Project Summary/Abstract Fuchs Endothelial Cornel Dystrophy (FECD), a common age-related dystrophy, which is more prevalent in women and smokers, is of unknown etiology. In FECD, corneal endothelial (CE) cell loss is accompanied by abnormal extracellular matrix (ECM) deposition in the form of guttae. Our laboratory was the first to link oxidative DNA damage and mitochondrial dysfunction in FECD pathogenesis. Specifically, we have shown that ultraviolet-A (UVA) light, by triggering estrogen oxidizing enzyme, CYP1B1, induced greatest FECD phenotype in female and estrogen-treated male mice due to mitochondrial DNA (mtDNA) damage. Estrogen and UVA induced ATM-driven G2/M cell cycle arrest; however, deficient DNA repair system, led to net DNA damage resulting in senescent and endothelial mesenchymal transition (EMT) phenotypes seen in FECD. Still, a unifying mechanism of how UVA and smoking induce greater FECD phenotype with guttae formation in aging females is unknown. Building upon our previous findings, we propose to investigate if UVA, age, and smoking– induced oxidant-antioxidant imbalance leads to polyploidy and fibrosis by causing cell cycle arrest-driven ECM deposition; and if this imbalance causes activation of aryl hydrocarbon receptor (AhR), the upstream inducer of CYP1B1, which leads to melatonin breakdown to N-acetylserotonin (increased NAS/melatonin ratio) and estrogen oxidation affecting mitochondrial DNA repair and biogenesis resulting in senescence seen in FECD. Our study is significant, as understanding the effect of environmental stressors on sex-dependent mechanisms involved in CE cell loss will provide new treatment targets for FECD. To achieve these aims, we will use our newly developed non-genetic mouse model of FECD along with immortalized human CE cell lines, aqueous humor, and ex vivo specimens of genotyped FECD donors. Our Specific Aims are: Aim 1: Determine whether UVA, age, and smoking lead to cell cycle-dependent polyploidization and subsequent senescence that results in profibrotic phenotype in FECD. This aim is based on the hypothesis that G2/M phase arrest leads to polyploidization of CE, which protects from acute injury, but with prolonged stress promotes senescence and fibrosis seen in guttae formation. Aim 2: Determine whether AhR and CYP1B1 activation, from UVA and smoking, increase NAS/melatonin ratio causing greater DNA damage and CE cell loss in females. This aim is based on the hypothesis that co-stimulatory effect of estrogen on AhR activates the CYP-family of enzymes that cause greater estrogen and melatonin metabolism in females in FECD. Aim 3: Determine the role of mitochondrial melatonin metabolism on DNA repair and mitochondrial biogenesis during the cell cycle arrest. This aim is based on the hypothesis that mitochondrial translocation of CYP1B1 increases NAS/melatonin ratio and leads to cell cycle-dependent DNA repair deficiency and mitochondrial dysfunction in FECD and aging.