PROGRESS REPORT 2020 Interdisciplinary Center for Clinical Research of the RWTH Aachen Faculty of Medicine Progress Report IZKF Aachen 2020 Preface Preface

Prof. Dr. Peter Walter (Speaker of the IZKF)

Dear Colleagues and Friends,

Our world has changed dramatically since last year, and we all are facing new challenges well-functioning and supportive unit, helping many researchers at our faculty to generate after the appearance of the SARS CoV2 virus. This is the second annual report of IZKF important data from their experiments. Aachen during the coronavirus pandemic, and although we have learned so many facts In 2020 we also established some new technologies within the IZKF units, such as the about the virus and its disease and although vaccines were developed in record-breaking CellenOne Single Cell Analysis in the Genomics Facility, fluorescence lifetime imaging time, there are still many restrictions in place that negatively impact scientific and clinical in the Two-Photon Imaging Facility, and the CRISPR/Cas technology in the Transgenic work as well as social life. Many researchers are still working in home offices together Service Facility. with their children who cannot attend classes in person. Our social life is limited to videoconferences. We cannot visit our parents or grandparents because we must prevent Also in 2020, the terms of some of the members of the IZKF Steering Committee came to transmission of the virus to those at risk. International travel, in-person conversations, and an end and new colleagues joined the board. In addition, the speaker’s term ended and the meetings with friends and colleagues are not possible. Even those of us who have been Medical Faculty decided that Professor René Tolba, Professor Marc Spehr and I would be strictly following the rules, convinced that it is the right thing to do, are getting tired after allowed to continue with our work for IZKF Aachen. more than a year of physical distancing. Throughout this difficult time the staff of the IZKF administrative office, Karen De Bruyne, But life is not standing still. Somehow things are moving forward and progress is being Sünje Frahm and Sandra Majewski, did a fantastic job to keep the wheels turning and to made. IZKF Aachen has funded two new collaborative projects: Professor Lampert find creative solutions even when the road was blocked and the exit was not visible. I am is managing the project on Sodium channel-related pain disorders, representing the deeply thankful. Neuroscience focus of the Medical Faculty, and Professor Wolf is leading the project on Regulatory networks controlling the soft tissue-alveolar bone crosstalk in periodontal It is my sincere hope and strong wish that my next report, describing IZKF Aachen’s disease, representing the Organ Crosstalk focus. achievements in 2021, will start with something like… “It is over…”

IZKF Aachen is now running eight core facilities. Each of the core facilities was successfully Fingers crossed, take care, and stay healthy. reviewed by several external experts, making it possible to continue their funding. However, Yours faithfully, organizational changes were necessary in the Genomics Facility, and a new concept was developed by Professor Ingo Kurth. This concept is now under evaluation to prepare for a new funding period. When mentioning the Genomics Facility at this point, it is also my sad, sad duty to announce that Dr. Bernd Denecke, the core facility manager of the Genomics Facility, a part of IZKF right from the beginning, passed away after suffering from Professor Dr. Peter Walter a serious illness. He developed the Genomics Facility from the early days of the IZKF into a on behalf of the Steering Committee of IZKF Aachen

4 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 5 Imprint Contents

Contents

Preface p. 4 Imprint p. 6

1. Annual Report p. 8 1.1 The IZKF in 2020 p. 8 1.2 Funding Measures in a Nutshell p. 11 1.3 Funding and Financial Overview p. 12 1.4 Participating Institutes and Clinics p. 14 1.5 Output and Evaluation p. 17

2. Function and Goals p. 18

3. Core Facilities p. 20 3.1 Using Institutes and Clinics p. 22 Progress Report 2020 3.2 Core Laboratory p. 26 3.3 Genomics Facility p. 29 3.4 Immunohistochemistry Facility p. 32 Publisher 3.5 Confocal Microscopy Facility p. 34 Interdisciplinary Centre of Clinical Research (IZKF Aachen) 3.6 Brain Imaging Facility p. 37 Speaker: Prof. Peter Walter 3.7 Two-Photon Imaging Facility p. 43 Pauwelsstraße 30 3.8 Transgenic Service p. 48 52074 Aachen 3.9 Proteomics Facility p. 52 3.10 Flow Cytometry Facility p. 56 Phone: +49 241 80 80034 Email: [email protected] www.izkf-aachen.de 4. Projects p. 60 Thematically based on the Main Research Focus Areas of the Faculty of Medicine Editor 4.1 Phase Transition in Disease p. 60 Karen De Bruyne M.A. (administration office) 4.2 Translational Neurosciences p. 94 4.3 Organ Crosstalk p. 120 Layout Nicole Miehlke, Stolberg 5. Research Groups p. 138 Cover 5.1 J. Di Russo: Retinal Epithelium Mechanobiology and Disease p. 140 Peter Winandy, Aachen 5.2 E. van der Vorst: Immune-Lipid Crosstalk p. 143 5.3 B. Namer: Different functional roles of nociceptor subclasses in human p. 146 Printing frank druck+medien GmbH & Co. KG, Aachen Appendix p. 152 Print Run 180 copies Articles of Association

Aachen, April 2021

The project leaders are responsible for the content of their reports and for the information on their external funding, publications, etc.

6 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 7 1.1 | The IZKF in 2020 1.1 | The IZKF in 2020

The hiring of Dr. Poznansky, a physicist with significant experience in developing MR The Interdisciplinary Center for Clinical sequences, will greatly improve the quality of methodological services in the Brain Imaging Research (IZKF) in 2020 Facility. One very pleasing development was the approval of investment funds from the DFG to purchase a multiphoton laser scanning microscope with FLIM for the Two-Photon Imaging Start of two Joint Research Project Proposals (2020-2023) Facility. Thanks to this approval, the facility will boast state-of-the-art equipment in 2021. On the basis of the external review process of the previous year the Steering Committee Prof. Tolba, Dr. Vogt, Prof. van Zandvoort and Prof. Martin were responsible for the unanimously decided to fund the following two joint projects for three years: successful grant application. With the hiring of Dr. Kapsokalyvas, we were able to expand our methodological expertise in the area of highly developed light microscopes, especially in the area of in vivo imaging in Funding start Joint Research Project Research Focus Area Speaker small animals and fluorescence lifetime imaging. 1.3.2020 Sodium channel-related pain disorders Translational Neurosciences Prof. A. Lampert We were also very pleased about the good news that the funding application for an 1.11.2020 Regulatory networks controlling the soft tissue- Organ Crosstalk Prof. M. Wolf investment in a 5-laser cell sorter for the Flow Cytometry Facility, organized by Prof. Pabst, alveolar bone crosstalk in periodontal disease was approved by the DFG. This high-end sorter will allow us to sort human cells and undertake important research projects. This further development of our equipment and methodology would not have been possible External evaluation process and further funding for existing Core Facilities without the department’s active support, since the IZKF budget does not cover the The Core Facilities were evaluated by two external reviewers with experience in the Core financing to acquire research equipment. Facility management. The comprehensive, individual reviews contained constructive The new large research devices will be put into operation throughout the year in 2021. criticism with suggestions for improvement, and also certified the facility‘s technical equipment with a good to very good rating. After the external reviews, the steering Election of the Steering Committee committee confirmed further funding for the Core Facilities for another three years The Speakers for the faculty research focus areas were requested to nominate members for according to their applications. The significance of the central services for faculty research the new IZKF Steering Committee. A Focus Area Coordinator and Deputy were proposed was reaffirmed: It is increasingly important to promote cooperation between groups within for each research focus area. Focus Area Coordinators do not receive IZKF funding the faculty which make use of the same equipment and techniques. themselves.

Restructuring of the Genomics Facility Core Facility Managers were also requested to select a Deputy from among their colleagues During the last year the Genomics Facility faced substantial managing difficulties. The Core to be assigned to the Steering Committee. Prof. Müller-Newen was re-elected as the Core Facility has been led provisionally by Prof. Ingo Kurth, Director of the Institute of Human Facilities Representative. Genetics. The scientific position and one technical position in the wet-lab remained vacant for a very long time. Due to an unclear perspective of the Facility the Steering Committee decided to postpone the external evaluation for a year, in order to rethink the direction of the Facility within the faculty. After intense discussions by the faculty and the users, the importance of Next Generation Sequencing technologies was emphasized and a restructuring of the Genomics Facility has been decided. In order to establish continuity and to make use of the expertise of human genetics, Prof. Kurth is to be appointed continuously as head of the Genomics Facility. The external evaluation is taking place in 2021.

Further development of equipment and methodology in the Core Facilities In the past year, the Core Facilities have seen very positive further development thanks to the hiring of important methodology experts and the prospect of acquiring advanced equipment.

The Genomics Facility has purchased the CellenOne F4.1 System, that can be used in combination with the iCell8 system (Takara) which is already available at the faculty. The Genomics Facility will now be able to cover the entire methodical spectrum of the genome wide single cell analyses.

Dr. Marks joined Transgenic Service as an experienced expert who can establish the high- demand CRISPR/Cas9 technology on site.

8 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 9 1.1 | The IZKF in 2020 1.2 | Funding Measures in a Nutshell

The newly elected Steering Committee consists of: Funding Measures in a Nutshell

Prof. Dr. P. Walter IZKF Speaker Project Funding Prof. Dr. R. Tolba IZKF Deputy Speaker 35 projects Prof. Ruth Knüchel-Clarke Focus Area Coordinator for Medicine Technology & Digital Life Sciences (40 % of the total expenses during the report period) Prof. Dr. N. Marx Focus Area Coordinator for Organ Crosstalk

Prof. Irene Neuner Focus Area Coordinator for Translational Neurosciences Research Groups

Prof. Dr. M. Hornef Focus Area Coordinator for Phase Transition in Disease 3 Research Groups

Prof. Dr. M. Spehr Representative of the Faculties of Engineering and Natural Sciences (14 % of the total expenses during the report period)

Prof. Dr. G. Müller-Newen Core Facilities Representative Core Facilities / Laboratory Prof. Dr. Julia C. Stingl Deputy Focus Area Coordinator for Medicine and Technology Core Laboratory Prof. Dr. O. Pabst Deputy Focus Area Coordinator for Organ Crosstalk/ Speaker SFB 1382 Genomics Facility Prof. Dr. J. Schulz Deputy Focus Area Coordinator for Translational Neurosciences Immunohistochemistry Facility Prof. Dr. E. Stickeler Deputy Focus Area Coordinator for Phase Transition in Disease Confocal Microscopy Facility Prof. Dr. S. Uhlig Dean of the Faculty of Medicine Brain Imaging Facility Prof. Dr. C. Trautwein Speaker TRR (guest member of the Steering Committee 2009-2020) Two-Photon Imaging Facility Prof. Dr. J. Jankowski Speaker SFB/TRR219 (guest member of the Steering Committee 2018-2021) Transgenic Service

Proteomics Facility

Election of the Speaker and the Deputy Speaker of the IZKF by the Faculty Board, Flow Cytometry Facility December 7th During the faculty meeting on December 7th, Prof. Peter Walter was re-elected as the Speaker of the IZKF by the Faculty Board for another three-year period. Prof. René Tolba was re-elected as the Deputy Speaker for the next three years. Prof. Spehr was (42 % of the total expenses during the report period) unanimously re-elected as the Representative of the Faculties of Engineering and Natural Sciences. 4 % of the total expenses during the report period was spent for the scientific coordinating office and central costs.

10 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 11 1.3 | Funding and Financial Overview 1.3 | Funding and Financial Overview

Research Groups Funding and Financial Overview In 2020, 40 % of the overall expenses of € 582,906 the IZKF was spent on project funding, 14 % 14 % on funding research groups and 41 % Project Funding In 2020 the IZKF received € 3,175,380 from the State Grant for Research and Teaching as on core facilities. A further 4 % was spent € 1,589,733 fixed in the faculty budget. It also received a faculty grant of € 1,227,289. The total budget on the administration office and 1 % on the 40 % amounted to € 4,402,669. IZKF core laboratory.

Core Facilities € 1,670,024 Budget and Expenses 2020 41 % Administration Office € 151,080 Budget 2020 € 4,402,669 4 %

Core Laboratory Total Expenses 2020 € 4,026,682 € 32,939 1 %

Projects € 1,589,733 Spreading of Expenses € 4,026,682 Phase Transition in Disease € 757,282 Translational Neurosciences (from 01.03.2020) € 649,459 Organ Crosstalk (from 01.11.2020) € 131,329 Organ Crosstalk, Single Project € 51,663

Flow Cytometry Facility Proteomics Facility Research Groups € 582,906 € 117,286 € 214,796 Immune-Lipid Crosstalk (van der Vorst) € 191,717 7 % Roles of Nociceptor Subclasses (Namer) € 189,812 Core Facilities were funded with 13 % Genomics Facility Retinal Epithelium Mechanobiology and Disease (Di Russo) € 201,377 € 1,670,024 in total. € 294,400 Two-Photon Imaging Facility 18 % € 177,398 Core Facilities € 1,670,024 10 % Immunohistochemistry Genomics Facility € 294,400 Facility € 227,631 Immunohistochemistry Facility € 227,631 Transgenic Service 14 % Confocal Microscopy Facility € 75,270 € 132,625 Brain Imaging Facility € 430,618 8 % Transgenic Service € 132,625 Confocal Microscopy Facility Two-Photon Imaging Facility € 177,398 € 75,270 Proteomics Facility € 214,796 Brain Imaging Facility 4 % Flow Cytometry Facility € 117,286 € 430,618 26 %

Administration Office and Core Laboratory € 184,019 Administration Office € 151,080 Core Laboratory € 32,939 Funding Core Facilities € 1,670,024

12 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 13 1.4 | Participating Institutes and Clinics 1.4 | Participating Institutes and Clinics

Teaching and Research Area Experimental Neuropsychiatry Participating Institutes and Clinics Teaching and Research Area Psychopathology Teaching and Research Area Experimental Behavioral Psychobiology Teaching and Research Area Structural Functional Brain Mapping Clinical Departments 2020 Teaching and Research Area Structure of Cortical Functional Units Department of Anaesthesiology Teaching and Research Area Functionality of Cortical Circuits Internal Medicine I Teaching and Research Area Neuropsychological Gender Studies Department of Cardiac and Thorax Surgery Department of Oral Maxillofacial and Plastic Facial Surgery X Teaching and Research Area Child Cardiac Surgery Department of Radiotherapy Department of Child and Adolescent Psychiatry Department of Surgery Teaching and Research Area Clinical Child and Adolescent Neuropsychology Department of Urology Department of Dental Preservation Department of Vascular Surgery Teaching and Research Area Oral Microbiology and Immunology X Internal Medicine II X Department of Dermatology Internal Medicine III X Department of Diagn. and Interv. Radiology Internal Medicine IV X Department of Diagn. and Interv. Neuroradiology Internal Medicine V Department of General, Visceral and Transplant Surgery Internal Medicine VI Department of Gynaecology and Obstetrics Surgical Intensive Care Teaching and Research Area Prenatal Medicine Institutes Department of Gynaecological Endocrinology and Reproductive Medicine Institute of Aerospace Medicine Department of Neurosurgery Institute of Applied Microbiology Department of Neurology X Institute of Anatomy and Cell Biology X Teaching and Research Area Clinical Cognition Institute of Biochemistry and Molecular Biology X Teaching and Research Area Neuropsychology Institute of Biochemistry and Molecular Cell Biology Department of Nuclear Medicine X Institute for Biochemistry and Molecular Immunology Department of Ophthalmology Helmholtz-Institute for Biomedical Engineering Teaching and Research Area Experimental Ophthalmology (Applied Medical Engineering) Department of Orthodontics X Teaching and Research Area Rehabilitation and Prevention Engineering Department of Orthopaedics X Teaching and Research Area Cardiovascular Engineering Department of Trauma Surgery X Teaching and Research Area Tissue Engineering and Textile Implants Department of Otorhinolaryngology, Plastic Head, Neck Surgery Helmholtz-Institute for Biomedical Engineering X Department of Paediatric Medicine X (Experimental Molecular Imaging) Teaching and Research Area Neonatological Intensive Care Helmholtz-Institute for Biomedical Engineering (Cell Biology) X Department of Paediatric Cardiology Teaching and Research Area Stem Cell Biology X Department of Palliative Medicine X Helmholtz-Institute for Biomedical Engineering (Biointerface) X Department of Phoniatrics, Pedaudiology and Communication Disorders Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry X Department of Plastic, Hand and Burns Surgery X Institute for Computational Genomics X Department of Prosthodontics and Dental Materials X Institute of History, Theory and Ethics in Medicine Teaching and Research Area Dental Materials and Biomaterials Research X Institute of Human Genetics X Department of Psychiatry X Institute for Hygiene and Environmental Medicine

14 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 15 1.4 | Participating Institutes and Clinics 1.5. | Output and Evaluation

Institute of Immunology Institute of Laboratory Animal Science Output and Evaluation Institute of Medical Psychology und Medical Sociology

Institute of Medical Informatics 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 Institute of Medical Statistics Publications* Articles 60 81 49 61 101 73 147 93 144 170 140 67 114 118 Institute of Medical Microbiology Scientific Diploma degrees** theses Teaching and Research Area Virology 6 3 7 10 15 8 18 13 14 26 19 7 4 2 Bachelor/ Institute of Molecular and Cellular Anatomy Master Institute for Molecular Cardiovascular Research X Doctoral 5 12 9 9 15 10 12 15 13 14 28 25 4 9 Institute of Molecular Medicine theses Institute of Neuroanatomy Postdoctoral Lecture 2 0 3 4 3 2 1 - - - 3 0 1 0 Institute of Neuropathology Qualification Institute of Pharmacology and Toxicology X Teaching and Research Area Pharmacology Teaching and Research Area Pharmacology of Inflammation Institute of Pathology X Teaching and Research Area Tumour Pathology Institute of Physiology X Institute of Medical Psychology and Medical Sociology External Cooporations DWI – Leibniz Institute for Interactive Materials AICES - Aachen Institute for Advanced Study in Computational Engineering Science Biology II, Faculty 1, RWTH Aachen University X

* IZKF support must be mentioned in publications and presentations. Publications are relevant to the IZKF if the support is mentioned in the acknowledgement and/or in the affiliation. The number of publications fluctuates year to year and depends on which stage the project is in during the funding period.

** Information about diploma theses and doctoral theses can be found in the project reports.

16 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 17 2 | General Information | Function and Goals 2 | General Information | Function and Goals

Joint Projects Function and Goals of the IZKF Thematically directed towards the faculty‘s research focus areas The IZKF places utmost importance on funding high-quality innovative joint research projects. All project applications are subject to an external peer review process, that guarantees the credibility of the underlying science and fosters a transparent and verifiable allocation of funds. The review process results in a high level of acceptance among the applicants and in the faculty of medicine. Furthermore, the funded projects are subject to a result-oriented internal evaluation.

Research Groups Group management for young, excellent researchers The IZKF offers an attractive funding possibility for scientists with outstanding achievements through its research groups: The research groups are assigned to an institute or department and independently work there. The heads of the groups possess excellent scientific qualifications and experience in acquiring external funding. They are as well externally reviewed on the basis of their scientific excellence, strategical fitting accuracy and their potential to strengthen the faculty´s research focus areas.

Core Facilities Technologies, equipment and expertise for ambitious research goals Making research infrastructure available to the community presents an enormous challenge to both facility managers and administration. The faculty-wide platforms overcome institutional boundaries, adhere to high quality standards and provide access to state-of-the-art technology by centralized acquisition and clearly defined user agreements and operator regulations. Function and goals of the Interdisciplinary Centre for Clinical Research

• Strengthening Research

• Funding Excellence

• Networking by Infrastructure

• Convincing by Quality

The Interdisciplinary Center for Clinical Research (IZKF) is the development and strategy program of the RWTH Aachen faculty of medicine, with the task to strengthen translational medical research that stems from its basic research and clinic: By supporting top-quality projects, it strives to greatly improve the chances for solving major and highly challenging problems in biomedicine and life science.

The IZKF functions as an initiator with vision: funding tools are modified in accordance with current requirements, thereby exploring different approaches. The IZKF has a major influence on the research portfolio of the faculty of medicine. By specifically defining the research focus areas, the IZKF concentrates expertise and provides the basis for the acquisition of thirdparty-funded collaborations. It represents well established structures that are in an excellent position to face the current and future challenges of clinical research.

18 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 19

Using Institutes and Clinics p. 22

Core Laboratory p. 26

Genomics Facility p. 29 Immunohistochemistry Facility p. 32 CORE FACILITIES Confocal Microscopy Facility p. 34

Brain Imaging Facility p. 37

Two-Photon Imaging Facility p. 43

Transgenic Service p. 48

Proteomics Facility p. 52

Flow Cytometry Facility p. 56

20 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 21 Core Facilities | Using Institutes and Clinics Core Facilities | Using Institutes and Clinics

Using Institutes and Clinics

Clinical Departments Core Facilities Department of Prosthodontics and Dental Materials Immunohistochemistry Facility, Confocal Microscopy Facility, Two-Photon Imaging Facility, Flow Cytometry Facility Clinic of Accident and Reconstructive Surgery Immunohistochemistry Facility, Flow Cytometry Facility Teaching and Research Area Dental Materials and Biomaterials Research Department of Anaesthesiology Immunohistochemistry Facility, Two-Photon Imaging Facility Department of Psychiatry Brain Imaging Facility Clinic for Cardiology, Pneumology, Angiology and Internal Intensive Medicine (Med I) Genomics Facility, Immunohistochemistry Facility, Confocal Microscopy Facility, Two-Photon Imaging Facility, Proteomics Facility, Flow Cytometry Facility Teaching and Research Area Experimental Neuropsychiatry Department of Cardiac and Thorax Surgery Teaching and Research Area Psychopathology Teaching and Research Area Child Cardiac Surgery Teaching and Research Area Experimental Behavioral Psychobiology Department of Child and Adolescent Psychiatry Brain Imaging Facility Teaching and Research Area Structural Functional Brain Mapping Teaching and Research Area Clinical Child and Adolescent Neuropsychology Teaching and Research Area Structure of Cortical Functional Units Department of Dental Preservation Brain Imaging Facility Teaching and Research Area Functionality of Cortical Circuits Teaching and Research Area Oral Microbiology and Immunology Teaching and Research Area Neuropsychological Gender Studies Department of Dermatology Genomics Facility, Immunohistochemistry Facility, Transgenic Service Department of Oral Maxillofacial and Plastic Facial Surgery Immunohistochemistry Facility Department of Diagn. and Interv. Radiology Immunohistochemistry Facility, Brain Imaging Facility Department of Radiotherapy Department of Diagn. and Interv. Neuroradiology Brain Imaging Facility Department of Surgery Department of General, Visceral and Transplant Surgery Immunohistochemistry Facility, Transgenic Service, Proteomics Facility Department of Urology Department of Gynaecology and Obstetrics Genomics Facility, Confocal Microscopy Facility, Two-Photon Imaging Facility, Department of Vascular Surgery Immunohistochemistry Facility Flow Cytometry Facility Internal Medicine II Genomics Facility, Immunohistochemistry Facility, Confocal Microscopy Facility, Teaching and Research Area Prenatal Medicine Two-Photon Imaging Facility, Transgenic Service, Flow Cytometry Facility Department of Gynaecological Endocrinology and Reproductive Medicine Internal Medicine III Genomics Facility, Immunohistochemistry Facility, Transgenic Service, Proteomics Facility, Flow Cytometry Facility Department of Neurosurgery Immunohistochemistry Facility, Brain Imaging Facility, Two-Photon Imaging Facility Internal Medicine IV Genomics Facility, Immunohistochemistry Facility, Confocal Microscopy Facility, Proteomics Facility, Flow Cytometry Facility Department of Neurology Confocal Microscopy Facility, Brain Imaging Facility, Transgenic Service, Proteomics Facility Internal Medicine V Teaching and Research Area Clinical Cognition Internal Medicine VI Teaching and Research Area Neuropsychology Surgical Intensive Care Confocal Microscopy Facility Department of Nuclear Medicine Immunohistochemistry Facility, Brain Imaging Facility Institutes Department of Ophthalmology Genomics Facility, Confocal Microscopy Facility, Two-Photon Imaging Facility Institute of Aerospace Medicine Teaching and Research Area Experimental Ophthalmology Institute of Applied Microbiology Department of Orthodontics Flow Cytometry Facility Institute of Anatomy and Cell Biology Immunohistochemistry Facility, Two-Photon Imaging Facility, Transgenic Service Department of Orthopaedics Immunohistochemistry Facility Institute of Biochemistry and Molecular Biology Genomics Facility, Confocal Microscopy Facility, Proteomics Facility, Flow Cytometry Facility Department of Trauma Surgery Institute of Biochemistry and Molecular Cell Biology Confocal Microscopy Facility, Proteomics Facility Department of Otorhinolaryngology, Plastic Head, Neck Surgery Institute for Biochemistry and Molecular Immunology Genomics Facility, Immunohistochemistry Facility, Transgenic Service, Department of Paediatric Medicine Genomics Facility, Immunohistochemistry Facility, Flow Cytometry Facility Proteomics Facility Teaching and Research Area Neonatological Intensive Care Helmholtz-Institute for Biomedical Engineering Immunohistochemistry Facility, Two-Photon Imaging Facility, Proteomics Facility Department of Paediatric Cardiology (Applied Medical Engineering) Department of Palliative Medicine Teaching and Research Area Rehabilitation and Prevention Engineering Department of Phoniatrics, Pedaudiology and Communication Disorders Brain Imaging Facility, Teaching and Research Area Cardiovascular Engineering Department of Plastic, Hand and Burns Surgery Immunohistochemistry Facility, Two-Photon Imaging Facility, Proteomics Facility, Teaching and Research Area Tissue Engineering and Textile Implants

22 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 23 Core Facilities | Using Institutes and Clinics Core Facilities | Using Institutes and Clinics

Helmholtz-Institute for Biomedical Engineering Genomics Facility, Confocal Microscopy Facility, Two-Photon Imaging Facility, External User (Experimental Molecular Imaging) Flow Cytometry Facility DWI – Leibniz Institute for Interactive Materials Immunohistochemistry Facility Helmholtz-Institute for Biomedical Engineering (Cell Biology) Genomics Facility, Two-Photon Imaging Facility, Transgenic Service, Flow AICES - Aachen Institute for Advanced Study in Computational Engineering Science Cytometry Facility Department of Ceramics and Refractory Proteomics Facility Teaching and Research Area Stem Cell Biology Genomics Facility, Confocal Microscopy Facility, Two-Photon Imaging Facility Materials, Faculty 5, RWTH Aachen University Helmholtz-Institute for Biomedical Engineering (Biointerface) Immunohistochemistry Facility, Confocal Microscopy Facility, Two-Photon Biology I, Faculty 1, RWTH Aachen University Genomics Facility Imaging Facility, Transgenic Service Biology II, Faculty 1, RWTH Aachen University Genomics Facility, Proteomics Facility Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Genomics Facility, Immunohistochemistry Facility Chemistry Biology III, Faculty 1, RWTH Aachen University Proteomics Facility Institute for Computational Genomics Biology V, Faculty 1, RWTH Aachen University Genomics Facility, Immunohistochemistry Facility, Proteomics Facility Institute for Geriatric Medicine Immunohistochemistry Facility Fraunhofer Institute for Molecular Biology and Applied Ecology Proteomics Facility, Flow Cytometry Facility Institute of History, Theory and Ethics in Medicine Institute of Human Genetics Genomics Facility, Immunohistochemistry Facility, Two-Photon Imaging Facility, Flow Cytometry Facility Institute for Hygiene and Environmental Medicine Institute of Immunology Flow Cytometry Facility Institute of Laboratory Animal Science Immunohistochemistry Facility Institute of Medical Psychology und Medical Sociology Institute of Medical Informatics Institute of Medical Statistics Institute of Medical Microbiology Genomics Facility, Immunohistochemistry Facility, Transgenic Service, Flow Cytometry Facility Teaching and Research Area Virology Institute of Molecular and Cellular Anatomy Transgenic Service, Flow Cytometry Facility Institute for Molecular Cardiovascular Research (IMCAR) Genomics Facility, Immunohistochemistry Facility, Two-Photon Imaging Facility, Transgenic Service, Flow Cytometry Facility Institute of Molecular Medicine Genomics Facility, Immunohistochemistry Facility, Transgenic Service, Flow Cytometry Facility Institute of Neuroanatomy Genomics Facility, Flow Cytometry Facility Institute of Neuropathology Transgenic Service Institute for Occupational and Social Medicine Proteomics Facility Institute of Pharmacology and Toxicology Genomics Facility, Immunohistochemistry Facility, Transgenic Service, Proteomics Facility Teaching and Research Area Pharmacology Teaching and Research Area Pharmacology of Inflammation Institute of Pathology Genomics Facility, Immunohistochemistry Facility, Two-Photon Imaging Facility, Transgenic Service, Proteomics Facility, Flow Cytometry Facility Teaching and Research Area Tumour Pathology Institute of Physiology Genomics Facility, Immunohistochemistry Facility Institute of Medical Psychology and Medical Sociology

24 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 25 Core Facilities | Core Laboratory Core Facilities | Core Laboratory

Equipment and contact persons Core Laboratory All equipment and laboratories can be used upon request and after having consulted the IZKF administration office and the responsible contact person.

Head of the Core Laboratory: Materials: € 23,330 De Bruyne, K. Investments/Equipment: € 9,609 Microscopes Wüffel, J. / Ensslen S. Safety delegate / assistant project supervisor for genetic engineering security: Preisinger, C. Western Blot Documentation System LAS Preisinger, C. / Gostek, S. 3000 Project supervisor for genetic engineering security: Preisinger, C. Agilent Bioanalyser 2100 Hübner, J. / Rudzinski, A.

7300 Real Time PCR Taq Man Hübner, J. / Rudzinski, A. Services In the Core Laboratory various equipment is provided, that is not correlated to any Core Fluorostar Optima Gostek, S. / Johnen, S. Facility. Like the Core Facilities, the equipment is not only available for IZKF project heads or project personnel, but for all the members of the Faculty of Medicine. The equipment Nanodrop Hübner, J. / Rudzinski, A. also can be used upon request by RWTH scientists. The Core Laboratory provides infrastructure, lab space and know-how. The personnel of the Core Laboratory provide Photometer Tappe, M. / Wüffel, J. services for research, assistance to use the equipment and advises in all scientific issues. The concept aims at handling organizational and technical-methodical challenges and HPLC/FPLC Preisinger, C. / Gostek, S. assisting the project personnel. Furthermore the centralization of equipment and expertise aims at conserving resources. Synergetic effects emerge from the collaboration of MALDI-TOF System Preisinger, C. / Gostek, S. researchers and Core Facilities and these effects are useful for the two-way technical- methodical assistance that keeps research and service at the highest level. Geldoc – DNA and Protein Ensslen, S. / Gan, L.

Autoclaves Tappe, M. / Wüffel, J. Users of the Core Laboratory In 2020 the Core Laboratory was used by 308 employees of 32 institutes and clinics. MilliQ Equipment Wüffel, J.

