Joseph D. Buxbaum - US grants

DESCRIPTION A preponderance of evidence suggests that neurons are a major source of Abeta in the brain. Little is known about the subcellular localization and processing of APP in central neurons, although it is known that APP undergoes both anterograde and retrograde transport. It is also known that APP is not found in appreciable amounts in neuronal plasma membranes. The fate of transported APP is therefore still a mystery. In preliminary studies the PI has been able to demonstrate that a majority of APP in synapses is found in a novel, rab5-rich, vesicular organelle. Rab5 is a small GTP-binding protein which is required for endocytosis in fibroblasts and is involved in the recycling of small synaptic vesicles in nerve terminals. The major focus of this application will be the further purification and characterization of this organelle, the determination of the role of rab5 (and other APP-associated proteins) in APP trafficking and processing, and the characterization of the novel organelle as a potential site of APP metabolism. Specific Aim 1: To localize APP within highly purified synaptosomes (containing nerve terminals and some postsynaptic elements), and purify and characterize the APP-containing synaptic organelles by conventional and/or immunological methods. Specific Aim 2: To determine the ability of specific proteins, associated with APP- containing synaptic organelles, to regulate the localization, trafficking, and processing of APP in cultured cells, including polarized epithelial cells and primary neuronal cultures. Specific Aim 3: To test the purified APP-containing synaptic organelles for their ability to metabolize APP and for the presence of proteases, including the a-secretase that the PI has recently identified, as well as for the presence of the Alzheimer-associated proteins, presenilin-1, presenilin-2, and apolipoprotein E.

The presenilins are proteins of little known function which are involved in the etiology of Alzheimer's disease (AD). These proteins appear to be involved in development (particularly neural progenitor cell survival and neurogenesis), survival forms of apoptosis, and Abeta formation. There is evidence indicating that at least some of the effects of presenilins are mediated by the interaction of the COOH-terminus of these proteins with a cytoplasmic protein. For this reason, we have used the two-hybrid system to identify proteins interacting with the COOH- terminus of the presenilins and have found a novel protein which we call "calsenilin". Our results indicate that the presenilins associate with calsenilin in situ and that calsenilin regulates the level of presenilin fragments. These data implicate calsenilin in the biology of the presenilins. Calsenilin is a novel member of the recovering family. Members of this family are calcium binding proteins which appear to play a role in modulating signaling transduction cascades in response to calcium signals. Our broad, long-term goal is to understand the normal function of the presenilins and to understand the molecular of their role in AD. As step towards this goal, we have identified a protein (calsenilin) which interacts with the domain of the presinilins which has been postulated as interacting with other proteins in order to exert biological effects. The next step would be to characterize the function of calsenilin, to study its role in presenilin-mediated effects, and to examine calsenilin in AD. From these studies, the role of calsenilin in presenilin biology and AD will be clarified. In the current proposal, we will take advantage of the unique resources of the ARDC to accomplish these goals. The specific aims are as follows: 1. To localize calsenilin in the rat and primate nervous system. 2. To determine the relationship between the distribution of calsenilin, presenilin, neurofibrillary pathology, Abeta deposition, glutamatergic receptor subtypes and dying neurons in AD brain. 3. To characterize neural development of nice in which the calsenilin gene has been disrupted. 4. To characterize presenilin levels, Abeta formation and Abeta accumulation in mice in which the calsenilin gene has been disrupted.

disease /disorder onset; enzyme linked immunosorbent assay; mass spectrometry; pathologic process

The majority of early-onset familial Alzheimer disease (AD) cases are caused by mutations in the highly related genes presenilin-1 (PS1) and presenilin-2 (PS1) and presinilin-2 (PS2) which encode what are predicted to be integral membrane proteins with six or eight membrane spanning domains. Because the presenilin mutations account for the majority of cases of inherited early onset forms of AD, understanding the normal function of the presenilins and how mutations in these proteins lead to Alzheimer disease are central questions in Alzheimer's research. In terms of AD, two aspects of presinilin activity have received particular attention. First, mutant presenilin has been shown to alter the relative levels of the longer and potentially more pathogenic amyloid Abeta peptide variants (i.e., Abeta1-42/43). Second, mutant presenilin has been shown to promote apoptosis. With regard to the role of presenilins in apoptosis, there is evidence suggesting that a protein-protein interaction between the COOH-terminus of PS2 and as yet unidentified protein(s) is responsible for activation for apoptosis. This proposal focuses on identifying and characterizing protein(s) which interacts with the COOH- terminus of the presenilins, mediating their effects on apoptosis and, possibly, on altering the relative levels of the longer Abeta variants. The specific aims are as follows: 1) To identify and characterize proteins that interact with the COOH-terminus of the presenilins; 2) To study the role of interactor protein(s) in presenilin-mediated apoptosis; 3) To dissect the role of interactor protein(s) in presenilin-mediated increases in longer Abeta variants; and, 4) To elucidate the relationship between presinilin-mediated apoptosis and presenilin-mediated increases in longer Abeta variants.

Autism has been shown to be highly heritable, with heritability estimated at over 90%. Estimates for the number of genes underlying autism range from 3 to over 15 and there is evidence for genetic heterogeneity. Although there are several genome-wide linkage analysis studies that have been completed, there is a disappointing concordance in linked regions between the studies. Chromosomal regions where there is more agreement include 7q and 2q. The evidence for linkage to chromosome 2 was observed in prior studies of ours as well as in other studies in the field. The current application represents a focused attempt to identify an autism susceptibility gene on 2q. Our preliminary data also shows evidence for linkage on chromosome 1 and other sites when the population is stratified based on repetitive behaviors. This study aims to identify an additional 150 families with at least two family members affected with autism, to genotype samples and analyze the data for linkage to autism and repetitive behaviors. In addition, we will look for overlap between areas of linkage for repetitive behaviors and candidate genes of the serotonin system.

