James W. Simpkins - US grants

We propose to evaluate the physiochemical and pharmacokinetic parameters of psychoactive drugs which influence their distribution to the fetus and the consequences of fetal drug exposure on development and aging of brain catecholamine neurons and neuroendocrine systems. We would conduct an extensive pharmacokinetic analysis of fetal disposition of the antidepressants desipramine, bupropion and its metabolite 2-hydroxy-bupropion to access the effects of lipid solubility of the drugs and maternal plasma protein binding on extent of exposure of the fetus to the maternally-administered drugs in the rat. These data will be correlated with our previous results for other antidepressants and barbiturates. In a second project, the "subclinical" teratogenic effects of fetal drug exposure would be evaluated for each of these drugs and correlated with our data on other tricyclic antidepressants. We will evaluate a variety of parameters of development, reproductive function and brain catecholamine neurotransmission. In view of our observation that a common teratological consequence of in utero exposure to both antidepressants and barbiturates is a persistent hyperactivity of hypothalamic catecholaminergic neurons in adult progeny, we will evaluate the ontogeny of noradrenergic neurons and the number and affinity of noradrenergic receptors in drug-treated progeny. Finally, we have observed that the teratogenic effects of psychoactive drugs on several organ systems are similar to those which are normally observed during the aging process. Thus, we will evaluate the intriguing hypothesis that a consequence of in utero drug exposure is an acceleration in the rate of aging of organ system or of the organism itself. Thus, we would monitor a population of imipramine- (or phenobarbital-) exposed progeny for its life-long mortality rate, and the functional capacity of several systems for which normal age-related alterations are well defined. Collectively, the results of these experiments should allow us (i) to further define the relationships among drug lipid solubility, plasma protein binding, the extent of fetal drug exposure and the subclinical teratogenic effects, and (ii) hence to test the validity of using in vitro tests of lipid solubility and plasma protein-drug binding as predictors of fetal drug exposure and potential teratogenic effects. Finally, this project would allow us to evaluate our hypothesis that "teratogenic aging" is a consequence of fetal exposure to some psychoactive drugs.

This grant application proposes to determine the role of age-related alterations in catecholamine (CA) metabolism in the initiation and maintenance of reproductive senescence in the rat. We would further extensively evaluate the consequences of the hormonal alterations associated with senescence on the rate of aging of CA neurons. All proposed experiments would utilize young (3 to 4 months), sexually mature, and middle-aged (11 to 12 months) and old (22 to 24 months) constant estrous (CE) rats of the Long Evans strain. For some experiments, the female Fischer 344 rat, which maintains a long reproductive life would be used for comparison. Based upon our observation that both norepinephrine (NE) and dopamine (DA) turnover decreases substantially in nuclei along the preoptico-tuberal pathway prior to the loss of luteinizing hormone (LH) secretory capacity at the onset of constant estrus, we would assess the effects of acutely and chronically administered alpha-adrenergic and dopaminergic receptor agonists on pulsatile LH release and steroid-induced surges of LH. The effects of these treatments on Luteinizing hormone-releasing hormone (LHRH) neurons would be evaluated by "push-pull" cannulae methods. The role of chronic hyperprolactinemia (Hyp) in the aging of DA neurons would be evaluated by measuring the sequence of alteration in DA metabolism following the onset of hyperprolactinemia in animals which exhibit early (Long-Evans rats) or late reproductive senescence. Thus, the rate of DA neuronal dysfunction would be correlated with length and/or severity of the Hyp, rather than age per se. Parallel studies would evaluate the effects of chronic suppression of serum prolactin (PRL) on DA metabolism in middle-aged and old CE rats. The role of opioid neurons in reproductive would be evaluated (i) by estimating release rates of enkephalins and endorphins using "push-pull" cannulae and (ii) assessing the effects on LH secretory capacity of chronic blockade of the opioid receptor through sustained release naloxone pellets. Finally, the effects of chronic Hyp on opioid neuronal function in aging rats would be evaluated using similar methods. These studies will (i) advance our knowledge of the neuronal system involved in the initiation and persistence of reproductive senescence (ii) provide an initial assessment of the role of chronic hormonal alteration, associated with reproductive senescence, in determining the rate of aging of CA neurons.

