Stephen Salton, MD, PhD - US grants

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of availability data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

DESCRIPTION: In addition to a role in growth, development and maintenance of normal brain structure and function, neurotrophic growth factors may also be involved in neurodegenerative disorders and in the normal response to injury in the nervous system. The investigator's research program has focused on identifying the mechanisms by which neurotrophic factors regulate both neuronal differentiation and the response of the adult nervous system to injury, using the PC12 pheochromocytoma cell line as a model. Upon addition of neurotrophic factors such as nerve growth factor (NGF), PC12 cells differentiate into neurite-bearing cells that share many properties with sympathetic neurons of the peripheral nervous system. Treatment of PC12 cells with non-neurotrophic factors such as epidermal growth factor (EGF) does not result in neuronal differentiation. The investigator employed subtractive hybridization techniques to clone cDNAs that were relatively rapidly and selectively induced by NGF in comparison to EGF, reasoning that these gene products might play a role in neuronal differentiation and further that study of the regulation of these genes might provide insight into the mechanism of neurotrophic factors action. The VGF clone is more robustly regulated by neurotrophic factors and is localized almost exclusively in neurons in the central and peripheral nervous systems. Recent studies demonstrate that VGF is rapidly regulated in vivo in response to seizure and injury and in addition, approximately 10 days following cortical injury VGF mRNA is upregulated in a region of the recovering brain in which compensatory regrowth of axons or sprouting occurs. Since VGF protein is stored in neurons and released from secretory vesicles, effects on surrounding cells may result from a rapid increases in vgf gene expression, suggesting that VGF may function n the formation of synapses during development and after injury. The specific aims of this application are designed to (1) characterize the transcription factors that are activated by neurotrophin treatment, leading to neuronal differentiation and induction of vgf gene expression in vitro, and (2) determine the specific mechanisms that restrict vgf gene expression to particular subsets of neurons in the CNS and PNS. Finally, the investigator will (3) determine the function of VGF and/or peptides derived from it in the developing, adult and injured nervous system through analysis of vgf-/vgf-knockout mutant mice. The proposed studies will clarify how neurotrophic factors and gene products they regulate support neuronal development and regeneration in the aging and injured nervous system.

Proteins must be directed to different places within a cell, particularly in nerve cells (neurons), where long processes like the axon can extend far from the cell body. This protein 'traffic' is thought to be done by packaging proteins into distinct vesicles, which then are targeted to the correct destination. Virtually nothing is known about this process. Two principal types of vesicles are the large dense-core vesicles (LDCVs) containing neuropeptides or monoamines, and small clear vesicles (SCVs) that contain neurotransmitters like gultamate and gamma-amino butyric acid (GABA). Neurons and neuroendocrine cells both must correctly sort peptide precursors into LDCVs, away from the neurotransmitter-containing SCVs that release and reload their contents through very different mechansims. This project uses molecular and immunocytochemical approaches to identify polypeptide sequences that target VGF to LDCVs and to characterize the novel sorting mechanism or receptor molecule involved.
Results will be important for defining the fundamental mechanisms that underly the sorting of proteins into distinct vesicle populations, ultimately transporting them to specific regions in highly polarized neurons, and to regions related to regulative or constitutive secretory pathways in these complex cells. Graduate training is included in this project.

DESCRIPTION: (Adapted from the applicant's abstract) The expression of VGF, a secreted polypeptide that is synthesized by neurons and neuroendocrine cells, is induced rapidly by neurotrophins in vitro and is regulated by electrical activity, injury and the circadian clock in vivo. Although found throughout the adult brain, VGF is particularly abundant in the hypothalamus. To gain insight into the function of VGF in vivo, the applicant has generated mice with a null mutation of the Vgf gene. Mice lacking VGF are indistinguishable at birth from normal littermates, but gain weight very slowly prior to weaning and remain 50-70 percent the size of normal or heterozygous littermates throughout adult life. Ad lib fed adult homozygous mutant mice are hyper metabolic, hyperactive, and relatively infertile with markedly reduced peripheral fat stores. Altered hypothalamic POMC, NPY, and AGRP expression and low peripheral leptin levels suggest that ad lib fed VGF mutant mice have the neuroendocrine profile of a fasted animal. Furthermore, in situ hybridization studies demonstrate induction of VGF mRNA in the hypothalamic arcuate nucleus of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that study of lean VGF mutant mice may provide insight into wasting disorders and obesity. This proposal seeks support to identify the neuroanatomic distribution of VGF in the hypothalamus (Aim 1), functional mechanisms responsible for resistance to obesity in VGF mutant mice (Aim 2), and mechanisms of regulation of VGF in the hypothalamus (Aim 3). The applicant further propose to determine the results of over expression of VGF in transgenic models and to examine the ability of these mice to rescue the phenotype of VGF mutant mice in genetic cross experiments (Aim 4), to elucidate the contribution of the autonomic nervous system and brown adipose tissue to cachexia and the phenotype of VGF mutant mice (Aim 5), and to examine the role that VGF plays in processing and regulated release of co-expressed neurotransmitters (Aim 6).

