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Yiannis Koutalos, Leopold Adler, Chunhe Chen; All-trans retinal levels and the generation of lipofuscin precursors in rod photoreceptors from wild type and Abca4-/- mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):37.
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© ARVO (1962-2015); The Authors (2016-present)
Following bleaching of rod photoreceptor outer segments, all-trans retinal is released by photoactivated rhodopsin. All-trans retinal is reduced to all-trans retinol and can also react with outer segment components to form lipofuscin precursors. The reduction of all-trans retinal to retinol requires metabolic input in the form of NADPH. We tested whether metabolic limitations that suppress the reduction of all-trans retinal also result in increased formation of lipofuscin precursors. Along with wild type we tested Abca4-/- mice, which show much higher accumulation of RPE lipofuscin.
Experiments were carried out with dark-adapted isolated photoreceptor cells from 129/sv and Abca4-/- mice. Animals were 2-3 months old. Isolated rods in physiological solution were placed on the stage of a fluorescence microscope at 37 0C. Experiments were carried out with physiological solutions containing different concentrations of glucose and glutamine as metabolic substrates. For an experiment, a cell was selected and bleached with >530 nm light (1 min exposure). The levels of all-trans retinal and all-trans retinol after bleaching were measured from the fluorescence excited by 380 and 340 nm light (em: >420 nm). Levels of rod outer segment lipofuscin precursors were determined from their fluorescence (ex: 490 nm; em: >520 nm).
Before bleaching, the levels of lipofuscin precursors were much higher in the outer segments of Abca4-/- compared to wild type rods. In both wild type and Abca4-/- rods all-trans retinal levels increased after bleaching, without any significant difference between the two strains. In both wild type and Abca4-/- rods bleaching in the presence of lower metabolic substrate concentrations resulted in higher all-trans retinal levels, which correlated with increased lipofuscin precursor formation.
Lack of Abca4 does not affect the clearance of the all-trans retinal generated by light in rod outer segments. Lipofuscin precursors can form in rod outer segments from the all-trans retinal released by light exposure. Conditions that limit the reduction of all-trans retinal result in increased formation of lipofuscin precursors.
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