Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2014 The Molecular Components of Estrogen Receptor Beta (ERβ) Signaling in Neuronal Sytems Natasha Mott Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Molecular and Cellular Neuroscience Commons Recommended Citation Mott, Natasha, "The Molecular Components of Estrogen Receptor Beta (ERβ) Signaling in Neuronal Sytems" (2014). Dissertations. 906. https://ecommons.luc.edu/luc_diss/906 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2014 Natasha Mott LOYOLA UNIVERSITY CHICAGO THE MOLECULAR COMPONENTS OF ESTROGEN RECEPTOR BETA (ERβ) SIGNALING IN NEURONAL SYTEMS A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN INTEGRATIVE CELL BIOLOGY BY NATASHA N. MOTT CHICAGO, IL MAY 2014 ACKNOWLEDGEMENTS To my advisor/mentor/friend/confidant, Dr. Toni Pak: I could not have asked for a better mentor. You have given me all the tools that I needed to succeed in grad school and hopefully beyond. I’ve experienced first-hand how your advice and guidance has gone far beyond expectations of a graduate mentor. I look forward to the future, where I imagine we will sit together and relive great memories, discuss experiments and debate our craziest vortex-of-insanity ideas over a martini – or two. There aren’t enough pages in this document for me to thank you for all of the insight, laughs and motivation you have given me. To my dissertation committee: Dr. Clodia Osipo, Dr. Nancy Zeleznik-Le, Dr. Wendy Kartje, and of course, our prized male member, Dr. James Roberts: Thank you all for having faith in me and most of all for pushing me to the pinnacle of my intellect, for this has shown me that I’m capable of more than I thought. Each meeting, I felt more and more that you all were the perfect committee: Giving me valuable recommendations, reminding me what I need to do to succeed and most importantly – never letting me off too easily! The only problem with being the best possible combination of people for a committee is that I will certainly call on you in iii the future for advice, letters and to read my grant proposals. I am beyond grateful for the support and guidance I have received from each of you. To my lab mates, past and present: You all are the best and brightest and more importantly, some of my very best friends. The amazing dynamic of our group could never be replicated. Finally, thank you to the ICB program, and the Physiology department: Dr. Le, thank you for convincing the rest of the CBNA admissions committee that I was a candidate worthy of acceptance into the program! Dr. DeTombe, thank you for allowing me to use your playground of equipment. Kay and Joe, thank you for your help with things I couldn’t figure out on my own. Last and certainly not least, thank you to Kim Stubbs for every single thing you’ve done to help me, as an administrator, but moreso as my dear friend. iv To Ken, Nova, Mom, Dad, FAS, Deanna, Pearl and TJ v Science is a way of thinking more than it is a body of work. Carl Sagan vi TABLE OF CONTENTS AKNOWLEGDEMENTS iii LIST OF TABLES x LIST OF FIGURES xii LIST OF ABBREVIATIONS xvi ABSTRACT xviii CHAPTER I: STATEMENT OF THE PROBLEM 1 CHAPTER II: INTRODUCTION - ESTROGEN SIGNALING AND THE AGING BRAIN: CONTEXT-DEPENDENT CONSIDERATIONS FOR POSTMENOPAUSAL HORMONE THERAPY (MOTT, NN ET. AL., ISRN ENDOCRINOLOGY, 2013) 4 Literature Review 4 The menopausal transition: E2 decline and health concerns 6 Estrogen receptor signaling 8 Structural contributions to ER signaling 10 Expression of ERs in the brain: a complex story 14 ERβ alternative splice variants 19 Novel protein interactions for E2-mediated nuclear processes 23 HSPs and Chaperone proteins 23 Transcriptional proteins and ERs 24 Nuclear actin: setting the stage 27 Post-translational modifications of ERβ 31 Estrogens and cognition 33 Estrogens and mood regulation 35 Summary 39 Hypothesis and Specific Aims 40 CHAPTER III: C-TERMINAL-INDEPENDENT STRUCTURAL REQUIREMENTS FOR HUMAN ESTROGEN RECEPTOR BETA (ERβ) TRANSCRIPTIONAL REGULATION IN NEURONAL CELLS (MOTT NN. ET AL., J. NEUROENDOCRINOLOGY, 2012) 45 Introduction 45 Results 50 Discussion 68 vii CHAPTER IV: AGE ALTERS THE DYNAMICS OF ERβ PROTEIN (ESTROGEN SIGNALING (MOTT, NN ET. AL., MOL. CELL. PROTEOMICS, 2014) Introduction 74 Results 78 Discussion 122 CHAPTER V: FINAL DISCUSSION Summary 133 Key Findings (Table 3) 134 Final Thoughts 137 Menopause and the ERβ-dominated brain 137 Supplements to nuclear receptor signaling are relevant during menopause 138 ERβ, mood and cognition during menopause: proposed novel mechanisms involving neuroprotection and the stress response 142 Correlations between changes in ERβ:protein interactions and changes in neuroprotection