Carol Colton - US grants

An atomic absorption spectrophotometer with furnace atomizer is essential for ultra-micro elemental measurements in three different research projects. It will be used to determine metal content (particularly zinc and iron) in hippocampal slices. The objective is to investigate the relationship of tissue metal content to neuronal damage in the brain during exposure to oxidative stress. Another project will use this equipment to measure sodium and potassium concentrations in nanoliter samples of tubular fluid from neonatal rat kidneys. The measurements will be used to assess the role of atrial natriuretic factor in water and electrolyte homeostasis in the immature rat. For the third project, the atomic absorption spectrophotometer will also be used to determine sodium and potassium concentrations in nanoliter samples of renal tubular fluid. The role of neurohumoral factors, particularly dopamine, regulation of sodium excretion in neonatal rats will be examined. The atomic absorption spectrophotometer and graphite furnace atomizer offers the unique capability of measuring the content of various metals and ions in extremely small samples of tissue and fluid at a reasonable cost.

Redox regulation of the NMDA channel has been demonstrated for primary neurons and recombinant proteins transfected into cells. Reduction of the channel increases whole cell currents while oxidation reduces currents. Site directed mutagenesis demonstrated the presence of critical cysteine groups on the NMDA channel proteins. Our recent data confirms the regulation of NMDA channel function by redox factors and further demonstrates that nitric oxide (NO) alters NMDA channel function increasing whole cell currents by increasing channel open probability. Since the NMDA channel is involved in a putative cellular form of learning and memory, long term potentiation (LTP), and has also been implicated in the excitotoxicity associated with various neuropathological disease states, alteration of the NMDA channel by the local tissue redox state may be of significance to the functional changes associated with these processes. The local tissue redox state, in turn, may be regulated by the generation of reactive oxygen species (including NO) into the environment of the NMDA channel. Microglia, the CNS macrophage, produce superoxide anion when stimulated as part of the respiratory burst and NO is produced when the microglia are stimulated with the appropriate cytoactive factors. Microglia also have cytoplasmic processes which abut synaptic endings and are involved in synaptic stripping. Thus, microglia are in the appropriate location to alter NMDA channel function by the production of reactive oxygen species. We will test the hypothesis that microglia alter NMDA channel function by using a sniffing patch technique. Outside-out patches will be prepared from HEK cells transfected with recombinant NMDA channel proteins. These patches will then be used to determine the effect of microglial-produced superoxide anion or NO on NMDA single channel parameters. Changes in single channel conductance, open probability, open duration and burst duration will be assessed in untreated microglia and microglia that have been induced to produce either superoxide anion or NO. The microglia will then be pretreated with lipopolysaccharide, (LPS), an inflammatory mediator, with interferon, an immunological mediator; or with Alphabeta peptide, a a microglial activator relevant to Alzheimer s disease. These factors are known to increase the activation of the microglia, promoting their cytotoxicity. Specificity of the effect will be determined using known antioxidants and a cell line engineered to generate NO.

DESCRIPTION (Applicant's Abstract): The distinguishing neuropathological feature of Alzheimer's disease (AD) is the presence of amyloid-containing neuritic plaques in the patient's brain. Activated microglia, the CNS-specific macrophage cells, are a prominent feature of these plaques where they function to release reactive free oxygen radicals, reactive nitrogen species, cytokines and proteases which may contribute to plaque generation. Recent evidence indicates that plaque density and the prevalence of Alzheimer's disease is positively associated with the APOE4 genotype. Apolipoprotein-E (apoE) , the protein component of lipoproteins, promotes the transport of lipids and cholesterol required for cell survival. Our preliminary data indicate that apoE may have another action in promoting the production of nitric oxide (NO) by microglia. In the cardiovascular system, apoprotein- containing LDL (low density lipoproteins) induced NO production and as a consequence reduced macrophage-mediated LDL oxidation. Our finding that apoE promotes NO production may signal a similar role for NO in the CNS. Our preliminary data also indicate that the 4 kDa amyloid-beta peptides (A beta 1-40 and A beta 1-42) alters microglial function by stimulating superoxide production. Thus, Dr, Colton proposes that A beta may disrupt the regulatory effect of apoE on NO production shifting the superoxide/NO balance in favor of oxidative stress. Using primary cultures of microglia from neonatal hamster CNS, a human clonal microglial cell line and human monocyte derived macrophages, Dr Colton will examine the effect of apoE on resting and immune-activated cells. Both superoxide and NO production will be measured in response to the common 2, 3, and 4 isotypes of apoE protein and pre-oxidized forms of apoE. Interaction of a po E with membrane receptors will be studied. Interference of this regulatory pathway by A beta alone, apoE alone or A beta plus apoE will be tested since AB forms stable complexes with apoE. The possible involvement of superoxide reacting with NO to form peroxynitrite and the role of A beta/apoE complexes in A beta-mediated dysregulation will be explored. Finally, the consequences of this regulatory pathway will be examined by determining the level of lipoprotein oxidation and cell survival in the presence and absence of apoE alone, AB alone or A beta/apoE complexes.

