cancer therapies. Studying the mode of action of known anti-glycolytic metabolites revealed unexpected results. Using the pSIRM-technology we compared the anti-glycolytic effects of 2-deoxy glu- Group Leader Nadine Royla cose and 3-bromo pyruvate. 3-bromo pyru- Dr. Stefan Kempa Christin Zasada vate inhibits glycolysis very effectively but Scientist Associated PhD Students on CCM. These results demonstrate that Dr. Chris Bielow Olga Demidova we2-deoxy have glucoseto carefully has only evaluate a minor the influence current Dr. Raphaela Fritsche Tobias Opialla knowledge in order to design and test met- Dr. Guido Mastrobuoni abolic anti-cancer therapies. Dr. Marica Orioli Technical Assistants Julia Diesbach In future, we aim to better understand the PhD Alina Eisenberger events of metabolic reprogramming during Fabian Bindel Jenny Grobe stem cell differentiation and oncogenesis. Henning Kuich - Amit Kumar Adminstrative Assistance tabolism and epigenetic regulation needs Matthias Pietzke Sabrina Deter toSpecifically be disentangled. the relationship between me

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BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY Photo: David Ausserhofer/MDC Photo:

Markus Landthaler

RNA Biology and Posttranscriptional Regulation

Our main interest is the understanding of dependent manner. The association of these posttranscriptional regulatory networks con- proteins with transcripts regulates the biogenesis and translation of mRNA. For a large trolling gene expression in humans. Nascent number of RNA-binding proteins the target RNA stably associates with RNA-binding pro- mRNAs and their function in RNA metabolism teins, RNA helicases and nucleases to form are unknown, limiting our understanding of ribonucleoprotein complexes. These complex- post-transcriptional regulatory processes. In particular, we are interested in RNA-bind- es play a key role in the regulation of spatial ing proteins that positively and negatively and temporal changes in protein synthesis by modulate the activity of microRNAs. By com- controlling transport, storage and translation bining maps of functional RNA-protein interactions with cell-based and biochemical of mRNAs. Deregulation and failed coordina- assays, we determine the dynamic assembly tion of these mechanisms contribute to patho- of RNA-binding proteins and microRNAs on physiologicial development and conditions. their target mRNAs as well as the elements A prerequisite for a systems level under- and mechanisms guiding mRNA maturation, localization, turnover and protein synthesis. standing of posttranscriptional regulation is a transcriptome-wide high-resolution map of Specificity and function of RNA the RNA-protein contacts that allows us to helicases study how these interactions control the fate Lea Gregersen, Mathias Munschauer of cytoplasmic RNA. To achieve this goal we RNA helicases are a family of highly conserved use a novel crosslinking approaches (PAR- proteins that utilize NTP hydrolysis to unwind CLIP [Hafner & Landthaler et al. Cell 2010] RNA structures and/or remodeling of ribo- and protein occupancy profiling on mRNA nucleoprotein complexes. RNA helicases participate in all biological processes that involve [Baltz & Munschauer et al. Mol Cell 2012) in RNA metabolism, including transcription, combination with next-generation sequenc- splicing and translation and have been impli- ing to identify functional RNA-protein interac- cated in disease states such as tumorgenesis and the replication of viruses. We are using tions at single-nucleotide resolution. - teractions of helicases, their helicase-inactive ourvariants PAR-CLIP and RNAapproach that areto define typically functional transient in Post-transcriptional regulation by RNA-binding proteins and sites provides the foundation for biochemical microRNAs andin nature. reverse The genetic identification approaches of to RNA investigate target Yasuhiro Murakawa, Alexandra Vasile the remodeling mechanism of ribonucleoprotein complexes by helicases. These studies Mammalian genomes encode several hun- give insights into the determinants of target dred RNA-binding proteins, each containing RNA selection, functional interactions with one or multiple domains able to recognize other RNA-interacting proteins and the physi- target mRNA in a sequenceand/or structure- ological role of RNA helicases.

178 MDC Research Report 2014 BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY

A) PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced B) Identification of the mRNA-bound proteome and protein Crosslinking and Immunoprecipitation). occupancy profiling on mRNA.

Global Changes in the mRNA- We use the described methods to monitor protein interactome upon stress the dynamic changes of the mRNA-protein and differentiation interactome to capture differentially binding Alexander Baltz, Miha Milek, Mathias proteins and occupied cis-regulatory mRNA Munschauer, Emanuel Wyler regions as a consequence of intra- and extracellular signals. These data provide insights We have developed a photoreactive nucleo- into the mechanisms that lead to changes in tide-enhanced UV crosslinking and oligo(dT) mRNA turnover and protein synthesis in response to stress and during differentiation. bound proteome using quantitative pro- teomicspurification and approachto display tothe identify protein theoccupancy mRNA- Selected Publications on mRNA transcripts by next-generation se- Gregersen LH, Schueler M, Munschauer M, Mastrobuoni G, Chen W, Kempa S, Dieterich quencing. Application to a human embryonic C, Landthaler M. (2014) MOV10 is a 5’ to 3’ RNA helicase contributing to UPF1 mRNA - target degradation by translocation along 3’UTRs. Molecular Cell 54, 1-13 teins. Nearly one-third were not previously Schueler M, Munschauer M, Gregersen LH, Finzel A, Loewer A, Chen W, Landthaler annotatkidney ed cell as line RNA identified binding. closeProtein to 800occupan pro- M*, - scriptome. Genome Biology 15:R15. (* equal contribution) Dieterich C*. (2014) Differential protein occupancy profiling of the mRNA tran catalog of potential cis-regulatory regions on N*, Landthaler M mammaliancy profiling mRNAs provides and a transcriptome-wideshowed that large targetGraf R*, recognition Munschauer and M*, regulation. Mastrobuoni RNA G, Biology Mayr F, 10:1146-1159 Heinemann U, (* Kempa equal contribution)S, Rajewsky *. (2013) Identification of LIN28B-bound mRNAs reveals features of stretches in 3’ UTRs can be contacted by the Memczak S, Jens M, Elefsinioti, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, mRNA-bound proteome, with numerous pu- Gregersen LH, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C, le Noble - tative binding sites in regions harboring dis- tory potency. Nature 495:333-338 F, and Rajewsky N (2013) Circular RNAs are a large class of animal RNAs with regula ease-associated nucleotide polymorphisms. Baltz AG, Munschauer M, Schwanhausser B, Vasile A, Murakawa Y, Schueler M, Youngs Our observations indicate the presence of a N, Penfold-Brown D, Drew K, Milek M, Wyler E, Bonneau R, Selbach M, Dieterich C, and large number of mRNA binders with diverse Landthaler, M on Protein-Coding Transcripts. Molecular Cell 46, 674-90 molecular functions participating in combi- (2012) The mRNA-Bound Proteome and Its Global Occupancy Profile natorial posttranscriptional gene-expression networks. More recently we described a global compari- Group Leader Karol Rogowski transcriptomes from different cell types, and Dr. Markus Landthaler Alexandra Vasile sonprovided of protein evidence occupancy for altered profiles mRNA on metabo mRNA- lism as a result of differential protein-mRNA Scientist Technical Assistants contacts. Additionally, in this work we in- Dr. Miha Milek Ouidad Benlasfer Dr. Emanuel Wyler - Secretariat ments.troduced Our a bioinformaticswork demonstrated workflow the value for the of PhD students Sabrina Deter analysis of protein occupancy profiling experi Marcin Kolinski cis-regulatory mRNA sequence space and its Mathias Munschauer Other prdynamicsotein occupancy in growth, profiling development for assessing and disthe- Yasuhiro Murakawa Jakob Trendel ease.

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BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY Photo: David Ausserhofer/MDC Photo:

Alexander Loewer

Signaling Dynamics in Single Cells

Regulation of proliferation and differentiation old of damage exists for its activation. Using is a fundamental challenge for multicellular for following in individual cells the number of organisms. Decisions between alternative cell damagedfluorescent sites reporters on DNA, we the developed kinetics a of system dam- fates are therefore tightly controlled by ex- age repair and the p53 response (with Galit ternal and internal signals. Through genetics, Lahav, Harvard Medical School). We found a genomics and proteomics, many molecular large variation in the initial number of DNA break sites and the rate of repair between players that process these inputs have been individual cells. Cells with higher number identified, providing topological maps of most of breaks had higher probability of showing signaling networks. We are now challenged a p53 pulse. However, there was no distinct to understand how these networks act dy- threshold for inducing a p53 response. Our results further suggest that the decision to ac- namically in living cells and how they intersect with each other to control the physiological - response of a cell. As individual cells within a encedtivate p53by both given cell-intrinsic a specific number factors and of breaks previ- ousis not exposure entirely to stochastic, DNA damage. but instead is influ population often show distinct responses to To understand the function of p53 signaling the same signals depending on their internal - state, we focus on the analysis of single cells acterized relationships between atomic, mo- and investigate the properties that unite them lecularon a system’s and cellular level, events we identified that steer and cell char fate decisions. In collaboration with Phil Selenko and the sources of variation that make them (FMP Berlin) and Gary Daughdrill (University different. We use time-lapse microscopy of of South Florida), we showed that the extent living cells to measure signaling dynamics to which a regulatory domain of p53 folds with high temporal and spatial resolution and combine the resulting quantitative data with Byinto introducing an ordered structuresingle point defines mutations, the binding we mathematical modeling to gain a predictive enhancedaffinity to itsMdm2 major binding negative without regulator affecting Mdm2. understanding of cellular decision processes. demonstrated that this leads to altered cel- lularthe remainingp53 dynamics, network target configuration gene expression and P53 dynamics and function in and ultimately cell fate decisions. Our results health and disease provide evidence that the biophysical proper- Elena Cristiano, Dhana Friedrich, Ana Finzel ties of p53 have been evolutionary selected Perez The tumor suppressor p53 is a central regula- protein interactions without compromising tor of cellular proliferation. It is activated by to fine-tune the affinities of its many protein- cellular stress and controls target genes that tumor suppressor to function as a hub in a mediate response programs such as cell cycle centralon binding cellular specificity, signaling thereby network. allowing this regulation, repair and apoptosis. In response To gain a better understanding of the p53 to genotoxic stress, p53 acts as a sensitive mediated cellular response, we investigate signaling system that responds even to low how p53 controls the temporal activation of amounts of DNA damage with a pulse of p53 its target genes. We developed reporter cell protein accumulation in the nucleus. Howev- lines to simultaneously quantify p53 dynam- - ics and expression of selected target genes.

er, it was debated whether a defined thresh 180 MDC Research Report 2014 BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY

We are now perturbing the network by introducing mutations, applying pharmacological interventions or altering interacting signaling pathways and determine how these changes affect the cellular response. This will allow us to better understand how modulating the p53 response during disease and therapy changes cell fate decisions and, potentially, clinical outcome.

Dynamics of the TGFβ pathway in single cells Henriette Strasen, Derya Günes The TGFb pathway interacts closely with the p53 network to induce a cytostatic response. We established reporter cell lines for central - acterized pathway activation in individual cellscomponents in response of the to TGFβdifferent network inputs. and One char ma-

mechanisms that determine the long-term dynamicjor goal is behavior to identify of the the network. dominant To feedback this end, we apply siRNA- or small molecule inhibitor- mediated perturbations of the signaling network and use mathematical modeling (in collaboration with Stefan Legewie, IMB Mainz) A) A fluorescent reporter cell line allows simultaneous measurement of DNA to gain a systematic understanding of the un- double strand breaks (53BP1-mCherry) and p53 dynamics in individual liv- derlying signaling mechanisms. ing cells. DNA damage was induced by γ-irradiation, followed by time-lapse microscopy for 24h. B) Using automated image analysis, both the number of Information flow in mammalian DNA breaks and p53 levels are quantified over time. P53 protein accumulates signal transduction pathways in uniform pulses during repair of the damage. C) There is no defined thresh- Ilias Nolis old of DNA breaks for inducing a p53 pulse, however, cells with higher num- Signal transduction networks relay infor- ber of breaks have higher probability of showing a response (from Loewer mation about external or internal inputs to et al. (2013) BMC Biol). D) Using fluorescent reporters and live cell imaging, the nucleus, and alter cellular behaviour by we quantify the dynamics of various signaling pathways in individual living changing gene expression. As they exhibit cells to understand how the topology of the underlying molecular networks striking similarities to typical communica- determine the outcome of cellular signaling processes. tion systems, the powerful framework of information theory can be applied to better Selected Publications understand these systems. In collaboration Loewer, A.#, Karanam, K., Mock, C., Lahav, G.# (2013). The p53 response in single cells with Nils Blüthgen (Charité Berlin), we sys- is linearly correlated to the number of DNA breaks without a distinct threshold. BMC # tematically investigate the information trans- Biol 11:114 corresponding author Karanam, K., Kafri, R., Loewer, A., Lahav, G. (2012). Quantitative Live Cell Imaging mission properties of the MAPK cascade and Reveals a Gradual Shift between DNA Repair Mechanisms and a Maximal Use of HR in compare them to properties of other signal- Mid S Phase. Mol. Cell 47(2): 320-9 - Purvis, JE., Karhohs, KW., Mock, C., Batchelor, E., Loewer, A., Lahav, G. (2012). p53 ways. We focus on understanding how infor- dynamics control cell fate. Science 336(6087): 1440-4 mationling systems about suchligand as concentration the p53 or TGFβ and ligand path Orth, JD., Loewer, A., Lahav, G., Mitchison, TJ. (2012). Prolonged mitotic arrest triggers type is encoded and decoded in the response partial activation of apoptosis, resulting in DNA damage and p53 induction. Mol Biol Cell. 23(4): 567-76 of the pathways, how large the capacity of Loewer, A., Batchelor, E., Gaglia, G., Lahav, G. (2010). Basal dynamics of p53 reveals the pathways is as information transmitting transcriptionally attenuated pulses in cycling cells. Cell 142(1): 89-100 channels and which design principles in the systems attenuate noise and therefore increase the amount of information that can be transmitted. Group Leader Graduate and Undergraduate Dr. Alexander Loewer Dhana Friedrich Genomic engineering Derya Günes Caibin Sheng Scientist In recent years, powerful new tools emerged Dr. Ilias Nolis Technical Assistants to engineer endogenous gene loci. We are op- Gitta Blendinger timizing the CRISPR/Cas9 system to enable PhD Silke Dusatko - Elena Cristiano Andrea Katzer sions at genes encoding critical mediators of Ana Finzel Perez cellularefficient signaling, insertion allowing of fluorescent us to generate protein ad fu- Caibin Sheng Secretariat vanced reporter cell lines with high through- Henriette Strasen Ines Krock put.

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Benedikt Obermayer

Evolutionary Modeling and Inference in Computational Systems Biology

Efforts to decode genomic functions benefit quences are largely detrimental, which led to from understanding the constraints on ge- the evolution of error-control strategies on the one hand and the evolution of a substantially nome evolution. These constraints result from increased error tolerance on the other. Our different processes and work on different previous work was concerned with quantify- scales. For instance, biochemical limitations ing the extent to which simple non-enzymatic of the cellular machinery drive the evolution in vitro systems are constrained by the thermodynamics of RNA and DNA duplex forma- of robust control strategies. On a large scale, tion. Our observations suggested simple con- regulatory dependencies between genomic trol strategies that could be advantageous in elements couple their respective evolution- hypothetical prebiotic (enzyme-free) environments. Current work explores technological ary dynamics. Importantly, different types applications of these ideas that could help to of evolutionary constraints leave different types of sequence fingerprints. However, with the lab of Michael Gotthardt, we also improve the fidelity of PCR reactions. Together these signals need to be distinguished from study physiological implications of the evolved a large background of more or less random context of cardiovascular gene expression. sequence variation. Our approach to detect balance between fidelity and robustness in the and organize informative evolutionary signals Evolution of post-transcriptional combines computational tools and modeling regulatory networks Post-transcriptional gene regulation is a ma- techniques from bioinformatics and statistical physics. With our methods, we address Messenger RNAs (mRNAs) are under tight a variety of questions, from the accuracy controljor determinant by various of regulatory cellular gene factors expression. during their entire life cycle. Among these factors are of transcription and translation reactions to microRNAs (miRNAs), acting on target sites high-level inference of post-transcriptional located in the 3’ untranslated regions (UTRs) regulatory networks and the detection of of mRNAs. Globally, this mode of post-tran- novel coding regions in the genome. scriptional gene regulation has been found to depend less on individual regulatory links between a single miRNA and a single target Modeling polymerization errors and site. More often, it is conveyed through the col- control strategies lective and concerted regulation by multiple Many biological functions across all domains miRNAs of entire target gene sets. Although of life fundamentally depend on template-di- individual target sites often have minor regu- rected polymerization processes. Prime exam- latory or phenotypic consequences and are ples are DNA replication, RNA transcription therefore typically not widely conserved, the and protein translation. Physical constraints collective function of the entire network ap- limit the accuracy of these reactions leading pears to remain much more stable during the to occasional misincorporations. These errors course of vertebrate genome evolution. This can be tolerated when rare, and sometimes implies that individual sites do not evolve in- they even serve as a source for evolutionary dependently of each other. We have developed innovations. However, in general their conse- computational methods to detect such non-

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Evolutionary correlations in the miRNA regulatory network: preferential conservation of miRNA target sites is evaluated by means of a phylogenetic background model trained on 3’UTR conservation in vertebrate whole-genome alignments. Comparing site pairs, significant similarity of dissimilarity of their conservation patterns is then detected as positive or negative evolutionary correlation, respectively. This analysis suggests that miRNAs with many positively correlated site pairs in the same 3’UTR share common functions in the regulatory network.

independent evolution (i.e., evolutionary cor- are often hosted by transcripts so far anno- relations) in the vertebrate miRNA regulatory tated as non-coding. The abundance of mi- network. Our results show that combinatorial cropeptides and their genomic features and regulation via an ensemble of multiple target biological functions remain largely unex- sites in the same 3’UTR is not only apparent plored. We develop and use computational from consistent co-occurrence of such target tools to detect conserved small open read- sites, but also from their co-conservation. Fur- ing frames in multiple species alignments for a number of different model organisms. for the coordinated targeting of protein com- We collaborate with the lab of Antonio Giral- plexesther, we differs find that from the those conservation for the regulation signatures of dez (Yale) to combine our computational signaling pathways, possibly suggesting differ- pipeline with experimental detection of ent control strategies. Our approach combines translated ORFs via ribosome footprinting phylogenetic modeling with ideas from statistical physics, and we subsequently extended widely conserved small ORFs are translated it in order to distinguish direct and indirect in zebrafish. This revealed that numerous evolutionary correlations. One application of that they give rise to functional products. these inference techniques has recently prov- Weduring further zebrafish work development,with several labs suggesting on the en very useful in aiding computational protein MDC campus to identify and characterize folding with distance constraints derived from micropeptides in other model organisms via correlated substitutions in protein sequence mass spectrometry, by means of structural alignments. By our theoretical analysis and analysis, and via genetics. with our generalized methods we aim to transfer these techniques to other biological Selected Publications contexts. Bazzini A.A.; Johnstone T.G.; Christiano R.; Mackowiak S.D.; Obermayer B.; Fleming Small open reading frames coding small ORFs in vertebrates using ribosome footprinting and evolutionary conservation for conserved micropeptides E.S.; Vejnar C.E.; Lee M.T.; Rajewsky N.; Walther T.C.; Giraldez A.J. Identification of Leu K.; Kervio E.; Obermayer B.; Turk-MacLeod R.M.; Yuan C.; Luevano J.M.; Chen E.; (PhD student: Sebastian Mackowiak) The EMBO journal (doi: 10.1002/embj.201488411; published online 2014-04-04) Gerland U.; Richert C.; Chen I.A. Cascade of reduced speed and accuracy after errors The coding potential of the genomes of most in enzyme-free copying of nucleic acid sequences Journal of the American Chemical Society 135 (1): 354-366 (2013-01-09) model organisms has been thought to be comprehensively explored. Hence, the re- - search focus recently often shifted to the vantage of transferring genetic information from RNA to DNA Nucleic Acids Research Leu39 (18): K.; Oberma 8135-8147yer B.; (2011-10) Rajamani S.; Gerland U.; Chen I.A. The prebiotic evolutionary ad discovery and characterization of putatively Obermayer B.; Krammer H.; Braun D.; Gerland U. Emergence of information transmis- non-coding mRNA transcripts. However, tra- sion in a prebiotic RNA reactor Physical Review Letters 107 (1): 018101 (2011-07-01) ditional methods for discovery of functional open reading frames (ORFs) relied on pre- conceptions about typical ORF characteristics. These have recently been shown to be rather incomplete with the discovery of Group Leader Graduate student functional micropeptides in various animals. Dr. Benedikt Obermayer Sebastian Mackowiak* Such peptides are not produced by cleaving (Delbrück Fellow) larger precursors but translated from small * jointly supervised with ORFs that can be as short as 11 codons and Nikolaus Rajewsky

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Uwe Ohler

Computational Regulatory Genomics

The expression of genes is a multi-step Computational Biology and Gene process that is tightly controlled on several Regulation levels — a large number of protein and RNA Understanding gene regulation emerged as factors and DNA and RNA sequence ele- a crucial challenge after the initial sequenc- ments enable the precise regulation of inter- ing of complex eukaryotic genomes, and acting gene products. Our lab develops and misregulation of gene expression is at the heart of many developmental defects and dis- uses computational approaches to under- eases. In this context, the particular strength stand the biology of gene regulation in eu- of a multi-disciplinary group like ours lies in karyotic organisms. the combination of genomics-oriented data According to our current understanding, generation and analysis with computational modeling. We frequently frame questions complexity in higher organisms is not achieved by a more complex repertoire of learning approaches to make testable predic- parts, i. e. genes, but instead by the more tions.as classification These predictions problems are and often use validated machine experimentally, either by us or our friends complex regulation of the parts. It is a key and collaborators. Computational techniques challenge to decipher these complex networks of players and interactions, and to complex approaches such as graphical mod- move biology from case studies to an in- elsrange and fromBayesian simple (non-parametric) (sparse) classifiers statistics, to to predict, annotate and analyze regulatory tegrated, global approach. Large initiatives regions and the encoded expression patterns such as the ENCODE consortium have set at a genome-wide scale. At the same time, in- out to map all functional elements in the hu- spection of model parameters frequently al- man genome, and in consequence, compu- lows us to draw conclusions, or at least make tational biology has become indispensable to particular biological features. analyze and ultimately make sense of large- specific suggestions, about the importance of scale data sets that look at the phenomenon Transcriptional control: from proxi- of gene regulation from different angles. Our mal initiation to distal regulation long term goal is to investigate how regulatory networks enable the correct development In eukaryotes, transcriptional regulatory el- of complex organisms, with their multitude ements are located in different regions relative to the gene they control (Figure 1). The of cell types that carry out different functions basal or core promoter, a ~100 base pair long despite the same genome. region centered on the transcription start site (TSS), has been traditionally assumed to perform the function to recruit the basal transcription complex to the correct genomic location, and thus to largely share the same sequence patterns across all genes. In contrast, distal non-coding regulatory regions such as enhancers contain a combination of sequence

factors (TFs) and thus convey condition-spe- patterns recognized by specific transcription

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Modeling of transcriptional regulation. A number of different deep sequencing protocols make it possible to identify putative control regions within the large space of non-coding sequence in the human genome. As example, regions of open chromatin that are accessible to regulatory factors can be identified at a genomic scale via DNase-seq. (Top) In this way, both the proximal promoters around transcription start sites of genes, as well as distal regulatory regions, can be identified. (Left) A subset of open chromatin regions are highly condition specific; knowing the location of these regions makes it possible to identify underlying sequence features and candidate regulators of specific expression patterns. (Right) At high resolution, DNase-seq data exhibits characteristic “footprints” at the sequence motifs that a transcription factor binds to. Using computational approaches, we can identify the subset of candidate sites that are occupied in a specific condition.

- by integrating heterogeneous data sources ment 2010). from sequence, occupancy, expression and cific activity (Ohler and Wassarman, Develop chromatin features. For a long time, the large - non-coding genomic sequence space of high- tributions towards an increased understand- er eukaryotes has posed severe limitations as ingWe of have the madecore promoter particularly as “model significant system” con for complex regulatory regions. Our studies degenerate sequence elements that are tar- showed that core promoters do not show a getedit made by it TFs, difficult and toto distinguishpinpoint the background short and single stereotypic arrangement and serve sequence variation from changes with func- different functions (Rach et al, Genome Biol tional consequences. Several recent genomic - approaches now provide “global regulatory tion patterns, which have become apparent - via2009). new This sequencing is reflected protocols in different that gener initia- ate libraries of millions of short transcripts orprofiles”, RNA. which reflect where regulatory fac pinpointing the locations of transcription tors targeted specific regions andsites in DNA initiation (Ni et al, Nature Methods 2010). - This diversity is observed across all eukary- vide coarse-resolution maps of nucleosome- otes; different types of core promoters dis- freeSpecifically, sites or DNaseI “open hypersensitive chromatin”, which sites proare universal hallmarks of all types of transcrip- also by conserved chromatin features (Rach tional regulatory elements. DHS site mapping ettinguish al, PLoS themselves Genetics not2011). just Eukaryotesby sequence thus but has been widely used to identify regulatory elements that control gene transcription, and but exhibit widely conserved chromatin pat- has been implemented in genome-wide pro- ternshave some for different species-specific expression sequence programs features at tocols (Figure 1). In an ENCODE companion the level of basal initiation (housekeeping paper, we used DNase-seq open chromatin genes, developmental regulators, translation machinery, germline, etc). In ongoing work, that provide a successful baseline for the pre- we examine connections of transcription ini- dictiondata from of diverseexpression cell typesfrom sequenceto build classifiers features tiation with pervasive non-coding/antisense transcription that has emerged as a feature of The resulting models contained factors with mammalian promoters. known(Natarajan as well et as al, testable Genome new Research functions, 2012). and illustrated the game-changing nature of ge- In complementary work, we have made prog- nome-wide annotations of distal regulatory regions for decoding expression patterns. We

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Computational approaches for investigating regulatory protein-RNA interactions. Recent sequencing protocols make it possible to quantify levels of transcripts as well as to identify how they are regulated at the RNA level, and we have developed a number of computational methods for appropriate genome-wide analyses. (Left) Cross- linking and immunoprecipitation data can be used to identify specific locations on transcripts to which regulators are binding. (Middle) Motif finding algorithms can identify the prevalent sequence features present at the binding sites. (Right) Integrating multiple data types, such as sequence, binding, and conservation, allows for highly specific predictions of regulatory events such as targeting by microRNAs.

Finding regulatory network motifs. To study how transcription factors and microRNAs together control target genes within regulatory networks, we have developed a permutation strategy that enables the identification of significant “network motifs”, i.e. preferential subnetwork structures that involve both TFs and miRNAs. Across different organisms, this algorithm identified three main types of such motifs.

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are now investigating models for DNase-seq - data at nucleotide resolution, to make it pos- tion algorithm which properly accounts for sible to pinpointing the individual TF binding factorsto develop at different a new networklevels of motifgene expression identifica events within regulatory regions. (Figure 3). Across different organisms, we - tifs, such as the frequent co-regulation of a TF RNA regulation: identifying target Zidentified by TF X aand subset miRNA of conserved Y, where Y network also targets mo sites of regulatory RNAs and pro- Z (Megraw et al, Genome Biology 2013). This teins static network analysis will be complemented by dynamic gene expression data to quantify The last decade has seen an outstanding the function of different network motifs in increase in appreciation of the role of ex- different conditions. pression regulation at the RNA level and its importance for the correct function of vir- The group moved from Duke University in tually all biological processes. RNA-binding late 2012. The described work had the sup- proteins and small, regulatory RNAs such as port from a number of excellent colleagues, including Pavel Tomancak (MPI Dresden); James Kadonaga (UC San Diego); David Was- microRNAs (miRNAs) recognize specific sites sarman (University of Wisconsin); Tom Tus- throughin target the RNA invention transcripts of new and deepinfluence transcrip their- chl (Rockefeller University); Jun Zhu (NHLBI); tomefate. Thesequencing field has protocols made dramatic that make advances it pos- Philip Benfey, Blanche Capel, Greg Crawford, sible to quantify the levels of protein-coding Jack Keene (Duke University). We are now messenger-RNAs and non-coding RNAs at looking forward to extending these collabo- different stages of post-transcriptional regu- rations to the new possibilities at BIMSB and lation (Figure 2). We are now also able to map MDC. the precise target sites of post-transcriptional regulators (including RNA-binding proteins and regulatory, non-coding RNAs) via crosslinking followed by deep sequencing. Selected Publications

These new data types pose intriguing com- and microRNAs are central building blocks of eukaryotic gene circuits. Genome Biol putational challenges. Over the past few M14:R85, Megraw, 2013. S Mukherjee, U Ohler. Sustained-input switches for transcription factors - proaches for reliable, high-resolution identi- Munschauer, S Dewell, M Hafner, Z Williams, U Ohler, T Tuschl. FMRP targets distinct years, we have implemented specific new ap mRNAM Ascano, sequence N Mukherjee, elements P Bandarto regulateu, JB protein Miller, JDexpression. Nusbaum, Nature DL Corcoran, 492:382-386, C Langlois, 2012 M up prediction of regulatory sequence motifs, includingfication of miRNA binding target sites, sitesas well as aas special for follow- case WHNature Majoros, Methods P Lekprasert, 10:630-633, N Mukherjee,2013 RL Skalsky, DL Corcoran, BR Cullen, U Ohler. MicroRNA target site identification by integrating sequence and binding information. approaches have been used in multiple genomic(Majoros studies et al, Naturewith collaborators Methods 2013). to identify These A1722, Natarajan, 2012. GG Yardimci, NC Sheffield, GE Crawford, U Ohler. Predicting Cell-Type the genes regulated by disease-implicated Specific Gene Expression from Regions of Open Chromatin. Genome Res 22:1711- RNA binding proteins such as the Fragile X mental retardation protein family (Ascano et initiation patterns indicate divergent strategies for gene regulation at the chromatin al, Nature 2012) or the cellular targets of hu- Elevel.A Rach, PLoS DR Genet. Winter, 7:e1001274, AM Benjamin, 2011. DL Corcoran, T Ni, J Zhu, U Ohler. Transcription

Microbe 2011). man viral miRNAs (Gottwein et al, Cell Host & Start of the Group: September 2012 Putting the pieces together: net- Alina-Christina Munteanu works involving different regulatory Group Leader Hans-Hermann Wessels mechanisms Prof. Dr. Uwe Ohler Graduate and Undergraduate Our work on DNA and RNA-based regulation Scientist Merve Öztürk Dr. Scott Lacadie Michael Rauer the quantitative contributions of individual Samta Malhotra regulatoryconverges inlayers, projects and where how they we aimneed to to dissect work Technical Assistants - - PhDDr. Neelanjan Mukherjee Nicholas Charles Jacobs opmenttogether as to compared define correct to rapid expression responses at to dif a Lorenzo Calviello SigneAntje HirsekornKnespel changingferent scales environment). (e.g. during In predefineda recent example, devel Dina Hafez Mahmoud Ibrahim Secretariat involving regulation of target genes by both Aslihan Karabacak Michaela Liemen TFswe have and identifiedmiRNAs. significantFor this aim, network we needed motifs

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BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY Photo: David Ausserhofer/MDC Photo:

Ana Pombo

Epigenetic Regulation and Chromatin Architecture

Our laboratory aims to understand mecha- transcript. Upon transcription termination, nisms of gene expression which act at addition of a protective polyA tail to the multiple levels, from the local action of tran- transcript’s 3’ end still occurs on chromatin. scription factors, to long-range chromatin These co-transcriptional events are medi- looping events that connect regulatory DNA ated by exquisite post-translational modifi- sequences with the genes they regulate, to cations of the RNAPII enzyme itself, which how whole chromosomes are positioned coordinate the timely recruitment of ap- within cell nuclei (Figure 1). propriate machineries at each stage of the Binding of transcription factors at gene transcription cycle. Chromatin modification promoters helps recruit chromatin remod- of histone tails are also often coordinated elers and the transcription machinery to by RNAPII itself, marking active promoters transcription sites. Upon recruitment, RNA and their coding regions for further cycles polymerases either remain primed for sub- of transcription. sequent activation (e.g. after environmental stress, during differentiation or disease), or directly transcribe the DNA template into RNA molecules, which code for proteins or Mechanisms of RNAPII and Poly- for structural RNAs. comb regulation in pluripotent and An increasingly important aspect of gene differentiated cells regulation are the mechanisms by which João Dias, Ana Miguel Fernandes, Carmelo distant regulatory DNA sequences physi- Ferrai, Alexander Kukalev, Kelly Morris, Kedar Natarajan, Tiago Rito, Marisa Saponaro, cally interact with gene promoters to recruit Elena Torlai-Triglia or activate RNA polymerase II (RNAPII), the enzyme responsible for transcribing Appropriate recruitment of the RNA processing machinery and chromatin remod- protein-coding genes. Much is known about elers to chromatin is mediated by post- the transcription cycle of protein-coding genes, which entails a fascinating and domain (CTD) of the largest RNAPII sub- highly coordinated sequence of processes translational modification of the C-terminal Rep. 10, 1213). The CTD is an unusual that promote nascent transcript maturation disorderedunit, Rpb1 (Brookesdomain composed& Pombo 2009,of the EMBO hep- concomitant with transcription. As the na- tapeptide consensus sequence, Tyr1-Ser2- scent RNA emerges from the polymerase, Pro3-Thr4-Ser5-Pro6-Ser7, repeated 52 it is capped on its 5’ end. Later, as the times in mammals. All aminoacids are tar- polymerase progresses through the coding of which we know most about Serine phos- region, introns are spliced from the nascent phorylation.gets for post-translational modifications,

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Our main aim is to understand the interplay between gene regulation and genome architecture, with the goal of defining rules and principles of genome function in mammals. We study local chromatin states using genome- wide methods to profile the occupancy of RNA polymerase II and epigenetic regulators, such as Polycomb repressor complexes. This information is used to interpret 3D chromatin folding, and to explore the role of gene positioning in the cell nucleus in gene expression

After recruitment of unphosphorylated In 2012, we showed that Polycomb and RNAPII to promoters, the CTD becomes RNAPII co-associate on chromatin at devel- phosphorylated on Ser5 residues (S5p), opmental regulator genes (Brookes et al. followed by sequential phosphorylation on 2012). We used chromatin immunoprecipi- Ser7 (S7p) and Ser2 (S2p) in the transition tation (ChIP) coupled with next generation to elongation. S5p recruits the capping en- sequencing, to map the phosphorylation zyme and H3K4 histone methyltransferases state of RNAPII and Polycomb occupancy (HMTs), which mark active promoters. S7p across the genome (Figure 2). Surprisingly, is thought to promote the transition to elon- - gation, whereas S2p recruits both H3K36 mental regulator genes under Polycomb HMT, to mark coding regions, and the splic- repression,not only did but we its find presence RNAPII was at positively develop ing and polyadenylation machineries. Our correlated with the amount and occupancy unbiased mass spectrometry analyses of of Polycomb. To characterize the extent of RNAPII complexes in mitosis (Moeller et transcriptional activity at poised genes, al. 2012; in collaboration with M. Selbach) which are targets of Polycomb repression, and in association with chromatin (ongoing we are analyzing chromatin-bound RNAs work) identify a growing number of nuclear using genome-wide approaches, and CAGE- activities coordinated with RNAPII, many of seq data (the latter in collaboration with which with roles in disease. FANTOM5).

Regulation of RNAPII by phosphoryla- Our unbiased genome-wide analyses of tion extends beyond the changes asso- RNAPII and Polycomb occupancy also iden- ciated with active transcription cycles. - Phosphorylated RNAPII has been reported lation in mouse ES cells, which were more at inducible genes, before activation, e.g. unexpectedtified novel gene(Brookes targets et ofal. Polycomb 2012). In regu par- - ticular, we found a cohort of genes which el poised form of RNAPII at silent develop- are active in ES cells but whose expression mentalheat-shock. regulator Our group genes has in identifiedmouse ES a cells,nov is dampened by Polycomb repression (Poly- which are repressed by Polycomb complex- comb-Active genes; Figure 2). This group of es, but become activated through early dif- genes includes many genes important for ferentiation and cell commitment (Stock et cell cycle, cancer, signaling and energy me- al. 2007, Nature Cell Biol. 9, 1428). Two ma- tabolism. Our current studies aim to better understand their regulation in the transi- and PRC2, are important for maintaining tion from pluripotency to differentiated thejor Polycombpoised state Repressor of primed Complexes, developmen PRC1- cellular states. tal regulator genes. Polycomb complexes modify histone tails; PRC2 contains Ezh2, To probe for the dynamic changes in poly- which methylates H3K27, and PRC1 con- merase and chromatin conformation that tains Ring1A and Ring1B, which monoubiq- accompany re-programming of gene ex- uitinylate H2AK119, but the mechanisms of pression during cell commitment, we have repression are not well understood. extended our analyses to neural progenitor

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Model of RNA polymerase II (RNAPII) and Polycomb regulatory states at Active and Repressed genes in mouse ES cells (Brookes et al. 2012 Cell Stem Cell). Active genes are associated with RNAPII-S5p,S7p during initiation, with S5p,S7p,S2p during elongation, and with productive expression but no Polycomb association. Inactive genes are not associated with RNAPII or Polycomb. Polycomb Repressed genes are not expressed, have high CpG content, high RNAPII-S5p+S7p-S2p- and Polycomb recruitment. An unexpected cohort of genes, which we call Polycomb- Active, show a mixture of Active and Polycomb repressed chromatin states. They are expressed at the mRNA and protein levels, yet they are up-regulated upon Polycomb knockout. These genes display different configurations at independent gene copies in the same or different cells across the ES cell population. Some alleles are repressed and associated with Polycomb and RNAPII-S5p with similar properties to PRC repressed genes. Other alleles are active and associate with active RNAPII complexes characterized by the presence of S5p and S2p (pink) but not Polycomb.

cells and differentiated neurons (co-funded point for the synthesis of transcripts. Genes by BBSRC and MRC, UK) and to cardiac lin- that regulate early development are often eages (co-funded by MRC-BHF, UK). Bioin- expressed at different stages of differentia- formatics analyses are ongoing to further tion and different cell types, and are accord- understand how Polycomb repression acts to establish poised states of RNAPII occu- with different regulatory regions. In addition pancy at genes with roles in pluripotency toingly contacts regulated with through their specificrespective interactions enhanc- and differentiation. ers (which can be located up to 1 Mbp from the gene promoter), genes also co-associate with other genes in 3D space. Our group has Genome architecture: roles of RNA- shown that Active and Polycomb-repressed PII and Polycomb in 3D chromatin genes associate, respectively, with ‘Active folding transcription factories’ (containing RNAPII- Mariano Barbieri, Robert Beagrie, Inês de S5p, S2p) and with ‘Poised transcription Castro, Dorothee Krämer, Liron Mark Lavitas, factories’ (containing RNAPII-S5p and Poly- Markus Schueler comb; Ferrai et al. 2010 PLoS Biol.; Brookes et al. 2012 Cell Stem Cell), suggested that Long-range gene regulation works through - chromatin looping mechanisms that bring tin topologies that are important for silenc- together the promoters of genes with distant ingsuch in associations the primed establishstate, or for specific robust chroma activa- regulatory regions (e.g. enhancers). During tion upon induction. this contact, mediated by transcription factors and other chromatin associated compo- We currently use polymer physics modeling nents, the transcriptional machinery is de- to study the mechanisms underlying com- livered to the gene, which acts as the starting plex chromatin folding patterns associated

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Using the String & Binders Switch (SBS) model (Barbieri et al. 2012), we can study how epigenetic information coating mammalian chromosomes can fold into increasingly higher levels of chromatin architecture. Using computer simulations, we can test how chromatin factors that bind to their specialized sites along the DNA molecule can promote higher order chromatin folding. Over time (moving from left to right and down in this picture), different floating factors (red and green) bind their respective sites on the DNA to create loops. As more proteins attach, loops become wound closer together, forming dynamic topological chromatin domains. Interfering with specific binding sites, by changing some red binding sites into blue, promotes passive looping of extended chromatin fibres, phenomena observed in vivo.

with long-range regulation, and how they Selected Publications might be established through different chromatin epigenetic states. With Prof. Mario Ni- Ferrai C, Xie SQ, Luraghi R, Munari D, Ramirez F, Branco MR, Pombo A, Crippa MP (2010) Poised transcription factories prime silent uPA genes prior to activation. codemi (Univ. Naples, Italy), we have shown PLoS Biology 8, e1000270. that available data on chromatin contacts, - Möller A, Xie SQ, Hosp F, Lang B, Phatnani HP, James S, Ramirez F, Collin GB, Naggert JK, Babu MM, Greenleaf AL, Selbach M, Pombo A (2012) Proteomic analysis of mitotic rescence in situ hybridization (FISH), can be RNA polymerase II complexes reveals novel interactors and association with proteins both originated by 3C approaches and fluo dysfunctional in disease. Molecular Cellular Proteomics 11(6):M111.011767.

(SBS) model (Barbieri et al. 2012, 2013). Brookes E, de Santiago I, Hebenstreit D, Morris KJ, Carroll T, Xie SQ, Stock JK, Heide- Currently,explained withwe are the usingStrings the & BindersSBS model Switch to mann M, Eick D, Nozaki N, Kimura H, Ragoussis J, Teichmann SA, Pombo A (2012) explore the 3D topology of the HoxB locus regulates metabolic genes in ES cells. Cell Stem Cell 10, 157-170. in mouse ES cells by simulating chroma- Polycomb associates genome-wide with a specific RNA polymerase II variant, and tin occupancy maps of epigenetic features Barbieri M, Chotalia M, Fraser J, Lavitas LM, Dostie J, Pombo A, Nicodemi M (2012) as interaction sites. The HoxB polymer is Proc. Natl. Acad. Sci. USA 109, 16173-16178. Complexity of chromatin folding is captured by the Strings & Binders Switch model. Barbieri M, Fraser J, Lavitas LM, Chotalia M, Dostie J, Pombo A, Nicodemi M (2013) A mixed in silico with binders with affinity to polymer model explains the complexity of large-scale chromatin folding. Nucleus 4, epigenetic states across the locus. Binding 267-273. inducesthe binding polymer sites folding defined based according on univer to the- sal thermodynamics principles. Our current work combining polymer modeling using the SBS model with single-cell imaging by cryo- FISH shows that the 3D chromatin folding of Start of the Group: April 2013 the HoxB locus in ES cells can be explained by homotypic associations between Active Group Leader PhD and Polycomb-target genes. We will extend Prof. Dr. Ana Pombo Robert Beagrie (MRC) our analyses to understand changes in chro- Dorothee Krämer matin topology during cell commitment by Scientist Elena Torlai Triglia modeling chromatin folding in neuronal Dr. Mariano Barbieri* João Dias precursor cells and terminally differentiated Dr. Inês de Castro* Ana Miguel Fernandes** neurons. To this end, we are analyzing HiC Dr. Carmelo Ferrai datasets produced in collaboration with Dr. Dr. Alexander Kukalev Graduate and Undergraduate Josée Dostie (McGill University, Montreal). Dr. Liron M. Lavitas* Marisa Saponaro** Dr. Kelly J. Morris To investigate the relevance of nuclear po- Technical Assistants sitioning for gene expression outcomes, we Dr. Tiago Rito (MRC-BHF) Signe Knespel have also explored changes in gene posi- Dr. MarkusKedar N. Schüler* Natarajan (MRC)* Adrienne Naporra* tioning that occur in Huntington’s disease and the nuclear positioning of the stochas- * Part of the period reported, Secretariat tically-expressed Ifnb gene in response to ** From January 2014 Michaela Liemen viral infection.

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Nikolaus Rajewsky

Systems Biology of Gene Regulatory Elements

My lab uses experimental (molecu- regulatory information is to a large degree lar biology, biochemistry) together with hardwired into the non-coding parts of the genome. Our lab focuses on decoding tran- computational methods (bioinformatics, computational biology, etc) to dissect, characterization of targets of transcription systems-wide, function and evolution of factorsscriptional in non-coding regulation DNA) (identification and post-tran and- gene regulation in metazoans. One major scriptional control mediated by RNA binding proteins and small, non-coding RNAs, in par- focus is to understand more about post ticular microRNAs. microRNAs are a recent- transcriptional gene regulation exerted by ly discovered large class of regulatory genes, small RNAs, in particular microRNAs. We present in virtually all metazoans. They have are developing predictive models for targets sites in 3’ untranslated regions (3’ UTRs) of of microRNAs. We also investigate general protein-encodingbeen shown to bind mRNAs to specific and, cis-regulatory by unknown mechanisms of gene regulation by microR- mechanisms, to repress protein production NAs and RNA binding proteins in cell lines of their target mRNAs. Our understanding of the biological function of animal microRNAs and in vivo. For example, we are studying function and mechanisms of post-transcrip- that microRNAs are regulating or involved tional gene regulation during early develop- inis a just large beginning variety of to biological emerge, butprocesses it is clear and ment in C. elegans. Furthermore, we have human diseases, such as developmental timing, differentiation, signalling, homeostasis established planaria as a model system in of key metabolic gene products such as cho- our lab. These freshwater flatworms are lesterol, cardiovascular diseases and cancer. famous for their almost unlimited ability to Overall, however, it is clear that miRNAs are regenerate any tissue via pluripotent, adult only a small part of the entire post transcriptional gene regulation apparatus used by stem cells. We investigate molecular mech- cells, and we are beginning to systematically anisms of pluripotency and the role of post- explore the largely unknown function of transcriptional gene regulation in planarian RNA binding proteins. stem cell biology and regeneration. It is clear that a better understanding of gene regulation and in particular of the Introduction can only come by integrating various data sourcesjust emerging (comparative universe ofsequence non-coding analysis, RNAs metazoans share to a large degree the same mRNA expression data, protein-protein in- repertoireA major lesson of protein-encoding from recent genomics genes. is Itthat is teractions, mutant phenotypes from RNAi thought that differences between cells with- screens, polymorphism data, experimentally in a species, between species, or between healthy and diseased animals are in many data, etc) since each data source alone is only cases due to differences in when, where adefined partial gene description regulatory of networks,how cells ChIP-chipfunction. and how genes are turned on or off. Gene For example, to understand microRNA func-

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Figure 1: Possible functions of circRNAs: (a) binding miRNAs or RNA binding proteins (RBPs) to change their local free concentration (b) transport of miRNAs/RBPs inside the cell (c) acting in trans (d) involved in assembly of other molecules (e) involved in regulating splicing (f) it is also a possibility that most circRNAs, after production, have no function.

tion, we not only need to identify their tar- put sequencing data (Friedlaender et al., gets but also to decode how microRNAs are Nature Biotechnology 2008). We have also pioneered approaches that allowed to exper- of the lab is therefore in developing meth- imentally assay, genome-wide, the impact of odstranscriptionally that integrate regulated.different data A majorsources focus and miRNAs on protein synthesis (Selbach et al., - predictions about how, when, and where rently to use and develop several key high- genesmethods are toregulated. produce This global will and ultimately yet specific lead throughputNature 2008). technologies A major ongoing for in effortvivo studies is cur - in C. elegans and planaria: high-throughput tion of gene regulatory networks. We will proteomics (SILAC), RNA sequencing, and continueto the identification to test, develop and functional and “translate” descrip new methods that allow the genome-wide these methods and their predictions using - proteins. Part of this work involved collabo- lism in mammals, regeneration in planar- rationsidentification with the of bindingStoffel lab sites (Rockefeller of RNA binding Uni- ians,specific early biological embryogenesis systems, in such C. elegans. as metabo and Selbach labs (MDC). Regarding miRNAs weversity, are currently Piano & workingGunsalus on labs advanced (NYU), meth Chen- ods to biochemically ligate miRNAs to target couldSpecifically, later on we show have developedthat microRNAs one of very the sites and thus to be able to obtain context- likelyfirst microRNA regulate targetthousands finding of algorithms genes within and dependent, in-vivo maps of genome-wide miRNA:target interactions (Grosswendt et Nature Genetics 2005; Lall et al., Current Bi- al. submitted). These maps must be inter- ologyvertebrates, 2006; Gruenflies, and et al.,nematodes PloS Computational (Krek et al., preted by Systems Biology methods to un- Biology 2006). We have further helped to derstand or predict phenotypes. elucidate the function of microRNAs in pancreatic beta cells (insulin secretion), in liver (cholesterol level), and other systems. More Circular RNAs recently, we have shown that microRNAs can - We have recently shown that single-strand- natures on hundreds of genes (Sood et al., ed circular RNAs (circRNAs) are a wide- PNASleave cell2006), type and specific that human mRNA genotypedexpression SNP sig spread class of animal RNAs with tissue and data can be used to explicitly demonstrate and quantify the contribution of microRNA (Memczak et al. Nature 2013). circRNAs are - usuallydevelopmental expressed stage from specificexons, are expression found in sky, Nature Genetics 2007). We have further the cytoplasm, and are unusually stable. For developedtargets to humancomputational fitness (Chenmethods and (miRD Rajew- a particular circRNA (CDR1as) we could eep) to predict miRNAs from high through- show that this circRNA can function as a

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Figure 2: By computational clustering of our data, we identified ~866 genes with cycling mRNA expression in C. elegans. The Figure shows qPCR data for randomly selected genes from this cluster (upper left panel) and negative controls (lower left panel). Orthologous genes were measured in C. briggsae (right upper and lower panels). Gruen et al., Cell Reports 2014.

sponge to sequester miR-7, a neuronal spe- throughput technologies to study differential gene expression and networks during very possible functions of circRNAs and mecha- early embryogenesis (for example the oocyte nismscific miRNA. of circRNA We are currentlybiosynthesis. investigating Besides to one-cell embryo transition upon fertiliza- circRNAs potentially acting as sponges, we tion). To overcome this problem, we have de- are studying a potential role of circRNA veloped a novel method (“eFACS”) that allows production in regulating splicing of mRNAs us to sort embryos at precise stages during (Collaboration with the Kadener lab, He- embryogenesis via FACS sorting (Stoeckius, brew University, Jerusalem). However, it is Maaskola et al., Nature Methods 2009). For ex- also possible that circRNAs are largely inert ample, we can now routinely obtain ~60,000 by-products of splicing (Fig. 1). Even in this one-cell stage embryos (at a purity of >98%) case, because of their unusual stability and in one FACS run, enough to apply virtually differential expression, it will be extremely any high-throughput method of interest. We interesting to study circRNAs as possible have used eFACS to assay the dynamics of biomarkers for processes. small RNA expression during embryogenesis. We discovered a wealth of orchestrated, spe-

Development in C. elegans thecific doorchanges for betweenmany computational and within virtually and func all- Although C. elegans is one of the most fa- tionalclasses follow of small up studies. RNAs. For These example, findings we openhave mous model systems for developmental biol- shown that sperm transmits RNA into the zy- ogy, it has been impossible to use most high- gote, and that hundreds of paternal mRNAs/

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small RNAs are present in the one-cell embryo (Stoeckius et al., submitted). We are also studying RNA binding proteins and microR- interested in understanding more global pat- NAs(miR-7). together. This findingUltimately illustrates we need the toneed study for terns of developmental gene expression. For regulatory relationships not in cell lines but example, in a recently published large-scale in vivo. In a proof of principle experiment, we - have shown that it is possible to biochemi- cording of RNA and protein levels during de- cally identify targets of RBPs in C. elegans velopment.study we described We recorded the these first systematiclevels in both re and at nucleotide resolution (Jungkamp et elegans and briggsae species to be able to in- al., Molecular Cell 2011). This new method terpret our results. One surprising discovery (“iPAR-CLIP”) allows us to systematically was the existence of ~866 genes which cycle identify the targets and to study the function every ~8hrs in expression the animal. This of any RBP in C. elegans (Fig. 2). One immedi- cycling is conserved between species (Fig. 2) ate goal is to identify the targets of DICER and and thus likely functional. There is currently other central components of small RNA path- no explanation for this effect. ways in vivo (Rybak et al., submitted). Part of this work involved collaborations with the Landthaler, Selbach, and Kempa labs (MDC). Stem cell biology

We used massive next generation sequencing Selected Publications to identify miRNAs and piRNAs in S. mediter- (2008). Widespread changes in protein synthesis induced by microRNAs. Nature 455, - Selbach58-63 M; Schwanhaeusser B; Thierfelder N; Fang Z; Khanin R; and Rajewsky N berranea. of these We also miRNAs identified are conserved miRNAs inthat humans seem specifically linked to stem cell biology. A num N (2011). Transcriptome-wide analysis of regulatory interactions of the RNA binding We have further developed experimental and Lebedeproteinva HuR. S, Jens, Molecular M, Theil Ce K,ll Schwanhaeusser 43, 340-352. B, Selbach M, Landthaler M, Rajewsky computational(Friedlaender & methods Adamidi that et allowedal., PNAS us 2009). to assemble the planarian transcriptome via next generation sequencing of mRNAs (Adamidi JungMolecularkamp AC, Cell Stoeckius 44, 828-40. M, Mecenas D, Mastrubuoni G, Kempa S, Rajewsky N (2011). In vivo and transcriptome wide identification of RNA binding protein target sites. et al., Genome Research 2011). We have used Onal P, Grün D, Adamidi C, Rybak A, Solana J, Mastrobuoni G, Wang Y, Rahn HP, FACS and subsequent RNA-seq and shotgun . Gene expression of pluripotency proteomics to obtain planarian stem cells determinants is conserved between mammalian and planarian stem cells. EMBO J 31, 2755-69.Chen W, Kempa S, Ziebold U, Rajewsky N (2012) - pressed in them. Comparison to mammalian Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, embryonicand to define stem which cells genes revealed are specifically that molecu ex- Gregersen LH, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C, le Noble lar mechanisms for pluripotency are deeply potency. Nature 495, 333-338. conserved throughout life and that planarian F, Rajewsky N (2013). Circular RNAs are a large class of animal RNAs with regulatory stem cells are indeed informative for mammalian stem cell biology (Oenal et al. EMBO J 2012). Part of this work involved collaborations with the Sanchez lab (Utah), Chen, Di- Group Leader Marvin Jens eterich, and Kempa labs (MDC). Currently we Prof. Dr. Nikolaus Rajewsky Toshiaki Kogame are studying the role of alternative splicing in Sebastian Mackowiak pluripotency. Scientist Sebastian Memczak Catherine Adamidi * Pinar Oenal Dr. Anna Elefsinioti * Panagiotis Papavasileiou * Towards systematic decoding of Dr. Dominic Gruen * the “post-transcriptional regulatory Dr. Filippos Klironomos * Marcel Schilling * code” Dr. Christine Kocks (Associated ChristinMarta Rodriguez-Orejuela Stottmeister * * PI) Kathrin Theil We have started to use biochemical methods Dr. Markus Meyer * Francesca Torti such as PAR-CLIP (Hafner et al., Cell 2010) to Dr. Agnieszka Rybak-Wolf investigate mechanisms and function of post- Dr. Benedikt Obermayer (Del- Technical Assistants transcriptional gene regulation. For example, brück Fellow) Salah Ayoub we have studied the human RNA binding Dr. Jordi Solana-Garcia Margareta Herzog - Dr. Marlon Stoeckius Luisa Maier ally relevant targets of HuR and could show Dr. Ulrike Ziebold Lena von Oertzen * thatprotein HuR HuR. regulates We identified mRNA processing >4000 function and alternative splicing (Lebedeva et al., Molecular Graduate Students Secretariat Cell 2011), previously unknown functions of Andrei Filipchyk * Alexandra Tschernycheff HuR. Very interestingly, we also found that Stephanie Grosswendt HuR directly and strongly controls the ex- Andranik Ivanov (* part of the time reported) pression of one conserved human microRNA

