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Development of a Cellular Model for C9ORF72-Related Amyotrophic Lateral Sclerosis

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Development of a Cellular Model for C9ORF72-Related Amyotrophic Lateral Sclerosis Development of a Cellular Model for C9ORF72-related Amyotrophic Lateral Sclerosis By Matthew John Stopford Sheffield Institute for Translational Neuroscience University of Sheffield Submitted for the degree of Doctor of Philosophy (PhD) May 2016 Acknowledgements Firstly, I would like to acknowledge and thank my supervisors Dr. Janine Kirby, Prof. Dame Pamela Shaw, and Dr. Adrian Higginbottom for their continued patience and support throughout my PhD. I am incredibly grateful to Janine for providing both academic and personal support during my PhD. I am thankful to Pam for her guidance, but also for pushing me to develop personally and as a scientist. Also, I am very thankful to Adrian, who has been a great mentor, helping me to develop my technical skills, my ability to think critically, and has also been incredibly patient when I have found the PhD hardest. I would also like to thank the Sheffield Hospitals Charity for funding my PhD project. There are several other individuals who have been incredibly helpful during my time in SITraN as well, who deserve acknowledgment. Dr. Guillaume Hautbergue has been optimistic and inspirational, and has taught me many techniques in the lab. Dr. Matthew Walsh has taught me several biochemical techniques, and has been incredibly supportive personally. Dr. Jonathan Cooper-Knock has been very helpful in explaining protocols, array analysis and generally supporting my work. Also, I would like to thank Dr. Paul Heath and Mrs. Catherine Gelsthorpe for their technical assistance and support with the transcriptomic work. This PhD has genuinely been the greatest challenge of my life, and without the support of my parents and family, the Sims family, my friends and other PhD students, I would have failed. Never again. i Abstract Background: ALS is an incurable late onset neurodegenerative disease that is characterised by progressive loss of motor neurons. A (G4C2)n repeat expansion in C9ORF72 is the most common genetic cause of ALS, but it is unknown how the repeat causes pathogenesis, although a gain of toxic function is likely. Aims and Objectives: 1) Generate stable, isogenic motor neuron-like NSC34 cellular models that have tetracycline-inducible (G4C2)n repeat expression. 2) Characterise the cell models for C9ORF72-ALS pathology and biochemical alterations. 3) Identify biological functions and pathways that may be transcriptionally dysregulated by (G4C2)n repeat expression early in C9ORF72-ALS pathogenesis. 4) Identify and interrogate potential therapeutic targets for C9ORF72-ALS. Results: Stable, isogenic NSC34 cell models with tetracycline-inducible (G4C2)n expression were successfully generated. Sense RNA foci and RAN translation products were detected in the cell models. No antisense derived RNA foci or RAN translation products were detected. Expression of the (G4C2)102 caused subtle toxicity and recapitulated some aspects of C9ORF72-ALS pathology in the NSC34 (G4C2)102. The (G4C2)102 expression caused transcriptomic dysregulation in RNA metabolism, protein transport, the PI3K/Akt signalling pathway, and also caused splicing alterations. Transcriptomic dysregulation in the PI3K/Akt signalling pathway was also detected in LCM motor neurons from C9ORF72-ALS patients. Pten knock-down provided a rescue effect against the (G4C2)102 induced toxicity in the NSC34 (G4C2)102. Conclusions: Stable, isogenic motor neuron-like cellular models that had tetracycline- inducible (G4C2)n expression were successfully generated, and allowed interrogation of the early biochemical effects associated with sense only (G4C2)n expression in a reductionist manner. Transcriptomic analysis of the NSC34 (G4C2)102 identified dysregulation in RNA splicing and the PI3K/Akt signalling pathway, which was corroborated by transcriptomic data from C9ORF72-ALS patient CNS tissue. This suggests dysregulation in these biological functions and pathways is disease relevant and an early biochemical event in C9ORF72-ALS pathogenesis. Pten is a potential therapeutic target that deserves further study. ii Table of Contents Acknowledgments – i Abstract – ii Table of Contents – iii List of Figures – xi List of Tables – xiii Abbreviations – xiv Chapter 1. Introduction .......................................................................................................... 1 1.1. Background ........................................................................................................................ 1 1.2. Clinical Presentation .......................................................................................................... 1 1.3. Epidemiology ...................................................................................................................... 3 1.4. Neuropathology ................................................................................................................. 3 1.5. Genetics ............................................................................................................................. 6 1.5.1. SOD1 ............................................................................................................................ 8 1.5.2. TARDBP ....................................................................................................................... 9 1.5.3. FUS ............................................................................................................................ 10 1.5.4. C9ORF72 .................................................................................................................... 11 1.5.5. Genes Involved in RNA Metabolism.......................................................................... 14 1.5.6. Genes Involved in Protein Transport and Degradation ............................................ 15 1.5.7. Genes Involved in Axonal Transport and Cytoskeleton ............................................ 18 1.6. General ALS Pathomechanisms ....................................................................................... 19 1.6.1. Oxidative Stress ......................................................................................................... 19 1.6.2. Glutamate Excitotoxicity ........................................................................................... 19 1.6.3. Mitochondrial Dysfunction ....................................................................................... 20 1.6.4. Impaired Axonal Transport ....................................................................................... 20 1.6.5. Dysregulated RNA Metabolism ................................................................................. 21 1.6.6. Impaired Protein Homeostasis .................................................................................. 21 1.6.7. Non-Cell Autonomous Toxicity and Neuroinflammation .......................................... 22 1.7. C9ORF72 (G4C2)n Repeat Expansion Specific Pathomechanisms ................................... 22 1.7.1. C9ORF72 Haploinsufficiency ..................................................................................... 22 1.7.2. RNA Toxicity .............................................................................................................. 24 1.7.3. Dipeptide Repeat (DPR) Protein Toxicity .................................................................. 25 1.8. C9ORF72-ALS Cellular and Animal Models ...................................................................... 27 iii 1.8.1. Loss of Function Models ........................................................................................... 32 1.8.2. Gain of Function Models ........................................................................................... 32 1.8.2.1. Toxicity associated with the (G4C2)n ................................................................. 32 1.8.2.2. Patient Derived Cellular Models ........................................................................ 34 1.8.2.3. DPR Toxicity Models........................................................................................... 35 1.8.3. Other Genetic Phenomena Associated with the (G4C2)n Repeat ............................ 36 1.9. Overall Aims and Objectives ............................................................................................ 36 Chapter 2. Materials and Methods ....................................................................................... 38 2.1. Materials .......................................................................................................................... 38 2.1.1. General Materials ..................................................................................................... 38 2.1.2. General Buffers and Solutions .................................................................................. 38 2.1.2.1. Phosphate Buffered Saline (PBS) ....................................................................... 38 2.1.2.2. 1X Tris Acetate EDTA (TAE) Buffer ..................................................................... 38 2.1.2.3. 20X Saline Sodium Citrate (SSC) Buffer .............................................................. 39 2.1.2.4. 1M Sodium Phosphate Buffer pH 7.0 ................................................................ 39 2.1.2.5. Diethylpyrocarbonate (DEPC) Treatment of Solutions ...................................... 39 2.1.3. Molecular Biology Materials ..................................................................................... 39 2.1.4. Cell Culture Materials ..............................................................................................
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