Meharvan Singh - US grants

6. Health-related Consequences of Female Reproductive Aging. The menopause represents the end of a woman's reproductive years and is associated with a precipitous decline in circulating gonadal hormones, including estrogen and progesterone. Since women have live well past the average age at which the menopause occurs, a substantial portion of a woman's life is spent in a hormone-deprived state. Also, many neuronal populations are normal targets of hormones such as progesterone, and thus, it is likely that the depletion of such hormones would greatly impact brain function and may contribute age-related brain dysfunction. While considerable emphasis has been placed on estrogen protective actions on brain function, the involvement and mechanisms for progesterone action have been less well studied, but whose impact on brain function may be no less significant than that implicated for estrogen. In this proposal, we postulate that the GABAA receptor is a central player in progesterone's neuroprotective actions. Classically, progesterone modulates GABAA receptor function through its 5alpha-reduced metabolite, allopregnanolone, which enhances GABA-gated chloride currents. However, our preliminary data show that progesterone, and not ?allopregnanolone, rapidly and reversibly inhibits GABA-gated currents. Further, we offer preliminary data to suggest that this effect of progesterone may involve the activation of signal transduction pathways. Recently, we identified that progesterone elicits the activation of such neuroprotection-related signal transduction pathways as the Mitogen-activated protein kinase (MAPK) pathway. Since the GABAA receptor is a phosphoprotein, and that phosphorylation of the GABAA receptor results in altered channel function, we propose that progesterone inhibits the GABAA receptor by phosphorylating discrete sites within the GABAA receptor complex, through the activation of the MAPK signaling pathway. Furthermore, we hypothesized that progesterone-induced inhibition of the GABAA receptor is consistent with progesterone's neuroprotective actions. These hypotheses will be tested using recombinant GABAA receptors expressed in HEK298 cells, where we will evaluate: 1) The importance of the MAPK pathway in mediating progesterone's effects on GABAA receptor function, 2) If phosphorylation of specific sites within GABAA receptor subunits are critical fro progesterone's actions, and 3) If such phosphorylation events are critical for progesterone cytoprotective actions.

