Vahram Haroutunian, Ph.D. - US grants

The amyloid deposits in senile plaques of Alzheimer's disease brain are derived from a family of polypeptides termed Alzheimer's amyloid precursor proteins (beta-APP). In addition to the excessive deposition of amyloid proteins, Alzheimer's disease is characterized by a variety of neurochemical deficits including decreases in forebrain cholinergic, noradrenergic and serotonergic markers. These neurotransmitter deficits are accompanied by the loss of the cortically projecting cholinergic cells of the nucleus basalis of Meynert (nbM) noradrenergic cells of the locus coeruleus, and serotonergic cells of the raphe nuclei. Lesions of these transmitter systems lead to significant impairment of cognitive function. We have found that lesions of the nbM in the rat lead to a greater than 2.5 fold increase in the synthesis of, and mRNA for, the 695 form of the Alzheimer's amyloid precursor protein. The induction is rapid (within 1 hour of lesioning), and persists for at least 45 days post lesion. This induction exhibits specificity to beta-APP in that neither overall protein synthesis nor GFAP synthesis are altered by the lesion. We have observed similar increases in beta-APP synthesis following lesions of the forebrain noradrenergic and serotonergic systems. In addition, preliminary results suggest that modulation of beta-APP synthesis is specific to decreased presynaptic neurotransmitter activity since augmentation of cholinergic activity by chronic (2 weeks) physostigmine administration, or some other perturbations of the CNS fail to affect cortical beta-APP synthesis. These results demonstrate that the synthesis of beta-APP can be modulated and studied by specific manipulations of the CNS in animal model systems which mimic many of the neurochemical changes in AD. The study of neurotransmitter modulation of beta-APP synthesis in vivo may shed light on the mechanisms involved in the accumulation of amyloid proteins and senile plaque biogenesis in Alzheimer's disease. The studies proposed in this application aim to describe the cellular and molecular mechanisms responsible for neurotransmitter modulation of beta- APP synthesis in vivo. We hypothesize that increased beta-APP synthesis may be a natural response to neurotransmitter deficits and may be involved in neurotrophism. We propose different studies to describe (a) whether the beta-APP induction is temporary (due to degenerative process) or permanent (due to the absence of innervation) (Specific Aim 1); (b) whether mature beta-APP ever accumulates (Specific Aim 1); (c) whether the beta-APP response to these lesions is generalized over all regions of the cortex, or whether it occurs in specific cell groups of specific cortical laminae and regions (Specific Aims 2 and 4); (d) whether the beta-APP induction response is differentially regulated in young vs aged rats, and cognitively impaired vs cognitively intact aged rats (Specific Aim 3); (e) whether multiple lesions will result in an elevated induction of cortical beta-APP and lead to other secondary AD-like neuropathologies (Specific Aim 4). In addition, these studies will establish the conditions for maximal induction of beta-APP.

The specific aims of the Brain Bank Core are to obtain, characterize, describe, dissect, preserve and distribute to different researchers engaged in Alzheimer's disease research, brain tissues obtained from normal control, and Alzheimer's disease subjects. The Brain Bank Core will obtain and bank brain tissues from as many of the subjects enrolled by the Recruitment and Diagnosis Core and studied by the Neurophyschology project as possible. The Brain Bank Core will perform autopsies with the lease possible post-mortem delay, and will maintain permanently all pertinent specimen related information in a manner accessible to all past, present and future investigators using the Brain Bank. The acquisition and banking of the projected cohorts will provide a unique resource to only for the proposed studies, but for generations of studies and investigators to come. The brain banking procedures outlined in this application have been designed to preserved the tissues optimally for as broad a set of experimental methodologies as possible. After extraction, brain specimens will be photographed, weighted and any gross lesions and abnormalities note. The right half of the brain will be preserved in paraformaldehyde for neuropathological and neuroanatomical studies. The left half will be dissected in 0.5-0.8 cm coronal slabs, and after identification of the structures in each slab they will be snap frozen in liquid freon. Frozen slabs will be kept at -80C until sub-dissected for use by specific studies. The Brain Bank Core will serve all five projects proposed in this application either directly or indirectly. The Brain Bank Core will contribute to the Neuropsychology, Family History and Acute Phase Reactants projects (Projects 1, 2 and 4) by performing autopsies of subjects who have been studied in life. The neuropathological information obtained (Project 5) on these cases will enhance the Neuropsychology project by providing data on the neuropathological of cognitive decline in the aged. The Neurochemistry project (Project 3) will use the tissues banked by this core to investigate the neurochemical correlates of cognitive decline in the aged. The procedures used by the Brain Bank Core have been optimized to enhance and facilitate multiple neurochemical studies of each case banked. In addition the acquisition and banking of brain tissues from this unique cohort of subjects will augment the utility of the Brain Bank for projects funded by other grants (eg., ADRC, Cellular and Molecular Markers in Schizophrenia-PPG), by providing specimens from normal elderly controls whose cognitive status has been confirmed by rigorous ante- mortem testing.

Advances in the treatment and understanding of AD are hampered by the lack of appropriate animal model systems. None of the model systems studied have led to the development of any of the neuropathological hallmarks of AD, such as neuritic plaques and neurofibrillary tangles. The human neurofilament mid-sized transgenic mouse (NF-M) has been neuropathologically characterized and found to possess some of the pathological features of AD. Whether or not these animals represent a more adequate model of the cognitive deficits of AD than the models currently available remains to be determined. The goal of this project is to determine the degree to which the NF-M mouse models the cognitive and pharmacological features of AD relative to operationally defined criteria for an adequate animal model system. We will test the hypotheses that a) the NF-M transgene produces learning and memory deficits; b) these cognitive impairments follow a time course similar to the time course of development of neuropathologic lesions; c) the NF-M transgenic mice are hypersensitive to anticholinergic agents and hyposensitive to cholinomimetic agents; and d) the in vivo cognitive deficits are reflected in deficits in the hippocampal long term potentiation (LTP) model of learning and memory. Pilot studies have been conducted to determine the appropriate parameters and procedures for the proposed behavioral studies. The locomotor activity and open field behavior of non-transgenic mice have been studies and parameters for assessing short and long-term habituation have been established. Passive avoidance acquisition and retention parameters have been outlined in non-transgenic mice with and without cognitive deficits caused by lesions of the cholinergic forebrain. Animals transgenic for the human vasopressin gene have been behaviorally characterized and studied using a subset of the assessment procedures proposed for the study of NF-M mice. Finally, fifteen DBA/J2 female mice have been mated with 10 NF-M transgenic males and a mating schedule has been worked out to provide the requisite numbers of animals for the proposed studies. Longitudinal studies of learning and memory will begin within the next 4 weeks when the first of the NF-M transgenic litters are weaned.

