The Role Of Transcription Factor EB In Oxidative Stress Induced Cell Autophagy

Background: Amyotrophic lateral sclerosis(ALS) is an adult-onset and progressive neurodegenerative disorder with no cure. Autophagy plays an important role in the efficient quality control of cytotoxic protein aggregates which is critical for the pathogenesis of ALS. Transcription factor EB(TFEB) is the master regulator of autophagy.Object: Investigate how TFEB regulates autophagy in cells exposed to hydrogen peroxide and whether the process depends on the oxidative modification of TFEB itself.Moreover, indentify the oxidative modification site on TFEB and the mechanism underlying TFEB nuclear translocation in cells under oxidative stress.Method: HEK293 cells were transfected with plasmids of wild-type and mutant of TFEB that fused to green fluorescent protein. These cells were then subjected to exposure to hydrogen peroxide, Torin-1(mTOR inhibitor) and starvation. Observe and detect the distribution of TFEB in cells and the level of autophagy using fluorescent microscopy,nucleo-cytoplasmic separation assay and Western blotting.Results: Under the oxidative stress, wild-type TFEB moved into the cell nucleus rapidly while most mutant TFEB stayed in the cytoplasm and the level of autophagy was enhanced distinctly in cells with wild-type TFEB while the autophagy of cells with mutant TFEB almost kept at the same level. Both of wild-type and mutant TFEB needed a relatively longer time to move into the nucleus under the condition of being exposed to Torin-1 or nutrient deprivation.Conclusion:(1) Under the oxidative stress, TFEB moves into the nucleus to enhance autophagy, and this process depends on the oxidation of TFEB itself at the very beginning.This approach is significantly different from what in cells exposed to Torin-1 or starvation.(2) The designed mutant TFEB successfully confirmed the locus of the oxidative modification of TFEB.(3)H2O2 causes dissociation of the TFEB-Rag GTPases complexthen leading TFEB transport to the nucleus.