Kristen J. Brennand, Ph.D. - US grants

DESCRIPTION (provided by applicant): Schizophrenia (SCZD) is a debilitating psychiatric disorder. While 1.1% of the population suffers from SCZD, the molecular mechanisms underlying the disease state remain unclear. Though its characteristic symptoms typically appear late in adolescence, SCZD is believed to result from abnormal neurodevelopmental processes that begin years before the onset of symptoms. We previously reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons; SCZD hiPSC neurons have reduced neuronal connectivity and altered gene expression relative to controls. Because gene expression profiles of our hiPSC-derived neural cells most resemble first trimester neural tissue, we believe that hiPSC neural cells are best used to study the embryonic developmental effects that contribute to disease initiation. Childhood-onset SCZD (COS) is a rare and particularly severe form of the disorder. Because COS patients present with symptoms much earlier than adult-onset cases of SCZD, our hypothesis is that neural cells derived from patients with COS will share cellular phenotypes with those we have already reported for adult-onset SCZD, but that the phenotypes may be accelerated and/or more severe. We believe that hiPSC studies of COS are an ideal platform from which to glean mechanistic insights into the early cellular and molecular factors responsible for disease initiation in SCZD. We have four primary goals for this BRAINS R01. First, we will generate hiPSC-based models of COS. Second, the cellular phenotypes of COS neural cells will be characterized across a panel of existing and validated assays. Third, mRNA and microRNA expression of COS neural cells will be integrated through causal network interference analysis in order to identify key microRNA regulators. Finally, we will begin mechanistic studies of candidate microRNAs altered in COS. We hope to use our novel hiPSC based platform to identify molecular insights into COS which may be generalizable across SCZD.

The large majority of genomic loci linked to schizophrenia heritability by genome-wide association harbor regulatory non-coding DNA, including enhancers and repressors, that are not bound to the nearest TSS but instead tethered via chromosomal contacts to genes located elsewhere on the chromosome. Therefore, unsurprisingly, `linear genome' based approaches such as gene expression quantitative trait loci (eQTL) and SNP prioritization algorithms have very limited success in assigning specific target genes to risk loci. Guided by recent Hi-C genome-scale chromosomal contact mapping studies by us and others, we will test in this proposal whether the genetic risk architecture of schizophrenia is associated with cell-type specific vulnerabilities as it pertains to the developmental reorganization of the chromosomal connectome. We predict that neuronal specification into glutamatergic, GABAergic and dopaminergic lineages associated with cell-type specific genome-scale prunings of chromosomal contacts and loss of smaller-scaled chromatin domains. This includes the domain protoype of many of the smaller self-folded `topologically-associated domain' (TAD), with the developmental dissolution of many subTADs nested into larger and megadomain TADs. Furthermore, we predict that differentiating neurons show a disproportionate increase in chromosomal contacts anchored in sequences conferring heritable risk for schizophrenia and related cognitive disorders and traits. We will monitor developmentally regulated shufflings of intranuclear positions for specific GWAS loci and predict mportant differences between the various neural cell types isogenically generated from hiPSCs. If so, then the `functional epistasis', or least co-regulation, of subsets of risk loci sharing the same nuclear sub-territory could be highly dependent on cell type. Last but not least, we predict that targeted mobilization of specific chromatin domains by CRISPR-Genome Organization (CRISPR-GO) approaches can be harnessed for simultaneous targeting of multiple GWAS locis to to specific nuclear compartments such as the nuclear lamina or Cajal body, resulting in multi-layered transcriptome and epigenome changes and cell-type specific phenotypic alterations.

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.

