Nenad Sestan - US grants

[unreadable] DESCRIPTION (provided by applicant): Language is a specialized mental faculty that enables humans to arrange and communicate thoughts through the use of different modalities such as sounds or symbols. Numerous studies have shown that several regions in the neural system might be responsible for the processing and production of speech and language. However, studies on the development and organization of the complex underlying neuronal circuitry at the cellular and molecular levels have been technically challenging. Recent reports by other groups and preliminary results generated in my laboratory provided us with an opportunity to undertake such an analysis. We have identified a small population of cortical pyramidal neurons present in the human fetal speech-related cortical region. These neurons are uniquely identified by the combinatorial expression of Forkhead Box P2 (FOXP2), haploinsufficiency in which can lead to a severe speech disorder, Nitric Oxide Synthase 1 (NOS1), and ZNF312, a novel layer V-specific transcription factor. Based on the areal and laminar position of these neurons, as well as their gene expression, we hypothesize that they are a central component in the circuitry that mediates language processing and speech production. In this grant application, we propose to characterize the functional roles of transcription factors FOXP2 and ZNF312 in the development, refinement and maintenance of that corticostriatal and corticothalamic circuits. In the first specific aim of this proposal, we propose to analyze the organization and distribution of FOXP2-positive corticofugal projections in the primate ventrolateral frontal cortex. In the second aim, we propose to determine the role of FOXP2 in the refinement and maintenance of corticostriatal and corticothalamic projections. In the third specific aim, we propose to determine the role of zinc finger protein, ZNF312, in the formation and corticostriatal and corticothalamic projections. The elucidation of how FOXP2 and ZNF312 may regulate development of neural pathways involved in spoken language as outlined in this proposal will help in understanding normal brain development and the neurobiological foundations of speech and language. This research may further facilitate the identification of additional disease genes and the development of new therapeutic strategies for the treatment of speech and motorrelated disorders [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Radial glial cells play a critical role in the construction of the mammalian cerebral cortex by first giving rise to neurons during early development, then providing guidance for neuronal migration, and at later stages, generating astrocytes. Abnormalities in radial glial development, differentiation, and guidance of neuronal migration lead to aberrant placement and connectivity of neurons. The decision of cortical radial glial cells to either multiply, differentiate or remain quiescent depends on an integration of multiple signaling mechanisms. The Notch signaling pathway is a key regulator of radial glial cell establishment and maintenance in the developing cerebral cortex. However, the molecular mechanisms underlying the specificity and context dependence of Notch signaling remain unclear. In this study, we will investigate the roles of a number of molecules in mediating Notch-dependent regulation of radial glial cell function. First, we will determine the specific and complementary roles of Delta-like 1 and Jagged 1 in regulating radial glial cell differentiation. Second, we will characterize putative interaction between Numb and Numb-like, and E-Cadherin in maintaining apicobasal polarity of radial glial cells. Finally, we will characterize the mechanism and function of oscillation in Notch activity during cortical neurogenesis. We will employ in vitro and in vivo systems and use both loss- and gain-of-function techniques to determine the functions and mechanisms of the above molecules. The elucidation of the molecular mechanisms of how Notch signaling and related molecules regulate cortical radial glial cell function as outlined in this proposal will advance the understanding of normal and abnormal brain development, and the stem cell biology.

DESCRIPTION (provided by applicant): Our understanding of genetic mechanisms controlling the evolution, formation, and pathological disruption of human neuronal circuits is impeded by a lack of comprehensive data on the developing brain transcriptome. The main goal of the two interrelated Grand Opportunity applications is to conduct a spatiotemporally comprehensive survey of the human brain transcriptome and create a unique multimodal atlas encompassing all pre- and postnatal developmental stages, including adolescence and adulthood. To achieve this ambitious goal in two years, we propose to bring together expertise in human and non-human primate brain development, large-scale transcriptional profiling, industrial scale histological data and atlas generation, MRI and DTI imaging, and the resources of the Allen Institute for Brain Science and Yale University in bioinformatics and information technology. This consortium will work together to create a series of large-scale data sets that will be integrated through a powerful public access web-based portal for visualizing, searching and sophisticated mining of spatiotemporal gene expression patterns in the anatomical context of human brain development. Specifically, the research planned to take place at Yale University will use high-throughput massively parallel RNA-sequencing (RNA-seq) methodology to gain a sensitive, specific and comprehensive view of the developing transcriptome across multiple regions of the human brain. We will analyze the whole transcriptome in up to 15 cerebral cortical and subcortical regions from both left and right sides of normal postmortem female and male brains at 12 stages of pre- and postnatal development. RNA-seq will provide a unique resource for comprehensive mining and functional analysis of the whole transcriptome. Expert- annotated MRI/DTI and reference atlases generated by the Allen Institute for Brain Science will provide the anatomical backbone of the resource, and the framework for informatics-based statistical analysis and generation of search and mining capabilities. Finally, direct links will be made to other non-human primate and rodent transcriptomes and cellular-level gene expression data sets available in participating laboratories to allow cross-species comparative analyses, both through web-based linking and parallel analysis of orthologous gene sets. The generated data will be available to the research community at-large via a user-friendly, web-based informatics framework. The expected results of the proposed studies will have a transformative effect on the field by cataloguing variations in gene regulation and alternative splicing across brain regions and developmental time-points for all coding and non-coding transcripts, enabling a better understanding of how specific susceptibility genes affect human brain development and potentially contribute to psychiatric disorders. PUBLIC HEALTH RELEVANCE: The identification of genes involved in formation and maturation of the human brain as outlined in this proposal will help in understanding normal human brain development and plasticity as well as the neurobiological foundations of mental disorders such as schizophrenia and autism. This research may further facilitate the identification of disease genes and the development of new therapeutic strategies for the treatment of these disorders.

