Macular degeneration is a heterogeneous group of disorders characterized by photoreceptor degeneration and atrophy of the retinal pigment epithelium (RPE) in the central retina. An autosomal dominant form of Stargardt macular degeneration (STGD) is caused by mutations in ELOVL4, which is predicted to encode an enzyme involved in the elongation of long-chain fatty acids. We generated transgenic mice expressing a mutant form of human ELOVL4 that causes STGD. In these mice, we show that accumulation by the RPE of undigested phagosomes and lipofuscin, including the fluorophore, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hyydroxyethyl)-4-[4-methyl-6-(2,6,6,-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E) is followed by RPE atrophy. Subsequently, photoreceptor degeneration occurs in the central retina in a pattern closely resembling that of human STGD and age-related macular degeneration. The ELOVL4 transgenic mice thus provide a good model for both STGD and dry age-related macular degeneration, and represent a valuable tool for studies on therapeutic intervention in these forms of blindness.phagosome ͉ Stargardt disease ͉ photoreceptor ͉ retinal pigment epithelium M acular degeneration involves the death of photoreceptor cells in the central retina, which is responsible for fine-detail vision. Age-related macular degeneration (AMD) affects Ϸ30% of people over the age of 75 (1, 2), and is becoming a greater health problem with the rapidly growing elderly population of developed countries. There is no treatment to halt or reverse the disease for the dry form, which comprises Ϸ90% of AMD cases. Moreover, there is a lack of suitable animal models for experimentation on therapies for dry AMD. Stargardt macular dystrophy (STGD) shares pathological features with AMD, except that it occurs at a young age. Both AMD and STGD are characterized by the accumulation of high levels of lipofuscin in the retinal pigment epithelium (RPE), which precedes degeneration of the photoreceptors in the macula and RPE atrophy.The gene responsible for an autosomal dominant form of STGD, STDG3, was identified recently as ELOVL4 (3, 4). It is predicted to encode an enzyme involved in the elongation of very long-chain fatty acids (hence the name, ELOVL), and is highly expressed in rod and cone photoreceptor cells (5, 6). Sequence analysis of human ELOVL4 cDNA predicts a protein of 314 aa that shares homology with members of the yeast Elo (elongation of long chain fatty acid) family and the human ELO1 homolog (HELO1) (3). HELO1 and the ELO family members possess biochemical features that suggest their participation in reduction reactions occurring during fatty acid elongation (7,8). Mutational analysis of the ELOVL4 gene in five large STGD-like macular dystrophy pedigrees revealed a 5-bp deletion, resulting in a frame-shift and the introduction of a stop codon, 51 codons from the end of the coding region (3). Subsequently, two 1-bp deletions, 789delT and 794delT, in ELOVL4 were identifi...

The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.The di-retinal conjugate A2E forms as a consequence of light related vitamin A cycling in the retina. This orange-emitting fluorophore is formed synthetically as the condensation product of all-trans-retinal and ethanolamine (1-3). NMR and corroborative total chemical synthesis revealed A2E to be a pyridinium bisretinoid consisting of an unprecedented pyridinium polar head group and two hydrophobic retinoid tails (4, 5). A2E, its slightly less polar photoisomer, iso-A2E, and other minor cis-isomers together constitute the most prominent age-related hydrophobic pigments (lipofuscin) in retinal pigment epithelial (RPE) 1 cell extracts assayed by reverse phase HPLC (3, 6). In vivo, A2E is generated by phosphate hydrolysis of the fluorophore phosphatidylpyridinium bisretinoid, the latter precursor forming from reactions between all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane (3,7,8).Although certain levels of A2E are clearly tolerated by RPE cells, adverse effects of its accumulation have also been reported. Thus, not only can A2E mediate detergent-like effects on cell membranes (9), its accumulation can also lead to the alkalinization of lysosomes (10) and to the detachment of proapoptotic proteins from mitochondria (11). A2E also bestows a sensitivity to blue light damage (11-13) that is proportional to t...

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...