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A Genome-Wide Rnai Screen for Modifiers of Polyglutamine-Induced Neurotoxicity in Drosophila

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A Genome-Wide Rnai Screen for Modifiers of Polyglutamine-Induced Neurotoxicity in Drosophila A Genome-Wide RNAi Screen for Modifiers of Polyglutamine-Induced Neurotoxicity in Drosophila Doctoral Thesis In partial fulfilment of the requirements for the degree “Doctor rerum naturalium (Dr. rer. nat.)” in the Molecular Medicine Study Programme at the Georg-August University Göttingen submitted by Hannes Voßfeldt born in Zerbst/Anhalt, Germany Göttingen, January 2012 FÜR MEINE FAMILIE - IM GEDENKEN AN NADINE DU FEHLST. … IT MATTERS NOT HOW STRAIT THE GATE, HOW CHARGED WITH PUNISHMENTS THE SCROLL, I AM THE MASTER OF MY FATE: I AM THE CAPTAIN OF MY SOUL. … Invictus – William Ernest Henley Members of the Thesis Committee: Supervisor Prof. Dr. med. Jörg B. Schulz Head of Department of Neurology University Medical Centre RWTH Aachen University Pauwelsstrasse 30 52074 Aachen Second member of the Thesis Committee Prof. Dr. rer. nat. Ernst A. Wimmer Head of Department of Developmental Biology Johann Friedrich Blumenbach Institute of Zoology and Anthropology Georg-August University Göttingen Justus-von-Liebig-Weg 11 37077 Göttingen Third member of the Thesis Committee Dr. rer. nat. Till Marquardt Research Group Developmental Neurobiology European Neuroscience Institute Göttingen Grisebachstrasse 5 37077 Göttingen Date of Disputation: 2 April 2012 Affidavit I hereby declare that my doctoral thesis entitled “A Genome-Wide RNAi Screen for Modifiers of Polyglutamine-Induced Neurotoxicity in Drosophila” has been written independently with no other sources and aids than quoted. Göttingen, January 2012 Hannes Voßfeldt LIST OF PUBLICATIONS IV List of Publications Parts of this work have already been published with authorisation of Prof. Jörg B. Schulz, Head of the Department of Neurology, University Medical Centre of the RWTH Aachen University, on behalf of the thesis committee. Poster “A genome-wide screen for modifiers of Ataxin-3-induced neurotoxicity in Drosophila melanogaster”, Regional Drosophila Meeting 2009, Münster/Germany (28 August 2009) Talk “A genome-wide screen for modifiers of ATXN3-induced neurotoxicity in Drosophila melanogaster”, PhD Retreat Molecular Medicine, Göttingen/Germany (17 June 2010) Talk “A genome-wide RNAi screen for modifiers of polyglutamine-induced neurotoxicity in Drosophila”, ScieTalk 2011, Göttingen/Germany (8 June 2011) Poster “A genome-wide screen for modifiers of polyglutamine-induced neurotoxicity in Drosophila melanogaster”, 23rd Biennial Meeting of ISN-ESN 2011, Athens/Greece (30 August 2011) Paper Voßfeldt H, Butzlaff M, Prüßing K, Ní Chárthaigh R-A, Karsten P, et al. (2012) Large-Scale Screen for Modifiers of Ataxin-3-Derived Polyglutamine-Induced Toxicity in Drosophila. PLoS ONE 7(11): e47452. doi:10.1371/journal.pone.0047452 ACKNOWLEDGEMENTS V Acknowledgements The work for this PhD thesis was conducted at the Department of Neurodegeneration and Restorative Research, University Medicine Göttingen (Germany) and the Department of Neurology, University Medical Centre of the RWTH Aachen University, Aachen (Germany), both headed by Prof. Dr. Jörg B. Schulz. I would like to thank Prof. Schulz for giving me the opportunity to produce my thesis in his department and for being my supervisor, furthermore for his intellectual input, constructive criticism and his helpfulness. I would like to thank the members of my thesis committee, Prof. Ernst A. Wimmer and Dr. Till Marquardt, for their intellectual and professional support and for accompanying the process of my promotion. I am deeply indebted to Dr. Aaron Voigt for being my advisor throughout the course of my PhD work, for being a great source of ideas and inspiration, for his intellectual and practical support and for his dedication and efforts in the past years. I owe special thanks to my dear colleagues at the Department of Neurology of the UK Aachen, above all my lab mates Dr. Malte Butzlaff, Dr. Peter Karsten, Sabine Hamm, Anne Lankes, Katja Prüßing, Jane Patricia Tögel, Kavita Kaur, Róisín-Ana Ní Chárthaigh, Li Zhang, Xia Pan, Antje Hofmeister and Marion Roller for invaluable help, support and not least their friendship. I thank Natalie Burdiek-Reinhardt and Isabel Möhring for their immense helpfulness and their pleasant company. I thank the people at the Department of Neurology for creating a friendly and enjoyable working and interpersonal atmosphere. I am indebted to Daniela Otten (Department of Biochemistry, Prof. Lüscher, UK Aachen) for technical support, ideas and discussion and to Fabian Hosp (MDC for Molecular Medicine Berlin, Prof. Selbach) for mass spectrometry experiments, ideas and discussion. I thank Dipl.