The lipofuscin component A2E selectively inhibits phagolysosomal degradation of photoreceptor phospholipid by the retinal pigment epithelium

Abstract

Daily phagocytosis of spent photoreceptor outer segments is a critical maintenance function performed by the retinal pigment epithelium (RPE) to preserve vision. Aging RPE accumulates lipofuscin, which includes N-retinylidene-N-retinylethanolamine (A2E) as the major autofluorescent component. We studied the effect of physiological levels of A2E in RPE cultures on their ability to phagocytose outer segments. A2E localized to lysosomes in cultured RPE as well as in human RPE in situ. A2E-loaded RPE cells in culture bound and internalized identical numbers of outer segments as control RPE indicating that A2E does not alter early steps of phagocytosis. A2E-loaded RPE degraded outer segment proteins efficiently but, strikingly, failed to completely digest phospholipids within 24 h. Because of the circadian rhythm of RPE phagocytosis in the eye, a delay in lipid degradation would likely result in a build up of undigested material in RPE that could contribute to the development of age-related macular degeneration.A ge-related macular degeneration (ARMD) is a degenerative disease that causes severe visual impairment in one fourth of the population over 65. Some predisposing genetic and environmental factors, such as oxidative stress, low eye pigmentation, excessive light exposure, and smoking, have been identified (1-3). However, the mechanisms underlying the pathogenesis of the disease remain largely obscure.Clinical and histopathological analysis of diseased eyes suggests that alterations in the retinal pigment epithelium (RPE) are key determinants of the disease (4). The most profound change of human RPE with age is the accumulation in the cytoplasm of lipofuscin granules, storage bodies with an autofluorescent mixture of lipids arising from incomplete digestion of phagocytosed photoreceptor (PR) outer segment (OS) membranes (5-7). One of the major components of RPE lipofuscin has been characterized as N-retinylidene-N-retinylethanolamine (A2E), a pyridinium bis-retinoid and photoinducible free-radical generator (8, 9). While human RPE in general accumulates lipofuscin with age, excessive lipofuscin accumulation is associated with a variety of hereditary retinal degenerations (10-12) and may correlate with ARMD (4).The identification and efficient synthetic production of A2E have enabled studies on the consequences of lipofuscin accumulation for RPE function (8, 9, 13). The chemical structure of A2E indicates that it may act as a lysosomotropic agent, abolishing the acidic pH gradient required for normal lysosome function. Indeed, Holz et al. reported that A2E accumulation alone is sufficient to inhibit the lysosomal turnover of endogenous proteins in cultured RPE (14). On the other hand, A2E did not directly reduce activities of a wide range of lysosomal hydrolases in in vitro assays (15). A2E can initiate apoptotic cell death in RPE and other cell types (16, 17) when present at high levels. None of the studies mentioned above measured cytoplasmic levels of A2E. In fact, at concentrations similar to those found in a...

The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells

“…We have previously shown that A2E affects neither the binding nor internalization of OS by RPE cells (8). Immediately after phagocytosis, total cholesterol levels in both control and A2E-loaded cells increased 3-fold relative to cells that were not fed OS.…”

“…We have previously demonstrated that A2E specifically delays the metabolism of phagocytosed OS lipids by the RPE, whereas lysosomal proteolysis is unaffected (8). Progressive lipid and cholesterol accumulation in the Bruch's membrane beneath the RPE has been identified as a contributing factor to age-related macular degeneration (AMD) (5).…”

The unconventional myosin-VIIa associates with lysosomes

“…Accumulation of undigested ROS material within the RPE, whether due to mutations in lysosomal proteases (Rakoczy et al 2002) or due to accumulation of natural non-degradative substrates (e.g. lipofuscin, Finnemann et al 2002), similarly causes photoreceptor cell death. Therefore, a tight coupling between photoreceptor outer segment shedding and RPE phagocytosis is necessary for retinal health.…”

Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt's macular degeneration

“…A2E has several potential cytotoxic effects on RPE cells, including destabilization of membranes (19)(20)(21), release of proapoptotic proteins from mitochondria (22,23), sensitization of cells to blue-light damage (24)(25)(26), impaired lysosomal acidification (27), and impaired degradation of phospholipids from phagocytosed outer segments (28). Irradiation of A2E with blue (430-nm) light resulted in a series of oxirane products containing up to nine epoxide rings, formed by addition of singlet oxygen to double bonds along the polyene chains (29).…”