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）对氧化应激的易感性的影响。对RPE的氧化应激被认为会减少RPE对邻近视网膜的支持，从而导致年龄相关性黄斑变性等疾病。具有抗氧化特性的黑色素在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，它可以成为促氧化剂。因此，目前尚不清楚黑色素是否真的保护RPE细胞免受氧化损伤或相反。此外，在细胞内，黑色素被隔离在颗粒细胞器中，即黑素体，其可以限制黑色素和破坏性物质之间的相互作用。因此，无论黑色素发挥什么样的功能，它都可能局限于颗粒的近域。此外，RPE黑素体是长寿的细胞器，可以经历改变其氧化还原功能的老化变化。该建议的假设是，黑素体可以减少或增加对RPE的氧化损伤。它执行的功能是由老化决定的，这使得黑素体由于其主要成分黑色素的物理化学性质的变化而更具促氧化性。在这个项目中，一个细胞生物学家和一个生物药理学家将使用创新的策略来测量亲和抗氧化剂的作用，在直接域的色素颗粒，无论是孤立的和RPE细胞内。“年轻的”和“老化的”RPE黑素体或对照颗粒将用于实验中，以解决以下具体目标：（1）确定分离的RPE黑素体是否以及如何改变附近的颗粒相关蛋白质和脂质的氧化态。黑素体将被特定的分子包裹，然后在区分黑素不同作用机制的条件下经受光敏化和其他氧化系统。(2)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，通过测量细胞存活、细胞铺展或颗粒运动来检测。运动性至少部分取决于应激敏感细胞骨架的完整性，并提供了在颗粒的亚细胞结构域中发生的事件的功能性测量。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，扫描EM和原子力显微镜的黑素体处理，以模拟老化，复制衰老协议，以诱导细胞老化的RPE，和新的应用程序的延时成像定量的反应活RPE细胞致死或亚致死的氧化应激诱导的可见光或化学氧化剂。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，它们被认为部分由于氧化应激的寿命而功能降低，包括由于光暴露引起的应激。RPE细胞含有吸光色素黑色素，理论上也可以作为促氧化剂和抗氧化剂。在这个项目中，将研究黑色素的性质，黑色素存在于称为黑色素体的颗粒中，以确定颗粒是否增加或减少RPE细胞对氧化损伤的敏感性。年龄对黑素体的影响也将被研究，以确定它们保护RPE细胞免受压力的能力是否会随着时间的推移而下降，这可能导致视网膜疾病，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.