Hongjun Song - US grants

DESCRIPTION (provided by applicant): The existence of neural stem cells in the adult central nervous system (CNS) of all mammals, including humans, raises the possibility to self-replace damaged or lost neurons by activation of endogenous neural stem cells or transplantation of in vitro expanded stem cells and their progeny derived from the same person. For the full potential of adult neural stem cells to be realized, we need to identify the origin and source of adult neural stem cells, to understand the mechanisms underlying their proliferation, fate determination, and importantly in the case of neuronal lineages, to characterize their functional properties. In the intact mammalian CNS, active neurogenesis only occurs in discrete regions with mostly gliogenesis in other regions. Multipotent neural progenitors, however, can be isolated from both neurogenic and non-neurogenic regions. Since neurological diseases and injury occur in diverse regions of the CNS, including non-neurogenic regions, it is important to determine whether endogenous adult neural progenitors only exist in neurogenic regions or they are widely distributed and their neurogenic potentials are limited by their local environment. It is also important to determine whether in vitro expanded neural progenitors derived from different regions of the adult CNS behavior exhibit similar properties. Specifically, we need to understand whether cell-intrinsic differences between neural progenitors from different regions of the adult CNS, especially from non-neurogenic regions, (1) will influence their response to extrinsic stimulations for differentiation and maturation, and (2) will limit their capacity to acquire full spectrum of functional properties of mature CNS neurons. We have established multipotent clonal lines of adult neural progenitors derived from different regions of the adult CNS and developed methods to investigate their fate determination and measure their functional and electrophysiological properties. In this project, we propose to use adult neural progenitors from hippocampus (neurogenic region) and spinal cord (non-neurogenic region) as examples to examine the fate specification and maturation of in vitro expanded neural progenitors and the underlying molecular mechanisms. In particular, we will characterize the functional properties of neuronal progeny of different adult neural progenitors with immunocytochemistry, electron microscopy, FM imaging and electrophysiology. Finally, we will determine the intrinsic neurogenic potentials of endogenous progenitors of adult spinal cord by a set of transplantation studies

DESCRIPTION (provided by applicant): The existence of neural stem cells in the adult central nervous system (CNS) of all mammals, including humans, raises the possibility to self-replace damaged or lost neurons by activation of endogenous neural stem cells or transplantation of in vitro expanded stem cells and their progeny derived from the same person. For the full potential of adult neural stem cells to be realized, we need to identify the origin and source of adult neural stem cells, to understand the mechanisms underlying their proliferation, fate determination, and importantly in the case of neuronal lineages, to characterize their functional properties. In the intact mammalian CNS, active neurogenesis only occurs in discrete regions with mostly gliogenesis in other regions. Multipotent neural progenitors, however, can be isolated from both neurogenic and non-neurogenic regions. Since neurological diseases and injury occur in diverse regions of the CNS, including non-neurogenic regions, it is important to determine whether endogenous adult neural progenitors only exist in neurogenic regions or they are widely distributed and their neurogenic potentials are limited by their local environment. It is also important to determine whether in vitro expanded neural progenitors derived from different regions of the adult CNS behavior exhibit similar properties. Specifically, we need to understand whether cell-intrinsic differences between neural progenitors from different regions of the adult CNS, especially from non-neurogenic regions, (1) will influence their response to extrinsic stimulations for differentiation and maturation, and (2) will limit their capacity to acquire full spectrum of functional properties of mature CNS neurons. We have established multipotent clonal lines of adult neural progenitors derived from different regions of the adult CNS and developed methods to investigate their fate determination and measure their functional and electrophysiological properties. In this project, we propose to use adult neural progenitors from hippocampus (neurogenic region) and spinal cord (non-neurogenic region) as examples to examine the fate specification and maturation of in vitro expanded neural progenitors and the underlying molecular mechanisms. In particular, we will characterize the functional properties of neuronal progeny of different adult neural progenitors with immunocytochemistry, electron microscopy, FM imaging and electrophysiology. Finally, we will determine the intrinsic neurogenic potentials of endogenous progenitors of adult spinal cord by a set of transplantation studies