DESCRIPTION (provided by applicant): Tight control of serum glucose levels is critically important in reducing long-term complications of diabetes mellitus. Accumulating evidence suggests that these long-term benefits are not risk-free, and that recurrent episodes of hypoglycemia, or severe hypoglycemic coma secondary to incorrect insulin dosing, have significant morbidity. Damage to the hippocampus and cerebral cortex has been noted, as have cognitive defects in humans and in animal models. However, the mechanisms that underlie hypoglycemic damage to the nervous system, particularly during embryogenesis, remain largely unknown. We propose to better define how neurons are damaged by hypoglycemia in the nervous system, whether synaptic connectivity and neuronal development are affected, and whether hypoglycemia triggers expression of particular genes within the brain, providing insight into the type of damage that is sustained and identifying possible avenues of therapeutic intervention. Using two complementary hippocampal primary culture models, we plan to identify the pathways that lead to neuronal injury in response to hypoglycemia (Specific Aim I). Neuronal vulnerability to cell death will be determined; type of cell death, death pathways and active intermediates, and the transmitter phenotypes of the affected hippocampal neurons will be defined. In addition, effects of hypoglycemia to delay or disrupt neuronal polarization, axonal and dendritic outgrowth, selective axonal and dendritic protein transport, and synaptogenesis will be analyzed. Since recurrent bouts of hypoglycemia in utero are associated with postnatal cognitive impairment, we propose to examine whether cell death pathways are activated in the hippocampus through exposure to hypoglycemia in utero (Aim II). In addition, we propose to identify genes that are regulated in the embryonic and early post-natal hippocampus by hypoglycemia, which we hope will offer insight into the mechanism(s) by which the hippocampus is damaged.

DESCRIPTION (provided by applicant): Complex neural circuits and a large number of peptide hormones and neuropeptides control feeding and energy expenditure. The VGF (non-acronymic) gene encodes a highly conserved mammalian polypeptide that is differentially cleaved in a tissue-specific manner and secreted from endocrine, neuroendocrine and neuronal cells. We have shown that targeted deletion of VGF results in profound alterations in the regulation of feeding and energy balance; VGF mutant mice are lean, hyperactive, hypermetabolic and highly resistant to obesity and diabetes. Interestingly, a number of independent genetic linkage studies have consistently shown strong evidence of a subregion linked with obesity over the VGF locus on 7q22.1 but the causative gene has not been identified. Based on its consistent linkage in independent studies and strong biological candidacy, we hypothesize that VGF is an excellent candidate gene for human obesity and leanness, and propose to investigate this using combined clinical and basic science approaches. One of us (JAM) will perform genetic association studies using a high density panel of single nucleotide polymorphisms (SNPs) in the Quebec Family Study (QFS) patient cohort who represents well-characterized samples from 950 individuals and 223 families. Several nonsynonymous, protein-altering SNPs are already known and additional SNPs across the VGF locus will be developed and fully characterized as potential functional variants and/or biomarkers. These SNPs and haplotype blocks will then be validated in a second, independent cohort of 1,425 individuals. Concurrent biologic studies (SRJS) will investigate the function of select human VGF SNPs in mouse models using gene 'knockout' and 'knock-in' strategies, and in various in vitro cell culture models where VGF expression, processing, and regulated release can be quantified. Since targeted VGF deletion generates mice that are lean and resistant to diet-induced and some forms of genetically-induced obesity, tissues from these mice, including adipose and muscle, will be used to identify additional gene products by high-density gene expression array analysis. These physiologically linked, target-tissue genes may themselves play a functional role in obesity resistance or susceptibility, and thus become excellent candidates for future investigation.