This is a renewal of a program previously entitled "Cholinergic Treatment of Memory Deficits in the Aged". That program helped to develop the rationale, methods, and preliminary data for many of the large multi-site clinical trials, of potential treatments for Alzheimer's Disease (AD) which are now being supported by NIH and the pharmaceutical industry. The current proposal is designed to fill a major gap in our understanding of AD resulting from the fact that the great majority of clinical and biologic studies of AD involve patients with established and often advanced disease. Further advances in the diagnosis, treatment and prevention of AD will benefit greatly from a better understanding of the earliest biological and clinical changes in AD. The current proposal includes 5 scientific projects investigating persons at high risk for AD and comparing them with demographically matched groups of normal controls and AD patients. The groups to be studied longitudinally are: cognitively normal elderly, AD, AD, and elderly first degrees relatives of AD probands. A high proportion of persons in all groups except the first degree relatives will coke to autopsy during the 5 years of the proposed program. Project 1 will involve all groups and will test hypotheses about the neuropsychological changes in early AD will be tested using autopsy material in projects 3 and 5, respectively. Project 2 will test hypotheses about demographic factors, particularly age of onset, that may be associated with a greater genetic contribution to the development of AD. Project 4 will determine whether acute phase reactants, which are elevated in serum of some AD patients and some first degrees relatives are early indicators of AD and whether they are specific for amyloidogenic conditions. The ultimate aim of the program is to improve the diagnosis and treatment of AD through a better understanding of it's earliest manifestations.

DESCRIPTION (provided by applicant): Glutamatergic/GABAergic system abnormalities in the cerebral cortex and morphological/anatomical/biochemical abnormalities in the thalamus are present in schizophrenia. Some of the evidence comes from studies conducted in our laboratories showing changes in the expression of mRNAs encoding for ionotropic glutamate receptor genes in both the thalamus and the cortex, and for the GABA synthesizing enzyme glutamic acid decarboxylase (GAD-65 and GAD-67). Studies on the same brain specimens derived from extremely well characterized neuropsychiatrically and neuropathologically assessed schizophrenic subjects has shown that GAD mRNA level changes are reflected in functional abnormalities in the activity of GAD. The studies proposed aim to determine whether glutamatergic/GABAergic abnormalities in the neocortex are associated with glutamatergic/GABAergic abnormalities in the thalamus. Brain tissue specimens will be derived from: Dorsolateral prefrontal cortex (DLPFC) and the mediodorsal nucleus (MDN) of the thalamus; Cingulate cortex and the anterior group of thalamic nuclei; Precentral cortex (Brodmann 4/6) and the ventrolateral nuclear group of the thalamus; Inferior temporal gyrus (Brodmann 20) and the pulvinar; and Striate / Visual cortex (Brodmann 17) and the lateral geniculate nucleus of the thalamus. These specimens will be derived from 37 normal controls, 32 antemortem assessed and diagnosed schizophrenics who have been neuropathologically characterized to be free of any confounding neuropathological lesions, and 15 Alzheimer disease cases for comparative purposes. We will test hypotheses designed to determine whether glutamatergic and GABAergic abnormalities (mRNA and protein) in specific thalamic nuclei are associated with glutamatergic and GABAergic abnormalities in specific cortical regions and vice versa. In addition, studies in rats will test the hypothesis that the glutamatergic and GABAergic abnormalities in the prefrontal cortex are a direct result of lesions in the MDN.

DESCRIPTION (provided by applicant): The proposal has been revised to respond to the concerns raised by the reviewers. The overarching aim of the studies proposed in this application is to continue to define some of the major neurobiological and cognitive features associated with early dementia and early (prodromal) Alzheimer's disease (AD) by examining the influence of cardiovascular risk factors (CvRF) in the initiation of cognitive impairment and progression of dementia and its neurobiology. The long-term objectives of the program are to provide a solid neurobiological basis for the consideration of certain CvRF as risk factors for AD and dementia in order to improve future identification and treatment of persons at risk for dementia. The projects in this application therefore continue the theme of exploring the cognitive and neurobiological bases of cognitive compromise and early AD established previously by examining the role of prominent CvRFs and their relationships to cognitive and neurobiological features of AD. The studies proposed combine exploration of CvRF influences on early dementia and its neurobiology in animal model systems with studies in well-defined elderly subjects. The study of elderly veterans allows diversification of characterization and risk-factor assessment to a well defined and relatively homogeneous population. The contribution of a common set of known or hypothesized CvRF to the onset of cognitive impairment and progression to dementia will be evaluated and related to neurobiological indices of AD. The neurobiological mechanisms through which prevalent CvRFs may influence cognitive impairment and dementia are studied. One project explores the extent of micro-cerebrovascular pathology. The studies of animal model systems focuses on the role of two known and well documented CvRF; hypercholesterolemia and high fat diet induced insulin resistance, and explore their influences on neurobiological indices of AD and on micro-cerebrovascular and neuronal pathology. The aims of the proposed cores are to support the specific studies and to provide and maintain a rich research infrastructure to sustain these projects and their derivatives in the future. The subjects participating in will be initially recruited and characterized by the Clinical Diagnosis and Assessment Core. These extensive medical and diagnostic assessments that are repeated yearly include CvRF assessment and neuroimaging studies where appropriate. The results derived from these core assessments and diagnoses, including neuropathological findings from study cases who come to autopsy are integrated by the Data management and Statistics Core with the data generated by the projects and are made available to all projects and cores. The Administrative Core provides the administrative oversight and governance infrastructure to maintain and enhance this tight integration.

The data management approach of the Program Project is an integrated statistics, data management and data protection solution. The Data Management and Statistics Core receives, stores, catalogues, tracks and integrates all data generated by the Cores and Projects in this Program Project Grant and provides expert advice for the statistical analysis of data components or integrations. A new data management plan has been developed, and its components have been deployed, during the past 18 months. At present, in excess of 2,400,000 data points are warehoused for this Program Project. Other components will be developed and integrated on a continuing basis. Because the data warehouse is implemented across multiple projects department-wide, it provides for data access not only to data derived by the specific projects and cores within this Program Project, but also to previously acquired legacy data and to data being derived by all related projects within the Department of Psychiatry network. A concrete example is data derived from subjects who were antemortem assessed by the Clinical Diagnosis Core, the Brain Bank Core, and data from experiments performed on Brain Bank distributed tissue specimens. Using the resources of the data warehouse, investigators in the current Program Project will be able to draw on these results to integrate antemortem clinical and neuropsychological assessment data, results of neuropathological diagnosis and lesion density, ApoE genotyping, data relating to cardiovascular risk factors (CvRF) and data obtained by specific studies to formulate their future hypotheses and to compare and contrast results obtained from identically processed specimens across multiple disease states. All of the projects in this proposal will also require considerable statistical data analysis and a high level of statistical analytic sophistication. This statistical analysis advice and support is provided by Dr. Schmeidler who has been the statistical expert for this Program Project Grant for over 14 years. Dr. Schmeidler is not only familiar with all of the data collected and analyzed by members of the Program in the past, but he has been actively involved in the planning of the currently proposed studies and in their experimental design. In this revised application, Dr. Schmeidler has helped each proposed project to reformulate their statistical approaches to more fully address the concerns raised by the reviewers. The specific aims of the Data Management and Statistics core are: 1) to continue development of an integrated data warehouse with normalized data warehousing from all projects and cores; 2) to integrate data resulting from the Program Project cores with data emerging from the specific projects into a centralized resource that enhances cross-fertilization of projects while at the same time providing for ready access to all relevant data to all investigators; 3) to provide for data security at the patient and donor level, as well as guarding against data loss and data corruption by centralized integrity checking and backup; and 4) to provide highly sophisticated statistical consultations that involve not only statistical analysis of specific data sets, but integrate data analysis from multiple studies to test hypotheses spanning across projects.

