Peter Penzes - US grants

DESCRIPTION (provided by applicant): Dendritic spines are highly dynamic and undergo activity-dependent morphological changes which involve the actin cytoskeleton. There is intense interest in the molecular mechanisms that regulate actin cytoskeletal dynamics in spines, because they are believed to be associated with plasticity. Since we have previously shown that the synaptic GDPIGTP exchange factor (GEF) kalirin-7 regulates spine morphogenesis, we hypothesized that mechanisms involved in synaptic development and plasticity also regulate kalirin-7. The short-term goal of the proposed research is to understand the mechanisms that regulate Kalirin-7 in dendritic spines, and thus the mechanisms that regulate spine morphogenesis. The overall goal of the proposed research is to provide a better understanding of how synapses are generated and change, and on the long run, to expand our understanding of the processes of brain development, learning and memory, mental retardation, and aging. We will examine the role of synaptic activity in regulating localization of kalirin-7 to spines and its GEF activity in primary cultures of cortical and hippocampal neurons. Because kalirin-7 may be associated with glutamate receptors, which are involved in synaptic plasticity, we will focus on the role of glutamate receptors in regulating kalirin-7. In addition, we will examine the association of glutamate receptors in complexes with kalirm-7 in neurons and brain. We will also examine the role of phosphorylation in regulation of kalirin-7 GEF activity and localization, with the goal to provide a general outline of the role of phosphorylation in kalirin-7 function. We will delineate the role of tyrosine vs. serine/threonine phosphorylation, and generate initial information about the types of protein kinases involved. Together, these experiments will provide a basis for future studies of regulation of kalirin-7 and spine morphogenesis.

DESCRIPTION (provided by applicant): Dendritic spines are highly dynamic and undergo activity-dependent morphological changes which involve the actin cytoskeleton. There is intense interest in the molecular mechanisms that regulate actin cytoskeletal dynamics in spines, because they are believed to be associated with plasticity. Since we have previously shown that the synaptic GDPIGTP exchange factor (GEF) kalirin-7 regulates spine morphogenesis, we hypothesized that mechanisms involved in synaptic development and plasticity also regulate kalirin-7. The short-term goal of the proposed research is to understand the mechanisms that regulate Kalirin-7 in dendritic spines, and thus the mechanisms that regulate spine morphogenesis. The overall goal of the proposed research is to provide a better understanding of how synapses are generated and change, and on the long run, to expand our understanding of the processes of brain development, learning and memory, mental retardation, and aging. We will examine the role of synaptic activity in regulating localization of kalirin-7 to spines and its GEF activity in primary cultures of cortical and hippocampal neurons. Because kalirin-7 may be associated with glutamate receptors, which are involved in synaptic plasticity, we will focus on the role of glutamate receptors in regulating kalirin-7. In addition, we will examine the association of glutamate receptors in complexes with kalirm-7 in neurons and brain. We will also examine the role of phosphorylation in regulation of kalirin-7 GEF activity and localization, with the goal to provide a general outline of the role of phosphorylation in kalirin-7 function. We will delineate the role of tyrosine vs. serine/threonine phosphorylation, and generate initial information about the types of protein kinases involved. Together, these experiments will provide a basis for future studies of regulation of kalirin-7 and spine morphogenesis.

DESCRIPTION (provided by applicant): Activity-dependent changes in the number and shape of dendritic spines during development and synaptic plasticity are essential for the formation of neuronal circuits, in learning and memory, and in the etiology of mental retardation and mental illness. Our long-term objective is to understand the signaling mechanisms which regulate activity-dependent plasticity of dendritic spines. Rho-like small GTPases are central regulators of the actin cytoskeleton and spine morphogenesis. We previously identified the Rac1-guanine- nucleotide exchange factor (GEF) kalirin-7 as a key regulator of spine morphogenesis in neurons. However, it is not clear whether kalirin-7 and Rac1 are regulated by synaptic activity and whether they regulate activity-dependent spine structural plasticity. In the Preliminary data section, we show that: 1) kalirin-7 recruitment to synapses is regulated by tyrosine phosphorylation and potentially synaptic activity;2) kalirin-7 tyrosine phosphorylation is regulated by synaptic activity;3) structural plasticity can be induced in cultured neurons by an NMDA receptor-dependent mechanism;4) NMDA-receptor-induced spine structural plasticity is mediated by the PDZ domain-containing protein AF-6, which interacted with kalirin-7 in a yeast 2-hybrid screen. Based on these observations, we hypothesize that kalirin-7 and Rac1 regulate activity-dependent synaptic structural plasticity. To test this hypothesis we propose the following specific aims: 1) to examine the synaptic activity-dependent regulation of kalirin-7 enzymatic GEF activity and GTP-binding by Rac1;2) to examine the association of kalirin-7 with NMDA receptors and the regulation of synaptic translocation of kalirin-7 and Rac1 by synaptic activity;3) to visualize the real-time translocation of EGFP-tagged kalirin-7 and Rac1 in neurons by time-lapse imaging and fluorescence recovery after photobleaching (FRAP);4) to assess the role and requirement of kalirin-7 and Rac1 in activity-dependent spine morphogenesis. These studies will use primary cultures of cortical and hippocampal neurons.

