Abstracts

Rationale: We investigated possible mechanisms used by the stress of sleep deprivation to produce awakening seizures. The Importin8 (IPO8) variant, R638W, extremely rare (frequency 0.00000921 at ExAC browser) localized on chr. 12p11.21, and identified by whole exome sequencing, co-segregates with all eight clinical and EEG affected members of a large family with CAE and eyelid myoclonia evolving to JME. Importin 8 is a member of the Importin β family. R638W is absent in 777 controls matched for ethnicity/ancestral origin. Five other rare missense mutations of IPO8, one rare 5' UTR SNP and one novel 3' UTR deletion of transcript variant2 of IPO8 is present in nine of 295 families with CAE/JME (3%). Importin 8 is involved in the nuclear transport of the transcription factor EB (TFEB), the master regulator of autophagy and lysosome biogenesis. Methods: HEK 293 cells were transiently transfected with GFP tagged plasmids carrying mutated human full length IPO8 or the wild IPO8. Three days and 15~20 hours after cell seeding without changing media, western blots assessed expression levels of TFEB and autophagy marker, LC3B, and phospho MTOR at 10 min after stresses produced by hydrogen peroxide (160uM). Additionally, immunoprecipitation by GFP coupling magnet beads, and lysosomal localization was examined using Lysosomal Associated Membrane Protein 1 (LAMP1) antibody by confocal microscopy SP5. This study also sets stresses on cells by depleting nutrients. Total protein normalization was applied using Biorad stain-free gels and Chemidoc XRS+ System with Image Lab.  Results: IPO8 variants found in JME patients significantly increased TFEB (R638W N=6 P=0.019, Q322K N=3 P=0.0000004, S486Y N=3 P=0.014) and LC3B expression. This results in upregulation of autophagy 10 min after administration of hydrogen peroxide (160uM) under starvation in HEK cells transfected with recombinant IPO8. In spite of upregulation of autophagy, no significant differences in phospho-mTOR (Ser2448) expression in western blotting were observed. However, immunoprecipitation for the cytoplasmic fraction showed recombinant IPO8 interacted with phospho-MTOR (Ser2448). IPO8 variant iR638W increased p-MTOR expression compared to wild IPO8. Confocal microscopy showed increased lysosomes diffusion within cytoplasms in HEK cells transfected with plasmids containing pathogenic variant R638W. In contrast, lysosomes are clustered in perinuclear regions in HEK cells transfected with wild-type IPO8. Conclusions: IPO8 regulates autophagy and pathogenic IPO8 variants increases autophagy by aberrant interaction with p-MTOR. Upregulated autophagy and breakdown of lysosomal function during autophagy could be the trigger of JME seizures induced by stresses of sleep deprivation.  Funding: No funding