Abstracts

Rationale: Malformations of cortical development (MCD) are common causes of neurological deficits in children including seizures and intellectual disability. Recent studies have found loss-of-function mutations in genes encoding the DEPDC5 and NPRL3 subunits of the amino acid regulatory complex of mTOR, GATOR 1. These mutations have been linked to various MCD-subtypes associated with seizures. However, the cellular consequences of these mutations remain undefined. We hypothesize that knockdown (KD) of DEPDC5 or NPRL3 leads to enhanced cell size, altered cell polarity, and altered subcellular localization of mTOR. Methods: Multiple cells lines- HEK 293FT, N2A and mouse neural progenitor cells (mNPCs)- were used to define the morphological consequences of shRNA KD of DEPDC5 or NPRL3 compared to control shRNA. Soma diameter was assessed by FACS or direct measurement of the soma in digital images. The number of actin immunolabeled filopodia in mNPCs were assayed in digital images. Soma size was assessed in neurons exhibiting ribosomal protein S6 phosphorylation (PS6+) in resections from patients with known NPRL3 mutations vs. control brain. The mTOR-dependent effects of DEPDC5 or NPRL3 KD was determined using rapamycin or a p70S6 kinase inhibitor (PF-4708671). Localization of mTOR to the lysosomal membrane or cytoplasm following DEPDC5 or NPRL3 KD was determined by colocalization analysis examining the proximity of mTOR to the lysosomal surface (LAMP2) with or without rapamycin or PF-4708671. DEPDC5 or NPRL3 KD was coupled with amino acid starvation in HEK 293FT and N2A cells to assess the functional consequences of KD on mTOR pathway activation and whether or not activation is attenuated with rapamycin or PF-4708671. Results: HEK 293FT and N2A cells transfected with DEPDC5 or NPRL3 shRNA were larger than control or wildtype cells based on FACS analysis (n=100,000 cells per group; p < 0.05). Direct measurements in mNPCs revealed a larger soma size in DEPDC5 or NPRL3 KD cells compared to control (n=50; p< 0.05). PS6+ pyramidal neurons from patients with NPRL3 mutations were 50% larger than those in control brains. DEPDC5 or NPRL3 shRNA transfected mNPCs exhibited an increased number of filopodia versus controls (n=50 per group; p< 0.05). Rapamycin or PF-4708671 treatment prevented cell soma enlargement following DEPDC5 or NPRL3 KD as measured by both FACS (N2A and HEK 293FT; n=100,000) and direct measurement (mNPCs, n=50). There was a higher degree of mTOR and lysosomal protein colocalization in KD versus rapamycin treated cells following DEPDC5 or NPRL3 KD in mNPCs. An increase in PS6 levels was observed in HEK 293FT and N2A cells after DEPDC5 or NPRL3 KD followed by amino acid deprivation; this was suppressed by rapamycin or PF-4708671 application. Conclusions: We demonstrate changes in cellular morphology in multiple cell lines after DEPDC5 or NPRL3 KD that are mTOR dependent. These findings recapitulate what is observed in patients with DEPDC5 or NPRL3 mutations. These findings may suggest avenues for treatment of MCDs associated with GATOR complex subunits with mTOR inhibitors. Funding: NIH NINDS Grant 1R01NS082343-01-A1 to PBC and a Shriners Hospitals Postdoctoral Fellowship to PHI