DESCRIPTION (From the Applicant's Abstract): Neurodegenenerative diseases like AD include brain inflammation as a major component of the disease process. The brain's own macrophage, the microglia, participate in the chronic inflammatory response, releasing cytoactive factors such as reactive oxygen species. Our goal is to understand the brain's immune response during disease in the context of oxidative stress. To accomplish this goal we have focused on the control of microglial production of ROS in models of human neurodegenerative disease. Our new data show that release of NO from microglia depends upon APOE genotype. Since APOE genotype is the major risk factor for the development of AD and since AD is associated with an increased presence of oxidative stress, then NO as an oxyradical species probably plays a direct role. Additional new data demonstrate that apoE treatment alters specific Cationic Amino acid Transporters (CATs) in an isoform specific manner. Since CAT transporters regulate arginine entry into cells, which is then converted by the action of NOS to NO, then this may be one important mechanism regulating NO release. We hypothesize that regulation of microglial nitric oxide production is mediated by specific "restriction" points in the arginine utilization pathway. Our preliminary data demonstrate that regulating the level of substrate for iNOS by altering the cellular uptake of arginine via CAT transporters is one such point. A reciprocal path regulated by arginase competes for arginine and may also serve as a restriction point for NO production. Immune activation of the microglia by extrinsic factors alters the restriction points, thereby modulating NO. Intrinsic genes and their products relevant to AD (APOE) also affects the restriction point regulation and thus predispose the microglia to overproduction of NO and promote tissue redox imbalance. We propose to examine arginine utilization pathways in microglia and the regulation of these pathways by extrinsic immune factors (viral mediators, bacterial components and cytokines) and by APOE. Since our preliminary data demonstrate that microglial immune activation promotes a gene switching process, whereby mENA for CAT1 appears repressed and CAT2b mRNA appears enhanced, we will investigate the gene induction process and the regulation of the gene switching by extrinsic vs intrinsic factors.

DESCRIPTION (Adapted from applicant's abstract): Loss of synaptic contacts, formation of neurofibrillary tangles and neuronal death are characteristic features of Alzheimer's disease. Oxidative stress causes neurodegeneration and affected neurons in AD demonstrate biochemical footprints of the past presence of oxidative stress. We reported that apolipoprotein E (apoE) regulates NO production, a key reactive oxygen/nitrogen species that causes oxidative stress. Our additional new data demonstrate that apolipoprotein E alters the neuronal uptake of L-arginine, the substrate* converted by nitric oxide synthase (NOS) into NO. We hypothesize that apoE regulates the function of cationic amino acid (CAT) transporters in an isoform specific manner, thereby altering the entry of L- arginine into the neuron which is then converted to NO by the action of NOS. We will test this hypothesis by measuring the functional activity of the CAT transporter in murine primary neuronal cultures exposed to human apoE and its isoforms and in primary neuronal cultures from mouse models expressing isoforms of human APOE and its gene products. Using semi-quantitative and quantitative measures, we will determine the effect of apoE and APOE genotype on CAT transporter mRNA and protein expression. Translation to NO production will be measured both directly by determining supernatant nitrite levels produced by NOS -containing neurons and indirectly by measuring indices of past-presence of NO (nitrotyrosine and hemeoxygenase 1). The validity of the mouse model will be tested by determining the apoE isoform dependency of arginine transporters in post-mortem human brain from individuals with and without previous clinical signs of dementia due to Alzheimer's disease.

