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Systematic Interaction Mapping Reveals Novel Modifiers of Neurodegenerative Disease Processes

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Systematic Interaction Mapping Reveals Novel Modifiers of Neurodegenerative Disease Processes Systematic interaction mapping reveals novel modifiers of neurodegenerative disease processes D i s s e r t a t i o n zur Erlangung des akademischen Grades d o c t o r r e r u m n a t u r a l i u m (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von M.Sc. Jenny Russ Präsident der Humboldt-Universität zu Berlin Prof. Dr. Jan-Hendrik Olbertz Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Stefan Hecht, Ph.D. Gutachter/in: 1. Prof. Dr. Hanspeter Herzel 2. Prof. Dr. Erich Wanker 3. Prof. Dr. Thomas Sommer Tag der mündlichen Prüfung: 12. September 2012 Abstract Neurodegenerative diseases (NDs) such as Alzheimer’s disease (AD), Parkinson’s disease (PD) or amyotrophic lateral sclerosis (ALS) are progressive brain disorders characterized by the accumulation of insoluble protein aggregates in neuronal cells or the extracellular space of patient brains. To elucidate potential common pathological mechanisms in different NDs, I created comprehensive interaction networks for various known and predicted neurodegenerative disease proteins (NDPs). I identified 18,663 protein-protein interactions (PPIs) for 449 bioinformatically selected wild-type target proteins and 22 mutant variants of 11 known NDPs by using an automated yeast two-hybrid (Y2H) system. The functional analysis of the interaction partners of corresponding wild-type and mutant NDPs revealed strong differences in the case of all 11 NDPs and especially for the ALS protein TDP-43. The identified PPIs were used to generate networks for individual NDs such as AD or PD and to identify proteins that are connected to multiple NDPs. For example, I found that five neurodegenerative diseases are connected by four proteins (APP, ZMAT2, ZNF179 and IQSEC1) that link known NDPs such as huntingtin, TDP-43, parkin, ataxin-1 and SOD1. Analysis of publicly available gene expression data suggested that the mRNA expression of the four proteins is abnormally altered in brains of ND patients. Moreover, the knock-down of IQSEC1, ZNF179 or ZMAT2 aggravates pathogenic disease mechanisms such as aggregation of mutant huntingtin or TDP-43 as well as hyperphosphorylation of tau. Additionally, I identified 22 modifiers of TDP-43 aggregation, which are members in 7 protein complexes. These complexes were predicted based on combined data from PPI as well as siRNA screenings. Finally, I found that the proteins HDAC1, pRB, HP1, BRG1 and c-MYC, which form one of the predicted complexes, influence TDP-43 aggregation by altering its mRNA expression. Keywords: neurodegenerative diseases, protein-protein interactions, modifiers, aggregation, toxicity, TDP-43 Zusammenfassung Neurodegenerative Erkrankungen (NDs) wie Alzheimer (AD), Parkinson (PD), und amyotrophe lateral Sklerose (ALS) sind Hirnerkrankungen, die durch unlösliche Proteinaggregate in Neuronen oder im Extrazellularraum charakterisiert sind. In dieser Arbeit habe ich für verschiede bekannte und vorhergesagte neurodegenerative Krankheitsproteine (NDPs) Proteininteraktionsnetzwerke erstellt, um mögliche gemeinsame Krankheitsmechanismen genauer zu studieren. Mit Hilfe eines automatisierten Hefe-Zwei-Hybrid-Systems (Y2H) konnte ich 18.663 Protein- Protein-Interaktionen (PPIs) für 449 wildtyp und 22 mutierte Proteine identifizieren. Eine genaue funktionelle Analyse der Interaktionspartner von korrespondierenden wildtyp und mutierten Proteinen ergab deutliche Unterschiede zum einen im Fall von allen untersuchten Proteinen und insbesondere im Fall vom ALS Krankheitsprotein TDP-43. Die identifizierten PPIs wurden außerdem verwendet um krankheitsspezifische Netzwerke zu erstellen und um Proteine zu identifizieren, die mit mehreren NDPs verbunden sind. Ich habe auf diese Weise vier Proteine (APP, IQSEC1, ZNF179 und ZMAT2) gefunden, die mit bekannten NDPs with Huntingtin, TDP-43, Parkin und Ataxin-1 interagieren und so fünf verschiedene NDs miteinander verbinden. Die Reduktion der mRNA Expression von IQSEC1, ZNF179 oder ZMAT2 mit Hilfe von siRNA führte zu einer Verstärkung von pathogenen Mechanismen wie der Aggregation von mutiertem Huntingtin und TDP-43 sowie der Hyperphosphorylierung des Proteins Tau. Außerdem habe ich 22 Proteine entdeckt, die die Aggregation von TDP-43 deutlich verändern und außerdem Mitglieder in sieben vorhergesagten Proteinkomplexen sind. Die Proteinkomplexe habe ich durch Kombination von Interaktionsdaten und Daten eines siRNA Screenings vorhergesagt. Zusätzlich habe ich herausgefunden, dass die Proteine eines vorhergesagten Komplexes, nämlich HDAC1, pRB, HP1, BRG1 und c-MYC, die Aggregation von TDP-43 durch Veränderung von dessen Genexpression