Giulio M. Pasinetti, M.D., Ph.D. - US grants

There is evidence that the complement (C), a major mediator of inflammatory responses may play an important role in Alzheimer's disease (AD) neuropathology. The proposed studies focus on brain experimental lesions to model select aspects of AD and aim at the understanding the role of C in mechanisms of hippocampal neuron death/survival and synaptic plasticity. In preliminary studies, we found that subsets of hippocampal pyramidal neurons in congenic mice genetically deficient of C component C5 (C5-) are more susceptible to glutamate neurotoxicity when compared to C5 sufficient (C5+) mice. We also found that the C5 derived anaphylatoxin C5a, diminished glutamate mediated neurotoxicity in hippocampal neuron cultures. We will attempt to further dissect the mechanisms which may underlie the neuroprotective role of the pro.inflammatory anaphylatoxin C5a. Specifically, we will attempt: (l.) characterize the role of C5a in hippocampal responses to experimental lesions using a C5a-receptor (C5aR) knockout mouse model, (2.) identify the actions of C5a on hippocampal neurons in intact adult brain and with experimental lesions, (3.) characterize the trophic /neuroprotective actions of hrC5a on hippocampal neuron in culture, (4.) characterize in C5-, C5+ and C5aR knockout mice altered glutamate receptors gene expression which we hypothesize might be under C5a control. These studies will clarify the role of C in brain and will lead to better understanding the mechanisms which may underlie the neuroprotective role of the anaphylatoxin C5a. They may lead to a better understanding of C mediated pathophysiology in AD.

The development of the transgenic mouse that expresses high levels of mutant APP v717-F with consequent development of diffuse and neuritic plaques is the most promising animal model of AD, and represents a breakthrough in the investigation of the inflammatory hypothesis of AD. Using this animal model it is now feasible to determine the extent and sequence of activation of inflammatory pathways by both diffuse and neuritic amyloid plaques. The proposed Pilot Project explores the role of complement system and other immuno-inflammatory molecules in brain of the amyloid precursor protein (APP)-v717-F transgenic mice bearing AD-type neuropathology and control littermates. If it can be shown that C-system activation, similar to that seen in AD is present in the brain of these mice, it will be feasible to propose future studies of anti-inflammatory interventions in the mice. These pilot studies may provide rationales, potential sites of action and timing for controlling immuno-inflammatory mechanisms in AD brain.

DESCRIPTION The epidemiological data showing NSAIDS reduce risk for AD demands further investigation at the animal model level. One major target of NSAIDs is Cox-2. Pasinetti and others have found that neuronal COX-2 is elevated in experimental neurodegeneration and in AD brain and that overexpression of Cox-2 potentiates Abeta-mediated oxidative stress in vitro. Based on this and other data, they propose to: Aim 1. Study the role of Cox-2 in neurodegeneration using experimental Abeta and kainic acid damage in transgenic mice with neuronal, human C0X-2 (NHC) overexpression and in COX-2 knockout mice. They hypothesize COX-2 overexpression will increase neurodegeneration. Aim 2. To identify the role of COX-2 in primary cultures of NHC transgenic and COX-2 KO mice in the response to Abeta and glutamate neurotoxicity evaluated by MTT and neuronal counts. This parallels aim 1. Aim 3. To identify mechanisms involved in COX-2-mediated potentiation of Abeta toxicity using organotypic hippocampal slice cultures derived from NHC transgenic and COX-2 KO mice. The hypothesis that COX-2 overexpression amplifies free radical mediated DCF fluorescence and lipid peroxidation will be tested. Aim 4. To study the role of COX-2 overexpression in bigenic NHC X APPsw (Hsiao mice) which are hypothesized to have accelerated neurodegeneration and cognitive impairment.

Non steroidal anti-inflammatory drugs are among the most promising classes of drugs for the prevention and possibly treatment of Alzheimer's disease (AD). A rapidly increasing number of large-scale therapeutic trials of such drugs are being initiated. The most likely target of NSAIDs in the brain is cyclooxygenase (COX)-2. We found that COX-2, but not COX-1 expression, is elevated in the neurons of AD brain, where it correlates with amyloid plaque density and neuronal atrophy. In this revised application, using a combination of in situ hybridization and immunocytochemical techniques, we will further study the regional distribution and cell-type expression of COX-2 and other inflammatory markers in the AD brain. To determine the relationship between COX-2 expression in the AD brain and clinical measures of disease activity, COX-2 expression will be correlated with antemortem assessment of dementia. Because therapeutic trials of potential disease-modifying regimens select patients at one or more stages of clinical disease, these studies will determine the relationship between AD clinical stage and COX-2 expression. In parallel studies the effect of COX inhibitors on COX-2 mediated responses in the brain will be explored using a transgenic mouse model of human (h)COX-2 overexpression in neurons. In preliminary studies using primary neuron cultures derived from these transgenic mice, we found that hCOX-2 overexpression potentiates beta amyloid (Abeta) neurotoxicity in vitro through potentiation of oxidative stress mechanisms. We will use this model system to compare the neuroprotective activity of various COX inhibitors on Abeta toxicity in vitro, and to study the mechanism of such neuroprotection. Based in part on the outcome of these studies, we will then test the brain activity of NSAID regimens administered systemically. We have established in our preliminary studies that transgenic mice with neuronal overexpression of hCOX-2 show increased lipid peroxidation in brain as measured by levels of malondialdehyde (MDA) along with elevated prostaglandin (PG)F2alpha. Preliminary studies also indicate increased expression of components of the complement cascade in the brain of hCOX-2 transgenics. Based on our evidence that COX-2 in neurons is indeed the appropriate target for NSAID regimens in AD, this transgenic model provides a unique method of measuring relevant brain activity of COX inhibitors. The outcome of the proposed studies will be immediately relevant to the design of human trials.

DESCRIPTION (adapted from applicant's abstract): There is evidence that the complement system, a major component of inflammatory responses, may play an important role in neurodegenerative conditions such as Alzheimer's disease (AD). The proposed studies focus on elucidating the role of the anaphylatoxin CSa in mechanisms of neurodegeneration. During the past grant period, the investigators have demonstrated that mice genetically deficient in the complement component CS are more susceptible to excitotoxic neurodegeneration, and that the C5-derived anaphylatoxin C5a may protect against glutamate and p-amyloid (Ap) mediated neurotoxicity in part through inhibition of caspase 3, an executioner of apoptotic dell death. This neuroprotective role of CSa complicates current theories that complement proteins may be involved in the pathogenesis of AD neurodegeneration. For the next grant period, they propose to extend this novel finding with a focus on CSa mediated signal transduction mechanisms involved in neuroprotection. Ongoing studies suggest that human recombinant (hr) CSa may neuroprotect against glutamate and Abeta 1-42 mediated neurotoxicity through mechanisms that involve inhibition of caspase and activation of mitogen activated protein (MAP)- kinase pathways. Further characterization of the complex role of complement proteins in neurodegeneration is essential especially in view of the possibility that the complement system represents a target for therapeutic interventions in AD.

