Christian J. Pike - US grants

DESCRIPTION (from applicant's abstract) Female gender is a risk factor for the development of Alzheimer's disease (AD). Accumulating evidence suggests that the massive reduction in estrogen levels that occurs in women following menopause appears to be the primary variable underlying this risk factor. Importantly, the clinical use of estrogen replacement therapy in postmenopausal women has been demonstrated to both delay the onset of AD and slow its progression. Because estrogen has many cellular effects potentially relevant to a protective role against AD, currently it is unclear what specific estrogen action(s) are salient to its inhibition of AD pathology. In this grant application, the applicants propose a novel neuroprotective mechanism of estrogen and predict that it functions to increase neuronal resilience against degenerative stimuli implicated in AD neurodegeneration. Based upon recent advances in the fields of endocrinology and oncology, they theorize that in estrogen responsive brain regions (e.g., hippocampus, entorhinal cortex, amygdala), estrogen activates its receptors, which initiates a genomic pathway that alters the expression of apoptosis- related proteins. In particular, their preliminary data suggest that estrogen significantly increases expression of the anti-apoptotic protein Bcl-XL. As a consequence of its regulation of apoptosis-related proteins, they theorize that estrogen sways the balance of neuronal apoptotic pathways toward enhanced viability, thereby increasing the resistance of estrogen-responsive neurons to apoptotic degeneration. Thus, the loss of estrogen following menopause is predicted to decrease levels of an important endogenous modulator of neuronal viability, rendering estrogen-responsive brain regions vulnerable to apoptotic challenge. The applicants propose three aims to investigate this novel theory: (1) Using cell culture and in vivo paradigms, they will evaluate estrogen's ability to regulate neuronal expresssion of apoptosis-related proteins. They will identify estrogen target proteins, determine the role of receptor activation and differential receptor subtype response, and examine possible synergism with other apoptosis modulators; (2) They will investigate predictions that functional consequences of estrogen's regulatory actions include decreased activation of specific apoptotic pathways (e.g., caspase-mediated proteolysis) and increased neuronal viability; (3) They will use quantitative image analysis techniques to correlate findings made in experimental systems (Aims 1 and 2) to the normal aged and AD brain. The applicants anticipate that their novel hypotheses will generate new insight into the ability of estrogen to modulate neuronal viability throughout life, from neural development through age-related neurodegenerative disorders.

DESCRIPTION (from applicant's abstract) Female gender is a risk factor for the development of Alzheimer's disease (AD). Accumulating evidence suggests that the massive reduction in estrogen levels that occurs in women following menopause appears to be the primary variable underlying this risk factor. Importantly, the clinical use of estrogen replacement therapy in postmenopausal women has been demonstrated to both delay the onset of AD and slow its progression. Because estrogen has many cellular effects potentially relevant to a protective role against AD, currently it is unclear what specific estrogen action(s) are salient to its inhibition of AD pathology. In this grant application, the applicants propose a novel neuroprotective mechanism of estrogen and predict that it functions to increase neuronal resilience against degenerative stimuli implicated in AD neurodegeneration. Based upon recent advances in the fields of endocrinology and oncology, they theorize that in estrogen responsive brain regions (e.g., hippocampus, entorhinal cortex, amygdala), estrogen activates its receptors, which initiates a genomic pathway that alters the expression of apoptosis- related proteins. In particular, their preliminary data suggest that estrogen significantly increases expression of the anti-apoptotic protein Bcl-XL. As a consequence of its regulation of apoptosis-related proteins, they theorize that estrogen sways the balance of neuronal apoptotic pathways toward enhanced viability, thereby increasing the resistance of estrogen-responsive neurons to apoptotic degeneration. Thus, the loss of estrogen following menopause is predicted to decrease levels of an important endogenous modulator of neuronal viability, rendering estrogen-responsive brain regions vulnerable to apoptotic challenge. The applicants propose three aims to investigate this novel theory: (1) Using cell culture and in vivo paradigms, they will evaluate estrogen's ability to regulate neuronal expresssion of apoptosis-related proteins. They will identify estrogen target proteins, determine the role of receptor activation and differential receptor subtype response, and examine possible synergism with other apoptosis modulators; (2) They will investigate predictions that functional consequences of estrogen's regulatory actions include decreased activation of specific apoptotic pathways (e.g., caspase-mediated proteolysis) and increased neuronal viability; (3) They will use quantitative image analysis techniques to correlate findings made in experimental systems (Aims 1 and 2) to the normal aged and AD brain. The applicants anticipate that their novel hypotheses will generate new insight into the ability of estrogen to modulate neuronal viability throughout life, from neural development through age-related neurodegenerative disorders.