This application proposes to evaluate a novel chemical delivery system for estradiol (E2-CDS) which we have demonstrated is "locked-into" the brain and there serves as a depot for the sustained release of the steroid. The delivery system is based upon the chemical attachment of estradiol (E2) to a lipoidal dihydropyridine carrier. This E2-CDS crosses the blood-brain barrier (BBB) and the carrier is rapidly oxidized to the charge, hydrophilic quaternary pyridinium salt (E2-Q+), thus reducing its rate of egress from the brain. The hydrolysis of E2 from the carrier results in the sustained release of the steroid in the brain and the rapid clearance of the non-toxic carrier. In vivo, the E2-CDS causes elevations in brain E2-Q+ and E2 and there both of these metabolites are cleared slowly. As an apparent result, long-lasting effects of E2-CDS are observed, including chronic suppression of LH, suspension of estrous cycles, stimulation of masculine sexual behavior suppression of testosterone secretion and of weight of androgen-responsive tissues and reductions in food intake and body weights. In the present application, we propose to use rats as an animal model (1) to determine the regional, cellular and subcellular distribution in the brain over time of the two major metabolites of the delivery system, E2-Q+ E2 using quantitative autoradiography; (2) to determine the mechanism by which E2-CDS causes chronic suppression in LH secretion; (3) to determine the mechanism by which E2-CDS causes chronic suppression of testosterone secretion; (4) to determine the effects of E2- CDS on the growth of androgen-dependent prostatic adrenocarcinomas; and (5) to compare the efficacy of E2-CDS with LHRH agonists and LHRH agonists plus antiandrogens on the growth of prostatic tumors. The proposed evaluation will provide needed information of the pharmacokinetics, metabolism and pharmacodynamics of this novel E2-chemical delivery system. This knowledge may lead to (1) a useful tool for dissociating central from peripheral effects of estradiol; (2) a potential method for preferentially treating brain estradiol deficiencies in women (i.e., brain-mediated components of the post-menopausal syndrome); (3) the selective reduction in gonadotropin secretion, which would be useful in fertility regulation and (4) a useful new approach for the therapy of certain androgen-dependent diseases, such as prostatic carcinomas.

health science research support; university;

biomedical equipment resource; biomedical equipment purchase;

densitometry; brain metabolism; biomedical equipment resource; biomedical equipment purchase;

biomedical equipment purchase;

biomedical equipment purchase;

Alzheimer's disease (AD) is a progressive neurodegenerative disorder whose cause is unknown and for which an effective therapy dose not exist. The present application proposes 5 years the funding of the University of Florida Drug Discovery Group for Alzheimer's Disease (UF-DDG) to continue the process of hypothesis-driven research into the mechanism(s), models and discovery of efficacious therapies that affect the underlying neuropathologies of AD. Four research projects and 4 supportive cores will continue the discovery of drug in 4 different therapeutic classes with efficacy in protecting neuron in vivo and in vitro and in enhancing memory- related behaviors in learning/memory deficient animal models. For a drug to emerge from our preclinical assessment and enter animal toxicity assessments, it must show neuroprotective and memory-enhancing activity. During the first funding period, this process has resulted in 3 drugs in 2 therapeutic classes entering clinical trial for efficacy assessment in AD. The present program of research proposes the assessment of the mechanism of action of the neuroprotective and cognitive-enhancing effects of our compounds, and the synthesis of novel compounds that are indicated from our studies. In Project 1, James W. Simpkins we will assess the neuroprotective activities of the estratrienes, a group of estrogen-like compounds that we have shown to be neuroprotective in a variety of in vitro and in vivo models and cognitive-enhancing in animals. Project 2, Edwin Meyer will determine the neuroprotective mechanism of novel alpha7 nicotinic agonists and we will synthesis new agonist, based upon discovered structure-activity relationships. In Project 3, Jeffrey Hughes/Edwin Meyer will continue their work on non-viral methods for the delivery of a nerve growth factor gene into brain neurons, using novels cationic liposomes that incorporate pH sensitive surfactants, designed to increase the transfection rate of the gene. The 4 cores are i) Administration, ii) Tissues Culture, iii) Animal Care and Behavioral Assessment, and iv) Neurochemistry. Collectively, these core will provide organization, reagents and services that are vital to the success of the program. In summary, this program of research will continue to discover novel drugs (and drug mechanisms) that are of potential efficacy in the treatment of the neurodegeneration and cognitive decline associated with AD.