[unreadable] DESCRIPTION (provided by applicant): The existence of neural progenitor/stem cells in the adult central nervous system (CNS) of all mammals, including humans, raises the possibility to replace damaged or lost neurons by activation of endogenous neural progenitor/stem cells or transplantation of in vitro expanded neural stem cells and their progeny. Since most degenerative neurological diseases and injuries occur in the aged population, better understanding of how neural stem cells interact with their local environment in the aged brain will be essential to develop strategies for stem cell based therapies. Adult neurogenesis in the dentate gyrus of the hippocampus, a region involved in learning and memory, has been shown to decrease with increasing age. Whether this reduction reflects changes in the intrinsic properties of neural progenitor/stem cells and/or changes of the local environment during aging is unknown. While neural stem cells of young adult rodents can generate functional neurons with essential properties of mature CNS neurons both in culture and in vivo, it is not known if aged neural stem cells can generate functional neurons and whether the aged brain can support functional neurogenesis. Aging is associated with increases of brain inflammation and oxidation. CNS inflammation was recently shown to negatively regulate adult neurogenesis. We have previously shown that inflammation modifies the sphingolipid and sterol content of neural cells and disrupts the structure and function of lipid rafts, which in turn can modify neural cell function by perturbation of cellular signaling. We have isolated neural progenitors from the hippocampus of both young adult and aged rats and developed methods to investigate their proliferation, fate determination, functional and electrophysiological properties both in culture and in vivo. In this project, we propose to determine the roles of intrinsic vs. extrinsic mechanisms in regulating the sequential steps of functional adult neurogenesis in a rodent model of aging. In particular, we will use pharmacological and dietary manipulations to modify redox balance, sphingolipid and sterol metabolism and quantitatively compare the properties of neural progenitor/stem cells of young adult and aged hippocampus both in vitro and in vivo. [unreadable] [unreadable]

One of the recent excitements in the schizophrenia field is the identification of multiple susceptibility genes from human genetic association studies. Recent studies also reinforce the emerging view that schizophrenia is a disease of neuronal development in nature with an adult onset. How specific schizophrenia susceptibility genes regulate different aspects of neuronal development, however, is largely unknown. The hippocampus and hippocampal neurogenesis are stronly implicated in schizophrenia and depression, and in the therapeutic actions of many anti-depressants. Within the hippocampus, granule cells in the dentate gyrus are continuously generated from neural progenitors throughout life in all mammals examined, including humans. They follow stereotypic patterns of neuronal morphogenesis, migration, axon and dendritic targeting and become synaptic integrated into the exiting neuronal circuits within one month after birth. Such stereotypic and prolonged neuronal development of a single CMS neuronal subtype in a relative steady-state of mature CNS environment offers a unique opportunity to investigate molecular and cellular mechanisms of neuronal development in vivo. We have developed a retrovirus-mediated "single-cell genetic" approach for study of the development of newborn granule cells in the brain in vivo using a combinational approach of immunocytochemistry, multiphoton confocal microscopy, electron microscopy and electrophysiology. Using these approaches, we have recently examined the function of Disrupted-in-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, and shown that DISC1 regulates multiple phases of neuronal development of granule cells in vivo. In the current project, we aim to extent these preliminary findings and characterize the functions of DISC1 in conjunction with its interactors, such as NDEL1 and FEZ1, in regulating neurogenesis and neuronal development in vivo. We will also examine whether defects of neurogenesis from DISC1 dysfunction will lead to changes in animal behaviors. Furthermore, we will determine the molecular mechanisms underlying DISC1 in neurogenesis and neuronal development. We will also examine whether defects of neurogenesis from DISC1 dysfunction will lead to changes in animal behaviors. We envision our study will fill the gap of our basic knowledge about the functions of schizophrenia susceptibility genes in neuronal development and may reveal the etiology and pathogenesis of schizophrenia.

