Formation of lipofuscin-like material in the RPE Cell by different components of rod outer segments

Abstract

The mechanisms that control the natural rate of lipofuscin accumulation in the retinal pigment epithelial (RPE) cell and its stability over time are not well understood. Similarly, the contributions of retinoids, phospholipids and oxidation to the rate of accumulation of lipofuscin are uncertain. The experiments in this study were conducted to explore the individual contribution of rod outer segments (ROS) components to lipofuscin formation and its accumulation and stability over time. During the period of 14 days incubation of ROS, lipofuscin-like autofluorescence (LLAF) determined at two wavelengths (530 and 585 nm) by fluorescence-activated cell sorting (FACS) was measured from RPE cells. The autofluorescence increased in an exponential manner with a strong linear component between days 1 and 7. The magnitude of the increase was larger in cells incubated with 4-hydroxynonenal (HNE-ROS) compared with cells incubated with either bleached or unbleached ROS, but with a different spectral profile. A small (10–15%) decrease in LLAF was observed after stopping the ROS feeding for 14 days. The phagocytosis rate of HNE-ROS was higher than that of either bleached or unbleached ROS during the first 24 h of supplementation. Among the different ROS components, the increase of LLAF was highest in cells incubated with all-trans-retinal. Surprisingly, incubation with 11-cis-retinal and 9-cis-retinal also resulted in strong LLAF increase, comparable to the increase induced by all-trans-retinal. Supplementation with liposomes containing phosphatidylethanolamine (22: 6-PE) and phosphatidylcholine (18:1-PC) also increased LLAF, while incubation with opsin had little effect. Cells incubated with retinoids demonstrated strong dose-dependence in LLAF increase, and the magnitude of the increase was 2–3 times higher at 585 nm compared to 530 nm, while cells incubated with liposomes showed little dose-dependence and similar increase at both wavelengths. Very little difference in LLAF was noted between cells incubated with either unbleached or bleached ROS under any conditions. In summary, results from this study suggest that supplementation with various ROS components can lead to an increase in LLAF, although the autofluorescence generated by the different classes of components has distinct spectral profiles, where the autofluorescence induced by retinoids results in a spectral profile closest to the one observed from human lipofuscin. Future fluorescence characterization of LLAF in vitro would benefit from an analysis of multiple wavelengths to better match the spectral characteristics of lipofuscin in vivo.

Studying melanin and lipofuscin in RPE cell culture models

“…Over the past two and a half decades there have been a plethora of studies reporting that repeated phagocytosis of isolated photoreceptor outer segments leads to the progressive accumulation of autofluorescent granules within cultured primary RPE cells and RPE cell lines (Boulton et al, 1989; Guha et al, 2013; Kaemmerer et al, 2007; Lei et al, 2013; Nilsson et al, 2003; Thampi et al, 2012; Wihlmark et al, 1996). The first such report was by Boulton and colleagues who fed cultured primary human RPE cells purified bovine rod outer segments daily for up to 3 months (Boulton et al, 1989).…”

Ultrastructural changes in the melanocytes of aging human choroid

“…The presence of many lipid droplets in aged CM, as seen in this study, would indicate a possible age-related decrease of lysosomal capacity to degrade them, which may then trigger formation of lipofuscin-like granules in CM, as was noted in the choroid of 80-year old donors and beyond (Table 2). A similar process might also be operative in human RPE (Lei et al, 2013). RPE lipofuscin sequesters the remnants of proteins and lipids of shed photoreceptor outer segments in lysosomes.…”

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

“…Hence, understanding the effects of light and related oxidative damage may require POS challenge of RPE cells 18,26 . Finally, repeated feeding with normal 27 or oxidized 25,28 POS can be used to induce cumulative effects in RPE cells in culture in order to understand pathological mechanisms that develop in vivo over longer periods of time. Drawing showing the different steps of the eye dissection in order to collect and homogenize the retina before the ultracentrifugation step on continuous sucrose gradients, starting top left.…”

“…The alphavbeta5 integrin receptor located at the RPE apical cell surface, in coordination with its ligand MFG-E8, binds specifically to POS , myosin II 20 and myosin VIIA 21,22 . Native or oxidized POS in vitro are also utilized to understand aging phenotypes of RPE cells in vivo linked to accumulation of poorly digested oxidized POS [23][24][25][26][27][28] . The generation of RPE cells derived from stem cells has initiated a new application for isolated POS that are used to prove functionality of cells before they are transplanted to animals or patients 29,30,27 .…”