Abstracts

Rationale: Loss-of-function (LOF) mutations in KPTN, encoding KAPTIN, cause megalencephaly, autism, and seizures. KPTN has been shown to form part of the KICSTOR complex recruiting GATOR1 to the lysosomal surface, resulting in mTOR inhibition when amino acids are scarce. We hypothesize that Kptn knockout (KO) will result in enhanced mTOR signaling, irrespective of nutrient conditions, and altered neuronal morphology in vitro and in vivo.

Methods: Kptn KO cell lines were generated using CRISPR/Cas9 knockout targeting exon 5 of Kptn in Neuro2a cells (N2aC). Kptn KO cells were treated with rapamycin, torin1, vehicle (DMSO) or left untreated. Lysates from these cells were probed for Kptn and phosphorylated ribosomal S6 protein (Ser 240/244; PS6) via Western assay. Soma size was assessed (Ammis FlowSight imager) and changes in process outgrowth were defined in digital images of KO, scramble, or WT N2aC with or without rapamycin application. Colocalization of mTOR on the lysosomal surface was defined in N2aC probed with primary antibodies targeting Kptn, mTOR, and Lamp2 after incubation in complete or amino acid free (AAF) media. Images were taken on a spinning disk confocal microscope, and the degree of colocalization between Kptn, mTOR, and lysosomes was quantified. Brains from Kptn KO mice (Wellcome Sanger Institute) were fixed in 4% PFA, paraffin embedded, and probed with primary antibodies targeting KPTN, PS6, Ki67, PCNA, and NeuN. Brain samples (KO and WT) were assessed for differences in cell size, proliferation, and process outgrowth between mice (ImageJ). Lysates from whole brain and liver were assessed for mTOR hyperactivation and Kptn expression by Western assay.

Results: : We observed reduced Kptn and enhanced PS6 in Kptn KO vs. scramble and WT N2aCs. Kptn KO cells incubated in AAF media still displayed increased PS6 vs control N2aCs. Kptn KO cells were larger than control cells based on direct measurement in digital images. There was a higher degree of mTOR/lysosome colocalization in KO versus scramble and WT cells in AAF media but not complete media conditions. Kptn KO brain specimens showed enlarged neurons and increased PS6 levels in the cortex and hippocampus. There was no significant difference in NeuN or Ki67 expression in KO vs WT sections. KO tissue lysates showed increased PS6 and decreased Kptn compared to WT.

Conclusions: We demonstrate changes in cellular morphology in Kptn KO both in vitro and in vivo. Kptn KO was associated with increased cell size and enhanced mTOR signaling. Pharmacological application of mTOR inhibitors rescued abnormal neuronal morphology and mTOR hyperactivation in vitro. Under nutrient deprived conditions, Kptn remains associated with the lysosome. These findings suggest that loss of KPTN in humans is associated with mTOR activation and that mTOR inhibition may be a novel strategy to treat KPTN associated malformations of cortical development.

Funding: Please list any funding that was received in support of this abstract.: NINDS 5R01NS099452-04.