DESCRIPTION (Adapted from applicant's abstract): The goal of the proposed studies is to further explore the mechanisms by which cyclooxygenase (COX)-2, an enzyme involved in inflammatory mechanisms as well as neuronal activities, may promote neurodegeneration in the brain of a mouse model of AD-type neuropathology. Using cDNA microarray high-throughput technique, we have being exploring changes in gene expression caused by over expressing human (h)COX-2 in neurons of APPswe/PSI-A246E/hCOX-2 triple transgenic mice, obtained by crossing APPswe/PSI-A246E with mice over expressing hCOX-2 in neuron. Genes that are differentially up- or down- regulated in the brain of APPswe/PSI-A246E or APPswe/PSI-A246E/ hCOX-2, relative to wild type (WT) control littermates (and which are not regulated in hCOX-2 single transgenics), were identified and organized into functional clusters based on similarities in biological functions. One cluster of genes whose expression was selectively influenced by hCOX-2 over expression in the brain of APPswe/PS I-A246E/hCOX-2, was found to correlate with variations in cell-cycle activity and Wingless/Int-I (Wnt) pathway function. The Wnt (the name is derived from mouse Int-I and drosophila wingless) signal transduction pathway is a large family of signaling molecules including e.g. Disheveled (Dvl), Glycogen synthase kinase (GSK-3beta), and beta-catenin among others, with well-established roles in regulating cell cycle, embryonic patterning, cell proliferation and cell determination. Based on the cDNA microarray evidence we continued exploring the potential role ofhCOX-2 on indexes of cell-cycle activity and Wnt signal transduction. We found that over expression of exogenous hCOX-2 in neuron of APPswe/PSI-A246E/hCOX-2 triple transgenics leads to >3 fold elevation in the number of S795-phosphorylated tumor suppressor (pRb) protein and active caspase 3 immunopositive neurons, which are indexes of cell-cycle-activity during response to Abeta1-42 mediated apoptotic death, confirming cDNA array evidence. This evidence was confirmed in in vitro studies showing that over expression of hCOX-2 in primary cortico-hippocampal neurons derived from hCOX-2 transgenics potentiates Abeta1-42 mediated apoptotic neuron death which could be attenuated prevented by treatment of cultures with the cell-cycle dependent kinase inhibitor (CDK) flavoperidol, suggesting that COX-2 may potentiate Abeta1-42 apoptotic neuron death through activation of cell-cycle. We continued exploring these mechanisms and found that the hCOX-2 mediated potentiation of A13 1-42 apoptotic death coincided with an inhibition of Y216 -GSK-3beta phosphorylation and intracellular redistribution of beta-catenin, which is an index of activation of the Wnt signal transduction pathway, as early as four hours after Abeta1-42 treatment. The proposed studies continuing the exploration of the mechanisms by which COX-2 may promote apoptotic neuron death in a model of AD type neuropathology will clarify the role of COX-2 in inflammatory neurodegeneration during Alzheimer's disease.

Despite decades of intense global research, initiatives to slow the clinical progression of AD have largely failed. This is attributable not only to the complexity inherent to AD, but also to the fact that AD is believed to be the result of myriad chronic, and perhaps even lifelong, physiological and genetic alterations that may not be immediately reversible. To evaluate the potential for early intervention for AD in pre-clinical populations, "lifestyle factors" known to increase the risk of cardiovascular diseases (cardiovascular risk factors (CvRFs)), have recently been evaluated as potential risk factors of AD. For example, recent evidence indicates that late life insulin resistance (a feature of non-insulin dependent diabetes mellitus), known to increase the risk of cardiovascular disease, increases the risk of AD dementia by >2-4 fold. Additionally, recent studies have reported decreased insulin insensitivity in the AD brain, and shown that insulin-sensitizing drugs in AD can improve cognition. To identify biological markers of the CvRF insulin resistance, and to further explore the relationship of insulin resistance to AD and early pre-clinical AD, we have studied insulin resistance in vivo using as experimental diabetogenic mouse model. Encouragingly, in recent high throughput genomic studies we identified several families of genes whose expression were altered under conditions of diabetogenic diet induced insulin resistance, and found these genes to be comparably regulated in the AD brain as a function of the clinical progression of AD dementia. This novel discovery provides a critical biological link between AD and insulin resistance, and supports further study of insulin resistance in the context of AD etiology. Further, in preliminary studies using a transgenic mouse model of AD, we found that diet induced insulin resistance significantly promoted beta-amyloid peptide generation in the brain of a transgenic mouse model of AD (Tg 2576 mice). In this application we propose to continue to investigate the regulation of these novel biomarker gene products of "insulin resistance" in AD and in AD model systems, and explore their specific relationship to AD-type neuropathology and cognition. At the completion of the funding period it is expected that we will develop an understanding of the biological relationship of insulin resistance to AD, yield feasibility data for ongoing and future anti-diabetic drug clinical trials in AD, and generate biological criteria for diagnosing/monitoring this CvRF in AD dementia and populations at high-risk of AD.

DESCRIPTION (provided by applicant): The overall goal of the studies proposed in this revised exploratory R21 application is to further explore the anti-amyloidogenic role of D-pinitol, a traditional insulin sensitizer herbal agent, in Tg2576 mice that model Alzheimer's disease (AD) amyloid neuropathology. The studies are supported by the feasibility evidence suggesting that a mechanism by which D-pinitol may prevent AD neuropathology in the brain of Tg2576 mice is through the inhibition of beta-amyloid (A(B) peptide generation. This may occur through a mechanism that prevents the generation of the gamma-CTF from amyloid precursor protein (APR), which is an index of gamma-secretase activity, without influencing normal Notch processing. Most interestingly, we also found that D-pinitol may prevent AB peptide generation and gamma-secretase at concentrations as low as 10 uM without evidence of cellular toxicity. Thus, it is possible that D-pinitol, in addition to its potential anti-amylolidogenic role as insulin-sensitizing agent, may also prevent AB peptide generation through direct modulation of a mechanism involved in gamma-secretase activation. Based on this new exciting evidence, the studies proposed in this revised application were designed to test the hypothesis that D-pinitol may alleviate AD amyloidosis in Tg2576 mice through mechanisms 1) dependent on the reversal of insulin-resistance and/or 2) through direct modulation of mechanisms ultimately resulting in decreased generation (or increased degradation) of amyloidogenic AB peptides. In view of the evidence that the amyloid hypothesis of AD maintains that the accumulation of the AB in the brain is a critical event in disease pathogenesis, the proposed studies will provide a unique opportunity for evaluating a novel therapeutic role of D-pinitol in AD.