beeinflussen. Schlagwörter: neurodegenerative Erkrankungen, Protein-Protein Interaktionen, Aggregation, Toxizität, TDP-43 Für Margerate und Angie, deren Kampf um ihr Leben mich jeden Tag neu inspiriert und die mir gezeigt haben jedes Hindernis mit einem Lächeln zu überwinden Danksagung Zuerst möchte ich Prof. Dr. Erich Wanker für die Themenstellung und besonders für die Unterstützung bei der Ideenfindung um wieder neue Aspekte aus unserem riesigen Netzwerk zu zaubern. Dank ihm bin ich zu einer reiferen und selbstständigen Wissenschaftlerin geworden bin, die ohne viele Schwierigkeiten in die weite Welt hinausziehen kann. Ganz herzlich bedanke ich mich auch bei meinen Kollegen im Labor ohne deren Hilfe ich nie so viel geschafft hätte und ohne die es im Labor echt langweilig gewesen wäre. Mein Dank gilt vor allem Kirstin Rau und Martina Zenkner nicht nur in Sachen Laborarbeit, sondern besonders was lange Diskussionen, tolle Kinoabende und aufregendes Eishockey angeht. Ich danke auch Alexandra Redel dafür, dass sie mich in „ihrem“ Labor so gut aufgenommen hat und mal wieder einen Plan hatte, wenn ich nicht so genau wusste, wie ein Experiment jetzt am Besten von statten geht. In diesem Zusammenhang möchte ich auch Angeli Möller und Katja Mühlenberg für ihre Unterstützung vor und während des Schreibens meiner Doktorarbeit danken. Besonders Danken möchte ich auch Maliha Shah. Dank ihr kann ich jetzt jeden Frust und Stress einfach davonlaufen und habe auch kein Problem bei einem Wettkampf im Schlussfeld ins Ziel zu kommen, denn schließlich ist der Weg das Ziel. Ich hatte viel Spaß dabei mit dir die schönsten Ecken Europas zu erkunden und schätze dich als eine meiner besten Freundinnen. Außerdem danke ich meinem Bruder für stundenlange Gespräche am Telefon über Gott und die Welt, aber doch meistens über Computer und andere technische Geräte. Ich verdanke ihm meine Liebe zu Mathematik, Informatik und Science Fiction, ohne die diese Arbeit niemals entstanden wäre. Ganz besonders danken möchte ich natürlich auch meinen Eltern, besonders für ihre jahrelange Unterstützung aller Art und dass sie mir die Freiheit und Möglichkeit gegeben haben, schon ganz früh die Welt zu erkunden. Dank euch bin ich zu dem Mensch geworden, der ich heute bin, und habe meine Liebe für die Wissenschaft entdeckt. Besonderen Dank gilt auch meiner großen Liebe Patrick, für sein Verständnis, das Aushalten meiner kleinen Vulkanausbrüche, aufbauende Worte während aller Krisenzeiten, die tollen Kurzreisen um mal wieder Stress abzubauen und dass er mich auffängt, wenn ich es brauche. Contents 1 Introduction .................................................................................................. 1 1.1 Neurodegenerative diseases ................................................................... 1 1.1.1 Common pathogenic characteristics and molecular mechanisms of neurodegenerative diseases ..................................................... 1 1.1.2 Alzheimer’s disease ...................................................................... 8 1.1.3 Amyotrophic lateral sclerosis....................................................... 11 1.2 Protein-protein interaction studies for neurodegenerative disease proteins.................................................................................................. 14 1.2.1 Single protein interaction studies ................................................ 14 1.2.2 Protein-protein interaction studies for multiple NDPs .................. 15 1.2.3 Studies analyzing PPI networks for proteins causing several different neurodegenerative diseases ......................................... 16 1.3 Aims of the thesis .................................................................................. 17 2 Results ........................................................................................................ 18 2.1 Generating protein-protein interaction networks for neurodegenerative diseases using an automated yeast two-hybrid system......................... 18 2.1.1 Prediction of 3,711 neurodegenerative disease-related target genes .......................................................................................... 18 2.1.2 Analysis of genes selected for systematic interaction screens.... 21 2.1.3 Selection of mutant variants for yeast two-hybrid screens .......... 23 2.1.4 Identification of protein-protein interactions with the Y2H method ........................................................................................ 26 2.1.5 Predicting high confidence Y2H interactions ............................... 29 2.1.6 Validation of Y2H interactions in mammalian cells with LUMIER 31 2.1.7 PHF19 specifically interacts with mutant TDP-43 in mammalian cells............................................................................................. 33 2.1.8 Validation of high-confidence
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