The Identification of Transcriptional Signatures of Steroid Response in Human Neurons and Microrna Networks As a Contributor to Reproductive Steroid Pathology
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THE IDENTIFICATION OF TRANSCRIPTIONAL SIGNATURES OF STEROID RESPONSE IN HUMAN NEURONS AND MICRORNA NETWORKS AS A CONTRIBUTOR TO REPRODUCTIVE STEROID PATHOLOGY A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Global Infectious Diseases By Allison Courtney Goff, M.A. Washington, DC April 16, 2021 Copyright 2020 by Allison Courtney Goff All Rights Reserved ii THE IDENTIFICATION OF TRANSCRIPTIONAL SIGNATURES OF STEROID RESPONSE IN HUMAN NEURONS AND MICRORNA NETWORKS AS CONTRIBUTORS TO REPRODUCTIVE STEROID PATHOLOGY Allison Courtney Goff, M.A. Thesis Advisors: David Goldman, M.D. and Peter J. Schmidt, M.D. ABSTRACT Ovarian steroids change the brain in utero, during puberty, and in adulthood. At the neuronal level, ovarian steroids affect structure, gene networks, and physiological outputs, and at the neurocircuitry level they modulate working memory, reward, and emotion. In reproductive related affective disorders such as premenstrual dysphoric disorder (PMDD), alterations in mood and affect are linked to changes in ovarian steroid levels. However, the pathophysiology of these disorders is just beginning to be characterized. For PMDD, previous work demonstrated intrinsic differences in the ESC/E(Z) complex, a major epigenetic modifier. However, this work showed a discrepancy between transcripts and protein: while transcripts in this complex tend to be upregulated in PMDD, protein quantities were downregulated. The work in this thesis builds directly on this knowledge by investigating the role of microRNAs, a unique class of regulatory molecules that dynamically regulate gene networks and are capable of resolving seemingly paradoxical relationships between transcript and protein levels. Congruently, several of the leading microRNA gene networks that were identified by this objective targeted ESC/E(Z) transcripts. This thesis also uncovered evidence for a key role of the Vascular Epithelial Growth Factor A gene (VEGFA) in PMDD and began the foundational work of identifying transcriptional signatures of estradiol, progesterone, and allopregnanolone on human iii neuronal cells across maturation stages, which can be used as markers for typical neuronal response to sex steroids. iv “None of it was real; nothing was real. Everything was real; inconceivably real, infinitely dear. These and all things started as nothing, latent within a vast energy broth, but then we named them, and, in this way, brought them forth.” ~George Saunders “And there would be quite a number of things to chat about: around you there is a wild people, provoking one’s curiosity, there is danger every day, extraordinary incidents happen; and no wonder there is occasion to regret that so few of us take notes.” ~Mikhail Lermontov “The only way to guarantee an outcome is to do nothing.” ~Mark Goff The research and writing of this thesis are dedicated to my family, my friends, and everyone else who helped make me who I am today. Many thanks, Allison Goff v TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ........................................................................................1 1.0 Sex Steroid Hormone Synthesis and Action ..............................................................1 1.1 Physiology of the Ovarian Cycle .............................................................................. 4 1.2 Sex Steroid Hormone Effects on the Developing Brain ............................................5 1.3 Sex Steroid Hormone Effects on the Adult Brain ......................................................8 1.4 Sex Steroid Hormone Effects on Mood and Behavior.............................................10 1.5 Pathophysiology of Premenstrual Dysphoric Disorder ............................................12 1.6 Cellular Differences in PMDD ................................................................................14 1.7 MicroRNAs ..............................................................................................................16 1.8 Lymphoblastoid Cell Lines ......................................................................................18 1.9 Human Neuronal Cell Lines ....................................................................................20 1.10 Thesis Focus...........................................................................................................21 1.11 Figures....................................................................................................................23 CHAPTER 2: GENERAL METHODS FOR ALL CHAPTERS ......................................27 2.0 Differential Gene Expression Analysis ....................................................................27 2.1 Pathway Analyses ....................................................................................................27 CHAPTER 3: TRANSCRIPTIONAL EFFECTS OF SEX STEROID HORMONES IN HUMAN FEMALE NEURONAL CELLS AT DIFFERENT STATES OF CELLULAR MATURATION .................................................................................................................29 3.0 Introduction ..............................................................................................................29 3.1 Specific Methods .....................................................................................................33 3.2 Results ......................................................................................................................39 vi 3.3 Discussion ................................................................................................................43 3.4 Tables and Figures ...................................................................................................50 CHAPTER 4: MICRORNAS IN PREMENSTRUAL DYSPHORIC DISORDER AND THE ROLE OF VASCULAR ENDOTHELIAL GROWTH FACTOR ...........................62 4.0 Introduction ..............................................................................................................62 4.1 Specific Methods .....................................................................................................64 4.2 Results ......................................................................................................................69 4.3 Discussion ................................................................................................................71 4.4 Tables and Figures ...................................................................................................78 CHAPTER 5: CONCLUDING DISCUSSION .................................................................92 5.0 Additional Thoughts on Chapter 3 ...........................................................................93 5.1 Long-Term Use of h-iPSCs in PMDD Research .....................................................94 5.2 Additional Thoughts on Chapter 4 ...........................................................................95 5.3 Future Work Involving the Results of Both of the Presented Studies .....................96 5.4 Final Summary .........................................................................................................96 BIBLIOGRAPHY ..............................................................................................................98 vii LIST OF FIGURES Figure 1.1: Sex steroid hormone synthesis pathway .....................................................23 Figure 1.2: Recurrence of symptoms of premenstrual syndrome during the addition of estradiol or progesterone to the Leuprolide regimen ....................................................24 Figure 1.3: ESC/E(Z) transcript/protein quantity disparity in PMDD LCLs ................25 Figure 1.4: microRNA biosynthesis in mammals ..........................................................26 Figure 3.1: Principal component analyses show distinctions between cell lines, subtle responses to sex steroid hormone .................................................................................51 Figure 3.2: Most expressed genes were expressed across NSC, UL, SH, and DL cell lines ...............................................................................................................................53 Figure 3.3: Top neuron-related pathways are present in genes that are shared between NSC, SH cells and NSC, SH and DL cells ...................................................................54 Figure 3.4: UL and SH cells share the highest number of upregulated and downregulated ALLO-responsive genes (FDR<0.1) .....................................................56 Figure 3.5: UL and SH cells share the highest number and most significant upregulated and downregulated ALLO-responsive genes (p<0.05) ..................................................58 Figure 3.6: UL and SH cells share the highest number and most significant upregulated and downregulated E2-responsive genes (p<0.05) ........................................................60 Figure 3.7: Few P4-responsive (p<0.05) genes shared across NSC, UL, SH, and DL cell lines .........................................................................................................................61 Figure 4.1: miRNA WGCNA modules are enriched for ESC/E(Z)-associated transcript targets .............................................................................................................................81 Figure 4.2: Differentially expressed miRs in PMDD and mRNA targets in PMDD vs control LCLs .................................................................................................85