DESCRIPTION (provided by applicant): The overall goal of the studies proposed in this revised exploratory R21 application is to further explore the anti-amyloidogenic role of D-pinitol, a traditional insulin sensitizer herbal agent, in Tg2576 mice that model Alzheimer's disease (AD) amyloid neuropathology. The studies are supported by the feasibility evidence suggesting that a mechanism by which D-pinitol may prevent AD neuropathology in the brain of Tg2576 mice is through the inhibition of beta-amyloid (A(B) peptide generation. This may occur through a mechanism that prevents the generation of the gamma-CTF from amyloid precursor protein (APR), which is an index of gamma-secretase activity, without influencing normal Notch processing. Most interestingly, we also found that D-pinitol may prevent AB peptide generation and gamma-secretase at concentrations as low as 10 uM without evidence of cellular toxicity. Thus, it is possible that D-pinitol, in addition to its potential anti-amylolidogenic role as insulin-sensitizing agent, may also prevent AB peptide generation through direct modulation of a mechanism involved in gamma-secretase activation. Based on this new exciting evidence, the studies proposed in this revised application were designed to test the hypothesis that D-pinitol may alleviate AD amyloidosis in Tg2576 mice through mechanisms 1) dependent on the reversal of insulin-resistance and/or 2) through direct modulation of mechanisms ultimately resulting in decreased generation (or increased degradation) of amyloidogenic AB peptides. In view of the evidence that the amyloid hypothesis of AD maintains that the accumulation of the AB in the brain is a critical event in disease pathogenesis, the proposed studies will provide a unique opportunity for evaluating a novel therapeutic role of D-pinitol in AD.

(+)-Cyanidanol; 2-Phenyl-3-Hydroxy-Benzopyran-4-Ones; 2H-1-Benzopyran-3,5,7-triol, 2-(3,4-dihydroxyphenyl)-3,4-dihydro-, (2R-trans)-; 3,3',4',5,7-Flavanpentol; 3,4',5-stilbenetriol; 3,5,4'-trihydroxystilbene; 3-hydroxy-4-keto-Flavonoids; APP Secretase; APP processing; Absorption; ActiVin (Grape Seed Extract); Adopted; Alzheimer; Alzheimer disease; Alzheimer sclerosis; Alzheimer syndrome; Alzheimer's; Alzheimer's Disease; Alzheimer's disease risk; Alzheimers Dementia; Alzheimers disease; Amyloid; Amyloid Precursor Protein Secretase; Amyloid Substance; Animals; Anthocyanidin Polymers; Anthocyanins; Archives; Assay; Bioassay; Bioavailability; Biologic Assays; Biologic Availability; Biological; Biological Assay; Biological Availability; Blood; Blood Plasma; Blood Sample; Blood specimen; Body Tissues; Botanicals; Brain; Catechin; Catechinic Acid; Catechuic Acid; Cell Culture System; Chemical Fractionation; Chemicals; Chemistry; Cianidanol; Cognitive; Complex; Consumption; Data; Dementia, Alzheimer Type; Dementia, Primary Senile Degenerative; Dementia, Senile; Deterioration; Dietary Polyphenol; Disease; Disorder; Drug Kinetics; Drug or chemical Tissue Distribution; Encephalon; Encephalons; Ensure; Esters; FRACN; Flavanol; Flavonols; Fractionation; Fractionation Radiotherapy; Goals; Grapes; H+ element; Hydrogen Ions; Hydrolyzable Tannins; Illinois; In Vitro; Individual; Intermediary Metabolism; Investigators; Juice; Knowledge; LC/MS; Label; METBL; Mammals, Mice; Measurement; Membrane; Memory Deficit; Memory impairment; Metabolic Processes; Metabolism; Methods; Methods and Techniques; Methods, Other; Mice; Molecular; Murine; Mus; Nervous System, Brain; Outcome; Pharmacokinetics; Physiologic; Physiologic Availability; Physiological; Pigments; Plant Taxonomy; Plasma; Preparation; Primary Senile Degenerative Dementia; Proanthocyanidins; Procedures; Process of absorption; Production; Programs (PT); Programs [Publication Type]; Property; Property, LOINC Axis 2; Protein Cleavage; Proteolysis; Protocol; Protocols documentation; Protons; Purpose; Quality Control; Range; Research Personnel; Research Resources; Researchers; Resources; Resveratrol; Reticuloendothelial System, Blood; Reticuloendothelial System, Serum, Plasma; Risk; Rodent Model; Role; Sampling; Scheme; Science of Chemistry; Serum, Plasma; Services; Source; Specific qualifier value; Specified; Standards; Standards of Weights and Measures; Tannin, Condensed; Techniques; Tg2576; Tissue Distribution; Tissues; Transgenic Organisms; Universities; Validation; Vitis; Wine; absorption; accelerator mass spectrometry; age dependent; age related; amyloid precursor protein processing; bioavailability of drug; dementia of the Alzheimer type; design; designing; disease/disorder; dosage; experiment; experimental research; experimental study; grape seed; grape seed extract; grape seed preparation; grapeseed; grapeseed extract; improved; in vivo; liquid chromatography mass spectrometry; membrane structure; mouse model; neuropathology; novel; phenolic acid; polyphenol; prevent; preventing; primary degenerative dementia; programs; research study; secretase; senile dementia of the Alzheimer type; social role; transgenic

Concordant epidemiological studies have demonstrated that moderate consumption of wine reduces the risk of developing Alzheimer's disease. Several antioxidant polyphenols occur in abundance in wine and are suspected to contribute to the beneficial effect of wine consumption in Alzheimer's disease. Despite skepticism concerning the bioavailability of these polyphenols, recent in vivo data have clearly demonstrated the neuroprotective properties of the naturally occurring polyphenol resveratrol in rodent models for stress and diseases, and consequently, a safety-efficacy study in Alzheimer's disease patients treated with resveratrol is currently conducted at the Mount Sinai School of Medicine. However, the exact molecular mechanisms involved in the beneficial properties of resveratrol and other natural polyphenols on the neurodegenerative process in Alzheimer's disease brain, remain to be clearly defined. Our recent data have revealed that resveratrol (frans-3,4',5-trihydroxystilbene) markedly lowers the levels of amyloid-p peptides in cell culture systems (see appendix 1). The long-term goal of this application is to elucidate the bioavailability of resveratrol and its metabolites in rodents and to characterize their anti-amyloidogenic properties in vitro and in vivo as a necessary prerequisite to the identification of novel complementary and alternative medicines for the prevention of the neurodegenerative mechanisms associated with Alzheimer's disease.