The overall goal of this grant application is to determine the mitochondrial mechanism and relative neuroprotective potency of a panel of non-feminizing estrogen-like, estratrienes, using a structure-activity relationship (SAR) approach. These compounds are selected from novel compounds synthesized in our Drug Synthesis Core as well as more than 60 estratrienes discovered during the previous funding period to be potent neuroprotectants, but to lack interaction with either ERalpha or ERbeta. The estratrienes will include compounds more potent and compounds less potent than 17 beta-E2. This goal will be achieved by addressing 5 specific aims. Specific Aim will determine the SAR of neuroprotection and mitochondrial membrane potential (delta-psi) in two cell types in vitro. Specific Aim 2 will determine the SAR of neuroprotection and Ca 2+ increase in cytosol and mitochondria of two cell types in vitro. Specific Aim 3 will determine the SAR of neuroprotection and blockade of activation of cyclin dependent kinases in an in vivo model for cerebral ischemia-reperfusion. Specific Aim 4 will determine the SAR of neuroprotection and tau hyperphosphorylation in an in vivo model for cerebral ischemia-reperfusion. Transient middle cerebral artery (MCA) occlusion will be used to activate tau hyperphosphorylation and the potency of estratrienes in neuroprotection and blockade of tau hyperphosphorylation will be determined. Finally, Specific Aim 5 will determine the SAR of neuroprotection and tau hyperphosphorylation/ neurofibrillary tangle (NFTs) in an in vivo model using local cortical transduction of human mutated tau (p301L) and cerebral ischemia-reperfusion. Collectively, achieving these specific aims requires coordination with the Vector Core (Core B), the Neuroimaging Core (Core C), and the Drug Synthesis Core (Core D) and will substantially advance our understanding of the potential for clinical development of estratrienes for the prevention/treatment of Alzheimer's disease. The need for this research program is critical, given the recent WHI reports of prothrombic and carcinogenic toxicities of PremPro(R) in elderly women. There is a critical need for the discovery of potent, non-feminizing estrogens in the management of post-menopausal hormone-deprivation syndrome.

DESCRIPTION (provided by applicant): We propose to create a multi-disciplinary pre-doctoral training program in the neurobiology of aging that addresses the training of Ph.D. and D.O./Ph.D. students as future researchers in an area of critical need due to the graying of America. The training program emanates from the recent creation of the Institute for Aging and Alzheimer's Research (IAADR) at the University of North Texas Health Science Center-Fort Worth (UNTHSC-FW), which was formed to enhance and focus research, training, and outreach in preventable brain aging. Under the direction of Dr. James W. Simpkins, this training program will recruit outstanding Ph.D. and D.O./Ph.D. students into training that includes a rigorous, existing didactic curriculum as well as new training activities, including a neurobiology of aging course, a neurobiology of aging seminar series, a neurobiology of aging journal club, and an annual Trainee Symposium. The proposed preceptors have a strong record of training undergraduate and graduate students and postdoctoral fellows, and are well funded and well published in the neurobiology of aging. The training program will complement and enhance the already excellent record of the institution in pre-doctoral training, particularly in the recruitment of minorities into pre-doctoral training. Finally, we will create a 10-component program of monitoring the success of our trainees and the training program. The program envisioned is a vital part of an overall effort of the UNTHSC-FW to enhance research and training into the causes, treatments and prevention of brain aging and its consequential diseases.

DESCRIPTION (provided by applicant): Proposed is a continuation of a productive program of research that has the overall goal of elucidating the mechanism(s) underlying cognitive decline with aging. To achieve this goal, we originally organized and will continue a program of research that includes 4 research projects, 3 essential core facilities and a group of talented investigators. The research program is driven by the now strongly supported hypothesis that oxidative stress in the brain leads to age-related oxidative damage and is a major determinant of the rate of cognitive aging. As such the 4 research projects focus on a systematic assessment of these issues. Project 1 will determine the role of now identified age-related oxidation sensitive proteins and highly significant shifts in the redox state of most brain regions in cognitive decline with age. Project 2 will determine the role of and mechanism by which insult-induced decline in protein phosphates results in persistent activation of a number of kinases, and oxidative stress that leads to AD-neuropathology and cognitive decline. Project 3 will determine the mechanisms underlying the function of immediate early gene products that control intracellular Ca2+ channels and intracellular Ca2+ homeostasis during cognitive aging and oxidative stress in the CNS. Project 4 will assess the signaling mechanism(s) by which the important ovarian steroid, progesterone, enhances LTP and reduces cognitive decline with aging, with a focus on its effects on NMDA receptors. All of these research projects are interactive in their mechanistic focus on the overall hypothesis of the program, the sharing of ideas, tissues, and the use of behavioral assessments following insults or interventions as important functional readouts in animal models shared and employed by all projects. This is achieved through an Administration Core (Core A) that will oversee the program and provide biostatistical support, an Animal Resources and Behavioral and Assessment Core (Core B) that will provide care for and behaviorally characterize all animal models and an Electrophysiology Core (Core C) that will provide assessment of the effects of age, genotypes, insults or interventions on an electrophysiological signature of memory and learning, LTP. This mechanistically driven and statistically-validated, multidisciplinary program of research, that focuses on determining critical molecular and functional mechanisms that underlie cognitive aging, will enhance our understanding of the role of oxidative stress in cognitive aging, and generate potential targets for effective intervention.