[unreadable] DESCRIPTION (provided by applicant): New neurons are generated from neural progenitors throughout life in discrete regions of the adult mammalian central nervous system (CNS), including humans. Adult neurogenesis recapitulates the whole process of neuronal development in a mature CNS environment, including proliferation and fate specification of adult neural progenitors, neuronal differentiation, maturation, migration, guidance and synaptic integration of newborn neurons. The molecular mechanisms that regulate the distinct steps of adult neurogenesis are largely unknown. Accumulating evidence suggests that adult neurogenesis is involved/altered in many physiological and pathological conditions, such as learning and memory, epilepsy, mental disorders and degenerative neurological diseases. Understanding the molecular mechanisms for adult neurogenesis may provide clues into the etiology and pathology of these mental disorders and diseases, and more importantly, may lead to novel therapies. A better understanding of adult neurogenesis may also lead to novel strategies to functionally replace damaged or lost neurons after injury or degenerative neurological disease using neurons derived from stem cells, including both embryonic stem cells and adult neural stem cells. My laboratory has been investigating neurogenesis in the dentate gryrus of adult mice as a model system to understand mechanisms regulating neural development in the adult CNS environment. We have established a "single-cell genetic approach" to specifically label and genetically manipulate newborn cells during adult neurogenesis in vivo. We have characterized the neuronal development of newborn granule cells in the hippocampus of adult mice with immunocytochemistry, electron microscopy, live imaging and electrophysiology. We also made the original discovery that GABA, a major neurotransmitter in the adult brain, depolarizes newborn neurons during their initial development in the adult hiippocampus and, furthermore, such depolaization appears to be essential for the inetgration of new neurons. In the current project, we propose to use the "singe-cell genetic approach" to investigate the functional roles and underlying mechanisms of GABA in regulating distinct steps of adult neurogenesis, including differentiation, maturation, survival, migration, axon and dendritic development, formation of synaptic inputs and outputs, and synaptic plasticity of neuronal progeny in the adult brain in vivo. These studies will provide the groundwork for future goals in understanding the mechanism and function of adult neurogenesis under physiological and pathological conditions and may lead to novel strategies for potential neuronal replacement therapies in the adult CNS. [unreadable] PUBLIC HEALTH RELEVANCE: The project aims at understanding how new neurons derived from neural stem cells become integrated into the existing neuronal ciruitry in the adult brain. Findings from these studies may also lead to novel strategies to functionally replace damaged or lost neurons after injury or degenerative neurological disease either using neurons derived from endogenous stem cells or transplantation of neurons from exogenous stem cells. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Severe psychiatric illnesses, such as schizophrenia and bipolar affective disorder, are chronic and generally disabling brain diseases in need of effective treatments. They affect a large portion of the world's population and have devastating consequence for the sufferers, their families and for the society as a whole. While their etiology is largely unknown, accumulative evidence support the view that schizophrenia is a disease of neuronal development. A number of susceptibility genes have recently been identified from human genetic association studies. One gene named disrupted-in-schizophrenia 1 (DISC1), was identified at the breakpoint of a balanced (1;11)(q42;q14) translocation that co-segregates with schizophrenia and other major affective disorders in a large Scottish family. Recently we have provided evidence to support a critic role of DISC1 in multiple aspects of adult neurogenesis in rodents, including neuronal morphogenesis, migration, axon and dendritic development, and synapse formation of newborn neurons in the adult brain. However, DISC1 gene is not well conserved between humans and rodents and the function of DISC1 in human embryonic neurogenesis and neural development is currently unknown. Recent success in reprogramming human skin fibroblasts into pluripotency (induced-pluripotent stem cells or iPSCs) paves the way to study human development and to discover the molecular basis of human diseases in more relevant experimental systems using human cells. Differentiation of iPSCs derived from schizophrenia patients into neurons and glia may revolutionize our ability to investigate molecular and cellular mechanisms underlying schizophrenia, including both genetic and sporadic origins. We have generated human iPSCs from patients with genetic disorders. We have also developed a battery of methodologies to examine the differentiation, functional maturation and integration of neurons derived from stem cell both in vitro and in vivo, including immunocytochemistry, confocal and electron microscopy, electrophysiology, and transplantation. In the current project, we aim to establish a platform using patient-derived iPSCs and their neural progeny to identify neurodevelopmental defects associated with schizophrenia and to understand functions of schizophrenia susceptibility genes in human neuronal development. During the R21 phase, we will derive and characterize iPSCs from both genetic (with DISC1 mutations) and sporadic schizophrenia patients and control subjects. During the R33 phase, we will examine the neuronal developmental defects of patient-derived iPSCs using established in vitro and in vivo analysis. Our proposed study may provide novel insight into mechanisms underlying these susceptibility genes in affecting human neurodevelopment and a better understanding of the etiology and pathogenesis of schizophrenia and other related major mental illnesses. Our study may lead to generation of new experimental models of human psychiatric diseases to be tested with potential therapeutic molecules. PUBLIC HEALTH RELEVANCE: Recent advances in human stem cell biology offer exciting possibility to investigate functions of disease genes and to model human diseases using relevant human cell types. We will establish a platform using patient-derived iPSCs to understand functions of schizophrenia susceptibility genes in regulating human neurogenesis under normal and disease state, a topic not accessible with traditional animal model systems. Our study will provide fundamental information on functions of susceptibility genes of mental disorders in human neurodevelopment and may reveal the etiology and pathogenesis of these disorders.

DESCRIPTION (provided by applicant): One striking form of plasticity in the adult brain is the ongoing process of neurogenesis in discrete brain regions of almost all mammals examined to date, including humans. In the adult hippocampus, new neurons arising from resident neural stem cells play important roles in many adaptive behaviors, including learning, memory, homeostatic stress responses and mood regulation. Accumulating evidence also suggests that adult neurogenesis is involved or altered in many pathological conditions, such as epilepsy, developmental psychiatric disorders and degenerative neurological diseases. Therefore, understanding the basic mechanisms of adult neurogenesis may provide clues regarding the etiology and pathology of these mental disorders, and potential novel therapies. While the field has made tremendous progress during past decades, there are major gaps in our understanding of adult neurogenesis that must be addressed to harness the endogenous plasticity and regenerative capacity of the adult brain for functional enhancement or repair after injury or diseases. One fundamental question in stem cell biology is whether and how niche signals calibrate the number of functional progeny based on local tissue demands. As adult hippocampal neurogenesis occurs within a dynamic neuronal network, local circuit activity could serve as an effective readout of current tissue demands and provide a signal to fine tune the neurogenic process. Our central hypothesis is that the neurotransmitter GABA is a dynamic niche factor that couples activation of specific neuronal circuitry to regulation of distinct stepsof adult hippocampal neurogenesis. Previous studies have established a critical role for depolarizing GABA signaling in regulating development of post-mitotic newborn neurons during adult neurogenesis. However, whether GABA also regulates neural stem cells and proliferative neural progeny is largely unknown and there is little data on the function of inhibitory GABA signaling during adult neurogenesis. Aided by new technologies for clonal analysis of individual quiescent neural stem cells and optogenetic manipulation of specific interneuron subtypes, we will investigate the niche source of GABA and the role of local interneuron circuitry in regulating three critical steps of adult hippocampal neurogenesis in vivo, including individual stem cell activation, survival of proliferating progeny, and integration of GABAergic and glutamatergic signaling as newborn neurons mature. Our proposed studies will address fundamental questions in adult neural stem cell biology and neurogenesis. Basic principles learned from these studies may be generalizable to the fields of stem cell and developmental biology, neural plasticity and regenerative medicine.

