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Nicholas Boyer, Daniel Higbee, Zsolt Ablonczy, Rosalie K. Crouch, Yiannis Koutalos; A2E and Lipofuscin Accumulation in the Retinal Pigment Epithelium of wild type and Abca4-deficient mice does not require Light Exposure. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3346.
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To test the effect of light exposure on the levels of lipofuscin and A2E in the retinal pigment epithelium (RPE). Current models of the formation of lipofuscin require photoactivation of rhodopsin and subsequent release of all-trans-retinal leading to the formation of major fluorophores such as A2E.
129/sv and Abca4-/- mice were reared in cyclic light or in darkness for up to 12 months. The Abca4-/- animals were on a 129/sv background. Dark-reared animals were exposed to dim red light only for monitoring and cage changes. A2E was measured from chloroform-methanol extracts of RPE-choroid samples with HPLC-UV/VIS spectroscopy. The identity of A2E was confirmed with tandem mass spectrometry. Lipofuscin fluorescence was measured from whole flattened eyecups with a 10× lens (NA = 0.3) on a SP2 Leica Laser Scanning Confocal microscope (excitation, 488 nm; emission, 565-725 nm) after removal of the retina. Samples were prepared under dim red light.
Both lipofuscin and A2E levels increased with age in both Abca4-/- and 129/sv mice. For each strain, there was no difference in the absolute levels and the rates of increase of lipofuscin and A2E between dark-reared and cyclic-light-reared animals. In 129/sv animals, A2E accumulated at a rate of ~1 pmol/eye/month, while in Abca4-/- animals at ~6 pmol/eye/month. In Abca4-/- animals, lipofuscin accumulated at a rate ~2x higher than in 129/sv.Fluorescence emission spectra (excitation, 488 nm) of lipofuscin granules from dark-reared 129/sv and Abca4-/- mice were very similar to each other and to those from cyclic-light-reared animals, with a broad peak at max ~ 610 nm.
The formation of lipofuscin and A2E in the RPE do not require the generation of all-trans-retinal through exposure to light. A major source of lipofuscin and A2E may be 11-cis-retinal, either free or as part of rhodopsin. The results also suggest the need to reexamine the role of the Abca4 protein.Support: NIH grants R01 EY014850 (YK), R01 EY004939 (RKC), R21 EY020661 (ZA/RKC), EY019065 (ZA), R24 EY14793 (MUSC vision core), Foundation Fighting Blindness, Inc. (Owings Mills, MD) (RKC); and unrestricted awards to the Departments of Ophthalmology at MUSC from Research to Prevent Blindness (RPB; New York); RKC is an RPB Senior Scientific Investigator.
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