Biallelic mutations in lead to abnormalities associated with cortical tubers in human iPSC-derived neurons.

Tuberous Sclerosis Complex (TSC) is a genetic disorder caused by mutations in or , and patients frequently have epilepsy, autism spectrum disorder, and/or intellectual disability, as well as other systemic manifestations. In this study, we differentiated human induced pluripotent stem cells (iPSCs) from a female patient with TSC with one or two mutations in into neurons using induced expression of NGN2 to examine neuronal dysregulation associated with the neurological symptoms in TSC. Using this method, neuronal differentiation was comparable between the three genotypes of iPSCs. We observed that +/- neurons show mTORC1 hyperactivation and associated increased cell body size and process outgrowth, as well as exacerbation of the abnormalities by loss of the second allele of in -/- neurons. Interestingly, iPSC-derived neurons with either a single or biallelic mutation in demonstrated hypersynchrony and down-regulation of FMRP targets. However, only neurons with biallelic mutations of demonstrated hyperactivity and transcriptional dysregulation observed in cortical tubers. These data demonstrate that loss of one allele of is sufficient to cause some morphological and physiological changes in human neurons but that biallelic mutations in are necessary to induce gene expression dysregulation present in cortical tubers. Finally, we found that treatment of iPSC-derived neurons with rapamycin reduced neuronal activity and partially reversed gene expression abnormalities, demonstrating that mTOR dysregulation contributes to both phenotypes. Therefore, biallelic mutations in and associated molecular dysfunction, including mTOR hyperactivation, may play a role in the development of cortical tubers.In this study, we examined neurons derived from iPSCs with two, one, or no functional alleles, and we found that loss of one or both alleles of results in mTORC1 hyperactivation and specific neuronal abnormalities. However, only biallelic mutations in resulted in elevated neuronal activity and up-regulation of cell adhesion genes that is also observed in cortical tubers. These data suggest that loss of heterozygosity of or may play an important role in the development of cortical tubers and, potentially epilepsy, in patients with TSC.