DESCRIPTION (provided by applicant): FE65 is an adapter protein composed of three protein-protein interaction domains. It contains a WW domain and two PI domains. FE65 binds to APP through its carboxy-terminal PI domain (PID2). FE65 was initially described as a transcriptional activator, which can be found both in the nucleus and the cytoplasm. It was later shown to bind the cytoplasmic domain of APP and to profoundly alter APP processing and trafficking. Recent reports have shown that FE65 can localize to the nucleus and that full-length APP serves as a cytosolic anchor for FE65. FE65 PID1 can bind the transcription factor CP2/LSF/LBP1 and can also bind the histone acetyl transferase Tip60 in the nucleus and modulate transcription. In analogy to the notch intracellular domain (NICD), it is suggested that FE65 binds to the gamma-cleaved cytoplasmic tail of APP (gamma-CTF) and translocates to the nucleus where it binds Tip60 and activates transcription. The central hypothesis of this proposal is that a complex, including the gamma-CTF fragment of APP and the adaptor FE65, is a regulator of transcription. The specific aims of this proposal are: Specific Aim 1. To identify the nuclear macromolecular complex that involves FE65 and the gamma-cleaved cytoplasmic tail of APP. Specific Aim 2. To discover genes whose transcription is regulated by the FE65/gamma-CTF complex. Specific Aim 3. To elucidate signals that regulate the formation and nuclear translocation of the FE65/gamma-CTF complex.

Vascular disease can cause multi-infarct dementia. However in addition vascular disease and its associated risk factors such as hypercholesterolemia may also hasten the progression of Alzheimer's disease (AD). The long-range goal of this project is to determine the effect that systemic hypercholesterolemia and its associated vascular disease have on series of parameters of relevance to AD pathogenesis using hypercholesterolemic mice that are now widely used in atherosclerosis research. Specifically, we will utilize mice with a targeted disruption of the low-density lipoprotein receptor gene (LDLR -/- mice). By modulating the amount of cholesterol in the diet, plasma cholesterol in these mice can be widely varied and the degree of associated vascular disease reproducibly varied from minimal to severe. As biochemical markers of the AD process we will examine Abeta (Ab) production and tau phosphorylation. We will examine the functional consequences of hypercholesterolemia in these animals in two models of neural plasticity and repair, namely repair after entorhinal cortex lesioning and effect on neurogenesis in the adult hippocampus. A further functional correlate will be obtained through behavioral testing and since little is known about the effects of systemic hypercholesterolemia on brain cholesterol metabolism we will examine a series of parameters of cholesterol metabolism including 24S-hydroxycholesterol production in brain. To distinguish the effects hypercholesterolemia from its vascular consequences animals will be examined after short-term dietary manipulations when hypercholesterolemia is present but little vascular disease and after longer treatment intervals when significant vascular disease will be present. In each case both systemic and cerebral microvascular changes will be quantitatively assessed. Effects in LDLR -/- mice will be extended by determining if systemic hypercholesterolemia and vascular disease influence Ab production, plaque load and tau phosphorylation in the Tg2576 mouse model of AD by breeding the Tg2576 transgene onto an LDLR -/-background. Finally, we will examine Ab levels and tau conformational changes by ELISA in patient samples that have been scored for cerebrovascular pathology and amyloid pathology. Collectively these studies give us the opportunity to examine the effects of hypercholesterolemia and vascular disease on a series of AD related changes in both mouse and human material.

Low density lipoprotein-receptor like protein 1 (LRP) is a transmembrane protein that has been directly and indirectly implicated in Alzheimer's disease (AD). LRP binds the longer splice variants of the Alzheimer amyloid protein precursor (APP) (APP751 and APP770), which contain a Kunitz protease inhibitory (KPI) domain. LRP also binds apolipoprotein E (Apo E), and a2-macroglobulin (A2M). APP, Apo E and A2M have all been genetically and biochemically implicated in AD. Polymorphism in LRP may also be associated with AD, although with weak effect. The cytoplasmic domain of LRP binds the adaptor FE65, a protein that also binds APP and may also show weak genetic association with AD. Finally, recent evidence indicates that, like APP, LRP undergoes a presenilin mediated intramembranous cleavage in a process now known as regulated intramembranous proteolysis (RIP). Presenilin is genetically and biochemically linked to AD and is directly involved in the formation of Abeta. In spite of the genetic and biochemical evidence for a role for LRP in AD-related proteins and processes, several questions remain unanswered. First, it is not clear whether the interactions between APP, Apo E or A2M and LRP stimulate a signal-transduction cascade through RIP that may affect cell function. Second, the degree to which LRP-interacting proteins can modulate APP processing and function is not fully understood. Finally, it remains obscure as to whether modulating such interactions may represent a therapeutic target in AD. In this Project, we will examine these questions, primarily focusing on dissecting the functional significance of the interactions between LRP and APP, Apo E, A2M, and FE65. Understanding the functional significance of these interactions will allow us to better understand the biochemical (and perhaps genetic) role of LRP in AD. Furthermore, such studies may contribute to our understanding of the biochemical (and perhaps genetic) role of the LRP interactors in AD. Finally, such studies may identify therapeutic targets in AD. We will also work with other Projects to elucidate the role of LRP in Abeta clearance and in statin effects. The central hypotheses of the current project are that LRP modulates APP metabolism, Abeta formation and clearance, and cell function contributing to Alzheimer's disease, and that LRP ligands mediate these effects. The specific aims of this proposal are: To characterize the effects of ligands of LRP on LRP localization and processing;To elucidate the effects of LRP on APP localization and processing, as well as Abeta formation;To dissect the cytoplasmic/nuclear complex of LRP that is involved in signaling and in modulating APP processing.;To identify genes that are regulated by the cytoplasmic/nuclear complex of LRP;To examine the function of LRP in neuronal differentiation in vivo;and, To define the role of LRP in the clearance and catabolism of Abeta in vivo.