[unreadable] DESCRIPTION (provided by applicant): Alcohol abuse causes increased mortality, neurological deficits and a huge cost to society to treat alcoholism, its social and medical consequences. Currently, there are no effective therapies for alcoholism and its neurological consequences. The present grant application proposes a research program to assess the efficacy of estrogens for treatment of the behavioral and neurological consequences of ethanol withdrawal (EW). Based upon our extensive preliminary data that indicate that estrogens reduce withdrawal signs, improve cerebellar-mediated behavioral outcomes and protect cerebellar Purkinje cells of ethanol withdrawn rats, we propose studies to further determine the efficacy and mechanisms of estrogen protection against EW-related neurobehavioral toxicity. We will achieve 5 specific aims in this grant. Specific Aim 1 will determine estrogen effects on the ethanol dependence and the EW phase. Male and female rats will be exposed to 17beta-estradiol (E2) during the dependence versus the withdrawal phase to determine the stage of dependence/withdrawal that is most responsive to estrogens. Specific Aim 2 will evaluate protective effects of nonfeminizing estrogens against neuronal and behavioral deficit in ethanol withdrawn rats. We will employ a novel estrogen, enatiomer-E2 that we have demonstrated to be neuroprotective in vitro and in vivo, but to lack estrogen receptor activity. Specific Aim 3 will determine if estrogens antagonize the pro-oxidant effects of EW by assaying an end product of lipid peroxidation product, malondialdehyde, in cerebellar tissue. Specific Aim 4 will determine if estrogen prevents oxidant-dependent nuclear factor-kappa B (NFrkappaB) activation in ethanol withdrawn rats. Specific Aim 5 will determine the role of estrogen-induced reduction in protein kinase activity and ERKI/2 phosphorylation in the estrogen blockade of nuclear translocation of NFrkappaB during EW. Collectively, the proposed studies will provide new knowledge on the mechanism of estrogen protection from the consequences of EW and determine if estrogens are potential pharmacotherapies for alcoholism and its consequences [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to perfect and apply a proteomic approach to supplement ongoing attempts (P01-AG022550-01) to determine the mechanisms responsible for age-related decrements in brain function. A significant body of evidence supports the hypothesis that oxidative stress may be a causal factor in such age-related changes. In this proposal, we will identify the specific protein targets of oxidation in the aging mouse brain and determine which of them are related to the severity of the age-associated attenuations in cognitive function. The main experimental design of this study w ill entail scoring groups of aged and young mice on a specific cognitive task (Morris water maze). The degree of association will be determined between performance in the cognitive task and the degree of oxidation, measured as carbonylation, associated with individual proteins in different regions of the brain. The specific brain regions and subcellular fractions to be targeted for identification of the aging-associated oxidation-sensitive proteins (AOSPs) will be those showing age-associated oxidative stress as measured by various other parameters described in Project 1 of AG022550. A positive correlation between the degree of loss in a specific cognitive or psychomotor domain and an AOSP will suggest a functional involvement of the protein in brain aging. Moreover, the identity of the AOSP will provide highly specific and testable hypotheses about the causes of age-associated functional decline. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This is the first competitive renewal of a training program in the Neurobiology of Aging at the University of North Texas Health Science Center. This training program builds on a successful predoctoral training program that in the first funding cycle trained 31 trainees and now proposes both a predoctoral and a post- doctoral training component for the next phase of training. Eighteen potential preceptors will provide their expertise, laboratory facilities and extensive pre- and post-doctoral training experience to this program. The existing programs for enhancement of the quality of training that will be continued are (1) our Neurobiology of Aging Course; (2) our Neurobiology of Aging Seminar Series; (3) our Neurobiology of Aging Journal Club; and (4) our Neurobiology of Aging Trainee Symposium. Several new features of the training program have been added. These new features of the proposed training program are (1) an associate fellows program that substantially increased the number of trainees in the first funding cycle from 2-6 per year to 15-19 per year, allowing the critical mass of trainees needed for an outstanding training program; (2) an institutional commitment of more than $200,000 to enhance this training effort; (3) a Preceptor-in-Training Program and a Mentoring-the-Mentor Program for our new faculty preceptors; (4) a Techniques in Neuroscience Program that use 6 excellent core laboratory facilities; and (5) a Training-in-Grant-Reviews Program that provides needed skills in grant reviewing to our post-doctoral trainees. As in the first funding cycle, a 10-metric evaluation program will be used to assess the quality of the training program and of the trainees. The success of the training program during the first funding cycle is indicated by the high productivity of our trainees, their placement in academic fellowships or faculty lines, their recognition for research achievements, their leadership roles among peers, the high minority involvement in the training program and the broad distribution of trainees among preceptors. In summary, a program of training of pre- and post-doctoral fellows is proposed that now has demonstrated success and includes a variety of new programs for training enhancement. This program will help fill the need for the next generation of scientists to deal with the prevention, treatment and cure of age-related diseases of the nervous system. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long term goal of this project is to develop gene transfer procedures to decrease the progression of Alzheimer's disease (AD). The underlying premise is that gene transfer offers a unique manner to impact on different aspect of the pathology of AD. The therapeutic potential of gene transfer for neurological disease is promising, yet substantial technical and theoretical problems remain to be solved before this technology can seriously be considered for clinical application. In this application, safe and efficient plasmid based gene transfer systems will be studied. Plasmid based gene delivery vectors are attractive because these vectors avoid potential problems associated with the use of viruses. In this proposal a plasmid coding for the human protein gelsolin, given by the hydrodynamic gene administration for localized expression into the liver will be used. This gene therapy results in an increase in circulating gelsolin concentrations. Gelsolin is one of a class of proteins known as amyloid peptide binding agents. These proteins can reversibly bind with beta amyloid fragments and serve as a peripheral sink thus increasing the clearance of amyloid fragments from the body and should result in decrease amyloid plaque and resulting sequel. One such effect is the blockade of neuronal alpha 7 nicotinic receptors from beta amyloid fibrils. In this proposal four specific aims are proposed the first aim will address the mechanism of gelsolin mediated amyloid fragment removal from the brain, the second aim will optimize peripheral liver gene expression, the third aim will address duration of action and the fourth aim will focus or returning the neuronal protection ability of an alpha 7 agonist hi a lesion model of neurodegeneration. These novel hypotheses will be investigated using an approach focusing on plasmid based vectors; if supported by our results, they may lead to a new type of vectors for researchers in the gene therapy of AD. [unreadable] [unreadable] [unreadable]

