Project 1: Structural determinants and pharmacological modulation of synaptic GEFs and GAPs
Project 1 will investigate the biology of synaptic GEFs and GAPs at the molecular/biochemical and cellular levels. GEFs and GAPs are complex multidomain proteins with multiple splice variants. Their protein domains perform enzymatic catalysis, subcellular targeting, and autoregulation. Such domain combinatorial complexity can further specialize function to various subcellular compartments, neuronal subtypes, and developmental time-points. Project 1 will test the hypothesis that a structure-function analyses of isoforms, domains, and clinical variants will reveal mechanisms of protein regulation that underlie neuronal morphogenesis, synapse formation, and synapse plasticity. We recently developed novel high- and medium-throughput assays to characterize the modulation of GEF/GAP catalytic activity and neuronal function by single nucleotide variants and chemical probes. Using these, we will delineate GEF and GAP spatiotemporal complexity by dissecting the roles of splice variants, enzymatic domains, and non-catalytic domains at the molecular and cellular levels.
Project 2: Biology of synaptic GEFs and GAPs in human neurons
The goals of Project 2 are to use iPSC-derived neuron models and cerebral organoids to investigate the contributions of gene expression and function to human neuron axon/dendrite growth, migration, morphology, synaptic structure and function, and brain development. We will perform studies in iPSC models that parallel the common themes explored in the other four projects. These common Center themes include: 1) how Trio and SynGAP function within specific cell types regulate circuit substrates (migration/morphogenesis, synapse connectivity, neural function); 2) how specific protein domains in Trio and SynGAP regulate circuit substrates; and 3) how small molecule targeting of GEF/GAP domains regulate circuit substrates. We will test the hypotheses that gene dosage, functional domains, catalytic activity, and genetic variants in Trio and SynGAP affect the ability of human neurons to undergo normal morphogenesis, synaptogenesis, and connectivity.
Project 3: Mechanisms underlying cortical local circuit regulation by synaptic GEFS and GAPS
In Project 3, we will address the broader goals of The Center by analyzing the roles of these complex signaling molecules in the formation and refinement of cortical microcircuits. Using ex vivo functional electrophysiological and optogenetic mapping approaches we will dissect the effect of disrupted Trio and SynGAP signaling on cortical architecture. The project will assess (1) the spatial cell-type and temporal functions of Trio and SynGAP, (2) the importance of enzymatic and non-enzymatic functional domains to their roles in cortical connectivity, and (3) the action of novel small molecule inhibitors of these GTPase regulators in synaptic function and connectivity at acute and chronic timescales. These studies will transform our understanding of how Trio and SynGAP function in developing cortical circuits and along with collaborative and integrated efforts in the Center will create a foundational understanding of their roles in neurodevelopment.
Project 4: Regulation of in vivo cortical circuit function and behavior by snypatic GEFS and GAPS
Project 4 will assess the function of these genes at the highest levels of brain function – how they regulate the functional integration of neuronal subtypes into circuits required for behavioral adaptations. Project 4 will address the unifying themes in the Center by measuring neuronal subtype functional circuit integration, circuit plasticity, and behavioral adaptions, by utilizing three classes of mouse perturbation models that target expression or function of these genes. The first set of perturbation models will assess how the cell-autonomous expression of each gene — within unique neural subtypes and at different periods of development — impact cortical circuit function and behaviors these circuits are known to guide. The second set of models will express point mutations within known functional domains of the proteins encoded by these genes. Finally, the third set of models will include acute and/or chronic pharmacological manipulations of Trio and Syngap1 GEF/GAP domains. The impact of this research is that it will highlight, when, where, and how these genes regulate circuit assembly and circuit plasticity required for transforming sensory signals into decisions that drive behavior.
High-Content Analysis Core
Neurodevelopment Stem Cell core
Mouse Resource Core
Administrative Core