The data management approach of the ADRC is an integrated statistics, data management and data protection solution. The Data Management Core receives, stores, catalogues, tracks and integrates data generated by the Cores and Projects in the ADRC and provides expert advice for the statistical analysis of data components or integrations. At the center of the data management core for the ADRC is the overarching aim to meet the needs of NACC at every level, from data acquisition to data storage, formatting, and reporting. Implementation of this data management system is a dynamic process that aims to meet the growing and diverse needs of the ADRC. These dynamic changes include readiness to respond to changes in not only national standards for research data acquisition and maintenance (e.g., HIPAA) but also to the necessary changes and growth that occurs to NACC. Every effort has been made to systematize and unify all data collection by the clinical and neuropathology cores. This unification serves to enhance the activities of the cores, the access to the generated data by the projects, and the accuracy and ease with which data from the cores can and is reported to NACC. Thus, all ADRC clinical core sites collect a common data set in a common and identical format. This data (Standardized Clinical Dementia Evaluation (SCDE)) is completed by investigators at each site and completed forms are then input into the Data Warehouse by the Data Management core personnel. The Data Warehouse provides for multiple levels of data integrity checking, including range and logic. Once the SCDE results have been input, generating a NACC report is designed to be a turn-key operation. Similarly, all data from the neuropathology core are coded onto CERAD neuropathology battery forms that are enhanced to guarantee inclusion of all NACC neuropathology variables. Again these data are input into the Data Warehouse where data integrity is highly controlled allowing for accurate data for research use and for NACC reporting. The Data Warehouse is also designed to accommodate more idiosyncratic data sets such as those generated by Projects 1-3. By design, all data input into the Warehouse MUST include a set of common identifier fields. The use of these common identifier fields allows for the integration and cross-fertilization of project and core based data. All of the projects in this proposal will also require considerable statistical data analysis and a high level of statistical analytic sophistication. This statistical analysis advice is provided by Dr. Schmeidler who has been the statistical expert for the ADRC for over 14 years. Dr. Schmeidler is not only familiar with all of the data collected and analyzed by members of the ADRC in the past, but he has been actively involved in the planning of the currently proposed studies and in their experimental design.

The Clinical Core established earlier has been combined with the Brain Bank Core. This newly formed core will recruit new subjects for brain tissue and neuroimaging studies. It will clinically follow previously recruited subjects and will perform a single neuropsychological and diagnostic assessment of newly recruited subjects who are likely to come to autopsy. Those subjects who come to autopsy without the benefit of antemortem diagnosis and assessment will receive expert review of medical records and informant interviews to complete a structured psychological autopsy. The Mount Sinai School of Medicine (MSSM) / Bronx Veterans Affairs Medical Center Brain Bank (BB) has been operating for approximately 23 years. The schizophrenia component of the bank has been in operation since 1989. Over 1380 brain tissue specimens have been banked and include 147 confirmed schizophrenia cases with no significant neuropathology or psychiatric comorbidity. In addition, 145 control cases are also available. In the past 18 months this cohort has been expanded to include 21 cases of schizophrenia under the age of 61 and 25 similarly aged controls. In addition, a cohort or 48 cases with major depression or bipolar disease has been added. Additional schizophrenic and control cases are accrued on a continual basis and the enrollment projections and objectives of the Clinical Assessment Core indicate that the number of schizophrenic cases available for autopsy will continue or grow during the next 5 years. All specimens are collected with the absolute minimum postmortem delay as possible (mode PMI = 6 hours for cases with legal next of kin present (65%)). Each brain specimen is banked in both flash-frozen and fixed form and receives a full state-of-the-art neuropathology assessment. Clinical records are searched for every case and all medical conditions and medications received during at least the last 12 months of life are recorded. This core has collaborated with Dr. Dwark (Columbia) and has confirm the initial schizophrenia-associated myelin gene expression deficit findings of the CCNMD in the brains of persons dying at younger ages (mean age 51). The aim of this core is to continue to perform state-of-the-art clinical assessments and brain banking to meet the needs of projects 1 (Hof), 2 (O'Donovan), 3 (Buxbaum) and 4 (Buchsbaum) of the CCNMD, the schizophrenia-associated funded projects that are dependent on the CCNMD, as well as to meet the needs of future, as yet, unspecified studies.

The data management approach of the CCNMD is a two tiered plan. Tier one, involves the implementation and maintenance of a data warehouse system that has recently been developed within the Department of Psychiatry center-wide. This system provides access to the contributing projects and cores within the CCNMD system integrated data storage and data query capacity through a secure web-based portal. A 3 server computer cluster and a 2 terabyte mass storage system positioned behind the Mount Sinai fire-wall and further protected by 128 bit encryption constitutes the physical structure for data management. All of the data tables generated by the Brian Bank Core and the Clinical Core during the past 22 years have been integrated into this browser based data warehouse which now contains over 3100 variables and more than 2 million data points. These data sets are available for interrogation and mining to all CCNMD participants through a secure intranet. Also included as part of the electronically available data sets are macro and microscopic images of Brain Bank tissues and PDF files of source documents on which the research data are based. Tier two, involves statistical support for all CCNMD projects and cores. All of the CCNMD projects require considerable statistical data analysis, and because of the complexity of some of the hypotheses addressed a high level of statistical analytic sophistication. While all of the CCNMD investigators are well versed in the statistical approaches that are applicable to their specific hypotheses and projects, this core will provide them with statistical expertise for not only state-of-the-art analysis of their specific data sets, but also for the integration of each projects data with the data derived from the other projects. The integration of cross-project data for statistical analysis will not only be aided by the data management system described, but also by the fact that statistical testing of hypotheses has been an integral part of the development of the aims and hypotheses of each CCNMD project form the time of their inception.