The major objective of the Administration Core is to manage the progress of the Program Project with the ultimate goal of discovering novel mechanisms and drugs which are effective in the therapy of cognitive decline associated with normal aging and AD. The specific aims of the Administration Core are: 1To hold overall responsibility for the administration of the program project. This goal would be achieved through frequent meetings of the program director with project and core directors, a group of external advisors and an Internal (Heath Science Center-wide) Advisory Committee and with the able assistance of an administrative assistant. 2. To manage the fiscal aspects of the Program Project. The program director will supervise and the administrative assistant will carry out the fiscal aspects of the program of research. 3. To manage the interactions between projects and cores. The design of this program of research requires thoughtful coordination of the interactions of research projects with two essential cores, the Animal Care and Behavioral Assessment Core (Core B) and the Electrophysiology Core (Core C). We will coordinate activities to achieve delivery of behaviorally characterized, aged mice to research projects in a manner that represents our historical population of aged animals as a whole. 4.To coordinate tissue sharing among research projects. Procedures are developed and implemented to ensure that all valuable animals and tissues generated as a result of the activities of this program of research are used or stored. 5. To foster an environment for the intellectual interactions of program investigators and between program investigators and the UNT Health Science Center community. 6. To Provide Biostatistical Support to the Project and Cores. The biostatistics component of this core will continue to conduct power analyses as part of the design of proposed studies, will provide advise to investigators on proposed analytic methods for both planned and post-hoc analyses (univariate or multivariate analyses for group difference) and management of the data that results from the various projects. Finally, the biostatistician will perform analysis of results of our behavioral assessment of mice to insure appropriate distribution of mice to each of the projects. These Adminisition Core functions has served this progress well during the first funding cycle and will continue to do so.

