Glucocorticoid Regulation of the G-Protein Coupled Estrogen Receptor (GPER) in Mouse Hippocampal Neurons by Kate Colleen Eliza Nicholson A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Biomedical Sciences Guelph, Ontario, Canada © Kate Colleen Eliza Nicholson, August, 2019 ABSTRACT GLUCOCORTICOID REGULATION OF THE G-PROTEIN COUPLED ESTROGEN RECEPTOR (GPER) IN MOUSE HIPPOCAMPAL NEURONS Kate Colleen Eliza Nicholson Advisor: University of Guelph, 2019 Dr. Neil J. MacLusky The most prevalent estrogen, 17β-estradiol, binds the non-classical G-protein coupled estrogen receptor (GPER) with high affinity resulting in rapid activation of the c- jun N terminal kinase (JNK) pathway. GPER activation mediates some of the rapid neurotrophic and memory-enhancing effects of 17β-estradiol in the female hippocampus. However, exposure to stressful stimuli may impair these beneficial effects. This thesis characterizes neurosteroid receptor expression in murine-derived mHippoE cell lines that are subsequently used to investigate the glucocorticoid regulation of GPER protein expression and functional activation. This thesis demonstrates that 24-hour treatment with a glucocorticoid receptor agonist reduces GPER protein expression and activation of JNK in female-derived mHippoE-14s. Using an in vivo model, treatment with glucocorticoids significantly reduces hippocampal activation of JNK in female ovariectomized CD1 mice. Collectively, this thesis uses in vitro and in vivo models to characterize glucocorticoid regulation of GPER expression and signalling in female murine hippocampal neurons. ACKNOWLEDGEMENTS To Dr. MacLusky: I would like to thank you for inspiring my passion for science and pursuit of knowledge. Over the past 2 years, you have provided me with countless opportunities to grow as a young researcher and I am tremendously grateful for this. Your ongoing support, guidance, and encouragement has given me the confidence to share my own thoughts, questions, and opinions with other researchers. You have challenged me to improve my scientific communication and critical thinking skills while inspiring enthusiasm for the implications of our research. This opportunity has been uniquely rewarding and I am so very grateful for your dedication as a mentor. To my advisory committee members, Dr. Bettina Kalisch and Dr. Jon LaMarre: I would like to thank you for your guidance, support, and insight throughout the course of my MSc degree. I feel very fortunate to have been able to approach both of you for friendly advice and opinions in shaping the design and interpretations of this project. To my MacLusky lab parents, Dr. Ari Mendell and Dr. Carolyn Creighton: I would like to sincerely thank you for your ongoing patience in training me while sharing your wisdom and mentorship throughout graduate school. Carolyn, I am very grateful for your kindness while introducing me to the foundations of lab work and research. Ari, thank you for supporting and challenging me to grow as a graduate student. You have always been the calm in the storm and I am incredibly grateful for your leadership and friendship throughout graduate school. I feel very fortunate to have worked alongside inspiring mentors and friends like you two. To my MacLusky lab siblings: E-Marti and Lo-dawg, thank you for being my rocks through it all. Thank you for making fun of me when I deserve it and putting up with me when I don’t. Hayl-storm, Dry Rice Lawton, and E-Craig, thank you for all of the lab chats, support, and humour. To all other MacLusky lab members, thank you for making the long lab days seem shorter. To the faculty and students of the Department of Biomedical Sciences: You have provided me with many rewarding opportunities to grow in and outside of the lab and classrooms. Thank you for making this a welcoming, enriching, and collaborative environment to learn in. Finally, thank you to my family for all of the love, long phone calls, and support while cheering me on throughout the ups and downs of graduate school. Thank you for always reminding me to look at the bigger picture. To my friends, thank you for keeping me grounded with your love, encouragement, humour, and kindness. iii DECLARATION OF WORK PERFORMED I declare that I have performed and completed all of the work presented within this Master of Science thesis, with the exception of the following: All ovariectomy surgical procedures, subcutaneous drug injections, cervical dislocations, and brain extractions in Chapter 5 were performed by Emily Martin (Master of Science candidate supervised by Dr. Neil MacLusky and Dr. Elena Choleris, Department of Psychology, University of Guelph). All Sanger sequencing was performed by the University of Guelph’s Advanced Analysis Centre Genomics facility. iv TABLE OF CONTENTS ABSTRACT ................................................................................................................................ ii ACKNOWLEDGEMENTS .......................................................................................................... iii DECLARATION OF WORK PERFORMED ............................................................................... iv LIST OF TABLES .................................................................................................................... viii LIST OF FIGURES .................................................................................................................... ix LIST OF ABBREVIATIONS ....................................................................................................... xi LIST OF APPENDICES ........................................................................................................... xiv CHAPTER 1: INTRODUCTION AND REVIEW OF THE LITERATURE ..................................... 1 Sex Differences in Neuropsychiatric and Neurodegenerative Disorders ............................ 2 The hippocampus………………………………………………………………………………..……...4 The Hypothalamic-Pituitary-Gonadal (HPG) Axis .................................................................. 6 Steroid and Estradiol Biosynthesis........………………...………………………………………….8 Steroid Receptors ...................................................................................................................13 Estrogen Receptors in the Hippocampus .............................................................................14 Functional Effects of Hippocampal Estrogens and GPER Activation .................................20 Stress and the Hypothalamic-Pituitary-Adrenal (HPA) Axis…………………………………...28 Glucocorticoid Biosynthesis ..................................................................................................30 Glucocorticoid Receptor Activation in the Hippocampus ....................................................31 Functional Effects of GR Activation in the Hippocampus ....................................................31 Stress and Estrogen Interactions in the Hippocampus ........................................................34 Rationale ..................................................................................................................................36 Hypothesis and Objectives ....................................................................................................37 CHAPTER 2: CHARACTERIZATION OF RECEPTOR MRNA EXPRESSION IN MHIPPOE-14 AND HIPPOE-18 IMMORTALIZED HIPPOCAMPAL NEURONS .............................................39 Introduction .............................................................................................................................40 Experimental Procedures .......................................................................................................47 Cell Culture Conditions ......................................................................................................47 RNA Isolation .....................................................................................................................48 Reverse Transcription and cDNA Synthesis .......................................................................48 Conventional Polymerase Chain Reactions and Visualization……………………..…………49 Results .....................................................................................................................................53 Discussion ...............................................................................................................................57 v Conclusion ..............................................................................................................................63 CHAPTER 3: GLUCOCORTICOID REGULATION OF GPER PROTEIN STEADY STATE LEVELS IN MHIPPOE-14 AND MHIPPOE-18 IMMORTALIZED HIPPOCAMPAL NEURONS 65 Introduction .............................................................................................................................66 Experimental Procedures .......................................................................................................68 Chemical and Stock Solutions ............................................................................................68 Cell Culture Conditions ......................................................................................................68 Treatment and Protein Collection .......................................................................................69 Western Blotting.................................................................................................................70