Abundant evidence suggests that the depletion of estrogen in postmenopausal women is a significant risk factor for the development of Alzheimer's disease (AD). As a normal consequence of aging, men also exhibit depletion of their primary sex steroid hormone, testosterone. The reduction in men's androgen levels is manifested clinically as impaired function in numerous androgen-sensitive tissues throughout the body, including the brain. Based on recent evidence from our laboratory, we propose that two neural functions of androgens are promotion of neuron viability and regulation of beta-amyloid protein (A-beta). We predict that impairment of these androgen functions occurring as a result of normal, age-related androgen depletion will place the brain at increased risk for the development of Alzheimer's disease. To investigate this hypothesis, we propose three Specific Aims that utilize complementary cell culture, animal model, and human subjects paradigms. In the Aim 1, we will investigate our hypothesis that androgens are endogenous regulators of neuron viability. Proposed studies will assess the role of androgen receptor in neuroprotection as well as elucidate the responsible downstream signaling cascades. Further, we will examine how age-related androgen depletion affects neuronal vulnerability to injury. In Aim 2, we investigate the hypothesized role of androgens as endogenous modulators of AE, levels. By experimental manipulation of androgen status in animal models, we will evaluate our hypothesis that androgen depletion will result in increased levels of A-beta. Mechanistic studies will evaluate the contributions of androgen receptor activation and androgen regulation of A- beta-catabolizing enzymes. Together, we anticipate that Aims 1 and 2 will establish that androgens have beneficial, protective actions in brain and that androgen depletion places the brain at risk for degeneration and disease. In Aim 3, we will further evaluate this hypothesis by both investigating how manipulation of androgen status affects progression of AD-like neuropathology in a transgenic mouse model of Alzheimer's disease. and examining the relationships between human aging, brain levels of A-beta and androgens, and AD status. Together, we believe these novel and timely studies will begin an important evaluation of interactions between normal male aging events, neuroprotection, A-beta regulation, and the risk of developing Alzheimer's 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.

DESCRIPTION (provided by applicant): In recent years, several conditions have been identified as risk factors for the development of Alzheimer's disease (AD). Two such risk factors are age-related testosterone loss in men and type 2 diabetes (T2D). It is unclear how these two conditions increase the risk for AD. Further, it is not known whether these conditions function as independent or related risk factors. In this application, we will evaluate the hypothesis that normal, age-related testosterone loss increases AD pathogenesis not only by direct effects on brain but also by promoting T2D. Similarly, we will investigate the complementary hypothesis that T2D increases AD pathology directly as well as by reducing testosterone levels. Thus, testosterone loss and T2D are postulated to be interrelated conditions that, in combination, cooperatively increase development of AD. Mechanistically, both low testosterone and T2D independently affect key features of AD neuropathology, including beta-amyloid accumulation, tau hyperphosphorylation, and inflammation. We hypothesize that testosterone and T2D cooperatively increase AD pathogenesis by interactions in the cell signaling pathways that regulate these three aspects of AD pathology. To investigate these hypotheses, we propose three specific aims. In Aim 1, we will investigate the effects of experimentally induced T2D on levels of testosterone and AD in animal models of aging and AD. In Aim 2, we will investigate the effects of experimentally-induced low testosterone on measures of T2D and AD in animal models of T2D. In both Aims 1 and 2, we will also examine the role of aging on identified relationships interactions between low testosterone and T2D. Finally, in Aim 3 we will evaluate candidate mechanisms underlying interactions between testosterone, focusing on signaling pathways that regulate beta-amyloid accumulation, tau hyperphosphorylation, and inflammation. Completion of our studies will characterize and mechanistically define relationships between testosterone and T2D and how they cooperatively act to promote development of AD, knowledge that will be invaluable in understanding and preventing AD in aging men.