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BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY Photo: David Ausserhofer/MDC Photo:

Baris Tursun

Gene Regulation and Cell Fate Decision in C. elegans

Cell fate decisions are fundamental during reprogramming, remains poorly understood. development of multicellular organisms. While A straight strategy to identify factors counteracting induction of cell fates is to perform ge- a few decisions can be reversed, most differ- netic screens. Forward genetics can be easily entiated cells are protected against induced applied to C. elegans enabling us to systemati- conversion of their identity [1]. The molecular cally interrogate the entire worm genome for mechanisms required for safeguarding dif- inhibitory factors. We are making use of transgenic worms that allow broad mis-expression ferent cell fates are not fully understood. We of TFs (cell fate master regulators), which are use C. elegans as a genetic model organism to systematically study the molecular basis of use a glutamatergic neuron-inducing TF, and thesufficient myogenic to induce TF MyoD distinct (HLH-1 tissue in worms), types. We as cell fate maintenance. Specifically, we are in- well as GATA TFs that induce the intestinal cell terested in mechanisms counteracting experimental attempts to induce an ectopic identity in mature cells. C. elegans provides genetic monitoringfate [2]. Also, cell these conversions worms express in vivo fluorescent (Fig.1C). Ongoingreporters mutagenesisthat label specific screenings cell types delivered thereby tractability and the instant visualization of cell several mutants that allow the ectopic induc- fate conversions in vivo. This allows unbi- tion of neuronal fates in non-neuronal tissues. ased large-scale interrogation of the genome These are currently being characterized to as- for genes playing a role in safeguarding cell - ronal characteristics in non-neuronal cells. fates. Our findings could help to overcome sess the efficiency and depth of inducing neu limitations in induced cellular transdifferentia- Reverse genetic screens to identify tion – often referred to as direct reprogram- inhibitors of inducing ectopic fates E. Kolundzic, M. Hajduskova, ML. Beato Del ming – and thereby support the development Rosal, M. Kazmierczak of future biomedical approaches. Complementary to our forward genetic screens we are performing whole-genome RNAi screens (knock-down of 20K genes). Forward genetics to identify Since evidence from previous studies suggest inhibitors of inducing ectopic that repressive chromatin signatures counter- cell fates act the induction of ectopic cell fates, we have O. Kaplan, S. Bulut, A. Ofenbauer created an RNAi sub-library that targets all C. In early C. elegans embryos, ectopic expres- elegans chromatin factors. The sub-library allows expedite examination of reprogramming can convert most cells into, e.g., neurons. regulation by epigenetic factors using different Insion contrast, of specific differentiated transcription cells factors in adults (TFs) conditions. It enables us to systematically apply a variety of parameters in order to genetically mis-expressed TFs [2]. Partly, limitations in enhance partial cell fate conversions. Manual directcannot reprogramming be reprogrammed can asbe efficientlybypassed by screens can be laborious and slow but sys- mis-expressing a cocktail of several different tematic and combinatorial genetic interroga- TFs [3]. But the actual issue, namely the mo- tions to reveal multigenic phenotypes require lecular signature causing limitations in direct high-throughput approaches. We are using

196 MDC Research Report 2014 BERLIN INSTITUTE FOR MEDICAL SYSTEMS BIOLOGY

A: We are studying limitations of directly converting cellular identities in vivo. B: The genetic model organism C. elegans provides genetic tractability and instant in vivo visualization of cell types. C: Transgenic worms allow broad mis-expression of master regulatory transcription factors (TFs) that induce glutamatergic neurons (CHE-1), the muscle fate (HLH-1/MyoD), and GATA TFs that induce the intestinal or epidermal cell fates. D: The BioSorter® (Union Biometrica) allows high-throughput scanning of 20 worms/sec and simultaneous detection of three fluorescent channels. An auto-sampling robot allows using multi-well plates for automated RNAi screening. Mutants derived from forward genetic screens are subject to whole genome sequencing to identify mutated genes.

the BioSorter® which screens 20 worms per second thereby reducing the time for a whole Reference: [1] Graf T. Cell Stem Cell 2011 genome RNAi screen from 250 (manual) to 40 [2] Tursun B. Curr Opin Genet Dev 2012 days (Fig.1D). Moreover, it provides simulta- [3] Ladewig J, Koch P, Brüstle O. Nat Rev Mol Cell Biol 2013 [4] Tursun B, Patel T, Kratsios P, Hobert O. Science 2011 [5] Patel T, Tursun B, Rahe DP, Hobert O. Cell Reports 2012. with high sensitivity. Ongoing manual screens yieldedneous detection a number of of three factors, fluorescent whose depletionchannels Selected Publications allow ectopic induction of neuronal reporters Cochella L*, Tursun B*, Hsieh YW, Galindo S, Johnston RJ, Chuang CF, Hobert O#. in epidermal, intestinal or germ cells. Such Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis - Genes Dev. 2014 Jan 1;28(1):34-43.

ing mutants for the respective genes (the Cae- elegans zinc finger transcription factor die-1. norhabditisRNAi based Geneticsphenotypes Center will provides be confirmed mutants us Patel T*, Tursun B*, Rahe DP, Hobert O#. Removal of Polycomb repressive complex 2 makes C. elegans germ cells susceptible to for more than 90% of all worm genes). Cell Rep. 2012 Nov 29;2(5):1178-86.

direct conversion into specific somatic cell types. The histone chaperone RBBP4/7 is Tursun B#. Cellular reprogramming processes in Drosophila and C. elegans. required for muscle maintenance Curr Opin Genet Dev. 2012 Oct;22(5):475-84. S. Seelk, M. Hajdusjova Tursun B*#, Patel T, Kratsios P, Hobert O#. The histone chaperone LIN-53 (human Science. 2011 Jan 21;331(6015):304-8. RBBP4/7) was previously shown to protect Direct conversion of C. elegans germ cells into specific neuron types. germ cells from direct conversion into neu- Tursun B*#, Cochella L*, Carrera I, Hobert O#. rons or muscles [4,5]. Genetic data indicate A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans. an interaction of Notch signaling with LIN-53 PLoS One. 2009;4(3):e4625. regulation for safeguarding germ cells. We * First Author#Corresponding Author are currently characterizing the nature of this intriguing interaction. LIN-53 is expressed in all tissues but its function in different cell types is not clear. Tissue-

to characterize LIN-53 in detail. Initial mo- Start of the Group: February 2012 lecularspecific andprotein cell biochemistry biological analysis is being revealed applied mis-regulation of critical TFs required for Group Leader muscle differentiation and maintenance in Dr. Baris Tursun lin-53 mutants. Hence, mutants show strong Graduate and Undergraduate muscle and motility defects and, additionally, Scientist Anuprabha Bhargava a short lifespan. Selman Bulut We expect that our efforts will yield a number Dr. Oktay Ismail Kaplan Samuel Katz of different factors and mechanisms to bet- Dr. Martina Hajduskova Maria Lena Beato del Rosal ter understand differentiation, maintenance PhD Technical Assistants and safe-guarding of cell fates. The C. elegans Marlon Kazmierczak Alina Schenk genome is ~60% homologous to those of Ena Kolundzic mammals such that our large-scale system- Andreas Ofenbauer Secretariat Stefanie Seelk Ines Krock of conserved mechanisms. atic approaches might lead to identification

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Robert P. Zinzen

Systems Biology of Neural Tissue Differentiation

How cells acquire and maintain their identity lar gene is directed by how that gene’s cis-reg- remains a fundamental question in biology. We ulatory elements (CRMs) interpret the cellular environment, e.g. which transcription factors aim to gain a detailed understanding of the (TFs) can be recruited to enhancers. Several molecular mechanisms and paths that undif- TFs crucial for NS patterning are known and ferentiated embryonic cells take to organize we are utilizing chromatin immunoprecipita- into distinct cell types and tissues. With the tion (ChIP) followed by massively parallel sequencing (Seq) to identify the genome-wide advent of modern ‘-omics’ approaches, it is - becoming increasingly possible to investigate tify the target genes of neurogenic TFs, the this problem systematically and on a genome- CbindingRMs through profiles which of these they TFs. act, Weand aimhow to TFs iden act combinatorially. This is a crucial step for un- wide scale. No longer limited to asking how derstanding a TF’s global regulatory potential, individual genes are regulated across devel- how TF combinations coordinate regulatory opment, we can now start asking how the networks, and how these networks change genome at large is regulated to coordinate across development. A caveat of ChIP-Seq studies is that binding development. We approach this problem in the context of early embryonic development in or predict transcriptional responses. To assess the fruit fly Drosophila melanogaster (D.mel.), thedata effect cannot that definitively individual identify TFs have target on globalgenes because of its ideal genetic tractability and transcription in vivo, we are utilizing the genetic tractability of D. mel. experimental accessibility. We are primarily marking embryos depending on the mutations interested in the nervous system (NS), which they carry. We will use a large-particle by fluorescently sorter derives from a large population of cells speci- to isolate pools of staged, homozygous-mutant embryos to analyze the effect of key neurogen- fied very early. This pluripotent primordium ic TFs on the developmental transcriptomes of quickly subdivides into columnar domains that give rise to specific neuroblasts. Our aim is to understand the molecular mechanisms Whilespecific some NS compartments. of the key TFs we are interested underlying NS primordium specification, NS others are employed more broadly. For ex- subdivision, as well as the precisely regulated inample, are expressed opposing inmorphogen defined subsets gradients of the signal NS, spatio-temporal delamination of differentiating via effector molecules to shape transcription neuroblasts. across the NS in a concentration-dependent manner. To understand how the morphogen gradients are interpreted at the DNA level Gene- and cis-regulatory networks across the developing NS in vivo, we are ana- for NS development L. Dämpfling, A. Klawiter, P. Wahle - lyzinglated from the developing genome-wide embryos binding – such profiles tissue of in development is driven to a large degree via effector TFs in specific NS components iso tightCoordinated spatio-temporal specification regulation and differentiation of gene ex- possible due to methodological advances (e.g. pression. Whether a cell will express a particu- BiTS-ChIP,specific analyses see below). have only recently become

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Patterning and dissection of the early Drosophila nervous system. A) Early signs of NS specification and subdivision in the cellularizing embryo: sog mRNA expression demarks the extent of the neurogenic ectoderm (NE) from which the NS derives, vnd demarks the ventral NE that gives rise to the ventral column of the NS, sim marks the ventral- most row of cells that will form the ventral midline upon gastrulation. Embryos are shown anterior left and dorsal up. B) Schematic representation of early NE subdivision; marker genes for the individual columns and their known genetic interactions are indicated. C) Workflow of the BiTS procedure for the isolation of tissue specific nuclei from staged, fixed embryos. D) Three transgenic lines for BiTS-ChIP-Seq, which direct expression of an SBP-tagged nuclear protein (visualized in red) in different parts of the developing NS.

we are assembling transgenic constructs that - tion of individual NS CRMs together with the allowproteins for thatthe tissuebind to specific them in biochemical vivo. Using isolamass spectrometry to identify the bound proteins, we are particularly interested in how CRM activity is affected by both the temporal dynamics of CRM-protein interactions across de-

of CRM-protein complexes. With this method, Tissue specific chromatin dynamics wevelopment, are not asonly well approaching as by the tissue a mechanistic specificity A. Glahs, J. Rossius understanding of individual CRMs, but once A more encompassing view of genome regulation will be obtained by investigating chroma- their broader neurogenic roles. tin state over the course of NS development. identified,Overall, canour aim probe is to novel establish interactors a predic for- We and others have shown that enrichment tive model of NS development: we want to understand the regulatory interactions driv- is not only predictive of genome features (ening tissue development to a degree that al- ofhancers, specific promoters, histone modification etc.), but also combinations of their reg- lows modeling the global effects of systematic ulatory state (active, repressed, etc.). However, perturbations. Additionally, we will assay the since chromatin features are employed dif- general applicability of regulatory principles learned in the context of D. mel. NS development to other tissues and organisms. ferentially across the embryo, tissue specific signaturinterest. esBiTS can (Batch only be isolation identified of ifTissue chromatin Spe- Selected Publications is first isolated from the cell populations of Bonn S, Zinzen RP, Perez-Gonzalez A, Riddell A, Gavin AC, Furlong EE (2012). Cell

cific chromatin) allows just that by leveraging using BiTS-ChIP. Nature Protocols. 7 (5): 978-994. type-specific chromatin immunoprecipitation from multicellular complex samples FACS,tissue whichspecific are transgenic then used markers for ChIP-Seq. to isolate We Zinzen RP, Bonn S, Girardot C, Gustafson EH, Perez-Gonzalez A, Delhomme N, havefixed been nuclei establishing of defined transgenic cell populations D.mel. lines by - Ghavi-Helmdevelopment. Y, NatureWilczynski Genetics. B, Riddell 44 (2): A, Furlong148-156. EE (2012). Tissue-specific analysis of chromatin state identifies temporal signatures of enhancer activity during embryonic - Zinzen RP, Girardot C, Gagneur J, Braun M, Furlong EE (2009). Combinatorial binding thatnar components,allow for the as identification well as in non-NS of tissue tissues. spe predicts spatio-temporal cis-regulatory activity. Nature. 462 (7269): 65-70. Thiscific chromatinallows determination signatures in of the chromatin NS, its colum state Zinzen RP, Cande J, Ronshaugen M, Papatsenko D, Levine M (2006). Evolution of the ventral midline in insect embryos. Developmental Cell. 11 (6): 895-902. Zinzen RP, Senger K, Levine M, Papatsenko D (2006). Computational models for regulatory features, and their activity dynam- neurogenic gene expression in the Drosophila embryo. Current Biology. 16 (13): icsin NSacross components, development. identification An important of genomic goal is 1358-1365. the functional integration of TF binding and chromatin state data to understand how TFs shape their chromatin environment and how the chromatin environment regulates the interaction of TFs with the genome. Start of the Group: March 2013 Sabrina Krüger Philipp Wahle Network extension Group Leader S. Krüger Dr. Robert Patrick Zinzen Technical Assistants An central aim is to gain a mechanistic under- Cristal Jane Peck standing of how individual CRMs translate Scientists Jana Rossius their cellular environment into regulatory (in)activity; however, it is clear that our pro- Alexander Glahs Secretariat visional networks of NS development are still AgnieszkaLea Dämpfling Klawiter Ines Krock very incomplete. To extend the NS networks,

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200 MDC Research Report 2014 The Experimental and Clinical Research Center (ECRC)

Coordinator: Friedrich C. Luft

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The Experimental and Clinical Research Center (ECRC)

Friedrich C. Luft

ry space and infrastructure through compe- and the Charité. The mission of the TECRChe ECRC is to is pursue a joint basic project science of the direct MDC- and the contract can be renewed, again on ly relevant to clinical medicine. The notion tition. The space is provided for five years of bringing novel ideas to patient care more is measured in publications and grant sup- quickly is termed “translational” research. port.a competitive Not all, but basis. most Scientific ECRC groups productivity are led The concept is not new. For instance insulin by clinicians who also have patient-care re- was discovered in 1921 and given to a dia- sponsibility at one of the Charité campuses betic patient in 1922. The Nobel Prize was and/or in our outpatient facility. Two of our awarded for this work in 1923. The ECRC group leaders are clinicians at the HELIOS- is there to keep that goal alive, but we are Klinikum-Berlin, a large private hospital not naïve. The 2005 international review panel founding the ECRC recognized that salary support for clinician-scientists. In the ECRC needed dedicated well-trained thisadjacent way, towe the address campus. the The problem ECRC provides of “pro- clinicians heading research groups. These tected time”, which is not generally avail- clinicians deserved a state-of-the-art infra- able for persons on a clinical salary line. structure. Equally important, the clinicians Recruitment, mentoring, and interactions required world-class basic scientists, who are our main tools at the ECRC. To entice are committed to working on clinical prob- - lems with clinicians that are generally less ated a research fellowship for clinicians at knowledgeable in the science. These mar- theclinicians MDC years into aago. scientific This fellowship career, we for initi two riages are seldom made in heaven. Who years with extension enables clinicians to remembers Bertram Collip, the biochemist additional degree. The program has been - veryaccrue popular scientific and expertise has been and adapted possibly insti an- outwho him, purified no treatment, insulin so nothat prize, the compound and more tution-wide by the Charité. Subsequent importantlycould actually no be cure. given to a human? With career development is a problem; however, two possibilities are attachment to an exist- The ECRC was designed as an interface being ECRC group or establishing a new inde- tween MDC scientists and Charité clinician/ pendent group. Another challenge is bring- scientists. We are located in a previous hos- ing MDC scientists and clinician/scientists pital building named after Robert Rössle, a together. We have developed a partnership Charité pathologist in the Virchow line of granting system to that end to foster such succession. ECRC groups acquire laborato- collaborations.

202 MDC Research Report 2014 ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER Photo: David Ausserhofer/MDC Photo:

Fig. 1. Philipp Du Bois and Jens Fielitz explain their project to Angela Merkel, while Friedrich Luft looks on.

The ECRC has a facility that exists nowhere Ideas outside the MDC could be channeled else in Berlin, although that state-of-af- via that source. Our medical school has the fairs may change in the future. We have a entire city of Berlin at its disposal. Thus, designated clinical research unit (CRU) to patient recruitment (from 4 million inhab- study humans, outside of the health-care itants) is possible as is nation-wide recruit- system and solely for the purposes of sci- ment. We view “translational research” as ence. This unit is operative since 2000 and has been extremely productive in fostering beating the diabetes team of Banting, Best, investigator-initiated mechanistic research. McLeod,our job. We and admit Collip. that But we this cannot goal is guarantee a worth- Much very valuable clinical research can be while aim. performed in a patient-care setting. Most

this venue. For instance, comparing tablet Research on human subjects in Aclinical with tablet research B, or viewed new cancer by the treatment public fits X the Clinical Research Unit (CRU) compared to old cancer treatment Y, or coated stent Q versus bare-metal stent R needs Patient/proband-oriented research is the no special unit because extra resources to test these questions are minimal. More dif- to humans or in revealing physiological orfinal pathological step in bringing mechanisms medical in innovations man. This suppose we develop a new stem-cell thera- bench-to-bedside and bedside-to-bench pyficult for are a genetic studies or involving acquired “new human ideas”. disease. Just process is today conducted under the trendy The commercial application is not obvious catchword “translational” research. Our and the health-care system is not enamored CRU is modeled after the US general clinical with the idea of supporting this endeavor. research centers and has functioned well That is where the CRU steps in. since 2000. We have succeeded in making the CRU independent of any patient-care The uniqueness of the ECRC is the mission. endeavors that concern third-party payers We are at the source of medically related - basic science. The MDC is the best broad- cians, nurses, scientists, technical experts based institute that Germany has to offer. and(cross students. financing). We have recruited physi

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Fig. 2. Our CRU organizational concept is adapted from the general clinical research center idea formulated by NIH. We supply infrastructure and various services for any investigator approved by the Institutional Advisory Committee.

Our CRU is located in a former hospital and glucose-clamp studies, and indirect building and relies on excellent facilities calorimetry. We also conduct detailed body included the former new bone-marrow composition measurements. We adapt our transplant unit. We are well equipped for capabilities to the investigator’s needs. Any cardiovascular and metabolic studies with clinical investigator at the Charité is eligible resources not available elsewhere, in- to use our facility and its infrastructure af- cluding a normobaric hypoxia chamber, a ter applying to and receiving approval by total-body metabolic chamber, and a large our Institutional Advisory Committee. gamut of techniques including microdialy- sis, spiro-ergometry, muscle nerve record- We have not focused on inpatient studies ings, lower-body negative pressure, insulin but could do so if required. As a result, for

Fig. 3. Michael Boschmann introducing notable guests to the CRU metabolic chamber. Photo: David Ausserhofer/MDC Photo:

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pharmacological investigations we currently are not doing phase 1 studies but conduct Recent CRU Publications phase 2 and phase 3 clinical trials, although Haufe S, Haas V, Utz W, Birkenfeld AL, Jeran S, Böhnke J, Mähler A, Luft FC, Schulz- our focus has been primarily on physician- Menger J, Boschmann M, Jordan J, Engeli S. Long-lasting improvements in liver fat investigator initiated mechanistic research. and metabolism despite body weight regain after dietary weight loss. Diabetes Care. We have cooperated actively with the MDC 2013;36:3786-92. magnetic resonance imaging center, par- Haufe S, Utz W, Engeli S, Kast P, Böhnke J, Pofahl M, Traber J, Haas V, Hermsdorf M, ticularly with Jeanette Schulz-Menger. Our Menger J, Jordan J. Left ventricular mass and function with reduced-fat or reduced- mission is also directed at supporting our Mähler A, Busjahn A, Wiesner S, Otto C, Mehling H, Luft FC, Boschmann M, Schulz- outpatient clinics. In the outpatient clinics, 2012;59:70-5. we have excellent ultrasound capabilities carbohydrate hypocaloric diets in overweight and obese subjects. Hypertension. Boschmann M, Nussberger J, Engeli S, Danser AH, Yeh CM, Prescott MF, Dahlke M, Jor- including state-of-the-art echocardiogra- dan J. Aliskiren penetrates adipose and skeletal muscle tissue and reduces renin-an- phy and a laboratory aimed at biobanking giotensin system activity in obese hypertensive patients. J Hypertens. 2012;30:561-6.

samples appropriately. Singer E, Elger A, Elitok S, Kettritz R, Nickolas TL, Barasch J, Luft FC, Schmidt-Ott KM. Urinary neutrophil gelatinase-associated lipocalin distinguishes pre-renal from intrinsic renal failure and predicts outcomes. Kidney Int. 2011;80:405-14.

Human Data Management Lechner SG, Markworth S, Poole K, Smith ES, Lapatsina L, Frahm S, May M, Pischke S, Suzuki M, Ibañez-Tallon I, Luft FC, Jordan J, Lewin GR. The molecular and cellular For data management of all human data, we identity of peripheral osmoreceptors. Neuron. 2011;69:332-44. use REDCap, a clinical data management system (also called electronic case report form) developed at Vanderbilt University in 2004. The REDCap Consortium is composed of 873 active institutional partners in 71 countries and is currently used in more Clinical Research Unit staff - ers. Thus, REDCap is one of the most fre- Friedrich C. Luft, MD quentlythan 89,000 used projects clinical datawith management over 116,000 sys us- Michael Boschmann, MD Heidrun Mehling, MD REDCap consortium. The entire program Natalia Rakova, PhD tems worldwide. Our group has joined the Jochen Steiniger, PhD org. We will now manage all our outpa- Verena Haas, PhD tientcan be contacts inspected with at: an www.project-redcap. electronic medical Nadine Krüger records system that we will install in 2014. Gabriele Rahn The Regenstrief Institute is an internation- Doris Merkert ally respected informatics and healthcare Anke Strauss research organization, recognized for its role in improving quality of care, increasing Anna Pakula - LarsAnja KlugMähler ing medical errors and enhancing patient Robert Preissner MD (Bioinformatics consultant) safety.efficiency The of Regenstrief healthcare Institute, delivery, preventInc., an internationally recognized informatics and healthcare research organization, is dedicated to the improvement of health through Outpatient clinics research that enhances the quality and cost- effectiveness of health care, by conducting Psychiatry (Memory and cognition) health-services research (HSR). HSR works Psychiatry (Post-stroke depression) to improve healthcare delivery and health Psychiatry (Fragile X syndrome) outcomes of patients through research on Neurology (Skeletal muscle disorders) patient safety, quality of care, patient-pro- vider communication strategies, applied Cardiology (Cardiomyopathy) health information technologies, system NephrologyNeurology (Inflammatory (Diabetes registry) CNS disorders) redesign, and medical education research. Metabolism (Diabetes and lipids) This healthcare information technology can Pediatrics (Pulmonary/allergic disease) be inspected at: www.regenstrief.org.

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ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER Photo: Peter Himsel/BBB GmbH Himsel/BBB Peter Photo:

Simone Spuler

Muscle Research Unit and Outpatient Clinic for Muscle Disorders of the Charité

Simone Spuler directs the skeletal muscle Dysferlinopathies disease program project (DFG_KFO192) Dysferlin, a large protein located at the sar- for the entire Charité. Much of the re- colemma, is involved in membrane repair search activities are located at the ECRC. - The outpatient clinic at the ECRC cares for quence of normal muscle activity. Mutations >1000 patients with genetic and acquired inafter dysferlin microinjury, lead to a muscular physiological dystrophy. conse Sarcolemmal repair is a complex multicom- skeletal muscle diseases. In 2010, KFO192 ponent process. We demonstrated that the was complemented by the international membrane repair process always involves research-training grant for myology, “Myo- Grad” (GK1631), recently recommended for the dynamin-related ATPase EHD2 as a new componentthe accumulation of the ofmembrane F-actin and repair identified com- renewal 2014 - 2019. This worldwide unique plex (Marg et al., 2012). In future studies training grant crosses national boundaries Andreas Marg and Tobias Timmel will dis- and unites the Université Pierre et Marie sect the repair process further. Curie in Paris, Université St. Quentin en Dysferlinopathies not only lead to faulty Yvelines in Versailles (French Speakers, membrane repair but appear to lead to Thomas Voit and Helge Amthor), with the Berlin located institutions: Free University, muscle. We found accumulation of lipids in specific metabolic alterations in skeletal Charité Medical Faculty and Max Delbrück MD et al., Am J Path 2014, in press). Stefan Center for Molecular Medicine (German Kempa’sdysferlin-deficient group at the type BIMSB 2 fibers and our (Grounds group Speaker, Simone Spuler). A binational de- are cooperatively investigating the mecha- gree (cotutelle de thèse) can be achieved nism. Verena Schöwel demonstrated that missense-mutated dysferlin can be rescued by graduation within MyoGrad. to function in vitro if the mutated protein The scientific focus of Simone Spuler’s is reallocated to the sarcolemma (Schoewel group is the pathophysiology and metabolic et al., 2012). She generated a new mouse model harboring a missense mutant in Dysf consequences of muscular dystrophies and will continue her studies in vivo. caused by genes encoding sarcolemmal proteins (dysferlin, caveolin 3) and related Susanne Philippi, Jakub Malcher and Hele- acquired disorders (statin-myopathy, critical na Escobar repair dysferlin at pre-mRNA or illness myopathy). Gene repair is a cooperative interest in MyoGrad subproject P6, TheseDNA level are closewith the cooperations overall aim with to find Luis a cureGar- headed by Simone Spuler and Luis Garcia. cia,for dysferlin-deficientUniversité St. Quentin muscular en Yvelines dystrophy. and

Zsuzsanna Iszvák (MDC). 206 MDC Research Report 2014 ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER

Annexin A1- and F-actin accumulation at the wounding area after laser irradiation (A) Annexin A1 immunostaining of a human myotube shows an enrichment of the protein after laser wounding. (B) Phalloidin-atto 565 staining demonstrates the accumulation of F-actin at the same membrane area. (C) Overlay of A and B.

Satellite cells Selected Publications Two separate repair mechanisms ensure Marg A, Schoewel V, Timmel T, Schulze A, Shah C, Daumke O, Spuler S. Sarcolemmal -

the survival of muscle fibers: Membrane re repairSchoewel is a V, slow Marg process A, Kunz and S, Overkampincludes EHD2. T, Siegert Traffic. Carrazedo 2012;13:1286-94. R, Zacharias U, Daniel PT, as outlined above and new formation of Spuler S. Dysferlin-peptides reallocate mutated dysferlin thereby restoring function. musclepair after via minute activating injury the to stem the sarcolemmacells proper PloS One. 2012; 7: e49603. of skeletal muscle, the satellite cells. Muscle Weber-Carstens C*, Schneider J*, Wollersheim T, Assmann A, Bierbrauer J, Marg A, Al satellite cells have the full potential to pro- Hasani H, Chadt A, Wenzel K, Koch S, Fielitz J, Kleber C, Faust K, Mai K, Spies CD, Luft liferate, differentiate, and conduct repair. We are exploring the potential of human and muscle contraction. Am J Resp Crit Care Med 2013; 187: 387-96. *equal contribu- tionFC, Spranger J, Spuler S. Critical illness myopathy and GLUT4 – significance of insulin satellite cells derived from adult donors to regenerate muscle. These investigations Timmel T, Schuelke M, Spuler S. Identifying dynamic membrane structures with atomic-force microscopy and confocal imaging. Microsc Microanal. 2014:1-7. [Epub also aim towards a therapy of muscular ahead of print] dystrophies. Grounds MD, Terrill J, Radley-Crabb H, Robertson T, Papadimitriou J, Spuler S, Shav-

[Epub ahead of print] PubMed PMID: 24685690. Acquired myopathies: Statin lakadze T. Lipid accumulation in dysferlin-deficient muscles. Am J Pathol. 2014, induced myopathy and metabolic aspects of critical illness myopathy

Inhibitors of 3-hydroxy-3-methylglutaryl- coenzym-A-reductase (statins) are pre- scribed to millions of patients of whom 10% develop muscle related side effects. Group Leader Joanna Schneider (M.D. student, We are exploring genetic and structural Prof. Dr. med. Simone Spuler until 4.2013) aspects of this common myopathy. Critical Dr. med. Verena Schöwel illness myopathy occurs as a severe side Scientists Dr. Ing. Tobias Timmel effect of intensive care unit treatment. In a Maximilian Berger (M.D. student) large translational and interdisciplinary ap- Dr. med. Ulrike Grieben Technicians proach that involved several ECRC groups Dr. rer. nat. Stefanie Grunwald Stefanie Belz we have shown that the failure to activate Leonie Heidt (M.D. student) Kornelia Gräning AMPK and to translocate GLUT4 to the sar- Séverine Kunz (Ph.D. student) Stephanie Meyer-Liesener (ma- - Jakub Malcher (Ph.D. student) ternity leave) cular atrophy. Dr. rer. nat. Andreas Marg Adrienne Naporra colemma contributes significantly to mus Tobias Opialla (Ph.D. student, together with Stefan Kempa, BIMSB) Manager of sponsored Primary funding sources Vinco Palava (Ph.D. student) programs Deutsche Forschungsgemeinschaft Susanne Philippi (PhD student) Susanne Wissler (KFO192 and MyoGrad IGK1631).

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Ralph Kettritz

Nephrology and Inflammatory Vascular Diseases

The group studies neutrophil functions in cause the glomerular lesions. Several leuko- health and disease and molecular mechanism cyte effector functions are implicated in NCGN. Most of this information is derived from in that control important neutrophil responses. vitro experiments and the in vivo role needs In addition to protecting the host from infec- to be established. For example, active neutro- tions, the neutrophil participates in autoimmu- phil serine proteases (NSPs) may modulate nity, cardiovascular diseases, and cancer. Our cytokines, growth factors, surface receptors, main clinical interest deals with patients who thesignaling inflammatory molecules, responseand extracellular by processing matrix suffer from systemic necrotizing vasculitis. proteins. It is also conceivable that active NSPs This highly inflammatory vasculitis is caused - phil elastase (NE) and cathepsin G (CG) are by autoantibodies directed against neutrophil participatethe most abundant in IL-1β NSP generation.s residing PR3, in neutro- proteins. Patients develop either anti-neutrophil granules and monocyte lysosomes. NSP phil cytoplasmic autoantibodies (ANCA) to activation requires proteolytic processing by proteinase 3 (PR3) or antibodies directed at the lysosomal cysteine protease dipeptidyl peptidase I (DPPI). Using our experimental myeloperoxidase (MPO). ANCA bind to their model, we showed that DPPI-/- target antigens on neutrophils and monocytes marrow (BM) protected from NCGN. Mice re- and these activated cells then interact with constituted with NE-/-xPR3-/- BMdeficient were equally bone protected against NCGN and this effect was the endothelium leading to vascular damage. The disease is truly systemic in that each and -/- BM-reconstituted every organ can be involved. Kidney and lung accompaniedanimals. Compared by a significantlyto WT monocytes, reduced DPPI IL-- /- involvement is most frequent and associated 1β generation in DPPI when stimulated with anti-MPO antibodies. with high mortality. Standard treatment con- monocytes produced less IL-1β in vitro sists of steroids, cytotoxic, anti-CD20 directed dependent as exogenous addition of PR3 cor- drugs, and plasma exchange. With these The reduction in IL-1β production was NSP- treatments patient and kidney survival has rectedanakinra, the protected defect in DPPI-deficient animals against monocytes. anti-MPO improved. However, treatment-related side Finally, the specific IL-1 receptor antagonist, effects are life-threatening. Improved understanding of disease mechanisms will result in Ab-induced NCGN suggesting that IL-1β is a novelcritical therapeutic inflammatory targets mediator for ANCA-mediated in this model. better treatment. diseasesWe believe in humans.that NSPs and IL-1β may provide

Endothelial cells acquire functional Studying disease mechanisms in a neutrophil proteins murine ANCA disease model We have developed an ANCA model for necro- leave the blood stream and migrate towards tizing crescentic glomerulonephritis (NCGN). During inflammation activated neutrophils- In this model, anti-MPO antibodies activate trophils interact with endothelial cells (EC). neutrophils and monocytes and these cells theWe speculated inflammatory that site. during In thisthis process,process neu -

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trophil proteins are transferred to the EC. MPO is one of the most abundant neutrophil proteins. Active MPO has the unique capacity to oxidize chloride ions and utilizes hydrogen peroxide formed during the respiratory burst to generate hypochlorous acid (HOCl), a potent microbicide that contributes to host defense. However, MPO was implicated in the promotion of cardiovascular disease and elevated plasma MPO provides an independent

increased cardiovascular morbidity and mor- vascular inflammation marker that is linked to vascular functions and promotes atherosclerosis.tality. Precisely MPO adversely how the endothelium influences important acquires active MPO from neutrophils was not clear. EC could capture soluble MPO released into the bloodstream by activated neutrophils. We established an additional mechanism where MPO is transferred at intimate neutrophil-EC contacts. This effect was dependent on neutrophil number, exposure time, and incubation temperature. Transfer occurred in several EC types, increased with endotoxin, and was not accompanied by MPO release into the medi-

MPOum. Addition transfer. of EC-acquired blocking antibodies MPO was against enzymat β2- icallyintegrins active, and as β2 demonstrated integrin deficiency by its abrogated ability to - ence of a hydrogen peroxide source and might An endothelial cell has acquired MPO (green) from an theroxidizeefore aminophenyl participate in fluorescein vascular damage in the pres and adherent neutrophil. The EC nucleus is in blue and the cell membrane in red. could be therapeutically relevant in athero- dysfunction.sclerosis and Wevasculitis. believe that these findings Selected Publications Genetic regulation of the PR3 pre- Jerke, U, Rolle, S, Purfurst, B, Luft, FC, Nauseef, WM, Kettritz, R: b2 integrin-mediated senting neutrophil surface receptor cell-cell contact transfers active myeloperoxidase from neutrophils to endothelial CD177 cells. J Biol Chem, 288: 12910-12919, 2013. Schreiber, A, Pham, CT, Hu, Y, Schneider, W, Luft, FC, Kettritz, R: Neutrophil serine pro- granulomatosis with polyangitis (formerly - Wegener’sPR3 is the majorgranulomatosis). autoantigen PR3 in patients is harbored with phritis. J Am Soc Nephrol. 23: 470-482, 2012. teases promote IL-1βeta generation and injury in necrotizing crescentic glomerulone by the entire neutrophil population but is bi- Jerke, U, Rolle, S, Dittmar, G, Bayat, B, Santoso, S, Sporbert, A, Luft, F, Kettritz, R: modally presented on the neutrophil mem- Complement Receptor Mac-1 Is an Adaptor for NB1 (CD177)-mediated PR3-ANCA brane yielding membrane PR3 (mPR3)low and Neutrophil Activation. J Biol Chem, 286: 7070-7081, 2011. high (mPR3) subsets. We found in our patient Bontscho, J, Schreiber, A, Manz, RA, Schneider, W, Luft, FC, Kettritz, R: Myeloperox- cohort that the larger the mPR3high subset the worse the clinical disease course. From a twin cytoplasmic autoantibodies-induced glomerulonephritis. J Am Soc Nephrol. 22: 336- idase-specific348, 2011. plasma cell depletion by bortezomib protects from anti-neutrophil study and a study in HLA matched siblings we learned that the percentage of mPR3high Schreiber, A, Rolle, S, Peripelittchenko, L, Rademann, J, Schneider, W, Luft, FC, Kettritz, R: Phosphoinositol 3-kinase-gamma mediates antineutrophil cytoplasmic autoanti- neutrophils is genetically determined with body-induced glomerulonephritis. Kidney Int. 77: 118-128, 2010. large effects coming from the HLA region. We - ously as a membrane receptor for PR3. CD177 rdescribedeceptor protein neutrophil-specific and full-length CD177 mRNA previ are expressed by the CD177positive/mPR3high popu- Group Leader lation, but not by the CD177negative/mPR3low Prof. Dr. Ralph Kettritz neutrophil population. We are now studying the genetic and epigenetic mechanisms that Graduate students and clinical Associated scientists and clini- control the restricted CD177 gene expression. fellows cal scientists We perform DNA and cDNA analysis of the PD Dr. Adrian Schreiber Prof. Friedrich C. Luft CD177 gene and genome-wide, copy-number PD Dr. Mira Choi variation analysis, haplotype studies in trios, Dr. Julia Bontscho Technical Assistants Dr. Uwe Jerke Susanne Rolle and non-coding RNAs. We will assess blood- Dr. Claudia Eulenberg Sylvia Krüger andstudies stem on cell-derived methylation, human histone neutrophils. modifications,

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Jeanette Schulz-Menger

Cardiac MRI

The CMR group at the Experimental Clinical During the last two years we were installing a Research Center (ECRC) and Clinics for Car- post-processing lab (CMR reading and development) giving us the opportunity to speed diology and Nephrology of the HELIOS Clinics up processes. In 2011 we founded an aca- Berlin Buch has focused research on in vivo demic outpatient department of cardiology assessment of functional and structural myo- focused on myocardial disease at the ECRC cardial abnormalities related to non-ischemic and established a research database dedicated on patient oriented research. diseases and ischemic heart disease. Our The group has several outreach activities. Jea- main hypothesis is, that the in-vivo differentia- nette Schulz-Menger is one of the Principal tion of myocardial structural injury is possible Investigators of the Charité within the Ger- man Center of Cardiovascular Research and in preserved ejection fraction and will help to is co-heading the Focus Group for Cardiovas- explain remodeling processes. Furthermore, cular Imaging of the German National Cohort. we understand cardiovascular disease as Furthermore, she was elected as member of systemic disorders and aim to understand the Executive Committee of the Society for Cardiovascular Magnetic Resonance. Florian the mechanism and the interaction of vascu- von Knobelsdorff and she were members of lar and cardiac disorders. In many projects, the writing group in societal position state- we are treading the path from the phantom/ ments. model including development of techniques Myocardial tissue injury into clinical research. Developing and es- and integrity tablishing innovative imaging biomarker will allow for quantification of structural integrity The assessment of myocardial tissue changes during follow-up is an ongoing challenging and in-vivo evaluation of prognostic relevant task in cardiovascular research and clinical processes. cardiology and has been our main focus since As Cardiovascular Magnetic Resonance years. The in vivo differentiation of myocardi- (CMR) has the unique possibility to differenti- Figure 1. Quantitative assessment of focal fibrosis ate myocardial injury, we are focusing on clini- in hypertrophic cardiomyopathy. cal research using a 1.5 clinical MRI-scanner. The group was able to expand the research activities by developing translational research tools applying advanced imaging modalities in close collaboration with the Berlin Ultra-High- Field Facility (B.U.F.F.). As we were a part of the application and establishment process one of the key aspects is cardiovascular research. This could only be established fast, because the head of the Experimental MR- Group (Prof Niendorf) has a strong interest and experience in CMR-research.

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(patho-)physiological mechanism is the driv- ingal structure, force of our cell research. integrity CMR and identificationhas the capabil of- ity to differentiate between the various forms

fraction.of myocardial During injuries the last (e.g. years edema, we hyperemia have pre- paredand fibrosis) the ground already to the in preservednext step towards ejection - ing assessment of extracellular volume. The underlyingquantification approach of structural is parametric changes mapping. includ

areAs a currentlyfirst step weworking published on therecently assessment (2014) Figure 2. In-vitro 4D flow imaging of heart valve prostheses ofnormal non-ischemic values at differentheart diseases. field strengths We expect, and

wash-out kinetics of contrast-media, but this that cell-integrity can be quantified using the- Selected Publications

publishedcan be influenced in 2013. by their specific character von Knobelsdorff-Brenkenhoff F, Tkachenko V, Winter L, Rieger J, Thalhammer C, Hezel F, Graessl A, Dieringer MA, Niendorf T, Schulz-Menger J.Assessment of the right istics. We have faced its potential influence as ventricle with cardiovascular magnetic resonance at 7 Tesla.J Cardiovasc Magn Reson. 2013 Mar 14;15:23.

engaged in myocardial steatosis measured by Utz W, Engeli S, Haufe S, Kast P, Böhnke J, Haas V, Hermsdorf M, Wiesner S, Pofahl Since 2010 (first publication) we have been- M, Traber J, Luft FC, Boschmann M, Jordan J, Schulz-Menger J.Int J Cardiol. 2013 Aug encing factor on myocardial remodeling. We 10;167(3):905-9. Epub 2012 Apr 10. investigatedmyocardial triglyceridesobese otherwise (MTG) healthy as an female influ Dieringer MA, Hentschel J, de Quadros T, von Knobelsdorff-Brenkenhoff F, Hoffmann W, Niendorf T, Schulz-Menger J. Design, construction, and evaluation of a dynamic MR 2011 we could publish an inverse relation compatible cardiac left ventricle model. Med Phys. 2012 Aug;39(8):4800-6. with preserved ejection fraction. Already in Wassmuth R, Prothmann M, Utz W, Dieringer M, von Knobelsdorff-Brenkenhoff F, Greiser A, Schulz-Menger J.Variability and homogeneity of cardiovascular magnetic resonance myocardial T2-mapping in volunteers compared to patients with edema.J MTGbetween and overweightthe relation andbetween fitness, LVH followed and MTG. by Cardiovasc Magn Reson. 2013 Mar 27;15:27. 2012 publication on the influence of diet on Vascular MR Rudolph A, von Knobelsdorff-Brenkenhoff F, Wassmuth R, Prothmann M, Utz W, ahead of print] As mentioned, we are understanding cardio- Schulz-Menger J.J Magn Reson Imaging. 2013 Oct 22. doi: 10.1002/jmri.24264. [Epub vascular diseases as systemic disorders and so far we have also started work an vascular changes of the great vessels including quan- Group leader Univ-Prof. Dr. Jeanette Pierre-Lars Hennig arterialtification hypertension, of wall alteration. aortic Aortic valve dilatation diseases Schulz-Menger Josephine Kermer andis a frequentfollowing finding aortic valvein different replacement. diseases Apart like Anna-Katharina Müller from diseases of the aortic wall itself, hemo- Scientists Johanna Richau dynamic factors are supposed to promote Dipl.-ing Matthias Dieringer Johannes Schüler aortic remodeling. We could establish con- Privatdozent Dr. Florian v. Etsham Shahid trast-media free techniques and applying 4D Knobelsdorff-Brenkenhoff Ralf Felix Trauzeddel Marcel Prothmann Felix Wanke and remodeling in different aortic valve dis- Dr. Andre Rudolph Lennart Wurche easesflow imagingcould be interaction published in of 2013. hemodynamics Currently - Dr. Agnieszka Töpper Technical Assistants nostic factors for further remodeling. Dr.Valerij Julius Tkachenko Traber Denise Kleindienst we are working on the identification of prog Dr. med. Dipl.-Phys. Wolfgang Kerstin Kretschel Cardiac MR at Ultra Highfield Utz Franziska Neumann (Koop Bayer) The close collaboration with B.U.F.F. at MDC Graduate students/student co- Evelyn Polzin - worker - Sana El-Mahmoud Study nurse/Project assistance acallowed function to publish of the right several ventricle joint paperin volunteers includ Henriette Gruettner Annette Koehler ating 7.0 the Tesla worldwide in 2013. first This paper challenging assessing research cardi Luisa Grosse Elke Nickel is ongoing and we expect further results soon.

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Maik Gollasch

Registry Diabetic Nephropathy / Blood Vessel Function and Target-Organ Damage

Current projects towards identifying a role of TMEM16a in Our group focuses on ion channels, primarily collateral arterial networks. In collaboration in vascular smooth muscle cells (VSMC), to with Ferdinand Le Noble, we have examined clarify mechanisms contributing to hyperten- the role of the Notch ligand Delta-like 4 (Dll4) sion and cardiovascular disease. Potassium in formation of collateral arterial networks channels, chloride channels and transient and in peripheral ischemia. We found that loss receptor potential (TRP) channels have re- of Dll4 in mice stimulates collateral formation ceived special attention. In collaboration with and angiogenesis, but in ischemia such ben-

smooth muscle TMEM16a chloride channels. functionality. We have also examined TRPV1 WeBjörn showed Schroeder, that weVSMC investigated TMEM16 the channels role of andeficial TRPV4 effects channels are overruled in regulating by adverse renal vesselblood function as regulator of agonist-dependent - arterial constriction and systemic blood pres- vator 20-HETE may contribute to ischemia/ sure. We are currently studying angiotensin flow. TRPV1 channels and their natural acti (Ang) II type 1a receptors (AT1aR) and ryanodine receptor isoforms (Figure 1) in VSMC reperfusionEicosanoids (I/R) and -induced inflammation kidney injury. potassium channel gating and myogenic tone. EETs serve as endothelial-derived hyperpo- Michael Bader and Thomas Jentsch are help- larizing factors (EDHF), but may also affect ing us here. We also collaborate with Huang Yu, Hong Kong, China. We found that protein mechanisms. Our current research in this kinase Cdelta plays a pivotal role in angioten- areacardiovascular is directed function towards byidentifying anti-inflammatory red blood sin II-induced endothelial cyclooxygenase-2 cells as source of vasodilatory EETs and me- expression, which represents a novel path- tabolism of eicosanoids by soluble epoxide hydrolase (sEH). We found that variations in the human sEH gene are associated with wayadipose to limit tissue vascular as a source inflammation. for relaxing We alsofac- tors.have Here, a project we collaborate focusing on with the perivascularWolf-Hagen ablation. Hypertension and vascular dysfunc- Schunck, who has peaked our interests in tionrecurrence result ofin atrial the increasedfibrillation expressionafter catheter of eicosanoids. Finally, in collaboration with the German Institute of Human Nutrition (DifE), angiotensin II-induced endothelial COX-2 we are studying human diabetic nephropathy upregulationCOX-2. We identified and release a novel of roleproatheroscle of PKCδ in- with a focus on genetics. rotic cytokine monocyte chemoattractant

TMEM16a and TRP channels curtailing endothelial COX-2 expression as a TMEM16a, TRPC6, and TRPV1 channels are protein-1. The findings raise the possibility of expressed in the vasculature. We used smooth also found that CLDN3 plays an important rolemean in of the limiting passage vascular of leukocytes inflammation. across the We found that TMEM16a downregulates agonist-inducedmuscle specific vasoconstrictions TMEM16a deficient and mice thereby and to new therapies for multiple sclerosis and contributes to blood pressure regulation. brain vasculature. These findings may lead- Our current research in this area is directed mation is common. Alzheimer’s disease, in which neuro-inflam

212 MDC Research Report 2014 ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER

Figure 1. Intracellular release of calcium ions by ryanodine type II receptor calcium channels in mesenteric artery smooth muscle cells. Intracel- lular calcium release is visualized by calcium sensitive dyes and Nipkow spinning disc microscopy. Three freshly-isolated cells are shown in this figure: The upper two cells are isolated from wild- type (WT) mice; the lower cell is isolated from a ryanodine receptor type 2 deficient (KO) mouse. The stimulator of intracellular ryanodine receptors caffeine (10 mM) induced a strong calcium release signal in WT, but not in KO cells.

Vasodilator signals from Primary funding perivascular adipose tissue The Deutsche Forschungsgemeinschaft (GO766/12-1; GO766/13-1; GO766/15-1; produced in the perivascular adipose tissue GO766/17-1; GO766/18-1, FOR1368), DAAD (ADRF).We have Our identified recent work a vasorelaxing showed that KCNQ factor and cooperative grants between the MDC

channels could represent the subtype of Kv and ECRC have funded this work. channels involved. The “third gas”, namely

H2S, could represent ADRF. However, other

alterations in the paracrine control of arterial toneadipokines by periadventitial may also play adipose a role. Wetissue identified in ani- Selected Publications mal models of hypertension and metabolic - disease. KCNQ and cystathionine gamma-ly- enbaum J, Vitzthum H, Gollasch M, Ehmke H, Schroeder BC, Hübner CA. Disruption of Heinzevascular C, Ca2+-activated Seniuk A, Sokolov chloride MV, Huebner currents AK, lowers Klementowicz blood pressure. AE, Szijártó J Clin Invest.IA, Schleif 2014 Feb 3;124(2):675-86. the role of Kv channels and H2S. ADRF and its aseputative deficient targets mice (KCNQ are available channels) to us might to clarify represent exciting new targets for the develop- Kooij G, Kopplin K, Blasig R, Stuiver M, Koning N, Goverse G, van der Pol SM, van Het Hof B, Gollasch M, Drexhage JA, Reijerkerk A, Meij IC, Mebius R, Willnow TE, Müller ment of drugs for treatment of cardiovascular D,PubMed Blasig PMID:IE, de Vries24356983. HE. Disturbed function of the blood-cerebrospinal fluid barrier aggravates neuro-inflammation. Acta Neuropathol. 2013 Dec 20. [Epub ahead of print] and metabolic disorders. Cristofaro B, Shi Y, Faria M, Suchting S, Leroyer AS, Trindade A, Duarte A, Zovein AC, Iruela-Arispe ML, Nih LR, Kubis N, Henrion D, Loufrani L, Todiras M, Schleifenbaum Genetic renal diseases J, Gollasch M, Zhuang ZW, Simons M, Eichmann A, le Noble F. Dll4-Notch signaling determines the formation of native arterial collateral networks and arterial function An outgrowth of Maik Gollasch’s clinical re- in mouse ischemia models. Development. 2013 Apr;140(8):1720-9. sponsibilities has been a focus on clinical Zavaritskaya O, Zhuravleva N, Schleifenbaum J, Gloe T, Devermann L, Kluge R, Mlad- genetics related to renal diseases. We have enov M, Frey M, Gagov H, Fésüs G, Gollasch M, Schubert R. Role of KCNQ channels in performed functional analyses of mutations skeletal muscle arteries and periadventitial vascular dysfunction. Hypertension. 2013 Jan;61(1):151-9.

emphasis on TRPC6 channels in focal and - segmentalcausing familiar glomerulosclerosis kidney diseases with(FSGS). specific We - Köhntial vasoregulation C, Schleifenbaum of rat J, andSzijártó mouse IA, aortas.Markó L,PLoS Dubrovska One. 2012;7(8):e41951 G, Huang Y, Gollasch M. Dif found that the proteoglycan syndecan 4 regu- ferential effects of cystathionine-γ-lyase-dependent vasodilatory H2S in periadventi lates TRPC6 channels in podocytes via RhoA/ Rho-associated protein kinase signaling. We are also studying human diabetic nephropathy with a focus on genetics. For these pur- Group Leader Graduate students poses, we recently established an Outpatient Univ.-Prof. Dr. med. and clinical fellows Kidney Clinic at Charité Campus Buch and the Dr. rer. nat. Maik Gollasch Chen Lan Registry of Diabetic Nephropathy (http:// Lena Löhr www.charite-buch.de/rdn/). We are continu- Associated Scientists Dr. Marwan Mannaa ously recruiting patients for our Registry and Dr. Galyna Dubrovska Dmitry Tsvetkov (ERA-EDTA fellow) - Dr. Mario Kaßmann ies. Through these studies, we hope to iden- Dr. Johanna Schleifenbaum tifyrenal/vascular novel mechanisms disease-specific leading to family increased stud Dr. Jean-Yves Tano (Alexander TechnicianDr. Istvan A. Szijarto cardiovascular risk and target-organ damage von Humboldt fellow) Yoland-Marie Anistan and novel treatment targets.

