Abstract
The age-dependent accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been associated with the development of retinal diseases, particularly age-related macular degeneration and Stargardt disease. A major component of lipofuscin is the bis-retinoid N-retinylidene-N-retinylethanolamine (A2E). The current model for the formation of A2E requires photoactivation of rhodopsin and subsequent release of all-trans-retinal. To understand the role of light exposure in the accumulation of lipofuscin and A2E, we analyzed RPEs and isolated rod photoreceptors from mice of different ages and strains, reared either in darkness or cyclic light. Lipofuscin levels were determined by fluorescence imaging, whereas A2E levels were quantified by HPLC and UV-visible absorption spectroscopy. The identity of A2E was confirmed by tandem mass spectrometry. Lipofuscin and A2E levels in the RPE increased with age and more so in the Stargardt model Abca4(-/-) than in the wild type strains 129/sv and C57Bl/6. For each strain, the levels of lipofuscin precursor fluorophores in dark-adapted rods and the levels and rates of increase of RPE lipofuscin and A2E were not different between dark-reared and cyclic light-reared animals. Both 11-cis- and all-trans-retinal generated lipofuscin-like fluorophores when added to metabolically compromised rod outer segments; however, it was only 11-cis-retinal that generated such fluorophores when added to metabolically intact rods. The results suggest that lipofuscin originates from the free 11-cis-retinal that is continuously supplied to the rod for rhodopsin regeneration and outer segment renewal. The physiological role of Abca4 may include the translocation of 11-cis-retinal complexes across the disk membrane.
Highlights
Toxic components of lipofuscin in the retina are proposed to arise from all-trans-retinal, a by-product of light detection
We found no significant differences of lipofuscin or A2E levels between animals reared in the dark and those reared in cyclic light
Lipofuscin Is Present in the retinal pigment epithelium (RPE) of Dark-reared Animals— Fluorescence images of RPE from dark-reared wild type and Abca4Ϫ/Ϫ animals reveal the presence of the characteristic orange lipofuscin granules (Fig. 1, A and B)
Summary
Toxic components of lipofuscin in the retina are proposed to arise from all-trans-retinal, a by-product of light detection. In the context of this model, ABCA4 is proposed to function as a transporter of N-retinylidene-PE that has been formed from the all-trans-retinal released by photoactivated rhodopsin [17]. The proposed models for lipofuscin and A2E accumulation in normal and Abca4-deficient retinas posit free all-trans-retinal as a necessary precursor, the generation of which requires light. Lipofuscin levels were considerably lower, and A2E was undetectable in Rpe65Ϫ/Ϫ animals These results demonstrate that light activation of the visual pigment is not necessary for the formation of lipofuscin or A2E, suggesting that other forms of retinal may be involved. Pursuing the potential identity of these forms of retinal, we have detected lipofuscin-like fluorophores in the outer segments of dark-adapted isolated rod photoreceptors, including those from dark-reared animals. The results are consistent with ABCA4 working in the same direction as other ABCA transporters [30], translocating the substrate away from the cytoplasm, i.e. from the cytosolic to the intradiscal leaflet of the disk membrane
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