Schahram Akbarian - US grants

As a trained psychiatrist and neuroscientist, the candidate has an ongoing commitment to increase knowledge about the molecular biology of complex behavior and to improve the treatment options for substance abuse disorders and related major psychiatric illness. The candidate intends to apply innovative methods of genetic recombination and large scale gene expression screening to animal models of substance abuse and psychosis. The long-term goal of these studies is to clarify the role of nerve growth factor molecules for drug-induced neuronal plasticity and addiction behavior at different stages of postnatal and adult life and to describe, on a large scale, neurotrophin- and drug-of-abuse mediated changes in genomic expression patterns. At the core of the candidate's project is the controlled manipulation of gene expression for two neurotrophic factors that are thought to be key regulators of synaptic plasticity in the reward structures: neurotrophin-3 (NT-3) and brain derived neurotrophic factor (BDNF). These growth factors and their receptors are expressed at high levels in some parts of the reward circuitry such as the nucleus accumbens (NAc) and the monoaminergic cell groups of the mid- and hindbrain. This proposal will induce controlled deletions of NT-3 and BDNF in the developing and in the adult brain, by using the P1 phage derived Cre/lox recombination system and a tetracycline-regulatable gene expression system. The resulting functional alterations in the brain's reward circuitry will be assessed on the molecular, the cellular and the behavioral level. Comparative studies will be conducted in mice with a controlled overexpression of NT-3 in the reward circuitry. Is orderly function of the reward circuitry dependent on neurotrophic factors? Are neurotrophins involved in the molecular mechanisms mediating addiction behavior and drug-related reinforcement? What are the molecular and cellular adaptations in the brain's reward systems in response to the induced changes in neurotrophin gene expression? The answer to these questions will be of major importance for a further understanding of the molecular biology of substance abuse disorders.

DESCRIPTION (provided by applicant): Depression is considered one of the most serious disorders in today's society, with a lifetime prevalence as high as 16.2% in the US adult population. The introduction of the first pharmacological antidepressant medications in the 1950s, and subsequent development of drugs with lower side-effect profiles has greatly improved the therapeutic outlook for depressed patients. Notably, all the antidepressant drugs now in use modulate monoamine neurotransmission and take six to eight weeks to exert their effects. Still, treatment-resistant depression, which typically refers to inadequate response to at least one antidepressant trial of adequate dose and duration, affects up to 50-60% of patients. Therefore, it will be necessary to test and develop antidepressants with conceptually novel mechanisms of actions and a more rapid therapeutic response. The focus of this application is on pre-clinical studies to assess the antidepressant potential of a novel class of therapeutic drugs, historic deacetylase inhibitors (HDACi). Our central goals are to 1) find out if HDACi's that cross the blood-brain barrier alter test performance in rodent models for anxiety, behavioral despair and learned helplessness and 2) to study HDACi-induced chromatin-remodeling, including histone acetylation, at proximal promoter sequences of genes that are involved in neurotrophin and/or angiogenic-endothelial signaling pathways. The behavioral experiments (Aim #1) are guided by preliminary data demonstrating that mice treated for 14-16 days with the HDACi, sodium butyrate, show lower levels of behavioral despair, in comparison to controls. The chromatin studies (Aim #2) will rely on immunoprecipitation of hippocampal extracts with site- and modification-specific anti-histone antibodies, in conjunction with quantitative real time PCR for proximal promoter sequences and RT-PCR for coding sequences of 10 neurotrophic and angiogenic factors that are thought to play a role in the neurobiology or treatment of depression. It is expected that these novel approaches will provide promising first insights into the antidepressant potential of HDACi's and will establish the epigenetic modification of hippocampal chromatin as an important molecular mechanism for the neurobiology of depression.

DESCRIPTION (provided by applicant): Rett syndrome is a neurological disease of early childhood. It is associated with deleterious mutations of the gene encoding methyI-CpG-binding protein 2 (MECP2) but it remains unclear how MECP2-deficiency results in neuronal disease. MECP2 is thought to regulate acetylation, methylation and other post-translational modifications of the core histones, that together with DNA wrapped around them comprise the fundamental structural unit of chromatin and thus regulate gene expression, DNA repair and chromosome segregation. Our central goals are 1) to test the hypothesis that histone hyperacetylation contributes to the Rett syndrome phenotype (Specific Aim 1). We will monitor in developing cerebral cortex of wildtype and mutant mice developmentally regulated changes in H3 and H4 covalent modifications at the site of regulatory sequences of ionotropic glutamate receptor subunit genes, at the beta-globin locus and at methyI-CpG-rich regulatory sequences of the imprinted gene Necdin and of the Xist gene (Specific Aim 2). We will examine in primary cortical cultures if drug-induced changes in histone acetylation affect neuronal growth and survival (Specific Aim 3). Finally, we will study histone methylation in postmortem cerebral cortex of subjects diagnosed with Rett syndrome and confirmed MECP2 mutations, in comparison to subjects diagnosed with autism spectrum disorder and to non-neurological controls. Based on preliminary data, our central hypotheses are: 1) Treatment with drugs that inhibit histone deacetylases (HDACs) will accelerate development of disease in MECP2-deficient mice; 2) HDAC inhibitors impair growth and survival of cultured MECP2-deficient neurons; 3) Histone methylation is dysregulated in cerebral cortex of Rett subjects; 4) developmentally regulated histone acetylation and methylation at defined genomic regions is altered in brain of MECP2 mutant mice. Our experiments include in vivo and vitro studies with HDAC inhibitor and activator drugs, using conditional mutant mice with cre/IoxP mediated Mecp2 excision in cerebral cortex at different stages of development, complemented by a collection of human postmortem tissue. Our experiments will rely on chromatin immunoprecipitation assays, immunoblotting and immunohistochemistry with antibodies against H3 and H4 epitopes defined by site-specific modifications. It is expected that these approaches will provide a clear and comprehensive picture on the developmental regulation of histone modifications in cortical neurons, including potential changes in MECP2-deficient brain.

