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Functions of the Mineralocorticoid Receptor in the Hippocampus By Functions of the Mineralocorticoid Receptor in the Hippocampus by Aaron M. Rozeboom A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in The University of Michigan 2008 Doctoral Committee: Professor Audrey F. Seasholtz, Chair Professor Elizabeth A. Young Professor Ronald Jay Koenig Associate Professor Gary D. Hammer Assistant Professor Jorge A. Iniguez-Lluhi Acknowledgements There are more people than I can possibly name here that I need to thank who have helped me throughout the process of writing this thesis. The first and foremost person on this list is my mentor, Audrey Seasholtz. Between working in her laboratory as a research assistant and continuing my training as a graduate student, I spent 9 years in Audrey’s laboratory and it would be no exaggeration to say that almost everything I have learned regarding scientific research has come from her. Audrey’s boundless enthusiasm, great patience, and eager desire to teach students has made my time in her laboratory a richly rewarding experience. I cannot speak of Audrey’s laboratory without also including all the past and present members, many of whom were/are not just lab-mates but also good friends. I also need to thank all the members of my committee, an amazing group of people whose scientific prowess combined with their open-mindedness allowed me to explore a wide variety of interests while maintaining intense scientific rigor. Outside of Audrey’s laboratory, there have been many people in Ann Arbor without whom I would most assuredly have gone crazy. Former housemates, friends from neighboring labs, and friends I have made through these people, a network that has kept me sane and happy throughout graduate school. I also need to thank my family whose love and support throughout my life gave me the courage to go back to school and pursue my dreams. Last, but most certainly not least, I need to thank my wife, Lina. Her amazing love, encouragement, and patience made it possible for me to finish this thesis. ii Table of Contents Acknowledgements ii List of Figures v List of Tables vii Abstract viii Chapter 1 Introduction 1 . HPA axis activity: 6 . HPA axis activation and mood disorders: 10 . Corticosteroid Receptors: Mineralocorticoid Receptor (MR) and Glucocorticoid Receptor (GR) 14 . Mechanisms of Corticosteroid Receptor-Mediated Gene Regulation: 18 . Corticosteroid-Mediated Regulation of HPA axis Activity. 25 . MR and GR Regulation of HPA Axis Activity: Pharmacological 27 . Corticosteroid and MR/GR Regulation of Anxiety-Related Behaviors: Pharmacological 30 . Genetic Manipulations of MR and GR: Effects on HPA axis and Anxiety 33 . Corticosteroids and Hippocampal Structure and Function 38 . Thesis Summary: 43 Chapter 2 Mineralocorticoid Receptor Overexpression in Forebrain Decreases Anxiety-Like Behavior and Alters the Stress Response in Mice 47 . Introduction: 47 . Materials and Methods: 50 . Results 58 . Discussion: 73 . Appendix 1 The Effects of Forebrain Overexpression of MR on Neurogenesis 79 . Appendix 2 The effects of overexpressionof MR in the forebrain on learning and memory 94 Chapter 3 Role of mineralocorticoid receptor in protection from glucocorticoid endangerment of HT-22 hippocampal cells 104 . Introduction: 104 . Materials and Methods: 107 . Results: 111 . Discussion: 120 iii Chapter 4 Comparison of corticosteroid-responsive genes in mouse hippocampal cell lines that either express the glucocorticoid receptor alone or both the glucocorticoid and the mineralocorticoid receptor 125 . Introduction: 125 . Materials and Methods: 129 . Results: 133 . Discussion: 166 Chapter 5 Conclusions and future directions 172 Bibliography 184 iv List of Figures Figure 1-1 Overview of the HPA axis, including principal classes of regulatory afferents and corticosteroid actions. 9 Figure 1-2 Disturbed feedback in patients with major depressive disorder. 13 Figure 1-3 Coronal sections of mouse forebrain depicting anatomical distribution of GR and MR mRNA. 17 Figure 1-4 Different mechanisms of transcriptional regulation of corticosteroid responsive genes by MR and GR. 20 Figure 1-5 Schematic of HPA axis including sites of negative feedback provided by GR and MR. 26 Figure 2-1 Transgene construct and properties of Flag-MR. 59 Figure 2-2 Forebrain specific transgene expression in MRov mice. 61 Figure 2-3 Transgene expression is not found in the hypothalamus of MRov mice. 62 Figure 2-4 Increased expression of MR mRNA in hippocampus of MRov mice. 64 Figure 2-5 Normal basal HPA axis activity but sexually dimorphic suppression of corticosterone release in response to restraint stress. 66 Figure 2-6 Reduced anxiety-like behavior in forebrain MRov mice. 68 Figure 2-7 Assessment of anxiety-like behavior on EPM in line 709 MRov female and male mice. 70 Figure 2-8 Altered expression of GR and 5HT1a in MRov mice. 71 Figure 2-9 Immunohistochemistry for BrdU in the dentate gyrus of the hippocampus. 86 Figure 2-10 Immunohistochemistry for Ki67 labeled cells in the dentate gyrus of the hippocampus. 