DESCRIPTION (provided by applicant): Although study methodologies have been intensely debated, a consensus has recently emerged that Alzheimer's disease (AD) is more likely to develop in women than in men. This gender-based difference has focused research on estrogen, and specifically, the lack of estrogen during aging in women as a contributing factor to the neuropathology of Alzheimer's disease. Estrogen is known to be beneficial to the CNS at many levels including direct effects on neurons. However, estrogen can also modify microglia activation and suppressing inflammation. Neurodegenerative diseases like Alzheimer's disease feature brain inflammation as a major component of the disease process. The brain's own macrophage, the microglia, participate in this chronic inflammatory response by releasing cytoactive factors such as reactive oxygen species (ROS), cytokines and proteases. Our previously published data demonstrate that the level of macrophage immune responsiveness is dependent on APOE genotype such that a higher level of activation was observed in human Alzheimer's disease patients expressing an APOE4 gene compared to Alzheimer's disease patients that do not express an APOE4 gene. The APOE 4 gene is a well-known "risk" factor for Alzheimer's disease and the observed increase in immune activation is consistent with the increased severity of neurodegeneration associated with APOE4 in Alzheimer's disease. The same pattern of enhanced macrophage activation is observed in mouse models that express human APOE 4 compared to those expressing human APOE3. Importantly, a gender differenced is observed in the APOE-mediated regulation of immune function suggesting that the presence of estrogen overrides the regulatory effects of the APOE4 gene. Our overarching goal of this research program is to understand brain inflammation in disease in the context of factors such as APOE genotype and gender that regulate the sensitivity of the immune system's responsiveness. Thus, we will examine the role of hormones in the regulation of microglial and peritoneal macrophage immune activation in mice expressing only human apoE3 protein or expressing only human apoE4 protein.

DESCRIPTION (provided by applicant): The overarching goal of this research project is to understand how the innate immune system in the brain participates in the generation of Alzheimer's disease. To accomplish this goal, we will use novel mouse models of AD that we have developed. These mice show AD-like pathology including (1) amyloid deposits, (2) hyperphosphorylated and aggregated native mouse tau in the somatodendritic neuronal compartment, (3) neuronal loss, 4) robust cognitive deficits and 5) neurovascular unit damage. The AD-like disease phenotype was generated by crossing mice that express mutated human APP with mice that lack a functional nitric oxide synthase 2 (NOS2) gene to produce a bigenic hAPP/NOS2-/- mouse. By reducing NO levels in mice during an immune response to those levels more equivalent in human, these mice express a full spectrum of AD-like pathology. Preliminary data from the APPSw/NOS2-/- mice that show a full spectrum of AD-like pathology demonstrate an inflammatory gene profile highly reminiscent of the immune profile in brains of humans with AD. In both bigenic mice and AD brain, a complex immune activation state is observed that includes genes that code for classical pro-inflammatory factors and genes that code for anti-inflammatory factors, repair factors (alternative activation) or down-regulatory responses (acquired deactivation). Our overarching hypothesis is that the immune state plays a causal role in the disease process in AD. We hypothesize that resident immune cells undergo complex changes in immune properties in response to A[unreadable] that vary throughout the life cycle of the disease. We also hypothesize that these immune changes alter the levels of specific AD pathology or alter disease progression. We will study the brain's immune status by 1) identifying changes in the brain's innate immune system as a function of the level of AD pathology and of disease progression using specific candidate markers of immune activation states (classical, alternative and acquired deactivation), 2) investigating the causal role of the innate immune activation state in disease pathogenesis by using interventions that will modify activation profiles and 3) investigating the cytotoxic potential of TNFa, the most likely immune-regulated cytokine to damage neurons in AD. PUBLIC HEALTH REVELANCE: This project will examine the role of the brain's innate immune state in generating the neuropathology associated with chronic neurodegenerative diseases such as Alzheimer's disease.