DESCRIPTION (provided by applicant): The ability to accomplish and develop complex cognitive and motor tasks depends on the accuracy and intricacy of synaptic connections both within the cerebral cortex and between the cortex and other regions of the brain. Axonal projections of excitatory projection (pyramidal) neurons constitute the motor output of the entire cortex and directly influence behavior. Molecular mechanisms regulating the molecular identity and connectivity of distinct cortical projection neurons are being unraveled. Our goal is to identify mechanisms that are important for the migration, molecular identity and connectivity of pyramidal neurons, and to investigate their functional roles using a variety of molecular and genetic approaches. Our published and preliminary studies, supported by this grant in the last four years, have functionally characterized several genes encoding transcription factors and axon guidance that control different aspect of cortical projection neuron development, such as their molecular identity, laminar position, dendritic arborization, and axonal projections. In this application we intend to further characterize the cellular and molecular mechanisms by which some of these genes function. Specifically, the proposed experiments are designed to determine how Sox5 controls early-born subcortical projection neurons migration to their proper laminar positions and extend axonal projections (Aim1); how layer-specific expression of Fezf2 and subsequently the molecular identity of projection neurons are controlled cell-intrinsically by upstream transcriptional regulators (Aim2); and how formation of axonal connections with subcortical targets, such as the thalamus, controls gene expression cell-extrinsically in cortical projection neurons during late embryogenesis (Aim3).

DESCRIPTION (provided by applicant): The human frontal cortex displays an accelerated phenotypic evolution in structural and functional complexity, and arguably contributes more than any other brain region to our cognitive and behavioral attributes. Consequently, the frontal cortex represents the most critical region in which to study the molecular and cellular bases of many psychiatric disorders. Our preliminary studies show that distinct areas of the human frontal cortex are distinguished from other cortical regions by the combinatorial expression of specific genes during the second trimester of gestation, and that microarray expression profiling can be used to identify further candidate genes for patterning of these areas. Our main hypothesis is that the patterning and development of the frontal cortex are associated with changes in the activity of genes, from embryonic stages to adolescence, and that these differentially expressed genes play critical roles in the formation and refinement of frontal cortical circuits. To identify such genes, we will use whole- genome exon microarrays and state-of-the-art bioinformatics to analyze temporal and spatial patterns of gene expression and alternative mRNA splicing in the developing human brain. Our studies will encompass all crucial stages of prenatal and postnatal development, including the first trimester, during which cortical neurons are generated; the second trimester, when cortical connections are starting to form; and the late fetal and early postnatal periods, when connections are organized to create functional circuits. Just as importantly, our studies will survey a comprehensive selection of brain regions for high spatial resolution of gene expression mapping, especially within the frontal lobe. PUBLIC HEALTH RELEVANCE: The identification of genes involved in formation and maturation of the frontal cortex as outlined in this proposal will help in understanding normal human brain development and plasticity as well as the neurobiological foundations of disorders such as schizophrenia, autism, and language impairment. This research may further facilitate the identification of disease genes and the development of new therapeutic strategies for the treatment of these disorders.