DESCRIPTION (provided by applicant): Dysfunction of the prefrontal cortex (PFC) is thought to play a key role in the neurobiology of schizophrenia. In particular, the deficit syndrome which includes negative symptoms such as anhedonia, amotivation, apathy and poverty of thought content, is attributed to PFC malfunction. Dysregulated gene expression may contribute to impaired cellular function of prefrontal neurons and glia, but the underlying molecular pathology remains unclear. Chromatin remodeling at gene promoter regions is increasingly recognized as a key control point of gene expression and may, therefore, contribute to altered gene expression in PFC of schizophrenics. Presently, nothing is known about chromatin regulation in normal and in diseased human brain. Chromatin immunoprecipitation assays (ChIP), in combination with DNA microarrays for promoter and other 5'regulatory gene sequences (chip-on-ChlP), are a powerful tool to map chromatin function on a comprehensive, genome-wide scale. Recently, chip-on-ChIP was used to study transcriptional regulation in human liver and pancreas. However, it is not known if chromatin immunoprecipitation techniques and chip-on-ChlP are applicable to human postmortem brain. Here, we will use chip-on-ChIP in order to examine posttranslational histone modifications, a major form of chromatin remodeling important for epigenetic control of gene expression, in PFC of schizophrenics and matched controls. Our experiments will rely on custom-made promoter microarrays and on our modified chromatin immunoprecipitation assay specifically designed for postmortem brain. It is expected that this novel approach will (i) provide first insights into posttranslational chromatin imprints regulating gene expression in human brain and (ii) test the hypothesis that dysregulated gene expression in PFC of schizophrenics is associated with altered chromatin remodeling and epigenetic modifications of the promoter. Finally, the proposed studies will provide the framework for future proposals designed to map histone modification patterns across entire chromosomes in human brain, including alterations in major psychiatric disease.

[unreadable] DESCRIPTION (provided by applicant): Dopaminergic signaling in striatum is involved in neuropsychiatric disease, including drug abuse and psychosis. Stimulants, antipsychotics and other drugs targeting the dopaminergic system regulate transcription in striatal neurons, but the underlying molecular mechanisms are not completely understood. Gaining a better understanding of how dopaminergic drugs induce transcription of early response and other genes should broaden the range and efficacy of treatments. The proposed experiments are designed to examine the dopaminergic regulation of covalent histone modifications in striatum, using acute and chronic paradigms. Histones are, together with DNA that wraps around them, the fundamental structural unit of chromatin and thus regulate gene expression, DNA repair and chromosome segregation, among others. Specifically, a "histone code" has been established, associating the sitespecific acetylation, methylation and phosphorylation of the amino-terminal tails of two core histones, H3 and H4, either to open chromatin and active gene expression or to silenced, inactive and condensed chromatin. Our central goals are 1) to examine on a molecular and cellular level dynamic changes in histone acetylation, methylation and phosphorylation after single or repeated administration of stimulants, DI agonists and D2-like antagonists (Specific Aims 1 and 2) and to identify intracellular messenger pathways that couple dopamine receptor signaling to the chromatin-remodeling complex in the nucleus (Specific Aim 3). These experiments are guided by preliminary data demonstrating that DI agonist- and D2-like antagonist-induced striatal transcription is accompanied by dynamic changes in open chromatin-associated histone acetylation, phospho-acetylation and methylation at the corresponding gene promoters. Therefore, our central hypothesis is that histone modifications defining open and closed chromatin are differentially regulated by stimulant drugs, D1 agonists and D2-like antagonists. Our experiments will rely on chromatin immunoprecipitation assays and immunoblotting, and laser capturemicrodissections of cells labeled with anti-histone antibodies that selectively recognize site-specific modifications at the NH2-terminal tails of H3 and H4. It is expected that these novel approaches will provide a clear picture on the dopaminergic regulation of the "histone code" in striatal neurons and will establish the epigenetic modification of striatal chromatin as a novel mechanism of action for stimulant drugs and also for conventional antipsychotics acting as D2-like antagonists. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dysfunction of the prefrontal cortex (PFC) is thought to play a key role in the neurobiology of schizophrenia. In particular, the deficit syndrome which includes a subset of negative symptoms such as anhedonia, amotivation, apathy and poverty of thought content, is thought to result from PFC malfunction. The neurobiology of PFC dysfunction in schizophrenia remains enigmatic, but it is now thought that, among other factors, functional hypometabolism and deficits in NMDA receptor-mediated signaling play a crucial role. It is thought that dysregulated gene expression in PFC and other brain regions of schizophrenics contributes to these defects, but the molecular pathology of transcriptional dysregulation in schizophrenia, including the underlying genetic and epigenetic mechanisms, remain unclear. In eukaryotes, including humans, changes in gene expression are typically accompanied by dynamic alterations in chromatin structure and function. Epigenetic control of gene expression, in particular, is mediated through a combinatorial set of covalent modifications at N-terminal residues of the core histones. Our central goals are 1) to find out, if in human brain, region-specific expression of NMDA receptor subunits is regulated through differential histone methylation and acetylation in chromatin surrounding 5' regulatory sequences of NMDA receptor subunit genes; and 2) to determine if, in PFC of schizophrenics, decreased expression of metabolic or NMDA receptor signaling-related mRNAs is accompanied by a deficit in open chromatin-associated histone methylation at the corresponding genes; and 3) to determine if chronic treatment with antipsychotic drugs induces epigenetic chromatin modifications at defined genomic regions in cerebral cortex. Our experiments will focus on human prefrontal and cerebellar cortex and will rely on a modified chromatin immunoprecipitation technique specifically designed for postmortem tissue. It is expected that this novel approach will provide (i) a clear picture on covalent chromatin imprints regulating gene expression in human brain and (ii) clarify if histone modifications as epigenetic regulators of gene expression contribute to transcriptional dysregulation affecting metabolism and NMDA receptor signaling in PFC of schizophrenics.

DESCRIPTION (provided by applicant): Dysfunction of the prefrontal cortex (PFC) is thought to play a key role in the neurobiology of schizophrenia. In particular, the deficit syndrome which includes negative symptoms such as anhedonia, amotivation, apathy and poverty of thought content, is attributed to PFC malfunction. Dysregulated expression of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), and other molecules regulating neuronal growth and plasticity in PFC may contribute to impaired cellular functions in schizophrenia, but the underlying molecular pathology remains unclear. Post-transcriptional and translational regulation by small RNA molecules, including microRNAs, is increasingly recognized as a key control point of gene expression and cellular functions. Presently, nothing is known about the role of microRNA-mediated regulation of gene expression during normal development of the human PFC and in chronic psychiatric disease conditions, including schizophrenia. Our preliminary data show that a subset of microRNAs predicted in silico to interact with the 3'UTR of BDNF are (i) expressed at robust levels in PFC pyramidal neurons, which comprise the primary cellular source of BDNF in cerebral cortex, (ii) are dynamically regulated during the extended course of PFC maturation and (iii) potentially dysregulated in some subjects diagnosed with schizophrenia. Guided by these preliminary findings, Aim #1 of this proposal will examine the developmental regulation and laminar and cellular expression pattern of over 20 microRNAs in human prefrontal cortex, including potential alterations in schizophrenia and regulation by antipsychotic drugs. Aim #2 will further explore the regulation of BDNF expression by the RNAi pathway in cultured cortical neurons with lentivirus-based transfection assays. Aim #3 will use a population genetics-based approach to search for BDNF regulating microRNA haplotypes that (i) might confer genetic risk for schizophrenia and related disease or (ii) affect levels of microRNA expression in PFC. It is expected that this integrative proposal, which combines postmortem studies with animal and cell culture models and clinical genetics, will provide first insights into the role of small RNAs for normal prefrontal development and chronic psychiatric disease. PUBLIC HEALTH RELEVANCE: Schizophrenia is a major psychiatric disease, but our understanding of the underlying molecular biology and genetics remains incomplete. This research project is focused on a family of molecules called microRNAs . Very little is known about the importance of these recently discovered molecules for brain functions. We will study a subset of microRNAs that might regulate a nerve growth factor protein, in postmortem brain of subjects diagnosed with schizophrenia and controls, these studies might advance our knowledge on the biological basis of schizophrenia, and perhaps, may lead to the development of new treatment strategies for the disorder.

