Inhibition of mitochondrial GSH carrier proteins leading to mitochondrial dysfunction and restoration of function with αB crystallin peptide in human RPE cells

Abstract

Purpose : We recently characterized two carrier proteins OGC (SLC25A11) and DIC (SLC25A10) for mitochondrial GSH (mGSH) in human RPE cells (Wang et al. IOVS 2019). Our aim in the present study is to investigate a) the effect of inhibition OGC and DIC on mitochondrial bioenergetics and function and, b) whether αB crystallin-mini chaperone peptide (αB Cry peptide) restores mitochondrial function.

Methods : Early passage confluent hRPE cells were used. OGC and DIC were inhibited with 5 mM-phenylsuccinic acid (PS) and 5 mM-butylmalonic acid (BM) as well as siRNA in the presence or absence of 75µg/ml αB Cry peptide for 24 h. Cell death was analyzed by TUNEL and caspase 3 staining. GSH was measured in mitochondrial and cytosolic fractions. Mitochondrial bioenergetics (Seahorse) and the regulation of total OXPHOS proteins were determined. Effect of treatment of polarized RPE monolayers with inhibitors alone, PS + αB Cry peptide and BM + αB Cry peptide was studied.

Results : Inhibition of GSH carrier proteins increased cell death as compared to the controls; PS and BM caused a significant (P < 0.01) increase over control and co-treatment with αB Cry peptide significantly decreased caspase 3 activation and cell death. Gene silencing of OGC yielded similar results; cell death was significantly (P<0.05) reduced in OGC silenced cells treated with αB Cry peptide. OGC and DIC inhibition caused a decline in mGSH vs untreated groups (P<0.05). αB Cry peptide co-treatment significantly upregulated mGSH to that of controls. However, there was no significant difference in GSH levels in the cytosol. Mitochondrial bioenergetics parameters were significantly (P<0.01) reduced with OGC and DIC inhibition and αB Cry peptide significantly (P<0.01) enhanced mitochondrial respiration. Further, OXPHOS proteins (I &V) were attenuated with OGC and DIC inhibition and upregulated by αB Cry peptide. In polarized RPE monolayers, inhibition of OGC and DIC decreased TER (Ω.cm2) by 50% and αB Cry peptide treatment restored TER to that of control.

Conclusions : Inhibition of OGC and DIC decreased mGSH, and augmented apoptosis in hRPE. αB Cry peptide significantly improved bioenergetics and cellular function. Our studies suggest that modulation of mGSH via its carrier proteins can be of therapeutic value.

This is a 2020 ARVO Annual Meeting abstract.