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ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER Photo: David Ausserhofer/MDC Photo:

Silke Rickert-Sperling

Cardiovascular Genetics

The Sperling Lab focuses on understanding cardiac transcription factors (Gata4, Mef2a, the molecular basis of cardiac dysfunction and - tions (H3ac, H4ac, H3K4me2, and H3K4me3) cardiovascular disorders, in particular congen- andNkx2.5, micr andoRNA Srf), expression.activating histone However, modifica it is ital heart malformations. Our team combines widely unknown which impact the exact po- molecular biology, bioinformatics and clinical sition of histone marks (relative to the tran- knowledge in systems biology approaches scription start site) has on cardiac and skeletal muscle gene expression. To elucidate this to study cardiac development and muscle question, we determined the genomic binding maturation in human and mice. We investigate disease-associated genes and epigenetic RNA expression in murine myoblasts and dif- profilesferentiated of three myotubes. histone Computational marks as well analyas the- modulators in the genomic, transcriptomic sis and integration of the data showed that a and proteomic context. We have identified - the transcription factor CITED2 as a human disease gene and characterized the chroma- wposition-dependenthich will hopefully enhanceprofile of our histone understand modi- ficationsing of muscle marks maturation. muscle tissue specific genes, tin factor DPF3, the first protein shown to bind Chromatin remodeling is essential for regulat- histone acetylation and methylation marks via ing the access of the transcription machinery its PHD domain. Moreover, we study tran- to the DNA. We characterized the epigenetic scription networks underlying heart devel- transcription factor DPF3 (BAF45c), which recruits the BAF chromatin remodeling complex opment and showed the interplay between to acetylated as well as methylated histones. multiple cardiac transcription factors and his- Thus, it connects distinct regulatory signals of tone modifications. Finally, we demonstrated read-out based on its neural, cardiac and mus- the polygenic origin of Tetralogy of Fallot by the histone code and enables a tissues-specific introducing a novel computational approach protein’s function, we generated Dpf3 knock- to identify disease-related genes. Our long- outcle specificmice. The expression. animals Toare further viable analyzeand fertile; the term goal is to combine knowledge of mo- they now await in-depth characterization by long-term monitoring, cardiac imaging and ex- lecular etiologies and mechanisms to improve pression analysis of Dpf3 downstream targets preventive and therapeutic opportunities for by RNA sequencing. This will complement our patients with congenital heart diseases. dpf3 is essential for cardiac function and so- knowledgemite structuring, already and gained give innovel zebrafish, insights where into Cardiac transcription networks the transcription networks underlying heart Huanhuan Cui, Vikas Bansal, Katherina Bell- and muscle development. mann, Marcel Grunert, Kerstin Schulz Congenital Heart Disease Cardiac and skeletal muscle development is Marcel Grunert, Cornelia Dorn, Vikas Bansal, Andreas Perrot, Sophia Schönhals, Sandra control of gene expression, which is regulated Schmitz, Andrea Behm bya finely the interplay tuned process of transcription that requires factors, a precise his- Congenital heart diseases (CHD) are the most complexes. Studying murine cardiomyocytes, common birth defect in human and affect wetone could modifications show the and interdependency chromatin remodeling of key nearly 1% of all newborns. They are a het-

214 MDC Research Report 2014 ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER

Interaction and functions of significantly over-mutated TOF genes identified from targeted resequencing. (A) TOF genes (dark red) interact in a molecular network with other genes of potential interest (light red and gray). Several genes show individual expression disturbances (red and green arrows). (B) Multiple TOF genes are affected by rare and/or private variations in TOF patients. The GMF is indicated in comparison to control individuals (European Americans from the Exome Sequencing project of the NHLBI).

erogeneous group of disorders ranging from data, which will hopefully enable a more com- minor alterations to complex malformations prehensive understanding of essential disease mechanisms. CHDs are probably caused by a combination ofrequiring multiple multiple genetic, surgeries. epigenetic The and majority environ of- Selected Publications related genes has been a great challenge and Grunert M*, Dorn C*, Schueler M, Dunkel I, Schlesinger J, Mebus S, Alexi-Meskishvili V, ismental complicated factors. by The the identification fact that every of disease-healthy Perrot A, Wassilew K, Timmermann B, Hetzer R, Berger F, Sperling SR (* contributed human carries hundreds of probably damag- equally). Rare and Private Variations in Neural Crest, Apoptosis and Sarcomere Genes ing genomic variations that seem to be toler- Jan 23. [Epub ahead of print] Define the Polygenic Background of Isolated Tetralogy of Fallot. Hum Mol Genet. 2014 ated in the individual context. Bansal V*, Dorn C*, Grunert M, Klaassen S, Hetzer R, Berger F, Sperling SR (* contrib- We aimed to unravel the complex genetics of Tetralogy of Fallot (TOF), the most common Resequencing in a Small Cohort of Patients with Tetralogy of Fallot. PLoS One. 2014 Jan 6;9(1):e85375. cyanotic form of CHD, and to develop novel uted equally). Outlier-Based Identification of Copy Number Variations Using Targeted Dorn C*, Grunert M*, Sperling SR (* contributed equally). Application of high-throughput sequencing for studying genomic variations in congenital heart disease. Brief related genes. Focusing on single nucleotide Funct Genomics. 2014 Jan;13(1):51-65. variations,methods forwe thedeveloped identification the novel of disease-concept Schlesinger J*, Schueler M*, Grunert M*, Fischer JJ*, Zhang Q, Krueger T, Lange M, of the gene mutation frequency (GMF), which - considers all deleterious variations in a gene PLoSTönjes Genet M, Dunkel. 2011 I,Feb;7(2):e1001313. Sperling SR (* contributed equally). The cardiac transcription net and can determine over-mutated genes in a work modulated by Gata4, Mef2a, Nkx2.5, Srf, histone modifications, and microRNAs. patient cohort in comparison to control in- Dunkel I, Krueger T, Mebus S, Lehrach H, Lurz R, Gobom J, Rottbauer W, Abdelilah- dividuals. This provided strong evidence for LangeSeyfried M, S, Kaynak Sperling B, S.Forster Regulation UB, Tönjes of muscle M, Fischer development JJ, Grimm by C,DPF3, Schlesinger a novel J,histone Just S, acetylation and methylation reader of the BAF chromatin remodeling complex. Genes Dev. 2008 Sep 1;22(17):2370-84.

combinationsthe polygenic originof deleterious of TOF and private identified and rare 16 mutations.significantly The over-mutated genes interact genes in a affected molecular by network and show sustained expression in the adult heart, which might help to understand Group Leader differences in long-term clinical outcomes of Prof. Dr. Silke Rickert-Sperling Cornelia Dorn TOF patients. Moreover, we developed a novel Sophia Schönhals* method to identify copy number variations Scientists (CNVs) that can be applied to small cohorts Dr. Marcel Grunert Technical Assistants and could identify CNVs affecting important Dr. Sandra Schmitz* Kerstin Schulz cardiac regulators in TOF patients. Andrea Behm* Graduate students To further study the underlying etiology of Vikas Bansal Staff Katherina Bellmann Andreas Perrot induced pluripotent stem cells (iPSCs) and Huanhuan Cui theirTOF, differentiation the establishment into cardiomyocytes of patient-specific will Secretary be a valuable model. Moreover, we aim to *Part of the period reported. Martina Luig integrate additional genetic and epigenetic

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ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER Photo: private Photo:

Joachim Spranger

Endocrinology, Diabetes and Nutritional Medicine

Physical inactivity contribute to the projec- endocrine response, including several adi- tion that cardiovascular disease but also pokines and myokines like leptin and myo- statin, and numerous other hormones like cancer will remain among the major causes insulin, thyroxin and inkretines like GLP-1 of death in Germany. Against the back- - factors crucially involved in the regulation ground of an individual genetic make-up, of food intake, muscle mass and metabolism, environmental risk factors interact with met- energy expenditure, and physical activity. To understand the role of this complex net- abolic/endocrine pathways to finally modify disease risk. Apparently, the endogenous We aim to elucidate the relevance of those reaction to environmental stimuli depends neuroendocrinework is a major researchcircuits byissue delineating of the group. the effects on metabolism and cardiovascular on numerous non-modifiable factors such risk. J Spranger is speaker of a DFG-funded as age, gender, but also stage of disease clinical research group (KFO218/2) being and existing co-morbidities. Thus, an age- devoted to investigate the endocrine regu- related metabolic inflexibility to exogenous lation of body weight maintenance. This research group was excellently evaluated and stimuli is likely to contribute to metabolic is currently in its second funding period. and cardiovascular ageing. We therefore Among others we currently perform a large aim to identify novel and further elucidate human intervention trial investigating the known endogenous metabolic modifiers of relation of lifestyle to body weight maintenance. In collaboration with other partners cardio-metabolic health. We analyze the from the MDC and Charité, we study hor- interrelation between environment, specific monal regulators of energy metabolism by molecular targets and cardiometabolic risk analyzing the relevance of novel hormones such as thyronamines, but also the impact in relation to age and comorbidities to finally of estrogen receptor signalling and the role translate those findings into preventive prac- of miRNAs. Together with partners from the tice. The group interacts with several other Charité Center for Advance Neuroimaging research groups in Berlin and outside and and the Bernstein Institute of Computation- al Neuroscience, we study behavioural mea- is partner of several joint research projects sures associated to central-nervous reward from the DFG, BMBF and EU. systems using fMRI technology.

Metabolism and peripheral tissues Hormonal regulation of energy expenditure and metabolism We are particularly interested in tissue spe-

Weight loss substantially improves several and muscle tissue is well established re- gardingcific processes. selected Evencytokines, if the the role complex of adipose reg- appears to persist as long as weight reduc- ulation and interaction in adipose and mus- tioncardiovascular is maintained. risk However, factors and mostly the benefitweight cle tissue is still not fully understood. Using regain is observed during follow-up. This in vivo and ex vivo approaches we analyse seems to be mediated by a coordinated -

tissue specific hormonal and metabolic pro 216 MDC Research Report 2014 ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER

The group investigates weight loss induced changes of hormonal circuits and gene expression in different tissues and analyses the impact on long term body weight and health. Perturbation of neuroendocrine axes during weight loss (A). Differentially expressed genes in skeletal muscle (B) and adipose tissue (C) during a 12-weeks weight loss. logFC indicates log fold change of differentially expressed genes (red points), logCPM indicates log of counts per million, black points indicate not differentially expressed genes. (D) Pathway analyses: weight loss induced changes of insulin signaling pathway in adipose tissue of obese subjects after weight loss (n=87). Green color reflects downregulation of gene transcription and red color reflects upregulation of gene transcription during weight loss. Age dependend regulation of gene expression and the impact on metabolic and cardiovascular health is adressed by the group. Exemplarily weight loss induced changes of transcription of gene involved in lipid metabolism is different in old and young individuals in adipose tissue (E).

cesses in human adipose and muscle tissue and their role in body weight regulation and Selected Publications energy metabolism. Exemplarily, we per- Dimas AS, Lagou V, Barker A, Knowles JW, Mägi R, Hivert MF, Benazzo A, Rybin D, Jackson AU, Stringham HM, Song C, Fischer-Rosinsky A, Boesgaard TW, Grarup N, Ab- formed a RNASeq analyses of fat and muscle basi FA, Assimes TL, Hao K, Yang X, Lecoeur C, Barroso I, Bonnycastle LL, Böttcher Y, biopsies of about 80 individuals before and Bumpstead S, Chines PS, Erdos MR, Graessler J, Kovacs P, Morken MA, Narisu N, Payne after dietary weight loss. These individu- PE, Häring HU, Smith U, Boehnke M, Bergman RN, Collins FS, Mohlke KL, Tuomilehto J, als were observed for another two years F,Quertemous Stancakova T, A, Lind Swift L, AJ,Hansen Tönjes T, A,Pedersen Bornstein O, WalkerSR, Cauchi M, Pfeiffer S, Froguel AF, P,Spranger Meyre D, J, SchwarzStum- to evaluate the predictors of body weight voll M, Meigs JB, Wareham NJ, Kuusisto J, Laakso M, Langenberg C, Dupuis J, Watanabe maintenance. We found a weight loss in- RM, Florez JC, Ingelsson E, McCarthy MI, Prokopenko I; on behalf of the MAGIC investigators. Impact of type 2 diabetes susceptibility variants on quantitative glycemic traits duced regulation of numerous signalling reveals mechanistic heterogeneity. Diabetes. 2013 Dec 2. pathways in fat but also muscle. Whether Weygandt M, Mai K, Dommes E, Leupelt V, Hackmack K, Kahnt T, Rothemund Y, those weight loss mediated changes are Spranger J, Haynes JD. The role of neural impulse control mechanisms for dietary suc- essential for long term outcome and car- cess in obesity. Neuroimage. 2013 Dec;83:669-78. diovascular health is addressed in our cur- Habegger KM, Stemmer K, Cheng C, Müller TD, Heppner KM, Ottaway N, Holland J, rent studies. The impact of weight loss on Hembree JL, Smiley D, Gelfanov V, Krishna R, Arafat AM, Konkar A, Belli S, Kapps M, skeletal muscle is addressed by the group, Itoh N, Kharitonenkov A, Spranger J, DiMarchi RD, Tschöp MH. Fibroblast growth fac- as decrease of muscle mass is regularly ob- Woods SC, Hofmann SM, D’Alessio D, Pfluger PT, Perez-Tilve D, Seeley RJ, Konishi M, Weber-Carstens S*, Schneider J*, Wollersheim T, Assmann A, Bierbrauer J, Marg A, Al served during weight reduction and might tor 21 mediates specific glucagon actions. Diabetes. 2013 May;62(5):1453-63. Hasani H, Chadt A, Wenzel K, Koch S, Fielitz J, Kleber C, Faust K, Mai K, Spies CD, Luft be important for long term failure of most FC, Boschmann M, Spranger J*, Spuler S* (*shared authorship). Critical illness myopa-

Med. 2013 Feb 15;187(4):387-96. thy and GLUT4: significance of insulin and muscle contraction. Am J Respir Crit Care bodylifestyle composition interventions. and of Within acute negative this project en- Bobbert T, Schwarz F, Fischer-Rosinsky A, Pfeiffer AF, Möhlig M, Mai K, Spranger J. Fibroblast growth factor 21 predicts the metabolic syndrome and type 2 diabetes in ergywe also balance aim toon separate local and effects systemic of modified metabo- Caucasians. Diabetes Care. 2013 Jan;36(1):145-9.

a collaborative research effort of the ECRC onlism. skeletal This project muscle. is Wealso are investigated especially within inter- Group Leader ested in lifestyle induced changes and the Prof. Dr. Joachim Spranger impact on metabolism and long term car- Graduate Students diovascular health. We currently investigate Scientists Maria Schlöcker the role of salt (sodium) on metabolism to- PD Dr. Knut Mai gether with a number of ECRC researchers. Dr. Lukas Maurer Study Nurse - Dr. Reiner Jumpertz Liane Franz derstand which environmental parameters Dr. Dierk Hampel Annett Pinta affectThose cardio-metabolic findings may finally risk. help to better un

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ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER Photo: David Ausserhofer/MDC Photo: David Ausserhofer/MDC Photo:

Dominik N. Müller Ralf Dechend

Hypertension-induced Target Organ Damage

The group’s major research interests are The immune system, salt and the renin-angiotensin system, the immune hypertension-induced target organ damage system, and how both systems cause hyper- Hypertension induces target-organ damage; tension-induced and Ang II-related target- however, the mechanisms are unclear. We hy- organ damage. Recent work has extended pothesize that immunological processes are the concept analyzing how epigenetic factors important. In general, the immune system is traditionally considered to be a complex such as a high-salt microenvironment influ- biological system that wards off disease, typi- ences immune cells, hypertension-induced - target organ damage, and autoimmunity. In organisms. Angiotensin (Ang) II induces in- cally by fighting the invasion of foreign micro a translational approach, our group focuses via the AT1 receptor. Innate immunity is piv- primarily on the placenta, heart and kidneys. otalflammation, in Ang II-related fibrosis, andtarget-organ growth remodeling damage. T The group has also been a resource for cells, macrophages, and dendritic cells all har- young clinicians and doctoral students begin- bor the AT1 receptor. Ang II stimulates T cell proliferation and dendritic cell migration. We ning their careers in experimental cardiovas- found that regulatory T cells modulate Ang cular research II-induced target-organ damage to a point of therapeutic utility. We also have shown that IFN-g signaling plays a pivotal role in cardiac We hypothesise that and renal damage.1 the immune system Together with Jens Titze and his laboratory affects the electrolyte (Erlangen/Vanderbilt), we provided evidence composition in the that sodium, a known risk factor for cardio- milieu interior and vascular disease, interacts with the immune vice versa. system. Recent evidence indicates that cells of the immune system may also have an important function in regulating the electrolyte content of the interstitium, or “milieu intérieur”. Current teaching suggests that Milieu Immune electrolyte balance in the microenvironment Interieur System bathing all cells is controlled by isosmotic Electrolytes ? T cells passive equilibration with plasma. However, Lymph vessels Macrophages recent evidence suggests that this assump- tion should be revised. Using dietary salt (NaCl) loading studies, it has been shown that water free Na+ was stored in the skin, resulting in a hypertonic microenvironment in the skin despite stable isotonic conditions in the circulation.2 Mononuclear phagocytic system (MPS) cells play a pivotal role in regulating the homeostatic processes necessary to allow the body to store Na+ in the skin.2 Upon excess

218 MDC Research Report 2014 ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER

dietary sodium, Na+ accumulates in the skin in trophoblasts. We are also currently investi- and activate the osmotic stress transcription gating the macrophage – trophoblast interac- factor, TonEBP (also known as NFAT5) in tion, the role of uterine NK cell depletion and MPS cells.2 This activation subsequently re- upregulation of regulatory T cells on the pre- sults in vascular endothelial growth factor-C eclamptic phenotype. In collaboration with (VEGF-C) mediated lymphatic vessel hyper- Michael Bader we investigated an inducible transgenic rat model for diabetes mellitus from the interstitium, and thus, a return to based on shRNA-mediated gene knockdown normalplasia, promoting levels of interstitial clearing of salt. hypertonic Disturbance fluid of the insulin receptor during pregnancy. Us- Ralf Dechend in this macrophage-driven regulatory axis ing different crosses we can differentiate be- therefore results in the accumulation of Na+ tween placental and fetal insulin resistance bound in part to glycosaminoglycans within with our without maternal insulin resistance. the microenvironment.2 Subsequently, the Various degree of IUGR is present in the fetus. consequence of this increased concentration In clinical studies, we began to investigate of interstitial Na+ may be a disturbance in the physiological parameters and novel biomark- function of cells residing within this compart- ers during and after preeclampsia, which help to explain the increased cardiovascular risk of Increased NaCl concentrations induced a 10- former preeclamptic patients. foldment, increase including in the infiltrating cytokine-driven immune TH17 cells. activation in human and murine naïve T cells.3 High salt activated the p38/MAPK pathway Patents / Patent applications during cytokine-induced TH17 polarization Use of renin inhibitors for the prevention or treatment of diastolic dysfunction or in these cells, involving TonEBP and the se- diastolic heart failure. 10008427.6-1216, 2005. Inventors: Feldman DL, Luft FC, Muel- rum- and glucocorticoid-inducible kinase 1 ler DN, Webb R. Novel Eicosanoid Derivatives. EP-1874, 2009. Inventors: Schunck WH, Wallukat G, (SGK1). The outcome of this increased TH17 Schmidt C, Fischer R, Muller DN (MDC), Puli N and Falck JR (UT Southwestern, Dal- activation resulted in an accelerated and las); WO 2010/081683 A1 more severe autoimmune disease (multiple sclerosis) in mice on a high-salt diet.3 The Selected Publications unique global research hypothesis of our laboratory is that: (i) immune cells are essential 1. Marameliorateskó L, Kvakan Ang II-inducedH, Park J-K, cardiac Qadri F, damage Spallek Hypertension B, Binger KJ,Bowman 2012;60(6):1430-6. EP, Kleinewietfeld for the normal regulation of electrolyte con- M, Fokuhl V,Dechend R, Muller DN. Interferon gamma (Ifn-γ) signaling inhibition 2. Wiig H, Schröder A, Neuhofer W, Jantsch J, Kopp C, Karlsen TV, Boschmann M, Goss J, centrations within the microenvironments of Bry M, Rakova N, Dahlmann A, Brenner S, Tenstad O, Nurmi H, Mervaala E, Wagner the body and that (ii) disturbance of this axis results in an accumulation of interstitial so- cells control skin lymphatic electrolyte homeostasis and blood pressure. J Clin Invest. dium, subsequently affecting the function of H,2013;123(7):2803-15 Beck FX, Müller DN, Kerjaschki D, Luft FC, Harrison DG, Alitalo K, Titze J. Immune

these same immune cells (Figure). Sodium Chloride Drives Experimental Autoimmune Disease by the Induction of Patho- 3. Kleinewietfgenic Th17 eldCells. M, Nature Manzel 2013; A, Titze 496(7446):518-22 J, Kvakan H, Linker (* RA,shared Müller authorship) DN*, Hafler DA*. Pathogenesis of preeclampsia Preeclampsia (PE) is a leading cause of ma- Schulz H, Wallukat G, Klapp BF, Nevers T, Sharma S, Staff AC, Dechend R and Blois 4. FSM.reitag Interfering N, Tirado-Gonzalez with Gal-1-mediated I, Barrientos angiogenesis G, Herse F, contributes Thijssen VL, to Weedon-Fekjaer the pathogenesis SM, of ternal death, and perinatal morbidity and preeclampsia. Proceedings of the National Academy of Sciences of the United States of mortality resulting from premature delivery America. 2013;110:11451-6. and intrauterine growth restriction (IUGR). 5. Herse F, Lamarca B, Hubel CA, Kaartokallio T, Lokki AI, Ekholm E, Laivuori H, Gauster New hypertension and proteinuria arises M, Huppertz B, Sugulle M, Ryan MJ, Novotny S, Brewer J, Park JK, Kacik M, Hoyer J, Verlohren S, Wallukat G, Rothe M, Luft FC, Muller DN, Schunck WH, Staff AC and secondary to diffuse maternal endothelial Dechend R. Cytochrome P450 subfamily 2J polypeptide 2 expression and circulating - epoxyeicosatrienoic metabolites in preeclampsia. Circulation. 2012;126:2990-9. ied the role of immunological dysregulation inand the inflammatory cause of preeclampsia. dysfunction.4 WeWe foundhave stud that activating autoantibodies against the AT1-receptor are implicated in the pathogenesis of preeclampsia. Recently, we showed that they Group Leader PhD are involved in dysregulating various aspects Priv. Doz. Ralf Dechend, MD Matthias Gebhardt of the immune system in preeclampsia. We Priv. Doz. Dominik N. Lukasz Przybyl performed gene arrays from preeclamptic Müller, PhD Julia Rugor

subfamily 2J polypeptide 2 (CYP2J2) as a nov- Scientists Technical Assistants eland candidate control placentas gene.5 CYP2J2 and identified metabolites, the CYP5,6 Andras Balogh, MD Juliane Anders epoxyeicosatrienoic acids (5,6-EET), were Katrina Binger, PhD (lab manager) Jana Czychi upregulated before the onset of the disease. Nadine Haase, PhD Ute Gerhard Cyp P450 inhibition reduced preeclamptic Florian Herse, PhD (lab manager) Ilona Kamer phenotype in two rat models of preeclampsia. May-Britt Köhler 5,6-EET can be metabolized to a thrombox- Maren Wellner, PhD (part time) Gabriele N’diaye ane analog, making it an interesting candidate Lajos Marko, MD, PhD Jutta Meisel for mediating preeclampsia. Together with Clinical Scientists Zsuzsanna Izsvak we are using the sleeping- Michaela Golic, MD Study Nurse beauty transposon technique to overexpress Nicola Wilck, MD Heike Schenck

candidate genes, such as CYP2J2 specifically MDC Research Report 2014 219

ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER Photo: private Photo:

Ulrike Stein

Translational Oncology of Solid Tumors

Our translational concepts in cancer research MACC1 function and prognostic for CRC pa- aim at the identification of key molecules in tumor progression and metastasis for im- inhibitorstients. We foralso restriction identified kinasesof MACC1-induced for MACC1 provement of prognosis and therapy of solid metastasisphosphorylation in mice. and MACC1 used is a specific biomarker kinase for tumors. We found novel genes, revealed their disease prognosis and prediction of therapy potential as biomarkers and gained insights - ety of solid cancers, linked to patient survival. in their function within cancer signaling net- response for CRC and is confirmed for a vari- works. Our main focus is on the metastasis inducers MACC1 and S100A4 as prognos- therapyA first clinical was recently study for completed MACC1-based successfully strati fication of stage II CRC patients for adjuvant tic and predictive biomarkers for colorectal (with Hoffmann-La Roche). cancer (CRC) and further solid tumors. Our S100A4: Inhibition of CRC metasta- knowledge on these molecular targets is sis by Wnt/ß-catenin-pathway-ori- employed for innovative signaling-based ap- ented small molecules targeting the metastasis progressor S100A4 proaches to intervene in metastasis. Pre- U. Stein, W. Walther, U. Sack, M. Dahlmann, clinically validated therapeutic strategies are P.M. Schlag; in cooperation with I. Fichtner currently translated into clinical trials to treat (MDC), R.H. Shoemaker, P. Pandalai (NCI/NIH, patients with metastatic disease. Bethesda) as transcriptional ß-catenin target. S100A4 MACC1: A driver for tumorigenesis stronglyWe identified increased the metastasiscell motility gene in vitro S100A4 and and metastasis and a therapeutic metastasis in mice. In clinical specimens, we target for metastasis restriction in demonstrated the prognostic value of S100A4 solid cancers in primary CRC for metastasis and patient U. Stein, W. Walther, M. Juneja, C. Lemos, survival. We targeted S100A4 for metastasis P.M. Schlag; in cooperation with W. Birch- restriction in mice, e.g. by systemic applica- meier, G. Dittmar, I. Fichtner, B. Jerchow, H. tion of shRNA acting on S100A4. We identi- Schmidt, (MDC), J. von Kries (FMP), U. Rohr (Hoffmann-La Roche) (e.g. niclosamide, calcimycin) from high- We discovered the gene metastasis-associat- throughputfied small molecule screens transcriptionalof pharmacologically inhibitors ac- ed in colon cancer 1 (MACC1) able to induce tive compounds that inhibit S100A4 by inter- proliferation and motility in vitro and tu- vening in the Wnt/ß-catenin cascade. We also mor growth and metastasis in mice. MACC1 used compounds known to interfere with this controls transcriptionally target genes, e.g. pathway (e.g. sulindac). These S100A4 inhibi- Met, promoting cell motility and metasta- tors reduced migratory and invasive abilities, sis. shRN A acting on MACC1, Met, or further and restricted metastasis in mice. Now we are MACC1 targets decreased metastases formation. Transgenic vil-MACC1 and vil-MACC1/ metastasis inhibition into clinical practice. APCmin mice have been generated; MACC1 Ourtranslating assay for our circulating findings onS100A4 S100A4-specific transcripts knock out models are in progress. in patient blood is used to monitor treatment success. Currently, FDA-approved inhibitory employed it in high throughput screens for compounds are being tested in clinical phase We identified the MACC1 gene promoter, and II trials for metastasized CRC patients: for su- inhibitors. We found MACC1 protein binding lindac, with the NCI/NIH(NCT01856322); for partnersfirst identification by mass spectrometry of FDA-approved essential MACC1 for niclosamide, with the Charité, Berlin.

220 MDC Research Report 2014 ECRC – EXPERIMENTAL AND CLINICAL RESEARCH CENTER

Figure 1. We demonstrated the prognostic Circulang S100A4 RAGE is essenal for S100A4 metastasis Phase II Clinical Trial transcripts in cancer NCT01856322 value of the metastasis inducer S100A4 for paent blood NCI/NIH USA, CRC metastasis linked to patient survival Charité, MDC when determined in tumors or blood. We Recruing since 2013 discovered S100A4 as target of the Wnt/ß- S100A4 in primary tumors catenin pathway, identified the receptor Sulindac RAGE to be essential for S100A4-induced cell motility, and linked inflammation and metastasis with SAAs as targets of S100A4. Target- ing S100A4 with S100A4-shRNA or small S100A4 is a target of Calcimycin molecule inhibitors intervening in Wnt/ß- Wnt/β-catenin catenin signaling such as sulindac, calcimycin signaling in CRC Niclosamide and niclosamide restricted S100A4-induced metastasis in mice. Clinical phase II trials translating this knowledge for sulindac Phase II Clinical Trial Systemic S100A4 and niclosamide to treat metastasized CRC shRNAinhibits CRC Charité, MDC metastasis in mice SAA1 and SAA3 are S100A4 targets Iniated 2014 patients are currently underway.

Imaging of carcinomas using Patents / Patent applications a novel RNAi-based detection Stein U, Lederer A, Schlag PM, Herrmann P, Seehofer D. MACC1 as a prognostic bio- mechanism marker in hepatobiliary tumors Patent application 2013; US 61/829,669 W. Kemmner; in cooperation with B. Ebert Stein U, Schwabe H, Walther W, Schlag PM. 7a5/Prognostin and use thereof for the (PTB), S. Lin (Guangzhou University, China), R. diagnostic and therapy of tumors Zeisig (EPO), R. Haag (Free University Berlin) Patent, granted 2013 EP 1646649B1; 2013 CA 2,526,240; 2011 JP 51-9895; 2011 AU 2004259281; 2010 US 7,851,168 B2 Stein U, Walther W, Sack U, Scudiero D, Schlag PM, Shoemaker RH. Niclosamide for down-regulation of Ferrochelatase (FECH) treatment of cancer metastasis expressionIn previous in studies, various we carcinomas found a significant leading Patent application 2013 PCT/US 14/112,521; 2012 PCT/EP2012/057049; 2011 EP - 11162875.6 tabolite of heme synthesis protoporphyrin IX Walther W, Kobelt D, Schlag PM, Schmidt M. Chemosensitivierung von Melanomen mittels Gentransfer eines TNF-alpha exprimierenden minimalistischen MIDGE-hTNF Vektors. (PpIX).to the accumulationFECH silencing of by the folate-PEG fluorescent cationic me Patent application 2010 EP 10195929.4 lipoplex-encapsulated FECH-siRNA enabled a Stein U, Herrmann P, Burock S, Schlag PM. Use of metastasis progressor S100A4 tran- sensitive in vivo tumor imaging. In the future, Patent application 2010 PCT/EP2010/001891; 2009 US 61/261,017 FECH-siRNA nanocarriers could be used for scripts in body fluids of colorectal and gastric cancer patients. sensitive detection of small metastatic tumor Selected Publications clusters. At the same time this technology can Stein U, Walther W, Arlt F, Schwabe H, Smith J, Fichtner I, Birchmeier W, Schlag PM. be used for photodynamic depletion of tumor cells of primary carcinoma and metastases. metastasis. Nature Med. 2009, 15:59-67. MACC1, a newly identified key regulator of HGF/Met signaling, predicts colon cancer - tion of the metastasis gene MACC1 in colorectal cancer. Mol Oncol. 2013, 7:929-43. Nonviral gene therapy for solid SackJuneja U ,M, Walther Ilm K, SchlagW, Scudiero PM, Stein D, Selby U. Promoter M, Kobelt identification D, Lemm M, Fichtnerand transcriptional I, Schlag PM, regula tumors Shoemaker RH, Stein U. Novel effect of anti-helmintic niclosamide on S100A4-induced W. Walther, D. Kobelt, F. Chiola, P. M. Schlag, U. metastasis in colon cancer. J Natl Cancer Inst. 2011, 103:1018-36. Stein; in cooperation with F. Kiecker, U. Keil- Dahlmann M, Sack U, Herrmann P, Lemm M, Fichtner I, Schlag PM, Stein U. Systemic shRNA mediated knock down of S100A4 in colorectal cancer xenografted mice re- holz, J. Piontek (Charité), I. Fichtner (MDC), B. duces metastasis formation. Oncotarget 2012, 3:783-97. Wittig (Free University Berlin), M. Schroff, M. Kobelt D, Aumann J, Schmidt M, Wittig B, Fichtner I, Behrens D, Lemm M, Freundt Schmidt (MOLOGEN) G, Schlag PM, Walther W. Preclinical study on combined chemo- and nonviral gene A new phase I clinical trial is conducted to therapy for sensitization of melanoma using a human TNF-alpha expressing MIDGE DNA vector. Mol Oncol. 2014 [Epub ahead of print]

minimalisticevaluate safety MIDGE-vector and efficiency in patients of nonviral with in-transitgene therapy skin usingmetastasis the TNF-α from expressingmelanoma (EudraCT: 2007-000423-18). This dose-esca- Group Leader - Prof. Dr. Ulrike Stein Maria Haustein until 30.4.2013: Katharina Ilm lation study will analyze TNF-α expression ef Prof. Dr. Peter M. Schlag andficiency of the after vector intratumoral DNA will be jet-injection evaluated. gene Harikrishnan Radhakrishnan Ourtransfer. efforts The also safety aim at of development the expressed of TNF-αnovel Scientist FelicitasManisha SchmidJuneja therapeutic genes. The use of bacterial toxins Dr. Mathias Dahlmann is an applicable option for treatment of solid Dr. Clara Faria Alvares de Lemos Technical Assistants tumors. The Clostridium perfringens entero- Dr. Wolfgang Kemmner Jutta Aumann toxin (CPE) is a pore-forming bacterial toxin, Dr. Dennis Kobelt Sabine Grigull, part time Prof. Dr. Wolfgang Walther Pia Herrmann and triggers membrane pore formation lead- Gudrun Koch, part time ingwhich to rapid specifically cell death. binds We to successfully claudin-3 exploit and -4 PhD/MD students Claudia Roeefzaad, part time this for selective tumor cell killing by CPE Francesco Paolo Chiola Janice Smith gene therapy of human claudin-3 and -4 over- Markus Hardt Cynthia Voss expressing tumors in mice.

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222 MDC Research Report 2014 Technology Platforms

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Central Technology Platforms

Central Technology Platforms he Max Delbrück Center has several and Core Facilities dedicated Core Facilities and Technol- Togy Platforms providing MDC groups (and external users by special request) with facilities and support for a wide range Advanced Light Microscopy of different techniques. Modern technol- Anje Sporbert Bettina Purfürst ogy and services which would otherwise Electron Microscopy demand an extremely long and intensive period of training, as well as large invest- Gunnar Dittmar ments, are the mainstay of the MDC’s cen- Mass Spectrometry tral Technology Platforms. These facilities provide all users with the opportunity to Arnd Heuser acquire both up-to-date service support Pathophysiology Hans-Peter Rahn Preparative Flow Cytometry Additionally,and the latest scientificthere are know-how. technology platforms associated with research groups Ralf Kühn (from May 2014) providing services for Interactomics, mag- Transgenics netic resonance (B.U.F.F.), Microarrays and Protein Sample Production Facility. On the

Research group associated descriptions of our central Technology Technology Platforms Platformsfollowing pagesand Core you willFacilities, find more the researchin depth group associated technology platforms as well as of BIMSB Technology Platforms. Erich E. Wanker Interactomics Thoralf Niendorf Magnetic Resonance (B.U.F.F.) Norbert Hübner Microarrays * Udo Heinemann Protein Sample Production Facility *

* See group pages N. Hübner (Cardiovascular and Metabolic Disease) and U. Heinemann (Cancer Research)

BIMSB Technology Platforms

Altuna Akalin Bioinformatics Wei Chen Genomics Protein fragments of colon bacteria, analyzed by mass spectrometry. Stefan Kempa Proteomics / Metabolomics Proteinfragmente eines Darmbakteriums, analysiert mithilfe eines Massenspektrometers.

224 MDC Research Report 2014 TECHNOLOGY PLATFORMS © M. Sury from the Selbach Group Selbach the © M. Sury from

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TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Anje Sporbert

Advanced Light Microscopy

The Advanced Light Microscopy platform Applications ranging from observing subcel- offers scientists access to state of the art, lular structures in cells to investigating the morphology of small organisms and large tis- high-end imaging techniques and supports sue areas at high optical resolution are avail- the research projects of scientific groups with able (Fig.A/B). Optical clearing of biological customised, methodological expertise. We are currently supporting about 150 re- thick specimen by confocal, 2-Photon or light- sheetsamples microscopy is often (Fig.B). beneficial In TIRF for microscopy, imaging of searchers from more than 35 groups mainly of the MDC, BIMSB and ECRC. be excited, making this an ideal technique In addition to teaching scientists in the theo- toonly image fluorophores dynamic processesclose to the close coverslip to the willcell membrane, e.g. vesicle transport, endocytosis retical aspects of light microscopy, we offer and exocytosis, even providing single mol- them profound practical training and assure ecule sensitivity. 2-Photon (2-P) microscopy the optimal performance of the provided inis used to image thick, turbid specimen, e.g. strumentation and infrastructure. - bryos. 2-P systems are also able to measure In collaborative imaging projects we offer label-freetissue preparations, SHG signals, mouse emitted or zebrafish by highly em or- individual user support covering a wide range dered biological structures, e.g. collagen. of expertise, e.g. project planning, optimisa- Customised image analysis and modelling tion of sample preparation and image acquisi- based on microscopy-related information reveal how differences in cell motility can tion conditions, as well as a customised image induce a directional migration of cardiac analysis. Cseresnyes/AG Seyfried, ref.2). Available imaging techniques Photoswitchingtissue in zebrafish and -bleaching heart development techniques (Z.of and selected projects structures labelled with genetically-encoded - We currently host 10 high-end microscope bility of proteins inside living cells, as well as systems, covering confocal laser scanning, thefluorescent tracking proteins of cells inallow whole to monitor live organisms, the mo e.g., to follow morphogenetic processes in light-sheet based microscopy, as well as laser- - assisted2-photon, micro-dissection. wide-field fluorescence, This instrumenta TIRF and- travital and in vivo imaging of small animals tion offers advanced imaging techniques for iszebrafish invaluable (A. Sporbert/AGfor validating Seyfried, results ref.4).obtained In by standard techniques in a physiological cells, tissue sections and preparations to environment. The DFG-supported Joint In- smallboth fixedorganisms and liveand specimen,live animals. ranging from travital Microscopy and Imaging platform Several computer workstations and software between R. Niesner, A. Hauser (DRFZ, Berlin) tools are also made available for advanced and the Advanced Light Microscopy provides image analysis and processing. Regular semi- individual, customized support from sample nars covering various aspects of light and preparation/animal surgery and imaging using standard techniques to intravital mi- - croscopy and dedicated image analysis (V. fluorescence microscopy are offered. Confocal microscopy with efficient separa include the imaging of whole mount brain tion of spectrally overlapping fluorophores slicesMatiash/Z. (AG Kettenmann) Cseresnyes). Supportedand spleen projects tissue biologicaland optical structures. z-sectioning is beneficial for (AG Höpken). multi-color 3D fluorescence imaging of many

226 MDC Research Report 2014 TECHNOLOGY PLATFORMS

A) Tile scan of murine fibrosarcoma tissue: green – tumor cells, red – stromal cells, yellow – endothelial cells, blue – T cells, courtesy of M. Leisegang, AG Uckert B) 3D reconstruction of a cleared whole- mount E11.5 mouse embryo, courtesy of H. Schmidt, AG Rathjen C) Quantum dot tri-exciton imaging (QDTI): sub-diffraction resolution imaging of nestin filaments in human neurospheres (ref.1) D) 2-Photon imaging of mouse spleen tissue with improved axial resolution by multi-beam striped-illumination (ref.3)

Method development Development of a platform management software Observing subcellular structures is limited by the spatial resolution of light microscopy. A customised management software was de- A number of new microscopy techniques veloped to make the access and use of the pro- (termed “superresolution microscopy”) have vided infrastructure easy, as well as to ensure been developed to overcome this limit. How- a fast communication for support and trouble- ever, these techniques require complex mi- shooting (5). “ShareMCF” integrates online croscopy arrangements. We aim at improv- - ing the spatial resolution by using available croscope booking calendar as well as mailing techniques and implementing novel imaging listsuser- and and tools project for reporting databases, news registration, and incidents mi concepts. (M. Richter). The MS Sharepoint software can In collaboration with M. Heilemann (Goethe be adapted to the needs of other Core Facilities. University, Frankfurt) we improved a method for 3D sub-diffraction limit imaging based on the ability of quantum dots for three-photon Selected Publications absorption (QDTI), resulting in a smaller (1) Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots. point spread function. In combination with Sporbert A, Cseresnyes Z, Heidbreder M, Domaing P, Hauser S, Kaltschmidt B, deconvolution, up to 1.9-fold improved spa- Kaltschmidt C, Heilemann M, Widera D. PLoS One. 2013 May 21;8(5) tial resolution can be achieved (ref.1). Since (2) Unilateral dampening of Bmp activity by nodal generates cardiac left-right asym- QDTI is realized on a confocal microscope metry. Veerkamp J, Rudolph F, Cseresnyes Z, Priller F, Otten C, Renz M, Schaefer L, Abdelilah-Seyfried S. Dev Cell. 2013 Mar 25;24(6) it can also be applied to thick, multi-color (3) High-resolution intravital microscopy. Andresen V, Pollok K, Rinnenthal JL, Oehme samples of extended size, i.e. neurospheres L, Günther R, Spiecker H, Radbruch H, Gerhard J, Sporbert A, Cseresnyes Z, Hauser (Fig.C). Based on a collaboration with the AE, Niesner R. PLoS One. 2012;7(12) groups of R. Niesner and A. Hauser (DRFZ, - Berlin), a method for high-resolution intra- bardo VA, Sporbert A, Abdelilah-Seyfried S. J Vis Exp. 2012 Sep 28;(67) (4) Cell tracking using photoconvertible proteins during zebrafish development. Lom vital microscopy is further developed us- (5) A Coustomised web-based tool box for microscopy core facility management. Cseresnyes Z, Kriegel F, Sporbert A. Imaging & Microscopy. 2011, Issue 4, V ing a multi-beam 2-photon microscope (A. Margineanu/Z. Cseresnyes). This novel approach uses striped-illumination, thus apply- Group Leader Dr. Vitali Matiash (part-time, DFG ing the principle of structured illumination Dr. Anje Sporbert grant, since 01/2014) and spatial modulation to achieve improved contrast and better axial resolution. This ap- Scientist Technical Assistant proach was successfully used to visualise cel- Dr. Anca Margineanu (since Matthias Richter lular interactions in spleen tissue (Fig.D) and 05/2013) in live lymph nodes (ref.3). Dr. Zoltan Cseresnyes (part-time, Student Helper DFG grant) Malte Hilsch (part-time)

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TECHNOLOGY PLATFORMS Photo: Anna Domaska-Akalin Anna Photo:

Altuna Akalin

BIMSB Bioinformatics

Research preview ing of the disease. However, our previous work has shown that certain types of can- Bioinformatics is the application of compu- cers share mutations on pathways relating tational methods to the management and analysis of biological data. By using bioin- - formatics, high-dimensional data can be tionsto DNA are modifications, previously shown such to as be methylation associated translated to biological knowledge. With withand hydroxymethylation.gene regulation. These Such new modifica class of the advent of high-throughput technologies cancers are distinguished by certain abnor- in biology, computational analysis needs of the labs are on the rise. This creates a chal- paves way for different approaches in pa- lenge in method development, application malities in their epigenomic profiles, which and also a demand in training new indi- analyze high-throughput sequencing data viduals. setstient to stratification investigate epigenomic and drug design.abnormali We- ties and differences in various normal and BIMSB Bioinformatics platform creates and disease cell types. maintains bioinformatics tools and databases, and studies biology using those in- house or publicly available computational Computational analysis of techniques. In addition, the platform pro- multi-level gene regulation vides collaboration opportunities, help- desk, and training for MDC scientists. The Gene regulation is a complex event where platform aims to help MDC scientists ad- multiple partners interact with each other dress their biological questions using and to achieve gene regulation. The level of developing computational methods. expression of a gene is initially controlled by transcriptional regulatory mechanisms. On top of these, we have a broad interest in This is followed by post-transcriptional mechanisms, which can further modify regulation and association of transcrip- transcript levels. In order to have full pic- tionalgene regulation, regulation specifically with epigenomics. transcriptional We are ture on gene-expression, one must take aiming to use data intensive computational into account all the different players in methods to uncover patterns in gene regu- these processes. The lab will aim to work lation relating to cell differentiation and on multi-level gene regulation by utilizing complex diseases. bioinformatics tools with high-throughput data sets such as CAGE, RNA-seq, ChIP-seq and PAR-CLIP. DNA modifications and disease

Discovering which abnormalities lead to Data-integration and machine complex diseases such as cancer remains learning approaches as a challenge. To complicate the matters, most cases are unique to their selves and do Most successful approaches in modeling not allow for simple all-embracing model- complex phenomena, such as complex

228 MDC Research Report 2014 TECHNOLOGY PLATFORMS

a) Examples of DNA methylation abnormalities in two different subtypes of acute myeloid leukemia. b) Examples of data integration and visualization techniques using in-house software.

diseases or gene regulation, make use of different types of data. These data sets Selected Publications should not only be limited to high-through- Lu C, Venneti S, Akalin A, Fang F, Ward PS, Dematteo RG, Intlekofer AM, Chen C et al. put sequencing based data sets mentioned Induction of sarcomas by mutant IDH2. Genes Dev. 2013 Sep 15;27(18):1986-98 above, but also should include proteomics Akalin A*, Kormaksson M, Li S, Garrett-Bakelman FE, Figueroa ME, Melnick A, Mason CE. methylKit: a comprehensive R package for the analysis of genome-wide DNA meth- through integration of multi-layered data, Akalin A, Garrett-BakelmanFE, KormakssonM, BusuttilJ, et al. Base-pair resolution ylation profiles. Genome Biology, 2012 Oct 3;13(10):R87* (Co-corresponding author) complex& metabolomics biological data processes sets. We believecan be onlyun- DNA methylation sequencing reveals profoundly divergent epigenetic landscapes in derstood in a system-wide level. This re- acute myeloid leukemia.. PLoS Genetics 2012;8(6):e1002781. PlessyC*,Pascarella G*, Bertin N*, Akalin A*, CarrieriC, VassalliA, LazarevicD, et al. analysis and visualization of diverse data Promoter architecture of mouse olfactory receptor genes. Genome Research. 2011 Dec 22. * (Equal Contribution) sets.quires efficient preprocessing, integration, Akalin A, Fredman D, Arner E, Dong X, Bryne JC, Suzuki H, Daub CO, Hayashizaki Y, Lenhard B. Transcriptional features of genomic regulatory blocks. Genome Biol. In principal, integration and analysis 2009;10(4):R38 problem of big data sets can be solved by translating computer science and statistics research to biology, and developing those Group Leader System administrators methods further where necessary. In col- Dr. Altuna Akalin David Graham Arnold laboration with other platforms and groups Dan Munteanu at BIMSB and MDC, we are aiming to use statistics and machine-learning techniques to Administrative Assistant uncover relationships in hig h-dimensional Sabrina Deter biological data from diverse sources.

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TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Wei Chen

BIMSB Genomics

The recent introduction of novel sequencing ~10Gb per exome sample can be achieved technology (NGS) has revolutionized genomic research. As one of the leading NGS the HiSeq2000 system and the SureSelect V5on aenrichment thoroughly optimizedsystem, generating workflow ~200using facilities in Europe, our lab has been provid- million single end 100nt reads for each ing NGS supports for both the researchers probe of a 2-plex pool on one lane. within MDC and external collaborators. The services ranges from whole genome Whole-genome sequencing (re)sequencing, targeted genome sequencing, transcriptome sequencing to various The Illumina Nextera whole-genome li- epigenomic profilings. Apart from offering brary preparation kit features the rapid ‘just’ the services to the institute as a core creation of gDNA libraries from very low input sample concentration (> 50ng ge- facility, my lab has been developing various nomic DNA). During library creation, a pro- NGS-based genomic assays (see BIMSB). cess termed ‘tagmentation’ simultaneously fragments and tags DNA, making fragments

NGS instrument and applications sequencing output of ~80Gb per genome sample,amenable which to direct is required PCR amplification. to cover the A BIMSB genomics platform is now equipped whole genome with >20x, can be achieved with most of the currently available next generation sequencing systems, includ- 2 lanes on the HiSeq2000 system, generating one 454 FLX (Roche), two SOLiD 4 (life ingon a~200 thoroughly million optimized paired-end workflow 2x100nt readsusing technologies), one GAIIX, two HiSeq2000 on one lane. and two Hiseq 2500 (Illumina), and one PacBio SMRT sequencing (PacBio). With these resources, a totle of 4,313 samples have been sequenced between 2011-1-1 and 2013-12-31. The raw sequencing data amounts to >52 Terabases. Our current NGS applications include:

Exome sequencing David Ausserhofer/MDC Photo:

The Agilent SureSelect V5 exome enrichment system is used to prepare exome sequencing libraries. With a sequencing output of ~10Gb, an average of ~80% of all reads fall within the targeted regions +/- 100bp and ~95% of all targeted bases Visit of H.E.Shi Mingde (2nd from right), Am- are covered >= 20x. A sequencing output of bassador of the People’s Republic of China.

230 MDC Research Report 2014 TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Wei Chen (left) with the PacBio RS System sequencer in his lab.

RNA-seq enrichment. After quality control of the se- Long RNA samples are processed in quencingligation, gellibrary, purification up to three and sample a final can PCR be labeled with unique barcodes and pooled paired-end libraries will be created using for sequencing, 1x50nt single-end, in one content-defined batches, standardized lane with a minimum output of 50 million Chemistry). up to three samples can be la- reads for each sample. beledestablished with different protocols barcodes (modified and Truseqpooled for sequencing of 100 nt from single end in one lane, with a minimum of 50 million DNA methylation analysis reads for each sample. Methylome–wide analysis is currently performed using Reduced Representation Small RNA-seq method was selected because DNA meth- Starting from 1µg of total RNA, a size se- ylationBisulfite pattern Sequencing can be (RRBS).analyzed The on the RRBS- nu- lection is performed for RNAs between 18 cleotide level in CpG island and CpGI shores and 30 nt to enrich for small RNAs, includ- with a coverage of around 10 % of all CpG ing miRNAs. Subsequent library prepara- positions. Moreover this method is feasible tion steps include: adapter ligation and with a very low amount of DNA. Up to three - RRBS samples can be labeled with different brary is obtained by a further size selection barcodes and pooled for sequencing of 100 step.PCR amplification.Up to 12 samples The can final be labelled smallRNA with li nt from single end in one lane, with a mini- unique barcodes and pooled for sequenc- mum of 50 million reads for each sample. ing, 1x50nt single-end, with a minimum of 10 million reads per sample.

Group Leader ChIP-seq Dr. Wei Chen

Starting from 10-20ng of ChIP-DNA, the Complete list of team members Biology, Group “Novel sequencing following library preparation steps are can be found in chapter Berlin technology, Functional and medical performed: end-repair, a-tailing, adapter Institute for Medical Systems genomics”

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TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Stefan Kempa

BIMSB Proteomics & Metabolomics

Within the past decades biochemical data of single processes, metabolic and sig-

Overview about the mass spectrometers in- naling pathways were collected and ad- stalled at BIMSB and related methods. During vances in technology led to improvements the last years we have installed a GC-ToF-MS, a of sensitivity and resolution of bioanalytical triple-quadrupole-MS and a LTQ-Orbitrap-Velos. techniques. These achievements build the The scheme shows the mass spectrometers, established methods and related metabolic bases for the so called ‘genome wide bio- pathways that can be analyzed. chemistry’.

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Overview about the metabolomics and proteomics analyses performed by the group. Visualized are the analyses performed within projects related to the associated institutions. In total 18,000 metabolomics and 4,500 proteomics samples were processed.

Single level study of a living organism cannot give a complete understanding of the mechanism regulating biological functions. The integration of transcriptomics, proteomics and metabolomics data with existing knowledge allows connecting biological processes which were treated as independent so far. In this context the aim of our group is to establish and apply metabolomics and proteomics techniques for Selected Publications - Synthetic lethal metabolic targeting of cellular senescence in cancer therapy. over rates of proteins and metabolites us- Doerr, J.R. and Yu, Y. and Milanovic, M. and Beuster, G. and Zasada, C. and Daebritz, J.H.M. and Lisec, J. and Lenze, D. and Gerhardt, A. and Schleicher, K. and Kratzat, S. and ingabsolute stable quantification isotopes. and to analyze turn Purfuerst, B. and Walenta, S. and Mueller-Klieser, W. and Graeler, M. and Hummel, M. and Keller, U. and Buck, A.K. and Doerken, B. and Willmitzer, L. and Reimann, M. and In order to decode molecular mechanisms Kempa, S. and Lee, S. and Schmitt, C.A. Nature 501 : 421-425. 19 September 2013 causing metabolic disorders (a) a dynamic Deregulated MYC expression induces dependence upon AMPK-related kinase 5. view on metabolism and (b) information Liu, L. and Ulbrich, J. and Mueller, J. and Wuestefeld, T. and Aeberhard, L. and Kress, T.R. and Muthalagu, N. and Rycak, L. and Rudalska, R. and Moll, R. and Kempa, S*. and about the expression levels of proteins, eg. Zender, L. and Eilers, M. and Murphy, D.J. Nature 483 (7391): 608-612. 29 March 2012 metabolic enzymes and regulatory pro- (corr. for metabolomics/proteomics) teins, is required. Using the established Proteome dynamics and early salt stress response of the photosynthetic organism techniques we are able to measure the met- Chlamydomonas reinhardtii. Mastrobuoni, G. and Irgang, S. and Pietzke, M. and Wen- abolic activity of cells, organs and organ- zel, M. and Assmus, H.E. and Schulze, W.X. and Kempa, S. BMC Genomics 13 (1): 215. isms in a dynamic manner in vitro and in 31 May 2012 vivo. We support BIMSB and MDC groups in De novo assembly and validation of planaria transcriptome by massive parallel sequencing and shotgun proteomics. Adamidi, C. and Wang, Y. and Gruen, D. and Mastrobuoni, G. and You, X. and Tolle, D. and Dodt, M. and Mackowiak, S.D. and Gogol- complex biological questions by applying Doering, A. and Oenal, P. and Rybak, A. and Ross, E. and Alvarado, A.S. and Kempa, S*. cuttingframe of edge scientific metabolomics collaborations and proteomics to address techniques: July 2011 (*co-corresponding author) and Dieterich, C. and Rajewsky, N. and Chen, W. Genome Research 21 (7): 1193-1200. Muscle-type creatine kinase interacts with central domains of the M-band proteins • myomesin and M-protein. Hornemann, T. and Kempa, S. and Himmel, M. and Hayess, K. and Fuerst, D.O. and Wallimann, T. Journal of Molecular Biology 332 (4): 877-887. 26 September 2003 • StableMetabolic isot opeprofiling resolved (targeted, metabolomics non- (intar geted,cell cultures lipid profiling) and in vivo) • Proteome analyses (SILAC, in vivo SILAC and targeted proteomics) Group Leader At present the integrative proteomics and Dr. Stefan Kempa metabolomics platform at BIMSB supports the setup of the BIH metabolomics core by Complete list of team members Biology, Group “Metabolic implementing the technologies and work- can be found in chapter Berlin Regulation” - Institute for Medical Systems ing the last years. flows that were established at BIMSB dur

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TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Bettina Purfürst

Electron Microscopy

The EM facility supports all research groups Negative contrast of the MDC with expertise and technology to study biological probes like macromol- This method allows a direct visualization of ecules, isolated cell organells, cells, tissue at high resolution. Here, we detected for the and clinical samples. In the period reported, purified proteins and other macromolecules- a new 11 Megapixel high speed CCD cam- tion of 150 – 200 nm vesicles released from era was installed on the Zeiss EM 910, re- humanfirst time myotubes a nearly (Figure homogeneous C, Zacharias popula et al., 2011), originating from patients with sulting in a much faster generation of large muscular dystrophies. Similar membrane picture series for phenotyping and quantifi- microvesicle fractions, called exosomes, are cation. Here are some selected topics from secreted by various cell types and may have the last two years: a promising diagnostic and therapeutic potential. In another approach, we successfully applied immunolabeling to negative con-

Figure D (collaboration with A. Böddrich). trast probes, as shown for amyloid fibrils in

Structure of heart and skeletal muscle under normal and Quantification of subcellular pathological conditions compartments We have characterized a large range of In two cases, we could detect and even heart and skeletal muscle probes for ultra- quantify endoplasmatic reticulum (ER) structural changes in different phenotypes stress in different model systems in cancer (collaborations with L. Thierfelder, M. Got- research. High-grade astrocytoma cells of- thard and B. Schröder from the MDC, S. ten contain a proliferated, parallel ER un- Spuler and J. Fielitz from the Charite and der normal cultivation conditions (Figure ECRC). Normal features of this type of tis- A). Vanilloid receptor agonists released by sue are easy to quantify, like sarcomere neural precursor cells induced a strong di- length, desmosome structure, and shape, number and inner membrane structure of these astrocytoma cells (Stock et al., of mitochondria, followed by secondary 2012).lation of A thesimilar ER lumen proliferated and finally ER with the death large effects like an increased amount of lipid droplets. In strong phenotypes, the Z-line lymphoma cell system (Figure B), where structure is heavily disturbed (minicore thecisternae ER stress could was be closely quantified related in to a autoph mouse- disease) and may result in a completely dis- agy and senescence (Dörr et al., 2013). In integrated, amorphous sarcomere. In some cases, we successfully immunolabeled and area of mitochondria in smooth muscle proteins in cell-cell contacts (Desmopla- cellsfurther and projects, the density we quantified of insulin the granula number in kin, Plakophilin 2 and Desmin) and in Ca2+ beta cells of the pancreas (collaborations channels of the heart (collaborations with with F. Le Noble and C. Birchmeier). A. Heuser and H. Haase).

