Abstract Genetic alterations directly or indirectly affecting the function of the tuberous sclerosis complex (TSC) protein complex or its downstream target the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) underlie a myriad of neurological disorders, including, but not limited to, epilepsy, autism spectrum disorder, brain overgrowth (megalencephaly) and focal cortical dysplasia, and neurocognitive deficits. We identified the protein TBC1D7 as a third stochiometric component of the TSC complex that is scaffold together with TSC2 via direct binding to TSC1. TBC1D7 is a component of the TSC complex in all mammalian cells and tissues examined, but also exists as a free, more unstable pool outside of the complex. While the sequence of TBC1D7 predicts that it serves as a GTPase-activating protein (or GAP) for Rab family GTPases, the molecular and cellular functions of TBC1D7, both within the TSC complex and outside of it, are unknown. In addition to its undefined role in the TSC complex, which is functionally disrupted in the severe neurological disorder TSC, independent human genetic studies have now found that TBC1D7 is genetically disrupted in a rare autosomal recessive megalencephaly syndrome associated with intellectual disabilities and neuropsychiatric manifestations. Genome-wide association studies have also implicated the TBC1D7 locus in Tourette’s syndrome, attention deficit/hyperactivity disorder, and migraines. Thus, it is important to understand the role of TBC1D7 in both normal brain development and physiology and in neurological disorders, a problem best addressed through the generation of novel genetic models. We have recently used CRISPR/Cas9 gene editing to generate a line of mice lacking TBC1D7. Initial characterization reveals that these mice exhibit megalencephaly and wide-gait phenotypes and that cultured Tbc1d7-/- primary neurons have elevated mTORC1 signaling, increased soma size, defective axon specification, and longer primary cilia relative to Tbc1d7+/+ neurons. In the two aims of this R21, we will develop a detail characterization of the cerebral and neuronal alterations caused by TBC1D7 loss. In Aim 1, we will focus on signaling, brain growth, cortical layering, cerebellar architecture, seizure susceptibility, and the effects of mTOR inhibitors on these phenotypes. In Aim 2, we use primary neuronal cultures to characterize neuron-intrinsic changes in neuronal signaling, growth, polarity, and the formation and function of primary cilia. We will use these phenotypic endpoints to mechanistically determine the role of both the TSC complex-associated and free pools of TBC1D7, its putative Rab-GAP activity, specific candidate Rab protein targets, and mTORC1 signaling. This new mouse model and study under this proposal will lay an essential foundation for defining the molecular function of TBC1D7 in brain development and the myriad of neurological disorders in which it and the processes and pathway that it regulates hav...