Microtubule Defect Involved in 'Mitophagy Resistance' Under Subacute Oxidative Stress - Potential Mechanism for Cellular Inflammation

Abstract

IntroductionOxidative stress is considered an essential mechanism in ICU-acquired weakness. The roles of oxidative stress in autophagy/mitophagy dysfunction remains elusive. Microtubule serves as an essential guide rail for auto/mitophagosome trafficking required for proper maturation of auto/mitophagosomes in normal circumstances, and microtubules network formation is regulated by signal transduction mechanisms involving Akt, GSK3{beta}, and the microtubule plus-end tracking molecule, EB1. We have investigated (1) whether oxidative stress affects this pathway, leading to the defective mitophagy response, and (2) whether trehalose, an auto/mitophagy modulator, can ameliorate these pathological conditions. MethodsBy stably transfecting markers for auto/mitophagy or MT synthesis, we have established a few new C2C12 myocyte cell lines, expressing, GFP-LC3, EB1-GFP, and/or tandem-fluorescence LC3 (tfLC3). To monitor microtubule network, the cells were stained by SiR-tubulin. The cells were cultured in the presence or absence of oxidative stress by hydrogen peroxide (H2O2) and treated with or without trehalose. The response of mitophagy parameters including vesicle motion and the maturation status was monitored by stimulating the cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an established mitophagy inducer, under a time-lapse confocal microscopy. Signal transduction mechanisms linking mitophagy to microtubule formation was analyzed by Western Blotting against Akt and GSK3{beta}. ResultsCells under the oxidative stress, showed abolished MT network formation, decreased microtubule synthesis by EB1, and a decrease in CCCP-invoked response of mitophagosome motion, perturbed mitophagosome maturation, and increased superoxide production. Signal resistance of Akt/GSK3{beta} pathway to mitophagic stimulation, was documented. Trehalose treatment reversed signal resistance, diminished MT synthesis, ameliorated the disturbed MT network, and improved maturation defects, suppressing the production of superoxide. ConclusionsOxidative stress decreases the response of mitophagy and abolishes microtubule network. Trehalose improves the synthetic ability of microtubule and normalized the disturbed microtubule network, resulting in the improvement of the perturbed mitophagosomes maturation under the oxidative stress.