DESCRIPTION (provided by applicant): The development of human brain is an immensely complex process, which is likely reflected in the complexity of the underlying transcriptional processes. Gene expression and its precise spatio-temporal regulation, particularly by histone modifications and non-coding RNAs, are crucial for normal human brain development and are thought to be altered in major developmental psychiatric disorders, such as autism spectrum disorders (ASD). Moreover, changes in the developmental brain transcriptome are likely the major contributors to the evolution of the most distinctly human aspects of cognition, some of which are also affected in ASD and other psychiatric disorders However, our understanding of transcriptional and epigenetic processes involved in the development, evolution and dysfunction of the human brain is still elusive. Furthermore, most of our knowledge of transcriptional processes in the human brain is limited to the expression of protein coding genes. Given that the genomes of humans and other mammals have approximately the same protein-coding complexity, there is likely an additional reservoir of transcriptional complexity, especially in organs such as the brain, which has many structurally and functionally distinct regions in humans. This view is corroborated by recent findings of the ENCODE consortium, which found many cis-acting regulatory regions and that 60% of the human genome is transcribed, with a majority of the transcripts belonging to non-coding RNAs. Moreover, these and other studies have also uncovered pervasive involvement of regulatory DNA variations in common human diseases and evolution. However, how these findings on non-coding elements in cell lines relate to the complexity of human brain development and dysfunction is still largely unknown. The objective of this proposal is to employ unbiased and genome-wide approaches to (1) discover and characterize developmentally regulated and human-specific non-coding functional genomic elements in multiple regions of the developing human and non-human primate brains, (2) and elucidate their role(s) in the molecular pathophysiology of ASD, by using genomic analyses of post-mortem ASD brains, by screening for de novo mutations in ASD quartets, and by modeling functional consequences of ASD-associated elements in the developing mouse brain.

? DESCRIPTION (provided by applicant): The human brain is arguably the most complex biological structure. Understanding how many different cell types exist in the human brain and mapping neural connections are critical tasks to better understand the development and function of the brain. This is particularly challenging in the human brain due to inherent limitations of working with postmortem tissue. This grant is specifically addressing these tasks in the human brain as well as a closely related non-human primate, Rhesus macaque, and a commonly studied mammalian organism, the mouse. The objective of this proposal is to employ novel methods and approaches to generate a systematic inventory/census of cell types and connections in the developing and adult human, macaque monkey and mouse prefrontal cortex (PFC). We have chosen PFC for this project due both to its importance in higher cognitive functions as well as for the alterations observed in PFC in certain psychiatric and neurological disorders. To generate a census of component cells in the PFC, we will use single cell RNA-seq to profile the transcriptomes of single cells isolated in a cell-type specific way through the use of viral-mediated tagging of ribosomes and axonal tracing methods. To apply these advanced techniques to post-mortem human brain, we will implement a novel tissue processing protocol [Hibernation-Cryopreservation combined with a Pulsatile Perfusion Hibernation System] to keep post-mortem brains in prolonged hibernation. This allows for the stabilization of nucleic acids, the concurrent collection of live single cells, and the applicationof classical and advanced methods for the identification of neuronal pathways. We will also develop agnostic and integrative computational methods to create a taxonomy of cell types based on molecular identity and connectivity. These features will be compared across species, ages, and sexes. There are four major distinguishing aspects of this application: (1) implementation of novel approaches developed to extend tissue integrity and viability of the postmortem human brain such that we can (2) perform single cell RNA-seq, (3) trace connections and examine cell morphology in the postmortem human brain, which is not amenable to classical experimentations~ and (4) develop novel analytical tools and approaches. This pilot project and methodologies directly address the goals of this BRAIN Initiative RFA and are designed to demonstrate their utility and scalability to ultimately complete a comprehensive cell census of the entire human brain in healthy and disease states.

DESCRIPTION (provided by applicant): Understanding the molecular processes involved in the development and functional organization of biological systems, as well as their alterations in disease states, requires precise measurements of nucleic acid- and protein-level properties of cellular machinery across different cell types and developmental time points. This is particularly difficult to achieve in the human brain due to its cellular complexity and inaccessibility for experimentation. Here, we propose a Center with a multi-disciplinary group of investigators that will develop upon several cutting-edge genomics approaches in a unique and innovative way to elucidate molecular networks underlying human brain development and evolution. This will be achieved through the generation and exploration of integrated multi-dimensional genomic scale data generated from single cells and tissues of developing and adult human and non-human primate (NHP; chimpanzee and macaque) brains. We will also use these new sources of information to facilitate the identification of regulatory mutations in autism spectrum disorders (ASD) as well as to elucidate common and cell type specific molecular networks compromised in ASD. Finally, we implement approaches to model and functionally characterize of human-specific and ASD-associated regulatory mutations in the context of mouse brain development. Our proposed Center couples these research efforts with extensive training opportunities in human and comparative genomics. This organizational structure combines the expertise of each individual key investigator and establishes a CEGS that is capable of much more than each individual working alone and whose resources will create capabilities that are much more than the sum of its parts. Our work will pave the way for reconstructing molecular networks in human development and disease states, and provide a clear path to new and more effective treatments of major disorders.

