Harley I. Kornblum - US grants

The aim of the current proposal is to determine if transforming growth factor alpha (TGFalpha) plays an important role in the development of the neostriatum and the formation of its connections with the ventral midbrain and neocortex. The neostriatum is a clinically important region as its neurons and connections are selectively vulnerable in neurodegenerative diseases. TGFalpha and EGF, which at the EGF receptor (EGF-R) have been shown to exert trophic activities on striatal, cortical, and substantia nigra neurons in vitro, and also to induce the proliferation of striatal neural stem cells. TGFalpha mRNA has been localized in the postnatal rat neostriatum. EGF-R mRNA has been localized in postnatal rat neostriatum, cortex and substantia nigra. If these molecules play a role in early neuronal proliferation, migration and differentiation of the neostriatum and connecting areas, then one would expect them to be expressed in these brain regions at appropriate stages of development. The ontogeny of EGF-R mRNA and protein and that of TGIalpha mRNA, in the embryonic and postnatal rat striatum, midbrain and cortex will be examined in Specific Aim 1. The cell types that produce TGFalpha and athe EGF-R in these brain areas will be determined by double-labelling in situ hybridization and immunocytochemistry in Specific Aim 2 in order to make specific predictions as to what the functional role of this ligand-receptor pair is. In Specific Aim 3 and 4 the hypotheses that TGFalpha is required for the normal production and differentiation of cells in the striatum, midbrain and neocortex and the formation of connections between these structures will be tested in mice that do not synthesize TGFalpha. Specific Aim 3 will test whether cell types that normally express the EGF-R are present in their usual distribution in these brain areas of TGFalpha-deficient mice. Specific Aim 4 will assess whether athe neostriatum possesses normal anatomical connections in these mice. Finally, in Specific Aim 5, in vitro experiments will be performed to determine if TGFalpha exerts trophic effects on the neuronal phenotypes that normally express HGF-R, in vivo. The goal of the proposed research is to provide information as to the trophic mechanisms by which the neostriatum develops and secondarily, to provide insight as to potential mechanisms of selective neuronal vulnerability in degenerative processes.

An integrated microarraying facility will be initiated in the Department of Molecular and Medical Pharmacology (DMMP) in the Center for Health Sciences (CHS) at UCLA. The facility will consist of a DNA processing robot, a microarrayer, a scanner, a DNA/clone resource, and a computational facility. The microarray facility has been designed to provide the highest practicable throughput of this powerful technology.

The research will cover the whole range of modern biology including studies of retroviruses, differentiation of neural stem cells and other cells, the cellular response to metals, synaptic transmission, neural growth factors, nitrous oxide physiology, immune system differentiation, neural aspects of behavior and glucocorticoid signaling. An important part is to improve the bioinformatics of microarray technologies using the data provided by the facility.

This facility will provide researchers and their students the ability to visualize gene expression patterns for thousands of genes at once and have a major impact on research in cell and molecular biology at UCLA.

