Alterations in Mitochondria-Associated Endoplasmic Reticulum Membranes Under Oxidative Stress in R28 Cells

Abstract This study aimed to investigate alterations in mitochondria-associated endoplasmic reticulum membranes (MAMs) under oxidative stress conditions in R28 retinal precursor cells. Hydrogen peroxide (H2O2) and hypoxia (0.2% oxygen) were used to induce oxidative stress in R28 cells. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential, mitochondrial calcium concentration, MAMs, and autophagy were evaluated. Mitofusin-2 (MFN2), a protein that plays a role in the structural regulation of MAMs, was either upregulated or downregulated in R28 cells to further explore the potential role of MFN2 in modulating MAMs. Oxidative stress not only modulated MAMs but also induced mitochondrial membrane potential (MMP) and increased mitochondrial calcium concentration. H2O2 and hypoxia also induced oxidative stress-associated autophagy in R28 cells. Additionally, autophagy inhibition increased MAM signaling. MFN2 silencing increased MAM expression and induced MMP collapse under normal conditions, whereas MFN2 upregulation rescued hypoxia-induced MAM alterations and alleviated MMP collapse. However, Mfn2 overexpression further enhanced H2O2-induced MAMs and failed to rescue MMP collapse. Our study indicates that MAMs possibly serve as a membrane source for oxidative stress-associated autophagy in R28 cells. MFN2 appears to play a negative role in maintaining MAM biogenesis under hypoxic conditions; however, its role in H2O2-associated MAM dysfunction remains unclear.