DESCRIPTION (provided by applicant): Growth factors in general, and brain-derived neurotrophic factor (BDNF) in particular, play critical roles in the nervous system to regulate neuronal development and survival, axonal outgrowth, synaptogenesis, and synaptic plasticity. BDNF signaling modifies depressive behavior, and a large number of studies demonstrate additional roles for BDNF/TrkB pathways in contextual fear conditioning and spatial memory, as well as in the regulation of synaptic plasticity and the induction of long term potentiation (LTP). These functions likely depend on genes or gene products that BDNF regulates at the transcriptional, translational or post-translational levels, several via activation of the transcription factor CREB, a critical molecular regulator of memory in a number of different species. However, few candidate genes downstream of neurotrophins and CREB that contribute to memory formation have been identified. Recent studies suggest that VGF, a neuronal secreted protein and peptide precursor that is rapidly induced by the neurotrophins BDNF, NGF and NT3, plays a role in memory formation. VGF-derived peptides are known to regulate synaptic plasticity, reproductive behavior and energy balance. Recent human genetic SNP mapping has identified a VGF polymorphism that results in premature VGF termination after amino acid 524, eliminating several bioactive VGF peptides and an alpha-helical region required for regulated secretion. Preliminary data included in this proposal show that VGF C-terminal peptides have anti-depressant efficacy and also regulate hippocampal neuronal electrical excitability and synaptic plasticity in hippocampal slices, consistent with impaired performance of VGF knockout mice in spatial memory tasks (e.g. Morris water maze) and contextual fear conditioning. In Aim 1 of the R21 phase, we will develop two mouse models that knock human VGF alleles into the mouse Vgf locus, encoding either full length human VGF (amino acids 1-615) or SNP-truncated VGF (amino acids 1-524). In Aim 1 of the R33 phase, we will test expression and function of human VGF in these mouse models, determining whether carriers of this polymorphism could be predisposed to depression and impaired cognition, much as those with aberrant BDNF expression and/or signaling are at risk for memory and behavioral disorders. Performance of VGF knock-in mice will be examined in spatial and contextual fear memory tasks, and in depression and anxiety testing. Overall, the proposed experiments will investigate the roles that VGF, VGF-derived peptides, and a specific polymorphism in the human VGF gene, play in the regulation of hippocampal synaptic plasticity, depression, and hippocampal-dependent memory. Relevance to Public Health: Characterization of the molecules and mechanisms that control cognition and behavior will lead to increased understanding of brain function and memory, both of which are clearly impacted by aging, by degenerative diseases such as Alzheimer's or ALS, and by mood disorders such as depression. to Public Health: Neurotrophic growth factors play critical roles in the nervous system to regulate brain development and function, but few candidate genes have been identified that are induced by neurotrophins and contribute to memory formation and behavior. Characterization of the molecules and mechanisms that control cognition and behavior will lead to increased understanding of brain function and memory, both of which are clearly impacted by aging, by degenerative diseases such as Alzheimer's or ALS, and by mood disorders such as depression.

DESCRIPTION (provided by applicant): Mount Sinai's Neuroscience Training Program offers Year 1 and Year 2 predoctoral students an exciting curriculum taught by a nationally and internationally recognized faculty, and a laboratory experience that builds on expertise in translational neuroscience, basic neurobiology, psychiatry and neurology, all uniquely 'interfaced' with one another due to close apposition of clinical and basic neuroscience research at Mount Sinai Hospital and the Icahn School of Medicine at Mount Sinai. At the heart of the Neuroscience Training Program are Mount Sinai's Neuroscience Ph.D., the school's second Ph.D. granting program, and a superb training faculty who share a common thematic interest: study of the function and plasticity of specific neural circuits, during development, in he adult, and in the aged, diseased or degenerating nervous system. During the initial funding period of this T32 training program, research space and Neuroscience training faculty number substantially expanded with the founding of the Friedman Brain Institute and opening of the Hess Center for Science and Medicine. Varied laboratory opportunities at Mount Sinai take advantage of strengths in translational neuroscience, notably in developmental neurobiology, neural aging and degeneration, mechanisms of neuropsychiatric disease, cognitive neuroscience, computational neuroscience and neuroimaging, sensory signal transduction, neuroendocrinology and synaptic and behavioral plasticity. The nervous system is studied in diverse model systems, from 'simple' invertebrates such as the sea snail Aplasia, the fruit fly, or the worm C. elegans, all the way to complex vertebrates including nonhuman primates and humans. Students will receive a strong foundation in basic neuroscience and the neurobiology of disease, in a collaborative environment that actively promotes multidisciplinary, integrative research. Using this interdisciplinary approach, the Neuroscience Training Program will provide students with the essential knowledge and experimental tools to initiate productive, independent careers in the laboratories of our training faculty.