DESCRIPTION (provided by applicant): The objective of this proposal is to characterize the mechanisms mediated by the protein kalirin that control synaptic structural and functional plasticity in pyramidal neurons. Building upon data produced in the previous grant period, and using a novel mouse model we have recently generated, we will examine the role of an important molecular regulator of dendritic spine plasticity. As both spine density and kalirin expression are reduced in schizophrenic patients' brains, these studies are expected to provide important insight into the mechanisms of spine pathology in mental disorders. Modifications in spiny excitatory synapse structure and function modulate synaptic transmission and plasticity, and underlie cognitive functions. Conversely, altered spine plasticity contributes to the pathogenesis of several mental disorders. Hence, understanding the molecular mechanisms that control spiny synapse plasticity and pathology will provide essential insight into the neurobiology of cognitive functions and mental disorders that affect cognition. Synapse structure and function are controlled by a complex network of interactions between numerous proteins. Our previous studies have established the postsynaptic protein kalirin as an important regulator of synaptic structural plasticity. Importantly, kalirin has recently been implicated in several mental disorders including schizophrenia. Kalirin is a brain-specific guanine-nucleotide exchange factor which activates the small GTPase Rac1 and its most abundant form, kalirin-7, is highly enriched in spines. In the previous funding period we demonstrated that kalirin-7 plays an important role in activity-dependent synaptic structural and functional plasticity downstream of NMDA receptors and CaMKII. We have shown that kalirin also regulates AMPA receptors in spines, and mediates N-cadherin-dependent synaptic adhesion signaling. We have also generated a full knockout of the KALRN gene (KALRN-/-) in mice, and found that this results in a robust and cortex-specific reduction in Rac1 activation and in the number of functional spiny excitatory synapses. KALRN-/- mice have impairments in specific cognitive functions. In this proposal we will dissect the functional roles of kalirin signaling in spiny synapse morphogenesis and plasticity. We hypothesize that kalirin signaling plays crucial and specific roles in synapse function and spine stability/dynamics. We propose the following Specific Aims: 1) To characterize the mechanisms underlying kalirin-dependent regulation of AMPA receptor-mediated transmission and plasticity. 2) To chart the time course and characterize the mechanisms of kalirin-dependent spine stability and dynamics. 3) To characterize the role of kalirin signaling in N-cadherin-dependent spine morphogenesis in vivo. 1

DESCRIPTION (provided by applicant): Multiple lines of evidence support a key role for abnormal synaptic connectivity in schizophrenia, but the molecular mechanisms underlying its pathogenesis are not known. Understanding these mechanisms may allow us to identify new targets for therapeutic intervention, especially early in the course of illness. The application wil focus on dendritic spines as cellular substrates of brain connectivity, because the majority of excitatory synapses are located on spines, and reduced spine density has been extensively documented in schizophrenia. Mounting evidence indicating that known schizophrenia susceptibility genes regulate spines and that regulators of spine plasticity are implicated in schizophrenia, strongly support the model that perturbations in the molecular network underlying spine plasticity are critically involved in the pathogenesis of schizophrenia. However, the mechanisms through which genetic alterations in this network underlie specific neurobiological phenotypes related to schizophrenia are not known. Recent data indicates that rare variants (including amino acid mutations) cumulatively account for a significant fraction of the missing heritability in schizophrenia, and cluster in gene networks that control synapses. Because a large fraction of such mutations are estimated to impair protein function, many are expected to cause brain circuit alterations. Thus, we propose that by identifying, testing for association, and characterizing rare variants enriched in schizophrenia, we will provide critical new insights into disease pathogenesis, because such mutations provide detailed knowledge about the affected molecular and cellular functions. Based on our preliminary data, we hypothesize that rare coding variants in genes that control dendritic spine plasticity, cumulativel enriched in subjects with schizophrenia, disrupt cortical connectivity and impact neuromorphological and cognitive measures in carriers. Using a multidisciplinary translational approach that combines human genetics, molecular and electrophysiological studies in cellular models, functional validation in mice, and cognitive assessment and structural brain imaging in patients, we will pursue these specific aims: 1) To assess the cellular impact of mutations in spine plasticity genes identified in schizophrenia subjects. 2) To determine the impact of mutations in spine plasticity genes on glutamatergic synaptic transmission. 3) To determine the impact of mutations in spine plasticity genes on cortical ultrastructure and functional connectivit in mice. 4) To assess the relationships between mutations in spine plasticity genes and phenotypic measures in patients.