? DESCRIPTION (provided by applicant): Two major goals of developmental neurobiology are to identify the germinal cells which form the central nervous system and to characterize the cellular and molecular mechanisms by which these cells generate the proper numbers and types of neurons. This proposal represents our ongoing efforts to precisely define the molecular and morphological identity of each neural precursor type present in the mammalian neocortical wall. In this project, the Haydar lab at Boston University teams with the Sestan lab at Yale University to perform genetic fate mapping of precursor populations, followed by next generation RNA sequencing at both the bulk and single cell level. Several differentially expressed genes have already been identified which modulate proliferation or neuronal output from specific precursor groups, and one new gene target has been engineered into a new fate mapping tool useful for further subdivision of cell lineages. Altogether, experiments in this project will identfy precursor cell types and subtypes with unprecedented clarity and isolate key differentially expressed genes. These cell type- specific target genes will then be tested in a series of in vivo experiments for their role(s) in neurogenesis and neuronal positioning.

? DESCRIPTION (provided by applicant): Somatic mutations are de novo mutations that occur after fertilization. Once a cell has acquired a somatic mutation, all of its progenitors will also carry that mutation. Thus, if a cell acquires a mutation early in embryonic development, the mutation will be carried by many of the cells in the body. However, if the mutation occurs late in development, then only a few cells might carry it. Thus, it is possible to have mutations that only occur in the brain, or a small region of the brain. It has been known for a while that somatic mutations can cause cancer, and recent studies are showing that somatic mutations are associated with neurodevelopmental disorders resembling autism spectrum disorders (ASDs) both in terms of their high de novo mutation rate and in terms of their associated symptoms such as intellectual disability and epilepsy. We hypothesize that somatic mutations represent a significant cause of (ASDs) because of the high rate of de novo mutations associated with ASDs, the importance of somatic mutations in some genes known to cause ASDs, and the importance of somatic mutations in other developmental brain disorders with features that overlap ASDs. The technical and resource limitations that had prevented a systematic study of the role of somatic mutations in ASDs have now been overcome thanks to 1] Next-Generation Sequencing (NGS), which allows for the deep sequencing of genes and their transcripts with the ability to analyze each sequence, and 2] tissue banks that have collected brain specimens from individuals who had ASD. In this collaborative UO1 we will employ complementary approaches to systematically identify and functionally characterize somatic brain mutations associated with ASD. For causative somatic mutations identified in ASD brain, we will use techniques developed in our labs to examine individual brain cells for the presence of somatic mutation. This will provide us with a map of what regions of the brain, and what cells types in the brain carry these somatic mutations. We will also model and functionally characterize ASD- associated brain mutations in induced pluripotent cells and mice. This study could 1] improve the genetic diagnosis of ASD; by assessing the prevalence of somatic mutations as a cause of ASD, 2] provide a paradigm that may apply to other complex neuropsychiatric diseases (such as schizophrenia), and 3] improve our understanding of the mechanisms underlying ASD by creating a map of brain regions and cell types involved in ASD.