DESCRIPTION (provided by applicant): Our goal is to generate a mouse model that closely resembles Alzheimer's Disease. In our first attempt we have developed two mouse models of AD that progress from A[unreadable] production and amyloid deposition to hyperphosphorylated native mouse tau at AD-associated epitopes, redistribution of tau to somatodendritic regions of neurons, aggregated tau, significant neuronal loss, robust behavioral changes and neurovascular unit involvement (see preliminary data). These new models express human APP mutations on a mouse nitric oxide synthase 2 (NOS2) knockout background. NOS2 and its gene product, iNOS play an important role in neuroinflammation by generating nitric oxide (NO), a critical signaling and redox factor in the brain. Neuroinflammation is an invariant feature of chronic neurodegenerative disease. Importantly, critical differences exist in the NOS2 gene that impact the production of NO during an immune response in humans compared to rodent. Genetic deletion of the NOS2 gene in mouse in the presence of mutated human APP has provided important insights into the pathology of AD. However, we hypothesize that a targeted replacement mouse where the human NOS2 gene including the promoter replaces the mouse NOS2 gene and that also expresses mutated human APP will be a defining model for AD. This R21/R33 project proposes to develop such a mouse in the R21 phase and then to phenotype the resulting pathology and test the amyloid cascade hypothesis in the R33 phase. PUBLIC HEALTH RELEVANCE: Our goal is to generate a mouse model that closely resembles Alzheimer's Disease. We hypothesize that a mouse with a more human-like immune response (in which the NOS2 mouse gene is replaced by a human NOS2 gene) and expresses human mutated amyloid precursor will promote progression of the disease beyond amyloid deposition alone. This will provide a realistic mouse model of AD to study and to use in the development of rational and effective therapeutics for AD.

DESCRIPTION (provided by applicant): Chronic diseases of the brain such as Alzheimer's disease (AD) impact an estimated 5 million individuals in the US. While progress has been made in identifying specific mutated genes that initiate a neurodegenerative disease process, we have limited information on etiology of many neurodegenerative diseases when a specific gene or agent cannot be identified. Importantly, most mouse models on neurodegenerative diseases use mutated genes, despite the fact that human mutations account for a very low percentage of affected individuals. Chronic diseases of the brain feature an innate immune response that contributes to the disease process. Since essential differences exist between innate immune function in mice and in humans, we have generated a novel "humanized" mouse model that expresses the human NOS2 gene in place of the mouse NOS2 gene (HuNOS2/mNOS2-/-). We have also crossed this mouse to an APP transgenic mouse that expresses mutated human amyloid precursor protein (APPSwDI/huNOS2/mNOS2-/-). We show that by reducing NO levels in mice to levels more typical of people, we now observe features of AD-like disease progression that are not found in other mouse models of AD. Murine and human conditions also differ by the presence of environmental factors such as stress and over- nutrition that initiate/accelerate chronic diseases including metabolic syndrome (MS). We hypothesize that metabolic syndrome will similarly accelerate the development of neurodegeneration. Using our novel "humanized" mice, we propose to expose mice to an environmental stressor that is known to lead to metabolic syndrome in mice. This "humanized" environment incorporates high fat/high sugar diet with cold-water stress. Use of the HuNOS2/mNOS2-/- mouse will allow us to potentially demonstrate for the first time in a mouse model that full AD-like pathology can be induced by environmental stress in the absence of mutated human genes known to produce AD in humans. Use of the APPSwDI /huNOS2/ mNOS2-/- will allow us to determine if onset and/or disease progression are altered in mice that express human disease genes. PUBLIC HEALTH RELEVANCE: The proposed research will study the action of environmental factors on the onset and progress of chronic neurodegenerative disease in a novel "humanized" mouse model.