? DESCRIPTION (provided by applicant): Recent evidence implicates glutamatergic synapses as key pathogenic sites in psychiatric disorders. Common and rare variants in the ANK3 gene, encoding ankyrin-G, have been associated with bipolar disorder (BD), schizophrenia (SZ), autism spectrum disorders (ASD) and intellectual disability (ID). While a number of studies suggested that ankyrin-G plays a role in neuronal function beyond its well-characterized actions at the axon initial segment, its functions in mammalian glutamatergic synapses have not been investigated. Our preliminary studies show for the first time that ankyrin-G is integral to AMPAR-mediated synaptic transmission and to the maintenance of spine morphology. Using super-resolution microscopy we found that ankyrin-G forms distinct nanodomain structures within the spine head and neck. At these sites, it differentially modulates mushroom spine structure and function. Neuronal activity promotes ankyrin-G accumulation in distinct spine subdomains, where it differentially regulates activity-dependent spine structural plasticity. Our preliminary findings implicate subsynaptic nanodomains containing a major psychiatric risk molecule as having location- specific functions, and opens novel directions for basic and translational investigation of psychiatric risk molecules. The functions of ankyrin-G in spines of glutamatergic synapses in the brain have not yet been investigated. In this proposal we will use super-resolution and in vivo two-photon microscopy, in combination with biochemical, electrophysiological, molecular, and mouse model approaches, to test the hypotheses that different ankyrin-G isoforms play differential and integral roles in dendritic spine maintenance and glutamatergic synaptic transmission and plasticity. We will test these hypothesis in the following Aims: 1) Regulation of glutamatergic postsynaptic structure and function by ankyrin-G isoforms; 2) Mechanisms of regulation of postsynaptic ankyrin-G in spiny synapses; 3) Regulation of spiny synapse remodeling and function by ankyrin-G isoforms in the intact brain.

? DESCRIPTION (provided by applicant): The majority of excitatory synapses in the mammalian brain are located on dendritic spines. Changes in spine structure and function play critical roles in brain development and plasticity. Conversely, alterations in spine morphogenesis occur in psychiatric disorders, including schizophrenia. Consequently, understanding the molecular mechanisms underlying spine plasticity and their alterations in psychiatric disorders could lead to the rational development of novel therapeutic strategies in mental disorders. Kalirin, a neuronal guanine-nucleotide exchange factor (GEF) for small GTPases, is emerging as a regulatory hub of structural and functional plasticity in spines. Recent genetic and neuropathological studies in human subjects have also implicated kalirin and its signaling partners in psychiatric disorders, including schizophrenia. Recent evidence has shown that kalirin isoforms are differentially affected in the brains of human subjects with mental disorders, carry missense mutations enriched in mental disorders, and have differential impacts on dendrite and spine morphology. Moreover, kalirin isoforms interact with several important trafficking proteins. However, the roles of these isoforms and their interactions in spines have not been examined. Based on these findings, we hypothesize that kalirin isoforms are distributed in distinct postsynaptic microcompartments, where they play specific functions in coordinating synaptic glutamate receptor trafficking with spine morphogenesis. Recent developments in superresolution imaging have opened up novel directions for functional analysis of synaptic proteins. Here we will utilize superresolution imaging in combination with molecular manipulations, biochemistry, immune-electron microscopy, and electrophysiology to pursue the following aims: 1) To map the nanoscale organization of kalirin isoforms in postsynaptic microcompartments. 2) To characterize the molecular mechanisms underlying the interactions of kalirin isoforms with Arf6, SNX1/2 and dynamin. 3) To determine the consequences of the interaction of kalirin isoforms with Arf6, SNX1/2, and dynamin on postsynaptic structure and function. The proposed studies will be the first to investigate at nanoscopic resolution the sub-synaptic distribution and functions of isoforms of an important regulator of spine plasticity and pathology, providing insight into the roles of protein isoforms i synapses. This proposal will also investigate novel roles for kalirin isoforms in coordinating synaptic trafficking with spine remodeling, which could implicate altered synaptic trafficking as a candidate pathophysiological mechanism in some mental disorders. The proposal will therefore shed light on key mechanisms of synaptic plasticity and pathology, and can facilitate the rational development of novel therapeutic strategies aimed at reversing synaptic deficits in mental disorders.