Alzheimer's disease is a devastating disorder that strikes 1 in 10 Americans over age 65 and almost half of Americans over 85. The odds of developing Alzheimer's disease double every five years after age 65. While it is increasingly common to meet heart attack or cancer survivors, it is impossible to meet an Alzheimer's disease survivor because there are none. The Center of Excellence for Research on Complementary and Alternative Medicine (CERC) in Alzheimer's disease is a joint effort from investigators at Mount Sinai School of Medicine (MSSM), the Purdue University Botanical Research Center and collaborating institutions defines our vision for an integrated multidisciplinary program of preclinical research projects all linked by a unifying scientific theme related to the understanding of the potential protective roles of grape-derived polyphenols in Alzheimer's disease. There is mounting evidence that grape-derived polyphenols may beneficially influence Alzheimer's disease. However, because polyphenolic compositions and bioactivities vary considerably due to plant-growth environments, there are problems with the preparation of grape-derived polyphenols (and other dietary polyphenols). These limitations have prompted us to assemble groups of interdisciplinary scientists with expertise in Alzheimer's disease and nutritional-botanical sciences to design a series of studies with the ultimate goal of isolating and identifying, from dietary grape sources, bioactive and bioavailable polyphenolic compounds that are capable of providing beneficial Alzheimer's disease-modifying activities. Significant discoveries on the beneficial role of dietary polyphenolics in the prevention of Alzheimer's disease through the attenuation of (3-amyloid pathogenesis is providing new knowledge in novel pathogenic mechanisms and the possibility of novel potential solutions. The studies and the information that would be gleaned from this proposal are precisely on target to provide these insights. We are confident that the information that will come from this work will have a major impact on protective roles of grapederived polyphenols in Alzheimer's disease.

[unreadable] DESCRIPTION (provided by applicant): A large body of evidence indicates that impaired energy homeostasis has a causative role in motor neuron death in amyotrophic lateral sclerosis (ALS). Recently we have found that human G93A human superoxide dismutase (SOD1) mutant transgenic male mice administered prophylactically with a high caloric ketogenic diet have an overall significant slower decline in ALS type motor impairment and lower mortality rate compared to SOD1-G93A mice fed a standard laboratory diet. Coincidentally we found a significant increase in levels of ketone bodies and notably D-3-beta-hydroxybutyrate (D-beta-HB) in the blood of transgenic ALS mice fed the ketogenic diet. We hypothesize that the protective effect of the ketogenic diet is due to increased utilization of ketone bodies in mitochondrial metabolism which is defective in SOD1-G93A mice. We propose 1) to determine the effects of D-beta-HB as a supplement, on motor function, muscle strength and survival in SOD1-G93A mice, and 2) to demonstrate that a mechanism of action of D-beta-HB is alleviating the blockage of mitochondrial function and consequent glutamate excitotoxicity associated with SOD1-G93A ALS type neuropathology. While the studies proposed in Aim 1, will provide immediate preclinical evidence on the beneficial role of D-beta-HB in ALS-type motor impairment in SOD1-G93A mutant mice, the studies proposed in Aims 2-3 will clarify the molecular and cellular mechanism(s) involved in D-beta-HB- mediated neuroprotection underlying ALS-type neurological improvement in vivo. Collectively, the proposed studies will provide impetus for larger preclinical studies testing the neuroprotective role of D-beta-HB in ALS-type neurodegeneration and will provide precious information about the possibility of developing the application of D-beta-HB as a potential novel alternative therapeutic agent in ALS. The studies proposed in this application will provide immediate preclinical evidence on the beneficial role of ketogenic diet and keton bodies, notably D-3-beta-hydroxybutyrate (D-beta-HB) in amyotrophic lateral sclerosis (ALS)- type motor impairment in SOD1-G93A mutant mice. Moreover, the studies will clarify the molecular and cellular mechanism(s) involved in ketones mediated neuroprotection underlying ALS-type neurological improvement in vivo. Collectively, the proposed studies will provide impetus for larger preclinical studies testing the neuroprotective role of D-beta-HB in ALS-type neurodegeneration and will provide precious information about the possibility of developing the application of D-beta-HB as a potential novel alternative therapeutic agent in ALS. [unreadable] [unreadable] [unreadable]

A major hallmark of Alzheimer's disease (AD) is the accumulation of neurotoxic p-amyloid (A(3) peptide in the brain. Inappropriate oligomerization of Ap peptides into soluble high molecular weight (HMW) species and the eventual deposition of A(3 peptides into extracellular A(3 plaques in the brain are major events involved in AD neuropathology and associated dementia. Accumulating evidence indicates a central role for soluble A|3 oligomers (with molecular weight higher than tetramers but lower than decamers) in preclinical AD cognitive deterioration as well as in AD dementia. The overall goal of the proposed studies in Project 3 is to identify bioactive grape seed extract (GSE)- polyphenolic compounds that can prevent AD-type amyloid neuropathology and cognitive deterioration in the Tg2576 mouse model of AD. The proposed studies are supported by preliminary evidence showing that a commercially available GSE (herein defined as NS-MA2 GSE) containing a variety of polyphenolic compounds, prevents the oligomerization of Ap peptides into soluble-extracellular HMW species and eventually AD - type Ap neuropathology, coincidental with the attenuation of spatial reference memory impairment in Tg2576 mice. Based on this consideration, the proposed studies are specifically designed to further identify and document how NS-MA2 GSE polyphenolic compounds are absorbed, metabolized and ultimately prevent AD-type cognitive deterioration in Tg2576 mice. The proposed studies will be the first to systematically identify GSE polyphenolic compounds in vivo using a combination of LC-tandem mass spectrorhetry and NMR techniques. Furthermore, this approach will make use of 14C labeled grape fractions and Accelerator Mass Spectrometry to accurately determine the pharmacokinetics of NS-MA2 GSE polyphenolic compounds in plasma and in the brain. Data from the proposed studies will provide impetus for immediate application to prevention of cognitive deterioration in AD, and potentially also in mild cognitive impairment (MCI), whose subjects are at high risk of developing AD dementia.