? DESCRIPTION (provided by applicant): We are submitting Integrated Multiscale Networks in Schizophrenia in response to RFA-MH-16-300. Schizophrenia (SCZ) is a generally devastating neuropsychiatric illness with considerable morbidity, mortality, and personal and societal cost. Genetic factors have been strongly implicated via family and twin data, and more recently directly through genome-wide association studies (GWAS) and sequencing studies. The primary objective of our project is to develop and apply advanced integrative methods for computational and functional analysis of networks, including but not limited to Bayesian network reconstruction and prediction algorithms of variant causality to identify key drivers of SCZ pathology for potential therapeutic intervention. To achieve this in Aim 1 we will construct single tissue and multi- tissue probabilistic causal network by applying a novel top-down and bottom-up or hypothesis-driven probabilistic causal network approaches in RNA sequencing key drivers of networks, novel pathways, and new mechanisms in SCZ pathology data from the CommonMind consortium, incorporating prior information. In Aim 2 we will use network models derived in Aim 1 in order to improve the predictive SCZ networks that could be used to identify SCZ-relevant transcription-based features that can be useful in therapeutic screening. Finally, in Aim 3 we will use modified RNA and cellular models to validate the network models, key drivers and investigate their phenotype effects.

PROJECT SUMMARY Schizophrenia (SZ) is a common and debilitating neurodevelopmental disorder that affects nearly three million Americans. Despite more than fifty years of research, no cures exist and the standard of treatment remains unsatisfactory. Genome wide association studies (GWAS) indicate that SZ risk reflects both highly penetrant rare copy number variants as well as common single nucleotide polymorphisms with small effect sizes. By overlapping GWAS and post-mortem expression analyses, common variants with expression quantitative trait loci (eQTL) that may contribute to altered gene expression and liability in SZ have been identified; however, demonstrating which risk loci are the causal contributors to disease risk remains an intractable problem. Consequently, we propose to apply a human induced pluripotent stem cell (hiPSC)-based approach to manipulate the genotype and/or expression levels of putative causal SZ risk variants, focusing largely on genes implicated in synaptic formation, maturation and function. Through isogenic comparisons, we propose to examine the molecular and functional effects of perturbing five putative causal eQTLs and ten SZ GWAS- associated genes, testing the impact on cis-gene expression, global network expression patterns and synaptic function. Our hope is that this work may identify novel therapeutic points of intervention in order to improve the disease course in individuals with SZ.

PROJECT SUMMARY Schizophrenia, bipolar disorder and autism are common and debilitating neurodevelopmental disorders that together affect more than 5 million Americans. Despite more than fifty years of research, no cures exist and the standard of treatment remains unsatisfactory. Heterozygous mutations of neurexin-1 (NRXN1) have been repeatedly associated with schizophrenia (SZ) and autism spectrum disorder (ASD). The clinical presentations of NRXN1+/- mutations (including diagnosis, severity, prognosis and age-of-onset) in affected patients are diverse and the genetic mechanism affecting the penetrance of these mutations remains unknown. Moreover, mouse models do not permit researchers to study how and why some NRXN1+/- deletions have more deleterious effects in patients. Our objective is to resolve how NRXN1+/- deletions perturb the NRXN1 isoform repertoire and impact neuronal maturation and synaptic function. Our preliminary data defined the NRXN1 alternative splice repertoire in control fetal and adult cortical tissue, and compared this to human induced pluripotent stem cell (hiPSC)-derived neurons from NRXN1+/- cases and controls. Here, we propose to evaluate the effect of experimental manipulation of the NRXN1 isoform repertoire in both control and NRXN1+/- patient-derived excitatory and inhibitory neurons. Ultimately, we hope to directly correlate genomic and functional deficits across increasingly refined populations of NRXN1+/- patient-derived neurons.