DESCRIPTION (Applicant's abstract): Understanding the intricacies of CNS cell lineage is a major goal of current work in developmental neuroscience. The fundamental mechanisms underlying the processes whereby CNS progenitors are generated and the factors guiding them from multipotent undifferentiated cells to unipotent, terminally differentiated neurons and glia have not been elucidated. Central to the current proposal is that an understanding of the gene expression patterns of CNS progenitor cells and their progeny at different stages is a critical step in forwarding our understanding of these processes. In our preliminary experiments we have used representational difference analysis coupled to microarray screening to identify novel markers of early differentiative events in the embryonic brain, and developed relatively high throughput methods for cataloging expression patterns of these genes. The experiments set forth here will: 1. Create a large cDNA microarray enriched for genes expressed by CNS progenitor cells as well as committed cells at early stages of differentiation. 2. Determine, in a rapid manner, the developmental expression pattern of those mRNAs found to be the most interesting. 3. Test the ability of this newly created resource (the microarray) to determine the differences in gene expression amongst different populations of CNS and other progenitor cells. We anticipate that these studies will not only directly enhance the understanding of CNS progenitor cell biology, but that they will also pave the way for other experiments using similar technology by showing the feasibility of the methodology and provide a microarray resource of early progenitor enriched genes for others to use. The following Specific Aims will be achieved: 1. A 6000 gene microarray will be constructed using cDNAs derived from an RDA subtraction designed to enrich for genes expressed by CNS progenitor and precursor cells at various stages of commitment. 2. Differentially expressed genes will be prioritized for further study based on a stepwise screen designed to confirm microarray expression data and efficiently characterize genes of interest. This screen includes high throughput sequencing, Northern blot, and determination of developmental expression patterns by in situ hybridization. 3. The "neurodevelopmental" microarray will be used to compare gene expression among progenitor populations derived from different brain regions, time points and different growth factor conditions. We hypothesize that differences in gene expression patterns will identify similarities and differences between these conditions that will have important functional consequences. The proposed research will significantly improve our understanding of CNS gene expression in two ways. First, we estimate that we will determine the expression pattern of nearly 500 novel or previously uncharacterized genes in the developing brain by screening in situ hybridization. Second, our newly created microarray will allow us to test gene expression patterns in a variety of experimental conditions including various cultures containing CNS progenitors, as well as in models of CNS disease or injury.

DESCRIPTION (provided by applicant): Neural stem cells (NSC) have tremendous therapeutic potential in the repair of central nervous system injury and disease. Knowledge of NSC biology will also aid in the understanding of developmental brain disorders as well as brain tumors, which may result from abnormal NSC proliferation. A fundamental property of neural and other stem cells is their ability to undergo self-renewing proliferation. This application focuses on NSC proliferation and self-renewal and is based on our previous discovery-driven work, identifying genes enriched in neural progenitors compared to their more differentiated progeny. It is hypothesized that genes expressed in multiple stem cell populations will play important roles in NSC self-renewal. The first two aims of this proposal focus upon three genes that share the characteristics of restricted expression in CNS germinal zones and expression in multiple stem cell-containing cultures: MELK (maternal embryonic leucine zipper kinase), PSP (phosphsoserine phosphatase), and TOPK (TLAK cell originating protein kinase). Expression analysis will be performed to determine whether these genes are synthesized by multipotent, self-renewing stem cells. This will consist of standard localization methods along with study of gene-specific, promoter-driven expression of enhanced green fluorescence protein. Progenitor cells transfected with these constructs will be sorted using FACS and assayed for their ability to serve as self-renewing, multipotent stem cells. The hypothesis that MELK, TOPK and PSP regulate neural stem/progenitor self-renewal will be directly tested by determining whether knockdown and/or over expression influences self-renewal of primary progenitors. The cell cycle mechanisms underlying any changes observed will be determined using a combination of FACS analysis of DNA content, and assay of cell cycle regulatory proteins. In addition to the study of individual genes, further experiments will test broader hypotheses with respect to gene expression in self-renewing NSCs. First, the hypothesis that genes expressed in multiple stem cell populations will regulate neural stem/progenitor cell proliferation will be more generally tested using 38 genes identified in a previous study. These genes will be further stratified and then screened for function in stem cell self-renewal using primary CNS progenitors. PTEN-deficient NSC have a greater capacity for self-renewal and genes enriched in these cells will be candidates to play important roles in NSC self-renewal. In another set of experiments, microarray analysis will be used to compare gene expression in PTEN-deficient and wild type neurosphere cultures to discover genes and gene networks regulating self-renewal.