? DESCRIPTION (provided by applicant): Autism Spectrum Disorder (ASD) is characterized by impairments in social communication and restricted or repetitive behavior or interests. The application of genomic technologies has led to the identification of many of the genes underlying ASD, presenting the opportunity to assess the insight these risk genes can give into the etiology of ASD. In this proposal we aim to: 1) Generate a list of ASD-associated genes; 2) Identify points of convergence between these genes in biological data (e.g. gene regulation and expression); and 3) Validate these points of convergence in model systems. Since ASD is a human neurodevelopmental disorder we will prioritize biological data that is collected longitudinally across development from human brain tissue. In our prior work we have demonstrated that de novo mutations, specifically copy number variants (CNVs) and loss of function (LoF) point mutations, are strongly associated with ASD. Furthermore, these mutations cluster at ASD risk genes and loci in cases but not in controls. By comparing the distribution of these mutations between cases and controls we can identify the points of mutational clustering that represent ASD risk loci (e.g. CNVs at the 500kbp 16p11.2 locus and LoFs at the gene CHD8). We have developed a statistical framework to assess this clustering as well as incorporating evidence from inherited variants and case-control data. This framework is called the Transmitted and De novo Associated Test (TADA). In Aim 1 we will develop this test further to incorporate all the available CNV, exome, genome, and targeted sequencing data into a single ASD gene list, ranked by the degree of ASD association. Previously we used the top nine ASD risk genes as seeds for gene co-expression networks and assessed the validity of these networks by their ability to incorporate 120 independent ASD risk genes. By limiting the co- expression input data to narrow windows of development and specific brain regions we could identify the spatiotemporal networks with the greatest enrichment, for example pre-frontal cortex in mid-fetal development. In Aim 2, we propose a similar approach, but using the DAWN (Detecting Association With Networks) method developed by our group. DAWN uses the narrow windows of co-expression data as before, but is able to incorporate evidence from other datasets such as gene regulation, and protein-protein interaction (PPI). By seeding the DAWN networks with the highest confidence genes we will assess the spatiotemporal networks that best predict other ASD genes. ASD shows a significant sex bias implicating an interaction between ASD etiology and sexually dimorphic factors. Building on our work of identifying sexually dimorphic transcripts in the developing human brain we will test their enrichment within specific networks identified by DAWN. To validate the ASD-associated networks, in Aim 3 we will identify the gene that best represents each network and assess if disrupting it also disrupts the other genes within the network. We will disrupt each gene using CRISPR/Cas9 in both mice and human-derived iPSCs and assess the genes disrupted using RNA-Seq.

ABSTRACT Genetic and genomic investigations have yielded important findings as to the genetic contributions to major psychiatric illnesses, illustrating significant etiological heterogeneity, as well as cross-disorder overlap. It has also become clear that understanding how this genetic variation leads to alterations in brain development and function that underlies psychiatric disease pathophysiology will be greatly advanced by a roadmap of the transcriptomic and epigenetic landscape of the human cerebral cortex across key developmental windows. Here, we propose, via a highly collaborative group of investigators, each with distinct areas of expertise and research focus, to create a scaffold of genomic data for understanding ASD pathophysiology, and psychiatric disorders more broadly. The work proposed here represents an ambitious multi-PI project (Yale, UCLA, and UCSF) that brings together three principal investigators and collaborators with strong publication records and expertise in all approaches necessary to perform this work using state-of-the-art and novel methodologies. We will perform time-, region-, and cell type-specific molecular profiling of control and ASD brains (Aim 1), including RNA-seq based transcriptomics, identifying cis-regulatory elements via ChIP-seq, and use Hi-C to determine the 3D chromatin architecture and physical relationships that underlie transcriptional regulation in three major regions implicated in neuropsychiatric disease (frontal and temporal cortex and striatum) across five major epochs representing disease-relevant stages in human brain development. This will include complementary genomic analyses in controls and matched post mortem ASD brain to identify genetic mechanisms underlying processes altered in ASD brain. We will address cellular heterogeneity via fluorescence-activated nuclear sorting (FANS) so as to profile neurons and non-neural cells separately, which will complement the whole tissue analyses. We will analyze and integrate these datasets to identify regional, developmental, and ASD-related processes to gain insight into underlying mechanisms, harmonizing these multi-omic data with other psychENCODE studies, as well as other large scale data sets, such as BrainSpan, ENCODE, GTEx and Roadmap Epigenomics Project (Aim 2). We will perform integrated analysis of germ-line ASD variations identified in more than 1000 families from the Simons Simplex Collection to characterize causal enrichments in developmental periods, brain regions, and cell types to better characterize the mechanisms by which genetic variation in humans alters brain development and function in health and disease (Aim 3). Completion of these aims will lead to a well-integrated resource across major periods in human cortical and striatal development that will permit generation of concrete testable hypotheses of ASD mechanisms, and inform our pathophysiological understanding of other related neuropsychiatric disorders.