PROJECT SUMMARY: Supplement to Functional genomic resource and integrative model of dopaminergic circuitry associated with psychiatric disease In response to PA-20-272, ?Administrative Supplements to Existing NIH Grants and Cooperative Agreements (Parent Admin Supp Clinical Trial Optional),? we propose to validate a small number of additional target genes identified by parent U01 (U01DA048279: Functional genomic resource and integrative model of dopaminergic circuitry associated with psychiatric disease). The parent award aims to construct transcriptome and epigenome (incl. 3D genome/chromosomal conformation) maps for midbrain dopaminergic neurons and for their surrounding nonneuronal cells, and to assess the relationship to known genetic risk factors for complex mental illness, including psychosis with substance abuse co-morbidity. We will apply integrative methods for functional analysis of genetic variation and networks, including but not limited to Bayesian network reconstruction and prediction algorithms of variant causality to identify key drivers of schizophrenia and bipolar disease pathology, and drug addiction co-morbidity. These methods will simultaneously integrate multiple different dimensions of data: DNA variation, RNA expression, chromatin accessibility, 3D structure of the genome, and known pathway and gene network information in the context of clinical phenotype data. The fundamental source of data for the project comes from the current studies on human midbrain functional omics, the CommonMind and PsychENCODE consortia (whole genome sequencing and cortical functional omics data), the Psychiatric Genomics Consortium, and the Million Veterans Project (genetic variation and disease phenotypes). The Million Veterans Project (MVP) has collected genotyping and phenotypic data from ~700,000 individuals, including a subgroup of 50,000 veterans diagnosed with SCZ and BD and a larger group of individuals diagnosed with other neuropsychiatric traits (recurrent depression, suicide and substance abuse). We will make our newly generated transcriptome and epigenome datasets from adult midbrain, as well as the network and predictive models, available to the research community in accordance with NIMH data sharing policies.

PROJECT SUMMARY/ABSTRACT DNA methylation contributes to epigenetic regulation of many important biological processes. The prevailing dogma is that DNA methylation almost exclusively occurs at the fifth position of cytosine (5mC) in eukaryotes. This dogma has been revised in the past five years as multiple studies reported the existence of N6- methyladenine (6mA) across eukaryotes including the mouse and human genome. A few studies have found evidence that suggests the diverse functions 6mA plays in mammals. Some studies have also reported increased 6mA level in certain neuronal cells and upon social stress, suggesting 6mA may play important roles in health and diseases. However, several studies have challenged the presence of 6mA in mammalian genomes, highlighting multiple sources of confounding factors. The active debate has created unusual confusions in the epigenetic community. Yet, tens of studies continued to report new discoveries about 6mA in the human genome including some papers at high-profile journals. A few ongoing research studies have set out to examine the functional roles of 6mA in brain cells and brain disorders, especially in Alzheimer?s disease. To unambiguously assess the abundance and prevalence of 6mA in the human genome, it is imperative to employ a reliable and sensitive 6mA mapping method. However, past and ongoing studies mostly employ antibody-based methods, which are associated with non-specificity. Although Single Molecule Real-Time sequencing (SMRT-seq) has been widely used to map 6mA at base resolution in bacteria, recent work by us and others have found that existing methods are not sensitive enough for eukaryotic genomes with low 6mA abundance. To address this fundamental technological gap, we will build on our >10-yr experience in SMRT- seq to develop a new method for sensitive 6mA detection, and take a neutral perspective to critically examine 6mA in the human genome and in brain tissues from patients with Alzheimer?s disease (AD), Primary age- related tauopathy (PART, another type of dementia) and controls. This project is significant and very timely because (1) if the novel method supports the existence of 6mA in the human genome, it will provide ongoing and future studies with a much-needed tool to detect 6mA in certain cell types and Alzheimer?s diseases; (2) if the novel method does not support a significant level of 6mA as reported, it will serve as a much-needed direct evidence to clarify the current debate.