DESCRIPTION (provided by applicant): Schizophrenia is defined by a lack of a straightforward genetic cause for a very large majority of affected individuals. The prefrontal cortex (PFC), among other brain regions, is thought to be frequently in subjects in schizophrenia, as reflected by its functional hypoactivity and dysregulated expression for a diverse set of genes. The underlying molecular mechanisms remain unknown but there is evidence that the prolonged maturation of the PFC, extending into or even beyond the second decade of life, plays a crucial role for normal human development and the neurobiology of schizophrenia. Recently, we presented the first evidence that a subset of epigenetic markings, including trimethylated histone H3-lysine 4 at sites of gene promoters, is involved in the dynamic regulation of PFC chromatin during throughout pre- and postnatal development, and may be altered in some cases with schizophrenia. This challenge grant proposal is "shovel ready" and create new jobs as well as retain existing ones. Specifically, we will combine two of the most innovative approaches in the field of neurosciences as it pertains to epigenetics, by selectively sorting neuronal chromatin followed by massively parallel sequencing of immunprecipitates (ChIP-seq) to obtain insight into the epigenomic landscape of prefrontal neurons. We will profile developmental and disease-related changes in histone methylation markings associated with active promoters (trimethyl-H3K4) or transcription (trimethyl-H3K36) selectively in PFC neurons. Furthermore, we will study in conditional mutant mice the role of a candidate histone lysine methyltransferase, Mll1 (mixed-lineage leukemia 1) for normal and diseased prefrontal development, by expressing Cre recombinase selectively in weanling PFC and then monitor behavioral and molecular alterations in the adult animal. The experiments proposed here, which are designed to be accomplished within the challenge grant's award period of 24 months, will clarify, for the first time, whether or not chromatin of human PFC neurons is developmentally regulated and altered in disease. The answer to this question on a genome-wide level will be critical in order to find out whether or not chromatin-based mechanisms are part of a final common pathophysiology underlying prefrontal dysfunction in the substantial portion of subjects diagnosed with schizophrenia. PUBLICH HEALTH RELEVANCE: For the majority of patients diagnosed with schizophrenia, no straightforward genetic cause has been identified. One of the important theories about schizophrenia implies that in some brain regions, such as the "prefrontal cortex", a number of genes are not switched on properly during normal development, as they are in healthy subjects. However, until now the molecular techniques to study these phenomena have been lacking. This challenge grant proposal is "shovel ready", will create new jobs and retain existing ones. It is also based on extremely innovative techniques that were recently developed in our laboratories. We were able, for the first time, to selectively isolate chromosomal materials and chromatin from the nerve cells of the human brain obtained postmortem, and study "epigenetic markings" (basically, chemical modifications that regulate gene expression and function without altering the genetic code) on a genome-wide level. In this proposal, we plan to examine epigenetic gene activation patterns during normal prefrontal cortex development and search for potential alterations in schizophrenia and in mutant mice designed to model the disease on a molecular level. This research should clarify whether or not epigenetic markings are dynamically regulated in human neuronal chromatin , and if major psychiatric diseases such as schizophrenia are associated with changes in the neuronal epigenome.

DESCRIPTION (provided by applicant): Currently available antidepressant drugs typically modulate serotonergic, noradrenergic or dopaminergic neurotransmission and take 6-8 weeks to exert their effects. In addition, for each drug, up to 50-60% of patients show inadequate responses in treatment trials. Therefore, it will be important to explore novel antidepressant treatment strategies in pre-clinical studies. It has been suggested that chromatin remodeling mechanisms, including histone modification changes, play an important role for the neurobiology of depressive disorders. The foundations of this hypothesis are built on two observations in the pre-clinical model, (i) commonly used treatments, including antidepressant drugs and electroconvulsive seizures, induce dynamic changes in histone acetylation, and (ii) drugs that inhibit histone deacetylases (HDACs) exhibit antidepressant-like effects, or enhance the therapeutic effect of a conventional antidepressant such as fluoxetine. Based on these findings, it is assumed that drug induced hyperacetylation of histones and the resulting antidepressant effect is linked to a loosening of chromatin structures and transcriptional activation. However, little is known about the neurological and behavioral phenotypes resulting from experimental manipulation of other histone modifications and histone modifying enzymes, particularly those defined by a genome-wide enrichment pattern that is highly divergent as compared to acetylation. To address these issues, we generated mice with a sustained increase in neuronal expression of Setdb1, encoding a developmentally regulated histone H3-lysine 9 specific histone methyltransferase associated with Sin3a-deacetylase, KAP-1 and NuRD transcriptional repressor complexes. Contrary to our original hypothesis, our preliminary data show that a sustained increase in Setdb1 levels in the forebrain is associated with antidepressant-like changes in behavioral despair and learned helplessness paradigms. Unexpectedly, Setdb1 occupancies in the genome were highly restricted overall, while enriched at several loci encoding glutamate receptor genes. This resulted in decreased levels of the NMDA receptor subunit Grin2b (NR2B) in prefrontal cortex and hippocampus. This finding is interesting because the non-specific NMDA receptor antagonist, ketamine, and the NR2B selective antagonist, CP101,606 elicit rapid antidepressant action in depressed subjects, including a subset of cases who had failed to respond to conventional treatments . The focus of this preclinical proposal is to explore the link between antidepressant-like phenotypes of Setdb1 transgenic mice, and the transcriptional regulation of NMDA receptor subunit Grin2b, and other gene expression changes, and to uncover novel treatment strategies for affective disorders. Our experiments utilize a comprehensive toolbox of in vivo and ex vivo approaches, including (a) multiple lines of genetically engineered mice to target the regulation of histone (H3-lysine 9) methylation in neuronal chromatin, (b) procedures to isolate neuronal chromatin from brain tissue, (c) chromatin immunoprecipitation followed by genome-wide profiling, (d) behavioral assays measuring despair and learned helplessness, anxiety, and learning and memory and (e) functional characterization of NMDA receptors in hippocampus and other brain regions implicated in the neurobiology of depression. This proposal will explore the antidepressant potential of Setdb1-regulated histone methylation, including the potential link to NMDA receptor-mediated signaling, thus offering the perspective of introducing a radically new treatment principle for a major psychiatric disorder.

