Abstract
Abstract: :
Purpose: Aging retinal pigment epithelium (RPE) accumulates lipofuscin, which includes N-retinylidene-N-retinylethanolamine (A2E) as its major autofluorescent component. We incorporated synthetic A2E into RPE derived cells in culture and studied their ability to perform phagocytosis of photoreceptor outer segment fragments (OS) in vitro. Methods: A2E was fed to RPE cultures that serve as model systems for OS phagocytosis. We used fluorescence microscopy to determine subcellular A2E distribution, fluorescence quantification to measure A2E load, and TUNEL apoptosis assays to confirm cell viability. We challenged RPE (loaded with A2E at concentrations similar to those detected in aged human RPE) with bovine outer segment fragments to measure kinetics and capacity of OS binding and internalization. Furthermore, we determined the degradation rates of phagocytosed fluorescent OS, phagocytosed OS proteins, OS phospholipids, and synthetic phospholipids by RPE in culture. Results: Human (d407) and rat (RPE-J) cells accumulated A2E at levels comparable to those observed in aged human RPE without apparent toxicity or loss of lysosomal integrity. A2E co-localized with the lysosomal marker protein lamp-1 in cultured RPE cells, as well as in human RPE in situ. RPE cells in culture loaded with A2E bound and internalized identical numbers of OS as control RPE indicating that A2E does not alter early steps of phagocytosis. A2E-loaded RPE degraded OS proteins efficiently but, unlike control RPE, failed to completely digest OS phospholipids within 24 hours. Conclusion: Our results suggest that levels of A2E accumulation similar to those observed in the aged human eye do not affect OS uptake or protein degradation but selectively delay the processing of photoreceptor lipids. Due to the circadian rhythm of OS phagocytosis in the eye, such delay would likely result in a build-up of undigested material in RPE, that could contribute to the development of age-related macular degeneration.
Keywords: 567 retinal pigment epithelium • 308 age-related macular degeneration • 458 lipids