This project is dedicated to the development and characterization of mouse model systems that best reflect the myelin and oligodendrocyte related (OMR) gene expression deficits in persons with schizophrenia. Several different genetically modified mouse model systems will be evaluated (e.g., Quaking, MAG, PTPRZ1, and Olig2. Each of these mouse model systems will be screened for deficits in the expression of OMR genes using a panel of OMR genes that we have shown to be differentially affected in schizophrenia. Those that evidence gene expression deficits on at least 3 OMR genes known to be affected in schizophrenia will then be assessed for behavioral deficits. The behavioral phenotyping test battery will include screening tests of simple (e.g., reflexes, locomotion, balance, sensation) as well as complex (learning, memory, startle, prepulse inhibition of startle, social interaction, anxiety) behaviors. Coupled with these purely behavioral tests will be pharmacological probes to ascertain whether pharmacological profiles commonly viewed as prototypical for rodent models of schizophrenia are also evidenced by the OMR gene deficient mice. Once best-fit model systems have been identified, they will be studied longitudinally to ascertain the evolution of gene expression and behavioral deficits from 3 months of age through to 18 months of age. In addition, laser capture microdissection techniques will be employed to investigate gene expression in identified cell groups. In collaboration with Project 1, the best-fit mouse model system will be systematically imaged by DTI in vivo at ages corresponding to those for behavioral testing. In collaboration with Project 3, brain tissue specimens from additional mice will be studied for changes in oligodendroglial proliferation, differentiation and survival. Significant progress has already been made in this regard. All of the behavioral test paradigms have been piloted and parameters have been optimized for use in mice in our phenotyping facility (results and descriptions appended). Three of the 4 animal model systems proposed for use (Quaking, Olig2, and MAG) have been obtained. Some studies have already been completed in these mice and colonies have been established to enable more large scale studies.

DESCRIPTION (provided by applicant): The parent grant of this supplement application is focused on testing the mechanistic hypothesis that oligodendrocyte and myelin related (OMR) gene and protein expression deficits in schizophrenia (SZ) are due, at least in part, to abnormalities in the execution of the cell-cycle program by oligodendrocytes. However, despite considerable theoretical and translational interest in the developmental hypothesis of schizophrenia and the known close temporal relationship between myelination and the age of onset of SZ, the parent grant was not designed or resourced to address the fundamental question of whether the OMR and cell cycle abnormalities in schizophrenia arise from developmental challenges or influences. Similarly, although the parent grant goes to considerable lengths to minimize the potential influence of antipsychotic treatment on the results from studies of persons with SZ, it does not attempt to determine the influence, beneficial or detrimental, of antipsychotic medication on the observed OMR and cell-cycle abnormalities directly. Published results from us and others suggest that antipsychotic agents can influence OMR gene expression, but the direction of effect is unclear and how these pharmacological agents affect the execution of the OMR associated cell cycle programs is unknown. Thus, it is not known how age and developmental state modulate OMR gene and protein expression and whether oligodendrocytes are more or less vulnerable to stress within their microenvironment at different postnatal ages. Similarly, it is not clear whether and how the OMR and cell cycle related (CCR) gene and protein expression abnormalities noted in SZ are influenced by antipsychotic medications. The studies proposed in this Supplemental/Revision application are intended to extend the concepts developed in the parent application to address these questions using a well-established animal model of myelin damage and recovery. Mice will be treated with cuprizone for 4 weeks at different ages and the OMR and CCR processes affected by demyelination and remyelination will be studies by quantitative gene and protein expression assays in cortical grey and white matter. Similar procedures will be employed to study the beneficial and/or detrimental effects of typical and atypical antipsychotic agents on OMR and CCR gene and protein expression during demyelination and remyelination. Scientifically, the proposed studies represent an extension of those in the parent grant with direct relevance to the analysis and understanding of the role of myelin, oligodendrocytes and cell-cycle processes in schizophrenia. At the same time, the proposed studies are responsive and faithful to the NIMH topic areas of interest, addressing the "Translational Science" and the "Understanding Postnatal Brain Development" and "critical periods" requirements of the RFA (NOT-OD-09- 058). In addition, this application is responsive to the ARRA in that it will employ two BA-level scientists and ensure the continued employment of a Ph.D. scientist. Furthermore, the award of this application will result in the indirect additional employment of one animal care technician. PUBLIC HEALTH RELEVANCE: Recently, genetic, neuroimaging and postmortem neurobiology studies from multiple laboratories have consistently found abnormalities associated with myelination in the brain of persons with schizophrenia. The parent grant of this supplement application is investigating gene and protein expression changes in specific populations of neurons and oligodendrocytes in postmortem tissue from persons with schizophrenia. How myelin associated gene and protein deficits are affected by age and development and how antipsychotic drugs affect demyelination and remyelination is not addressed by the parent grant. The current proposal aims to address both these questions using an animal model system.

