PROJECT SUMMARY Age-related macular degeneration (AMD) is the leading cause of irreversible blindness involving gradual dysfunction and degeneration of photoreceptors and the retinal pigment epithelium (RPE). Mitochondrial dysfunction represents a hallmark of AMD. Excessive and persistent reactive oxygen species (ROS) damages mitochondrial DNA (mtDNA), resulting in fewer mitochondria. Impairments in respiratory chain activity and reduced oxidative phosphorylation (OXPHOS) impacts ATP synthesis and further drives ROS production. Our prior work has shown that exogenous mitochondrial transplantation to cells can induce a bioenergetic shift towards increased OXPHOS and ATP production, and reduced ROS. Mitochondrial cell-to-cell transfer in response to stress rescues aerobic respiration to reduce deleterious cell dynamics. We aim to create a sustainable source of mitochondria proximal to the RPE in the form of mesenchymal stem cells (MSCs) transfected with nuclear respiratory factor 1 (NRF1), a driver of mitochondrial biogenesis, for enhanced cell-to- cell transfer of mitochondria. Our objectives are to determine how NRF1 transfection changes MSC transcriptional activity, mitochondrial production and trafficking, and extracellular vesicle (EV) contents, and test the strategy in two rodent models of retinal degeneration. We hypothesize that our cell therapy using NRF1- overexpressing MSCs will not only provide the needed energetic boost via mitochondrial mass transfer to retinal cells, but also induce profound cellular reprogramming through the secretion of EV-containing factors, thereby counteracting and potentially reversing the onset of late-stage dry AMD. We will use a combination of molecular and biochemical methods, including transcriptomics, to characterize the potency of mitochondrial transfer to impact AMD. We will first investigate the impact of NRF1 on mitochondrial transfer mechanisms in MSCs. We will elucidate the role of NRF1 on mitochondrial transfer mechan