PROJECT 2 SUMMARY 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. In Aim 1, we will determine the expression and function of Trio and SynGAP in diverse neural subtypes within developing human neurons and organoids. In Aim 2, we will determine the physiological importance of functional domains in Trio and SynGAP in human neurons and organoids. Finally in Aim 3, we will determine the effects of pharmacological inhibition of Trio and SynGAP in human neurons and organoids. These proposed studies will have a broad, long-lasting impact on the field by determining the physiological importance of individual isoforms and domains, as well as enzymatic and non-enzymatic functions of Trio and SynGAP in human neurons. Demonstrating effects of pharmacological modulators of Trio and SynGAP will further yield a valuable toolbox with which to study other GEF and GAP superfamily members. Findings from Project 2 will provide fundamental information about the neurobiology of these important regulators of GTPase signaling in the brain.