88 Figure 2-11 Estimated volumes of the dorsal hippocampus. 89 Figure 2-12 MRov mice are not impaired in the acquisition phase of the Morris water maze. 97 Figure 2-13 Spatial memory is not altered in MRov mice 99 Figure 2-14 There are no significant changes in long-term memory in MRov mice 100 Figure 3-1 HT-22 Parent and HT-22/MR clones express either GR alone or MR and GR in different ratios. 112 Figure 3-2 HT-22 Parent, HT-22/MR16, and HT-22/MR24 clones exhibit different transactivation profiles in response to different doses of corticosterone. 114 Figure 3-3 Receptor specificity of corticosteroid transactivation properties. 116 Figure 3-4 Glucocorticoid exacerbation of glutamate toxicity is attenuated in Clone HT-22/MR16. 119 Figure 3-5 Corticosterone pre-treatment enhances α-Fodrin cleavage in HT-22 Parent but not HT-22/MR16 cells. 121 Figure 4-1 Array integrity is equivalent between samples. 134 Figure 4-2 Gene Ontology of genes regulated by 100nm corticosterone in HT-22 Parent cells. 144 v Figure 4-3 Gene Ontology of genes regulated by 1nm corticosterone in HT-22/MR16 cells. 147 Figure 4-4 Gene Ontology of genes regulated by 100nm corticosterone in HT-22 MR16 cells. 160 Figure 4-5 Venn diagram representing the overlap between cell treatment groups of genes that are regulated by corticosterone. 161 Figure 4-6 Quantitative RT-PCR confirmations of microarray data 165 vi List of Tables Table 1-1 Hypothalamo-pituitary-adrenocortical (HPA), adrenomedullary hormonal system (AHS), and sympathetic nervous system (SNS) responses to different stressors 4 Table 1-2 Stimuli triggering “reactive” vs. “anticipatory” HPA stress responses 7 Table 1-3 HPA system dysregulation and behavioral symptoms in mice with targeted mutations of GR and MR 34 Table 2-1 HPA axis gene expression profiles 72 Table 4-1 Corticosterone responsive genes of HT-22 Parent cells activated or repressed following GR induction. 136 Table 4-2 Ten genes most highly induced or repressed in response to corticosterone in HT-22 Parent and HT-22/MR16 cells. 142 Table 4-3 Corticosterone responsive genes activated or repressed following MR induction in HT-22/MR16 cells treated with 1nM corticosterone. 146 Table 4-4 Corticosterone responsive genes activated or repressed following MR and GR induction with 100nM corticosterone in HT-22/MR16 cells. 149 Table 4-5 Corticosterone-responsive genes per group from venn diagram in Figure 5. 162 vii Abstract Functions of the Mineralocorticoid Receptor in the Hippocampus by Aaron M. Rozeboom Chair: Audrey F. Seasholtz In the central nervous system, glucocorticoids influence neuroendocrine function, cognition, neurogenesis, neurodegeneration, and cell survival. Glucocorticoid hormones signal through the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), closely related members of the steroid hormone receptor superfamily. Their expression profiles and modes of action suggest both overlapping and distinct functions in mediating glucocorticoid effects. As GR function has been widely examined, research in this thesis focuses on the roles of MR action in the central nervous system using both in vivo and in vitro approaches. First, we generated a transgenic mouse model that overexpresses MR in the forebrain (MRov). Relative to wild-type littermate controls, MRov mice display reduced anxiety-like behaviors and exhibit suppressed HPA axis activity in response to stress. These data demonstrate that functions of forebrain MR can both overlap (regulation of neuroendocrine function) and oppose (modulation of anxiety-like behavior) viii GR-mediated actions. Second, we utilized the mouse hippocampal cell line, HT-22, to address corticosteroid receptor-mediated effects on both cell survival and regulation of transcription in an in vitro system. HT-22 cells express GR but not MR, and have been shown to be sensitive to glutamate toxicity in a manner that is exacerbated by activation of GR. To address the role of MR in this “glucocorticoid endangerment”, we generated stable clones of HT-22 cells that express MR in addition to GR. Using these cell lines, we confirmed that while GR activation enhanced glutamate toxicity, the co-activation of MR and GR in the HT-22/MR clone attenuated the glucocorticoid endangerment of the cells. Finally, MR- and GR-mediated regulation of glucocorticoid responsive genes was monitored at the transcriptome level in HT-22/Parent and HT-22/MR cells. This research demonstrated that co-activation of MR and GR resulted in the regulation of a substantially larger set of genes relative to GR activation alone, including classes of genes known to regulate cell survival and proliferation, suggesting that changes in the balance of receptor levels may result in functionally significant alterations in global transcriptome regulation. Together, these data reveal important overlapping and distinct roles for hippocampal MR and GR. ix Chapter 1 Introduction When an organism is confronted with a situation that is perceived as threatening, a myriad of events occur that prepare the organism for a response that is typically characterized as “fight-or-flight”.
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