SUMMARY: Age-related macular degeneration (AMD) is marked by retinal pigment epithelial (RPE) lipid dys- homeostasis, including pathologic extracellular lipid accumulation, impaired mitochondrial lipid metabolism, and increased lipid peroxidation, a toxic form of lipid oxidative damage. The RPE normally plays an essential role in retinal lipid homeostasis, consuming and secreting prodigious amounts of lipid. Lipid consumption comes in the form of a large daily lipid load, including both lipoprotein uptake from the choroid and phagocytosis of lipid-rich photoreceptor outer segments (OS). Some of this lipid is oxidatively metabolized in the mitochondria, producing metabolites that energetically support photoreceptors. Lipid oxidative metabolism also minimizes glucose use by the RPE, allowing glucose to pass unused from the choroid to highly glycolytic photoreceptors. Lipid secretion is primarily through lipoprotein, some of which supports regeneration of OS and some of which accumulates as the extracellular deposits typical of AMD. A characteristic of cells facing large lipid loads is the formation of lipid droplets (LD), intracellular lipid storage organelles that dynamically regulate lipid trafficking, metabolism, and lipid peroxidation. Specific LD function varies widely between different cell types. RPE LD have been studied in the context of visual cycle biology, but their physiology and role in maintaining non-retinoid lipid homeostasis in the RPE is essentially unexplored. This proposal examines the composition, physiology, and role of LD in human RPE lipid homeostasis. The over-arching hypothesis is two-fold: a) LD can temporarily store a wide-range of lipids derived from the RPE’s daily lipid load, preventing their lipid peroxidation; and b) lipid released from LD by specific enzymes is preferentially metabolized in mitochondria rather than secreted. By facilitating lipid oxidative metabolism over lipid secretion, RPE LD may enhance metabolic support for photoreceptors and decrease the accumulation of pathologic extracellular lipid seen in AMD. To test the hypothesis, various lipid loads will be fed to primary- and induced pluripotent stem cell (iPSC)-derived human RPE cultures, with LD composition assessed by mass spectrometry. The fate of lipids released from LD will be tracked via microscopy, biochemical methods, and lipidomics. The principal investigator’s long-term goal is to develop the expertise to define intracellular RPE lipid trafficking pathways that enhance RPE lipid homeostasis and decrease pathologic extracellular lipid deposition, opening up new therapeutic avenues for AMD. To achieve this goal, the principal investigator’s career development plan (CDP) focuses on RPE lipid biology, establishing expertise in iPSC-RPE culture, radiolabeled lipid tracing, high-resolution microscopy, bio-energetic profiling, and lipidomics. The CDP’s mentorship team takes advantage of deep expertise in lipid biology and lipidomics at University ...