Roberta Diaz Brinton - US grants

Neuromodulation provides a mechanism whereby the nervous system can expand the range of both its responsiveness to afferent signals and its repertoire of efferent responses. Modulation of both input and output properties of the nervous system significantly contributes to the complexity of mammalian behavior. The primary goal of this research project is to use the vasopressin (AVP) neuromodulation of norepinephrine (NE)-induced accumulation of cAMP interaction in the hippocampus as neuromodulatory model to test the hypothesis that in some instances neuromodulatory can serve as a biochemical analog of the associative processes which appear to govern learning and memory. If this postulate is true, then the factors which govern electrophysiological associative processes, namely long-term potentiation, should also hold true for the biochemical analog. To test this hypothesis, five specific aims related to associative processes will be determined over a five year period: (1) Determination of the anatomical and spatial relationship between recognition sites for AVP and NE in the hippocampus? (2) Determination of the temporal relationship between activation of receptors for AVP and NE and the subsequent expression of neuromodulation? (3) Determination of the mechanism underlying the calcium/calmodulin dependency of AVP-induced neuromodulation? (4) Determination of the influence of AVP and NE singly and in combination on the induction of long-term potentiation in the dentate gyrus of the hippocampus? (5) Determination of the effect of AVP and NE singly and in combination on the growth and morphology of cultured hippocampal and dentate gyrus neurons. Information derived from this project will lead to insights which will have significance from basic science, theorectical and applied perspectives.

minority institution research support; secondary schools;

Neuromodulation provides a mechanism whereby the nervous system can expand the range of both its responsiveness to afferent signals and its repertoire of efferent responses. Modulation of both input and output properties of the nervous system significantly contributes to the complexity of mammalian behavior. The primary goal of this research project is to use the vasopressin (AVP) neuromodulation of norepinephrine (NE)-induced accumulation of cAMP interaction in the hippocampus as neuromodulatory model to test the hypothesis that in some instances neuromodulatory can serve as a biochemical analog of the associative processes which appear to govern learning and memory. If this postulate is true, then the factors which govern electrophysiological associative processes, namely long-term potentiation, should also hold true for the biochemical analog. To test this hypothesis, five specific aims related to associative processes will be determined over a five year period: (1) Determination of the anatomical and spatial relationship between recognition sites for AVP and NE in the hippocampus? (2) Determination of the temporal relationship between activation of receptors for AVP and NE and the subsequent expression of neuromodulation? (3) Determination of the mechanism underlying the calcium/calmodulin dependency of AVP-induced neuromodulation? (4) Determination of the influence of AVP and NE singly and in combination on the induction of long-term potentiation in the dentate gyrus of the hippocampus? (5) Determination of the effect of AVP and NE singly and in combination on the growth and morphology of cultured hippocampal and dentate gyrus neurons. Information derived from this project will lead to insights which will have significance from basic science, theorectical and applied perspectives.

IBN-9511423 Brinton, Roberta F. Learning and memory are complex functions that are dependent upon many brain regions of which the cerebral cortex is one of the principal players. Growth in the projections emanating from cortical neurons is a structural feature shown to be associated with learning and memory. In fact, a chemical messenger, vasopressin has been shown to enhance memory functions and numerous recognition sites have been found for vasopressin in the cerebral cortex. However, there is little functional information on the role of vasopressin within the cortex. Dr. Brinton will determine whether activation of vasopressin recognition sites leads to an increase in the growth of nerve cells from the cerebral cortex. Additional studies will determine whether vasopressin modulates the function of critical support cells in the brain, which markedly influence the growth and survival of cortical nerve cells. These studies will provide a functional basis for understanding how messenger molecules influence learning and memory by altering cellular physiology in the cortex.

The broad goal of this research is to discover estrogenic steroids with neurotrophic activity that will promote the growth and survival of neurons derived from brain regions involved in cognitive function. Germination this proposal began when we discovered several estrogenic steroids that exhibit relatively low affinity for the uterine estrogen nuclear receptor (122-126), which could reduce the risk of breast and uterine cancer, but which exerted a neurotrophic effect similar to what we had observed for the endogenous human female estrogen, and beta-estradiol. (3) Of particular interest was the neurotrophic activity of the equine estrogen, equilin. Equilin is a major component of Premarin, a complex formulation of at least 10 different conjugated equine estrogens, that is currently the leading prescribed pharmaceutical for estrogen replacement therapy in postmenopausal women and is currently under study in 8,000 women participating in the multicenter Women's Health Initiative Memory Study. (59) The proposed studies are designed to test the overall hypothesis that steroids which are estrogenic in chemical structure will influence neuronal outgrowth and survival with varying efficacies depending on their molecular structure. Further, the basis for the different efficacies and potencies of estrogen steroids on neuronal outgrowth will be reflected in their ability to regulate receptor channel function. We further hypothesize that mechanisms that promote an increase in intracellular calcium will underlie estrogenic steroid-induced neurotropism. Lastly, we hypothesize that estrogenic steroids that exert a neurotrophic effect will also promote neuronal survival by enhancing viability and thereby will enhance the capacity of neurons to resist a oxidative stress by beta-amyloid peptide and other free radical generators. To test these hypotheses morphological analyses of dissociated cortical and hippocampal neurons in culture in response to estrogenic steroids and to pharmacological agents that block nuclear estrogen receptors, plasma membrane glutamatergic receptors and protein kinases will be conducted. In addition, whole cell recording will conducted in cultured neurons and slice preparations to analyze the electrophysiological effects of neurotrophic estrogens. Results of these studies will have a direct impact on the use and design of estrogen replacement therapy for the amelioration of cognitive deficits in postmenopausal women and for the prevention of Alzheimer's Disease in this population.

