Flora Vaccarino - US grants

9514238 Vaccarino The early embryonic stages of brain development in mammmals are regulated by the influence of growth factors which stimulate neuroblasts, the source of nerve cells, to proliferate in a coordinated manner. While the presence of this control is well established, the mode of action of these growth factors is not well undertood. The objective of this proposal is to explore the distribution of the molecular receptors for these growth factors on neuroblasts. Experimental administration of specific growth factors by microinjection will be used to examine effects on the proliferation and death of neuroblasts. Parallel studies will be performed on neuroblasts maintained in cell culture. The results of these experiments will enhance our understanding of how the cerebral cortex develops.

0083104
Vaccarino

Fibroblast Growth Factors (FGF) are thought to increase the generation of neurons in the Central Nervous System. These factors elicit their effect by binding to FGF receptors on the surface of neural progenitor cells. The function of each of the four existing FGF receptors during normal brain development is unclear. This Project investigates whether FGF receptor 1 (FGFR-1) controls the growth of the mammalian brain. To study the function of FGFR-1 in brain development, mice will be generated that lack the FGFR-1 gene only in the brain. These mice are produced by targeting a DNA recombinase called Cre to cells of the developing brain. The Cre recombinase will excise critical segments of the FGFR-1 gene in these cells. The resulting brain-specific FGFR-1 mutants will be studied to elucidate the role of FGFR-1 in cell proliferation and fate.
Cognitive functions strongly correlate with an increase in the relative size and complexity of the brain. Studying the basic mechanisms that construct the brain during early development may shed light on the underlying basis for cognitive functions. In addition, because neural progenitor cells persist in the adult mammalian brain and may continue to use FGF-regulated mechanisms, this research may help understand the role of this receptor in brain cell renewal and repair.

The size of the cerebral cortex increases during mammalian evolution, underlying an increased complexity of cognitive operations and specialization of functions. Little is known of the mechanisms that control the number of neurons generated in this region. Basic Fibroblast growth factor (FGF2) is expressed by cortical progenitor cells and increases their proliferation both in vitro and in vivo. Adult rats microinjected with FGF2 in the cerebral ventricles during embryogenesis have an increased number of cortical neurons, whereas mice lacking the FGF2 gene product have fewer neurons and glia in the cerebral cortex. The mode of action of FGF2 during corticogenesis is not clear. We hypothesize that FGF2 increases the number of rounds of cell divisions leading to an expansion in the population of cortical progenitor cells, and that FGF1, another FGF ligand, exerts a similar function. In the first specific aim, we test whether FGF2 regulates the number of neurons and glia, whether this action is restricted to the cerebral cortex and what stage of development FGF2 is required. This will be accomplished by morphometric and immunocytochemical analyses at different stages of development of FGF2 null (FGF2 -/-) and FGF2 transgenic mice, which, respectively, lack and over-express the FGF2 gene. We will also examine the phenotype of the FGF2 and other genes. In Specific Aim 2, we study the mechanism of action of FGTF2 on progenitor cells in vivo. We will compare FGF2-/- and FGF2 transgenic mice with regard to volume, cell number and apoptosis within histogenic domains of the telencephalon containing neuronal and glial progenitor cells. In another experiment, the fraction of progenitor cells that re-enters the cell cycle and the fraction that exits the cycle will be measured over the neurogenetic interval within the cortical neuroepithelium. In Specific Aim 2, the role of FGF2 in the regulation of cortical connectivity will be studied. Neurite outgrowth will be measured in neurons grown from FGF2-/- and wild type mice in vitro. To assess the proportion of synapses, dendrites and axons in FGF2-/- and wild type mice in vitro. To assess the proportion of synapses, dendrites and axons in FGF2-/- and wild type mice and in vivo, electron microscopic studies will be carried out. In Specific Aim 4, we will determine FGF1, like FGF2, regulates neuron number in the brain and whether it acts redundantly with FGF2. FGF1 null mutant mice will be compared with the FGF2 null mutants and with FGF1 and FGF2 null compound homozygotes and heterozygotes. Morphometric and immunocytochemical analyses will include areas where FGF1 and FGF2 are jointly expressed during development.

DESCRIPTION (provided by applicant): Missing description had to be pieced together by reviewer. This is the second time that no abstract was provided; the application should have been considered incomplete). The long-term objective is to identify mechanisms that are critical for regeneration and repair following hypoxia. Chronic sublethal hypoxia (CSH) in neonates may induce a regenerative response that is attempting to replace cells lost through hypoxia-induced decreased proliferation and increased apoptosis. Several gene products involved in brain growth and cell proliferation during embryogenesis are re-activated under hypoxic conditions, including basic Fgf (Fgf2), Fgf receptor-1 (Fgfgr-1), and Fgf target genes. Because Fgf2 and Fgfr-1 are also necessary for neural stem cell/progenitor cell proliferation in the postnatal subventricular zone (SVZ), this pathway is hypothesized as being critical for an effective post-hypoxic regenerative response. This project will measure proliferation and apoptosis, as well as expression of Fgfr- 1 and other growth factor receptors during and after hypoxia in the SVZ and the hippocampal subgranular layer (HSL). Two mouse strains differing in their sensitivity to hypoxia will be used. Fgf2 knockout mice, as well as mice with a conditional Fgfr-1 deletion will be studied. The experiments are expected to determine whether Fgfr-1 signaling is critical for cell genesis and /or cell survival after hypoxia.