? DESCRIPTION (provided by applicant): Schizophrenia (SZ) genome-wide association studies (GWAS) have identified >100 genome-wide significant (GWS) risk loci. However, risk loci typically span 1 gene and multiple GWS index and proxy SNPs in linkage disequilibrium, leaving causal genes/variants largely unknown, which hinders translating GWAS findings into disease biology and drug targets. It is thus imperative to identify at each locus the functional risk variants, the affected risk gene/s and the associated cellular phenotype changes. Most risk variants are noncoding, and likely influence gene expression through modulating chromatin accessibility to transcription factors (TF). Accessible (open) chromatin overlaps with cis-regulatory sequences and is enriched for common disease risk variants. Our pilot study in neurons derived from induced pluripotent stem cells (iPSCs) also showed an enrichment of SZ GWS index and proxy SNPs in open chromatin flanking TF footprints. We hypothesize that many causal variants at SZ loci modulate TF binding in open chromatin, thereby altering transcriptional and neuronal phenotypes relevant to SZ pathophysiology. We present an innovative approach to identify chromatin-modulating variants at each SZ locus, for both GWS index SNPs and proxy variants. We will directly compare the allele-specific effect of a heterozygous SNP on the quantitative measurements of open chromatin (i.e., ASoC) within the same individual, which minimizes experimental variation and increases assay sensitivity, allowing an effective study using a smaller sample size. We have identified a set of 20 super-heterozygous (super-het) subjects with >80% power to detect ASoC at 70 SZ loci. We found that chromatin openness correlated with gene expression changes from iPSC?neurons at loci of interest, and ASoC was prevalent in iPSC-neurons of a single subject. We propose to extend the ASoC assay to the 20 super-het subjects, and pursue three specific aims: (1) We will map open chromatin by ATAC-seq (Assay for Transposase-Accessible Chromatin by sequencing) in pathophysiologically relevant iPSC-derived neuronal stem cells, dopaminergic and glutmatergic neurons, and search for regulatory SZ-risk variants that present ASoC in sequences flanking TF footprints. (2) We will use multiplex CRISPR-Cas9 genome editing to generate pairs of isogenic iPSC lines that differ only for every 6~7 regulatory SZ-risk variants, and identify the gene(s) cis regulated by each variant by comparing expression differences between the pair of isogenic iPSC-derived neuronal cells. (3) We will prioritize synaptic genes cis-regulated by a regulatory SZ-risk variant and generate isogenic iPSCs differing only for the regulatory variant, and compare neuronal morphological, biochemical, and electrophysiological phenotypes. SZ is a devastating disorder afflicting 1% of the population without cure. This project will help move the field beyond SZ GWAS to deciphering causal mechanisms which will aid in the development of more effective treatments, and will also create a rich research resource of iPSCs carrying SZ GWS risk alleles.