The deposition of beta-amyloid (Abeta) in the Alzheimer's brain has long been regarded as a potential causative agent in the progression of Alzheimer's disease (AD) pathology. Over the past 10 years, characterization of the proteolytic events leading to Abeta generation has been the object of rigorous and exhaustive investigation. It is now understood that processing of the amyloid precursor protein (APR) by a group of enzymes known as the secretases ultimately underlies the liberation of AS. As more is known about these enzymes, it becomes increasingly clear that they likely have many substrates and inhibiting them may have multiple biological effects. The central question of this proposal is which intercellular signals regulate regulate the cleavage of APP. The processing of APP resembles that of other transmembrane proteins. The most notable among these is that which exists between APP and Notch. Binding of Notch to any of its cognate ligands (ie delta, jagged, serrate) results incleavage of the Notch receptor and shedding of the extracellular domain. This regulated step is followed by cleavage by through regulated intramembranous processing (RIP), leading to the liberation of the Notch Intracellular Domain (NICD). NICD is then translocated to the nucleus where it acts as regulator of transcription. With Notch, it is binding of ligand that activates the processing secretases. It appears likely that specific activation of APP cleavage may be induced by the binding of ligand to APP (and, perhaps ligand binding to heterologous receptors). The Specific Aims are as follows: 1) Determine the identity of ligands that bind APP and regulate cleavage in a manner consistent with Notch processing; 2) Determine the potential for growth-factor receptors to regulate cleavage of APP; 3) Elucidate the potential molecular pathways through which APP ligands or growth-factor receptors regulate APP cleavage; and, 4) Determine the levels and distribution of first messenger pathways (including APP ligands and heterologous receptors and their ligands) that regulate APP processing in control and Alzheimer tissue.

DESCRIPTION (provided by applicant): The proposed renewal of the Mount Sinai Center for the Neuroscience of Mental disorders (CCNMD) is designed to remain a highly focused effort to elucidate the role of white matter, oligodendrocytes and myelin in schizophrenia. This proposal is informed by increasing evidence of white matter, myelin and oligodendrocyte abnormalities in schizophrenia in a variety of areas of scientific exploration. A failure in connectivity has been demonstrated to have a role in schizophrenia. Myelination and those factors that affect myelination, such as the function of oligodendrocytes, are critical processes that could profoundly affect neuronal connectivity, especially given the diffuse distribution of oligodendrocytes and the widespread distribution of brain regions that have been implicated in schizophrenia. Multiple lines of evidence now converge to implicate oligodendrocytes and myelin in schizophrenia. Imaging, neuroanatomical, genetic and gene expression studies have all supported abnormalities in these cells and processes and contribute to our hypothesis that oligodendrocyte dysfunction and even death, with subsequent abnormalities in myelin maintenance and repair, contribute to the schizophrenic syndrome (see overview and specific project for detailed references). A broad set of methodologies and expertise will be brought to bear on the questions the CCNMD will pursue, including neuroanatomy, neuroimaging, molecular biology, molecular genetics, animal models neuropsychology, phenomenology, statistics, and data management. The CCNMD is comprised of 5 Cores: Core A, Administrative; Core B, Clinical; Core C, Brain Bank; Core D, Data Management and Statistics; and, Core E, Mouse Phenotyping. The projects of the CCNMD include: Project 1: (Hof/Tang) Oligodendrocyte and neuron pathology in cingulate cortex; Project 2: (O' Donovan/Owen) Genetic dissection of abnormal oligodendrocyte and myelin function in schizophrenia; Project 3: (Buxbaum/Sakurai) Neuregulin signaling in oligodendrocytes; Project 4: (Buchsbaum) Structure and function of white matter in schizophrenia; and, Project 5: (Friedman/Davis) White matter imaging correlates of functional outcome in schizophrenia.

This proposal is based on several key findings, described in detail in Background and Significance and in Preliminary Results: 1) The genes for Neuregulin 1 (NRG1) and the NRG1 receptor ERBB4 appear to represent susceptibility loci for schizophrenia, and their gene products demonstrate abnormalities in postmortem studies;2) We have identified an ERBB4 signaling complex comprised of ERBB4, a MAGI scaffolding protein, and a receptor phosphotyrosine phosphatase (RPTP);3) We have evidence that PTPRZ1, the gene coding for RPTPb (as well as for the secreted phosphacan splice variants), represents a schizophrenia susceptibility locus (gene-wide P=0.007;best P=0.0002): RPTPb plays an important role in oligodendrocyte development and in neuron-glia signaling;and, 5) Analysis of mRNA expression data indicates that RPTPb and RPTPb-binding proteins show expression abnormalities in schizophrenia. These findings lead to our overarching hypothesis: alterations in PTPRZ1/RPTPb signaling cause dysfunction of oligodendrocyte development and/or function with resultant myelin deficits, and can thereby contribute to schizophrenia susceptibility. In this project, we will characterize the role of PRPTZ1 and RPTPb in schizophrenia, using genetic and postmortem expression studies, as well as cell biological and animal models. Through these analyses, our aim is to establish a causal involvement of PTPRZ1/RPTPb signaling abnormalities in aspects of the schizophrenia phenotype. Our specific aims are: 1. To further determine whether PTPRZI/RPTPb, and associated signaling components, are altered in schizophrenia;2. To characterize the function of PTPRZI/RPTPb, and associated signaling components, in oligodendrocyte development and myelin formation in cell culture systems;and, 3. To analyze the effects of perturbation of the molecular components of PTPRZ1 signaling on oligodendrocyte development and function, white matter coherence, and behavior in animals. We will work closely with Administrative Core, Clinical Core/Brain bank, and Statistics Core, and Project 1, 2, 4, and 5, to accomplish these aims. Through these analyses, we hope to establish the causal involvement of PTPRZ1 -signaling abnormalities in oligodendrocytes in aspects of the schizophrenia phenotype.

