THE PRODUCTION AND LOCALIZATION OF LUTEINIZING HORMONE IN THE BRAIN A thesis submitted to the Kent State University Honors College in partial fulfillment of the requirements for University Honors by Ya’el Courtney May, 2019 Thesis written by Ya’el Courtney Approved by _____________________________________________________________________, Advisor ____________________________ _________, Chair, Department of Biological Sciences Accepted by ___________________________________________________, Dean, Honors College ii TABLE OF CONTENTS LIST OF FIGURES AND TABLES …..……………………….………………………..iv ABBREVIATIONS………..…….……………………………………………….………v ACKNOWLEDGMENTS……………………………………………...……………..vi-vii CHAPTER I. INTRODUCTION……………………………………………….………1 II. METHODS…………..………………………………………….….……16 III. RESULTS………………………………………………………………..26 IV. DISCUSSION………………………………………….…….…………..39 REFERENCES……………………………………………………………...…………...47 iii LIST OF FIGURES AND TABLES Figure 1. Normal and Dysfunctional HPG Axis Feedback Mechanism…………...………..4 Figure 2. Single Cell RNA Sequencing Data Processing Pipeline…………….…………..21 Figure 3. Single Cell RNA Sequencing Quality Assurance Metrics…………..….……22-23 Figure 4. LHβ Probe Validation in Rat Pituitary………………………..………….……...27 Figure 5. LHβ In Situ Hybridization in Cortex………….………….……….…….……….29 Figure 6. LHβ In Situ Hybridization in the Hippocampal Formation……………………..30 Figure 7. LHβ In Situ Hybridization in the Hypothalamus………………………………..31 Figure 8. LHβ In Situ Hybridization in the Amygdala………………………….…………33 Figure 9. Sex Differences in LHβ RNA Expression……………………………….………35 Figure 10. SHAM vs. OVX Differences in LHβ RNA Expression…………………..……37 Table 1. LHβ TPM in Cortex Cells..……………...……………………………….……….29 Table 2. LHβ TPM in Hippocampal Cells…………………………………………………30 Table 3. LHβ TPM in Hypothalamic Cells………………………...………………………31 Table 4. LHβ TPM in Amygdalar Cells…………………………...………………………33 Table 5. Wilcoxon Signed Ranks Test for M v. F LH………………………..……………35 Table 6. Wilcoxon Signed Ranks Test for SHAM v. OVX LH…………………………...37 Table 7. LHβ TPM in Non-Neuronal Cells…………………………………………..……38 iv ABBREVIATIONS Abbreviation Meaning HPG Hypothalamic-pituitary-gonadal GnRH Gonadotropin-releasing hormone LH Luteinizing hormone FSH Follicle-stimulating hormone hCG Human chorionic gonadotropin GnRHR Gonadotropin-releasing hormone receptor LHR, LHCGR Luteinizing hormone and human chorionic gonadotropin receptor AD Alzheimer’s disease HRT Hormone replacement therapy CGA Glycoprotein hormone alpha polypeptide LHB Luteinizing hormone, beta subunit OVX Ovariectomy HCR-FISH Hybridization chain reaction fluorescence in situ hybridization scRNAseq Single-cell RNA sequencing CTX Cortex HPF Hippocampal Formation HYP Hypothalamus AMYG Amygdala IT Intratelencephalic PT Pyramidal Tract GLU Glutamatergic GABA Gamma-aminobutyric acid PVN Paraventricular nucleus RSC Retrosplenial cortex CNS Central nervous system CSF Cerebrospinal fluid v ACKNOWLEDGMENTS With my deepest gratitude, I owe my thanks to everyone who has guided me through my undergraduate scientific career and enabled me to reach my goals. First, I am immensely thankful for my thesis advisor, Dr. Gemma Casadesus. She took me as an undergraduate in her lab when I possessed zero wet-lab skills and has fostered my growth as a scientist through her continuous encouragement, honesty, and unrelenting standards for robust science. I would particularly like to thank Dr. Casadesus for the trust she placed in me and the freedom she gave me to explore novel methods as we seek to understand Luteinizing Hormone in a deeper way. My time in her lab has refined my skills, both personal and scientific, in ways that will benefit me greatly throughout my pursuit of my Ph.D. I also thank the members of my thesis committee for their time and counsel: Dr. Timothy Meyers, Dr. Wilson Chung, and Dr. Joel Hughes. I would like to thank Megan Mey, a graduate student in the Casadesus lab, for her mentorship and constant sunny encouragement. She taught me wet-lab techniques from square one with unwavering patience and kindness and sacrifices her time on many occasions to help me. I thank Sabina Bhatta for sharing her knowledge and insightful questions throughout the process. I also thank the other members of the Casadesus lab, Rachel, John, and Spencer for welcoming me into the lab community and helping me with my countless inane questions. I also owe my thanks to mentors outside of the Casadesus lab who opened for me the door into a world of science. I thank my first undergraduate mentor, Dr. Joel Hughes, vi and his cardiovascular psychophysiology lab for taking a chance on a freshman and enabling me to get my first summer research experience at Washington University in St. Louis. I thank the BP-ENDURE program, Dr. Diana Jose-Edwards, and