Biological Properties of RPE Melanin

描述（由申请人提供）：拟议研究的长期目标是确定年龄对视网膜色素上皮（RPE）对氧化应激易感性的影响。RPE的氧化应激被认为会减少RPE对邻近视网膜的支持，导致年龄相关性黄斑变性等疾病。黑色素具有抗氧化特性，理论上可能是一种重要的RPE抗氧化剂。然而，当黑色素被可见光照射时，它会变成一种促氧化剂。因此，尚不清楚黑色素究竟是保护RPE细胞免受氧化损伤，还是起相反的作用。此外，在细胞内，黑色素被隔离在一个颗粒状的细胞器中，即黑色素小体，它可以限制黑色素与有害物质之间的相互作用。因此，无论黑色素发挥什么作用，它都可能局限于颗粒的近区域。此外，RPE黑素小体是长寿的细胞器，可以经历老化变化，改变其氧化还原功能。该提议的假设是黑素体可以减少或增加RPE的氧化损伤。它的功能是由衰老决定的，由于其主要成分黑色素的物理化学性质的变化，衰老使黑素体更容易氧化。在这个项目中，一名细胞生物学家和一名生物物理学家将使用创新的策略来测量色素颗粒的亲氧化和抗氧化作用，包括分离的和在RPE细胞内的色素颗粒。“年轻”和“年老”的RPE黑素小体或对照颗粒将用于实验，以解决以下具体目标：(1)确定分离的RPE黑素小体是否以及如何改变附近颗粒相关蛋白和脂质的氧化状态。黑色素小体将被特定的分子包裹，然后在区分不同黑色素作用机制的条件下进行光敏化和其他氧化系统。(2)通过测量细胞存活、细胞扩散或颗粒运动来确定活的RPE细胞内的黑素体是否以及如何影响细胞对氧化应激的反应。运动性至少部分取决于应力易感细胞骨架的完整性，并提供颗粒亚细胞结构域中发生的事件的功能测量。方法将包括电子自旋共振（ESR）光谱，蛋白质氧化和脂质过氧化的生化分析，黑素体的扫描电镜和原子力显微镜处理来模拟衰老，复制衰老方案来诱导RPE细胞衰老，以及延时成像的新应用来量化活的RPE细胞对可见光或化学氧化剂诱导的致死或亚致死氧化应激的反应。公共卫生相关性：RPE细胞支持视网膜光感受器的存活，但随着RPE细胞的老化，它们的功能被认为效率较低，部分原因是氧化应激，包括光暴露引起的应激。RPE细胞含有吸收光的色素黑色素，理论上也可以作为促氧化剂和抗氧化剂。在这个项目中，将研究黑色素的特性，黑色素存在于称为黑色素小体的颗粒中，以确定这些颗粒是增加还是减少RPE细胞对氧化损伤的易感性。年龄对黑素体的影响也将被研究，以确定它们保护RPE细胞免受压力下降的能力是否会随着时间的推移而下降，这可能会导致视网膜疾病，如年龄相关性黄斑变性。

DESCRIPTION (provided by applicant): 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 measures of cell survival, cell spreading or particle motility. Motility depends at least partly on the integrity of the stress-susceptible cytoskeleton and provides a functional measure of events occurring in the sub-cellular domain of the particle. Methods will include electron spin resonance (ESR) spectroscopy, biochemical analyses of protein oxidation and lipid peroxidation, scanning EM and atomic force microscopy of melanosomes treated to simulate aging, replicative senescence protocols to induce cellular aging of the RPE, and novel applications of time-lapse imaging to quantify the responses of living RPE cells to lethal or sub-lethal oxidative stress induced by visible light or chemical oxidants. PUBLIC HEALTH RELEVANCE: RPE cells support the survival of retinal photoreceptors, but as RPE cells age they are believed to function less efficiently due in part to a lifetime of oxidative stress, including stress due to light exposure. RPE cells contain the light-absorbing pigment melanin, which can theoretically also act as both a pro-oxidant and an antioxidant. In this project, the properties of melanin, which is found inside granules called melanosomes, will be studied to determine whether the granules increase or decrease RPE cell susceptibility to oxidative damage. The effects of age on melanosomes will also be studied to determine whether their ability to protect RPE cells from stress declines over time, which could contribute to diseases of the retina such as age-related macular degeneration.