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A: Endoplasmatic reticulum (ER) in high- grade astrocytoma cells grown in control Selected Publications medium. The ER is proliferated and arranged U Zacharias, B Purfürst, V Schöwel, I Morano, S Spuler, and H Haase. Ahnak1 abnor- in parallel stacks, but the ER lumen remains mally localizes in muscular dystrophies and contributes to muscle vesicle release.J thin, see Stock et al., 2012.Cryosection accord- Muscle Res Cell Motil 32:271-280 (2011) ing to Tokuyasu, membranes appear white, K Stock, J Kumar, M Synowitz, S Petrosino, R Imperatore, ESJ Smith, P Wend, B Purfürst, UA Nuber, U Gurok, V Matyash, J-H Wälzlein, SR Chirasani, G Dittmar, BF bar = 500 nm Cravatt, S Momma, GR Lewin, A Ligresti, L De Petrocellis, L Cristino, V Di Marzo, H B: ER in lymphoma cells from a mouse model Kettenmann, R Glass. Neural precursor cells induce cell death of high-grade astrocyto- system, treated with adriamycin. The ER is mas through stimulation of TRPV1.Nature Medicine 18: 1232-1238 (2012) proliferated and shows highly dilated cister- Wende, C Birchmeier. Colonization of the satellite cell niche by skeletal muscle progen- nae (arrowheads), see Dörr et al., 2013.Epon- Ditor Br cellsöhl, E depends Vasyutina, on notchMT Czajkowski, signals.Developmental J Griger, C Rassek, Cell 23: H-P 469-481 Rahn, B (2012)Purfürst, H embedding, membranes appear dark, with H Waiczies, S Lepore, S Drechsler, F Qadri, B Purfürst, K Sydow, M Dathe, A Kühne, T black ribosomes attached, bar = 500 nm - 19 1 C: A population of microvesicles released tion with a shingled-leg radio-frequency head probe for F/ 3:Lindel, 1280 W (2013) Hoffmann, A Pohlmann, T Niendorf, S Waiczies. Visualizing brain inflamma from human myotubes, see Zacharias et al., H MRI.Scientific Reports JR Dörr, Y Yu, M Milanovic, G Beuster, C Zasada, JHM Däbritz, M Reimann, J Lisec, K 2011.Negative contrast, bar = 200 nm Schleicher, S Kratzat, B Purfürst, S Walenta, W Müller-Klieser, U Keller, AK Buck, B D: Amyloid fibrils, immunolabeled with 12 nm Dörken, L Willmitzer, S Kempa, S Lee, CA Schmitt. Synthetic Lethal Metabolic Targeting colloidal gold particles.Negative contrast, bar of Cellular Senescence in Cancer Therapy.Nature 501: 421-25 (2013) = 200 nm

Group Leader Dr. Bettina Purfürst Secretariat Iris Rasch Technical Assistants (part-time, until December 2013) Marianne Vannauer Manuela Brandenburg Margit Vogel (part-time) (from January 2014)

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TECHNOLOGY PLATFORMS Photo: Peter Sonnabend/NGFN Peter Photo:

Erich Wanker

Interactomics

Interactome ing proteins visible. The system is particu- The relevance of interactions between proteins for biological inquiry is beyond transient interactions. This makes it prone dispute. Several methods to identify pro- tolarly identifying suited to detectingalso false alsopositives, low-affinity which oris tein-protein interactions (PPIs) are well-es- why we have established comprehensive tablished. One method that has been devel- quality scoring and validation techniques. oped into an automated high-throughput The membrane yeast-two-hybrid sys- system is the yeast-two-hybrid method. It tem. MYTH is a split-ubiquitin based sys- allows screening of binary protein-protein tem to detect interactions between integral interactions and compilation of large inter- membrane proteins and membrane-asso- action network maps. These networks are the raw material for many disciplines: Ex- 1998, PNAS). perimental biologists select subsets of in- Theciated/proximal cytosolic yeast-two-hybrid proteins (Stagljar system. et al., teractions to form and test new hypotheses CytoY2H is a split-ubiquitin-based cytosolic on protein function and molecular mecha- screening system to detect interactions be- nisms, computational biologists use them tween transcriptionally active proteins and to predict biologically relevant interactions, enables the detection of PPIs in the cyto- systems biologists derive an understanding plasm (Mockli et al., 2007, Biotechniques). of how the proteome-wide interactome is The LUMIER assay. This is a luminescence- organised. based mammalian interactome screening technology (Barrios-Rodiles et al., Science, Background - The research group of Prof. Erich Wanker is malian cells, which we have established for2005) validation for the identification of PPIs detected of PPIs in thein mam Y2H - system. Like the Y2H techniques, it has manone ofdisease, the pioneering Huntington’s labs disease in the field.(Goehler We been automated and can be applied to high- ethave al., put 2004, forward Molecular the first Cell) PPI as map well of as a huthe throughput testing of interactions. The clone library. For the interaction for human (Stelzl et al., 2005, Cell). In the screenings, a comprehensive prey-library coursefirst proteome-wideof these studies we interactome have established map arrayed in microtiter plates is available. It a platform of automated screening and vali- contains ~23,000 entry-clones, of which dation techniques which we offer for use to ~14,000 are unique full-length cDNAs. This all scientists at the MDC and outside. library covers more than 75% of the human genome. We are able to re-array the library Platform technologies according to the needs and interests of di- The automated yeast-two-hybrid system. Song (1989, Nature) and is based on the verseIdentification research projects. of Human Proteins principleIt was of entering first described bait and byprey Fields plasmids and that Modify Misfolding and Proteo- toxicity of Pathogenic Ataxin-1 strain. If the protein products interact, a Petrakis et al., 2012, PLoS Genetics reporterof interest gene in ais geneticallyactivated that modified enables yeast the In this study, we sought to identify proteins yeast’s growth on selective medium. A co- that interact with Ataxin-1, causative to lour reaction makes clones with interact- spinocerebellar ataxia type 1, a rare neu-

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Schematic representation of a cell-based LUMIER co-immunoprecipitation assay. To investigate the Selected Publications interaction between the proteins ATXN1Q30 (bait) H. Goehler, M. Lalowski, U. Stelzl, S. Waelter, M. Stroedicke, U. Worm, A. Droege, K. S. and Pum1 (prey) the fusion protein pairs (A) PA-RL- Lindenberg, M. Knoblich, C. Haenig, M. Herbst, J. Suopanki, E. Scherzinger, C. Abraham, ATXN1Q30/FL-Pum1, (B) PA-RL/FL-Pum1 and (C) B. Bauer, R. Hasenbank, A. Fritzsche, A. H. Ludewig, K. Büssow, S. H. Coleman, C.-A. PA-RL-ATXN1Q30/FL were co-produced in HEK293 Gutekunst, B. G. Landwehrmeyer, H. Lehrach, and E. E. Wanker, “A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington’s disease.,” cells. After 48 h the cells were lysed and protein Mol. Cell, vol. 15, no. 6, pp. 853–865, 2004. extracts were applied to IgG coated magnetic beads U. Stelzl, U. Worm, M. Lalowski, C. Haenig, F. H. Brembeck, H. Goehler, M. Stroedicke, (Dynabeads; Invitrogen) for co-immunoprecipitation M. Zenkner, A. Schoenherr, S. Koeppen, J. Timm, S. Mintzlaff, C. Abraham, N. Bock, S. (co-IP) of interacting proteins. Following the co-IP Kietzmann, A. Goedde, E. Toksöz, A. Droege, S. Krobitsch, B. Korn, W. Birchmeier, H. the firefly luminescence signal was determined for Lehrach, and E. E. Wanker, “A human protein-protein interaction network: a resource for annotating the proteome.,” Cell, vol. 122, no. 6, pp. 957–968, 2005. all three samples (A–C) using a luminescence plate reader. In sample A, as a result of the interaction K. Venkatesan, J.-F. Rual, A. Vazquez, U. Stelzl, I. Lemmens, T. Hirozane-Kishikawa, T. between PA-RL-ATXN1Q30 and FL-Pum1 a high Hao, M. Zenkner, X. Xin, K.-I. Goh, M. A. Yildirim, N. Simonis, K. Heinzmann, F. Gebreab, J. M. Sahalie, S. Cevik, C. Simon, A.-S. de Smet, E. Dann, A. Smolyar, A. Vinayagam, H. Yu, luminescence signal was detected. In comparison, D. Szeto, H. Borick, A. Dricot, N. Klitgord, R. R. Murray, C. Lin, M. Lalowski, J. Timm, K. relatively low signals were obtained in control Rau, C. Boone, P. Braun, M. E. Cusick, F. P. Roth, D. E. Hill, J. Tavernier, E. E. Wanker, A.-L. samples B and C. Using the measured values we cal- Nat. Methods, vol. 6, no. 1, pp. 83–90, 2009. culated the binding ratios R-op and R-ob, which are Barabási, and M. Vidal, “An empirical framework for binary interactome mapping.,” a measure for the specificity of the ATXN1Q30 and A. Vinayagam, U. Stelzl, R. Foulle, S. Plassmann, M. Zenkner, J. Timm, H. E. Assmus, M. A. Andrade-Navarro, and E. E. Wanker, “A directed protein interaction network for Pum1 interaction. Based on previous empirical stud- investigating intracellular signal transduction.,” Sci. Signal., vol. 4, no. 189, p. rs8, 2011. ies with well-characterized interaction test pairs, we define an interaction as positive when the calculated - berg, A. Möller, A. Reinhardt, A. Vinayagam, M. H. Schaefer, M. Boutros, H. Tricoire, M. R-op and R-ob ratios are >1.5. S. Petrakis, T. Raskó, J. Russ, R. P. Friedrich, M. Stroedicke, S.-P. Riechers, K. Muehlen misfolding and proteotoxicity of pathogenic ataxin-1.,” PLoS Genet., vol. 8, no. 8, p. e1002897,A. Andrade-Navarro, Jan. 2012. and E. E. Wanker, “Identification of human proteins that modify rodegenerative disease that belongs to the polyglutamine repeat disorders. We investigated a selection of interactions with Atax- Group Leader in-1 in our LUMIER assay (Figure 1), identi- Prof. Dr. Erich Wanker fying PUM1 as an interactor that promotes misfolding and aggregation of the Ataxin-1 Engineers protein. Using a PPI screening and valida- Ronny Kalis Technical Assistant tion strategy, biologically relevant proteins Martina Zenkner Susanne Köppen

and pathways can be identified. MDC Research Report 2014 237

TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Thoralf Niendorf

Magnetic Resonance (Berlin Ultrahigh Field Facility, B.U.F.F.)

B.U.F.F. has been implemented on the Cam- our data base BUFFportal, which is used for pus Berlin-Buch. B.U.F.F. builds on collabo- - agement, data handling, publication tracking, rations with our strategic partners from the etc.safety To training,push the comprehensivecapabilities of magnetic project manreso- Charité, the Leibniz Institute for Molecular nance and to drive next generation MR meth- Pharmacology (FMP), the Physikalisch-Tech- odology and MR hardware B.U.F.F. provides infrastructure for radiofrequency (RF) coil nische Bundesanstalt (PTB), Siemens Health- design, for MR pulse sequence development, care, Bruker Biospin and other (inter)national for development, validation and application institutions. To learn more about the mission of ancillary hardware and for rapid prototyp- of B.U.F.F. please visit ing (Figure 2, right). https://www.mdc-berlin.de/37752989/en/ecrc/ Research focus Ultrahigh_Field_MR/B_U_F_F_ Research at B.U.F.F. concentrates on the development of MR methodology and MR technology with a focus on new ways of mapping and Equipment and infrastructure probing anatomy, morphology, microstruc- B.U.F.F. provides state-of-the-art infrastructure, function, physiology and metabolism to-

and is equipped with small animal (9.4 T) ture for interdisciplinary imaging projects Thesegether efforts with explorations are designed into to spatiallythe benefits resolve and scanners (Figure 1). For experimental and andchallenges characterize of ultrahigh (patho)physiological field (UHF) imaging. and andpreclinical whole-body imaging human B.U.F.F. (7.0 runs T &an 3.0 S1 T) certi MR- biophysical processes to promote a transfer - from basic research to (pre)clinical studies mal housing. To foster translational research and vice versa. To highlight the anatomical andfied laboratoryexplorations and into supports clinical smallstudies scale B.U.F.F. ani detail and spatial resolution capabilities of is equipped with 2 reception areas to comfort MRI Figure 3 surveys examples derived from and guide volunteers and patients. B.U.F.F. in vivo mice phenotyping and from in vivo hu- comprises four dressing rooms, all connected man imaging. to scanner rooms and reception areas (Figure 2, left). Several web interface based reading Contributions to major research stations are available; imaging data can be ac- initiatives cessed from every desktop available at B.U.F.F. Principle investigators of B.U.F.F. form a key/ All technical prerequisites needed for clinical studies are setup including emergency equip- the Helmholtz Alliance for Imaging and Cur- - ingintegral Environmental part of major Diseases initiatives (ICEMED), including the population MRI program of the German Na- usedment, forextra research patient monitoringstaff are separated units, defibril from tional Cohort (GNC), the DFG research group thelators scanner and extra areas MR to safe meet patient the needs beds. for Offices data FOR 1368 on Hemodynamics of Acute Kidney safety and volunteer/patient convenience. To meet all data safety requirements B.U.F.F. Center for Cardiovascular Research (DZHK), is equipped with a commercial PACS system Injury, the imaging program of the German used for data storage, data archiving and data SPECT/MR imaging, the BMBF initiative on mining. The PACS system is connected with sodiumthe EU funded MRI (NAMRIS) FP7 project and INSERT the German on hybrid Ul-

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Fig. 1: Picture photographs of the whole-body human Fig. 2: Overview of the volunteer/patient preparation areas, (7.0 T & 3.0 T) and small animal (9.4 T) MR scanner the clinical imaging setup and the MR hardware develop- environments at B.U.F.F. ment laboratories available at B.U.F.F.

trahigh Field Imaging (GUFI) network. B.U.F.F. Basic research and clinical science is an integral part of the imaging core facility opportunities of the Berlin Institute of Health (BIH). The B.U.F.F. team is always looking for collaborators. For further details about opportu- How to get to there nities for collaboration, access, resources and B.U.F.F. is easily accessible via public transportation as well as by car. Go green and use https://buffportal.mdc-berlin.de or public transport. Public transportation is di- www.mdc-berlin.de/BUFFprojects please visit . rectly arriving/leaving on campus Berlin-Bu- The user guidelines can be found under ch (line 353 from S-Bahn station Berlin-Buch https://buffportal.mdc-berlin.de./procedure. to station Lindenberger Weg-Süd). The bus For further details please contact the Head of station is located right in front of B.U.F.F. Free B.U.F.F. (Thoralf Niendorf, parking space is available in front of B.U.F.F. [email protected], +49 30 9406 4505).

Fig. 3:Illustration of the anatomical detail and spatial resolution provided by MR imaging. Top: In vivo MR images of Patents / Patent applications the mice heart (in-plane spatial resolution 50 micrometer), please see the chapter on Ultrahigh Field Magnetic Resonance (UHF-MR) of the mice brain (in-plane spatial resolution 30 micrometer), of the vessel tree of the mice brain (in-plane spatial Selected Publications resolution 30 micrometer) and of the mice kidney (in-plane Pohlmann A, Cantow K, Hentschel J, Arakelyan K, Ladwig M, Flemming B, Hoff spatial resolution 150 micrometer) obtained with the 9.4 U, Persson PB, Seeliger E, Niendorf T. Linking non-invasive parametric MRI with T small animal MR scanner at B.U.F.F. Bottom: In vivo MR invasive physiological measurements (MR-PHYSIOL): towards a hybrid and inte- images of the human heart (in-plane spatial resolution 1.0 2013;207(4):673-689. grated approach for investigation of acute kidney injury in rats. Acta Physiol (Oxf) mm), of the human brain (in-plane spatial resolution 0.5 Sinnecker T, Dorr J, Pfueller CF, Harms L, Ruprecht K, Jarius S, Bruck W, Niendorf T, mm), of the left hemisphere vessel tree of the human brain Wuerfel J, Paul F. Distinct lesion morphology at 7-T MRI differentiates neuromyelitis (in-plane spatial resolution 0.5 mm) and of the human liver optica from multiple sclerosis. Neurology 2012;79(7):708-714. (in-plane spatial resolution 1.0 mm) obtained with the 7.0 Niendorf T, Muller DN, Neininger M, Cavallaro A, Eckardt KU, Schmieder RE, Luft FC, T whole body human MR scanner at B.U.F.F. KoppUder M,C, LinzTitze P, J. Wachsmuth (23)Na magnetic L, Dahlmann resonance A, Horbach imaging T,of Schofl tissue C,sodium. Renz W, Hypertension Santoro D, 2012;59(1):167-172. von Knobelsdorff-Brenkenhoff F, Tkachenko V, Winter L, Rieger J, Thalhammer C, Hezel F, Graessl A, Dieringer MA, Niendorf T, Schulz-Menger J. Assessment of the right ventricle with cardiovascular magnetic resonance at 7 Tesla. J Cardiovasc Magn Reson 2013;15:23. Ku MC, Wolf SA, Respondek D, Matyash V, Pohlmann A, Waiczies S, Waiczies H, Niendorf T, Synowitz M, Glass R, Kettenmann H. GDNF mediates glioblastoma-induced microglia attraction but not astrogliosis. Acta Neuropathol 2013;125(4):609-620.

Structure of the Group please see the chapter on Ultrahigh Field Magnetic Resonance (UHF-MR)

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TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Gunnar Dittmar

Mass Spectrometry

Cellular signaling proteomic techniques in different collabora- Cells interact with their environment and tions with groups at the MDC (Figure 1). We react to different stimuli and changes in the are working closely with these groups to op- environment. These reactions can either be based on transcriptional activity or chemical timizeTargeted the methods proteomics for the different projects. Monitoring the regulation of proteins under gainmodifications insight into of the existing regulation proteins, of these so called pro- different conditions can provide deeper in- tein-interactionpost-translational networks modifications. it is necessary In order to sight in the regulatory network, which underlies a signal transduction cascade. For many measure the protein expression levels within identify proteins, their modifications and to- eral hundreds or thousands proteins and knowledgecascades the into major a targeted players strategy in these allowspathways fo- a cell. This requires the identification of sev cusingare already to monitor identified. changes Incorporating in the concentra this- addition the information has to be collected tion of these players, avoiding the sequencing their quantification in a complex mixture. In of unrelated, not regulated proteins in the are matched by modern mass spectrometry cell. A method that allows the selection of a andin a timeresult efficient in the way.methods All these rise requirementsto be the de- limited number of proteins is selected reac- - tion monitoring (SRM). The technique has cation in life sciences. the advantage of a high sensitivity combined faultThe cormethode facility for large-scalemass spectrometry protein identifi offers with short run times on the liquid chromatog- a wide range of mass spectrometry meth- raphy systems. This opens the possibility of measuring large quantities of different sam- of proteins and peptides. Besides the iden- ples in a short time period and quantifying all ods for the identification and quantification components of the cascade. spectrometry core facility uses a number of Several collaborations of the core facility tification of proteins in gel slices, the mass within the MDC are now based on multiple Fig 1: Electro spray setup (ESI) for the analysis of peptides. Peptides reaction monitoring experiments. The core are ionized in front of the orifice of the mass spectrometer. The charged facility has access to two different mass- peptides are measured in the mass spectrometer. spectrometers capable for this type of experiments (Q-Trap 5500 and Q-Trap 4000).

Non-targeted proteomic approaches Metabolic labeling of cells offers great ad-

The cells of interest are therefore cultured in media,vantages which for thecontain quantification isotopic labeled of proteins. (non radioactive) amino acids (Figure 2). The additional mass of the amino acids can later be detected by mass spectrometry. Comparison of two different samples is possible by analyzing the sample on a high accuracy mass spectrometer (LTQ-Orbitrap). The results of these measurements are relative ratios for each protein detected in the two different samples. This techniques is currently used by the core

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Fig 3: Analysis of digestion patterns generated by the proteasome a 2 MDa protease. The digestion of foreign proteins is important for the cellular immunity. The proteasome as one of the largest cellular proteases generates peptides from proteins, which can then presented by the immun system. The analysis of the peptides generated by the proteasome in vitro were analysed and revealed new interesting digestion patterns

Fig 2: Metabolic labelling of proteins using isotope containing amino acids allows the quantification of several thousand proteins in one experiment. The isotope containing amino acids are fed to cells in cell culture (SILAC) leading to a homoge- niously labelled population of proteins. After mixing the differently labelled proteins and preparing them for the analysis the Selected Publications peptide mixtures are analyzed using a high resolution mass Bagola, K., von Delbrück, M., Dittmar, G., Scheffner, M., Ziv, I., Glickman, M., Ciecha- spectrometer. The spectra contain characteristic pairs of mass nover, A. and Sommer, T. Ubiquitin binding by a CUE domain regulates ubiquitin chain peaks which can be used by the analysis software for the quan- formation by ERAD E3 ligases, Molecular Cell, 50, 528-539 (2013). tificaiton of the samples. The SILAC technique can be applied to Anton, F., Dittmar, G the analysis of complex protein mixtures (lower panel) or the mitofusin and regulate mitochondrial fusion along independent pathways. Molecular analysis of the proteins interactome (upper panel). Cell, 49, 487-498 (2013).., Langer, T. & Escobar-Henriques, M. Two deubiquitylases act on

Müller-Rischart, A.-K., Pilsl, A., Beaudette, P., Patra, M., Hadian, K., Funke, M., Peis, R., Deinlein, A., Schweimer, C., Kuhn, P.-H., Lichtenthaler, S., Motori, E., Hrelia, S., Wurst, W., Trümbach, D., Langer, T., Krappmann, D., Dittmar, G., Tatzelt, J., and Winklhofer, K.. The E3 ligase parkin maintains mitochondrial integrity by increasing linear ubiquitination of NEMO. Molecular Cell, 49, 908-921 (2013).

Stock, K., Kumar, J., Synowitz, M., Petrosino, S., Imperatore, R., St J Smith, E., Wend, P., Purfürst, B., Nuber, U.A., Gurok, U., Matyash, V., Wälzlein, J.-H., Chirasani,, S.R., Dittmar, - G., Cravatt, B.F., Momma, S., Lewin, G.R., Ligresti, A., De Petrocellis, L., Cristino, L., Di - Marzo, V., Kettenmann, H., and Glass, R. Neural precursor cells induce cell-death of facility for the quantification of immuno-pre high-grade astrocytomas via stimulation of TRPV1. Nature Medicine, 18, 1232–1238 tions. (2012). Fcipitationsor samples, and which large scalecannot protein be cultured identifica in media containing heavy amino acids, other Schwanhäusser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J., Chen, W., and Nature 473, techniques are available. This techniques ei- 337–342 (2011). ther rely on a highly reproducible liquid chro- Selbach, M. Global quantification of mammalian gene expression control. matography (Figure 3) or the peptides are labelled post-lysis using a chemical reaction.

Identification of post-translational Group Leader modifications Dr. Gunnar Dittmar Besides the translational regulation of proteins, another layer of regulation exists, which Scientist Tamara Kanashova - Dr. Julia Kikuchi Sylvia Zerath Guerevich tions. For the understanding of the molecular Dr. Oliver Popp mechanisms,is mediated bywhich post-translational regulate these proteins, modifica it Dr. Daniel Perez-Hernandez Engineer is necessary to gain insight into the different Alina Dagane PhD The core facility actively pursues the develop- Günther Kahlert Students post-translational modifications of proteins. Patrick Beaudette Felix Kallenberg

ment of new methods for the identification of modification sites by ubiquitin-like proteins. MDC Research Report 2014 241

TECHNOLOGY PLATFORMS Photo: Peter Himsel/MDC Peter Photo:

Arnd Heuser

Pathophysiology

Despite numerous advances in research techniques such as in vitro methods and intially the sacrificenumber of animals animals at used different per study. time advanced computer modeling that avoid points, allowing for significant reductions the use of animals, there is still the need procedures and husbandry to minimize ac- for a whole-system approach to preclinical tualWe continuouslyor potential pain, try tosuffering, improve distress scientific or research and to obtaining data from con- lasting harm and/or improve animal welfare in situations where the use of animals scious and unconscious animals. For exam- is unavoidable. While the central focus of ple, understanding the complex physiologi- the facility is cardiovascular research, the cal interactions between the cardiovascular, techniques that are employed may also be respiratory and central nervous systems will as cancer, neurobiology and developmental be critical for the development of many hu- biology.useful to investigators in other fields such man pharmaceuticals and therapies. High Frequency Ultrasound Ultrasonography is a powerful imaging method that permits a noninvasive, real- The Pathophysiology Platform provides time visualization of organs and tissues. We researchers with a central resource for the perfom sonographic studies of small ani- study of animal disease models. We use mals using a High-Frequency Ultrasound validated approaches and state-of-the-art system. Its transducers produce frequen- instrumentation that allow for the sensitive cies as high as 70MHz, permitting the iden- screening of phenotypic variations. We focus on techniques which minimize animal with a maximum temporal resolution of use and enable researchers to obtain com- 740fps.tification This of structuresmakes the asplatform small asideal 30µm for parable levels of information from fewer pre-clinical research because it permits animals or to obtain more information from the same number of animals, thereby The Methodological Spectrum of the reducing animal use in the future. With this Pathophysiology Platform. aim, we use a variety of non-invasive in vivo test tools that allow the monitoring of physiological and bioelectrical variables (e.g. blood pressure, heart rate, ECG and respiration) in conscious animals. The non-invasive, whole-body imaging of small animals using techniques such as High-Frequency Ultrasound and Time-Domain-Nuclear- Magnetic-Resonance imaging is helping to reduce the number of animals used in basic research and drug development. The same animal can be imaged multiple times in order to monitor visually, often in real time, the progression or regression of infections or disease. This avoids the need to sequen-

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the study of many organs, tissues, and the behavior of biological systems that need to be studied at this tiny scale. The non- invasive nature of ultrasound means that it can be used without harming animal or hu-

variety of protocols to analyze the pheno- typesman subjects. of mice andWe haverats: establishedFor cardiovascular a wide studies, ultrasound imaging can be used to detect the presence of localized or gen- High Frequency Ultrasound Imaging: eralized hypertrophy or a thinning of the (A) Parasternal Long Axis View of a Murine Heart, myocardium of the left ventricle (LV) and (B) Micro-Ultrasound Imaging of a Murine Embryo at E12.5, the presence of regional or global wall mo- (C) Contrast Imaging of a Melanoma Tumor, tion abnormalities associated with systolic (D) 3D-Color Doppler of a Murine Testicle. dysfunction. The application of transmitral Doppler analysis permits the detection of

LV diastolic dysfunction. Strain analysis providesabnormal a fillinghighly patterns sensitive associated speckle-track with- ing-based echocardiographic imaging technique that offers a quantitation of the veloc- Selected Publications ity of the walls, displacement, strain, strain alpha(1A)-adrenergic receptor-directed autoimmunity induces left ventricu- rate, and time to peak analysis. Because of lar damage and diastolic dysfunction in rats.Wenzel, K. and Wallukat, G. and Qadri, F. and Huebner, N. and Schulz, H. and Hummel, O. and Herse, F. and Heuser, its non-invasive nature, ultrasound can be A. and Fischer, R. and Heidecke, H. and Luft, F.C. and Mueller, D.N. and Dietz, R. and used in longitudinal studies for tumor di- Dechend,R. PLoS ONE 5 (2): e9409. 24 February 2010 agnosis and monitoring during therapies. Growth arrest specific protein 6 participates in DOCA-induced target-organ 3D-Ultrasound imaging allows for a reli- damage.Park, J.K. and Theuer, S. and Kirsch, T. and Lindschau, C. and Klinge, U. and able delineation of tumor boundaries and Heuser, A. and Plehm, R. and Todiras, M. and Carmeliet, P. and Haller, H. and Luft, F.C. an assessment of tumor heterogeneity. In and Mueller, D.N. and Fiebeler, A. Hypertension 54 (2): 359-364. August 2009 addition, ultrasound imaging is ideal for Role of the multidomain protein spinophilin in blood pressure and cardiac studying pregnancy and development in function regulation.da Costa-Goncalves, A.C. and Tank, J. and Plehm, R. and Diedrich, A. and Todiras, M. and Gollasch, M. and Heuser, A. and Wellner, M. and Bader, M. and mice and rats. Jordan, J. and Luft, F.C. and Gross, V. Hypertension 52 (4): 702-707. October 2008

Body Composition Analysis Dietary n-3 polyunsaturated fatty acids and direct renin inhibition improve electrical remodeling in a model of high human renin hypertension.Fischer, R. Time-Domain-Nuclear-Magnetic-Reso- and Dechend, R. and Qadri, F. and Markovic, M. and Feldt, S. and Herse, F. and Park, nance imaging (TD-NMR) is a powerful J.K. andGapelyuk, A. and Schwarz, I. and Zacharzowsky, U.B. and Plehm, R. and Safak, E. and Heuser, A. and Schirdewan, A. and Luft, F.C. and Schunck, W.H. and Mueller, D.N. Hypertension 51 (2): 540-546. February 2008 Withouttool for theanesthesia non-invasive or other quantification preparations, of Soluble epoxide hydrolase is a susceptibility factor for heart failure in a rat model of human disease.Monti, J. and Fischer, J. and Paskas, S. and Heinig, M. and animalssolids and such liquids as mice in and objects rats can under be tested study. Schulz, H. and Goesele, C. and Heuser, A. andFischer, R. and Schmidt, C. and Schird- - ewan, A. and Gross, V. and Hummel, O. and Maatz, H. and Patone, G. andSaar, K. and ids and lean tissue within a few minutes. Vingron, M. and Weldon, S.M. and Lindpaintner, K. and Hammock, B.D. and Rohde, K. and Dietz, R.and Cook, S.A. and Schunck, W.H. and Luft, F.C. and Huebner, N. Nature for body composition of fat, free body flu Genetics 40 (5): 529-537. May 2008 particle spins within a high energetic mag- After a stimulated adjustment of nuclear times of spin rearrangements are recorded netic field, specific patterns of relaxation relaxation patterns can be correlated to the Group Leader densityupon removal of body of compounds, the magnetic i.e. field.lean tissue,These Dr. Arnd Heuser

their proportional composition. The NMR- Technical Assistants Secretariat basedfat and bodyfree fluids, composition giving concrete analyzer values allows of Stefanie Schelenz Manuela Brandenburg Martin Taube whole-body components of small animals. non-invasive and reliable quantifications of

MDC Research Report 2014 243

TECHNOLOGY PLATFORMS Photo: David Ausserhofer/MDC Photo:

Hans-Peter Rahn

Preparative Flow Cytometry

Fluorescence-activated cell sorting (FACS) of tumor and primary murine hematopo- is a specialized type of flow cytometry. It etic cells in order to analyse the function of ectopically expressed genes in comparison was designed in 1971 by Len Herzenberg to to endogenous expression levels in lympho- provide a fast physical separation method ma cells. to fractionate heterogeneous mixtures of cells into distinct subsets, based generated various mouse models of human upon the light scattering and fluorescent lymphomasThe research and group fatal of Klauslymphoproliferative Rajewsky has characteristics of different cell types. disorders to elucidate underlying mecha- The FACS Core Facility at the MDC mainly nisms of pathogenesis as well as genetically manipulated mice for conditional loss- or provides assistance in operating cell sorters gain-of-function studies to discover the role with a speed up to 20,000 cells per second for large-scale cell isolation; for more developing a new genome editing technol- analytical purposes, single cells can also be ogy,of specific the CRISPR/Cas9 gene elements. system. They The have FACS been analyzer and sorter are indispensable equip- spotted very precisely into microtiterplates ment to characterize, quantify, and purely or onto slides. In addition, the core facility staff assists in the design and set-up of sophisticated experiments on additional Fortessaisolate specific equipped immune with 14-channel cell population param as- eterswell asenables genetically them modified to analyze cells. extremely The BD state of the art analyzers. The facility oper- rare cell population such as haematopoietic ates one analyzer of the type “BD LSR”, one stem cells, memory cells, and cancer pro- 4-colour laser line sorter BD Fortessa, and genitors.

three high-capacity “BD Aria” digital sorters. Stromal cells are pivotal for the onset and survival of lymphoma B cells; however, which stromal cell subpopulations within lymph nodes contribute to the formation The facility has been used over the last few of a lymphoma growth-promoting niche remained elusive. The group of Armin Rehm from the MDC, BIMSB, ECRC and the FMP. has applied a combination of magnetic Sortedyears bycells many are often different prepared scientific for microar groups- bead cell enrichment and FACS sorting to ray- or sequencing-based gene expression isolate rare stromal subsets of mesenchy- analysis, quantitative real-time PCR, DNA mal origin which could be used for gene ex- sequencing, live-cell imaging or adoptive pression analysis. Thus, the establishment transfer experiments in animals. of this method was a prerequisite to the

The greatest number of requests for all preparative cell sorting at the MDC arise from stromalanalysis cell of atypes. specific tumor-induced gene primary human and murine cells, which are expression profile in endothelial and other characterized by their immunophenotypic In addition, the group of Martin Lipp focus- parameters. For example, the group of Ber- es on distinct memory and effector T cell nd Dörken routinely uses FACS enrichment populations which are important for the

244 MDC Research Report 2014 TECHNOLOGY PLATFORMS

development of antibody-based adaptive immune responses. In this context central memory, effector/memory and follicular B helper T cells are enriched by FACS us-

these cells. ing specific patterns of surface markers on More recently, FACS technologies have gained substantial importance for the group of Walter Birchmeier; in this lab, the focus has been on the analysis and isolation of tumor and benign stem cells. For example, it is possible to separate somatic stem cells of the skin, the mammary gland, the heart, and neuronal stem cells. Reli- able cell-surface biomarkers are becoming Left: Inside view of a FACSAria (flow cell with sort block more widely available. This holds true also and deflection plates), Right: Schematic view of the separa- for the area of cancer stem cells or cancertion principle initiating cells.

Sorting is also used by the lab from Carmen Birchmeier to study how different signaling pathways control the development and Selected Publications function of satellite cells, the stem cells of Mathas, S., S. Kreher, K.J. Meaburn, K. Jöhrens, B. Lamprecht, C. Assaf, W. Sterry, M.E. adult muscle. These cells are needed for Kadin, M. Daibata, S. Joos, M. Hummel, H. Stein, M. Janz, I. Anagnostopoulos, E. Schrock, T. Misteli, and B. Dörken. 2009. Gene deregulation and spatial genome reorganiza- muscle growth and also for the regeneration near breakpoints prior to formation of translocations in anaplastic large cell - lymphoma. Proceedings of the National Academy of Sciences of the United States of ellite cells account for only a small fraction America. 106:5831–6. doi:10.1073/pnas.0900912106. oftion muscle of injured tissue, muscle they tissue.can be Although isolated andsat Bröske, A.-M., L. Vockentanz, S. Kharazi, M.R. Huska, E. Mancini, M. Scheller, C. Kuhl, A. enriched by FACS, permitting an analysis Enns, M. Prinz, R. Jaenisch, C. Nerlov, A. Leutz, M. a Andrade-Navarro, S.E.W. Jacobsen, and F. Rosenbauer. 2009. DNA methylation protects hematopoietic stem cell multipo- of gene expression changes in mouse mu- tency from myeloerythroid restriction. Nature genetics. 41:1207–15. doi:10.1038/ tants and a more detailed characterization ng.463. of these cells using in vitro assays. Klaus, A., M. Muller, H. Schulz, Y. Saga, J.F. Martin, and W. Birchmeier. 2012. Wnt/ -catenin and Bmp signals control distinct sets of transcription factors in cardiac FACS technology has also gained impact at progenitor cells. Proceedings of the National Academy of Sciences. 109:10921–10926. the MDC in the generation of iPS (induced doi:10.1073/pnas.1121236109. pluripotent stem) cells. For example, the group of Mathias Treier labels ES cells with Wende, and C. Birchmeier. 2012. Colonization of the Satellite Cell Niche by Skeletal Bröhl,Muscle D., Progenitor E. Vasyutina, Cells M.T. Depends Czajkowski, on Notch J. Griger, Signals. C. Rassek,Developmental H.-P. Rahn, Cell. B. 23:469–481. Purfürst, H. proteins to enrich different germ layers duringvarious differentiation reporters containing to understand fluorescent how doi:10.1016/j.devcel.2012.07.014.Stoeckius, M., J. Maaskola, T. Colombo, H.-P. Rahn, M.R. Friedländer, N. Li, W. Chen, F. cell fate decisions are made. dynamics of small RNA expression. Nature methods. 6:745–751. Piano, and N. Rajewsky. 2009. Large-scale sorting of C. elegans embryos reveals the To study early embryogenesis employing high-throughput genomics as well as biochemistry assays, the lab of Nikolaus Ra- Group Leader Technical Assistants amounts of precisely staged C. elegans em- Dr. Hans-Peter Rahn Kirstin Rautenberg bryosjewsky by devised FACS. This a method method to collectwill make large a contribution towards a more complete un- Secretary derstanding of gene regulatory networks Manuela Brandenburg during early C. elegans development.

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(From May 2014): Ralf Kühn

Transgenics

Transgenic Core Facility (TCF)

The Transgenic Core Facility (TCF) offers expertise in all kinds of mouse Assisted Re- productive Technologies (ART). Starting at the planning phase, we support scientists from the MDC and their external collaborators with the design and layout of their -

Thisprojects. is accomplished One focus of either our work by gene is the target gen- ingeration in embryonic of genetically stem modified (ES) cells mouse and lines. subsequent generation of chimeric mice from recombinant ES cell clones or by micro-in-

into the pronuclei of fertilized oocytes. jection of plasmid or BAC type transgenes A second focus of our work is the rederivation of lines for the population of the new very clean facility with highest hygienic demands that allows the breeding

the burden caused by opportunistic infections.of immune Moreover deficient we cryo-preserve mouse lines precious without mouse lines and bring frozen lines back to life. Since most groups choose to conserve mouse sperm this is the main form of germ plasm we freeze down and store in liquid nitrogen. Due to the growing number of mouse models worldwide, we see an increase in the number of organizations who offer to send frozen material instead of live mice.

Pronuclear microinjection. When a zygote is fixed to the holding capillary, both the injection needle filled with DNA solution as well as one of the pronuclei are brought into focus. Then the zona pellucida is penetrated with the injection capillary aiming at one of the pronuclei (a). The injection capillary is moved further to the far side of the pronucleus and then slightly pulled back to place the capillary’s tip in its middle (b). When the injection pressure is applied a swelling of the injected pronucleus has to be clearly visible (comparearrowheads in a to those in c).

246 MDC Research Report 2014 TECHNOLOGY PLATFORMS

These lines are revitalized at the MDC following a variety of protocols depending on the method of cryo-preservation. There are

the above that can be supported by the TCF andindividual we will projects help whenever that do not it comesfit into anyto the of production, isolation, manipulation, culture or retransfer of pre-implantation embryos. Moreover, we can give advice on cloning and targeting strategies, BAC preparation, ES cell culture, ES cell strain background, coat color genetics, ES cell derivation, and help with the selection of targeted ES cell clones from international knock out consortia for the generation of the corresponding gene targeted mouse lines. A future focus of the TCF will lie on the utilization of

via enzyme mediated induction of double strandnew technologies breaks and forsubsequent genome modificationhomologous recombination either in cells or in zygotes directly.

Selected Publications

Transposon-mediatedHiripi L, Moreno C, Lazar transgenesis, J, Bashir S, transgenicZidek V, Popova rescue, E, andJerchow tissue-specific gene expressionA, Walter I, inGrzybowksi rodents and M, rabbits.Corbett KatterM, Filho K, AR,Geurts Hodges AM, MR,Hoffmann Bader O,M, Mátés Ivics Z, L, Jacob Landa V, B, Becker K, Devaraj

HJ, Pravenec M, Bosze Z, Rülicke T, Izsvák Z. FASEB J. 2013 Mar;27(3):930-41. doi: 10.1096/fj.12-205526. Epub 2012 Nov 29.

Record 2013: Group Leader Administrative Assistance PD Dr. rer. nat. Ralf Kühn Christine Krause • 13 lines from recombinant ES cells (From May 2014) • 52 sperm freezings Technical Assistants • 6 in vitro fertilizations • 13 hygienic rederivation Andrea Leschke Katja Becker

MDC Research Report 2014 247

248 MDC Research Report 2014 Technology Transfer

MDC Research Report 2014

Technology Transfer

echnology transfer at the Max Del- European Molecular Biology Laboratory brück Center aims to identify, protect, (EMBL) and further Helmholtz institutes. T - These activities draw together expertise ings and technologies resulting from re- from the partners to establish new plat- search.manage This entails, and market for example, promising supporting find forms for drug screening, to improve hits the MDC’s scientists in drafting patenting through medicinal chemistry methods, and and marketing strategies and consulting provide new animal models for in vivo vali- them on whether an invention would be dation. best advanced by licensing, by establishing a cooperation agreement with industry, or Several funding schemes exist to help by founding a start-up company. The MDC bridge the gap between basic research and has established technology transfer activi- commercialization of inventions, e.g. the ties and relations with industry partners. BMBF Validation Fund, the BMBF Go-Bio- Program or the Helmholtz Validation fund. At the MDC, technology transfer is coordi- In addition, the MDC has set up an internal nated by the legal and technology transfer department and carried out in collabora- this scheme demonstrate innovative com- tion with an external company, Ascenion mercialfunding or program. clinical potential Projects fundedand are underlikely to obtain external funding once further inventions and the patenting and commer- developed. Also, MDC funding can be used cializationGmbH, which process. provides Ascenion scientific is the advice daugh for- to follow up expired Go-Bio awards, thus ter company of the German Life Science supporting later implementation phases. Foundation, which the MDC founded to- Hence, the MDC’s program strengthens the gether with the other Helmholtz research institute’s own applied and spin-off ori- centers, i.e. the German Research Center for ented research and facilitates moving from Environmental Health, the Helmholtz Cen- research into industry. tre for Infection Research and the German Cancer Research Center. The MDC technology transfer strategy re- fers to international benchmarks such as As is the case with most basic research, invention disclosures, patent applications, the MDC’s inventions and discoveries of- patent assignments, cooperation with inten need substantial further development dustry, license agreements and license before they can enter the market. Despite proceeds as a measure of its success. On av- - disease pathways and novel drug targets, a significant progress in the elucidation of aerage, patent. the Currently, MDC files 10-15the MDC invention holds around disclo producing substances that can potentially 70sures patent per year, families the majority with over of which300 patents. lead to bemajor used bottleneck for therapies. still lies To thisin identifying end, the MDC and The MDC’s most successful area of technol- builds collaborations between the MDC, the ogy transfer is the commercialization of an- Leibniz-Institut für Molekulare Pharmakol- imal models and of biological materials. At ogie (FMP) on the Berlin-Buch campus, the

present, 8 active projects are funded by the

250 MDC Research Report 2014 TECHNOLOGY TRANSFER

internal MDC technology transfer program. ery decisive point (such as wording of the Key to successful licensing of technologies patent application for rule example; the are an effective patent strategy, excellent decision to nationalize the application), it publications and well-established contacts must be considered who can be a potential to the industry. It is very important that the licensee. scientists are sensitized and supported to The MDC also supports scientist-initiated spin-offs. In this area the cooperation with thatfile patents the patenting before procedure they submit does their not paper delay Ascenion is close. Ascenion develops a spe- theto journals. publication. It has At to every be ensured, point in however, the pat- cial program “Spinnovator”, where private enting process the question of the potential licensee needs to be addressed. During the offs. The successful MDC spin-off OMEICOS whole patenting process, especially at ev- and public money together finance spin-

is the beneficiaries of the program.

Figures for Technology Transfer 2013

Patent applications per year 4

Patent rights 305

License agreements (new in 2013) 2

License revenues (in TEUR) 195

Company foundings 1

MDC Research Report 2014 251

TECHNOLOGY TRANSFER

Annett Böddrich Adeeb Eldahshan

Enabling Technologies for Drug Development of inhibitors of intra- Discovery in Alzheimer’s and Fur- cellular protein-protein interactions ther Protein Misfolding Diseases for the treatment of heart failure

Annett Böddrich, Thomas Wiglenda, Adeeb Eldahshan, Sylvia Niquet Sandra Neuendorf, Lydia Brusendorf, Nancy Schugardt, Sigrid Schnögl, Erich E. Wanker

Alzheimer’s, Parkinson’s and Huntington’s Protein-protein interactions represent a disease are neurodegenerative disorders, new class of drug targets and their inhi- for which no effective treatments exist. The bition may lead to the discovery of novel diseases are believed to be caused by mis- drugs that are more effective and have few- folding and aggregation of disease-relevant er side effects than conventional drugs. In polypeptides. We have started to establish this context, we focus on A-kinase anchor- - ing protein (AKAP)-dependent protein- tion and characterisation of chemical and protein interactions that play a crucial role biologicala technology substances platform that for interact the identifica with the in heart failure. AKAPs comprise a family protein misfolding and aggregation cascade of about 50 scaffolding proteins that tether of different disease proteins. In the last - year, we successfully established a small partments. All AKAPs bind protein kinase A molecule screening assay for the amyloid- (PKA)signalling and proteins additionally to defined bind variouscellular othercom beta target in Alzheimer’s disease. The as- kinases, their substrates, phosphatases and say is based on the time-resolved Förster’s phosphodiesterases. Thus, they form sig- resonance energy transfer (TR-FRET) tech- nalling hubs and coordinate cellular signal- nology. This technology relies on a quan- ling spatially and temporally. Our aim is to - when amyloid-beta forms aggregates in cule inhibitors of AKAP-dependent protein- vitro.tifiable Applying FRET signal it, compound that can effectsbe measured can be proteindevelop interactions previously identifiedtowards smalldrug candi mole- - dates for the treatment of heart failure. ity. To validate the assay, parameters like thedetected Z-factor with were high sensitivitydetermined, and indicating specific In our medicinal chemistry laboratory we suitability for high-throughput screen- optimise these molecules with regard to ing of large compound libraries. The assay - was successfully automated and applied to velop diversity-oriented synthesis concepts a focussed library of 1,200 small molecule andspecificity test the and affinity.structure-activity-relation Therefore, we de- compounds, which consisted of about 500 ships (SAR) of the molecules in vitro. We use compounds we previously discovered to act a variety of techniques, including molecu- on aggregation of various misfolding-prone lar modelling, Nuclear Magnetic Resonance proteins and of the library of FDA-approved 1H-15N heteronuclear compounds. Currently, we are identifying single quantum correlation (HSQC), com- new funding sources and industry-based putational(NMR), specifically screening as well as biacore and collaboration partners to broaden the technology platform and continue characterisa- (HTRF) assays. These efforts are strongly tion of hit compounds. connectedhomogenous to the time-resolved research of the fluorescence Anchored Signalling group of Prof. W. Rosenthal and PD Dr. E. Klußmann that tests novel molecules with regard to biological activity.

252 MDC Research Report 2014 TECHNOLOGY TRANSFER

Christiane Wetzel and David Miles Carter Gary R. Lewin

Exploitation of novel transduction Development of novel compounds targets for pain relief that inhibit growth of prostate cancer cells

Christiane Wetzel, Gary R. Lewin, David Carter, Udo Heinemann, Ulrich Kathleen Barda Gohlke (MDC), Edgar Specker, Jessica Przygodda, Marc Nazaré (FMP)

There is an unmet need for more effective Histone lysine demethylase (KDM) en- analgesics and novel strategies for analge- zymes are an emerging class of therapeutic sic drug development. With Stomatin-like targets, known for their ability to regulate the growth of various cancer cells. The potential molecular target that directly par- KDM4 family of isoenzymes comprises six protein-3ticipates in (STOML3) the transduction we have of noxious identified and a members denoted KDM4A-F. Both KDM4A innocuous mechanical stimuli in sensory and KDM4C isoforms are known to regulate the growth of prostate cancer (PCa) cells mice have many mechanoreceptors and no- via an epigenetic mechanism. The enzymes ciceptorsneurons. Wewhich found are thatessentially STOML3 insensitive deficient stimulate gene transcription by demethyl- to mechanical stimulation. Interestingly, ating tri- and di-methylated forms of lysine 9, within the core histone H3. This process minor symptoms of neuropathic pain and initiates the expression of various growth thisSTOML3 may be deficient due to the mice impairment show only of touch very factors, which facilitate the growth of can- reception in these mice. We are developing cer cells. Knockdown of both isoforms was novel high throughput screens for small shown to halt growth of PCa cell lines and molecules that disrupt STOML3 function. demonstrated their novelty as targets for development of small molecule inhibitors, For further validation and lead optimiza- which may prove useful in a clinical setting. tion of small molecules we employ a set of To address this possibility, we performed experimental paradigms. For example we a high-throughput screening campaign in can test whether candidate compounds order to identify compounds that inhibit block mechanosensitive currents in iso- the most active KDM4 isoform, KDM4E. We lated sensory neurons using the whole-cell patch-clamp technique. We can further test KDM4E with IC50 values in the mid- to low- if local application of our compounds to the micromolaridentified several range. compounds Importantly, that the inhibit com- skin will block mechanosensitivity at the - receptive endings of single primary affer- ily of enzymes and show reduced activity ents in the skin using the in vitro skin-nerve towardspounds specificallyother representative inhibit the membersKDM4 fam of technique. Eventually we will perform be- the KDM superfamily. Currently we are em- havioral tests in order to determine if the ploying methods of structure-based drug application of novel, potentially analgesic design to increase the potency and selectiv- compounds lead to a lack of hypersensitivity of these compounds. In parallel, we are ity to touch evoked pain in an animal model establishing cell-based assays to monitor of neuropathic pain. the ability of our compounds to halt or slow the growth of PCa cell lines. Ultimately we

in a mouse xenograft model of PCa and to steeraim to the test compounds the efficacy towards of our compoundscompletion of pre-clinical studies.

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TECHNOLOGY TRANSFER

Michael Radke

Alternative Splicing as a Therapeutic Target for Cardiac Disease A

Michael Radke (Scientist), Carmen Judis (Technician), Michael Gotthardt, Martin Liss, Florian Hinze, Jens Peter von Kries (FMP Berlin – Screening Unit) (Associ- ated Scientists)

Cardiovascular diseases are the main cause of death in developed countries. While several pharmacological agents have been devel- B oped that help improve cardiac contraction (systolic function), there is no targeted ther- -

fractionapy to improve (alias diastolic cardiac fillingdysfunction) (diastolic is morefunc prevalenttion). Heart in diseasefemales. with Risk preserved factors are ejection hypertension, diabetes and increased age of the patient, but the molecular basis of diastolic heart disease is only poorly understood.

The elastic properties of the heart and thus

the extracellular matrix, the sarcomere, diastolic filling are largely determined by these proteins are alternatively spliced to and proteins regulating ion traffic. Most of of select factors that regulate splicing can resultconcertedly in genetic adapt forms cardiac of filling. cardiac Deficiency disease. Furthermore, both cardiomyopathies and coronary artery disease of unknown cause have been associated with differential iso- fom expression of sarcomeric proteins such Reporter assay to screen for splice as titin. Therefore alternative splicing could activators and inhibitors. be a suitable therapeutic target. A) Titin based splice reporter assay. Without RBM20, both firefly (Fluc) and renilla luciferase (Rluc) are included in We use in silico analysis, cell based reporter the resulting isoform. With RBM20, the exon containing assays, and animal models to identify splice Fluc is excluded. B) Small molecule screen to identify splice factors that regulate cardiac isoform expres- effectors. Potential splice inhibitors (red), splice activators sion. Among them is RBM20, which regulates (blue), or compounds that do not influence splice activity alternative splicing of titin as well as other (green) are derived from a screen of 40.000 compounds in sarcomeric and signaling proteins related the 384-well format to diastolic function1. Using an RBM20 dependent reporter assay and high throughput screening we aim to identify small molecules that modulate alternative splicing. These are evaluated for toxicity, and validated in tissue culture and in animal models, such as a genetic model with diastolic dysfunction, as a 1. Guo, W. et al. RBM20, a gene for hereditary cardiomyopathy, regulates prerequisite for future clinical studies. titin splicing. Nat. Med. 18, 766–773 (2012).

254 MDC Research Report 2014 TECHNOLOGY TRANSFER

Crystalized proteins in a drop under the microscope. The picture was taken by Song Gao from the Daumke group.

