Generation of NADPH and the formation of lipofuscin fluorophore precursors in human rod photoreceptors

With photoreceptor cell death being one of the major causes of vision loss, we undertook a study of photoreceptor metabolic competence by measuring the generation of NADPH, which is used in synthetic reactions and the reduction of all-trans retinal to all-trans retinol. All-trans retinal is released within rod photoreceptors during light detection and can form lipofuscin fluorophore precursors, which accumulate in the form of the cytotoxic pigment lipofuscin in the adjacent cells of the retinal pigment epithelium. We have used fluorescence imaging to measure the levels of all-trans retinal, all-trans retinol, and lipofuscin fluorophore precursors in single living rod photoreceptors isolated from human donor eyes. We supplied isolated rods with exogenous all-trans retinal and used its reduction to all-trans retinol to measure their capacity to generate NADPH. Although the exogenous all-trans retinal loads were much higher than those of the endogenous released during light detection, the cells were able to sustain the reduction of a steady 80 to 90% proportion to all-trans retinol, similar to that maintained for the reduction of the endogenously generated. Generation of NADPH required the presence of extracellular glucose. The capacity to generate NADPH deteriorated with time after donor death and isolation of the retina, resulting in increased formation of lipofuscin fluorophore precursors by the supplied all-trans retinal. This suggests that in the living eye, an initial deterioration of rod photoreceptor metabolism would result in increased lipofuscin accumulation and impairment of retinal pigment epithelium function, which would lead to further deterioration of photoreceptor cell health and eventual death.