Despite decades of intense global research, initiatives to slow the clinical progression of AD have largely failed. This is attributable not only to the complexity inherent to AD, but also to the fact that AD is believed to be the result of myriad chronic, and perhaps even lifelong, physiological and genetic alterations that may not be immediately reversible. To evaluate the potential for early intervention for AD in pre-clinical populations, "lifestyle factors" known to increase the risk of cardiovascular diseases (cardiovascular risk factors (CvRFs)), have recently been evaluated as potential risk factors of AD. For example, recent evidence indicates that late life insulin resistance (a feature of non-insulin dependent diabetes mellitus), known to increase the risk of cardiovascular disease, increases the risk of AD dementia by >2-4 fold. Additionally, recent studies have reported decreased insulin insensitivity in the AD brain, and shown that insulin-sensitizing drugs in AD can improve cognition. To identify biological markers of the CvRF insulin resistance, and to further explore the relationship of insulin resistance to AD and early pre-clinical AD, we have studied insulin resistance in vivo using as experimental diabetogenic mouse model. Encouragingly, in recent high throughput genomic studies we identified several families of genes whose expression were altered under conditions of diabetogenic diet induced insulin resistance, and found these genes to be comparably regulated in the AD brain as a function of the clinical progression of AD dementia. This novel discovery provides a critical biological link between AD and insulin resistance, and supports further study of insulin resistance in the context of AD etiology. Further, in preliminary studies using a transgenic mouse model of AD, we found that diet induced insulin resistance significantly promoted beta-amyloid peptide generation in the brain of a transgenic mouse model of AD (Tg 2576 mice). In this application we propose to continue to investigate the regulation of these novel biomarker gene products of "insulin resistance" in AD and in AD model systems, and explore their specific relationship to AD-type neuropathology and cognition. At the completion of the funding period it is expected that we will develop an understanding of the biological relationship of insulin resistance to AD, yield feasibility data for ongoing and future anti-diabetic drug clinical trials in AD, and generate biological criteria for diagnosing/monitoring this CvRF in AD dementia and populations at high-risk of AD.

The overall objective of this Center of Excellence for Research on Complementary and Alternative[unreadable] Medicine in Alzheimer's disease application is to identify bioactive grape-derived polyhenols in AD[unreadable] prevention and/or therapy in the Tg2576 mouse model of Alzheimer's disease-type Ap neuropathology and[unreadable] cognitive deterioration. The proposed studies in this application are based on our hypothesize that selective[unreadable] bioactive grape-derived polyphenols protects against the onset and progression of AD-type cognitive[unreadable] deterioration by inhibiting the generation of A/7peptides from the amyloid precursor protein. We will test this[unreadable] hypothesis by using bio-guided fractionation methodologies to generate polyphenol preparations with[unreadable] progressively less complexity from Cabernet Sauvignon and from Concord purple grape juice that will exert A0-[unreadable] lowering activity in the Tg2576 AD mouse model. We will continue to explore the efficacy of ApMowering[unreadable] polyphenol preparations to attenuate the onset and progression of AD-type cognitive deterioration in Tg2576[unreadable] mice and to clarify the mechanisms of action.[unreadable] The proposed studies in this application will complement the other Research Projects in this Center[unreadable] application and elucidate general mechanisms by which grape-derived polyphenolics may blunt age-related[unreadable] Alzheimer's disease, especially in a growing population affected by mild cognitive impairment that is at high[unreadable] risk for AD. Outcomes of the proposed studies will likely result in the development of selective grape-derived[unreadable] polyphenolic compounds as lead compounds for clinical testing in Alzheimer's disease[unreadable]

DESCRIPTION (provided by applicant): Recent evidence suggests that treatment with certain antihypertensive drugs may decrease the incidence of Alzheimer's disease (AD), while other work has failed to support this finding. We hypothesized that the apparent inconsistency could be due, in part, to unknown pharmacological features exerted by subsets of antihypertensive drugs. Based on this consideration, we initiated a high throughput screening of 55 antihypertensive drugs, which encompass almost all of the prescribed antihypertensive drugs representing all the clinically relevant antihypertensive pharmacological classes available. Excitingly, we identified 7 clinically prescribed antihypertensive drugs that significantly reduce the accumulation of total AD-type [unreadable]-amyloid (A[unreadable]) in vitro and, as recently found, also in vivo in response to treatment with propranolol-HCI, nicardipine-HCI, losartan in Tg2576 mice, even when these drugs were delivered in a short term dosing regimen at concentrations ~2-3 folds lower than the recommended dose for hypertension in the absence of hypotensive side effects. Moreover, consistent, in part with a central role of high-molecular-weight (HMW)- soluble oligomeric A[unreadable] species in the development of AD-type cognitive impairment, we found that coincidental with attenuation of memory deterioration, long-term valsartan, another antihypertensive drug with A[unreadable] lowering properties identified in our high-throughput drug screening, significantly reduces ApMo/Api-42 and HMW soluble A[unreadable] oligomeric content in the brain and plasma when delivered at subclinical doses. Finally, in molecular topological studies assessing the structural basis of Ap-lowering activity amongst the originally 55 antihypertensive-A[unreadable] lowering agents screened, allowed us to identify 32 novel molecules that we plan to further characterize as novel Ap-lowering lead compounds. Collectively, the proposed studies in this revised U01 application will allow to continue the preclinical characterization of a select group of seven antihypertensive-A[unreadable] lowering drugs in vivo for the treatment of AD (Aims 1-2) while in molecular topological studies we will continue the refinement of 32 refined A[unreadable] lowering lead compounds as novel AP lowering agents for the treatment of AD.

