Oxidative stress is implicated in virtually every disease of aging, including age-related macular degeneration. However, exactly how oxidative stress contributes to age-related disease in different tissues is poorly understood because little is known about the cellular effects of chronic, sub-lethal stress. Lethal stress is commonly studied, but results of these studies have questionable relevance for aging, which is characterized by declining functional efficiency, not overt cell death. The long-term objective of the proposed research is to determine how chronic, sub-lethal oxidative stress to the retinal pigment epithelium (RPE), especially photic stress, contributes to functional impairments in the tissue that limit its ability to support the adjacent retina. Sub-lethal photic stress to the RPE was recently shown to have an unexpected functional consequence: organelle movement is impaired. The potential significance of this observation cannot be overstated. Organelle translocation is an ongoing cellular process essential for maintaining the polarized epithelial phenotype on which the RPE depends to efficiently support the function and survival of the retina. In the proposed project the mechanisms underlying light-induced organelle slowing in the RPE will be probed using melanosomes as a model. These pigment granules are an ideal model: they can be readily tracked for motility analysis by imaging technologies, they can be isolated from cells and used to retrieve the organelle- associated proteins that mediate motility, and they are themselves photoreactive due to the properties of their major constituent melanin, which can be experimentally manipulated to identify oxidative mechanisms. In the proposed project an RPE cell biologist and melanin biophysicist will collaborate to test three hypotheses: (1) Sub-lethal photic stress impairs RPE organelle movement by affecting the oxidation or phosphorylation state of organelle-bound proteins implicated in actin- or microtubule-based motility;(2) Motility slowing induced by sub-lethal photic stress is due to locally generated reactive oxygen species, including reactants produced by organelle contents that are themselves photoreactive;(3) The consequence of chronic photic stress to the RPE, with its attendant impairment of organelle translocation, is the loss of a functional polarized phenotype. Methods proposed for use include electron spin resonance (ESR) spectroscopy, biochemical analyses of protein oxidation and phosphorylation, immunostaining and confocal imaging of cultured RPE monolayers, and live cell imaging analysis of organelle translocation.
RPE cells support the survival of retinal photoreceptors, but as RPE cells age they function less efficiently due in part to oxidative stress, including photic stress from a lifetime of light exposure, which is believed to contribute to diseases of the retina such as age-related macular degeneration. In this project photic stress will be studied to determine how it interferes with the ability of RPE cells to move sub-cellular components called organelles, which must be effectively and continuously moved to achieve the most efficient cell architecture. Antioxidants will also be tested to see if binding them to organelles helps the RPE sustain normal organelle motility despite ongoing photic stress associated with aging.
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