DESCRIPTION (provided by applicant): The proposed investigations focus on the role of Fibroblast Growth Factors (Fgf) receptors on the morphogenesis and function of the cerebral cortex, and particularly frontal regions, whose development is disrupted in schizophrenia. We hypothesize that Fgf receptor 1 (Fgfr-1) play essential roles in inducing or maintaining the specification of neural stem cells to anterior cortical fates and in promoting stem cell expansion within these regions. To test these hypotheses, we will use mice lacking the Fgfr-1 gene product in neuroepithelial cells of the dorsal telencephalon by site-specific recombination (conditional knockout) beginning at either E9.5 or E13.5 stages of development. Aim 1 will test that Fgfr-1 is necessary to either induce or maintain anterior cortical fates, and that the disruption of this gene produces a "posteriorization" of the VZ and cortical mantle, as tested by region specific molecular markers. Aim 2 asks whether Fgfr-1 expands the number of radial glial progenitor cells during cortical development. This will be approached by examining cell proliferation and radial glial markers in the cortical ventricular zone (VZ) in Fgfr-1 recombinant mice and littermate controls, both in vivo and in tissue explants. Aim 3 analyzes the mechanisms of callosal dysgenesis in Fgfr-1 conditional knockout mice. Tract-tracing experiments will assess the trajectories of long-range neocortical projections in Fgfr-1 recombinant animals as compared to controls. We will test whether Fgfr-1 regulates the expression of receptors for guidance cues by callosal pyramidal neurons or the patterning of midline cellular structures that must be traversed by their axons. Aim 4 examines whether aberrant cortical development leads to impairments in learning and memory and the inhibitory control of behavior. This question will be approached by testing Fgfr-1 recombinant mice and littermate controls in a battery of sensorimotor and learning tasks. We predict that recombinant mice will show impairments in working memory and will have difficulty in suppressing previously conditioned responses (reversal learning). We further predict that the degree of these impairments will be predicted by the degree of neuronal hypoactivity (as assessed by c-fos immunostaining) within regions of the frontal cortex.

[unreadable] DESCRIPTION (provided by applicant): Tourette's Syndrome (TS) is a childhood-onset neuropsychiatric illness characterized by motor and vocal tics. The cerebral cortex-basal ganglia circuitry regulates motor habits and goal-directed behavior. A question still unanswered is whether there are cellular abnormalities in the basal ganglia and cerebral cortex of TS. We have conducted preliminary postmortem brain tissue studies involving four cases of TS and four normal control (NC) subjects using unbiased stereological techniques. A higher number of parvalbumin (PV)-containing neurons was found in a major output nucleus of the basal ganglia, the internal segment of the globus pallidus (GPi), in TS brains as compared to NC subjects. These are inhibitory neurons projecting to the thalamus. By contrast, PV neuron number and density were decreased in the Caudate (Cd), Putamen (Pt) and insular cerebral cortex in the same TS subjects. The PV neurons are born in the embryonic medial ganglionic eminence (MGE) and tangentially migrate to the striatum and cerebral cortex. The imbalance in PV neuron distribution between cortex, striatum and GPi suggests altered migration or survival of inhibitory neurons in severe, persistent TS. In this proposal, we will use the four TS brain specimens already in our collection plus additional TS brains collected by two different sources to investigate the number and distribution of PV as well as other types of inhibitory neurons in the basal ganglia and cerebral cortex of TS individuals. In the first specific aim we will ascertain whether the number and relative proportion of PV, calretinin (CR), calbindin (CB) and cholinergic (Ach) neurons is altered in striatum and globus pallidus of TS brains as compared to matched NC. In the second specific aim we will estimate the number and proportion of three main cortical interneuron subtypes, containing PV, CB or CR, in TS as compared to NC brains. We will analyze motor and premotor regions and the insula, areas that are overactive during tics, as compared to primary visual cortex, an area not activated by tic behavior. We hypothesize that the altered distribution of PV neurons and possibly of other interneurons will be correlated with TS diagnosis. If confirmed, this hypothesis may represent a catalyst for further investigations on the developmental mechanisms of TS and a step forward in devising future treatment for this disabling condition. [unreadable] [unreadable] [unreadable]

Astroglial cells proliferate in the subventricular zone (SVZ) in the recovery period after perinatal hypoxic injury. While we know a great deal about the astroglial precursor cells/stem cells in the normal developing brain, and how these cells generate neurons for the olfactory bulb, we are still far from understanding whether and how these astroglial precursors contribute to brain repair after injury. This proposal addresses this clinically relevant issue by asking whether reactive astroglial cells give rise to new cortical neurons and oligodendrocytes in the cerebral cortex after chronic perinatal hypoxia, what types of neurons arise from these cells in the normal and injured cerebral cortex, and whether these neurons are electrophysiologically active and normally integrated in the cortical circuitry. This will be accomplished by genetically marking astroglial cells with reporter genes using a transgenic mouse carrying a drug-inducible Cre recombinase gene expressed under the GFAP promoter. Second, we will investigate whether the astroglial precursor cells that give rise to cortical neurons are located in the SVZ and subsequently migrate to the cerebral cortex or whether they are homed within the cortex. For this, we will activate reporter gene expression in local populations of Gfap+ cells via microinjection of a viral vector, or, in a different approach, we will FACS-purify marked Gfap+ cells from cortex or SVZ and follow their fate after transplantation. Third, we will ascertain the gene expression profile that characterizes Gfap+ cells of the SVZ after hypoxia, as opposed to Gfap+ cells present in the cortical parenchyma. The resulting candidate genes will be overexpressed in mouse pups to assess whether they are sufficient to induce cortical neurogenesis. One of these candidate genes is the Fibroblast Growth Factor Receptor 1 (Fgfr1), which is greatly upregulated after perinatal hypoxia. To test whether Fgfr1 is required for the regeneration of cortical neurons that we have observed in hypoxically reared mice, this gene will be deleted in postnatal Gfap+ cells using our inducible Cre transgenic mice. Our results should suggest novel approaches involving exogenous or endogenous astroglial precursors to improve recovery in pediatric patients suffering from hypoxic encephalopathy. Hypoxia¿low oxygen¿is a relatively frequent cause of brain injury in premature birth or other birth-related complications. Hypoxia often results in devastating consequences, from spasticity to mental disability, for which there is no available therapy. Emerging clinical data suggest that a portion of the prematurely-born children undergo remarkable cognitive improvements over the years. This is mirrored by our observations that mice can regenerate cortical neurons following a perinatal hypoxic insult. We believe that this recovery is due to neural stem cells naturally present in the brain, which react to hypoxia and regenerate the neurons and glia that are lost. Our proposal focuses on the cells that are involved in this regenerative process. By targeting these cells with a genetic marker we will follow their fate and investigate the molecular factors that allow this remarkable cellular replacement to occur. Thus, using our clinically-relevant mouse model, this proposal will permit us to enhance these growth-promoting responses and it will define the crucial cell population and molecular ingredients that are required for appropriate reconstitution of neurons and glial cells in cortex. Importantly, the regenerative processes investigated in this proposal could be mimicked in adult and aged individuals, in which unfortunately cortical neuron loss is not recovered.

