Abstracts

Rationale: Malformations of cortical development associated with variants in mTOR pathway genes are common causes of epilepsy and are associated with mTOR pathway hyperactivation. Several studies have reported the efficacy of the mTOR inhibitor Sirolimus in reducing, but not eliminating, seizures in patients with the Tuberous Sclerosis Complex and Pretzel Syndrome. Recently, modulatory control of the mTOR pathway by protein complexes known to regulate amino acid signaling, e.g., GATOR1, GATOR2, and KICSTOR, has emerged as key regulators of mTOR pathway activation with amino acid starvation diminishing mTORC1 kinase activity in non-neuronal cells. We hypothesize that amino acid deprivation in vitro will attenuate mTOR pathway hyperactivation induced by knockdown of Tsc2 or Strada or constitutive activation of mTOR in neuronal cell types. Methods: Mouse neuroblastoma cells (N2aC) were co-transfected with shRNA targeting Tsc2, Strada or a constitutively active mTOR plasmid- each coupled to RFP- and a separate plasmid containing the FRET (CFP/YFP) biosensor TORCAR targeted to the lysosomal membrane. TORCAR permits the assessment of mTOR pathway activation in live cells via a FRET pair coupled to 4E-BP1 (a downstream target of mTORC1). 72 hrs after transfection, successfully co-transfected live cells were imaged on a point scanning confocal microscope. Baseline 4E-BP1 phosphorylation was assessed in N2a cells transfected only with TORCAR. Cells were imaged in serum-containing media (complete media) with or without rapamycin (150 nM) or in amino acid free (AAF) media. Images were taken every two minutes for up to 60 minutes and phosphorylation of 4E-BP1 was assessed by measuring the CFP/YFP ratio. Results: In complete media, a statistically significant increase (n=10 cells per group; P < 0.05) in CFP/YFP ratio was observed in N2aC transfected with Tsc2 or Strada shRNA or constitutively active mTOR plasmid compared to N2aC transfected only with TORCAR in the same culture dish (n= 10). After washing on complete media containing rapamycin, a gradual reduction in CFP/YFP ratio was observed in co-transfected and TORCAR-only transfected N2aC (n=10 per group). TORCAR-only transfected N2aC exhibited a greater reduction in CFP/YFP ratio than co-transfected cells (P <0.05). In N2aC incubated in complete media followed by washing on AAF media, a dramatic decrease in CFP/YFP ratio was observed in co-transfected N2aC and in TORCAR-only transfected N2aC (n= 10 per group). There was no statistical difference in CFP/YFP ratio between co-transfected and TORCAR transfected cells after incubation in AAF media. Further, the reduction in CFP/YFP ratio was greater in co-transfected AAF incubated cells vs. co-transfected rapamycin treated cells Conclusions: We demonstrate that mTOR pathway hyperactivation induced by KD of Tsc2 or Strada or constitutively active mTOR is attenuated by amino acid deprivation and that amino acid deprivation exerts a stronger effect on 4E-BP1 phosphorylation than does rapamycin, in vitro. These findings may provide new strategies to modulate mTOR signaling in MCD. Funding: R01NS099452