[unreadable] DESCRIPTION (provided by applicant): Androgens influence much more than parameters related to reproductive function. Among the increasingly diverse functions regulated by androgens is their ability to influence cell survival, although the precise mechanism and receptor(s) that are involved remain unclear. Classically, the effects of androgens like dihydrotestosterone (DHT) are attributed to the regulation of gene transcription through the androgen receptor, a member of the nuclear steroid hormone receptor superfamily. However, recent evidence supports the ability of androgens to elicit "non-genomic" actions as well, including the ability to elicit signal transduction pathways. Here, we provide preliminary data that not only support the ability of androgens to influence cell signaling pathways relevant to promoting cell survival, but also offer evidence for a functional membrane-associated androgen receptor. Moreover, the consequences of activating the membrane- associated androgen receptor were different from activating the intracellular androgen receptor. We infer that cells or tissue that have relative differences in one receptor type over another may respond differently to androgens or their antagonists. As such, inconsistent findings as to whether androgens are protective or damaging may be due to the existence of two competing mechanisms, one via the intracellular receptor and the other via the classical intracellular receptor. Our hypothesis is that preferential activation of the membrane androgen receptor influences parameters related to cell protection differently than when intracellular receptors are activated. In this application, we will differentiate the consequences of activating the classical receptor versus the membrane-associated receptor in four complementary experimental systems that model neurons and glia. Specifically, we will determine whether neurons and glia have the same level of expression of the putative membrane androgen receptor. Secondly, we will assess how activation of this membrane androgen receptor influences 1) signal transduction relevant to neuroprotection, and 2) cell survival, in both the neuronal and glial models. The results obtained from the proposed experiments will provide new and important mechanistic details about the neurobiology of androgens that we believe will be pertinent to the development of novel androgen-based therapeutic strategies for such age- related diseases like Alzheimer's Disease. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The indications for androgen therapy has expanded such that today, androgens are used in both men and women, and in addition to their use in treating primary (or secondary) hypogonadism the potential benefit of increasing bone mineral density, inducing greater muscle mass and strength, enhancing sexual function, and improving mood. Androgens are also being considered as a treatment for rheumatoid arthritis, wound healing, and male contraception. More than 1.7 million prescriptions for testosterone were written in the United States in 2002, representing a 30% increase over that prescribed in 2001 and a 170% increase from that prescribed in 1999. Further, Frost & Sullivan report that that the market for androgens is growing at a 25% compound annual growth rate (CAGR). Of specific relevance to this proposal, it should be noted that though the aging population is clearly an important demographic for which androgen therapy may be indicated, this same population is also at a higher risk for stroke or developing such neurodegenerative diseases as Alzheimer's disease. Given the discrepancy between reports in the literature as to whether androgens are beneficial or detrimental, there is a critical need to devise an effective screening tool to better define the population most likely to benefit from androgen therapy. Currently there is no known diagnostic available to define risk in a patient who is considered for androgen therapy. As such, this Phase I application outlines a strategy, based on our preliminary data, to develop a diagnostic tool for the relative assessment of two pharmacologically distinct androgen receptors, each of which appear to mediate different effects on cell survival. One receptor (the classical, intracellular/nuclear androgen receptor), subserves a beneficial (cell survival promoting) effect, while the other, a membrane androgen receptor (mAR), promotes cell death. Such potentially "competing" mechanisms could explain the discrepancy in the literature as to whether androgens are protective or promote cell dysfunction and death. Importantly, these data serve as the basis for our hypothesis, which states that the relative abundance (or ratio) of these two competing receptor mechanisms predicts whether individuals will respond favorably to androgens or are more prone to the negative consequences of androgens. This hypothesis will be tested within the confines of two specific aims. The first will develop and validate a methodology to simultaneously assess the levels of the classical AR and the mAR from both brain tissue and blood (white blood cells). The latter will address whether simple blood measurements of these receptors may reliably predict the mAR:AR ratio in androgen's target tissue. The second aim will test the predictability of the mAR:AR ratio on the therapeutic outcome of androgens by assessing if the negative outcome associated with androgens in an animal model of stroke is correlated with a relatively higher abundance of mAR in both the affected brain regions and in white blood cells (the latter serving as a surrogate marker for the brain). Successful completion of this project will provide the necessary foundation for a Phase II STTR application and the development of a diagnostic tool to define those individuals for whom androgen therapy is not only indicated, but also effective and safe. [unreadable] [unreadable] [unreadable]

The discrepancy between numerous basic science, epidemiological and clinical studies that supported the potential benefit of hormone therapy in preventing age-associated cognitive impairment, and the recently published results from the Women's Health Initiative Memory Study (WHIMS), underscores the fact that our understanding of hormone neurobiology is incomplete. Since the mechanisms of gonadal steroid hormones in reproductive tissue may not be identical to that of the brain, a better understanding of how these hormones work in the brain is needed. To this end, we will systematically address the role of the newly discovered membrane progesterone receptor (mPR) in mediating the neuroprotective effects of progesterone. Further, we propose that the failure of the WHIMS to show positive effects of hormone therapy was due, in part, to the synthetic progestin used in the hormone therapy regimen, medroxyprogesterone acetate (MPA), and hypothesize that its lack of efficacy is due to important differences in its mechanism of action, relative to that of progesterone. Our hypotheses will be tested in primary cortical cultures (explants and dissociated neurons) in which we have confirmed the expression of the classical progesterone receptor (PR) and the mPR as well. We will use complementary pharmacological (cell membrane impermeable progestins), molecular (siRNA) and genetic tools (PRKO mice) to address the following aims: Aim 1: deterimine if the mPR mediates the effects of progesterone on the ERK/MAPK pathway (a pathway that we show is required for progesterone-induced protection);Aim 2: determine if PR-specific patterns of ERK phosphorylation may explain the difference between progesterone's and MPA's ability to protect;Aim 3: determine if the mPR mediates the effect of progesterone on cell survival;and Aim 4: determine if progesterone protects in an in vivo model of stroke, specifically, in animals whose expression of the classical PR has been disrupted (PRKO). The ovariectomized, "stroked" mouse will serve as a common model used by all projects within this Program of research and will enhance and facilitate the conceptual and experimental integration among projects. Collectively, the studies proposed in this project may identify the mPR as an important molecule to which future drug discovery efforts can target for the treatment or prevention of brain dysfunction resulting from age- or age-associated diseases like Alzheimer's Disease.