PROJECT SUMMARY A complex interplay of genetic variation underlies predisposition for autism spectrum disorder (ASD). There is now strong evidence from large consortia studies that mutations in genes involved in chromatin modification, transcriptional regulation, and synaptic proteins confer substantial risk for ASD; however, the extent to which these genes are interconnected and ultimately converge on a small number of functional deficits is largely unknown. A critical need therefore exists to model new gene discoveries, to directly evaluate their functional impact, and to determine their points of convergence. Innovations from our team and others in high-throughput CRISPR-engineering have now made parallelized mechanistic studies tractable, and human induced pluripotent stem cell (hiPSCs) derived neurons are well-suited to test the impact of ASD risk variants predicted to exert their influence during fetal cortical development. Here, our multi-PI proposal will undertake an ambitious, systematic isogenic loss-of-function (LoF) mechanistic screen in a compendium of 48 of the most robust ASD risk genes discovered from the largest genetic studies to date. Moreover, our exciting preliminary results suggest that transcriptional signatures shared across neuronal models of ASD genes converge on critical regulatory nodes that result in synaptic deficits. Aim 1 will characterize isogenic glutamatergic and GABAergic neurons with highly penetrant LoF mutations in 48 genes associated with ASD risk at genome-wide significant thresholds and that are expressed in neurons. These analyses will identify transcriptional and functional signatures of individual ASD genes through RNAseq and a series of high-throughput phenotyping assays in both neuronal sub-types. Aim 2 will expand our Preliminary Results to discover convergent genes downstream of ASD risk loci, characterize the synaptic consequences of the ten most compelling discoveries from individual genes and/or convergent signatures, and integrate these data to explore the druggability of the convergent networks. Our overarching goal is to define any commonalities among diverse genes, pathways and networks that underlie ASD etiology, and to dramatically expand the list of possible therapeutic targets for ASD. These studies will generate an unprecedented isogenic resource of CRISPR-edited ASD genes, and matched RNAseq and cellular phenotyping in glutamatergic and GABAergic neurons, that will be provided for open distribution to the broader community through the NIMH RUDCR resource to yield new insights into neuropsychiatric disorders.

Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by progressive cognitive decline and dementia. Despite more than fifty years of research, no cures exist and the standard of treatment remains unsatisfactory. Genome wide association studies (GWAS) indicate that AD risk reflects both highly penetrant rare variants as well as common single nucleotide polymorphisms with small effect sizes. By overlapping GWAS and post-mortem and myeloid cell expression analyses, we have identified common variants with expression and splicing quantitative trait loci that may contribute to altered gene expression or alternative splicing; however, demonstrating which risk loci are the causal contributors to disease risk remains an intractable problem. Here, we will apply statistical approaches to prioritize putative causal variants in AD-associated loci by incorporating expanded AD GWAS functional annotations, single cell chromatin profiles and histone modification and microglia gene expression datasets. We will then apply a human induced pluripotent stem cell (hiPSC)-based approach to manipulate the genotype of prioritized putative causal AD risk variants that alter gene expression or alternative splicing of mRNAs, focusing largely on genes implicated in phagocytosis and lysosomal-autophagy function. Through isogenic comparisons of neurons, microglia, and astrocytes, we propose to examine the molecular and functional effects of perturbing five putative causal SNPs separately testing their cell autonomous and non-cell autonomous impact on cellular function. Our hope is that this work may identify novel therapeutic points of intervention in order to prevent or slow disease course in individuals with AD. This project will have a large overall impact by providing a mechanistic interpretation of genetic variants associated with AD susceptibility.