DESCRIPTION (provided by applicant): To date, the majority of chromatin studies on brain are focused on the regulation of DNA methylation and post-translational histone modifications. However, replication-independent nucleosome remodeling and chromatin assembly, a mechanism associated with the process of gene expression, is likely to be of crucial importance for neurons and other postmitotic cells. Presently, next to nothing is known about its regulation in mature CNS. This is mainly due to methodological barriers, due to the difficulty to selectively sort neuronal chromatin from brain tissue and the lack of reliable antibodies to study the exclusive substrate for replication independent deposition, which is the histone H3 variant H3.3. The goal of this exploratory proposal is to overcome these critical shortcomings for brain-related chromatin assays. Specifically, we propose to develop a toolbox that includes (i) a transgenic mouse line expressing green fluorescent protein (GFP)-tagged histone H2B in a select set of forebrain neurons, which is complemented by (ii) adeno-associated virus (AAV)-based delivery of an expression cassette for FLAG/HA-epitope-tagged H3.3 under control of the c-fos promoter and (iii) acute challenge with dopamine D1 receptor agonist and D2-like antagonists, a well known trigger for early response gene expression and other transcriptional activity in prefrontal cortex and striatum. The proposed experiments will, for the first time, explore the phenomenon of replication-independent chromatin assembly and histone H3.3 deposition in neuronal nuclei of adult mice on a genome-wide scale, by combining chromatin immunoprecipitation techniques with massively parallel sequencing (Chip-seq). These pioneering studies will provide first insights into the role of replication-independent histone deposition in the mature nervous system, including the potential association with neuronal gene expression in response to changes in dopaminergic input. PUBLIC HEALTH RELEVANCE: The majority of common neurological and major psychiatric diseases (Alzheimer's, schizophrenia, autism, depression, druga, just to name a few examples) are thought to involve a combination of genetic and non-genetic factors. The latter includes "epigenetic" mechanisms, which typically are defined by a variety of chemical and molecular modifications of the genomic DNA and of the surrounding chromatin, without a change in the DNA sequence. One interesting layer of epigenetic regulation involves the exchange of certain histones (a type of protein closely attached to the genomic DNA) during the process of gene expression. The role of this mechanism, also called replication independent nucleosome exchange and histone deposition, is at present totally unexplored in the normal and diseased brain. The goal of this grant application is to overcome this critical shortcoming for brain-related epigenetic studies. Specifically, we propose to generate mice in order to assay replication independent histone deposition in specific types of nerve cells in the mouse brain.

DESCRIPTION (provided by applicant): The two major goals of this project is to 1) provide the research community and public domain for the first time with a comprehensive genome-wide atlas of the histone methylation landscape in selected subpopulations of cortical GABAergic interneurons and other cells residing in mouse cerebral cortex;and 2) To gain first insights into chromatin remodeling mechanisms of GABAergic neurons during the transition from juvenile to mature age. Previous work on chromatin extracted from human and mouse cortex indicated that histone methylation at "GABAergic gene" promoters is dynamically regulated during the extended period of maturation at least until early adulthood, thereby linking chromatin remodeling mechanisms to the developmental clock. The rationale to focus on the epigenome of cortical interneurons goes beyond mere academic curiosity. Dysregulated gene expression in GABAergic interneurons is considered a hallmark of the molecular pathophysiology and a major factor for the synchronization deficits in neural networks that affect widespread areas of the cerebral cortex in subjects on the psychosis or autism spectrum. These GABA related gene expression deficits include distinct cell types such as the class of fast spiking interneurons commonly recognized by expression of the calcium binding protein, Parvalbumin. Therefore, in the context of this proposal, we plan to generate 4 isogenic lines of BAC (bacterial artificial chromosome) transgenic mice to express green fluorescent protein (GFP)-tagged histone H2B in GABAergic interneurons overall, and in 3 specific subpopulations defined by differential expression of calcium buffering proteins Parvalbumin (PARV) or Calbindin (CALB) or Calretinin (CALR). Recent methodological advances enable us to separate and sort with high efficiency GFP-tagged nuclei from brain tissue for the purposes of chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). Focus will be on tri- and mono-methyl-histone H3-lysine 4 (H3K4me3, H3K4me1) which are enriched at transcription start sites (H3K4me3) and enhancer sequences including those further removed from proximal promoters (H3K4me1), and a mark associated with RNA polymerase II activity and transcriptional elongation across coding and non-coding regions (H3K36me3). We expect that the various interneuron subpopulations, which differ in terms of function and developmental history, will show cell type specific chromatin signatures in many portions of the genome. When analyzed in conjunction with cell-specific transcriptomes and other datasets, histone methylation mapping of specific interneuron types is likely to provide radically novel insights into the developmental history and (epi)genomic architecture of cells ascribed a key role in schizophrenia and related disease. PUBLIC HEALTH RELEVANCE: For the majority of patients diagnosed with schizophrenia, no straightforward genetic cause has been identified. One of the important theories about schizophrenia implies that in some brain regions, such as the "prefrontal cortex", a number of genes are not switched on properly during normal development, as they are in healthy subjects. Many of these genes related to a type of cell called the 'GABA neuron'which comprise less than 10% of all cells (nerve cells and others) in the cortex, but are very powerful because in effect they regulate synchronization of large neural networks in the brain. To further understand the role of the GABA neurons in psychiatric disease, and to clarify why gene expression is abnormal in these cells, it will be important to explore their genomes and chromatin architectures at high resolution. This grant proposal is based on extremely innovative techniques that were recently developed in our laboratories. We will be able, for the first time, to selectively isolate chromosomal materials and chromatin from the GABA neurons of the mouse brain for the study of "epigenetic markings" (basically, chemical modifications that regulate gene expression and function without altering the genetic code) on a genome-wide level. We expect that the work resulting from this project will provide a valuable resource /chromatin atlas for the neuroscience research community, and will shed light on some of the developmental mechanisms that govern proper gene expression activity in mature GABA neurons.