The National Institute of Mental Health (NIMH), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Neurological Diseases and Stroke (NINDS), National Institutes of Health, seek to make human brain tissue and biospecimen samples more widely available for research on neurological, psychiatric, and neurodevelopmental disorders. The objective of this initiative is to increase availability of human disease and control biospecimens, increase proactive involvement of the disease advocacy and patient communities, and more efficiently acquire, curate, store, and distribute these tissues with the ultimate goal of reducing the burden of nervous system disorders. In order to do so, the NIH intends to establish multiple contract collection sites (i.e., the NIH Brain and Tissue Repositories (NBTR)) to provide services that will actively acquire, receive, process, store, curate, preserve, and distribute central nervous system and related biological specimens (including fetal tissues) to qualified investigators. These services were previously provided through grants that have expired and/or will be expiring in the coming years. The NIH will now continue these existing services through competitively awarded contracts. New sources are also encouraged to compete.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) affects half of the US population over the age of 85 and causes destruction of select networks and cell groups within the brain. AD manifests initially as mild cognitive decline, but gets progressively worse and is always fatal. Despite significant progress identifying susceptibility loci for AD in genome-wide association and whole exome sequencing studies, to date, a predictive risk score for AD that achieves clinical utility on an individual basis given DNA variation information alone has been elusive. This proposal aims to develop a multiscale-network approach to elucidating the complexity of AD. Multiscale network models causally linked to AD will be developed based on existing AD-related large scale molecular data and the high-impact, high-resolution complementary datasets generated through this application. Using brain slice cultures, iPS-cell-derived mixed cultures of human neuronal, oligodendroglial, and astrocytic cell systems, and fly models of AD, we seek to reconstitute the AD-related networks discovered in the multiscale analysis in these living systems and then employ high-throughput molecular and cellular screening assays to not only validate the actions of individual genes on molecular and cellular AD-associated processes, but also validate the molecular networks we implicated in the disease. Our initial multiscale studies have implicated the microglial protein TYROBP as one key driver of AD pathogenesis, a hit we have partially validated, but that we will further validae along with other hits using iPSC-derived mixed cultures of different brain cell types, murine brain slices and AD fly models. We will analyze the potential ability for network-derived hits like TYROBP to modulate standard AD pathology involving A¿ and tau as well as its ability to shift networks in those same systems in such a way as to reflect the behavior of networks discovered in the multi-scale analysis. Importantly, the model building and validation will be iterated to produce updated/refined models based on validation results that, in turn, will be mined to generate updated lists of prioritized targets for validation. In this way, through the course of th grant, as new knowledge accumulates externally and as we generate increased amounts of data including validation data, our models will take into account the most up to date information to produce the most predictive models of AD. As a service to the AD research community, we will provide dramatically improved general access to large-scale, multidimensional datasets, together with systems level analyses of these datasets.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) affects half of the US population over the age of 85 and causes destruction of select networks and cell groups within the brain. AD manifests initially as mild cognitive decline, but gets progressively worse and is always fatal. Despite significant progress identifying susceptibility loci for AD in genome-wide association and whole exome sequencing studies, to date, a predictive risk score for AD that achieves clinical utility on an individual basis given DNA variation information alone has been elusive. This proposal aims to develop a multiscale-network approach to elucidating the complexity of AD. Multiscale network models causally linked to AD will be developed based on existing AD-related large scale molecular data and the high-impact, high-resolution complementary datasets generated through this application. Using brain slice cultures, iPS-cell-derived mixed cultures of human neuronal, oligodendroglial, and astrocytic cell systems, and fly models of AD, we seek to reconstitute the AD-related networks discovered in the multiscale analysis in these living systems and then employ high-throughput molecular and cellular screening assays to not only validate the actions of individual genes on molecular and cellular AD-associated processes, but also validate the molecular networks we implicated in the disease. Our initial multiscale studies have implicated the microglial protein TYROBP as one key driver of AD pathogenesis, a hit we have partially validated, but that we will further validae along with other hits using iPSC-derived mixed cultures of different brain cell types, murine brain slices and AD fly models. We will analyze the potential ability for network-derived hits like TYROBP to modulate standard AD pathology involving A¿ and tau as well as its ability to shift networks in those same systems in such a way as to reflect the behavior of networks discovered in the multi-scale analysis. Importantly, the model building and validation will be iterated to produce updated/refined models based on validation results that, in turn, will be mined to generate updated lists of prioritized targets for validation. In this way, through the course of th grant, as new knowledge accumulates externally and as we generate increased amounts of data including validation data, our models will take into account the most up to date information to produce the most predictive models of AD. As a service to the AD research community, we will provide dramatically improved general access to large-scale, multidimensional datasets, together with systems level analyses of these datasets.

Mount Sinai ADRC: Core D (Haroutunian/Purohit) | PROJECT SUMMARY The chief function of the Neuropathology Core is to provide state of the art diagnostic services, collection, characterization and preservation of well-prepared brain material, and distribution of samples for cutting edge research. As a core that has been functioning continuously for the past 29 years, the personnel and infrastructure to provide the functions necessary to fulfill this overarching aim are well in place and have operated efficiently and productively over the entire history of the Mount Sinai ADRC. This Neuropathology Core application will review how this has been accomplished and describe the large, well characterized specimen collection and its associated rich database of clinical and neuropathologic information. We also wish to point out that this Neuropathology Core has been and continues to be very pro-active in stimulating research that employs both our tissue repository as well as our very complete databases. We have a long record of encouraging researchers in becoming involved in these activities. Encouraging the wide use of our facilities and core resources has resulted in considerable research productivity that is outside of what is specifically required for a neuropathology Core or is specified in this Core's Specific Aims. However, this record of productivity further emphasizes the Center environment of the ADRC and provides and how the Core stimulates research among ADRC participants and the greater AD and aging research community.

The National Institute of Mental Health (NIMH), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the National Institute of Neurological Diseases and Stroke (NINDS), National Institutes of Health, seek to make human brain tissue and biospecimen samples more widely available for research on neurological, psychiatric, and neurodevelopmental disorders. The objective of this initiative is to increase availability of human disease and control biospecimens, increase proactive involvement of the disease advocacy and patient communities, and more efficiently acquire, curate, store, and distribute these tissues with the ultimate goal of reducing the burden of nervous system disorders. In order to do so, the NIH intends to establish multiple contract collection sites (i.e., the NIH Brain and Tissue Repositories (NBTR)) to provide services that will actively acquire, receive, process, store, curate, preserve, and distribute central nervous system and related biological specimens (including fetal tissues) to qualified investigators. These services were previously provided through grants that have expired and/or will be expiring in the coming years. The NIH will now continue these existing services through competitively awarded contracts. New sources are also encouraged to compete.

Project Summary Alzheimer's disease (AD) is a debilitating neurodegenerative disorder. Pathologically, AD is characterized by amyloid plaques and neurofibrillary tangles. Clinically, AD patients present with progressive memory decline followed by deterioration of other cognitive domains and activities of daily living. Advanced age is the greatest risk factor. No effective method is available for preventing and/or treating this devastating disease. However, certain individuals of the elder population (? 85 years) remain cognitively intact, including some with substantial plaques and neurofibrillary tangle burdens, the two pathological hallmarks for fully symptomatic AD. The mechanisms of cognitive resilience and protection against AD in these elderly persons remain elusive. This proposal brings together scientists and postmortem human brain tissue samples from two major AD-research centers (the Icahn School of Medicine at Mount Sinai and the Rush University Medical Center) and aims to systematically identify and validate genetic variants, genes, proteins, and molecular networks underlying cognitive resilience to AD risk and proposes to build a comprehensive unbiased signaling pathway map underlying cognitive resilience to AD. Towards this end, we will develop an AD resilient cohort comprised of genetic, transcriptomic and proteomic data in the prefrontal cortex from a large number of brains in four categories: 1) very old (age of death (AoD) ? 85) AD-resilient, 2) young (AoD < 85) healthy, 3) very old (AoD ?85) AD and 4) young (AoD < 85) AD. We will perform systems genetics and integrative network biology analyses on the large-scale high-dimensional molecular profiling data to identify genetic variants, genes, proteins, and molecular networks underlying cognitive resilience to AD risk. We will systematically validate key drivers of the molecular networks underlying the cognitive resilience to AD using two diverse (C. elegans and mouse) model systems. We will validate the structures of AD-resilient molecular networks for building a data- driven, comprehensive signaling pathway map underlying cognitive resilience to AD risk. In particular, we will test the hypotheses that enhanced mitochondrial function and immune competence as well as their underlying molecular networks confer cognitive resilience. Our study will not only present a global landscape of the interplays among genetic variants, mRNAs and proteins responsible for cognitive resilience to AD but also pinpoint critical network structures and key drivers that can potentially lead to development of novel prevention strategies in combating AD.

