Abstracts

Rationale: Variants in mTOR pathway genes including TSC2, DEPDC5, and STRADa have been linked to malformations of cortical development (MCD) and epilepsy (‘mTORopathies’). Histopathological analysis of mTORopathy specimens reveals cytomegalic dysmorphic neurons in close physical apposition and an in vitro model of Tuberous Sclerosis Complex revealed cellular aggregates in astrocytes. We hypothesized that knockout of Nprl3, Tsc2, STRADa, or Kptn would result in cellular aggregation in vitro.

Methods: CRISPR/Cas9 knockout N2a cell (N2aC) lines were generated for Nprl3, Tsc2, STRADa, and Kptn and used in conjunction with WT and scramble control N2a cells. N2aC were passed through a cell strainer and plated at equal density into video dishes or chamber slides. After 24 hrs, N2aC were incubated in complete media or treated with complete media containing rapamycin (50 nM; 48 hrs) or torin1 (50 nM; 48 hrs). Video dishes were maintained in an imaging incubator. Three regions of interest were selected in each dish, images were taken every 15 minutes for 48 hours, and images were compiled into videos for analysis. Cells in chamber slides were fixed in PFA and probed for F-actin using fluorescent secondary antibodies to visualize aggregates. Aggregate number, volume, and cell density were measured in digital micrographs and statistically analyzed using ANOVA with a p value < 0.05 deemed significant. Three biological replicates of each cell line were sent for mass spectrometry analysis of isolated membrane and cytosolic fractions and RNAseq to define changes in proteins associated with cellular aggregation. Finally, all cell lines were subjected to cell death, proliferation, and cell-free DNA assays.