The Age Lipid A2E and Mitochondrial Dysfunction Synergistically Impair Phagocytosis by Retinal Pigment Epithelial Cells

Abstract

Accumulation of indigestible lipofuscin and decreased mitochondrial energy production are characteristic age-related changes of post-mitotic retinal pigment epithelial (RPE) cells in the human eye. To test whether these two forms of age-related impairment have interdependent effects, we quantified the ATPdependent phagocytic function of RPE cells loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis either pharmacologically or genetically. We found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibited oxidative phosphorylation (OXPHOS) of RPE cells. Furthermore, in media with physiological concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis. Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes. Because mitochondrial mutations accumulate with aging, we generated novel genetic cellular models of RPE carrying mitochondrial DNA point mutations causing either moderate or severe mitochondrial dysfunction. Exploring these mutant RPE cells we found that, by itself, only the severe but not the moderate OXPHOS defect reduces phagocytosis. However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE with moderate OXPHOS defect and cause cell death of RPE with severe OXPHOS defect. Taken together, RPE cells rely on OXPHOS for phagocytosis when the carbon energy source is limited. Our results demonstrate that A2E accumulation exacerbates the effects of moderate mitochondrial dysfunction. They suggest that synergy of sub-toxic lysosomal and mitochondrial changes in RPE cells with age may cause RPE dysfunction that is known to contribute to human retinal diseases like agerelated macular degeneration. Retinal pigment epithelial (RPE)5 cells form a polarized monolayer epithelium between the photoreceptors of the neurosensory retina and the choroidal capillary bed. Daily phagocytosis of outer segment (OS) tips shed by adjacent photoreceptors is a vital task of the RPE (recently reviewed by Strauss (1)). RPE cells are post-mitotic and face each ϳ30 photoreceptor outer segments in the human eye, all of which shed their distal tip containing stacked membrane disks once a day. Diurnal phagocytosis and digestion of thousands of OS disks for life renders RPE cells the most active phagocytes in the body. Photoreceptor function strictly depends on efficient RPE phagocytosis of spent OS. Complete failure of RPE cells to engulf OS causes rapid photoreceptor degeneration in the Royal College of Surgeons rat (2-4). Impaired RPE phagocytosis also contributes to human retinal disease such as retinitis pigmentosa and, likely, age-related macular degeneration (5, 6).The continuous nature of outer segment renewal implies that any delay in OS removal by aged or damaged RPE will gradually cause OS components to accumulate. RPE cells are at risk for oxidative damage due to their location in the highly oxygenated environment of the outer retina and th...

Toll-like Receptor 3 Is Required for Development of Retinopathy Caused by Impaired All-trans-retinal Clearance in Mice

“…Toxicity of all-trans-retinal as a reactive aldehyde includes increased cell permeability and mitochondrial impairment (29). Accumulated conjugates of all-trans-retinal, A2E, and alltrans-retinal dimer that can induce RPE death by oxidative stress and inhibit RPE phagocytosis (58,59) were observed in vitro and in animal investigations. Amounts of accumulated A2E directly reflect the quantity of all-trans-retinal formation during the retinoid cycle.…”

Disrupted in schizophrenia 1 (DISC1) is a constituent of the mammalian mitochondrial contact site and cristae organizing system (MICOS) complex, and is essential for oxidative phosphorylation

“…Oxygen consumption was measured in intact cells using 1 mM pyruvate as substrate, with or without 1 mM of the uncoupler FCCP, and 1.5 mM KCN as the terminal inhibitor, in an Oxygraph chamber equipped with a Clark-type electrode (Hansatech) at 37 C, as described in (57). For complex-driven respiration we proceeded as described (59), with minor modifications.…”

“…Live imaging was performed as described previously (57). The uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone (3 mM) was added to the cells to depolarize mitochondria and measure residual, nonDWm-dependent fluorescence, which was subtracted from the initial fluorescence values.…”

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

“…Indeed, during normal aging and/or when POS are not digested properly, POS degradation products accumulate gradually as lipofuscin deposits and can lead to pathologies due to oxidation mechanisms 24,27 . 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.…”

“…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 .…”