DESCRIPTION (provided by applicant): Growth factors in general, and brain-derived neurotrophic factor (BDNF) in particular, play critical roles in the nervous system to regulate neuronal development and survival, axonal outgrowth, synaptogenesis, and synaptic plasticity. BDNF signaling modifies depressive behavior, and a large number of studies demonstrate additional roles for BDNF/TrkB pathways in contextual fear conditioning and spatial memory, as well as in the regulation of synaptic plasticity. These functions likely depend on genes or gene products that BDNF regulates at the transcriptional, translational or post-translational levels, several via activation of the transcription factor CREB. However, few candidate genes downstream of neurotrophins and CREB that contribute to depression and memory formation have been identified. Several recent studies indicate that VGF, a secreted neuronal protein and peptide precursor that is rapidly induced by the neurotrophins BDNF, NGF and NT3, plays a role in depression and the response to antidepressant treatment. VGF-derived peptides are known to regulate synaptic plasticity, reproductive behavior and energy balance. Preliminary and recently published studies show that VGF C-terminal peptides have antidepressant efficacy, and also regulate hippocampal neuronal electrical excitability in slices via a BDNF-dependent mechanism, consistent with impaired performance of VGF knockout mice in spatial and contextual fear memory tasks and depressed behavior in the forced swim and tail suspension tests. To better understand VGF function in the nervous system we have generated a mouse line with a loxp-flanked (floxed) Vgf gene, allowing us to conditionally ablate VGF expression in a tissue-specific manner. In Aim 1, we will investigate how VGF regulates depressive behavior, using VGF knockout mouse models, and paradigms of depression that include social defeat and novelty induced hypophagia, which are responsive to chronic but not acute antidepressant treatment. In Aim 2 we will utilize a number of depression pardigms to determine whether VGF expression is required for antidepressant efficacy, studying responses in conditional and germline VGF knockout mice. Aim 3 will determine when in development and where in the CNS VGF functions to regulate depressive behavior, taking advantage of the new floxed VGF line, and either conditional VGF ablation or localized VGF ablation, the latter using targeted infusion of adeno-associated virus expressing Cre-recombinase. In Aim 4 we will determine whether VGF expression is required for hippocampal neurogenesis. Overall, the proposed experiments will investigate the roles that VGF and VGF-derived peptides play in the regulation of hippocampal synaptic plasticity, hippocampal neurogenesis, depressive behavior, and the response to antidepressants, using well- studied and newly generated mouse models. PUBLIC HEALTH RELEVANCE: Characterization of the molecules and mechanisms that control emotional behavior and cognition will lead to increased understanding of brain function, mood disorders such as depression, and memory, all clearly impacted by aging and by degenerative diseases including Alzheimer's.

DESCRIPTION (provided by applicant): The proposed Training Program in Mental Health Research will provide predoctoral and postdoctoral students in the Neurosciences with an integrated training experience in the laboratories of nationally and internationally recognized faculty. The predoctoral training program builds on an exciting, translationally relevant curriculum taught in years one and two of graduate school that has been recently awarded NIH support through the Jointly Sponsored Predoctoral Early Stage T32 Training Program mechanism. The postdoctoral program will draw together complementary pools of clinical and basic science fellows. The proposed new training program would be Mount Sinai's first to support research training for Ph.D. students in the Neurosciences, and would be unique at Mount Sinai in its approach to providing postdoctoral mental health research training to clinical and basic fellows. Outstanding training faculty share a common thematic interest: understanding how the function and plasticity of specific neural circuits impact, and are impacted by, neurodevelopmental and neuropsychiatric disease. Varied laboratory opportunities at Mount Sinai School of Medicine take advantage of particular strengths in translational neuroscience, notably in developmental neurobiology, mechanisms of neuropsychiatric disease, cognitive neuroscience, neuroimaging, signal transduction, and synaptic and behavioral plasticity. At Mount Sinai, the nervous system is studied in diverse model systems, from 'simple' invertebrates such as the sea snail Aplysia, the fruit fly, or the worm C. elegans, all the way to complex vertebrates including nonhuman primates and humans. Through their course work, predoctoral trainees will have received a solid foundation in basic neurobiology and the pathophysiology of neurological and psychiatric disease. Postdoctoral trainees will be admitted from residency or fellowship programs (e.g. Psychiatry), or following completion of Ph.D. or M.D./Ph.D. programs, and they will be offered a tailored didactic and research experience. Selection of a research mentor is made in a collaborative environment that actively promotes multidisciplinary, integrative research. The training program encourages participation of faculty mentors whose research grants directly focus on mental health research, while not excluding those whose research is critically important for the interdisciplinary training we seek to impart. Research training will also have a didactic 'work in progress' component, to foster these important interdisciplinary interactions, hone presentation skills, and improve awareness of ethical issues. Using this approach, the Training Program in Mental Health Research will provide predoctoral and postdoctoral students with the guidance and experimental tools, in the laboratories of our training faculty, to launch successful, productive, independent careers in mental health research.

