This abstract has been invited to present during the Investigators Workshop Platform poster session
Rationale: Overactivation of the mammalian target of rapamycin (mTOR) pathway causes “mTORopathies” that are characterized by cortical malformations and epilepsy. One such disease, polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is characterized by facial dysmorphism, macrocephaly, severe epilepsy, significant cognitive impairment, and focal cortical dysplasias. PMSE is caused by homozygous variants in the STRADA gene, an upstream regulator of the mTOR complex 1 (mTORC1). Truncating variants in STRADA result in dysregulation of AMP kinase and increased mTOR signaling. We aim to elucidate how mTOR overactivation in the developing nervous system results in structural brain abnormalities and epilepsy. By understanding the underlying pathogenesis, we can uncover therapeutic targets for PMSE as well as other mTORopathies including focal cortical dysplasia, tuberous sclerosis, and hemimegalencephaly. Methods: Fibroblasts from two subjects with PMSE and two controls were reprogrammed into induced pluripotent stem cells (iPSCs). iPSCs were differentiated into cortical organoids, three-dimensional neural cultures that resemble structural aspects of the developing human brain. Size and morphology of live organoids were measured over time. Organoids were periodically harvested up to 16 weeks in vitro for fixation, cryosectioning, and immunostaining using neural progenitor, neuronal, and glial markers. Results: During the second week of differentiation, PMSE organoids were 35% larger in size (p< 0.0001), displayed a 2.8-fold increase in budding of neuroepithelial tissue compared to controls (p< 0.0001), and had a delay in neurogenesis. Addition of the mTOR inhibitor rapamycin attenuated the neuroepithelial budding in day 8 PMSE organoids (0.3-fold, p< 0.0001), although this effect was diminished at day 10 and day 12. At 12 weeks of differentiation, PMSE organoids had increased expression of HOPX+ outer radial glia as well as GFAP+ glia. After 16 weeks of differentiation, PMSE organoids displayed abnormal large cells that resembled balloon cells (an abnormal cell type found in focal cortical dysplasia Type IIb) and that expressed nestin, GFAP, and neuronal markers. Conclusions: At early timepoints, PMSE organoids are larger with increased neuroepithelial budding, suggestive of the megalencephaly phenotype displayed by PMSE subjects. mTOR hyperactivity results in preservation of a neural stem cell fate and a shift toward an increase in outer radial glia, neural progenitors that contribute to the expanded human cortex, and this could also result in megalencephaly. These findings are consistent with a previous study on PTEN-/- organoids and the observation that mTOR pathway genes are enriched in outer radial glia. The finding of balloon cells in this model recapitulates one of the principal pathological findings seen in focal cortical dysplasia. Because balloon cells have not been previously demonstrated in animal models, our organoid model may better recapitulate some aspects of mTORopathies. Funding: Please list any funding that was received in support of this abstract.: This work was supported by NIH NS109289 and by a Ravitz Advancement Award from the Department of Pediatrics at Michigan Medicine (LTD).