Abstracts

Rationale: Loss-of-function mutations in genes encoding NPRL3- a subunit of the amino acid regulatory complex of mTOR, GATOR 1- have been linked to various MCD subtypes associated with seizures. The pathophysiological consequences of NPRL3 mutations have not been fully defined. We therefore hypothesize that knockout of Nprl3 will result in mTOR pathway hyperactivation producing enhanced cell size, altered process outgrowth, altered subcellular localization of mTOR in vitro, and neuronal migration defects in vivo. Methods: Mouse Neuro2a cells (N2aC) were transfected with one of two CRISPR/Cas9 plasmids targeting exon 7 of Nprl3 or scramble plasmid. Changes in soma size and process extension were defined in digital images of WT, scramble, or Nprl3 KO N2aC with or without rapamycin or torin1 (both 50 nM; 24 hrs). Changes in mTOR pathway activation were determined by Western assay probing for phosphorylated ribosomal S6 protein (240/244; PS6) with or without rapamycin (150 nM; 60 min), torin1 (100 nM; 60 min), or amino acid free (AAF) vs. complete media. The degree of colocalization of mTOR on the lysosome was defined using fixed N2aC probed with primary antibodies targeting mTOR and LAMP2 and fluorescent secondary antibodies (alexa 488 and alexa 594, respectively) after incubation in complete or AAF media. Images were taken on a spinning disk confocal microscope and the degree of mTOR/lysosome colocalization was quantified using a fluorescence colocalization assay. In utero electroporation was performed on E14 CD1 mouse embryos using Nprl3 CRISPR/Cas9 or scramble plasmids described above. A separate cohort of dams were injected with rapamycin (5 mg/kg) 24 hrs after surgery. Electroporated pups were dissected at P3, 5, and 10. Immunohistochemistry was performed on paraffin embedded sections probed with primary antibodies for mCherry (Cas9) or Cux1 and fluorescent secondary antibodies (alexa 594 or alexa 488, respectively) and analyzed by binning tissue sections into cortical plate, subventricular zone and ventricular zone as well as measuring cell size.  Results: Nprl3 KO N2aC were larger and had greater numbers of processes than scramble or wildtype cells (n=50 cells per group; p<0.05). Rapamycin or torin1 treatment prevented soma enlargement and process outgrowth in Nprl3 KO cells (n=50, p<0.05). Western blot demonstrated that Nprl3 KO cells displayed increased mTOR pathway activation as measured by increased PS6 (240/244) vs. scramble and WT cells. In Nprl3 KO cells, mTOR pathway hyperactivation was prevented by rapamycin or torin1 application but not by incubation in AAF media. There was a higher degree of mTOR/LAMP2 colocalization in Nprl3 KO vs. scramble and WT cells in AAF media (n= 10; p<0.05). Enhanced colocalization between mTOR and the lysosomal membrane was also observed in Nprl3 KO cells vs. scramble and WT cells in complete media (n= 10; p<0.05). P3 , 5, and 10 mouse pups electroporated with Nprl3 KO plasmid displayed migratory defects and increased cell size (n= 50; p<0.05) as evidenced by mCherry and CUX1 positive neurons dispersed throughout the electroporated region vs. scramble electroporated neurons where mCherry+ cells appeared only in the cortical plate (n=5 pups per group). Migratory defects and neuronal size were corrected in Nprl3 KO mouse pups from dams treated with rapamycin (n = 5). Conclusions: We demonstrate changes in cellular morphology and function in N2aC after Nprl3 KO that are mTOR-dependent. Further, Nprl3 KO results in migratory defects in vivo which were corrected with rapamycin. These findings suggest that mTOR inhibitors may be beneficial for the treatment of MCDs associated with GATOR1 complex subunit variants. Funding: R01NS099452 to PBC