NPRL3: Direct Effects on Human Phenotypic Variability, mTOR Signaling, Subcellular mTOR Localization, Cortical Lamination, and Seizure Susceptibility

Nitrogen Permease Regulator Like 3 (NPRL3) variants are associated with malformations of cortical development (MCD) and epilepsy. We report a large (n=133) founder NPRL3 (c.349delG, p.Glu117LysFS) pedigree dating to 1727, with heterogeneous epilepsy and MCD phenotypes. Whole exome analysis in individuals with and without seizures in this cohort did not identify a genetic modifier to explain the variability in seizure phenotype. Then as a strategy to investigate the developmental effects of NPRL3 loss in human brain, we show that CRISPR/Cas9 Nprl3 knockout (KO) in Neuro2a cells (N2aC) in vitro causes mechanistic target of rapamycin (mTOR) pathway hyperactivation, cell soma enlargement, and excessive cellular aggregation. Amino acid starvation caused mTOR inhibition and cytoplasmic mTOR localization in wildtype cells, whereas following Nprl3 KO, mTOR remained inappropriately localized on the lysosome and activated, evidenced by persistent ribosomal S6 and 4E-BP1 phosphorylation, demonstrating that Nprl3 loss decouples mTOR activation from metabolic state. Nprl3 KO by in utero electroporation in fetal (E14) mouse cortex resulted in mTOR-dependent cortical dyslamination with ectopic neurons in subcortical white matter. EEG recordings of these mice showed hyperexcitability in the electroporated hemisphere. NPRL3 variants are linked to a highly variable clinical phenotype likely as a consequence of mTOR-dependent effects on cell structure, cortical development, and network organization.