PROJECT SUMMARY There is a current lack of understanding of differential gene expression within the nervous system. Ideally one would like to know, across all neuron types, exactly how the genome is transcribed and processed into functional RNAs. This information is fundamentally important because differential gene expression defines the form and function of individual neurons, determines how individual neurons contribute to circuit physiology and behavior, and influences how individual neurons are affected by injury and disease. Further, detailed and complete knowledge of differential gene expression within the nervous system would help elucidate the logic and cellular mechanisms that generate neuronal diversity, including regulation of gene expression, alternative splicing, and miRNA function. Yet progress in this area has been limited: For most nervous systems, the exact number of distinct types of neurons is unknown and therefore a global map of neuron-specific gene expression is not achievable. Here we propose to address this problem in a project to discover and analyze the C. elegans Neuronal Gene Expression Map & Network (CeNGEN). The C. elegans nervous system contains precisely 302 total neurons comprising 118 classes of distinct neuronal types. We propose to exploit this unique attribute to analyze gene expression with high accuracy in every individual neuronal type. CeNGEN proceeds in four specific aims. Aim 1) Establish 118 transgenic strains, each one expressing fluorescent markers that uniquely label a single type of neuron. Aim 2) Use innovative cell dissociation and FACS methods to isolate each type of neuron from age-matched adults, and use RNA-seq approaches to assess global coding transcript and miRNA expression, as well as splicing diversity. Aim 3) Utilize single cell sequencing technology to precisely map gene expression over multiple parameter spaces. Aim 4) Build cell-centered and gene-centered expression maps, and seek connections with other uniquely known features of the C. elegans nervous system including the wiring diagram, the cell lineage, neurotransmitter identity, and function. CeNGEN represents a paradigmatic advance in neurogenetics, and provides a unique opportunity to elucidate the global control of neuron-specific gene expression and to relate gene expression to neuronal wiring and function. Expected significant outcomes include: Identification of conserved regulatory mechanisms that generate neuronal specificity and diversity; Detailed understanding of alternative splicing and miRNA function across the nervous system; Relationship of differential gene expression to neuronal lineage, anatomy, function and connectivity. CeNGEN will also serve as a resource for future studies in C. elegans neuroscience, and will provide a framework for addressing global differential gene expression in more complex nervous systems that are currently not amenable to this comprehensive approach.

PROJECT SUMMARY The mammalian brain is arguably the most complex biological structure. Investigating cellular functions and mapping neural connections in the brain are critical tasks to better understand the brain in health and disease. This is particularly challenging in vivo due to the inherent limitations in experimental latitude and simultaneous access to multiple brain regions within the same animal. These shortcomings hinder multimodal interrogation of multi-synaptic circuits and mesoscale connectomics. Of particular importance, these experimental inadequacies grow in proportion to the complexity of the brain and cranial anatomy, impeding translation to larger mammals. This grant addresses these tasks by optimizing and validating a first-in-class neurotechnology called BrainEx for the restoration of molecular and cellular functions of the postmortem large mammalian brain under ex vivo, normothermic conditions. We specifically propose to continue optimizing BrainEx in porcine brains, while validating the efficacy of the BrainEx system as a new experimental platform for electrophysiological, connectomic, and imaging studies in the fully isolated, intact, and functional large mammalian brain. There are four major distinguishing aspects of this application: (1) implementation of novel approaches developed to restore cerebral macro- and microcirculation and extend cellular viability of the postmortem brain under normothermic conditions such that researchers can (2) simultaneously trace connections and characterize cellular function and morphology by chemical and vector-based techniques across myriad brain regions, including areas inaccessible to in vivo surgical approaches; (3) investigate multisynaptic long-range circuitry and cortical network electrical activity; and (4) perform functional PET and CT imaging studies in the ex vivo large mammalian brain. This methodology represents a new tool for more thorough investigation of the structure and function of complex circuits and the cells within them. Wide distribution of this technology will grant investigators experimental advantages across species not afforded by tissue culture or in vivo approaches.

ABSTRACT Recent advances in genetics and genomics have identified hundreds of coding variants that increase risk for major neuropsychiatric disorders, such autism spectrum disorder. Work to clarify the contribution of non-coding variants is also underway and is expected to accelerate rapidly in the next few years. While these advances have considerably improved our understanding of the genetic landscape neuropsychiatric disorders, a deeper understanding of molecular pathophysiology is still missing. This knowledge gap is due to, in part, the heterogeneity of risk loci involved, their potential roles in regulating expression of a large number of genes, the pleiotropic nature of risk genes, and the high likelihood that neuropsychiatric disorders result from dysfunctional circuitry involving multiple cell types and brain regions, altogether making the identification of molecular and cellular mechanisms underlying a disease problematic, especially in the context of the protractive and complex nature of brain development. Therefore, the discovery and characterization of the full spectrum of functional genomic elements active in the human brain, as well as their activity/expression patterns across the spatiotemporal dimensions, is essential for clarifying when, where, and what cell types are relevant to the etiology and treatment of neuropsychiatric disorders. This is particularly so in the context of non-coding variants, which are difficult to annotate, yet potentially hold the promise of providing highly specific spatial, temporal, and cell type specific information. To address this knowledge gap and to continue our contributions to the PsychENCODE Consortium, we propose four specific aims that identify gene regulatory and cell type- specific mechanisms of human neurodevelopment. In Aim 1, we identify functional genomic elements across single cells (nuclei), cell types, regions and developmental time points of neurotypical human and macaque postmortem brains. In Aim 2, we map the spatio-temporal proteome of neurotypical human and macaque postmortem brains. In Aim 3, we perform integrative identification of functional genomic elements and proteomics in diseased brains and iPSC-derived neural cells. In Aim 4, we integrate results from Aims 1-3, as well as with independent genetic datasets of neuropsychiatric populations, to identify non-coding elements, genes, or molecular pathways that will lead to a better understanding of the underlying pathophysiological mechanisms of neuropsychiatric disorders. Finally, these mechanisms will be functionally characterized in model systems. Data from this proposal will also serve as a critical new resource for members of the community, with which they can intersect their results and draw deeper and more meaningful conclusions, especially as the wealth of genomic data from neuropsychiatric disorders continues to accumulate.

