Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss, primarily driven by oxidative stress–induced degeneration of retinal pigment epithelium (RPE). Erythropoietin (EPO), a hematopoietic cytokine with neuroprotective properties, has been shown to reduce apoptosis and retinal degeneration. In this study, we examined the cytoprotective role of a non-erythropoietic EPO variant, EPO-R76E, in suppressing oxidative stress and mitochondrial dysfunction related to oxidative stress in RPE cells. Stable ARPE-19 cell lines expressing EPO-R76E were generated via lentiviral transduction and exposed to paraquat to induce oxidative stress. Oxidative stress was induced using paraquat. EPO-R76E expression conferred increased cell viability and resistance to mitochondrial damage, as assessed by cytotoxicity assays. Western blot analysis revealed reduced expression of ferritin and p62/SQSTM1, diminished activation of p-AMPK and NRF2, and restoration of GPX4 levels, indicating enhanced antioxidant defenses. Moreover, intracellular iron accumulation and reactive oxygen species were significantly reduced in EPO-R76E-expressing cells exposed to paraquat. These findings suggest that EPO-R76E promotes mitochondrial homeostasis and modulates oxidative stress pathways. Our study positions EPO-R76E as a promising therapeutic candidate for halting RPE degeneration in AMD.

Retinal pigment epithelium (RPE) degeneration remains a formidable challenge in dry age-related macular degeneration (AMD) research, primarily due to the toxic interplay between iron overload and ferroptosis. We investigated whether EPO-R76E, a non-erythropoietic modified variant of erythropoietin, could effectively interrupt this destructive cycle. Using ARPE-19 cells challenged with ferric ammonium citrate (FAC) to model iron-induced toxicity, we show that EPO-R76E confers protection against ferroptosis. Our results demonstrate that this variant significantly reduces the intracellular labile iron pool, directly quenching the lipid peroxidation that drives ferroptotic cell death. This resilience is fueled by a robust upregulation of Glutathione Peroxidase 4 (GPX4) and the broad transcriptional activation of the NRF2 (Nuclear factor erythroid 2-related factor 2) NRF2 antioxidant axis. Furthermore, we found that EPO-R76E enhances autophagic flux, ensuring that cells maintain essential proteostasis and “housekeeping” functions even under metabolic crisis. By integrating iron sequestration with reinforced antioxidant signaling and cellular clearing mechanisms, EPO-R76E stands out as a potent candidate for preserving RPE health. These findings uncover a novel molecular framework for protecting the retina against iron-mediated injury, positioning EPO-R76E as a versatile and targeted gene-based therapeutic for addressing the fundamental causes of retinal degeneration.