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Characterisation of Novel Genetic Variants of Amyotrophic Lateral Sclerosis

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Characterisation of Novel Genetic Variants of Amyotrophic Lateral Sclerosis Characterisation of Novel Genetic Variants of Amyotrophic Lateral Sclerosis By Tobias Alexander Moll Sheffield Institute for Translational Neuroscience A thesis submitted for the degree of Doctor of Philosophy (PhD) June 2020 Abstract Background: Amyotrophic lateral sclerosis (ALS) is an invariably fatal and relatively common neurodegenerative disorder without effective therapy. Identified genetic variants cluster in biological pathways including RNA processing, axonal transport, and protein homeostasis. Discovery of new genetic variants within new biological pathways highlights new disease biology, and can lead to novel therapeutic targets. This project will focus on the development of cell and animal models to characterise novel ALS-associated mutations associated with GLT8D1, CAV1 and CAV2. Aims and objectives: i) To evaluate the relative toxicity of ALS-associated GLT8D1 mutations in neuronal and non-neuronal cell lines via MTT and lactate dehydrogenase assays. ii) To investigate the effect of mutations on the enzyme activity of GLT8D1 using a UDP-GloTM glycosyltransferase assay. iii) To test whether mutant GLT8D1 causes fragmentation of the Golgi network using immunocytochemistry. iv) To model GLT8D1 mutations in zebrafish larvae via RNA microinjection. v) To model CAV1/CAV2 enhancer mutations in neuronal and non-neuronal cells via CRISPR/Cas9 genome editing. vi) To measure ganglioside expression in human cells expressing GLT8D1, CAV1 and CAV2 mutations using live cell imaging. Results: I studied in detail two ALS-associated GLT8D1 mutations: R92C and G78W. The relative toxicity of the mutations in model systems mirrors the clinical severity. Mutated GLT8D1 exhibits in vitro cytotoxicity and induces motor deficits in zebrafish larvae consistent with ALS. Identified GLT8D1 mutations are proximal to the substrate-binding site; both R92C and G78W mutations impair GLT8D1 enzyme activity. An R92C mutation reduces membrane ganglioside expression, which is indicative of dysregulated neurotrophic signalling. Ganglioside biosynthesis occurs in the Golgi; GLT8D1 localises to the Golgi in neuronal and non-neuronal cells, and preliminary data suggests an R92C mutation causes Golgi fragmentation. The second stage of this project follows the identification of ALS-associated variation within an enhancer linked to expression of CAV1/CAV2. CAV1 and CAV2 encode major components of caveolae, which organise membrane lipid rafts (MLR) important for neurotrophic signalling. Gangliosides are a key component of MLR. Discovered enhancer mutations reduce CAV1/CAV2 expression and disrupt ganglioside expression within MLR in patient-derived cells; and CRISPR/Cas9 perturbation proximate to a patient-mutation is sufficient to reduce CAV1/CAV2 expression in neurons. Conclusions: These results place dysregulated ganglioside metabolism upstream in the pathogenesis of ALS. I propose that GLT8D1 and CAV1/CAV2 share a common pathway of pathogenesis in ALS via disruption of ganglioside recruitment to MLR and impaired neurotrophic signalling. I Declaration I, Tobias Moll, confirm that this thesis is my own work. I am aware of the University’s Guidance on the Use of Unfair Means (www.sheffield.ac.uk/ssid/unfair-means). This work has not been previously been presented for an award at this, or any other, university. Where stated, sections of this thesis are comprised of the following published papers: Chapter 1: Introduction – Moll et al., 2020. Disrupted glycosylation of lipids and proteins is a cause of neurodegeneration. Brain. awz358. In addition to Moll et al., 2020, there is further discussion in Chapter 1 that is not published. Chapter 3: ALS-linked G78W and R92C variants in GLT8D1 are toxic to HEK293 and N2A cells and impair enzyme activity – Cooper-Knock et al., 2019. Mutations in the glycosyltransferase domain of GLT8D1 are associated with familial amyotrophic lateral sclerosis. Cell Reports. 26(9):2298-2306. In addition to Cooper-Knock et al., 2019, further results are presented in Chapter 3 that are not published. Chapter 4: Knockdown of endogenous glt8d1 and overexpression of mutant GLT8D1 produces motor impairment in zebrafish larvae – Cooper-Knock et al., 2019. Mutations in the glycosyltransferase domain of GLT8D1 are associated with familial amyotrophic lateral sclerosis. Cell Reports. 