Neuronal hyperactivity becomes mTORC1 independent due to epigenetic changes in Tuberous Sclerosis Complex [RNA-seq]

Tuberous Sclerosis Complex (TSC) is caused by variants in either TSC1 or TSC2, which cooperate to inhibit the mechanistic target of rapamycin complex 1 (mTORC1). TSC is associated with neurological disorders that have been attributed to disinhibition of mTORC1, but mechanisms connecting dysregulation of mTORC1 to molecular and physiological changes in neurons remain unclear. In this study, we sought to understand transcriptional changes in TSC and identified down-regulation of the immediate early gene, EGR1, in TSC2-deficient excitatory neurons. Furthermore, we found that activity-dependent transcription is impaired in TSC due to abnormalities in epigenetic maturation. Finally, we determined that mTORC1 inhibition started late in neuronal maturation of human neurons is only partially effective in reversing gene expression changes and ineffective in reducing spontaneous neuronal hyperactivity in TSC. These data demonstrate a critical window in early brain development where mTORC1 dysregulation leads to epigenetic changes that contribute to persistent neuronal abnormalities. Overall design: iPSC neurons of three different genotypes (TSC2+/+, TSC2+/-, and TSC2-/-) were differentiated using induced neurogenin-2 induction. Neurons were re-plated with astrocytes on 48 well multielectrode arrays. Cells were treated with either rapamycin 20nM or DMSO starting at either day 10 or 30 of differentiation. Spontaneous neuronal activity was monitored, and then on day 40 of differentiation, cells were lysed and RNA was extracted.