DESCRIPTION (provided by applicant): Bisphenol A (BPA) is one of the most prevalent manufactured chemicals in the world. An organic compound with estrogenic effects, it is used in the production of plastic composites and epoxy resins that are used to make food containers, dental products, thermal receipt paper, and many other items that we encounter on a daily basis. Because of its pervasive presence in our environment and ecosystem, it is critical that we understand the physiological effects of our incidental contact with BPA. The objective in this proposal is to focus on the mature central nervous system and identify the role of BPA on neural stem cells and neuronal development in the adult brain. One of the most striking examples of adult neural plasticity is the de novo formation of neurons throughout life in the dentate gyrus of the hippocampus. Convergent data suggests that this phenomenon plays a critical role in cognition and the regulation of affective behavior. Many of these functional roles appear to be part of a feedback loop in which environmental factors can alter the levels of adult neurogenesis, which in turn may alter our ability to cope with environmental demands. It is therefore paramount that one identifies the nature and mechanisms underlying the interaction between this form of adult neural plasticity and the environment. One such area in need of systematic investigation is the neural impact of frequently encountered environmental chemicals. To begin to address this gap in knowledge, the focus here is on BPA as an entry point for a rigorous analysis of how a near ubiquitous chemical contaminant may affect the activation and fate choice of neural stem cells and the development of their neuronal progeny. To accomplish this objective, novel quantitative approaches developed and refined in this laboratory will be used to analyze how BPA may regulate stem cell behavior and the properties of newborn neurons in the adult hippocampus. Taking advantage of a recently developed clonal analysis approach, both the identifying and quantifying properties of individual stem cells and stem cell populations in the adult brain following systemic exposure to BPA will be tested (Specific Aim 1). Further quantification of the impact of BPA on the developmental trajectory of newborn neurons will be accomplished using retroviral-mediated fluorescent labeling to enable morphological and electrophysiological analyses (Specific Aim 2). The results of these experiments should provide critical new information on the potential health risks of exposure to BPA and its effect on the adult central nervous system. PUBLIC HEALTH RELEVANCE: BPA is one of the most prevalent chemicals in our environment but there are little data on its effect on the adult central nervous system. Our proposed experiments will provide critical new information regarding the effect of BPA on neural stem cells and development in the adult brain and provide a foundation for future regulatory evaluation of this chemical.

DESCRIPTION (provided by applicant): One striking form of plasticity in the adult brain is the ongoing process of neurogenesis in discrete brain regions of almost all mammals examined to date, including humans. In the adult hippocampus, new neurons arising from resident neural stem cells play important roles in many adaptive behaviors, including learning, memory, homeostatic stress responses and mood regulation. Accumulating evidence also suggests that adult neurogenesis is involved or altered in many pathological conditions, such as epilepsy, developmental psychiatric disorders and degenerative neurological diseases. Therefore, understanding the basic mechanisms of adult neurogenesis may provide clues regarding the etiology and pathology of these mental disorders, and potential novel therapies. While the field has made tremendous progress during past decades, there are major gaps in our understanding of adult neurogenesis that need to be fully addressed before we can harness the endogenous plasticity and regenerative capacity of the adult brain for functional enhancement or repair after injury or diseases. One fundamental question in stem cell biology is whether and how niche signals calibrate the number of functional progeny based on local tissue demands. As adult hippocampal neurogenesis occurs within a dynamic neuronal network, local circuit activity could serve as an effective readout of current tissue demands and provide a signal to fine tune the neurogenesis process. Our central hypothesis is that the neurotransmitter GABA is a dynamic niche factor that couples activation of the local circuitry to regulation of distinct stes of adult hippocampal neurogenesis. Previous studies have established a critical role for depolarizing GABA signaling in regulating development of post-mitotic newborn neurons during adult neurogenesis and GABA has recently been shown to affect activation of a specific population of quiescent neural stem cells. However, whether GABA regulates different types of quiescent neural stem cells and proliferative neural progeny is largely unknown and there are little data on the function of inhibitory GABA signaling during adult neurogenesis. Aided by new technologies for clonal analysis of individual quiescent neural stem cells and optogenetic manipulation of specific interneuron subtypes, we will investigate the niche source(s) of GABA and roles of local interneuron circuitry in regulating three critical steps of young adult mouse hippocampal neurogenesis in vivo, including activation and lineage choice of different quiescent neural stem cells, survival of proliferating progeny, and development of glutamatergic synaptic inputs as newborn neurons mature. Our proposed studies will address fundamental questions in adult neural stem cell biology and neurogenesis. Basic principles learned from these studies may impact the fields of stem cell and developmental biology, neural plasticity and regenerative medicine.