6. Abstract The overall aim of this project is to determine the mechanisms and long-term consequences of estrogen on mitochondrial function and metabolism in brain. Our target outcome is sustaining mitochondrial function to sustain neurological health for prevention of neurodegenerative diseases associated with aging and mitochondrial dysfunction. Results of our previous mechanistic analyses indicate that 17¿-estradiol (E2) protects against dysregulation of mitochondrial calcium homeostasis, enhances mitochondrial function, increases efficiency of oxidative phosphorylation, protects against oxidative damage and regulates the mitochondrial proteome. In this project, we are testing the global hypothesis that E2 sustains mitochondrial function to promote the energetic capacity of brain mitochondria by maximizing aerobic glycolysis (oxidative phosphorylation coupled to pyruvate metabolism). As a corollary to this hypothesis, we propose that in the female brain, E2-induced enhancement of mitochondria oxphos-coupled glycolysis can prevent decreased glucose utilization characteristic of aging and delay its expression in a female mouse model of Alzheimer's disease (AD). To test these hypotheses, in Specific Aim I we will determine the functional consequences of long-term E2 regulation of key enzymes required for aerobic glycolysis and oxidative phosphorylation in brain, the cellular selectivity of E2 regulation of mitochondria in primary neurons and astrocytes and impact of loss of ovarian hormones and E2 on brain metabolism using microPET imaging. In Specific Aim II we will investigate the estrogen receptor subtype and signaling mechanisms whereby E2 induces integrated regulation of the mitoproteome and mitochondrial function. In Specific Aim III we will determine the E2-induced signaling pathway(s) that regulate brain mitochondrial function by investigating (1) the role of estrogen receptor subtypes, the obligatory role of the (2) src/MAPK/CREB and (3) PI3K/Akt pathways and (4) nuclear versus mitochondrial site of action in the E2-induced regulation of the functional mitoproteome. In Aim III, we will determine the therapeutic efficacy of E2 to sustain mitochondrial function and brain metabolism during aging and reproductive senescence, ovarian hormone deprivation in a mouse model of Alzheimer's disease. Results of these discovery efforts will advance our knowledge of estrogen action in brain mitochondria and its impact on development of neurodegenerative pathology. Further, these analyses will generate insights into early events that lead to mitochondrial dysfunction and the compensatory responses that precede it. Together, these findings could identify biomarkers of mitochondrial function and elucidate strategies to intervene at the earliest stages of dysregulation to prevent mitochondrial dysfunction and subsequent neurodegenerative disease.

DESCRIPTION (provided by applicant): Results of the Women's Health Initiative Memory Study which indicated an increased risk of Alzheimer's disease (AD) in women treated with hormone therapy (HT) led to a reevaluation of the benefits versus harm of hormone interventions. As part of this reevaluation, disparities between the beneficial outcomes of estrogen (E2, ET) and progesterone (P4) found in basic science analyses and the adverse outcomes of clinical trials were clear as were the disparities between the generally positive outcomes of ET and HT in observational studies versus the deleterious outcomes of clinical trials. The Progesterone in Brain Aging and Alzheimer's Disease Program Project is designed to address key elements hypothesized to underlie these disparities as well as addressing the paucity of knowledge regarding the neurobiology of progesterone (P4) action in brain regions required for cognition and vulnerable to age associated degenerative disease such as Alzheimer's. The over arching hypothesis of our program is that the ovarian steroid hormone progesterone promotes the brain's molecular, synaptic, cellular, and behavioral plasticity and reduces its vulnerability to the development of Alzheimer's disease (AD). Specifically, we hypothesize that progesterone has direct effects via activation of progesterone receptors in hippocampus and indirect effects via interaction with estrogen pathways. We further hypothesize that the reproductive endocrine status, duration of ovariprivation and presence of AD related pathology regulates the plasticity response to ovarian steroids. We have designed the Program Project to investigate P4 action at 7 levels of function from molecular to behavioral to exacerbation of AD-like pathology in vivo. Embedded in each proposal is an assessment of: (1) P4 receptor expression;(2) direct effect of P4 on neural plasticity;(3) clinically relevant progestins for their comparability to P4;(3) change in response to E2 and P4 with reproductive endocrine status, age or duration of ovariprivation and (4) E2 and P4 regulation of neural plasticity or inflammatory responses in a triple transgenic mouse model of Alzheimer's disease. Our long-term goals are three fold: (1) To establish a foundation of P4 neurobiology in brain regions required for cognitive function and vulnerable to Alzheimer's disease. (2) To develop rodent models of human perimenopause and menopause for both our investigations and those within the field of women's health. (3) To provide insights into the basis of disparities between basic science outcomes and clinical trial outcomes as well as the disparities between observational studies and clinical trials of HT and AD.