Med III Cell Culture Laboratory Gostek, S. / Preisinger, C. Med II Pathology Med I Constant Temperature Laboratory Hübner, J. ZWBF IZKF Immunology Ophtalmology Other IBMT-Cellbiology Paediatrics IMCAR AME LFG ZMG Plastic Surgery Humangenetics Occupational Medicine Orthopaedics Orthodontics Dermatology General Surgery Biochemistry Med. Microbiology Helmholtz Stemcell Pharmacology & Toxicology Neuropathology Neurology Mol. Pathobiochemistry Med IV Vascular Surgery Radiology Neuroradiology

0 5 10 15 20 25 30 35 40

26 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 27 Core Facilities | Core Laboratory Core Facilities | Genomics Facility

Usage of the equipment Following overview shows a selection of the equipment of the Core Laboratory and their usage in 2020: Genomics Facility

Equipment Using Clinics/Institutes Total appointments Head of the facility Staff: Kurth, I. (Commissary) Abdallah, A. (Bioinformatics) Nanodrop 15 14.038 samples Gan, L. (Bioinformatics) Head of Bioinformatics Hübner, J. (Technician) Agilent Bioanalyzer 3 96 Abdallah, A. Rudzinski, A. (Technician)

Taq Man 6 216 Advisory Board Costa, I. (Institute of Computational Biology) Materials/Travel expenses 2020: € 194,493 Light Cycler 1 99 Eggermann, T. (Institute of Human Genetics) Zenke, M. (IBMT – Cell Biology) Investments/Equipment 2020: € 152,896 Autoclaves 11 493 Kramann, R. (Institute for Experimental Internal Medicine and Systems Biology) Revenues 2020: € 286,328 LAS3000 (Western Blot) 8 207 Skills/Services/Training courses/Consulting service Cell Culture Laboratory 4 448 The Genomics Facility provides wet lab and bioinformatics expertise to coordinate competencies and produce high-quality support. In the following, we list the essential Microscopes 20 373 services and fields of support:

Spectramax 9 302 A) Experimental design: We discuss the factors with our users, which could affect the experimental design and provide individual support and assistance. To ensure high- quality experiments, we also go through numerous steps with researchers and assess the advantages and disadvantages of concrete applications/ methods. B) Sample preparation: We use starting material from cell culture, tissue, or blood to extract total RNA, mRNA, miRNA, DNA, etc.. The sample preparation service enables scientists to implement the newest NGS applications and array technologies in their projects. For DNA fragmentation we use Covaris Technology. Quality control, quantification including analyses of DNA, RNA, and protein is done using the 2100 Bioanalyzer/Tape Station 4200 microfluidics-based platforms (Agilent), quantification using the NanoDrop technology and Quantus (Promega). During the amplification/labeling procedures, we monitor all steps by various analyses and controls. Finally, we offer DNA/RNA library preparation for NGS at various scales of input material. C) Data generation: We generate genomics data based on two technologies: (1) Array Hybridization Affymetrix microarray hybridization, washing steps, antibody-staining, and scanning the arrays according to SOPs. (2) NGS-Sequencing using the NextSeq500 Sequencing System from Illumina. D) Data analysis: We provide routine data analysis for established NGS applications and microarrays as optional direct support. Our primary analyses‘ scope depends on the nature of the application itself and the degree of automation. We also offer tailored solutions on a collaborative basis to project specific issues. Advanced analysis can be, e.g., new cutting- edge applications and technologies, advanced data analysis methods, data visualization, and method optimization. Additionally, we offer project consulting and support by assisting researchers through all research phases. We complete these services by managing raw data, results, tools, and procedures, which are especially essential for NGS data analysis, enabling access to data and analysis results through a web interface. Finally, we consult researchers in selecting and using established bioinformatics tools. E) Training and Seminars: We organize trainings for our user and co-organize the NGS user club.

28 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 29 Core Facilities | Genomics Facility Core Facilities | Genomics Facility

Users 115 users of 24 clinics/institutes of the medical faculty of the RWTH Aachen used the Genomics Facility. The Genomics Facilty had external user from the Institute of Biology II, III and V of Faculty 1 of the RWTH Aachen, the University Hospital Bonn, the UMC Maastricht and the Antbodies online GmbH.

Collaborations The Genomics Facility had project collaborations with the Medical clinic and polyclinic I - Laboratory for Innate Cellular Immunology and the Polyclinic for Orthodontics of the University of Bonn and the Biology II and V of the RWTH Aachen.

Publications Krenkel O, Hundertmark J, Abdallah AT, Kohlhepp M, Puengel T, Roth T, Branco DPP, Mossanen JC, Luedde T, Trautwein C, Costa IG, Tacke F (2020) Myeloid cells in liver and bone marrow acquire a functionally distinct inflammatory phenotype during obesity-related Status and Development steatohepatitis. Gut. 69(3):551-563. doi: 10.1136/gutjnl-2019-318382. [IF 17.943] In 2020, the Genomics Facility was able to further develop methodologically and in terms of equipment. We extended our wet lab infrastructure by the CellenOne F1.4 machine, Buhl EM, Djudjaj S, Klinkhammer BM, Ermert K, Puelles VG, Lindenmeyer MT, Cohen CD, a microplate-based single-cell isolation system with 4-fluorescent excitation channels, He C, Borkham-Kamphorst E, Weiskirchen R, Denecke B, Trairatphisan P, Saez-Rodriguez system-tailored single-cell application solutions, and compatible with other single-cell J, Huber TB, Olson LE, Floege J, Boor P (2020) Dysregulated mesenchymal PDGFR-β drives systems (iCell8) that are established at our Faculty. The synergy between these modern kidney fibrosis. EMBO Mol Med. 2(3):e11021. doi: 10.15252/emmm.201911021. [IF 10.293] systems is expected to greatly improve the support scope of cutting-edge genomics applications in the Faculty. This will contribute significantly to the performance of high- Baumeister J, Chatain N, Hubrich A, Maié T, Costa IG, Denecke B, Han L, Küstermann C, quality research including single-cell sequencing. Sontag S, Seré K, Strathmann K, Zenke M, Schuppert A, Brümmendorf TH, Kranc KR, Koschmieder S, Gezer D (2020) Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic Importantly, we are currently involved in many active collaborations in and outside of target in JAK2V617F-positive myeloproliferative neoplasms. Leukemia. 34(4):1062-1074. RWTH-Aachen. For instance, we are supporting projects involving data analysis of single- doi: 10.1038/s41375-019-0629-z. [IF 8.665] cell immune profiling and single-cell RNA-Seq with cell hash-tagging (HTO) from 10x, which are also applications at the cutting edge of the single-cell technology. Rose M, Maurer A, Wirtz J, Bleilevens A, Waldmann T, Wenz M, Eyll M, Geelvink M, Gereitzig M, Rüchel N, Denecke B, Eltze E, Herrmann E, Toma M, Horst D, Grimm T, We are also supporting a couple of additional NGS applications. For instance, the support Denzinger S, Ecke T, Vögeli TA, Knuechel R, Maurer J, Gaisa NT (2020) EGFR activity of 3’ mRNA-Seq started this year in productive mode and is anticipated to become a addiction facilitates anti-ERBB based combination treatment of squamous bladder cancer. constituent part of our NGS service repertoire. Due to the high sample multiplexing potential Oncogene. 39(44):6856-6870. doi: 10.1038/s41388-020-01465-y. [IF 7.791] of this application and the lower needed number of reads per sample, it has already been an option for several users. It is expected to ultimately replacing the corresponding Zechendorf E, O‘Riordan CE, Stiehler L, Wischmeyer N, Chiazza F, Collotta D, Denecke microarrays. Furthermore, we are now establishing CUT&RUN, a new NGS application, B, Ernst S, Müller-Newen G, Coldewey SM, Wissuwa B, Collino M, Simon TP, Schuerholz regarded as a cheap and more qualitative alternative to ChIP-Seq. T, Stoppe C, Marx G, Thiemermann C, Martin L. (2020) Ribonuclease 1 attenuates septic cardiomyopathy and cardiac apoptosis in a murine model of polymicrobial sepsis. JCI Regarding the quality of sequencing services, we have introduced the shallow sequencing Insight. 5(8):e131571. doi: 10.1172/jci.insight.131571. [IF 6.205] approach in a couple of use cases to optimize our single-cell sequencing workflow. Furthermore, we are now using sophisticated saturation analyses to compute needed reads Müller AK, Markert N, Leser K, Kämpfer D, Schwiy S, Riegraf C, Buchinger S, Gan L, for resequencing experiments accurately. The approach is saving project costs and time in Abdallah AT, Denecke B, Segnere H, Brinkmann M, Crawford SE, Hollert H. (2020) the long run. We are planning to extend the usage of this approach to most of our single- Bioavailability and impacts of estrogenic compounds from suspended sediment on rainbow cell projects. trout (Oncorhynchus mykiss). Aquat Toxicol. [In Press] [IF 4.344]

Consulting for NGS projects this year was mostly offered via virtual meetings. Often, we Vaughan D, Ritvos O, Mitchell R, Kretz O, Lalowski M, Amthor H, Chambers D, Matsakas have involved two scientists and sometimes one technical expert in these meetings. This A, Pasternack A, Collins-Hooper H, Ballesteros R, Huber TB, Denecke B, Widera D, allowed for an improved level of experimental design assistance. We could also speed up Mukherjee A, Patel K. Inhibition of Activin/Myostatin signalling induces skeletal muscle meetings appointment via MS TEAMS and have used the possibilities of virtual tools to hypertrophy but impairs mouse testicular development. (2020) Eur J Transl Myol. present our analyses and to better understand customer‘s study design. 30(1):8737. doi: 10.4081/ejtm.2019.8737. [IF 1.4]

30 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 31 Core Facilities | Immunohistochemistry Facility Core Facilities | Immunohistochemistry Facility

Status and Development Immunohistochemistry Facility Overall, services and the number of users have decreased in 2020 compared to 2019. We suspect that this is due to the Corona pandemic.

In 2020 (2019) 160 (213) researchers from 35 (32) different clinics/institutes of the medical Head of the facility Staff: faculty at the RWTH Aachen and 14 (18) researchers from 4 (5) extern institutes used the Ensslen, S. Ensslen, S. Immunhistochemistry Facility. The main users are from the Department of Internal Medicine Tappe, M. II and III. Advisory Board Wüffel, J. Ostendorf, T. (Medical Clinic II - Clinic for Renal and Hypertensive Disorders, A total of 18681 (20791) tissue samples were dehydrated and embedded. 14951 (16837) Rheumatological and Immunological Diseases) Materials/Travel expenses 2020: € 40,470 slices were produced and 3463 (4322) slides were stained. 551 (884) immunhistological Boor, P. (Pathology Institute) staining procedures were performed. Investments/Equipment 2020: € 6,969

Skills/Services/Training courses/Consulting service Revenues 2020: € 33,522 Histological studies are used in many fields such as research, diagnosis, and education to visualize the molecular and structural components of tissues and cells and to detect structural integrity or alterations of the integrity in diseased tissue. Besides classical histological staining, immunhistological staining is widely used in diagnosis and basic research to characterize cells and tissues.

Our Immunohistochemistry Facility offers competent guidance and support in planning and lead-through of histological and immunohistological experiments. The following service can be offered: • pre-experimental consulting • support to excision and preparation of samples and tissues (choice of proper fixatives) • dehydration and embedding of fixed tissues • preparation of tissue slices (paraffin and cryo) • histological staining (H&E, EvG, Giemsa, PAS etc.) • direct and indirect immunohistological staining • testing of antibodies (according to prior agreement) • microscopy and documentation (according to prior agreement)

The Team of the Immunohistochemistry Facility

32 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 33 Core Facilities | Confocal Microscopy Facility Core Facilities | Confocal Microscopy Facility

• 3-dimensional confocal images Confocal Microscopy Facility - Preparation of layered image stacks („z-stacks“) - Reconstruction of 3-dimensional objects

• Live-cell imaging under cell culture conditions (control of temperature and CO2).

Head of the facility Staff: Advanced microscopy Müller-Newen, G. (Institute of Biochemistry and Molecular Biology) Ernst, S. • Pre-experimental consulting and hands-on training • FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in Advisory Board photobleaching) to determine the mobility of fluorescently labeled molecules in living Lüscher, B. (Institute of Biochemistry and Molecular Biology) Materials/Travel expenses 2020: € 12,770 cells • FRET (Förster resonance energy transfer) to detect interaction of fluorescent Investments/Equipment 2020: € 9,290 molecules Skills/Services/Training courses/Consulting service • Use of photoconvertible and photoactivatable fluorescent proteins to determine Confocal microscopy generates detailed images from fluorescently labelled samples. Out- Revenues 2020 € 17,433 protein dynamics in living cells of-focus light that blurs the image in conventional microscopy cannot pass the confocal pinhole and is therefore eliminated. A 3-dimensional representation of a sample can be generated from serial optical sections. Confocal microscopy is performed on tissue Status and Development sections, fixed cultured cells or even with living cells containing an appropriate fluorescence With the beginning of 2021 the CMF has been part of the IZKF Core Facilities for more than label. Laser light is used for efficient excitation of fluorescently labelled samples. The nine years. In 2020, the CMF has been used by 17 institutes and clinics of the Medical various lasers of a confocal microscope can also be used as a precision tool to modulate Faculty. In most cases, more than one individual of the respective department was involved fluorescence in living cells with subcellular resolution. Based on this property, advanced as a user. Thus, the number of users exceeds 40. At least 11 papers have been published fluorescence techniques have emerged that can give access to intracellular dynamics and in 2020 with support of the CMF (refs. 1-11). interactions of fluorescently labelled biomolecules. In context of the Art. 91b GG application by Dr. Rafael Kramann and colleagues a new The Confocal Microscopy Facility (CMF) operates two confocal microscopes: Nikon A1-Ti2-N-STORM confocal & super-resolution microscope has been installed within the facility in May 2018. This microscope is used by the CMF (20%) and by the other • Zeiss LSM 710 (inverted) applicants of the proposal (80%). After some recurrent technical problems were solved - Laser lines 405, 458, 488, 514, 561 and 633 nm in 2019 by substitution of hardware components, the demanding technique of super- - Spectral detection unit resolution microscopy through single molecule localization will be further established in this - Motorized stage funding period (2020-2023). - Incubator for live-cell imaging Moreover, the installation of a new state-of-the art Zeiss LSM980 with AiryScan2 in addition • Nikon A1-Ti2-N-STORM (inverted) to the 11-year-old Zeiss LSM710 is a major goal for 2021. A proposal for the Zeiss LSM980 - Laser lines 405, 445, 488, 514, 561 and 647 nm has been submitted to the Dean of the Medical Faculty and will be approved by the - Spectral detection unit Planning and Allocation Committee of the Exzellenzinitiative at RWTH Aachen University. - Motorized stage - N-STORM unit for single molecule localization super-resolution microscopy (SMLM) Users within the faculty • All microscopes are located in an air-conditioned darkroom 40 users from 17 different clinics/institutes of the medical faculty of the RWTH Aachen used the Confocal Microscopy Facility.

The following services are offered by the CMF:

Confocal imaging Publications • Individual consulting on the application of confocal microscopy in research projects. Dohmen M, Krieg S, Agalaridis G, Zhu X, Shehata SN, Pfeiffenberger E, Amelang J, • Advice on sample preparation. Butepage M, Buerova E, Pfaff CM, Chanda D, Geley S, Preisinger C, Sakamoto K, Luscher • Individual hands-on training on the confocal microscope adapted to the depth of use. B, Neumann D, Vervoorts J (2020) AMPK-dependent activation of the Cyclin Y/CDK16 • 2-dimensional confocal images complex controls autophagy. Nat Commun 11, 1032 [IF 12.1] - Multi-channel images with up to 4 fluorophores - Exact overlay of fluorescence with differential interference contrast (DIC) images Hollmann J, Brecht J, Goetzke R, Franzen J, Selich A, Schmidt M, Eipel M, Ostrowska - Fluorescence intensity profiles A, Hapala J, Fernandez-Rebollo E, Muller-Newen G, Rothe M, Eggermann T, Zenke - Colocalization analysis M, Wagner W (2020) Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells. Stem Cell Res Ther 11, 105 [IF 5.1]

34 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 35 Core Facilities | Confocal Microscopy Facility Core Facilities | Brain Imaging Facility

Juhlen R, Martinelli V, Vinci C, Breckpot J, Fahrenkrog B (2020) Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts. Sci Rep 10, 19301 Brain Imaging Facility [IF 4.0]

Lin YY, Schuphan J, Dickmeis C, Buhl EM, Commandeur U, Fischer H (2020) Attachment of Ultralow Amount of Engineered Plant Viral Nanoparticles to Mesenchymal Stem Cells Head of the facility Staff: Enhances Osteogenesis and Mineralization. Adv Healthc Mater 9, e2001245 [IF 7.4] Rodriguez-Raecke, R. Rodriguez-Raecke, R. Poznansky, O. Moreno-Andres D, Yokoyama H, Scheufen A, Holzer G, Lue H, Schellhaus AK, Weberruss Advisory Board Schüppen, A. M, Takagi M, Antonin W (2020) VPS72/YL1-Mediated H2A.Z Deposition Is Required for Binkofski, F. C. (Department of Neurology) Ritter, C. Nuclear Reassembly after Mitosis. Cells 9 [IF 4.4] Mathiak, K. (Department of Psychiatry) Sijben, R. Habel, U. (Department of Psychiatry) Eder, G. Qiao Z, Wang W, Luo P, Hofman M, Horst K, Muller-Newen G, Greven J, Hildebrand F Wiesmann, M. (Department of Diagnostic and Interventional Neuroradiology) Richter, F. (2020) The Impact of Plasma-Derived Microvesicles From a Femoral Fracture Animal Model Nitzpon, L. on Osteoblast Function. Shock 53, 78-87 [IF 3.0] Fuchs, A.

Shi Y, Luo P, Wang W, Horst K, Blasius F, Relja B, Xu D, Hildebrand F, Greven J (2020) M1 But Not M0 Extracellular Vesicles Induce Polarization of RAW264.7 Macrophages Via the Materials/Travel expenses 2020: € 8,148 TLR4-NFkappaB Pathway In Vitro. Inflammation 43, 1611-1619 [IF 3.2] Investments/Equipment 2020: € 74,105 Woltje M, Brunler R, Bobel M, Ernst S, Neuss S, Aibibu D, Cherif C (2020) Functionalization of Silk Fibers by PDGF and Bioceramics for Bone Tissue Regeneration. Coatings 10 [IF 2.4] Revenues 2020: € 30,753

Yamoah A, Tripathi P, Sechi A, Kohler C, Guo H, Chandrasekar A, Nolte KW, Wruck CJ, Katona I, Anink J, Troost D, Aronica E, Steinbusch H, Weis J, Goswami A (2020) Aggregates of RNA Binding Proteins and ER Chaperones Linked to Exosomes in Granulovacuolar Degeneration of the Alzheimer‘s Disease Brain. J Alzheimers Dis 75, 139- 156 [IF 3.9]

Zaja R, Aydin G, Lippok BE, Feederle R, Luscher B, Feijs, KLH (2020) Comparative analysis of MACROD1, MACROD2 and TARG1 expression, localisation and interactome. Sci Rep 10, 8286 [IF 4.0]

Zechendorf E, O‘Riordan, CE, Stiehler L, Wischmeyer N, Chiazza F, Collotta D, Denecke The Team of the Brain Imaging Facility B, Ernst S, Muller-Newen G, Coldewey SM, Wissuwa B, Collino M, Simon TP, Schuerholz T, Stoppe C, Marx G, Thiemermann C, Martin L (2020) Ribonuclease 1 attenuates septic cardiomyopathy and cardiac apoptosis in a murine model of polymicrobial sepsis. JCI Insight 5 [IF 6.2] Skills/Services/Training courses/Consulting service The Brain Imaging Facility (BIF) is a central support unit for all neuroimaging research at the Faculty of Medicine of the RWTH Aachen University, providing technical and scientific support at all stages of research projects. The BIF offers these services to approximately 18 Applied and actual third-party funding different research groups from 10 clinical departments. The BIF works in tight collaboration with both the departments of Neurology and Psychiatry, who provide two research- S. Uhlig RWTH Life Sciences Core Facilities Exzellenzinitiative applied € 1,525,280 dedicated Siemens 3 Tesla MRI PRISMA scanners, equipped with 20-, 32-, and 64-channel G. Müller-Newen RWTH Aachen head coils as well as head coils for phosphorus and sodium imaging. The BIF provides C. Preisinger state-of-the-art MR compatible equipment such as visual (projectors, LC displays, goggle P. Walter systems) and auditory (both with and without active noise cancellation) stimulation devices, eye-trackers, response devices, physiological measurement hardware as well as hardware and software for real-time fMRI-based applications such as neurofeedback. In addition to assistance at the stage of project preparation, the BIF also supports data collection, storage, and analysis up to obtaining final results and publications.

36 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 37 Core Facilities | Brain Imaging Facility Core Facilities | Brain Imaging Facility

Data analysis and educational courses Thanks to their differing fields of expertise, BIF staff members can support data analysis within a broad spectrum of available methods and software packages. We also offer several theoretical courses related to MRI data analysis (SPM, Matlab, stimulation software). The premises of the BIF also comprise conference and office spaces as well as flexible research space for satellite experiments before and after MR imaging.

Status and development The previous year (2020) was difficult for our users, as many measurements had to be postponed due to the COVID-19 pandemic. During the first lockdown, no research MRI measurements could be performed and later on many restrictions were implemented, leading to a reduced number of measurements in 2020 compared to 2019 (Figure 1). The BIF supported various research projects and collaborative initiatives of the Faculty of Medicine at the RWTH Aachen University, including: Antipsychotic Induced Brain Changes (APIC), Psychiatric Imaging Network Germany (PING), International Research Training Group 2150 (IRTG2150), Human Brain Project (HBP), Jülich Aachen Research Alliance (JARA Brain), Enhancing Schizophrenia Prevention and Recovery through Innovative Treatments (ESPRIT), ERA-NET NEURON (MiGBAN and Cog-Stroke). The support included contribution to the setup of network hardware, MRI measurement protocols, design of experiments, data analysis, and data quality assessment.

We are very happy to announce that the BIF has now hired a very experienced MR physicist Preparation of neuroimaging projects to support MRI sequence optimsation. The BIF staff was further trained to support projects Prior to any measurements, all neuroimaging-related projects are presented and discussed using machine learning and big data, and related to this goal, the BIF implemented an at the “Neuroimaging Colloquium”, a seminar organized by the BIF that intends to facilitate XNAT entity for state-of-the-art research data management (RDM) to support Open Science collaboration and exchange of competences and ideas between diverse research groups. initiatives based on the FAIR principle (Findable – Accessible – Interoperable – Reusable). The BIF will provide all necessary infrastructure and services to integrate measured data as Data collection and data storage early as possible into a structured and supported RDM data cycle, which is consistent with The BIF provides a heterogeneous computer network for storage and analysis of research plans and adjustments of IRTG 2150 and several other users of the BIF, and established a data including 13 servers (physical and virtual) and 18 workstations for users (both Linux taskforce for RDM. The BIF also has a new website and a staff wiki for documentation. and Windows operating systems) with up-to-date software. The facility also offers secured Recently the BIF provided updated forms, terms and conditions. The MRI operators, the data storage including mandatory raw data storage and backup for at least 10 years as well advisory board and the BIF agreed on the possibility to apply for reduced fees for MRI as comprehensive data quality assurance, consistent with good scientific practice and data measurements. We intend to encourage young researchers with limited funding and protection regulations. projects in need of pilot measurements to be eligible to apply for external funding. The BIF now also offers support with writing proposals for research funding and facilitates Data quality assurance and optimization of sequence parameters cooperation by offering online meetings for researchers to explore possibilities for The BIF provides in-depth data quality assessment and advice for optimization of cooperation related to specific topics. Despite COVID-19 safety regulations, the BIF could sequence parameters. We regularly acquire data with different coils on both scanners and keep the bi-weekly neuroimaging colloquium by establishing an online format and also held analyse noise levels, also separately for different receive channels. When needed, our MR online educational courses throughout the year. physicist also provides information on how to optimise data acquisition, e.g. by finding the optimal flip angle for transmit coils to acquire diffusion weighted images (DWI). In order to The BIF purchased a third BIOPAC system to record psychophysiological parameters in minimize acquisition time, we started to use multi-band sequences for fMRI and DWI data the facility for behavioural experiments or in the MRI environment in addition to the other acquisition. These sequences are tested in combination with various acceleration factors systems. Moreover, storage extensions were purchased that are now being implemented to to find combinations which induce minimal image artefacts. We are currently developing further improve both the storage capacity for project data and an even more sophisticated new structural phantoms for gradient non-linearity correction. We also compared different backup regime. The stimulus presentation equipment at the MR scanners was also approaches for susceptibility based distortion correction (as implemented in FSL and SPM). updated with state-of-the art matrix switches to allow for seamless switching between different computers and screens. E.g., this facilitates feeding back physiological recordings to the screen inside the MR room.

38 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 39 Core Facilities | Brain Imaging Facility Core Facilities | Brain Imaging Facility

Publications Müschenich FS, Sichtermann T, Di Francesco ME, Rodriguez-Raecke R, Heim L, Singer M, Wiesmann M, Freiherr J (2020) Some like it, some do not: behavioral responses and central processing of olfactory-trigeminal mixture perception. Brain Structure and Function Online ahead of print. doi: 10.1007/s00429-020-02178-4. [IF 3.298]

Radke S, Jankowiak K, Tops S, Abel T, Habel U, Derntl B (2020) Neurobiobehavioral responses to virtual social rejection in females—exploring the influence of oxytocin. Social Cognitive and Affective Neuroscience Epub ahead of print. doi: 10.1093/scan/nsaa168. [IF 3.71] Figure 1: MRI measurements 2019 and 2020 Jungblut M, Mais C, Huber W, Binkofski FC, Schüppen A (2020) 5-year course of therapy-induced recovery in chronic non-fluent aphasia - Three single cases. Users within the faculty Cortex132:147-165. doi: 10.1016/j.cortex.2020.08.009. [IF 4.009] 39 users from 7 clinics/institutes of the medical faculty of the RWTH Aachen used the Brain Imaging Facility. The main user is the Clinic for Psychiatry, Psychotherapy and Sonderfeld M, Mathiak K, Häring GS, Schmidt S, Habel U, Gur R, Klasen M (2020) Psychosomatic Disorders. Supramodal neural networks support top-down processing of social signals. Human Brain Mapping Epub ahead of print. doi: 10.1002/hbm.25252. [IF 4.421]

External users Jo H-G, Wudarczyk O, Leclerc M, Regenbogen C Lampert A, Rothermel M, Habel U (2020) Effect of odor pleasantness on heat-induced pain: An fMRI study. Brain Imaging and Koten, J. Universität Graz, Institut für Psychologie, Universitätsplatz 2/DG, Behavior Epub ahead of print. doi: 10.1007/s11682-020-00328-0. [IF 3.391] 8010 Graz, Österreich Jütten K, Mainz V, Delev D, Gauggel S, Binkofski FC, Wiesmann M, Clusmann H, Na Jungblut, M. Institut für Interdisziplinäre Musik-und Sprachtherapie, C-H (2020) Asymmetric tumor-related alterations of network-specific intrinsic functional Am Lipkamp 14, 47269 Duisburg connectivity in glioma patients. Human Brain Mapping (16):4549-4561. doi: 10.1002/ hbm.25140. [IF 4.421] Merhof, D. Lehrstuhl für Bildverarbeitung RWTH Aachen Keller M, Pelz H, Perlitz V, Zweerings J, Röcher E, Baqapuri HI, Mathiak K (2020) Neural Kopernikusstraße 16 correlates of fluctuations in the intermediate band for heart rate and respiration are related 52074 Aachen to interoceptive perception. Psychophysiology 57(9):e13594. doi: 10.1111/psyp.13594. [IF 3.692]

Zvyagintsev M, Zweerings J, Sarkheil P, Bergert S, Baqapuri HI, Neuner I, Gaebler AJ, Collaborations Mathiak K (2020) Auditory mismatch processing: Role of paradigm and stimulus The Brain Imaging Facility had project collaborations with the Research Center Jülich, characteristics as detected by fMRI. Biological Psychology 154:107887. doi: 10.1016/j. the Universityhospital Essen, the University of Oxford (UK), the Human Brain Project, the biopsycho.2020.107887. [IF 2.763] University of Graz (AU), the Institut für Interdisziplinäre Musik-und Sprachtherapie (Duisburg, GER), the Institute for Psychology at the RWTH Aachen, Emerging TechTrans (USA), Hofhansel L, Regenbogen C, Weidler C, Habel U, Raine A, Clemens B (2020) CEDIMAT – Department of Neurology (DO) and the Department of Neurology - Hannover Stimulating the criminal brain: Different effects of prefrontal tDCS in criminal offenders and Medical School (GER). controls. Brain Stimulation 13(4):1117-1120. doi: 10.1016/j.brs.2020.03.022. [IF 6.565]

Joue G, Boven L, Willmes K, Evola V, Demenescu LR, Hassemer J, Mittelberg I, Mathiak K, Schneider F, Habel U (2020) Metaphor processing is supramodal semantic processing: The role of the bilateral lateral temporal regions in multimodal communication. Brain and Language 205:104772. doi: 10.1016/j.bandl.2020.104772. [IF 2.339]

Votinov M, Wagels L, Hoffstaedter F, Kellermann T, Goerlich KS, Eickhoff SB, Habel U (2020) Effects of exogenous testosterone application on network connectivity within emotion regulation systems. Scientific Reports 10(1):2352. doi: 10.1038/s41598-020- 59329-0. [IF 3.998]

40 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 41 Core Facilities | Brain Imaging Facility Core Facilities | Two-Photon Imaging Facility

Clemens B, Derntl B, Smith E, Junger J, Neulen J, Mingoia G, Schneider F, Abel T, Bzdok D, Habel U (2020) Predictive Pattern Classification Can Distinguish Gender Identity Subtypes from Behavior and Brain Imaging. Cerebral Cortex 30(5):2755-2765. doi: Two-Photon Imaging Facility 10.1093/cercor/bhz272. [IF 5.043]

Sarkheil P, Ibrahim CN, Schneider F, Mathiak K, Klasen M (2020) Aberrant functional connectivity profiles of brain regions associated with salience and reward processing in Head of the facility Staff: female patients with borderline personality disorder. Brain Imaging and Behavior 14(2):485- Vogt, M. Vogt, M. 495. doi: 10.1007/s11682-019-00065-z. [IF 3.391] Decker, L. Advisory Board Kapsokalyvas, D. Creyaufmüller M, Heim S, Habel U, Mühlhaus J (2020) The infuence of semantic Martin, C. (Institute of Pharmacology and Toxicology) associations on sentence production in schizophrenia: an fMRI study. European Archives of van Zandvoort, M. (Institute for Molecular Cardiovascular Research) Materials/Travel expenses 2020: € 43,055 Psychiatry and Clinical Neuroscience 270(3):359-372. doi: 10.1007/s00406-018-0936-9. Tolba, R. (Institute of Laboratory Animal Science) [IF 3.288] Investments/Equipment 2020: € 3,364

Holtbernd F, Romanzetti S, Oertel WH, Knake S, Sittig E, Heidbreder A, Maier A, Krahe J, Advanced optical microscopy is a highly valued methodology in biomedical research with Revenues 2020: € 10,500 Wojtala J, Dogan I, Schulz JB, Schiefer J, Janzen A, Reetz K (2020) Convergent patterns rapid development dynamics in recent years. The potential for a variety of research projects of structural brain changes in rapid eye movement sleep behavior disorder and Parkinson’s with high originality and excellent quality is undisputed. The Core Facility offers scientists of disease on behalf of the German rapid eye movement sleep behavior. Sleep. Epub ahead of the RWTH Aachen University and also external users the opportunity to use the innovative print. doi: 10.1093/sleep/zsaa199. [IF 4.805] microscopic method of two-photon laser scanning microscopy (TPLSM). TPLSM is based on the principle of 2-photon excitation, in which the simultaneous Wagels L, Schneider I, Menke S, Ponge AK, Kohn N, Schneider F, Habel U (2020) absorption of two near-infrared photons (total energy corresponds to that of a single Autism and Reactions to Provocation in a Social and Non-social Context. Journal of Autism photon at half the wavelength used in classical fluorescence microscopy) excite fluorescent and Developmental Disorders 50(2):402-414. doi: 10.1007/s10803-019-04257-w. [IF 3.047] molecules in the sample. Since the probability of simultaneous absorption (within 0.5 fs) of two photons is very low, the excitation occurs only in the focus of a pulsed near-infrared Sarkheil P, Odysseos P, Bee I, Zvyagintsev M, Neuner I, Mathiak K (2020) Functional laser. Out-of-focus absorption and excitation are missing, resulting in the imaging of an connectivity of supplementary motor area during finger-tapping in major depression. optical slice. In addition, near-infrared light penetrates deeper into scattering tissues, Comprehensive Psychiatry 99:152166 https://doi.org/10.1016/j.comppsych.2020.152166 allowing excitation deep in the sample. Scanning a stack of images at a certain depth [IF 2.567] range enables 3D reconstruction and spatial visualization of fluorescent structures in the volume. Therefore, TPLSM has an improved depth penetration with good resolution in three dimensions. The combination of all these properties makes TPLSM advantageous over classical fluorescence microscopic techniques, for visualizing structures in three dimensions Applied and actual third-party funding that are deeper in viable tissues. The Core Facility is currently equipped with Sijben, R. Mapping of inter-brain synchronicity using fMRI based START applied € 24,900 a TPLSM system (Olympus FV1000MPE) hyperscanning and offers the possibility for in vitro, ex vivo, in situ imaging and in vivo experiments in small animals or isolated whole mount samples or 3D cell cultures. In addition, an In Vivo Optical Imaging System is integrated into the Core Facility (Perkin Elmer IVIS Lumina XR). The In- Vivo Optical Imaging system enables the recording of bioluminescence and fluorescence signals from cell cultures, ex vivo samples and non-invasively in living small animals for extended time periods in the same animal. The overlay with a Figure 1: HUVSMC (Human umbilical vein smooth muscle photographic or radiographic image brings cell) in a fibrin gel. Smooth muscle actin (red), collagen the bioluminescence and/or fluorescence (green), and nucleus (blue). (V. Kučikas, IMCAR). signal into an anatomical context. The Core Facility „Two-Photon Imaging“ is located in the Institute for Laboratory Animal Science, thus creating optimal experimental conditions for intravital fluorescence imaging

42 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 43 Core Facilities | Two-Photon Imaging Facility Core Facilities | Two-Photon Imaging Facility

in rodents and other animal models using TPLSM. In addition to the microscope systems, Status and Development the facility offers an S1 laboratory with basic equipment for experimental work and sample The IZKF Core Facility currently is equipped with only one TPLSM system. After ongoing preparation. A stereo microscope with work space is available for small animal surgery. technical problems the LaVision TriMScope I microscope system is out of order since May The generation of big imaging data is followed by the need of hard- and software to 2019 and cannot be repaired due to missing spare parts at a reasonable price. visualize and process the data. Therefore, the Core Facility offers two high-performance In 2019 the Core Facility submitted a DFG proposal for a major research instrumentation workstations and image processing software (eg. ImageJ/FIJI, ImagePro, Bitplane Imaris, (according to Art. 91b GG) with the participation of nine working groups from the faculty AutoQuantX3) for data processing and analysis. The image processing software Imaris of medicine. In May 2020 the DFG proposal for a state-of-the-art multiphoton microscope (Bitplane) was upgraded in 2020. system was granted (GZ: INST 222/1345-1 FUGG) and the microscope system was In addition to local storage, the workstations of the Core Facility are connected to a data ordered end of December 2020. server (IZKF-Cloud) for temporal storage. The users of the Core Facility have decentralized access to their microscopic data for data processing and transfer. The use of the Core Facility is regulated by an operating and usage concept and organized with an online booking calendar. The imaging is usually carried out by the staff of the Core Facility together with the user. For projects with long-term use, the researchers are trained to operate the systems independently. The goal of the Core Facility is to provide a modern and advanced microscopic platform to overcome limitations for current projects and to enable future collaboration projects within the RWTH Aachen. The Core Facility also envisions itself as a platform for further methodological qualification and the promotion of scientific education for students, doctoral candidates, and researchers in medicine and natural sciences at the RWTH.