? DESCRIPTION (provided by applicant): Adult hippocampal neurogenesis is a dynamic process in which new neurons are continuously generated in the adult brain and integrated into the dentate gyrus, a region that is critical for learning, memory and mood regulation. Dysregulation of this process has been implicated in various psychiatric and neurological disorders, including major depression and epilepsy. Characterizing how these adult- born neurons develop and acquire signaling properties that can affect the local circuitry is important to understand the role of this phenomenon in brain function and pathology. Much of what is known about the electrophysiological properties of these newborn dentate granule cells as they develop has been derived from ex vivo preparations of hippocampal slices. These studies revealed a critical period of plasticity when the cells are around 4 to 6 weeks of age in which they exhibit enhanced synaptic plasticity. This striking observation suggests that there may be a unique, developmentally-regulated role of adult born neurons during a specific time window of maturation. Consequently, a prevalent hypothesis is that newborn granule cells of a particular age exhibit signature patterns of activity in response to environmental stimuli. A direct test of this hypothesis through extracellular single unit recordings has not been possible, however, due to technical limitations that prohibited determining the age of the recorded cell in vivo. To overcome this obstacle, this project is designed to produce a highly specific genetic mouse model (Aim 1) that is amenable to optogenetically-guided tetrode recordings in the dentate gyrus to birthdate, identify and record from single adult-born neurons in freely moving animals (Aim 2). Developing and validating an inducible genetic strategy to target highly proliferative neural progenitors within a narrow time window (i.e. 2 - 4 days) will generate a model in which cohorts of newborn cells can be identified and manipulated with unprecedented precision. This will be a novel resource for the neurogenesis research community to investigate the endogenous function of this population and how its dysregulation may contribute to neural pathology. For the current project, this model will be used to express light-activated opsin channels (channelrhodopsin) in newborn neurons to allow for stimulation and recording of light-responsive putative adult-born granule cells in vivo, via an implanted optical fiber. Completion of these experiments will result in the first description of the firing properties of adult-born granue cells in vivo and the first direct evaluation of whether the critical period of plasticity observedin slice recordings translates to changes in behaviorally relevant neural activity. This innovative approach to address one of the most critical outstanding questions in the field will provide a new genetic model, technique, and dataset to facilitate investigations into the function and dysfunction of adult neurogenesis.

ABSTRACT Development of the central nervous system requires orchestrated interactions among several regulatory elements that determine the fate, properties, and functions of cells at any given time, ultimately leading to complex neural networks that control our most basic behaviors and complex cognitive processes. As an intermediate regulatory domain between DNA sequences and gene expression, epigenetic mechanisms can exert considerable influence on brain development on a scale that we are only beginning to appreciate. One major advance in the field of epigenetics in recent years is the discovery of novel modifications of genomic DNA, such as 5-hydroxymethylcytosine (5hmC), and molecular pathways to install, remove, and interpret these modifications, which are highly enriched in the nervous system and are dynamically regulated by neuronal activity under physiological and pathological conditions. The overarching goal of this P01 is to take a systematic approach to understand how global and specific changes in the epigenome and transcriptome regulate stem cell behavior, neuronal development and neuronal integration using hippocampal neurogenesis as a model system. Hippocampal neurogenesis is a constitutive phenomenon in the adult mammalian brain and is a well- established model for neural development that is comprised of defined stages, which originate with neural stem cell activation and result in the maturation and integration of a single neuronal subtype in an anatomically restricted region of the brain. This phenomenon also represents striking structural plasticity and has been shown to contribute to critical brain functions, whereas its dysregulation has been implicated in various neurological and degenerative disorders. Characterization of neurogenic processes in hippocampus may eventually inform cell transplantation-based therapeutic strategies to repair the central nervous system after stroke, injury or neurological disorders. Integrating results from adult hippocampal neurogenesis in rodents with human induced pluripotent stem cell (iPSC)-based models will allow for the identification of fundamental epigenetic principles governing neural development at the molecular, cellular, and systems levels. Our team includes experts in epigenetics, hippocampal neurogenesis, rodent stem cell biology, human iPSCs, chemical biology, high-throughput sequencing, bioinformatics, electrophysiology and transplantation. Successful completion of the research projects will guide future investigations into the role of dysregulated DNA modifications in neurodevelopmental disorders and facilitate the development of new technological approaches to identify epigenetic marks with high resolution.