The overall objective of this Center of Excellence for Research on Complementary and Alternative Medicine (CERC) in Alzheimer's disease application is to identify bioactive grape-derived polyhenols in AD prevention and/or therapy in the Tg2576 mouse model of Alzheimer's disease-type Ap neuropathology and cognitive deterioration. The overall research activities in this Center will explore grape-derived polyphenolic compounds for their potential roles of reduce Alzheimer's disease type cognitive deterioration in Tg2576 mice by reducing the accumulation of soluble extracellular oligomeric A|3 species in the brain. Based on this consideration, the purpose of Core C (Mouse Phenotyping Core) is to directly support the overall Center effort by facilitating the characterization of cognitive functions in Tg2576 mice in response to treatments with polyphenolic preparations from grape juice/wine (Project 1), the grape-derived polyphenolic, resveratrol (Project 2) and polyphenolic preparations from grape seed extract (Project 3). In recognition that multiple cognitive and behavioural modalities contribute to the overall cognitive functional status in higher animals, including human and AO mouse models, Core C will use multiple behavioural paradigms to provide a more comprehensive and sensitive measure of changes cognition function in response to polyphenolic treatments. Thus, the activity of Core C will integrate the specific goals of individual projects with the overall objective of this CERC in Alzheimer's disease application. The collective effort from the CERC in Alzheimer's disease will provide the rational basis for developing individual or a combination of grape-derived polyphenolic compounds for Alzheimer's disease prevention and/or therapy by modulating one or more of the AD-related mechanisms.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most prevalent neurodegenerative disease of aging and the cause of major burden for AD patients, caregivers, and the health care system. Current FDA-approved AD treatments have modest symptomatic effects at best and do not significantly modify disease course. In a population-based sample of incident AD, we observed that use of ¿-adrenergic antagonists was associated with slower functional decline. Our data combined with other epidemiologic data point to the potential therapeutic effect of ¿-adrenergic antagonists in patients with AD. This observation prompted us to screen ¿-adrenergic antagonists for effects on amyloid-¿ (A¿) synthesis in cell culture. One agent that passed the screen was carvedilol, a ¿-adrenergic antagonist FDA-approved for several cardiac indications. In two different transgenic mouse models of AD chronic oral administration of carvedilol decreased brain monomeric and oligomeric A¿ content, attenuated cognitive deterioration, and improved basal neuronal transmission in the brain. The effect on lowering A¿ oligomers is especially relevant, since there is growing evidence that these soluble oligomers (rather than the insoluble A¿ fibrils in plaques) disrupt synaptic transmission early in AD prior to neuronal loss. Thus, a treatment that lowers brain A¿ oligomer levels may be of particular benefit in early AD. Additionally, carvedilol may have a beneficial effect on vascular risk factors for AD by stabilizing blood pressure and improving brain perfusion, since it is an approved treatment for hypertension and congestive heart failure and has been shown to be neuroprotective in brain ischemia models. These preliminary data suggests that carvedilol may have a dual mechanism of action, by decreasing brain A¿ oligomer levels and by having a beneficial effect on cerebrovascular conditions. Since carvedilol is a generic drug, already FDA approved, with a well understood, generally well tolerated safety profile and if carvedilol has a beneficial effect in AD it offers the advantages of being relatively safe and inexpensive. For these reasons, we propose to administer a target dose of 25 mg daily of carvedilol to 50 AD patients in a 6-month randomized, placebo-controlled, double-blind, single-site trial, with change in episodic recall as the primary outcome and biomarker change and safety/tolerability as secondary measures. Significance: The results of this proof-of-concept trial will underlie a Go-No-Go decision. If we observe a significant improvement in clinical outcomes, we will propose a definitive trial of carvedilol in AD. If we observe change only in biomarker outcomes, this will inform further studies of similar treatment mechanisms (whether carvedilol or alternative agents). Should carvedilol prove to be effective in AD, it has several advantages over novel agents in human trials since it has a well-characterized, generally well tolerated safety profile and is available as a generic drug.

The overall objective of this Center of Excellence for Research on Complementary and Alternative Medicine in Alzheimer's disease application is to identify bioactive grape-derived polyhenols in AD prevention and/or therapy in the Tg2576 mouse model of Alzheimer's disease-type Ap neuropathology and cognitive deterioration. The proposed studies in this application are based on our hypothesize that selective bioactive grape-derived polyphenols protects against the onset and progression of AD-type cognitive deterioration by inhibiting the generation of A/7peptides from the amyloid precursor protein. We will test this hypothesis by using bio-guided fractionation methodologies to generate polyphenol preparations with progressively less complexity from Cabernet Sauvignon and from Concord purple grape juice that will exert A0- lowering activity in the Tg2576 AD mouse model. We will continue to explore the efficacy of ApMowering polyphenol preparations to attenuate the onset and progression of AD-type cognitive deterioration in Tg2576 mice and to clarify the mechanisms of action. The proposed studies in this application will complement the other Research Projects in this Center application and elucidate general mechanisms by which grape-derived polyphenolics may blunt age-related Alzheimer's disease, especially in a growing population affected by mild cognitive impairment that is at high risk for AD. Outcomes of the proposed studies will likely result in the development of selective grape-derived polyphenolic compounds as lead compounds for clinical testing in Alzheimer's disease

? DESCRIPTION (provided by applicant): We recently identified, in preclinical studies, specific polyphenol-rich botanical dietary supplements [e.g., grape seed polyphenol extract (GSPE), Concord grape juice (CGJ), and resveratrol (RSV)] that are effective in promoting cognitive and psychological resilience under stress conditions. The overall goal of the proposed Botanical Dietary Supplement Research Center (BDSRC) is to identify the specific polyphenol components from these bioactive botanical supplements that underlie their bioactivities and to characterize specific cellular/molecular mechanisms contributing to the attenuation of physiological stress, such as that associated to stressful life events, that have detrimental impact on psychological health, cognitive functions, and ultimately wellbeing (Projects 1-2). In addition the proposed studies will, for the first time, clarify the role of the gastrointestinal (G) microbiome, particularly the human GI microbiome, in modulating the bioavailability of polyphenol components responsible for the cognitive and psychological health benefits of dietary polyphenol botanical supplements using humanized gnotobiotic mice (Project 3). Thus, the proposed studies, which (1) characterize botanicals, (2) assess, pre-clinically, their bioavailability and bioactivity in the periphery and in the brain, while defining molecular mechanisms of resilience, and (3) identify safety issues essential for future development into clinical application, are in full alignment with the goals of NCCAM and ODS, as indicated in the Research Objectives of the RFA. The proposed studies in this BDSRC will improve scientific knowledge and technical capabilities through the development of a rigorous scientific program, will provide training and career development opportunities, and, critically, will provide an intellectually rigorous examination of the role of dietary polyphenol botanical supplements in the maintenance and promotion of psychological and cognitive resilience.