Administrative Core of this P01 is composed of two components: 1) Scientific Program 2) Education. The Scientific Program component will be led by Flora M. Vaccarino, M.D., and will include Program, Internal Advisory and External Review Committees. The program Committee will meet regularly and is responsible for reviewing the research progress, ensure that the goals of the Program are met, assess significance of the data, and discuss future directions. Another major function of the Program Committee is to see that clinical and translational implications of the research are identified and separately pursued as appropriate. The Internal Advisory Committee is represented by a group of senior Yale investigators who will advise the Principal Investigator and the Project Directors on the quality of the ongoing research. In addition, it will help the Program Committee in the overall administration of the program by reviewing program progress at least once a year. The External Review Committee is a group of outside consultants that will periodically review the scientific findings of the Program and be available to discuss program issues with Dr. Vaccarino and members of the Program Committee. The educational component concerns the training of medical students, graduate students, postdoctoral associates, residents and clinical fellows in the Child Study Center and Departments of Pediatrics, Neurobiology, Pathology, Obstetrics and Gynecology, Comparative Medicine, Radiology and School of Epidemiology and Public Health. The Core supports monthly seminars of all investigators involved in the projects and Cores, Program Committee members, and occasionally invited internal speakers.

As many as 50% of low-birth-weight infants suffer cognitive deficits due to chronic hypoxia and circulatory complications. Longitudinal studies suggest recovery in both brain volume and cognitive functions by early adulthood in some children, however this recovery is variable and the neurobiological basis of this improvement are not understood. Our mouse model of chronic sublethal hypoxic injury reproduces the initial cortical volume deficit and ventriculomegaly, as well as the subsequent recovery, although some neuron loss and cognitive abnormalities persist long-term in these mice. Identifying the cellular and molecular events that underlie neuronal recovery and finding ways to enhance this process are the common goals of this program project. We hypothesize that recovery from hypoxic brain injury is due to a coupled neurogenic/angiogenic response. This includes (1) proliferation of neural stem cells and progenitors, which requires specific growth factors in neural stem cells and vascular endothelium;(2) survival of newly-born neuronal and glial populations, which requires improved energetic metabolism in the newly-generated cells and trophic influences from neural and vascular compartments. Specific projects in this program will test these hypotheses by generating tissue-specific and time-dependent loss- or gain-of function genetic models to test the function of several proliferative, survival and differentiation factors in neural stem cells, oligodendrocyte progenitors, and vascular endothelium. The factors under investigation include Fibroblast growth factor 2 (FGF2), the Epidermal growth factor receptor (EGFR), TrkB and its ligand Brain derived neurotropic growth factor (BDNF), the mitochondrial uncoupling protein 2 (UCP2) and the gut hormone ghrelin. Elucidating the role of these genes in specific cell types will reveal the reciprocal and dynamic interactions between vascular, neural and metabolic components as the animals recover from injury in standard or enriched environment. A multidisciplinary approach, which includes morphometric and immunocytochemical analyses, electron microscopy and electrophysiology, is used to assess whether the signaling systems under study contribute adaptive changes triggered by the enriched environment in hypoxic animals. Furthermore, the beneficial effect of enhancing specific components of these signaling systems will be tested at the cellular and behavioral level. The long-term goal of these studies is to identify new means of therapeutic intervention to decrease the developmental disability and neurobehavioral sequelae of preterm birth.

