(430) TFEB nuclear retention, mTOR activation by rare variants of IPO8 found in childhood absence epilepsy (CAE) evolving to Juvenile myoclonic epilepsy (JME).

Rationale: We have identified an extremely rare variant, R638W, in importin 8 (IPO8) on 12p11.21 through whole-exome sequencing in a large Mexican family with a 37-member on four-generation with CAE evolving to JME affected members. IPO8 is a nuclear-cytoplasmic transporter, a member of the Importin β family, contains a predicted CRM1 domain. R638W cosegregated with all eight clinical and EEG affected members and absent in 777 controls matched for ethnicity/ancestral origin. Six other rare missense mutations of IPO8 were found in ten of 295 families with JME. IPO8 is involved in the nuclear translocation of TFEB, a master regulator of lysosomal biogenesis and autophagy (PMID: 26168401)

Methods: HEK 293 cells were transiently transfected with plasmids carrying human full length IPO8 with/without a rare variant. Western blots assessed expression levels of TFEB, p-P70S6K, LC3B (autophagosome marker) and LAMP1 (Lysosomal-associated membrane protein 1), 10 min after administration of hydrogen peroxide (160uM). Simultaneously samples were fixed by Methanol and studied lysosomal localization by LAMP1 antibody using Leica confocal microscopy SP8. Total protein normalization was applied using Biorad stain-free gels and Chemidoc XRS+ System with Image Lab.

Results: TFEB significantly increased in the nucleus under hydrogen peroxide (160uM) administration on nutrient-depleted conditions in the variants. It increased LAMP1 and LC3B. Importantly, phospho-p70S6K also increased in the variants. Confocal microscopy showed diffuse LAMP1 with extension to the plasma membrane in IPO8 variants, but perinuclear accumulation in the wild-type. Under the administration of 200nM of Wortmaninn, the mTOR inhibitor and the PI3K inhibitor, the wild-type decreased TFEB expression in the nucleus context-dependently. But the nuclear export inhibitor leptomycin did not change. While IPO8 variants consistently increased TEFB concentration in the nucleus.

Conclusions: We conclude IPO8 functions nuclear export of TFEB, and regulation of mTOR. Nuclear retention by variants requires phosphorylation of TFEB by activated mTORC1 in the nucleus. Nuclear retention of TFEB with activated mTORC1 results in aberrant localization of increased lysosomes, and defective fusion with autophagosomes and lysosomes. We suggest this autophagy disruption under oxidative stress is involved with awakening seizure formation.

Funding: Please list any funding that was received in support of this abstract.: National Institutes of Health (1R01NS055057)
VA Merit Review (5I01CX000743)
Startup funds from Chapman University School of Pharmacy to Nguyen VH
Laragen Miseq sequencing grant