Figure 3: Two-photon laser scanning microscope: Olympus Figure 4: In-vivo optical imaging systems: IVIS Lumina XR Fluoview FV1000MPE

The ordered new microscope system is the Stellaris 8 DIVE FALCON, the new version of Multi-Photon Laser Scanning Microscopes offered by Leica, with an integrated module for fluorescence lifetime imaging (FLIM). The system enables fluorescence excitation with a dual line laser in the infrared range (up to 1300 nm), multiplexing using spectral information and video-rate FLIM. Crucial in this new set-up are also flexibility of beam routing to achieve a balance between resolution and penetration depth and use of the extended excitation wavelength range for optimal penetration, imaging of far red probes, three-photon excitation, and third harmonic imaging. The acquisition thus represents an institutionally relevant infrastructural focus for the Medical Faculty and the RWTH Aachen University. We thus are confident that the new system within the animal facility opens up new project possibilities including intravital imaging on various animal types for highly publicizable results and the associated acquisition of third-party funding. The new features and the provision of this technology should have a positive and tangible impulse Figure 2: Service/Workflow - Two-Photon Laser Scanning Microcopy on different fields of research. Last but not least, the microscope setup with Figure 5: FLIM image of a cat‘s eye. Simultaneous spectral the option for infrared excitation (up to 1300 fluorecscence (grey) and FLIM (colored) imaging shows the nm) would allow the unique establishment of contrast through the lifetime. Acquisition and visualization a dual imaging platform by combining in-vivo with STELLARIS 8 FALCON (source Leica Microsystems).

44 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 45 Core Facilities | Two-Photon Imaging Facility Core Facilities | Two-Photon Imaging Facility

whole-body optical imaging and multi-photon microscopy. This creates a platform from External users whole-body imaging to subcellular imaging deep in the tissue in the same animal and clearly enriches the methodological scope of the Core Facility. Tengström, F. MERLN The unused old LaVision microscope system was rebuilt by the addition of a RCM (re- Institute for Technology-Inspired Regenerative Medicine, Maastricht University scanning confocal microscope) module from the company confocal.nl. The transformed system is still in the establishing phase and will be used for high-resolution imaging in combination with high penetration in samples by a near infrared 785 nm diode-laser. Collaborations The Two-Photon Imaging Facility had project collaborations with the Advanced Microscopy Unit (CARIM) in Maastricht (NL) and with the Network “Advanced Light Microsocopy Users within the faculty Aachen” (ALMA). 56 users from 20 clinics/institutes of the medical faculty oft he RWTH Aachen used the Two-Photon Imaging Facility. The Main users are Biomedical Technologies - Institute of Applied Medical Technology, Institute for Biomedical Engineering - Division of Stem Cell Publications Biology and Cellular Engineering and Biomedical Technologies - Institute of Experimental Lin YY, Schuphan J, Dickmeis C, Buhl EM, Commandeur U, Fischer H. (2020) Attachment Molecular Imaging. of Ultralow Amount of Engineered Plant Viral Nanoparticles to Mesenchymal Stem Cells Enhances Osteogenesis and Mineralization Adv Healthc Mater 9(21):e2001245 [IF 7.367]

Zhang S, Thiebes AL, Kreimendahl F, Ruetten S, Buhl EM, Wolf M, Jockenhoevel S and Christian Apel C. (2020) Extracellular Vesicles-Loaded Fibrin Gel Supports Rapid Neovascularization for Dental Pulp Regeneration. Int J Mol Sci 21(12): 4226 [IF 4.556]

May JN, Golombek SK, Baues M, Dasgupta A, Drude N, Rix A, Rommel D, von Stillfried S, Appold L, Pola R, Pechar M, van Bloois L, Storm G, Kuehne AJC, Gremse F, Theek B, Kiessling F, Lammers T. (2020). Multimodal and multiscale optical imaging of nanomedicine delivery across the blood-brain barrier upon sonopermeation. Theranostics 10(4):1948- 1959 [IF 8.579]

Baues M, Klinkhammer BM, Ehling J, Gremse F, van Zandvoort MAMJ, Reutelingsperger CPM, Daniel C, Amann K, Bábíčková J, Kiessling F , Floege J, Lammers T, Boor P. (2020) A collagen-binding protein enables molecular imaging of kidney fibrosis in vivo. Kidney Int 97(3):609-614 [IF 8.945]

Aveic A, Janßen S, Nasehi R, Seidelmann M, Vogt M, Pantile M, Rütten S and Fischer H. (2020) A 3D printed in vitro bone model for the assessment of molecular and cellular cues in metastatic neuroblastoma. Biomater Sci (Epub ahead of print) [IF 6.183]

The Team of the Two-Photon Imaging Facility.

Applied and actual third-party funding

Tolba, R. H. Multi-Photonen Laser Scanning Mikroskop mit fast DFG 04/2020 € 1,275,000 Martin, C. FLIM Modul INST 222/1345-1 FUGG Van Zandvoort, M. Vogt, M.

46 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 47 Core Facilities | Transgenic Service Core Facilities | Transgenic Service

This includes the following services: 9. Import and export management of live animals and animals by-products Transgenic Service (e.g. Frozen Embryonic cells, Cryopreserved Sperm etc.) 10. Organization of our animal software database “Tierbase” and our new software database “Tick@lab” 11. Organization of animal capacity in our SPF-Facility and the quarantine Head of the facility Staff: 12. Organization of animal welfare relevant issues for new generated mouse lines by the Pfeffer, T. Pfeffer, T. TGS, e.g. the creation of severity assessment according to the Animal Welfare Act. Lingg, F. Scientific supervision Brehm, E. Tolba, R. (Institute of Laboratory Animal Science) Atanasova, D. Status and Development Kandil, N. The Core Facility „Transgenic Service“ continues to be an essential factor for the Medical Faculty of RWTH Aachen University. Materials/Travel expenses 2020: € 16,083 Skills/Services/Training courses/Consulting service One of the main tasks of the TGS in the last funding period - in parallel to the revitalization The Institute for Laboratory Animal Science and the Central Laboratory for Laboratory Animal-Housing Costs 2020: € 1,370 or rederivation of imported new mouse strains - is the cryopreservation of mouse lines in Animals have been operating the „Transgenic Service (TGS)“ since 2009 at the medical accordance with the 3Rs principle. faculty at the university hospital of the RWTH Aachen. The TGS is a service platform Investments/Equipment 2020: € 9,323 at the RWTH Aachen University for the generation of genetically modified mouse lines, furthermore, the offers in vitro techniques for the rederivation or revitalization of mouse lines Revenues 2020: € 35,854 and the cryopreservation of mouse lines.

Furthermore, we support the individual working groups in Breeding management and offered consulting service for experimental planning according to the 3R principle (Russel & Burch 1959; Refinement, Replacement and Reduction of animal experiments), which has become to a central focus in the Core Facility.

In addition, we offer the “Vasectomy of mice” of the surgical training of FELASA-B courses, to make the technique available to a larger group of researchers.

This service is preferably available to members of the medical faculty and other faculties of the RWTH Aachen University. Cooperation with external partners are also possible, e.g. in the context of collaborative projects with other universities or research institutions. To ensure the quality of our services and the claims of internal and external users, the core facility “Transgenic Service”, as a part of the institute of laboratory animal science, has been certificated according to DIN ISO 9001:2015 in 2012. In the last 3 years with the successful establishment of the cryopreservation of mouse In cooperation with the core facility „Two photon Imaging“, the skills of the TGS is an ideal sperm, we have been able to freeze over 280 strains faster and more cost-effectively than addition to extend the scope of biomedical research in the medical faculty. with the cryopreservation of mouse embryos.

The Services of the Transgenic Service contain following items. These are mouse lines that were mostly bred for preservation only and cannot be used for 1. Embryo transfer and rederivation of infected or imported mouse lines actual projects. In accordance with the 3Rs principle, the Transgenic Service will try to make 2. Cryopreservation of mouse embryos and mouse sperms users aware that their lines can be frozen for hygienic and animal welfare reasons. 3. Quality control of frozen mouse embryos and mouse sperms The number of genetically modified mouse lines generated has remained largely stable over 4. Generation of knockout animals the last few years and further projects are already being planned. 5. Generation of transgenic animals 6. In-vitro fertilization (IVF) An increasingly important and time-consuming issue for the Core Facility is the import and 7. Cell culture of embryonic stem cells export of live animals and animal by-products, which has increased in the last years. Due 8. Generation of embryonic stem cells to the considerable hygienic differences between the animal laboratories, as there are no binding international rules, all health certificates and import forms have to be checked The IZKF Core Facility is a central unit in the Institute for Laboratory Animal Science and carefully in order not to risk the health status in the Institute for Laboratory Animal Science. also coordinates existing work processes inside the institute to provide the hygiene. Alternatives may have to be found, for example by transporting cryogenic material.

48 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 49 Core Facilities | Transgenic Service Core Facilities | Transgenic Service

Furthermore, the bureaucratic effort in this area has increased considerably. Import permits 22 users of 15 institutes/clinics of the medical faculty of the RWTH Aachen University used by the veterinary office of the city region, internal market permit according to EU directive, the Transgenic Service. The main users are the Departments of Internal Medicine II and III. transport documents, transport of hazardous substances in the case of GenTg modified animals, IATA freight documents etc. are part of the documents have to be obtained.

Due to the Covid-19 pandemic and the resulting embargo in the import/export area, many mouse lines could not be imported and were postponed indefinitely. Even after the first lockdown, many import restrictions remained in place, so that there are still difficulties in this area. For safety reasons and on the advice of the institute, the capacity of the animal facilities was reduced to a minimum during this period in order to relieve the animal care staff and thus reduce contacts. As a result, the work of the TGS in the area of sanitation also had to be significantly reduced.

With the start of the new funding period, Dr. rer. nat. Matthias Marks has joined the Transgenic Service team as a scientific staff member, with a working time of 50%. Dr. Marks supports researchers, scientists and all employees of the Institute of Laboratory Animal Science with genetic questions, breeding problems and new projects such as the generation of ES cells and the generation of transgenic mice. Dr. Marks is already collaborating with some research groups to generate new mouse lines. Furthermore, we continued to work on the establishment of the CRISPR/Cas9 system in zygotes. In ES cells, the system has already been successfully established and could be included as a service in our method repertoire.

Users within the faculty Since the Core Facility was established in 2009, a total of 28 departments and 100 users have used the services of the TGS. The number of departments using the TGS annually remains constant.

50 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 51 Core Facilities | Proteomics Facility Core Facilities | Proteomics Facility

The facility provides advice and support for the generation of experimental setups and Proteomics Facility workflows based on the individual scientific question of the interested researchers. This includes help and suggestions for the process of appropriate sample generation (including proper controls). The facility offers experimental support in protein enrichment strategies such as co-IPs and affinity purification as well as PTM-specificotein pr and peptide enrichment strategies. These methods can be introduced and demonstrated to the Head of the facility Staff: respective groups. Preisinger, C. Preisinger, C. Gostek, S. Advisory Board Cramer, T. (Clinic for General, Visceral, and Transplantation Surgery) Materials/Travel expenses 2020: € 55,590 Lüscher, B. (Department of Biochemistry and Molecular Biology) Schulz, J. B. (Department of Neurology) Investments/Equipment 2020: € 45,733

Revenues 2020: € 57,167 Skills/Services/Training courses/Consulting service Sample preparation (depending on the “state” of the submitted sample) • Gel electrophoresis of protein samples • Lysis and proteolytic digestion of protein samples in-solution, in-gel or on-bead (proteases: trypsin, LysC, GluC, chymotrypsin…) • Sample purification and desalting (C18); small (zip-tips) to large scale (C18 cartridges) • Sample concentration/depletion (plasma/serum) • Sample labelling for protein quantification: isobaric tags (TMT or iTRAQ; maximum 8-plex), or peptides labelled using the dimethyl approach. However, the label-free quantification approach is usually the preferred choice of quantitation for most analyses. • Sample fractionation/enrichment – peptide chromatography: The PF provides peptide separation using high pH reversed phase chromatography or strong anion/cation exchange (SAX/SCX). • The PF also provides phosphopeptide enrichment: TiO2 (peptides phosphorylated The Team of the Proteomics Faciliy. on serine and threonine residues) or anti-phosphotyrosine antibody mediated immunoprecipitation, followed by subsequent mass spectrometry analysis and quantification of phosphorylation changes.

Mass spectrometry: • Analysis of desalted/purified samples by nanoLC-MS/MS using standardized methods and gradients on either the Q Exactive Plus or the Orbitrap Elite system. Both mass spectrometers are coupled to a nano-UPLC system (Dionex RSLCnano).

Data analysis: • Analysis of the raw data is performed by the PF in house using designated software packages including protein (and PTM) identification and quantification. Software includes MaxQuant, MSFragger, Perseus, R scripts. The resulting data is explained to and discussed with the researchers. Furthermore, the facility provides advice on data interpretation and possible follow-up experiments. • Storage of raw and analysis data in house (IZKF cloud) on separate hard drives for access at later time points.

Special services: • The PF can also provide Western Blot analysis and technical consulting, assistance and expertise for “unusual” biological questions in the field of protein biochemistry.

52 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 53 Core Facilities | Proteomics Facility Core Facilities | Proteomics Facility

Status and Development Publications The Proteomics Facility (PF) was launched in January 2012 in order to provide the medical Dohmen M, Krieg S, Agalaridis G, Zhu X, Shehata SN, Pfeiffenberger E, Amelang J, faculty of the RWTH Aachen with access to proteomic technologies in order to facilitate the Bütepage M, Buerova E, Pfaff CM, Chanda D, Geley S, Preisinger C, Sakamoto K, Lüscher analysis and investigation of protein function on a molecular level in health and disease. B, Neumann D, Vervoorts J. (2020) AMPK-dependent activation of the Cyclin Y/CDK16 Two high resolution/high mass accuracy mass spectrometers (Orbitrap Elite, Thermo complex controls autophagy. Nat Commun. 11(1):1032. [IF 12.12] Scientific; 2013 and Q Exactive Plus, Thermo Scientific; 2018) have been acquired and installed during this time period. The implementation of the Orbitrap Elite enabled the PF Mahameed M, Boukeileh S, Obiedat A, Darawshi O, Dipta P, Rimon A, McLennan G, to drastically increase the availability of experimental approaches that could be utilized Fassler R, Reichmann D, Karni R, Preisinger C, Wilhelm T, Huber M, Tirosh B. (2020) to analyse protein/proteome samples. It was successfully used for the analysis and Pharmacological induction of selective endoplasmic reticulum retention as a strategy for characterization of large-scale protein interactomes and the quantitative analysis of protein cancer therapy. Nat Commun. 11(1):1304. [IF 12.12] phosphorylation changes, amongst others, in collaboration with several groups within the medical faculty. The acquisition of the Q Exactive Plus in 2018 was a logical consequence Vieri M, Preisinger C, Schemionek M, Salimi A, Patterson JB, Samali A, H Brümmendorf of the ever increasing demand for high resolution/high mass accuracy mass spectrometry T, Appelmann I, Kharabi Masouleh B. (2020) Targeting of BCR-ABL1 and IRE1α induces within the faculty in recent years. Since then the new instrument has helped to significantly synthetic lethality in Philadelphia-positive acute lymphoblastic leukemia. Carcinogenesis. enhance the proteomic analysis possibilities within the faculty. This has helped the PF to 2020 Sep 11:bgaa095. doi: 10.1093/carcin/bgaa095. [IF 4.603] contribute important analytical results to several IZKF funded “Joint Research Projects” as well as established research consortia (SFB/TRR 57, SFB 1382, CRU644). Parlak ZV, Labude N, Rütten S, Preisinger C, Niessen J, Aretz A, Zybała R, Telle R, Neuss S, Schickle K. (2020) Toward Innovative Hemocompatible Surfaces: Crystallographic Plane In the last year, the PF has, similar to previous years, carried out analyses of a large range Impact on Platelet Activation. ACS Biomater Sci Eng. 6(12):6726-6736. [IF 4.152] of samples for an equally large range of clinics and institutes. Again, there was a significant increase in sample diversity. During this time period the PF has analysed, amongst others, both cell line (human and mouse), tissue/organ (brain, liver) proteomes from animal models (including transgenic mouse and zebrafish) and serum (various disease backgrounds) Applied and actual third-party funding on a much more in-depth level than previously possible (up to 6000 quantifiable protein groups as well as the identification and quantification of >5000 phosphorylation sites). A Uhlig, S. RWTH Life Sciences Core Facilities Exzellenzinitiative applied € 1,525,280 key focus in the forthcoming years will again be the analysis of small sample amounts (e.g. Müller-Newen, G. RWTH Aachen FACS-sorted cells with numbers in the sub-million range) as well as the investigation of cell Preisinger, C. culture/primary cell/tissue supernatants. Walter, P. Moreover, the PF has applied for a state-of-the-art mass spectrometer that has the ability of ion mobility separation. This technological advancement should boost the analytical capabilities of the PF and will aid in a much more sophisticated qualitative and quantitative analysis of complete proteomes (such as organs or tissues), subcellular proteomes (e,g mitochondria, exosomes), patient serum, protein interactomes, and investigation of post- translational modification throughout the following years.

Users within the faculty 46 users from 13 clinics/institutes of the medical faculty of the RWTH Aachen used the Proteomics Facility. The main users are Department of Internal Medicine III, the Institutes of Biochemistry and Helmholtz Institute for Biomedical Engineering.

External users The Proteomics Facility had external users from the Department of Ceramics and Refractory Materials – Faculty V, from the Institute of Biology II, III and V – Faculty I and from the Fraunhofer Institute.

Collaborations The Proteomics Facility had project collaborations with the FH Aachen, the Department of Biological Sciences/Xi‘an Jiaotong-Liverpool University and the Institute of Chemical Technologies and Analytics, Technical University of Vienna.

54 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 55 Core Facilities | Flow Cytometry Facility Core Facilities | Flow Cytometry Facility

• The FCF provides small instrumentation and bench space for sample handling including a laminar flow, centrifuges, pipettes etc., lab consumables and reagents to run the Flow Cytometry Facility instruments are provided (sheath fluid, plastic material, filters, etc.).

Status and Development 2020 has been the third year of full operation. Building on the encouraging user evaluation Head of the facility Staff: in 2019 and the positive review by external experts and the IZKF steering committee in Pabst, O. Steinke, N. January 2020, FCF has continued and further expanded its activities. Vaßen, S. FCF is headed by O. Pabst (not paid by FCF/IZKF). N. Steinke has continued to work full Advisory Board Schatanek, A. time as operator and coordinator in the FCF. Since July 2020, starting the second 3 year Berres, M.-L. (Department of Internal Medicine III) IZKF funding period, FCF recruited a second full time operator. The position was advertised in May 2020 and in September Silke Vaßen started to work as full time operator/technician Materials/Travel expenses 2020: € 62,610 in the FCF. S. Vaßen has been trained by N. Steinke and now the FCF has two full-time operators, organizing cell sorting services, user introductions, management of user fees, lab Skills/Services/Training courses/Consulting service Investments/Equipment 2020: € 2,135 organization and further development of the FCF. Additionally, the FCF employed a student The FCF supports Flow Cytometry (Fluorescence activated cell sorting - FACS) based assistant, A. Schatanek. A Schatanek mainly supported introduction of new user working cell purification and analysis. The FCF has been established within the IZKF and medical Revenues 2020: € 32,300 with the spectral cytometer Cytek Aurora. faculty and is open to all members of RWTH Aachen University. Depending on available capacities, scientists from other research institutions are also welcome to use our services. In April 2020, a funding proposal for a 5 Laser high-end cell sorter submitted by O. Pabst Support offered by the FCF comprises all stages of FACS based experiments, ranging from (INST 222/1347-1 FUGG) was approved by the DFG. FCF performed a search for potential consultation and experimental planning, access to well-maintained instruments, cell sorting instruments and defined key instrument requirements according to user needs. The official service and support in data analysis. announcement for this instrument was opened in November 2020 and in December the FCF received offers from two companies. We expect installation of the new cell sorter until A) Advice in experimental design: We advise in the design of flow cytometry based spring 2021. In 2021, a major focus will be on setting-up the new high-end cell sorter and experiments and sample preparation by routine sort of human cells and S2 material.

• personal consultation to precisely determine the needs of individual users, Since September 2020, FCF operates an additional device for determining cell counts and • providing state-of-the-art protocols for cell preparation from mouse and human tissues viability (ViCell XR Cellcounter, Beckman Coulter). The instrument is available to users 24/7. and cell staining and Decision of the IZKF steering committee to adjust user fees according to DFG • offering training and advice on the interpretation of experimental data and display of recommendations were implemented starting in July 2020. User fees for BD Canto were FACS experiments. raised from 5€ to 10€ per hour. Cost for cell sorting were charged differently for cell sorting • The “FACStastic day” is a seminar series of presentations and hand-on-training service and instrument usage only (Service operation: 50€ per hour; operation: 30€ per exercises to introduce FACS technology. This shall allow us to cover basic concepts and hour). Changes in user fees resulted in higher revenues from basic FACS analysis but had a forum to introduce more advanced applications. no major impact on revenues obtained from cell sorting service. As net effect of increased user fees and overall activity of the FCF, revenues increased from 29k€ in 2019 to 37k€ B) Cell sorting under safety level S1 conditions as a service according to user demands. in 2020. Revenues generated in the FCF are 69% of funds provided by IZKF for running This activity is the major and most time consuming service. costs (material, maintenance) and about 25% of the full costs covered by IZKF (full costs including staff). C) Access to instruments, reagents and software: • Hands-on training for FACS analysis using BD FACS Canto, BD Fortessa and for Regular information about new developments in the facility were sent to our users roughly spectral FACS analysis using Cytek Aurora. The use of the FACS Canto and Cytek on a monthly basis and the FCF has again organized the “FACStastic day” in two online Aurora is open to all users and allows complex multi-colour analyses of cell populations seminars to teach basics in Flow Cytometry and discuss more advanced approaches in with up to 8 colours (FACS Canto) or 30+ colours (Cytek Aurora). Upon completion multiparameter flow cytometry. of the training, the instrument is available to all users 24/7. Access to BD Fortessa is possible during regular working hours and on demand. Expectedly the Corona pandemic had a major impact on operation of the FCF. FCF • In 2020, the FCF installed a camera-based cell counting system (ViCell XR). Upon implemented strict hygiene regulations and limited the number of people allowed in the completion of the training, the Cell counter is available to all users 24/7. lab. Obligatory wearing of mask was introduced ahead of the general rules put forward by • FCF provides access to basic FACS reagents and antibodies to allow proper the university and administrative tasks were performed in the home office. Nonetheless, compensation and control of staining specificity. Moreover, FCF has been building up a FCF operated continuously in 2020. Reduced lab activity of our users resulted in only collection of anti-human antibodies, different viability markers and secondary reagents to slightly reduced total instrument usage of 2548 h in 2019 to 2333 h in 2020 (BD Canto, BD aid panel design and increase flexibility offered to users. Aria and Cytek Aurora, only counting effective operation). A major reason of why the FCF • The FCF purchased software licenses to analyze flow cytometry experiments that can be suffered from only modest reduction in instrument usage time is because of the increased rented by users against a small fee. Basic training in the use of the software is available use of the Cytek Aurora in 2020. In 2020, only 8 days had no active users working in/with upon request. the facility (out of 254 working days, see attachment I “FCF Geräteauslastung 2020”)

56 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 57 Core Facilities | Flow Cytometry Facility Core Facilities | Flow Cytometry Facility

Installation of a new high-end cell sorter in a biosafety level 2 environment will enable FCF to extend its cell sorting service. We hope that offering routine sort of human cells and potentially infective material will allow recruiting new users. With the help of two full-time operators, we are confident that FCF can service more users in the future.

To establish and demonstrate the seamless integration of human material available in the biobank and clinics, FACS-based analysis and data management of flow cytometry data, FCF is performing a small sized study to characterize changes in the B cell compartment of COVID-19 patients by spectral flow cytometry. Methods and approach established during this study will be available to all users.

External users The Flow Cytometry Facility had external users from the ITMC – Institute of Technical and Macromolecular Chemistry at the RWTH Aachen, Maastricht University – Department of Pathology and from the Frauhofer Institute of Production Technology IPT.

Publications Klemm, P., Ohl, K. (2020) Nrf2 expression driven by Foxp3 specific deletion of Keap1 results in loss of immune tolerance in mice. Eur. J. Immunol. 2020. 50: 515–524 DOI: 10.1002/eji.201948285 [IF 4.404]

Figure 1: Instruments and revenues in the FCF. A) Number of users for BD Canto, BD Aria and Cytek Aurora. In 2019 the Sternkopf, M., Noels, H. (2020) Native, Intact Glucagon-Like Peptide 1 Is a Natural number of user slightly dropped for BD Canto and Aria and increased for Cytek Aurora. B) Instrument usage in hours for BD Suppressor of Thrombus Growth Under Physiological Flow Conditions. Arterioscler Thromb Canto, BD Aria (Cell sorting) and Cytek Aurora. C) Revenues generated by use of the three instruments. Revenues generated Vasc Biol. 2020;40:e65–e77. DOI: 10.1161/ATVBAHA.119.313645 [IF 4.940] by cell sorting (BD Aria) slightly dropped in 2020 as compared to 2019. Revenues generated by use of the BD Canto slightly increased despite fewer user because of increased user fees since July 2020. D) Breakdown of user fees per month in 2020. Wessels, I., Grommes J. (2020) Zinc supplementation ameliorates lung injury by The sudden drop of revenues generated in the FCF in March/April coincides with the lockdown in spring. During summer reducing neutrophil recruitment and activity. Thorax 2020;0:1–9. DOI: 10.1136/ 2020 user revenues increased again but dropped at the end of 2020. thoraxjnl-2019-213357 [IF 10.844]

Kabbert, J., Pabst, O. (2020) High microbiota reactivity of adult human intestinal IgA requires somatic mutations. J Exp Med. 2020 Nov 2;217(11):e20200275. DOI: 10.1084/ Users within the faculty jem.20200275 [IF 11.743] In 2020 95 users of 21 institutes/clinics of the medical Faculty used the Flow Cytometry Facility. The main users had been coming from the Institute of Molecular Medicine (8.585€ of revenues) and the Institute of Cell Biology (6.885€ of revenues). All user were charged identical fees. Due to the Corona pandemic, FCF has largely stopped access of external Applied and actual third-party funding users in 2020. Pabst, O. 5-Laser Cellsorter INST 222/1347-1 FUGG Approval: Apr. 2020 € 565,250 Installation: Spring 2021 Outlook The Corona pandemic continues to be a major threat. FCF is in the process of creating online teaching material to help the introduction of new users until access to the labs can be fully opened again.

Additionally, FCF has no dedicated lab and office space. After intense discussion with the dean, we expect that this situation cannot be changed in the near future and the FCF will continue to rely on lab and office space within the Institute of Molecular Medicine. In 2021 we expect the FCF to move along with the Institute of Molecular Medicine to new labs, delay in the construction of this lab space may hinder installation of the new cell sorter. While the close interaction with the Institute of Molecular Mdicine creates relevant synergies, it also limits growth of the facility.