DESCRIPTION (provided by applicant): The neuropathological features of Alzheimer's disease are characterized by the presence of insoluble amyloid deposits in the brain and cerebrovasculature, the intra-neuronal accumulation of abnormally phosphorylated and aggregated forms of tau, a microtubule binding protein and neuronal loss. Although the exact mechanisms producing these pathological changes remain unknown, the amyloid cascade hypothesis states that the peptides (A?) that make up amyloid deposits are the cause of the disease process. Despite numerous supportive studies, discrepancies between animal models of AD and humans with AD remain an obstacle for full acceptance of A? as the primary causal agent for AD. By altering the nitric oxide in mouse brain, we have generated a novel mouse model that provides unique insights into mouse-human differences. Our bigenic mouse models of AD increase the expression of human A? on a murine nitric oxide synthase 2 (NOS2) knockout background. The resulting phenotype is highly reminiscent of the pathology observed in humans with AD including high levels of A? peptides, tau hyperphosphorylation, tau redistribution and tau aggregation, neuronal loss and behavioral deficits. A primary advantage of the APPSw/NOS2-/- mouse is the formation of tau pathology from normal, not mutated tau AND the presence of significant neuronal loss. Thus, our model provides a unique opportunity to fully test the amyloid cascade hypothesis in vivo under conditions of chronic disease. The first aim will confirm a pathological cascade in the APPSw/NOS2-/- mouse brain by measuring A?, tau pathology, neuronal loss and memory and learning in the APPSw/NOS2-/- mice brains at specific ages. To establish a direct, causal role for A? peptides in the cascade, we propose a) to reduce A? levels in the brains of APPSw/NOS2-/- mice by passive immunization and b) to increase A? levels in the brains of NOS2-/- mice using intrahippocampal injection of A? peptide mixtures. The second aim will examine the role of NOS2. We propose a) to reduce brain iNOS protein using lentivirus delivery of small interfering RNA (shNOS2 lentivirus) b) to test the ability of NOS2 and NO replacement to alter the pathological cascade mediated by A?. The third aim will examine a likely mechanism of NO's action, the regulation of caspase activity. PUBLIC HEALTH RELEVANCE: This project will examine the role of A beta peptides derived from the amyloid precursor protein in generating the neuropathology associated with chronic neurodegenerative diseases such as Alzheimer's disease. The method used will involve the generation of a novel mouse model for potential therapeutic value and will provide a useful to tool to fully investigate therapeutics in the pre-clinical stage.

DESCRIPTION (provided by applicant): The role of the immune system in neuronal death in chronic neurodegenerative diseases remains a critical unsolved question. One common view is that chronic neurodegeneration represents a form of immune pathology, in which the excessive production of inflammatory mediators such as TNFalpha, IL-1beta, and reactive nitrogen or oxygen species leads to neuronal cell death. There is a strong precedent for such immune pathology in diseases such as acute viral or bacterial infection. However new evidence from multiple sources argues that pro- inflammatory immune pathology is not the cause of neuronal loss in chronic brain disease. Here we propose that a different type of immune pathology, one most commonly associated with immune suppression, is responsible for neuronal cell death in chronic neurodegenerative diseases such as Alzheimer's disease. Immunosuppression is a feature of acquired immune privilege that protects critical cells but reduces the ability of the tissue to mount an effective toxic response that would clear immunogens including Abeta. Ineffectual clearance leads to persistent infection and hence to a chronic inflammatory disease. Our data on a mouse model of AD that shows full AD-like pathology (the CVN mouse) strongly suggest that Alzheimer's disease may represent an inappropriate immunosuppressive state, initiated or facilitated by Abeta production. Preliminary data from this mouse show increased expression of anti- inflammatory/repair genes and proteins at the onset of cellular Abeta production and parenchymal deposition. Pro-inflammatory gene expression occurs with age but is accompanied by increased expression of anti-inflammatory and tolerogenic genes and proteins. Thus, we believe that an immunosuppressive environment is maintained throughout the neurodegenerative process. Nutrient deprivation is a principal mechanism by which immune cells induce immune- suppression. By increasing arginine and tryptophan uptake, immune cells reduce the levels of these essential amino acids in the microenvironment. Surrounding cells may undergo amino acid starvation and increased autophagy leading to cell death. Our preliminary data suggest that amino acid starvation occurs in CVN mice brain. Increased activity of two enzymes are featured in immunosuppression; namely arginase (Arg) that uses arginine to make ornithine for polyamine and proline production and Indoleamine dioxygenase (IDO) that uses tryptophan to produce kyneurine. The CVN mouse model of AD demonstrates both decreased brain levels of arginine and increased arginase and IDO expression. This proposal will focus on the role of nutrient deprivation as a key factor in the induction of autophagy and neuronal loss in AD. The aims will establish, 1) the relationship between nutrient deprivation and the progression of AD- like pathology in the CVN mouse 2) if immunosuppressive immune cells contribute directly to disease progression through regional nutrient deprivation and 3) if immune-mediated nutrient deprivation plays a causal role in neuronal death and AD pathology. These proposed studies represent a novel approach to understanding the basic mechanisms of immune mediated disease in chronic neurodegenerative disease.