DESCRIPTION (provided by applicant): The overall goal of this program project is to identify and characterize new and alternative mechanistic targets by which estrogens and progestins are neuroprotective. This program of research is driven by a critical need to improve our understanding of steroid hormone neurobiology, a need that became evident following the results of the Women's Health Initiative Memory Study that identified effects of estrogen and/or progestins that were contrary to expectation. To address this need, we have organized a program of research consisting of 4 highly interactive research projects and 2 supportive cores. The studies proposed in these projects challenge the field to consider a novel membrane PR (Project 1), a mitochondria-localized estrogen receptor (Project 2), intracellular Ca2+channels, including IPS receptors and a novel mitochondrial ryanodine receptor (Project 3), and a previously ignored, naturally occurring estrogen, 17a-E2 (Project 4), as critical players in neuroprotection and/or neurogenesis. Supporting these projects will be the Administrative Core (Core A) that not only oversees the program of research, but will also provide biostatistical support and a common animal model (ovariectomized animals that have undergone transient cerebral ischemia) to the projects, the latter serving as a point of integration for the research performed in the individual projects. In addition, the Mass Spectrometry Core (Core B) will serve all 4 projects by providing powerful tools to assess brain steroid levels and for the routine identification and/or quantification of proteins and their posttranslational modifications, which will serve to enhance the Program's ability to define relevant mechanistic targets of estrogens and progestins. Through the successful completion of the proposed research, we expect to have identified key players in the neuroprotection cascade relevant to the protective effects of estrogen and progesterone. In particular, we will have identified the most protective hormone along with their important intracellular targets that are critical for protecting neural tissue, which can in turn, be exploited for the development of safer and more effective therapeutic strategies for treating the menopause and age associated disorders such as Alzheimer's disease, whose incidence and risk increases in the postmenopausal period.

Progesterone is protective against insults relevant to the aging process and neurodegenerative disease. However, the impact of age,and whether mechanisms implicated in progesterone's protective effects translate to the ability of progesterone to preserve cognitive function is still unclear. In the first period of funding, we determined that progesterone is neuroprotective and that this protection was dependent on the ERK/MAPK pathway and neurotrophin signaling. In addition, our data suggested that the NMDA receptor may be a relevant downstream target of progesterone's protective effects. The NMDA receptor is an important mediator of hippocampal long term potentiation (LTP). Since the ERK pathway and neurotrophin signaling are also implicated in the regulation of cognitive function, we hypothesized that progesterone by itself protects against age-associated cognitive impairment by regulating the function and/or expression of NMDA receptors and associated hippocampal LTP. Further, we predict that the intactness of progesterone- induced signaling, NMDA receptor phosphorylation and/or expression, and LTP will predict successful cognitive aging in old mice. These hypotheses will be tested through the completion of the following 4 aims: 1) To determine if progesterone prevents against ovariectomy-induced cognitive deficits in young adult, middle aged and old C57BI/6 mice, and if such effects are associated with effects of progesterone on hippocampal LTP;2) To determine if progesterone regulates NMDA receptor expression and/or phosphorylation as a potential mechanism underyling its effects on LTP and cognition;3) To determine if signaling mediators of progesterone-induced neuroprotection (such as ERK)mediate the effects of progesterone on NMDA receptor phosphorylation and hippcampal LTP;and 4) To determine if the "intactness" of progesterone-induced signaling, NMDA receptor phosphorylation (or expression) and regulation of LTP,predicts successful aging. The successful completion of this project will fill an important gap in our understanding of how progesterone alone regulates cognitive function, and will be achieved through extensive utilization of the the 3 Cores and substantial interaction with the other Projects. In addition to greatly expanding our understanding of progesterone neurobiology, the results may also impact how we consider hormones in treating such neurodegenerative diseases as Alzheimer's disease.

