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The Role of the Myotubularin Pseudophosphatase MTMR13 In

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The Role of the Myotubularin Pseudophosphatase MTMR13 In The Role of the Myotubularin Pseudophosphatase MTMR13 in Myelination and Disease by Danielle C. Robinson A DISSERTATION Presented to the Neuroscience Graduate Program at the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 5, 2016 Oregon Health & Science University ____________________________________ CERTIFICATE OF APPROVAL ____________________________________ This is to certify that the Ph.D. dissertation of DANIELLE C. ROBINSON has been approved on December 5, 2016 ____________________________________ Advisor, Fred Robinson, Ph.D ____________________________________ Member and Chair, Philip Stork, M.D ____________________________________ Member, Gary Westbrook, M.D. ____________________________________ Member, Ben Emery, Ph.D. ____________________________________ Member, Mary Logan, Ph.D The role of MTMR13 in myelination and disease List of Figures.............................................................................................................vi List of Abbreviations................................................................................................viii Acknowledgments ......................................................................................................x Table of Contents Chapter 1 — Introduction to the Mechanisms of Schwann Cell Myelination and Inherited Peripheral Neuropathy ........................................................................................................ 3 Forward ....................................................................................................................................... 3 1.1 — Introduction to the Peripheral Nervous System (PNS) .......................................... 4 1.1.1 —Schwann Cell Myelin ................................................................................................... 5 1.1.2 — Schwann Cell Differentiation and Development ........................................................... 5 1.1.3 —Regulation of Myelination ............................................................................................ 7 1.1.4 — Endosomal Trafficking is Critical to Schwann Cell Polarization and Myelination ...... 9 1.1.5 — PNS Myelin Sheath Assembly Requires Key Lipids and Proteins ........................... 11 1.1.6 — Myelin Sheath Wrapping Requires Phosphoinositide Membrane Lipids and Trafficking Via Non-Compact Regions .......................................................................................... 16 1.1.7 — Basal Lamina Enable Schwann Cells to Interact with Their Enviornment ............... 18 1.1.8 — Schwann Cells and Axons Engage in Bidirectional Signaling and Support ............... 19 1.2 — Myelin Dysfunction and Disease .............................................................................. 20 1.2.1 — Introduction to Charcot-Marie-Tooth Peripheral Neuropathy ................................... 21 1.2.2 — Diagnosis of CMT .................................................................................................. 23 1.2.3 — The CMT Family of Inherited Peripheral Neuropathies ........................................... 25 1.2.4 — CMT Patient Outcomes and CMT Therapies in Development ................................ 28 1.3 — Endosomal/Lysosomal Signaling in Demyelinating Forms of CMT ................. 31 1.3.1 — Phosphoinositides are Critical to Intracellular Membrane Trafficking ........................ 33 1.3.2 — Phosphoinositide Regulation by Kinases and Phosphatases ......................................... 34 i 1.3.3 — Regulation of the Early Endosome is Critical to Cell ............................................... 36 1.3.4 — Regulation of Autophagic Can Impact Schwann Cell Myelination ............................ 38 1.3.5 — The Myotubularin Phosphatase Family’s Role in Disease ......................................... 39 1.3.6 — MTMR13 Structure and Function .......................................................................... 42 1.3.7 — The Balance of Phosphoinositides, PI Kinases, and PI Phosphatases at the Early Endosome ....................................................................................................................................... 44 1.4 — Summary and Major Questions in the Field ........................................................... 48 Chapter 2 — An In Vitro Model of CMT2B2 Enables Exploration of the roles of Myotubularin Phosphatases in Myelin Disease .............................................................................. 54 Forward ..................................................................................................................................... 54 2.1 — Introduction ................................................................................................................. 55 2.2 — Materials and Methods .............................................................................................. 58 2.2.1 — Mice ........................................................................................................................ 58 2.2.2 — Antibodies .............................................................................................................. 58 2.2.3 — Myelinating Schwann Cell and Dorsal Root Ganglia Neuron Explant Cultures ..... 59 2.2.4 — Immunofluorescence ................................................................................................... 61 2.2.5 — Image Acquisition and Analysis ............................................................................... 62 2.2.6 — Image Quantification ................................................................................................ 63 2.2.7 — Electron Microscopy of SC-DRG Co-Cultures ......................................................... 64 2.2.8 — Lentivirus Production and Concentration .................................................................. 65 2.2.9 — Lentivirus Titring ..................................................................................................... 66 2.2.10 — Immunoblotting ...................................................................................................... 67 2.3 — Results ........................................................................................................................... 69 2.3.1 — Mtmr13 -/-Schwann Cell and Neuron Co-culture as an In Vitro Model of CMT4B2 ...................................................................................................................................................... 69 2.3.2 — Exogenous MTMR2 Supresses Myelination and Partially Rescues the Mtmr13-/- Outfolding Phenotype In Vitro ....................................................................................................... 71 ii 2.4 — Discussion..................................................................................................................... 72 Chapter 3. Mtmr13’s DENN Domain: New Tools and Approaches to Clarify its Function in Schwann Cell Myelination ............................................................................................ 85 Forward ..................................................................................................................................... 85 3.1 —Introduction .................................................................................................................. 85 3.1 — Materials and Methods ............................................................................................... 88 3.1.1 — Development of a GFP-Tagged Mtmr13 DENN Domain ...................................... 88 3.2.2 – Protein Expression and Lentivirus Production ............................................................ 93 3.3 — Results ........................................................................................................................... 94 3.3 — Discussion..................................................................................................................... 95 Chapter 4 — Discussion and Future Directions .................................................................. 110 4.1 — Summary and Discussion ........................................................................................ 110 4.1.3 — Future Directions for Investigation ......................................................................... 114 4.1.4 — Concluding Remarks ............................................................................................. 116 Appendix .................................................................................................................................... 118 I. Detailed protocol: Myelinating explant cultures from dorsal root ganglia .............. 118 II. Infusion cloning primer sequences ............................................................................... 120 References .................................................................................................................................. 122 Tables, Figures, and Abbreviations Figure 1.1 — Endosomal proteins are critical for PNS Schwann cell myelination. ................. 50 Figure 1.2 — Phosphoinositide structure localization, and regulation. ...................................... 51 Figure 1.3 — The myotubularin family of PI 3-phosphatases. ................................................... 53 Figure 2.1 — Mtmr13-/- Schwann cell and neuron co-cultures myelinate robustly in vitro. .... 77 iii Figure 2.2 — Mtmr13-/- Schwann cell and neuron co-cultures
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