Abstracts

Rationale: Loss-of-function mutations in KPTN, encoding the protein Kaptin, have been shown to cause megacephaly, neurodevelopmental delay, and seizures. Recently, KPTN was shown to form part of the pentameric KICSTOR complex. KICSTOR acts to recruit GATOR1 to the lysosomal surface resulting in mTOR inhibition when amino acids are scarce. However, the morphological and function consequences of KTPN mutations remain undefined in neurons. We hypothesize that CRISPR/Cas9-mediated knockout (KO) of Kptn will lead to enhanced cell size, altered process outgrowth, and altered subcellular localization of mTOR. Methods: Neuro2a (N2a) cells were used to define the characteristics of CRISPR/Cas9 mediated Kptn KO vs. scramble control and WT cells. N2a cells were transfected with a CRISPR/Cas9 Kptn guide RNA construct with an mCherry tag using lipofectamine LTX and Plus reagents. Transfected cells were incubated for 72 hours in serum-free media before being FAC sorted based on RFP fluorescence. FAC sorted cells were re-plated and grown to confluence.  Kptn KO cells were treated with rapamycin (150 nM, 60 min), torin1 (100 nM, 60 min), vehicle (DMSO), or left untreated. Lysates from these cells were probed for KPTN and phosphorylated ribosomal S6 protein (PS6 240/244) via Western assay. Soma size and changes in process outgrowth were defined in digital images of WT, scramble, or Kptn KO N2a cells with or without rapamycin (50 nM; 24 hr) or torin1 (50 nM, 24 hr) application (n=50 cells per group). Colocalization of mTOR on the lysosomal surface was defined in N2a cells probed with primary antibodies targeting KPTN, mTOR, and LAMP2 and fluorescent secondary antibodies (Alexa 488, Alexa 594, Alexa 647, respectively) after incubation in complete or amino acid free media. Images were taken on a spinning disk confocal microscope and the degree of colocalization between mTOR and the lysosome was quantified using a fluorescence colocalization assay. Results: N2a cells transfected with KPTN CRISPR/Cas9 were larger than control or wildtype cells based on direct measurement in digital images. Direct measurements of experimental N2a cells revealed an increased soma size and process outgrowth in KPTN KO compared to control (n=50 per group; p<0.05). Rapamycin or torin1 application prevented cell size enlargement following KTPN KO vs. scramble and WT cells (n=50 per group; p<0.05). Western assay revealed a decrease in KPTN with an increase in PS6 in KTPN KO vs. scramble and WT N2a cells. Rapamycin or torin1 application results in a decrease in PS6 in each group. However, Kptn KO cells incubated in amino acid free media displayed increased PS6 vs. scramble and WT cells. There was also a higher degree of mTOR/lysosomal colocalization in Kptn KO versus scramble and WT cells in AAF media (n=10; p<0.05). In scramble and WT N2a cells, there was increased colocalization between KPTN and LAMP2 in AAF media but not complete media conditions. Conclusions: We demonstrate changes in cellular morphology in CRISPR/Cas9-mediated KO of