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256 MDC Research Report 2014 Academics Akademische Aktivitäten

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International PhD Program

Internationales Doktorandenprogramm

he MDC considers the training of new genera- ie Ausbildung des wissenschaftlichen Nach- tions of researchers in Molecular Medicine to be wuchses in der Biomedizin ist eine grundlegende Ta prerequisite for sustainable development and DVoraussetzung für die nachhaltige Entwicklung - und den internationalen wissenschaftlichen Erfolg des bines biomedical research and interdisciplinary train- MDC. Das Internationale Doktorandenprogramm ver- ing,international complemented scientific by asuccess. career Thedevelopment PhD program program, com - biomedizinischer Forschung und bietet den Doktoran- tunities. denbindet Zugang die Weiterbildung zu High-End-Technologien junger Nachwuchstalente und interdiszip mit- and provides high-end technology and project oppor In 2003, the MDC and the Humboldt-Universität zu Berlin (HU) established the International PhD Program Daslinären Internationale Forschungsprojekten. Doktorandenprogramm von MDC to attract talented international graduate students. und Humboldt-Universität (HU) wurde 2003 ins Le- Receiving over 1000 applications annually, the Inter- ben gerufen, um Nachwuchswissenschaftlern eine national PhD Program invites around 100 candidates interdisziplinäre Ausbildung innerhalb eines struk- from all over the world for on-site assessment in Berlin. turierten Rahmenprogramms zu bieten. Jedes Jahr Every year 50-100 PhD students begin their thesis in gehen mehr als 1000 Bewerbungen ein, woraus etwa one of the research groups at the MDC or its partners 100 Kandidatinnen und Kandidaten zu Bewerbungs- HU, Freie Universität (FU), Charité –Universitätsmed- gesprächen nach Berlin eingeladen werden. Von den izin Berlin, or the Leibniz Institut für Molekulare Phar- - makologie (FMP). More than 40% of these doctoral - students come from abroad, representing 36 countries. sen50-100 Partnern, Doktoranden, der HU, der die Freien jährlich Universität ihr Dissertations (FU), der Charité-Universitätsmedizinprojekt in einer der Arbeitsgruppen oder dem am MDC Leibniz-Insti oder des- Following the success of the International PhD Pro- tut für Molekulare Pharmakologie (FMP) aufnehmen, gram, the MDC and its university partners were award- kommen mehr als 40% aus dem Ausland (verteilt auf ed a grant from the Helmholtz Association to establish 36 Länder). the Helmholtz Graduate School “Molecular Cell Biology” (HGS-MCB) in 2007. The HGS-MCB offers an interdisci- Auf Grund des Erfolgs des Internationalen Doktoran- denprogramms sprach die Helmholtz-Gemeinschaft outstanding opportunities for personal development dem MDC und seinen Universitätspartnern 2007 För- ofplinary doctoral platform students. for structuredCurrently, 350scientific students training work and to- dermittel für die Gründung der Helmholtz-Graduier- tenschule Molecular Cell Biology (HGS-MCB) und für die the academic curriculum. Training records of individ- weitere Unterstützung der strukturierten, interdiszip- wards their PhD degree at the MDC and benefit from- linären Ausbildung von Doktoranden zu. Gegenwärtig ests are documented in the HGS-MCB credit point sys- gibt es am MDC etwa 350 Promovierende, die das viel- ual students reflecting their personal needs and inter seitige Ausbildungsprogramm der Graduiertenschule receive upon completion of their degree. nutzen. Unter Berücksichtigung persönlicher Bedürf- tem, which is the basis of the PhD certificates students nisse und Interessen können die Doktoranden ihren The HGS-MCB offers: individuellen Ausbildungsplan zusammenstellen. Die • A Welcome Center to help international students während der Promotion erbrachten Leistungen wer- settle in Berlin den in einem Punktesystem dokumentiert und in ei- • A University interface • Supervision by a PhD Thesis Committee and regular meetings to evaluate students’ progress Dienem HGS-MCB Abschlusszertifikat bietet: zusammengefasst. • • Ein Welcome Center, das Promovierenden aus dem general molecular medicine and specialized areas, Ausland den Start in Berlin erleichtert coursesScientific and training: introduction lectures into and new seminar methodologies, series in • Eine Schnittstelle zu den Universitäten • Ein kollegiales Betreuungssystem: regelmäßige Tref- • Professional skills training and career development fen der Doktoranden mit einem Betreuungskomitee good scientific practice

complementing the scientific training zur Diskussion des Projektfortschritts 258 MDC Research Report 2014 ACADEMICS

International PhD Program • Annual Campus Symposium and PhD Retreat to pro- • Wissenschaftliche Weiterbildung: Vorlesungen, Semi- nare, Methodenkurse, Einführung in neue Technolo- co-organized by students gien, Seminare zur „Guten wissenschaftlichen Praxis“ Internationales Doktorandenprogramm • Tmoteravel networking and collaboration and scientific funds to exchange; support bothattendance are • Professionelle Weiterbildung und Karriereplanung of international conferences, workshops, and visits als Ergänzung zur wissenschaftlichen Ausbildung in collaborating laboratories • Reise- und Forschungsstipendien für die Teilnahme • Building an alumni network an internationalen Konferenzen und Methodenkur- sen oder Forschungsaufenthalten im Ausland Four specialized research schools and international ex- • Jährliches von den Doktoranden organisiertes Cam- change programs have been established and are inte- pus-Symposium und PhD-Retreat zur Förderung der grated within the HGS-MCB. These are smaller, themati- Vernetzung und des wissenschaftlichen Austauschs cally focused doctoral programs that provide tailored •

research areas at the MDC. SeitAufbau 2007 eines wurden Alumni-Netzwerks neben der Graduiertenschule klei- training and foster scientific networks within specific nere, thematisch fokussierte Doktorandenprogramme Helmholtz Research School gegründet und in die HGS-MCB integriert: “Molecular Neurobiology” (MolNeuro) Helmholtz-Kolleg The close collaboration between the MDC and the FU “Molecular Neurobiology” (MolNeuro) led to the establishment of the Helmholtz Research School in Molecular Neurobiology (MolNeuro) in 2007. Die enge Zusammenarbeit zwischen dem MDC und der The curriculum focuses on basic and advanced con- FU führte 2007 zur Gründung des Helmholtz-Kollegs cepts of Molecular Neurobiology. The Berlin Brain „Molecular Neurobiology“ (MolNeuro). Im Fokus des Days, a biannual conference for PhD students offer a Curriculums stehen Konzepte der Molekularen Neu- Berlin-wide platform for young scientists to meet and robiologie (Grundlagen und spezialisierte Konzepte).

„Berlin Brain Days“ bietet Doktoranden aus ganz Berlin discussHelmholtz their Researchresearch projects. School dieDie Möglichkeitalle zwei Jahre zum stattfindende Austausch und Graduiertenkonferenz zur Diskussion ih- „Translational Cardiovascular and Metabolic Medicine” (TransCard) Helmholtz-Kollegrer Forschungsprojekte. In 2009 the TransCard Research School was estab- „Translational Cardiovascular and Metabolic lished as a collaborative endeavor between the MDC, Medicine” (TransCard) the Charité, the HU, and the FU as well as the Hubrecht Laboratory in Utrecht, Netherlands, and the University Das 2009 gegründete Helmholtz-Kolleg (TransCard) of Arizona in Tucson AZ, USA as international partners. ist eine Kooperation zwischen dem MDC, der Charité, The school is embedded within the Cardiovascular and der HU und der FU sowie dem Hubrecht Laboratory in Metabolic Diseases Research Program, which covers a Utrecht, Niederlande, und der University of Arizona in diverse spectrum of research areas, ranging from cel- Tucson AZ, USA, als internationale Partner. Das Kolleg lular biology and organ development, to genetics and ist in das Programm Herz-Kreislauf- und Stoffwechse- pathophysiology. The program trains excellent young lerkrankungen eingebettet und deckt somit ein breites researchers at the interface of basic molecular, ge- Spektrum verschiedener Wissenschaftsgebiete von der netic, and clinical research with the goal to elucidate Zell- und Entwicklungsbiologie, über Genetik bis hin the molecular mechanisms causing cardiovascular and zur Pathophysiologie ab. Das Programm bildet heraus- metabolic diseases and to transfer this knowledge from molekularer Grundlagenforschung und klinischer For- a lecture and seminar series, annual PhD retreats, E- schungragende im junge Bereich Wissenschaftler translationaler an derHerz-Kreislauf- Schnittstelle undvon learningbench to units,bedside. and The a clinicalscientific visits curriculum program consists in coop of- Stoffwechselerkrankungen aus. Das wissenschaftliche eration with the HELIOS Klinikum Berlin-Buch that Curriculum umfasst eine Vorlesungs- und Seminar- provides students unique insight into the clinical perspective. ein Clinical Visits-Programm in Kooperation mit dem HELIOS-Klinikumreihe, jährliche Retreats, Berlin-Buch, E-Learning-Einheiten das den Studenten sowie hilft, International Exchange Program in Systems ihre eigenen Arbeiten in einen translationalen Kontext Biology (MDC-NYU PhD Exchange Program) einzuordnen.

In 2009, the Berlin Institute for Medical Systems Bi- MDC-NYU PhD Exchange Program ology and the Center for Genomics and Systems Bi- Das Berlin Institute for Medical Systems Biology PhD Exchange Program. The students are working on (BIMSB) und das Center for Genomics and Systems Bio- ology of the New York University started their joint logy der New York University riefen 2009 ihr gemein- research interests and technological approaches in sames PhD-Austauschprogramm ins Leben. Die Dokto- variouscollaborative topics projects of gene betweenregulatory two elements labs, bridging and medi the- cal systems biology. All students are under supervision wissenschaftliches Interesse und technologische Fra- randen arbeiten an gemeinschaftlichen Projekten, die

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of a MDC and a NYU faculty member and conduct their gestellungen auf dem Gebiet der Genregulation und der research in the respective laboratories, both in Berlin medizinischen Systembiologie miteinander verbinden. and New York. Zusätzlich zu ihrer Betreuerin oder ihrem Betreuer am MDC haben alle Studierenden ein Fakultätsmitglied German-Israeli Helmholtz Research School der New York University als Mentor und betreiben ihre “Frontiers in Cell Signaling and Gene Regu- Forschung in den entsprechenden Laboren sowohl in lation” (SignGene) Berlin als auch in New York.

Established in 2013, SignGene Deutsch-Israelisches Helmholtz-Kolleg between the MDC, the HU, and the Charité in Berlin, “Frontiers in Cell Signaling and Gene Regu- as well as the Technion, Israel Institute is a joint of Technology endeavor lation” (SignGene) in Haifa, and the Hebrew University of Jerusalem. Re- search and training within SignGene focus on the areas Das 2013 gegründete deutsch-israelische Austausch- of cellular signaling, the mechanisms of gene regulation, programm SignGene ist eine Kooperation zwischen and quantitative biology with the goal to understand dem MDC, der HU und der Charité in Berlin sowie dem the molecular basis of diseases such as cancer. Within Technion, Israel Institute of Technology in Haifa und this interdisciplinary framework, SignGene students der Hebrew University in Jerusalem. Die Schwerpunkte des Forschungs- und Ausbildungsprograms liegen auf - den Gebieten der zellulären Signalübertragung, den conductsor at the their home thesis institute projects and a in co-mentor bilateral abroad. cooperation Pro- Mechanismen der Genregulation und der quantitativen andlonged benefit exchange from visits the expertise at the co-mentor’s of their main laboratory supervi Biologie mit dem Ziel, die molekularen Grundlagen der are integral to the curriculum, as are biannual training Krankheitsentstehung wie z.B. Krebs zu verstehen. In and networking events, which bring together scientists diesem interdisziplinären Rahmen arbeiten SignGene- from the two countries. - ersDoktoranden am Heimatinstitut an bilateralen sowie einem Kooperationsprojekten Co-Mentor im Aus- land.und profitieren Längere Forschungsaufenthalte von der Expertise ihres im HauptbetreuPartnerlabor Inka Gotthardt from the PhD office explaining the program sind integraler Bestandteil des Curriculums, ebenso of the MDC’s Graduate School at the CareerDay. Inka Gotthardt vom PhD-Büro erläutert beim CareerDay Veranstaltungen, die die Kollegmitglieder aus beiden des MDC das Programm der Graduiertenschule. Ländernwie zweimal zusammenführen. pro Jahr stattfindende wissenschaftliche

MDC Research Report 2014 Photo: Guido Rottmann/MDC Photo: ACADEMICS

MDC Postdoctoral Program

here are more than 450 scientists and visiting re- ehr als 450 Forscherinnen und Forscher sowie searchers from more than 45 different countries Gäste aus mehr als 45 Ländern verfolgen ihre Tpursuing their academic career at the MDC, and Makademische Karriere am MDC, und ihr Beitrag their contribution to the MDC research excellence is zur Exzellenz des MDC ist kaum zu überschätzen. Unter - fellowship-holders supported by the Helmholtz Asso- schaft, der Alexander-von-Humboldt-Stiftung, des Ma- ciation,difficult theto overestimate. Alexander von Among Humboldt them Foundation,are quite a fewthe rie-Sklodowska-Curie-Programmsihnen finden sich Stipendiaten der Helmholtz-Gemeinder Europäischen - Kommission und des Fulbright Scholar Programms. bright Scholar Program, etc. Marie Skłodowska-Curie actions of the EC, the Ful Am MDC betrachten wir die Postdoc-Zeit als wesent- At the MDC we view the postdoctoral period as an essential part of professional training of our scientists Forschenden. Wir unterstützen sie bei der Erlangung and support them in gaining valuable knowledge and vonlichen Wissen Bestandteil und ergänzenden der beruflichen Fähigkeiten Ausbildung für vielfältiunserer- complementary skills important in pursuing a broad ge zukünftige Karrierewege: in Wissenschaft, Indust- range of future careers: in academia, industry, govern- - wie als Unternehmer, sowohl in Deutschland als auch entrepreneurship, both in Germany and abroad. To- imrie, Ausland.bei staatlichen Mit diesem und Non-Profit-Organisationen Ziel haben wir ein Weiterbil so- wardsmental this and goalnon-profit we are organizations developing a as Training well as throughand Ca- dungs- und Karriereentwicklungsprogramm entwi- reer Development (TCD) Framework and offer both - stützung durch: ckelt und bieten flexible und maßgeschneiderte Unter •flexible Researchers’ and tailored Training support Program through: (which includes lec- • Weiterbildungsprogramm für Forscherinnen und ture series, seminars and courses, broad range of soft Forscher (Vortragsreihen, Seminare und Kurse, eine skills training offers, language and IT courses, etc.) breite Palette von Soft-Skills-Trainingsangeboten, • Funding Opportunities and Support Service (via Sprach- und EDV-Kurse) dedicated funding schemes and information events • Fördermöglichkeiten und Support-Service (mit Hilfe on funding opportunities, individual advice and spezieller Förderprogramme und Informations- grant writing support, etc.) veranstaltungen zu Finanzierungsmöglichkeiten, • Mentoring and Career Services (including a dedicat- individueller Beratung und Unterstützung bei der ed Mentoring program for female scientists, annual Beantragung von Forschungsgeldern) Career Day, “Career Pathways” talks, career counsel- • Mentoring und Karriere-Services (einschließlich ing, etc.) einem speziellen Mentoring-Programm für Wissen- • Measures to ensure productive Work-Life Balance (e.g. Welcome Center taking care of all visa/work permit Day, “Career Pathways“-Veranstaltungen und Karri- formalities, on-campus childcare facility, diverse sport ereberatung)schaftlerinnen, dem jährlich stattfindenden Career activities, weekly “Beer-Hour” sessions). • Maßnahmen zur Gewährleistung einer produktiven Work-Life-Balance (z. B. Übernahme der Formali- Moreover, the MDC Postdoctoral Association promotes täten für Visum oder Arbeitserlaubnis durch das informal networking and stimulates closer interaction Welcome Center, Kindertagesstätte auf dem Campus, between postdoctoral scholars at the same career stage diverse Sportaktivitäten, informelle wöchentliche by organizing regular social events, monthly meetings Treffen (“Beer-Hour”).

tours, etc. Darüber hinaus fördert die MDC Postdoctoral Associa- over lunch, pizza evenings, joint excursions and theatre tion die informelle Vernetzung. Für eine engere Zusam- menarbeit zwischen Postdoktoranden in der gleichen Phase ihrer Karriere organisiert sie regelmäßige Ver- anstaltungen, monatliche Mittagstreffen, Pizza-Aben-

de, Ausflüge sowie Theaterbesuche.

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MDC International

he MDC is a truly international research institute: as MDC ist eine wahrhaft internationale For- more than a third of its scientists come from more schungseinrichtung: Mehr als ein Drittel der Tthan 50 countries and almost a half of our PhD DForscherinnen und Forscher stammen aus über students are international. EU-citizens form the larg- 50 Ländern und fast die Hälfte der PhD-Studenten sind est group of MDC researchers (with nationals of Italy, aus dem Ausland. Angeführt von den Nationalitäten France and Spain topping the list). Chinese scientists Italien, Frankreich und Spanien bilden EU-Bürger die form the largest single foreign-nationality community größte Gruppe unter den Forschenden am MDC. Als at the MDC, with more than 40 researchers and PhD einzelne Nationalität ragt China hervor: Mehr als 40 students working and visiting the MDC at any time, and Forscher und PhD-Studenten oder Gastwissenschaftler there are also large groups of nationals of India, Russia, kommen daher. Zusätzlich gibt es große Gruppen aus USA, Brazil, Turkey, etc. Indien, Russland, den USA, Brasilien und der Türkei.

MDC researchers successfully cooperate with their Forscher des MDC kooperieren erfolgreich mit Kolle- peers around the globe: more than half of the MDC’s ginnen und Kollegen aus aller Welt. Mehr als die Hälfte publications (2012-2013) appeared in co-authorship der Publikationen (2012 bis 2013) erschien in Koauto- with international partners from approximately 50 renschaft mit internationalen Partnern aus annähernd countries, with the USA, UK, and France leading the 50 Ländern, angeführt von den USA, Großbritannien list. Renowned international scientists are frequently und Frankreich. Das MDC lädt regelmäßig renommierte invited to the MDC to give a lecture or a seminar, for Wissenschaftlerinnen und Wissenschaftler ein, im Rah- sabbaticals and collaborative visits. Several distin- - guished international scientists were awarded presti- träge und Seminare zu geben. Um die Kooperationen gious prizes to strengthen their cooperation with the mitmen MDC-Wissenschaftlern eines Sabbatjahres oder zu verstärken, von Kooperationen haben etliche Vor MDC scientists: e.g. Prof. Michael Glickman from the hervorragende internationale Forscher prestigeträchti- Technion-Israel Institute of Technology received the ge Preise erhalten: Professor Michael Glickmann vom Friedrich Bessel Research Award of the Alexander Technion – Israel Institute of Technology erhielt bei- von Humboldt Foundation in 2012, and Prof. Jehu- spielsweise 2012 den Friedrich Wilhelm Bessel-For- 43+13976543 dit Bergman from the Hebrew University in Jerusalem schungspreis der Alexander von Humboldt-Stiftung was honored with the Helmholtz International Fel- und Professor Jehudit Bergman von der Hebräischen low Award in 2013. Universität Jerusalem wurde 2013 mit dem Helmholtz International Fellow Award ausgezeichnet. Jointly organized bilateral meetings provide an excel- Gemeinsam organisierte bilaterale Veranstaltungen bilden exzellente Plattformen, um aktuelle wissenschaft- lent platform to discuss the latest scientific findings lichen Erkenntnisse und vielversprechende Ideen für bothand identify at the MDC promising and abroad ideas forwith joint partners projects. from In Israel,2012- France,2013 seven China such and joint the symposia UK. The andMDC retreats also takes took an place ac- gemeinsamer Symposien und Klausurtagungen fanden tive part in the recently established Helmholtz-Israeli zwischengemeinsame 2012 Projekte und 2013 zu am diskutieren. MDC oder im Sieben Ausland solcher mit Cooperation in Personalized Medicine, Partnern aus Israel, Frankreich, China und Großbritan-

early 2014. Fruitful cooperation of individual with research the first (die Auswahl soll Anfang 2014 erfolgen), übernimmt grouptandem leaders research often projects translates to be into selected closer for collaborative funding in dasnien MDC statt. außerdem Mit den ersten eine aktive Tandem-Forschungsprojekten Rolle in der kürzlich eta- blierten Helmholtz-Israel-Kooperation in Personali- sierter Medizin. Erfolgreiche Kooperationen einzelner •links The in MDC-INSERM form of joint research(Institut groups, National e.g.: de la Santé Forschungsgruppenleiter münden oft in enge Verbin- et de la Recherche Médicale) group “Stem cell and dungen, in Form gemeinsamer Forschungsgruppen: macrophage biology” led by Dr Michael Sieweke at the Centre of Immunology Marseille-Luminy (CIML) • Die MDC-INSERM (Institut National de la Santé (since 2012), which is part of the Helmholtz-INSERM et de la Recherche Médicale)-Gruppe „Stammzell- Franco-German cooperation initiative; biologie und Makrophagen“. Sie wird seit 2012 von

262 MDC Research Report 2014 ACADEMICS

MDC International • Helmholtz-Russia Joint Research Group “Pos- Dr. Michael Sieweke vom Centre d‘Immunologie de Marseille-Luminy (CIML) in Südfrankreich geleitet ablation in carcinogen-induced and sporadic cancer, und ist Teil der französisch-deutschen Helmholtz- assible studied beneficial in mice” effects (2011-2014), of TNF and/or led bylymphotoxin Dr. Marina INSERM Kooperationsinitiative. Drutskaya (Engelhardt Institute of Molecular Biology, • Helmholtz-Russia Joint Research Group: In Russian Academy of Sciences, Moscow) in coopera- Kooperation mit Professor Blankenstein leitet Dr. tion with Prof. Thomas Blankenstein at the MDC; Marina Drutskaya (Engelhardt Institut für Moleku- • Helmholtz International Research Group “Metab- larbiologie, Russische Akademie der Wissenschaften olism and Neurodegeneration” (2013-2016) led by in Moskau) die Forschungsgruppe „Die Rolle von Dr. Vanessa Schmidt and Prof. Thomas Willnow (both TNF oder Lymphotoxin bei der Tumorentstehung” - Lundbeck Foundation, Aarhus University, Denmark); toxin ablation in carcinogen-induced and sporadic • MDC)Helmholtz in cooperation International with Dr.Research Mads Kjolby Group (MIND, “Role of cancer,(“Possible as studiedbeneficial in mice”,effects 2011 of TNF bis and/or 2014) lympho Kinins in Obesity” (2013-2016) led by Prof. Michael • Helmholtz International Research Group: Die Bader and Dr. Natalia Alenina (both MDC) in coop- internationale Helmholtz-Forschungsgruppe „Stoff- eration with Prof. João B. Pesquero (Universidade wechsel und Neurodegeneration“ (2013 bis 2016) Federal de São Paulo, Brazil), co-funded by the São leiten die MDC-Forscher Dr. Vanessa Schmidt und Paulo Research Foundation FAPESP. Professor Thomas Willnow in Kooperation mit Dr. - Another important area of the MDC’s activities, where on an der Universität Aarhus in Dänemark. • HelmholtzMads Kjolby In vomternational MIND-Labor, Research Lundbeck Group: Foundati Die the doctoral training of its PhD students. In addition internationale Helmholtz-Forschungsgruppe „Rolle tointernational hosting international cooperation undergraduate is of major importance,students from is der Kinine bei Fettleibigkeit“ (2013 bis 2016) leiten North America (e.g. within the MIT-Germany, DAAD die MDC-Forscher Professor Michael Bader und Dr. RISE and Fulbright programs) and Europe (e.g. within Natalia Alenina in Kooperation mit Professor João the EU Leonardo da Vinci Program SignGene), the MDC B. Pesquero (Universidade Federal de São Paulo, has established collaborative bilateral PhD programs with the USA, France and with Israel. Stiftung (Research Foundation, FAPESP). Brazil), kofinanziert von der São Paulo Forschungs- Working in an international context – through the su- Ein anderer wichtiger Bereich der Aktivitäten des MDC, pervision by two cooperating scientists from Germany bei denen internationale Kooperationen eine große and a cooperating country – and prolonged research Rolle spielen, ist die Ausbildung der PhD-Studentinnen stays abroad form an integral part of training and und -Studenten. Zusätzlich zu der Betreuung interna- equips the next generation of young researchers with tionaler grundständiger Studenten aus Nordamerika essential skills and international experience. In addi- (beispielsweise über die Programme MIT-Germany, tion to improving the level of training and increasing DAAD RISE und Fulbright Programs) und Europa (über the MDC’s international visibility, these programs play das „Leonardo da Vinci“ Programm) hat das MDC gemeinsame bilaterale PhD-Programme mit den USA, links between MDC researchers and their international Frankreich und Israel etabliert. partners.a significant role in stimulating further collaborative Die Arbeit in einem internationalen Umfeld und die Betreuung durch zwei kooperierende Wissenschaft- ler aus Deutschland und dem Partnerland sowie aus- gedehnte Forschungsaufenthalte im Ausland bilden einen wesentlichen Teil der Ausbildung und statten

Fähigkeiten und internationaler Erfahrung aus. Zusätz- lichdie nächstezur Verbesserung Generation des junger Ausbildungslevels Forscher mit wichtigen und der internationalen Sichtbarkeit des MDC, spielen diese Programme eine wichtige Rolle dabei, Anreize für weitere Kooperationen zwischen MDC-Forschern und ihren internationalen Partnern zu schaffen.

The concept of the German-Israeli Helmholtz Research School “SignGene” was finalized in Haifa, Israel. Das Konzept von SignGene wurde in Haifa diskutiert und verabschiedet. From left to right (v.l.n.r.): SignGene Faculty Aaron Ciechanover (Technion, Nobel Prize Winner 2004), Amit Meller (Technion, SignGene speaker), Claus Scheide- reit (MDC, SignGene Speaker), and Peretz Lavie, President Photo: SignGene Photo: of the Technion – Israel Institute of Technology.

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Appointments

Berufungen

2012

Prof. Uwe Ohler appointed to W3-Professorship at Prof. Uwe Ohler wird auf eine W3-Professur an Humboldt University Berlin die Humboldt-Universität zu Berlin berufen

Uwe Ohler studied computational science at Friedrich Uwe Ohler studierte Informatik an der Friedrich- Alexander University Erlangen where he also received Alexander-Universität in Erlangen. Nach der Promo- his PhD. Then he worked as a postdoctoral fellow in the tion arbeitete er als Postdoc in den USA, zunächst an der University of California in Berkeley, dann am Mas- at the Massachusetts Institute of Technology in Boston. sachusetts Institute of Technology in Boston. Von 2005 SinceUSA, first 2005, at theUwe University Ohler was of Assistant California Professor at Berkeley, at Duke then bis 2012 war Ohler als Assistant Professor an der Duke University in Durham (USA). In this function, he made - tembiologie beteiligt. Im September 2012 wurde Uwe biology at Duke University. In September 2012, Uwe OhlerUniversity auf indie Durham W3-Professur (USA) zentral für Systembiologie am Aufbau der Sysder Ohlermajor was contributions appointed to to a the full establishmentprofessorship for of Systems systems Genregulation an die Humboldt-Universität zu Berlin Biology of Gene Regulation at the Humboldt University. berufen. Seine Forschungsgruppe ist am Berliner Insti- His research group is part of the MDC’s Berlin Institute tut für Medizinische Systembiologie (BIMSB) des MDC of Medical Systems Biology (BIMSB) and uses compu- angesiedelt und nutzt computerbasierte Ansätze, um tational approaches to understand the biology of gene die Biologie der Genregulation in höheren Organismen regulation in higher organisms. zu verstehen.

BIMBS Junior Research Group for Dr. Baris Tursun BIMSB-Nachwuchsgruppe für Dr. Baris Tursun

Baris Tursun begann im Februar 2012 als neuer Nach- MDC’s BIMSB in February 2012. Dr. Tursun’s team uses wuchsgruppenleiter am BIMSB. Seine Gruppe verwen- theBaris roundworm Tursun began Caenorhabditis as a new juniorelegans group as a model leader or at- det den Fadenwurm Caenorhabditis elegans als Mo- ganism. Their aim is to identify genes and epigenetic dellorganismus für ihre Untersuchungen. Ziel ist es, mechanisms that are involved in regulating reprogram- die Gene und epigenetischen Mechanismen zu identi- ming and direct cell fate conversions.

Dr. Tursun studied biology at the University of Ham- Schicksalfizieren, die der in Zellen die Regulation nehmen. Nachder Reprogrammierung dem Studium der Biologievon Zellen an involviertder Universität sind und Hamburg somit Einflussmachte aufTursun das Center for Molecular Neurobiology (ZMNH), University seine Doktorarbeit am Zentrum für Molekulare Neu- ofburg. Hamburg, Then heas joineda PhD student. Ingolf Bach’s In this laboratorytime he was at sup the- robiologie (ZMNH) der Universität Hamburg. In dieser ported by a doctoral fellowship from the Boehringer Zeit wurde er durch ein Doktorandenstipendium der Ingelheim Foundation. He completed his PhD and con- Boehringer Ingelheim Stiftung unterstützt. Er blieb tinued his work as a postdoctoral fellow until 2006. bis 2006 als Postdoc am ZMNH. Dann wechselte er auf Baris Tursun then accepted a postdoctoral position in eine Postdoc-Position im Labor von Oliver Hobert an the laboratory of Oliver Hobert at Columbia Universi- der Columbia University/Howard Hughes Medical Ins- ty/Howard Hughes Medical Institute, New York. There titute, New York, USA. Hier wurde er mit dem angese- he also received a “Francis-Goelet” postdoctoral fel- henen “Francis-Goelet” Postdoktoranden-Stipendium lowship, a prestigious stipend only awarded to highly ausgezeichnet, welches nur an sehr vielversprechende promising scientists. Nachwuchswissenschaftler verliehen wird.

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Appointments Dr. Michael Sieweke joined the MDC Dr. Michael Sieweke schließt sich dem MDC an

The Sieweke group is based at the Centre of Immunol- Die Arbeitsgruppe von Michael Sieweke hat ihren Berufungen ogy Marseille-Luminy (CIML) in southern France, and Sitz am Centre d‘Immunologie de Marseille-Luminy (CIML) in Südfrankreich. Sie schloss sich dem MDC im INSERM Franco-German cooperation initiative. The Juli 2012 als Teil der Helmholtz-INSERM-Initiative zur groupjoined isthe co-funded MDC in July by the2012 MDC as part and ofINSERM the Helmholtz- (Institut deutsch-französischen Zusammenarbeit an. Die Grup- National de la Santé et de la Recherche Médicale). The pe wird von MDC und INSERM (Institut National de work of Dr. Sieweke’s team is interfacing immunology and stem cell research. Stem cells can renew them- Siewekes Gruppe arbeitet an der Schnittstelle zwischen selves and differentiate into many cell types. There Immunologiela Santé et de laund Recherche Stammzellforschung. Médicale) kofinanziert. Stammzellen Dr. are large hopes that stem cells may revolutionize fu- können sich selbst erneuern und in viele verschiede- ture medicine. These hopes are based on the idea that ne Zelltypen differenzieren. Es gibt große Hoffnungen, these immature, still ‘undecided’ cells can be coaxed dass Stammzellen die zukünftige Medizin revolutio- into making large numbers of any particular cell type nieren. Man hofft, dass die noch unreifen Stammzel- of the body to serve a therapeutic purpose. Dr. Sieweke len dazu gebracht werden können, große Mengen ei- studied biochemistry at Eberhard Karls University nes bestimmten Zelltyps zu produzieren, die dann für Tübingen and earned his doctorate at the University therapeutische Zwecke eingesetzt werden können. Dr. of California at Berkeley, USA. Then he moved to the Sieweke studierte Biochemie an der Eberhard-Karls- European Laboratory for Molecular Biology (EMBL), Universität Tübingen und erhielt seinen Doktortitel von der University of California, Berkeley, USA. Dann wechselte er an das Europäische Labor für Molekular- 1999,Heidelberg, Dr. Sieweke where accepted he stayed a group for 7 leader years, position first as at a biologie (EMBL) in Heidelberg. Dort arbeitete er die thepostdoctoral CIML. fellow, then as junior faculty member. In nächsten sieben Jahre, zunächst als Postdoc, dann als Nachwuchsgruppenleiter. 1999 nahm Dr. Sieweke eine Gruppenleiterposition am CIML an.

Bust of Max Delbrück (by Hans Scheib) in front of the MDC.C, the MDC’s communications center. Büste von Max Delbrück (Ausschnitt) vor dem Kongresszentrum des MDC (Bildhauer: Hans Scheib). Photo: David Ausserhofer/MDC Photo:

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ACADEMICS Photo: David Ausserhofer/MDC Photo:

The opening ceremony in the foyer of the Max Rubner House. Blick von oben in das Foyer des Max-Rubner-Hauses bei der Einweihung des Gebäudes. 2013

Prof. Ana Pombo recruited from the Imperial Prof. Ana Pombo wechselt vom Imperial College College London to BIMSB London ans BIMSB

With Ana Pombo, program head of the Molecular Sci- Mit Ana Pombo, Leiterin der Molecular Sciences Sec- ences Section of the Institute of Clinical Sciences, Im- tion des Institute of Clinical Sciences, Imperial College perial College London, and MRC group leader at the London, und MRC-Gruppenleiterin am MRC Clinical MRC Clinical Sciences Centre in London, UK, the MDC Sciences Centre in London, UK, konnte eine exzellente succeeded in recruiting an excellent female scientist to Wissenschaftlerin ans BIMSB rekrutiert werden. Die Berufung auf die W3-Professur für Transkriptiona- for Translational Regulation and Gene Architecture by le Regulation und Genarchitektur, die gemeinsam mit theBIMSB. MDC The and joint Humboldt appointment University to aBerlin full professorship is supported der Humboldt-Universität zu Berlin erfolgte, wird mit through additional funding from the Impulse and Coop- zusätzlichen Fördermitteln aus dem Impuls- und Ver- eration Fund of the Helmholtz President. Prof. Pombo’s netzungsfonds des Helmholtz-Präsidenten unterstützt. work is characterized by a high level of innovation and Prof. Pombos Arbeiten sind sehr innovativ und verbin- powerfully combines imaging methods with molecular den erfolgreich bildgebende Verfahren mit molekula- - ren und biochemischen Ansätzen. Sie haben wesentlich uted to the understanding of the relationship between zum Verständnis der Beziehung zwischen der Organi- theand organizationbiochemical approaches.of the cellular She nucleus significantly and transcrip contrib- sation des Zellkerns und der Transkriptionsregulation tional regulation. For her research achievements, Ana beigetragen. Ana Pombo wurde für ihre wissenschaftli- Pombo has been honored by the renowned Feulgen chen Leistungen mit dem renommierten Feulgen-Preis Award. Ana Pombo studied biochemistry at the Univer- ausgezeichnet. Nach einer biochemischen Ausbildung sity of Lisbon in Portugal. She continued her academic an der Universität Lissabon in Portugal setzte Ana career in Great Britain as a PhD student in the lab of Pe- Pombo ihre Karriere in Großbritannien fort. Im An- ter Cook. After her doctoral work she stayed as a post- schluss an ihre Doktorarbeit im Labor von Peter Cook doctoral fellow at the University of Oxford for two years. - Since 2000, Prof. Pombo has directed her own indepen- sität Oxford erhielt sie im Jahr 2000 bereits eine un- dent research group at the core-funded MRC Institute, abhängigeund einer zweijährigenMRC-Forschungsgruppe. Postdoc-Phase Ab an2002 der war Univer Ana Clinical Sciences Centre. Since 2002 she was honorary Pombo zunächst als Honorardozentin für Zellbiologie lecturer for Cell Biology at the Imperial College London. am Imperial College London tätig. 2011 wurde sie zur In 2011, she became professor for Cell Biology. Professorin für Zellbiologie ernannt.

BIMBS Junior Research Group for Dr. Marina BIMSB-Nachwuchsgruppe für Dr. Marina Chekulaeva Chekulaeva

In January 2013, Marina Chekulaeva came from the Marina Chekulaeva wechselte im Januar 2013 vom Friedrich Miescher Institute for Biomedical Research Friedrich-Miescher-Institut für Biomedizinische For-

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- schung in Basel an das MDC, um hier ihre neue Nach- search group. Dr. Chekulaeva’s team studies the mo- wuchsgruppe am Berliner Institut für Medizinische lecularin Basel mechanisms to the BIMSB that where regulate she built mRNA up atranslation, junior re Systembiologie (BIMSB) aufzubauen. Die Gruppe von localization and stability and the role of non-coding Dr. Chekulaeva untersucht die molekularen Mechanis- RNAs in this process. Dr. Chekulaeva studied biochem- men, die die Translation, Lokalisation und Stabilität der istry at the State University of Moldova in Kishinev (Re- mRNA regulieren, sowie die Rolle von nicht-kodieren- public of Moldova) and became a graduate student at den RNAs bei diesem Prozess. Dr. Chekulaeva studierte the Institute of Protein Research, Russian Academy of Biochemie an der Staatlichen Universität von Chisinau Sciences, in Pushchino. She did her doctoral work at (Moldavien) und machte ihre Diplomarbeit am Ins- the European Molecular Biology Laboratory (EMBL) in titut für Proteinforschung der Russischen Akademie Heidelberg, where she continued to work for another der Wissenschaften in Pushchino. Ihre Doktorarbeit year as a postdoc after completing her doctorate. In fertigte sie am Europäischen Labor für Molekularbio- 2006, she went on as a postdoctoral fellow to the Fried- logie (EMBL) in Heidelberg an. Dort setzte sie ihre For- rich Miescher Institute. schungsarbeiten für ein weiteres Jahr als Postdoc fort. 2006 wechselte sie für eine zweite Postdoc-Phase an das Friedrich-Miescher-Institut. Dr. Robert Zinzen starts BIMSB Junior Research Group Dr. Robert Zinzen startet BIMSB Nachwuchsgruppe Robert Zinzen from the EMBL in Heidelberg started as - Robert Zinzen vom EMBL in Heidelberg startete im cal Systems Biology in March 2013. A research focus März 2013 als Nachwuchsgruppenleiter am BIMSB. Die ofa junior Dr. Zinzen group is theleader investigation at the Berlin of the Institute nervous for system Medi Gruppe von Dr. Zinzen beschäftigt sich mit der Entwick- Drosophila melanogaster. Drosophila Dr. Zinzen did his graduate studies at the University of melanogaster. Dr. Zinzen studierte an der University of Georgiadevelopment in Athens, in the Georgia fruit fly (USA) and received his PhD Georgialung des in Nervensystems Athens, Georgia in der(USA), Fruchtfliege und promovierte im degree in Molecular and Cell Biology from the Univer- Fach Molekulare und Zellbiologie an der University of sity of California at Berkeley. Since 2007, he was post- California, Berkeley. Seit 2007 arbeitete Robert Zinzen doctoral researcher and later staff scientist in Eileen als Postdoc in Eileen Furlongs Gruppe am EMBL. Dort Furlong’s group at the EMBL, where he got additional wurde er zudem in der Genomik und Epigenomik aus- training in genomics and epigenomics. gebildet.

Prof. Oliver Daumke receives W3 professorship at W3-Professur an der FU für Prof. Oliver Daumke the FU Der Biochemiker und Proteinkristallograph Oliver The biochemist and protein crystallographer Oliver Daumke nahm den Ruf auf eine W3-Professur für Struk- Daumke accepted an appointment to the full profes- turbiologie des Membranverkehrs der Freien Univer- sität (FU) Berlin an. Prof. Daumke studierte an der Uni- at the Free University Berlin. Oliver Daumke studied versität Freiburg Biologie, ging als Austauschstudent biologysorship forat theStructural University Biology of Freiburg of Membrane and the Trafficking Univer- an die Universität von Sussex in Brighton, England, sity of Sussex, UK. He received his PhD degree from the und wechselte zur Promotion an die Universität zu University of Cologne and made his PhD thesis at the Köln. Seine Doktorarbeit verfasste er am Max-Planck- Max Planck Institute for Molecular Physiology in Dort- Institut für molekulare Physiologie in Dortmund bei mund under the supervision of Alfred Wittinghofer. Alfred Wittinghofer. Nach einer Postdoc-Phase am La- After a postdoctoral period at the lab for Molecular bor für Molekularbiologie in Cambridge, England, kam Biology in Cambridge, UK, he returned as a Helmholtz Prof. Daumke als Helmholtz-Nachwuchsforscher an das MDC. 2010 wurde er Juniorprofessor am Institut became Junior Professor at the Institute for Medical für Medizinische Physik und Biophysik an der Charité. Physicsjunior group and Biophysics leader to the at Charité.MDC. In He2010, has Prof. received Daumke nu- Er erhielt bereits zahlreiche Auszeichnungen, darun- merous awards, including the Bayer Early Excellence in ter 2010 den Bayer Early Excellence in Science Award, Science Award in 2010, a scholarship from the Human ein Stipendium des Human Frontier Science Program, Frontier Science Program, the Otto Hahn Medal of the die Otto-Hahn-Medaille der Max-Planck-Gesellschaft Max Planck Society and the Klaus Liebrecht Prize of the und den Klaus-Liebrecht-Preis der Universität Köln University of Cologne for the best dissertation. He con- für die beste Doktorarbeit. Prof. Daumke wird sein La- tinues to work with his group at the MDC during his full bor am MDC weiterführen. Hier untersucht er Struktur professorship. They will further investigate the struc- und Funktion von Proteinen, die an zelluläre Membra- ture and function of proteins which bind to cellular nen binden und diese verformen. Für die Entwicklung membranes and lead to structural changes. These pro- von zahlreichen Krankheiten wie Krebs und Diabetes teins play a crucial role in the development of several oder bei Infektionen spielen diese Proteine eine zen- diseases such as cancer, diabetes or infections. In 2013, trale Rolle. Ende 2013 erhielt er einen Consolidator Oliver Daumke received a Consolidator Grant from the Grant des Europäischen Forschungsrates (ERC). European Research Council (ERC).

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Awards

Preise

Martin SCHAEFER 2011 [Research Group / Arbeitsgruppe: Miguel Andrade] Participant at the 63th Lindau Nobel Laureate Meeting / Teilnehmer beim 63. Lindauer Nobelpreisträgertreffen November / December Thomas SOMMER (MDC) & Michael GLICKMAN (Technion, Israel Institute of Technology, Haifa) Preis der Deutschen Technion-Gesellschaft Michael BADER Deutsche Technion-Gesellschaft e.V. Franz-Gross-Wissenschaftspreis Deutsche Hochdruckliga (DHL)

Aaron CIECHANOVER Humboldt Research Award / Humboldt-Forschungspreis Alexander von Humboldt Foundation / Stiftung (for a research period at the MDC / für einen Forschungs- aufenthalt am MDC) 2013

Thomas WILLNOW Research Grant / Förderpreis Alzheimer Forschung Initiative (AFI) Soyoung LEE & Clemens SCHMITT Johann-Georg-Zimmermann-Forschungspreis 2013/2014 Förderstiftung der Medizinischen Hochschule Hannover

James POULET Paul-Ehrlich- und Ludwig-Darmstaedter-Nachwuchs- Preis 2012 Paul-Ehrlich-Stiftung Klaus RAJEWSKY José Carreras Award European Hematology Association Michael GLICKMAN, Technion, Israel Institute of Technology, Haifa Klaus RAJEWSKY Friedrich Wilhelm Bessel Research Award / STS/CCS Honorary Medal Forschungspreis Signal Transduction Society und Cell Communication and Alexander von Humboldt Foundation / Stiftung Signalling (for a research period at the MDC / für einen Forschungs- aufenthalt am MDC) Sandrine SANDER [Research Group / Arbeitsgruppe: Hua JING (MDC), Julia KASE (Charité) Curt-Meyer-Gedächtnispreis Curt-Meyer-Gedächtnispreis Berliner Krebsgesellschaft Klaus Rajewsky] Berliner Krebsgesellschaft e.V.

Nikolaus RAJEWSKY Kristin STOCK Gottfried Wilhelm Leibniz Prize 2012 [Research Group / Arbeitsgruppe: Helmut Kettenmann] Deutsche Forschungsgemeinschaft (DFG) / German Re- Nachwuchswissenschaftlerinnenpreis search Foundation Forschungsverbund Berlin-Brandenburg (FVB)

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European Research Council (ERC) Grants

ERC Advanced Grant ERC Starting Grant

BeyOND CardioSplice Metabolic basis of neurodegenerative disease A systems and targeted approach to alternative splicing Thomas Willnow (starts in 2014) in the developing and diseased heart: Translating basic cell biology to improved cardiac function TRANSPOSOstress Michael Gotthardt Impact of stress-induced transposon activities on human disease BrainStates Brain states, synapses and behaviour James Poulet CYTOVOLIONZsuzsanna Izsvák Ion homeostasis and volume regulation of cells and IsletVasc organelles Molecular Mechanisms Regulating Pancreatic Islet Thomas J. Jentsch Vascularization Matthew Poy EXTREMOPHILE MAMMAL Molecular exploitation of an extremophile mammal HEPATOPANCREATIC Gary Lewin Mechanisms underlying cell fate decision between pancreas and liver LYMPHOMA Francesca Spagnoli Modeling lymphoma pathogenesis in mice – from basic mechanisms to pre-clinical models ThermoReg Peripheral and Central Mechanisms of Temperature Detection and Core Body Thermoregulation Klaus Rajewsky Jan-Erik Siemens (since February 2013 Medical Faculty of Heidelberg) ERC Consolidator Grant

MitoShape Structural basis of mitochondrial inner membrane shape and dynamics Oliver Daumke

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Third-party Funding 2012-2013

Drittmittelprojekte

Selected projects (ausgewählte Projekte)

EU-Funding DFG Projects

Collaboration Intra-European Fellowship Research Grants / Sachbeihilfe Atherochemokine Health Investigation of the role of CXCL5/CXCR1 - EUROSPIN pathway in atherosclerosis European Consortium on Synaptic Protein A finite-element model of human phona Olig3-Lbx1 breathing Alternatives Spleißen in der perinatalen Networks in Neurological and Psychiatric tion with fluid-structure interaction Function of the transcription factors Olig3 Herz-Entwicklung und bei Kardiomyopathie Diseases and Lbx1 in brainstem respiratory nuclei - EURATRANS PostScribeDNAdamage ger alpha-Importine der Säuger European large-scale functional genomics Global characterization of post-transcrip- Analyse der spezifischen Funktion paralo in the rat for translational research tional regulatory interactions in DNA Charakterisierung der Funktion mem- branständiger Ubiquitin-Ligasen bei der SynSys damage response Dislokation fehlerhafter Proteine Synaptic Systems: dissecting brain function in health and disease Critical contribution of proneurotrophin- International Incoming receptor Sortilin to the pathology of Fellowship multiple sclerosis and experimental auto- Information and RNARegMap immune encephalomyelitis Communication Technologies Der EDA-A1/EDAR/NF-kappaB Signalweg TARGETAMD Condition specific RNA Regulatory Maps als multifunktioneller Regulator der Haar- Transposon-based, targeted ex vivo gene International Outgoing follikelinduktion therapy to treat age-related macular Diabetes, abdominal obesity, weight degeneration Fellowship Kinase crosslinking change, metabolic factors and risk of liver 3x3D Imaging Capturing kinase-substrate pairs in intact cancer in the European Prospective Investi- Fast two-photon in vivo imaging and mammalian cells by using unnatural amino gation into Cancer and Nutrition stimulation with simultaneous three- acid mutagenesis and photocrosslinking Die Bedeutung von Cytochrom P450 (CYP) dimensional random-access scanning in 2J2 und deren Metabolite in der Pathogen- multiple brain regions ese der Präeklampsie International Reintegration Grant - Nanotechnologies ENDOPANC works in mouse brain MARINA Function of panglial gap junctional net Novel signals guiding endodermal progeni- Genetic regulation of CD177 and the role of Managing Risks of Nanoparticles tor cells toward a pancreatic fate CD177-proteinase-3 interactions in ANCA- MuRF and hypertrophy associated vasculitis. Regulation and function of the E3 ubiquitin Genetics of endocardial-myocardial interac- Marie Curie cardiac hypertrophy Haarzyklusregulation durch NF-KB bei ligases Muscle RING finger 1 and 3 in tions during zebrafish heart development Career Integration Grant Mensch und Maus: Manipulation eines DYNASTIIC Initial Training Network epithelialen Schlüsselsignals als innovativer Ansatz zur Therapie von Haarerkrankungen Dynamic Signal Transduction in Individual EVONET Cells Evolution of gene regulatory networks in In vivo tracking of manipulated dendritic REPROL53U48 animal develpoment cells in tumor models Characterization of direct reprogramming- ATTRACT Investigating the interaction of high-grade regulating factors LIN-53 and USP-48 Advanced Teaching and Training for Adop- gliomas and microglial cells WNT/CALCIUM IN HEART tive Cell Therapy Investigations on the roles of Claudins dur- WNT/Calcium signaling in cardiovascular development JIMI – a network for intravital mikroscopy: Infrastructures ing zebrafish cardiovascular development

imaging to cell tracking in living organisms Industry-Academia EU-OPENSCREEN dedicated project supervision from cell Partnerships and Pathways European Infrastructure of Open Screening Mechanismen der Protein-Qualitätskon- InduStem Platforms for Chemical Biology trolle des endoplasmatischen Retikulums Comparative stem cell research in mouse ISBE Mechanisms of DNA damage induced IKK and humans Infrastructure for Systems Biology - Europe and NF-kB activation

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Third-party Funding 2012-2013 Mechanisms of the switch of stem cells to SFB 650: Clinical Research Units / cancer stem cells in head and neck cancer Klinische Forschergruppen Cellular Approaches for the Suppression of Molecular causes of primary angle-closure Unwanted Immune Reactions - From Bench to Bedside Drittmittelprojekte glaucoma KFO 192: Molecular characterization of cis- and Regulation und Fehlregulation von Muskel- SFB 665: trans-acting gene and ncRNA regulation wachstum Developmental Disturbances in the Ner- vous System during human chondrogenesis KFO 218: Selected projects (ausgewählte Projekte) Molekulare Charakterisierung cis- und Hormonal regulation of body weight SFB 740: trans-aktiver Gen- und ncRNA-Regulation maintenance Von Molekülen zu Modulen: Organisation während der humanen Chondrogenese und Dynamik zellulärer Funktionseinheiten SFB 958: C/EBPß and functional implications Scaffolding of Membranes: Molecular Post translational modifications (PTM) of Research Fellowships / Mechanisms and Cellular Functions Shaping a lymphoma survival niche within Forschungsstipendien secondary lymphoid organs Simulation of excitation contraction cou- Dissection and optogenetic manipulation of pling in ventricular cardiac myocytes - Research Training Groups / Sorting-Rezeptoren als Regulatoren des works in the control of pain and addiction Graduiertenkollegs Lipoproteinstoffwechsels und des kardio- the Habenula-IPN cell-specific neuronal net The molecular mechanism of Semaphorin vaskulären Risikos 3E-Plexin-D1 signaling in shaping vascular GRK 1772: Structural and functional analyses of pan- topology during development Computational Systems Biology glial coupling networks and its impact on brain signalling. Structural Studies of the Nucleocapsids of Negative-strand RNA Viruses as Targets Priority Programmes / CRC / Transregios of the Dynamin-like Myxovirus Resistance Schwerpunktprogramme Protein A TRR 19: Strukturelle und funktionelle Charakte- SPP 1395: Information and Communication Theory in Pathogenesis and Therapy risierung von ß-Amyloid Aggregations- Inflammatory Cardiomyopathy - Molecular Intermediaten Molecular Biology TRR 36: Targeting the secretory pathway in SPP 1356: Principles and Applications of Adoptive T cytotoxic T lymphocytes to modulate their Pluripotency and Cellular Reprogramming Cell Therapy cytolytic capacity in cancer immunotherapy SPP 1365: TRR 43: The (pro)renin receptor and cardiovascular The regulatory and functional network of ubiquitin family proteins processes disease The brain as a target of inflammatory The Coxsackievirus and Adenovirus Recep- SPP 1395: TRR 52: tor CAR in Cardiac Remodeling Informations- und Kommunikationstheorie Transcriptional Programming of Individual T Cell Subsets The role of NF-kB in an animal model of in der Molekularbiologie (InKoMBio) ANCA-associated vasculitis and therapeutic SPP 1230: options using NF-kB inhibition Mechanisms of gene vector entry and Titin in Biomechanik, Signaltransduktion persistence Clusters of Excellence / und Stoffwechsel Funktionelle Analyse der SPP 1463: Exzellenzcluster N2B-Region im Vergleich der Geschlechter Epigenetic regulation of normal hematopoiesis and its dysregulation in myeloid EXC 257: neoplasia NeuroCure – towards a better outcome of neurological disorders Research Units / Forschergruppen

FOR 667: Independent Junior Research Emmy Noether-Nach- Epithelial Mechanisms in Renal Volume Groups / Graduate Schools / Regulation wuchsgruppen Graduiertenschulen FOR 748: Die Rolle ß-Catenin/TCF-abhängiger tran- Neuronal and glial P2 receptors; molecular GSC 86: skriptioneller Netzwerke bei Morphogen- Berlin School of Mind and Brain ese und Regeneration renaler Epithelien FOR 806: basis and functional significance GSC 203: Interfering with intracellular protein- Berlin-Brandenburg School for Regenera- protein interactions - probing protein tive Therapies functions with small molecules Collaborative Research GSC 1091: FOR 1054: Centres / Sonderforschungsbereiche Berlin School of Integrative Oncology (BSIO)

Hypertrophy Sex-Specific Mechanisms in Myocardial SFB 618: FOR 1341: Theoretical Biology: Robustness, modu- Barrel Cortical Function larity and evolutionary design of living International Research FOR 1352: systems Training Groups / Internationale Graduiertenkollegs Structure, Function and Regulation of the SFB 633: Induction and Modulation of T-Cell Medi- FOR 1368: GRK 1631: Myofibrillar Z-disc Interactome ated Immune Responses in the Gastroin- Hemodynamic Mechanisms of Acute testinal Tract International Research Training Group for Myology

Kidney Injury

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Conferences and Scientific Meetings

Kongresse und wissenschaftliche Tagungen

2012

Date / Datum Event / Veranstaltung Organizer / Veranstalter

Green Campus – Lebendige Labore 30.03.2012 MDC, Abt. TFM Errichten Nachhaltige Wissenschaft

19.04. – 20.04.2012 9. Laborrunde – Konferenz MDC, Abt. TFM Errichten

Prof. Th. Blankenstein 03.05. – 04.05.2012 SFB Meeting: 2nd Symposium on Adoptive T Cell Therapy Prof. W. Uckert

International Spring Meeting Force 09.05. – 12.05.2012 Prof. G. Lewin Transduction & Emerging Ion Channels

Auftaktveranstaltung Deutsches Zentrum für Herz-Kreislauf- 10.05.2012 Dr. J. Krebser, DZHK Forschung

3rd Annual Scientific Symposium Prof. Th. Niendorf 08.06.2012 „Ultrahigh Field Magnetic Resonance: Clinical Needs, Research Dr. B. Ittermann Promises and Technical Solutions Prof. J. Schulz-Menger

5th Berlin Summer Meeting „Computational & Prof. N. Rajewsky 28.06. – 30.06.2012 Experimental Molecular Biology“ Prof. K. Rajewsky

Franz Volhard Symposium zu Ehren des 07.09. – 08.09.2012 Prof. D. Müller 70. Geburtstages von Prof. Luft

German French Symposium 25.10.2012 Dr. O. Seumenicht Frontiers of Cardiovascular Research

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2013

Date / Datum Event / Veranstaltung Organizer / Veranstalter

25.04. – 26.04.2013 10. Laborrunde – Konferenz MDC, Abt. TFM Errichten

23.04. – 24.05.2013 Brain Tumor Meeting Prof. H. Kettenmann

4th Annual Scientific Symposium ,,Ultrahigh Field Magnetic Reso- 31.05.2013 Prof. T. Niendorf nance Clinical Need, Research Promises, Technical Solutions”

6th Berlin Summer Meeting Computational & Prof. N. Rajewsky 13.06. – 15.06.2013 Experimental Molecular Biology Prof. K. Rajewsky

13.06. – 15.06.2013 Deutsch-Chinesisches Kardio- Meeting Prof. M. Bader

60 Jahre DNA,Kongress für Lehrkräfte, Dr. L. Bengtsson 13.09. – 14.09.2013 Studierende, Schüler und Interessierte Abt. Kommunikation

10.11. – 13.11.2013 Stem Cells in Development and Disease Dr. D. Besser, GSCN

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Seminars 2012-2013

2012

First name Last name Organization Talk title

University of Florida, Dept. of Neurosci- David R. Borchelt Why can't I remember ence, Gainsville

Novartis Institutes for Biomedical Research, Tewis Bouwmeester Drugging the Wnt pathway Basel

Reinhard Büttner Institute of Pathology, Cologne LSD1 and epigenetics in cancer

Chromatin remodelling during Schwann cell Bruce Carter Vanderbilt University, Nashville differentiation

Regulation of the assembly and function of Raymond Deshaies Howard Hughes Med. Institute, Pasadena cullin-RING ubiquitin ligases

Goethe University, Institute of Cardiovascu- MicroRNAs: therapeutic targets and diag- Stefanie Dimmeler lar Regeneration, Frankfurt nostics in cardiovascular disease

Otto-von-Guericke-Universität Magdeburg, Role of myeloid cell subsets in parasitic Ildiko Rita Dunay Institut für Medizinische Mikrobiologie infections

Reading FGF8 morphogenetic activity dur- Diego Echevarria University Miguel Hernandes, Alicante ing neural tube patterning

High content analysis of integrin alpha2 Anastasia Eskova BIOQUANT, Heidelberg endocytosis regulators

A novel role for neurotransmitters in sensory Laura Forero King's College London organ and forebrain development

DNA reactivity with formaldehyde and the Maxim Frank-Kamenetskii Boston University double helix’s breathing modes

National Research Institute for Child Health Humanized mouse models of Epstein-Barr Shigeyoshi Fujiwara and Development, Tokyo virus infection and associated diseases

Centre for Molecular and Structural Bio- Matthias Futschik medicine, University of Algarve

Institut du Fer à Moulin, INSERM U839, Deciphering the variety of serotonin subsys- Patricia Gaspar Paris tems

Diversity of Protein-Protein Interactions, Toby Gibson EMBL, Heidelberg Modular Protein Architecture, and the Nature of Cell Regulation

Christine Hartmann Institute of Molecular Pathology, Vienna WNTing embryonic stem cells

Multiple level interactions between neu- Klinik und Poliklinik für Neurologie, Michael T. Heneka roinflammation and neurodegeneration in Klinische Neurowissenschaften, Bonn Alzheimer´s disease

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Seminars 2012-2013 First name Last name Organization Talk title

The crystallization and characterization of Manuel Hessenberger Paris Lodron University, Salzburg NLRP14_PYD

“A Pipeline for MHC-II Ligand Identification Kie Kasuga Karolinska Institutet Stockholm, Sweden from a Single Patient”

Viral ion channel proteins in model mem- Ziad Khattari Hashemite University, Zarqua branes: a comparative study by X-ray reflectivity

Nuffield Department of Clinical Medicine at Selective targeting of protein interactions Stefan Knapp the University of Oxford mediated by acetyl lysine reader domains

Hörforschungszentrum der Univerität Marlies Knipper Molecular Basis of Tinnitus Tübingen

Florian Kohlhepp Dept. for Infectious Diseases, Heidelberg EHD proteins in Apicomplexa parasites

Attenuation of TORC1 suppresses replica- Marina Kolesnichenko The Scripps Research Institute, La Jolla tive and RAS-induced cellular senescence

Modeling Holoprosencephaly in the Mouse: Robert S. Krauss Mount Sinai School of Medicine, New York Regulation of Sonic Hedgehog Signaling by Co-receptors

Molecular dissection of inflammation in al- ECRC / Charité / Max Delbrück Center Young-Ae Lee lergic diseases - Towards novel interven- (MDC), Berlin tional targets

Biotech Research and Innovation Centre Role of miR-9 in RNA metabolism and Eleonora Leucci (BRIC), University of Copenhagen Lymphomagenesis

Max-Planck-Institut für Infektionsbiologie, Mosquito immune responses against Elena A. Levashina Berlin malaria parasites

MRC National Institute for Medical Re- Regulation of inflammation by the MAP Steve Ley search, London kinase kinase kinase TPL-2

Sialic acid residues as triggers of comple- Institut für Rekonstruktive Neurobiologie, Bettina Linnartz ment receptor-3 dependent removal by Universität Bonn microglia?

