High-Content Analysis Core
Our Conte Center proposes to study a large number of domain mutations and previously uncharacterized NDD- associated genetic variants in TRIO and SYNGAP1 using a high-throughput strategies (Project 1), to perform functional screens for newly developed chemical probes in mouse neurons (Project 1), and to generate novel iPSC-derived neuronal models (Project 2). All of these research endeavors will rely heavily upon efficient medium or high-throughput screening capabilities as well as the ability to analyze a multitude of phenotypes in dozens of conditions. Core A will provide the following services: 1) assist Projects 1 and 2 with the optimization of imaging strategies, MEA recordings, and analysis of cultured neuron systems; 2) perform high-content imaging (HCI) of neuroarchitectural phenotypes for Projects 1 and 2; 3) perform multielectrode array (MEA) analysis of neuronal network electrophysiological phenotypes for Projects 1 and 2; 4) provide instruction of Center users for HCI, MEA recordings, and analysis; 5) facilitate the offline analysis of data; 6) facilitate the storage of phenotypic data; 7) ensure quality control of data and analysis. To accomplish this work in an efficient, cost-effective, and rigorous manner, Core A will exploit standardized high-throughput workflows that unite innovative technologies with economies of scale. The Specific Aims of Core A are: Aim 1. To assess the impact of SNVs in TRIO and SYNGAP1 on neuroarchitecture and network activity in mouse neurons. Aim 2. To assess the impact of chemical modulators of GEF and GAP activity on neuroarchitecture and network activity in mouse neurons. Aim 3. To assess the impact of genetic mutations and chemical probes on human iPSC-derived neuronal models.
Neurodevelopment Stem Cell Core
Synaptic GEF and GAPs have emerged as significant players in neurodevelopmental disorders (NDDs). While a large number of clinical variants have been reported in these genes in patients with NDDs, their molecular and cellular consequences have not been systematically analyzed. The precise biological functions of synaptic GEF and GAP in human neuronal subtypes remain unclear, while there are no chemical tool compounds that can acutely manipulate their catalytic activity. Recent advances in the generation and differentiation of patient-specific induced pluripotent stem cells (iPSCs) offer new opportunities for elucidating the pathogenic mechanisms of NDDs. Certain unique capabilities of iPSC technologies are well suited for the study of NDDs, including providing a platform for examining the effects of genetic approaches or chemical probes on human neuron activity, morphology, and developmental processes, and enabling investigations of distinct human neuronal subtypes. Our Center proposes to study genetic variants in human synaptic GEF and GAP genes and their associated phenotypes in human iPSC-derived neurons. The overarching goal of this Core is to provide high quality, efficient and cost-effective services to Center members related to iPSC technologies. This core will have an exclusive focus on the neurobiology of NDDs, aligning with the goals of Projects 1 and 2 to interrogate NDD-relevant cellular and network phenotypes. All data and cell lines generated by the Neurodevelopmental Stem Cell Core will be shared with the members of our Center and the broader academic community. The specific aims of the Neurobiology Stem Cell Core are: 1) To generate, perform QC, expand and maintain high-quality iPSC lines. 2) To perform CRISPR mutagenesis to generate isogenic lines with high-priority missense and nonsense mutations. 3) To generate human iPSC-derived excitatory/inhibitory neuron co-cultures.
Mouse Resource Core
The principal goal of this Center is to understand how gene expression and associated function within key neuronal subtypes regulates neurobiological substrates required to form cortical circuits that enable decision-making and behavioral adaptations. Ras and Rho-like small GTPases play fundamental biological roles within neurons by controlling cell-autonomous growth-related signaling pathways and orchestrating neuronal circuit assembly. Consistent with the biological importance of GTPase signaling in brain development and function, several prominent neurodevelopmental disorder (NDD) risk genes are upstream regulators of neuronal small GTPase signaling. We will focus on the neurobiology of two major genetic regulators of GTPase signaling, TRIO, a top 10 risk factor for schizophrenia, and SYNGAP1, a top 10 risk factor for ASD and intellectual disability. TRIO/SYNGAP1 are molecular effectors that orchestrate neuronal migration, neuronal morphogenesis, synaptic connectivity, synaptic plasticity, in vivo neural circuit function, and associated behavioral adaptations. However, it is currently unknown how their expression and function within specific neuronal subtypes and defined developmental time windows refine cortical circuits to impact cognitive processing and associated behaviors. Our interdisciplinary, multi-institutional “Center for GTPase Regulation of Neuronal Cell Biology and Behavior” will study the contributions of TRIO/SYNGAP1 in cortical circuit assembly at cellular, synapse, circuit, systems, and behavioral levels in complementary model systems, including mouse models and human iPSC-derived neurons. The Mouse Resource Core will facilitate studies using mouse models. Mouse somatosensory cortex shares similar overall architecture and approximates human molecular pathways, neuronal function, and circuit organization. Thus, mouse models are a valuable system for studying cortical development at the level of neurons, synapses, circuits and systems, as well as for evaluating potential therapeutic strategies. The mouse genome is a tractable system for engineering mutations in orthologous genes to enable study of genetic variants in the context of brain tissue. Conditional deletion strategies allow for dissection of cell-type specific effects underlying synapse and circuit dysfunction. Combining genetic manipulation with electrophysiologic and optogenetic tools, allows for detailed, multilevel mechanistic studies in the context of an intact cortex, our Center will address gaps in knowledge about the contribution of small GTPase regulators to cortical circuit assembly. The primary objectives of the Mouse Resource Core are to provide centralized support of mouse model needs for the Center Projects, including generation of novel knock-in mouse lines; maintenance and specialized breeding of genetically modified mice; and facilitating sharing of mouse lines with the academic community. Our centralized mouse core will provide efficient generation of experimental mice to generate rigorous and reproducible data across projects in a cost-effective manner.
Administrative Core
This Core will function as the organizational and scientific hub of the Center to ensure success of the research projects and cores that make up the foundation of the Center. This will be accomplished by promoting effective governance, efficient management, good communication, and a strong culture of collaboration among all investigators that encourages creativity, knowledge sharing, and scientific rigor. This Core will be responsible for the overall organization of the Center including the scheduling of teleconferences and meetings, engagement of the External Scientific Advisors and interactions with NIH program staff. In addition, the Core will coordinate the community engagement activities. Finally, this Core will lead efforts to communicate Center activities and findings to the public and scientific community through maintenance of a website, strategic use of social media, scientific publications, presentations at national and international conferences, resource sharing and data dissemination. The Administrative Core will pursue the following Specific Aims: Aim 1) Manage and coordinate all administrative and collaborative activities of the Center. This Aim will involve organization and leadership of the Center, formation of an Executive Committee, interaction with the Internal and External Advisory Boards, writing a Center Charter, promoting scientific rigor and reproducibility, conflict resolution as well as student and postdoctoral training. Aim 2) Facilitate communication across participating institutions and community partners. This Aim will involve organization of project and core team meetings, of a seminar series and an annual meeting, community engagement, and reporting to NIH. Aim 3) Enable public awareness of Center activities and ensure timely resource sharing. This Aim will involve creating a Center website, social media outlets, as well as resource sharing and dissemination of results.