58 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 59 O3 I Joint Research Project I Weiskirchen/Schemionek p. 62 Mesenchymal interactions and fibrogenic signalling in cancer development: Molecular mechanisms in solid and hematologic neoplasia

O3-1 I Weiskirchen/Koschmieder p. 63 O3-7 I Boor/Brümmendorf p. 81 Lipocalin 2 controls efferocytosis and formation of PDGF signalling in myelofibrosis and myeloproliferative cancer-associated fibroblasts neoplasms PROJECTS

O3-2 I Lammers p. 69 O3-8 I Vervoorts-Weber/Vucur p. 84 Modulating macrophage-mediated microenvironmental The contribution of autophagy in HSCs for the remodelling to improve targeted anticancer herapy progression of HCC Phase Transition in Disease

O3-3 I Wagner, W./Gesteira Costa Filho p. 72 O3-9 I Lederle/Borkham-Kamphorst p. 86 Epigenetic signatures of fibroblasts in cancer Targeting stromal interactions in colon cancer and liver metastasis O3-4 I Liedtke p. 75 Impact of cell cycle proteins for the crosstalk between O3-10 I Ohl/Tenbrock p. 88 hepatocellular carcinoma and the tumor environment The role of Nrf-2 pathways in Myeloid derived suppressor cells, relevance for immunosuppression in the O3-5 I Schemionek/Huber p. 77 tumormicroenvironment Interplay of (malignant) mast cells and mesenchymal stroma cells in CML O3-11 I Kramann/Zenke p. 90 Dissecting the cross talk between megakaryocytes and O3-6 I Otto/Trautwein p. 79 Gli1+ stromal cells in bone marrow fibrosis and leukemic Role of IL6/gp130 signaling in hepatic stellate cells transformation in myeloproliferative neoplasms and its impact on HCC development in mice

60 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 61 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-1

Mesenchymal interactions and fibrogenic Lipocalin 2 controls efferocytosis and formation signalling in cancer development: Molecular of cancer-associated fibroblasts mechanisms in solid and hematologic neoplasia

Weiskirchen, R. Weiskirchen, R. (Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical (Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry) Chemistry) Schemionek, M. (Department of Internal Medicine IV) Koschmieder, S. (Department of Internal Medicine IV)

The consortium “Mesenchymal interactions and fibrogenic signalling in cancer Altered expression of Lipocalin 2 (LCN2) has been reported in many pathological conditions development: Molecular mechanisms in solid and hematologic neoplasms” has joint to and proposed to be significantly associated with tumor progression. In the present project, improve the networking structure, publication output and international visibility in the area Weiskirchen, R. Schemionek, M. we have determined transcriptional changes of LCN2 expression during tumorigenesis in Weiskirchen, R. Koschmieder, S. “Oncology“ at the RWTH Aachen University Hospital. The network was composed of 11 many cancer cell lines originating from the liver, pancreas, and the hematopoietic system. projects with 21 contributing scientists/clinicians belonging to 12 different institutes/clinics. We have delineated pathways and cytokines that provoke altered LCN2 expression during The projects were focused on the understanding of key drivers inducing a tumorigenic tumorigenesis. Recently, we established several LCN2-depleted monoclonal cell clones of environment in hepatocellular carcinoma and hematologic malignancies such as chronic human cancer cell line PC-3 using the CRISPR/Cas9 technology (Figure 1). The respective myeloid leukemia. cell lines show striking differences in the organization of the cytoskeleton and further Staff: During the three years of funding, research activities were bundled and a close networking lowered expression of connexin 43, IL-1β, and IL-8. Interestingly, the expression of IL-1β is Schröder, S. K. structure in the respective field “Oncology” was established, resulting in synergistic restorable by reconstitution of LCN2 expression or by adding conditioned media containing Tillmann, S. functioning. The main outcome of this initiative is documented in a large number of joint LCN2. Although we have not systematically tested the applicability of this read out system, publications, successful appointments of principal investigators to other universities, and we think that this system provides an effective tool to measure LCN2-dependent effects career advancements of involved staff such as the achievement of the PhD or the conferral and to quantify the biological activity of LCN2 mutants. Comparative analysis of respective of the Venia Legendi for individual researchers. In addition, beside the plan to improve clones and parental cell line PC-3 will now allow us to unravel tumor-stromal interactions research in the respective field, several members of the consortium were already highly of LCN2 in the pathogenesis of tumors, determine the capacity of LCN2 in promoting successful in third party fundraising. More important, several members of the consortium immunosuppressive effects in the tumor microenvironment, and to investigate the molecular helped to define a scientific “niche” and a coherent strategic agenda for setting a new SFB/ activities and pathways by which LCN2 regulate tumor-initiating cell plasticity. In the TRR initiative with a tentative title “Mutual dynamics between fibrosis and cancer”. For hematopoietic system, we identified endoplasmic reticulum (ER) stress as a main driver this purpose, common distinctive features and added values were identified that will be of of LCN2 expression in BCR-ABL- and JAK2V617F-positive myeloproliferative neoplasms fundamental importance to maximize the coherence and synergies between participating (MPN). By analyzing peripheral blood from MPN patients, we found Lcn2 gene expression work groups. to be increased, especially in patients diagnosed with chronic myeloid leukemia (CML) or with myelofibrosis (MF). Using the murine 32D cell model, we show that Thapsigargin and Brefeldin A, two well described inducers of ER stress and the associated unfolded protein response (UPR), lead to a drastic increase in LCN2 expression (Figure 2). Strikingly, In sum, the IZKF facilitated and fostered the the effect of ER stress and UPR was much stronger than the exogenous application of implementation of research structures and framework inflammatory cytokines such as TNF-α or IL-1β, and the induction of ER stress-induced LCN2 expression was independent of the respective oncogene. Further investigations using conditions that will also be helpful for long-term the chemical inhibitors Kira-6 and JNK-IN-8 (inhibiting the kinase activity of IRE1α and JNK, respectively) show a significant reduction in thapsigargin-induced Lcn2 expression both at governance and operational cooperation between the mRNA and protein levels. These findings strongly point towards the IRE1α – JNK axis as many basic scientists and clinicians in the area of a major driver of Lcn2 expression in hematopoietic malignancies, and provide a rationale for the development of innovative therapeutic approaches for MPN patients. “Oncology”.

62 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 63 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-1 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-1

Figure 2: Manipulation of Lcn2 expression in 32D cells. (A) RT-qPCR analysis of peripheral blood mononuclear cells (PBMC) from healthy donors (HD) or patients diagnosed with chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), or myelofibrosis (MF). (B) RT-qPCR analysis of Lcn2 mRNA, isolated from stably transduced 32D cells carrying the oncogenes BCR-ABL (blue), JAK2V617F (red) or the empty vector (grey). (C) Western blot analysis of 32D cell lysates following 8h treatment with 2 µg/ml Brefeldin A (BFA) show accumulation of LCN2 protein. (D) RT-qPCR analysis of 32D cells after 24h treatment with 20 ng/mL TNF-α, 50 ng/mL IL-1β, 100 nM thapsigargin (Tha) or 2 µg/ml BFA. (E) RT-qPCR analysis of 32D cells after 24h treatment with 30 nM Tha and 1 µM Kira-6 or 2 µM JNK-IN-8. (F,G) Western Blot analysis of 32D cell lysates following treatment with 30 nM Tha and/or 1 µM Kira-6 (F), or JNK-IN-8 (G). Significant differences were determined using one-way-ANOVA followed by a Tukey post-hoc test. Asterisks indicate p-values of: *=p<0,05; **=p<0,01; ***=p<0,001. Figure 1: CRISPR gene editing of LCN2 in PC-3 cells. (A,B) Using the CRISPR/Cas9 technology, monoclonal LCN2-depleted cell clones of human cancer cell line PC-3 were generated. (C) Western blot analysis of cell extracts (left) or supernatant (right) of respective clones show the deficiency in LCN2. (D) Phalloidin staining demonstrates striking differences in cytoskeletal organization in respective clones compared to their parental cell line PC-3. (E,F) Compared to PC-3, the LCN2-depleted cell clones show reduced expression of connexin 43, IL-1β, and IL-8 Publications in Western blot or RT-qPCR analysis. (G,H) The expression of IL-1β is restorable by reconstitution of LCN2 expression by adenoviral infection with Ad-hLCN2 or addition of conditioned media Originals that resulted from the IZKF funded project with IZKF acknowledgement containing LCN2. Asimakopoulou A, Engel KM, Gassler N, Bracht T, Sitek B, Buhl EM, Kalampoka S, Pinoé-Schmidt M, van Helden J, Schiller J, Weiskirchen R. (2020) Deletion of Perilipin 5 protects against hepatic injury in nonalcoholic fatty liver disease via missing inflammasome activation. Cells 9(6):1346 [IF 4.366]

Borkham-Kamphorst E, Haas U, Van de Leur E, Trevanich A, Weiskirchen R. (2020) Chronic carbon tetrachloride applications induced hepatocyte apoptosis in Lipocalin 2 null mice through endoplasmic reticulum stress and unfolded protein response. Int J Mol Sci. 21(15):5230 [IF 4.556]

Drescher HK, Schippers A, Rosenhain S, Gremse F, Bongiovanni L, Bruin A, Eswaran S, Gallage SU, Pfister D, Szydlowska M, Heikenwalder M, Weiskirchen S, Wagner N, Trautwein C, Weiskirchen R, Kroy DC. (2020) L-Selectin/CD62L is a key driver of non- alcoholic steatohepatitis in mice and men. Cells 9(5):1106 [IF 4.366]

Hahn L, Helmrich N, Herebian D, Mayatepek E, Drebber U, Domann E, Olejniczak S, Weigel M, Hain T, Rath T, Wirtz S, Mollenkopf HJ, Schmidt N, Ewers C, Baier A, Churin Y, Windhorst A, Weiskirchen R, Steinhoff U, Roeb E, Roderfeld M. (2020) IL-13 as target to reduce cholestasis and dysbiosis in Abcb4 knockout mice. Cells 9(9):1949 [IF 4.366]

64 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 65 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-1 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-1

Hermert D, Martin IV, Reiss LK, Liu X, Breitkopf DM, Reimer KC, Alidousty C, Rauen T, Xie J, Weiskirchen R. (2020) What does the „AKT“ stand for in the name „AKT kinase“? Floege J, Ostendorf T, Weiskirchen R, Raffetseder U. (2020) The nucleic acid binding Some historical comments. Front Oncol. 10:1329 [IF 4.848] protein YB-1-controlled expression of CXCL-1 modulates kidney damage in liver fibrosis. Kidney Int. 97:741-752 [IF 8.945] Publications that resulted from cooperation of the IZKF funded project with IZKF groups or core facilities with IZKF acknowledgement Kim P, Zhang CC, Thoröe-Boveleth S, Weiskirchen S, Gaisa NT, Buhl EM, Stremmel Azzam M, El Safy S, Abdelgelil SA, Weiskirchen R, Asimakopoulou A, de Lorenzi F, W, Merle U, Weiskirchen R. (2020) Accurate measurement of copper overload in an Lammers T, Mansour S, Tammam S. (2020) Targeting activated hepatic stellate cells using experimental model of Wilson disease by laser ablation inductively coupled plasma mass collagen-binding chitosan nanoparticles for siRNA delivery to fibrotic livers. Pharmaceutics spectrometry. Biomedicines 8(9):356 [IF 4.717] 12(6):590 [IF 4.845]

Meurer SK, Tezcan O, Lammers T, Weiskirchen R. (2020) Differential regulation of Lipocalin Baumeister J, Chatain N, Hubrich A, Maié T, Costa IG, Denecke B, Han L, Küstermann C, 2 (LCN2) in doxorubicin-resistant 4T1 triple negative breast cancer cells. Cell Signal Sontag S, Seré K, Strathmann K, Zenke M, Schuppert A, Brümmendorf TH, Kranc KR, 2020;74:109731 [IF 3.968] Koschmieder S, Gezer D. (2020) Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms. Leukemia 34(4):1062-1074 Schröder SK, Asimakopoulou A, Tillmann S, Koschmieder S, Weiskirchen R. (2020) TNF-α [IF 8.665] controls Lipocalin-2 expression in PC-3 prostate cancer cells. Cytokine 135:155214 [IF 3.488] Buhl EM, Djudjaj S, Klinkhammer BM, Ermert K, Puelles VG, Lindenmeyer MT, Cohen CD, Winkler I, Bitter C, Winkler S, Weichenhan D, Thavamani A, Hengstler JG, Borkham- He C, Borkham-Kamphorst E, Weiskirchen R, Denecke B, Trairatphisan P, Saez-Rodriguez Kamphorst E, Kohlbacher O, Plass C, Geffers R, Weiskirchen R, Nordheim A. J, Huber TB, Olson LE, Floege J, Boor P. (2020) Dysregulated mesenchymal PDGFR-β (2020) Identification of Pparγ-modulated miRNA hubs that target the fibrotic tumor drives kidney fibrosis. EMBO Mol Med. 12(3):e11021 [IF 8.8] microenvironment. Proc Natl Acad Sci U S A 117:454-463 [IF 9.412] Gasterich N, Wetz S, Tillmann S, Fein L, Seifert A, Slowik A, Weiskirchen R, Zendedel A, Reviews that resulted from the IZKF funded project with IZKF acknowledgement Ludwig A, Koschmieder S, Beyer C, Clarner T. (2020) Inflammatory responses of astrocytes Khurana A, Sayed N, Allawadhi P, Weiskirchen R. (2020) It’s all about the spaces between are independent from Lipocalin 2. J Mol Neurosci., in press. doi: 10.1007/s12031-020- cells: role of extracellular matrix in liver fibrosis. Ann Transl Med., in press. doi: 10.21037/ 01712-7 [IF 2.678] atm-20-2948 [IF 3.297]

Meurer SK, Karsdal MA, Weiskirchen R. (2020) Advances in the clinical use of collagen as biomarker of liver fibrosis. Expert Rev Mol Diagn. 20:947-969 [IF 4.096] Applied and actual third-party funding (DFG, BMBF, EU, foundations)

Tacke F, Weiskirchen R. (2020) Non-alcoholic fatty liver disease (NAFLD)/non-alcoholic Danielyan, L,. CellOpt, Opposite Cell Differentiation Program: a game EU, Horizon 2020; Call: NN rejected steatohepatitis (NASH)-related liver fibrosis: mechanisms, treatment and prevention. Ann Nguyen, H.P.H., changing cell-based therapeutic approach for H2020-FETOPEN-2018-2020 Transl Med ., in press. doi: 10.21037/atm-20-4354 [IF 3.297] Weiskirchen, R., Perrier, degenerative disorders A., Kirik, D., Ramos, I. ten Hove M, Pater L, Storm G, Sabine W, Weiskirchen R, Lammers T, Bansal R. (2020) The Weiskirchen, R., Fricker, New therapeutic approaches for the treatment of BMBF Ausschreibung NN rejected hepatic lipidome: From basic science to clinical translation. Adv Drug Deliv Rev. 159:180- G., Stremme,l W. Wilson disease „Gezielter Wirkstofftransport“ 197. [IF 13.300] Weiskirchen, R., Protective roles for Fructophilic lactic acid NRF-DAAD Partnership NN rejected Weiskirchen S, Weiper K, Tolba RH, Weiskirchen R. (2020) All you can feed: Some Kim, I. bacteria in the development of hypertension and Program; Germany-Korea comments on production of mouse diets used in biomedical research with special NAFLD after high-fructose intake Partnership Program (GEnKO emphasis on non-alcoholic fatty liver disease research. Nutrients 12(1):163 [IF 4.546] Program) Vural, H., Weiskirchen, R. Pathophysiological role of electronegative LDL in Philipp Schwartz-Initiative 02/2020-01/2022 € 120,000 Weiskirchen R. (2020) Special issue on „Cellular and molecular mechanisms underlying the nonalcoholic fatty liver disease der Alexander von Humboldt- pathogenesis of hepatic fibrosis“. Cells 9(5):1105. [IF 4.366] Stiftung mit Unterstützung des Weiskirchen R. (2020) Severity of coronavirus disease 2019 (COVID-19): Does surfactant Auswärtigen Amtes matter? Front Microbiol. 11:1905 [IF 4.235] Asimakopoulou, A., Die Schlüsselfunktionen von Perilipin Wilhelm Sander-Stiftung 01/2020-12/2022 € 117,210 Weiskirchen R. (2020) Commentary: Re-regulation of hepatic stellate cell contraction and Weiskirchen, R. 5 und Lipocalin 2 in der Pathogenese des nicht-alkoholischen Steatohepatitis- cirrhotic portal hypertension by Wnt/β-catenin signaling via interaction with Gli1. Br J Pharmacol, in press. doi: 10.1111/bph.15282 [IF 7.730] Hepatozellulärkarzinoms Khurana, A., Bansal, R., Lipoxygenases: From dissecting mechanism to DAAD-P.R.I.M.E. 04/2021-05/2022 € 127,375 Weiskirchen R, Penning LC. (2020) COMMD1, a multi-potent intracellular protein involved in Weiskirchen, R. targeted therapeutics for the treatment of non- Program-2019 copper homeostasis, protein trafficking, inflammation, and cancer. J Trace Elem Med Biol, alcoholic steatohepatitis (NASH) Projektkennziffer: 57573064 in press. [IF 3.245]

66 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 67 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-1 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-2

Geervliet, E., Bansal, R., Chemokine receptor CCR2 and CCR8 peptide EASL: PhD Studentship Juan 05/2020-04/2023 € 157,400 Weiskirchen, R. antagonists as a promising treatment for non- Rodes Programme 2020 Modulating macrophage-mediated alcoholic steatohepatitis (NASH)

Henis, Y. I., Ehrlich, M., TGF-β und Cholesterol: Wechselwirkende DFG Deutsch-Israelische 03/2021-02/2024 € 715,716 microenvironmental remodelling to improve Dooley, S., therapeutische Zielstrukturen in der Behandlung Projektkooperation (DIP) (WE Weiskirchen, R. von NASH 2554/13-1) targeted anticancer therapy Huber, M., Weiskirchen, Mechanismen zur Kontrolle von pro- DFG (WE 2554/15-1) 03/2021-02/2024 € 532,182 R., Meurer, S. K., inflammatorischen Mastzellfunktionen und ihre Liedtke, C. Auswirkungen auf chronische Lebererkrankungen und hepatozelluläres Karzinom Lammers, T. (Institute for Experimental Molecular Imaging) Koschmieder, S. Mechanismen von IFN-alpha (IFNa) bei der MPN- DFG (KO 2155/7-1) 1/2020-12/2022 €401.671 assoziierten Myelofibrose Nanomedicines are designed to improve the biodistribution of systemically administered Engel, K., Hopf, C., Untersuchung molekularer Mechanismen der DFG (WE 2554/16-1) Revision, in NN chemo-therapeutic drugs. In 2020, we completed efforts aiming to develop taxane-loaded Weiskirchen, R. Lipid-vermittelten Leberentzündung mittels Begutachtung polymeric micelles for gastrointestinal (GI) cancer therapy. GI cancers are among the most (bildgebender) massenspektrometrischer (23.7.2020) lethal malignancies and very difficult to treat. We demonstrated that Π electron-stabilized Verfahren polymeric micelles based on PEG-b-PHPMA-Bz can be loaded highly efficiently with the Weiskirchen, R. Lipocalin 2, ein wichtiger molekularer Schalter in DFG (WE 2554/17-1) Revision, in NN taxane drug docetaxel and potentiate chemotherapy responses in multiple advanced- Lammers, T. der Regulation des Leberstoffwechsels und der Begutachtung stage GI cancer mouse models (Fig. 1A-C). Complete cures and full tumor regression Leberimmunität (16.10.2020) were achieved in subcutaneous gastric cancer cell line derived xenografts (CDX), in Weiskirchen, R., Phytochemicals for the alleviation of steatosis, BMBF – DBT Cooperative newly submitted NN patient-derived xenografts (PDX), as well as in CDX models with intraperitoneal and lung Staff: Kumar, A. inflammation and fibrosis: an innovative approach Science Program (31.10.2020) metastases. Mechanistically, docetaxel-loaded micelles modulated the tumor immune Sun, Q. towards the management of nonalcoholic and microenvironment in CDX and PDX tumors, beneficially affecting the ratio between M1- Dadfar, S. alcohol-associated liver disease and M2-like macrophages. These findings exemplify that Π-conjugated polymeric micelles Bai, X. loaded with docetaxel hold significant potential for the treatment of advanced-stage GI Mauer, J., Cancer stem cells from triple negative breast DFG (WE 2554/18-1) newly submitted NN cancers. In parallel, using multimodal and multiscale optical imaging, we demonstrated that Weiskirchen, R. cancer utilize TGF-β signaling to rearrange tumor (19.11.2020) clinical-stage PEG-PHPMA-Lac polymeric micelles show a very favorable biodistribution, microenvironment and to metastasize to the liver with strong accumulation in tumors at the whole-body level and prominent engagement of immune cells at the cellular level, providing opportunities for to boost the immune system in case of cancer nanoimmunotherapy (Fig. 1D). We managed to obtain BMBF and DFG Promoting of young researchers funding to follow up on these findings, aiming to use nanomedicine to promote the efficacy Doctoral Theses of immunotherapy.

Schröder, S. K. Ongoing RWTH Aachen University, Faculty 1 Biological function of LCN2 in tumor initiation and progression Tillmann, S. Ongoing RWTH Aachen University, Faculty 1 The role of Lipocalin 2 (LCN2) in myeloproliferative neoplasms Cierpka, R. Ongoing RWTH Aachen University, Faculty 10 LCN2 and PLIN2 in initiation and progression of hepatocellular carcinoma

Figure 1: Polymeric micelles for anticancer drug delivery. A-C: PEG-PHPMA-Bz polymeric micelles enhance the in vivo efficacy of docetaxel in advanced-stage gastrointestinal cancer. D: Artistic impression of the multimodal and multiscale optical imaging work done with clinical-stage PEG-PHPMA-Lac polymeric micelles.

68 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 69 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-2 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-2

Publications Ten Hove M, Pater L, Storm G, Weiskirchen S, Weiskirchen R, Lammers T, Bansal R*. (IZKF-funded students and PI are underlined. # shared first author. * corresponding (2020). The hepatic lipidome: From basic science to clinical translation. Adv Drug Deliv Rev author) S0169-409X:30073-30079. [IF 13.3]

Liang C#, Bai X#, Qi C#, Sun Q, Han X, Lan T, Zhang H, Liang R, Jiao J, Zheng Z, Fang J, Dasgupta A, Biancacci I, Kiessling F, Lammers T*. (2020). Imaging-assisted anticancer Lei P, Wang Y, Möckel D, Metselaar J, Storm G, Hennink WE, Kiessling F, Wei H*, Lammers nanotherapy. Theranostics 10:956-967. [IF 8.6] T*, Shi Y*, Wei B*. (2021). Π electron-stabilized polymeric micelles potentiate docetaxel therapy in advanced-stage gastrointestinal cancer. Biomaterials 266:120432. [IF 10.3] Decuzzi P*, Peer D*, Mascolo DD, Palange AL, Manghnani PN, Moghimi SM, Farhangrazi ZS, Howard KA, Rosenblum D, Liang T, Chen Z, Wang Z, Zhu JJ, Gu Z, Korin N, Wang B, Van Herck S, Chen Y, Bai X, Zhong Z, Deswarte K, Lambrecht BN, Sanders NN, Letourneur D, Chauvierre C, van der Meel R, Kiessling F, Lammers T. (2020). Roadmap on Lienenklaus S, Scheeren H, David S, Kiessling F, Lammers T*, De Geest B*, Shi Y*. (2020). nanomedicine. Nanotechnology 32: 012001. [IF 3.5] Potent and prolonged innate immune activation by enzyme-responsive imidazoquinoline TLR7/8 agonist prodrug vesicles. J Am Chem Soc 142:12133-12139. [IF 14.5]

Sun Q#, Bai X#, Sofias AM, van der Meel R, Ruiz-Hernandez E, Storm G, Hennink WE, Applied and actual third-party funding (DFG, BMBF, EU, foundations) De Geest B, Kiessling F, Yu HJ, Lammers T*, Shi Y*. (2020). Cancer nanomedicine meets immunotherapy: opportunities and challenges. Acta Pharmacol Sin 41:954–958. [IF 5.1] Lammers, T. Meta-Targeting: Macro-nanomedicine to treat ERC Consolidator Grant - 04/2020-03/2025 € 1,979,000 metastatic cancer 864121 Biancacci I#, Sun Q#, Möckel D#, Gremse F, Rosenhain S, Kiessling F, Bartneck M, Hu Lammers, T. Penetration-promoting polymeric micelles for the DFG - LA2937/4-1 10/2020-10/2023 € 441,500 Q, Thewissen M, Storm G, Hennink WE, Shi Y, Rijcken CJF*, Lammers T*, Sofias AM*. treatment of fibrotic tumors (2020). Optical imaging of the whole-body to cellular biodistribution of clinical-stage PEG-b- pHPMA-based core-crosslinked polymeric micelles. J Control Release 328:805-816. [IF 7.6] Lammers, T. PP-TNBC: Paclitaxel-Picelles for the treatment of BMBF - 16GW0319K 02/2021-02/2024 € 1,988,000 Triple Negative Breast Cancer Dadfar SM, Camozzi D, Darguzyte M, Roemhild K, Varvarà P, Metselaar J, Banala S, Straub M, Güvener N, Engelmann U, Slabu I, Buhl M, van Leusen J, Kögerler P, Hermanns B, Schulz V, Kiessling F, Lammers T. (2020). Size-isolation of superparamagnetic iron oxide nanoparticles improves MRI, MPI and hyperthermia performance. J Nanobiotechnology 18: Promoting of young researchers 22 (2020) [IF 6.5] Doctoral Theses

Lammers T*, Ferrari M. (2020). The success of nanomedicine. Nano Today 31:100853 Bai, X. Ongoing RWTH Aachen University, Faculty 10 Pi-conjugated polymeric micelles for [IF 16.9] anticancer therapy Sun, Q. Ongoing RWTH Aachen University, Faculty 10 Nanomedicine formulations to improve May JN, Golombek S, Baues M, Dasgupta A, Drude N, Rix A, Rommel D, von Stillfried S, immunotherapy Appold L, Pola R, Pechar M, Bloois L, Storm G, Kuehne A, Gremse F, Theek B, Kiessling F, Lammers T*. (2020). Multimodal and multiscale optical imaging of nanomedicine delivery Dadfar, S. Ongoing RWTH Aachen University, Faculty 10 Iron oxide nanoparticles for imaging across the blood-brain barrier upon sonopermeation. Theranostics 10:1948-1959. [IF 8.6] and drug delivery

Baues M, Klinkhammer BM, Ehling J, Gremse F, van Zandvoort M, Reutelingsperger C, Daniel C, Amann K, Bábíčková J, Kiessling F, Floege J, Lammers T*, Boor P*. (2020). A Appointments to other universities collagen-binding protein enables molecular imaging of kidney fibrosis in vivo. Kidney Int 97:609-614. [IF 8.9] Tacke, F. Charite, Berlin W3 accepted

Metselaar JM*, Lammers T*. (2020). Challenges in nanomedicine clinical translation. Drug Deliv Transl Res 10: 721-725. [IF 3.0]

Meurer SK*, Tezcan O, Lammers T, Weiskirchen R*. (2020). Differential regulation of Awards Lipocalin 2 (LCN2) in doxorubicin-resistant 4T1 triple negative breast cancer cells. Cell Lammers, T: 2020 International Award of the Belgian Society for Pharmaceutical Sciences Signal 74:109731. [IF 4.0] (BSPS)

Moss JI*, Barjat H, Emmas SA, Strittmatter N, Maynard J, Goodwin RJA, Storm G, Lammers T, Puri S, Ashford MB, Barry ST. (2020). High-resolution 3D visualization of nanomedicine distribution in tumors. Theranostics 10:880-897. [IF 8.6]

70 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 71 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-3 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-3

Zeevaert K, Elsafi Mabrouk MH, Wagner W, Goetzke R. Cell mechanics in embryoid bodies. Epigenetic signatures of fibroblasts in cancer Cells (2020) 9(10): 2270. [IF 4.366]

Baumeister, J., Chatain, N., Hubrich, A., Maié, T., Costa, I.G., Denecke, B., Han, L., Küstermann, C., Sontag, S., Seré, K., Strathmann, K., Zenke, M., Schuppert, A., Brümmendorf, T.H., Kranc, K.R., Koschmieder, S., Gezer, D., 2020. Hypoxia-inducible Wagner, W. factor 1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative (Institute for Biomedical Engineering - Stem Cell Biology and Cellular Engineering) neoplasms. Leukemia 34, 1062–1074. [IF 8.665] Gesteira Costa Filho, I. (Institute for Computational Genomics) Leimkühler, N.B., Gleitz, H.F.E., Ronghui, L., Snoeren, I.A.M., Fuchs, S.N.R., Nagai, J.S., Banjanin, B., Lam, K.H., Vogl, T., Kuppe, C., Stalmann, U.S.A., Büsche, G., The aim of this project was to characterize the epigenetic differences between fibroblasts Kreipe, H., Gütgemann, I., Krebs, P., Banz, Y., Boor, P., Tai, E.W.-Y., Brümmendorf, T.H., and cancer-associated fibroblasts (CAFs) in different cancers. We successfully isolated Koschmieder, S., Crysandt, M., Bindels, E., Kramann, R., Costa, I.G., Schneider, R.K., and phenotypically analysed fibroblasts from liver tumours and healthy tissue. The DNA 2020. Heterogeneous bone-marrow stromal progenitors drive myelofibrosis via a druggable was sent for DNAm profiling with Illumina EPIC BeadArrays to Life and Brain (Bonn). The Wagner, W. Gesteira Costa Filho, I. alarmin axis. Cell Stem Cell, epub. [IF 20.860] first results of the data analysis showed significant epigenetic differences between CAFs and fibroblasts from healthy tissue (Figure 1, A). Furthermore, an Epigenetic-Fibroblast- Signature has been selected by using an in-house bioinformatics pipeline trained on public Staff: datasets to test the hypothesis that the proportion of fibroblasts correlates with clinical Schmidt, M. data from cancer patients. We could show for a selected number of TCGA datasets, such Maié, T. as kidney carcinomas, that the Epigenetic-Fibroblast-Signature is an indicator for cancer patient survival (Figure 1, B). With a similar approach we could also generate more cell- type-specific epigenetic signatures for different human cell types and performed cell type classification on independent datasets (Figure 1, C). By combining these cell-type-specific signatures with cellular deconvolution algorithms we have been able to show that we can estimate cellular proportions in different tissues even with a low number of CpG sites per signature. Further, our results show that we can achieve accuracy similar to that of methods whose signatures use thousands of CpG sites (Schmidt, M., Maié, T. et al., BMC Biology 2020). Using our generated methylation data from CAFs and by generating an epigenetic signature specifically selected for CAFs we aim to further improve the estimation of the amount of CAFs in a tumour sample and its correlation with clinical data.

Publications Schmidt, M., Maié, T., Dahl, E., Costa, I.G., Wagner, W. et al. Deconvolution of cellular subsets in human tissue based on targeted DNA methylation analysis at individual CpG sites. BMC Biology 18, 178 (2020). [IF 6.765]

Cypris O, Eipel O, Franzen J, Rösseler C, Tharmapalan V, Kuo CC, Vieri M, Nikolić M, Kirschner M, Brümmendorf TH, Zenke M, Lampert A, Beier F, Wagner W. PRDM8 reveals aberrant DNA methylation in aging syndromes and is relevant for hematopoietic and neuronal differentiation. Clinical Epigenetics (2020) 12: 125. [IF 5.028]

Fernandez-Rebollo E, Franzen J, Goetzke R, Hollmann J, Ostrowska A, Oliverio M, Sieben T, Rath B, Kornfeld J-W, Wagner W. Senescence-associated metabolomic phenotype in Figure 1: A – scatterplot of the mean methylation of all CpGs in isolated cancer-associated fibroblasts (CAF) versus primary and iPSC-derived mesenchymal stromal cells. Stem Cell Reports (2020) 2: 201- fibroblasts of neighbouring tissue (NAF), marked are all significant CpGs (limma adjusted p-values < 0.05). B – Hazards 209. [IF 6.032] ratios from Cox proportional hazards models for datasets from The Cancer Genome Atlas (TCGA). C – Selection of cell-type specific CpGs. The difference of meanβ values of each cell type versus all other cell types was plotted against the sum of Han Y, Franzen J, Stiehl T, Gobs M, Kuo CC, Nikolic M, Hapala J, Koop BE, Strathmann K, variances within both groups. Ritz-Timme S, Wagner W. New Targeted Approaches for Epigenetic Age Predictions. BMC Biology (2020) 18: 71. [IF 6.765]

72 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 73 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-3 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-4

Applied and actual third-party funding (DFG, BMBF, EU, foundations) Impact of cell cycle proteins for the crosstalk Costa, I.G., Kramann, R., Spatial Map of Fibrosis BMBF – Systems Medicine 01/2020 -12/2024 € 3,487,990 Schneider, R., Puelles, V. Consortia between hepatocellular carcinoma and the Costa, I.G. Untangling and Targeting Mechanisms of DFG Clinical Research Unit 2019-2022 € 246,000 Myelofibrosis in Myeloproliferative Neoplasms tumor environment (MPN) Wagner, W., Epi-Blood-Count BMBF VIP+ 02/2019-01/2022 € 1,459,238 Koschmieder, S., Isfort, S., Rink, L. Liedtke, C. (Department of Internal Medicine III) Wagner, W. TRACK-AML Krebshilfe 02/2019-01/2021 € 178,150

Wagner ,W. Human Epigenetics and Bio-informatics for DFG 07/2017-08/2020 € 278,390 Hepatocellular carcinoma (HCC) is one of the most severe tumor diseases developing Forensic Age Estimation during a multistep process involving chronic liver inflammation and liver fibrosis. The latter is M. Sociale, Wagner, W. Prick-Me Else Kröner-Fresenius Stiftung 01/2021-12/2023 € 328,900 characterized by accumulation of extracellular collagen produced by Hepatic Stellate Cells (HSCs). This process involves cell cycle re-entry and proliferation of the normally quiescent Wagner, W., Gillner, A. Topostem DFG 01/2019-12/2021 € 242,150 HSCs. The cell cycle is regulated by cyclins and associated cyclin-dependent kinases (Cdks). In the present study, we examined the role of Cyclin E1 (CcnE1) and Cdk2 in HSCs Liedtke, C. Promoting of young researchers for liver fibrogenesis and hepatocarcinogenesis. We generated conditional, HSC-specific Doctoral Theses Cyclin E1 (CcnE1∆HSC) and Cdk2 (Cdk2∆HSC) knockout mice using transgenic cre-expression under control of the L-rat promoter. Cdk2∆HSC and CcnE1∆HSC mice were challenged with Staff: Schmidt, M. Ongoing RWTH Aachen University, Faculty 1 Epigenetic characterization of cancer-associated CCl4 for 6 weeks and subsequently investigated for liver fibrosis. Induction of fibrosis and Hennings, J. ∆HSC fibroblasts. HCC in CcnE1 mice was performed using the DEN/CCl4 model. Genetic ablation of Cdk2 or Cyclin E1 specifically in HSCs significantly reduced collagen Maié, T. Ongoing RWTH Aachen University, Faculty 1 Epigenetic signatures of fibroblasts in cancer. accumulation and fiber formation in the liver after CCl4 treatment (Figure 1 left). Accordingly, Cdk2∆HSC and CcnE1∆HSC mice showed a significantly reduced HSC activation in the liver. This suggests that cell cycle re-activation of naïve HSCs in vivo requires functional Cdk2 presumably in complex with Cyclin E1. In order to translate these findings into a pre-clinical therapeutic model, we challenged human LX-2 and murine GRX HSC cell lines with the pan-Cdk inhibitor CR8 (Figure 1 left). CR8 treatment substantially reduced Cdk kinase activity in both cell lines. In addition CR8 inhibited proliferation, survival and pro-fibrotic activation in both LX-2 and GRX cells, and also triggered DNA damage and cell cycle arrest. Importantly, we identified effective CR8 dosages mediating anti-fibrotic effects in primary murine HSCs without affecting cell cycle activity and survival in primary hepatocytes. CcnE1∆HSC mice challenged with DEN/CCL4 showed a reduced number and size of dysplastic lesions when compared to cre-negative littermates, which was again associated with decreased HSC activation even after long-term observation (Figure 1 right). We conclude that the pro-fibrotic properties of HSCs depend on functional Cyclin E1 and Cdk2 in vitro and in vivo. In addition, pharmacological pan-Cdk inhibition reduced the fibrogenic functions of HSC lines and primary HSCs without affecting the regenerative capacity of hepatocytes in vitro. Therefore, early HSC-specific inhibition of Cyclin E1 in patients with chronic liver disease could be a novel therapeutic approach to treat liver fibrosis and prevent onset of HCC.

