Troy A. Roepke - US grants

[unreadable] DESCRIPTION (provided by applicant): The long range goal of the proposed research is to understand how estrogen affects energy homeostasis either through controlling potassium channel gene expression in hypothalamic neurons or by synergistically altering neuronal excitability with other neurotransmitters such as serotonin. Estrogens are anorectic leading to decreased food intake and body weight and are known to potentiate the anorectic effects of serotonergic drugs. One potential mechanism for the anorectic effects of estrogen and serotonin is inhibition of the M- current in POMC neurons. In order to determine if the M-current plays a role in these effects, changes in expression of KCNQ subunits (2, 3, & 5), which form the heteromultimeric channels responsible for the M- current in the brain, and their signaling modulators will be determined following estrogen treatment in the arcuate nucleus using qRT-PCR. Single cell RT-PCR will be used to determine the distribution pattern of KCNQ subunits and co-localization with cell type markers (POMC, NPY). The effects of estrogen pre- treatment on the electrophysiological properties of the M-current in arcuate neurons will be examined using whole cell patch recordings of POMC and NPY neurons from oil and estrogen-treated animals. Recordings will also determine any direct modulation of the M-current via a rapid response to estrogen through the putative estrogen membrane receptor using the M-current attenuation by a muscarinic receptor agonist and a selective 5HT2C receptor agonist. The inhibition of food intake by serotonin may involve modulation of the M-current through 5HT2C receptors. Agonist of the 5HT2C receptor will injected (icv) into ovariectomized mice treated with oil or estrogen. Weight and food intake will be measured for 48 hr after injection of a selective 5HT2CR agonist with or without pharmacological modulators of the M-current. If the modulation of the M current is important for the regulation of food intake by either serotonin or estrogen, the M-current activator should partially or completely attenuate the anorectic effects of 5HT and/or estrogen. Obesity is one of the major health issues facing the adolescents and adults in the developed world with few safe therapeutic drugs available to assist the population in maintaining a healthy weight. The mechanism through which peripheral and central signals control food intake must be examined to develop new strategies and therapies for one of the most serious health issues facing us today. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long range goal of this application is to understand the mechanisms by which estrogen and environmental estrogens affect hypothalamic functions such as energy homeostasis. There is an obesity epidemic in the US and understanding the etiology of any and all factors that contribute to expression of obesity is critical for treatment of this disorder. Therefore, the goal of the mentored projects is to understand how estradiol affects energy homeostasis through multiple membrane-initiated mechanisms that include the control of neuronal excitability through an estradiol-responsive membrane GPCR (mER) and control of gene expression in arcuate neurons through both ER?- and mER-mediated mechanisms. Estradiol is known to control energy homeostasis through ER? and mER because STX, a selective ligand for the mER, attenuates body weight gain post-ovariectomy. Estradiol also alters neuronal excitability of POMC arcuate neurons through the mER. One potential mER-mediated mechanism for the effects of estradiol is modulation of a non-inactivating, sub-threshold K+ current that tempers the excitability of POMC (M-current). To determine if modulation of the M-current by estradiol plays a role in these effects, I will first measure the expression and activity of the KCNQ/M-current in POMC and NPY neurons from oil- and estradiol-treated females using qRT-PCR in pooled single cells and electrophysiology. Second, I will measure the effects of acute (via mER) estradiol treatment on the electrophysiological properties of the M-current in POMC and NPY neurons using whole cell patch recordings. The independent phase will build on the techniques learned during the mentored phase and examine the role of membrane-initiated and ERE-independent estrogen signaling in hypothalamic functions and determine whether environmental estrogens activate these same pathways in their effects. The first aim will determine the role of ERE-independent signaling in the control of energy homeostasis and hypothalamic gene regulation by estradiol and bisphenol A using wild-type, ?ERKO and ERalpha KI/KO mice models. The final aim will determine the electrophysiological effects of bisphenol A on arcuate POMC and NPY neurons that control energy homeostasis. PUBLIC HEALTH RELEVANCE: The mentored phase may directly impact the health of women (esp. post-menopausal women) since STX, the selective ligand for the membrane estrogen receptor, is a potential lead compound for an non-traditional hormone replacement therapy and treatment of obsesity disorders. Finally, the independent phase will address the potential mechanisms that environmental estrogens can impact hypothalamic functions.