During the course of this first funding cycle, Project 2 made several important discoveries. Among these were the following: (1) Multiple signaling kinases are persistently activated by pro-oxidant insults in vitro and cerebral ischemia in vivo. (2) Pro-oxidant insults cause persistent reductions in at least three protein phosphatases. (3) Persistent activation of these signaling kinases converts them into death-inducing kinases that contribute to oxidative damage, AD-like neuropathology and neuronal cell death. (4) Both feminizing and non-feminizing estrogens prevent the pro-oxidant induced persistent down regulation of protein phosphatases and the persistent activation of signaling kinases. Also, estrogens ameliorate neuronal death and AD neuropathology causes by persistent kinase activation. (5) Cerebral ischemia causes phosphatase decline, persistent kinase activation, progressive AD neuropathology, hippocampal damage and cognitive decline in young rats. (6) Persistent inhibition of protein phosphatases causes cognitive decline and tauopathy in young rats. Having demonstrated these normally functioning signaling pathways become, with persistent activation, death-inducing kinase pathways, we propose to determine the causative factors by (Aim 1) determining the mechanism by which chronic phosphatase inhibition causes cognitive and LTP decline and AD neuropathology;(Aim 2) determining the role of persistent kinase activation in AD neuropathology;(Aim 3) determining the role of induction of cyclin-dependent kinases in AD neuropathology;and determining the effects of E2 and non-feminizing estrogens on insult-induced AD-like neuropathology;(Aim 4) determining the mechanism by which estrogens ameliorate these changes;and ( Aim 5) determining the role of L-type Ca2+ channels in the estrogen up regulation of protein phosphatases. Collectively, these studies will determine the mechanism(s) by which oxidative events initiate and contribute to the progression of AD neuropathology and the extent to which estrogen can antagonize these effects of oxidative stress.

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.

PROJECT SUMMARY (See instructions): During the previous funding period, we made a number of fundamental observations relative to the mechanism of action of estrogens on mitochondria that guide the aims of the present proposal. We have evidence that the bioenergetic crisis seen during normal brain aging and in AD is caused by mitochondrial structure, function and mobility dysfunctions that leads to a breakdown in synaptic integrity resulting in cognitive decline that characterizes both aging and AD. The present continuation of this grant will further assess the mechanism(s) of effects of estrogens on mitochondria and determine if these effects occur in vivo and in post-mortem samples from women. We will address 4 specific aims. Specific Aim 1 will determine if pharmacological antagonism or genetic reduction in the PKA/DRP1 pathway leads to a loss of synaptic integrity, mitochondrial fission and immobility, and bioenergetic decline in primary hippocamal neurons. Specific Aim 2 will determine if ovariectomy for 2, 12 or 20 weeks compromises the PKA/DRP1 pathway leading to synaptic loss and mitochondrial dysfunction and if these deficits can be restored by E2, an ER(3 agonist, DPN, or P4 treatment for 6 weeks, in vivo. Specific Aim 3 will determine if age and post-ovariectomy duration, changes the synaptoneurosome response to E2, DPN or P4. Specific Aim 4 will determine if therapy with DPN improves PKA/DRP1 pathway function, thereby ameliorating loss of synaptic integrity, mitochondrial immobility and fragmentation seen in a 5XFAD mice model. For all of the aims, we will assess DRPI phosphorylation state, a panel of pre- and post-synaptic markers, and a panel of bioenergetic measures. For aims 1 and 4, we will conduct a detailed assessment of mitochnodrial fragmentation and mobility. Successful completion of these proposed studies could lead to new understanding of estrogen targets in the brain as well as potential new therapies for age-related cognitive decline and AD.

Proposed is the West Virginia Stroke CoBRE (here after called the Stroke CoBRE) with a focus area of stroke. The Stroke CoBRE was Initiated as an essential part of the Center for Basic and Translational Stroke Research (CBTSR), a newly formed Center that Is focused on enhancing basic and translational stroke research on the West Virginia University (WVU) campus to reduce the burder of stroke. Our approach Is to address the mechanisms by which risk factors, particularly prevalent in our Appalachian population, affect stroke and thereby to decipher mechanisms of these risk factors and potential treatments that target these mechanisms. We envision that the CBTSR will become a national center of excellence In basic and translational stroke research that addresses bench to bed-side to community Issues in stroke. This will be accomplished through the development of junior Investigators to research Independence, and the development of core resources that support the WVU stroke research community, thereby enhancing resources and personnel devoted to stroke research at the WVU to achieve programmatic research activities. Three cores and five research projects are proposed. The Administrative Core will manage the various aspects of the Stroke CoBRE, including an intense junior faculty mentoring program, advisory committees and program evaluation. The Experimental Stroke Core will provide resources, expertise and experimental stroke procedures to junior Investigators as well as to the WVU neuroscience and cardiovascular research community. The Biostatisitic Core will provide critical biostatistical support during the design and conduct of the junior Investigator-Initiated projects. The research projects proposed eminate from 5 junior Investigators who are committed to developing independent research programs through the CoBRE mechanism. Collectively, their research projects address the linked areas of stroke prediction, causes, prevention, acute treatment and rehabilitation. The program-as-a-whole has a well-developed and Intensive mentoring program and a rigorous summative and formative evaluation program. Institutional support is also proposed, in the form of faculty hires, facilities construction and monitary contributions.

