Significant Role of Mitochondrial A and B Crystallins in Oxidative and ER Stress Induced Apoptosis in RPE

Abstract

Abstract: : Purpose: To investigate a) the subcellular changes of αA and αB crystallins in oxidative stress and endoplasmic reticulum (ER) stress to human RPE and b) the susceptibility of RPE from αA (–/–) and αB (–/–) mice to oxidative and ER stress. Methods: Human RPE in early passages (2–4) were starved in 1% FBS–containing DMEM for 6h and treated with 150µM H2O2 or 50ng/ml of the ER stressor tunicamycin (TM) for 24h. Four different methods were used to examine α–crystallin compartmentation: mitochondrial fractionation (BioVision), ultracentrifugation, confocal microscopy, and transmission electron microscopy (TEM). The effect of H2O2 and TM on apoptosis in RPE from αA (–/–), αB (–/–) and wild type (wt) mice was assessed by TUNEL assay. Expression of Bcl–2 was determined by western blot analysis. Results: Treatment of human RPE with H2O2 or TM resulted in a dose–dependent decrease in αB crystallin mRNA expression. Subcellular fractionation of RPE showed that H2O2 and TM treatments decreased cytosolic and mitochondrial pools of αA and αB crystallins significantly (p<0.02 vs controls). Immuno EM confirmed the mitochondrial depletion of α–crystallins with oxidative and ER stress. RPE from αA (–/–), αB (–/–) mice exhibited increased susceptibility to apoptosis induced by H2O2 and TM. Bcl–2 expression was significantly decreased in α–crystallin knockout RPE after oxidative and ER injury as compared to wt. Conclusions: The depletion of α–crystallins from mitochondria may be an important factor in increased susceptibility of RPE to oxidative events leading to cell death.

Keywords: crystallins • oxidation/oxidative or free radical damage • mitochondria