DESCRIPTION (provided by applicant): Neural progenitors and stem cells hold a great deal of promise as therapeutic tools in the treatment of neurologic and psychiatric disease. However, a great deal of knowledge regarding neural progenitor/stem cell biology needs to be obtained prior to their effective therapeutic utilization. For example, there are no reliable extracellular markers for neural stem cells to distinguish them from other cells in the mammalian brain. Previous studies have identified sets of genes that are uniquely expressed in neural progenitors and not more differentiated cells. However, the identification of mRNAs or mRNA fragments does not necessarily identify the sets of proteins that are actually expressed. Furthermore, very few of already identified genes predict proteins that are expressed at the cell surface, the most useful localization to serve as a cell-specific marker. The identification of cell surface proteins would not only facilitate the understanding of the biology of neural progenitors, but would also aid in the prospective identification of neural progenitors for studies of basic biology as well as therapeutic strategies. Modern proteomic approaches allow for the rapid identification of proteins differentially expressed in two populations of cells. It is the goal of this pilot study to determine whether a proteomic approach can be successfully applied to the study of neural progenitor cells. In the first set of experiments we will test the hypothesis that neural progenitors express specific sets of cell surface proteins. To achieve this, we will first determine whether neural progenitors express membrane proteins that are different from those expressed in more differentiated cells. Membrane proteins from neurosphere cultures derived from embryonic or postnatal mice containing stem and progenitor cells will be compared to proteins from more differentiated sister cultures. We will then compare results obtained with our previous results obtained using genomic methods. We will also determine whether neural progenitors derived from mice at different ages express different membrane proteins, as in vitro studies demonstrate they have different differentiation potentials. This study will aid in determining whether the stem/progenitor cells isolated at these different ages are different "kinds" of cells. After we identify candidate proteins, downstream studies will determine whether these molecules are potential cell surface markers for subsets of neural progenitors and whether they have important roles in neural progenitor biology using a combination of methods. It is anticipated that these pilot studies will pave the way for new avenues in the study of neural progenitor cell biology.

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is almost universally fatal. The discovery of tumor-initiating cells with the capacity to self-renew, sometimes termed "cancer stem cells", has created tremendous enthusiasm for the development of new avenues of therapy. These cells utilize familiar pathways for their proliferation, such as the PI3 Kinase pathway. Despite the hope raised by the discovery of brain tumor stem cell-like cells, numerous obstacles lie in the path of therapeutic development. One complication is that these cells have significant resistance to conventional therapies and to inhibition of pathways. Another is that there are differences amongst brain tumor stem-like cells that are present in the tumors of different patients and probably amongst the multiple types of such cells within individual patient's tumors. The goals of this study are to critically examine brain tumor stem cell-like cell biology in order to develop the means to attack them and to overcome their mechanisms of resistance. First, we will determine the genetic mechanisms underlying the dependence of GBM stem cell-like cells on the PI3K pathway. We will use a limited sequencing approach to determine the mutational spectrum of the stem cell-like cells derived from different patients and that of different clonal lines derived from individual patients with the goal of establishing the fundamental genotype-phenotype relationships in these biologically important sets of cells. We will test whether those cells with mutations that activate the PI3K pathway are more dependent on this pathway for proliferation and tumorigenesis than the stem cell-like cells with mutations in other pathways. We will next assess the role of the PI3K pathway in mediating the enhanced resistance to radiation observed in brain tumor stem cell-like cells.To identify potential mechanisms of this radioresistance, we will determine if PTEN modulates autophagy and also whether the PI3K pathway promotes survival through an antioxidant response following ionizing radiation in the same cells. We will then explore mechanisms of chemoresistance in GBM stem cell-like cells. We will use cell culture, in vivo assays and microfluidics-based immunocytochemical analysis (MIC) to determine whether rapamycin selects for stem cell-like cells with enhanced tumorigenicity and pathway activation. We will also determine whether resistance to rapamycin treatment can be overcome through inhibition of hyperactivated pathways. Then, we will identify pathways of resistance based on a completed phosphoproteomic screen to discover proteins that are phosphorylated or dephosphorylated during the development of rapamycin resistance. We will determine the potential role of the proteins identified by this screen in the development of resistance. Finally, we will use MIC to study the mechanisms of resistance to the EGF receptor inhibitor, erlotinib, a drug that has been proposed as a molecularly targeted therapy for a subset of GBM patients. We will determine whether erlotinib selects against EGFRvIII-positive calls, and whether these resistant cells have cancer stem cell properties. Furthermore, we will identify the means to overcome this resistance.

