The Mitochondrial-Targeted Peptide, SS31, Protects Murine 661W Cells from Oxidative Damage via Induction of Autophagy.

The goal of this study was to examine the impact of the mitochondrial-targeted antioxidant peptide, SS31, and its role in promoting autophagy in cone photoreceptor 661W cells that were subjected to oxidative damage. To do so, we examined the viability of 661W cells in the presence of increasing concentrations of H₂O₂ with or without SS31 pre-treatment using the MTT assay and by expression of autophagy and apoptosis-associated proteins LC3-II/I, P62, and caspase-3. Autophagy was evaluated by fluorescence microscopy in cells stained with monodansyl cadaverine (MDC). Autophagy was induced with rapamycin (Rap) and inhibited with bafamycin A1 (bafA1) followed by examination of Reactive oxygen species (ROS) levels in target 661W cells by fluorescence microscopy and flow cytometry. Annexin V/PI staining was used to evaluate the rate of apoptosis and mRNA sequencing (mRNA-seq) analysis (Illumina platform) was performed on H₂O₂-exposed 661W cells treated with SS31. Among our results, we observed a substantial and concentration-dependent decrease in 661W cell viability in response to H₂O₂-exposure; production of ROS, autophagy and apoptosis were induced at 8 h in response to exposure to 100 M of H₂O₂. Pre-treatment with 100 nM SS31 resulted in significant attenuation of H₂O₂-mediated cytotoxicity, together with reduced ROS production and enhanced autophagy observed in response to oxidative stress. Both Rap and bafA1 were used to modulate SS31-mediated autophagy; the impact of Rap was similar to that of SS31. By contrast, administration of bafA1 counteracted autophagy induced by SS31. Furthermore, mRNAseq analysis revealed that SS31 promoted significant alterations in gene expression in 661W cells and suggested that autophagy was induced via the mTORC1-mediated signaling. In conclusion, our results indicate that exposure to H₂O₂ resulted in reduced 661W cell viability via mechanisms associated with oxidative damage, apoptosis, and autophagy. Notably, we demonstrated that pre-treatment with SS31 protects 661W cells from H₂O₂-induced oxidative damage that may result in part from induction of autophagy via mTORC1-mediated signaling pathways.