Abstracts

Rationale: Malformations of cortical development (MCDs) and epilepsy, result from germline or somatic mutations in mTOR pathway genes (‘mTORopathies’). A common histological phenotype associated with these mutations is cytomegalic dysmorphic neurons that are pS6 positive within resected cortical tissue specimens harboring these variants. In addition, neurons are frequently found in close apposition to each other within the cortex and aggregates of cells are often observed in experimental models of tuberous sclerosis complex. We therefore hypothesized that knockout (KO) of Tsc2, Depdc5, Strada, or Kptn in N2a cells would exhibit mTORC1-, but not mTORC2-, dependent cellular aggregation.






Methods: CRISPR/Cas9 KO of N2a cell lines (N2aC) were generated for Tsc2, Depdc5, Kptn, and Strada, along with WT and scramble N2aC controls. N2a cells for each line were cultivated in video dishes for live cell imaging and chamber slides for immunocytochemistry. N2a cells for live imaging were passed through a cell strainer in complete media and plated in video dishes. Each cell line had a baseline control, and two treatment groups. Treatment groups were rapamycin (50 nM; 48hrs), torin1 (50 nM; 48 hrs), or an mTORC2 inhibitor (50 nM; 48 hrs). All cells were plated at equal density. Video dishes were maintained in an imaging incubator, and images were taken every 15 minutes for 48 hours. Images were then compiled for video analysis. Chamber slide samples were fixed in PFA and probed from F-actin with fluorescent secondary antibodies. Aggregate volume, density, and number were measured using digital micrographs and ANOVA statistical analysis was performed, with a p value < 0.05 deemed significant.