around the time of menopause 146 Nuclear actin aids traditional and non-traditional ERβ interactions that are altered by E2 in the aged brain 152 Implications for ERβ in the periphery 155 Future directions 158 Take home message 162 CHAPTER VI: GENERAL METHODS Chapter III Human tissue 164 Primer sequences 164 Cell culture 165 Transient transfections 165 Reporter constructs 166 Luciferase assays 167 EMSA: Oligonucleotides 168 EMSA: Gel electrophoresis 169 Autoradiography and analysis 169 Site-directed mutagenesis 170 Chapter IV Animals 170 E2 Enzyme-linked immunoassay 171 2D Sample preparation 171 CyDye labeling 173 Isoelectric focusing and SDS-PAGE 173 Imaging and analysis 174 Spot analysis and statistics 174 Spot picking 175 In-gel digestion 176 viii Identification of proteins with LC-ESI-MS/MS 177 Western blotting 178 APPENDIX A: SUPPLEMENTARY DATA 180 REFERENCES 202 VITA 231 ix LIST OF TABLES Page Table 1. Summary of proteins interacting with ERβ in the ventral hippocampus and the relative change in association with ERβ after E2 treatment in young and aged animals 88 Table 2. Summary of proteins interacting with ERβ in the ventral hippocampus that are not significantly more or less associated ERβ after E2 treatment in young and aged animals 90 Table 3. Summary of major findings 128 Table 4. Epigenetic enzymes co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 184 Table 5. Transcriptional proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 185 Table 6. DNA replication and repair proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 186 Table 7. Other DNA binding proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 187 Table 8. RNA binding/translational proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 188 Table 9. Post-translational modifying protein co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 189 Table 10. Chaperone proteins co-immunoprecipitated with ERβin the ventral hippocampus after DTBP crosslinking 190 Table 11. Cell signaling proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 191 x Table 12. Kinases & Phosphatases co-immunoprecipitated with ERβ in the ventralhippocampus after DTBP crosslinking 192 Table 13. GTPases & related proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 193 Table 14. Cell cycle & Cell death related proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 194 Table 15. Scaffolding proteins co-immunoprecipitated with ERβ in the Ventral hippocampus after DTBP cross linking 195 Table 16. Membrane associated proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 196 Table 17. Metabolic proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 198 Table 18. Multifunctional proteins co-immunoprecipitated with ERβ in the ventral hippocampus after DTBP crosslinking 199 xi LIST OF FIGURES Figure Page 1. Representative image of domains within human and rat ERβ splice variants 13 2. Timeline showing factors affecting ER gene expression throughout the female life span 15 3. Age and hormonal milieu exponentially increases the potential diversity of estrogen receptor signaling leading to context dependent gene regulation 30 4. Schematic representation of specific human estrogen receptor (hERβ) splice variants 49 5. Expression of hERβ splice variants in human brain tissue 51 6. Ligand-independent DNA binding activity of human estrogen receptor hERβ1, hERβ2, hERβ4 and hERβ5 53 7. Apo-human estrogen receptor (hERβ) splice variants on estrogen response element (ERE)-mediated promoter activity 55 8. Apo-human estrogen receptor (hER)β splice variants on activator protein-1 (AP-1)-mediated promoter activity 57 9. Effects of 17β-estradiol (E2), 5α-androstane-3β, 17β-diol (3β-diol) and ICI 182 780 on human estrogen receptor (hERβ) splice variant-mediated estrogen response element (ERE) and activator protein-1(AP-1) promoter activity 60 10. Effects of 17β-estradiol (E2), 5α-androstane-3β, 17β-diol (3β-diol) and ICI 182 780 on human estrogen receptor (hERβ) splice variant-mediated arginine vasopressin (AVP) promoter activity before and after deletion of an activator protein-1 (AP-1) site 62 11. Phosphoinositide 3-kinase (PI3K) and p38 kinase inhibition on human estrogen receptor (hER)β1-mediated repression of activator protein-1 (AP-1) promoter activity 64 xii 12A. Phosphoinositide 3-kinase (PI3K) inhibition on human estrogen receptor (hERβ) splice variant-mediated repression of human AVP (hAVP) promoter activity 66 12B. p38 kinase inhibition on human estrogen receptor (hERβ) splice variant -mediated repression of human AVP (hAVP) promoter activity 67 13. Hormone treatment paradigm 79 14. Serum 17β-estradiol levels from young and aged animals following treatment paradigm 80 15.