Direct regulation of DNA repair by NR4A Michal Malewicz MRC Toxicology Unit, Leicester nuclear orphan receptors

Max Planck Institute of Biochemistry, Application of proteomics to large scale Matthias Mann Munich protein sequencing and genetics

University of Utah School of Medicine, Salt Phophoselective PDE3 inhibition: a new Matthew Movsesian Lake City therapeutic strategy for heart failure?

Playing with RNA conformation: Hairpin Sabine Müller Ernst-Moritz-Arndt-Universität, Greifswald ribozyme variants for RNA repair, self- processing and ligand sensing

The selectivity of Hv1 and proton and elec- Boris Musset Rush University Medical Center, Chicago tron currents in human monocytes

Aarhus University, Dep. of Molecular Biol- The role of the oligomer in alpha-synuclein Daniel Otzen ogy and Genetics aggregation and Parkinson's Disease

Building the Rho-ome: An integrative study Evangelia Petsalaki Mount Sinai Hospital, Toronto of the role of RhoGAP/GEFs in the Rho protein signaling network

The impact of membrane remodelling on Friedrich-Schiller-University Jena Institute Britta Qualmann neuromorphogenesis, vesicle formation and of Biochemistry neuronal network activity

Tobias Reichenbach The Rockefeller University, New York Biophysics of hearing

Rho GTPase signaling in cell adhesion and Anne Ridley King's College London migration

MDC Research Report 2014 275

ACADEMICS

First name Last name Organization Talk title

EphB2 trans-endocytosis during contact Max-Planck Institute of Neurobiology, Maria Sakkou repulsion is mediated by Src/Dock180/Elmo Martinsried signaling

Twists and turns in ubiquitin conjugation Brenda Schulman St. Jude Children`s Res. Hospital, Memphis cascades

Cancer Research UK London Research Regulation of glucose and lipid metabolism Almut Schulze Institute by oncogenic signalling pathways in cancer

Beckman Research Institute Of City of The role of CEACAM1 in inflammation and John E. Shively Hope, Duarte cancer

Université Victor Segalen Bordeaux 2 Role of central 5-HT2B receptors in the Umberto Spampinato Centre Recherche Inserm U862 - Institut control of dopamine ascending Francois Magendie 146

Basic principles and potential applications Max Delbrück Center (MDC), Microscopy of superresolution microscopy. Part 2: Anje Sporbert Core Facility, Berlin Localisation microscopy methods (PALM, STORM, GSDM)

Grey matter damage and repair in inflam- Christine Stadelmann-Nessler Institut für Neuropathologie, Göttingen matory demyelination

Novel insights into evolutionary conserved Eduard Stefan University Innsbruck protein kinase A signalling circuits

C/EBPdelta: Dr. Jekyll and Mr. Hyde in Esta Sterneck National Cancer Institute, Faro breast cancer biology

Acute regulation of cell surface area by the Institute of Molecular and Cellular Biosci- Shiro Suetsugu balance between flattening and endocyto- ences, Tokyo sis of caveolae

Cerebral leukocyte recruitment: an essential Department of Physiology and Biophysics, Juliana Tavares crosstalk between immune and vascular Belo Horizonte system

Functional genetics using Drosophila mod- Aaron Voigt Universität Aachen els of human neurodegenerative diseases

Structural Insight into RNA Induced Gene Yanli Wang Institute of Biophysics Beijing Silencing

Cardiac electrical remodelling triggered Andreas Werdich Harvard Medical School, Boston by stretch: what can we learn from the zebrafish?

Stanford School of Medicine, Division of T cell effector mechanisms in atheroscle- Cornelia M. Weyand Immunology and Rheumatology rosis

Guidance of retinal ganglion cells by Andrea Wizenmann Institute of Anatomy, Tübingen Engrailed

Michael Yaffe (MIT): Post-translational Michael Yaffe MIT, Boston modification of proteins, PPIs, and cellular signaling

276 MDC Research Report 2014 ACADEMICS

2013

First name Last name Organization Talk title

Studying cardiac development using ze- Jeroen Bakkers Hubrecht Institute, Utrecht brafish genetics

New insights into RelB function and regula- Veronique Baud Institut Cochin- INSERM, Paris tion

Tom Becker Brain and Mind Research Institute, Sydney Human Genomics in transgenic Zebrafish

London Research Institute, Cancer Re- Stem cells, cell fate reprogramming, and Axel Behrens search UK cancer

Action at the junction: Distinct roles for Biozentrum, University of Basel, Dept. Cell Heinz-Georg Belting VE-cadherin during vascular network Biology formation

The role of the proteasome and resistasome Barshop Institute for Aging, Dept. of Physi- Rochelle Buffenstein in sustained good health in the long lived ology, Texas naked mole rat

From phenotypic information to molecular Monica Campillos Helmholtz Center, Munich information

New Gap Treatments in Biological Se- Ramu Chenna TU Dresden quence Alignment and a Tool for Exploring Functional Context of Protein Motifs

Striped-illumination deep-infrared Deutsches Rheumaforschungszentrum multiphoton microscopy: a novel tool for Zoltan Cseresnyes Berlin (DRFZ) and Max Delbrück Center high-resolution deep-tissue imaging at (MDC), Berlin the MDC

Department of Bioelectronics Institute of 3D-Ultramicroscopy of whole mouse brains Hans-Ulrich Dodt Solid State Electronics TU Vienna and mouse embryos

Computational Biology Center, Sloan- Accurate detection of differential RNA Phillip Drewe Kettering Institute, New York processing

Protein folding and aggregation kinetics Simon Ebbinghaus Ruhr-Universität Bochum probed in the cell

"Functional characterization of RNA binding Institute of Parasitology and Biomedicine, Sandra Fernandez Moya proteins DRBD3 and RBP33 in Trypano- Granada, Spain soma brucei"

Systematic dissection of roles for chromatin Nir Friedman Hebrew University of Jerusalem regulators in a yeast stress response

Physiologisches Institut, Lehrstuhl II, Eber- Olga Garaschuk High resolution in vivo imaging of microglia hard Karls Universität Tübingen

Exzellenzcluster NeuroCure, Synaptic Neural circuit repair by deep brain stimula- Rosemarie Grantyn Dysfunction Group and Experimental Neu- tion rology, Berliln

Splicing regulator PRMT5 links Alternative Institute of Molecular and Cell Biology Ernesto Guccione Splicing to the p53 response in mammalian (IMCB), Singapore development

Noninvasive perfusion measurement: basics Matthias Günther Fraunhofer Mevis, Bremen and not-so basics of arterial spin labeling

Seeing deeper: tissue clearing methods Julia Haseleu Max Delbrück Center (MDC), Berlin applied in thick-sample imaging

Mechanismen der Entstehung einer Über- Kristina Heinig Max Delbrück Center (MDC), Berlin lebensnische für murine CLL-Leukämiezel- len in sekundären lymphatischen Organen

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ACADEMICS

First name Last name Organization Talk title

Why does selection care about codon us- University of Bath, Dept. of Biology and Laurence Hurst age (or whatg really determines ribosome Biochemistry, Somerset velocity)?

Linear polyubiquitination: a new regulator of Kazuhiro Iwai Kyoto University NF-kappaB activation

Structural determinants of gating proper- Wanchana Jangsangthong University of Cologne ties of voltage-gated L-type Ca2+ channel as revealed by single-channel analysis

Albert Einstein College of Medicine, New Canonical and non-canonical Wnt signaling Andreas Jenny York in Drosophila

The molecular basis for chromosomal The Hebrew University of Jerusalem, Life Batsheva Kerem instability in early stages of cancer devel- Sciences Institute opment

Spatio-temporal control of cell adhesion Fraunhofer Institute for Biomedical Engi- Michael Kirschbaum and neurite outgrowth via microstructured neering, Potsdam thermoresponsive polymer coatings

MRC Laboratory of Molecular Biology, Mechanisms, roles, and uses of OTU do- David Komander Cambridge main deubiquitinases

Direct production of mouse mutants using Ralf Kühn Helmholtz-Zentrum München ZFNs, TALENs or Crispr/Cas in one-cell embryos

Dept. of Cell and Developmental Pancreas development and function: the Limor Landsman Biology,Sackler Faculty of Medicine, Tel role of mesenchymal cells Aviv

University of Southern California, Los Ralf Langen How proteins bend membranes Angeles

Tumor Suppressor Genes in the biology of Han Li Spanish National Cancer Centre, Madrid Stem Cells

Harvard Medical School, Brigham and Systems approaches in cardiac and vascu- Calum MacRae Women's Hospital, Boston lar development

Fred Hutchinson Cancer Research Center, Genetic conflicts: the usual suspects and Harmit Malik Seattle beyond

Identifying Protein Recognition Elements on Technion - Israel Institute of Technology, Yael Mandel-Gutfreund RNA by Combining Sequence and Struc- Haifa tural Information

Max-Perutz-Laboratories, University of Molecular Mechanisms of Autophagosome Sascha Martens Vienna Formation: Insights from Reconstitutions

Role Of DNA Triple Helical Structures In All India Institute of Medical Sciences, New Rajeswari R. Moganty Neurodegenerative Disorders, Friedreich's Delhi Ataxia

Calcium Signaling in human inducted pluripotent stem cell-derived Cardiomyo- Cardiac Signaling Center, Medical Univer- Martin Morad cytes from normal and Catecholaminergic sity of South Carolina, Clemson polymorphic ventricular tachycardia-afflicted subjects

Tales of stripes: deciphering cell fates in the Christian Mosimann University of Zürich lateral plate and the neural crest

Introduction to Microfluidics: Applications Yaakov Nahmias Hebrew University, Jerusalem in Biology

Howard Hughes Medical Institute, Chevy Teresa Nicolson Ribbon synapses in hair cells: size matters Chase, Maryland

Deutsches Rheumaforschungszentrum The state of JIMI: ongoing intravital imaging Raluca Niesner Berlin (DRFZ) projects in the MDC-DRFZ network

278 MDC Research Report 2014 ACADEMICS

First name Last name Organization Talk title

Deutsches Rheumaforschungszentrum Statistical methods applied in the analysis Laura Oehme Berlin (DRFZ) of in-vivo imaging data

Institute of Biotechnology and Biomedicine, Protein allosteric sites - a structural bioin- Alejandro Panjkovich Universitat Autónoma de Barcelona formatics perspective

Neuroglial Interactions in Cerebral Physio- Unraveling the many ways astroglial con- Nathalie Rouach pathology, Collège de France, Paris nexins control neurotransmission

Gene regulatory networks controlling pan- Maike Sander University of California San Diego creatic beta cell development

Deutsches Rheumaforschungszentrum Automated cell tracking in intravital imag- Jannike Sarmadi Berlin (DRFZ) ing: limitations and advantages

Weizmann Institute of Science, Rehovot, Actin Cables Guide Polarized Secretion in Eyal Scheijter Israel Tubular Organs

Molecular mechanisms of T cell tolerance Andrea Schietinger University of Washington, Seattle to self/tumor antigens

YAP1 and the Hippo Signaling Pathway Karin Schlegelmilch Boston Children's Hospital Regulate Progenitor Proliferation

RIKEN Brain Science Institute (BSI), Mechanisms regulating subcellular Ca2+ Alexey Semyanov Hirosawa dynamics in astrocytes

Real-time kinetic analysis of the in vitro invasion phenotype of the NCI60 tumor cell Robert H. Shoemaker National Cancer Institute, Bethesda line panel enables identification of genes associated with mesenchymal invasion and inhibitory compounds.

''Identification and expression profiling of Konstantinia Skreka Biocenter/Medical University Innsbruck novel ncRNAsinvolved in mESC in vitro neural differentiation''

The state of JIMI: ongoing intravital imaging Anje Sporbert Max Delbrück Center (MDC), Berlin projects in the MDC-DRFZ network

Mass Spectrometry supported determi- Florian Stengel ETH Zurich nation of protein complex structure – an integrated approach

Gastrulation in the mouse depends upon a Francis Stewart BIOTEC TU Dresden switch between histone 3 lysine 4 methyltransferases

Treating Alzheimer's Disease by using Charite - Universitätsmedizin Berlin CCR/ Franz Theuring Tau-Aggregation-Inhibitors: the time for Tau Institut für Pharmakologie is NOW

Deconstructing Metastasis - Epigenetic Sakari Vanharanta Sloan-Kettering Institute, New York Expansion of the VHL-HIF Signal Output Drives Renal Cancer Progression

Alf Wachsmann Max Delbrück Center (MDC), Berlin eduroam – wireless network everywhere

Novel approaches to target STAT3 in City of Hope National Medical Center, Hua Yu specific immune subsets: opportunities for Duarte, USA clinical use

Position, recognition, and the assembly of Niccolo Zampieri Columbia University, New York motor circuits

MDC Research Report 2014 279

280 MDC Research Report 2014 Annex Anhang

MDC Research Report 2014

ANNEX

Helmholtz Association

Helmholtz-Gemeinschaft

he Helmholtz Association is the largest science ie Helmholtz-Gemeinschaft ist mit ihren rund organization in Germany with its approximately 36.000 Mitarbeiterinnen und Mitarbeitern in T36,000 employees and collaborators in 18 re- D18 Forschungszentren sowie einem Jahresbudget search centers as well as with an annual budget of von rund 3,8 Milliarden Euro, davon ca. 30% Drittmittel about 3.8 billion Euro, ca. 30% of which are third-party aus dem öffentlichen und privatwirtschaftlichen Bereich, funds from the public and private sector. die größte Wissenschaftsorganisation Deutschlands.

The research centers of the Helmholtz Association are Die Forschungszentren der Helmholtz-Gemeinschaft arbeiten in sechs Forschungsbereichen: (1) Energie, (2) and Environment, (3) Health, (4) Key Technologies, (5) Erde und Umwelt, (3) Gesundheit, (4) Schlüsseltechno- Structureactive in sixof Matter fields ofas research:well as (6) (1) Aeronautics, Energy, (2) Space Earth logien, (5) Struktur der Materie sowie (6) Verkehr und Weltraum. Innerhalb dieser Forschungsbereiche sind - anderal Transport.centers. Scientists Within thesefrom theresearch respectively fields, thematicinvolved mehreren Zentren getragen werden. Wissenschaftler programs are defined that are supported by one or sev thematische Programme definiert, die von einem oder- they work together in a cross-center approach. This sam das Forschungsprogramm, in denen sie Zentren- structurecenters jointly of program-oriented develop the research funding program (POF) in was which in- übergreifendaus den jeweils zusammenarbeiten. beteiligten Zentren Dieseentwickeln Struktur gemein der troduced in 2001 following a suggestion by the German programmorientierten Forschung (POF) wurde 2001 Council of Science and Humanities. auf Vorschlag des Wissenschaftsrates eingeführt.

- Der Forschungsbereich Gesundheit konzentriert sich auf die großen Volkskrankheiten. Die Forschung erfolgt in programs:The research (I) fieldcancer Health research, focuses (II) oncardiovascular the major wide and fünf Programmen: (I) Krebsforschung, (II) Herz-Kreis- metabolicspread diseases. diseases, Research (III) nervous is being system carried disorders, out in (IV)five lauf- und Stoffwechselerkrankungen, (III) Erkrankungen des Nervensystems, (IV) Infektion und (V) Gen-Umwelt- on widespread diseases with a focus on diabetes mel- - litus,infection chronic and (V)lung genetic diseases and and environmental allergies. The influences MDC co- abetes mellitus, chronischen Lungenerkrankungen und ordinates the program “Cardiovascular and Metabolic Allergien.Einflüsse aufDas Volkskrankheiten MDC koordiniert mit das Schwerpunkt Programm auf„Herz- Di Diseases” and substantially contributes to the pro- Kreislauf- und Stoffwechselerkrankungen“ und beteiligt grams “Cancer Research” and “Functions and Dysfunc- sich substantiell an den Programmen „Krebsforschung“ tions of the Nervous System”. und „Erkrankungen des Nervensystems“.

Die wichtigsten Forschungsaktivitäten im Gesund- heitsbereich sind an fünf Zentren angesiedelt: am GermanThe most Cancer important Research research Centre activities (DKFZ) inin theHeidelberg, field of Deutschen Krebsforschungszentrum (DKFZ) in Heidel- athealth the Germanresearch Centerare concentrated for Neurodegenerative at five centers: Diseases at the berg, am Deutschen Zentrum für Neurodegenerative (DZNE) in Bonn, which was newly established in 2009, Erkrankungen (DZNE) in Bonn, das 2009 neu gegrün- at the Helmholtz Zentrum München – German Research det wurde, am Helmholtz Zentrum München für Ge- Center for Environmental Health (HMGU), at the Helm- sundheit und Umwelt (HMGU), am Helmholtz-Zentrum holtz Centre for Infection Research (HZI) in Braun- für Infektionsforschung (HZI) in Braunschweig und am schweig and at the Max Delbrück Center for Molecular Max-Delbrück-Centrum für Molekulare Medizin (MDC). Medicine (MDC) Berlin Buch. Moreover, other Helmholtz Darüber hinaus leisten weitere Helmholtz-Zentren centers make vital contributions: the Helmholtz-Zentrum wichtige Beitrage: Helmholtz-Zentrum Dresden-Ros- Dresden-Rossendorf (HZDR), the GSI Helmholtz Centre sendorf (HZDR), Helmholtz-Zentrum für Schwerionen- for Heavy Ion Research in Darmstadt and the Helmholtz forschung (GSI) in Darmstadt, und Helmholtz-Zentrum Centre for Environmental Research (UFZ) in Leipzig. für Umweltforschung (UFZ) in Leipzig.

Bei Themen von zunehmender Bedeutung, wie der per- as personalized medicine, collaboration takes place sonalisierten Medizin, wird Zentren- und Programm- When it comes to topics of increasing significance, such

282 MDC Research Report 2014 ANNEX

Helmholtz Association

Helmholtz-Gemeinschaft Photo: Heidi Scherm/Helmholtz-Gemeinschaft Photo:

A team of five researchers led by Klaus Butterbach-Bahl (2nd from right) of the Karlsruhe Institute of Technology (KIT) was awarded the “Wissenschaftspreis des Stifterverbandes – Erwin Schrödinger-Preis 2013”. This prize is jointly awarded by the Stifterverband für die Deutsche Wissenschaft and the Helmholtz Association. Ein fünfköpfiges Forscherteam um Klaus Butterbach-Bahl (2.v.r.) vom Karlsruher Institut für Technologie (KIT) wurde bei der Helmholtz-Jahrestagung 2013 mit dem Wissenschaftspreis des Stifterverbandes – Erwin Schrödinger-Preis 2013 ausgezeichnet. Den Preis vergeben der Stifterverband für die Deutsche Wissenschaft und die Helmholtz-Gemeinschaft jedes Jahr gemeinsam.

across centers and programs. To this end, partners are übergreifend zusammengearbeitet. Dazu werden Partner insbesondere aus der Hochschulmedizin ein- For strengthening translational research, the Helm- gebunden. Zur Stärkung der translationalen Forschung holtzinvited, Association especially fromhas establishedthe field of university partnerships medicine. with hat die Helmholtz-Gemeinschaft Partnerschaften mit - Universitäten, insbesondere der Hochschulmedizin, cine, other extramural research institutions and the weiteren außeruniversitären Forschungsinstitutio- industry.universities, These in particular partnerships the fieldare beingof university continuously medi nen sowie mit der Industrie etabliert. Diese werden further developed. Moreover, the health research cen- kontinuierlich weiter entwickelt. Darüber hinaus sind ters of the Helmholtz Association are partners of the die Gesundheitsforschungszentren der Helmholtz- German Centres for Health Research and of the German Gemeinschaft Partner der Deutschen Zentren der Ge- National Cohort. sundheitsforschung sowie der Nationalen Kohorte.

Program-oriented funding POF Die Programmorientierte Förderung POF

In autumn 2001, the entire research of the Helmholtz Im Herbst 2001 wurde die gesamte Forschung der Association was newly structured. The Helmholtz As- Helmholtz-Gemeinschaft neu strukturiert. Die Helm- sociation bundled its research activities within the six holtz-Gemeinschaft hat ihre Forschungsaktivitäten in den sechs oben genannten großen Bereichen gebün- - delt und innerhalb dieser Forschungsbereiche thema- sourcesmajor research are not allocated fields mentioned to individual above institutions, and defined but ratherthematic to cross-center programs within research these programs, research which fields. com Re- einzelnen Institutionen zuerkannt, sondern Zentren- pete with each other. übergreifendentische Programme Forschungsprogrammen, definiert. Ressourcen werden die sich nicht un-

- vides the basis for funding these research programs. Einetereinander strategische im Wettbewerb Begutachtung, befinden. die alle fünf Jahre statt- ThisA strategic task is review,carried carriedout by outwell-known every five experts years, from pro - across the globe. Their assessments provide the basis schungsprogramme. Diese Aufgabe übernehmen renom- for the decision to which amount the federal govern- miertefindet, Experten bildet die aus Basis aller fürWelt. die Ihre Finanzierung Gutachten bilden der For die ment and the federal states provide funding for the in- Grundlage für die Entscheidung, in welcher Höhe Bund dividual programs. und Länder die einzelnen Programme fördern.

Die erste Förderperiode lief von 2003 bis 2008, second program period covered the time between die zweite Programmperiode von 2009/2010 bis 2009/2010The first funding and 2013/2014, period ran depending from 2003 on to the 2008, time the of assessment. The assessments for the second funding Begutachtungen für die zweite Förderperiode erfolg- period took place in spring 2008 (Aeronautics, Space 2013/2014, je nach Zeitpunkt der Begutachtung. Die-

ten im Frühjahr 2008 (Verkehr und Weltraum, Ge MDC Research Report 2014 283

ANNEX

and Transport, Health, Earth and Environment) and in spring 2009 (Energy, Structure of Matter, Key Tech- (Energie, Struktur der Materie, Schlüsseltechnologi- nologies). The program assessments for the third fund- en).sundheit, Die Programmbegutachtungen Erde und Umwelt) und imfür Frühjahrdie dritte 2009 För- ing period took place in spring 2013 for the research derperiode erfolgten für die Forschungsbereiche Ver- kehr und Weltraum, Gesundheit, Erde und Umwelt im and Environment and are scheduled for spring 2014 fields Aeronautics, Space and Transport, Health, Earth Energie, Struktur der Materie, Schlüsseltechnologien Key Technologies. Frühjahr 2013 und stehen für die Forschungsbereiche for the research fields Energy, Structure of Matter and In both a national and international comparison, the Imim Frühjahrnationalen 2014 und an. internationalen Vergleich schätzten - die Gutachter die wissenschaftliche Qualität aller Pro- gramme im Forschungsbereich Gesundheit als herausra- andreviewers of extraordinary appraised strategic the scientific relevance quality for society, of all pro the gend und von außerordentlicher strategischer Relevanz economygrams in and the science. research field Health to be outstanding für die Gesellschaft, Wirtschaft und Wissenschaft ein.

In addition to the research programs, the Helmholtz Neben den Forschungsprogrammen betreiben die centers maintain large-scale research infrastructures Helmholtz-Zentren große Forschungsinfrastrukturen, that are used by scientists both at home and abroad. die national und international genutzt werden. Die- se werden einer separaten Begutachtung unterzogen. process. With its involvement in the German National Der Forschungsbereich Gesundheit etabliert mit seiner These infrastructures are subject to a separate review- Beteiligung an der Nationalen Kohorte erstmalig eine tablishes a large-scale infrastructure. The participation große Infrastruktur. Der Anteil der Helmholtz-Zentren ofCohort, the Helmholtz the research centers field inHealth the German for the firstNational time Coes- an der Nationalen Kohorte wurde einer separaten Be- hort was assessed separately. gutachtung unterzogen.

Assessment of centers Begutachtung der Zentren

The MDC performed successfully in all three program Das MDC hat in allen drei Programmbegutachtungen reviews carried out in the context of the third funding im Rahmen der dritten Förderperiode im März 2013 period in March 2013 and received the highest marks erfolgreich abgeschnitten und Höchstnoten von den Gutachtern für seine wissenschaftlichen Leistungen erhalten. Die Ergebnisse dieser Begutachtungen bestä- resultfrom the of reviewersthe center for review its scientific that took achievements. place in March The tigten eindrucksvoll das Ergebnis der Zentrumsbegut- 2012.results of these assessments impressively confirm the achtung vom März 2012.

Between the reviews in the context of program-orient- Zwischen den Begutachtungen im Rahmen der pro- ed funding, assessments of the individual centers take - place on the level of the research groups. The research tungen der einzelnen Zentren auf der Ebene der For- groups at the MDC were last reviewed in March 2012. schungsgruppengrammorientierten statt. Förderung Die Forschungsgruppen finden die Begutach des A total of 38 external reviewers assessed the work of MDC wurden zuletzt im März 2012 begutachtet. Insge- the research groups at the MDC directly on site. All in samt bewerteten 38 externe Gutachter vor Ort die Ar- all, the MDC was assessed to be “highly visible on an beit der Forschungsgruppen des MDC. Das MDC wurde international level” and received the highest mark of insgesamt als „highly visible on an international level“ “outstanding” since more than a third of all research und mit der Höchstnote „outstanding“ bewertet, da groups were given the highest mark. Of the 62 research mehr als ein Drittel aller Forschungsgruppen mit der groups, 29% received the highest mark “outstanding”, Höchstnote bewertet wurden. Von den 62 Forschungs- 26% were rated “outstanding to excellent” and 26% gruppen erhielten 29% die Höchstnote „outstanding, were rated “excellent.” This means that a total of 81% 26% „outstanding to excellent“ und 26% „excellent“. of research groups were rated to contribute excellent Damit wurden insgesamt 81% der Forschungsgrup- pen exzellente und herausragende wissenschaftliche Leistungen bescheinigt. Darüber hinaus haben die Gut- ofand the outstanding MDC with its scientific cross-disease achievements. approach Moreover, as a mark the of achter die besondere Ausrichtung des MDC mit seiner strengthreviewers and highlighted basis for success.the specific strategic orientation krankheitsübergreifenden Aufstellung als Stärke und Basis für den Erfolg herausgestellt.

The Helmholtz Association comprises 18 research centers in Germany. Die Helmholtz-Gemeinschaft umfasst 18 Forschungszentren in Deutschland.

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Berlin Institute of Health (BIH)

Berliner Institut für Gesundheitsforschung

n the Berlin Institute of Health (BIH) the Max Del- m Berliner Institut für Gesundheitsforschung/Berlin brück Center for Molecular Medicine (MDC) and the Institute of Health (BIH) bündeln das Max-Delbrück- I ICentrum für Molekulare Medizin (MDC) und die promote translational research. Within the overarch- Charité - Universitätsmedizin Berlin ihre Kräfte. Unter ingCharité theme –of Universitätsmedizin systems medicine the Berlin goal is join to accelerate forces to dem übergreifenden Forschungsansatz der Systemme- dizin wird am BIH translationale Forschung betrieben: (bench to bedside) and to increasingly address unmet Erkenntnisse aus der Grundlagenforschung sollen ziel- medicalapplication needs of in research basic research findings (bedside into clinical to bench). practice In gerichteter in die klinische Anwendung gebracht und an interdisciplinary systemic approach the BIH aims at klinische Fragestellungen verstärkt ins Labor getragen developing innovative concepts in diagnosis, therapy werden. Ziel ist es, durch interdisziplinäre und syste- mische Ansätze innovative Konzepte der Diagnostik, improved public health. Therapie und Prävention zu entwickeln und diese zur and prevention for the ultimate benefit of patients and Verbesserung der Gesundheit der Menschen umzuset- Founded in the beginning of 2013 as a cooperation of zen. MDC and Charité, the BIH is scheduled for early 2015 to become a corporation under public law (Körperschaft Das BIH wurde Anfang 2013 als Kooperation des MDC öffentlichen Rechts). This will lift the long-standing und der Charité gegründet. Bis 2015 soll es per Lan- desgesetz in eine Körperschaft des öffentlichen Rechts an institutional level and create a novel structure for - translationalproject-based research. collaboration Both partners, of MDC MDC and Charitéand Chari to- basierte Kooperation von MDC und Charité auf eine té, will keep their status as legal entities and thus retain institutionelleüberführt werden. Ebene Damit gebracht wird dieund langjährige eine neue Strukturprojekt their full independence. Responsibilities for fundamen- der Translationsforschung geschaffen. MDC und Cha- tal affairs of each institution, such as research in the rité als Gliedkörperschaften des BIH bleiben rechtlich framework of the Helmholtz Association at the MDC, selbstständig; sie können über eigene Belange, am will not be affected. MDC etwa die Forschung im Rahmenprogramm der Helmholtz-Gemeinschaft, weiter frei bestimmen. Until 2018 the BIH can draw on a total budget of about 280 million Euros for its set-up. In 2013 and 2014 the - designated volume of 38 million Euros will be provided onen Euro zur Verfügung. 2013 und 2014 stammen die by the Helmholtz Association. Thereafter the BIH will vorgesehenenFür den Aufbau 38 des Millionen BIH stehen Euro bis 2018Förderung rund 280 aus Milli Mit- receive institutional funding by the Federal Govern- teln der Helmholtz-Gemeinschaft. Danach erhält das ment and the State of Berlin in a ratio of 90:10. By 2018 BIH eine institutionelle Förderung durch den Bund und the institute will run on an annual budget of about 80 das Land in einem 90:10-Schlüssel. In der Endausbau- million Euros. Moreover, the BIH will be supported by phase in 2018 wird das BIH über einen Jahresetat von the Private Excellence Initiative (Private Exzellenzini- rund 80 Millionen Euro verfügen. Zusätzlich wird das tiative) of Johanna Quandt who provides another 40 BIH durch die „Private Exzellenzinitiative“ von Johan- million Euros over the next ten years. The funds of the na Quandt gefördert, die das Institut über die nächsten Private Excellence Initiative are managed by the Chari- zehn Jahre mit 40 Millionen Euro unterstützt. Diese té foundation (Stiftung Charité). Gelder werden von der Stiftung Charité verwaltet.

The institute will be operated by a four-member Ex- - ecutive Board: The Chair of the MDC, Walter Rosenthal, standsvorsitzende des MDC, Walter Rosenthal; der Vor- the Chair of the directorial board of the Charité, Karl standsvorsitzendeDem BIH steht ein dervierköpfiger Charité, KarlVorstand Max Einhäupl;vor: der Vor die Max Einhäupl, the Dean of the Charité, Annette Grüt- Dekanin der Charité, Annette Grüters-Kieslich; und ein ers-Kieslich, and an independent Chairman, Ernst Th. unabhängiger Vorstandsvorsitzender, Ernst Th. Riet- Rietschel. A Supervisory Board with members from schel. Ein Aufsichtsrat mit Vertretern von Land, Bund, the State of Berlin, the Federal Government, the Helm- Helmholtz-Gemeinschaft und Berliner Universitäten holtz Association and the Berlin Universities oversees beaufsichtigt das BIH. Die inhaltliche und strategische Begleitung des BIH erfolgt durch einen Beirat aus zwölf

the BIH. A Scientific Advisory Board consisting of 12 286 MDC Research Report 2014 ANNEX

Berlin Institute of Health (BIH)

Berliner Institut für Gesundheitsforschung Photo: David Ausserhofer/MDC Photo:

external and internationally recognized scientists ad- externen international anerkannten Wissenschaftle- rinnen und Wissenschaftlern. questions. vises and supports the BIH in scientific and strategic Im BIH werden biomedizinische Grundlagenforschung, At the BIH, basic biomedical research, clinical research klinische Forschung und ärztliche Praxis unter ei- and medical practice are consolidated under one roof. nem Dach zusammengeführt. Im Mittelpunkt des For- Systems medicine lies at the heart of the BIH research schungsprogramms steht dabei die Systemmedizin: program: Recent technological breakthroughs such as Die Entwicklung von Hochdurchsatzverfahren und high-throughput technologies and powerful omics tools leistungsfähigen Omics-Technologien (Genomics, Prote- (Genomics, Proteomics, Metabolomics) have enabled the omics, Metabolomics) hat eine umfassende molekulare comprehensive molecular characterization of diseases. Charakterisierung von Krankheiten ermöglicht. Zusam- Combined with advances in bioinformatics and clini- men mit Fortschritten in der Bioinformatik und der kli- cal data management this allows systems-oriented ap- nischen Datenverarbeitung erlaubt das die Anwendung proaches to be applied to medicine and makes the holis- systemorientierter Ansätze in der Medizin, bei der tic analysis of complex (patho-)physiological processes komplexe (patho-)physiologische Prozesse und deren and their interplay possible. Instead of looking at single dynamische Wechselwirkungen in ihrer Gesamtheit organs or pathologies, systems medicine focuses on untersucht werden. Statt um einzelne Organe oder spe- overarching aspects of disease. The BIH will thus primar- - ily address cross-cutting topics pertaining to multiple fender Aspekte von Krankheiten. Am BIH werden daher diseases such as immunology, cellular and sub-cellular verstärktzielle Pathologien Querschnittsthemen geht es um die wie Aufklärung Immunologie, übergrei zellu- processes, cell degeneration and regeneration, metabo- läre und subzelluläre Prozesse, Degeneration und Rege- lism, the genetics of disease and gender medicine. neration von Zellen, Stoffwechselvorgänge, die Genetik von Krankheiten und die Bedeutung von Geschlechts- The complementary strengths of MDC and Charité make unterschieden in Krankheitsprozessen adressiert. them ideal partners to pursue systems medicine-guided translational research. The MDC contributes its exper- Für diesen Forschungsansatz bilden MDC und Charité tise in molecular and systems biology and provides ac- mit ihren komplementären Stärken ideale Ausgangs- cess to animal models of human disease and state-of- partner. Das MDC bringt seine herausragende mole- the-art omics technologies. The Charité adds essential kulare und systembiologische Expertise ein und bietet knowledge of disease mechanisms and in clinical pheno- Zugang zu einer Vielzahl von Tiermodellen der Hu- typing. It provides access to a large number of well-char- manpathologie und zu neuesten Entwicklungen in den acterized patients over the whole range of common and Omics-Technologien. Die Charité verfügt über große rare diseases and has expert knowledge in the manage- Expertise im Bereich der Krankheitsmechanismen und ment of clinical trials, biobanks and clinical databases. der klinischen Phänotypisierung. Sie ermöglicht den Zugang zu einer großen Anzahl von gut charakterisier- - - cellent research in translational systems medicine, re- tenen Erkrankungen und verfügt über Expertenwissen The BIH will be active in five areas: It will support ex ten Patienten im ganzen Spektrum der häufigen bis sel

MDC Research Report 2014 287

ANNEX Photo: David Ausserhofer/BIH Photo:

At the opening ceremony of the BIH: Neuroscientist Alan Richardson-Klavehn accompanied a slide show on the piano. Back- ground: The BIH Board of Directors Walter Rosenthal, Ernst Rietschel, Karl Max Einhäupl and Annette Grüters-Kieslich. Bei der Eröffnungszeremonie des Berliner Instituts für Gesundheitsforschung begleitete der Neurowissenschaftler Alan Richardson-Klavehn eine Diashow am Piano. Im Hintergrund zu sehen ist der BIH-Vorstand mit Walter Rosenthal, Ernst Rietschel, Karl Max Einhäupl und Annette Grüters-Kieslich.

cruit outstanding scientists, provide cutting-edge tech- bei klinischen Studien und der Arbeit mit Biobanken nologies and infrastructures, build up Clinical Research und klinischen Datenbanken. Units and develop new career paths for early-career scientists. Das BIH wird in fünf Bereichen aktiv. Es wird exzellente systemmedizinisch-translationale Forschung fördern, To foster interdisciplinary collaboration of basic sci- herausragende Wissenschaftler rekrutieren, hochmo- entists and clinicians and to advance translational and derne Infrastrukturen und Technologie-Plattformen systems medicine, competitive BIH programs provide zur Verfügung stellen, Clinical Research Units BIH Collabora- und neue Wege in der Nachwuchsförderung beschrei- tive Research Grants ten. aufbauen consortiafunding for of excellentMDC and researchCharité scientists projects. at the interface will finance large interdisciplinary Um die interdisziplinäre Zusammenarbeit von Grund- have started in spring 2014. BIH Twinning Research lagenwissenschaftlern und klinischen Forschern zu Grantsof basic research and clinical practice. The first projects- fördern und im Sinne der Translation und Systemme- laborations. dizin weiter auszubauen, fördert das BIH im Rahmen will provide financial support for smaller col von kompetitiven Programmen exzellente Forschungs- Recruitment of complementary expertise in areas such BIH Collaborative Research Grants as stem cell biology and bioinformatics is crucial to the von Wissenschaftlern und Klinikern aus MDC und Cha- ritéprojekte. gemeinsam getragene, langfristig angelegte finanzieren Ver- end the BIH will create well-funded and internation- allyscientific competitive strategy BIH and Chair excellence positions. of theAdditionally, BIH. To thisthe 2014 gestartet. BIH Twinning Research Grants unter- institute will provide supporting funding for appoint- stützenbundprojekte. kleinere Die Kollaborationen. erste Förderrunde ist im Frühjahr

288 MDC Research Report 2014 ANNEX

ments at Charité or MDC with particular relevance to Entscheidend für die inhaltliche Ausrichtung und Ex- the BIH research program thus attracting top scientists zellenz des BIH wird die strategische Rekrutierung to Charité and MDC. ergänzender Expertise sein, etwa in den Gebieten Stammzellbiologie und Bioinformatik. Das BIH schafft Access to state-of-the-art technology platforms and in- dafür kompetitiv ausgestattete BIH Chair-Positionen. frastructures build the foundation for internationally Um die besten Köpfe zu gewinnen, bietet das BIH at- competitive research at the BIH. This includes service traktive Zusatzausstattungen für an beiden Partnerein- for omics technologies with a common service platform richtungen vorgesehene Berufungen mit besonderer mainly set up at the MDC site. Bioinformatics expertise Relevanz für das BIH. for systemic analysis of molecular and clinical data will be consolidated in the BIH Center of Bioinformatics and Der Zugang zu modernsten Infrastruktureinrichtungen supported by extension of IT infrastructures. Joint bio- und Technologie-Plattformen bildet die Basis für eine bank structures will provide access to patient samples international kompetitive Forschung am BIH. Dazu and imaging capacities for clinical phenotyping will be gehören die Omics-Technologien, die als zentrale Platt- expanded. Measures for advancing stem cell biology form vor allem am Standort MDC angesiedelt sein wer- in the BIH will include the set-up of a service unit for den. Die bioinformatische Expertise zur Auswertung induced pluripotent stem cells. In addition, a unit for der systemmedizinischen und klinischen Daten wird transgenic techniques will be established. Cooperation im BIH Center of Bioinformatics vereint und durch IT- with the FMP secures access to state-of-the-art tech- Infrastrukturmaßnahmen unterstützt. Gemeinsame nologies for high-throughput screening of small mol- Biobanken ermöglichen den Zugang zu Patientenpro- ecule libraries. ben. Für die klinische Phänotypisierung werden die Imaging-Plattformen ausgebaut. Ein weiteres Stand- To create an excellent framework for patient-oriented bein wird der Ausbau der Stammzellbiologie inklusi- research and clinical studies, Clinical Research Units ve einer Service-Einheit für induzierte pluripotente (CRU) will be established. As units independent of Stammzellen sein. Zudem wird eine Einheit für trans- regular patient care they offer top infrastructure for in- gene Techniken etabliert. Über eine Kooperation mit vestigator-initiated trials with patients and probands. dem FMP wird der Zugang zu neuesten Techniken im Following the example of the ECRC at the campus Buch, Wirkstoff-Screening sichergestellt. which already comprises a functional CRU, the BIH will create further CRU at other Charité sites. Um am BIH hervorragende Rahmenbedingungen für die patientenorientierte Forschung und für klinische Educational activities of the BIH include stipends for Studien zu schaffen, werden Clinical Research Units (CRU) etabliert: Das sind vom regulären ambulanten Clinical Scientists and funding opportunities for doc- und stationären Klinikbereich getrennte Einheiten, in toralmedical and students, postdoctoral programs students for with the qualificationa special inter of- denen Patienten und Probanden im Rahmen von wis- est in clinical research. The different educational pro- senschaftsinitiierten Studien außerhalb der Regelver- grams will be consolidated under the umbrella of the sorgung behandelt werden können. Nach Vorbild des BIH Academy of Biomedical Education and Training. The ECRC am Campus Buch, das bereits eine funktionsfähi- BIH Academy aims at interlinking career support and ge CRU umfasst, werden im BIH weitere CRU-Struktu- training in systems and translational research. It will ren an Standorten der Charité aufgebaut. coordinate and establish training opportunities for undergraduate students, doctoral students, postdoctoral Zu den Ausbildungsaktivitäten des BIH gehören Sti- fellows and clinical researchers. pendien für Studierende der Medizin, Programme für die Ausbildung von Clinical Scientists, und die Förde- Thus, the BIH will train a new generation of “transla- rung von Doktoranden und Postdoktoranden mit ei- tional” scientists that bridge the gap between clinicals nem besonderen Interesse an klinischer Forschung. and experimental research. Die Ausbildungsprogramme werden unter dem Dach der BIH Academy of Biomedical Education and Training vereint. Ziele der BIH Academy sind eine am gemeinsa- men Forschungsparadigma orientierte Vernetzung der Nachwuchsförderung und die Schaffung und Koordina- tion entsprechender Angebote für Studierende, Dokto- randen, Postdoktoranden und forschende Kliniker.

Damit wird das BIH eine neue Generation von „translationalen“ Wissenschaftlern ausbilden, die die Brücke zwischen Klinik und experimenteller Forschung bilden.

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ANNEX

German Centre for Cardiovascular Research Deutsches Zentrum für Herz-Kreislauf-Forschung

he Deutsches Zentrum für Herz-Kreislauf-Forsc- as Deutsche Zentrum für Herz-Kreislauf-For- hung (DZHK – German Centre for Cardiovascular schung (DZHK) ist eines von sechs Deutschen TResearch) is one of six German health research DZentren der Gesundheitsforschung und wurde centers and was established in 2011 by the German 2011 vom Bundesministerium für Bildung und For- Federal Ministry of Education and Research. The DZHK schung ins Leben gerufen. Das DZHK ist angetreten, has stepped up to bring together in a network cardio- Herz-Kreislauf-Forscher aus der Grundlagen und der - klinischen Forschung deutschlandweit zu vernetzen cal research from all over Germany in order to create und somit bessere Voraussetzungen für die Translation improvedvascular researchers conditions forfrom the the translation fields of basic of research and clini re- von Forschungsergebnissen in die klinische Praxis zu sults into clinical application practice. schaffen.

The MDC is one of 27 member institutions of the DZHK Das MDC ist eine von 27 Mitgliedseinrichtungen im and provides 9 principal investigators (PIs). Together DZHK und stellt 9 Principle Investigators (PIs). Es bil- with the Charité – Universitätsmedizin Berlin, the det mit der Charité – Universitätsmedizin Berlin, dem Deutsches Herzzentrum Berlin (DHZB, German Heart Deutschen Herzzentrum Berlin und dem Robert-Koch- Institute Berlin) and the Robert Koch Institute, it con- Institut den DZHK-Standort „Berlin“, einen von sieben stitutes the DZHK partner site “Berlin”, one of seven Standorten. such partner sites. Die MDC-PIs sind im Rahmen des DZHK an größe- In the context of the DZHK, the MDC PIs are involved - gen, erbliche und entzündliche Herzerkrankungen, Herzschwäche,ren Standortprojekten Prävention im und Bereich Bildgebung Gefäßerkrankun beteiligt. in larger site projects in the fields of vascular diseases, Darüber forschen sie gemeinsam mit anderen DZHK- conducthereditary research and inflammatory together with cardiac other DZHKdiseases, partners cardiac in Partnern in standortübergreifenden kooperativen Pro- insufficiency, prevention and imaging. In addition, they

Withincross-site the cooperative DZHK, MDC projects. PIs and other MDC scientists MDC-PIsjekten. und andere MDC-Wissenschaftler bieten in- offer their know-how and expertise in the form of so- nerhalb des DZHK ihr Knowhow und ihre Expertise called “shared expertise”. By means of this shared ex- als sogenannte „Shared Expertise“ an. Mit den Shared pertise, the DZHK promotes synergies and collabora- Expertise fördert das DZHK Synergien und die Zu- tion between the respective sites. The two most used sammenarbeit der Standorte. Die beiden im DZHK am - times) are “Generation and cardiovascular phenotyp- rierung und kardiovaskuläre Phänotypisierung trans- ingshared of transgenic expertise rats” fields and at “Genomics/Proteomics” the DZHK (each used nine and genermeisten Ratten“ genutzten und „Genomics/Proteomics“ Shared Expertise (je 9 mal)werden „Gene von are offered by the MDC PIs Prof. Michael Bader and MDC-PIs Prof. Michael Bader und Prof. Norbert Hübner Prof. Norbert Hübner. angeboten.

MDC Director Prof. Walter Rosenthal is a member of MDC-Vorstand Prof. Walter Rosenthal ist Vorstands- the executive board of the DZHK. The funding manage- mitglied des DZHK. Das Fördermittelmanagement für

at the MDC. am MDC abgewickelt. ment for the DZHK is handled by a team of five based das DZHK wird von einem fünfköpfigen Team mit Sitz

290 MDC Research Report 2014 ANNEX

German Centre for EU-Life Cardiovascular Research Deutsches Zentrum für Herz-Kreislauf-Forschung

esearch and innovation sit high on the agenda of orschung und Innovation haben einen hohen Stel- the European Union’s strategy for the next couple lenwert in der Strategie der Europäischen Union, Rof years. The fragmentation of the research policy Fund das wird auch in den kommenden Jahren so landscape, as well as the constant need to promote sci- sein. Zugleich ist die forschungspolitische Landschaft Europas fragmentiert. Vor diesem Hintergrund und mit of a pan-European alliance of research institutes. Thir- der Aufgabe vor Augen, wissenschaftliche Exzellenz in teenentific top excellence research ininstitutions Europe, has (including sparked manythe creation of the Europa voranzubringen, haben 13 der renommiertes- most prestigious) from thirteen European countries ten Forschungseinrichtungen aus 13 Ländern eine eu- ropäische Allianz zur Förderung von Spitzenforschung gegründet: EU-Life. have joined forces and created EU-Life with the aim of Thepromoting goals of scientific this alliance excellence. are many and multi-faceted. Die Ziele dieser Allianz sind vielfältig. Ein Hauptau- Primarily it concerns itself with sharing best practices genmerk liegt auf dem Austausch von „Best Practice“- on virtually every topic of interest to the thirteen par- Beispielen zu buchstäblich allen Fragen, die für die Mit- gliedseinrichtungen von Belang sind. Dieser Austausch umfasst die Bereiche Forschungsstrategie, Forschungs- andticipating many institutes.other areas These of interest. exchanges Sharing span strategies the fields förderung und Kommunikation sowie viele weitere The- andof scientific policies strategy,among the research most successful funding, communicationEuropean insti- men. Eine solche Zusammenarbeit erfolgreicher lebens- tutes provides a formidable advantage not only for the wissenschaftlicher Institute bietet nicht nur dem MDC MDC but also for the entire European Research area. Vorteile, sondern dient dem gesamten europäischen It will lead to a better understanding of the challenges Forschungsraum. Sie führt zu einem besseren Verständ- European life science institutes are currently facing, as nis der aktuellen Herausforderungen, vor die europäi- well as to better positioning and representing Europe- sche Life Science-Zentren heute gestellt sind. Zudem sollen europäische Interessen im Bereich der Wissenschaft besser positioniert und präsentiert werden. Thean interests main goal in scientificof the alliance domains. is to promote and con- - - cellence including cutting-edge technologies, research elemente zu fördern und zu festigen, die wissenschaft- andsolidate infrastructures, the core elements internationality, that constitute sound scientific adminis ex- licheDas wesentliche Exzellenz ausmachen. Ziel der Allianz Dazu ist,gehören diejenigen innovative Kern tration and ethics, and social responsibility. In order Spitzentechnologien, gute Forschungsinfrastrukturen, to establish stronger connections and collaborations Internationalität, eine solide Verwaltung sowie ethi- among researchers in its member institutions, EU-Life sche Grundsätze und soziale Verantwortung. Um einen - stärkeren Austausch und Kooperationen zwischen den sic and Translational aspects of Cancer Biology, sched- Forschern der verschiedenen Mitgliedseinrichtungen uledis planning to take its place first in scientific 2014. meeting on the topic of Ba zu etablieren, plant EU-Life sein erstes wissenschaftliches Symposium zu den Grundlagen und translationa- All these actions are creating a new powerful bloc that len Aspekten der Tumorbiologie. Das Treffen ist für das - Jahr 2014 geplant. lence and integrity in Europe at a variety of levels: fromcan advocate concrete for operations the advancement at the levels of scientificof institutes excel to All diese Aktivitäten bilden eine neue kraftvolle Bewe- the highest realms of policy and strategy at the EU. The gung, die für den Fortschritt wissenschaftlicher Exzel- MDC is thrilled to contribute its experience and vision lenz und Integrität in Europa auf verschiedenen Ebenen - eintreten kann: das reicht von konkreten Maßnahmen tutes in helping pave the way for a bright future of fun- auf Seiten der Institute bis hin zu den höchsten Ebenen damentalin EU-Life research and will in join Europe. the other participating insti der EU-Politik. Das MDC freut sich sehr, seine Erfah- rungen und Vorstellungen in EU-Life miteinbringen zu können und möchte gemeinsam mit den anderen teil- nehmenden Instituten den Weg bahnen für eine erfolgreiche Zukunft der Grundlagenforschung in Europa.