74 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 75 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-4 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-5

Interplay of (malignant) mast cells and mesenchymal stroma cells (MSCs) in CML

Schemionek, M. (Department of Internal Medicine IV) Huber, M. (Institute of Biochemistry and Molecular Immunology) Figure 1: Schematic representation of the key results gained within this project. Further explanations are given in the main text.

The implementation of Tyrosine Kinase Inhibitors (TKIs) that inhibit the oncogenic driver BCR-ABL induces cytogenetic and even molecular remission in the majority of chronic myeloid leukemia (CML) patients. However, specific challenges remain, such as the Publications persistence of leukemic stem cells (LSCs), as previously shown by several labs including our Hubbers, A, Hennings, J, Lambertz, D, Haas, U, Trautwein, C, Nevzorova, YA, Sonntag, R, own. This prevents a cure of the disease as even in a selected cohort of ideally responding Schemionek, M. Huber, M. Liedtke, C (2020) Pharmacological Inhibition of Cyclin-Dependent Kinases Triggers Anti- patients, CML reoccurs in about 50% upon TKI discontinuation. Our recent data show Fibrotic Effects in Hepatic Stellate Cells In Vitro. Int J Mol Sci 21(9):3267 [IF 4.556]. that this is at least partially mediated via an inflammatory microenvironment and targeting either microenvironmental-derived factors or the specific signaling pathway within the Staff: LSC population induced by these factors, significantly affects the CML-inducing stem Namasu, C. cell pool. In this project, we focused on the malignant microenvironment and studied the Jaquet, T. Applied and actual third-party funding (DFG, BMBF, EU, foundations) effect of CML development on mesenchymal stromal cells (MSC) as well as the biology of malignant mast cells (MCs) in the disease. Expression profiling of CML patient- vs healthy Liedtke, C. Therapy of Hepatocellular Carcinoma through targeted DFG LI 1045/4-2 07/2020 –06/2023 € 276,500 donor (HD)-derived MSCs revealed significant changes e.g. in the regulation of chemokine inhibition of the cell cycle mediators Cyclin E1 and and extracellular polysaccharide biosynthetic processes upon disease development Cdk2 in mouse models (Figure 1A+B). Interestingly, specific cytokines such as TGF-β, which had previously been implicated in LSC persistence, were shown to be increasingly expressed by the malignant Huber, M., Liedtke, C., Mechanisms controlling pro-inflammatory mast cell DFG LI 1045/6-1 01/2021 – 12/2023 € 535,182 MSC population. Likewise, the MSC biology was altered as CML MSCs showed increased Meurer, S., functions and implications for chronic liver disease Liedtke: adipogenic and osteogenic differentiation levels in CML samples (Figure 1C+D), whereas Weiskirchen, R. and cancer € 178,394 chondrogenesis was impaired in CML samples (Figure 1E). Moreover and intriguingly, we could show an increase in CML MC quantity. We also studied CML MC quality and here bone marrow-derived BCR-ABL-positive compared to wild-type MCs showed significantly Promoting of young researchers increased production of proinflammatory cytokines in response to IgE-mediated stimulation. Doctoral Theses The depletion of MC had no significant effect on the hematopoietic compartment, including stem cells (Figure 1F+G) using Cpa3Cre/+ mice. This will now allow us to test for the specific Hennings, J. Ongoing RWTH Aachen University, Faculty 1 Role of E-type cyclins and Cdk2 in cells of the hepatic effect of MC depletion on CML LSC persistence in future experiments in vivo. tumor environment (working title)

76 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 77 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-5 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-6

Role of IL6/gp130 signaling in hepatic stellate cells and its impact on HCC development in mice

Otto, T. (Department of Internal Medicine III) Trautwein, C. (Department of Internal Medicine III)

In this project, we proposed to investigate the role of gp130 signalling in hepatic stellate cells (HSCs) and its impact on tumour development in a chronically injured liver. We concentrated on the characterization of pro-tumorigenic alterations in the microenvironment of a chronically injured liver such as fibrogenic remodelling of the liver architecture. Otto, T. Trautwein, C. Using an established model of toxin-induced chronic liver injury (chronic CCl4 administration), we uncovered an important protective function of gp130 signalling in HSCs to prevent excessive liver fibrosis. This essential function of the IL-6/gp130 pathway in non-parenchymal Staff: hepatic stellate cells will likely protect from liver carcinogenesis in an injured liver. In contrast, Lynen, C. in a chronic liver injury model driven by persistent cholestatic damage (using surgical bile duct ligation, BDL), surprisingly, we did not observe any protective function of IL-6/gp130 signalling in HSCs with regard to the extent of liver damage or fibrogenesis (see Figure 1). This may indicate a mechanistically distinct course of disease progression during chronic cholestasis, which presumably involves portal fibroblasts rather than HSCs. Therefore, we expect that liver carcinogenesis will be affected particularly in models involving toxin-induced chronic liver injury such as the well-established DEN-CCl4 model of murine HCC development. Although preliminary results in this model point towards a protective function of HSC-specific IL-6/ Figure 1: A) Volcano plot showing differentially expressed genes in CML patient vs healthy donor MSCs (n=4 each). B) Gene ontology analysis revealed significant modifications upon gp130 signalling, we currently await validation from a second cohort of mice. disease development. C) Adipogenic D) Osteogenic, and E) Chondrogenic potential of CML patient vs HD MSCs were analysed by quantification of oil red-, alizarin-red or alcian blue-staining, respectively. F) Gating strategy to identify long-term repopulating stem cells (LT-HSCs) in Cpa3+/+ vs Cpa3Cre/+ mice. G) Mast cell (MC) depletion does not affect the LT-HSC pool in non-CML mice, identified by immunophenotyping.

Applied and actual third-party funding (DFG, BMBF, EU, foundations)

Huber, M., Liedtke, C., Mechanisms controlling pro-inflammatory mast cell DFG HU 794/14-1 01/2021 – 12/2023 € 535,182 Meurer, S., Weiskirchen, functions and implications for chronic liver disease Huber: R. and cancer € 178,394

78 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 79 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-6 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-7

Publications Mohs A*, Kuttkat N*, Otto T*, Youssef SA, De Bruin A, Trautwein C (2020). MyD88- PDGF signalling in myelofibrosis and dependent signaling in non-parenchymal cells promotes liver carcinogenesis. Carcinogenesis. 41(2):171-181. [IF 4.6] myeloproliferative neoplasms

Mohs A, Otto T, Schneider KM, Peltzer M, Boekschoten M, Holland CH, Hudert CA, Kalveram L, Wiegand S, Saez-Rodriguez J, Longerich T, Hengstler JG, and Trautwein C (2020). NRF2/KEAP1 in hepatocytes controls fibro- and carcinogenesis in chronic liver disease. J Hepatol. S0168-8278(20)33695-3. [IF 16.5] Boor, P. (Institute of Pathology and Department of Nephrology) Brümmendorf, T. (Department of Internal Medicine IV)

Promoting of young researchers Myeloproliferative neoplasms (MPNs) are a group of diseases characterized by increase Doctoral Theses hematopoietic stem cell (HSC) turnover resulting in over-production of myeloid cells in bone marrow. Increased Platelet-Derived Growth Factor (PDGF) signaling activity caused by Lynen, C. Ongoing RWTH Aachen University, Role of IL-6/gp130 signaling in non-parenchymal liver cells during liver genetic alterations contributes to defined subtypes of MPNs as well as some other types of Faculty 1 carcinogenesis. malignancies like gliomas, dermatofibrosarcoma, protuberans and gastrointestinal stroma Boor, P. Brümmendorf, T. tumors. Other than promoting malignancy, PDGF signaling is also known for its association with angiogenesis and fibrosis. However, the detailed mechanism of PDGF signaling in MPNs and myelofibrosis has not been well studied due to the lack of appropriate animal Staff: models. Therefore we study the role of PDGF signaling in MPNs and myelofibrosis by Tai, E. generation of cell-specific PDGF receptor mutation mice models.

The study aims to (1) Characterize PDGF and PDGFR expression in bone marrow cells: We have made use of the PDGFR-GFP reporter mice and immunohistochemistry/fluorescence and in situ hybridization to study the expression of PDGFR and the PDGF ligands in bone marrow in mice and in patient’s bone marrow biopsies. (2) Define the role of PDGF in stromal cells as a potential driver of myelofibrosis: We have studied the localization of Gli1 positive cells, which were shown to be crucial mediators of myelofibrosis, and established transgenic mice with constitutively active PDGFR mutations activated specifically in Gli1+ stroma cells. (3) Analyze the role of PDGF signaling in hematopoietic cells as a driver of myeloproliferative neoplasms using mice with constitutively active PDGFR mutations activated specifically in hematopoietic cells. The analysis of these mouse strains is ongoing and the protocol for isolation and immortalization of the hematopoietic cells from mice has been optimized. (4) Use of PDGFR specific small molecule probe to explore the possibility of direct, non-invasive Analyze the feasibility of PDGFR as a biomarker, in which first promising results were found in patients samples.

In addition, we have shown that in JAK2V617F-positive myeloproliferative neoplasms hypoxia-inducible factor 1 (HIF-1) might be a potential therapeutic target (Ref 1) and JAK2V617F mutations confer increased molecular responses to interferon-α (Ref 2). We also shown, the serum of myeloproliferative neoplasms stimulated hematopoietic stem and progenitor cells (Ref 3). In addition to the above, we have developed a unique and first model of pure renal fibrosis using transgenic mice expressing constitutively active PDGFRb in renal mesenchymal cells, which reflects several aspects of chronic kidney diseases in patients (Ref 4) and analyzed molecular and cellular mechanisms of disease transition of a rare crystal-induced nephropathy (Ref 5). Collectively, our data could bring novel insights into the cell-specific role of PDGF in myelofibrosis and myeloproliferative neoplasms. Our animal models could be potentially used for testing of novel treatments for these diseases.

80 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 81 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-7 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-7

Publications Applied and actual third-party funding (DFG, BMBF, EU, foundations) Baumeister J, Chatain N, Hubrich A, Maié T, Costa IG, Denecke B, Han L, Küstermann C, Sontag S, Seré K, Strathmann K, Zenke M, Schuppert A, Brümmendorf TH, Kranc KR, Brümmendorf, T. Cellular aging in clonal and normal hematopoiesis in DFG: KFO344, 1/2020-12/2022 € 220,100 Koschmieder S, Gezer D. Hypoxia-inducible factor 1 (HIF-1) is a new therapeutic target in Wagner, N. myelofibrosis BR1782/5-1 JAK2V617F-positive myeloproliferative neoplasms. Leukemia 2020, 34:1062-1074 Boor, P. The role of MIF signalling in FSGS BMBF: STOP FSGS 05/2019–04/2022 € 540,300 Djudjaj, S. SP1 (01GM1901A) Buhl EM, Djudjaj S, Klinkhammer BM, Ermert K, Puelles VG, Lindenmeyer MT, Cohen CD, He C, Borkham-Kamphorst E, Weiskirchen R, Denecke B, Trairatphisan P, Saez-Rodriguez Boor, P. Cells -specific role of PDGF system in renal fibrosis DFG: SFB/TRR57, P25 01/2017-12/2020 € 486,400 J, Huber TB, Olson LE, Floege J, Boor P (2020). Dysregulated mesenchymal PDGFR-β Floege, J. drives kidney fibrosis. EMBO Mol Med 2020, 12:e11021 Boor, P. Central platform: Histopathological Platform for DFG: SFB/TRR57, Q1 01/2017-12/2020 € 486,400 Longerich, T. Analyses of Hepatic and Renal Fibrosis Klinkhammer BM, Djudjaj S, Kunter U, Palsson R, Edvardsson VO, Wiech T, Franklin Thorsteinsdottir M, Hardarson S, Foresto-Neto O, Mulay SR, Moeller MJ, Jahnen-Dechent W, Floege J, Anders HJ, Boor P (2020). Cellular and molecular mechanisms of kidney injury Boor, P. PDGF- a mediator of CVD in CKD DFG: SFB/TRR219, M01 01/2018-12/2021 € 449,000 in 2,8-dihydroxyadenine nephropathy, J Am Soc Nephrol 2020, 31:799-816 Floege, J. Boor, P. Central platform for standardization and development DFG: SFB/TRR219, S02 01/2018-12/2021 € 630,400 Becker JU, Mayerich D, Padmanabhan M, Barrat J, Ernst A, Boor P, Cicalese PA, Hohl, C. of animal models Mohan C, Nguyen HV, Roysam B (2020). Artificial intelligence and machine learning in Boor, P. SINERGIA: advanced technologieS for drug dIscovery H2020-MSCA-ITN-2019 11/2019- 10/2023 € 252,780 nephropathology. Kidney int, 98:65-75 and precisioN mEdicine: in vitRo modellinG human No. 860715 physiology and diseAse Boor, P. Heisenberg Professor – Translational Nephropathology DFG: BO 3755/6-1 2017-2020 € 376,200 Awards Boor, P. Ecosystem for Pathology Diagnostics with AI BMWI: EMPAIA 2020-2022 € 228,000 2020 – Franz Volhard-Award of the Germany Society for Nephrology to Peter Boor Assistance Boor, P. The role of epithelial CD74 in kidney diseases DFG: BO 3755/9-1 2020-2022 € 442,050 Djudjaj, S. Boor, P. Deutsches Register von COVID-19 obduzierten Fällen BMG: ZMVI1- 2020-2022 € 923,250 2520COR201 Boor, P. (Coordinator) Deutsches Forschungsnetzwerk Autopsien bei BMBF: 01KX2021 09/2020-03/2021 € 6,882,255 Pandemien Boor: € 836,660 Boor, P. Deep Liver: Diagnosestellung und Risikostratifizierung BMG: ZMVI- 10/2020-09/2023 € 1,932,500 von Lebererkrankungen mittels Deep Learning anhand 2520DAT111 Boor: € 500,428 von klinischen Routinedaten

82 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 83 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-8 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-8

The contribution of autophagy in HSCs for the Applied and actual third-party funding (DFG, BMBF, EU, foundations) Vervoorts, J.. Investigation of the regulation of autophagy by the Cyclin DFG Applied submitted; pending progression of HCC Y/CDK16 kinase complex result

Promoting of young researchers Vervoorts-Weber, J. (Institute of Biochemistry and Molecular Biology) Bachelor/Master Theses Vucur, M. (Department of Internal Medicine III) Bronneberg, G. 2020 RWTH Aachen University, Functional characterization of the cyclin-dependent kinase CDK16 and its Faculty 1 substrates The aim of the study is to define the role of autophagy in hepatic stellate cells (HSCs) during liver cancer development and to understand the contribution of Cyclin Y/CDK16 for the induction of autophagy in a in vivo model. For this purpose, we performed the well- established DEN/CCl4 liver cancer model in mice in which autophagy was inhibited (ATG5- Doctoral Theses (within the scope of the project) knock-out) or hyper-activated (mTOR-Knock-out) specifically in stellate cells. In the case of Vervoorts-Weber, J. Vucur, M. inhibited autophagy in hepatic stellate cells, no difference in liver cancer development could Müller, K. Ongoing RWTH Aachen University, The relevance of autophagy in HSCs during liver cancer development be detected. Interestingly, however, in the case of hyper-activated autophagy, a significant Faculty 1 decrease in tumor development could be observed. RIPK1 was identified as a possible Staff: Amelang, J. Ongoing RWTH Aachen University, The contribution of Cyclin Y/CDK16 in HSC during liver cancer development target responsible for the observed effect. Further investigations are currently in progress to Amelang, J. Faculty 1 elucidate the phenotype. Unfortunately, the involvement of Cyclin Y/CDK16 and its influence Müller, K. on autophagy could not be investigated in this model yet, as the establishment of CDK16- /- mice had proven to be very complex and lengthy. However, we were able to complete and publish the studies on the regulation of Cyclin Y/CDK16 by AMPK during the induction of autophagy. Furthermore, we have established a BioID screen for Cyclin Y/CDK16, which allowed us to identify a number of autophagy regulatory factors as Cyclin Y/CDK16 substrates. Further investigation of the identified factors is the subject of a DFG proposal, which is submitted.

Publications Dohmen M, Krieg S, Agalaridis G, Zhu X, Shehata SN, Pfeiffenberger E, Amelang J, Bütepage M, Buerova E, Pfaff CM, Chanda D, Geley S, Preisinger C, Sakamoto K, Lüscher B, Neumann D ,Vervoorts J (2020) AMPK-dependent activation of the Cyclin Y/CDK16 complex controls autophagy. Nat. Commun., 16(1):1032. doi: 10.1038/s41467-020- 14812-0. [IF 12.1]

Müller K, Honcharova-Biletska H, Koppe C, Egger M, Schneider AT, Böhm F, Boege Y, Healy ME, Schmitt J, Comtesse S, Chan LK, Castoldi M, Preisinger C, Szydlowska M, Gaisa N, Tacke F, Boor P, Davis RJ, Longerich T, Heikenwalder M*, Weber A*, Vucur M*, Luedde T*. JNK signaling prevents biliary cyst formation through a CASPASE-8-dependent function of RIPK1 during aging. PNAS. 2021. in press. [IF 9.4]

84 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 85 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-9 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-9

Targeting stromal interactions in colon cancer and liver metastasis

Lederle, W. (Institute for Experimental Molecular Imaging) Borkham-Kamphorst, E. (Institute of Molecular Pathobiochemistry, Experimental Gene Therapy and Clinical Chemistry)

Advanced colorectal cancer (CRC) is associated with a poor prognosis due to distant metastases, most frequently occurring in the liver. Thus, novel therapeutic options are needed to improve patients’ survival. Lederle, W. Borkham-Kamphorst, E. Immunotherapy targeting both, CTL-4 and PD-L1, strongly inhibited colon cancer growth and prevented liver metastasis in a microsatellite-stable CRC model. In addition to the anti- tumorigenic T cell response induced by CTLA-4 blockade, anti-PD-L1 mediated polarization Staff: of macrophages to the pro-inflammatory M1 phenotype was shown to play an important Salimi, A. role in tumor growth inhibition (Fiegle et al., 2019). Cell culture experiments showed no clear effect of PD-L1 blockade on gene expression by macrophages in mono- and co-culture with colon cancer cells, indicating that complex interactions are responsible for the anti-PDL-1 induced alterations in macrophage function in vivo.

A stronger variation in tumor size was observed after single immunotherapy as compared to combination therapy. To study this heterogeneity in treatment response more closely, sole immunotherapy was performed in additional tumor bearing animals, and tumor growth and angiogenesis were investigated by longitudinal noninvasive imaging. Initial results revealed that in addition to varying tumor growth, the composition of the tumor microenvironment was Figure 1: Heterogeneities in the composition of the tumor microenvironment upon single immune checkpoint blockade. heterogeneous in response to therapy (Figure 1). Detailed gene expression analyses of the Left: Scheme of the orthotopic colon cancer model with primary colon cancer and liver metastases. Right: Individual tumors tumors are currently undertaken to gain deeper insights into the mechanisms that regulate treated with anti-PD-L1 antibodies differ in microvessel density (green) and the amount of FAP positive fibroblasts (red) (scale the response to immune checkpoint inhibition. bar:100µm).

Publications Applied and actual third-party funding (DFG, BMBF, EU, foundations) Borkham-Kamphorst E., Haas U, Van de Leur E, Trevanich A, Weiskirchen R. (2020) Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Lederle, W. Priming tumor blood vessels DFG, GRK2375 10/2018-09/2021 € 189,720 Null Mice through Endoplasmic Reticulum Stress and Unfolded Protein Response. Int J Mol and the microenvironment Sci. 21: 5230. [IF 4.55]

Promoting of young researchers Doctoral Theses

Fiegle, E. 2020 RWTH Aachen University, Dual CTLA-4 and PD-L1 blockade inhibits tumor growth and liver metastasis Faculty 10 in a highly aggressive orthotopic mouse model of colon cancer

86 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 87 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-10 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-10

The role of Nrf-2 pathways in Myeloid Applied and actual third-party funding (DFG, BMBF, EU, foundations) Ohl, K. Role for the Itaconate/Nrf2 axis in metabolic and DFG applied € 315,550 derived suppressor cells, relevance epigenetic reprogramming of neonatal macrophages submitted; pending result for immunosuppression in the tumor- Tenbrock, K. PERMIDRIAR: Personalized Medicine in refractory EU 07/2021-06/2024 € 1,030,000 microenvironment juvenile idiopathic arthritis

Promoting of young researchers Doctoral Theses Ohl, K. (Department of Pediatric Pneumology, Allergology and Immunology) Tenbrock, K. (Department of Pediatric Pneumology, Allergology and Immunology) Hebbar Subramanyam, S. Ongoing RWTH Aachen University, Faculty 1 NRF2 as regulator of immune suppression in the tumor environment

Oxidative stress within the tumor microenvironment can reprogram cancer cells but also immune cells. Among Immune cells, immunosuppressive myeloid derived suppressor cells (MDSCs) play a major role in tumor immunology. MDSCs accumulate in tumors, enhance expression of regulatory T cells and thus prevent T cell mediated anti-tumor activities and are therefore a potential target for cancer therapy. Oxidative stress conditions and high Ohl, K. Tenbrock, K. levels of reactive oxygen species (ROS) activate Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the main transcriptional regulator of the anti-oxidant stress defence. The aim of this project is to decipher the transcriptional and metabolic cues how Nrf2 regulates Staff: immunosuppression in human tumor-associated MDSCs, how Nrf2 activation affects Hebbar Subramanyam, S. hepato-intestinal tumorigenesis and further to uncover genes and pathways that are regulated in tumor MDSCs during carcinogenesis. In 2018, we could confirm that Nrf2 activation induces metabolic alterations in MDSCs in mice in vivo (Ohl et al., Frontiers in Immunology, 2018) and regulates human MDSC differentiation in vitro, while surprisingly, it prevents the expression of regulatory T cells resulting in autoimmunity in mice (Klemm et al. Eur J Immunol 2019). Moreover we could show that glucose metabolism is altered in neonatal macrophages (Dreschers et al. Nat. communications 2019), which might be related to Nrf2 mediated pathways. In in vivo models of cancer, mice bearing a constitutive activation of Nrf2 in immune cells revealed higher number of colorectal tumors in an in vivo model of colitis-associated cancer and a higher tumor size in a subcutaneous tumor model. Enhanced carcinogenesis involved higher numbers of MDSCs and regulatory T cells and a reduced infiltration of cytotoxic T cells into the tumor tissue. In cooperation with the Biobank, we are currently analysing Nrf2 expression in human tumour tissues. In addition, a MDSC breast cancer in vitro model in cooperation with Jochen Maurer, experimental gynecology is ongoing.

88 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 89 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-11 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-11

In summary, our mouse data indicate that CXCL4 in MPN HSPC induces migration of Gli1+ Dissecting the cross talk between stromal cells and myofibroblastic differentiation and thus represents a central chemokine in the fibrotic transformation. Our data suggest that CXCL4 released from megakaryocytes megakaryocytes and Gli1+ stromal cells in bone and platelets induces an inflammatory reaction including S100A8/S100A9 in hematopoietic cells but also stromal cells (Gleitz et al., 2020). CXCL4/CXCL4L1 targeting should be an marrow fibrosis and leukemic transformation in interesting therapeutic option for BM fibrosis in combination with JAK inhibitors. The patient specific JAK2 V617F iPS cells with and without CXCL4/CXCL4L1 knockout allows testing this in the human system and identifying compounds targeting this pathway, similar to a myeloproliferative neoplasms study on KIT D816V in mast cell leukemia (Toledo et al., 2020).

Kramann, R. (Department of Internal Medicine II) Zenke, M. (Department of Cell Biology, Institute for Biomedical Engineering)

The objective of the study was to determine the impact of dysplastic megakaryocytes (MK) on bone marrow (BM) fibrosis in myeloproliferative neoplasms (MPN) in both the mouse and the human system.

BM fibrosis refers to the replacement of blood forming cells in BM by scar tissue, which is Kramann, R. Zenke, M. associated with leukemic transformation. In this funding period we studied BM fibrosis (i) in the mouse model of thrombopoietin (ThPO)-induced fibrosis and (ii) in the human model of patient-specific JAK2 V617F induced pluripotent stem cells (iPS cells). Staff: Böhnke, J. Using genetic fate tracing of Gli1+ cells and CXCL4 knockout in hematopoietic stem/ Kabgani, N. progenitor cells (HSPC) in ThPO-induced BM fibrosis (Figure 1A), we examined whether CXCL4 upregulation is one of the early events responsible for the myofibroblast differentiation and migration of Gli1+ stromal cells. We found that higher CXCL4 expression in HSPC in MPN leads to increased JAK-STAT activation, has profibrotic effects and mediates the characteristic inflammation in the initiation and progression of fibrosis (Figure B and C). CXCL4 knockout did not entirely resolve BM fibrosis but restored the pathognomonic MPN phenotype, indicating that targeting CXCL4 might be an interesting therapeutic option in combination therapy with JAK inhibitors.

In the human model, iPS cells with monoallelic or biallelic JAK2 V617F mutation were generated from patients with JAK2 V617F mutation and differentiated into MK (Figure 1D). Figure 1: Schematic representation of ThPO-induced myelofibrosis mouse model (A). CXCL4 knockout in hematopoietic cells decelerates the progression of fibrosis (B) and leads to reduced iPS cells without JAK2 mutation of the very same patient were also obtained and used as expression of profibrotic pathways and reduced inflammation (C). Experimental design for iPS cell-derived in vitro MK / MSC coculture experiments (D). Repair of the biallelic JAK2 V617F controls. A patient with high JAK2 V617F allele burden (96%) yielded only biallelic JAK2 mutation in iPS cells (JAK2 V617Fhom) to generate monoallelic JAK2 V617F iPS cells and isogenic iPS cells without JAK2 V617F mutation (JAK2 V617Fhet and JAK2, respectively) (E) and V617F iPS cells and thus monoallelic JAK2 V617F iPS cells and isogenic iPS cells without schematic representation of CRISPR/Cas9 genome editing complex (F). Differentiation of JAK2 V617F iPS cells reveals increased production of CD235a/glycophorin+ erythroid cells due to JAK2 mutation were generated by JAK2 V617F mutation repair by CRISPR/Cas9 genome JAK2 V617F mutation. editing (Figure 1E).

JAK2 V617F iPS cells (and isogenic controls) were differentiated into JAK2 V617F dysplastic MK and cocultured with mesenchymal stem/stromal cells (MSC) to recapitulate the findings on CXCL4 of the mouse model in the human system (Figure 1D). The human genome contains CXCL4 and the CXCL4 isoform CXCL4L1, which is not present in mice. To this end CXCL4 and CXCL4L1 deficient JAK2 V617F iPS cells were generated by CRISPR/Cas9 genome editing. This collection of iPS cells puts us now in the position to determine the impact of the JAK2 V617F-CXCL4/CXCL4L axis on myelofibrosis.