26(9):2298-2306. In addition to Cooper-Knock et al., 2019, further results are presented in Chapter 4 that are not published. In addition, the following experimental chapter includes a paper currently in submission: Chapter 6: Characterisation of ALS-associated CAV1/CAV2 enhancer variants – Rare variant burden analysis within enhancers identifies CAV1 as a new ALS risk gene. Available at: https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3606796 At the start of each chapter, a full acknowledgment and reference of the publication is given, in addition to my contribution, as well as that of my co-authors and collaborators. Some chapters contain additional data and commentary that is not in the published/submitted manuscripts. II Dedication This thesis is dedicated to Mr Lee Newton. The generous contribution from the Newton family has made this work possible, and I hope that this research will aid in the fight against motor neurone disease. I would also like to dedicate this thesis to my father, Victor Moll. Your vision and work ethic inspired me from a young age. I hope I have made you proud. III Acknowledgements I am highly indebted to the enthusiasm, commitment, and above all, friendship of Dr Johnathan Cooper-Knock, who has provided me with every opportunity to develop as a researcher. His passion and dedication have driven me to achieve things I did not think were possible. A very special mention goes to Professor Dame Pamela Shaw, for her continual guidance and support throughout this PhD, and for believing in my ability as a scientist. I would also like to thank Dr Janine Kirby for providing me with so much academic and personal support over the past five years. This PhD would not have been as successful without the help of some very important people at SITraN, particularly Dr Adrian Higginbottom, who has been extremely generous with his time and has shown unprecedented levels of patience when helping me to develop my technical skills and my ability to think critically. I was extremely fortunate to have the support of the Hautbergue lab, who always found the time to explain protocols and help me with performing challenging experiments. I would like to extend my gratitude to Dr Tennore Ramesh for his help with the zebrafish work. To all my friends and colleagues who have given me support, laughter and happiness along the way: John, Sam, Ramya, Helia, Dave, Andre, Robin, Toby, Osman, Mirinda, Emily, Charlotte, Ale, Matt, Nikita, Erica, Cleide, Allan, Alex…I love you all! I would be in trouble if I did not mention my wonderful girlfriend, Anna, who is without question one of the most understanding and supportive people I have known. Thank you for putting up with my working evenings and weekends over the past three years! Finally, this PhD would not have been possible without the love and support of my amazing family, particularly my mother, who has provided me with every opportunity in life, and has continually tried her hardest to understand what I do for a living. I would not have achieved half of what I have without the support of my amazing sister and best friend, Poppy. To the rest of the Moll family: Victor, Max, and Leo – I have always looked up to you boys and I hope I have made you as proud as I am of you. IV Table of contents List of figures .......................................................................................................................... XII List of tables ......................................................................................................................... XIV List of abbreviations ............................................................................................................. XV Chapter 1. Introduction ........................................................................................................... 1 1.1. Introduction to amyotrophic lateral sclerosis ........................................................... 1 1.2. Clinical presentation and disease management ..................................................... 1 1.3. Genetics of ALS ........................................................................................................... 2 1.3.1. SOD1...................................................................................................................... 3 1.3.2. TARDBP ................................................................................................................ 3 1.3.3. FUS ........................................................................................................................ 4 1.3.4. C9ORF72 .............................................................................................................. 4 1.4. ALS pathological mechanisms .................................................................................. 5 1.4.1. Oxidative stress .................................................................................................... 5 1.4.2. Mitochondrial dysfunction ................................................................................... 6 1.4.3. Impaired axonal transport ................................................................................... 6 1.4.4. Dysregulated RNA metabolism .........................................................................
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