DESCRIPTION (provided by applicant): We are planning and have commitments of all invited presenters for a two-day conference on the Window of Opportunity for Estrogen and Progestin Intervention in Brain Aging and Alzheimer's disease. This meeting will be held in Fort Worth Texas at the University of North Texas Health Science Center (UNTHSC) and will feature many of the major participants currently engaged in the national discussion of the window of opportunity. The overall objective of this Window of Opportunity Conference is to provide an authoritative update on new science and reanalysis of the Women's Health Initiative Memory Study (WHIMS) to determine if data support or reject the hypothesis of a window of opportunity for hormone therapy in the treatment/prevention of cognitive decline in post-menopausal women. The specific objectives are: (1) To assemble a team of leading thinkers on the subject of widow of opportunity in order to consider new science and re-evaluate existing science on the subject. (2) To assemble an audience of endocrinologists, geriatricians, gynecologists, neuroscientists and others with an interest in hormone therapy and Alzheimer's disease (AD) treatment, delay and/or prevention. (3) To create a forum for free exchange of ideas on the subject of window of opportunity, using a novel format for the meeting that encourages exchange of ideas. (4) To publish the proceeding of the meeting in a high profile text or journal. (5) To evaluate the effectiveness of the conference in achieving its stated objectives. Five sessions are proposed, including a graduate student/postdoctoral fellow session at which young scientists will present their unpublished data that address the issue of a window of opportunity. Also, a data blitz session will allow all participants to present breaking news type data on the topic. This will be followed by three sessions that each address different facets of the window of opportunity issue.! As of this submission, all proposed presenters have confirmed their willingness to participate, session leaders have been identified and we are in final negotiations with a publisher of the proposed text of the proceeding of the meeting. In addition to the $30,000 herein requested, we have raised $20,000 locally to support this conference and are committed to raising more funds to support expanding the graduate student/post-doctoral fellow session(s). Finally, we have in place an evaluation plan for the assessment of the effectiveness of the conference and the presenters in achieving the goals of the conference. This conference is needed and timely, since menopausal age women will reach 41 million by 2020 and discovering the appropriate timing of the right hormones is much needed. Also, we are not aware of a window of opportunity meeting scheduled in 2012, despite that urgent need for a reconsideration of the use of hormones for brain protection in some post-menopausal women. PUBLIC HEALTH RELEVANCE: Menopausal age women will reach 41 million by 2020 and discovering the appropriate timing of the right hormones is much needed. Also, we are not aware of a window of opportunity meeting scheduled in 2012, despite that urgent need for a reconsideration of the use of hormones for brain protection in some post- menopausal women. We propose a conference that invites many of the leading thinkers in the field to consider if timing of post-menopausal hormone therapy affects the response of the brain to these hormones.

DESCRIPTION (provided by applicant): With the graying of America we are faced with the need to address the ever increasing number of individuals in our society who have age-associated nervous system diseases and conditions. It is estimated that by the year 2050, there will be up to 16 million Americans with Alzheimer's disease and an even larger number with additional nervous system-related diseases that are attributed to the aging of our population. To address this problem, we need multidisciplinary approaches to facilitate the discovery of the mechanisms, treatments and prevention of these diseases. Active, integrated research-based training of pre-doctoral students is a key to re- supplying the research personnel needed to address these biomedical health care issues. Herein we propose to continue a successful predoctoral program that focuses on the neurobiology of aging. The present application is a competing renewal of a very successful program of pre- and post-doctoral training at the University of North Texas Health Science Center (UNTHSC). This funding cycle we had 22 full fellows, of whom 10 completed their training and their Ph.D. and an additional 17 associate fellows for a total of 39 trainees in less than 5 years. Our trainees published 91 peer-reviewed papers; we trained 7 (of 22 = 32%) minority students as full fellows; 6 (of 17 = 35%), for a total of 33% minority participation in our training program. The proposed training program will enhance an already strong and successful training program in the neurobiology of aging. The continuation of our predoctoral training program is proposed, as our Institute for Aging and Alzheimer's Disease Research (IAADR), founded by the Training Program Director, has matured through the addition of faculty and the funding of three program project grants, and has initiated clinical research into the causes, treatment and prevention of brain aging and Alzheimer's Disease at the UNTHSC. Collectively, our new training initiatives create a stimulating environment for the training of predoctoral trainees in the neurobiology of aging. Unique features of the training program are (1) our Associate Fellows program, which increases the number of trainees participating fully in the training program; (2) our emphasis on diversity training, that resulted in a 33% minority participation in the training program; (3) our high institutional support which is proposed to amount in an institutional commitment to the training program of approximately $276,800 over the next 5 years; (4) our focus on the development of research excellence and leadership among our trainees; and (5) our Preceptor-in-Training Program, which trains our more junior preceptors in pre-doctoral training. Collectively, these attributes of our Neurobiology of Aging Training Program have produced an outstanding program of training that we wish to continue.