Project Summary Alzheimer's disease (AD) is the most common form of dementia and is characterized by progressive cognitive impairment and neurodegeneration. Despite decades of clinical, neuropathological and neurobiological research the molecular substrates and causal biological substrates of AD remain elusive. Most studies have focused on vulnerable brain regions defined by neuroimaging or neuropathological findings. However, our recent unbiased study of transcriptomic networks in 19 brain regions identified the parahippocampal gyrus as the brain region with the greatest transcriptomic changes associated with disease development and progression irrespective of whether disease development and progression were defined using functional measures of cognition or the canonical neuropathological lesions of AD (Wang MH et al, Genome Medicine 8:104). These observations have been reinforced further by our larger scale multi-Omics data from over 200 donors and 4 brain regions, for which we have generated whole exome, whole genome and RNA sequencing data through the current AMP-AD Consortium. These studies, and most others published to date, have not examined transcriptomic, epigenetic and proteomic changes in the same tissues and donors and by utilizing whole tissue homogenates have been unable to identify vulnerabilities holistically and with cell-type-specific fidelity. Here we propose to generate additional, matched large-scale proteomic and epigenetic data as well as cell type specific transcriptomic and epigenomic data from the parahippocampal gyrus and develop novel network inference and analysis approaches to integrate all these multi-Omics data as well as cognitive, pathological and physiological data to construct high-resolution, multiscale molecular networks in the parahippocampal gyrus in AD. To overcome some of the drawbacks of postmortem studies and to gain insight into causal mechanisms and networks we will experimentally validate networks identified in postmortem tissue by perturbing a number of key drivers in AD transgenic mouse primary brain cells and human iPSC derived brain cell cultures to determine the mechanisms underlying vulnerability of the parahippocampal gyrus. We expect that our data-driven and hypothesis-free multiscale network modeling of parahippocampal vulnerability in AD will have a large impact on the AD field and lead towards a more comprehensive and precise understanding of AD pathogenesis. More importantly, the proposed research will pave a path towards drug discovery for AD targeted at specific vulnerabilities in specific brain cell types.

PROJECT SUMMARY Genome wide association studies of complex neuropsychiatric diseases, including schizophrenia (SCZ) and bipolar disorder (BD), have identified numerous risk loci that are mostly situated in non-coding regions, necessitating a systematic study of non-coding regulatory elements. It has also been established that SCZ risk loci are preferentially located within promoter and enhancer regulatory sequences of neurons and that they co- localize with expression Quantitative Traits Loci (eQTL), thus implicating specific genes. However, work that has been performed to-date has limited spatiotemporal resolution as: (1) only a few cortical regions have been examined, (2) the effect of 3D genome on transcriptional regulation across the lifespan has never been examined, and (3) studies have been limited to homogenate brain tissue or include only broadly defined neuronal and non-neuronal populations. To address these limitations, we will generate cell type-, brain region- and age period-specific high-dimensional data that will inform us of the effect of 3D genome on the transcriptional regulation and will link regulatory elements with specific transcripts. In Aim 1, we will examine the impact of SCZ and BD risk variants on 3D genome structure and transcriptional regulation. We will use fluorescence activated nuclei sorting to isolate glutamatergic and GABAergic neuronal as well as oligodendrocyte and astrocyte nuclei from five human cortical and subcortical regions relevant to SCZ and BD across five postnatal age periods. We will then generate cell-type specific annotations for gene expression and enhancer RNA (RNA-seq and CAGE-seq), open chromatin (ATAC-seq), insulators (CTCF ChIP-seq), active enhancers and promoters (H3K27ac and H3K4me3 ChIP-seq), and chromatin loop interactions (HiC and Capture-C). Using the resulting data, we will delineate cis transcriptional regulation associated with the 3D genome (including promoter-enhancer loopings) and uncover the functional consequences of SCZ and BD risk loci on enhancer-transcript units. In Aim 2, we will examine the impact of SCZ and BD risk variants on cell type-specific gene expression and epigenome QTLs. We will map RNAseq and ATACseq at the single cell level and will use cell type-specific markers and deconvolution approaches to the existing large scale transcriptome and epigenome datasets, from CommonMind consortium, psychENCODE and other projects, in order to generate cell type-specific expression and epigenome QTLs. We will then co-localize SCZ and BD risk loci with expression and fine map epigenome QTLs to define disease-associated enhancer-transcript units. Finally, in Aim 3, we will validate disease-associated enhancer-transcript units by epigenomic editing of risk loci in iPCS-derived cells. We will apply the CRISPR/Cas9 to activate (p300) or inhibit (KRAB) enhancers of the disease-associated enhancer-transcript units (Aims1-2). Lastly, we will introduce epigenomic perturbations and characterize gene expression, chromatin accessibility and chromatin loop interactions in hiPSC-derived cells. It is our expectation that these integrated analyses will enable us to assign specific regulatory units within SCZ and BD risk haplotypes to specific cell types, brain regions and age windows, thereby providing insight into the mechanisms of genetic risk for SCZ and BD.

Project Summary Alzheimer's disease (AD) pathology is characterized by the presence of phosphorylated tau in neurofibrillary tangles (NFTs), dystrophic neurites and abundant extracellular ?-amyloid in senile plaques. However, the etiology of AD remains elusive, partly due to the wide spectrum of clinical and neurobiological/neuropathological features in AD patients. Thus, heterogeneity in AD has complicated the task of discovering disease-modifying treatments and developing accurate in vivo indices for diagnosis and clinical prognosis. Different approaches have been proposed for AD subtyping, but they are generally neither suitable for high-dimensional data nor actionable due to the lack of mechanistic insights. Increased knowledge and understanding of different AD subtypes would shed light on recently failed clinical trials and provide for the potential to tailor treatments with specificity to more homogeneous subgroups of patients. By integrating genetic, molecular and neuroimaging data to more precisely define AD subtypes, we may be able to better discriminate between highly overlapping clinical phenotypes. Furthermore, the identification of such subtypes may potentially improve our understanding of its underlying pathomechanisms, prediction of its course, and the development of novel disease-modifying treatments. In this application, we propose to systematically identify and characterize molecular subtypes of AD by developing and employing cutting-edge network biology approaches to multiple existing large-scale genetic, gene expression, proteomic and functional MRI datasets. We will investigate the functional roles of key drivers underlying predicted AD subtypes as well as three candidate key drivers from our current AMP-AD consortia work in control and AD hiPSC-derived neural co-culture systems and then in complex organoids by screening the predicted transcriptional impact of top key drivers in single cell and cell-population-wide analyses. Functional assays in each cell type will be used to build evidence for relevance to AD-subtype phenotypes. Single cell RNA sequencing data will be generated to identify perturbation signatures in selected drivers that will then be mapped to subtype specific networks to build comprehensive signaling maps for each driver. The top three most promising drivers of AD subtypes and the three existing AMP-AD targets will be further validated using a) an independent postmortem cohort, and b) recombinant mice, including amyloidosis, tauopathy and new ?humanized? models.