-Ing. Manfred Bovi (Department of Pathology, UK Aachen) for recording the scanning electron micrographs. I am thankful for the financing of this project by the Competence Network Degenerative Dementias (KNDD). TABLE OF CONTENTS VI Table of Contents List of Figures .......................................................................................................................................... X List of Tables ......................................................................................................................................... XI List of Abbreviations ......................................................................................................................... XII 1 Abstract ................................................................................................................................................. 1 1 Zusammenfassung ............................................................................................................................. 2 2 Introduction ......................................................................................................................................... 3 2.1 Overview: proteopathies and polyglutamine diseases .............................................................. 3 2.2 Pathogenic mechanisms of polyglutamine diseases ................................................................... 6 2.2.1 Cytotoxicity of polyglutamine structures .............................................................................. 6 2.2.2 Molecular pathways to polyglutamine disease................................................................. 10 2.3 Examples of polyglutamine diseases ............................................................................................. 13 2.3.1 Huntington’s Disease (HD) ....................................................................................................... 13 2.3.2 Spinocerebellar ataxias .............................................................................................................. 14 2.3.2.1 Spinocerebellar ataxia type 1 (SCA1) ............................................................................... 15 2.3.2.2 Spinocerebellar ataxia type 2 (SCA2) ............................................................................... 16 2.3.2.3 Spinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) .............. 18 2.4 Drosophila melanogaster as an animal model in research .................................................... 21 2.4.1 The UAS/GAL4 expression system ........................................................................................ 21 2.4.2 RNA interference (RNAi) ........................................................................................................... 22 2.4.3 Rough eye phenotype (REP) .................................................................................................... 24 2.4.4 Drosophila models of polyglutamine disease .................................................................... 24 2.4.5 Previously implemented modifier studies ......................................................................... 26 3 Aim of the Study ................................................................................................................................ 27 TABLE OF CONTENTS VII 4 Material and Methods ..................................................................................................................... 28 4.1 Chemicals, reagents and equipment .............................................................................................. 28 4.2 Fly experiments ...................................................................................................................................... 32 4.2.1 Transgenic flies and housing conditions ............................................................................. 32 4.2.2 Mating procedures ....................................................................................................................... 33 4.2.3 Evaluation of rough eye phenotype modification............................................................ 34 4.2.4 Documentation of eye phenotypes ........................................................................................ 35 4.2.5 Dissection and staining of eye imaginal discs ................................................................... 35 4.2.6 Longevity analysis ........................................................................................................................ 35 4.2.7 Protein collection from fly heads ........................................................................................... 36 4.2.8 Immunoblotting ............................................................................................................................ 36 4.2.9 Filter Retardation Assay ...........................................................................................................
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