Comparative mechanistic study of RPE cell death induced by different oxidative stresses
Age-related macular degeneration (AMD) is a degenerative disorder of the macula, the region of central retina responsible for the greatest visual acuity. Although its mechanism is still unclear, oxidative stress associated with advanced age and dietary/environmental factors could drive AMD progression. Geographic atrophy (GA), which occurs in late dry AMD, is characterized by scattered or confluent areas of degradation of retina pigment epithelial (RPE) cells. The mechanism whereby RPE cells die under oxidative stress and in GA is still controversial. The goal of the current study is to compare the features of RPE cell death induced by different oxidative stresses, to determine whether they have common or distinct underlying mechanisms, and to identify potential universal therapeutic targets for GA. In the study, RPE cell death was induced both in vitro and ex vivo by 4-Hydroxynonenal (4-HNE), a major product of lipid peroxidation, sodium iodate (NaIO3) that has been widely used to model RPE cell death in dry AMD, a ferroptosis inducer RAS-selective lethal 3 (RSL3) or a necroptosis inducer shikonin. We found that RPE necroptosis and ferroptosis show common and distinct features. Common features include necrosome activation and lipid reactive oxygen species (ROS) accumulation, although lipid ROS accumulation is much milder during necroptosis. This supports cross talk between RPE ferroptosis and necroptosis pathways and is consistent with the rescue of RPE necroptosis and ferroptosis by Receptor-Interacting Protein Kinase (RIPK)1 inhibitor Necrostatin-1 (Nec-1) or in Ripk3-/- RPE explants. Distinct features include activated mixed lineage kinase domain like pseudokinase (MLKL) that is translocated to the cell membrane during necroptosis, which is not happening in ferroptosis. This is consistent with the failure to rescue RPE ferroptosis by MLKL inhibitor Necrosulfonamide (NSA) or in Mlkl-/- RPE explants. Using this framework, we found that 4-HNE and NaIO3 induced RPE cell death likely through necroptosis based on the molecular features and the rescuing effect by multiple inhibitors. Our studies suggest that multiple markers and inhibitors are required to distinguish RPE necroptosis and ferroptosis, and that necroptosis inhibitor Nec-1 could be a potential therapeutic compound for GA since it inhibits necrosome activation and lipid ROS accumulation occurred in both necroptosis and ferroptosis pathways.