Body weight is controlled in large part by communication between the brain and peripheral metabolic tissues, including white and brown adipose tissue, via the sympathetic nervous system to regulate energy expenditure and lipolysis. Pharmacotherapeutic intervention to reduce adiposity, however, has been relatively unsuccessful. We have identified the neurotrophin-inducible neuronal protein VGF (non-acronymic), and one of its processed C-terminal peptides TLQP-21, as central and peripheral regulators of energy expenditure and lipolysis. TLQP-21 activates the Complement C3a Receptor 1 (C3aR1), an integral component of the innate immune system, and in adipocytes, enhances lipolysis mediated by the beta-adrenergic agonist isoproterenol. Mice with VGF ablated in the adult ventromedial hypothalamus (VMH) and arcuate (ARC) have increased adiposity and decreased energy expenditure, a phenotype that is consistent with a key physiological role for TLQP-21 in the adult CNS, one that is also congruent with many actions of brain-derived neurotrophic factor (BDNF) in the hypothalamus. Utilizing floxed (lox-p flanked) VGF and C3aR1 mouse models together with established transgenic Cre-driver lines and targeted AAV-Cre administration, we will test the hypothesis that in adults, VGF and its peptides, particularly TLQP-21, regulate energy expenditure, lipolysis, and glucose homeostasis via central modulation of sympathetic outflow from the VMH and paraventricular hypothalamus (PVH), which receives extensive VGF-containing projections from ARC/VMH, and provides essential BDNF- and VGF-containing sympathetic outflow pathways to brown adipose tissue (BAT). Two specific aims are proposed. Aim 1 will probe the roles of VGF in the CNS pathways that originate in the PVH and VMH, which can be activated by `designer receptors exclusively activated by designer drugs' (DREADD), and regulate energy expenditure, glucose metabolism and lipolysis via sympathetic outflow from hypothalamus. Aim 2 will define the site(s) of action and function(s) of the pivotal VGF-derived peptide TLQP-21, determining whether its actions in the adult CNS are dependent on C3aR1 that is expressed on neurons, microglia, and/or astrocytes. The complementary research expertise of the PIs will be essential for successful completion of our aims, providing fundamental insight into the mechanisms by which VGF, its peptide TLQP-21, and the TLQP-21 receptor C3aR1, contribute to hypothalamic-sympathetic circuits that control energy and glucose homeostasis, potentially identifying promising new drug targets for the treatment of obesity and diabetes.