90 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 91 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-11 Progress Report 2020 | Joint Projects | Phase Transition in Disease | O3-11

Publications Applied and actual third-party funding (DFG, BMBF, EU, foundations) Kuppe, C., Ibrahim, M. M., Kranz, J., Zhang, X., Ziegler, S., Perales-Patón, J., Jansen, J., Reimer, K. C., Smith, J. R., Dobie, R., Wilson-Kanamari, J. R., Halder, M., Xu, Y., Kabgani, Kramann, R. GLi2Fib MIWF NRW 2020-2022 € 560,000 N., Kaesler, N., Klaus, M., Gernhold, L., Puelles, V. G., Huber, T. B., Boor, P., Menzel, S., Kramann, R. ORGANOSTRAT BMBF 2020-2021 € 139,000 Hoogenboezem, R. M., Bindels, E. M. J., Steffens, J., Floege, J., Schneider, R. K., Saez- Rodriguez, J., Henderson, N. C., and Kramann, R. (2020). Decoding myofibroblast origins Kramann, R. COVID-EPI-Map DFG 2021-2022 € 140,000 in human kidney fibrosis. Nature, online ahead of print [IF 42.7] Kramann, R. Single cell resolution of chronic kidney disease DFG 2019-2021 € 570,000

Leimkühler, N. B., Gleitz, H. F. E., Ronghui, L., Snoeren, I. A. M., Fuchs, S. N. R., Nagai, Kramann, R. Development of novel therapeutics to target fibrosis MIWF NRW 2016-2021 € 1,25 Mio. J. S., Banjanin, B., Lam, K. H., Vogl, T., Kuppe, C., Stalmann, U. S. A., Buesche, G., Kramann, R. Targeting Cardiac Fibrosis in CKD ERC 2016-2021 € 1,5 Mio. Kreipe, H., Gütgemann, I., Krebs, P., Banz, Y., Boor, P., Wing-Ying, E., Brümmendorf, T. Kramann, R. Dissecting the bone marrow stromal contribution to DFG-KFO 344 2020-2022 € 370,000 H., Koschmieder, S., Crysandt, M., Bindels, E., Kramann, R.,* Costa, I. G.* and Schneider, myelofibrosis. R. K.* (2020). Heterogeneous bone marrow stromal progenitors drive myelofibrosis via a druggable alarmin axis. Cell Stem Cell, online ahead of print [IF 20.8] * shared senior Kramann, R. A 3D integrated map of the kidney and bone marrow BMBF 2020-2024 € 1,1 Mio. authors. interstitium Zenke, M. StemCellFactory: Automatic production, expansion and EU/Bio.NRW 2017-2020 € 1,013 Mio. Gleitz, H. F. E., Dugourd, A. J. F., Leimkühler, N. B., Snoeren, I. A. M., Fuchs, S. N. R., differentiation of induced pluripotent stem cells (iPS cells) Menzel, S., Ziegler, S., Kröger, N., Triviai, I., Büsche, G., Kreipe, H., Banjanin, B., Pritchard, Zenke, M., Rolke, Bio2Treat BMBF 2019-2022 € 1,193 Mio. J. E., Hoogenboezem, R., Bindels, E. M., Schumacher, N., Rose-John, S., Elf, S., Saez- R., Kurth, I. & A. Rodriguez, J., Kramann, R.,* and Schneider, R. K.* (2020). Increased CXCL4 expression Lampert in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN. Blood 136: 2051–2064 [IF 17.5] * shared senior authors. Zenke, M. The impact of human megakaryocytes in JAK2V617F and DFG-KFO 344 2020-2022 € 225,600 CALR mut induced myelofibrosis. Baumeister, J., Chatain, N., Hubrich, A., Maié, T., Costa, I. G., Denecke, B., Han, L., Küstermann, C., Sontag, S., Seré, K., Strathmann, K., Zenke, M., Schuppert, A., Brümmendorf, T. H., Kranc, K. R., Koschmieder, S., and Gezer, D. (2020). Hypoxia-inducible factor-1 (HIF-1) is a new therapeutic target in JAK2V617F-positive myeloproliferative neoplasms. Leukemia 34, 1062-1074. [IF 8.6] Promoting of young researchers Doctoral Theses Hollmann, J., Brecht, J., Goetzke, R., Franzen, J., Selich, A., Schmidt, M., Eipel, M., Ostrowska, A., Hapala, J., Fernandez-Rebollo, E., Müller-Newen, G., Rothe, M., Eggermann, Böhnke, J. Ongoing RWTH Aachen University, Faculty 1 Patient-specific iPS cells for modelling bone marrow T., Zenke, M., and Wagner, W. (2020). Genetic barcoding reveals clonal dominance in iPSC- fibrosis and leukemic transformation. derived mesenchymal stromal cells. Stem Cell Res. Ther. 11, 105. [IF 1.5] Flosdorf, N. Ongoing RWTH Aachen University, Faculty 1 The impact of human megakaryocytes in JAK2V617F induced myelofibrosis. Lampert, A., Bennett, D. L., McDermott, L. A., Neureiter, A., Eberhardt, E., Winner, B., and Zenke, M. (2020). Human sensory neurons derived from pluripotent stem cells for disease modelling and personalized medicine. Neurobiol. Pain 8, 100055. [IF 0.8]

Toledo, M. A. S., Gatz, M., Sontag, S., Gleixner, K. V., Eisenwort, G., Feldberg, K., Hamouda, A. E. I., Kluge, F., Guareschi, R., Rossetti, G., Sechi, A. S., Dufva, O. M. J., Mustjoki, S. M., Maurer, A., Schüler, H. M., Goetzke, R., Braunschweig, T., Kaiser, A., Panse, J., Jawhar, M., Reiter, A., Hilberg, F., Ettmayer, P., Wagner, W., Koschmieder, S., Brümmendorf, T. H., Valent, P., Chatain, N. *, and Zenke, M. * (2020). Nintedanib targets KIT D816V neoplastic cells derived from induced pluripotent stem cells of systemic mastocytosis. Blood, online ahead of print with Commentary by A. Dorrance “Mast”ering drug discovery by iPSCs” [IF 17.5] * shared senior authors.

92 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 93 TN1 I Joint Research Project / Lampert/Kurth p. 96 Sodium channel-related pain disorders: From molecular mechanisms towards personalized treatment

TN1-1 I Lampert p. 98 TN1-6 I Rolke p. 109 Sodium channel mutationinduced excitability changes of In-depth extended phenotyping and neuromodulation using human matrix stimulation PROJECTS TN1-2 I Kurth/Haag p. 101 TN1-7 I Rothermel/Spehr p. 111 Genetics of pain-related sodium channelopathies: From Sodium channel mutation-induced excitability changes of genetic variants to clinical pain phenotypes human nociceptors Translational Neurosciences TN1-3 I Machtens p. 102 TN1-8 I Habel p. 114 Decoding Nav dysfunction in pain at atomic resolution Central processing of sensory input in patients with neuronal sodium channel mutations TN1-4 I Rossetti p. 104 Correcting Channel Dysfunction using variants-selective drugs TN1-9 I Dohrn/Häusler p. 116 Phenotyping pain perception: the key to understanding TN1-5 I Schmalzing/Hausmann p. 107 sodium channelopathies Biochemical and Functional characterization of heterologously expressed pain-related NaV mutants

94 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 95 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1

Sodium channel-related pain disorders: From molecular mechanisms towards personalized treatment

Lampert, A. (Institute of Physiology - Neurophysiology) Kurth, I. (Institute of Human Genetics)

Voltage-gated sodium (Nav) channels are crucial for pain perception. This is illustrated by several human genetic conditions that all lead to either chronic pain or, vice versa, to con- genital painlessness. The type of mutation, its impact on neuron excitability as well Lampert, A. Kurth, I. as the affected Nav channel subtype delineates a complex picture of the disorders. A detailed un- derstanding of these pain disorders, however, has long been precluded given the lack of direct structural information on human Nav channels. Our consortium is now in an excellent position to revise and advance current knowledge on pain-related channelopathies. We will address the new concept of allosteric inactivation that replaces the long-standing ball-and- chain model of channel inactivation, investigate Nav channel dimerization and the impact of mutations on neuronal excitability. Moreover, basic principles of ion-channel function will provide a framework for translation to research in patients suffering from neuropathic pain. Within the first year of the funding period we initiated a prospective, longitudinal patient registry and recruited study patients which were subjected to detailed clinical examination. Multi-modal brain imaging studies in patients are in preparation. Blood samples were investigated by high-throughput molecular genetics and electrophysiological assessment of patient-associated sodium channel mutations was performed together with data integration for atomistic molecular dynamics simulations, machine learning, and computational drug design. Variants were selected for induced pluripotent stem cell (iPS-cell)-derived sensory neuron models, single-channel gating analyses, biochemical assessments of Nav channel proteins. We aim to generate unifying predictive models that directly link pain-associated Nav channel genetic variants with changes in molecular biophysics, altered electrophysiological function, pharmacological responsiveness and clinical phenotypes. Within the first year of the funding period the group of the consortium coalesced nicely leading to the foundation of the Sodium Channel Network Aachen (SCNAachen), which fosters the network and exchange between clinicians and scientists with an interest in sodium channels and pain research. We initiated the SCNAachen online seminar series, which features renowned speakers from around the world with topics related to a translational pain and sodium channel research. The network, as well as the scientific achievements of this IZKF consortium will form the basis for translational, patient-oriented insights and future grant applications.

96 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 97 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-1 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-1

Sodium channel mutation induced excitability changes of human nociceptors

Lampert, A. (Institute of Physiology - Neurophysiology)

Recent evidence suggests that mutations in voltage-gated sodium (Nav) channels and extent of myelination of nerve fibers by Schwann cells are contributing factors in neuropathic pain. During the first funding period we investigated the effect of Nav dimerization on pain-linked mutations in Nav1.7 (Fig. 1A). In collaboration with P3 and P5 were able to show that Figure 1: A: Impact of sodium channel Nav1.7 dimerization on pain-mutation induced persistent current. Co-transfection of the putatively disease causing persistent current is strikingly reduced monomers, adding Lampert, A. difopein in HEK cells interrupts Nav dimers and reduces persistent current as assessed by patch clamp. B: Human induced channel homodimerization to the list of factors potentially modifying the patients pain pluripotent stem cells (hiPSCs) from pain patients are differentiated into sensory neurons and show hyperexcitability in whole- symptoms (Rühlmann et al. 2020). We are in the process of performing a comprehensive cell current clamp recordings. Using CRISPR/Cas) repair of the disease causing mutation reverts the electrical phenotype. C: structure-guided alanine scanning mutagenesis together with P5 to identify assembly Staff: Co-culture of iPSC-derived Schwann-cells and sensory neurons shows their alignment. S100b: Schwann-cell marker. p75: domains involved in homodimerization of Nav1.8 subunits. We performed protein- Körner, J. sensory neuron marker. DAPI stains nuceli. Scale bar: 50µm. protein docking (RosettaDock) simulations of isoforms Nav1.7 and Nav1.8 in homodimer Le Cann, K. configurations. In ongoing work we aim to investigate candidate amino acids residing in the putative dimerization site using site-specific photoreactive cross-linking with unnatural aminoacids in heterologous expression systems. Publications Induced pluripotent stem cells of patients carrying a Nav-mutation were previously Rühlmann, AH, J Körner, R Hausmann, N Bebrivenski, C Neuhof, S Detro-Dassen, P generated (Fig. 1B). Differentiation of iPS-cells into nociceptors showed neuronal Hautvast, CA Benasolo, J Meents, JP Machtens, G Schmalzing, A Lampert. “Uncoupling hyperexcitability, which we could revert by CRISPR/Cas genetic engineering (with M. sodium channel dimers rescues the phenotype of a pain-linked Nav1.7 mutation.” British Zenke). We aim to apply these methods to cells collected from patients from the cohort of Journal of Pharmacology, 2020 Jul 14. doi: 10.1111/bph.15196. the consortium, whose visits are scheduled for the next few months in cooperation with P9 and P6. Kerth CM, Hautvast P, Körner J, Lampert A, Meents JE. “Phosphorylation of a chronic pain To asses the impact of myelination and Schwann cell support in neuropathic pain we have mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity.” J Biol in the last year implemented Schwann cell precursor differentiation from induced pluripotent Chem. 2020 Dec 22 stem cells (Fig 1C). Electrical activity of sensory neurons in the presence of Schwann cells will be investigated using multielectrode array compatible with microfluidic chambers, which Le Cann K, A Foerster, C Rösseler, A Erickson, P Hautvast, S Giesselmann, D Pensold, I are currently being developed in collaboration with Prof. Offenhäuser (FZ Jülich). Kurth, M Rothermel, VB Mattis, G Zimmer, S von Hörsten, B Denecke, T Clarner, J Meents, A Lampert. “The difficulty to model Huntington’s Disease in vitro using striatal Medium Spiny Neurons differentiated from human induced pluripotent stem cells” Scientific Reports, accepted

Israel MR, TS Dash, SN Bothe, SD Robinson, JR Deuis, DJ Craik, A Lampert, I Vetter, T Durek. “Characterization of Synthetic Tf2 as a NaV1.3 Selective Pharmacological Probe” Biomedicines 2020, 8(6), 155.

Lampert, A, DL Bennett, LA McDermott, A Neureiter, E Eberhardt, B Winner, M Zenke. „Human sensory neurons derived from pluripotent stem cells for disease modelling and personalized medicine.” Neurobiology of pain, 2020 Nov 18;8:100055.

98 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 99 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-1 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-2

Applied and actual third-party funding (DFG, BMBF, EU, foundations) Genetics of pain-related sodium Lampert, A. Sodium channelopathies as steppingstone for Nanion technologies, Munich, 10/2020-08/2021 9 month SynchroPatch Hausmann, R. personalized clinical pain therapies Germany for HTS patch-clamp/ channelopathies: From genetic variants to consumables UKA and Grünenthal Bio2Treat BMBF 13GW0334B 2019-2022 € 640,000 clinical pain phenotypes coordinator (€ 1,193,264 total)

Promoting of young researchers Kurth, I. (Institute of Human Genetics) Master Theses Haag, N. (Institute of Human Genetics)

Eswaran, V. Ongoing RWTH Aachen University, Molecular Basis for Permethrin Toxicity in Cats Faculty 10 and its Rescue by Riluzole Mutations in three voltage-gated sodium channels, NaV1.7 (SCN9A), NaV1.8 (SCN10A), and NaV1.9 (SCN11A) have been linked to heritable pain-related disorders. The P2 Albrecht, T. Ongoing RWTH Aachen University, Multielectrode arrays combined with microfluidics subproject focuses on the identification of additional NaV channel variants as well as novel Faculty 1 and 10 for the study of peripheral nerve activity genes implicated in monogenic pain disorders. To achieve this, patients are analysed using whole-exome sequencing and data are evaluated regarding variants in known pain-related Kurth, I. Haag, N. genes as well as novel genes whose function indicates a possible relation to pain disorders. Doctoral Theses During the first year of the funding period, we have sequenced and analysed the exomes of 51 patients who had been recruited from the Z-project. Within those patients, we identified Staff: Rühlmann, A. 2020 RWTH Aachen University, Uncoupling sodium channel dimers rescues the 8 variants in the relevant NaV channel genes of which 3 are of unclear significance and have Lischka, A. Faculty 10 phenotype of a pain-linked Nav1.7 mutation not or only rarely been reported in genetic databases, making them the most interesting candidates for further analyses. In patients who were negative for these mutations, the Kerth, C. 2020 RWTH Aachen University, Phosphorylation of a chronic pain mutation in the focus was broadened and additional variants in interesting genes (e.g. NAGLU, KCNQ5, Faculty 10 voltage-gated sodium channel Nav1.7 increases FBLN5, WDR48) were identified. As a next step, we will assess the impact of the most voltage sensitivity promising variants on protein structure and function and thus their role in the pathogenicity Trinler, C. 2020 RWTH Aachen University, iPSC-derived Schwann Cell Differentiation and of SFN. Furthermore, during the second year of the funding period, we will use long-read Faculty 10 Electrophysiological Analysis of Myelinated nanopore sequencing to decipher the transcriptome and methylome in iPSC-derived Nociceptor Co-cultures nociceptors from patients with NaV channel mutations. Kriegeskorte, S. Ongoing RWTH Aachen University, High-througput patch-clamp assessment of Faculty 10 temperature dependent gating of selected voltage-gated sodium channels Applied and actual third-party funding (DFG, BMBF, EU, foundations)

UKA and Grünenthal Bio2Treat BMBF 13GW0334B 2019-2022 € 60,000 (€ 1,193,264 total)

100 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 101 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-3 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-3

Decoding Nav dysfunction in pain at atomic resolution

Machtens, A. (Institute of Clinical Pharmacology)

Voltage-gated sodium channels (Navs) are responsible for the depolarizing phase of the action potential with essential roles in fast electrical signalling, including signal propagation from the periphery to the central nervous system. Various pain disorders, including Figure 1: Molecular dynamics simulation system of membrane-embedded Nav1.7 with the increased and decreased sensitivity to painful stimuli, have been linked to mutations in the β1 subunit. genes encoding Nav1.7–Nav1.9. We use state-of-the art molecular dynamics simulations to investigate the modulation of Nav1.7 by β subunits, the mechanisms of channel Machtens, A. dimerization, and fast-inactivation gating, which all play key roles in pain-associated channel dysfunction. In close collaboration with experimental studies, we aim at a validated understanding of Nav function at atomic resolution. Furthermore, our simulations will Staff: provide a basis for state-specific drug screening. Kostritskii, A. In 2020 we constructed all-atom molecular dynamics (MD) simulation systems of Kötter, I. C. membrane-embedded Nav1.7, and used extensive MD simulations in combination with Markov state modelling to study the modulation of Nav1.7 with the β1, β2, and β4 subunits and its functional consequences (Figure 1). In collaboration with P1 and P5, we investigated the human Nav1.7/A1632E mutant, which causes symptoms of erythromelalgia and paroxysmal extreme pain disorder (PEPD). Using structural modelling and MD simulations of wild-type and mutant Nav1.7, we uncovered how the mutation impairs binding of the inactivation motif leading to incomplete fast inactivation and so-called persistent currents. However, preventing functional interactions in Nav1.7 channel dimers using the 14-3-3 inhibitor difopein restored the functional phenotype of Nav1.7, indicating that Nav channel inactivation is modulated by channel dimerization. These findings demonstrate the critical implications of sodium channel dimerization in Nav-associated pain syndromes (Rühlmann et al., 2020). These studies are expected to advance our mechanistic understanding of Nav channel function and to promote variant-specific drug development for genetically identified patients as a first step towards personalized treatment of pain disorders.

Publications Rühlmann, AH, Körner, J, Hausmann, R, et al. Uncoupling sodium channel dimers restores the phenotype of a pain-linked Nav1.7 channel mutation. Br J Pharmacol. 2020; 177: 4481– 4496 [IF 7.73]

102 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 103 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-4 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-4

Correcting Channel Dysfunction using variants- selective drugs

Rossetti, G. (Department of Neurology, Faculty 10, RWTH Aachen and Forschungszentrum Jülich)

In this proposal, we aim to develop potent gating modifiers of Nav1.7 by decrypting the effect of small molecules on Nav1.7 gating mechanisms. This knowledge will be next exploited to selectively affect pain-associated Nav1.7 variants and in turn develop of gene- variants-tailored therapeutics in the direction of personalized medicine. Rossetti, G. To this purpose we have implemented three different approaches in parallel. The first approach deals with the understanding of Nav1.7 druggability, and, in particular, how the allosteric cross-talk across ligand binding to different protein’s binding site can Staff: impact on the Nav1.7 functioning. This is the case of Permethrin / Riluzole diverse interplay Albani, S. Figure 1: (A) Permethrin docked on the human, dog and cat Nav 1.7 channel. The binding poses display a similar orientation in human vs cat (Felis catus) vs dog (Canis lupus familiaris). Homology models of cat Eswaran, V. in the binding pocket (chlorine atoms towards the center of the pore), but they established different interactions with the and dog Nav1.7 were constructed and simulated for 2 microseconds by coarse grained channel. Mutation on some of these interacting residues (e.g., Phe 1445/1446 and Asn 955/956) were shown to be molecular dynamics using a modified Martini force field (ElNeDyn 2.2). Preliminary molecular responsible for Permethrin resistance in insects. (B) Dimensionality reduction plot (t-SNE) applied to 54 known inhibitors models of Permethrin differential binding to the Nav1.7 coming from different species were of the channel, showing that clustering molecules based on their chemical similarity, allows for prediction of the associated established (Fig. 1A). binding site. In this case, the considered sites were the Voltage Sensing Domain IV (VSD4), the local anesthetic site (LA) and In the second approach we started evaluating how the chemical features of known Nav1.7 the outer vestibule. (C) Analysis of the bending of the helices during the last microsecond of simulation of the cat channel. binders can impact the binding site selectivity. In a preliminary analysis, we identified three (D) Analysis of the bending of the helices S3 and S4 of the second domain as a function of time. This analysis highlights different sub-chemical spaces, each corresponding to a specific binding site of the Nav1.7 the structural differences of cat vs dog vs human. In both panel C and D, the bending ranges from 0 degrees (blue) to 60 channel: these are the local anaesthetics, the extracellular vestibule and the voltage-sensing degrees (red). domain 4 binding sites (Fig. 1B). In the third approaches we established a simulations protocol to model the effect of mutations on binding sites’ druggability. This was proved to be rather successful already to highlight the differences between cat, dog and human Nav1.7 (Fig. 1C and 1D).

104 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 105 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-4 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-5

Applied and actual third-party funding (DFG, BMBF, EU, foundations) Biochemical and functional characterization PIs from 8 EU universities,including RWTH, H2020-MSCA-ITN-2017 H2020-MSCA-ITN-2017 2018-2021 3 years PhD position plus Humboldt University of Berlin and University of -SimulaTIon in MUltiscaLe consumables of heterologously expressed pain-related NaV Wuppertal, in Germany physicAl and biological sysTEms (STIMULATE) mutants PIs from RWTH Aachen University, Universität Helmholtz School for Data Helmholtz Information 2019-2023 2 PhD positions for three zu Köln, DLR, and the Max-Planck-Institut fur Science in Life, Earth and & Data years plus consumables Eisenforschung Energy (HDS-LEE) Science School (HIDSS), within the framework of Schmalzing, G. and Hausmann, R. (Institute of Clinical Pharmacology) the Helmholtz Information Data Science Academy (HIDA) Our project aims to biochemically and functionally characterize pain-associated Nav Carloni, P. Innovative high performance Helmholtz European 2019-2021 2 PhD positions for three channel mutations at the molecular level to elucidate the underlying diverse mechanisms Rossetti, G. computing Partnering programme years plus consumables of specific disorders. To this end, we cloned basic molecular tools, such as GFP fusion Parrinello, M. approaches for molecular constructs of Nav1.2, Nav1.5, Nav1.7, and Nav1.8, all four Nav-ß subunits, and all De Vivo, M. neuromedicine members of the 14-3-3 family. To optimize our detection capability, we characterized the Concedda, L. influence of cRNA synthesis, fused GFP variants, and detergents suitable for extraction. Schmalzing, G. Hausmann, R. Neumaier, B. We found that post-transcriptional capping, compared with co-transcriptional capping, PIs from RWTH NHR4CES (National High DFG 2020-2030 1 PhD and 2 postdoc enhanced the expression of Nav-GFP constructs at both biochemical and functional levels. In cooperation with JARA-CSD and the Artificial Performance Computing position for 10 years Importantly, the fused GFP portion neither affects activation or inactivation gating. Hence, Staff: Intelligence Center (RWTH Aachen), as well as Center for Computational we can reliably use the same GFP-fusion constructs for both biochemical and functional Li, L. Technical University Darmstadt. Engineering Sciences) characterizations. Using high-resolution clear-native PAGE (hrCN-PAGE), we provided the Paul, P. first biochemical evidence that Nav1.7 and Nav1.8 share homodimerization ability with Nav1.5. In addition, we co-expressed the Nav1.7WT and the NaV1.7A1632E mutant in exact Promoting of young researchers stoichiometric ratios in our oocyte expression system, allowing us to record the relative Master Theses persistent current in heteromeric Nav1.7 channel dimers (see Fig. 1). We published these results together P1 and P3 (Rühlmann et al, 2020). Also together with P1 and P3, we are in Eswaran, V. Ongoing RWTH Aachen University, Molecular Basis for Permethrin Toxicity in Cats the process of performing a comprehensive structure-guided alanine scanning mutagenesis Faculty 10 and its Rescue by Riluzole to identify assembly domains involved in homodimerization of Nav1.8 subunits.

Doctoral Theses

Albani, S. Ongoing RWTH Aachen University, Identification of Voltage-gated sodium channels- Faculty 1 variants-selective inhibitors

Figure 1: Voltage dependence of relative persistent current in X. laevis oocytes expressing the indicated wt or mutant Nav1.7, alone or together.

106 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 107 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-5 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-6

Publications Rühlmann AH, Körner J, Hausmann R, Bebrivenski N, Neuhof C, Detro-Dassen S, Hautvast In-depth extended phenotyping and P, Benasolo CA, Meents J, Machtens JP, Schmalzing G, Lampert A (2020) Uncoupling sodium channel dimers restores the phenotype of a pain-linked NaV 1.7 channel mutation. neuromodulation using matrix stimulation Br J Pharmacol. 177(19):4481-4496 [IF 7.73]

Applied and actual third-party funding (DFG, BMBF, EU, foundations) Rolke, R. (Department of Palliative Medicine)

Lampert, A. Sodium channelopathies Nanion technologies, 10/2020-08/2021 9 month SynchroPatch for HTS patch-clamp/ Hausmann, R. as steppingstone for Munich, Germany consumables This subproject is organized in co-operation with IZKF group leader Prof. Dr. med. Barbara personalized clinical pain Namer. therapies Goals: Main goal of the subproject is the development of a test batterie for extended sensory phenotyping in patients with small fiber neuropathy (SFN) compared to healthy controls. This approach comprises assessment and stimulation paradigms focusing on smell detection and electrical detection and pain thresholds upon axonal stimulation. Smell Rolke, R. Promoting of young researchers detection thresholds are assessed using sniffing sticks with different concentrations of Bachelor/Master Theses n-butanol. Axonal stimulation allows direct activation of nerve fibers bypassing the skin level and intraepidermal nerve fiber endings with referring touch or pain receptors. For Staff: Neuhof, C. 2020 RWTH Aachen University, Electrophysiological characterization of mutated and fluorescence-tagged, assessing the complete pathway from the peripheral axon to the brain we perform pain- Wahl, R. Faculty 1 voltage-gated sodium channels of the subtype Nav1.5 and Nav1.7 related evoked potentials (PREPS). For a subgroup of SFN patients, we intend to use microneurography in order to assess single nerve fiber activity that might be impaired due to sodium channel mutations in some of the patients. In a second step from assessment to treatment, we intend to modulate the perceived ongoing pain intensity of SFN patients using a commercially available matrix stimulation bandage. Achievements: Within 2020, all assessment, stimulation and neuromodulation techniques have been implemented. Besides developing the experimental framework including acquisition and implementation of devices, we have started training of experimenters, elaborated and shaped protocols and agreed on a final axonal stimulation paradigm. Pilot data of healthy control subjects were measured including PREPS. Microneurography was performed in control subjects and 3 SFN patients. Currently, we suggest no modifications of the original study plan proposal.

Figure 1: Equipment and pain-related evoked potentials (PREPS) of hand and foot showing cerebral cortex activity upon electrical stimulation of A-delta fibers. A. EEG recording unit, B. evoked potential skin electrode, C. trigger unit, D. DS5 digitimer electrical current stimulator, E. PREPS after recording and data sampling reflecting normal activity of A-delta fiber function in a healthy subject.

108 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 109 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-6 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-7

Applied and actual third-party funding (DFG, BMBF, EU, foundations) Cellular and systemic effects of Nav in olfaction Lampert, A. & Rolke R. Bio2Treat BMBF 13GW0334B 2019-2022 € 181,700 (co-applicants) (€ 1,193,264 total) Rolke, R. PallPan - a national strategy for a palliative care BMBF 01 KX2021 08/2020-12/2021 € 75,000 (subproject PI) action plan during the COVID-19 pandemic (NUM-project) Rothermel, M. (Institute for Biology II) Rolke, R. OpTEAMal – palliative care interprofessional Robert Bosch Foundation No. 04/2019-01/2021 € 52,000 Spehr, M. (Institute for Biology II) learning strategies 01000039-008 Rolke, R. (subproject PI) EPCOG – early palliative care in glioblastoma BMBF 01GY1703 11/2018-10/2023 € 115,000 Voltage-gated sodium (Nav) channels are indispensable for the initiation of action potentials and neuronal signal propagation. A neuron’s individual firing properties are a direct Promoting of young researchers consequence of its Nav channel expression profile, which is assembled from a pool of nine Doctoral Theses different α-subunit isoforms as well as various β- and γ-subunits. Surprisingly, however, still relatively little is known about Nav channel subcomposition in several types of sensory Rothermel, M. Spehr, M. Calonec, A. Ongoing RWTH Aachen University, In-depth extended phenotyping: quantitative olfactory testing and electrical axonal neurons. Faculty 10 stimulation test in patients with small fiber neuropathy Interestingly, a direct link between two major sensory systems – pain and olfaction – was recently demonstrated for an inherited Nav channelopathy, involving the Nav1.7 channel Staff: Brammen, J. Ongoing RWTH Aachen University, Neuromodulation using matrix stimulation in small fiber neuropathy isoform. Loss-of-function mutations in SCN9A, the gene encoding Nav1.7, lead to Mayland, J. Faculty 10 complete inability to perceive pain (congenital indifference to pain or CIP) in humans and mice. In the peripheral nervous system, Nav1.7 expression extends from free nerve endings in the skin to terminals in the dorsal horn. Strikingly, the same loss-of-function mutation causes loss of the sense of smell (congenital general anosmia) in both mice and humans. Like several other Navs, Nav1.7 is almost undetectable in olfactory receptor neurons at the mRNA level. However, Nav1.7 was later shown to be specifically expressed only at olfactory receptor neurons presynaptic terminals thereby explaining its importance for olfactory processing. To shed light on potentially overlooked functions of other Nav channels in olfaction we started by investigating a transgenic NaV1.8-cre line generously provided by Prof. Rohini Kuner (Heidelberg University). In collaboration with Prof. Ingo Kurth and Dr. Natja Haag (TN1-2) we performed histology from P11 NaV1.8-cre mice crossed to a dt-tomato reporter line (Figure 1). Sparse but clear cellular marker protein expression was apparent at the level of the olfactory epithelium. Fiber terminals at the level of the olfactory bulb potentially argue for a transgene expression in olfactory receptor neurons. Motivated by this unexpected finding we are currently in the process of getting ready to test Nav1.8 /1.9 double knockout animal generated by Prof. Kurth for potential olfactory phenotypes. First, we aim to determine the KO animals‘ olfactory sensitivity. We, therefore, established a novel behavioral paradigm in which mice were trained to report the absence of an odorant by licking for no-odor (blank) trials (and refrain from licking to any odorants presented). In this paradigm, animals should signal indetectable odor concentrations by starting to lick randomly. In contrast to a classical odor concentration paradigm (where odorants are rewarded) this new paradigm has the advantage that a lack of motivation (no licking) cannot be misinterpreted as an inability to detect an odorant concentration. In parallel, we are pursuing our goal to investigate the potential influence of a gain-of- function mutation on odor perception in rodent models by generating a novel transgenic mouse model which will allow for specific Nav1.7 gain-of-function expression in olfactory sensory neurons. Together, this study is designed to enhance our understanding of Nav function in sensory signaling, benefitting from the unique molecular and cellular similarities between olfaction and pain.

110 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 111 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-7 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-7

Publications Brunert D, Rothermel M. Cortical multisensory integration-a special role of the agranular insular cortex? Pflugers Arch - Eur J Physiol. 2020 May 26. doi: 10.1007/s00424-020- 02400-6. PMID: 32458084

Jo HG, Wudarczyk O, Leclerc M, Regenbogen C, Lampert A, Rothermel M, Habel U. Effect of odor pleasantness on heat-induced pain: An fMRI study. Brain Imaging and Behavior 2020 DOI 10.1007/s11682-020-00328-0 PMID: 32770446

Böhm E, Brunert D, Rothermel M. Input dependent modulation of olfactory bulb activity by HDB GABAergic projections. Scientific Reports ,10, 10696 (2020). https://doi.org/10.1038/ s41598-020-67276-z PMID: 32612119

Medinaceli Quintela R, Bauer J, Wallhorn L, Le K, Brunert D, Rothermel M. Dynamic impairment of olfactory behavior and signaling mediated by an olfactory corticofugal system. J Neurosci. (2020) JN-RM-2667-19; DOI: https://doi.org/10.1523/ JNEUROSCI.2667-19.2020 PMID: 32817250

Promoting of young researchers Doctoral Theses

Mayland, J. Ongoing RWTH Aachen University, Faculty 1 Cellular and systemic effects of Nav in olfaction

Figure 1: A) Top: Nav1.8-tdTomato expression was observed in mature olfactory sensory neurons as indicated by co- localization with the Olfactory Marker Protein, OMP (anti-OMP staining). Cell body, axon, and apical dendrite with dendritic knob showed tdTomato expression. Bottom: Nav1.8-tdTomato also co-localizes with OMP at the level of the OB glomeruli (OMP labels OSN axons). B) Left: Schematic overview of the mouse training setup depicting a head-fixed mouse freely running on a styrofoam ball. Maus behavior was monitored during the session using an infrared camera. The odor-tube and lick sensor are positioned in front of the mouse. The schematic picture is courtesy of Jennifer Bauer. Bottom: experimental trial structure. S+ / S- presentations were randomized. Right: Representative part of one behavioral session (ordinate displays trial number in chronological order). Mice were trained in a go/no-go odor paradigm and discriminated rewarded no odor (blank) stimuli (S+, duration 4 s) from odorants presented at different concentrations (S–, shaded red area indicates an increase in odor concentration) by licking in response to the S+ and refraining from licking to the S–. Behavioral session subtrials are ordered by S+ and S–trials. Light dots represent licks. A response on a S+ trial (hit) and no response during an S– trial (correct rejection) were categorized as correct responses; no response on a S+ trial (miss) or a response during an S– trial (false alarm) was categorized as incorrect responses. Odor concentration was increased in the S- trials changing the animal‘s behavior from an expected random licking at concentrations indistinguishable from rewarded blank presentations, to correct rejections.