The overall goal of the Administrative Core of the West Virginia Stroke CoBRE (hereafter called the Stroke CoBRE) is to manage the development of a center of excellence in basic and translational stroke research at the West Virginia University. To achieve this goal, the follow specific aims will be achieved: Specific Aim 1: To create a vision for the Stroke CoBRE and implement goals, strategies and tactics to realize this vision. Speciflc Aim 2: To develop and implement fiscal, administrative and scientific policies and processes for the Stroke CoBRE. Specific Aim 3: To develop high quality research, research training and career development in the Stroke CoBRE. All five research projects emanate from the research activifies of bright assistant professors in a tenure track in their respective departments. The themafic focus area is stroke and the projects assess linked areas ofthe predicfion, causes, prevention, acute treatment and rehabilitafion of stroke. Speciflc Aim 4: To foster an environment for the intellectual interacfions of program investigators and between program invesfigators and the WVUHSC community. Numerous mechanisms are in place or under development to foster the intellectual curiosity of faculty, fellows and students at the West Virginia University. Specific Aim 5: To develop and implement a mentoring program for junior faculty that addresses the transifion to and attainment of independent research funding. The process involves a contract between mentor and mentee; frequent interaction, not just behween mentor and mentee, but also among the laboratory staffs; an identificafion of career goals; iterative composifion and internal reviews of both manuscripts and grant applicafions; high standards for research accomplishment, both in terms of number and quality of the research product; sensitivity to the need to balance professional and personal lives for a sustainable career in research; and professionalizafion of the junior faculty, including their participation in the most relevant nafional and international scientific meeting, participation in journal and grant review activities, and involvement in the scientific societies that represent our discipline. Specific Aim 6: To development and implement a formafive and summative evaluation strategy with specific milestones.

DESCRIPTION (provided by applicant): The Research Training Program in Behavioral and Biomedical Sciences (BBS) at West Virginia University provides advanced research training at the interface of behavioral and biomedical sciences to PhD students planning careers as independent investigators. The BBS Program draws on existing, high-level training in the Biomedical, Psychology, and Public Health PhD Programs and provides added value through a core curriculum consisting of behavioral neuroscience methods, biostatistics, bioinformatics, and epidemiology. Additional training in behavioral and biomedical research is provided through a highly interactive journal club and an extensive seminar series. Specific training and experiential activities also are provided to foster scientific leadership, communication skills, teaching excellence, responsible conduct of research, and cognizance of issues related to human health, physiology, and disease. Structuring the BBS Program to leverage the strengths of the Biomedical, Psychology, and Public Health PhD Programs ensures an experienced and productive preceptor group, a wide range of research training opportunities, and a highly qualified and diverse applicant pool. The BBS Program remains committed to successful recruiting of underrepresented minority, disabled, and disadvantaged students; programs and partnerships have been developed to address this issue. The BBS Program provides a vehicle for training the next generation of behavioral scientists with the intellect, knowledge, training ad courage to address complex research problems with significant

The overall objecfive of the Experimental Stroke Core (ESC) is to provide the instrumentation, expertise, resources and training for the incorporafion of animal models of stroke into the research of junior invesfigators in the Stroke CoBRE and researchers in the greater WVU neuroscience and cardiovascular research community. This overall objective would be reached by achieving the following specific aims: Specific Aim 1: To provide the instrumentafion, expertise and resources needed to produce strokes in rodent models. The ESC will be Director by James W. Simpkins (Core Director, CD) and he will be assessed by Dr. Xuefang Ren (Core Manager, CM), and James M. O'Donnell will oversee the behavioral assessment of animals. Specific Aim 2: To broadly notify the availability of these core services and resources to the greater WVU neuroscience and cardiovascular community. The availability ofthese services and resources are being and will confinue to be presented to the WVU neuroscience and cardiovascular research community through individual meefings, seminars, presentafions to neuroscience and cardiovascular group meetings and departmental seminars. Specific Aim 3: To provide processes by which users can access the core instrumentation, expertise and resources. Procedures for requesting use of core resources are described and include a brief applicafion describing the rationale for the proposed studies and the proposed design of the study to be conducted. Specific Aim 4: To provide training in experimental stroke research. Twice per year, the CD and CM will organize trainings session on the theory, practice and outcome interpretations for potenfial users of the core. Specific Aim 5: To develop and implement a business plan for the ESC that will insure its sustainability. A business plan for the ESC is in development by the CD and will be reviewed by the Core Resource Management Committee of the WVU Health Science Center. Specific Aim 6: To develop formafive and summative evaluafion processes for the ESC. Collectively, achieving these specific aims will insure a highly funcfioning ESC that will provide a vital sen/ice to the Stroke CoBRE junior invesfigators and to the greater WVU neuroscience and cardiovascular research community.