The Administrative Core will manage the overall progress of the Program Project with the ultimate goal of defining the neurobiological basis of the therapeutic window of opportunity for estrogen and progesterone. The Administrative Core will: 1) hold overall responsibility for the administration of the program project, 2) manage the fiscal aspects of the Program Project, including the set up of project and core accounts, the appointment of new employees and re-appointment of existing personnel to the program accounts, reconciling accounts, reporting budgets to investigators, ordering of reagents and supplies, tracking orders and reporting expenditures to the UNT Health Science Center as well as NIA personnel, 3) manage the interactions between projects and cores, provide statistical support for all projects, and procure and distribute human tissue to the projects, and 4) foster an environment for the intellectual interactions of program investigators and between investigators at the UNT Health Science Center community, and with the research community at large.

PROJECT SUMMARY (See instructions): During the first period of funding. Project 1 focused on addressing the role of progesterone receptors in mediating the cytoprotective effects of progesterone (P4). In particular. Project 1 challenged the dogma that classical progesterone receptors (PR) are the principal mediators of P4's protective effects in the brain by suggesting that recently described membrane-associated progesterone receptors may be equally important. Major findings included the observation that indeed both the classical PR and membrane-associated progesterone receptors, particularly Pgrmcl, cooperate to regulate key cytoprotective mediators, including effectors of the ERK/MAPK pathway (such as ERK1/2 and ERK5) and BDNF. In the continuation of this project, we propose to apply this knowledge to understand whether 1) there is a finite period (i.e., therapeutic window) following steroid hormone deprivation, or with age, where P4's effects on neuroprotection and key mediators of cytoprotection are maintained, and 2) if the relative abundance of PR and Pgrmcl predict sustained protective effects of P4. Further, we will determine whether specific patterns of progesterone receptor expression predict whether P4 complements or antagonizes estrogen's protective effects. Our hypothesis states that the combined expression of the classical PR and the membrane progesterone receptor, Pgrmcl, is required to maintain responsiveness of the brain to P4. Further, we propose that the relative abundance of these two receptors will also predict whether P4 complements estrogen's protective program or whether it antagonizes estrogen's effects. These hypotheses will be tested in cellular models, where the relative levels of PR and Pgrmcl can be manipulated (pharmacologically or using molecular tools), as well as in an animal model of steroid deprivation (ovariectomy), and finally, translated to a human model of ovariectomy (i.e., the surgical menopause). While the loss of estrogen's beneficial effects with increasing post-ovariectomy duration has been described, nothing is known with respect to the response to P4. Thus, the studies proposed herein will reveal a key piece of the therapeutic window puzzle by defining which progesterone receptors are critical determinants of P4's protective effects and importantly, may offer unique insight into how the therapeutic window may be expanded (i.e., through regulation of specific progesterone receptors). Together with the studies proposed in Projects 2 and 3, we expect this program of research to advance our understanding of the neurobiological basis of the critical window of therapeutic opportunity for estrogen and progesterone, a goal that could not be achieved by Project 1 alone.

