RPE Phenotype and Oxidative Stress

描述(申请人提供)：氧化应激与几乎所有的衰老疾病有关，包括老年性黄斑变性。然而，氧化应激在不同组织中究竟如何导致与年龄相关的疾病还知之甚少，因为人们对慢性亚致命应激对细胞的影响知之甚少。致命性应激通常被研究，但这些研究的结果与衰老的相关性值得怀疑，衰老的特点是功能效率下降，而不是明显的细胞死亡。这项拟议研究的长期目标是确定对视网膜色素上皮(RPE)的慢性亚致命氧化应激，特别是光应激，如何导致组织的功能损伤，限制其支持邻近视网膜的能力。亚致死性光应激对RPE的作用最近被证明具有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为过。细胞器移位是一个持续不断的细胞过程，对于维持RPE有效支持视网膜功能和生存所依赖的极化上皮表型至关重要。在拟议的项目中，将使用黑素体作为模型来探索RPE中光诱导细胞器减慢的机制。这些色素颗粒是一个理想的模型：它们可以很容易地通过成像技术进行跟踪以进行运动性分析，它们可以从细胞中分离出来并用于提取介导运动性的细胞器相关蛋白，而且由于其主要成分黑色素的特性，它们本身具有光反应能力，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检验三个假说：(1)亚致死性光应激通过影响与肌动蛋白或微管有关的细胞器结合蛋白的氧化或磷酸化状态而损害RPE细胞器的运动；(2)亚致死性光应激诱导的运动减慢是由于局部产生的活性氧物种，包括细胞器内容物自身光反应产生的反应物；(3)慢性光胁迫对RPE的后果是丧失功能极化的表型。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、培养的RPE单层的免疫染色和共聚焦成像，以及细胞器移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.