PROJECT SUMMARY Increasingly persuasive evidence suggests genomic variants driving derived features in humans and among primates are enriched in regulatory elements, but the vast majority of these evolutionarily relevant variants have yet to be discovered or characterized. This is unfortunate, as among the approximately 35 million single nucleotide substitutions (SNPs), 5 million insertions or deletions (indels), and 90 megabases of structural variants where the human and chimpanzee genomes differ are countless variants associated with development, function, or disease. Identifying evolutionarily relevant genetic variants, as well as those implicated in disease or function, can be guided by the analysis of species differences in intermediate molecular phenotypes (e.g., transcriptomic and epigenomic signatures), which are most likely the primary effects determined by genomic variation. In this proposal, we propose to perform primate comparative functional genomics to uncover genetic variants explaining lineage-specific phenotypes affecting the human and non-human primate (NHP) brain, an organ exhibiting pronounced molecular and functional differences between species. To do so, in our first aim we will develop a taxonomy of gene expression and open chromatin across primates, applying single nucleus RNA-seq and single nucleus ATAC-seq to study the mid- fetal and neonatal (late fetal and early infancy) development of the post-mortem human and NHP brain, as well as brain organoid co-cultures containing cells differentiated from multiple primate stem cells and fibroblasts and lymphoblastoid cell lines. In our second aim, we will complement this atlas of species differences in gene expression and open chromatin by cataloguing SNPs, indels, and large, complex structural variants in multiple primate species. This will allow us to differentiate between lineage-specific (i.e., human versus chimpanzee and macaque) and Hominidae-specific (i.e., human and chimpanzee versus macaque) genomic variants. Finally, in our third aim we will integrate and functionally validate, using the Massively Parallel Reporter Assay, CRISPR/Cas9 genome editing, human induced pluripotent stem cells, and mouse models of neural development, key regulatory elements and de novo genes identified through these experiments. Through these aims, we will identify and functionally validate genomic variants and patterns of gene expression and open chromatin potentially driving derived phenotypes in the human and non-human brain and consequently plausibly associated with human cognition, social behaviour, and neuropsychiatric disease.

ABSTRACT The human brain is a highly complex biological tissue organized into hundreds of regions composed of a myriad of cell types with distinct molecular, morphological, and physiological properties. These cells and their associated circuits underlie our mental abilities and, when dysfunctional, lead to neurological and psychiatric disorders. Consequently, developing an atlas of these cell types and how they differ from one another is essential for understanding the biological processes underlying human brain development and function. In addition, because both the general cytoarchitecture of the brain and its constituent cells are generally conserved across primates, knowledge of how these cell types differ between species is also essential for understanding uniquely human aspects of cognition and behavior. We therefore propose to use single cell transcriptomics to rigorously define cell types and states in ten brain regions involved in higher cognition and behavior and across five key developmental timepoints of human, chimpanzee (one of our closest extant relatives), rhesus macaque (the most commonly studied Old World non-human primate), and common marmoset (an emerging New World non- human primate model system). By generating, analyzing, and integrating these data with existing unpublished datasets, we propose to develop a human and non-human primate cell atlas and identify shared and divergent molecular and cellular features across species, regions, and ages. Furthermore, we will validate key aspects of this atlas, including molecular signatures, and create a data visualization and dissemination portal.

