Sustained mitochondrial GSH is critical for retinal protection: characterization of GSH carrier proteins in RPE and protection by increased expression with αB crystallin chaperone peptide.

Abstract

Purpose : Glutathione (GSH) is a well-known endogenous antioxidant important in maintaining the cellular viability of human RPE (hRPE). Mitochondrial GSH (mGSH) is derived from the larger cytosolic pool and plays a significant role in cellular defense against oxidative injury. The aim of the present study was to characterize two specific transporters of GSH to mitochondria viz. oxoglutarate (OGC) and dicarboxylate (DIC), and to investigate the role of αB crystallin chaperone peptide in their regulation and cellular protection.

Methods : The expression and localization of OGC and DIC proteins in confluent hRPE, polarized hRPE, and mouse retina was assessed using immunoblotting and confocal microscopy. Time- and dose-dependent expression of the two carriers in mitochondria was determined in hRPE after treatment with H₂O₂ for 24h. Effect of polarity on their expression was assessed in highly polarized RPE (TER: 380±36 Ω.cm²) vs non-polarized controls. The effect of specific inhibition with phenyl succinate (OGC) and butyl malonate (DIC) as well as siRNA knockdown on apoptotic cell death (TUNEL) and mGSH levels was studied. In addition, kinetics of caspase 3/7 activation with OGC and DIC inhibition and effect of co-incubation with αB crystallin peptide (50-200 µg/ml; 24h) on RPE cell death was followed using IncuCyte live cell imaging.

Results : OGC and DIC are expressed in hRPE mitochondria and their expression showed a time-and dose-dependent decrease in stressed RPE. Induction of polarity upregulated OGC and DIC expression significantly (2-3 fold over non-polarized control). αB crystallin peptide restored cellular integrity (ZO-1) in stressed polarized RPE. The downregulation of the two carrier proteins by specific inhibitors and siRNA knockdown resulted in significantly increased apoptosis (P<0.001 vs control) and in marked mGSH depletion (P<0.05 vs control). αB crystallin peptide co-treatment prevented apoptosis through restoration of mGSH in RPE (P<0.05).

Conclusions : Our studies demonstrate the importance of mitochondrial GSH in RPE health and suggest that regulation of mGSH transporters may be a valuable approach in devising therapeutic strategies for retinal disorders associated with mitochondrial damage, such as AMD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.