The goal of our Program Project is to discover the biological transformafions that occur in the brain during the perimenopausal transition that can result in phenotypes predicfive of risk for development of AD pathology. In Project 3, our specific emphasis is the neuroprotective acfions of ovarian hormones on AD pathogenesis. Menopause is characterized in part by deplefion 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, condifions 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 bioenergefics and increase infiammafion. Thus, perimenopause results in adverse changes to both ovarian hormones and adiposity, which we theorize interact cooperatively in the promofion on AD pathogenesis via their effects on bioenergefic, 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 regulafion of bioenergefic, 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. RELEVANCE (See instructions): Perimenopause results in ovarian hormone depletion and adiposity, which we theorize interact cooperatively to promote Alzheimer's pathogenesis via effects on bioenergetic, infiammatory, and Alzheimer-specific pathways. This proposal will define these relafionships and how they are affected by hormone interventions, basic science informafion that is essenfial for opfimizing hormone therapy in postmenopausal women.

DESCRIPTION (provided by applicant): In recent years, several conditions have been identified as risk factors for the development of Alzheimer's disease (AD). Two such risk factors are age-related testosterone loss in men and type 2 diabetes (T2D). It is unclear how these two conditions increase the risk for AD. Further, it is not known whether these conditions function as independent or related risk factors. In this application, we will evaluate the hypothesis that normal, age-related testosterone loss increases AD pathogenesis not only by direct effects on brain but also by promoting T2D. Similarly, we will investigate the complementary hypothesis that T2D increases AD pathology directly as well as by reducing testosterone levels. Thus, testosterone loss and T2D are postulated to be interrelated conditions that, in combination, cooperatively increase development of AD. Mechanistically, both low testosterone and T2D independently affect key features of AD neuropathology, including beta-amyloid accumulation, tau hyperphosphorylation, and inflammation. We hypothesize that testosterone and T2D cooperatively increase AD pathogenesis by interactions in the cell signaling pathways that regulate these three aspects of AD pathology. To investigate these hypotheses, we propose three specific aims. In Aim 1, we will investigate the effects of experimentally induced T2D on levels of testosterone and AD in animal models of aging and AD. In Aim 2, we will investigate the effects of experimentally-induced low testosterone on measures of T2D and AD in animal models of T2D. In both Aims 1 and 2, we will also examine the role of aging on identified relationships interactions between low testosterone and T2D. Finally, in Aim 3 we will evaluate candidate mechanisms underlying interactions between testosterone, focusing on signaling pathways that regulate beta-amyloid accumulation, tau hyperphosphorylation, and inflammation. Completion of our studies will characterize and mechanistically define relationships between testosterone and T2D and how they cooperatively act to promote development of AD, knowledge that will be invaluable in understanding and preventing AD in aging men.