DESCRIPTION (provided by applicant): Developmentally regulated histone modification and DNA methylation changes, shaping gene expression patterns and genome organization, are likely to be of fundamental importance for orderly ontogenesis and cellular differentiation. Therefore, comprehensive and high resolution mapping of cell type-specific epigenomes from brain bear enormous merit both from the viewpoint of developmental biology and translational medicine alike. To date, however, comprehensive and genome-wide maps of epigenomes for defined cell populations from brain, and their developmental trajectories, do not exist. This important gap in epigenetic information can finally be addressed with a recently introduced protocol that efficiently separates-in brain tissue-chromatin from different cell types. In this proposal, we will explore, for the first time, the histone methylation landscapes of ventral midbrain dopaminergic neurons of the human brain. These cells play a key role in the neurobiology of a wide range of neuropsychiatric disorders ranging from drug addiction to neurodegenerative (e.g., Parkinson's) conditions. Focus will be on tri-methyl-histone H3-lysine 4 (H3K4me3), a mark enriched at transcription start sites (H3K4me3) and a subset of CpG-rich sequences, and tri-methyl-H3K36me3, a mark associated with RNA polymerase II activity and transcriptional elongation across coding and non-coding regions. Beyond the goals of this R21 proposal, the long-term vision for the collaboration between our two groups at Wayne State and UMASS is to explore changes in the dopaminergic epigenome of subjects diagnosed with stimulant and other substance abuse.

The objective of Project 2 is to characterize the involvement of MBT (malignant brain tumor domain) proteins in prefrontal cortex (PFC) and nucleus accumbens (NAc) in mediating depression- and antidepressant-like responses in animal models. MBT proteins are the best known readers-i.e., effectors-for several key methylation states of histones, including repessive histone methylation at Lys 9 of histone H3 (H3K9me2). However, virtually nothing is known about the function of MBT proteins in brain. We have found that 3 MBT proteins, L3MBTL1, L3MBTL2, and SFMBT1, are highly expressed in PFC and NAc, where they display dramatic regulation in response to several forms of chronic stress. Depressed humans show similar altered levels of some of these same MBT proteins. Moreover, mice lacking L3MBTL1 show a pro-depression-like phenotype, consistent with findings in Project 1 that downregulation of H3K9me2 increases susceptibility to chronic stress. We have generated mutant lines of the other MBT proteins and now propose the comprehensive characterization of: 1) the regulation of L3MBTL1, L3MBTL2, and SFMBT1 in our Center's battery of depression models, and 2) the behavioral phenotypes of conditional and brain region-specific knockout, or overexpression, of these three MBT proteins. We will then use our novel method, which permits the genome-wide analysis of chromatin modifications specifically within adult PFC neurons, to map the binding of MBT proteins and their key target sites of histone methylation, including H3K9me2, in PFC neurons in chronic stress models, with parallel studies performed on PFC neurons from depressed humans (Project 4). We will also study conditional knockouts of several key histone methyltransferases, including G9a (with Project 1), which catalyze the methylated histone sites read by MBT proteins, based on the hypothesis that similar phenotypes will be observed. We are particularly excited about comparing preclinical and clinical chromatin datasets through which we will construct, specifically for PFC neurons of mouse and human, a genome-wide map of epigenetic risk loci highly relevant for depression. Together, this work provides a template for the analysis of the role played by other histone reader proteins in depression. RELEVANCE (See instructions): Depression has a lifetime risk of ~15% for the U.S. general population, yet available antidepressant therapies are based on serendipitous discoveries over 6 decades ago, and fully treat <50% of all affected individuals. An improved understanding of the molecular basis of depression will lead to improved treatments and diagnostic tests-a high priority for the National Institutes of Health.

DESCRIPTION (provided by applicant): In this proposal, we examine for the first time an epigenetic mechanism called nucleosome remodeling and how it regulates coordinate gene expression required for cocaine-induced memory formation. The nucleosome is the repeating unit of chromatin and fundamental to the compaction of genomic DNA. Nucleosome remodeling complexes modify chromatin structure and regulate expression by repositioning nucleosomes at the promoters of genes. Recent human exome sequencing studies have identified subunits of the polymorphic BAF complexes (mammalian SWI/SNF nucleosome remodeling complex) that are frequently mutated in sporadic mental retardation and sporadic autism. Moreover, de novo mutations in various subunits of neuron-specific Brg1- associated factor (nBAF) nucleosome remodeling complex have been implicated in Coffin-Siris and Nicolaides-Baraitser syndromes, both of which are associated with intellectual disability. Together, these studies suggest that nBAF function is necessary for normal cognitive function. Although an important topic in other fields (e.g. yeast genetics and cancer), nucleosome remodeling has received little attention in neuroscience. However, a major discovery was the identification of the first neuron-specific BAF complex, which was subsequently found to regulate gene expression required for the conversion of precursor cells into terminally differentiated neurons. Importantly, the nBAF complex has a subunit, BAF53b, which participates in making nBAF neuron- specific. This subunit is both neuron and nBAF complex specific, making it an ideal target for investigating the potential contributions of nBAF to synaptic physiology and behavior. Building on this point, we propose to test the hypothesis that BAF53b, after playing a key role in neuronal fate decisions during development, continues to regulate gene expression and does so in a manner critical to adult memory processes as well as cocaine-induced memory formation. We propose three specific aims to test this hypothesis. In Specific Aim 1, we will use genetically modified mice to examine the role of BAF53b in long-term memory. In Specific Aim 2, we will use next generation sequencing, RNA seq, and chromosomal conformation capture 3C to determine what gene expression profiles are being regulated by BAF53b and chromatin looping during memory consolidation. In Specific Aim 3, we will determine how cocaine regulates coordinate gene expression via BAF53b-dependent nucleosome remodeling and chromatin looping during cocaine-induced memory formation. Together, the work under these specific aims will elucidate the contributions of BAF53b and the nBAF complex in general, to memory processes, and more specifically to cocaine-induced memory formation as a precursor event to persistent drug-seeking behavior.