Oxygen deprivation is a major cause of neurodevelopmental impairments in preterm infants. Cortical gray matter loss and cognitive disability improve over time in some children, while others remain cognitively impaired. The mechanisms undertying this variable recovery are unknown. Our mouse model of chronic sublethal hypoxia reproduces the initial brain atrophy as well as the subsequent recovery in brain structure. In the recovery period following hypoxia, we observed increased proliferation of neural stem and progenitor cells (NSC/NPCs) as well as generation of new cortical neurons from these precursors. Despite this, longterm deficits in working memory persist in the hypoxia-reared animals and significant decreases in the number of GABA interneurons remain in the cerebral cortex. The goals of Project 1 are to investigate the mechanisms for the differential response of excitatory and inhibitory neurons to hypoxia and enhance anatomical and functional recovery following the insult. In Aim 1, we will study whether the long-term interneuron deficiency after hypoxia is due to a loss of cells or to a deficient maturation of GABAergic inhibitory properties by immunocytochemical and electrophysiological analyses in Gad1[GFP/+] mice. In Aim 2, we will examine whether environmental enrichment promotes the generafion of new excitatory and inhibitory neurons and glial cells from GFAP+ NSC/NPCs or their survival and synaptic integrafion into the circuitry. These studies will use genetic fate mapping in GFAP-CreERT2 mice and, in collaboration with Projects 3 and 4, electrophysiology and electron microscopy. In Aim 3 we will examine whether BDNF signaling is required for the beneficial effects of environmental enrichment on neuronal survival by comparing wild type and TrkB-null cell lineages in mice with inducible delefion of TrkB receptors in GFAP+ cells. In Aim 4 we will test whether exogenously increased FGF signaling, together with environmental enrichment, improves motor and cognitive funcfion by enhancing inhibitory interneuron development after hypoxic insult, including the maturation of their synaptic connecfions probed by ultrastructural studies. Identifying the crucial cell populations and molecular ingredients that are required for appropriate reconstitufion of neurons and glial cells in cortex will permit us to enhance these growth-promoting responses in critically ill children.

DESCRIPTION (provided by applicant): The brain is thought to have decreased neuronal turnover with aging, leading to a progressive decline in neuron number, as well as to increased susceptibility to disease. The hippocampal dentate gyrus and the olfactory bulb continue to generate new neurons postnatally, but this process becomes less prominent during adulthood and especially during aging. Other regions, like the cerebral cortex, can generate new neurons in infant and juvenile animals, but not in adulthood. However, the changes in neuronal turnover rates with age have never been assessed. In this project we will use the GFAP- CreERT2 (GCE) transgenic mice, in which GFAP+ cells express a constitutively inactive Cre recombinase that can be transiently activated by a tamoxifen injection in vivo, allowing to genetically "mark" a time-specific cohort of astroglial cells with EGFP to follow their fate over subsequent epochs of development. We will tag GFAP+ cells with permanent EGFP expression in juvenile, adult and aged mice, and test the specificity and efficiency of reporter gene expression in astroglial cells at these different ages. We will then use this genetic fate mapping system to quantitatively assess neural stem cell fates and neuronal turnover in juvenile and adult mice, with the aim of eventually extending these studies to the aged brain. The total number of cells arising from GFAP precursors, visualized via EGFP reporter gene expression, will be estimated by stereological unbiased counting techniques and characterized by immunocytochemical markers and electron microscopy (EM). Two lines of studies will be pursued;(1) a quantitative assessment of the number and proportions of neurons, oligodendrocytes and astrocytes arising from GFAP+ cells in juvenile as compared to adult brains, and (2) an estimation of the rate of neuronal replacement in three key brain regions, comparing juvenile versus adult brains. In Aim 1, we will characterize the fate attained by GFAP+ cells in the cerebral cortex, hippocampus and olfactory bulb of adult animals reared in standard or enriched environment. The hypothesis is that the ability of GFAP+ cells to give rise to neurons and oligodendrocytes decreases as a function of aging, and that this effect will be less marked in mice reared under environmental stimulation. In Aim 2, we will compare the rate of neuronal turnover in the cerebral cortex, hippocampal dentate gyrus and olfactory bulb of juvenile and adult animals. At different times after tagging (up to 6-7 months), the total number of new and pre-existing neurons and the proportion of new versus total NeuN+ neurons will be ascertained. The experiment will be done in standard and enriched rearing conditions to assess the ability of the brain to modify turnover rates as a result of increased sensorimotor stimulation and cognitive activity. PUBLIC HEALTH RELEVANCE: Decreased production of neurons and increased cell death are common in the aged central nervous system and contribute to increased susceptibility to brain injury and degenerative disorders. The goal of this project is to develop rigorous methods for assessing neuronal turnover and how this turnover is affected by environmental enrichment, with the intent of applying these methods to study the aging CNS. Understanding how neuronal turnover differs in the immature and aged brain tissue will provide new ideas to improve plasticity in the aged brain.

DESCRIPTION (provided by applicant): Our project capitalizes on recent procedures that allow derivation of pluripotent stem cells from fibroblasts obtainable through a small skin biopsy from living individuals. These induced pluripotent stem cells (iPSC) can differentiate into any cell types of the body, including neural stem cells (NSCs). We will use these methods to investigate neuronal differentiation in autism spectrum disorders (ASD). Our hypothesis states that increased brain size, a highly replicated biological phenotype in ASD, is attributable to altered dynamics of cell proliferation and/or differentiation intrinsic to NSCs, which, in turn, will correlate with specific changes in gene expression and the underlying changes in genomic sequence and/or epigenomic imprinting. To test this hypothesis, we have assembled a group of investigators with the range of expertise necessary for a multi-level and multi-dimensional approach to this problem. In Specific Aim 1, we will derive iPSC lines from patients with ASD exhibiting an increase in head size and from typically developing children. These iPSC lines will be differentiated into NSCs. The NSC lines from ASD individuals will be compared to those derived from typically developing individuals with respect to their proliferation, cell death and differentiation into different neuronal subtypes, as well as synaptic specification. In Specific Aim 2, we will use advanced genomics and epigenomics technologies to generate high-resolution and comprehensive datasets of variation in the genomic sequence, epigenetic marks, and transcript abundance at progenitor and mature stages of neuronal cell differentiation. We will integrate multi-level genomics and gene expression datasets with findings from cell biology and clinical phenotypes. In Specific Aim 3, we will transplant NSCs from control and patients into the ventricles of mouse embryos in order to determine their in vivo phenotype and their ability to contribute neurons to various brain regions. The potential impact of our research is to develop cell lines derived directly from patients that will recapitulate in vitro the biological steps that enable an embryonic stem cell to differentiate into multiple CNS cell types. This project will lay the foundations for beginning to correlate genomic sequence, regulation and intensity of gene expression, cellular (biological) consequences, and patient behavior, and thus understand the biological mechanisms of disease. Candidate genes and regions found in iPSC lines can be subsequently validated with statistical significance in targeted large-scale screens. The direct analysis of specific differences in gene expression and regulation that pertain to individual patients and their clinical phenotype may offer unique insights into disease pathogenesis. PUBLIC HEALTH RELEVANCE: This project will develop lines of pluripotent cells (iPSC) from individuals with autism spectrum disorders and typically developing children using cells obtained through a skin biopsy. These iPSC will be differentiated into neuronal cells, allowing us to investigate for the first time differences in neural cells proliferation, differentiation and survival in patients and controls, and to correlate such differences with underlying changes in gene expression and in the genomic sequence. The analysis of gene expression and regulation in neural cells that pertain to individual patients and their clinical phenotype may offer unique insights into disease pathogenesis.