Vascular disease can cause multi-infarct dementia. However in addition vascular disease and its associated risk factors such as hypercholesterolemia may also hasten the progression of Alzheimer's disease (AD). The long-range goal of this project is to determine the effect that systemic hypercholesterolemia and its associated vascular disease have on series of parameters of relevance to AD pathogenesis using hypercholesterolemic mice that are now widely used in atherosclerosis research. Specifically, we will utilize mice with a targeted disruption of the low-density lipoprotein receptor gene (LDLR -/- mice). By modulating the amount of cholesterol in the diet, plasma cholesterol in these mice can be widely varied and the degree of associated vascular disease reproducibly varied from minimal to severe. As biochemical markers of the AD process we will examine Abeta (Ab) production and tau phosphorylation. We will examine the functional consequences of hypercholesterolemia in these animals in two models of neural plasticity and repair, namely repair after entorhinal cortex lesioning and effect on neurogenesis in the adult hippocampus. A further functional correlate will be obtained through behavioral testing and since little is known about the effects of systemic hypercholesterolemia on brain cholesterol metabolism we will examine a series of parameters of cholesterol metabolism including 24S-hydroxycholesterol production in brain. To distinguish the effects hypercholesterolemia from its vascular consequences animals will be examined after short-term dietary manipulations when hypercholesterolemia is present but little vascular disease and after longer treatment intervals when significant vascular disease will be present. In each case both systemic and cerebral microvascular changes will be quantitatively assessed. Effects in LDLR -/- mice will be extended by determining if systemic hypercholesterolemia and vascular disease influence Ab production, plaque load and tau phosphorylation in the Tg2576 mouse model of AD by breeding the Tg2576 transgene onto an LDLR -/-background. Finally, we will examine Ab levels and tau conformational changes by ELISA in patient samples that have been scored for cerebrovascular pathology and amyloid pathology. Collectively these studies give us the opportunity to examine the effects of hypercholesterolemia and vascular disease on a series of AD related changes in both mouse and human material.

The Greater New York Autism Research Center of Excellence, a collaborative effort of the Mount Sinai School of Medicine, New York State Psychiatric Institute/Research Foundation for Mental Hygiene, University of Toronto, State University of New York at Stony Brook, and North Shore-Long-Island Jewish Health System, will aim to better understand the functions of the serotonin system in autism, specifically in its relation to repetitive behaviors, by combining methodologies from genetics, functional imaging and neuropsychopharmacology. By investigating core symptom domains of autism and linking this information to etiological factors, the multidisciplinary center's basic science and clinical research will facilitate development of novel or improved strategies for diagnosis, early detection and treatment of autism spectrum disorders. In achieving these goals, the center will be comprised of four integrated cores that will facilitate three interrelated research projects as well as multi-center collaborative endeavors. Project I: Identification of Autism Susceptibility Genes; Project II: Imaging Serotonin Function in Asperger's Disorder; Project Ill: Fluoxetine versus Placebo in Child/Adolescent Autistic Disorder. Core B: Clinical Core; Core C: Data Management and Statistics Core; Core D: Information Transfer Core.

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In response to the call of the STAART initiative to create Autism research centers of excellence, we are proposing to create The Greater New York Autism Research Center of Excellence (GNY-ARCE), which will be a collaborative effort of the Mount Sinai School of Medicine, NYSPI/Research Foundation for Mental Hygiene (NYSPI/RFMH), the University of Toronto and North Shore-Long Island Jewish Health System. This center will interweave clinical and research expertise in order to shed new light on the underlying mechanisms and treatment of autism. Its central purpose will be to find links between core symptoms, underlying neurobiological mechanisms and treatments, in order to better understand the syndrome of autism and to aid in the development of new and better treatments. The proposed three research projects will address this issue by combining methodologies from genetics, functional imaging and psychopharmacology. Another goal of the proposed STAART center of excellence will be to influence future research through interdisciplinary collaborations, as well as the infusion of new perspectives through a regular series of meetings, talks and conferences. At the same time, we are committed to disseminate information about ongoing research and treatments to the public.

DESCRIPTION (provided by applicant): This collaborative application is submitted in response to RFA MH-09-171. The root causes of autism remain unknown, limiting efforts to understand disease heterogeneity, diagnose cases, and prevent and treat disease. Epidemiological findings have repeatedly and unequivocally determined that heritable variation in DNA plays a substantial role in the etiology of autism and autism spectrum disorders, yet traditional efforts to identify the genetic basis of this striking heritability have met with very limited success to date and have therefore provided limited insight into disease biology. We propose here an unprecedented partnership between expert large- scale sequencing centers (at the Baylor College of Medicine and the Broad Institute of MIT and Harvard) and a collaborative network of research labs focused on the genetics of autism (brought together by the Autism Genome Project and the Autism Consortium). These groups will work together to utilize dramatic new advances in DNA sequencing technology to reveal the genetic architecture of autism, first through a detailed examination of 1000 genes implicated by previous genetic studies or postulated to be functionally relevant, and later, as the technology continues to advance, through unbiased whole-genome sequencing. The goal is to conclusively identify which genes harbor individual or collections of rare DNA variants that predispose to autism, and thus translate the abstract heritability into solid biological clues to disease pathogenesis that can be studied molecularly and approached therapeutically. These efforts and their follow-up, which will be performed on thousands of autism families collected by the autism research groups and being provided with phenotype data to NIMH repositories, will form the cornerstone of autism genetic research going forward.