PROJECT SUMMARY Neuropsychiatric symptoms (NPS) are core features of Alzheimer's disease (AD) and related dementias that are associated with major adverse effects on daily function and quality of life, and accelerate time to institutionalization. Of all the NPS, depression is the most frequently observed symptom in people with mild cognitive impairment and early AD. As the disease progresses, agitation, delusions and hallucinations become more common, whereas apathy is the most persistent and frequent NPS throughout all the stages of AD. AD-NPS share some clinical features with serious mental illnesses (SMIs), such as schizophrenia, bipolar disorder and major depressive disorder, but whether these conditions share similar aethiopathies is unclear. Given that reliable treatments for NPS in the context of AD and other dementias do not exist, a better understanding of the molecular mechanisms and pathways underlying NPS in AD and other neuropsychiatric illnesses is a critical next step to identify reliable biomarkers that could lead to novel therapeutics. There are two overarching goals of this proposal. First, we will identify the molecular mechanisms and neuropathological changes that are associated with the presence of NPS in patients with AD. Second, we will examine if the mechanisms of pathology associated with NPS are shared or distinct among AD and SMIs. More specifically, we propose to build multi-scale integrative models using phenomics and genomics data from 1,264 autopsy cases derived from a single brain bank. The bank includes detailed phenomics data such as well characterized NPS, clinical diagnosis (AD and other neurodegenerative or neuropsychiatric traits), severity of cognitive decline and neuropathology for each patient sample. From each case, we will apply innovative approaches that reduce the cost and technical biases associated with conventional methods, and capture gene expression signatures and epigenetic regulatory elements at the single-cell level. Novel deep-learning methods will be applied for the multi-scale integration of neuropathologic changes with genetic markers and functional genomic changes (such as changes in gene expression and enhancer sequences) within specific cell types, to predict various NPS in AD and other neuropsychiatric traits; we refer to these integrative models as genotype- marker-phenotype models. We expect that these models will enable us to assign genotypes and molecular markers to specific NPS within AD and other neuropsychiatric traits at the single-cell level, an unprecedented level of resolution. In addition, we will test the translational potential of the genotype-marker-phenotype models to predict AD-NPS using independent large-scale biobank datasets, in which genotypes and electronic health records are available. Successful completion of the proposed studies will have immediate utility by generating potential biomarkers for NPS diagnosis and prognosis and by providing predictive models for patient stratification in clinical trials. In the longer term, our models will help us create a blueprint for therapeutic strategies and interventions to treat NPS in AD.

PROJECT SUMMARY Great progress has been made in mapping the transcriptome and some its epigenomic determinants of the adult and developing human cerebral cortex (including alterations in common psychiatric disease) through the efforts of the PsychENCODE consortium. However, next to nothing, or very little, is known about genomic regulation in brainstem monoaminergic neurons and their ascending projections into the forebrain, a circuitry critically involved in the pathophysiology of mood and psychosis spectrum disorders and substance abuse disorders, among others. The goal of our project is to construct transcriptome and epigenome (incl. 3D genome/chromosomal conformation) maps for midbrain dopaminergic neurons and for their surrounding non- neuronal cells, and to assess the relationship to known genetic risk factors for complex mental illness, including psychosis with substance abuse co-morbidity. We will apply integrative methods for functional analysis of risk genetic variation and networks, including but not limited to Bayesian network reconstruction and prediction algorithms of variant causality to identify key drivers of schizophrenia and bipolar disease pathology, and drug addiction co-morbidity. These methods will simultaneously integrate multiple different dimensions of data: DNA variation, RNA expression, chromatin accessibility, 3D structure of the genome, known pathway and gene network information in the context of clinical phenotype data. The fundamental source of data for the project comes from the current studies on human midbrain functional omics, and the CommonMinds and PsychENCODE consortia (whole genome sequencing and cortical functional omics data), the Psychiatric Genomics Consortium and the Million Veterans Project (genetic variation and disease phenotypes). The Million Veterans Project (MVP) has collected genotyping and phenotypic data from ~700,000 individuals, including a subgroup of 50,000 veterans diagnosed with SCZ and BD and a larger group of individuals diagnosed with other neuropsychiatric traits (recurrent depression, suicide and substance abuse). We will make our newly generated transcriptome and epigenome datasets from adult midbrain, as well as the network and predictive models, available to the research community in accordance with NIMH data sharing policies.

PROJECT SUMMARY Despite extensive clinical and genomic studies, the mechanisms of development and progression of Alzheimer's disease (AD) remain elusive. Microglia and other myeloid origin cells (collectively called human brain immune cells, or HBICs) have recently emerged as crucial players in the pathogenesis of AD. This is supported through genetic association studies, where many of the common and rare risk loci affect genes that are preferentially or selectively expressed in HBICs, emphasizing the pivotal role of the innate immune system in AD. In addition, single cell RNA sequencing analysis in mouse models of AD has identified a microglia subpopulation that is present at sites of neurodegeneration. It is unclear if HBICs assume a protective or damaging role, but that might vary depending on the stage and progression of AD. Therefore, further analysis of microglia and other immune cells purified from human brains is needed to understand the state of HBIC activity in human AD at different stages of disease. As HBICs constitute a small proportion of total brain cells, homogenate-based studies in human brain tissue are unlikely to capture the full spectrum of HBIC molecular signatures, especially in light of the growing appreciation for the diversity of HBICs in the brain. The proposed work addresses some of the limitations of previous research and is focused on: (1) cell type specific and single cell studies in immune cells isolated from human brain tissue; and (2) a systematic study of the regulatory effects of non-coding DNA on gene and protein expression, which is necessary given that the majority of common risk variants are situated in non-coding regions of the genome. More specifically, our application is uniquely designed to: (1) apply innovative genomic approaches and generate multi-omics data from HBICs isolated from 300 donors, including whole genome sequencing, RNAseq, ATACseq, HiC chromosome conformation capture and proteomics; (2) perform state-of-the-art single cell analysis that will allow us to assess the diversity of HBIC subpopulations, as well as detect those that are associated with AD; (3) connect AD risk loci with changes in the regulatory mechanisms of gene and protein expression in HBICs; and (4) organize HBIC multiscale data in functional networks and identify key drivers for AD. Our overall hypothesis is that HBIC subpopulations assume a neuroprotective role during aging and early stages of AD, but as disease progresses, specific HBIC subpopulations transform to neuroinflammatory phenotype(s). This conversion is partially driven by AD risk genetic variants, which affect regulatory mechanisms of genes that are key drivers of neuroinflammatory HBIC subpopulations. Successful completion of the proposed studies will provide: (1) an increased mechanistic understanding of dysfunction in AD risk loci; (2) prioritization of significant loci and genes for future mechanistic studies; and (3) access to large-scale, multidimensional datasets, together with systems level analyses of these datasets for transcriptional regulation in HBICs, which is an urgently needed (and currently missing) resource.