DESCRIPTION (provided by applicant): A consistently replicated biological phenotype in autism spectrum disorders (ASD) is a larger head circumference (HC) in the first years of life. We hypothesize that increased brain size in ASD is attributable to altered dynamics of cell proliferation and/or differentiation due to genetic changes intrinsic to neural cells. In this application, we will derive induced pluripotent stem cells (iPSC) from skin fibroblasts in individuals with ASD and typically developing children with macrocephaly. Whole genome studies examining structural genetic variation in DNA isolated from iPSC as compared to lymphocytes of the same individuals will ensure genetic stability of the reprogrammed cells. In Specific Aim 1, iPSC lines obtained from 23 participants with ASD and 11 typically developing individuals with macrocephaly will be characterized with respect to cell proliferation, cell survival and genome wide structural variation such as copy number variations (CNVs) by paired end mapping (PEM) and array capture and sequencing. In Specific Aim 2, genome wide CNV as well as sequence variation datasets will be obtained in blood lymphocyte DNA taken from the same 23 participants with ASD and 11 typically developing individuals, plus a limited number of their family members. This will involve (1) PEM (2) array capture for exons and promoter regions with sequencing, and (3) genome-wide mapping of retroelement patterns. Genetic regions potentially important for ASD that will emerge from this study will be validated by targeted resequencing in two larger, independent cohorts of ASD probands and their family members, each comprising about 500 individuals. The immediate goal of our project is to create a new resource and analytical tool. The genetic studies comparing DNA sequence variation in iPSC and blood samples are essential to establish that the iPSC genomic structure corresponds to that identified in the patients. In future studies, iPSC lines generated in this project will be specifically differentiated along the neural lineage and further analyzed with respect their proliferation, differentiation and survival, allowing us to test whether increased brain size in ASD is attributable to altered dynamics of cell proliferation and/or differentiation. These neural cells derived from iPSC lines will be characterized at the transcript and epigenetic levels, for which the basic characterization proposed in this project will provide a necessary platform. Our ultimate goal is to link neurobiological phenotypes and changes in gene expression during the neural differentiation process, with the underlying genetic structure of the individuals to elucidate disease pathogenesis. Therefore, the proposed project will provide a resource for correlating, in future studies, genomic sequence, regulation and intensity of gene expression, cellular (biological) consequences, and patient behavior. PUBLIC HEALTH RELEVANCE: This project will develop lines of pluripotent cells (iPSC) from individuals with autism spectrum disorders (ASD) with macrocephaly and typically developing children, using cells obtained by a skin biopsy. We will produce several iPSC lines per individual and characterize them with respect to their biology and their structural genetic variation. The aim is create a resource and analytical tool, which will allow us to examine neuronal differentiation in autism spectrum disorders.

DESCRIPTION (provided by applicant): Drawing data from a variety of cell lines, the ENCODE project found that more than 60% of the human genome is transcribed, and that the majority of these messages are not translated into proteins and are likely to have regulatory functions. Over two thirds of protein non-coding RNAs are novel and specific to a particular cell type and developmental stage. In this project we will (1) provide a genome-wide catalogue of all known and novel transcripts and their regulatory elements in progenitors and early neurons from the embryonic human frontal cerebral cortex~ (2) understand whether their expression is recapitulated in vitro during neuronal differentiation from induced pluripotent stem cells (iPSC)~ (3) establish the brain specificity of novel and known transcripts nd their regulatory elements by comparing our dataset with those of the ENCODE project, and (4) identify and catalogue those transcripts and their regulatory elements that are in loci previously implicated in schizophrenia and autism. Gene expression of the embryonic brain is different from that of the postnatal brain. The systematic discovery and analysis of all active genomic elements that we propose here for the mid-gestational embryonic cerebral cortex has not yet been performed, neither is planned under the ENCODE tier 3 projects. The selected histone marks and transcription factors will identify a large fraction of enhancers/promoters active in any specific cell type. Only some were previously ascertained in the developing brain. Abnormalities in very early aspects of brain development, and specifically the developing cortex, are likely to underlie the pathogenesis of common neuropsychiatric disorders like schizophrenia and autism. Human genomic variants that have been linked to these disorders often lay in poorly annotated regions of the genome. These variants could play a direct role in disease pathogenesis by modifying the coding regions of novel, non- annotated transcripts and/or modifying transcription factor binding to their promoters/enhancers. Hence, our first priority is to discover, as well as provide a catalogue of such elements. Their functinal role in development must then be established. The iPSC model system offers an opportunity to begin answering the question of whether these novel transcripts may have an important biological effect. However, the validity of iPSCs as a true representational model of neurodevelopment needs to be established by performing a direct comparison of their transcripts and epigenetic regulators with those that are active in neural cells in vio at comparable stages of neuronal development, ideally in the same genetic background. Hence, in this project we will provide the first rigorous validation of the iPSC model by comparing all transcripts and chromatin marks of progenitors and neurons that are derived from iPSC with those that are present in the brain at comparable stages of development. This will allow the future use of iPSCs to elucidate the function of non-coding elements of the genome and their potential relevance to psychiatric disorders.