Summary/Abstract The Administrative - Biostatistics/Data Management Core A of this BDSRC has a two tier plan. The Administrative Tier I Plan Component is divided into three broad categories: scientific, academic, and organizational. The components of the administrative core will provide the day-to-day management of the BDSRC and will facilitate interactions within and between the participating research groups at the Icahn School of Medicine at Mount Sinai and the external participating institutions. The organizational support provided by the Administrative Tier I Plan Component of this Core will operate through three ?Work Groups? and will manage all administrative issues of the BDSRC, including scientific affairs, contractual agreements, allocation of funds/fiscal issues, personnel related issues, and fostering of new research. Overall the Work Groups in the Administrative Tier I Plan Component will be regulated by an Internal Steering Committee (ISC) composed of Research Project and Core Leaders to assess research progress and determine whether research goals are being met. The Administrative Tier I Plan Component of Core A will also be responsible for the creation and management of an External Advisory Committee (EAC) designed to provide oversight and assist the PDs/PI and Co-PD/PI in making scientific and administrative decisions. In parallel, the Biostatistics/Data Management Tier II Plan Component is organized into two Work Groups: Biostatistics Work Group and Data Management Work Group, which provide statistical support for all BDSRC Research Projects and Cores. While all of the BDSRC investigators are well versed in the statistical approaches that are applicable to their specific hypotheses and projects, Core A will provide statistical expertise, state-of-the-art analysis, and storage for all data sets, allowing integration of each Project's data with the data derived from the other Projects. The Tier II Plan Component has access to a data warehouse system that has recently been developed within Mount Sinai's Scientific Computing facility. This system will be available to each Research Project and Core for data integration/storage and data query through web interface. The integration of cross-project data for statistical analysis will not only be aided by the data management system described, but also through statistical testing of hypotheses, which will be an integral part of the development of each BDSRC Project from the time of its inception.

Summary Sleep is necessary for optimal cognitive function. There is broad consensus that insufficient sleep leads to a general slowing of response speed and increased variability in measures of alertness, attention, and vigilance. Accumulating evidence demonstrates that sleep deprivation (SD) negatively impacts higher level cognitive capacities, including memory and executive functions, and these cognitive disturbances are beyond those produced by impaired response and attention processes. Memory consolidation is known to be influenced by SD and our feasibility evidences strongly suggest dietary supplementation with specific botanical supplement mixtures (grape seed extract, Concord grape juice and resveratrol) significantly promoted resilience to cognitive dysfunction in the context of SD. We have also identified that a number of phenolic metabolites, derived from these bioactive botanical supplement mixtures that accumulate in brain tissues, are capable of modulating synaptic plasticity through two independent yet interrelated pathways, one mediated through activation of CREB signaling and the other through induction of immediate early genes (IEGs) expression. Our innovative proposal will test the overarching hypothesis that select brain-penetrating phenolic metabolites have a protective effect in promoting resilience to SD-induced cognitive dysfunction through comparative in vitro and in vivo assessments. The proposed project will mechanistically investigate the role of the grape-derived botanical supplement mixtures in modulating synaptic plasticity in a mouse model of SD with the goal of testing the ability of select brain-available phenolic metabolites in promoting resilience to SD-induced cognitive deficits through activation of the CREB signaling pathway (Aim 1) and modulation of IEG expression (Aim 2). In collaboration with Project 3, we will also test whether the application of novel probiotics specifically designed to promote conversion of metabolites identified in Aims 1-2 studies can maximize the effect of bioactive botanical supplement mixtures on promoting resilience to SD-induced cognitive dysfunction (Aim 3). The protective effect of the phenolic metabolites, evaluated through behavioral cognition tests in mouse models of sleep deprivation, will provide alternative translational approaches to promote cognitive integrity in the context of SD.

Project Summary/ Abstract The research scope proposed in this administrative supplement, in response to the funding opportunity PA-18-591 (Administrative Supplements to Existing NIH Grants), will leverage the resources and experience developed in our NIH/NCCAM-funded parent P50 Center Grant for advancing Research on Botanical and Other Natural Products (CARBON). To date, this parent grant has provided critical new insights on both the brain and plasma bioavailability of dietary polyphenols, and has initiated an understanding of the biomolecular mechanisms through which dietary polyphenol metabolites provide resilience against multiple neuropathogenic processes, in particular neuroinflammation. Using the extensive resources and in-depth experience accumulated from our P50 Center Grant, with a specific focus on the technical support from our CORE facilities, the proposed studies in this administrative supplement will investigate if the pharmacological application of brain-bioavailable polyphenol metabolites that we have identified to possess anti- inflammatory activity can preserve the integrity and function of the blood brain barrier (BBB), towards the development of a novel therapeutic against Alzheimer's disease (AD). The degeneration of the BBB is a notably neuropathological feature in neurodegenerative disorders, including AD. Likewise, the upregulation of inflammatory mediators and ensuing chronic neuroinflammation apparent in AD patients decreases the structural integrity of the BBB linking to two neuropathologies. However, to date, a direct mechanistic link between neuroinflammation and BBB integrity in the context of AD has not been elucidated and we believe that approaches using bioavailable polyphenols that suppress neuroinflammatory pathways represent a valuable approach towards preventing causative neuropathologies in AD involving BBB integrity. Of particular interest are the effects of IL-1? overexpression in the brain as IL-1? plays a central role in the progression of neuropathologies observed in AD. Particularly, research has demonstrated that IL-1? directly interferes with the expression of vascular endothelial tight junction proteins by releasing vascular endothelial growth-factor (VEGF)-A, which upon interaction with the VEFG-A receptor, leads to downstream iNOS-mediated down-regulation of junction proteins, which critically support the structural integrity of the BBB and maintain its homeostatic functions. The objective of our research is to, for the first time, comprehensively characterize, through innovative in vitro and in vivo paradigms, a unique panel of 16 brain-bioavailable polyphenol metabolites identified through our P50 parent grant for their anti-inflammatory capabilities and their capacity to buffer against BBB dysregulation. Overall, these studies will provide the mechanistic basis for developing novel strategies using bioactive, brain-bioavailable polyphenol metabolites to promote resilience against AD and other neurological disorders by protecting BBB functional integrity during pro-inflammatory insults.