Project Summary Stroke - like many brain diseases - is clearly associated with aging and a plethora of age-related co-morbid conditions, including cognitive decline, Alzheimer?s disease (AD), multi-infarct dementia, cardiovascular disease, cerebrovascular disease, hypertension, hypercholesterolemia, immune suppression, metabolic syndrome and obesity, sleep deprivation, and depression. However, age-related co-morbid conditions, as a variable in stroke occurrence, severity and long- term recovery is seldom studied experimentally, but is one of the identified factors in the lack of progress in the discovery of new therapies for acute stroke brain damage. To optimize the impact of our research on the discovery of new preventatives, acute treatments and rehabilitation methods for stroke, we need to train the next generation of stroke researchers to assess stroke in the context of the affected patent population: those who are elderly and have multiple co-morbid conditions. The Stroke and Its Co-morbidities Predoctoral Training Program will formalize and standardize our already strong training programs in the biomedical sciences and focus on stroke research. A number of innovative aspects of this training program are semester-long didactic courses in ?Stroke? and the ?Neurobiology of Aging?, a clinical immersion in our WVU Stroke Center, a Neuroscience Emerging Research Discoveries in Stroke (NERDS) journal club, an Associate Scholars Program, and experience in community engagement. The proposed training program will select the best PhD students from the participating Biomedical Sciences Training programs at the West Virginia University (WVU) Health Science Center (WVU HSC) and will prepare them with the skills, knowledge and acumen needed for a successful career in stroke research. The specific training for each of 6 mentee will be tailored based on their annually updated Individualized Development Plan (IDP), and an ?Exploring Career Paths? Program will help prepare them for their chosen career. Program training is expected to last 2-to-3 years. This pre-doctoral training program will create a new generation of young scholars who can address the need for innovative stroke research for the citizens of West Virginia and the nation.

Project Summary The overall goal of the Administrative Core of the WVU Stroke CoBRE is to provide leadership and manage the development of a center of excellence in basic and translational stroke research at West Virginia University (WVU). To achieve this goal, the following specific aims will be achieved: The thematic research focus area is stroke and the research projects assess linked areas of the prediction, causes, acute treatment and rehabilitation of stroke. To achieve this goal, the follow specific aims will be achieved: Specific Aim 1: Develop high quality mentored research and career development in the WVU Stroke CoBRE. All five research projects emanate from the research activities of assistant/associate professors in a tenure track appointment in their respective departments. Aim 1 be achieved by expanding our mentoring program for junior faculty that addresses the transition to and attainment of independent research funding, with an emphasis on supporting the professional development of junior faculty. Clear expectations and milestones for mentees and their respective mentors (both internal and external) are highlighted in the annually signed mentoring agreement. Specific Aim 2: To hire new faculty with a research expertise in stroke. The Program Director and Internal Advisory Committee members are well positioned to direct recruitment of stroke researchers into new positions at WVU. Additionally, mid-funding cycle graduation of the current group of junior investigators will free up funds to provide startup packages for new recruits. These strategic appointments have already resulted in the active recruitment to WVU of 13 new faculty in stroke research in Phase I. Specific Aim 3: To continue a formative and summative evaluation strategy with specific milestones. Achieving these aims will launch the JIs as independent scientists in stroke research fields and provide a foundation of professional development for their long-term academic career success. Accomplishing these goals will position the WVU Stroke CoBRE to become a national center of excellence in stroke research that addresses the issue of stroke and its co-morbidities in West Virginia (WV) and the nation. The CoBRE mechanism will increase the number of stroke researchers, expand our capacity to programmatically address important issues in stroke and enhance our vital research cores that serve the stroke, cardiovascular and neuroscience communities at WVU and across the state while meeting the nation?s need for innovative approaches to this devastating disease.