PROJECT SUMMARY/ABSTRACT The presence of lipid-rich deposits underneath the retinal pigment epithelium (RPE) is a pathologic feature of early age-related macular degeneration (AMD). Drusen development has been associated with RPE lipid metabolism, redox biology and extracellular matrix (ECM) degradation. Mutations in a gene affecting ECM degradation, tissue inhibitor of metalloproteinase 3 (TIMP3), results in a rare inherited retinal degeneration with similar clinical features to AMD, called Sorsby Fundus Dystrophy (SFD). The mechanism by which abnormal ECM turnover influences lipid metabolism and RPE redox resulting in the formation of sub-RPE deposits remains unknown. The goal of this proposal is to test the hypothesis that ECM degradation overloads the RPE with ECM-derived metabolites, resulting in the reprogramming of RPE towards lipid synthesis and mitochondrial oxidative metabolism. This in turn results in the deposition of excess lipids and reduced antioxidative capacity of the RPE. The proposed specific aims are: Aim 1. Determine the influence of ECM degradation on lipid metabolism. Our preliminary results show that increased ECM degradation in SFD RPE activates lipid synthesis and oxidation of branch-chain amino acids (BCAAs). BCAAs are ketogenic and abundant in the ECM. The goal of Aim 1 is to test the hypothesis that ECM degradation of protein-rich components reprograms RPE metabolism towards enhanced BCAA oxidation for lipid synthesis and lipid deposition. We will use quantitative proteomics, quantitative metabolomics, metabolic flux analysis, perifusion assays, CRISPR/Cas9 gene-editing, and single cell nuclear RNA-Seq of patient-derived iPSC RPE to comprehensively investigate the metabolic pathways in ECM remodeling and lipid deposition. Aim 2. Determine the influence of ECM degradation on redox metabolism. Our preliminary data show that both NADPH and glutathione are depleted in SFD RPE, and ECM-derived metabolites interfere with NADPH and glutathione metabolism. The goal of Aim 2 is to test the hypothesis that increased ECM turnover results in impaired NADPH and glutathione metabolism. We will quantify the metabolic flux of ECM degradation, determine the roles of ECM-related metabolites in NADPH production and glutathione synthesis, and restore cellular redox with different approaches to enhance antioxidative capacity. The proposed research will define the biochemical impacts of ECM turnover on RPE metabolism, including changes in lipid metabolism and oxidative stress, and identify the relationship between nutrient metabolism, protein synthesis and degradation, and redox biology in normal and disease-relevant RPE.