Current therapies designed to treat Alzheimer's disease based on a single disease mechanism have not changed this debilitating disorder in humans. While individual targets are critical to uncover, it is likely that interacting pathways are altered in AD. The immune system of the brain is a network of interdependent pathways whose regulation is not well understood but which is clearly implicated in AD. We know, however, that AD is a chronic disorder and results in slow deterioration of the brain over a number of years. Based on our recently published studies, we propose that loss of neurons in AD is not due to toxic pro-inflammatory mechanisms that rapidly kill cells. Our data suggest a different pathology; one that involves immune-mediated nutrient deprivation caused by prolonged immunosuppression. We propose that prolonged immunosuppression initiated by microglia changes the levels of arginine and methionine in the surrounding microenvironment, disrupting interrelated metabolic pathways either within microglia or other cells that are dependent on a maintained supply of these amino acids for normal function. We hypothesize that a patterned change in levels of the amino acids used during the prolonged immune response will lead to a consistent and definable profile of specific metabolic and functional outcomes within the brain's parenchyma. We further hypothesize these patterns will be different between normal (unaffected) and AD-like conditions and will change with progression of AD-like pathology. In Aim 1 we will use our novel mouse model of AD at early and late stage of disease to define patterns of specific amino acids, tissue metabolites, and gene and protein expression levels that characterize immune regulated arginine and methionine. Entry point into this immune regulated system is through activation of an anti-inflammatory related protein, arginase and through subsequent arginine utilization and its impact on other pathways that require arginine. We will stress the interlocking system by dietary reduction or supplementation of arginine and/or methionine. Aim 2 will measure specific functional outcomes that result from the immune-induced amino acid and metabolite changes in the brains of AD and normal mice including cell proliferation (via polyamines) and methylation and its impact on myelin (via methionine and methyl donors). Aim 3 will block the disrupted and restore normal patterns by blocking arginase to determine if a single target approach is viable at early or late stage disease.

? DESCRIPTION (provided by applicant): Microglial activation is considered to be a primary pathophysiology of Alzheimer's disease. While the exact outcomes of a microglial response during the disease process remain unclear in humans with AD, it is now evident from genome wide gene association studies as well as other studies that inflammation is a primary factor in neurodegeneration. Data from our mouse model of AD that presents characteristic pathological features of AD including amyloid deposition, disease progression to phosphorylated, aggregated tau, behavioral changes, and neuronal loss show a complex immune phenotype. Using flow cytometry we have isolated a sub type of CD11c+ microglia associated with areas of neuronal loss from our mouse model. We have used gene screening to identify genes expressed by this cell type and these data clearly indicate an immunosuppressive characteristic reminiscent of monocyte derived suppressor cells (MDSCs). The close regional association of these CD11c+ immunosuppressive microglia and AD pathology in our model suggest a causative role of immunosuppression in AD. A primary mechanism for tissue damage under these conditions is amino acid starvation of surrounding cells caused by the increased consumption of specific amino acids, primarily arginine, methionine and tryptophan. Data from our mouse model supports this hypothesis. In humans with AD, activated-disease microglia exhibit similar characteristic antigens used to identify immune activated-disease microglia in mouse brain including CD11c, major histocompatibility complex class 1 and 2 antigens and St6gal1 antigen. Using our mouse model data as a guide and brain autopsied tissue from normal, mild cognitive impairment (MCI), mild and severe AD we will determine if a population of immunosuppressive microglia is found in humans with MCI and if this immuno-phenotype gene expression differs with disease progression. Experiments will be carried out using laser capture microscopy and unbiased gene analysis . If true, this finding will provide a broader understanding of the complexity and timing of immune phenotypes during disease progression in humans with AD and provide insight into novel disease mechanisms. These data may also impact the design and application of anti-inflammatory therapeutics as treatment for individuals with AD