The neuronal circuits involved in energy homeostasis are being extensively explored; however, the intrinsic mechanisms underlying the regulation of these neurons are just beginning to be elucidated and will be critical for understanding disorders associated with energy homeostasis (i.e., obesity, anorexia, cachexia, etc.). These neuronal circuits are modulated by many different peripheral hormones including 17?-estradiol (E2). E2, which varies during the menstrual cycle, is anorectic leading to decreased food intake and body weight. E2 can alter homeostatic functions by activating ER? and novel G-protein coupled membrane estrogen receptors (GqmER) to alter the expression and activity of cation channels that control neuronal excitability. Hence, an emerging and significant field in neuroendocrinology within the past decade has been the convergence of membrane-initiated steroid signaling and physiological effects. The membrane-initiated events utilized by E2 involve activation of a host of known pathways that control neuronal excitability, gene expression and cellular functions. A novel transgenic strain of mice (ER? Ki/Ko) lack a functional DNA binding domain on the ER? protein and, thus exhibit no Estrogen Response Element-mediated transcription. Using these mice along with wild type and full ER? KO, we can determine the actions of ERE-dependent and ERE-independent transcription and cell signaling on energy homeostasis. Furthermore, there is growing evidence that both of these types of signaling events are potential targets for environmental estrogens (bisphenol A (BPA), alkylphenols, phytoestrogens, etc.). Therefore, environmental estrogens have a multiplicity of potential targets in the hypothalamus outside of altering normal reproductive capacity including other hypothalamic functions controlled by endogenous estrogen. Experiments outlined this application will examine the multiple receptormediated pathways that E2 (and environmental estrogens) impact energy homoeostasis and other hypothalamic functions in the hypothalamus. In the first aim, Specific Aim 3 of K99/R00, we will elucidate the effects of ERE-dependent and ERE-independent E2 signaling (ER? and/or Gq-mER) in the control of energy homeostasis and hypothalamic gene expression and if maternal exposure to environmental estrogens function through similar mechanisms. The second aim, Specific Aim 4 of K99/R00) will examine the electrophysiological effects of exposures to environmental estrogens on POMC and NPY neuronal activity and cation channel expression both in vivo and in slice preparations. The electrophysiological effects of environmental estrogens on hypothalamic neuronal activity has not been examine previously. Since recent evidence suggests a link between developmental exposure to BPA and adult obesity, the goals of this research will address basic neurological effects of these compounds and further enhance our knowledge of the impacts environmental estrogens have on human health using novel approaches (transgenic mouse models and electrophysiological techniques) and integration with whole animal studies.

Project Summary The prevalence of obesity, Type II diabetes, and metabolic syndrome has been attributed, in part, to nutritional, psychological, and lifestyle changes in the developed world over the past century. However, other factors may contribute to this epidemic and include influences of endocrine disrupting compounds (EDCs). One such group of EDCs is flame-retardants used in household products, furniture, clothing, toys, and electronics. These EDCs are a potential ?obesogens?, which may lead to an increase or susceptibility to obesity, metabolic syndrome, and Type II diabetes in children and adults. Organophosphate flame-retardants (OPFR) are increasing in usage due to the phase-out of PBDE flame-retardants. Three of the most common OPFR are triphenyl phosphate, tricresyl phosphate, and tris(1,3-dichloro-2-propyl) phosphate. Little is known about the effects of these compounds, at environmentally relevant concentrations, on adult energy or glucose homeostasis in humans or in rodent models. Therefore, the proposed research will be focused on the effects of adult exposure to these OPFRs, in a mixture and alone, on energy and glucose homeostasis in mice. The mechanisms behind these effects are unknown but potentially involve transcriptional effects in the hypothalamus mediated by the actions of the classical estrogen receptors (ER?) and nuclear receptors (PPAR?). Our hypothesis is that OPFR impinge on the activity of nuclear receptors, both steroid and metabolic, to exert their multi-focal effects on energy and glucose homeostasis in the hypothalamus. By investigating the effects of OPFRs on energy and glucose homeostasis, we can begin to understand the impacts of these environmental contaminants on human health. Experiments will compare the effects of OPFR in the hypothalamus using a combined approach of whole-animal physiology and behavior, gene and protein expression, electrophysiology, and peptide hormone analysis in transgenic mouse strains. In Aim 1, we will examine the effects of adult OPFR exposure on energy intake, energy expenditure, and glucose homeostasis in adults and on hypothalamic gene and protein expression via steroid and nuclear receptors. In Aim 2, we will examine the effects of adult OPFR exposure on NPY neuronal sensitivity to peripheral peptide hormones (leptin, insulin, ghrelin) and K+ channel activity and subunit expression using state-of-the-art electrophysiology coupled with single cell-type qPCR.

Project Summary Chronic exposure to stressful experiences can result in maladaptive affective states that yield behavioral disturbances in rodents and stress-related mood disorders in humans. Over the last decade, the neural circuits underlying these maladaptive effects of stress have become better defined. One region of importance is the bed nucleus of the stria terminalis (BNST), which is a major output pathway connecting the central amygdala to the ventromedial nucleus of the hypothalamus that also receives direct projections from other limbic areas. Therefore, BNST may be an integrative center for limbic information and valence monitoring. Psychological and physiological stressors affect males and females differently. The BNST is a sexually dimorphic structure that may contribute to distinct chronic stress responses in males and females because expression of aromatase and both estrogen receptors (ER) ??? differs in male and female BNST. Direct activation of neurons within the oval nucleus of the BNST (ovBNST) increases anxiety-associated negative valence behaviors in male rodents, and our preliminary data demonstrates that exposure of C57BL/6J male mice to chronic variable mild stress (CVMS) results in increased corticotropin releasing hormone (CRH/CRF) signaling, increased mEPSC amplitude, altered resting membrane potential, and diminished M-currents in ovBNST neurons. While these data suggest that ovBNST may be a nexus for the effects of chronic stress on affective states, many questions remain unanswered. Our overall hypothesis is that CRH-expressing ovBNST neurons are critical mediators of the chronic stress response and that sexual dimorphism in the BNST underlies the distinct chronic stress responses found in males and females. The proposed specific aims will directly answer these questions and increase our understanding of how ovBNST mediates the maladaptive effects of chronic stress. In Aim 1, we will identify the cell populations that are impacted by chronic stressors (CVMS, chronic nondiscriminatory social defeat stress [CNSDS]) and the neurophysiological consequences in male and female mice. In Aim 2, we will assess how chronic stress affects ER? signaling in BNST and whether optogenetic modulation of ER?-positive BNST neurons mimics and/or reverses the effects of chronic stress on behavior. In Aim 3, we will determine the necessity and sufficiency of CRH-signaling in ovBNST neurons in mediating the behavioral effects of CVMS and CNSDS.