DESCRIPTION (provided by applicant): The exploration of brain epigenomes, including DNA methylation and covalent histone modifications, has provided fundamentally new insights into the mechanisms of brain ontogenesis and maturation. Moreover, deleterious mutations and rare structural variants in more than 50 genes encoding various types of chromatin regulators have been linked to neurodevelopmental diseases, including autism spectrum disorders (ASDs). Therefore, it is now generally accepted that proper regulation of chromatin structure and function during pre- and early postnatal development is critically important for the proper unfolding of cognitive abilities and emotional states. ASDs are a group of neurodevelopmental conditions bound together by broad syndromic overlap, with key behavioral deficits in social interaction, communication, and motor behavior including stereotypies. There is a strong genetic contribution to ASDs, yet environmental influences may also be etiologically important. Only a few studies, however, studied chromatin structures in diseased tissue (i.e., postmortem brain tissues from ASD subjects). In addition, epigenetic regulations, including histone modification landscapes, are highly specific for cell type, which is a key challenge for the field given the enormous cellular heterogeneity of the brain tissue, with multiple sub-population of inhibitory and excitatory neurons and various types of non-neuronal cells residing in the same tissue blocks. In this exploratory proposal, we will develop and test radically novel approaches in the human brain research, including the sorting of multiple subtypes of cortical neurons and the cell type-specific charting of 3- dimensional chromosomal architectures at selected genomic loci, with focus on ASDs. Specifically, we will profile open chromatin-associated histone methylation and acetylation, and promoter-enhancer associated chromosomal loop formations in GABAergic interneurons derived from the medial ganglionic eminence (MGE), in comparison to other neurons from ASD and control brains. If successful, the experiments proposed here will push the existing frontiers in human brain research, and for the first time, draw a connection between regulatory non-coding DNA, chromosomal architectures, and histone methylation profiles in multiple neuronal subtypes in health and disease.

DESCRIPTION (provided by applicant): Great progress has been made in mapping functional units of the genome as well as the epigenome through the efforts of the ENCODE project, such work has mainly been carried out in cell lines and peripheral tissues. The goal of our PsychENCODE project is to construct detailed maps for multiple important chromatin modifications in a tissue of specific relevance to schizophrenia (SCZ), human neurons and glia, and subsequently assess the relationship of several of these marks to known genetic risk factors for SCZ. We will first broadly survey a series of important chromatin marks in two brain regions, the pre-frontal cortex and anterior cingulate cortex, in limited number of samples to provide brain and neuronal specific maps of the chromatin marks. We will then focus deeply on a single region, the pre-frontal cortex, and on an important promoter and enhancer mark, in a large sample of post-mortem human brain samples. Specifically, we will first, map cell-type specific transcriptome and epigenome components in cortical tissue homogenates and in neuronal and non-neuronal chromatin in nuclei from normal human adult brain (n=20) using fluorescence- activated cell sorting followed by ChIP-Seq. Second, we will map promoter (H3K4me3) and enhancer (H3K27ac) marks in neuronal chromatin of the prefrontal cortex of 328 cases with SCZ, and 315 controls. We will integrate genome-wide SNP, CNV and brain mRNA expression with the neuronal ChIP-seq promoter and enhancer marks to identify novel SCZ genes using a variety of analytic strategies including constructing weighted interaction and causal probabilistic SCZ networks to identify key drivers and subnetworks underlying SCZ. Finally, we will map distal enhancer elements using chromosome conformation capture and investigate the role of medication confounds on mapping results. Our PsychENCODE project will apply innovative techniques and analytic strategies including cell-type specific epigenome mapping, chromosome conformation capture, deconvolution mapping and construction of causal probabilistic network analyses. All data will be made available to the research community through the Sage Bionetworks Synapse Platform. There is a deep need to understand the epigenetic landscape in the human brain, and in particular in neurons and glia and to integrate this information with human SCZ genetics. We have assembled the critical key personnel, sample resources, technological know-how, and analytic strategies to be able to provide both useful maps for the field, as well as begin to unravel SCZ biology.

DESCRIPTION (provided by applicant): The genetics of schizophrenia (SCZ) is advancing at rapid pace. An increasing number of risk-associated polymorphisms and variants are found in intergenic, intronic and other non-coding sequence. However, it has been a major challenge to design testable hypotheses to elucidate the potential function of such types of disease-associated non-coding DNA. Many of these sequences are thought to exert regulatory functions, including long range enhancer and repressor elements physically interacting with transcription start sites (TSS) separated on the linear genome by many kilobases of interspersed DNA. We will first construct high-resolution Expression Quantitative Loci (eQTL) maps by analyzing jointly gene expression, and genotyping and epigenomic profiles from publicly available as well as in-house generated datasets and then integrate the eQTL maps with the most updated dataset from the Psychiatric Genomics Consortium (PGC) that provides with genome-wide SNP coverage for more than 35,000 SCZ cases and 47,000 controls, with replication look-up into 70,000 subjects. We will thereby identify SCZ associated non- coding regions that are positioned within regulatory regions tagged with combinatorial histone modification signatures indicative of poised or active enhancers and statistical (eQTL) evidence for long range TSS interactions. We then will employ innovative approaches in neuroepigenetics, including chromosome conformation capture (3C) to map long range enhancer-promoter interactions in human brain collected postmortem, complemented by functional assays with gene expression reporter systems and activity-induced paradigms in cultured neurons derived from reprogrammed skin cells. The multidimensional approach presented here provides a roadmap to unravel the neurological functions of the vast but in brain largely unexplored non-coding sequences of the human genome.

? DESCRIPTION (provided by applicant): The growing list of monogenic forms of neurodevelopmental disease includes mutations in at least 50 genes each encoding a different chromatin regulator. Remarkably, among these are five members of the lysine methyltransferase (KMT) and demethylase (KDM) family of molecules specifically targeting the K4 residue of the nucleosome core histone H3. These disease-associated mutations affect 3 KMTs, MLL1, MLL2, MLL3 and 2 KDMs, KDM5A and KDM5C/JARID1C/SMCX. However, very little is known about the molecular mechanisms by which H3K4-specific KMTs and KDMs regulate brain chromatin structure and function. MLL1, and its homologue, MLL2, are expressed at high levels in a large majority of neurons, but their specific effects on neuronal histone methylation landscapes and genome organization are from a genome-scale perspective still unclear. Here, we will apply conditional mutagenesis to explore the role of MLL1 and MLL2 for neuronal health and function, in conjunction with assays for working and long-term memory, social cognition and other behavioral paradigms. Furthermore, we will study synaptic plasticity, including spike timing-dependent short- and long-term potentiation and depression (sLTP and sLTD) in prefrontal and striatal circuitry of mice subject to neuron-specific methyltransferase deletion. We will apply some of the most innovative approaches in neuroepigenetics, including cell-type specific epigenome and transcriptome mappings in conjunction with chromosome conformation capture assays. We introduce radically novel hypotheses to the field. We propose that in neurons, H3K4 methylation is a mark essential for chromosomal loopings and physical interactions between gene-proximal promoters and distal enhancers, that are separated on the linear genome by thousands of kilobases. The work proposed here is expected to illuminate new principles of epigenetic regulation in the nervous system, and could pave the way for novel therapeutic approaches aimed at common neurodevelopmental and cognitive disorders, including autism and schizophrenia.