PROJECT SUMMARY (See instructions): During the first period of funding. Project 1 focused on addressing the role of progesterone receptors in mediating the cytoprotective effects of progesterone (P4). In particular. Project 1 challenged the dogma that classical progesterone receptors (PR) are the principal mediators of P4's protective effects in the brain by suggesting that recently described membrane-associated progesterone receptors may be equally important. Major findings included the observation that indeed both the classical PR and membrane-associated progesterone receptors, particularly Pgrmcl, cooperate to regulate key cytoprotective mediators, including effectors of the ERK/MAPK pathway (such as ERK1/2 and ERK5) and BDNF. In the continuation of this project, we propose to apply this knowledge to understand whether 1) there is a finite period (i.e., therapeutic window) following steroid hormone deprivation, or with age, where P4's effects on neuroprotection and key mediators of cytoprotection are maintained, and 2) if the relative abundance of PR and Pgrmcl predict sustained protective effects of P4. Further, we will determine whether specific patterns of progesterone receptor expression predict whether P4 complements or antagonizes estrogen's protective effects. Our hypothesis states that the combined expression of the classical PR and the membrane progesterone receptor, Pgrmcl, is required to maintain responsiveness of the brain to P4. Further, we propose that the relative abundance of these two receptors will also predict whether P4 complements estrogen's protective program or whether it antagonizes estrogen's effects. These hypotheses will be tested in cellular models, where the relative levels of PR and Pgrmcl can be manipulated (pharmacologically or using molecular tools), as well as in an animal model of steroid deprivation (ovariectomy), and finally, translated to a human model of ovariectomy (i.e., the surgical menopause). While the loss of estrogen's beneficial effects with increasing post-ovariectomy duration has been described, nothing is known with respect to the response to P4. Thus, the studies proposed herein will reveal a key piece of the therapeutic window puzzle by defining which progesterone receptors are critical determinants of P4's protective effects and importantly, may offer unique insight into how the therapeutic window may be expanded (i.e., through regulation of specific progesterone receptors). Together with the studies proposed in Projects 2 and 3, we expect this program of research to advance our understanding of the neurobiological basis of the critical window of therapeutic opportunity for estrogen and progesterone, a goal that could not be achieved by Project 1 alone.

Summary With the ?graying of America? we are faced with the need to address the ever increasing number of individuals in our society who have age-associated nervous system diseases and conditions. To address this problem, we need multidisciplinary approaches to facilitate the discovery of the mechanisms, treatments and prevention of these diseases. Active, integrated research-based training of pre-doctoral students is a key to re-supplying the research personnel needed to address these biomedical health care issues in a sustainable manner. Herein we propose to continue a successful pre-doctoral program at the University of North Texas Health Science Center (UNTHSC) that focuses on the neurobiology of aging. During this funding cycle we supported 33 fellows, of whom 22 completed their training and their Ph.D. The trainees were very productive and published 46 peer-reviewed papers to date; we also trained 7 (of 33 = 21.2%) minority students. Importantly, we increased our retention/graduation rate from 84.6% in the prior 5-year funding period to 100% in the most recent funding cycle. The proposed training program will enhance an already strong and successful training program in the neurobiology of aging. The continuation of our predoctoral training program is proposed, as our Institute for Healthy Aging has matured through the addition of faculty and continued funding of program project grants, such as that led by the PI, Dr. Meharvan Singh. Such growth has facilitated more basic, translational and clinical research into the causes, treatment and prevention of brain aging and Alzheimer's Disease at the UNTHSC. Collectively, these new initiatives create a stimulating environment for the training of predoctoral students in the neurobiology of aging. Unique and innovative features of the training program for the next period of funding include: (1) Several experiential learning opportunities to include the Geriatrics Skills Lab and the Translational Aging & Alzheimer's Disease Research Program; (2) our continued emphasis on diversity training, that resulted in greater than 20% minority participation in the training program; 3) Career counseling and interview skill development program, 4) techniques in neuroscience program; 5) our institutional support which will expand those students who will have access to some or all of this training program; 6) our focus on the development of research excellence and leadership among our trainees; and 7) our Mentoring-the-Mentor Program which trains junior faculty in mentoring trainees under the guidance of a senior faculty. Collectively, these attributes of our Neurobiology of Aging Training Program has produced an outstanding program of training that we wish to continue.