Project 3: Progesterone and Neurogenic Plasticity in Aging and AD. In this Program Project, we jointly hypothesize that the sex steroid hormone progesterone (P4) promotes the brain's molecular, synaptic, cellular, and behavioral plasticity and reduces its vulnerability to the development of Alzheimer's disease (AD). We further hypothesize that the reproductive endocrine status, duration of ovariprivation and presence of AD related pathology regulates the plasticity response to ovarian steroids. The potential neural benefits of hormone therapy is constrained, at least in part, by our currently limited knowledge of the (1) basic neurobiology of P4, (2) the neural response to clinically relevant progestogens, (3) the complex modulatory interactions between progesterone and estrogen and (4) the impact of extended ovarian hormone privation and AD neuropathology on the brain's responsiveness to progesterone and estrogen. The objectives of Project 3 are to determine 1) the neurogenic potential of progestogens; 2) their associated mechanism of neurogenic action; 3) the impact of age and endocrine status on ovarian hormone- induced neurogenesis; and 4) the impact of Alzheimer's disease pathology progression on gonadal hormone regulated neurogenesis. To achieve these objectives, we have developed an interactive research plan that capitalizes on the expertise throughout the program project. Consistent with the Specific Aim structure of all projects within the Program, Specific Aim 1 will determine the direct effect of P4 on neurogenesis and mechanisms of action. Specific Aim 2 will determine the proliferative efficacy of clinically relevant progestins. Specific Aim 3 will determine P4 regulation of 17 beta-estradiol (E2)-induced neurogenesis. Specific Aim 4 will determine the impact of E2/P4 on neurogenesis in rat models of human perimenopause and menopause. Specific Aim 5 will determine the impact of E2/P4 on neurogenesis in the SxTgAD mouse model of AD. To conduct these investigations, 5 technological approaches will be extensively utilized: in vitro culture of rat and mouse neural progenitors derived from adult dentate gyrus, fluorescent immunocytochemistry, biochemical analyses of kinase activation and protein expression, gene arrays for cell cycle gene expression and in vivo BrdU labeling followed by unbiased stereology. Because the proposed studies were designed to address issues of clinical relevance, results of these analyses should contribute to our (1) basic science understanding of progesterone regulation of cellular neurogenic plasticity in the hippocampus and the factors that control this 'response and (2) understanding the basis of disparities across clinical studies of hormone therapy and basic science analyses of E2/P4 in brain.

DESCRIPTION (provided by applicant): The long-range goal of our therapeutic development endeavors is to develop efficacious and safe agents to prevent and/or delay progression of age-related neurodegenerative disease such as Alzheimer's. Toward that end, we propose a preclinical project to develop the neurosteroid, allopregnanolone as a neurogenic regenerative therapeutic. The current proposal is a translational therapeutic development project to conduct preclinical analyses required for an Investigational New Drug (IND) application to the FDA to determine the efficacy of allopregnanolone as a neurogenic regenerative agent. Preclinical IND studies proposed herein build upon our foundation of basic science discovery and mechanistic understanding of allopregnanolone action in neural progenitor cells. To conduct the proposed project, we have assembled an interdisciplinary team of scientists with expertise in 1) neurobiology of allopregnanolone (Brinton USC research team), 2) behavioral analyses to determine therapeutic efficacy on learning and memory functions affected in mild cognitive impairment and Alzheimer's disease (PsychoGenics), and 3) pharmacokinetics, pharmacodynamics, toxicology, therapeutic formulation, regulatory affairs and IND document preparation (Stanford Research Institute International;SRI) 4). In addition, we have assembled a team of Consultants with expertise in development of therapeutics for AD within the following domains: 1) AD therapeutic development within the pharmaceutical industry, 2) regulatory affairs, 3) pharmacokinetics and dynamics, 4) therapeutic formulation, 5) design of clinical trials and 6) cognitive and neurological deficits diagnostic of mild cognitive impairment and AD. Consistent with preclinical analyses required for an FDA IND application, we propose four IND related Specific Aims and one project management Specific Aim. Together these aims are designed to [I] Determine therapeutic efficacy of allopregnanolone and impact of age and gender on efficacy in the triple transgenic mouse model of Alzheimer's;[II] Determine pharmacodynamics, pharmacokinetics (ADME) and toxicology of allopregnanolone;[III] Acquire cGMP quality allopreganaolone for Phase I Clinical Trial;[IV] Generation of FDA Investigational New Drug Application and Clinical Investigator Brochure documents;and [V] Project Management to achieve IND filing goal. Each Specific Aim is designed to address preclinical IND requirements and each is milestone driven with clearly articulated Go / no-Go decision criteria.

The mission of our Perimenopause in Brain Aging and Alzheimer's Disease Program Project is to discover the biological transformafions that occur in the brain during the perimenopausal transifion that can result in phenotypes predictive of risk for development of AD pathology. The mission of Project 1 is to determine the perimenopausal bioenergetic phenotypes most at risk for developing bioenergetics predictive of AD pathology;delineate the mechanistic pathways involved in development of the at-AD-risk phenotype;and assess the impact of ovarian hormone and nutritional interventions on expression of bioenergetic biomarkers of AD. Ufilizing rodent models of human perimenopause. Project 1 will achieve its mission by determining; (1) in Aim 1 the bioenergefic phenotypes induced by the perimenopausal transition, (2) in Aim 2 the mechanisms that generate the at-AD-risk phenotype and (3) in Aim 3 the window of opportunity to prevent bioenergefic at-AD-risk phenotype. To address our hypotheses, we will experimentally determine bioenergefic gene expression across the perimenopausal transifion and monitor indicators of mitochondrial function which will include: bioinformafics network analyses of gene expression, in vivo cerebral glucose metabolism and synapfic transmission. Mechanisfic analyses will address (1) hypometabolism and cell- specific metabolic profiles, (2) redox control of bioenergefic pathway, and (3) white matter loss. Project 1 collaborates with Projects 2, 3 and 4 to test the hypothesis that decline in brain bioenergetics leads to acfivafion of the infiammatory response in brain (Project 2) which exacerbates mitochondrial funcfion which leads to development of AD pathology (Project 3) and mitochondrial dysfunction is associated with cognifive decline in women assessed through ancillary analyses of the ELITE NIA sponsored clinical trial (Project 4). Outcomes of Project 1 research will include: (1) basic science discovery ofthe bioenergefics ofthe perimenopausal aging transifion in brain regions vulnerable to development of AD pathology;(2) mechanistic pathways that lead to decline in bioenergetics in the perimenopausal brain;(3) translational research discovery of the window of opportunity for ovarian hormone intervenfion to prevent decline in bioenergetics in the perimenopausal brain and (4) clinical associafions of bioenergefic gene expression, mitochondrial funcfion in peripheral blood cells and cognitive decline in postmenopausal women. RELEVANCE (See instructions): More than 60% of Alzheimer's disease (AD) patients are women. Project 1 of our Perimenopause Program Project will determine the impact of the perimenopause transition on the ability of the brain to generate the energy required for cognitive function and to prevent development of Alzheimer's. Project 1 research will also determine the window of opportunity for hormone therapy to prevent decline in brain energy to reduce the risk of Alzheimer's.

