Functions of the Mineralocorticoid Receptor in the Hippocampus by Aaron M. Rozeboom A ...

206 Pages·2008·1.34 MB·English

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.
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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

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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

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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

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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

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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)
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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.

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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”. Physiological manifestations of this fight-or-flight
response that are common to all mammals include but are not limited to, increased heart
rate, increased respiration, and increased vigilance. The body also mobilizes energy
sources and redirects the use of that energy to areas that most need it. These efforts are
energetically costly but serve one very important purpose, the maintenance of an internal
equilibrium that allows the organism to survive in a constantly changing environment.
This concept was formally proposed by Claude Bernard who noted in his 1865
work, Introduction to Experimental Medicine, “Constance of the internal environment is
the condition for a free and independent life” (Bernard 1865). It was over half a century
later, however, before Walter Cannon used the term “homeostasis” to describe the
“fundamental condition of stability” of an organism, and the ability of “various
physiological arrangements which serve to restore the normal state when it has been
disturbed” (Cannon 1932). Cannon was the first to describe the acute changes in
secretions from the adrenal gland that were associated with what he called the fight-or-
flight response. He found that any number of a variety of threats to homeostasis, such as
hypoglycemia, cold exposure, or emotional distress resulted in the activation of both the
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