Drusen proteins are released in association with exosomes

Abstract

Purpose : Despite our growing understanding of dry AMD, effective treatments remain elusive. In the early stages of AMD, drusen accumulate between the RPE and the Bruch's membrane, leading to RPE atrophy and ultimately, central vision loss. Exosomes, cell-derived extracellular vesicles containing proteins and RNAs have been found in drusen of AMD patients. Exosomes play a role in spreading toxic-aggregated proteins in other diseases. Our goal is to recapitulate key features of AMD in human RPE, including drusen-like deposits and, to characterize the cargo of secreted exosomes under cellular stress.

Methods : We established an efficient protocol to produce functionally RPE cells from hiPSC. We induced oxidative stress in the hRPE by exposure to cigarette smoke extract. To characterize exosomes secreted by hRPE cells, we analyzed the concentration, morphology, and proteomic content of released exosomes. We validated the proteins found in exosomes by western blot. For exosome uptake assays, exosomes released by GFP-hRPE were co-cultured with wt hRPE, followed by PCR and immunofluorescence analysis to evaluate internalized exosomes in the recipient cells.

Results : We have established an efficient strategy to derive functionally RPE monolayers from hiPSC. We introduced a model that combines several key aspects of AMD and then, characterize the directional exosomal protein cargo of hRPE. Proteomic profiling of hRPE exosomal cargo revealed that exosomes were selectively enriched in proteins involved in oxidative stress, immune response, inflammation, and drusen-associated proteins pathways. Interestingly, exosomes secreted by hRPE cells within an AMD environment significantly enhanced the release of AMD-associated proteins. Finally, we confirmed the uptake of exosomal proteins and mRNAs released from hRPE cells by recipient cells.

Conclusions : This work represents the first proteomic profile from exosomes released by hiPSC-derived RPE and the first demonstration that proteins linked to drusen are released in association with exosomes. The exosomal cargo suggests a role for exosomes in both, normal physiology of the retina and the progression of retinal disorders. The identification of well-known drusen proteins secreted in exosomes also suggests a potential involvement of exosomes during drusen development. The confirmation that exosomes released from hRPE are taken up by target cells opens new avenues for possible therapeutic interventions.

This is a 2021 ARVO Annual Meeting abstract.