PROJECT SUMMARY Neurodevelopmental psychiatric disorders including intellectual disability (ID), autism (ASD) and schizophrenia (SZ) are often comorbid with neurological disorders, such as epilepsy. However, the biological substrates of such comorbidity are poorly understood. Understanding these molecular substrates could provide insight into the pathogenesis of these disorders as well as into fundamental neurobiological processes. One promising strategy is to investigate inherited, monogenic neurodevelopmental syndromes comorbid with epilepsy. Mutations in a number of genes have recently been discovered which cause neurodevelopmental disorders comorbid with epilepsy. Interestingly, the majority of such genes have putative functions at synapses and in dendrites, prompting the hypothesis that synaptic connectivity dysfunctions could be key for both types of disorders. Notably, mutations in a disproportionately large number of synaptic adhesion molecules are associated with neuropsychiatric disorders, underscoring the importance of understanding their neuronal functions. A significant fraction of these genes encode members of the neurexin and contactin family. Here we propose to investigate the synaptic and dendritic functions of a prominent representative of this family, CNTNAP2, mutations in which cause monogenic syndromes of ID, ASD, and SZ comorbid with epilepsy. Because Cntnap2 modulates synapse structure and function, here we propose to investigate novel molecular mechanism underlying Cntnap2 functions newly discovered by us, such as glutamate receptor trafficking, interneuron dendrite arborization, and paracrine signaling by ectodomain shedding. Based on our preliminary studies we hypothesize that Cntnap2 plays crucial roles in AMPAR trafficking and in the maintenance of synapto-dendritic architecture through its protein interaction network. We will test this hypothesis by employing several novel and cutting-edge methodologies such as superresolution and multi-photon imaging, LC-MS/MS proteomics, and CRISPR/Cas9-engineered iPSC-derived neurons (iN), and by integrating mechanistic studies in neuronal cultures with human iNs and mouse models. We will pursue the following Specific Aims: 1) Regulation of AMPAR trafficking by Cntnap2 and its protein partners; 2) Control of spine architecture and interneuron dendrite arborization by Cntnap2 and its partners; 3) Mechanisms of paracrine signaling by Cntnap2 extracellular domain shedding. Data generated will provide novel insight into signaling by Cntnap2, the regulation of synaptic circuits in the brain shedding new light on glutamate receptor trafficking by adhesion molecules, formation of neurotransmitter receptor intracellular aggregates, nanoscopic localization of synaptodendritic molecules, interneuron-specific maintenance of dendritic arborization, control of cortical E/I balance, as well as paracrine signaling by ectodomain shedding. Proposed studies will also uncover mechanisms potentially relevant for the pathogenesis of neurodevelopmental disorders.

ABSTRACT Recent data emerging from large-scale genomic studies has revealed that copy number variations (CNVs) are a major class of mutations that play a key role in the etiology of psychiatric disorders, including autism (ASD) and schizophrenia (SZ), increasing risk up to 30 fold. However, the large number of genes in CNVs, and the wide variety of clinical phenotypes associated with them, has made understanding CNV-associated disorders and their genotype-phenotype correlations especially challenging. Duplications of 16p11.2 chromosomal region, occur in ASD, SZ, intellectual disability (ID), Rolandic epilepsy, and other disorders, and are among the top 2 most highly penetrant and frequent CNVs in SZ. Despite this progress in genomics, synaptic phenotypes in models of 16p11.2 CNV have not yet been thoroughly studied. The identification of robust synaptic phenotypes would result in experimentally approachable targets for treating common aspects of neuropsychiatric disorders such as cognitive dysfunction. Alterations in glutamatergic synapses and dendritic architecture have been implicated by genomic, neuropathological, and functional studies as key sites of pathogenesis in neurodevelopmental psychiatric disorders including SZ, ASD, and ID. However, the synaptic biology that contributes to the pathogenesis of CNV disorders remains largely elusive. In this renewal application we propose to investigate the impact of CNVs on synaptic and dendritic dysfunction in SZ, ASD and other neurodevelopmental disorders by focusing on the 16p11.2 duplication. We hypothesize that individual genes within the 16p11.2 locus drive distinct sub-phenotypes, often expressed as cellular compartment-specific alterations, by modulating localization of proteins encoded by genes outside the CNV. These phenotypes can be reversed by targeting network hubs. In this application, we will use an integrated approach spanning cultured neurons, mouse models, and patient-derived iNs, and a combination of cutting-edge technologies including SIM and two-photon imaging, in utero electroporations, slice electrophysiology, protemics, multi-array electrode recordings, and high-content imaging screens, to pursue the following Aims: 1) Mechanisms underlying synaptic sub-phenotypes in 16p11.2 microduplication disorder; 2) Mechanisms underlying dendritic sub-phenotypes in 16p11.2 microduplication disorder. 3) Pharmacological reversal of 16p11.2 duplication phenotypes. The proposed studies are novel and impactful, given that the 16p11.2 duplication is a major psychiatric risk factor and its synapto-dendritic impact has not yet been investigated. If successful, this proposal will be the first to demonstrate that cellular subcompartment-specific proteomics and highly penetrant monogenic disease genes within the CNV can be harnessed to identify novel mechanisms whereby a driver within the CNV can regulate a protein network outside of the CNV. Such cellular compartment-specific protein network alterations, not predicted by global mRNA profiling, could underlie specific disease sub-phenotypes. Such phenotypes could be be reversed globally by targeting network hubs, opening novel strategies for the treatment of psychiatric disorders.

