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Drivers of TDP43 Dyshomeostasis in Amyotrophic Lateral Sclerosis by Kaitlin T. Weskamp a Dissertation Submitted in Partial Fulfi

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Drivers of TDP43 Dyshomeostasis in Amyotrophic Lateral Sclerosis by Kaitlin T. Weskamp a Dissertation Submitted in Partial Fulfi Drivers of TDP43 Dyshomeostasis in Amyotrophic Lateral Sclerosis by Kaitlin T. Weskamp A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Neuroscience) in the University of Michigan 2019 Doctoral Committee: Assistant Professor Sami Barmada, Chair Professor Geoffrey Murphy Professor Jack Parent Associate Professor Peter Todd Professor Michael Uhler Kaitlin T. Weskamp [email protected] ORCID iD: 0000-0003-4474-2850 Dedication This dissertation is dedicated to John, who has offered unwavering love, support, and confidence in my abilities through every academic endeavor. ii Acknowledgements First, I thank my mentor Dr. Sami Barmada, for his expertise, guidance, and patience throughout my training. He has done a remarkable job of curating a safe and supportive environment in which his students can grow as scientists, and I am thankful for the opportunity to work with him. I thank members of the Barmada laboratory for their contributions to our hilarious, bizarre, and productive work environment, and for making it a joy to come to work every day. These relationships are among the most valuable things I gained during my time in graduate school, and I consider myself lucky to work among friends. I thank my support system, particularly John, Anita, and Megan, who kept me moving through the lows and reminded me to celebrate the highs. I thank my dissertation committee members Dr. Michael Uhler, Dr. Jack Parent, Dr. Geoffrey Murphy, and Dr. Peter Todd for their valuable suggestions that strengthened the quality of my work. I thank members of the Feldman, Paulson, and Todd labs for their generosity with expertise, reagents, and hallway conversations. I thank the staff of the Neuroscience Graduate Program, the Program in Biomedical Sciences, the Neurology Department, and the Biomedical Sciences Research Building janitorial staff for all their hard work over the years. Other collaborators, contributors, and sources of support are acknowledged throughout the dissertation iii Table of Contents Dedication ....................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x List of Appendices ....................................................................................................................... xiii Abstract ........................................................................................................................................ xiv Chapter 1. Introduction ................................................................................................................... 1 1.1 Overview ............................................................................................................................... 1 1.2 TDP43 Pathology in ALS ..................................................................................................... 2 1.3 TDP43 Structure .................................................................................................................... 3 1.4 Other TDP43 Isoforms .......................................................................................................... 3 1.5 Mechanisms of TDP43 Autoregulation................................................................................. 4 1.6 Disruption of TDP43 Autoregulation in ALS ....................................................................... 7 1.7 Downstream Consequences of Failed TDP43 Autoregulation: TDP43 Nuclear Exclusion and Cytoplasmic Accumulation .................................................................................................. 8 1.7.1 Disrupted TDP43 Autoregulation Interrupts Normal Protein Function ......................... 8 1.7.2 TDP43 Inclusions and Gain of Function Toxicity ........................................................ 14 1.8 Summary and Aims of the Dissertation .............................................................................. 15 1.9 Acknowledgements ............................................................................................................. 16 References ................................................................................................................................. 17 iv Figures ....................................................................................................................................... 27 Chapter 2. RNA Degradation in Neurodegenerative Disease ....................................................... 30 2.1 Introduction ......................................................................................................................... 30 2.2 Mechanisms to Maintain RNA Stability ............................................................................. 30 2.2.1 Polyadenylation ............................................................................................................ 31 2.2.2 Methylguanine Cap....................................................................................................... 32 2.2.3 Secondary Structure ...................................................................................................... 33 2.2.4 Stress Granules ............................................................................................................. 33 2.3 Mechanisms of RNA Decay ................................................................................................ 34 2.3.1 RNA Degradation Machinery ....................................................................................... 35 2.3.2 Nonsense-mediated Decay ........................................................................................... 35 2.3.3 Nonstop Decay ............................................................................................................. 38 2.3.4 No-go Decay ................................................................................................................. 38 2.3.5 Adenylate-Uridylate-Rich Elements............................................................................. 39 2.3.6 Staufen-mediated Decay ............................................................................................... 40 2.3.7 microRNAs ................................................................................................................... 41 2.3.8 Constitutive Decay Elements ........................................................................................ 42 2.3.9 Histone mRNAs ............................................................................................................ 42 2.3.10 Processing Bodies ....................................................................................................... 43 2.4 RNA Turnover in Neurodegenerative Disease.................................................................... 43 2.4.1 RNA Sequestration ....................................................................................................... 44 2.4.3 RNA Transport ............................................................................................................. 50 2.4.4 The RNA Exosome Complex ....................................................................................... 52 2.4.5 Alternative Splicing ...................................................................................................... 53 2.4.6 Retrotransposons .......................................................................................................... 55 2.5 Conclusions and Future Directions ..................................................................................... 57 2.6 Acknowledgements ............................................................................................................. 57 References ................................................................................................................................. 58 Figures ....................................................................................................................................... 76 Chapter 3. Neuronal Hyperexcitability Drives TDP43 Pathology via the Upregulation of Atypical, Shortened TDP43 Splice Isoforms ................................................................................ 79 3.1 Introduction ......................................................................................................................... 79 3.2 Results ................................................................................................................................. 81 v 3.2.1 TDP43 is regulated by neuronal activity ...................................................................... 81 3.2.2 Hyperexcitability drives TARDBP alternative splicing ................................................ 84 3.2.3 sTDP43 is cytoplasmically localized due to a putative NES in its C-terminal tail ...... 85 3.2.4 sTDP43 overexpression is neurotoxic .......................................................................... 87
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