DESCRIPTION (provided by applicant): Emerging evidence suggest that not all cells of the human body have identical DNA sequence, a phenomenon called somatic mosaicism. Dividing cells can accumulate single nucleotide variations (SNVs) as well as larger structural variants (SVs), such as copy number variations (CNVs). Our recent studies suggest that somatic mosaicism normally occurs in at least 30% of human skin fibroblasts. The human cerebral cortex displays a very high degree of mitotic expansion during ontogenesis and may be particularly susceptible to accumulating somatic variation during development. Somatic mosaicism could be an adaptive or maladaptive phenomenon, accounting for inter-individual human genetic variability and shaping individual susceptibility and resilience to neuropsychiatric disorders. Yet, the extent of somatic mosaicism in the normal human brain is unknown. In this proposal we will investigate the degree of somatic variation in the developing human brain, using postmortem fetal human tissue. The ideal way to study somatic mosaicism would be to sequence the genome of single cells, however, the extreme degree of amplification that is required creates inevitable artifacts. Our principal appoach will be to sequence the genome of clonal cell populations derived from single brain cells, identify genomic variants manifested in each clone, and verify the presence and frequency of these variants in the original brain tissue to verify that it is, indeed, mosaic Using this comprehensive dataset, we will then evaluate and refine variant calls obtained by whole genome amplification of single brain cells. In Aim 1, we will construct a map of somatic variations in human brain progenitor cells and estimate their frequency in the developing cerebral cortex and basal ganglia. We will compare the genomes of clonal cell populations and single cells extracted from brain tissue, followed by high resolution analyses to verify their presence and allele frequency in the original brain tissue as well as in th blood. In Aim 2, we will determine the impact of somatic mosaicism on gene expression by assessing whether clone-manifested genomic variants have consequences at the level of gene transcription and/or have effects on biological functions that may confer adaptve advantage to the cells. In Aim 3, we will investigate the most likely biological origin of somatic variants by analyzing sequence features at variation sites, correlating variants with recombination hotspots, CpG islands and histone marks. Together, these specific aims will provide the first comprehensive estimate of the number and allelic frequency of genomic variation in somatic cells of the brain and will yield hypotheses about mechanisms responsible for their creation as well as their significance for brain development.

? DESCRIPTION (provided by applicant): Tourette Syndrome (TS) is a disorder of the developing telencephalon for which no significant causative genetic variant has yet emerged through the examination of blood samples. In this proposal we investigate whether somatic mutations might underlie in part the pathogenesis of TS. Existing evidence suggests that cells accumulate somatic mutations after the formation of the zygote, implying that cells of the human body do not have identical DNA sequence. Besides single nucleotide variation (SNV) and small insertion/deletions (InDels), cells can accumulate copy number variations (CNVs, i.e., duplications and deletions), insertions of transposable elements, inversions and translocations, all involving from few hundred to several millions of nucleotides. Somatic mosaicism arising in brain cells could explain the failure to discover consistent, replicable genetic risk factors in neuropsychiatric disorders like TS, and underlie at least in part the frequently observed variability between blood genotype and overall phenotype. There is no estimate of somatic mosaicism in either normal development or in disease. To test the hypothesis that somatic mutations might underlie the emergence of TS, in this proposal we will discover and quantify somatic genome variation in TS and normal control brains, followed by exploration of potential functional consequences of this variation. In Aim 1, we will perform using advanced sequencing techniques comprehensive discovery of lineage-specific and region-specific somatic genomic variations: SNVs, InDels, CNVs, retrotransposon insertions, inversion and translocations. The analysis will involve 20 TS brains and matched 20 normal control brains. Mosaic variants will be discovered and validated in prefrontal cortex (PFC), premotor cortex (PMC) and striatum (STR), three regions strongly implicated in TS, as well as in specific cell lineages isolated from these regions, including pyramidal neurons, medium spiny neurons, interneurons and microglial cells. In Aim 2, we will select 10 genomic variants, engineer them into iPSCs and in transgenic mice using CRISPR technologies, and characterize their impact on the molecular, tissue and behavior level. Together, these specific aims will provide the first estimate of somatic genomic variation (number, type, frequency) in the brain of TS and will yield hypotheses about their significance for brain development.

