C9orf72 frontotemporal dementia and amyotrophic lateral sclerosis: investigating repeat pathology in cell culture models and human post-mortem brain. Charlotte Elizabeth Ridler A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy from University College London Department of Neurodegenerative Disease Institute of Neurology University College London 2016 1 Declaration I, Charlotte Ridler, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Acknowledgements My deepest thanks go to everyone who has helped and supported me during this PhD project, without whom it would not have been possible. Thank you firstly to my supervisor Dr. Adrian Isaacs for all of his guidance, encouragement and enthusiastic discussions during this exciting and unpredictable project. Thank you also to Dr. Sarah Mizielinska, who has been an incredible mentor and role-model during this PhD; I have learnt a huge amount from her endless dedication and generosity. I would also like to thank my secondary supervisor Prof. Elizabeth Fisher for all her wise words of advice. Thank you to all members of the Isaacs and Fisher labs past and present. Special thanks to Frances Norona, Dr. Karen Cleverly, Julian Pietrzyk, Dr. Ione Woollacott and Dr. Anny Devoy who were all great help during the cloning phase of this project; to Dr. Rubika Balendra for all her help and discussions in the nucleoli work; and to Dr. Emma Clayton and Dr. Roberto Simone for their constant support. A number of other people have lent me their valuable expertise for this project, for which I am very grateful. Thank you to Dr. Tammaryn Lashley for provision of histological samples and for her valuable discussions, to Dr. Teresa Nicolli for all her work and discussions regarding the fly project, and to Dr. Vincent Plagnol for his vital statistics advice. I am also extremely grateful to my funders Alzheimer’s Research UK for their support for this project. Thank you to fellow PhDs Dr. Xun Yu Choong and Angelos Armen with whom I have shared the entire rollercoaster ride of the last three years. Thank you also to everyone in the PhD office for all your support and shenanigans, James Miller, Laura Pulford, Justin Tosh, Ludmila Sheytanova, Dr. Billy West, Lucianne Dobson, Dr. Rachelle Saccon, Amy Nick, Julian Jaeger, Ruchi Kumari, and Alexandra Philiastides. Thank you also to all of the support staff at the Institute of Neurology. Finally, I would like to thank all of my wonderful family and friends outside of the lab who supported me, encouraged me and made me cups of tea: my partner Thomas Jennings, my parents Kay and Tony Ridler, my brother Samuel Ridler, all of my extended family, my supportive dance friends, and my housemates Thomas Beales Ferguson, Craig Buchanan, Jacob Spencer, Abigail Attrell, Adam Ladds, Oliver Mattos, and Otso Liikanen. 3 Abstract A GGGGCC repeat expansion in the first intron of chromosome 9 open reading fame 72 (C9orf72) is the most common known genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeats are transcribed into RNA in both sense and antisense orientations, which aggregates to form RNA foci in cells. Additionally, repeat RNA undergoes repeat-associated non-ATG initiated (RAN) translation producing dipeptide repeat (DPR) proteins in all six sense and antisense reading frames. This thesis aims to dissect RNA and DPR protein gain-of-function mechanisms operating in C9orf72 FTD/ALS to study their effects in isolation. To achieve this, three classes of DNA constructs were generated: 1) pure GGGGCC repeats that produce RNA foci and DPR proteins, 2) ‘RNA-only’ constructs designed to preclude RAN translation, and 3) ‘protein-only’ constructs coding for all five DPR proteins that do not produce GGGGCC repeat RNA. Cultured cells transfected with pure and RNA-only repeat constructs exhibited a positive correlation between repeat length and both sense and antisense RNA foci formation. Additionally, DPR protein subcellular localisation was examined in protein-only repeat construct transfected cells and compared to the pathology found in patient brain. A collaborative project expressing these constructs in Drosophila showed that arginine- containing DPR proteins were responsible for C9orf72 repeat toxicity in flies. To determine the relevance of the different DPR proteins to human disease, the frequency and distribution of DPR protein inclusions was studied in C9orf72 FTD patient brain, using a novel automated image analysis protocol. Poly(glycine-arginine) DPR protein inclusion- bearing neurons also exhibited an increased nucleolar volume compared to inclusion- negative neurons, confirming recent speculation implicating nucleolar stress in disease pathogenesis. These studies in cell culture, Drosophila and human post-mortem brain implicate poly(glycine-arginine) as a toxic species within C9orf72 FTD, which may aid in the targeting of future treatments for this condition. 4 Table of Contents Declaration ............................................................................................................. 2 Acknowledgements ................................................................................................. 3 Abstract ................................................................................................................... 4 Table of Contents ..................................................................................................... 5 List of Figures ......................................................................................................... 13 List of Tables .......................................................................................................... 17 CHAPTER 1: INTRODUCTION ......................................................................................... 22 1.1 Overview .......................................................................................................... 22 1.2 Clinical features of frontotemporal lobar degeneration and amyotrophic lateral sclerosis ................................................................................................................. 23 1.2.1 Frontotemporal dementia .............................................................................. 23 1.2.2 Amyotrophic lateral sclerosis ......................................................................... 24 1.2.3 Clinical overlap................................................................................................ 26 1.2.4 Treatment ....................................................................................................... 26 1.3 Pathological features of FTLD and ALS ............................................................... 27 1.3.1 FTLD-TDP and ALS-TDP ................................................................................... 27 1.3.2 FTLD-FUS and ALS-FUS .................................................................................... 29 1.3.3 ALS-SOD1 ........................................................................................................ 29 1.3.4 FTLD-tau .......................................................................................................... 30 1.3.5 FTLD-UPS ........................................................................................................ 31 1.4 Genetics ........................................................................................................... 31 5 1.5 The C9orf72 repeat expansion mutation ............................................................ 35 1.5.1 Identification of a hexanucleotide repeat expansion mutation in C9orf72 ... 35 1.5.2 Genetic characteristics of the C9orf72 repeat expansion .............................. 35 1.5.3 Epidemiology of C9orf72 FTD/ALS .................................................................. 38 1.5.4 Clinical features of C9orf72 FTD/ALS .............................................................. 39 1.5.5 Neuroanatomical features of C9orf72 FTD/ALS ............................................. 40 1.5.6 Neuropathology in C9orf72 FTD/ALS .............................................................. 41 1.6 Potential disease mechanisms in C9orf72 FTD/ALS ............................................. 43 1.6.1 Loss of C9orf72 protein function .................................................................... 44 1.6.2 RNA gain-of-function mechanisms ................................................................. 48 1.6.3 Gain of peptide function ................................................................................. 53 1.6.4 Proposed cellular functions affected in C9orf72 FTD/ALS .............................. 57 1.7 Thesis aims ....................................................................................................... 59 CHAPTER 2: MATERIALS AND METHODS ....................................................................... 61 2.1 Cloning Protocols .............................................................................................. 61 2.1.1 Annealing and ligation of oligonucleotides .................................................... 61 2.1.2 Preparation of bacterial culture solutions ...................................................... 61 2.1.3 Transformation of chemically competent E. coli ............................................ 63 2.1.4 Colony PCR for detection of GGGGCC repeats ..............................................