PROJECT SUMMARY Serious mental illness (SMI) that includes schizophrenia (SCZ) and bipolar disorder (BD) are common, complex and debilitating psychiatric disorders that together affect over 2% of the population and carry considerable morbidity, mortality, and personal and societal cost. Over the last decade, large-scale genome wide association studies (GWAS) have identified hundreds of loci contributing to the risk of SCZ and BD. Advancing these statistical associations to causal mechanisms for SMIs is very challenging due to incomplete understanding of the non-coding regulatory mechanisms in the human brain tissue and the local correlation of risk variants. Therefore, a systematic analysis that performs fine-mapping to jointly identify and validate a credible set of causal variants in SMI and molecular features that includes transcripts and regulatory sequences, in relevant tissues and cell types is a critical next step. The overarching goal of our proposal is to leverage genomics and multiscale functional omics (gene expression and epigenome regulation) data and perform fine mapping to detect and validate causal variants, transcripts and regulatory sequences in SMI. In Aim 1, we will perform large-scale trans- ancestry GWAS of SCZ and BD to expand the current repertoire of risk (and resilience) loci and refine the credible sets of causal variants underlying genome-wide significant associations. In Aim 2, we will integrate putative causal variants with multiscale functional omics data from human brain tissue that capture gene expression and epigenome regulation at the bulk, cell type-specific and single cell level to identify credible sets of transcripts and regulatory sequences. In Aim 3, we will functionally validate putative causal variants and regulatory sequences, by using novel approaches that combine massively parallel reporter assays and genome editing in excitatory and inhibitory neurons derived from human induced pluripotent stem cells. Our computational and experimental aims bridge the gap between the fine-mapping of causal variants, the molecular gene- regulatory effects of risk variants on enhancer activity and gene expression and their biological effects at the cellular level. If successful, our project can elucidate the genes, pathways, and mechanisms underlying SCZ and BD, and provide new insights and avenues for therapeutic development.

Project Summary Alzheimer's disease (AD), in particular la?te onset AD, is the most common form of dementia, accounting for about two thirds of all the dementia cases, and its pathogenesis may start decades early before its actual clinic manifestation. The search for disease modifying treatments is the primary objective of most rigorous therapeutic research efforts on AD. However, AD is currently incurable and available therapies are only effective in partially alleviating selected AD clinical symptoms, but not its onset and/or progression. The unmet need for the timely development of potent therapies for AD has been constantly growing with the heavy burden on our healthcare system reaching a critical level. There is an urgent need to reinvigorate AD drug development by utilizing systems biology, especially network biology approaches which have the potential to present not only global landscape of pathway-pathway interactions but also detailed molecular interaction/regulation circuits underlying AD. Network biology approaches to integrate large-scale multi-omics data in AD have demonstrated that differentially regulated subnetworks in AD, which regulate diverse AD pathogenic phenotypes, often include a large number of key regulators. Therefore, drugs and drug combinations that can modulate such subnetworks as a whole are the most pertinent for therapeutic intervention and have better chance to be successful. In this application, we propose to develop novel molecular network based drug repositioning approaches to identify individual FDA approved drugs as well as their combinations that can potentially reverse molecular signatures and network states of AD. A large number of predicted drugs and drug combinations will be tested in multiple model systems including mouse brain primary cells, human iPSC derived brain cells and AD mouse models. This project will establish an integrative platform comprised of highly innovative systems and experimental biology components for rapid drug discovery for AD.

PROJECT SUMMARY Schizophrenia (SZ), bipolar disorder (BD) and autism spectrum disorder (ASD) are common and debilitating neurodevelopmental disorders that together affect more than 5 million Americans. Despite more than fifty years of research, no cures exist and the standard of treatment remains unsatisfactory. Dysregulation of alternative splicing in the human brain has been implicated in SZ. Among genes with critical brain functions, many have a large number of exons, resulting in complex splicing patterns that can vary between neuronal subtypes; however, the isoform repertoires of most of these complex genes have not been resolved in a cell-type specific manner. Recent advances in long read sequencing have provided an unprecedented opportunity to resolve complex alternative splicing. Thus, to better understand the clinical impact of changes of alternative splicing in psychiatric diseases, it is critical to evaluate how isoform repertoire impact neuronal maturation and synaptic function in a cell-type specific manner. Here, we propose to catalog and functionally characterize the complexity of human isoform repertoires of SZ-associated genes using a single-cell long-read sequencing based approach, and to study how genetic variations influence alternative splicing and impact neuronal maturation and synaptic function in a subtype specific manner. This project will provide comprehensive catalogs of cell-specific full isoform repertoires of SZ genes that will broadly facilitate SZ research. It will also deepen the understanding of how genetic variations and alternative splicing contribute to SZ, help better predict the clinical outcome and identify novel therapeutic interventions. The single cell approach used in this project is generally applicable to study other mental disorders such as BD and ASD.