The mission of our Perimenopause in Brain Aging and Alzheimer's Disease Program Project (P3) is to discover the biological transformations that occur in the brain during the perimenopausal transition which can result in phenotypes predicfive of risk for development of Alzheimer's pathology. We seek to identify the mechanisms by which these changes occur, and translate these discoveries to determine the opfimal timing and strategies for preventing conversion to the perimenopausal at-risk phenotype. To achieve our Perimenopausal Program Project mission, we will determine the perimenopausal phenotypes most at risk for developing biomarkers of Alzheimer's disease; delineate the mechanistic pathways involved in development of these phenotypes; and assess the impact of ovarian hormone and nutritional interventions on expression of Alzheimer's disease biomarkers. The mission of Analytic Core is to provide standardized steroid hormone regimens, sample archiving, processing and distribution, as well as analytic support across the Perimenopausal Program Project. To achieve this mission. Analytic Core will provide two levels of support. The first level of support entails Program-wide standardized steroid hormone regimens and sample management, which includes sample procuring, banking, distribufing, sample information recording through the central Integrafive Data Management System, and protein and RNA preparations (Specific Aim 1). The second level of support entails sample analysis and data interpretation on three major studies: (1) LC- MS/MS steroid hormone analysis (Specific Aim 2); (2) custom Taqman low-density array gene expression analysis (Specific Aim 3); and (3) bioinformafic gene funcfional analysis (Specific Aim 4). Analytic Core was an instrumental and essential resource for the currentiy ongoing Progesterone Program Project. Through the infrastructure and analytic methods it has developed. Analytic Core will continue to play a crucial role in achieving the Perimenopausal Program Project's missions and aims. RELEVANCE (See instructions): Women have a higher lifetime risk of developing Alzheimer's disease (AD) and represent -60% of the AD population. Discovery of at-risk phenotypes and the underiying mechanisms of phenotype development at perimenopausal transition could potentially lead to the eariy identification of those at greatest risk of developing AD and intervenfions to prevent the disease.

The goal of our Program Project is to discover the biological transformations that occur in the brain during the perimenopausal transition that can result in phenotypes predictive of risk for development of AD pathology. In Project 3, our specific emphasis is the neuroprotective actions of ovarian hormones on AD pathogenesis. Menopause is characterized in part by depletion of ovarian hormones. We hypothesize that menopause induces neural changes that attenuate the established protective effects of estradiol and progesterone against pathways associated with AD pathogenesis. Menopause is also linked with increases in body weight and adiposity that often lead to obesity and metabolic syndrome, conditions that are established risk factors for the development of AD. Significantly, adiposity and obesity are not only regulated by ovarian hormones, but also are known to impair bioenergetics and increase inflammation. Thus, perimenopause results in adverse changes to both ovarian hormones and adiposity, which we theorize interact cooperatively in the promotion on AD pathogenesis via their effects on bioenergetic, inflammatory, and AD pathways. To investigate these relationships, we propose three specific aims that are highly collaborative across all cores and projects. Specific Aim 1: Prodromal phenotypes in rat and mouse models of human perimenopause/menopause. We will characterize the effects of reproductive aging on AD genes and pathways using rodent models of perimenopause. Specific Aim 2: How does obesity interact with perimenopause in the regulation of bioenergetic, inflammatory, and Alzheimer pathways? We will determine the effects of diet-induced obesity on AD pathways and how they interact with reproductive aging. Specific Aim 3: Perimenopausal hormone intervention: timing and efficacy for protection against Alzheimer pathways. We will define the window of opportunity for delivering estradiol and progesterone hormone therapy that effectively protects against AD pathways in our rodent models of perimenopause using both rats and the 3xTg-AD mice.

The mission of our Perimenopause in Brain Aging and Alzheimer's Disease Program Project (P3) is to discover the biological changes that occur in the brain during the perimenopausal transition which can result in phenotypes predictive of risk for development of Alzheimer's pathology. We seek to identify the mechanisms by which these changes occur, and translate these discoveries to determine the optimal timing and strategies for preventing conversion to the perimenopausal at-risk phenotype. To achieve our mission, we will determine the perimenopausal phenotypes associated with different cycle transition states; delineate the mechanistic pathways involved in cJevelopment of these phenotypes; and assess the impact of ovarian hormone and high-fat diet induced obesity (DIO) on expression of Alzheimer's disease biomarkers. The mission of Animal Core (Core B) is to ensure the success ofthe Perimenopause Program Project through provision of animals as neecled to Projects 1-3. To achieve its mission. Animal Core will develop rodent models of perimenopause and menopause; maintain and track animals from acquisition or birth, though determination of cycling status, study enrollment, experimental manipulation, to fissue collection across the entire Perimenopause Program Project; and obtain tissue samples for storage and genetic analyses by Analytic Core. RELEVANCE (See Instructions): The complexity of the perimenopausal process has been a major barrier to both basic and clinical research of this aging transition in women. Development of rodent models ofthe human perimenopause is critical to advancing knowledge ofthe effects ofthe perimenopause on the brain, identifying phenotypes at risk for age-associated neurological diseases, and developing strategies that promote healthy neurological aging in women to prevent age-associated diseases such as Alzheimer's.

