Abstracts

Rationale: Overactivation of the mechanistic target of rapamycin (mTOR) pathway in the developing nervous system results in developmental abnormalities such as focal cortical dysplasia (FCD), hemimegalencephaly (HME), and tuberous sclerosis complex (TSC), often causing severe epilepsy and cognitive impairment. While mTOR inhibitors have been effective for brain, cardiac, and other tumors, they have not been as successful in controlling epilepsy. Studies of FCD, HME, and TSC have been challenging because of genetic and clinical heterogeneity, with poor genotype-phenotype correlation. We decided to focus on polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) because of its clinical and genetic homogeneity. PMSE is caused by a homozygous deletion in the STRADA gene. STRADA serves as an upstream regulator of AMP kinase and inhibits mTOR signaling. Children with PMSE have facial dysmorphism, macrocephaly, severe epilepsy, significant cognitive impairment, and focal cortical dysplasias. We aim to elucidate how mTOR pathway overactivation results in structural brain abnormalities and epilepsy, with the goal of developing more effective therapies for FCD, HME, TSC, and PMSE. Methods: Skin biopsy-derived fibroblasts from two subjects with PMSE were reprogrammed into induced pluripotent stem cells (iPSCs). iPSCs were then differentiated into three-dimensional cortical organoids using protocols from the Pasça (cortical spheroid protocol) and Song (spin-omega protocol) laboratories, both involving dual-SMAD inhibition for cortical patterning. The size of the organoids was measured over time, and periodically harvested for fixation, cryosectioning, and immunostaining. Results: Cortical-like organoids were successfully grown from two subjects with PMSE and two control subjects. Organoids displayed multiple ventricular zone-like neural rosettes that were positive for neural stem cell markers Pax6 and Sox2.  MAP2- and Tuj1-positive neurons, as well as HOPX-positive outer radial glial cells, surrounded the rosettes. During the second week of differentiation using the Song Lab protocol, PMSE organoids were more numerous and larger in size than controls. PMSE organoids also displayed more robust budding of neuroepithelial tissue. However, when using the Pasça Lab protocol, organoids that were grown to later stages of development (11 weeks) did not appear to display any difference in size. Conclusions: Using patient-derived iPSCs differentiated into cortical organoids, we are able to examine the effect of abnormal mTOR signaling on early human forebrain development. At 1-2 weeks of development, PMSE organoids appear to be larger and more numerous, concordant with the megalencephaly phenotype displayed by PMSE patients. Further studies are in progress to examine effects of STRADA mutation on more differentiated cortical organoids, and to confirm that altered mTOR pathway activation is the cause of the early phenotype. Funding: This work was supported by NIH HD028820 (LTD).