Grant: $380,000 - Department of Health and Human Services - Jul. 20, 2009
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Award Description: The long-term objective of the proposed research is to determine the effects of age on the susceptibility of the retinal pigment epithelium (RPE) to oxidative stress. Oxidative stress to the RPE is believed to decrease the support that the RPE provides to the adjacent retina, contributing to diseases like age-related macular degeneration. The pigment melanin, which has antioxidant properties, could theoretically be an important RPE antioxidant. However, when melanin is irradiated with visible light it can become a pro-oxidant. It is therefore not clear whether melanin actually protects RPE cells from oxidative damage or does the reverse. Further, within cells melanin is sequestered in a granular organelle, the melanosome, that can restrict interaction between melanin and damaging species. So whatever function melanin performs it is likely limited to the near domain of the granule. Additionally, RPE melanosomes are long-live organelles that could undergo aging changes that alter their redox function. The hypothesis of this proposal is that the melanosome can either decrease or increase oxidative damage to the RPE. Which function it performs is determined by aging, which makes the melanosome more pro-oxidizing due to changes in the physicochemical properties of its major component melanin. In this project a cell biologist and a biophysicist will use innovative strategies developed to measure pro- and antioxidant effects in the immediate domain of pigment granules, both isolated and within RPE cells. ‘Young’ and ‘aged’ RPE melanosomes or control particles will be used in experiments to address the following specific aims: (1) To determine whether and how isolated RPE melanosomes modify the oxidation state of nearby, granule-associated proteins and lipids. Melanosomes will be coated with specific molecules and then subjected to photosensitized and other oxidizing systems under conditions that discriminate different mechanisms of action of melanin. (2) To determine whether and how melanosomes within living RPE cells affect cellular responses to oxidative stress detected by a dynamic and sensitive measure of cell survival. Methods will include electron spin resonance (ESR) spectroscopy, biochemical analyses of protein oxidation and lipid peroxidation, and novel applications of time-lapse imaging to quantify the responses of living RPE cells to lethal oxidative stress induced by visible light or chemical oxidants
Project Description: We have conducted experiments relating to the project's second specific aim in which one goal is to determine whether RPE melanosomes protect cells against oxidative stress from the chemical oxidant hydrogen peroxide (H2O2). We are pursuing two types of investigations. In one series of studies we are refining methods for exposing cultured cells to H2O2. Usually the agent is delivered in a single pulse, followed by assaying cells for toxicity at pre-selected intervals thereafter. However, H2O2 rapidly decays in culture medium so the starting concentrations are often unphysiologically high and are sustained for only a short time. We are testing methods in which H2O2 is delivered to cultures not as a single high pulse, but by continuously generating the agent from glucose in the culture medium by adding the enzyme glucose oxidase. When this protocol is developed we plan to use it to more effectively simulate the type of stress that is likely relevant for aging in which the oxidant environment is chronically rather than acutely elevated. In a second series of studies we are directly testing the cytoprotective (or cytotoxic) effects of RPE melanosomes on H2O2 – induced stress by first loading cells by phagocytosis with pigment granules or with control latex particles in known numbers. After H2O2 treatment we use a newly-developed dynamic assay for cell death in which the nuclei of damaged cells become fluorescent with a DNA-staining dye (propidium iodide [PI]); the time of onset of nuclear PI fluorescence is quantified in real time by live cell imaging. Our preliminary data suggest that melanosomes slow the onset of RPE cell death after oxidant treatment, that cells with more granules are better protected, and that experimentally aged granules (extensively light treated) are less protective and may even be cytotoxic. These preliminary data suggest that with aging RPE melanosomes may change from an anti-oxidant to a pro-oxidant.
Jobs Summary: None (Total jobs reported: 0)
Project Status: Less Than 50% Completed
This award's data was last updated on Jul. 20, 2009. Help expand these official descriptions using the wiki below.