Rationale: Biomedical research at the present time is dominated by the paradigm of linking genes and environment to function. Recent advances in human genetics through genome-wide association analyses have greatly accelerated the disease gene discovery process. However, in this post-genome sequencing era, we are faced with the challenge of determining the cellular and organismal functions of these genes and how gene dysfunction leads or contributes to the phenotype of the disease (i.e. functional genomics). In the past, most functional genomics work was carried out through using genetic manipulations to build animal models that carry the same mutations in genes as in human diseases. However, this approach is often laborious and, more importantly, how much of the human disease can be recapitulated in those animal models remains a huge uncertainty. However, until recently, no viable alternative approaches were available. The establishment of human pluripotent embryonic stem cells (hESCs) and human induced pluripotent stem cells (h-iPSCs) is beginning to revolutionize the way to approach functional genomics, disease modeling, disease mechanistic studies, drug screening, and development of novel therapeutic interventions. Particularly, with iPSC technology, where patient-specific cells are utilized as research objects, we are finally able to utilize the genetic manipulations that nature has already generated, as well as taking into account the enormous genetic predispositions/variations that exist in the population, to develop population stratified or even personalized effective therapies. To utilize this expanding technology, IDDRC investigators have expressed the need for centralized expertise, coordination, and help with stem cell/iPSC generation, maintenance, lineage differentiation, and standardization, with the aims of building novel cellular and molecular models relevant to IDD. A strong internal consensus within the UCLA IDDRC community about the importance of these cells has become the driving force for the establishing of this new Stem Cell Core, and we have all the required expertise in place at UCLA to provide such a sen/ice. A number of IDDRC investigators are studying pediatric brain tumors with the goal of alleviating the mortality and developmental disability associated with them. In an analogous fashion to the explosion in knowledge of the genotype/phenotype relationship in genetically-based developmental disorders, similar breakthroughs are being made in the study of cancer. The Cancer Genome Atlas (TCGA) project is delineating the spectrum of mutations present in human brain tumors (http://cancergenome.nih.gov/), and there has been a large increase in the understanding of oncogenic pathways in brain tumors. However, similar to genetic developmental disorders, the study of pediatric brain tumors has been hampered by the lack of appropriate in vitro models. The recent discovery of stem cell-like cells in brain tumors (Hemmati et al., 2003), including pediatric brain tumors and the ability to propagate these highly relevant, tumorigenic cultures permits the study of molecular processes that drive these cells, the correlation of genotype and phenotype, and the development of novel potential therapies. The purpose of this new Core is to provide excellent technical support and expertise in the generation, characterization, maintenance, expansion, and lineage differentiation of human pluripotent stem cells including primarily IPSCs from patients as well as previously established hESCs (as controls and for comparative studies). In addition, due to the additional joint interest among our IDDRC investigators on brain tumors, methodologies of growing brain tumor stem cells, together with prepared tumor stem cell cultures from resected tumor specimens will also be provided by the core. In addition to the rationale outlined above, there are additional reasons for establishing a Stem Cell Core within the IDDRC. Previously, based on the consensus among scientists conducting hESC work, researchers worid-wide submitted RNA samples from their brew of cultured hESCs and a large scale gene expression array analysis was carried out. The results indicated that the most important element that accounts for variation among the different samples depended upon who had been handling the cells. Different investigators handle cells differently, which probably changed the molecular/cellular properties of the cells. Therefore, a centralized effort for stem cell production, characterization, maintenance, and expansion is very beneficial for subsequent research. This Core will provide standardization and quality control of the cells to ensure reproducibility and stability of the cell sources. In addition, based on many years of experience in studying neural stem cell (NSC), differentiation from various sources including NSCs derived from developing mouse, rat, and human embryos and adult, NSCs derived from mouse and human ESCs, as well as NSCs derived from mouse and human iPSCs, Drs. Sun and Zeng are well-situated to provide expertise concerning how to effectively differentiate human iPSCs and human ESCs first into expandable NSCs, and then subsequently into functional neurons that form synaptic network and glial cells (i.e., astrocytes and oligodendrocytes). Finally, Dr. Kornblum is among the earliest investigators studying brain tumor stem cells. He and Dr. Le Belle are very familiar with the sample (brain tumor) collection as well as the subsequent derivation of brain tumor stem cell cultures. It would be difficult for an average scientist in the IDDRC to interact with the clinicians and to have access to clinical samples in a regulated manner. Drs. Kornblum and Le Belle represent an enormous resource for the IDDRC community and will be able to handle the technical or scientific issues related to brain tumor stem cells, as well as distribution of brain tumor stem cells for many types of studies.