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Biotechnology Park with Innovation and Founding Center

Biotechnologiepark mit Innovations- und Gründerzentrum

asic research and clinical research on the campus rundlagenforschung und klinische Forschung as well as at neighboring hospitals provide a stimu- auf dem Campus sowie benachbarte Kliniken Blating environment for the development of the Bio- Gbilden ein stimulierendes Umfeld für die Ent- techPark with its Innovation and Founding Center (IGZ). wicklung des BiotechParks mit seinem Innovations- und Gründerzentrum (IGZ). The BiotechPark Berlin-Buch is one of the largest biotechnology parks in Germany. On about 31,000 square Der BiotechPark Berlin-Buch ist einer der größten Biotechparks in Deutschland. Auf rund 31.000 Quad-

companymeters, attractive founders and industry-specific enterprises. Start-ups laboratories can grow and - atoffices the Innovation with state-of-the-art and Founding equipment Center; additionalare available sites to dernsterratmetern Ausstattung. finden Gründer Start-ups und Unternehmen können im günstigeInnova- for building on the campus open up options for devel- tions-branchenspezifische und Gründerzentrum Labor- und wachsen, Büroflächen Baufelder mit modes opment. The operating company BBB Management Campus eröffnen Entwicklungsoptionen. Die Betrei- GmbH Campus Berlin-Buch supports the enterprises in bergesellschaft, BBB Management GmbH Campus Ber- all issues and provides a comprehensive service. lin-Buch, unterstützt die Unternehmen in allen Belan- gen und bietet einen umfassenden Service. Currently, 56 small and medium-sized enterprises conduct research and development at the BiotechPark. Im BiotechPark forschen und entwickeln gegenwärtig 56 kleine und mittelständische Unternehmen. 41 da- - von sind im Bereich der Biomedizin tätig. Im Spektrum calOf these,devices, 41 molecular are active biologyin the field diagnostic of biomedicine. products Theand treatments,spectrum of pre-clinical their fields pharmacological of business comprises and pharma medi- Produkte, molekularbiologische Diagnostika und The- cogenomic tests, RNA technologies, search for pharma- rapien,ihrer Geschäftsfelder präklinische pharmakologische finden sich medizintechnische und pharmacologically relevant target molecules, production and ko-genomische Tests, RNA-Technologien, Suche nach assessment of pharmaceuticals as well as analysis and pharmarelevanten Zielmolekülen, Herstellung und synthesis of biomolecules. Prüfung von Arzneimitteln sowie Analytik und Synthe- se von Biomolekülen. Since 1998, the number of employees working for the biotechnology enterprises has tripled. Currently (sta- Seit 1998 hat sich die Mitarbeiterzahl der Biotech- tus 2014), the companies employ about 775 members Unternehmen verdreifacht. Die Firmen beschäftigen of staff. The companies achieve a total annual turn- derzeit (Stand 2014) etwa 775 Mitarbeiter. Die Unter- over of 140 million Euro. Their capital stock includes approximately 150 patents, which often derive from Euro. Zum Kapitalstock zählen ca. 150 Patente, die oft - ausnehmen Kooperationen erzielen jährliche mit Arbeitsgruppen Umsätze von 140in Forschung Millionen search and clinic applications. und Klinik stammen. cooperations with working groups in the fields of re In favor of a sustainable campus development, the BBB Zugunsten einer nachhaltigen Campusentwicklung hat has once again actively and intensively promoted the des Stadtteils Berlin-Buch zu einer Gesundheitsregion health region and to this end has attracted funding to- eingebrachtsich die BBB erneutund dafür intensiv Fördermittel in die weitere in Höhe Profilierung von ca. talingfurther about profiling 500,000 of the Euro city for district the years Berlin-Buch 2011 to 2014. as a 500.000 Euro für die Jahre 2011 bis 2014 eingewor- Together with the Buch-based industry stakeholders ben. Gemeinsam mit den Bucher Wirtschaftakteuren - - ing, an umbrella brand has been developed for the enting eine Dachmarke für den gesamten Gesundheits- tireand healthin the locationcontext of Buchthe part-project and numerous for communicasite market- standortwurden imBuch Rahmen entwickelt des Teilprojekts und zahlreiche Standortmarke Kommuni- tion and marketing measures have been implemented, kations- und Werbemaßnahmen realisiert, wie z.B. ein such as a new web portal, an image brochure, a regu- neues Webportal, eine Imagebroschüre, ein regelmäßig larly published site magazine, advertising campaigns erscheinendes Standortmagazin, Anzeigenkampagnen and a signposting system for Berlin-Buch. und ein Beschilderungssystem für Berlin-Buch.

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Biotechnology Park with Innovation - and Founding Center for select areas on the health location Berlin-Buch as well wicklung eines Nutzungskonzepts für ausgewählte asThe recommendations part-project “Development for a marketing of a utilization strategy and concept con- FlächenErfolgreich am abgeschlossen Gesundheitsstandort wurde das Berlin-Buch Teilprojekt sowie „Ent tinuation of the master plan for Berlin-Buch” has been Empfehlungen für eine Vermarktungsstrategie und ein successfully completed. As a result, detailed utilization Fortschreiben des Masterplans für Berlin-Buch“. Im Biotechnologiepark mit Innovations- und Gründerzentrum - Ergebnis wurden detaillierte Nutzungskonzepte mit keting recommendations for numerous areas and build- Wirtschaftlichkeitsberechnungen und Vermarktungs- ingsconcepts in Berlin-Buch including calculationshave been developed of profitability for companies and mar empfehlungen für zahlreiche Areale und Gebäude in at the BiotechPark that are interested in establishing Berlin-Buch für ansiedlungsinteressierte und wachs- themselves and that are experiencing strong growth. tumsstarke Unternehmen des BiotechParks entwickelt.

At present, the BBB Management GmbH Campus Berlin- Die BBB Management GmbH Campus Berlin-Buch be- Buch employs 34 members of staff. It was established schäftigt derzeit 34 Mitarbeiter und wurde 1995 aus in 1995 as a spin-off company of the Max Delbrück dem Max-Delbrück-Centrum für Molekulare Medizin Center for Molecular Medicine (MDC) Berlin-Buch. Co- (MDC) Berlin-Buch ausgegründet. Mitgesellschafter partners are the Leibniz-Institut für Molekulare Phar- sind das Leibniz-Institut für Molekulare Pharmakolo- makologie (FMP) and the Bayer HealthCare Pharma- gie (FMP) sowie die Bayer HealthCare Pharmaceuticals, ceuticals, Bayer Pharma AG. Economically independent Bayer Pharma AG. Wirtschaftlich selbstständig, nicht Gewinn orientiert und mit öffentlichem Mandat ist die BBB is a neutral and intermediary partner. It manages BBB ein neutraler und intermediärer Partner. Sie be- theand interfacesa non-profit between company research, with a publicclinical mandate, application, the dient die Schnittstellen zu Forschung, Klinik, Business, business, administration, politics and the public and is Verwaltung, Politik und Öffentlichkeit und engagiert active in competence, support and service networks on sich in Kompetenz-, Unterstützungs- und Servicenetz- both a regional and international level. werken auf regionaler und internationaler Ebene.

The Gläsernes Labor Das Gläserne Labor

In 2013, in its capacity as the best-known non-school Das von der BBB Management GmbH betriebene Gläser- learning site in Berlin, the Life Science Learning Lab ne Labor hat als bekanntester außerschulischer Lernort “Gläsernes Labor”, operated by the BBB Management Berlins im Jahr 2013 ca. 36.500 Kindern, Schülern, Lehr- GmbH, offered some 36,500 children, school students, kräften und einem breiten Laienpublikum anspruchs- teachers and a broad swathe of the general public de- volle Labor- und Mitmachkurse zu den Themen Genetik, Neurobiologie, Zellbiologie, Ökologie, Radioaktivität und of genetics, neurobiology, cell biology, ecology, radio- Chemie angeboten. Davon besuchten 12.500 Schüler die activitymanding and laboratory chemistry. and Of join-in this number, courses 12,500 on the schooltopics Laborkurse auf dem Campus. Fast 24.000 Kita-Kinder students attended the laboratory courses on the cam- und Schulkinder besuchten Experimentierkurse des

MDC Research Report 2014 293 Guido Rottmann/MDC Photo: ANNEX Photo: Werner Huthmacher/MDC Werner Photo:

pus. Almost 24,000 day care center and school children Forschergartens in Berliner Kitas, Grundschulen sowie attended experimentation courses of the “Forscher- im Gläsernen Labor. Das Gläserne Labor beteiligte sich garten” hosted at Berlin-based day care centers, pri- mit Experimentierangeboten an zahlreichen Veranstal- mary schools as well as at the Gläsernes Labor. The tungen, wie dem Tag der offenen Tür der Bundesregie- Gläsernes Labor contributed experimentation attrac- rung, den TSB-Schülertagen sowie dem Sommerfest der tions to numerous events, such as the federal govern- HOWOGE, und erreichte so eine breite Öffentlichkeit. ment’s open house day, the TSB Schülertage student days and the summer party of the HOWOGE building 2013 hat das Gläserne Labor den Weiterbildungsbe- society, and thereby achieved widespread publicity. reich für technische Angestellte und Wissenschaftler umfangreich ausgebaut. Neben Kursen für MDC-Mit- In 2013, the Gläsernes Labor comprehensively expand- arbeiter wurde dies durch Kooperationen mit Weiter- ed its continuing education provision for technicians and scientists. In addition to courses for MDC employees, this was achieved by cooperations with continu- Gläsernebildern wie Labor C&Q erfolgreich Haberhauffe, einen Prometheus eintägigen undKongress dem fürLette-Verein technische erreicht. Angestellte Im Frühjahrdurch. Im 2013November führte 2013 das Prometheus and the Lette-Verein. In spring 2013, the wurde für den Weiterbildungsbereich in einem Labor- Gläsernesing education Labor providers successfully such organized as C&Q a one-day Haberhauffe, con- gebäude des BiotechParks zusätzlich ein viertes Labor ference for technicians. In November 2013, an addi- in Betrieb genommen. tional fourth laboratory was taken into operation for the continuing education sector, housed in a laboratory Das MDC hat diese Aktivitäten maßgeblich unterstützt, building of the BiotechPark. z.B. durch die Finanzierung eines Schülerlabors sowie von Angeboten zur naturwissenschaftlichen Ferienbe- The MDC has substantially supported these activities, treuung für Mitarbeiterkinder zur Verbesserung der for example, by providing funding for a student labora- Vereinbarkeit von Beruf und Familie. tory as well as for attractions for the natural science holiday camps for children of employees for improving Auf Grundlage einer langfristigen Vereinbarung mit the compatibility of family and work. den Campus-Einrichtungen zur Öffentlichkeitsarbeit des Campus betreute das Gläserne Labor im Jahre In 2013, on the basis of a long-term agreement with the 2013 über 20 Besuchergruppen mit Vertretern aus campus institutions regarding the public relations of Wissenschaft, Wirtschaft, Politik und Medien. Dafür or- the campus, the Gläsernes Labor supervised more than ganisierte und begleitete es entsprechende Veranstal- 20 visitor groups of representatives from the worlds of tungsprogramme. Das Gläserne Labor unterstützte das

294 MDC Research Report 2014 ANNEX Photo: David Ausserhofer/MDC Photo:

science, industry, politics and the media. To this end, Max-Delbrück-Centrum und das Naturkundemuseum it organized and supervised corresponding event pro- auch bei der Organisation und Durchführung des Kon- grams. The Gläsernes Labor also supported the Max gresses „60 Jahre DNA“ für Lehrer. Die Campus-Öffent- Delbrück Center and the Natural Science Museum in lichkeitsarbeit war verantwortlich für die Organisation organizing and carrying out the “60 Years DNA” confer- und erfolgreiche Durchführung von Veranstaltungen

was responsible for the organization and successful re- und der äußerst erfolgreichen und besucherstarken alizationence for of teachers. events such The as campus the New public Year’s relations Eve reception office „Langenwie dem NeujahrsempfangNacht der Wissenschaften.“ der Campus-Einrichtungen Ein Veranstal- of the campus institutions and the extremely successful tungshöhepunkt war die Durchführung des 19. Pan- and much attended “Long Night of Sciences”. One event kower Wirtschaftstags in Zusammenarbeit mit dem highlight was the realization of the 19th Pankower Bezirksamt Pankow und unter Einbeziehung von Part- Wirtschaftstag (Pankow Economy Day) in cooperation nern aus dem Bezirk. with the Pankow district authority and including the involvement of partners from the urban district. 2012 wurde die Mitarbeiter- und Imagezeitschrift - In 2012, the employee and image magazine “Campus- terentwickelt. Diese Publikation bezieht neben den Campusthemen„CampusNews“ zumauch Standortjournal Themen von Bucher „buchinside“ Partnern wei ein, “buchinside”. In addition to campus-related topics, this die die Entwicklung des gesamten Gesundheitsstand- publicationNews” was further includes developed also matters to become relating the to sitepartners journal in orts betreffen. 2013 erschienen drei Ausgaben. Darü- Buch that concern the development of the entire health ber hinaus bringt sich die BBB aktiv in die Aktivitäten location. In 2013, three issues were published. More- und Kommunikationsplattformen des Berlin-Branden- over, the BBB actively contributes to the activities and burger Technologie-Clusters sowie des Netzwerkes communication platforms of the Berlin-Brandenburg von Berlin Partner ein. technology cluster and the Berlin Partner network.

MDC Research Report 2014 295

ANNEX

Health Care Industry Cluster

Cluster Gesundheitswirtschaft

he life sciences and health care industry play a lead- ie Lebenswissenschaften und die Gesundheits- ing role in the Berlin-Brandenburg region. More wirtschaft spielen in der Region Berlin-Branden- T Dburg eine führende Rolle. Mehr als 300.000 Men- health care; the industry sector achieves an annual turn- schen arbeiten im Bereich der Gesundheitswirtschaft; over ofthan about 300,000 16 billion people Euro. work The respective in the wider federal field state of die Branche macht einen Jahresumsatz von rund 16 Milliarden Euro. Die Landesregierungen von Berlin und Brandenburg haben die Bedeutung der „Gesundheits- andgovernments approved of a Berlinmaster and plan Brandenburg in October 2007. have identifiedThis plan region“ sehr früh erkannt und im Oktober 2007 einen wasthe significance evaluated and of the reviewed “health in care 2012. region” The verymanagement early on Masterplan verabschiedet. Er wurde 2012 evaluiert und board of the “Health Care Industry Cluster” is responsible überarbeitet. Mit der Umsetzung des Masterplanes ist for implementing this master plan. The aim of the clus- das Management des „Clusters Gesundheitswirtschaft“ ter and its stakeholders is to develop the Berlin-Bran- betraut. Ziel des Clusters und seiner Akteure ist es, die denburg region to become the leading site for managed Region Berlin-Brandenburg zum führenden deutschen health care in Germany. In late 2013, Walter Rosenthal, Gesundheitsstandort zu entwickeln. Sprecher des Clus- ters ist seit Ende 2013 der wissenschaftliche Direktor the MDC, was appointed spokesman for the cluster. und Stiftungsvorstand des MDC, Walter Rosenthal. Scientific Director and Chair of the Board of Directors of For the MDC in its capacity as a center for basic research, Für das MDC als Zentrum der Grundlagenforschung the fast transfer of knowledge from the world of science ist ein schneller Transfer von der Wissenschaft in die to clinical and industrial applications is of great impor- Klinik und die Wirtschaft wichtig. Dabei geht es um tance. This comprises far more than merely the devel- weit mehr als nur die Entwicklung neuer Wirkstoffe opment of new active agents and the establishment oder die Gründung von Spin-offs. Es geht um neue Be- of spin-off enterprises. This is about new treatment handlungsstrategien, andere Diagnose-Werkzeuge und strategies, different tools for diagnosis and prevention Prävention von Krankheiten bis hin zur Rehabilitation. of diseases right up to medical rehabilitation. The MDC Das MDC und seit 2013 das Berliner Institut für Ge- and, since 2013, the Berlin Institute of Health (BIH – a sundheitsforschung/Berlin Institute of Health sind an cooperation of the Charité – Universitätsmedizin Berlin einem solchen Transfer im Sinne „translationaler Me- with the MDC) are both interested in such a transfer in dizin“ interessiert. the sense of providing “translational medicine”.

Einige Zahlen zur Gesundheitsregion Berlin-Brandenburg • 19 Berlin-based and 6 Brandenburg-based universi- • 19 Berliner und 6 Brandenburger Hochschulen mit Someties figures with some for the 170 Berlin-Brandenburg health-related study health courses; care region: rund 170 gesundheitsbezogenen Studiengängen • more than a dozen extramural research institutions • mehr als ein Dutzend außeruniversitäre For- focusing on the life sciences; schungseinrichtungen mit Schwerpunkt in den • approximately 30 pharmaceutical companies with a Lebenswissenschaften total of about 10,000 employees; • ca. 30 Pharmaunternehmen mit rund 10.000 Be- • about 280 medical engineering companies with a schäftigten total of more than 11,600 employees; • etwa 280 Medizintechnikunternehmen mit mehr als • about 220 biotechnology companies with a total of 11.600 Beschäftigten about 4,300 employees; • etwa 220 Biotech-Unternehmen mit ca. 4.300 Be- • approximately 5,900 health care industry enterprises; schäftigten • 132 hospitals with a total of more than 35,000 beds; • ca. 5.900 Unternehmen der Gesundheitswirtschaft • the largest university hospital in Europe: the Charité • 132 Kliniken mit mehr als 35.000 Betten – Universitätsmedizin Berlin; • größte Uniklinik Europas: die Charité – Universitäts- • more than 3,000 clinical studies per year. medizin Berlin • mehr als 3.000 klinische Studien pro Jahr More information: www.healthcapital.de (available only in German language) Mehr: www.healthcapital.de

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Health Care Industry Cluster

Cluster Gesundheitswirtschaft Source / Quelle: „Life Sciences Brochure“, Berlin Partner für Wirtschaft und Technologie für Wirtschaft Partner Berlin Brochure“, Sciences „Life / Quelle: Source

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ANNEX

Press Department

Pressestelle

uring 2012/2013 the MDC Press Department ie MDC-Pressestelle informierte 2012/2013 in published 135 press releases in German and 135 Pressemitteilungen (Deutsch/Englisch) die DEnglish and informed the media about research DMedien über Forschungsergebnisse, Entwick- results, developments and events. These resulted in lungen und Ereignisse. Sie waren Anlass für rund 90 about 90 interview requests. MDC news were covered Interviewanfragen. Die Informationen der Pressestelle by newspapers, weeklies, magazines, radio and televi- fanden Widerhall in hunderten von Beiträgen von Zei- sion in hundreds of articles and reports both in Ger- tungen, Wochenzeitungen, Magazinen, Funk- und Fern- many and abroad. sehen im In- und Ausland.

Among them were the BBC World Service, La Chaîne Unter ihnen waren BBC World Service, La Chaîne Info Info (French TV group), La Pagina (El Salvador). El (französische TV-Gruppe), La Pagina (El Salvador). El Correo (Bilbao, Spain), El Heraldo, El Mexicano (Mex- Correo (Bilbao, Spanien), El Heraldo, El Mexicano (Me- ico), Le Monde (Paris, France), German public service xiko), Le Monde (Paris, Frankreich), öffentlich-recht- broadcasters such as ARD, 3Sat, Hessischer Rundfunk liche Rundfunkanstalten wie ARD, 3Sat, Hessischer (HR), Mitteldeutscher Rundfunk (MDR), Rundfunk- Rundfunk (HR), Mitteldeutscher Rundfunk (MDR), Berlin-Brandenburg (rbb), Deutschlandfunk (DLF), Rundfunk-Berlin-Brandenburg (rbb), Deutschlandfunk Deutschlandradio Wissen, Deutschlandradio Kultur, (DLF), Deutschlandradio Wissen, Deutschlandradio Westdeutscher Rundfunk (WDR5), private broadcast- Kultur, Westdeutscher Rundfunk (WDR5), Privatsen- ers such as N24, RTL and magazines like Focus, Spie- der wie N24 und RTL, Magazine wie Focus, Spiegel, gel, Stern (here all online), and newspapers such as Stern (alle online), Tageszeitungen darunter Frankfur- the Frankfurter Allgemeine Zeitung (FAZ), Der Stan- ter Allgemeine Zeitung (FAZ), Der Standard (Wien, Ös- dard (Vienna, Austria), Süddeutsche Zeitung, Die Welt, terreich), Süddeutsche Zeitung, Die Welt, sowie Deut- the German newswire service DPA as well as Die Zeit, - die Wissenschaftsmagazine Science und Nature. Im tioned period the Press Department published seven genanntensche Presse-Agentur Zeitraum (dpa),gab die Die Pressestelle Zeit, Laborjournal sieben Pres und- pressLaborjournal, reviews documenting Science and Nature.part of this In the coverage. above-men sespiegel heraus, in denen ein Teil der Medienbericht- erstattung dokumentiert ist. Three topics of particular importance in 2012 were Von besonderer Bedeutung 2012 waren der Leibniz- the founding of the German Center for Cardiovascular Researchthe Leibniz (DZHK) Award and for the Professor plans of the Nikolaus Federal Rajewsky, Ministry Deutschen Zentrums für Herz-Kreislaufforschung of Education and Research (BMBF) at that time on the (DZHK)Preis für und Prof. die Nikolaus Pläne des Rajewsky, Bundesforschungsministe die Eröffnung des- future cooperation of MDC and Charité – now Berlin In- riums (BMBF) über die künftige Kooperation von MDC stitute of Health (BIH), see below). Especially for the - latter two topics, the MDC Press Department organized forschung (BIG) (s.u.). Besonders zu den beiden zuletzt many interviews with the MDC director as well as with genanntenund Charité Themen – jetzt Berlinervermittelte Institut die MDC-Pressestelle für Gesundheits the designated BIH Chairman. Also notable was the 20th zahlreiche Interviews mit dem MDC-Vorstand sowie anniversary of the MDC. dem designierten Vorstand des Berliner Instituts für Gesundheitsforschung. Beachtet wurde auch das Broad media coverage was given to the MDC press - ers. Among these was the research of Professor Gary R. Auf20-jährige ein breites Bestehen Medienecho des MDC. stieß 2012 die MDC-Pres- Lewin,work about who showed scientific that publications a gene mutation of MDC leads research to the searbeit über wissenschaftliche Publikationen von - MDC-Forscherinnen und -Forschern. Darunter war ings published in PLOS Biology. Also of great interest eine in PLOS Biology veröffentlichte Arbeit von Prof. toimpairment the media ofwas two the senses research – touch of Marion and Bimmler, hearing, findwho Gary R. Lewin, der zeigte, dass eine Genmutation zum showed in a study published in PLOS ONE that autoan- Verlust sowohl des Gehör- als auch des Tastsinns führt. tibodies can damage blood vessels in the brain, an im- Großes Presseecho fand die Forschungsarbeit von Ma- portant factor in the development of Alzheimer’s dis- rion Bimmler, die in einer in PLOS ONE veröffentlichten

298 MDC Research Report 2014 ANNEX

Opening ceremony of the Berlin Institute of Health (BIH) on June 18, 2013 in Berlin-Mitte (from left) Eröffnungsfeier für das Berliner Institut für Gesundheits Press Department forschung (BIG) am 18. Juni 2013 in Berlin-Mitte (v. l.): Prof. Karl Max Einhäupl (CEO of the Charité/Vorstandsvorsit- zender der Charité), Dr. Georg Schütte (State Secretary of the Federal Ministry of Education and Research/Staatssekretär im Pressestelle Bundesforschungsministerium), Prof. Ernst Rietschel (Chair- man, BIH/BIG-Vorstandsvorsitzender), Prof. Johanna Wanka (Federal Minister of Education and Research/Bundesforschungs ministerin), Prof. Walter Rosenthal (Chairman of the Board and Scientific Director of the MDC/Vorstandsvorsitzender und wissenschaftlicher Stiftungsvorstand des MDC) and/und Prof. Annette Grüters-Kieslich (Dean of the Charité Medical Faculty/Dekanin der Medizinischen Fakultät der Charité). Photo: David Ausserhofer/BIG Photo:

Studie nachwies, dass Autoantikörper Blutgefäße im Kettenmann and colleagues of the Charité, published Gehirn schädigen können, ein wichtiger Faktor für die inease Nature and dementia.Medicine, Thealso findingsreceived ofmuch Professor media Helmut cover- Entstehung von Alzheimer und Demenz. Von großem age. They showed that neural precursor cells induce Interesse für die Medien war eine von Prof. Helmut cell death in certain brain tumors. Also well covered by Kettenmann und Kollegen von der Charité in Nature the media was a new approach to treat life-threatening Medicine publizierte Arbeit, in der er zeigte, wie neuro- acute liver failure, developed by Dr. Stefan Donath, a nale Stammzellen des Gehirns Hirntumoren zerstören. physician and researcher, published in Hepatology. Auch die Forschung von Dr. Stefan Donath über einen Therapieansatz gegen das lebensbedrohliche akute Le- The outstanding event in 2013 was the opening of the berversagen, publiziert in Hepatology, fand Beachtung. BIH, which received high media attention. In coordination with the (then designated) Chairman of the BIH, Herausragendes Ereignis 2013 war die Eröffnung des Berliner Instituts für Gesundheitsforschung/Berlin In- Corporate Communications Division of the Charité, the stitute of Health (BIG/BIH), über das die Medien groß MDCthe Office Press of Departmentthe Scientific carried Director out of the MDCentire and press the berichteten. In Abstimmung mit dem (damals desi- work for the opening of the BIH, wrote the invitations gnierten) Vorstandsvorsitzenden des BIG, dem Büro for the media, the press release and the background in- des wissenschaftlichen Vorstands des MDC und der formation, and arranged numerous interviews. Charité-Unternehmenskommunikation koordinierte die MDC-Pressestelle die gesamte Pressearbeit für die Well covered by the media in 2013 was the MDC press Eröffnung, verfasste Presseeinladung, Pressemittei- Nature, of Professor lung sowie Hintergrundinformationen und vermittelte Dominik Müller and colleagues from Yale, Harvard, and zahlreiche Interviews. workMIT in on the the USA, findings, who showed published that in increased dietary salt intake may drive the development of autoimmune diseases. Broad media coverage, especially in France, was Pressearbeit über die Forschung von Prof. Dominik also given to Dr. Michael Sieweke’s (Inserm and MDC) MüllerAuf große und MedienresonanzKollegen von Yale, fiel Harvard 2013 und auch MIT die in MDC- den Nature publication about the ability of blood stem cells USA, die in einer in Nature-Publikation zeigten, dass zu to respond to emergencies, and to Dr. James Poulet, viel Kochsalz die Entstehung von Autoimmunerkran- who received the Paul Ehrlich and Ludwig Darms- kungen fördern könnte. Auf ein breites Presseecho, vor taedter Prize for Young Researchers in St. Paul’s Church allem in Frankreich, stieß die Nature-Publikation von in Frankfurt am Main. Dr. Michael Sieweke (INSERM, MDC), der einen kör- pereigenen Notfallmechanismus für die Blutbildung Furthermore, the MDC Press Department was respon- bei Infektionen entdeckte. Breit berichtet wurde auch sible for the press work for the opening of the new über Dr. James Poulet, der in der Frankfurter Paulskir- laboratory building of the MDC, the Max Rubner House, che den Paul Ehrlich- and Ludwig Darmstaedter-Preis with an accompanying photo exhibition there. Togeth- für Nachwuchswissenschaftler erhielt. er with the Press Department of the German Cancer Research Center (DKFZ), the MDC Press Department Weiter machte die MDC-Pressestelle die Pressearbeit invited media representatives to a press conference in zur Eröffnung des Max-Rubner-Hauses des MDC mit Berlin on the occasion of the founding of the German einer begleitenden Fotoausstellung. Anlässlich der Stem Cell Network (GSCN) and moderated this confer- Gründung des deutschen Stammzellnetzwerks lud die ence together with a DKFZ colleague. MDC-Pressestelle gemeinsam mit der Pressestelle des Deutschen Krebsforschungszentrums (DKFZ) die Me- dien zu einer Pressekonferenz in Berlin ein, die sie gemeinsam mit einer DKFZ-Kollegin moderierte.

Press Department staff: Mitarbeiter der Pressestelle: Barbara Bachtler (head) and Barbara Bachtler (Leitung),

Ann-Kathrin Schöpflin (team assistant) Ann-Kathrin Schöpflin (Teamassistenz) MDC Research Report 2014 299

ANNEX

Communications Department

Kommunikationsabteilung

n 2012 and 2013, the communications department ie Kommunikationsabteilung des MDC organi- of the MDC organized about two dozen events. These sierte in den Jahren 2012 und 2013 rund zwei I DDutzend Veranstaltungen. Dazu zählten 13 wis- the Berlin Institute of Health (in cooperation with the senschaftliche Konferenzen, die Eröffnung des Berliner eventincluded management 13 scientific department conferences, of the Charité) the opening and the of Instituts für Gesundheitsforschung/Berlin Institute ceremony for celebrating the 20-year anniversary of of Health (in Kooperation mit dem Veranstaltungsma- the Max Delbrück Center with more than 600 guests. - The guests invited to the anniversary celebrations in- gen Bestehen des Max-Delbrück-Centrums mit mehr als cluded the then Federal Minister of Education and Re- 600nagement Gästen. der Unter Charité) den Gästen und der des Festakt Festaktes zum waren 20-jähri die search, Annette Schavan, the Berlin Science Senator, damalige Bundesforschungsministerin Annette Scha- Cornelia Yzer, as well as many members of the German van, die Berliner Forschungssenatorin Cornelia Yzer Bundestag and House of Representatives. In the years sowie viele Abgeordnete des Bundestags und des Ab- under review, the department also supervised visits from high-ranking politicians and guests of state, in- von der Abteilung betreute Besuche von hochrangigen cluding the President of the Republic of Singapore,Dr. Politikerngeordnetenhauses. und Staatsgästen, Hinzu kamen darunter in den der Berichtsjahren Staatspräsi- Tony Tan, and the Chinese Ambassador to Germany, dent von Singapur, Dr. Tony Tan, und der chinesische Shi Mingde, and it organized a parliamentary evening Botschafter in Deutschland, Shi Mingde, sowie ein as well as an open house day. An aspect of the depart- Parlamentarischer Abend und ein Tag der offenen Tür. ment’s work that holds extraordinary public appeal is Ein außerordentlich publikumswirksamer Aspekt der the organization of the annual Long Night of Sciences comprising guided tours of the laboratories, science Langen Nacht der Wissenschaften mit Laborführungen, slams and other events. Each year, these events are at- ScienceArbeit ist Slams die Organisation und weiteren der Aktionen. jährlich Zustattfindenden diesen Ver- tended by several thousands of people visiting the cam- - pus. The communications department organizes also the CareerDay, which takes place once a year. anstaltungen kommen jeweils mehrere Tausend Men- ganisiert.schen auf den Campus. Auch der CareerDay, der jährlich In early 2012, the MDC became the target of a cam- stattfindet, wird von der Kommunikationsabteilung or paign launched by protesters against animal experi- Das MDC wurde Anfang 2012 Ziel einer Kampagne ments. The trigger for this were the plans for a re- von Tierversuchsgegnern. Auslöser waren die Pläne placement building for two old animal houses, where für einen Ersatzbau von zwei alten Tierhäusern, in the keeping of animals and laboratories are to be dem Haltung und Labore nach modernsten Standards combined according to the latest standards (IPL – In kombiniert werden (In-vivo-Pathophysiologie-Labor, vivo Pathophysiology Laboratory). In consequence, the MDC further increased its already proactive bereits pro-aktive Kommunikation zu der Arbeit mit communication regarding the work with laboratory Versuchstieren.IPL). Daraufhin Die verstärkte Kommunikationsabteilung das MDC seine bis dahinstellt animals. The communications department provides umfangreiche Informationen zum Thema Tierversuche comprehensive online information on the topic of online zur Verfügung, sie organisiert Führungen dazu animal experimentation, it organizes guided tours bei den Langen Nächten der Wissenschaften und ko- on the topic during the Long Nights of Science and it ordiniert die mediale und politische Kommunikation coordinates the communication with the media and zum Thema Tierversuche. So gab es dazu zahlreiche politics regarding the topic of animal experimenta- Treffen mit Landes- und Bundespolitikern, einen Par- tion. To this end, for instance, numerous meetings lamentarischen Abend und Laborbesuche durch Medi- with federal state and federal government politicians en und Politik. Das Thema wird auch aktiv in Schulen have been organized as well as a parliamentary eve- getragen, wo Mitglieder der Kommunikationsabteilung ning and laboratory visits for media and political rep- und Wissenschaftler mit Schülerinnen und Schülern resentatives. The topic is also actively addressed at diskutieren. schools, where staff members of the communications department and scientists enter into discussion with Zu den Aufgaben der Abteilung gehören Print- und the students. Onlinepublikationen (Corporate Publishing) aller

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Communications Department

Kommunikationsabteilung Photo: Guido Rottmann/MDC Photo:

Networking at the MDC Careerday.

The department’s tasks include print and online publica- Art, darunter auch der vorliegende Research Re- tions (corporate publishing) of all kinds, including also port. Im Berichtszeitraum erschienen insgesamt 20 the research report at hand. During the period under Publikationen, vom Mitarbeitermagazin imdc über review, a total of 20 publications were published, rang- Familie) bis hin zu einer Fotobroschüre und einem thematic brochures, to a photo brochure and a 350-page populärwissenschaftlenFlyer und Themenbroschüren 350-Seiten-Buch (Ausbildung, über Beruf eine & ing from the employee magazine imdc, over leaflets and mehr als 20 Jahre währende Studie zu genetischen the genetic fundamentals of high blood pressure that Grundlagen von Bluthochdruck („The Case of the wentpopular on sciencefor more book than about 20 years a scientific (“The Case investigation of the Short- of Short-fingered Musketeer“). Die Abteilung Kommuni- kation koordinierte auch eine 44-seitige Sonderver- coordinated also a 44-page special issue of the “bild der öffentlichung von „bild der wissenschaft“ anlässlich wissenschaft”fingered Musketeer”). magazine The on communications occasion of the department MDC’s 20- year anniversary. In addition, it supervised the confer- Tagungsbände zu wissenschaftlichen Konferenzen wurdendes 20-jährigen betreut. Bestehens des MDC. Auch mehrere

Inence the proceedings context of ofa severalstrategic scientific realignment conferences. of the com- Im Zuge einer strategischen Neuausrichtung der Kom- munication strategy, the employee magazine imdc was munikation wurde Mitte 2013 das Mitarbeitermagazin discontinued by the middle of 2013 so as to instead imdc eingestellt und dafür eine verstärkte Online-Prä- place more emphasis on the online presence. Since May senz geschaffen. Das Intranet des MDC verfügt seit Mai 2013, the MDC’s intranet includes a news center and, since spring 2014, the online service includes MDC In- 2014 gibt es MDC Insights, das Nachrichtenzentrum sights, the MDC’s news center for external visitors to des2013 MDC über für ein externe Nachrichtenzentrum, Besucher unserer und Webseite. seit Frühjahr Auch our website. We offer also the service of an electronic einen elektronischen Newsletter bieten wir an. Das newsletter. The MDC maintains a presence on the most MDC unterhält Präsenzen auf den wichtigsten Social important social media platforms Facebook, Twitter, Media-Plattformen Facebook, Twitter, LinkedIn und LinkedIn and Xing and is particularly active on Face- Xing und engagiert sich insbesondere auf Facebook book and Twitter. und Twitter.

In its capacity as a publicly funded institution for ba- Als öffentlich geförderte Einrichtung der Grundlagen- sic research, the MDC is aware of its obligation to contribute to an improved understanding of science in the besseren Verständnis von Wissenschaft in der Öffent- public, in particular amongst school students. To this lichkeit,forschung insbesondere sieht sich das bei MDC Schülerinnen in der Pflicht, und Schülern,zu einem end, the MDC offers a continuing education program beizutragen. Hierzu bietet das MDC ein bundesweit for teachers that has received much notice throughout viel beachtetes Lehrerfortbildungsprogramm an: La- Germany: Labor trifft Lehrer (LTL – Laboratory Meets bor trifft Lehrer (LTL). 13 Lehrerfortbildungen, 7 Schü- Teacher). On occasion of the 60th anniversary of the lerworkshops und eine große Konferenz anlässlich des publication of the structure of DNA, the LTL and coop- 60. Jahrestages der Veröffentlichung der DNA-Struktur eration partners organized 13 continuing education wurden von LTL und Kooperationspartnern organi- courses for teachers, 7 school student workshops and a siert. large-scale conference. Um das Gemeinschaftsgefühl am MDC zu stärken To strengthen the community spirit at the MDC (com- (Community Building), engagiert sich die Kommu- munity building), the communications department is nikationsabteilung bei den wöchentlichen sozialen involved in the weekly social evenings (beer hour), or- Abenden (Beer Hour), mit einem Glühweinstand zur ganizes a glühwein stall during the Christmas season - - gruppe Beruf und Familie. Auch die GreenCampus- ily. The GreenCampus campaign for the economical use KampagneAdventszeit zum und sparsamendurch die MitarbeitUmgang mitin derRessourcen Projekt ofand resources cooperates likewise in the derives project fromgroup an Career initiative and of Fam the geht auf die Initiative der Kommunikationsabteilung communications department zurück.

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302 MDC Research Report 2014 ANNEX

MDC Research Report 2014 303

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Organizational Structure

Organisationsstruktur

he Max-Delbrück Center for Molecular Medicine as Max-Delbrück Centrum für Molekulare Medi- (MDC) Berlin-Buch is a foundation under public zin (MDC) Berlin-Buch ist eine Stiftung des öf- Tlaw of the State of Berlin with the purpose of pur- Dfentlichen Rechts des Landes Berlin. Ihr Zweck ist suing medical research at the molecular and cellular es, medizinische Forschung auf molekularer und zellu- levels and implementing its clinical and practical ap- lärer Ebene zu betreiben und ihre klinische Anwendung plication. und praktische Umsetzung zu voranzubringen.

Board of Trustees Kuratorium

The Board of Trustees is the supervisory body of the Das Kuratorium ist das Aufsichtsgremium des MDC. Es MDC and monitors the conduct of operations with re- überwacht die Rechtmäßigkeit, Zweckmäßigkeit und Wirtschaftlichkeit der Geschäftsführung. Es entschei- - det über die allgemeinen Forschungsziele und über spect to legality, appropriateness, economic efficiency - and financial viability. The Board decides on general re genheiten der Stiftung. search objectives as well as important research policy wichtige forschungspolitische und finanzielle Angele and financial matters of the Foundation Members of the Board of Trustees Mitglieder des Kuratoriums

Ministerial Director/ Ministerialdirektorin Prof. Dr. Magdalena Götz * Helmholtz Zentrum München Bärbel Brumme-Bothe (since 2010) Federal Ministry of Education and Research Prof. Dr. Roger Goody * (BMBF), Berlin (Chair) Max Planck Institute of Molecular Physiology, Bundesministerium für Bildung und Forschung Dortmund (BMBF), Berlin (Vorsitzende) Max Planck Institut für Molekulare Physiologie, Dortmund Dr. Jutta Koch-Unterseher Senate Administration for Education, Science and Ministerial Counselor Dr. Jan Grapentin Research, Berlin (Vice-Chair) (since August 2010) Senatsverwaltung für Wirtschaft, Technologie und Federal Ministry of Education and Research Forschung, Berlin (Stellv. Vorsitz) (BMBF), Berlin Bundesministerium für Bildung und Forschung Dirk Rothenpieler (BMBF), Berlin Senate Administration for Health and Social Affairs, Berlin Prof. Dr. Eckart Gundelfinger * Senatsverwaltung für Gesundheit und Leibniz-Institut for Neurobiology, Magdeburg Soziales, Berlin Leibniz-Institut für Neurobiologie, Magdeburg

Prof. Dr. Peter A. Frensch (since April 2011) Prof. Dr. Annette Grüters-Kieslich Vice-President for Research, Humboldt University, Dean, Charité – Universitätsmedizin Berlin Berlin Dekanin, Charité – Universitätsmedizin, Berlin Vizepräsident für Forschung, Humboldt-Universi- tät zu Berlin

* Also members of the Scientific Committee

304 MDC Research Report 2014 ANNEX

Prof. Dr. Matthias Hentze * Regierungsdirektorin Marianne Pyrczek EMBL Heidelberg Federal Ministry of Finance, Berlin Bundesministerium der Finanzen, Berlin Prof. Dr. Nancy Hynes * (since August 2010) Friedrich Miescher Institute for Biomedical Re- Prof. Dr. Monika Schäfer-Korting search, Basel Freie Universität Berlin Friedrich Miescher Institut für Biomedizinische Forschung, Basel Dr. Birgit Schnieders Federal Ministry of Health, Bonn Prof. Dr. Nine Knoers * Bundesministerium für Gesundheit, Bonn University Medical Center Utrecht Dr. Baris Tursun Prof. Dr. Thomas Lüscher * (since August 2010) MDC Berlin-Buch University Hospital Zurich, Head of Cardiology Universitätsspital Zürich, Klinik für Kardiologie, Dr. Jana Wolf Zürich MDC Berlin-Buch

Prof. Dr. Renato Paro * Center of Biosystems, Swiss Federal Institute of Technology Zurich, Basel

* Also members of the Scientific Committee

Scientific Wissenschaftlicher Committee Ausschuss

- Der Wissenschaftliche Ausschuss des Kuratoriums be- pares the decisions of the Board of Trustees in scien- reitet die Entscheidungen des Kuratoriums in wissen- The Scientific Committee of the Board of Trustees pre schaftlichen Fragen vor. Er trägt die Verantwortung für for the ongoing evaluation of the results of the re- die fortlaufende Ergebnisbewertung der Forschungsar- tific matters. The Scientific Committee is responsible beiten des MDC durch wissenschaftliche Begutachtung. Dem Wissenschaftlichen Ausschuss gehören neben den Trustees,search work up toof seventhe MDC external through specialists scientific sit assessment. on the Sci- wissenschaftlichen Mitgliedern des Kuratoriums bis zu Together with the scientific members of the Board of sieben externe Fachwissenschaftler an.

entific Committee.

Members of the Scientific Committee Mitglieder des Wissenschaftlichen Ausschusses

Prof. Dr. Matthias Hentze * Prof. Dr. Martin Eilers EMBL Heidelberg (Chair / Vorsitzender) University of Würzburg, Theodor Boveri Institute, Biocenter Prof. Dr. Renato Paro * Center of Biosystems, Swiss Federal Institute of Prof. Dr. Magdalena Götz * Technology Zurich, Basel (Vice-Chair)* Helmholtz Zentrum München Center of Biosystems ETH Zürich, Basel (Stellv. Vorsitzender) Prof. Dr. Roger Goody * Max Planck Institute of Molecular Physiology, Prof. Dr. Frederico Caligaris-Cappio Dortmund Max Planck Institut für Molekulare Physiologie, Department of OncoHematology Dortmund University Scientific Institute San Raffaele, Prof. Dr. Anna Dominiczak University of Glasgow, Cardiovascular Research Centre, Glasgow, United Kingdom * Also members of the Board of Trustees

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Prof. Dr. Eckart Gundelfinger * Prof. Dr. Kerstin Krieglstein (since April 2014) University of Freiburg, Institute of Anatomy and Leibniz-Institut for Neurobiology, Magdeburg Cell Biology Leibniz-Institut für Neurobiologie, Magdeburg Albert-Ludwigs-Universität Freiburg, Institut für Anatomie und Zellbiologie Prof. Dr. Nancy Hynes * Friedrich Miescher Institute for Biomedical Re- Prof. Dr. Thomas Lüscher * search, Basel University Hospital Zurich, Head of Cardiology Friedrich Miescher Institut für Biomedizinische Universitätsspital Zürich, Klinik für Kardiologie, Forschung, Basel Zürich

Prof. Dr. Elisa Izaurralde Prof. Dr. Frauke Melchior Max Planck Institute for Developmental Biology, Ruprecht-Karls-University Heidelberg Tübingen Max Planck Institut für Entwicklungsbiologie, Prof. Dr. Anna Tramontano Tübingen University of Rome “La Sapienza”

Prof. Dr. Nine Knoers * (since April 2014) University Medical Center Utrecht * Also members of the Board of Trustees

Executive Board Stiftungsvorstand

The Executive Board manages the Max Delbrück Cen- Der Stiftungsvorstand leitet das Institut und besteht aus einem wissenschaftlichen Mitglied, Prof. Dr. Wal- Rosenthal, and an administrative member, Dr. Heike ter Rosenthal, und einem administrativen Mitglied, Dr. Wolke.ter and Theconsists Chair of ofa scientific the Executive member, Board Prof. is Dr. Prof. Walter Dr. Heike Wolke. Vorsitzender des Stiftungsvorstandes ist Walter Rosenthal. Prof. Dr. Walter Rosenthal.

Scientific Council Wissenschaftlicher Rat

Der Wissenschaftliche Rat berät den Stiftungsvorstand in den Angelegenheiten von grundsätzlicher wissen- The Scientific Council advises the Executive Board in schaftlicher Bedeutung. matters of fundamental scientific importance.

Members of the Scientific Council Mitglieder des Wissenschaftlichen Rates

Prof. Dr. Gary Lewin Prof. Dr. Norbert Hübner (Chair / Vorsitzender) Dr. Zsuzsanna Izsvak Prof. Dr. Oliver Daumke Prof. Dr. Thomas Jentsch (Vice-Chair / Stellv. Vorsitzender) Dr. Stefan Kempa Prof. Dr. Michael Bader Prof. Dr. Helmut Kettenmann Prof. Dr. Walter Birchmeier Dr. Markus Landthaler Prof. Dr. Carmen Birchmeier-Kohler Prof. Dr. Ferdinand Le Noble Prof. Dr. Thomas Blankenstein Prof. Dr. Young-Ae Lee Dr. Marina Chekulaeva Prof. Dr. Achim Leutz Dr. Wei Chen PD Dr. Martin Lipp Prof. Dr. Bernd Doerken Dr. Alexander Loewer Dr. Iduna Fichtner Prof. Dr. Friedrich C. Luft Prof. Dr. Michael Gotthardt Dr. Henrike Maatz Prof. Dr. Udo Heinemann Prof. Dr. Jochen Meier Dr. Uta Hoepken Prof. Dr. Ingo Morano

306 MDC Research Report 2014 ANNEX

Dr. Thomas Mueller Dr. Ruth Schmidt-Ullrich Prof. Dr. Thoralf Niendorf Dr. Björn Christian Schroeder Prof. Dr. Uwe Ohler Prof. Dr. Matthias Selbach Dr. Daniela Panáková Dr. Michael Sieweke Prof. Dr. Tobias Pischon Prof. Dr. Thomas Sommer Prof. Dr. Ana Pombo Dr. Francesca Spagnoli Dr. James Poulet Prof. Dr. Ludwig Thierfelder Dr. Matthew Poy Prof. Dr. Mathias Treier Prof. Dr. Nikolaus Rajewsky Dr. Baris Tursun Prof. Dr. Klaus Rajewsky Prof. Dr. Erich Wanker Prof. Dr. Fritz Rathjen Dr. Christina Westphal Dr. Armin Rehm Prof. Dr. Thomas Willnow Dr. Oliver Rocks Dr. Jana Wolf Prof. Dr. Walter Rosenthal Dr. Susanne Wolf Prof. Dr. Claus Scheidereit Dr. Robert Patrick Zinzen

Staff Council Personalrat

The Staff Council is the collective representation of the Der Personalrat ist auf der Grundlage des Personal- employees of the MDC, based on the Staff Representa- vertretungsgesetzes des Landes Berlin die kollektive tion Act of Berlin. Interessenvertretung der Mitarbeiterinnen und Mitar- beiter des MDC. The Staff Council is involved in decisions regarding personnel and social matters and monitors the observance Der Personalrat ist an Entscheidungen zu personellen of labor rights, which are regulated in laws, collective und sozialen Belangen beteiligt und überwacht die Ein- agreements, internal agreements and administrative haltung von Arbeitnehmerrechten, die in Gesetzen, Ta- provisions. The Staff Council receives suggestions and rifverträgen, Dienstvereinbarungen und Verwaltungs- - vorschriften geregelt sind. Er nimmt Anregungen und er with the responsible departments or the Executive Beschwerden der Mitarbeiter entgegen und klärt diese Board.complaints of the employees and clarifies these togeth in Zusammenarbeit mit den zuständigen Abteilungen oder dem Vorstand. The Staff Council currently has 13 members from all employee groups at the MDC. Der Personalrat hat dreizehn Mitglieder aus allen Be- schäftigtengruppen des MDC.

Members of the Staff Council Mitglieder des Personalrates

Ingo Kahl (Chair / Vorsitzender) Siegne Knespel Jana Droese (Vice-Chair / Stellv. Vorsitzende) Dennis Kobelt Lutz Else (Vice-Chair / Stellv. Vorsitzende) Rainer Leben Dagmar Gerhard Alexander Loewer (Vice-Chair / Stellv. Vorsitzende) Dennis Siuchninski Manuela Adloff Corinna Volkwein Robby Fechner Brigitta Wedekind (Secretary / Sekretariat) Elke Guettler

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Representatives & Mediators Beauftragte

- Am MDC gibt es eine Anzahl von Beauftragten für be- budsmen for different topics, who are here to answer stimmte Fragestellungen und Themenbereiche. Sie un- questions,The MDC has provide various information, representatives, and to officers help with and prob om- den Mitarbeitern Informationen zu Ihrem Sachgebiet an. terstützen in Problem- oder Konfliktfällen und bieten lems or in cases of conflict. Animal Welfare Officer Tierschutzbeauftragte Anne Zintzsch Anne Zintzsch

Anti-Corruption Officer Antikorruptionsbeauftragte Ina Herrmann Ina Herrmann

Data Protection Officer Datenschutzbeauftragte Christine Rieffel-Braune Christine Rieffel-Braune

Disabilities Officer Schwerbehindertenbeauftragte Marina Winterfeld Marina Winterfeld

IT Security Officer IT-Sicherheitsbeauftragte Ute Wackernagel Ute Wackernagel

Research Ombudsman (Ombudsman for Ombudsmann für Fragen guter Good Scientific Practice) wissenschaftlicher Praxis Prof. Dr. Jens Reich Prof. Dr. Jens Reich

PhD Student Ombudspersons Vertrauenspersonen für Doktoranden Prof. Dr. Thomas Sommer Prof. Dr. Thomas Sommer Dr. Daniela Panáková Dr. Daniela Panáková

Women’s Representative Frauenvertreterin Dr. Christiane Nolte Dr. Christiane Nolte

Youth and Trainee Representatives Jugend-und Auszubildendenvertretung Lisa Huegel Lisa Huegel Lisa Mallis Lisa Mallis Vivien Rabke Vivien Rabke Martina Kneiseler Martina Kneiseler

308 MDC Research Report 2014 ANNEX

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Publications

MDC Publications 2012/2013 Total number of publications High Impact Papers (IF>=10) (articles and reviews)

2013 404 57*

2012 379 48

Source: MDC Repository; As of 31.01.2014 * IF as of 2012

Ten most cited MDC publications 2012 Rank ID (Web of Science) Document type Reference

Gherardi, E.*, Birchmeier, W.*, Birchmeier, C., and Woude, G.V. (2012). Targeting 1 WOS:000300261700009 Review MET in cancer: rationale and progress. Nat Rev Cancer 12, 89-103. *Joint first authors

Baltz, A.G.*, Munschauer, M.*, Schwanhaeusser, B., Vasile, A., Murakawa, Y., Schueler, M., Youngs, N., Penfold-Brown, D., Drew, K., Milek, M., Wyler, E., Bon- 2 WOS:000305095400016 Article neau, R., Selbach, M., Dieterich, C., and Landthaler, M. (2012). The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell 46, 674-690. *Joint first authors

Friedlaender, M.R.*, Mackowiak, S.D.*, Li, N., Chen, W., and Rajewsky, N. 3 WOS:000298733500013 Article (2012). miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res 40, 37-52. *Joint first authors

Nickolas, T.L., Schmidt-Ott, K.M., Canetta, P., Forster, C., Singer, E., Sise, M., Elger, A., Maarouf, O., Sola-Del Valle, D.A., O’Rourke, M., Sherman, E., Lee, P., Geara, A., Imus, P., Guddati, A., Polland, A., Rahman, W., Elitok, S., Malik, N., 4 WOS:000299115700008 Article Giglio, J., El-Sayegh, S., Devarajan, P., Hebbar, S., Saggi, S.J., Hahn, B., Kettritz, R., Luft, F.C., and Barasch, J. (2012). Diagnostic and prognostic stratification in the emergency department using urinary biomarkers of nephron damage a multicenter prospective cohort study. J Am Coll Cardiol 59, 246-255.

Bieschke, J.*, Herbst, M.*, Wiglenda, T., Friedrich, R.P., Boeddrich, A., Schiele, F., Kleckers, D., Lopez Del Amo, J.M., Gruening, B.A., Wang, Q., Schmidt, M.R., Lurz, R., Anwyl, R., Schnoegl, S., Faendrich, M., Frank, R.F., Reif, B., Guenther, S., 5 WOS:000298414100017 Article Walsh, D.M., and Wanker, E.E. (2012). Small-molecule conversion of toxic oligomers to nontoxic β-sheet-rich amyloid fibrils. Nat Chem Biol 8, 93-101. *Joint first authors

Poulet, J.F.A.*, Fernandez, L.M.J.*, Crochet, S.*, and Petersen, C.C.H. (2012). 6 WOS:000300793100009 Article Thalamic control of cortical states. Nat Neurosci 15, 370-372. *Joint first authors

Anders, G., Mackowiak, S.D., Jens, M., Maaskola, J., Kuntzagk, A., Rajewsky, 7 WOS:000298601300027 Article N., Landthaler, M., and Dieterich, C. (2012). doRiNA: a database of RNA interactions in post-transcriptional regulation. Nucleic Acids Res 40, D180-D186.

Sander, S., Calado, D.P., Srinivasan, L., Koechert, K., Zhang, B., Rosolowski, M., Rodig, S.J., Holzmann, K., Stilgenbauer, S., Siebert, R., Bullinger, L., and Rajew- 8 WOS:000307686000006 Article sky, K. (2012). Synergy between PI3K signaling and MYC in Burkitt lymphomagenesis. Cancer Cell 22, 167-179.

Zhelyazkova, P., Hammani, K., Rojas, M., Voelker, R., Vargas-Suarez, M., Boerner, T., and Barkan, A. (2012). Protein-mediated protection as the predominant mecha- 9 WOS:000303164400031 Article nism for defining processed mRNA termini in land plant chloroplasts. Nucleic Acids Res 40, 3092-3105.

Guo, W.*, Schafer, S.*, Greaser, M.L., Radke, M.H., Liss, M., Govindarajan, T., Maatz, H., Schulz, H., Lincoln, S.E., Parrish, A.M., Dauksaite, V., Vakeel, P., Klaas- sen, S., Gerull, B., Thierfelder, L., Regitz-Zagrosek, V., Hacker, T.A., Saupe, K.W., 10 WOS:000303763500044 Article Dec, G.W., Ellinor, P.T., MacRae, C.A., Spallek, B., Fischer, R., Perrot, A., Ozcelik, C., Saar, K., Hubner, N., and Gotthardt, M. (2012). RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med 18, 766-773. *Joint first authors

Source: ISI Web of Science; As of 14.04.2014

310 MDC Research Report 2014 ANNEX

Ten most cited MDC publications 2013

Rank ID (Web of Science) Document type Reference

Memczak, S.*, Jens, M.*, Elefsinioti, A.*, Torti, F.*, Krueger, J., Rybak, A., Maier, L., Mackowiak, S.D., Gregersen, L.H., Munschauer, M., Loewer, A., Ziebold, U., 1 WOS:000316650500035 Article Landthaler, M., Kocks, C., le Noble, F., and Rajewsky, N. (2013). Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333-338. *Joint first authors

Kettenmann, H., Kirchhoff, F., and Verkhratsky, A. (2013). Microglia: new roles for 2 WOS:000313404600004 Review the synaptic stripper. Neuron 77, 10-18.