The purpose of the Administrative Core is to coordinate and intergrate all activities of the CCNMD. In order to provide the necessary cooperation and coordination to maximize the productivity of the center, the Administrative Core provides a forum for formal, structured interactions between CCNMD investigators, between participating scientific and clinical institutions, and between administrative elements of the center.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 11/28/2008 The principle objective of the Autism Genome Project (AGP) is to identify genes that underlie susceptibility to autism to ultimately understand the biological mechanisms underlying the disorder. The AGP will accomplish this goal by capitalizing on the scientific expertise and sample collections from consortia in North America and Europe: the Autism Genetics Cooperative (AGC), the Autism Genetics Resource Exchange (AGRE), Collaborative Programs of Excellence in Autism (CPEA) and International Molecular Genetic Study of Autism Consortium (IMGSAC), and from additional individual groups and experts. This is the largest collaborative effort of its kind representing 13 different institutions - see table at the end. Hypothesis: Central to the AGP's strategic plan is to combine genome-wide analysis of multiplex families, targeted analysis of functional candidate genes, and detection of copy number variations (CNVs) or polymorphisms (CNPs) to identify genetic risk factors, using data analytic techniques of linkage and association.

DESCRIPTION (provided by applicant): The long-term goal of our group is to identify therapeutic targets in autism spectrum disorders (ASDs) and this proposal will focus on SHANK3 where it has been shown that deletions and mutations lead to ASDs. The overall objective of the proposal is to identify therapeutic targets for SHANK3-haploinsufficiency and our central hypothesis is that haploinsufficiency of SHANK3 leads to alterations in synapse development, glutamate transmission and synaptic plasticity in vivo that lead to observable behavioral phenotypes. The rationale for this proposal is that as we begin to understand the molecular, cellular, and regional impact of SHANK3 haploinsufficiency in vivo, we will have new targets that can form the basis of novel therapies for ASDs and associated disorders. In Aim 1, we will measure excitatory synaptic function in Shank3-deficient mice by electrophysiology and synaptic biochemistry. In Aim 2, we will quantify neuronal morphology and synapse structure and density in these animals. In Aim 3, we will access social and learning and memory behaviors in the Shank3-deficient mice. In Aim 4 will assess research compounds for effects in the mice using molecular deficits in synapses (Aim 1) to define targets for experimental interventions, with electrophysiological (Aim 1), morphological (Aim 2), and behavioral changes (Aim 3) as endpoints to assess the efficacy of a given intervention. The studies are significant because they represent a first step towards ultimate therapies for SHANK3-haploinsufficiency syndromes in that they will (a) identity molecular targets for therapies and (b) define preclinical outcome measures to be used for the assessment of novel therapeutics. In addition, as SHANK3 is such a central player in the synapse, these studies will also advance our understanding of the basic neurobiology of the synapse. The studies will also provide important data on the molecular, cellular and network components that underlie cognition and social behaviors. The proposed research is innovative, in our opinion, because it for the first time makes use of Shank3-deficient mice to study Shank3 function in situ, as a model for SHANK3-haploinsufficiency. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health and to the NIH mission because the studies will lead to a molecular and systems level understanding of Shank3 function and will identify molecular targets for novel therapeutics in human SHANK3-haploinsufficiency and in autism spectrum disorders. In addition, these studies will advance our understanding of the basic neurobiology of the synapse and of brain components that underlie cognition and social behaviors.

Alterations in connectivity among brain regions such as the frontal lobe, basal forebrain and limbic system have been proposed as network deficits in schizophrenia. The multiregional aspects of the hypothesized problems in connectivity implicate a possible deficit in white matter that could lead to the rerouting or interruption of a number of specific brain circuits. A global deficit in myelin in schizophrenia may also produce a pattern of distributed multiregional deficits compatible with the complex, not clearly localizing, behavioral and cognitive disorganization in schizophrenia. Alteration in numbers, distribution, and ultrastructural integrity of oligodendrocytes, key white matter components, has recently been reported in the prefrontal cortex in schizophrenia, consistent with our original diffusion tensor findings of diminished anisotropy in frontal white matter and our replication of this in the first funding period of this project. To extend our findings of white matter abnormalities in schizophrenia we plan four projects: 1) we will complete a longitudinal sample study with follow-up scans 3 yrs in a cohort of patients with schizophrenia and controls where we have already acquired diffusion tensor and structural images from the already acquired sample of 3T longitudinal sample (240 subjects - 125 patients with schizophrenia and 115 matched controls);2) We will also acquire FDG-PET with absolute glucose quantification on a cohort of 32 unmedicated patients with schizophrenia and 32 age- and sex-matched controls to further develop our initial finding of increased white matter relative metabolic rate in schizophrenia, we will develop exploratory voxelby- voxel correlations between anisotropy and glucose metabolic rate;3) We will exploit our recently developed tract tracing programs to assess the specific tract directions and termination points for cingulate, thalamic, striatal, and callosal fibers in the prefrontal cortex;4) We will share white matter anisotropy and volumetric measures with Projects 1, 2, 3 and 5 and Core B in order to facilitate and inform their potential choice of brain areas to be examined. Taken together, these aims will allow us to obtain the most reliable, valid, functionally different, and informative white matter assessments to confirm specific thalamo-frental, fronto-striatal and cingulate pathway abnormalities in schizophrenia.