Summary/Abstract The Administrative - Biostatistics/Data Management Core A of this Botanical Dietary Supplement Research Center (BDSRC) has a two tier plan. The Administrative Tier I Plan Component is divided into three broad categories: scientific, academic, and organizational. The components of the administrative core will provide the day-to-day management of the BDSRC and will facilitate interactions within and between the participating research groups at the Icahn School of Medicine at Mount Sinai and the external participating institutions. The organizational support provided by the Administrative Tier I Plan Component of this Core will operate through three ?Work Groups? and will manage all administrative issues of the BDSRC, including scientific affairs, contractual agreements, allocation of funds/fiscal issues, personnel related issues, and fostering of new research. Overall the Work Groups in the Administrative Tier I Plan Component will be regulated by an Internal Steering Committee (ISC) composed of Research Project and Core Leaders to assess research progress and determine whether research goals are being met. The Administrative Tier I Plan Component of Core A will also be responsible for the creation and management of an External Advisory Committee (EAC) designed to provide oversight and assist the PD/PIs in making scientific and administrative decisions. In parallel, the Biostatistics/Data Management Tier II Plan Component is organized into one Work Group: Biostatistics and Data Management Work Group, which provide statistical support for all BDSRC Research Projects and Cores. While all of the BDSRC investigators are well versed in the statistical approaches that are applicable to their specific hypotheses and projects, Core A will provide statistical expertise, state-of-the-art analysis, and storage for all data sets, allowing integration of each Project's data with the data derived from the other Projects. The Tier II Plan Component has access to a data warehouse system that has recently been developed within Mount Sinai's Scientific Computing facility. This system will be available to each Research Project and Core for data integration/storage and data query through web interface. The integration of cross-project data for statistical analysis will not only be aided by the data management system described, but also through statistical testing of hypotheses, which will be an integral part of the development of each BDSRC Project from the time of its inception.

Summary Abstract The studies proposed in this U19 application titled ?The Influence of Dietary Botanical Supplements on Biological and Behavioral Resilience? represent a cohesive program of integrated and interdisciplinary research approaches that comprehensively address the objectives and purpose of RFA-OD-19-001. In particular, the principal objective of this botanical dietary supplement research center (BDSRC) is to provide valuable insight, through both pre-clinical and clinical lines of investigation that may inform a future clinical trial designed to determine if dietary polyphenol supplements can provide resilience against stress-induced psychological impairment. We have shown through rigorous feasibility studies utilizing stress-induced models of depression that supplementation with BDPP promotes resilience to depression-like behaviors. We have identified biomolecular systems associated with immune function and neuronal activity that specific bioavailable metabolites of BDPP influence to promote resilience to stress. We note that bioavailable metabolites suppressed production of peripheral leukocytes derived inflammatory cytokines, in particular IL-6, which is important to consider given studies that find production of IL-6 is a critical response that confers susceptibility to stress. Whether or not metabolites of BDPP suppress the downstream pathophysiological effects of stress-induced IL-6 that directly affect neuron function and behavior has yet to be established. Therefore, Project 1 of this BDSRC will characterize if our botanical supplement provides resilience against physiological pathways elicited by stress that are associated with increased IL-6 activity and that confer susceptibility to the onset of depressive-like behavior. Project 1 will also identify biological targets in microglia, interneurons, and blood brain barrier cells influenced by BDPP metabolites by state-of-the-art cell-specific RNA-sequencing and imaging techniques. Project 2 will directly synergize with Project 1 by first providing an assessment of the clinical properties of BDPP, and whether they parallel those observed in rodents. We will conduct a pharmacokinetic and steady-state profile to define bioavailable metabolites found in human plasma, and to confirm the presence of metabolites that exert biological effects against IL-6 production. Project 2 will also utilize a multivariate adaptive regression splines model to identify specific metabolites or combinations of BDPP metabolites responsible for modulating IL-6 expression. In addition, Project 2 will validate plasma IL-6 as a marker of biological resilience in response to BDPP treatment by testing if BDPP promotes resilience against upregulation of plasma IL-6 in response to the Trier Social Stress Test in humans. The proposed Projects are designed and optimized to synergize with each other, and to integrate seamlessly with the two Scientific Cores. Together, this proposal offers to provide critical information of the mechanism of action and the clinical properties of BDPP and its metabolites that ?ll the most critical gaps in the existing body of data needed to optimally design a future clinical trial to test resilient properties of BDPP in response to stress.

Summary/Abstract The goal of Project 1 is to establish that a botanical supplement provides resilience against stress-induced pathophysiological responses that confer susceptibility to depressive behavior. This proposal will investigate how polyphenol metabolites of a bioactive dietary polyphenol preparation (BDPP) engage biomolecular and genetic targets in microglia, medium spiny neurons (MSNs), and blood brain barrier (BBB) cells to promote resilience of neuronal function and behavior in response to stress. Recent studies show that dysfunctional immune activity confers susceptibility to stress by affecting activity of these aforementioned cells. Systemic upregulation of inflammatory cytokines, in particular interleukin-6 (IL-6), by stress is observed in model systems of depression, as well as in patients with MDD. From preliminary studies involving a validated model of social stress, we show persistent IL-6 expression from leukocytes affects the BBB function only in stress- susceptible mice. We present evidence that BDPP bioavailable metabolites may promote resilience to social stress by suppressing production of cytokines, which is associated with maintenance of BBB integrity. Our first goal is therefore to establish if BDPP provides resilience against stress-induced depression by targeting mechanisms associated with BBB function. This aim will determine whether a botanical supplement prevents BBB permeability and infiltration of peripheral immune factors through the neuroendothelium, and will characterize biomolecular targets of BDPP metabolites in endothelial cells. Our preliminary studies show BDPP supplementation suppresses stress-induced microglia hyper-ramification and upregulation of inflammatory genes, which are associated with stress-susceptibility. Our second goal is to conduct an unbiased screen to identify molecular bioactivities of BDPP metabolites in microglia in response to stress. This aim involves a cell-specific RNA-sequencing technique termed translating ribosomal affinity purification (TRAP). Moreover, Aim 2 will characterize how increased activity of toll/nod-like receptors in microglia confer susceptibility to stress-induced depression, and act as proximate biological targets for BDPP metabolites. Finally, we show how BDPP metabolites prevent maladaptive glutamatergic synapse generation in the NAc in response to stress by regulating expression of synaptic genes in MSNs. We also show that peripheral IL-6 production by peripheral leukocytes in response to stress is critical for facilitating aberrant synapse formation in the NAc. The final goal will therefore be to conduct an unbiased screen to identify molecular bioactivities of BDPP metabolites in D2 MSNs using the TRAP method and to investigate if peripheral IL-6 diffusion into the brain activates microglia via the IL-6R to effect regulation of NAc synapse densities. This would provide a mechanism for why suppression of peripheral IL-6 by a botanical supplement can promote resilient behaviors for a future clinical trial. Together, Project 1 will validate that pathophysiological responses associated with IL-6 can be suppressed by a botanical supplement to promote resilience to stress.