PROJECT SUMMARY HIV and methamphetamine (MA) use are global health problems with devastating human and societal consequences. HIV and methamphetamine use also produce independent and additive impairments in neurocognition, and current clinical and basic science research suggest complex and currently inadequately understood interactions between HIV and MA pathophysiologies. We therefore propose to conduct comprehensive characterization, at the single cell/nuclear level, of human brain tissue and regionally specified organoids derived from human induced pluripotent stem cells. For these single nuclear (sn)RNA-seq and snATAC-seq analyses, we will sample 3 brain regions (prefrontal cortex, ventral striatum, and basolateral amygdala) critical for the neurobiological response to MA use in 20 brains from each of two donor groups, HIV+MA+ and HIV-MA+. These data generation efforts will complement ongoing efforts in these same brain regions from HIV-MA- and HIV+MA- donors and allow us to elucidate differences in gene expression and key biological pathways that occur in response to MA use, HIV, or the combination of the two. In addition, we will assay brain cell types for HIV transcripts, allowing us to identify cellular reservoirs of HIV in donor brains. These efforts will be aided by the use of human cortical organoid and medial ganglionic eminence organoid cultures, which offer complex, region-matched model systems recapitulating in vivo-like cellular diversity and microenvironments without potentially confounding factors including patient history, varying co-morbidities, prolonged postmortem intervals, or tissue degradation. We will then apply cutting edge and novel data analysis pipelines to integrate snRNA-seq and snATAC-seq data and identify cell population and gene expression differences between cell clusters (i.e., putative cell types) in different conditions. These data will also be integrated with external datasets from the SCORCH Consortium and other multi-omic data including genotype, RNA-seq, HiC, ChIP-seq, and ATAC-seq data from both healthy subjects and subjects with HIV infection, neurological disease, or a history of drug abuse. Finally, we will use multiplexed immunohistochemistry and single molecular fluorescent in situ hybridization to validate the cell type specific expression and co-expression of candidate genes, biological processes, and gene regulatory networks implicated in the etiology of HIV or MA pathophysiology. All data generation protocols and data analysis tools will be made freely available to the research community, and all data generated will be provided as both raw and processed resources. Taken together, the proposed experiments will generate invaluable resources and offer new biological insights into the human brain and its disorders.

ABSTRACT Alzheimer?s disease (AD) is the leading cause of dementia (60-80%), affecting tens of millions of people globally and, due to longer lifespans and aging populations, perhaps hundreds of millions more by 2050. AD pathology is first observed in allocortical and limbic areas within the cerebrum, in particular medial temporal cortical regions critical for learning and memory including the hippocampal formation and entorhinal cortex (HIP-EC). Within these areas, pathology exhibits subregional and cell type specificity, with layer 2 of entorhinal cortex and the hippocampal CA1 field (Sommer?s sector) exhibiting pathological hallmarks before dentate gyrus granule cells and other major hippocampal neuronal subtypes. Understanding the molecular basis of this selective vulnerability (and conversely the resilience of other cell types) will provide new insights into the etiology of AD, but to date only limited efforts have been made to understand the molecular signatures differentiating neuronal and non-neuronal cells in HIP-EC. We therefore propose to conduct single nuclear RNA sequencing (snRNA-seq) and single nuclear Assay for Transposase Accessible Chromatin (snATAC- seq) in 5 regions of HIP-EC of AD brains, young/mid adult and aged neurotypical ?control? human brains, and young adult and aged rhesus macaque brains. This will allow us to develop a high resolution cell census of HIP-EC which will in turn allow us to identify enriched genes, gene expression patterns, gene regulatory networks, and biological processes potentially mediating cell type specific differences in the AD and aged HIP-EC.

ABSTRACT Functional genomic analyses of the developing human brain have revealed highly dynamic spatiotemporal patterns of gene expression and epigenetic changes during prenatal and early postnatal development and across brain regions. Disruptions of these developmentally dynamic processes have been implicated by numerous complementary analyses in the etiology of multiple neurodevelopmental and neuropsychiatric disorders. Expression quantitative trait loci (eQTLs), along with splicing quantitative trait loci (sQTLs) and structural variant quantitative trait loci (svQTLs), are genomic variants that differ between individuals, with these differences correlating with functional changes to gene expression or splicing behavior. Many of these QTLs show specificity to tissues, brain regions, developmental stages, or cell types, and a proportion overlap with known genetic risk factors of human disorders. Here, we propose to pursue three integrated Aims, including whole-genome sequencing and both bulk tissue and single-nuclei RNA sequencing, to identify genomic variants, eQTL/sQTL/svQTLs, and patterns of gene expression and co-expression in two regions of the human brain across mid-fetal development through to adolescence. In addition, we will apply novel and newly developed computational tools to associate these QTLs with specific cell types and loci or genes implicated in neuropsychiatric disorders. By so doing we will augment, and dramatically expand upon, earlier efforts to understand QTLs and their roles in neural development, function, and neuropsychiatric disorders.