Metabolic control of sensory neuron survival by the p75 neurotrophin receptor in Schwann cells By Rose M. Follis Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Neuroscience February 28, 2021 Nashville, Tennessee Approved: Dr. Ronald Emeson, Ph.D. Dr. Jun Li, M.D., Ph.D. Dr. Ned Porter, Ph.D. Dr. Bruce Carter, Ph.D. Dedication To family, friends, and the world’s baristas. ii Acknowledgements No science is ever done in isolation. As I wrote this, I was constantly reminded of how many people have contributed to the experiments within these pages. However, since this monster is already clocking in at 170 pages, I’ll spare you a full account and just go for a few shout outs. Starting at home, the Carter lab, I’d like to thank our head honcho, Bruce, for his leadership, guidance, and pumpkin porter. Also, all my awesome Carter lab coworkers who joined me in hours long debates on how to figure out if your randomization is random, if mice think we are alien overlords or don’t think about us at all, and why on the heck every incubation in science is 5 minutes (I vote superstition). I’d also like to thank those that kept us feed and funded (the VBI, Biochemistry Department, and NIH). As well as those that had to put up with my horrible handwriting (the staff of the EM and TPSR cores), horrible paperwork skills (Roz, Jen, and Beth), and endless questions when this project went in down unexpected paths (too many to name, if I’m honest). Also, my committee past and present. Lastly, I would like to thank all friends and family. May they all be less tormented from here on out by my grumpy mutterings detailing cannibal mice and core computers with Windows Vista installed. iii Table of Contents Page Dedication .......................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii List of Tables ................................................................................................................................... vii List of Figures ................................................................................................................................. vii List of Abbreviations ....................................................................................................................... x Chapter I. Introduction .................................................................................................................................... 1 Overview ......................................................................................................................................... 1 Introduction .................................................................................................................................... 2 The Impact of Neuropathy.................................................................................................. 2 Cells of the Peripheral Nervous System .......................................................................... 3 A Timeline of Nerve Development .................................................................................... 6 Schwann Cell Lipid Dysregulation and Neuropathy ..................................................... 14 Overview of Diseases Linked to Neurotoxic Lipid Accumulation in the PNS ........... 19 Implications in Other Disease Pathologies .................................................................... 35 Project Aims ................................................................................................................................. 36 II. Deletion of p75 in Schwann Cells Leads to Uniform DRG Neuron Loss and Hypoalgesia .................................................................................................................................... 39 Introduction .................................................................................................................................. 39 Methods ......................................................................................................................................... 40 Results ........................................................................................................................................... 42 Discussion .................................................................................................................................... 51 III. Deletion of p75 leads to the Accumulation of Neurotoxic 7-DHC, Resulting in Neuronal Loss During Development ........................................................................................ 54 Introduction .................................................................................................................................. 54 Methods ......................................................................................................................................... 55 iv Results ........................................................................................................................................... 59 Discussion .................................................................................................................................... 69 IV. P75 Regulates SC Cholesterol Biosynthesis in Coordination with the ErbB2 Receptor ........................................................................................................................................... 73 Introduction .................................................................................................................................. 73 Methods ......................................................................................................................................... 74 Results ........................................................................................................................................... 76 Discussion .................................................................................................................................... 81 V.Deletion of P75 Results in Significantly Altered Nerve Lipid Profiles ......................... 83 Introduction .................................................................................................................................. 83 Methods ......................................................................................................................................... 84 Results ........................................................................................................................................... 85 Discussion .................................................................................................................................... 91 Project Discussion ..................................................................................................................... 93 Conclusions and Future Directions ................................................................................. 93 Significance ........................................................................................................................ 96 Appendix I. The Role of NFκB in Inherited Neuropathy .......................................................................... 98 Introduction .................................................................................................................................. 98 Charcot-Marie-Tooth Disease ......................................................................................... 98 CMT1 Pathology ................................................................................................................ 99 NFκB Signaling in Neuropathy ...................................................................................... 101 Methods ....................................................................................................................................... 104 Results ......................................................................................................................................... 106 Discussion and Future Directions ....................................................................................... 113 II. Supplementary Tables and Bibliography .......................................................................... 117 Table of Reagents ..................................................................................................................... 117 UPLC-MS/MS Tables .................................................................................................................. 121 Bibliography ............................................................................................................................... 133 v List of Tables Table Page 1. Diseases associated with toxic lipid accumulation. ....................................................38 2. Quantification (nmol/mg protein) of cholesterol and cholesterol precursor levels.. .68 3. 7-DHC production is elevated in p75 null Schwann cells ............................................69 4. P75 null nerves have few large-scale changes in lipid abundance .............................88 5. Comprehensive List of Reagents ................................................................................ 120 6. Lipid Compounds with Altered Abundance Recorded in Negative