Project Summary/Abstract Growing evidence suggests that patients with Tuberous Sclerosis Complex (TSC), like other neuro- developmental disorders, have dysfunctional synaptic connections. These defects in neuronal connectivity likely contribute to symptoms of TSC, such as cognitive deficits, autism and epilepsy. However, defective synaptic development and function by human neurons has only been suggested from animal model studies and a few recent studies using human neurons, which have led to conflicting reports. To directly address these fundamental questions and help resolve conflicting findings, we will compare the development and function of human cortical neuron synapses of TSC patient neurons with their corrected control counterparts. Cortical neurons are differentiated from human induced pluripotent stem cells (hiPSCs) generated from TSC patient cells and their engineered isogenic counterparts. Using a series of morphometric and molecular signaling assays, we will examine the structure and function of pre- and post-synaptic compartments of control and TSC2 mutant synapses in mixed genotype cultures. Our surprising recent findings suggest that TSC2 functions independent of mTORC in growth cones to directly regulate the cytoskeleton and control axon guidance, prompting us to consider similar mechanisms may function in dendritic spine development. We will also test how synaptic transmission and network activity may be altered in TSC2 patient neurons. Over the long term, we believe our research may help identify key druggable targets in patients with TSC.