While autism spectrum disorders (ASD) are highly heritable, it is clear that there is also a strong environmental component to ASD pathogenesis. ASD is frequently associated with brain enlargement, which is often present at birth and affects multiple cell types, suggesting that dysfunction in an early stem or progenitor population contributes to ASD etiology. We hypothesize that brain enlargement is related to enhanced self-renewal of neural stem cells (NSCs) leading in turn to increased neurogenesis and abnormal connectivity that has been observed in ASD. Mutations in the tumor suppressor PTEN that are observed in association with the autism macrocephaly phenotype are almost all heterozygous (HET), although HET mutations in mice produce few or subtle brain abnormalities. PTEN HET mutations in humans may or may not contribute to autism with brain overgrowth on their own but could act as a genetic susceptibility in combination with environmental factors that affect their function. One known environmental risk factor for ASD that might interact with genetic risk factors is the Maternal Inflammatory Response (MIR). Although MIR has been linked to both autism and to brain overgrowth, the biologic mechanisms for its potential pathological effects remain undefined. We will test the hypothesis that reactive oxygen species (ROS) generated by MIR exposure can increase stem cell self- renewal and neurogenesis in human neural stem cells through the reversible oxidative inactivation of PTEN protein and subsequent enhancement of PI3K pathway activation and that this effect is enhanced by heterozygous PTEN mutation. To do this we will use state of the art methods to generate lymphocyte-derived induced pluripotent stem cells (iPSCs) from our unique clinical population with identified PTEN HET mutations, brain overgrowth, and autism and from unaffected relatives. We will then derive forebrain NSCs from the iPSCs to test the hypothesis that PTEN mutations interact with ROS to promote an abnormal degree of self- renewing proliferation and neurogenesis. This will be done by directly exposing cells to ROS as well as to candidate inflammatory cytokines that are known to be produced by MIR, and, which, in turn could activate ROS production. We will determine the molecular mechanisms underlying the altered cellular phenotypes that we may observe through the analysis of the PI3K and other pathways that may interact with the PI3K pathway. We will also determine whether different forms of PTEN HET mutations which may result in different levels of residual PTEN function respond differently to ROS/cytokine stimulation. These studies will elucidate the relationship between genetic susceptibility and exposure to MIR that could inform the development of novel interventions by identifying mechanisms of susceptibility to a common environmental risk factor. The findings obtained in this study will also have broader implications for susceptibility to environmental autism risk factors due to the fact that there are many different genetic susceptibilities that may interact with MIR through final common pathways which lead to altered neural stem cell function during critical periods in brain development. .