? DESCRIPTION (provided by applicant): 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. Epigenetic modifications play a well-accepted role in a variety of medical and neurological illnesses, and are also implicated in SCZ. Somatic mosaicism is an underexplored, but potentially very important contributor to SCZ. There have been some intriguing hints that somatic mosaicism may play a role in SCZ, but assessment of this possibility awaits rigorous experiments, and that is the overarching goal of this proposal. The primary objective of our project is to identify and characterize the extent of somatic variation in post-mortem human brain samples from individuals with SCZ and controls. Following on work of members of our team, we will rigorously assess the somatic mosaicism in a large cohort of post-mortem human brains from the Common Mind Consortium, which members of our group are already analyzing for genotype, mRNA-seq and epigenome mapping. These brains are from individuals with SCZ (250) and controls (50+). We will look for retrotransposition events, copy number variants (CNVs) and single nucleotide variants (SNVs). All data will be made available to the research community through the Sage Bionetworks Synapse Platform. We have assembled the critical personnel, sample resources, technological know-how, and analytic strategies to be able to assess the role of somatic variation in the brain as well as begin to unravel SCZ biology.

Many genetic, internal and external risk factors impact the immature brain, resulting in cognitive and behavioral deficits at much later periods, with the delayed onset of symptoms often not before adulthood. Therefore, longitudinal modeling is critical in the context of neuropsychiatric disease. However, even after hundreds of studies published to date, functional genomics in the nervous system still faces a formidable barrier: Virtually all transcriptomic and epigenomic approaches currently available are cross-sectional, providing snapshots of genome function only for the time point of tissue harvest. The field is in urgent need of a toolbox that makes it feasible to pursue `retrospective functional genomics'. This would allow, for example, direct correlation of an animal's behavior in adulthood with the status of its neuronal (or glial) genomes in early life. This exploratory proposal will develop technology to map neuronal genome organization in a longitudinal context, starting with the exposure of juvenile and young adult mice to risk factors associated with long-lasting impairments in cognitive function, including subchronic treatment with NMDA receptor antagonist drugs and social isolation stress. We will map 3-dimensional genome organization and function in specific subtypes of cortical neurons, including excitatory projection neurons and inhibitory fast-spiking interneurons, and develop technology to map neuronal genome organization in a longitudinal context. Our retrospective 3D genome mapping approach is based on transient, Herpes-based vectors to express, in prefrontal cortex, chimeric constructs of bacterial Dam methyl-adenine methyltransferase, fused to synthetic DNA binding proteins anchored to cis-regulatory sequences of neuronal genes. These include Gad1 encoding GABA synthesis enzyme, Arc encoding an activity-regulated cytoskeletal protein and Bdnf encoding brain-derived neurotrophic factor. Our approach, as retrospective 3D-genome mapping, will allow us to directly correlate the animal's behavior and cognition with the spatial architectures of its neuronal genomes during the period of risk exposure dating back 3 months prior. The approaches presented here, if successful, could offer critical opportunities to explore the molecular and neuro-epigenetic underpinnings of mechanisms associated with long-lasting inter-individual variations in resilience to early life exposures and childhood adversity.  

PROJECT SUMMARY ? PROJECT 2 Project 2 made considerable progress over the past four years in defining the influence of several histone methyltransferases and demethylases, acting in prefrontal cortex (PFC) neurons, in controlling depression- related phenomena. A major finding from this work is that genome regulation is often ?punctate? as opposed to continuous and affects certain clusters along the genome. Consistent with this notion is our evidence that depression-related epigenetic regulation partly bypasses the linear genome and affects the 3D structure of chromatin. One of the most dramatically affected genomic regions is the Pcdh locus, which expresses numerous isoforms of protocadherins, strongly implicated in neuronal growth and synapse formation, but poorly understood in adult brain, let alone depression. Importantly, the Pcdh locus also displays robust regulation in the Center's genome-wide datasets, including dramatic induction in PFC of mouse models and depressed humans, effects seen in males and females, and Pcdh genes are among the most highly induced in PFC by early life stress. We will now further characterize the influence of the 3D genome, with particular attention to Pcdh, in depression in several key ways. We will study how manipulation of specific proteins which control 3D chromatin structure in PFC neurons influence depression-related behavioral abnormalities. We will use gene-editing tools, with the Chromatin and Gene Analysis Core, to modify the Pcdh locus and study directly its influence in depression models. This is a required approach, since the large number of Pcdh genes and compensatory changes in other isoforms when a single isoform is manipulated has made it difficult to characterize protocadherin function in adult brain. We will also map how chronic stress alters the 3D genome in PFC neurons, both focusing on Pcdh as well as the global genome, with parallel studies of depressed humans performed in Project 4. Finally, we will employ a novel method to retrospectively map how the 3D genome is altered by early life stress when examining adult mice based on their susceptibility to subsequent stress. Together, these studies will reveal how Pcdh genes influence depression-related phenomena and provide fundamentally new information about the 3D genome in adult neurons.

The genome of brain cells is organized into thousands of `topologically associated domains' (TADs), with the linear genome folded upon itself across hundreds of kilobases. A deeper understanding of TAD regulation and function will advance knowledge about epigenomic and genetic risk architectures of neuropsychiatric disease. However, to date regulatory mechanisms governing TAD structure and function in brain cells remain completely unexplored. In this proposal, we will study neuronal maintenance of a subset of very large, mega-base scale `superTADs' that critically depend on Set-domain-bifurcated 1 (Setdb1/Eset/Kmt1e), encoding a histone H3-lysine 9 methyltransferase. This includes the 1.2 megabase-spanning topologically associated domain at the clustered Protocadherin (cPcdh) locus, encompassing >70 Pcdh and non-Pcdh genes important for neuronal connectivity. We propose to dissect, in vivo, the regulatory layers governing the neuronal 3D genome, including SETDB1- sensitive neuronal superTADs. Aim #1 will test the hypothesis that SETDB1 shields neuronal genomes from excess binding by the multifunctional chromatin organizer CCCTC binding factor (CTCF). To this end, we will map, by in situ Hi-C assays, the 3D genomes of glutamatergic projection neurons in adult cerebral cortex and inhibitory projection neurons of cerebellar cortex, comparing wildtype with Setdb1 and Ctcf deficient neurons. Aim #2 will explore single cell-stochastic constraint of cPcdh genes, including potential alterations after Setdb1 and Ctcf ablation and after (epi)genomic editing of loop-bound non-coding sequences within the local superTAD. Furthermore, we will study of functional connectivity after neuron- specific deletion of Setdb1 and Ctcf. We will assess changes in synaptic drive onto top-down rostromedial frontal-to-visual cortex projection neurons between adolescence and adulthood, with projection-specific whole-cell patch clamp recordings of miniature excitatory (mEPSC) postsynaptic currents at multiple developmental time points, together with dendritic spine characterization. Taken together, the experiments proposed here will provide deep insights into regulatory mechanisms governing the maintenance and function of large megabase-scale higher order chromatin structures in mature neurons. This includes an intriguing role of the chromosomal connectome inside neuronal nuclei shaping the brain's connectome, by regulating expression of the cPcdh genes.

