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)对氧化应激的敏感性的影响。RPE的氧化应激被认为会减少RPE对邻近视网膜的支持，从而导致老年性黄斑变性等疾病。黑色素具有抗氧化性能，理论上可能是一种重要的RPE抗氧化剂。然而，当黑色素受到可见光照射时，它会成为一种促氧化剂。因此，目前尚不清楚黑色素实际上是保护RPE细胞免受氧化损伤，还是相反。此外，在细胞内，黑色素被隔离在颗粒细胞器--黑素小体中，该细胞器可以限制黑色素与有害物种之间的相互作用。因此，无论黑色素起什么作用，它很可能仅限于颗粒的近端区域。此外，RPE黑素小体是长寿命的细胞器，可能会经历老化变化，从而改变其氧化还原功能。这一建议的假设是，黑素小体可以减少或增加对RPE的氧化损伤。它的功能是由衰老决定的，这使得黑素小体由于其主要成分黑色素的物理化学性质的变化而更容易氧化。在这个项目中，一位细胞生物学家和一位生物物理学家将使用开发的创新策略来测量RPE细胞中分离的和RPE细胞内的色素颗粒的直接区域中的促氧化和抗氧化效果。“年轻的”和“老化的”RPE黑素小体或对照颗粒将用于实验，以解决以下特定目的：(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的氧化状态。黑素小体将被特定的分子覆盖，然后在区分黑色素不同作用机制的条件下受到光敏和其他氧化系统的影响。(2)确定活的RPE细胞内的黑素小体是否以及如何影响细胞对氧化应激的反应，通过测量细胞存活、细胞扩散或颗粒运动来检测。运动性至少部分依赖于应力敏感细胞骨架的完整性，并提供了发生在颗粒亚细胞区域的事件的功能测量。方法包括电子自旋共振(ESR)光谱，蛋白质氧化和脂质过氧化的生化分析，模拟衰老处理的黑素体的扫描电子显微镜和原子力显微镜，诱导RPE细胞衰老的复制衰老方案，以及时间推移成像的新应用，以量化活的RPE细胞对可见光或化学氧化剂诱导的致命或亚致命氧化应激的反应。公共卫生相关性：RPE细胞支持视网膜光感受器的生存，但随着RPE细胞年龄的增长，它们的功能效率被认为降低了，部分原因是终生氧化应激，包括光暴露造成的应激。RPE细胞含有吸收光的黑色素，理论上它既可以作为促氧化剂，也可以作为抗氧化剂。在这个项目中，将研究黑色素的性质，这种黑色素存在于被称为黑素小体的颗粒中，以确定颗粒是增加还是降低RPE细胞对氧化损伤的敏感性。还将研究年龄对黑素小体的影响，以确定它们保护RPE细胞免受应激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，如老年性黄斑变性。

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.