Project 4: Novel epigenetic treatment of IDH mutant gliomas SUMMARY/ABSTRACT Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are found in several cancer types, including the majority of low-grade gliomas and secondary glioblastomas (GBM). Although their survival is relatively prolonged relative to patients with wild-type IDH, patients with IDH mutant gliomas still almost invariably succumb to their disease. Mutant IDH causes the aberrant production of the oncometabolite D-2-hydroxyglutarate (2HG). How 2HG contributes to glioma formation is not well-understood, but it is postulated that 2HG interferes with a number of ?-ketoglutarate dependent enzymes, including those involved in DNA demethylation. A number of lines of evidence indicate that inactivation of the demethylator TET2 could result in the DNA hypermethylation observed in many IDH mutant tumors. Treatment with selective inhibitors of mutant IDH have shown promise in acute myelogenous leukemia (AML), but results of pre-clinical studies in glioma have been mixed. Our preliminary data indicate that the transcription factor OLIG2 may be responsible for downregulating TET2 mRNA which, in combination with 2HG, potentially renders TET2 activity virtually non-existent in IDH1-mutant gliomas. As such, inhibition of mutant IDH alone would be insufficient to recoup TET2 function. It is our fundamental hypothesis that IDH mutant gliomas are dependent on repression of TET2 expression and function, and that a combined approach of inhibition of the enzymatic function of mutant IDH along with the suppression of OLIG2 will have a beneficial effect on the treatment of IDH mutant gliomas. In Aim 1, we will validate the importance of OLIG2 in IDH mutant gliomas, using CRISPR-based gene editing in vitro and in vivo. These experiments will also determine whether IDH mutant gliomas with different background mutations, e.g., P53 mutation or 1p/19q deletion, will have different dependency on OLIG2. In Aim 2, we will then determine whether disruption of OLIG2 alone and in combination with inhibition of mutant IDH1 function -- using the investigational compound AG-881 (a novel brain-penetrant pan-IDH mutant inhibitor) -- disrupts TET2 function and inhibits tumor growth. Since direct small molecule inhibitors of OLIG2 have not been developed, our clinical strategy will focus on the use of the FDA-approved histone deacetylase (HDAC) inhibitor, panobinostat to downregulate OLIG2. In pre-clinical studies, we will test the effects of panobinostat and other HDAC inhibitors with and without AG-881 on OLIG2 expression and TET2 function, as well as on growth of IDH mutant tumors in vitro and in vivo. In Aim 3, we will then proceed with a 2-stage clinical study. In the first stage, we will perform a pharmacokinetic/pharmacodynamic clinical trial to verify the effects of panobinostat on OLIG2 expression in patients with IDH mutant tumors. In the second stage, we will conduct a Phase II randomized clinical trial comparing the effects of AG-881 plus panobinostat versus AG-881 alone on tumor response rate and progression-free survival (PFS). By the end of the project period, we will have verified whether our therapeutic strategy is a viable option for patients with IDH mutant glioma.

CORE C: Abstract The need for physiological assessments was generated by UC-TRaN faculty who required electrophysiological studies beyond collaborations. These electrophysiological assessments consisted of experiments performed in brain slices, acutely isolated neurons or in cultures, providing a functional analysis of changes in neurons, local circuits and microcircuits induced primarily by genetic alterations in cellular, mouse or rat models. The first objective of Core C will be to continue these analyses because the need remains and the Core will assist investigators with functional analysis at the cellular, circuit, and systems level using state-of-the-art electrophysiology and optogenetic recording methods in in vitro and in vivo preparations. Recent advances in both genomic and stem cell technologies, particularly hESC and hiPSC, have opened the door to new approaches in IDD research based on human cells. For example, cortical neurons harboring genetic mutations with a given disorder can in principle be readily produced from hiPSC generated from patients. A significant challenge remains to translate this promise into new discoveries about the basis of IDD and therapies. The second objective of this Core will be to facilitate this process by assisting UC-TRaN investigators to produce, propagate, and differentiate hESC and hiPSC into neural and glial cell types of interest to create in vitro models of IDD. Another function of the Core is to work with investigators and molecular screening resources available at UCLA to harness the potential of these in vitro models for drug discovery.