A substantial number of the more than 45 million postmenopausal US women will develop dementia or cognitive impairment. Our Perimenopause in Brain Aging and Alzheimer's Disease Program Project seeks to discover the biological transformations occurring in the brain during the perimenopausal transition which can result in phenotypes predictive of risk for development of Alzheimer's disease (AD) pathology and to identify the mechanisms by which these changes occur, and translate these discoveries to determine the optimal timing and strategies for preventing conversion to the perimenopausal at-risk phenotype. Project 4 will characterize biological profiles from the perimenopausal and postmenopausal periods, and evaluate the association of these profiles with cognition in women who are in eariy vs. late menopause. Project 4 will (1) determine whether biomarkers hypothesized to be important in the perimenopausal period and in AD characterize the postmenopausal human female in eariy vs. later menopause; (2) determine whether and to what extent these biomarkers are associated with cognition; and (3) evaluate whether administration of menopausal hormone therapy will modify these markers. Project 4 will use data and stored tissue samples from the NIA-funded Eariy versus Late Intervention Trial with Estrogen (ELITE) (R01AG-024154) trial to test four hypotheses: (1) The perimenopause transition results in the bioenergetic and inflammatory phenotype consistent with biomarkers of Alzheimer's disease risk; (2) the perimenopause transition will result in multiple phenotypes, subgroups of which will predict risk of developing biomarkers of Alzheimer's disease; (3) the perimenopause transition is a critical window that determines ovarian hormone response and their role in prevention vs. increased risk of developing eariy biomarkers of AD; and (4) ovarian hormone and bioenergetic interventions can modify development of biomarkers of Alzheimer's disease in the perimenopausal at-risk phenotype. This Project will work closely with Analytic Core and Administrative Core on processing samples and data, and with Projects 1-3 on bioenergetic, inflammatory, and dietary analyses. RELEVANCE (See instructions): More than 60% of Alzheimer's disease (AD) patients are women. This phase of our Perimenopause Program Project seeks to evaluate, identify and prevent perimenopause-related brain changes predictive of increased AD risk. Project 4 will determine whether flushing and the timing and duration of perimenopause are associated with increased risk of cognitive decline. Project 4 will evaluate the impact of hormonal and dietary interventions on cognitive function, and identify a treatment window to reduce risk of cognitive decline.

Alzheimer disease (AD) involves inflammatory processes at many levels. Because ovarian steroids show many anti-inflammatory activities, hormone therapy (HT) in AD may involve inflammatory pathways activated during the menopause transitions when hormonal deficits emerge progressively. We propose rodent models of perimenopausal stages to analyze inflammatory changes in brain regions vulnerable to AD, with a focus on aging Sprague-Dawley rats. The 3xTg-AD mouse model will be used to examine specific hypothesis about relationships of inflammation during aging to human familial AD. These models will be examined for responsiveness to HT, according to the KEEPS clinical model of E2 + cyclic P4. We will examine E2-P4 effects on microglial-astrocyte interacfions in inflammation and in neurotrophic effects. Specific Aim 1: Inflammatory profile in rat and mouse models of human perimenopause and menopause stages (Animal Core B). 1.1. Inflammatory gene expression and bioinformatics analysis in hippocampus and cerebral cortex of aging rats and mice from different menopausal stages (Analytic Core). 1.2. In vitro modeling of perimenopausal inflammatory changes, using primary glial cultures originated from perimenopausal rats. 1.3. Bioenergetics of glia cultured from perimenopausal rats (with Project 1). Specific Aim 2: Neurodegenerative mechanisms in perimenopausal inflammatory changes, using glial- neuronal co-cultures. 2.1. Role of inflammatory genes in glial-mediated neuronal support. 2.2, Role of estrogen receptors (ERa:ER|3) in regulating perimenopausal glial inflammatory phenotypes. Astrocytes from aging rats are deficient in neurotrophic support, in which one factor is altered ratio of estrogen receptors ERa:ERp (pilot data). 2.3. Role of progesterone receptors (PR) in regulating perimenopausal glial inflammatory changes, with a focus on Pgmrc1 (pilot data). 2.4. Role of glial N0S2 in neuronal mitochondria dysfunction (with Project 1). 2.5. Obesity-inflammation interactions (with Project 3). Specific Aim 3: Effects of perimenopausal HT on brain cell inflammation. Following the KEEPS model of E2 + cyclic P4, rats and 3xTg-AD mice will be given HT during perimenopausal cycling stages. 3.1. Inflammatory gene expression and bioinformatics (Analytic Core). 3.2. In vitro models of inflammatory changes, using primary glial from aging rat cerebral cortex. 3.3. Bioenergetics of glial cultures (with Project 1).