Comorbidity of Alzheimer's disease (AD) and Major Depressive Disorder (MDD) is frequent but unexplained by common genetic variants. Members of the Accelerating Medicines Partnership-Alzheimer's Disease (AMP-AD) program have exhaustively profiled gene expression in multiple brain regions from AD and control subjects through multiple cohorts and then performed systems biology analyses to identify molecular networks and drivers implicated in late onset AD. VGF (non-acronymic) is one of the top ranked AD drivers conserved in multiple cohorts. We show that VGF overexpression in hippocampus reduces neuropathology and cognitive impairment in the 5xFAD mouse model of amyloidosis (Beckmann et al., under review), and VGF is already known to have a role in depression. Its AD network includes the dual-specificity phosphatases DUSP4 and DUSP6 (MAP Kinase Phosphatases 2 and 3, respectively), all reduced in level in AD, connected via their network to Amyloid Precursor Protein/Abeta and Tau, and also previously identified by our group to be part of a network that contributes to MDD in females only. Our published and preliminary studies further demonstrate that VGF levels are reduced in MDD, in hippocampus and PFC, and that VGF overexpression in these regions has antidepressant efficacy in mice. Preliminary network analysis further identifies (1) an immune module with colony stimulating factor 1 receptor (CSF1R), a protein required for adult microglial survival, as a driver down- regulated in AD plus MDD, but up-regulated in AD alone, and (2) aquaporin-4 (AQP4), a brain water channel, which is down-regulated in AD plus MDD vs AD, is expressed in astroglial endfeet, and is implicated in AD. We hypothesize that members of our identified VGF, CSF1R, and AQP4 causal networks contribute to cognitive decline, depression-like behavior, and neuropathology in mouse models and patients with AD and MDD. In Aim 1, high throughput transcriptomics, proteomics, and multiscale network molecular modeling will be carried out on dorsolateral prefrontal cortex (DLPFC) from a new cohort of AD patients with and without comorbid MDD, MDD patients without AD, and control subjects, to identify additional shared and distinct molecular mechanisms that regulate these two diseases. In Aim 2, we propose to determine the role(s) that the VGF/DUSP shared network plays in comorbid MDD plus AD, by determining the underlying pathways by which VGF, DUSP4, and DUSP6 block or delay cognitive dysfunction, depression-like behavior, and the development of neuropathology, including microglial changes, utilizing AAV-mediated overexpression strategies in APP/PS1 mice. In Aim 3, we will validate the novel subnetworks and key drivers identified in Aim 1 that differentiate AD plus MDD from AD alone. Initially, we will investigate CSF1R/immune/microglial and AQP4/astroglial network function in depression-like behavior, neuropathology, and the regulation of gene expression (transcriptomics), in APP/PS1 mice overexpressing either CSF1R or AQP4, and also for CSF1R, in APP/PS1 mice that lack TYROBP, resulting in a normalized immune module and rescued cognitive impairment.

Late onset Alzheimer's disease (AD) is the most common form of dementia, and is characterized by initial memory loss and then a progressive decline in cognitive function. Members of the Accelerating Medicines Partnership-Alzheimer's Disease (AMP-AD) program have exhaustively profiled gene expression in multiple brain regions from multiple cohorts of AD and control subjects, and have then performed systems biology analyses to identify molecular networks and drivers implicated in late onset AD. VGF (non-acronymic) is one of the top ranked AD drivers identified by several groups. Moreover, biomarker studies have consistently identified reduced VGF levels in the brains and CSF of patients with neurodegenerative disease including AD, and show that VGF is also a strong candidate biomarker of AD progression, with an estimated 10% decrease in CSF levels of VGF per year in diseased patients but not in age-matched controls. We show that VGF overexpression in hippocampus or chronic intracerebroventricular (icv) infusion of the VGF-derived neuropeptide TLQP-21 (named by its N-terminal 4 amino acids and length) reduces cortical and hippocampal amyloid deposition, microgliosis, and astrogliosis, and cognitive impairment in the 5xFAD mouse model of amyloidosis (Beckmann et al., under review). TLQP-21 activates the complement C3aR1 G-protein coupled receptor (GPCR), a regulator of AD pathogenesis that is expressed in the CNS on neurons, microglia, and astrocytes. The mechanism(s) of action of TLQP-21 with respect to AD neuropathology will be further investigated in our proposed studies, and differences between C3a and TLQP-21 in the regulation of microglial function via C3aR1 will be identified in vitro and in vivo. Three specific aims are proposed: (1) To determine the mechanism(s) by which VGF-derived peptide TLQP-21 activates C3aR1 and modulates function in microglia, and to investigate how these signaling outcomes differ compared to C3a/C3aR1; (2) To determine the underlying pathways by which VGF and VGF-derived C-terminal peptides TLQP-21 and TLQP-62, a C- terminally extended peptide overlapping TLQP-21 that does not activate C3aR1, block or delay development of neuropathology and cognitive impairment in mouse PS19 tauopathy and 5xFAD amyloidopathy models; (3) To determine whether C3aR1 expression on microglia, astroglia, and/or neurons is required for VGF/TLQP-21 efficacy to reduce amyloid load, microgliosis, and/or astrogliosis in the 5xFAD mouse model. In addition to measuring the progression of amyloid and tau pathology, we will employ microglial-specific and bulk RNA seq approaches throughout our studies to construct signaling networks that will help us determine how altered VGF or TLQP-21 levels impact microglial function and gene expression, in 5xFAD and PS19 mouse models with germline or conditional ablation of C3aR1. Comparison of these networks to integrative genomic efforts (AMP- AD) that map networks underlying the onset and progression of human AD may identify novel targets and pathways for pharmacotherapeutic intervention in AD.