This proposal requests 5 years of additional funding for the UCLA Intellectual and Developmental Disabilities Research Center (IDDRC). For over 40 years, our mission has been to provide an optimal environment for conducting multidisciplinary research into the mechanisms underlying intellectual and developmental disabilities (IDDs), to translate these findings into effective treatments for IDDs, and disseminate these findings to the scientific community and the public. This submission expands on the translational focus that we began 5 years ago with an added emphasis on human research and clinical trials, and closer ties to the community. Each of the 5 cores are structured to facilitate interdisciplinary collaborations, following four thematic goals: 1) providing state of the art infrastructure for IDD related research; 2) encourage innovation by supporting technical development and providing financial incentives for new projects; 3) promote integration across disciplines, by encouraging interdisciplinary research among faculty and between cores; and 4) to disseminate advances in technology to other scientists, train new IDD investigators, and convey findings to the scientific community and stakeholders in community outreach efforts. We propose 5 interacting cores: A: Administration and Dissemination, which oversees core functions and usage, assures quality and accountability, and promotes outreach and dissemination; B: Clinical Translation, supporting clinical trials, recruitment, diagnosis and deep phenotyping, and biosample collection; C: Genetics, Genomics and BioInformatics, which performs genetic analysis, sequencing, expression, and provides resources for planning and executing analysis of genomics data; D: Cells, Circuits and Systems, supporting human iPSC and 3-dimensional organoid development, in- and ex- vivo electrophysiology and optogenetics; and E: Structural and Functional Visualization, which provides training, access, and analysis services for in vitro microscopy, mini-cameras for in vivo visualization, animal and human structural and functional MRI and spectroscopy. Our model research project focuses on mechanisms underlying sleep impairments in two IDDs, building on new findings from our last submission: a near absence of slow-wave sleep in Dup15q syndrome. We will examine mechanisms underlying sleep impairments in Dup15q and Rett's Syndrome using a multidisciplinary approach that includes a clinical component, animal models, and brain organoid model using patient-derived IPSCs, to elucidate how mechanisms underlying altered sleep physiology lead to cognitive dysfunction. Results from this project will directly inform next steps for developing interventions that may modulate sleep and, in turn, neurodevelopment in IDDs.

CORE D: Abstract The purpose of The Cells and Circuits Core is to provide investigators with tools needed to understand the cellular and physiological basis of intellectual and developmental disabilities (IDDs) and the role of potential therapeutics in IDD biology. Electrophysiological assessment is a vital to the understanding of both cellular and circuit alterations in models of IDD and cell culture is a vital tool in the understanding of cellular and molecular basis of ID. The core will consist of two components, the Neurophysiological Assessment component and the Cellular and Organoid Modeling component. In the Neurophysiological Assessment component, we will provide functional assessments at the cellular, circuit and systems level. The use of state-of-the-art electrophysiological approaches will aid IDDRC investigators in uncovering and understanding basic mechanisms causing the disorders being studied. These electrophysiological approaches consist of experiments performed in brain slices, acutely isolated neurons or cultures providing functional analyses of changes in neurons, local circuits and microcircuits induced primarily by genetic alterations in cellular, mouse or rat models. New techniques include optogenetics, EEG and local field potential (LFP) recording in vivo, and the use of miniscopes to image neuronal calcium transients in freely-behaving rodents to permit and facilitate analyses of developmental neurological functions at the cellular, circuit, and systems levels. In the Cellular and Organoid Modeling component, we will develop and provide models of IDD using human pluripotent stem cells, tissue-derived neural stem cells, and three-dimensional organoid cultures. Because human central nervous system cells are inherently different from rodent cells, we developed the facilities and capabilities to propagate and distribute them 2 cycles ago. In the previous cycle, we expanded the human cell core greatly, with a focus on pluripotent stem cell-derived cultures, including building the basis for the study of human cerebral organoids. We continue to provide these tools and expand our ability to deliver cells and expertise to ID researchers serving as a proxy to estimate neuronal activity in more ethological conditions. The use of stem cell technology provides a novel approach to modeling disease and developing rationale therapies based on utilization of human cells. The Core will provide facilities and expertise to propagate human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), neural stem and progenitor and other cerebral cell types to create and study cellular models of IDD. A major function of the Core will be to aid investigators in culturing and studying cerebral organoids derived from hESCs and hiPSCs. In addition to its training and service functions, the Core will continue to develop novel methodologies in physiological assessment and human cell culture and analysis. The two components of the Core will interact seamlessly. The Core will interact regularly with the other Cores of the IDDRC and with the Research Project and will also aid investigators in drug discovery and development through interactions with Molecular Screening Shared Resource in the Broad Stem Cell Research and Jonsson Comprehensive Cancer Centers.