Project 3: Role of a psychiatric disease risk factor in synaptic function and gene transcription regulation ABSTRACT Genetic complexity underlying the vast majority of mental disorders has made the study of these diseases exceptionally challenging. Many risk-associated genes have been identified but the biological role is largely unknown. Dysregulated neurodevelopment with altered structural and functional connectivity is believed to underlie many neuropsychiatric disorders and ?a disease of synapses? is the major hypothesis for the biological basis of schizophrenia and other major psychiatric disorders. However, little is known about pathophysiology of synapses in patient neurons, underlying molecular and cellular mechanisms, and to what extent psychiatric disorders may share these mechanisms. Disrupted in Schizophrenia 1 (DISC1) is a gene in which mutations have been associated with increased risk for schizophrenia, bipolar disorder, and other major psychiatric disorders. A large number of animal studies have shown that DISC1 affects multiple neurodevelopmental processes, including synapse formation. Understanding synaptic dysfunction in major psychiatric disease requires direct investigation of synapse properties in human neurons derived from patients with these disorders. Reprogramming patient somatic cells enables recapitulation of normal and pathological human tissue developmental properties in defined conditions and a new way to identify the cellular processes underlying complex human diseases, which can lead to mechanism-based drug discovery. A rare mutation of a 4 base-pair frame-shift deletion at the C-terminus of DISC1 was discovered to co-segregate with major psychiatric disorders in a smaller American family (Pedigree H). The current project is built upon our initial results showing that forebrain neurons derived from iPSCs with the DISC1 mutation exhibit significant synaptic defects and RNA-seq analysis showed significant dysregulation of a large number of neuronal genes related to synaptic function and psychiatric disorders in patient neurons. Recent studies using iPSCs from idiopathic schizophrenia patients also showed similar defects in synaptic function and share multiple dysregulated genes. Project 3 will test the hypothesis that a psychiatric disorder risk gene modulates synaptic function of human neurons via biochemical and transcriptional dysregulation, a core defect that may also be present in idiopathic schizophrenia and bipolar patient-derived neurons. Aim 1 will characterize cellular phenotypes of human cortical neurons differentiated from Pedigree H and idiopathic schizophrenia patient iPSCs. Aim 2 will determine the role of mutant DISC1 in iPSC-derived astrocytes. Aim 3 will evaluate neuronal subtype specificity of mutant DISC1 effects on neuronal development, synaptic function and transcription. Each aim requires the involvement of at least one academic and one industrial partner as well as Core B at Janssen and is designed to incorporate cross-validation across labs and establish protocols that can be disseminated for further validation. Cellular phenotypes will be compared to other iPSC lines in the NCRCRG and will be further developed into miniaturized assays via Core C at SBMRI for future drug screens.

? DESCRIPTION (provided by applicant): The overarching goal of this NCRCRG is to develop a robust, scalable and generalizable platform to investigate the pathophysiology of psychiatric disease using induced pluripotent stem cells (iPSCs). The recent discovery that adult human somatic cells can be reprogrammed to a pluripotent state raises the exciting possibility that human neurons can be generated with the same genetic profile as patients, and disease mechanisms can now be investigated in relevant cell types. Because this new field is rapidly expanding and evolving, this is a critical time to bring together academic and industrial partners committed to standardizing the process of iPSC generation, cell type-specific differentiation, phenotypic assay development, and preparation for eventual high-throughput diagnostic and drug discovery. Formal partnerships and open communication between commercial entities and academic institutions will greatly facilitate this effort by ensuring that every stage of the proces is validated and amenable to industrial process control and standardization. This NCRCRG is composed of two scientific cores, an administrative core, and three highly integrated projects that will be performed across four nonprofit and two industrial sites. Hypothesis-driven projects are focused on two of the most common psychiatric disorders, bipolar disorder (BP) and schizophrenia (SZ), using well- characterized and carefully chosen BP or SZ patient cohorts that were selected for lithium responsiveness and/or the presence of a genetic risk factor. Each project systematically evaluates differentiation protocols and cellular assays in at least two patient cohorts. Projects are designed to test and extend our preliminary results, which show robust and partially overlapping phenotypes in neuronal excitability, mitochondrial function, synaptic function, calcium signaling, and gene expression. Through systematic investigation of convergent and divergent cellular signatures of BP and SZ in multiple cohorts, by multiple investigators, this group will be able to assay reliability and reproducibility of differentiation protocols and extensively validated cellular phenotypes. Scientific cores will work with each project to perform the following: 1) Validation of consistency of differentiation protocols for fou cell types relevant for psychiatric disease; 2) Single-cell RNA-sequencing and transcriptomic, proteomic, and metabolomic analyses for pathway discovery; and 3) Assay miniaturization for high-throughput preparation, phenotype validation, discovery, and transition to screening platforms. Cumulatively, completion of these experiments will further our understanding of the mechanistic similarities and differences between BP and SZ, and establish a model for iPSC-based investigations of psychiatric disorders.

