Brain organoid models of SZT2-related disease reveal an overproduction of outer radial glial cells through mTORC1 activation

Biallelic loss-of-function variants of Seizure Threshold 2 (SZT2) cause neurodevelopmental diseases with developmental delay, epilepsy, and macrocephaly. SZT2 forms the KICSTOR complex, which represses the mechanistic target of rapamycin complex 1 (mTORC1) amino acid-sensitive pathway. SZT2 dysfunction is thought to cause abnormal activation of the mTOR pathway, underlying the pathogenesis of SZT2-related diseases. We previously reported constitutive activation of mTORC1 in lymphoblastoid cell lines derived from patients with SZT2-related disease. However, the impact of SZT2 dysfunction on human brain development remains unclear. In this study, we examined the effects of SZT2 dysfunction on brain development using human brain organoids. We generated pluripotent stem cell-derived brain organoids and found a significantly greater number of outer radial glial cells (oRGCs) in the subventricular zone-like layer (SVZ) of SZT2 mutant (MT) brain organoids compared to control (WT) brain organoids. The number of upper-layer neurons, which generally originate from oRGCs, was also significantly greater in SZT2 MT brain organoids. Mechanistically, SZT2 MT brain organoids showed higher mTORC1 activity in the SVZ, where neural stem/progenitor cells amplify for cortical expansion in response to mTORC1 activity. Our data suggest that SZT2 dysfunction may cause macrocephaly through dysregulation of mTORC1 in early neural development.