112 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 113 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-8 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-8

Publications Central processing of sensory input in patients Jo, H. G., Wudarczyk, O., Leclerc, M., Regenbogen, C., Lampert, A., Rothermel, M., & Habel, U. (2020). Effect of odor pleasantness on heat-induced pain: An fMRI study. Brain with neuronal sodium channel mutations Imaging and Behavior, 1-13. [IF 3.391]

Promoting of young researchers Doctoral Theses Habel, U. (Department of Psychiatry, Psychotherapy and Psychosomatics) Scheliga, S. Ongoing RWTH Aachen University, Faculty 10 Central processing of sensory input in patients with neuronal sodium channel mutations This subproject proposes a systematic brain imaging approach that provides insight into which brain structures and functions are significantly involved in pain processing in inherited pain disorders associated with Nav1.7, Nav1.8, and Nav1.9 mutations. In order to achieve this goal, we will choose pain patients identified in the consortium carrying rare sodium channel mutations. Patients will undergo a combined EEG-fMRI recording while being presented with pain and odor stimuli simultaneously. Particularly pain disorders with Habel, U. Nav1.7 mutations are of interest since Nav1.7 has also been shown to be the main sodium channel in olfactory sensory neurons and their processes. Previous studies examining the interplay of odor and pain in peripheral sensory neurons at the cell level suggest, there is an Staff: interaction between these two sensory domains. Therefore, we also want to find out how Scheliga, S. this interaction works in mutation carriers at the system level by using neuroimaging. For 2020, the successful implementation of the experiment and pilot testing were achieved. From the pilot phase, we received a first data set, which we will include in the group of healthy controls. We are currently programming the scripts for the pre-processing and for the analysis of task-fMRI data. First analysis of these data has shown significantly higher brain activity in pain-related structures (amygdala and thalamus) under hot temperatures compared to warm temperatures (see figure 1). Notably, amygdala and thalamus, are known to be part of the pain network. Furthermore, we organized the recruitment of patients. We will conduct another pilot test for the combined EEG-fMRI measurement with simultaneous presentation of pain- and odor-stimuli. After successful testing, we plan to start clinical examinations on mutation carriers.

Figure 1: Brain activation under painful thermal stimulation

114 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 115 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-9 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-9

Phenotyping pain perception: the key to understanding sodium channelopathies

Dohrn, M. (Department of Neurology) Häusler, M. (Department of Pediatric Neurology) Maier, A. (Department of Neurology)

In relation to our overall project aims, the main goals for the first year were A) to screen as many neuropathic pain patients as possible, B) to characterize these patients and thereby assess the detailed sub-phenotypes, and C) to enter these phenotype data in a newly set- up integrative registry to enable further patient recruitment for other co-projects. Additionally Figure 1: Z project recruitment overview to the 100 patients with idiopathic small fiber neuropathies (SFN) that we had characterized Dohrn, M. Häusler, M. Abbreviations: WES, whole exome sequencing; NRS, numeric pain scale; painDETECT, standardized neuropathic pain before 2020 already, we were now able to invite 98 pre-screened adults and 32 children questionnaire; NCS, nerve conduction studies; QST, quantitative sensory testing; fMRI, functional magnet resonance imaging; for a detailed study visit. Out of the above, we fully enrolled (aim A, B, C) 50 patients iPSCs, induced pluripotent stem cells. (15 male, 35 female) in the department of neurology and 8 patients (5 male, 3 female) in the department of neuropediatrics in the first year of this project. In 8 further adults and Staff: two further children, some examination results are pending, whereas enrolment is still Berch, R. incomplete in another 14 individuals. A non-foreseen limitation was the Covid-19 pandemic Quade, A. Promoting of young researchers that rendered us unable to conduct study visits between March and May 2020. Later on, Peschke, G. Maike F. Dohrn is a postdoctoral researcher and a resident physician at the Department 12 patients were not able to travel to our center for their scheduled study visit due to high of Neurology at the RWTH Aachen University Hospital. As PI of the Z project, this project regional Covid-19 case levels. We conducted a Covid-19 questionnaire study assessing contributes significantly to her scientific visibility. Further young researchers in this group the individual worries and needs in a mixed adult patient cohort (n = 100) with rare are Regina Berch, likewise a resident physician, and Christina Dumke, medical doctoral neuromuscular diseases, one third of whom (n = 33) represented by patients with idiopathic student and now junior resident. Both young women pursue a clinical and scientific career SFN. In order to develop a specific questionnaire on risk constellations in childhood and in parallel. Within the frame of the Z project, they do not only have the chance to learn novel adolescence, we interviewed 13 adult patients with familiar or genetic SFN, assessing scientific methods, but also to incorporate own ideas. Greta Peschke is a young medical exposures and events that preceded disease onset. Likewise to establish specific features student with great ambitions for a scientific career. In her position as a “Studentische of genetic SFN subtypes, we examined 30 children/ adolescents with diabetes, 15 with Hilfskraft” for the Z project, she gets the chance to become acquainted with study protocols suspected psychosomatic symptoms, and 8 that receive chemotherapy and compared and methods. For all young researchers in this group, this is a unique and indispensable them with our idiopathic SFN cohort. Through aggregation and clustering of detailed experience and chance to learn from senior scientists in the field. phenotype data, we continue to aim at characterizing the entire spectrum of potentially Annegret Quade is a postdoctoral researcher and works as a paediatrician at the hereditary neuropathic pain syndromes at any age. Although not being able to travel, we still Department of Neuropediatrics at the RWTH Aachen University Hospital. Her aim is had the chance to promote our work at several virtual conferences (invited oral presentation to further investigate primary and secondary small fiber neuropathy in children and on “small fiber neuropathies” in the Richard-Jung-Kolleg, DGKN congress 2020; poster adolescents and their symptoms in this age group. on “hopes and needs of 100 idiopathic small fiber neuropathy patients”, DGKN congress Three medical doctoral students assist her at doing so. In this connection, all of them get in 2020; 3 further abstracts accepted at the DGM and AAN conferences 2021). We enlarged touch with research and learn new methods. our international collaborative network by becoming project partners with Professor Stephan Züchner at the University of Miami and Professor Anne Louise Oaklander at the Harvard University in Boston, USA. These recently established collaborations are important opportunities for the exchange of experimental methods as well as unsolved patient cases that might be of value to gain further knowledge on voltage-gated sodium channel mutations in the context of pain. Within the frame of the Z project, three original papers are currently in preparation: one on the phenotypic spectrum and biomarkers of idiopathic small fiber neuropathies, one on our Covid-19 questionnaire study, and one on a Brazilian patient cohort with hereditary sensory neuropathies and congenital insensitivity to pain, partially caused by loss-of-function mutations in voltage-gated sodium channels.

116 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 117 Progress Report 2020 | Joint Projects | Translational Neurosciences | TN1-9

Promoting of young researchers Doctoral Theses

Dumke, C. 2020 RWTH Aachen University, Faculty 10 Characterization of idiopathic small fiber neuropathies considering phenotypic subgroups and potential risk factors Berch, R. 2020 RWTH Aachen University, Faculty 10 Questionnaire study on thoughts and needs in the pandemic, focussing on and comparing individuals with rare neuromuscular diseases (Charcot-Marie- Tooth disease, small fiber neuropathies, and chronic inflammatory demyelinating polyradiculoneuropathy) Bruell, A. Ongoing RWTH Aachen University, Faculty 10 Abnormal sensory perception due to secondary neuropathies and psychsomatic disorder. A pediatric comparative clinical trial.

Study Population 1: oncological patients (N=15) Havenith, J. 2020 RWTH Aachen University, Faculty 10 Abnormal sensory perception due to secondary neuropathies and psychsomatic disorder. A pediatric comparative clinical trial.

Study Population 1: patients with DM Typ 1 (N=30) Müller, K. 2020 RWTH Aachen University, Faculty 10 Abnormal sensory perception due to secondary neuropathies and psychsomatic disorder. A pediatric comparative clinical trial.

Study Population 1: patients suffering from psychosomatic disorder (N=15)

118 IZKF Aachen Progress Report 2020 OC1 I Joint Research Project / Wolf p. 122 Regulatory networks controlling the soft tissue-alveolar bone crosstalk in periodontal disease: From a comprehensive mechanistic understanding to novel diagnostic and therapeutic strategies

OC1-1 I Pufe p. 123 OC1-9 I Wolfart/Schneider p. 131 The influence of the soft tissue´s Nrf2-Keap1 system on Liver transplantation and its effects on the oral microbiome PROJECTS alveolar and long bone regeneration and periodontal health in combination with different dental prosthetic restoration OC1-2 I Wolf/Mottaghy p. 124 Dissecting the role of TLR4 dependent signaling networks in OC1-10 I Götzl/Beier p. 132 Organ Crosstalk vitro and quantitative molecular imaging of periodontal bone ASC-based in vitro and in vivo bone tissue engineering for remodelling in vivo. bone defect reconstruction

OC1-3 I Neuss-Stein/Jahnen-Dechent p. 125 OC1-11 I Jankowski/Wolf p. 133 Impact of stem cells on wound healing and integration of Impact of chronic renal failure on periodontal bone disease tissue engineered alveolar bone and evaluation of potential treatment by recently identified and yet unknown calcification mediators OC1-4 I Lüscher/Sechi p. 126 Influence of the PCTAIRE Kinase CDK16 on osteoclast OC1-12 I Braun/Wenzler p. 134 differentiation and biology in alveolar bone remodelling Evaluation of endodontic periodontal interface and impact on alveolar bone OC1-5 I Schneider-Kramann/Crysandt p. 127 ALARMin MDS and periodontal diseas OC1-13 I Eschweiler/Tingart p. 135 In vitro and in silico investigation of mechano-regeneration OC1-6 I Abdelbary/Conrads p. 128 of subchondral bone using an artificial neuronal network for Porphyromonas gingivalis – from genomics to inference cartilage - alveolar bone vs. long bone of virulence signals on both local and systemic level in periodontal bone diseases OC1-14 I Fischer/Aveic p. 136 Unraveling the effect of dynamic forces on regeneration OC1-7 I Ludwig/Babendreyer p. 129 capacity of periodontal ligament stem cells embedded in 3D Osteoclast activation by soluble mediators derived from bone-like ECM inflamed mucosa

OC1-8 I Modabber/Hildebrand p. 130 Bone regeneration of typical morphological bone defect in periodontitis by mesenchymal stem cells of the pulp based on the regulatory influence of trauma related release of extracellular vesicles

120 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 121 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-1

Regulatory networks controlling the soft The influence of the soft tissue´s Nrf2-Keap1 tissue-alveolar bone crosstalk in periodontal system on alveolar and long bone regeneration disease: From a comprehensive mechanistic understanding to novel diagnostic and therapeutic strategies

Wolf, M. (Department of Orthodontics and Craniofacial Orthopedics) Pufe, T. (Institute for Anatomy and Cellbiology)

Inflammatory diseases and degenerative processes can cause a loss of alveolar bone mass Reconstituting lost bone is a subject that is germane to many orthopedic conditions and periodontal/peri-implant degeneration, resulting in healing defects, tooth destabilization, including non-union, bone tumors, osteoporosis, periprosthetic infection, or inflammatory loss of orthodontic treatment option and ultimately tooth or implant loss. Often, alterations arthritis. While acute inflammation appears to be beneficial for bone repair, chronic in oral and periodontal conditions can be a sign of severe systemic disease. However, inflammation is not and dysregulated inflammation increases bone resorption, causing net the molecular mechanisms underlying this sequence of events are currently not well bone loss. Periodontitis is also characterized by destruction of connective tissue through understood, and therefore at present no targeted therapies are available that can efficiently Wolf, M. an inflammatory host response secondary to infection by periodontal bacteria, ultimately Pufe, T. prevent and treat periodontal diseases and tooth destabilization. causing excessive alveolar bone resorption, which forms a challenge to maxillofacial This IZKF joint project aims at replacing the conventional tissue centered view on the surgeons. pathophysiology of periodontal disease by an integrated and interdisciplinary approach that Crosstalk among inflammatory cells and cells related to bone healing is essential to bone takes into account that dental diseases are influenced not only by complex interactions formation, repair and remodeling. Reactive oxygen species (ROS) have a broad role as within oral cavity, but also by systemic changes and diseases. Therefore, scientific groups intra- and intercellular messengers in diverse cell signaling processes and the Keap1-Nrf2 with a focus on dental diseases and working groups with expertise and strong scientific system is a master regulator of the cytoprotective defense. track records in systemic diseases will join forces to explore mechanisms of organ We were the first to demonstrate a crucial role of Nrf2 in bone regeneration. cross-talk in periodontal disease in this proposal. In the individual projects of this joint We now aim at demonstrating potential differences of Keap1-Nrf2 signaling during the proposal, several mechanistic and methodological approaches will be combined. We aim regeneration of two different types of bone (alveolar vs. long bone) as well as elucidating its at generating synergies between projects that will allow a more comprehensive view on role in the periodontal ligament. mechanisms of organ interactions in periodontal disease by bringing these distinct areas of expertise together. The biological findings and clinical implications of this scientific approach will help to identify novel biomarkers, develop innovative diagnostic tools and therapeutic strategies in this clinical setting.

122 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 123 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-2 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-3

Dissecting the role of TLR4 dependent signaling Impact of stem cells on wound healing and networks in vitro and quantitative molecular integration of tissue engineered alveolar bone imaging of periodontal bone remodelling in vivo

Wolf, M. (Department of Orthodontics and Craniofacial Orthopedics) Neuss-Stein, S. Mottaghy, F. (Department of Nuclear Medicine) (Helmholtz Institute for Biomedical Engineering, Biointerface Group and Institute of Pathology) Jahnen-Dechent, W. Clinical evidence refers to a fundamental role of periodontal micro-environment for (Helmholtz Institute for Biomedical Engineering, Biointerface Group) regulating tissue remodeling, tooth movement and progression of tooth root resorption to periodontal breakdown, the most frequent reason for loss of teeth. Recently, toll-like- receptor 4 (TLR4) signaling networks were proposed as potential modulators of benign Clinical evidence indicates that wounds in alveolar bone heal faster and more efficiently in periodontal remodeling as well as in diseases but the exact roles of TLR-signaling in Wolf, M. Mottaghy, F. the maxilla (upper jaw) than in the mandible (lower jaw). Alveolar bone differs in composition, Neuss-Stein, S. Jahnen-Dechent, W. periodontal tissue for regeneration and degeneration are still unclear. with 23 % bone marrow and 46 % lamellar bone in the upper jaw, and 16 % bone marrow The project consists of 3 parts: Part (I) addresses the regulation of TLR4 in an in vitro and 63 % lamellar bone in the lower jaw. Differences concerning the endogenous stem model. We therefore study periodontal ligament (PDL) cells isolated from mice upper cells (dental pulp stem cells, gingiva stem cells, mesenchymal stem cells and periodontal molars and compare them with murine tooth root cementum cells (OCCM) in a model of ligament cells), and their recruitment towards wounded areas and their differentiation mechanical cell stress in order to simulate periodontal remodeling. In part (II) we investigate potential may also regulate regeneration. physiological relevance for dental tissue remodeling in an in vivo model with TLR4-knockout mice via μPET/CT imaging using tracer for apoptosis, bone metabolism and inflammation, Human mesenchymal stem cells (MSC) are beneficial for wound healing and tissue including their time dependent regulation in early and late phase of remodeling. In part regeneration of mesodermal tissue. MSC differentiate into the cementoblastic and (III), we intend to define the correlation between findings from in vitro cell signaling, murine osteoblastic lines, and exert their trophic function by secreting a variety of cytokines periodontal stimulation and mediator expression in human crevicular fluid samples. In and growth factors. They are well-characterized and frequently used cells in bone tissue line with our findings, we intend to map a tissue specific TLR4 signaling cascade for engineering approaches. periodontium and cementum to define potential therapeutic targets for periodontal remodeling and disease. We propose to study periodontal ligament (PDL) cells isolated from third molars of the upper (u-PDL cells) and lower jaw (l-PDL cells) and compare them with MSC. We will analyze (i) their stem cell characteristics (ii), their trophic phenotype (via transcriptome and proteome analyses), (iii) their migration capacity and (iv) their cell fate within three- dimensional, hydrogel-based bone constructs. To trace PDL cell fate, we will generate stable, fluorescently labeled cell lines from the primary cells. Finally, we will improve established 3D models by using hydrogel-based, ECM containing co-culture systems using PDL cells and endothelial cells to build in vitro constructs with osteoblasts and capillary-like structures for future alveolar bone tissue engineering approaches.

124 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 125 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-4 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-5

Influence of the PCTAIRE Kinase CDK16 on ALARMin MDS and periodontal diseas osteoclast differentiation and biology in alveolar bone remodeling

Lüscher, B. (Institute of Biochemistry and Molecular Biology) Schneider-Kramann, R. Sechi, A. (Institute of Biomedical Engineering / Department of Cell Biology) (Institute for Biomedical Engineerung, Department of Cell Biology) Crysandt, M. (Department of Medicine IV)

Orthodontic movement of teeth and periodontal diseases are associated with alveolar bone remodeling, which is executed by bone rebuilding osteoblasts and bone resorbing Descriptive studies demonstrated a link between periodontal inflammatory conditions and osteoclasts. For the latter osteoclasts attach onto the bone surface and create an isolated cancer. However, the underlying mechanisms remain unclear and haven’t been investigated resorptive microenvironment. This requires the reorganization of the actin cytoskeleton using functional models. In this grant application, we will shed light on the functional to assemble a sealing zone around the resorptive microenvironment composed of Lüscher, B. Sechi, A. association between periodontal inflammation and pre-leukemic conditions (MDS). Schneider-Kramann, R. Crysandt, M. podosomes. Moreover, membrane trafficking has to be reorganized to form bone-facing Preliminary evidence from our collaborative work with Michael Wolf supports a link actin-based ruffled borders enriched in lysosomal membrane proteins. The coordination of between mutations in hematopoietic cells (pre-leukemia) and alterations in the oral cavity. these processes is still elusive. MDS mutations led to degrading periodontal remodeling processes with an increase of inflammatory cells in the periodontal tissue. CDK16 is a cytoplasmic cyclin-dependent kinase that is postulated to affect cytoskeletal Based on these data we hypothesize that the inflammation caused by MDS mutations in processes. CDK16 is involved in the terminal steps of spermatogenesis to generate hematopoietic cells fuels the local inflammation in the periodontium. We propose that this fertile sperms. In the brain CDK16 is suggested to regulate neurite outgrowth and in leads to increased local but also systemic inflammation and a vicious cycle of periodontal muscle CDK16 is involved in terminal differentiation. All these differentiation processes are and systemic inflammation and potentially clonal selection of mutant (pre-leukemic) accompanied by a massive reconstruction of the cellular content, shape and architecture, hematopoietic cells which are potentially more resistant to inflammation than wild-type cells. which requires reorganization of the actin cytoskeleton and membrane transport. To In aim 1, we thus plan to analyze the effect of chronic inflammation caused by periodontal understand CDK16 at the molecular level, we performed a substrate screen and found inflammatory conditions on clonal selection and malignant transformation. In aim 2, we will that one quarter of the 41 identified potential CDK16 substrates have been described determine the influence of mutated hematopoietic cells on periodontal inflammatory bone to regulate the actin cytoskeleton. In this proposal, we will focus on the regulation degeneration. Finally, in aim 3, we will determine if “inflammatory mediators” (in saliva/ of actin cytoskeleton remodeling by the CDK16 substrates. We will investigate their crevicular fluid can be used as a biomarker to detect pre-leukemic conditions. phosphorylation-dependent regulation by CDK16 during osteoblast differentiation and bone resorption. To verify our findings in vivo we will use CDK16 and available substrates knock out mice to investigate their function for alveolar bone remodeling during orthodontic tooth movement. This project provides a deeper understanding of the molecular mechanisms by which osteoclasts resorb bone and may lead to the development of new antiresorptive drugs targeting CDK16 or their substrates.

126 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 127 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-6 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-7

Porphyromonas gingivalis – from genomics to Osteoclast activation by soluble mediators inference of virulence signals on both local and derived from inflamed mucosa systemic level in periodontal bone diseases

Abdelbary, M. (Division of Oral Microbiology and Immunology) Ludwig, A. (Institute for Pharmacology and Toxicology) Conrads, G. (Division of Oral Microbiology and Immunology) Babendreyer, A. (Institute for Pharmacology and Toxicology)

Periodontitis is a biofilm-induced chronic inflammatory disease, which affects the tooth- Infection or damage of oral mucosa triggers release of various mediators from epithelial supporting tissues including the alveolar bone. This continuing inflammation exerts a cells. Soluble mediators become released either upon induction of biosynthesis, secretion major impact on systemic health, as it increases e.g. a patient’s risk for atherosclerosis or of stored vesicular content or proteolytic shedding of surface expressed molecules. In diabetes. It is primarily the host inflammatory response that inflicts the irreversible damage this project we want to investigate the crosstalk between the soft mucosa and the hard to the periodontal tissues, but it is triggered by the sulcus-associated biofilm “red complex” Abdelbary, M. Conrads, G. jawbone. For this purpose, the nature and release mechanism of epithelial cell derived Ludwig, A. Babendreyer, A. bacterial species: Treponema denticola, Tannerella forsythia and Porphyromonas gingivalis. soluble mediators relevant for the modulation of osteoclast activity in the jawbone will The latter species transforms the normally symbiotic microbiota into a dysbiotic state. P. be identified and investigated. We will study conditioned media of epithelial cell lines and gingivalis has evolved sophisticated strategies to evade or subvert the host immune system. primary oral epithelial cells undergoing inflammatory or mechanic stress due to treatment We will isolate 20 strains of P. gingivalis from high-stage, high grade cases of periodontitis with bacterial toxins, stretching or wounding. Release mechanisms for soluble mediators and compare these strains with 20 isolates from low-stage, low-grade cases. This will be differentiated by inhibition of gene transcription, protein secretion or protease activity. comparison will be done on whole-genome level. We will determine e.g. the role of mobile We will study the potential of the epithelial supernatants to induce activation of cultured genetic elements and core-gene mutations in the adaption of P. gingivalis to the inflamed osteoclast cell lines and primary osteoclasts. Selected molecules will be investigated in subgingival sulcus or periodontal pocket leading to a vicious circle. Information on species detail, in terms of their induction, release mechanism, their receptor interaction and their interaction in the corresponding microbiota will be achieved by metagenome analysis. pathway of osteoclast activation. This includes the transmembrane chemokine CX3CL1 Together with genome-information from publicly available databases we will generate the that is induced in epithelial cells and shed by the proteolytic activity of ADAM family world’s largest genome database of this key-pathogen embedded in corresponding clinical metalloproteinases. CX3CL1 mediates recruitment of monocytes and macrophages via and metagenome information. The main expected result is the elucidation of processes its receptor CX3CR1 on osteoclasts. We hypothesize that this pathway is relevant for involved in P. gingivalis evolution to enhance our understanding of bacterial mechanisms osteoclast activation, which will be further studied in tissues of periodontitis patients and a and signaling underlying the development of virulence, both local and systemic. murine model of ligation-induced gingivitis and periodontitis using knockout mice for ADAM proteases or CX3CR1.

128 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 129 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-8 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-9

Bone regeneration of typical morphological Liver transplantation and its effects on the bone defect in periodontitis by mesenchymal oral microbiome and periodontal health in stem cells of the pulp based on the regulatory combination with different dental prosthetic influence of trauma related release of restoration extracellular

Modabber, A. (Department of Oral and Maxillofacial Surgery) Wolfart, S. Hildebrand, F. (Department of Trauma and Reconstructive Surgery) (Department of Prosthodontics and Biomaterials, Center of Implantology) Schneider, K. M. (Clinic for Gastroenterology, Metabolic Diseases and Internal Intensive Care Mesenchymal stem cells (MSCs) have a positive effect on bone regeneration in Medicine) experimental bone defect models. Therefore, dental pulp stem cells (DPSCs) and their extracellular vesicles (DPSC-EVs) are being considered as suitable therapeutic options in cases of impaired bone healing. These disturbances of bone regeneration are especially Modabber, A. Hildebrand, F. Emerging evidence suggests that there is a possible link between a dysbiotic oral Wolfart, S. Schneider, K. M. found in the context of relevant inflammatory reactions, as typically found in periodontitis microbiota and liver disease. While it is already well established that a dysbiotic oral and after severe trauma. This can result in bone loss or impaired fracture healing, both of microenvironment promotes oral diseases such as dental caries or periodontitis, recent which have a significant negative impact on the clinical outcome of the affected patients data also highlight unfavorable oral microbiota composition and translocation of certain oral and represent a major therapeutic challenge for the treating physician. Therefore, the bacteria as drivers of liver disease progression. potential benefit of the therapeutic use of the DPSCs and DPSC-EVs under the above- Still, data on the pathophysiological crosstalk between oral and liver diseases and its mentioned conditions will be investigated in this study. interrelation with the oral and gut microbiota remain scarce. Liver transplant patients offer the unique opportunity to study how restoration of normal liver function affects The aim is to analyze the influence of DPSCs and their EVs on the egenerationr of bone oral microbiota, mucosal immunologic homeostasis and periodontal health. These defects and fracture healing. Furthermore, a potential inflammation modulating effect of patients provide valuable information on the impact that replacement of a diseased liver DPSCs and EVs under inflammatory conditions in periodontal disease, isolated femur with a healthy liver has on the oral microbiota, gut microbiota and periodontal health in fractures, and polytrauma will be investigated. For this purpose, different scaffolds coated combination with different prosthetic restorations. with DPSCs or DPSC-EVs will be used. The analyses include ELISAs, histological staining, Based on a translational approach including a clinical human study with liver transplant μ CT scans and biomechanical tests. patients as well as mechanistic preclinical studies in mouse models, the proposed project is designed to unravel clinically relevant pathophysiological connections between liver disease, oral microbiota and periodontal homeostasis. The premise of this proposal is that a better understanding of the molecular circuits linking oral microenvironment with liver disease will guide efforts to identify future diagnostic and therapeutic targets.

130 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 131 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-10 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-11

ASC-based in vitro and in vivo bone tissue Impact of chronic renal failure on periodontal engineering for bone defect reconstruction bone disease and evaluation of potential treatment by recently identified and yet unknown calcification mediators

Götzl, R. (Department of Plastic Surgery, Hand Surgery, Burn Center) Jankowski, J. (Institute for Molecular Cardiovascular Research) Beier, J. P. (Department of Plastic Surgery, Hand Surgery, Burn Center) Wolf, M. (Department of Orthodontics and Craniofacial Orthopedics)

In an aging society, periodontal soft and hard tissue crosstalk, addressed by regenerative The loss of kidney function leads to a reduction in kidney’s ability to filter metabolites, medicine and tissue replacement approaches, so called Tissue Engineering, plays an causing accumulation of uremic toxins in the blood. Uremia is a result of chronic kidney increasingly important role in periodontal disease. The incidence of a loss of alveolar bone disease (CKD), and adverse outcomes include kidney failure, cardiovascular disease, and mass and periodontal/ peri-implant degeneration will increase in an aging population. The premature death. In addition, reduction of kidney function impairs regulation of acid-base use of human adipose derived stem cells (hASC) in Tissue Engineering is a promising Götzl, R. Beier, J. P. balance, water and electrolyte balance, blood pressure and bone metabolism. Several Jankowski, J. Wolf, M. approach in particular for alveolar bone tissue engineering for alveolar bone defect reports demonstrated significant changes in soft and hard tissue homeostasis in the maxilla reconstruction. hASC of subcutaneous adipose tissue can be easily harvested in large and mandibular facial system such as delayed tooth eruption, morphological alterations in quantities via liposuction, e.g. Human ASC are capable to differentiate into various cell the periodontium and alveolar bone, and deformation of tooth roots. Despite all evidences types, including osteoblasts. In this study, we aim to attain a better understanding of the that CKD is closely associated with the onset and progression of periodontal degeneration, behavior of ageing primary hASC in alveolar bone tissue engineering. a detailed molecular understanding and potential therapeutically targets are missing. In particular, we will analyze the donor´s age influence on viability, proliferation and The overall aim of this project is an assessment of morphological structure and chemical osteogenic differentiation capacity of hACS and investigate the influence of different composition of changes in periodontal soft and hard tissue homeostasis in the maxilla biophysical impacts on ageing ASC in vitro. Therefore, we will assess the impact of and mandibular facial system in patients suffering from CKD and the effectiveness centrifugal gravity, hyperbaric therapy and hyperbaric oxygen (HBO) therapy as well as of remineralizing therapy. A major issue in this context are extra skeletal calcification hypoxia. Furthermore, we will analyze the impact of pharmacological stimulation with processes, which indicate dysregulated mineral metabolism. We are analyzing periodontal different endocannabinoids on ageing hASC. ligament cells and cementoblasts in their ability to mineralize in presence and absence of Subsequently, we will transfer the in vitro results in a subcutaneous in vivo, implanting already known and yet unknown mediators, isolated from hemofiltrate of CKD patients. dynamic pre-cultured hASC with an established osteogenic scaffold over 2 and 4 weeks in Furthermore, we are analyzing their effect on the structure of alveolar bone and periodontal a small animal model as a first proof-of-concept study, in which we will analyze new bone ligament in a mouse model and thus determine a potential role on periodontal degeneration. formation and its vascularization.

132 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 133 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-12 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-13

Evaluation of endodontic/periodontal interface In vitro and in silico investigation of mechano- and impact on alveolar bone regeneration of subchondral bone using an artificial neuronal network for cartilage - alveolar bone vs. long bone

Braun, A. Eschweiler, J. (Department of Orthopaedic Surgery) (Clinic for Operative Dentistry, Periodontology and Preventive Dentistry) Tingart, M. (Department of Orthopaedic Surgery) Wenzler, J.-S. (Clinic for Operative Dentistry, Periodontology and Preventive Dentistry) The aim of this approach is to answer if there can be any optimization of substrate stiffness and mechanical loading in order to induce the mechano-regeneration of the subchondral The development of new medical therapies increasingly involves the use of biocompatible bone of the alveolar bone in comparison to the long bone for cartilage. materials to replace destroyed or lost natural tissues. In addition to a possible defense We improve an in-house bioreactor system with parallel cultivation chambers which enables reaction to the material used, the unavoidable interface areas between biological tissue and to perform dynamic experiments with different parameters. Based on functionalized artificial material has a major impact on possible healing disorders. Especially an insufficient Braun, A. Wenzler, J.-S. membranes with a diversity in material stiffness, cells will be seeded on them. Human Eschweiler, J. Tingart, M. endodontic/periodontal interface can cause a penetration of bacteria, remnants of necrotic alveolar bone (HAB), human long bone (HLB), and human periodontal ligament (HPL) cells tissue or inflammatory mediators remaining in the dentinal tubules in periapical tissues will be cultured on these artificial silicone membranes which possess different stiffness and thus causing alveolar bone inflammation. Therefore, the objective of the project is the and will be exposed to a different combination of mechanical loading. After mechanical development of a universal analysis system for testing interfaces for leaks, diffusion and stimulation, differentiation markers will be analyzed by real-time polymerase chain reaction emanation, suitable to assess the endodontic/periodontal interface. (PCR) in order to control the osteogenic behavior and differentiation, respectively, of the The test system uses a high vacuum to quantitatively assess even smallest particles and cells. Cell density and arrangement of cells will be determined using histological methods. analyze them by mass spectrometry. In the diffusion test, a measuring chamber filled with Withal, substrate stiffness of scaffolds will be measured prior to dynamic experiments as test gas shall be sealed by a test specimen, consisting of a hybrid structure of root dentin well as after dynamic experiments in order to determine the alteration in stiffness. Finally, and endodontic sealing material. The measuring arrangement is then connected to the the data gathered from experiments will be used to train an artificial neuronal network high-vacuum chamber and evacuated. The test gas diffusing through the test specimen is (ANN) which can be used to predict and improve the mechanical properties of bone tissue. then quantitatively detected by a mass spectrometer. During the emanation test, the gases Sensitiveness analysis method in ANN will be used to determine the significance of different and evaporable chemical compounds emitted from the material sample can be fed to a input parameters on the mechanical properties of the scaffold. The major question here is, mass spectrometer and determined both qualitatively and quantitatively. if it will be possible to create an ANN model which includes cellular process, in our case, is The study intends to generate a standardized simulation and test set-up for endodontic/ osteogenesis, as an input values. periodontal interfaces to assess and optimize hybrid structures of root dentin and endodontic sealing material to avoid affecting surrounding alveolar bone tissue.