PROJECT SUMMARY ? PROJECT 2 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. Our focus is on the primary genetic risk factor for late-onset AD, the ?4 allele of apolipoprotein E (ApoE4), and how it is disproportionally affects AD risk in women. In particular, we will investigate interactions between several significant risk factors for AD: age, the ApoE4, and female sex. In Project 2, our specific emphasis is how ApoE4 interacts with female sex, perimenopause and adiposity to cooperatively drive development of AD pathology. Perimenopause initiates the age-related depletion of ovarian hormones in women. Perimenopause is also linked with increases in body weight and adiposity that often lead to obesity, an established risk factor 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 creates a dangerous convergence of AD risk factors in middle-aged women that we hypothesize is exacerbated by ApoE4. We hypothesize that the central unifying link between AD, ApoE, obesity and estrogen is inflammation. Pro- inflammatory pathways can function as critical driving forces in AD pathogenesis, are elevated by both ApoE4 genotype and obesity, and are inhibited by estrogen. To investigate these relationships, we propose three specific aims that are highly collaborative across all Cores and Projects. Specific Aim 1: ApoE genotype interactions in the regulation of inflammation and Alzheimer's pathways. We will compare the effects of ApoE alleles on expression of AD- and inflammation-related genes, AD neuropathology, metabolism, and cognition Specific Aim 2: Obesity and ApoE genotype interactions with female sex in the regulation of inflammation and Alzheimer's pathways. We will determine how ApoE status interacts with obesity in the regulation of AD- and inflammation-related pathways, AD neuropathology, metabolism and cognition. Specific Aim 3: Hormone interventions for protection against inflammation and Alzheimer's pathways associated with ApoE genotype, obesity and perimenopause. We will evaluate the efficacy of estrogen-based hormone therapies in attenuating the effects of ApoE4, in the presence and absence of obesity, during both perimenopause and late menopause.

The primary genetic risk factor for Alzheimer?s disease (AD), the apolipoprotein E ?4 allele (APOE4), disproportionally affects women. Studies in mice have shown that APOE4 promotes cognitive deficits and AD- like pathology that are significantly more pronounced in females. Despite this strong association between female sex and APOE4, there is a striking gap in our knowledge on how APOE4 imparts this female bias for AD risk. One mechanism underlying neural sex differences is developmental sexual differentiation, which results in permanent structural and functional alterations in the brain. Our central hypothesis is that sexual differentiation yields a female brain that is inherently more vulnerable to the AD-promoting actions of APOE4, a relationship that increases with age. How the female brain affects APOE4 function is unknown, though at least two critical components of AD pathology likely contribute to the increased AD risk in female APOE4 carriers. Specifically, we posit that interactions among APOE4, female sex, and AD pathology are modulated by: (i) microglia-induced neuroinflammation, and (ii) isoform-specific apoE lipidation and consequent changes in amyloid-? (A?) solubility. We propose 4 Specific Aims to investigate these hypotheses. In Aim 1, we will answer the critical questions of whether women are inherently more vulnerable to the effects of APOE4 and whether sexual differentiation contributes to this increased risk. We will investigate these issues using a large consortium of longitudinal human twins studies. In Aim 2, we propose rodent studies to complement the human twin studies. To address our central hypothesis, we will manipulate the sexual differentiation of EFAD mice (a transgenic mouse model of AD that contains both AD transgenes and the human APOE genotypes 3/3, 3/4, and 4/4) by creating masculinized females and feminized males to understand how cognitive impairment and the development of AD-like neuropathology are altered across the female ? male continuum in adulthood and aging. In Aims 3 and 4, we evaluate two mechanisms that are hypothesized to significantly contribute to the interactions among APOE4, sex, and AD pathology. In Aim 3, we investigate the hypothesis that sexual differentiation results in a female brain that is more vulnerable to the pro-inflammatory phenotype associated with APOE4, which promotes AD pathogenesis. In Aim 4, we investigate the hypothesis that APOE4 and female sex interact to reduce apoE lipidation, impairing clearance of soluble A?, resulting in memory/cognitive deficit and accelerated AD pathology. This proposal will provide novel insights into the critical link between female sex and APOE4-induced risk for AD and define the role of sexual differentiation in this relationship. Our complementary approach compares the female ? male continuum and the influences of 0,1, and 2 APOE4 alleles in both human twins and EFAD mice. In addition, we evaluate two potential mechanisms that may underlie these effects. Collectively, these studies promise to add significant depth to our understanding of the relationship between sex and APOE.