The timing of onset and the severity of the disease process in Alzheimer?s disease (AD) are varied and involve risk factors that include the expression of gene alleles of APOE4. Biological female sex is also implicated as a strong risk factor for AD. However, despite accumulating evidence supporting this association, recent large population-based analyses indicate that this association is complex. Immunity, like sex and APOE genotype, is a primary disease factor in AD and impacts both the onset and pathological features of neurodegenerative events. The impact of sex hormones on immunity suggests that this additional interaction may contribute to the role APOE4 plays in AD. How sex, APOE genotype and immunity interact to initiate or amplify AD pathology, however, remains essentially unknown. Our preliminary data point to a shift in metabolic pathways that is directly controlled by immunity, is impacted by sex and by APOE genotype and which may serve as an underlying and unifying mechanism defining the disease process. In this proposal we will identify these metabolic pathways and test if changing estrogen levels as found in menopause alters these metabolic outcomes to worsen disease and in an APOE genotype dependent manner. To accomplish these goals we have developed a series of mouse models that permit direct analysis and comparisons of changes in brain metabolism under conditions where APOE genotype and female sex interact in a more-human like immune background. To fully characterize these models, we will use advanced phenotyping techniques that include unbiased proteomics, transcriptomics and metabolomics The use of laser capture microdissection combined with genomic or proteomic analysis will permit regional and/or cellular localization of changed pathways. Measuring pathway flux with heavy labeled isotopes allows us to directly confirm specific pathway differences based on biological sex, genotype or age. Initiating estrogen depletion in our mouse models using ovarian chemical disruption allows us to determine and compare the effect of lack of estrogen (menopause) on the pathological phenotypes. Overall, our research plan will allow us to identify critical pathways and to widen our knowledge of the impact of AD-like pathology on multiple brain functions, including immunity. Importantly, using bioinformatics tools we will be able to compare across the experimental data sets generated from our analyses of the mice models, to human tissue metabolites and finally to corresponding available human data sets outside of Duke, enabling us to confirm and extend our results.

Biological sex (genetic and hormonal), and lifestyle are major risk factors for cognitive decline during normal aging and the onset and progression of Alzheimer?s disease (AD). Thus, being female and physically inactive increases the susceptibility for age-related cognitive impairments and AD, particularly following menopause. Physical exercise has emerged as a powerful strategy for maintaining brain health and resilience during aging and for staving off the cognitive decline associated with neurodegenerative disease. However, despite strong evidence for sex differences, hormonal effects, and lifestyle factors in cognitive aging and disease, studies to unravel the how interactions among these factors affect female vulnerability and the mechanisms that mediate these effects are lacking. Here we propose to address these fundamental issues that are of significant consequence for the cognitive health and well-being of more than half of the human population: the potential neuroprotective and cognitive benefits of physical exercise on the female brain, with a specific focus on the greater susceptibility of post-menopausal females to age-related cognitive decline and AD. To address these questions, we will combine two powerful mouse models: 1) our CVN-AD mouse model of AD, which exhibits neuropathological features similar to those of humans with AD as well as exacerbated AD-like neuropathogenesis and resistance to therapeutic intervention in females; and 2) a mouse model of menopause, in which a chemically-induced progressive loss of ovarian hormones mimics the human menopausal transition. Our focus on building an AD mouse model for investigating female physiology has the potential to enhance health-related research on female aging and AD. We will first determine the effects of transitional menopause on memory, neuropathogenesis, and gene expression in CVN-AD and control mice. We will then investigate the extent to which exercise training at different stages of AD-like disease and the menopausal transition can attenuate the decline in cognition and brain function in menopausal and hormonally- intact CVN-AD females and the molecular pathways shared by these processes. Together, these studies will serve as an exploratory first step to answer fundamental questions relevant to healthy brain aging and neurodegenerative disease in females and may lead to the discovery of novel mechanisms that mediate these processes and the development of more effective intervention strategies to sustain and protect the cognitive health and well-being of aging females with and without AD.