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

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

Although many cells and neural circuits clearly contribute to opiate and other substance abuse disorder, the path to drug addiction travels through midbrain dopaminergic neurons. Though a rare cell type (it is estimated that a mere 1 of every 200,000 neurons in the human brain is of a dopaminergic phenotype), changes in dopaminergic neurotransmission are thought to play a role in various stages of addiction, from acute reward mechanisms and goal-directed actions, to the development of habitual behavior and increased salience of cues associated with drug use, as well as the anhedonia and dysphoria associated with drug withdrawal. Surprisingly little is actually known about persistent changes in gene expression that presumably underlie the dysfunction of dopamine systems in brain exposed to opiates and other drug of abuse. Our project is centered on three Specific Aims. In Aim #1,we will extract chromatin from immunotagged midbrain dopaminergic neuron nuclei collected by fluorescence-activated sorting from 150 controls and 150 cases diagnosed with opiate abuse and then profile, on a genome-wide scale, the transcriptome and open chromatin landscapes and promoter-enhancer loopings and other types of chromosomal conformations (the ?3D genome?) in cell type-specific manner. In Aim #2, we will apply integrative genomics approaches and leverage Aim #1 postmortem brain data with population-scale genotypes and phenotypes provided by the Million Veterans Project and the Psychiatric Genomics Consortium to build causal probabilistic networks and predict key drivers within the regulatory non-coding DNA space of the dopaminergic system. In Aim #3, we will validate addiction-relevant cis-regulatory sequences (from Aim #1, #2) with small RNA-guided epigenomic editing systems in cultured human dopaminergic neurons. Collectively, our midbrain dopaminergic neuron- focused project will fill critical voids in the field of human addiction research and human neurogenomics and embark, for the first time, on a deep epigenomic assessment of one of the key cell populations in reward and addiction circuitry.

HIV-associated neurocognitive disorders (HAND) persist in the era of combination antiretroviral therapy (cART). Proof of HIV latency in human CNS is currently lacking, despite continued high prevalence of HIV- associated neurologic disease and increasing recognition of CNS viral escape in people stably suppressed with cART. One of the major issues regarding CNS HIV in need for study is HIV integration. With other words, whether CNS HIV integration has biologically significant impact, contributing to pathogenesis? Issues of CNS functional deficit are further complicated by the co-registered epidemic of opiate and other substance use disorders (SUD) in people living with HIV/AIDS (PLWHA), as SUD also have profound impact on CNS function, and potentially on HIV latency. Nowhere in the CNS is this more evident than in the neuroanatomic overlap of HIV and SUD in striatonigral dopaminergic circuitry and frontostriatal projections, sites of predilection for functional and neurobiologic disease as well as for increased burden of HIV infection. Accordingly, directly utilizing brain tissues in these regions, from neurologically well-characterized HIV-infected individuals with and without SUD, the goal of this application will be: (i) to replicate for brain some of the emerging genomic mechanisms recently discovered in peripheral cells, linking HIV host genome integration and virus latency to nuclear topography and open chromatin; (ii) to explore whether HIV signatures in transcriptomes and epigenomes in dopaminergic circuitry is associated with prospectively monitored neurological status in the years before death and exposure to drug of abuse; (iii) explore HIV expression and integration in potential reservoir cells of the brain, including microglia and astrocytes derived from HIV+ brain at autopsy and (iv) explore the impact of substance history on HIV integration and activity in primary microglial cultures derived from HIV- brains at autopsy and (vi) expose primary microglia in culture to drugs of abuse and HIV. The innovative experiments proposed here are expected to offer novel insights into epigenomic landscapes in specific brain cells and explore potential links between neurogenomic status of the infected brain and neurological and cognitive symptoms and substance abuse. While recognizing the high-risk aspects, these analyses will nevertheless have predictable, high gain benefits in understanding the complex neurobiology underlying HIV-associated CNS disease in PLWHA and SUD.

HIV-associated neurocognitive disorders persist in the era of combination antiretroviral therapy (cART) while HIV latency, and cell-specific expression of HIV transcript in human CNS remains incompletely understood. There is high prevalence of HIV-associated neurologic disease and increasing recognition of CNS viral escape in people stably suppressed with cART, often further complicated by the co-registered epidemic of substance use disorders (SUD) in people living with HIV/AIDS (PLWHA), as SUD also have profound impact on CNS function. Ongoing work in our laboratory is providing first assessments of cell-type specific HIV 'molecular signatures', including genome integration patterns and alterations on the level of the transcriptome and epigenome in reward- and addiction circuitry of the human postmortem brain. As described in detail in the Preliminary Data section, we found dramatically high levels of HIV expression in a subset of microglia from postmortem specimens, with HIV transcript levels ranking among the top 5 highest expressed RNAs in microglia, or the 99.9% percentile of all microglial transcript. Correspondingly, HIV genome integration sites in addiction circuitry are dominated by microglia-specific genes, with strong preference for active chromatin compartments. However, lingering effects of latent infection that persist during cART have not been well characterized?in part because of fundamental challenges in identifying the extent to which microglial cells contribute to the latent reservoir. Our preliminary studies also provide a model system whereby we can track and isolate persistently infected cells which can be applied to the microglial compartment and will allow us to define the genomic perturbations that persist during cART. By studying HIV genomics in human microglia residing in the mouse brain and linking this with technology to track persistently infected microglia, we will be able to model, for the first time, experimental therapies and interventions to complement our descriptive work in human postmortem brain. Specifically, our Cre-reporter based HIV-induced lineage tracing (HILT) marking system will allow us to quantify and isolate the rare latently infected microglia that persist during cART, and map transcriptomic and epigenomic alterations separately both for infected, and non-infected microglia, both collected from the same mouse brain. With focus on addition circuitry, we will study neuroinflammation, cognition and reward behavior in mice treated with standard cART regimens and an experimental therapy involving Cannabinoid receptor 2 agonist drugs that, according to our preliminary data, are linked to anti-inflammatory activity limiting the extent of HIV infection in tissues.