Delayed forebrain excitatory and inhibitory neurogenesis in STRADA-related megalencephaly via mTOR hyperactivity

Biallelic pathogenic variants in STRADA, an upstream regulator of the mechanistic target of rapamycin (mTOR) pathway, result in megalencephaly, drug-resistant epilepsy, and severe intellectual disability. This study seeks to understand how mTOR pathway hyperactivity alters cell fate specification in dorsal and ventral forebrain development using STRADA knock-out human stem cell derived brain organoids. In both dorsal and ventral forebrain STRADA knock-out organoids, neurogenesis is delayed, with a predilection for stem cell maintenance and an increase in outer radial glia. Ventrally, interneuron subtypes shift to an increase in neuropeptide-Y expressing cells. Inhibition of the mTOR pathway with rapamycin results in rescue for most phenotypes. When mTOR pathway variants are present in all cells of the developing brain, overproduction of interneurons and altered interneuron cell fate may underlie mechanisms of megalencephaly, epilepsy, and cognitive impairment. Our findings suggest mTOR inhibition during fetal brain development as a potential therapeutic strategy in STRADA deficiency. The four iPSC lines used for this study (C1, C2, M1, M2) were derived from a commercially available source of male foreskin fibroblasts using CRISPR gene editing of STRADA. C1 and C2 lines were unedited and carried the wild-type sequence of STRADA (NM_001003787.4). M1 was a compound heterozygote, with c.632_636del (p.Leu211Argfs*45) on one allele and c.636del (p.Gly213Valfs*8) on the other. M2 was also a compound heterozygote, with two variants, c.635_636del (p.Ser212Trpfs*45) and c.631C>G (p.Leu211Val) on one allele, and c.636_637insA (p.Gly213Argfs*45) on the other allele.