DESCRIPTION: Therapeutics to prevent, delay and treat Alzheimer's disease (AD) remains to be achieved. Currently, over 5 million Americans are diagnosed with AD and the number is projected to increase to 11-16 million within two decades unless therapeutic advances are made. Proposed herein is a regenerative medicine, systems biology approach that targets the regenerative system of the brain while simultaneously activating systems to reduce AD pathology. Allopregnanolone (Allo) is a pleiotropic regenerative therapeutic that promotes neurogenesis and restores cognitive function in both a preclinical AD model and wild type aged mice and reduces pathology in a preclinical AD model. Further Allo promotes regeneration of human neural stem cells. Allo is a neurosteroid endogenous to the brain of low molecular weight and blood brain barrier penetrant with abundant existing safety data in animals and humans. Its mechanisms of neural stem cell proliferation and restoration of cognitive function are well characterized and consistent with well-described neurogenic mechanisms in brain. Allo reduces AD pathology via well-established pathways upstream to Abeta generation to prevent the generation of Abeta while also decreasing inflammation and increasing myelin generation. Based on a foundation of preclinical discovery (ADDF), translational research (NIA U01), clinical development with NIA USC ADRC and FDA assessment, we propose a Phase 1 multiple ascending dose clinical trial of four Allo doses administered in a regenerative regimen of once-per-week for twelve weeks to establish a safe and tolerated dose of Allo necessary to advance to a Phase 2 efficacy trial. To achieve this goal, two specific aims are proposed. Aim 1 is designed to conduct a Phase 1 multiple ascending dose trial of Allo in participants diagnosed with MCI due to AD and early AD. Primary safety objectives are to determine: 1) maximally tolerated dose; 2) incidence and severity of treatment emergent adverse events; 3) designated medical events; 4) clinically important changes in safety assessments including amyloid related imaging abnormalities (ARIA). Aim 2 is designed to conduct exploratory safety and feasibility analyses regarding the effect of once- per-week-exposure for 12 weeks to Allo at 4 doses on cognitive function and MRI-based biomarkers. Secondary objectives are to: 1) assess potential short-term effects of Allo dosing on cognition and MRI indicators of AD; 2) inform subsequent phase 2 proof of concept trial with MRI-based biomarkers of regenerative efficacy. A multidisciplinary team of investigators with expertise in Allo systems biology and translational research and clinical trials for AD therapeutics are committed to the project. Trial outcomes will provide: 1) an estimated safe and well-tolerated dose of Allo; 2) parameter estimates for cognitive efficacy to advance to a Phase 2 proof of concept trial of Allo; and 3) parameter estimates for MRI-based biomarkers. This proposal meets the objectives of RFA-AG-13-016.

? DESCRIPTION (provided by applicant): The urgency for therapeutics to prevent, delay and treat Alzheimer's is evident national and global as the epidemic of this disease grows with every passing minute. The overarching goal of the proposed planning grant is to address this need by building the foundation of intellectual, technological and infrastructure resources necessary to create an Alzheimer's Disease Translational Center for Predictive Drug Development. To capture the function of this future center, we refer to it as SysPharmRx-AD (Systems Pharmacology Therapeutics for Alzheimer's Disease). The SysPharmRx-AD vision is a portfolio of highly effective therapeutics personalized to etiology and stage of Alzheimer's disease. Ultimately, the mission of SysPharmRx-AD will be to provide expertise and resources that enable Alzheimer's researchers at all levels of translation from discovery to translational to clinical to effectively and efficiently develop therapeutics for AD. Goals of the planning grant wil be achieved through three Specific Aims designed to successively build the network of enabling technologies, infrastructure and organizational management required for SysPharmRx-AD, an Alzheimer's Disease Translational Center for Predictive Drug Development. Specific Aim 1. Determine the enabling technologies and expertise required for identifying phenotypes of Alzheimer's disease risk, etiology of disease, stage of progression and systems based therapeutic targets. Objective of this aim is to develop the strategies required to know whom to treat, when to treat and what outcomes to measure as per NIA Alzheimer's Disease Summit recommendations. Specific Aim 2: Determine the enabling technologies and expertise required for translating systems biology targets into systems pharmacology based therapeutics. Objective of this aim is to develop quantitative systems pharmacology strategies for predictive drug development, whether novel or repurposed, that address the complex etiologies and progressive continuum of Alzheimer's disease, i.e. whom to treat, when to treat and with what to treat NIA Alzheimer's Disease Summit recommendations. Specific Aim 3. Develop the organizational structure and function required to network enabling expertise, resources and infrastructure to create SysPharmRx-AD. Objectives of this aim are to: a) integrate capacities and resources to ensure seamless functionality and communication within SysPharmRx-AD that enable rapid and broad public sharing of data, analytical and research tools, and models prior to publication and b) address barriers to public private partnerships between academic, pharmaceutical / biotech and Alzheimer's philanthropic organizations to enable SysPharmRx-AD mission.