DESCRIPTION (provided by applicant): While there has been great progress in understanding the risk architecture of autism, there are still unanswered questions about the nature of the genetic and non-genetic risk for autism. Many of these questions can be best addressed with a population-based epidemiological sample with detailed demographic and environmental information. To date, almost all studies on the etiology of autism relied on convenience samples, which are subject to biases in capturing genetic and, possibly even more so, environmental risk. Epidemiologically based samples provide a unique resource to identify genetic and non-genetic causes of autism, while allowing for a precise estimate of risk in the population attributed to each source of risk. Sweden benefits from a centralized medical system that has been the foundation of large-scale epidemiological studies in psychiatric disorders, particularly schizophrenia and bipolar disorder. In our opinion, the significance of this proposal lies in the value of a unique, population-based epidemiological sample, analyzed in such a way as to address several outstanding issues in autism. These include: 1) better estimates of heritability and environment in autism; 2) assessing the rate of recurrent risk CNV in autism; 3) discovery of rare standing single nucleotide variation in autism; 4) dissection of mechanisms underlying the association of non-genetic findings with autism-and the discovery of novel environmental associations; and, 5) cross-disorder analyses to better understand shared liability to autism and schizophrenia. The aims are: 1) To ascertain and biobank at least 1300 cases with autistic disorder and 1000 additional controls, to develop an international resource for ASD, and to assess selected, putative risk factors; 2) To genotype all samples using high-density SNP arrays, including dense exome coverage, and sequence all trios using whole-exome approaches; and, 3) To use novel methods to assess the role of inherited and de novo variants in autism and to evaluate rare standing variation in autism, while integrating key environmental variables. In later years the relationship between autism risk and risk for schizophrenia will be assessed. The proposed research is innovative, in our opinion, because it ascertains autism samples in an epidemiologically valid manner, targeting a genetically homogenous population, for which schizophrenia and bipolar samples have already been collected. The proposal is also innovative in the use of novel methods to estimate heritability and to identify rare, standing variation conferring risk to autism, while providing an integrated model for genetics and environment in autism. Finally, the application is innovative, in our opinion, in that it provides he groundwork for understanding shared risk across autism and schizophrenia, making use of a homogenous group to have better power to identify shared risk. This new and substantively different approach to studying autism, compared to studies carried out in convenience samples, addresses many of the open questions in autism research and provides a path towards a better understanding of the risk factors for autism and ultimately to better interventions in autism.

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): While there has been great progress in understanding the genomic architecture of autism, only a moderate number of the hundreds of genes and genomic regions thought to be involved in ASD have been identified. Next-generation sequencing (NGS) has proven its utility to rapidly identify variants underlying ASD, and this approach is being carried out in ca. 6,000 independent ASD samples through multiple studies. There is an urgent need to develop a framework to integrate and expand these current studies, and to jointly analyze emerging data to maximize the identification of valid ASD loci, because validated risk variants present opportunities for genetic counseling, understanding pathogenesis, and drug development. The Autism Sequencing Consortium (ASC) represents a coordinated effort by more than 20 independent groups to rapidly identify and validate ASD risk genes, which represent lead targets for neurobiological analyses and drug discovery. The long-term goal of the ASC is to make use of genetics to identify therapeutic targets in ASD, while contributing to translating such research findings to clinical practice. The overall objective of tis proposal is to rapidly identify ASD genes representing lead targets for high impact neurobiological studies and drug discovery. Our central hypothesis - formulated based on data with SNV, indels, and CNV, as well as review of medical genetic conditions in ASD and targeted sequencing in ASD - is that multiple independent rare variants account for a very significant proportion of risk to ASD. Our rationale for this proposal is that the identification of genetic variants conferring high-risk risk to ASD and associated neurodevelopmental disorders can form the bases of studies to understand pathogenesis as well as the bases for novel therapies. Moreover, such variants have direct implications for patients and their families in terms of etiological diagnosis, genetic counseling and patient care. These objectives will be accomplished with the following Specific Aims: 1) Maintain the infrastructure to support the ASC objectives; 2) Deploy pipelines for data cleaning and harmonization and variant calling; 3) Implement novel statistical methods for identifying ASD-associated genes; and, 4) Carry out whole-exome sequencing of 3,000 ASD subjects and parents. This contribution is significant because it represents the first step in research to understand pathogenesis of ASD and to the development of pharmacological strategies for treatment of core symptoms of ASD and etiologically related neurodevelopmental disorders. The research proposed in this application is innovative, in our opinion, because it involves an entirely new model of sharing data before publication, uses state-of-the-art methods for calling diverse types of variants in NGS data, incorporates novel methods for updating variant calling and sharing data, and includes highly innovative statistical methods to identify risk loci. This is a new and substantively different approach to gene discovery in ASD that departs significantly from the status quo and provides the means to achieve these important goals.

DESCRIPTION (provided by applicant): Our central hypothesis is that developmental delay syndromes including autism spectrum disorders lead to alterations in synaptic function in integrative brain regions that result in aberrant behavioral phenotypes. We will explore this hypothesis in a genetically modified rat model. Haploinsufficiency of SHANK3 leads to neurodevelopmental changes that include autism spectrum disorders, attentional disorders, absent or delayed speech, mild to moderate intellectual disability, and motor alterations. The SHANK3 protein forms a key structural part of the postsynaptic density. Because of the closer physiology between rats and humans as compared to mice, rats remain the primary choice of the pharmaceutical industry for studying pharmacokinetic (PK) properties of novel drugs. In addition, rats provide a far more tractable experimental model system for neurobiological, electrophysiological and behavioral studies, and it is of course advantageous, when considering drug development, that the biological assays be done in the same species where the PK studies are carried out. We have used zinc-finger nucleases to develop a genetically engineered rat with a disruption in the full-length rat Shank3 gene. This represents a first-ever genetically modified rat model for ASD and permits us to carry out detail studies in the prefrontal cortex, an area of great importance in autism, not easily studied in mouse models. We propose to carry out a detailed analysis of this model. We plan to test our central hypothesis with the following specific aims: 1) Behavioral assessment of prefrontal function in Shank3-deficient rats; 2) Electrophysiological analysis of prefrontal function in Shank3-deficient rats; and, 3) Neuropathological and neurochemical investigation of prefrontal function in Shank3-deficient rats. 3) The research is innovative, in our opinion, because it will make use of a first-ever rat model of ASD. In addition, it is innovative in the use of state-of-the art approaches to understanding the role of PFC in ASD, a key region not yet studied in detail in ASD model systems. The focus on PFC also allows for studying neuronal pathways that feed into the PFC, including the first-ever behavioral neurophysiological assessment of hippocampal-prefrontal circuitry in a rodent model for ASD. Our approach to high-resolution 3D imaging and analysis of neuronal morphology down to the level of single spine is notably novel. This form of analysis will allow us to identify molecular targets that are affected in Shank3-deficient rats, in particular, te distribution of excitatory receptors and synaptic proteins known to be linked to spine and synapse size and maturity. Finally, our behavioral analyses will make use of novel touchscreen chambers for detailed analysis of PFC function.

