Histone deacetylase inhibitors for the epigenetic therapy of proximal spinal muscular atrophy Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Lutz Garbes aus Köln Köln 2010 The Doctoral Thesis "Histone deacetylase inhibitors for the epigenetic therapy of proximal spinal muscular atrophy“ was performed at the Institute of Human Genetics, Institute of Genetics and Centre for Molecular Medicine Cologne (CMMC) of the University of Cologne from November 2006 to 2010. Berichterstatter/in: Prof. Dr. rer. nat. Brunhilde Wirth Prof. Dr. rer. nat. Thomas Wiehe Tag der letzten mündlichen Prüfung: 22.11.2010 Für meine Eltern Acknowledgements First, I would like to thank my supervisor Brunhilde Wirth for giving me the opportunity to work on various interesting and challenging projects, for sharing her scientific knowledge and enthusiasm, and for allowing me to work independently. Furthermore, I woud like to thank her for motivating discussions and encouragement, for her generous support to attend scientific meetings, and for bringing me in touch with various scientist all over the world. I greatly appreciate her dedication. I thank my examiners Prof. Dr. Thomas Wiehe and Prof. Dr. Günter Schwarz. Of course a big “Thanks!” to all past and present members of the SMA group, and the whole Institute of Human Genetics in Cologne. A very big “extra thank you” to Irmgard Hölker for her excellent technical support during the last years, for countless triplicates and for staying at my side during the VPA odyssey. I thank the Markus Rießland for good advice whenever needed, valuable discussions about science, all the world and his wife and all the interesting news out there. Furthermore, I like to thank Bastian Ackermann and Sandra Kröber for keeping a hilarious atmosphere in our lab and all those funny Fridays. In the end, I salute the navigator for leading the quarterhorse cowboys on the right track. Moreover, I thank Miriam Jakubik and Ylva Mende for “introducing me to the mice”, and how to put syringes in mice without making kebap. Of course, I thank all the people, who carefully went through this manuscript, for their helpful suggestions and stamina: Markus (the big one), Julia (always positive – I merely bought it), Basti, Sabrina and Markus (the small one). Furthermore, I thank also thank Karin Boß reading the draft, sharing her English skills and donating me some extra commas. I thank Eric Hahnen helpful discussions (400nM!!) and for bringing me in touch with his buddies in Erlangen and Bonn. I would to thank Ingmar Blümcke and Roland Coras for the NSCs and OHSCs and Christian Tränkle for the HDAC inhibition assay. I thank Carsten Müller and Tim Bauer for the coffee meetings and their help and patience with the LC-MS/MS. Especially, I think Tim Bauer for his willingness to shorten his weekends for some additional sample measurements. I would like to thank Jürgen-Christoph von Kleist-Retzow for his help with polarography experiments. I thank my parents for their constant support and motivation, their confidence and everything they taught me. This work wouldn´t have been possible without you!! Furthermore, I give props to Maurits and Niklas for distraction whenever needed . Finally, this list wouldn´t be complete without Sabrina Rosenbaum: Thank you for just being you - you are more than beautiful. Table of contents i Table of contents LIST OF ABBREVIATIONS ........................................................................................................................... VI 1. INTRODUCTION ............................................................................................................................... 1 1.1 AUTOSOMAL RECESSIVE PROXIMAL SPINAL MUSCULAR ATROPHY (SMA) ........................................................... 1 1.1.1 Clinical diagnosis ...................................................................................................................... 1 1.1.2 Classification of proximal SMA ................................................................................................. 3 1.1.2.1 SMA type I (Werdnig-Hoffmann disease), MIM #25330 ...................................................................... 3 1.1.2.2 SMA type II, MIM #253550 .................................................................................................................. 4 1.1.2.3 SMA type III (Kugelberg-Welander disease), MIM #253400 ................................................................ 4 1.1.2.4 SMA type IV (adult SMA), MIM #271150 ............................................................................................. 4 1.2 THE MOLECULAR BASIS OF SMA ................................................................................................................ 4 1.2.1 SMN1 and SMN2 ...................................................................................................................... 5 1.2.2 SMN splicing ............................................................................................................................. 6 1.3 THE SMN PROTEIN................................................................................................................................. 9 1.3.1 The SMN complex ................................................................................................................... 10 1.3.2 Functions of SMN .................................................................................................................... 10 1.3.2.1 Housekeeping functions .................................................................................................................... 10 1.3.2.2 Neuron-specific functions .................................................................................................................. 12 1.3.2.3 Muscle-specific functions .................................................................................................................. 13 1.4 ANIMAL MODELS OF SMA ..................................................................................................................... 13 1.4.1 Non-vertebrate SMA models .................................................................................................. 13 1.4.2 Vertebrate SMA models .......................................................................................................... 14 1.5 SMA: WHY DO SPECIFICALLY MOTOR NEURONS DIE? .................................................................................. 17 1.6 THERAPEUTIC APPROACHES IN SMA ........................................................................................................ 18 1.6.1 Epigenetic therapy targeting SMN2 expression ..................................................................... 19 1.6.1.1 The concept of epigenetic therapy .................................................................................................... 19 1.6.1.2 Histone Acetyl Transferases (HAT) ..................................................................................................... 22 1.6.1.3 Histone Deacetylases (HDAC) ............................................................................................................ 23 1.6.1.3.1 Class I HDACs .............................................................................................................................. 23 1.6.1.3.2 Class II HDACs ............................................................................................................................. 23 1.6.1.3.3 Class III HDACs (sirtuins) ............................................................................................................. 24 1.6.1.3.4 Class IV HDACs ............................................................................................................................ 24 1.6.1.4 HDAC inhibitors ................................................................................................................................. 24 1.6.1.5 Epigenetic SMA therapy with HDACi ................................................................................................. 27 1.6.1.6 VPA in SMA therapy ........................................................................................................................... 27 1.6.2 Further therapies targeting SMN2.......................................................................................... 28 1.6.2.1 RNA-based therapy ............................................................................................................................ 28 1.6.2.2 Gene therapy ..................................................................................................................................... 29 ii Table of contents 1.6.2.3 Drug therapy ...................................................................................................................................... 29 1.6.3 SMN2-independent therapies ................................................................................................. 30 1.6.3.1 Neurotrophic agents .......................................................................................................................... 30 1.6.3.2 Stem cell therapy ............................................................................................................................... 31 2. STUDY AIMS .................................................................................................................................. 33 3. MATERIAL