DESCRIPTION (provided by applicant): Therapeutics to prevent, delay and treat Alzheimer's disease (AD) remains to be achieved. Currently, over 5 million Americans are diagnosed with AD and the number is projected to increase to 11-16 million within two decades unless therapeutic advances are made. Proposed herein is a regenerative medicine, systems biology approach that targets the regenerative system of the brain while simultaneously activating systems to reduce AD pathology. Allopregnanolone (Allo) is a pleiotropic regenerative therapeutic that promotes neurogenesis and restores cognitive function in both a preclinical AD model and wild type aged mice and reduces pathology in a preclinical AD model. Further Allo promotes regeneration of human neural stem cells. Allo is a neurosteroid endogenous to the brain of low molecular weight and blood brain barrier penetrant with abundant existing safety data in animals and humans. Its mechanisms of neural stem cell proliferation and restoration of cognitive function are well characterized and consistent with well-described neurogenic mechanisms in brain. Allo reduces AD pathology via well-established cholesterol clearance pathways upstream to prevent the generation of Abeta while also decreasing inflammation and increasing myelin generation. Proposed herein is a program of translational IND-enabling toxicological and safety analyses required to advance to a Phase 2 clinical trial of the neurosteroid, allopregnanolone (Allo), for the treatment of persons with MCI due to Alzheimer's disease (AD) or early AD. FDA IND #113,772 is approved for a Phase 1 clinical trial with additional chronic exposure safety analyses required to advance to a Phase 2 clinical trial. Aims proposed within this U01 application specifically address FDA guidance as well as an Aim focused on regulatory documentation and design of the Phase 2 trial. Each specific aim is milestone driven with clearly articulated Go / no-Go decision criteria. Aims I and II will be conducted in two species. Specific Aim I is designed to conduct a nine-month chronic toxicology study to determine the toxicokinetic and safety profiles for intramuscularly administered allopregnanolone. Specific Aim II is designed to conduct a six-month chronic toxicology to determine the toxicokinetic and safety profiles for intramuscularly administered allopregnanolone. Specific Aim III is designed to determine the risk of cerebral micro-hemorrhages after exposure to once per week allopregnanolone in aged mouse model of AD. Specific Aim IV is designed to conduct regulatory assessments and generate documentation for submission to FDA and to generate Phase 2 clinical trial design and plan. A multidisciplinary team of investigators with expertise in Allo systems biology, translational research and clinical trials for AD therapeutics are committed to the project. Outcomes of these analyses will support and advance therapeutic development of Allo to a Phase 2 clinical trial in persons with MCI due to AD and early AD.

PROJECT SUMMARY-ADMINISTRATIVE CORE A The mission of our Perimenopause in Brain Aging and Alzheimer's Disease Program Project (P3) is to discover the biological transformations that occur in the brain during the perimenopausal transition, which can result in phenotypes predictive of risk of development of Alzheimer's pathology. We seek to identify mechanisms by which these changes occur and translate these discoveries to determine optimal timing and strategies for preventing conversion to the perimenopausal at-risk phenotype. Success in achieving the mission of Perimenopause program project demands a high degree of coordination, intellectual synergy, collaboration and communication across and among all Projects and Cores. Administrative Core A (Admin Core A) provides a structural organization to facilitate timely and efficient communication and integrative links among Projects and Cores. Admin Core A will ensure the success of P3 through effective leadership, the provision of data management and biostatistical resources, and the forging of partnerships to synergize efforts and maximize resources. Admin Core A specific aims are: (1) Specific Aim 1: Lead and administer Perimenopause in Brain Aging and Alzheimer's Disease Program Project (manage and steward intellectual, technological and financial resources, identify and overcome barriers to progress, create liaisons with similarly-focused research and training programs); (2) Specific Aim 2: Provide organizational systems for data management and communication within the program project members and external collaborators (maintain a web-based data entry, data management, animal- and specimen tracking system, and transition experimental results to REDCap database); (3) Specific Aim 3: Program-wide integrated statistical analysis and resource sharing plan (provide biostatistical consulting in the design, coordination, and analyses of projects, oversee gene array and bioinformatics data management on program data management system, integrate gene expression profile across program; implement resource sharing plan).

DESCRIPTION (provided by applicant): Therapeutics to prevent, delay and treat Alzheimer's disease (AD) remains to be achieved. Currently, over 5 million Americans are diagnosed with AD and the number is projected to increase to 11-16 million within two decades unless therapeutic advances are made. Proposed herein is a regenerative medicine, systems biology approach that targets the regenerative system of the brain while simultaneously activating systems to reduce AD pathology. Allopregnanolone (Allo) is a pleiotropic regenerative therapeutic that promotes neurogenesis and restores cognitive function in both a preclinical AD model and wild type aged mice and reduces pathology in a preclinical AD model. Further Allo promotes regeneration of human neural stem cells. Allo is a neurosteroid endogenous to the brain of low molecular weight and blood brain barrier penetrant with abundant existing safety data in animals and humans. Its mechanisms of neural stem cell proliferation and restoration of cognitive function are well characterized and consistent with well-described neurogenic mechanisms in brain. Allo reduces AD pathology via well-established cholesterol clearance pathways upstream to prevent the generation of Abeta while also decreasing inflammation and increasing myelin generation. Proposed herein is a program of translational IND-enabling toxicological and safety analyses required to advance to a Phase 2 clinical trial of the neurosteroid, allopregnanolone (Allo), for the treatment of persons with MCI due to Alzheimer's disease (AD) or early AD. FDA IND #113,772 is approved for a Phase 1 clinical trial with additional chronic exposure safety analyses required to advance to a Phase 2 clinical trial. Aims proposed within this U01 application specifically address FDA guidance as well as an Aim focused on regulatory documentation and design of the Phase 2 trial. Each specific aim is milestone driven with clearly articulated Go / no-Go decision criteria. Aims I and II will be conducted in two species. Specific Aim I is designed to conduct a nine-month chronic toxicology study to determine the toxicokinetic and safety profiles for intramuscularly administered allopregnanolone. Specific Aim II is designed to conduct a six-month chronic toxicology to determine the toxicokinetic and safety profiles for intramuscularly administered allopregnanolone. Specific Aim III is designed to determine the risk of cerebral micro-hemorrhages after exposure to once per week allopregnanolone in aged mouse model of AD. Specific Aim IV is designed to conduct regulatory assessments and generate documentation for submission to FDA and to generate Phase 2 clinical trial design and plan. A multidisciplinary team of investigators with expertise in Allo systems biology, translational research and clinical trials for AD therapeutics are committed to the project. Outcomes of these analyses will support and advance therapeutic development of Allo to a Phase 2 clinical trial in persons with MCI due to AD and early AD.

