Ageing of Melanin in Retinal Pigment Ephitelial Cells

Melanin granules are a prominent structural feature of retinal pigment epithelial cells (RPE), but the biological functions of the pigment in RPE are incompletely understood. Aside from its accepted function of maximizing visual acuity by absorbing stray light that is not captured by retinal photoreceptors, RPE melanin has been postulated to protect cells from oxidative stress by acting as an efficient cellular antioxidant. For example, under some conditions melanin exhibits the antioxidant properties of scavenging reactive free radicals, quenching singlet oxygen and inhibiting lipid peroxidation. However, when irradiated with visible light, melanin has been shown to generate potentially damaging superoxide anion and hydrogen peroxide. Thus it remains speculative whether melanin has antioxidant properties, and, more importantly, whether melanin has antioxidant properties within the RPE cells that may protect cells from oxidative stress, especially photic stress. The question of the presumed antioxidant and cytoprotective properties of RPE melanin becomes even more important when considering the consequences of age. Unlike melanin in most pigmented tissues, RPE melanin is synthesized during embryogenesis and shows little if any turnover throughout life. Age-dependent modifications of RPE melanin are therefore not only possible, but also likely. How aging affects the antioxidant and cytoprotective properties of RPE melanin is unknown. A major goal of the project proposed in this application is to refine and use RPE cell culture models, including a model of experimentally aged cells, to study melanin`s properties in intact cells. We plan, therefore, to extend sophisticated biophysical analyses of isolated RPE melanin to include additional studies of melanin that has been aged in situ. To determine the biological relevance of the properties of melanin that are identified or induced, the same preparations of melanin will also be introduced by phagocytosis into cultured RPE cells. We will therefore analyze young and old melanin, intact and modified melanin within young and aged RPE cells. Finally, our long- term goal is to determine the effects of age on antioxidant systems that protect the RPE from oxidative stress.