Abstracts

Rationale: Pathogenic genetic variants in the mechanistic target of rapamycin (mTOR) pathway cause neurodevelopmental disorders known as “mTORopathies”, with early-onset epilepsy, cortical malformations, and cognitive impairment. To study how mTOR hyperactivity affects the earliest stage of brain development, including cell fate determination and interneuron migration, we focused on PMSE syndrome (Polyhydramnios, Megalencephaly, Symptomatic Epilepsy), a rare mTORopathy caused by inherited biallelic loss-of-function (LOF) mutations in the STE20-related kinase adaptor alpha (STRADA) gene, an upstream inhibitor of mTOR Complex 1 (mTORC1). Our prior studies demonstrated STRADA loss expanded the neural stem cell pool and outer radial glial (oRG) cells in dorsal forebrain (DF) organoids, but how it affects development of inhibitory interneurons that arise from the ventral forebrain (VF) remains unclear.

Methods: We used human pluripotent stem cell (hPSC)-derived cerebral organoids (hCOs). Biallelic frameshift mutation in exon 9 of STRADA was introduced via CRISPR editing to generate LOF mutant and isogenic control iPSCs. We adapted a directed differentiation protocol for growing dorsal (dhCOs) and ventral (vhCOs) hCOs in parallel. To explore cell fate determination, we used scRNA-seq in weeks 5 and 11 control/mutant hCOs to identify shifts in cell types with differentially expressed genes. Changes were further analyzed at the protein level via immunocytochemistry at weeks 5, 8, and 11. To understand interneuron migration, week 4 vhCOs were plated on a Geltrex layer and imaged every 20 min in the phase-contrast channel for 36 hours. We also virally labeled dhCOs with CAG::tdTomato, and vhCOs with mDLX::GFP (mDLX as the GABAergic interneuron promoter) at week 4, fused WT or mutant d/vhCOs at week 5, and compared migration dynamics via live imaging. To examine network activity changes, we plated fusions on multi-electrode arrays (MEA) for longitudinal recordings from week 10 to 17. We evaluated the role of GABAergic synaptic transmission by pharmacological assays on MEA recordings.

Results: By comparing the transcriptome and protein landscapes between STRADA mutant and control hCOs, we found that mTOR hyperactivity resulted in profound changes to interneuron subtype specifications: There were significantly fewer GABAergic, but increased somatostatin (+) and calretinin (+) interneurons in mutant vhCOs. STRADA mutant vhCOs exhibited GABAergic interneuron migration deficits, partially rescued by rapamycin (mTORC1 inhibitor) treatment. Phalloidin staining further indicated that the migration deficits were associated with decreased F-actin polymerization. When we treated fusions on MEA with GABA_A receptor antagonist, bicuculline, at week 17, we saw augmented firing rates and burst frequencies in control, but not mutant fusions, indicating a decrease of endogenous GABA level in mutant fusions.


Conclusions: Our findings support a mechanistic link between hyperactive mTORC1 and aberrant interneuron development in the VF associated with epileptogenesis, including altered subtype specification, migration, and endogenous GABA activity in PMSE syndrome.

Funding: This work was supported by NIH R01NS127829 (to LTD).