DESCRIPTION (provided by applicant): Therapeutics to prevent, delay and treat Alzheimer's disease (AD) remains to be achieved. Currently, over 5 million Americans are diagnosed with AD and the number is projected to increase to 11-16 million within two decades unless therapeutic advances are made. Proposed herein is a regenerative medicine, systems biology approach that targets the regenerative system of the brain while simultaneously activating systems to reduce AD pathology. Allopregnanolone (Allo) is a pleiotropic regenerative therapeutic that promotes neurogenesis and restores cognitive function in both a preclinical AD model and wild type aged mice and reduces pathology in a preclinical AD model. Further Allo promotes regeneration of human neural stem cells. Allo is a neurosteroid endogenous to the brain of low molecular weight and blood brain barrier penetrant with abundant existing safety data in animals and humans. Its mechanisms of neural stem cell proliferation and restoration of cognitive function are well characterized and consistent with well-described neurogenic mechanisms in brain. Allo reduces AD pathology via well-established cholesterol clearance pathways upstream to prevent the generation of Abeta while also decreasing inflammation and increasing myelin generation. Proposed herein is a program of translational IND-enabling toxicological and safety analyses required to advance to a Phase 2 clinical trial of the neurosteroid, allopregnanolone (Allo), for the treatment of persons with MCI due to Alzheimer's disease (AD) or early AD. FDA IND #113,772 is approved for a Phase 1 clinical trial with additional chronic exposure safety analyses required to advance to a Phase 2 clinical trial. Aims proposed within this U01 application specifically address FDA guidance as well as an Aim focused on regulatory documentation and design of the Phase 2 trial. Each specific aim is milestone driven with clearly articulated Go / no-Go decision criteria. Aims I and II will be conducted in two species. Specific Aim I is designed to conduct a nine-month chronic toxicology study to determine the toxicokinetic and safety profiles for intramuscularly administered allopregnanolone. Specific Aim II is designed to conduct a six-month chronic toxicology to determine the toxicokinetic and safety profiles for intramuscularly administered allopregnanolone. Specific Aim III is designed to determine the risk of cerebral micro-hemorrhages after exposure to once per week allopregnanolone in aged mouse model of AD. Specific Aim IV is designed to conduct regulatory assessments and generate documentation for submission to FDA and to generate Phase 2 clinical trial design and plan. A multidisciplinary team of investigators with expertise in Allo systems biology, translational research and clinical trials for AD therapeutics are committed to the project. Outcomes of these analyses will support and advance therapeutic development of Allo to a Phase 2 clinical trial in persons with MCI due to AD and early AD.

ABSTRACT: Numerous clinical trials failed to modify progression of Alzheimer's Disease (AD) highlighting our incomplete knowledge of the underlying molecular mechanisms. Increasing evidence suggests that AD is a metabolic dis- order with perturbations in pathways essential for cellular energetics occurring early in the disease process: changes in glucose utilization in the brain, a hallmark of AD, correlate well with early clinical disease manifestation; the APOE £4 allele, the strongest genetic risk factor for sporadic AD, is known to affect lipid and cholesterol metabolism; over twenty additional genes linked to AD are implicated in cellular metabolism. Sex has also been demonstrated to play a major role in risk, pathogenesis, progression, and clinical manifestations of AD: women with AD tend to exhibit a broader spectrum of dementia-related behavior and experience greater cognitive deterioration compared to men; female brains are more predisposed to develop AD, and APOE £4, among other genetic risk factors, confers stronger risk in women compared to men. The identification of the key metabolic alterations and pathways underlying differential progression of AD in males vs. females could aid the development of efficacious and disease-modifying therapeutic strategies for AD. The AD Metabolomics Consortium (ADMC) led by one of the PIs in partnership with the AD Neuroimaging Initiative (ADNI) has embarked on mapping metabolic changes in the periphery and in the central nervous system (CNS) across the trajectory of the disease, placing them within a pathway and network context. Our preliminary data demonstrated that among multiple metabolic alterations in blood, changes in lipid metabolism were most prominent in females with AD while changes in branched-chain amino acids (BCAAs) were prevalent in AD males. Predictive networks accompanied by correlations with CSF and imaging biomarkers identified lipids as important drivers of disease in women while BCAAs were marked as drivers in men. Moreover, studies in healthy cohorts in Europe led by one of the PIs demonstrated that women have much higher levels of lipids in blood, while men show significantly higher blood levels of most amino acids, including BCAAs. Based on these observations, we propose the hypothesis that early changes associated with alternative substrate utilization to compensate for altered brain glucose metabolism discriminate mechanistic pathways involved in the disease progression in males vs. females. To test this hypothesis, we assembled a team of investigators with complementary expertise in systems biology, statistics, network analysis, cellular energetics, and animal models to define sex-specific differences in the etiology of AD on a metabolic level. Our team, that has been working collaboratively over the past several years, brings the power of the large, deeply phenotyped ADNI cohorts, state of the art methods in metabolomics, metabolic network reconstruction, imaging and CSF biomarker analysis, and translational studies in animal models to further define molecular mechanisms of disease in men and women with AD in order to develop and test novel therapeutic strategies.