? DESCRIPTION (provided by applicant): Autism spectrum disorders (ASDs) affect 1%-2.5% of children worldwide. We suggest that etiological and genetic heterogeneity might converge in a few neurobiological downstream pathways. We have been investigating the pathobiology of ASD with large brain volume (macrocephaly), a phenotype which confers poorer prognosis. Ongoing studies have shown that telencephalic organoids differentiated in vitro from induced pluripotent stem cells (iPSC) derived from patients with ASD and macrocephaly have increased cell proliferation, increased synaptic growth and overproduction of GABAergic inhibitory neurons, indicating an early imbalance in glutamate/GABA neuron ratio. RNA interference experiments suggested that the overproduction of GABAergic cells is attributable, at least in part, to an increase in expression of FOXG1, a master regulatory transcription factor crucial for telencephalic development. Major goals of this application are (1) to expand our analysis of the developmental pathways that are dysregulated in ASD to a larger number of families and (2) to understand to what extent developmental alterations we identified in ASD with macrocephaly also apply to ASD in general. To this end, we will obtain data on neurobiological measures, transcriptome and chromatin active regions in organoids derived from ASD patients with enlarged brain size and ASD patients with normal brain size. The altered gene regulatory network will be inferred and the two networks will be compared to understand similarities and differences in the two subgroups of ASD. To begin to understand the upstream causes of these developmental alterations, we will then investigate whether patients with ASD carry an increased burden of rare genomic variations in regions of the genome that participate in this regulatory network. Finally we will perform overexpression and RNAi knockdown experiments to examine the specific role of our current best candidate transcription factor, FOXG1, in the constellation of neurobiological and transcriptome alterations found in ASD-derived progenitors. We will assess the impact of perturbing FOXG1 gene expression on neurobiological functions (cell proliferation, glutamate/GABA neuron fate, synaptic growth), transcriptome and activity of transcription regulatory regions by RNA-seq and ChIP-seq, respectively, to gain insights into the role of a FOXG1-driven transcriptional program in the aberrant neuronal differentiation of ASD-derived neural progenitors. In summary, in this application we delineate strategies for (1) identifying gene networks and biological pathways that characterize altered development in two subgroups of ASD; (2) testing the causal role of one crucial node in such networks, the transcription factor FOXG1, which is over- active in ASD with macrocephaly; and (3) identifying regulatory factors, both genetic and epigenetic, upstream from neurobiological and gene expression abnormalities. The impact of these experiments will be the definition of a number of biological functions and molecular markers that are implicated in the neurobiology of ASD.

Autism spectrum disorder (ASD) is a disorder of prenatal brain development. While syndromic forms of ASD have received considerable attention, to what extent findings in these heterogeneous disorders apply also to the broader or idiopathic form of ASD with no identified single genetic risk is unclear. In this project, we will study how the normal trajectory of prenatal neurobiological development of the brain is disrupted in idiopathic ASD. To identify neurobiological factors that are associated with risk or protection from ASD during prenatal development, we will recruit participants from a well-characterized cohort of younger siblings of children with ASD, who were followed longitudinally. The siblings will be either concordant for ASD diagnosis (ASD:ASD; n=12 pairs) or will be discordant (ASD:TYP; n=12 pairs). We will use induced pluripotent stem cells (iPSC) derived cortical organoids, 3D cellular structures which model in vitro the fetal development of the human cerebral cortex. Organoids will be analyzed by high resolution imaging approaches, molecular tools and transcriptomics. In Aim 1 we will obtain sets of biological measures (excitatory/inhibitory neuron fate, density of synapse, and neuronal arborization), comparing and contrasting phenotypes between ASD:ASD concordant sibs ASD:TYP discordant sibs. This comparison will refine our ability to isolate risk/protective factors that will be exclusively at work in the discordant pairs. In Aim 2 we will perform global gene expression analysis by RNA-seq and network analyses, aiming at finding differences in gene expression and network organization between the ASD:ASD concordant network and the ASD:TYP discordant network. We will perform correlation analyses where neurobiological measures and gene expression will be correlated with each other and with clinical severity scores. The correlations between neurobiological and gene expression measures with symptoms severity may help discriminate between risk and protection. In Aim 3, in collaboration with Project 2, we will obtain structural MRI and BOLD-based functional connectivity data on the concordant (ASD:ASD) and discordant (ASD:TYP) sib pairs that participate in Aim 1 and Aim 2 studies. We will then make correlations between imaging and neurobiological and gene expression measures. We hypothesize that increased inhibitory neuron fate in ASD may be correlated with less efficient cortical network connectivity and that increased synaptogenesis and neuronal arborization may be correlated with higher gray matter ratio, and also to altered connectivity. This project will feed data to the statistical core, where imaging and neurobiological measures can be used to predict clinical severity, allowing a more powerful analysis of risk factors for ASD. In summary, our in vitro ASD risk human cellular model will allow, in principle, to develop future biomarkers for early diagnosis and the exploration of new treatment options based on the underlying biology.  

Autism spectrum disorder (ASD) is a developmental disorder that emerges in the prenatal period, likely during the first weeks of brain development. Chromatin regulatory events in early brain development have been repeatedly implicated in ASD. Chromatin regulation in prenatal development differs in fundamental ways from chromatin regulation in adulthood, which has been an obstacle to understand ASD pathogenesis. Here, we will use telencephalic organoids derived from human iPSCs to assess the functional activity of regulatory elements we identified through the PsychENCODE project to begin to unravel chromatin and gene regulation during early stages of cortical development, including stages that are not commonly accessible using postmortem brain tissue. We will longitudinally map the activity of these elements at critical developmental transitions in both normal organoids and ASD organoids, fractioned in different cell types (progenitors and neurons), examine their functional disruption in ASD by assessing their enrichment in disease-associated variants and determine their target genes from chromatin conformation capture experiments. In Aim 1, we will use STARR- seq to map the activity of H3K27ac histone-associated putative enhancers in organoids mimicking early cortical development and will compare the STARR-seq enhancers with histone-based enhancers active in stem cells, prenatal and adult postmortem brain identified through PsychENCODE and Epigenome Road map projects. In Aim 2, we will use ATAC-seq and STARR-seq to identify and compare enhancer activity in organoids from ASD patients and controls across early development and in different cell types. For this, we will use a collection of iPSC lines we generated from families with ASD. In Aim 3, we will use capture Hi-C and RNA-seq to study the 3D chromatin organization and promoter-enhancer interactions and their effect on gene expression in ASD neural cells. We will then explore whether ASD-implicated enhancers harbor disease- associated mutations by intersection with Simons and MSSNG whole genome public databases sequence variants. Finally, in Aim 4, we will carry out detailed functional analyses on ASD-associated mutations found in the implicated enhancers. We will engineer mutations in control iPSC lines, compare pairs of isogeneic organoids with or without the mutations, and perform capture Hi-C to identify their target genes and RNA-seq to confirm their effect on gene expression. These studies will chart gene regulation in human prenatal forebrain, across stages and cell types, map enhancers that are differentially active in early neural development in autism and identify mutations that are putatively responsible for these alterations. The end results will be the identification of a network of interacting genes involved in the pathophysiology of ASD, and the genetic/epigenetic mechanism responsible for their altered function in the disorder.

