Abstract The retinal pigmented epithelium (RPE) functions to maintain the outer blood-retinal barrier and to support pho- toreceptor function, including regeneration of visual pigment and turnover of outer segments. Dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among older Americans. Given that a major function of the RPE is to process photorecep- tor outer segments, the proper functioning of the RPE endosomal pathway is important for retinal health. Multi- ple lines of evidence indicate that one of the major culprits observed in RPE dysfunction is dysregulation in the endosomal pathway. It is thought that in AMD this dysregulation in RPE cells is at least in part responsible for the formation of drusen (protein- and lipid-rich extracellular deposits) between the basal lamina of the RPE and the pentalaminar collagen- and elastin-rich Bruch’s membrane (BrM). At present, the exact mechanisms for drusen formation are unknown. Since RPE-released exosomes and other extracellular vesicles (EVs) are essential parts of the endosomal pathway, we hypothesize that exosomes released from stressed RPE cells are distinct from those released from unstressed RPE cells, and that these exosomes are involved in the pathognomonic deposit formation and ECM changes that underlie the early and late stages of AMD. Accordingly, approaches to characterize these vesicles and modulate their release have high potential to give important insight to disease mechanisms and new treatment targets. Significantly, very little is known about RPE-released exosomes and other EVs. By evaluating two complementary in vitro and ex vivo AMD models, our overall goal for this project is to determine the role of exosome secretion in sub-RPE deposit for- mation and in ECM changes under conditions relevant to AMD, and whether pharmacological or gene therapy/biological therapeutic interventions are possible. In the first aim, we will characterize and quantify the protein and lipid composition of sub-RPE deposits, ECM and basal-side exosomes in RPE stressed by patho- physiological conditions implicated in AMD (oxidative stress, complement dysregulation, and age), while modulating exosome release by both pharmacological and genetic approaches. In the second aim we will characterize the protein and lipid content in drusen and BrM from human post-mortem eyes from normal aged and AMD donors, by exhaustive proteomic and lipidomic analyses with cutting-edge instrumentation and analysis methodologies. This work serves as a preclinical study and proof of principle to probe the potential of modulating exosome secretion and target exosome composition as a therapeutic approach for treating AMD.