RPE Phenotype and Oxidative Stress

描述（由申请人提供）：氧化应激几乎与所有衰老疾病有关，包括年龄相关性黄斑变性。然而，氧化应激究竟是如何在不同组织中导致与年龄相关的疾病的，人们知之甚少，因为人们对慢性、亚致死应激的细胞效应知之甚少。致死性应激通常被研究，但这些研究结果与衰老的相关性值得怀疑，衰老的特征是功能效率下降，而不是明显的细胞死亡。本研究的长期目标是确定视网膜色素上皮（RPE）的慢性亚致死氧化应激，特别是光应激，如何导致组织功能损伤，从而限制其支持邻近视网膜的能力。亚致死光胁迫对RPE有一个意想不到的功能后果：细胞器运动受损。这一观察结果的潜在意义怎么强调都不过分。细胞器易位是一个持续的细胞过程，对于维持极化上皮表型至关重要，RPE依赖于极化上皮表型有效地支持视网膜的功能和存活。在拟议的项目中，将以黑素体为模型探讨光诱导RPE中细胞器减慢的机制。这些色素颗粒是一种理想的模型：它们可以很容易地通过成像技术跟踪运动分析，它们可以从细胞中分离出来并用于检索介导运动的细胞器相关蛋白，并且由于其主要成分黑色素的特性，它们本身具有光反应性，可以通过实验操作来确定氧化机制。在拟议的项目中，RPE细胞生物学家和黑色素生物物理学家将合作测试三个假设：(1)亚致死光胁迫通过影响与肌动蛋白或微管运动相关的细胞器结合蛋白的氧化或磷酸化状态，损害RPE细胞器运动；(2)亚致死光胁迫引起的运动减缓是由于局部产生的活性氧，包括由本身具有光反应性的细胞器内容物产生的反应物；(3)慢性光胁迫对RPE的影响，及其伴随的细胞器易位损伤，是功能性极化表型的丧失。建议使用的方法包括电子自旋共振（ESR）光谱，蛋白质氧化和磷酸化的生化分析，培养RPE单层的免疫染色和共聚焦成像，以及细胞器易位的活细胞成像分析。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.