Authors :
Presenting Author: Miyabi Tanaka, MD – GLAVREF
Viet-Huong Nguyen, PharmD – Chapman university
Iris Martinez Juarez, MD – Instituto Nacional de Neurología y Neurocirugía/ National Institute of Neurology and Neurosurgery Mexico
Reyna Duron, MD – Universidad Tecnológica Centroamericana | UNITEC
Aurelio Jara-Prado, phD – Instituto Nacional de Neurología y Neurocirugía
Toshimitsu Suzuki, phD – Nagoya city university
Adriana Ochoa, MS – Instituto Nacional de Neurología y Neurocirugía
Laura Guilhoto, MD – Universidade Federal de São Paulo, São Paulo
Jenny Wight, MPH – University of Southern California
Maria Alonso, MD – Tegucigalpa; National Institute of Neurology and Neurosurgery Manuel Velasco Suarez
Marco Medina, MD – National Autonomous University of Honduras
Kazuhiro Yamakawa, phD – University of Nagoya City University
Julia Bailey, phD – UCLA
Richard Olsen, phD – UCLA
Antonio Delgado-Escueta, MD – UCLA
Rationale:
A whole genome scan following whole exome sequencing for the large family of drug-resistant Juvenile myoclonic epilepsy (JME) patients identified a rare variant, R638W (Allele frequency 0.000009599 in the gnomAD browser v4.1.0), in IPO8 on 12p11.21, which is a nuclear-cytoplasmic transporter, a member of the importin β family, and contains a predicted CRM1 (Exportin1) domain. Heterozygous KO IPO8 mice demonstrated seizures by vaporized Isoflurane. JME patients have awaking seizures triggered by sleep deprivation, various stresses, or alcohol drinking, which cause oxidative stress in a cell. Therefore, our investigation targeted autophagy-related genes on oxidative stress.
Methods:
Using HEK cells transfected the plasmids containing an entire IPO8 sequence with/without a rare variant, protein quantification for nuclear and cytoplasmic fractions was performed by immunoblotting and immunoprecipitation (IP) by GFP beads. The terminal organelle of autophagy, lysosomes, was analyzed by confocal microscopy. We simulated the cellular condition as a JME seizure occurrence. ie, cells were analyzed 10 min after the addition of 160 uM of hydrogen peroxide on nutrient depletion by no change culture media.
Results:
Protein expression depends on the degree of the nutrient deficiency, ie, when the time to cell harvest from cell seeding is less than 96 hours (early starvation), nuclear TFEB and mTOR retention occurred, and LAMP1(lysosomal membrane protein1) increased significantly in cells with IPO8R638W (R638W cells). However, after 96 hours (progressive starvation), nuclear TFEB and mTOR were no longer prominent. During the whole starvation, cytoplasmic p-AKT (S473), total mTOR and p-p70S6K (T389) increased significantly. However, Rictor increased dominantly. Similarly, nuclear PARP1, required for autophagy induction, increased in R638W cells during early starvation, while in progressive starvation, it did not. According to the progression of starvation, nuclear and cytoplasmic parylated PARP1 increased upon oxidative stress. Cytoplasmic PARyation significantly increased, and abnormal PARyated proteins were observed in R638W cells. Microscopic analysis showed that nuclear PAR expression in IPO8 transfected cells was eliminated compared to that of untransfected cells, but it was retained in R638W cells under oxidative stress at early starvation. Moreover, oxidative stress caused lysosomal dispersion to the plasma membrane in R638W cells, while the wild cells showed perinuclear accumulation. Preincubation of PARP1 inhibitor (talazoparib) suppressed lysosomal peripheral translocation completely.Conclusions:
Our study clarified IPO8 functions in the nuclear-cytoplasmic transport of PARP1, mTOR, and parylated substances and regulation of PARylation. A rare variant, R638W, impaired export function at early starvation. However, PARP1 activation to create PARylation under oxidative stress continues during starvation in R6398W cells. PARP1 response against oxidative stress involves lysosomal translocation to the plasma membrane in these cells. This data strengthens our hypothesis that lysosomal exocytosis affects the cell membrane and triggers seizures.Funding:
NIH, Chapman startup fund,