ABSTRACT Copy number variations (CNVs) are a major cause of neurodevelopmental disorders, but their biological investigation and pharmacological targeting pose many challenges. Deletions locus are among the most frequent causes of autism spectrum disorder and duplications at the 16p11.2 (ASD). However, alterations in the corresponding protein networks, especially at key cellular sites for pathogenesis, have not been investigated in this or other CNVs. We propose to use compartment-specific neuroproteomics, combined with bioinformatics, super-resolution microscopy, and drug repurposing, to understand and alter dendritic excitability phenotypes in 16p11.2 mouse and induced pluripotent stem cell (iPSC) models. Based on our extensive preliminary data, we hypothesize that altered expression of PRRT2, which likely regulates the trafficking of a subset of ion channels and receptors, drives and abnormal complement of ion channels and receptor on the plasma membrane, leading to abnormal excitability, excitatory/inhibitory (E/I) balance, and network properties in 16p11.2 models and patients. These phenotypes may be reversed by targeting ion channel function using FDA- approved anti-epileptic drugs or ERK signaling using repurposed cancer drugs. Our collaborative team, which includes experts in neurodevelopmental disorders (Penzes), neuroproteomics (Savas), molecular pharmacology (Barbolina), and ion channel physiology (George) will employ a powerful and multidisciplinary combination of highly innovative methodologies to pursue the following Specific Aims: (1) To chart the developmental regulation and determine molecular mechanisms underlying abnormal excitability in dup and del mice and human neurons. (2) To chart the developmental profile and determine the molecular mechanisms underlying the role of PRRT2 as a driver of excitability and seizure phenotypes. (3) Pharmacological reversal of 16p11.2 del and dup phenotypes. This proposal will be the first to demonstrate that cellular subcompartment-specific proteomics combined with super-resolution microscopy, informed by highly penetrant monogenic disease genes within a CNV, can identify novel disease mechanisms. Such phenotypes could be reversed globally by targeting network hubs using repurposed drugs, opening novel strategies for the treatment of neurodevelopmental disorders.

ABSTRACT Dendritic and spine plasticity plays key roles in brain development, function, behavior, and disease. Indeed, spine and dendrite pathology is a common feature of many neuropsychiatric disorders (NPDs), including autism spectrum disorder (ASD), schizophrenia (SZ), and bipolar disorder (BPD). Rho-like small GTPases, including Rac1, are a family of regulatory proteins with central roles in dendrite and spine plasticity. Their extensive implication in NPDs suggests that these pathways can serve as therapeutic targets in NPDs. The activity of small GTPases is enhanced by guanine-nucleotide-exchange factors (GEFs), among which the Rac1- GEF kalirin is highly enriched in spines, and is perhaps the best-characterized GEF in the brain. Kalirin is a central regulator of dendrite arborization, spine plasticity, glutamatergic transmission, neuronal connectivity, and cognitive behavior. While small-molecule pharmacological modulators have been invaluable tools for studying the biological functions of kinases, receptors, or ion channels, no such tools exist for Rho-GEFs, including kalirin. Kalirin is an optimal drug target for several reasons: its expression is largely restricted to the CNS, it is highly enriched in spines, it is a signaling hub in a synaptic network including many NPD risk factors, its enzymatic activity can be modulated, and the 3D structure of its GEF domain has been determined. Here we outline a novel and innovative hit validation cascade that will allow us to develop small-molecule tools to investigate a previously unapproachable target relevant to NPDs. The brain-specific expression of kalirin and its highly compartmentalized subcellular localization at synapses suggests that regulation of Rac1 signaling, through pharmacological interventions targeting kalirin, may allow neuron- and synapse-specific effects. This is key to developing tools that produce cell type-specific and context-dependent Rac1 modulation. Using a combination of high-throughput screening (HTS) and in silico screening against the target proteins kalirin/Rac1, we produced a hit list of potential regulators of kalirin activity suitable for follow-up analysis in well-characterized and optimized assays. We hypothesize that small-molecule compounds isolated in HTS and in silico screens modulate kalirin's GEF and biological activity in rodent and human iPSC-derived neuron models, and reverse neuroarchitectural abnormalities in models of NPDs. We will test this hypothesis in the following aims: 1) Hit validation and in vitro characterization, selection, and prioritization. 2) Prioritization of mouse and human neuronal model systems for testing validated hits. 3) Characterization of validated hit compounds in mouse and iPSC models.