Biallelic Mutations in TSC2 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 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 mTOR complex 1 (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 downregulation 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 induced pluripotent stem cells with two, one, or no functional (tuberous sclerosis complex 2) alleles and 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 upregulation 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.