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): Autism is the fastest growing developmental disorder in the United States with that affects about 1 in 68 children. Thus, not surprisingly, the cost and burden on families, patients, and caregivers is enormous. Associated with autism spectral disorder are other pervasive developmental disorders that include Rett syndrome, Phelan-McDermid syndrome and Fragile X syndrome. These ASD-associated syndromes are classified as rare genetic diseases and have limited or no treatment options. As such, there is an immediate need to develop more effective and better- tolerated medications for these patients. The overall goal of this Phase I SBIR is to further characterize and test the efficacy of our novel first-in-class pro-drugs that target the orphan Fragile X and Rett syndromes; with the long-term objective of improving the lives of individuals suffering from autism. Recently, a 12-week double- blind, placebo controlled treatment regimen of N-acetylcysteine (NAC), a glutamatergic modulator and an antioxidant, resulted in a marked decrease in irritability in children with autism with few side effects. While the promise of NAC in autism is great, its ability to cross the blood- brain barrier is low. To address this, Promentis has developed several lead small molecules that successfully deliver NAC to the brain (far superior to NAC itself) and have further confirmed their preclinical proof-of-efficacy in rodent models of mental illness. Ultimately, NAC's early success in treating a major symptom of autism removes substantial risk from the project and increases the chances that Promentis will be successful in preclinical studies and subsequent Phase I clinical trials. We propose to conduct proof-of-efficacy experiments of our novel pro- drugs in rodent animal models of autism with the intention of developing a lead molecule to meet FDA requirements for IND filing.

? DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) affects 1 in 68 children (CDC, 2014) with known genetic causes accounting for 10-15% of cases (Devlin & Scherer, 2012; Gaugler et al., 2014). Approximately 2% of severely affected children with ASD have deletions or point mutations in the SHANK3 gene, which results in Phelan- McDermid syndrome (PMS) (Betancur & Buxbaum, 2013; Leblond et al, 2014). SHANK3 is a scaffolding protein in glutamate synapses (Bodzdagi et al., 2013; Yang et al., 2012). Over 80% of children with PMS meet Diagnostic and Statistical Manual of Mental Disorders (DSM) criteria for ASD and intellectual disability and are minimally verbal (Soorya et al., 2013). Sensory reactivity abnormalities such as hypo- and hyper-reactivity represent a new DSM-5 criterion for ASD. While sensory reactivity issues have been shown in idiopathic forms of ASD (Tomcheck & Dunn, 1997; Tavassoli et al., 2012, 2013), no studies have investigated sensory reactivity abnormalities in severely affected children, including those with PMS. Identifying sensory reactivity abnormalities is especially important in such children as they cannot verbally describe their sensory experiences. In idiopathic forms of ASD, vision is the most researched sensory modality (Simmons et al., 2009; Tavassoli et al., 2011; Weinger et al., 2014). Moreover our pilot studies suggest that hyporeactivity in the visual domain is common in children with PMS. Preliminary data also show that behavioral and electrophysiological measures of sensory reactivity may have the capacity to objectively differentiate between children with (1) PMS, (2) idiopathic ASD, and (3) typically developing controls. The aim of the proposed project is to develop behavioral and neural biomarkers of sensory reactivity in ASD, which are feasible in severely affected individuals (including PMS) and can be translated to other genetically defined subtypes and across sensory modalities. This project aims to develop reliable measures of behavioral and neural sensory reactivity within the visual system of children with PMS and ASD more broadly. The knowledge gained will provide an enhanced understanding of how one sensory system is affected in PMS and ASD, while assessing the relationship with neural functioning. This study is important because it seeks to develop biomarkers that can be applied to other clinical populations of severely affected individuals.

Project summary Schizophrenia is a chronic, severe and disabling brain disorder that affects an estimated 1 in 100 persons. Though its key symptoms generally appear late in adolescence, schizophrenia is a neurodevelopmental condition with a strong genetic component and heritability estimated to be as high as 80%. Although therapeutic treatments do exist, they target few putative mechanisms and are not effective in all the patients and/or do not address all the symptoms of the disease. While there have been improvements in the understanding of the biological systems implicated in the pathogenesis and pathophysiology of schizophrenia, progress has been slow and limited both by the difficulty in obtaining relevant tissues from patients and the inadequacy of animal models to deal with the level of genetic complexity involved in this disease. To date, most of the molecular and cellular studies of schizophrenia have been performed on postmortem tissues or on genetically defined mouse models that do not fully recapitulate the human genetic risk or neural phenotype. The rapid advances in induced pluripotent stem cell (iPSC) methodology provide new opportunities to overcome some of the obstacles inherent to the modeling of neurodevelopmental diseases. As a consequence of the groundbreaking work of the Yamanaka laboratory, somatic cells from a simple patient biopsy can be reprogrammed into pluripotent stem cells that can be differentiated into other cell types, including neural cells. Because the resulting neural cells retain that individual's genetic information, this approach has tremendous potential as a tool for understanding genes and pathways that are dysregulated in schizophrenia and can provide a platform for in vitro screening assay for novel therapeutics. The first aim of the project is to apply revolutionary robotic methods to generate pluripotent stem cells from a large cohort of patients and carefully matched controls. We will then use this sample, as well as two existing samples of iPSCs with child onset schizophrenia and/ or known, rare, highly penetrant genetic lesions, to generate excitatory neurons. This will create the first large scale, highly standardized library of iPSC and neurons derived from patients with schizophrenia. The second aim of the project is to perform gene expression profiling on the schizophrenia and control neurons and use innovative systems biological analyses to identify dysregulated pathways in schizophrenia and key molecular drivers that underlie these pathway changes. These key molecular drivers represent potentially high-impact targets for drug development. Altogether, the completion of the aims will provide new insight into the neuronal pathways disrupted in schizophrenia, and identify potential drug targets. The study will also provide the community with a large schizophrenia iPSC cohort and a neuronal RNA sequencing dataset, and will lay the foundation towards establishing a high-throughput platform useful for drug screening and accelerating drug development processes.