134 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 135 Progress Report 2020 | Joint Projects | Organ Crosstalk | OC1-14

Unraveling the effect of dynamic forces on regeneration capacity of periodontal ligament stem cells embedded in 3D bone-like ECM

Fischer, H. (Dental Materials and Biomaterials Research) Aveic, S. (Dental Materials and Biomaterials Research)

Periodontal tissue is a complex structure composed of alveolar bone, gingiva, cementum, and per-iodontal ligament (PDL). Multipotent periodontal ligament stem cells (PDLSC) can be isolated from the PDL and have been exploited for differentiation towards the alveolar bone. Their proper func-tion is important for the periodontal homeostasis maintenance during orthodontic tooth movement (OTM). During this movement, mechanical stresses are Fischer, H. Aveic, S. applied to the teeth and diffused to the al-veolar bone through the PDL. Mechanical forces, including compression and stretching, continu-ously occur during jaw movement and occlusal forces. However, a study of the molecular back-ground during dynamic interaction between multicellular components of the periodontal tissue is hardly achievable due to a lack of adequate in vitro models able to mimic physiological events on-going during OTM. Therefore, we propose a novel tissue-engineered approach to explore the effect of dynamic load-ing on PDLSC. For that purpose, we will apply three-dimensional (3D) hydrogel structures laden with PDLSC, fibroblasts, and endothelial cells. By applying stretching mechanism forces we will simulate conditions found in vivo. This approach will allow us to examine the effects of mechanical forces on cell proliferation, differentiation, and interaction between the cells and their extracellular matrix. The expected findings will contribute to a better understanding of how physiological forces influence PDLSC, deriving osteoblast and fibroblast cells and will improve our knowledge of the importance of endothelial cells for the intercellular crosstalk inside the alveolar bone.

136 IZKF Aachen Progress Report 2020 Di Russo p. 140 Retinal Epithelium Mechanobiology and Disease van der Vorst p. 143 Immune-Lipid Crosstalk

Namer p. 146 Different functional roles of nociceptor RESEARCH GROUPS subclasses in human

138 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 139 Progress Report 2020 | Research Group | REMeD Progress Report 2020 | Research Group | REMeD

along with the retina’s radial distance, which strongly correlates with the RPE Retinal Epithelium Mechanobiology and mechanobiological properties. Our working hypothesis is that this heterogeneity is responsible for some of the region-dependent functions of the cells. In the future, we will Disease Research Group systematically address our questions using specific in-vitro assays.

Di Russo, J. (Institute of Anatomy and Cell Biology)

Jacopo Di Russo was born in Florence (Italy) on September 26th 1984 and after undergraduate studies in Molecular and Cell Biology at the University of Florence, in 2010 he joined an Initial Training Network of Marie Skłodowska-Curie’s action at the University of Muenster, Germany. During that period, he worked in the laboratory of Prof. Lydia Sorokin to understand how extracellular matrix (ECM) influences the mechanical proprieties of

endothelium and its function of shear stress sensor in small arteries. In 2015, after obtaining © Adam Breitscheidel the PhD in Natural Sciences, he joined the group of Prof. Joachim Spatz at the Max Di Russo, J. Planck Institute of Medical Research in Heidelberg. There he kept studying the effect of cell adhesion in epithelia, enlarging his interdisciplinary background in biophysics and surface chemistry. Since January 2019 he is the independent research group leader of the Medical Technology and Digital Life Sciences research focus area. Funding Period: 01.01.2019 – 31.12.2021 Understanding the role of Bruch’s membrane biochemical and physical properties in controlling the Staff: Steins, T. retinal pigment epithelium functions. Kozyrina, A.

The last year’s main activity was to characterize Bruch’s membrane’s biochemical heterogeneity in relation to retinal pigment epithelium (RPE) mechanics. The characterization was carried out in adult BALB/c mice by whole-mount immunofluorescence stainings and image analyses. The quantification of the different Bruch’s membrane components revealed a heterogeneous distribution of some specific matrix proteins. For the basement membrane, we detected a significant reduction of laminina 5 in correlation with the radial distance from the optic nerve. In contrast, the general laminin staining (PanLaminin) showed a different distribution. Finally, collagen type IV is homogenously distributed along the retina, supporting the hypothesis of the crucial role of laminins in the differential biochemical signaling to the RPE. We further investigate the interstitial matrix components finding a linear increase of elastin and collagen type I from center to periphery which confirms the literature’s data. To characterize the mechanobiological features of the epithelia in-vivo, we used two different image-based force inference analyses. Firstly, we quantified RPE cells’ shape using an adimensional index that has been developed to describe epithelial monolayer mechanics and stress. The indices’ distribution shows an increase in value from the center to the periphery, indicating a gradual monolayer jamming in the opposite direction. Secondly, we used the cell force-inference algorithm (CellFIT) to measure the monolayer’s stress heterogeneity. The algorithm calculates only relative values to the specific area of the monolayer, thus we took into consideration their standard deviations (SD) as a measurement of the mechanical heterogeneity. The calculation revelated a Figure 1: Mechanical characterization of RPE in relation to its basement membrane heterogeneity. The graphical representation of RPE cells shape-index and CellFIT are overlaid on a flat- significant increase of the standard deviation of “intracellular-stresses” from the center to mount murine retina (not-to-scale). On the right are the graphs showing the quantification of the image-based force inference analyses and basement membrane components. * P < 0.05, ** the periphery in line with the quantification of the shape indices. P < 0.01, *** P < 0.001, **** P < 0.0001, n.s.= not significant, resulting from Mann-Whitney test (immunofluorescence quantifications) and unpaired t-test (cell shape index and standard Altogether we revealed a biochemical heterogeneity of Bruch’s membrane components deviation of intracellular pressure).

140 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 141 Progress Report 2020 | Research Group | REMeD Progress Report 2020 | Research Group | Immune-Lipid Crosstalk

Publications Kozyrina A. N., Piskova T. and Di Russo J. “Mechanobiology of Epithelia from the Immune-Lipid Crosstalk Research Group Perspective of Extracellular Matrix Heterogeneity”. Frontiers in Bioengineering and Biotechnology, 8:596599, doi: 10.3389/fbioe.2020.596599, 2020 [IF 3.644]

van der Vorst, E. (Institute for Molecular Cardiovascular Research) Applied and actual third-party funding (DFG, BMBF, EU, foundations) Dr. Emiel van der Vorst was born on 14.08.1987 in Sittard, The Netherlands. He completed Di Russo, J. Generation of a retinal organoid system to dissect Pro Retina Stiftung – 09/20 - 08/23 € 46,800 a B.Sc. (2008) in Molecular Life Sciences and M.Sc. (2010) in Cardiovascular Biology and the biochemical and mechanical contribution of “Promotionsstipendium“ rejected Medicine at the University of Maastricht, The Netherlands, and a M.Sc. (2015) in Management Bruch‘s membrane in the retinal pigment epithelium Sciences at the Open University Netherlands. In 2010 he joined the Department of Pathology pathophysiology. at the Cardiovascular Research Institute Maastricht (CARIM), The Netherlands, and completed his PhD in 2015. Before moving to Aachen, he was an Postdoctoral Fellow at the Di Russo, J. Mechanobiology of Plastic Adaptation in Retinal Pigment DFG applied € 212,630 Institute for Cardiovascular Prevention (IPEK) in Munich, Germany. van der Vorst, E. Epithelium submitted

The main focus of our work is to identify novel © Peter Winandy Promoting of young researchers Funding Period: Doctoral Theses approaches to develop new therapies for 01.01.2019 – 31.12.2021 Kozyrina, A. Ongoing RWTH Aachen University, The mechanobiology of retinal pigment epithelium heterogeneity Faculty 1 cardiovascular diseases, especially those driven by chronic kidney disease. Staff: Nazir, S. Bonnin-Márquez, A. Peters, L. (externally funded) Acquired Affiliation High-Density lipoprotein (HDL) plays an important role in the lipid metabolism by Sundararaman, S.S. (externally funded) 2020 – present Associated scientist at the DWI - Leibnitz-Institute for Interactive Material contributing to the reverse cholesterol transport pathway. Over the last years it became Fluonia, Z. (externally funded) clear that HDL also has major effects on immune-modulation. Initially it was believed that HDL-cholesterol levels were the main determinant of the beneficial effects mediated by HDL. However recent studies, highlighted in two recent reviews from my group, demonstrated that HDL function rather than concentration might be the most important determinant. HDL function can be modulated by a wide variety of mechanisms, like for example post-translational modifications.

Recently, we were able to demonstrate that patients with chronic kidney disease (CKD) have an increased degree of modification of ApoA-I, the main protein which is present in HDL. In ongoing experiments in collaboration with Prof. Vera Jankowski (IMCAR) we could already show that the modification of ApoA-I has immune-modulatory effects on vascular cells as well as myeloid cells. Unmodified ApoA-I clearly demonstrated to have anti-inflammatory effects, as shown before, while the modification of ApoA-I almost completely abolished this effect. Strikingly, in myeloid cells we could even observe that modified ApoA-I induces cell death. Although we could clearly demonstrate that modification of ApoA-I as observed in CKD patients has obvious cellular effects, the underlying mechanisms remain elusive.

Recently, we established a Kinase Array platform in our laboratory enabling us, in addition to the regular RNA and protein quantifications, to screen the effects of modified ApoA-I on kinase activity. This platform can thereby give us clear indications which signalling pathways are involved in the observed immune-modulatory and cell survival effects. Finally, we are also investigating novel mechanisms of Immune-Lipid Crosstalk that play a role in cardiovascular disease and chronic kidney disease. Initial studies have identified two interesting novel candidates that seem to be key mediators of crosstalk between lipids and the immune system. In-vivo experiments are currently ongoing to validate their role in immune-lipid crosstalk and especially cardiovascular disease.

142 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 143 Progress Report 2020 | Research Group | Immune-Lipid Crosstalk Progress Report 2020 | Research Group | Immune-Lipid Crosstalk

Van der Vorst EP, Daissormont I, Aslani M, Seijkens T, Wijnands E, Lutgens E, Duchene J, Santovito D, Döring Y, Halvorsen B, Aukrust P, Weber C, Höpken UE, Biessen EA. (2020) Interruption of the CXCL13/CXCR5 chemokine axis enhances plasma IgM levels and attenuates atherosclerosis development. Thombosis & Heamostasis, 120(2):344-347 [IF 4.4]

Döring Y, Jansen Y, Cimen I, Aslani M, Gencer S, Peters LJ, Duchene J, Weber C, Van der Vorst EP. (2020) B-cell specific CXCR4 protects against atherosclerosis development and increases plasma IgM levels. Circulation Research, 13;126(6):787-788 [IF 14.5]

Van der Vorst EP, Biessen EA, Donners MM. (2020) Letter by van der Vorst et al regarding article “Anti-inflammatory effects of HDL (High-Density Lipoprotein) in macrophages predominate over proinflammatory effects in atherosclerotic plaques. Arteriosclerosis, Thrombosis, and Vascular Biology, 40(2):e31-e32 [IF 6.6]

Applied and actual third-party funding (DFG, BMBF, EU, foundations) Figure 1: Crosstalk between high density lipoproteins and inflammation (Source: Nazir S et al. Adv Drug Deliv Rev 2020) van der Vorst, E. Targeting immune-lipid crosstalk in cardio- Else Kröner-Fresenius- 01/2020 – 12/2022 € 396,000 metabolic diseases: focus on HDL dependent Stiftung - 361089 Publications regulation of chemokine-receptor signaling Van der Vorst EP, Döring Y. (2020) Tracing endothelial CXCR4 may pave the way for van der Vorst, E. The aryl hydrocarbon receptor (AhR), a friend that Deutsches Zentrum für Herz- 10/2020 – 09/2021 € 80,000 localized lesional treatment approaches. Arteriosclerosis, Thrombosis, and Vascular Biology, turns out to be a foe? Kreislauf-Forschung (DZHK) In Press [IF 6.6] – 81X3600220

Saar-Kovrov V, Donners MM, Van der Vorst EP. (2020) Shedding of Klotho: Functional Döring, Y./ Rieben, R./ Unraveling the role of SARS-CoV-2 on endothelial Schweitzer Nationalfond (SNF) 01/2021 – 21/2022 1,950,00 CHF (total) implications in chronic kidney disease and associated vascular disease. Frontiers in Mercader, N./ Longnus, dysfunction in Covid-19 related vascular 4078P0_198297 10,000 CHF Cardiovascular Medicine, In Press [IF 3.9] S./ Odening, K./ Leib, S./ inflammation and thrombosis (for Van der Vorst) Kleinbongard, P./ Bonnin-Márquez A, Nazir S, Van der Vorst EP. (2020) High-density Lipoprotein van der Vorst, E. modifications: A pathological consequence or cause of disease progression? Biomedicines, van der Vorst, E. Surface Plasmon Resonance Instrument DFG Forschungsgroßgeräte Applied € 260,000 In Press [IF 4.7] nach Art. 91b GG

Nazir S, Jankowski V, Bender G, Zewinger S, Rye KA, Van der Vorst EP. (2020) Interaction van der Vorst, E. MiRNA26b, a novel mediator and therapeutic Volkswagen Stiftung Applied € 120,000 between high-density lipoproteins and inflammation: Function matters more than target of viral infections like Influenza A and concentration! Advanced Drug Delivery Reviews, 159:94-119 [IF 13.3] SARS-CoV2 van der Vorst, E. / Van Euregional Health Education; Empowering young Interreg Euregio Meuse-Rhine Applied € 3,375,000 (total) Döring Y, Noels H, Van der Vorst EP, Weber C. (2020) Seeing is repairing – how image Schayck, O. / Wolters, adolescents and their families to engage in a € 1,300,000 based timely interference with CXCR4 could improve myocardial repair after MI. European D. / Georis, A-L. / Op ‘t healthy lifestyle (for Van der Vorst) Heart Journal, Editorial, 1;41(37):3576-3578 [IF 22.7] Eijnde, B. / Simons, A.

Peters LJ, Floege J, Biessen EA, Jankowski J, Van der Vorst EP. (2020) MicroRNAs in chronic kidney disease: Four candidates for clinical application. International Journal of Molecular Sciences, 7;21(18):6547 [IF 4.6] Promoting of young researchers Doctoral Theses Peters LJ, Biessen EA, Hohl M, Weber C, Van der Vorst EP, Santovito D. (2020) Small things matter: relevance of microRNAs in cardiovascular disease. Frontiers in Physiology, 7;11:793 Sundararaman, SS. Ongoing RWTH Aachen University, The calcium-sensing receptor as novel mediator of atherosclerosis [IF 3.2] Faculty 10 Peters, L. Ongoing RWTH Aachen University, Role of miRNA26b in CVD and CKD Kiouptsi K, Pontarollo G, Todorov H, Braun J, Jäckel S, Koeck T, Bayer F, Karwot C, Faculty 10 Karpi A, Gerber S, Jansen Y, Wild P, Ruf W, Daiber A, Van der Vorst EP, Weber C, Döring Y, Reinhardt C. (2020) Germ-free housing conditions do not affect aortic root and aortic Bonnin-Márquez, A. Ongoing RWTH Aachen University, Effects of post-translational modifications on HDL function arch lesion size of late atherosclerotic low-density lipoprotein receptor-deficient mice. Gut Faculty 10 Microbes, 1;11(6):1809-1823 [IF 7.7]

144 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 145 Progress Report 2020 | Research Group | Roles of Nociceptor Subclasses Progress Report 2020 | Research Group | Roles of Nociceptor Subclasses

In a newly established cooperation with the field of theoretical mathematics (Dr. Kutafina, Different functional roles of nociceptor Biosignals group, Dept. for medical informatics) we aim to scrutinize discharge patterns of these two nociceptor classes as recorded by microneurography healthy and diseased subclasses in human patients.Using machine learning techniques, we model the dependency between conduction velocity of action potentials with preceding nerve activity i.e. the short term memory of single axons (manuscript submitted to “Medical informatics Europe Conference “) (fig. 1C). We submitted an DFG grant application with the topic of scrutinizing the patterns of spontaneous activity of C-nociceptors in neuropathic pain patients via these Namer, B. (Institute of Physiology) models.

Barbara Namer was born 1976 in Nürnberg, Germany. She earned her medical degree Currently we are expanding the range of our electrophysiological techniques for scrutinizing 2002 at the Friedrich-Alexander-University of Erlangen-Nürnberg, Germany. After working peripheral nerve fiber function by establishing the “sucrose gap model” which enables us clinically at the Department of Neurology at the University Erlangen-Nürnberg she joined the to record mean membrane potential and compound action potential of peripheral nerves in Institute of Physiology and experimental Pathophysiology in Erlangen as research assistant. animal and human (Cooperation with Prof. Beyer) (see fig. 1D). There she completed her “Habilitation” in 2010. Before moving to Aachen, she was group leader of her DFG funded research group at the Institute of Physiology and experimental To elucidate further the role of sodium channels in neuropathic pain, I am active cooperation Pathophysiology in Erlangen with regular research stays at the Riskhopital in Oslo, Norway. Namer, B. partner of the IZKF consortium “SCNAachen” on sodium channels by recording from single nerve fibers from patients with neuropathic pain with and without sodium channel mutations (Cooperation with Dr. Dohrn, Dept for neurology). And I also serve as member of the “managing team” to steer and prepare the DFG application for a research unit arising from My vision is to help solving the pathophysiological Funding Period: the consortium “SCNAachen”. enigma of chronic pain and itch by scrutinizing the 01.01.2019 – 31.12.2021 functional role of different nociceptor classes in human from the skin to the brain Staff: Namer, B. Fiebig, A. (externally funded) Chronic pain and itch are major health burdens with no specific treatment, because DeCol, R. underlying mechanisms specifically in human are still poorly understood. Within the Koulchitsky, S. peripheral nervous system signaling via so called mechano-sensitive C-nerve fibers (CM- Prast, A. nociceptors) is more important for spatial and temporal aspects of acute pain whereas Charan das Mundigonda, G. signaling via so called mechano-insensitive C-nerve fibers (CMi-nociceptors, “sleeping Popovic, R. nociceptors”) is involved in sensitization, inflammation and neuropathic pain. In our project we focus on the role of these two nociceptor classes in neuropathic pain and itch. We could develop electrical stimulation paradigms to stimulate these different nociceptor classes separately (Jonas R., Eur. J Pain, 2020; Rukwied R., Pain, 2020) (fig. 1B). This is the basis for a DFG application in cooperation with Prof. Lampert (Dept. for Physiology) and Prof Kurth (Clinic for Human genetics) scrutinizing the molecular identity of CM and CMi nociceptors (submitted 2020).

Via assessing the maximal firing frequencies of these different nociceptor classes we could Team of the research group. identify for the first time a new nociceptor subclass in pig and human, which is responsible for transmitting high intense mechanical pain (Werland F, J physiol, 2020).

Both nociceptor classes, CM and CMi are involved in signalling “itch”. We could show for the first time in human that Lsyophosphatidic acid, a potential mediator of cholestatic itch and neuropathic pain, can cause pain as well as itch dependent on the mode of application (focal versus injection). Application dependent differential activation of CM and CMi contributes to this phenomenon. Itch may arise from activation of single nerve fibers with high spatial contrast to unexcited surrounding afferents and a specific combination of activated fiber subclasses may contribute. (manuscript under revision in “Pain”).

146 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 147 Progress Report 2020 | Research Group | Roles of Nociceptor Subclasses Progress Report 2020 | Research Group | Roles of Nociceptor Subclasses

ones). Note the different shape of the red marked action potentials, which are definitely coming from one single nerve fiber and the similar shape between the yellow marked action potential from another nerve fiber and the red marked one of the last lines. Currently we work on new analyzing algorithms including machine learning techniques to be able to identify and sort single action potentials to single nerve fibers reliably for analyzing discharge patterns in healthy and diseased peripheral nerve fibers.

1B) CM fibers (action potential latencies marked in red) are strongly activated by half sine stimulation whereas CMi nociceptors (action potential latencies marked in blue) are not activated by half sine stimuli with the same intensity.

1C) Modern machine learning techniques show a good prediction of latency changes due to activity dependent conduction velocity slowing. Both short term and long term (60seconds) effects combined show the best results showing that different molecular mechanisms with distinct short and long term kinetics are involved into the “short term memory” of the axon.

1D) The sucrose gap technique is used to study the membrane potential of axons. The method creates conduction block in a section of the nerve by introducing a gap of high resistance. A nonionic sucrose solution is used to increase resistance in the extracellular area in this section. One side of the gap (the cut end) is depolarized by using intracellular solution. The other side contains an extracellular solution (normal membrane potential). The sucrose solution allows all of the current originating on one side of the gap to flow to the other side only through the interior of the nerve or axons. Thus, the potential between the gaps reflects the membrane potential formed by all axons of the nerve. With the same setup and an additional section (for electrical stimulation), also compound action potentials can be measured. Pilot experiments on a mouse skin nerve show that BAM 8-22 and beta-Alanin application lead to hyperpolarisation of the resting membrane potential whereas capsaicin depolarises the resting membrane potential

Figure 1: 1A) Microneurography: The recording needle is inserted into the nervus peronäus at ancle level. In the inset the recording electrode (white flag) is inserted into the peripheral nerve which is marked in blue with a felt pen. The reference electrode (blue flag) is inserted into the skin. The second inset shows an electron microscopic picture with the tip of the recording needle in a peripheral nerve containing myelinated and unmyelinated peripheral nerve fibers. Electrical stimulation is performed in the receptive field of the recorded fibers. After each electrical pulse one action potential of one single nerve fiber can be observed with a stable individual latency (marked by light red ovals). The signal is amplified, digitized and stored on a computer. The amplified raw signal is made hearable via a loudspeaker. Note the typical pattern of a stable latency, followed by a significant increase in latency in response to the additional stimulus which is called “marking”, and then followed by a gradual recovery period back to the stable latency. It is however momentarily not possible to determine how many action potentials with which frequency are preceding the delayed test action potential or if other action potentials (the yellow marked one in recoding line 3 originates from the same fiber as the red marked

148 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 149 Progress Report 2020 | Research Group | Roles of Nociceptor Subclasses

Publications Jonas R, Namer B, Schnakenberg M, Soares S, Pakalniskis J, Carr R, Schmelz M, Rukwied R. (2020) Sympathetic efferent neurons are less sensitive than nociceptors to 4 Hz sinusoidal stimulation. Eur J Pain. 2020;24(1):122-133. doi: 10.1002/ejp.1467. [IF 3.5]

Rukwied R, Thomas C, Obreja O, Werland F, Kleggetveit IP, Jorum E, Carr RW, Namer B, Schmelz M. (2020) Slow depolarizing stimuli differentially activate mechanosensitive and silent C-nociceptors in human and pig skin. Pain 4. doi: 10.1097/j.pain.0000000000001912. [IF 6.0]

Werland F, Hirth M, Rukwied R, Ringkamp M, Turnquist B, Jorum E, Namer B, Schmelz M, Obreja O. (2020) Maximum axonal following frequency separates classes of cutaneous unmyelinated nociceptors in the pig. J Physiol. 28. doi: 10.1113/JP280269. [IF 4.6]

Applied and actual third-party funding (DFG, BMBF, EU, foundations)

Namer, B. Methylglyoxal - Wirkungen auf nozizeptive DFG 970- 3-1 01.01.2018 € 342,000 Neurone, Mechanismen der Sensibilisierung, - 30.06.2022 Übererregbarkeit bzw. Inaktivierung Namer, B. Pain in the brain: development of selective nerve OPSF568 2019: rejected fiber stimulation in the magnetic resonance 12 months environment Namer, B. Machine learning approaches to neuronal DFG NA 970 3 years € 337,000 de-coding of spontaneous nociceptor activity in submitted patients with neuropathic pain. Namer, B. A biomarker for neuropathic DFG 3 years € 482,000 pain: Determining the molecular identity of silent submitted nociceptors.

Promoting of young researchers Doctoral Theses

Schwarzenauer, M. Ongoing, manuscript in preparation RWTH Aachen University, The effect of neuromodulation via electrical Faculty 10 matrixstimulation on itch Frömbgen, J. Ongoing RWTH Aachen University, Modulation von Juckreiz durch Bradykinin Faculty 10 Hornberg, A. Ongoing RWTH Aachen University, Geschlechtsunterschiede in der Funktion von Faculty 10 Nozizeptoren des Menschen. Fiebig, A. Ongoing RWTH Aachen University, Functional Characterization of mechanically Faculty 1 sensitive C-fibers in human.

150 IZKF Aachen Progress Report 2020 Appendix | Articles of Association Appendix | Articles of Association

3. Participants Articles of Association 3.1. The participants are the managers of the projects, core facilities (appointed by the steering committee) and research groups funded by the IZKF.

3.2. The participants commit • to submit interim and final reports on the progress and results of their work to the steering committee in due compliance with predetermined deadlines, • to reference the support of the IZKF in publications and presentations, • to demonstrate appropriate involvement in public relations tasks.

3.3. The participation in the IZKF concludes The Interdisciplinary Center for Clinical Research (IZKF) • upon termination of the employment agreement with the RWTH of the RWTH Aachen Faculty of Medicine • upon decision of the steering committee, if the relevant person severely impairs the activities of the IZKF or fails to meet his/her duties and obligations within the IZKF • at request, to be communicated to the steering committee in writing.

1. Preamble 4. The steering committee The Interdisciplinary Center for Clinical Research (IZKF) is – pursuant to Section 70 (2) of the 4.1. The steering committee manages the IZKF and consists of the following members German University Act (HG), Section 10 University Hospital Regulations (UKVO) and Section with voting rights: 7 (1) of the articles of association of the university hospital – a funding program of the • the speaker and deputy speaker, who are elected by the faculty board upon Faculty of Medicine of RWTH Aachen University for outstanding and strategically important proposal by the dean for a period of three years. A re-election is possible. research projects as well as for maintaining core facilities. • a research area coordinator and deputy research area coordinator from each faculty For the fulfilment of its duties, the IZKF receives funds granted by the German State of research area who may not be funded by the IZKF and is/are appointed by the North Rhine Westphalia (NRW) in accordance with the budget proviso in the budget of relevant faculty research areas for three years. A re-election is possible. The deputy the competent ministry. The Dean’s office decides on the amount of additional funding in research area coordinator will be eligible to vote only in the absence of the research coordination with the faculty board. area coordinator. • a representative of the core facilities, who is appointed by the core facility managers 2. Purpose and objectives for a period of three years. A re-election is possible. The IZKF has developed and established structures and processes that continuously • a member of the natural science or engineering faculties of RWTH Aachen who is enable highly-qualified research at RWTH Aachen, incorporating the structural conditions appointed by the faculty board for a period of three years upon proposal by the of the Faculty of Medicine which offer a combination of health care services, research and speaker. A re-election is possible. teaching. The primary objective is the realisation and funding of joint projects and individual projects with a thematic relevance to the core research areas of the Faculty of Medicine, as The dean, the vice-dean for research as well as the speakers of the Collaborative well as the funding of core facilities for the improvement of research opportunities. Research Centers (SFBs), German Research Foundation (DFG) research groups and research training groups of the Faculty of Medicine and the research coordinator of 2.1. The structural objectives are the IZKF as well as the medical director and the commercial director can attend the • to further develop processes for promoting basic, translational and clinical research, meetings of the steering committee in an advisory capacity. • to support the research profile of the university and faculty, If a member of the steering committee resigns prior to the expiry of the three year • to use funds pursuant to quality criteria, period or if the research area coordinator belonging to the steering committee is funded • the transparent financing of research supplementary to the other programs of the by the IZKF, a re-election will be carried out in the faculty research area. If a faculty faculty of Medicine research area is not pursued further, the steering committee membership of the person • the establishment and maintenance of core facilities. coordinating the research area shall also be terminated.

2.2. The scientific objectives are 4.2. The speaker represents the IZKF and manages the daily operations with the • the funding of research projects in accordance with the core research areas of the assistance of the IZKF administrative office. The speaker will submit an annual activity Faculty of Medicine, report to the Faculty of Medicine and the Rector of RWTH Aachen. • the planning, coordination and implementation of interdisciplinary research projects with the aim to achieve optimised research results through the joint use of the spatial, material and personnel resources, • the encouragement of cooperation and information exchange between the Faculty of Medicine and the other faculties and institutions of RWTH Aachen, • the assurance of scientific quality in the IZKF by means of internal and external evaluation.

152 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 153 Appendix | Articles of Association Appendix | Articles of Association

4.3. Tasks of the IZKF steering committee: 6.4. Termination of project funding • overall responsibility for the IZKF including its further development, Project funding ends when the employment agreement of the person acting as project • management of the business operations not included in daily operations, manager is terminated or interrupted, if no successor or interim arrangement has been • responsible for the distribution of the funds granted to the IZKF, determined by the steering committee of the IZKF. If in this event sub-projects should be • decides within the scope of the available funds on the launch, termination and carried out at a different site in order to successfully complete the project, this is possible further funding of projects classified as eligible for funding, for a fixed period of time. This course of action must be applied for at the steering • carries out internal quality controls, committee by the new project manager. The funds released through the termination of • advises IZKF participants with regard to project-specific prospects, a sub-project shall flow back to the IZKF. The investments in the relevant projects are – • coordinates the use of the central laboratory space and core facilities allocated to subject to approval requirements of third-parties – owned by RWTH; the IZKF is entitled the IZKF, to these investments. • approves the rules of procedure. 7. External experts 4.4. Research coordination External experts are appointed by a work group consisting of the speaker, deputy speaker, The steering committee is assisted by the research coordinator. He/she manages the dean, vice-dean for research and member of the steering committee representing the business operations of the administrative office in a target and esource-orientedr way in natural science and engineering faculties. In the case of a conflict of interests these can close cooperation with the administration of the university hospital. Within the scope of appoint additional work group members. External experts are reimbursed for their activities. organisation development he/she adapts processes and develops an interdisciplinary Any incurring travel expenses will be refunded in accordance with the provisions of the personnel and process management under special consideration of quality aspects in travel allowance regulations. accordance with the specifications of the steering committee. External experts are responsible for the following tasks: 5. Research funding • They assess the quality of proposals and cast votes on the eligibility of the projects The following funding instruments are available: to receive funds. Negative votes are binding. Positive votes are to be implemented in • Joint projects: Joint projects are projects from several applicants working on one joint accordance with the financial means of the IZKF. topic. • They evaluate research groups. • Individual projects: Individual projects are projects with a maximum of two applicants on • They assess proposals of the core facilities for (follow-up) funding and give a fixed topic. recommendations for increased funding, establishment or termination of core facilities. • Research groups: Research groups serve to support highly qualified junior scientists. • Core facilities: Facilities in which equipment, expertise and methods are made available 8. Procedures which are generally not accessible to individuals, but are nonetheless in the interest of Unless special regulations have been agreed in these articles of association, the rules of the faculty. Core facilities are available to all members of the Faculty of Medicine. procedure of the IZKF - as amended - shall apply should any procedural questions arise.

6. Application 9. Amendments 6.1. Eligibility If individual provisions of these articles of association should be invalid or amended Eligible to apply are all post-doctoral scientists of the Faculty of Medicine who are by resolution of the faculty board of the Faculty of Medicine, this shall not affect the employed at the UKA or RWTH on at least a part-time basis (50% of the regular working effectiveness of the remaining provisions. hours) and who can submit a declaration by the clinic or institute management, which is confirmed by the HR and Finance departments, that the financing of their position 10. Entry into force is guaranteed for the duration of the funding program. Project proposals by applicants These articles of association replace the version dated 6 May 2010. They come into force who belong exclusively to other faculties of RWTH Aachen, may be admitted after prior after being approved by the faculty board of the Faculty of Medicine and by the Rector of request by the steering committee, if this is in the interest of the Faculty of Medicine. RWTH Aachen. Project managers may not submit an application for their own position. The articles of association dated 6 May 2010 apply to all projects in the current funding phase, ending on 30 June 2017. The regulations of the amended version shall exclusively 6.2. Procedure apply for the new funding phase. The calls for proposal are published to all eligible applicants in accordance with 6.1 by the speaker of the steering committee in coordination with the dean of the Faculty of Medicine.

6.3. IZKF staff The employment of IZKF-financed employees is applied for by the speaker upon proposal by the project manager for a period not exceeding the duration of the approved funding.

154 IZKF Aachen Progress Report 2020 IZKF Aachen Progress Report 2020 155