Dr. Erik Herzog for mentoring me through two summers of research and teaching me the basics of scientific communication. I thank Dr. Todd Braver, Dr. Joset Etzel, and Debbie Yee and the Cognitive Psychopathology Lab at WashU for teaching me how to learn challenging and frustrating new skills, and when to ask for help. I thank Dr. Sara Newman and Dr. Josh Pollock and the Electrophysiological Neuroscience Lab at Kent State for letting me lead the development of my own experiment and for facilitating and understanding the evolution of my scientific interests. I thank the Broad Institute Summer Program, especially Dr. Bruce Birren and Francie Latour, for unabashedly helping me dive into my weaknesses as a person and as a scientist and seek a growth mindset. I thank Dr. Beth Stevens and Dr. Matthew Johnson at Harvard for their mentorship over the summer of 2018, and for their advocacy in my graduate school applications. Each of these mentors has been an indispensable piece in the puzzle of experiences that allowed me to achieve my dream of admission to a top-tier neurobiology Ph.D. program. I thank BP-ENDURE, SACNAS, ABRCMS, and Kent State University for their monetary support of conference travel. It is through these avenues that I have been able to accrue experience presenting my research at an international level, and I have not taken these opportunities for granted. vii My trajectory into, through, and out of Kent State has been unconventional and uniquely challenging. Strong enough words do not exist to convey my gratitude for those named and unnamed who have supported me in every imaginable way. I am thankful for my grandma, Linda Powlison, and her unconditional open arms and open ears. I am thankful for my boss at Bellacino’s of Stow, Dave Segen, who has been supportive and flexible when I take time off for summer research, for conferences, and for graduate school interviews. I am thankful for the customers I serve and bartend for, who ask me about my science and let me re-kindle my excitement for my pursuits with every explanation. I am consistently in awe that science is a real career, and that I can get paid to think about questions that are boundlessly intriguing and exciting. I am thankful for roommates who, throughout the years, have made my home environment a safe, relaxing, and accepting space. Lastly, I thank all the scientists who have surrounded me in each research experience I’ve had. I thank those who have taken time to answer my questions, to encourage me, to inspire me. I have seen the value in a diverse body of scientists and learned that collaboration will foster better science than competition ever will. These are lessons I will hold in my heart for the rest of my life and implement at every turn in my career. viii 1 Chapter 1: Introduction The Importance of Studying Age-Related Cognitive Decline Over the last 200 years, the world has achieved impressive progress in health that has led to dramatic increases in life expectancy. Since 1900 the global average life expectancy has more than doubled and is now approaching 70 years, and in some countries is as high as 85-90 years. Although this increasing life expectancy generally reflects positive human development, it brings new challenges. These challenges stem from the fact that growing older is still inherently associated with biological and cognitive degeneration, although the progression of cognitive decline, physical frailty, and psychological impairment varies between individuals. Degenerative aging processes underlie a host of diseases including cancer, ischemic heart disease, type 2 diabetes, Alzheimer's disease, and others (Atwood & Bowen, 2011; Prasad, Sung, & Aggarwal, 2012). Mental health deterioration due to chronic neurodegenerative diseases represents the largest cause of disability in the world. There are well documented, common patterns of negative effects of aging in the brain. These especially relate to learning and memory that are regulated by brain regions that comprise the memory portion of the limbic system (Rolls, 2015). These areas include the cingulate, entorhinal, and parahippocampal cortices as well as the hippocampal formation. Generally, visuospatial capabilities, psychomotor speed, and general intelligence decrease with age 2 (Kolanowski et al., 2017; Li et al., 2011; Lindeboom & Weinstein, 2004; Martin, Wittert, & Burns, 2007; Salthouse, 1996; Shock, 1984). Spatial memory is also impaired with age, especially the ability to form a cognitive map. This ability is highly dependent on the hippocampus, indicating that hippocampal function decreases with age (Bryan et al., 2010; Jeffery, 2018; Packard & McGaugh, 1996; Ziegler & Thornton, 2010b). Human aging is a complex process with multiple driving factors. Many