Tourette syndrome (TS) is a common disorder that afflicts as many as 1 in 150 children. Despite the high familiar recurrence rate, no significant causative or predisposing factor has yet emerged in TS. Neuroimaging and anatomical studies have implicated the striatum within the basal ganglia in TS. In postmortem brain tissue of patients with severe TS we found a decrease in striatal cholinergic neurons (CH/TAN) and two types of GABA interneurons, the parvalbumin+ (PV) and Somatostatin/Nitric Oxide Synthase /Neuropeptide Y+ (SST/NOS/NPY) by immunocytochemistry. Transcriptome profiling by RNA sequencing highlighted a decrease in synaptic neurotransmission and metabolism-related biofunctions in TS, as well as a prominent increase in inflammatory transcripts, as compared to matched normal controls (NC) brains. However, these signatures are an average of a complex cellular mixture and most likely miss changes occurring in cell subpopulations, particularly interneurons. We now seek to identify the transcriptome of striatal medium spiny neuron (MSN), interneuron (INT), astrocytes & microglia (AST/MICR) and oligodendrocytes (OLIG) cell subpopulations by fluorescence-activated nuclear sorting (FAN) as well as single neuronal nuclei in TS and NC postmortem brain tissue. Correspondingly, the epigenome of these cell types will be characterized by chromatin immunoprecipitation and sequencing (ChIP-seq) in the same cellular fractions. Differentially active enhancer regions will be mapped in the striatum of TS vs NC and a gene regulatory network encompassing changes in gene expression and corresponding enhancer activities will be built. Network modules differentially active in TS will be used to construct a model of dysfunctional striatal circuitry in TS. To understand the origin and potential causes of this network dysfunction, we will recapitulate early telencephalic development in vitro using a human induced pluripotent stem cell (iPSC) model of the disorder. Basal ganglia and cortical organoids from chronic TS patients, recovered TS patients and NC will be longitudinally analyzed and compared on the cellular, transcriptomic, epigenomic and electrophysiological levels to reveal cell fate, neuronal differentiation, molecular and functional abnormalities that underlie the disorder and its outcome. These complementary experiments will define the likely time of origin, pathophysiology, and molecular underpinnings of TS and provide a disease model where genetic and epigenetic changes can be perturbed to assess their neurobiological effects.

The human induced pluripotent stem cell (hiPSC) technology promises major advances in disease modeling and personalized medicine. Using hiPSCs, organoid systems have been generated in recent years that resemble the identity of several brain regions, including cortex, basal ganglia, cerebellum and spinal cord. Major shortfalls of these models are the lack of a reproducible topography of the cell types and tissue architecture that are generated, and the failure to recapitulate the full range of cellular and molecular diversity that characterizes in vivo systems. Thus, our goal is to generate a more accurate and reproducible model for formation of human brain regions and their interactions in vitro. During early development, gradients of diffusible morphogens program cellular identity along the major dimensions of the vertebrate body, the antero- posterior (A-P) and dorso-ventral (D-V) axes, conveying positional information by inducing specific genetic programs. Recreating these morphogen gradients in vitro promises to increase the diversity of the organoid's cellular repertoire and its reproducibility. We focus on two signaling cues, WNT and Sonic Hedgehog (SHH), which, respectively, caudalize and ventralize the early neural tube in mammals. Naïve neural organoids tend to generate dorsal forebrain if not exposed to any patterning signals, and indeed cerebral cortical (CTX) fate is the default identity for the nervous tissue. In Aim1, we will use specially designed mesofluidic chambers to create stable concentration gradients of the posteriorizing morphogen WNT to generate organoid identities along the A-P axis (cortex-diencephalon-mesencephalon-brainstem) from 10 biologically different hiPSC lines. In parallel, we will test that hiPSC exposed to a concentration gradient of SHH will generate organoids identities along the D-V axis (hypothalamus- caudal- lateral-medial ganglionic eminences-cortex). Regional and cellular fates will be assessed by immunocytochemistry (ICC), single cell RNASeq and DBiT-seq, a novel spatial in situ transcriptomics approach. We will then test whether morphogen-induced initial specification achieved through the methodology proposed here will result in accurate and reproducible connections by developing multi-organoid aggregates (i.e., assembloids). In Aim 2, we will assemble region-specific organoids to form components of the cortico-basal ganglia-thalamo-cortical circuit. By labeling neurons with specific reporters, we will examine their projections to the adjacent regions and will test the functional activity and synaptic development of those projections using optogenetics. Generation of a series of differentially induced regions in close spatial proximity is important to allow subsequent migration and appropriate wiring of the CNS. Our approach promises to deliver a new system for modeling neuronal fate and circuitry development in humans and testing its functionality on the cellular, molecular and genomic level.