Project Summary/Abstract The past decade has seen outstanding advances in the genetics of autism spectrum disorder (ASD), however only a moderate number of the hundreds of genes and genomic regions thought to be involved in ASD have been identified. Advances have come largely from the study of rare genetic variants, especially de novo variation, including single nucleotide variation (SNV), insertion/deletions (indels), copy number variation (CNV), and larger chromosomal imbalances. A portion of the progress for ASD has come through the efforts of the Autism Sequencing Consortium (ASC), which represents a coordinated effort by more than 40 independent groups to rapidly identify ASD risk genes. Here we propose to continue the work of the ASC, largely by continued production and analysis of sequence data from ASD subjects and their families. The ASC benefits from substantial leveraging of resources, including the Exome Aggregation Consortium (ExAC) centered at the Broad Institute (BI) and whole-exome sequencing (WES) of ASC samples, supported by an NHGRI Center Grant to BI, to make this renewal as low cost as possible. We also plan new avenues of research, such as integrating whole genome sequence (WGS) data and building on ideas that have emerged from the study of common variants to understand the interplay of common and rare variants to impact risk. Through this new research we will accelerate our overall objective, which is the identification of ASD genes, thereby facilitating our long-term goal of building the foundation from which therapeutic targets for ASD emerge. Our rationale is that the identification of genes conferring significant risk to ASD and associated neurodevelopmental disorders can form the basis of studies to understand pathogenesis, as well as the basis for novel therapies. Moreover, such variants have direct implications for patients and their families in terms of etiological diagnosis, genetic counseling and patient care. Our central hypothesis ? formulated based on results over the past decade ? is that rare and common variation contributes additively to risk for ASD, but only certain rare variants confer substantial risk. The objectives will be accomplished with the following Specific Aims: 1) Produce and/or analyze WES of 30,000 new ASD subjects, parents and other controls, for a total of more than 50,000 samples; 2) Develop and apply approaches to find ?hidden? risk variants, and, 3) Use results from common and rare variant studies to describe the interplay of such variation in ASD risk. This contribution is significant because it represents the first step in research to understand pathogenesis of ASD and to the development of pharmacological strategies for treatment of core symptoms of ASD and etiologically related neurodevelopmental disorders. The research proposed is innovative, in our opinion, because it uses groundbreaking and novel statistical methods for identifying risk variants and for integrating rare and common variation. This is a new and substantively different approach to gene discovery in ASD that departs significantly from the status quo and provides the means to achieve these important goals.

While enormous progress has been made in elucidating genetic factors underlying autism spectrum disorder, it is largely unknown how genetic and non-genetic risk factors integrate and how they shape severity of social communication and cognitive deficits. This gap can be addressed by developing comprehensive liability models using a population-based epidemiological sample with dense genetic and phenotypic data. To fill this gap, we have developed the Population-based Autism Genetics and Environment Study (PAGES), involving a Swedish epidemiological cohort obtained by ascertaining samples with DSM-IV autistic disorder (AD), which captures more severely affected individuals, chosen from a national, population-based sample of over 7,000 living individuals. Modeling liability in the epidemiological sample of AD has provided accurate estimates of the risk conveyed by common and rare genetic variation. This study has also revealed that ~40% is still unaccounted for. Combining critical environmental variables (paternal and maternal age, gestational history) and phenotyping data (IQ, autism severity, family psychiatric history) with measures of heritability is key to fully understand autism liability. We now propose to strengthen PAGES by pursuing the following specific aims: 1) To recruit, genotype and sequence at least 1,500 additional cases, including 1,350 less severely affected individuals; 2) To study common and rare genetic variation in relation to ASD severity and cognitive function; 3) To determine how other sources of putative risk for ASD are distributed in relation to ASD severity and cognitive function, and, 4) To discover risk genes for ASD by analysis of whole-exome sequence data and identify common risk variation by genome-wide association study (GWAS). We expect to contribute liability models that integrate genetic and environmental risk factors and take into account the phenotypic complexity along two core dimensions: severity of social deficits and cognitive function. In our opinion, this is significant because it allows us to: 1) study rare genetic variation at all scales across phenotypic groups; 2) understand the interplay between polygenic risk and highly penetrant rare variants across phenotypic groups; 3) measure heritability and environmental influences in light of phenotypic variability; and, 4) define the familial burden, both genetic and non-genetic. In our opinion, our study is innovative because it probes specific components of risk, both genetic and non-genetic, in a population-based cohort and introduces phenotypic variability as an additional dimension. It is also innovative because it combines genetic (additive and rare inherited) variation, parental age, and family history of psychiatric disorder to assess the familial burden, while introducing novel tools and approaches to genetic analyses. This radically new way of tackling ASD liability, compared with current studies, will provide novel insights into autism risk factors and their interactions in determining phenotype, thus opening new avenues for clinical assessment of risk, prevention and clinical care.