Core B: Integrated cellular profiling and pathway discovery ABSTRACT The advent of cellular reprogramming and the evolution of high-throughput sequencing and omic methodologies have converged to enable unbiased genome-wide profiling of human cells relevant for the study of genetically complex psychiatric disorders. This confluence of technological advances holds tremendous promise for transforming our understanding of the biological bases of these disorders. Realizing this promise, however, requires substantial material resources, expertise that spans multiple disciplines, and well-designed experiments. Our NCRCRG proposal has been organized to meet these demands and exploit state-of-the-art techniques to establish a robust iPSC-based discovery platform. The Integrated Cellular Profiling and Pathway Discovery Core (Core B) at Janssen and JHU is critical to meeting three key goals of this NCRCRG. The first is to develop reliable and robust differentiation protocols for four neural cell types implicated in major psychiatric diseases. Core B will validate the efficacy and reproducibility of differentiation to different cell types across different sites through quantitative transcriptomic analysis. The second is to identify cellular phenotypes of four disease-relevant cell types derived from iPSCs of patients with major psychiatric disorders and explore underlying molecular mechanisms. To meet this objective, Core B will perform single-cell RNA-seq analysis for each project and, further, hypothesis-driven proteomic and metabolomics analysis. The third goal is to integrate all information generated from the NCRCRG for biological pathway discovery, identification of potential biomarkers, and to reveal similarities and differences of bipolar disorder (BP) and schizophrenia (SZ) at the cellular and molecular levels. Core B will develop a bioinformatics pipeline and interact with each Project and core to achieve these goals. Core B will also work with the Administrative Core (Core A) to facilitate data sharing among all members of the NCRCRG in real time, and with Core C to contribute to high- throughput platform development.

PROJECT ABSTRACT ? Core A N/A per PAR-14-183

PROJECT ABSTRACT ? Project 2 N/A per PAR-14-183

? DESCRIPTION (provided by applicant): The mammalian thalamus is uniquely positioned to regulate diverse functions of the cerebral cortex including sensation, motor control, learning and memory and emotion. These functions critically depend on the proper development of dozens of thalamic nuclei, each of which exhibits characteristic patterns of afferent and efferent connections to and from different areas and layers of the cerebral cortex. Understanding how thalamic neurons are generated from neural stem cells and assembled to form specific nuclei during early development is needed to determine the underlying principles of thalamic organization. Despite recent advances in the study of developmental mechanisms responsible for laminar cytoarchitecture such as that of the neocortex, little is known about the formation of nuclear organization that is representative of many brain structures. Clonal lineage analysis has shown that in the neocortex, cohorts of neurons generated from individual neural stem cells form a radial column that spans the entire cortical wall and acts as a functional unit. In contrast we do not know whether thalamic nuclei are each composed of progeny derived from distinct neural stem cell populations, or if there is a spatial or temporal organization that determines the fate of neural stem cell progeny, which is not aligned with the anatomical borders of individual nuclei. The goal of the proposed research is to combine the expertise of two investigators to explore this important question. The Song laboratory has long been a leader in investigating the cellular and molecular mechanisms underlying hippocampal adult neurogenesis and has extensively used genetic lineage tracing at the clonal level to determine the patterns of proliferation and differentiation of adult stem cells. The Nakagawa laboratory characterized the molecular diversity of progenitor cell domains of the embryonic thalamus and has made significant contributions to understanding the mechanisms of patterning and neurogenesis in the developing thalamus. Based on preliminary data using a genetically-based single cell lineage tracing technique that employs the MADM (mosaic analysis of double marker) method, we hypothesize that individual neural stem cells in the thalamus produce postmitotic neurons that are distributed in radial columns that span several thalamic nuclei and that the repertoire of the thalamic nuclei that are generated from individual stem cells is determined by the initial position of the originating cell. Completion of the proposed experiments will be a critical first step towar understanding fundamental mechanisms governing the development and organization of nuclear structures in the brain. This knowledge will have important implications for future investigations of the functional and structural consequences of dysregulation within thalamic progenitor domains in early neural development.

Core A: Administrative Core ABSTRACT Key components of any successful collaborative effort are transparency, communication, and organization. For this NCRCRG, the goal of which is to cross-validate protocols and assays for iPSC-based research across multiple academic and industrial sites, these objectives become even more critical. Core A, the Administrative Core, is designed to facilitate all of these through a comprehensive plan to ensure frequent communication, ease of data sharing, assistance with logistics of material transfer, and recourse to resolve any disputes. There are four major goals of Core A: to facilitate sharing of resources and collaborations among NCRCRG investigators; to maintain a centralized data repository for information transfer among NCRCRG investigators; to coordinate external oversight and timely progression toward milestones; and to disseminate all results and methodology to the public in a timely manner. Core A will provide additional support to the NCRCRG by keeping team members apprised of the latest technological advances in data collection, storage, sharing, and analysis. This will not only enhance our collaborative efforts and data sharing among investigators within the NCRCRG, but will facilitate our eventual deposition of all iPSC lines and associated data into public repositories. Core A will assume responsibility for ensuring that all investigators are adhering to our strict standards for data validation and documentation. Through these activities, Core A will be the foundation for a successful collaborative effort through centralized coordination of the proposed Projects and Cores and the leveraging of existing resources within NCRCRG partner institutions.