Mount Sinai ADRC (Sano): Neuropathology Core (Core D) ? Research Summary The overarching aim of the Neuropathology core is to promote research in AD by providing exceptionally well characterized postmortem human brain tissue and related specimens to ADRC and non-ADRC researchers within and outside the Mount Sinai neuroscience community and to support the ADRC cores, including the Clinical, Biomarker, Genetics, Education and Data Management cores. The Core's enduring goal and practice of providing accurate assessments and well-characterized brain tissues and derivatives to the neuroscience community is precisely aligned with the National Plan to Address Alzheimer's Disease. Emerging and potentially paradigm-shifting neuropathological concepts, such as AD subtypes, depend on the availability of well- characterized brains from extensively phenotyped donors for research. Yet, there is an alarming shortage of well annotated specimens and neuropathologists with expertise to support the research community. The overall aim of this core is to continue to maintain and operate the Brain Bank in such a way as to meet the dementia research needs of neuroscience laboratories optimally while providing state-of the art neuropathologic characterization of all brain specimens referred to the bank by the clinical core. In addition, Core D endeavors to anticipate the research needs of the future by banking multiple non-CNS specimens to enable: a) the translation of findings in the CNS to readily accessible biomarkers; and b) studies of systemic and environmental influences on dementia neuropathology. The specific aims of the Brain Bank/Neuropathology Core are to: maintain, manage and expand a large dementia brain biorepository; enable and facilitate state-of-the-art translational research; determine and record quantitatively the extent and distribution of relevant lesions present within each brain specimen; provide training opportunities for new core leaders and neuropathology researchers.

Project summary Alzheimer's disease (AD) pathology is characterized by the accumulation of neurofibrillary tangles, dystrophic neurites, and abundant extracellular fibrils of amyloid-? peptide. However, the etiology of typical late onset AD remains elusive. Over 20 genes have been associated with late onset AD, and this heterogeneity complicates the task of discovering disease modifying treatments. The parent application proposed to: (i) identify robust molecular subtypes of AD and their characteristic molecular signatures across different layers of Omics data; (ii) characterize molecular subtypes of AD by molecular signatures, multiscale regulatory networks and key drivers; (iii) evaluate genomic and functional impact of key drivers using human iPSC derived neurons and glia; and (iv) validate key drivers of molecular networks underlying AD subtypes. Recently, efforts by the investigators in the parent grant led to the identification of the VGF gene as a key driver of the network predicted to be altered in AD. However, the molecular mechanism by which VGF modulates the network altered in AD is not well understood. It is possible that receptor systems activated by peptides derived from VGF play a crucial role in this process. Support for this comes from our previous studies of another key driver, PREPL, where we found that decreases in PREPL expression leads to decreases in levels of secreted VGF- derived peptides. Also, several VGF-derived peptides have been detected in the cerebro-spinal fluid of AD subjects and many of these peptides exhibit distinct biological activities. This suggests the existence of receptors for the VGF-derived peptides and an important role for them in AD. To date receptors for the majority of these peptides have not been definitively identified. In this supplement we propose to carry out studies to identify neuronal receptors to 18 VGF-derived peptides using the PRESTO-TANGO® assay system that contains 302 G protein-coupled receptors including 135 listed as ?orphan? receptors. Identification of these receptors is a prerequisite to studies investigating the physiological significance of VGF-derived peptides to AD as well as to identifying small molecules targeting these receptors, which could become potential therapeutics for the treatment of AD.

PROJECT SUMMARY Alzheimer?s disease (AD) stands out as notable in two respects??in not having a cure and in affecting women more than men. While declining estrogen has been thought to underpin post?menopausal AD, there is a clear clinical correlation of AD with rising levels of follicle?stimulating hormone (FSH). Most notably, there is a ?spike? in cognitive decline in women in the early years of the menopausal transition, when serum estrogen is normal and FSH levels begin to rise. Collaborative studies between the Mount Sinai and Emory groups have identified FSH as a potential driver for AD?and suggest that rising FSH levels may contribute to the disproportionate increase of AD in aging women. Notably, we find that FSH receptors (FSHRs) are expressed in both mouse and human brain, and that the injection of recombinant FSH or ovariectomy (that elevates serum FSH) aggravates AD pathology and cognitive decline in 3xTg mice. Inhibiting the action of FSH in 3xTg or APP/PS1 mice by an FSH?blocking antibody or downregulating Fshr expression in the hippocampus prevents onset of the AD phenotype. The Emory group also provides strong preliminary evidence that FSH upregulates C/EBP?, which activates asparagine endopeptidase (AEP), a ??secretase that cleaves amyloid precursor protein (APP) and Tau??resulting in neuritic plaques and neurofibrillary tangles, respectively. The goal of the transdisciplinary collaboration between the disciplines of endocrinology and neuroscience is to fully understand the mechanism of FSH action on AD?vulnerable brain regions. Thus, in Specific Aim 1, we will map the distribution and cellular localization of the FSHR and its signaling partners CEBPB and LGMN in human and mouse brain using single?transcript technologies. In Specific Aim 2, we will examine the function of the brain FSHR in driving AD pathology and cognitive decline. For this, we will downregulate or overexpress the Fshr in specific brain areas of 3xTg mice by stereotaxically injecting AAV expressing siFshr or Fshr. We will also study the effect of high FSH in 3xTg mice rendered haploinsufficient in Cebpb, and delineate the transcriptomic architecture of FSH?treated human neuronal cells by RNA?seq. In Specific Aim 3, we will determine whether deleting the Fshr or inhibiting FSH action by our murine FSH blocking antibody, Hf2, injected over the lifespan of 3xTg mice can prevent the onset of cognitive decline. To contemporaneously replicate our data, the Emory group will study the effect of treating established cognitive impairment with Hf2 in 18?month?old APP knock?in (KI) mice. In all, our proof?of?concept studies??conducted using our Good Laboratory Practices (GLP) Platform??should not only establish a role for high FSH in driving AD, but also provide a framework for the future testing of our humanized FSH?blocking antibody, Hu6, in aging women.