Santos, R.A.S., Ferreira, A.J., Verano-Braga, T., and Bader, M. (2013). Angiotensin- 3 WOS:000315733400001 Review converting enzyme 2, angiotensin-(1-7) and Mas: new players of the renin angiotensin system. J Endocrinol 216, R1-R17.

Holland, J.D., Klaus, A., Garratt, A.N., and Birchmeier, W. (2013). Wnt signaling in 4 WOS:000317886100016 Article stem and cancer stem cells. Curr Opin Cell Biol 25, 254-264.

Floegel, A., Stefan, N., Yu, Z., Muehlenbruch, K., Drogan, D., Joost, H.G., Fritsche, A., Haering, H.U., Hrabe de Angelis, M., Peters, A., Roden, M., Prehn, C., Wang- 5 WOS:000314263600035 Article Sattler, R., Illig, T., Schulze, M.B., Adamski, J., Boeing, H., and Pischon, T. (2013). Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach. Diabetes 62, 639-648.

Carlo, A.S., Gustafsen, C., Mastrobuoni, G., Nielsen, M.S., Burgert, T., Hartl, D., Rohe, M., Nykjaer, A., Herz, J., Heeren, J., Kempa, S., Petersen, C.M., and 6 WOS:000313046500034 Article Willnow, T.E. (2013). The pro-neurotrophin receptor sortilin is a major neuronal apolipoprotein E receptor for catabolism of amyloid- peptide in the brain. J Neu- rosci 33, 358-370. β Scholz, C.W., Pinto, A., Linkesch, W., Linden, O., Viardot, A., Keller, U., Hess, G., Lastoria, S., Lerch, K., Frigeri, F., Arcamone, M., Stroux, A., Frericks, B., Pott, C., 7 WOS:000313793700010 Article and Pezzutto, A. (2013). 90yttrium-ibritumomab-tiuxetan as first-line treatment for follicular lymphoma: 30 months of follow-up data from an international multicenter phase II clinical trial. J Clin Oncol 31, 308-313.

Mossadegh-Keller, N., Sarrazin, S., Kandalla, P.K., Espinosa, L., Stanley, E.R., Nutt, 8 WOS:000318558200036 Article S.L., Moore, J., and Sieweke, M.H. (2013). M-CSF instructs myeloid lineage fate in single haematopoietic stem cells. Nature 497, 239-243.

Krabbe, G.*, Halle, A.*, Matyash, V., Rinnenthal, J.L., Eom, G.D., Bernhardt, U., Miller, K.R., Prokop, S., Kettenmann, H.#, and Heppner, F.L.# (2013). Functional 9 WOS:000317898000084 Article impairment of microglia coincides with beta-amyloid deposition in mice with Alzheimer-like pathology. PLoS ONE 8, e60921. *Joint first authors;# Joint senior authors

Montonen, J., Boeing, H., Fritsche, A., Schleicher, E., Joost, H.G., Schulze, M.B., Steffen, A., Pischon, T. Consumption of red meat and whole-grain bread in relation 10 WOS:000313812100034 Article to biomarkers of obesity, inflammation, glucose metabolism and oxidative stress. Eur J Nutr 52(1): 337-345, 2013.

Source: ISI Web of Science; As of 14.04.2014

MDC Research Report 2014 311

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Facts and Figures

Kennzahlen

Personnel / Personal

Personnel (as of December 31, 2013) Personalstand in Köpfen (Stichtag 31.12.2013)

in-house third-party limited financed financed Total contract permanent grund drittmittel Gesamt befristet unbefristet finanziert finanziert

Scientists (not including PhD students) 286 229 57 194 92 Wissenschaftler/-innen

PhD Students 237 237 0 128 109 Doktoranden/-innen

Technical Assistants (TAs) 218 100 118 180 38 Technische Angestellte im wissenschaftlichen Bereich

In Training 60 60 0 60 0 In Ausbildung

Other* 323 112 211 303 20 Wissenschaftsunterstützendes Personal*

SUMME 1.124 738 386 865 259

Other* Scientists (not including PhD students) Wissenschaftsunterstützendes Personal* Wissenschaftler/-innen

26% 29%

In Training 5% 21% In Ausbildung 19% PhD Students Doktoranden/-innen

Technical Assistants (TAs) Technische Angestellte im wissenschaftlichen Bereich

* Infrastructure, administration, technology26+ transfer, DZHK grant management at the MDC, BIH management21+19529

312 MDC Research Report 2014 ANNEX

Type of Employment Contract Art der Verträge

limited contract permanent befristet unbefristet Scientists Wissenschaftler/-innen Assistants Technical TAs Other* Andere* PhD Students Doktoranden/-innen In Training In Ausbildung 250 Scientists 229 57 Wissenschaftler/-innen

PhD Students 200 237 0 Doktoranden/-innen

Technical Assistants 100 118 150 TAs

In Training 60 0 100 In Ausbildung

Other* 112 211 Andere* 50

SUMME 738 386 0 * Infrastructure, administration, technology transfer, limited contract / befristet DZHK grant management at the MDC, BIH management permanent / unbefristet

Type of Financing Art der Finanzierung

third-party in-house financed financed drittmittel Scientists Wissenschaftler/-innen Assistants Technical TAs PhD Students Doktoranden/-innen In Training In Ausbildung grundfinanziert finanziert Other* Andere* 350 Scientists 194 92 Wissenschaftler/-innen 300 PhD Students 128 109 Doktoranden/-innen 250

Technical Assistants 180 38 200 TAs

In Training 150 60 0 In Ausbildung 100 Other* 303 20 Andere* 50 SUMME 865 259 0 * Infrastructure, administration, technology transfer, in-house financed / grundfinanziert DZHK grant management at the MDC, BIH management third-party financed / drittmittelfinanziert

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Financing / Finanzierung

Costs of research programs in 2012 in TEUR Kosten der Forschungsprogramme in 2012 in TEUR (Vollkosten)

Programs & Categories / Programme und Kategorien Costs / Kosten

Cancer Research / Krebsforschung 16.499

Cardiovascular and metabolic diseases / Herz-Kreislauf- und Stoffwechselerkrankungen 27.473

Function and dysfunction of the nervous system / Funktion und Dysfunktion des Nervensystems 10.960

Portfolio topics (Wirkstoffforschung / Metabolisches Syndrom) 924

Total Program Costs / Gesamt 55.856

Function and dysfunction of the nervous system Cardiovascular and metabolic diseases Funktion und Dysfunktion des Nervensystems Herz-Kreislauf- und Stoffwechselerkrankungen 20% 2% Portfolio topics 49% (Wirkstoffforschung / Metabolisches Syndrom)

29%

Cancer Research 20+Krebsforschung2+2949

Extramural funding in 2012 in TEUR Drittmittelfinanzierung 2012 in TEUR

Extramural funds / Drittmittelgeber Amounts / Drittmittelausgaben

Federal Ministry of Education and Research / Bundesministerium für Bildung und Forschung (BMBF) 4.207

German Research Foundation / Deutsche Forschungsgemeinschaft 6.738

Helmholtz Association / Helmholtz-Gemeinschaft 5.167

EU / EU 3.014

Industry and other organizations / Industrie und andere Organisationen 2.382

Total Program Costs / Gesamt 21.508

Industry and other organizations Industrie und andere Organisationen Federal Ministry of Education and Research 11% Bundesministerium für Bildung und Forschung (BMBF)

EU 20% EU 14%

24% 31%

Helmholtz Association German Research Foundation Helmholtz-Gemeinschaft Deutsche Forschungsgemeinschaft

314 20+31241411 MDC Research Report 2014 ANNEX

MDC Research Report 2014 315

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Index

A Bansal, Vikas...... 215 Barbieri, Mariano...... 191 Adamidi, Catherine...... 195 Barros, Carlos...... 35 Adami, Eleonora...... 39 Bartels-Klein, Eireen...... 47 Akalin, Altuna �� 224, 228, 229 Bartolini, Laura...... 15 Akilli-Öztürk, Özlem...... 77 Bartusch, Marko...... 35 Albert, Tobias...... 149 Bashammakh, Saleh �� 35 Albrecht, Sebastian...... 141 Bauer, Christof...... 91 Alenina, Natalia...... 35 ...... 39 Alvares de Lemos, Clara Faria...... 221 Baumann, Matti...... 115 Alves de Melo, Arthur...... 87 Bauerfeind,Baumert, Philipp Anja ...... 13 Anders, Juliane...... 219 Bauml, Katharina...... 31 Anders, Kathleen...... 81 Beagrie, Robert...... 191 Andrade, Miguel ��72, 73 Beato del Rosal, Maria Lena...... 197 Andrée, Christel...... 47 Beaudette, Patrick �� 119, 241 Andrich, Kathrin...... 163 ...... 247 Anistan, Yoland-Marie...... 213 Becker, Michael...... 95 Annamalai, Karthika...... 173 Becker, MikeKatja...... 35 Apostel-Krause, Iris �� 23, 25, 35 Begay-Mueller, Valerie...... 149 Arakelyan, Karen...... 51 Behm, Andrea...... 215 Aranda Chale, Paulina...... 63 Behrens, Diana...... 95 Arkhipova, Valeriya...... 25 Behrmann, Heide...... 99 Arnold, David Graham...... 229 Beis, Daniel...... 35 Arslan, Seda Çöl...... 119 Bellmann, Katherina...... 215 Arumughan, Anup...... 163 Belz, Stefanie...... 207 Aschmann, Mehrshad...... 67 Benary, Uwe...... 31 Aue, Annekatrin...... 21 Beniazza, Meryam...... 123 Aumann, Jutta...... 221 Benlasfer, Ouidad...... 179 Aydin, Atakan...... 47 Berger, Maren...... 127 Ayoub, Salah...... 195 Berger, Maximilian...... 207 Bernert, Carola...... 151 Berruyer-Pouyet, Carole...... 123 Beuster, Gregor...... 121 B Bhargava, Anuprabha...... 197 Biedermann, Markus...... 91 Babic, Ana...... 175 Bielow, Chris ��177 Bach, Karola...... 145 Bikou, Maria...... 63 Backhaus, Carolin...... 141 ...... 41 Bähring, Sylvia...... 47 Billig, Gwendolyn...... 141 Baddack, Uta...... 107 Bindel,Bilic, Ilija Fabian ��177 Bader, Michael ��32, 35 Binger, Katrina...... 219 Bagola, Katrin...... 127 Birchmeier, Carmen �� 134, 137 Balasubramaniyam Sivanandam, Arasu...... 87 Birchmeier, Walter ��74, 77 Balke, Annika...... 35 Bit-Avragim, Nana...... 23 Balogh, Andras...... 219 Blachut, Susanne...... 39 Balueva, Kira...... 137 Blankenstein, Lydia...... 119 ...... 99 Blankenstein, Thomas ��78, 81

Banchenko, Sofia 316 MDC Research Report 2014 ANNEX

Blaschke, Florian �� 57 Chu, Van Trung...... 113 Blendinger, Gitta...... 181 Chuykin, Ilya...... 35 ...... 141 Cirovic, Branko...... 103 Block, Franziska...... 67 Clauß, Julian...... 129 Blessing,Blum, Maximilian Anja ...... 47 Cloos, Birgit ��87, 99, 145 Böddrich, Annett �� 163, 252 Coin, Irene...... 119 Böhm, Julia Wiebke...... 103 ...... 57 Böttger, Adelheid...... 35 Conrad, Lisa...... 25 Bogdanow, Boris...... 53 Cozzitorto,Conrad, Anja Corinna...... 25 Boiani, Sara...... 109 Cristiano, Elena...... 181 Bolgehn, Daniela...... 113 Cruz e Silva, Andreia...... 141 Bollen, Sander...... 73 ...... 15 Bonkowski, Jasmin...... 19 Cseresnyes, Zoltan...... 227 Bontscho, Julia...... 209 Cui,Csályi, Huanhuan Kitti ...... 215 Borgwald, Kathrin...... 81 ...... 137 Born, Gabriele...... 39 Czychi, Jana...... 219 Boschmann, Michael...... 205 Czajkowski, Maciej Brandenburg, Manuela �� 149, 235, 243, 245 Brand, Franziska...... 107 Brandt, Bettina...... 137 D Braunschweig, Maria...... 149 Brendebach, Holger...... 127 da Costa Goncalves, Susanne...... 35 Bridger, Charmaine...... 57 Däbritz, Henry...... 121 ...... 163 ...... 199 Broecheler, Bettina...... 61 Dagane, Alina...... 241 Briese,Broehl, AnjaDominique...... 137 Dämpfling,Dahlmann, MathiasLea ...... 221 ...... 67 Dalda, Malgorzata Anna...... 41 Brümmer, Juliane...... 115 Damm, Henning...... 61 Brusendorf,Brouwer-Lehmitz, Lydia...... Antje 163 Daniel, Peter ��82, 83 Buchert, Sven...... 137 da Silva, Tiago Fernando Ferreira...... 51 Bukowiecki, Raul...... 163 Dauksaite, Vita...... 7 Bulut, Selman...... 197 Daumke, Oliver ��84, 87 Bunse, Mario...... 129 Deak, Veronika...... 19 Buntru, Alexander...... 163 de Almeida Sassi, Felipe...... 159 Burbach, Nadine...... 121 de Castro, Inês...... 191 Burgert, Tilman...... 29 Dechend, Ralf �� 218, 219 Busse, Dorothea...... 31 Dema, Alessandro...... 19 ...... 159 Demidova, Olga ��177 Derudder, Emmanuel...... 113 Butzek, Tatjana Deter, Sabrina �� 175, 177, 179, 229 ...... 81 C Dias, João...... 191 Dhamodaran,...... Arunraj 87 Caglayan, Safak...... 29 Dieringer, Babette...... 51 Caglio, Giulia...... 9 Dick,Dieringer, Alexej Matthias ��51, 211 Cai, Huiqiang...... 41 Diesbach, Julia ��177 Cakmak, Nese...... 109 Dietrich, Ann-Christin...... 23 Calviello, Lorenzo...... 187 Diez, Lisa Maria...... 163 Carlo-Spiewok, Anne-Sophie...... 29 Dittmar, Gunnar �� 224, 240, 241 Casado, Victoria...... 115 ...... 159 Cavalcanti, Diogo...... 35 Dörken, Bernd ��88, 91 Cerda-Esteban, Nuria...... 25 Djodari-IrDörr, Jan...... ani, Anais 121 Chekulaeva, Marina �� 172, 173 Domaing, Petra...... 13 Chen, Jiaxuan...... 53 Donat, Stefan...... 23 Chen, Jie-Shin...... 137 Dornblut, Tracy Alice...... 99 Chen, Tao...... 175 Dorn, Cornelia...... 215 Chen, Wei �� 174, 175, 224, 230, 231 ...... 155 ...... 81 dos Santos Periquito, Joao...... 51 Cheret, Cyril...... 137 Dorrn,Drenckhahn, Anja Luise Joerg-Detlef...... 57 Chiola,Chen, Xiaojing Francesco Paolo...... 221 Driesner, Madlen...... 145 Choi, Mira...... 209 Dubrovska, Galyna...... 213 Christa, Anna...... 29 Duchene, Johan...... 35 Christ, Annabel...... 29 Du, Hang...... 175

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ANNEX

Dumanis, Sonya...... 29 Franz, Liane...... 217 Dumoulin, Alexandre...... 145 Frauke Kosel...... 77 Dunham, Hannah Rose...... 113 Frenzel, Henning...... 149 Dusatko, Silke...... 181 Friedrich, Dhana...... 181 Dusek, Petr...... 51 Friedrich, Mathias...... 81 Friese, Christian...... 81 Fritsche, Raphaela ��177 Froehler, Sebastian...... 175 E Fröhlich, Chris...... 87 Fröhlich, Janine...... 7 Eccles, Rebecca...... 115 Fröse, Sabine ��31, 53 Eckert, Klaus...... 95 Fuchs, Katharina Maria...... 51 Eckhard, Jamina...... 43 Fuchs, Nina...... 41 Edes, Inan...... 129 Fuerl, Stephanie...... 81 Eichelbaum, Katrin...... 53 Eigenbrod, Ole...... 149 Eisenberger, Alina ��177 Eisenmann, Andra �� 107, 151 G Eisermann, Beate...... 19 ...... 73 Gärtner, Angelika...... 81 Eldahshan, Adeeb ��252 ...... 91 ElElef Amrani,sinioti, KhadijaAnna...... 195 Gao, Qingsong...... 175 ...... 21 Garcia-Perez,Gätjen, Marcel Angelica...... 41 El-Mahmoud, Sana...... 211 Garg, Ankur...... 99 Elger, Antje...... 51 Gavvovidis, Ioannis...... 81 Escobar, Helena...... 41 Gebhardt, Marie...... 73 Escot,Els, Antje Anne Sophie...... 25 Gebhardt, Matthias...... 219 Esparza-Gordillo, Jorge...... 43 Geelhaar, Andrea...... 19 Estebanez, Luc...... 155 Geirsdottir, Laufey...... 123 Eulenberg, Claudia...... 209 Genehr, Carolin...... 129 Ewald, Dominik...... 57 Gentek, Rebecca...... 123 Georgieva, Petya...... 159 Gerhard, Cathrin...... 35 Gerhard, Mathias...... 39 F Gerhard, Ute...... 219 Gerlach, Kerstin...... 91 ...... 87 Gerloff, Michael...... 77 Falak, Samreen...... 39 Gibson, Meino Alexandra...... 159 Falcke,Fälber, KatjaMartin ��4, 5 Gillissen, Bernd...... 83 Fang, Liang...... 77 Glahs, Alexander...... 199 Farah, Hanad...... 153 Glöde, Julia...... 61 Faust, Doerte...... 19 Gloger, Marleen...... 91 Favret, Jérémy...... 123 Gödde, Kathrin...... 141 Fazekas, Dora...... 41 Gök, Caglar...... 149 Fechner, Robby...... 23 Göritz, Petra...... 141 ...... 175 Goetz, Frank...... 19 Felsenberg, Shirley-Ann Jennifer...... 19 Gogol-Doering, Andreas...... 175 Fendler,Feldkamp, Annika Mirjam...... 77 Gohlke, Ulrich...... 99 Fernandes, Joao Miguel Parente...... 19 Goldbrich-Hannig, Beate...... 7 Ferrai, Carmelo...... 191 Golic, Michaela...... 219 Ferrarese, Leiron...... 155 Gollasch, Maik �� 212, 213 Fichtner, Iduna ��92, 95 Gomez, Lorena...... 35 Filipchyk, Andrei...... 195 ...... 73 Fink, Claudia...... 7 Gopal Tattikota, Sudhir...... 63 Finzel Perez, Ana...... 181 Gotthardt,González López, Michael Adrián ��6, 7 Fishman,Veniamin...... 35 Gottschling, Karin...... 137 Fleischer, Raluca...... 149 ...... 7 Flinders, Michael...... 35 Grabowski, Piotr...... 7 Förster, Susann...... 103 Govindarajan, Thirupugal...... 41 ...... 151 Gräning, Kornelia...... 207 Fontaine, Jean-Fred...... 73 Graessl,Grabundzija, Andreas Ivana...... 51 Fournier,Förstera, BenjaminDavid...... 73 Graf, Robin...... 113 Fraga, Carlos...... 35 Graf, Yasmine...... 35

318 MDC Research Report 2014 ANNEX

Grelle, Gerlinde...... 163 Henning, Mechthild...... 145 Grieben, Marlies...... 115 Hensel, Markus...... 81 Grieben, Ulrike...... 207 Herda, Stefanie...... 91 Gries, Margarete...... 109 Hergeselle, Martha...... 53 Griger, Joscha...... 137 Herget, Regina �� 149, 167 Grigoryan, Tamara...... 77 Hernandez Miranda, Luis Rodrigo...... 137 Grigull, Sabine...... 221 Herrmann, Pia...... 221 Grobe, Jenny �� 107, 177 Herrmann, Sarah Joana...... 173 Grohmann, Maik...... 35 Herse, Florian...... 219 Grosse, Claudia...... 113 Hertel, Olivia...... 25 Grosse, Luisa...... 211 ...... 29 Groß, Isabel...... 145 Herzog, Margareta...... 195 Grosskopf, Stefanie...... 77 Herzog,Hesse Christin Katja ��77, 163 Grosswendt, Stephanie...... 195 Hesse, Evelyn...... 81 Gruen, Dominic...... 195 Hesse, Franziska...... 163 Gruettner, Henriette...... 211 Hessenberger, Manuel...... 87 Grüger, Sabine...... 35 Hetsch, Florian...... 145 Grunert, Marcel...... 215 Heuberger, Julian...... 77 Grunwald, Stefanie...... 207 Heuer, Miriam...... 77 Grunwald, Stephen...... 87 Heuser, Arnd ��57, 224, 242, 243 Grunz, Katharina...... 39 Hezel, Fabian...... 51 Günes, Derya...... 181 Hill, Undine...... 103 Guenther, Martin...... 51 Hilsch, Malte...... 227 Guimaraes, Paola...... 35 Hinze, Christian...... 21 Gupta, Ritesh...... 77 Hinze, Florian...... 7 Hinz, Michael...... 119 Hiren Thakkar, Meghna...... 7 Hirsch, Christian...... 127 H ...... 187 Hoedke, Daniela...... 61 Haase, Hannelore...... 13 Hirsekorn, Antje ...... 141 Haase, Nadine...... 219 Hoehne, Marko...... 51 Haas, Verena...... 205 Hönig,Hoegg-Beiler, Katrin...... Maja 81 Haehn, Kristina...... 23 Höpken, Uta...... 107 Hänig, Christian...... 163 Hofstätter, Maria...... 103 Hafez, Dina...... 187 Hoherz, Judith...... 159 Hagen, Anne...... 159 Holland, Jane...... 77 Hahmann, Anne...... 163 ...... 47 Hainer, Cornelia...... 35 Homfeld, Johanna...... 149 Haink, Petra �� 15, 31, 53, 115 Hollfinger,Horn, Sabrina Irene...... 81 ...... 197 Hoser, Dana...... 81 Hall, James...... 149 Hou, Jingyi...... 175 HHammes,ajduskova, Annette Martina �� 164, 165 Hsu, Meng-Tung...... 81 Hampel, Dierk...... 217 Hübner, Norbert ��36, 39, 224 Hampel, Martina...... 83 Hübner, Ulrike...... 163 Hampf, Dirk...... 19 Huebener, Nicole...... 67 Hardt, Markus...... 221 Hügel, Stefanie...... 35 Harm, Monika...... 81 Huelnhagen, Till...... 51 Hartmann, Nina...... 145 Hu, Feng...... 159 Haseleu, Julia...... 149 Hummel, Franziska...... 91 Hauchwitz, Janina...... 129 Hummel, Kordelia...... 129 Haupt, Irene...... 159 Hummel, Oliver...... 39 Haustein, Maria...... 221 Hu, Yuhui...... 175 Heidenreich, Matthias...... 141 Heidt, Leonie...... 207 Heinemann, Udo ��96, 99, 224 Heinemann, Vera...... 159 I Heinig, Kristina...... 107 Heinig, Matthias...... 39 Ibrahim, Mahmoud...... 187 Heinze, Daniel...... 119 Ifuku, Masataka...... 159 Hellmig, Nicole...... 109 Ilm, Katharina...... 221 Hennig, Maria...... 57 Imami, Koshi...... 53 Hennig, Pierre-Lars...... 211 Imperatore, Francesco...... 123

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ANNEX

Ipenberg, Inbal...... 119 Kaßmann, Mario...... 213 Ivanov, Andranik...... 195 Katzer, Andrea...... 181 Izsvak, Zsuzsanna ��40, 41 Katz, Samuel...... 197 Kazmierczak, Marlon �� 145, 197 Keil, Albrecht...... 35 Keil, Marlen...... 95 J Kemmner, Wolfgang...... 221 Kempa, Stefan �� 176, 177, 224, 232, 233 Jabs, Sabrina...... 141 Kermer, Josephine...... 211 Jacobs, Nicholas Charles...... 187 Kerscher, Tamara...... 43 Jacoby, Ben...... 61 Kettenmann, Helmut �� 156, 159 Jaeschke, Lina...... 61 Kettritz, Ralph �� 208, 209 Järve, Anne...... 35 Keyner, Daniela �� 107, 119 Jaksch, Sarah...... 103 Kieback, Elisa...... 129 Janke, Jürgen...... 61 Kienitz, Tina...... 35 Janz, Martin...... 91 Kikuchi, Julia...... 241 Jarchow, Birgit...... 159 Kirchner, Marieluise...... 53 Jarosch, Ernst...... 127 ...... 57 Jedamzick, Johanna...... 141 Klahn, Sylvia ��81, 127 Jens, Marvin...... 195 KingaKlasen, Dziegelewska, Christian...... Maja 67 Jentsch, Thomas J. �� 138, 141 Klaus, Alexandra...... 77 Jeran, Stephanie...... 61 Klaus, Anna...... 9 Jerke, Uwe...... 209 Klawiter, Agnieszka...... 199 Jia, Shiqi...... 137 Kleckers, Daniela...... 163 Jimenez Orgaz, Ana...... 41 Kleindienst, Denise...... 211 Jin, Sha...... 163 Klementowicz, Agnieszka...... 153 Ji, Yiyi...... 51 Klempin, Friederike...... 35 Jordan, Philipp...... 159 Klems, Alina...... 9 Jose, Dinto...... 31 Klinghammer, Konrad...... 95 Jouhanneau, Jean-Sebastien...... 155 Klippert, Julia...... 29 Judis, Carmen...... 7 Klironomos, Filippos...... 195 Jüttner, René...... 145 Klix, Sabrina...... 51 Jumpertz, Reiner...... 217 Klockmeier, Konrad...... 163 ...... 221 Kluessendorf, Thies...... 63 Jung, Josephine...... 81 Klug, Lars...... 205 Jungmann,Juneja, Manisha Sabine...... 119 Klußmann, Enno ��16, 19 Knespel, Andreas...... 99 Knespel, Signe �� 187, 191 Knispel, Rosita...... 51 K Knoblich, Ilse-Maria...... 103 Kobelt, Dennis...... 221 Kaada, Manuela...... 13 Koch, Gudrun...... 221 Kärgel, Eva...... 119 Kocks, Christine...... 195 Käsk, Lauri...... 109 ...... 113 Kahlert, Günther �� 103, 241 Koehler, Annette...... 211 Kaiser, Hermann-Josef...... 141 Koehler,Köhler, Anett Anja...... 127 Kalb, Birgit...... 43 Köhler, May-Britt...... 219 Kaldrack, Joanna...... 7 Köhn, Anne...... 145 Kalis, Ronny �� 163, 237 Koehncke, Clemens...... 13 Kallenberg, Felix...... 241 König, Janett...... 155 Kamarys, Mareen...... 129 Köppen, Susanne �� 163, 237 Kamer, Ilona...... 219 Koestner, Ulrich...... 137 Kammertoens, Thomas...... 81 Kofahl, Bente...... 31 Kampf, Kristin ��29, 165 Kofent, Julia...... 25 Kampfrath, Branka...... 107 Kogame, Toshiaki...... 195 Kamprath, Maria...... 91 Kokot, Karoline...... 57 Kanashova, Tamara...... 241 Kolesnichenko, Marina...... 119 Kandalla, Prashanth...... 123 Kolinski, Marcin...... 179 Kaplan, Oktay Ismail...... 197 Kolundzic, Ena...... 197 Karabacak, Aslihan...... 187 Konigorski, Stefan...... 61 Karamatskos, Karin...... 81 Korthals, Stefanie...... 159 Karczewski, Karin...... 13 Korvers, Laura Maria...... 159 Kase, Julia...... 121 Kosizki, Liana...... 149

320 MDC Research Report 2014 ANNEX

Kovalchuk, Tetiana...... 149 Leisle, Lilia...... 141 Kowenz-Leutz, Elisabeth...... 103 Lemm, Margit...... 95 Kox, Stefanie...... 51 le Noble, Ferdinand ��8, 9 Ko, Young-In...... 163 Lepore, Stefano...... 51 Krämer, Dorothee...... 191 Leschke, Andrea...... 247 Kraft, Sabine...... 23 Leu, Romy...... 103 Krahn, Inge...... 119 Leutz, Achim �� 100, 103 Krause, Christine...... 247 Lewin, Gary R. �� 146, 149, 253 Krause, Claudia...... 107 Le Wuehrmann, Lara...... 81 Kraus, Oliver...... 51 Liebold, Janet...... 141 Kreher, Stephan...... 91 Liekweg, Melanie...... 29 Kressin, Franziska...... 149 Liemen, Michaela �� 187, 191 Kressner, Susanne...... 127 Lietz, Maria...... 129 Kretschel, Kerstin...... 211 Lipp, Martin �� 104, 107 Krieger, Karsten...... 119 Li, Shuang...... 91 Krivokharchenko, Alexander...... 35 Liskovykh, Michael...... 35 Krock, Ines �� 173, 181, 197, 199 Liss, Martin...... 7 Krönke, Nicole...... 141 Listopad, Joanna...... 81 Krüger, Janna...... 9 Liu, Na...... 35 Krüger, Kerstin...... 107 Liu, Qingbin...... 103 Krüger, Nadine...... 205 Loewer, Alexander �� 180, 181 Krüger, Sabrina...... 199 Löhr, Lena...... 213 Krüger, Sylvia...... 209 Lombardo, Veronica...... 23 Kruse, Christine...... 29 Longmuss, Enya...... 67 Kühn, Ralf �� 224, 246, 247 Lopez-Aranguren Blazquez, Blanca...... 51 Kuhle, Verona...... 29 Lorenz, Carmen...... 163 Kuich, Henning ��177 Lorenz, Felix...... 129 Kukalev, Alexander...... 191 Lossie, Janine...... 13 Kumar, Amit ��177 Ludwig, Carmen...... 141 Ku, Min-Chi...... 51 Luft, Friedrich C. ��44, 47, 205, 209 Kunz, Séverine...... 207 ...... 21 Kurths, Silke...... 99 Luig, Martina...... 215 Kutz, Kamila...... 53 Lusatis,Luganskaja, Simone Tatjana...... 91 Lutter, Darius...... 141 Lutter, Steffen...... 13 L Lysiak, Darius Georg...... 51

Labi, Verena...... 113 Lacadie, Scott...... 187 M Lahmann, Ines...... 137 Lakshmipathy, Sathish...... 127 Maass, Philipp...... 47 ...... 91 Maatz, Henrike...... 39 Lan, Chen...... 213 Mackowiak, Sebastian �� 183, 195 Landthaler,Lamprecht, MarkusBjörn �� 178, 179 ...... 205 Langanki, Reika...... 35 Madai, Vince...... 51 Lange, Doris...... 119 MählerMadel, ,Anette Anja ...... 81 Langhorst, Hanna...... 145 Mahmoodzadeh, Shokoufeh...... 13 Langner, Patrick...... 57 Mah, Nancy...... 73 Langnick, Claudia...... 175 Maier, Luisa...... 195 Lan, Linxiang...... 77 Mai, Knut...... 217 Lapatsina, Liudmilla...... 149 Makowska, Zuzanna...... 67 Lapidous, Irina...... 35 Malcher, Jakub...... 207 Larcheveque, Oriane...... 13 Malchin, Victoria...... 103 Latz, Meike...... 35 Malhotra, Samta...... 187 Lauhkonen-Seitz, Riikka...... 113 ...... 107 Lavitas, Liron M...... 191 Mallis, Lisa...... 35 Lechner, Stefan G...... 149 Mallem,Mallow, KevenNedjoua...... 35 Lee, Soyoung...... 121 Mall, Sabine...... 61 Lee, Young-Ae ��42, 43 Mannaa, Marwan...... 213 Leicht, Roman...... 51 Marenholz, Ingo...... 43 Leip, Beatrice...... 57 Marg, Andreas...... 207 Leisegang, Matthias...... 129 Margineanu, Anca...... 227

MDC Research Report 2014 321

ANNEX

Marino, Stephen...... 87 Motta, Daisy...... 35 ...... 219 Mühl, Astrid...... 47 Markus, Carolin Elfriede...... 21 Mühlstedt, Silke...... 35 MarMartin,ko, LajosConrad...... 51 Müller, Andrea...... 35 Maßwig, Sven...... 121 Müller, Anita...... 39 Mastrobuoni, Guido ��177 ...... 83 ...... 43 Müller, Anna-Katharina...... 211 Mathas, Stephan...... 91 Müller,Müller, AnjaDominik N. �� 218, 219 MatanoMath, Isabellvic, Anja...... 109 Müller, Gerd...... 107 Mattei, Daniele...... 159 Müller, Jürgen-Joachim...... 99 Matthaeus, Claudia...... 145 Müller, Marion...... 77 Matthes, Susann...... 35 Müller, Paul Markus...... 115 Matyash, Marina...... 159 Müller, Thomas...... 137 Matyash, Vitali �� 159, 227 Münch, Jonas...... 141 Maul, Alena...... 153 Muhle, Maximilian...... 51 Maurer, Lukas...... 217 Munschauer, Mathias...... 179 Mauri, Marta...... 173 Munteanu, Alina-Christina...... 187 Maurizio, Julien...... 123 Munteanu, Dan...... 229 McShane, Erik...... 53 Murakawa, Yasuhiro...... 179 Mecklenburg, Nora...... 165 Megahed, Douaa...... 7 Mehling, Heidrun...... 205 Mehnert, Martin...... 127 N Meier, Jochen �� 150, 151 ...... 9 Nagy, Eniko Eva...... 41 Meisel, Jutta...... 219 Naporra, Adrienne �� 207, 191 MeierMemczak,, Katja Sebastian...... 195 Narayanavari, Suneel...... 41 Menyhert, Judit...... 41 Naschke, Michaela...... 129 Mer, Arvind...... 73 ...... 191 Merkert, Doris...... 205 Naumann, Heike...... 25 Merks, Anne �� 7, 15 N’diaye,Natarajan, Gabriele Kedar ...... N. 219 Merle Viol, Ricarda...... 21 ...... 187 Meyer, Alexander...... 15 Neuendorf, Sandra...... 41 Meyerhuber, Peter...... 129 Neumann,Neelanjan, FranziskaMukherjee...... 211 Meyer, Katrina...... 53 Nguyen, Tam Hoai...... 109 Meyer-Liesener, Stephanie...... 207 Nickel, Elke...... 211 Meyer, Markus...... 195 Niedobitek, Antonia...... 91 Michaelis, Edward G...... 107 Niendorf, Thoralf ��48, 51, 224, 238 Middleton, Steven...... 149 Nimptsch, Katharina...... 61 Mieth, Christin...... 39 Nitschke, Norma...... 141 Miguel Fernandes, Ana...... 191 Nitschke, Ute...... 91 Mikuda, Nadine...... 119 ...... 145 Mikutta, Janine...... 9 Nitz, Katrin...... 35 ...... 121 Nolis,Nitze, IliasKatja...... 181 Milek, Miha...... 179 Nolte, Christiane...... 159 Milanovic, Maja ...... 155 Nowak, Marcel...... 127 Miles Carter, David ��253 Nunes, Valeria...... 35 MillMilenkovic-Zujko,ward, Jason...... Nevena 51 ...... 81 Ming, Qianqian...... 99 Milojkovic,Möller, Angeli Ana Kishor...... 163 O Moenke, Gregor...... 5 Mohr, Till-Yong...... 43 Obenaus, Matthias...... 81 Molawi, Kaaweh �� 113, 123 Oberacker, Eva Irene...... 51 ...... 77 Oberheide, Karina...... 141 Morais, Rafael...... 35 Obermayer, Benedikt �� 182, 183, 195 Morano,Momeny, Ingo Majid ��10, 13 Oden, Felix...... 107 Moroni, Mirko...... 149 Özerdem, Celal...... 51 Morris, Kelly J...... 191 Öztürk, Merve...... 187 Moshourab, Rabih...... 149 Oenal, Pinar...... 195 Mosienko, Valentina...... 35 Ofenbauer, Andreas...... 197 Mossadegh, Noushine...... 123 Ohler, Uwe �� 184, 187 Mothes, Janina...... 31 Olal, Daniel...... 87

322 MDC Research Report 2014 ANNEX

Olbrich, Sylvia ��7, 63, 73, 153 Popova, Elena...... 35 Oliveira, Aline...... 35 Popp, Oliver...... 241 Omerbasic, Damir...... 149 Poulet, James �� 154, 155 Opialla, Tobias �� 177, 207 Poy, Matthew N. ��62, 63 Orioli Marica ��177 Preissner, Robert...... 205 Orozco, Ian...... 141 Preston, Patricia...... 145 Otten, Cecile...... 23 Prigione, Alessandro...... 163 Overkamp, Tim...... 83 Prothmann, Marcel...... 211 Przybyl, Lukasz...... 219 Purfürst, Bettina �� 224, 234, 235 P Päseler, Claudia...... 137 Q Pakula, Anna...... 205 Pakula, Hubert...... 77 Qadri, Fatimunnisa...... 35 Palava, Vinco...... 207 ...... 77 ��14, 15 Qiao, Tianwu...... 35 ...... 107 Quedenau,Qi, Jingjing Claudia...... 175 Pankonien,Panáková, Daniela Ines...... 13 Pannell,Panjideh, Maria Hossein...... 159 Pannwitz, Daniel...... 109 ...... 29 R Papavasileiou, Panagiotis...... 195 Pantzlaff, Tatjana ...... 141 Rabelo, Luiza...... 35 Patone, Giannino...... 39 Radhakrishnan, Harikrishnan...... 221 Paul,Pareja Fabian Alcaraz, Alexander Ruth ...... 165 Radke, Michael ��7, 254 Paul, Florian...... 53 Räbel, Katrin...... 141 Peck, Cristal Jane...... 199 Rätzer, Jenny...... 67 ...... 77 Raghunathan, Vaishnavi...... 41 Peper, Eva...... 51 Rahn, Gabriele...... 205 Perets,Penzvalto, Ekaterina Zsofia...... 19 Rahn, Hans-Peter �� 224, 244, 245 Perez-Hernandez, Daniel...... 241 �� 110, 113 Perrin, Pierre...... 123 �� 183, 192, 195 Perrot, Andreas...... 215 Rakova,ajewsky, Natalia Klaus ��47, 205 Persicke, Kathleen...... 57 Ramillon,ajewsky, VincentNikolaus...... 145 Persicke, Michael...... 173 Ramirez, Cinthia Claudia Amaya...... 173 Peter, Franziska...... 107 ...... 35 Petermann, Markus...... 35 Rasch, Iris...... 235 ...... 99 Rasko,Ranjan, Tamas Ashish...... 41 Petsch, Kerstin...... 113 Rassek, Claudia...... 137 Pezzutto,Petrović, SašaAntonio �� 108, 109 �� 142, 145 Philippi, Susanne...... 207 ...... 63 Philipp, Regina �� 9, 41 Rathjen,Rauer, Michael Fritz G...... 187 Picci, Christina...... 149 RRau,athjen, Kirstin Thomas...... 163 Pietzke, Matthias ��177 Rautenberg, Kirstin...... 245 Pinta, Annett...... 217 Razoux, Florence...... 51 Pippow, Mathias...... 7 Reckzeh, Laura...... 115 Pisch, Erika...... 163 Redel, Alexandra...... 163 Pischon, Tobias ��58, 61 Rehm, Armin...... 91 Piske, Regina...... 159 Reimann, Henning...... 51 Planells-Cases, Rosa...... 141 Reimann, Maurice...... 121 Plehm, Ralph...... 35 Reincke, Momsen...... 141 Pluska, Lukas...... 127 Rendon, Damaris Anell...... 121 Pohl, Baerbel...... 19 Renz, Marc Andreas...... 23 Pohlmann, Andreas...... 51 Retzlaff, Kristin...... 81 Polack, Christopher...... 7 Reusch, Sebastian...... 115 Polzin, Evelyn...... 211 Reuss, Simone...... 109 Pombo, Ana �� 188, 191 Reznick, Jane...... 149 Poncette, Lucia...... 81 Rharass, Tareck...... 15 Pongrac, Igor...... 25 Richau, Johanna...... 211 Pontes de Oliveira, Kivía A...... 119 ...... 83 Poole, Kathryn �� 149, 166, 167 Richter, Annelie...... 61 Richter, Anja

MDC Research Report 2014 323

ANNEX

...... 83 Scheffner, Carolin...... 23 Richter, Jana...... 23 Scheidereit, Claus �� 116, 119 RichtRichter,er, AntjeMatthias...... 227 Schelenz, Stefanie...... 243 Richter, Nadine...... 159 Schenck, Heike...... 219 Rickert-Sperling, Silke �� 214, 215 Schenk, Alina...... 197 Riemer, Carolin...... 115 Scheu, Susanne ��87, 107 Riepenhausen, Thorsten...... 35 Schiele, Phillip...... 35 Rimpler, Ute...... 57 Schiffner, Ivonne...... 137 Ringler, Mario...... 141 Schiller, Johanna...... 103 Rintisch, Carola...... 39 Schilling, Marcel ...... 195 Rito, Tiago...... 191 Schlag, Peter M...... 221 Rivera Markelova, Maria...... 95 Schlegel, Jeanette...... 87 Rocha Lourenco, Marta...... 23 Schleifenbaum, Johanna...... 213 Rocio Servin Vences, Martha...... 167 Schleussner, Nikolai...... 91 Rocks, Oliver �� 114, 115 Schlöcker, Maria...... 217 ...... 195 Schmeisser, Maria...... 29 Rodriguez-Seguel, Elisa...... 25 Schmid, Felicitas...... 221 RRoeefzaad,odriguez-Orejuela, Claudia...... Marta 221 Schmidt, Hannes...... 145 Roel, Giulietta...... 119 Schmidt, Karin...... 81 Rösch, Marion...... 81 Schmidt, Kristin...... 113 Rogowski, Karol...... 179 Schmidt-Ott, Kai ��20, 21 Rohde, Klaus...... 39 Schmidt, Sabine...... 39 ...... 67 Schmidt-Ullrich, Ruth...... 119 Rolff, Jana...... 95 Schmidt, Vanessa...... 29 Rolle,Rojas, SusanneFabio ...... 209 Schmitt, Clemens A. �� 120, 121 Romaker, Daniel...... 57 Schmitz, Sandra...... 215 Rosenbaum, Eva...... 87 Schneidereit, Marit...... 61 Rosenthal, Walter ��16, 19 Schneider, Joanna...... 207 Roske, Yvette...... 99 Schnögl, Sigrid...... 163 Rossius, Jana...... 199 Schnuppe, Kristin...... 141 Rothe, Michael...... 81 Schön, Christian...... 81 Rother, Franziska...... 35 Schönhals, Sophia...... 215 Rothe, Victoria...... 159 Schönheit, Jörg...... 103 ...... 35 Scholz, Florian...... 107 Rousselle, Anthony...... 35 Schormann, Eileen...... 163 Royla,Rotteveel, Nadine Martijn ��177 Schöwel, Verena...... 207 Rüschendorf, Franz...... 39 Schrade, Katharina...... 19 Rudolph, Andre...... 211 Schreiber, Adrian...... 209 Rudolph, Franziska...... 7 Schreiber, Maya...... 81 Ruffert, Janett...... 21 Schröder, Philip...... 121 Rugor, Julia ��41, 219 �� 152, 153 Ruhe, Larissa...... 173 Schroeter, Micha...... 19 Rybak-Wolf, Agnieszka...... 195 SchülerSchroeder,, Johannes Björn Christian...... 211 Schüler, Markus...... 191 ...... 99 Schütz, Laura...... 145 S Schütze,Schütz, Anja Sebastian...... 141 Schugardt, Nancy...... 163 Saar, Kathrin...... 39 Schulte, Kathrin...... 87 Sack, Ulrike...... 113 Schulz, Herbert...... 39 Sakel, Petra...... 13 Schulz, Julia...... 103 Salazar Thula, Oriana...... 149 Schulz, Kerstin...... 215 Sanchez, Enrique Manuel Muro...... 73 Schulz-Menger, Jeanette �� 210, 211 Sander, Marie Sandrine...... 113 Schulz, Sylvia...... 95 Saponaro, Marisa...... 191 Schulz, Vivian...... 87 Sarrazin, Sandrine...... 123 Schunck, Wolf-Hagen...... 47 Schabe, Ariane...... 35 Schwarz, Monika...... 63 Schaefer, Martin...... 73 Schwarz, Sabine...... 103 Schäfer, Anne...... 151 Schwede, Heike...... 107 Schäfer, Sebastian...... 39 Schwedhelm, Carolina...... 61 ...... 35 Schweiker, Rachel...... 63 Scharek, Nadine...... 159 Schwerter, Michael...... 51 Schalow,Scheer, Anke Tanja...... 149 Schwochow, Melanie...... 129

324 MDC Research Report 2014 ANNEX

Sczakiel, Henrike...... 91 Swolinsky, Jutta Sybille �� 21 Seeger-Zografakis, Michaela...... 159 ...... 213 Seelk, Stefanie...... 197 Szmitkowska, Agnieszka...... 109 Seidler, Patrick...... 141 SzijartSzulzewsky,o, Istvan Frank A...... 159 ...... 141 Selbach, Matthias ��52, 53 Seja,Semtner Patricia, Marcus...... 151 Servin Vences, Martha...... 149 Seyfried, Salim ��22, 23 T Shah, Claudio...... 87 Shahid, Etsham...... 211 Tafazzoli, Aylar...... 23 Sheean, Maria Elzbieta...... 137 Tano, Jean-Yves...... 213 Sheng, Caibin...... 181 Tarak, Diana...... 81 Sibilak, Sylvia...... 103 Taube, Martin...... 243 Siegert, Romie...... 13 Tausch, Sebastian...... 151 Sierra, Yinth Andrea Bernal...... 149 Ter-Avetisyan, Gohar ��145 Sieweke, Michael �� 122, 123 Terne, Mandy...... 137 Sihn, Gabin...... 35 Textor, Ana...... 81 Silvestrov, Maxine...... 95 Thalhammer, Christof...... 51 Simaite, Deimante...... 39 Theil, Kathrin...... 195 Singer, Eugenia...... 21 Thiede, Katerina...... 81 Singh, Manvendra...... 41 Thierfelder, Ludwig ��54, 57 Skorna-Nussbeck, Madeleine...... 35 Thränhardt, Heike...... 149 Smith, Janice...... 221 Timmel, Tobias...... 207 Sohn, Madlen...... 175 Tischer, Janett...... 99 Sokolov, Maxim...... 153 ...... 211 Solana-Garcia, Jordi...... 195 Todiras, Mihail...... 35 Sommer, Christian...... 53 Töpper,Tkachenko, Agnieszka Valerij...... 211 Sommermann, Thomas...... 113 Tomann, Philipp...... 119 Sommer, Thomas �� 124, 127 Torlai Triglia, Elena...... 191 Souza, Laura...... 35 Torti, Francesca...... 195 Spagnoli, Francesca M. ��24, 25 Traber, Julius...... 211 Sparta, Karine...... 99 Trauzeddel, Ralf Felix...... 211 �� 224, 226, 227 Treier, Anna-Corina...... 67 Spranger, Joachim �� 216, 217 Treier, Mathias ��64, 67 SpulerSporbert,, Simone Anje �� 206, 207 Trendel, Jakob...... 179 Stache, Vanessa...... 107 Trepte, Philipp...... 163 Staerck, Lilian...... 129 Treutler, Jana...... 61 Stallerow, Petra...... 137 Trnka, Philipp...... 115 Stauber, Tobias...... 141 Tröster, Philip...... 145 Stauch, Maren...... 39 Tschernycheff, Alexandra...... 195 Stauß, Dennis...... 107 Tsvetkov, Dmitry...... 213 Stear, Jeffrey �� 149, 167 Tursun, Baris �� 196, 197 Stecklum, Maria...... 95 Steinborn, Melanie...... 83 Steinbrecher, Astrid...... 61 Steinhof, Anne...... 163 U Steiniger, Jochen...... 205 Stein, Simone...... 77 Uckert, Wolfgang �� 128, 129 Stein, Ulrike �� 220, 221 Uhlenhaut, Nina Henriette...... 39 Stendal, Manuela...... 61 Uhlenhut, Ines...... 15 Stoeckius, Marlon...... 195 Ullrich, Florian...... 141 Stottmeister, Christin...... 195 Ulrich, Katrin...... 91 Strasen, Henriette...... 181 Utz, Wolfgang...... 211 Strauss, Anke...... 205 Strehle, Michael...... 137 Stricker, Sarah...... 163 Stuhlmann, Till...... 141 V Subkhangulova, Aygul...... 29 Suhr, Frank...... 13 Valenti, Giovanni...... 77 Sun, Chuanbo...... 41 van den Bruck, David...... 173 Sun, Wei...... 175 Vannauer, Marianne...... 235 Swinarski, Marie...... 15 Vargas Aguilar, Stephanie...... 123

MDC Research Report 2014 325

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Vasile, Alexandra...... 179 Winkelmann, Aline...... 151 ...... 53 Winkler, Annika...... 67 Veauthier, Anette...... 61 Winkler, Wiebke...... 91 Vierheller,Vasiljevic, DjordjeJanine ...... 5 Winter, Lukas...... 51 Vigolo, Emilia...... 21 Wirtz, Tristan David...... 113 Vilianovitch, Larissa...... 35 Wissler, Susanne...... 207 v. Knobelsdorff-Brenkenhoff, Florian...... 211 Wittstruck, Angelika...... 127 Vogel, Margit...... 235 Wiznerowicz, Irmgard...... 77 Vogel, Regina...... 77 Woehlecke, Janine...... 113 Vogt, Janis...... 141 Woischwill, Christiane...... 13 Voigt, Birgit...... 155 Wolf, Jana ��30, 31 Volkwein, Corinna...... 127 Wolf, Susanne...... 159 von Bock, Anyess...... 141 Wollert-Wulf, Brigitte...... 91 von Buehler, Erika...... 129 Wrackmeyer, Uta...... 7 von Delbrück, Maximilian...... 127 Würfel, Jens...... 51 von Hoff, Linda...... 119 Wulf-Goldenberg, Annika...... 95 von Oertzen, Lena...... 195 Wurche, Lennart...... 211

Voss, Cynthia...... 221 Wurster, Kathrin...... 91 Voß, Felizia...... 141 Wyler, Emanuel...... 179 Voß, Karolin ��51, 107 X W Xiao, Mei-Sheng...... 175 Wabo, Arsene...... 57 Wahle, Philipp...... 199 Waiczies, Sonia...... 51 Walcher, Jan...... 149 Y Walentin, Katharina...... 21 Walther, Wolfgang...... 221 Yasuda, Tomoharu...... 113 Wang, Jichang...... 41 Yin, Xiushan...... 67 Wang, Xi...... 175 You, Xintian...... 175 Wang, Yongbo...... 175 Yesim Yurtdas, Zeliha...... 21 Wanke, Felix...... 211 Yu, Yong...... 121 Wanker, Erich E. �� 160, 163, 224, 236, 237 ...... 119 Weber, Annika...... 127 Weber, Timm Peer Christopher...... 113 Yιlmaz, Zekiye Buket Wefeld-Neuenfeld, Yvette...... 47 Wegener, Sandra...... 121 Z Weidlich, Andrea...... 141 Weigt, Martina...... 175 Zaniewska, Magdalena...... 35 Weinert, Stefanie...... 141 Zappulo, Alessandra...... 173 Wellner, Maren...... 219 Zarmstorff, Ruth...... 103 Wende, Hagen...... 137 Zasada, Christin ��177 Wendt, Stefan...... 159 Zauber, Henrik...... 53 Werner, Carsta...... 95 Zeisig, Reiner...... 95 Werner, Sabine...... 87 Zenkner, Martina �� 163, 237 Wessels, Hans-Hermann...... 187 Zerath Guerevich, Sylvia...... 241 Westen, Christel...... 81 Zhang, Juan...... 25 Westermann, Jörg...... 109 Zhao, Wen-Jie...... 155 Westphal, Christina...... 47 Zhu, Qionghua...... 77 Wethmar, Klaus...... 103 Ziebold, Ulrike...... 195 Wetzel, Christiane �� 149, 253 Zillmann, Silke...... 141 Wichner, Katharina...... 107 ...... 9 Wiedemann, Franziska...... 163 Zimmermann, Juliane...... 5 Wiedmer, Petra...... 67 Zimmer,Zinke, Robert Anja...... 57 Wiglenda, Thomas...... 163 Zinzen, Robert P. �� 198, 199 Wilck, Nicola...... 219 Zühlke, Kerstin...... 19 Wild, Raphael...... 9 Zummach, Ramona...... 47 Willenbrock, Michael...... 119 Willimsky, Gerald...... 81 Willnow, Thomas ��26, 29

326 MDC Research Report 2014

Common Facilities A 8 Gate House with Café Max and apartments A 9 Reception gate A 13 Gläsernes Labor / Life Science Learning Lab; Campus Info Center A 14 Cafeteria

MDC Guesthouses B 54 Hans Gummel House B 61 Salvadore Luria House with kindergarden Research Max Delbrück Center for Molecular Medicine (MDC) C 27 Walter Friedrich House C 31.1 -2 Max Delbrück House C 31.3 -5 Energy and Infrastructure Center South (EZS) C 83 MDC.C / Max Delbrück Communications Center C 84 Hermann von Helmholtz House B 63, 64 Research Services B 88 Ultrahigh Field Facility B 89 Max Rubner House A10 Library

Leibniz-Institut für Molekulare Pharmakologie (FMP) C 81 FMP

Facilities shared by MDC and FMP C87 Timoféeff-Ressovsky-Haus Clinical Research B 42-53 Experimental and Clinical Research Center (ECRC) Companies A 7 Eckert & Ziegler AG, Eckert Wagniskapital und Frühphasenfinanzierung (EWK) A 15 Car mechanics, EZAG, Gardener B 55 OCVH - Oskar-und-Cécile-Vogt-Haus BBB-post office, 8sens.biognostic, FILT, MRI, ConGen, E.R.D.E., ART-CHEM, LIPIDOMIX, Roboklon, Myelo, Fresenius, Patent lawyers Dr. Baumbach, L.O.T., WISE, Octreopharm, Institut E & G, NIKON, Hermes-Analytik, Steinbeis-Transfer-Institut, I.M.S.M., AJ Innuscreen B 64 epo D 16/23 Eckert & Ziegler AG D 79 Erwin Negelein House Akademie der Gesundheit, BioTeZ, imaGenes, AviTop, GlycoUniverse D 80 Otto Warburg House ALRISE, Silence Therapeutics, Celares, OMEICOS Therapeutics D82 Karl Lohmann House EPO GmbH D85 Arnold Graffi House BBB, stratec, aokin, Biosyntan, emp, ascenion, MerLion, KFFB, Prof. Wanker, Nutrineu, AMAL, pi-PharmaImage, GLYCOTOPE, S. Lang- macker accountant www.mdc-berlin.de