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.

Figure 1

A2E accumulates in lysosomes of human RPE. Transverse cryosections of human eyes from healthy donors were immunolabeled with lamp-1 (A) or nonimmune antibody (B) and FITC-conjugated secondary antibody. Nuclei were labeled with DAPI (blue). Confluent d407 cells were fed with ethanol solvent (C) or A2E (D–F) for 6 h and either fixed immediately (D) or chased with fresh medium for 16 h and fixed (C, E, F). Samples were labeled with lamp-1 (C–E) or nonimmune antibody (F) and FITC-conjugated secondary antibody. Images show A2E signals detected in the rhodamine channel (A1–F1) and FITC signals (A2–F2). A3–F3 show merged red and green signals. Representative fields for each condition are shown.

Figure 2

A2E does not affect binding or internalization of OS. Confluent d407 cells were fed with increasing concentrations of A2E as indicated for 6 h followed by incubation in fresh medium for 16 h. All cells then received FITC-OS for 5 h during which RPE cells bind and internalize, but do not degrade OS. Binding and internalization indices are given as averages ± SD (n = 4).

Figure 3

A2E impairs degradation of FITC-OS components other than protein. Confluent d407 cells (A, C, D) or RPE-J cells (B) were challenged with FITC-OS (A–C) or apoptotic HL-60 cells (D). After 2 h, when control and A2E-treated cells carried identical levels of FITC-OS (data not shown), unbound OS were removed and cells further incubated. At different time points up to 24 h following the initial particle addition, samples were fixed and the total fluorescence was quantified and compared with the total particle count at 2 h, which was set as 100%. All values are averages ± SD, with n = 5 (A–C) and n = 3 for (D). Control d407 (A, ●) and RPE-J (B, ■) cells completely lost FITC-OS fluorescence within 24 h after initial OS challenge. In contrast, in the presence of A2E both d407 (A, ×) and RPE-J (B, ▾) cells significantly delayed FITC-OS clearance (Student's t test P < 0.002 for both cell types). (C) OS protein disappeared from both control d407 (●) and A2E-treated d407 cells (×) between 8 and 16 h following initial OS challenge. (D) DNA derived from phagocytosed apoptotic cells was efficiently degraded by control (●) and A2E-treated d407 cells (×) between 8 and 16 h after initial challenge with apoptotic cells.

Figure 4

FITC-OS lipid accumulates in RPE cells fed with A2E. (A) FITC-OS were extracted with organic solvents without treatment (lane 1), after mock treatment with methanol (lane 2), or after treatment with NaOH in methanol, which destroys glycerophospholipids (lane 3). Fluorescence scanning revealed fluorescent components of FITC-OS extracts separated by TLC. Note that the major fluorescent components of FITC-OS completely disappeared after treatment with NaOH. OS preparations that were not labeled with FITC did not contain fluorescent components (data not shown). (B) Control (empty bars) and A2E-treated d407 cells (filled bars) were harvested either immediately following 2 h of FITC-OS challenge, to determine total FITC-lipid uptake, or following further incubation in fresh culture medium until 16, 20, or 24 h following the initial addition of OS. Values represent relative amounts of remaining FITC-OS lipid compared with total FITC-OS lipid uptake, which is set at 100%. All values were normalized to cell numbers. Values given are averages ± SD of three independent experiments, each performed in triplicate. Absolute differences among triplicates were less than 10%. At 20 and 24 h, A2E-treated cells retained significantly more FITC-OS lipid than control cells (Student's t test, P < 0.05 at 20 h, P < 0.005 at 24 h).

Figure 5

A2E impairs the ability of d407 cells to degrade NBD-phosphatidylcholine (PC). d407 Cells in the absence (empty bars) or presence of A2E (filled bars) were subjected to lipid extraction at different times of chase following a 2 h incubation with liposomes containing fluorescent NBD-PC. In each of four independent experiments, cells treated with A2E retained significantly larger amounts of the fluorescent lipid than control cells (Student's t test for each P < 0.005). Values shown represent averages ± SD of four parallel samples from one experiment. After 4 and 6 h, control cells retained 29% and 15% of their initial NBD-phosphatidylcholine load, whereas A2E-treated cells retained 47% and 30%, respectively.