Project Summary/Abstract Alzheimer's disease (AD) is a devastating complex neurological degenerative disorder affecting 10% of people over 65 with no cure. The overarching goal of the proposed study is to identify and functionally characterize AD-associated SNPs utilizing novel functional genomic approaches and iPSC-derived cellular models. Our plans include: (1) Determine the functional significance of candidate SNPs in three iPSC-drived 2D AD relevant cell types. (2) Identify genes regulated by distal non-coding SNPs in three iPSC-drived 2D AD relevant cell types. (3) Test the biological consequences of high confidence AD rSNPs from (1) and (2) in isogenic iPSC- derived 2D cell cultures and 3D minibrain organoids. The designed study will be very first comprehensive investigation of AD associated SNPs, thus will shed light on how non-coding genetic variations contribute to AD. Obtaining knowledge for the fundamental genetic mechanisms of AD will expand our horizons to develop improved preventative and diagnostic methods, and also yield targets for novel therapeutic interventions, ultimately leading to a cure for AD.

SUMMARY Adult hippocampal neurogenesis has garnered significant interest over the past two decades as a robust and unique form of plasticity in a region critical for learning and memory. It has also proven to be fertile ground for understanding fundamental principles of stem cell biology, neuronal development, as well as illustrating the capacity of the mature brain to integrate immature neurons, which has important implications for regeneration and transplantation efforts for neural repair following injury or diseases. Despite considerable progress in understanding the molecular and cellular mechanisms underlying adult neurogenesis, there are still critical outstanding questions in the field that have not been addressed due to the technical limitations of traditional experimental approaches. In the proposed series of studies, we will use several cutting-edge techniques that we have developed or adapted to investigate the developmental origin of adult neurogenesis, its functional impact in the adult brain, and the fidelity of rodent models to human neuronal development. First, we will characterize the origin and properties of embryonic neural precursor cells that give rise to the largely quiescent pool of neural stem cells that maintain neurogenesis throughout life in a rodent model. Building on our recent findings that Hopx-expressing neural progenitors in the embryonic dentate gyrus can generate the constitutive populations in the dentate gyrus before adopting a quiescent state indicative of adult neural stem cells, we will identify the molecular mechanisms regulate this precursor population and its transition into quiescence. These studies will provide novel insight into the intrinsic and extrinsic signaling cues that establish a long-term pool of stem cells in the developing and adult brain. Second, we have developed a 3D organoid model of dentate gyrus development using human induced pluripotent stem cells to investigate the properties of neural progenitors, neurogenesis and fate specification. These studies could lead to the potential identification of human-specific markers of neural stem cells and new granule neurons in the dentate gyrus and mechanistic differences and similarities with rodent models, which would inform the current debate over the extent of postnatal neurogenesis in the human dentate gyrus. Third, we will investigate the functional properties of adult neurogenesis in adult behaving mice using an optogenetic strategy to identify and record electrophysiological activity of single newborn granule cells at different stages of maturation. We will also investigate the circuit- level impact of silencing these cells at the population level. These data would provide novel information to evaluate the hypothesis that adult-born granule cells make a unique contribution to information processing in the hippocampus using techniques with high temporal resolution. Together, these studies combine an array of approaches to answer fundamental questions about the origin, impact, and plasticity of neural stem cells and their progeny in the dentate gyrus using both rodent and human models.

PROJECT SUMMARY A fundamental question in biology is to understand how genetic variation affects genome function to influence phenotypes. The majority of genetic variants associated with human diseases are located within non-coding genomic regions and may affect genome functions and phenotypes through modulating the activity of cis- regulatory elements and cell-type specific gene regulatory networks (GRNs). However, our knowledge about the impact of genomic variants (alone or as combinations) on gene expression, GRN activity and ultimately cellular phenotypes are rather limited. Further, because transcription factors (TFs) and related cis-regulatory elements are known to have distinct functions based on cell-type and state, how genomic variants influence cell-type/state-specific activity of functional elements and phenotypes remains to be characterized in much greater details. This proposal aims to leverage a panel of multi-ethnic, gender-balanced human induced pluripotent stem cell (hiPSC) lines (European, African American and African hunter gatherers) as well as recent advances in single- cell time-resolved or multi-omics technologies, predictive modeling of regulatory networks by machine learning and high throughput single-cell perturbation methods to study the functional impact of genomic variations on regulatory network, cellular phenotypes. First, we will establish a robust experimental framework of deploying advanced time-resolved and multi-omic single-cell technologies for detecting functional genetic variants at single-cell level. Next, we will develop novel computational methods for integration of single-cell data across different modalities and for accurate reconstruction and predictive modeling of GRNs driving cellular identify, developmental dynamics (cardiac and neural lineage cell fate transition). Finally, we will apply high-throughput combinatorial genetic or epigenetic perturbation approaches to modulate activity of key genes or putative cis- regulatory elements at single-cell levels to improve our understanding of network level relationships among genomic variants and phenotypes.