Abstract
Oxidative stress in the retinal pigment epithelium (RPE) can cause mitochondrial dysfunction and is likely a causative factor in the pathogenesis of age-related macular degeneration (AMD). Under oxidative stress conditions, some of the RPE cells become senescent and a contributory role for RPE senescence in AMD pathology has been proposed. The purpose of this study is to 1) characterize senescence in human RPE; 2) investigate the effect of an αB Crystallin chaperone peptide (mini Cry) in controlling senescence, in particular by regulating mitochondrial function and senescence-associated secretory phenotype (SASP) production and 3) develop mouse models for studying the role of RPE senescence in dry and nAMD. Senescence was induced in human RPE cells in two ways. First, subconfluent cells were treated with 0.2 μg/ml doxorubicin (DOX); second, subconfluent cells were treated with 500 μM H2O2. Senescence biomarkers (senescence-associated beta-galactosidase (SA-βgal), p21, p16) and mitochondrial proteins (Fis1, DRP1, MFN2, PGC1-α, mtTFA) were analyzed in control and experimental groups. The effect of mini Cry on mitochondrial bioenergetics, glycolysis and SASP was determined. In vivo, retinal degeneration was induced by intravenous injection of NaIO3 (20 mg/kg) and subretinal fibrosis by laser-induced choroidal neovascularization. Increased SA-βgal staining and p16 and p21 expression was observed after DOX- or H2O2-induced senescence and mini Cry significantly decreased senescence-positive cells. The expression of mitochondrial biogenesis proteins PGC-1 and mTFA increased with senescence, and mini Cry reduced expression significantly. Senescent RPE cells were metabolically active, as evidenced by significantly enhanced oxidative phosphorylation and anaerobic glycolysis, mini Cry markedly reduced rates of respiration and glycolysis. Senescent RPE cells maintain a proinflammatory phenotype characterized by significantly increased production of cytokines (IFN-ˠ, TNF-α, IL1-α IL1-β, IL-6, IL-8, IL-10), and VEGF-A; mini Cry significantly inhibited their secretion. We identified and localized senescent RPE cells for the first time in NaIO3-induced retinal degeneration and laser-induced subretinal fibrosis mouse models. We conclude that mini Cry significantly impairs stress-induced senescence by modulating mitochondrial biogenesis and fission proteins in RPE cells. Characterization of senescence could provide further understanding of the metabolic changes that accompany the senescent phenotype in ocular disease. Future studies in vivo may better define the role of senescence in AMD and the therapeutic potential of mini Cry as a senotherapeutic.
Keywords:
Mitochondrial dysfunction; Oxidative stress; Retinal pigment epithelium; SASP; Senescence; Senolytic drugs; Subretinal fibrosis; αB crystallin peptide.