Age-related Changes in Epithelial Microvilli

描述（由申请人提供）：在长视网膜色素上皮（RPE）微绒毛和成熟的光感受器外节之间建立密切的相互作用对视力至关重要。RPE执行高度专业化，独特的功能至关重要的神经视网膜稳态。这包括光感受器外段的吞噬作用、营养物质进入光感受器细胞的定向运输和废物的清除以及视觉色素的运输和再生。RPE的顶端微绒毛在介导这些活动中起关键作用。该提案的假设是，对年轻和年老的视网膜色素上皮（RPE）的顶端微绒毛进行蛋白质组学比较，将有助于了解受衰老影响的RPE的基本功能。本研究探讨了通过研究RPE顶端微绒毛来了解RPE中发生的关键年龄相关变化的概念。目的1的实验将使用基本方案从年轻和老年大鼠中获得完整的RPE微绒毛。最先进的蛋白质组学方法和几种生化分析将确定蛋白质组成和与每个年龄组的样品相关的重要翻译后修饰。共聚焦和免疫电镜分析将进一步量化相关蛋白表达的变化。目的2的实验将表征ezrin在衰老RPE微绒毛中的作用。Ezrin是一种肌动蛋白结合蛋白，参与细胞衰老和信号转导等过程。在RPE中，我们已经表明ezrin是顶端微绒毛和基部包络发育的关键。因此，本研究还将对年轻和老年RPE顶端微绒毛和-MV片段中的ezrin结合伙伴进行比较，以更好地了解该细胞的衰老相关变化。我们还将研究老年RPE微绒毛中ezrin的翻译后差异修饰是否与RPE顶端微绒毛的年龄相关减少有关。这项研究的长期目标是确定受正常和病理衰老过程特异性影响的蛋白质。老年人视力下降是神经视网膜和RPE变化的直接后果，这是一个有充分证据的现象。本研究项目将描述RPE生理老化，并将其与眼部年龄相关疾病联系起来。

DESCRIPTION (provided by applicant): The establishment of an intimate interaction between the long retinal pigment epithelium (RPE) microvilli and the mature photoreceptor outer segments is essential for vision. The RPE performs highly specialized, unique functions essential for homeostasis of the neural retina. These include phagocytosis of photoreceptors shed outer segments, directional transport of nutrients into and removal of waste products from photoreceptor cells and visual pigment transport and regeneration. The apical microvilli of the RPE play a key role in mediating these activities. The hypothesis of this proposal is that proteomic comparison of young and old apical microvilli of the retinal pigment epithelium (RPE) will provide insight into fundamental RPE functions specifically affected by aging. This proposal explores the concept that the RPE apical microvilli can be studied to understand key age-related changes taking place in the RPE. Experiments in aim 1 will use a basic protocol to obtain intact RPE microvilli from young and aged rats. State-of-the-art proteomic methodologies and several biochemical assays will determine the protein composition and significant post- translational modifications associated with the samples in each age group. Confocal and immunoelectron microscopy analysis will further quantify the changes in the expression of relevant proteins. Experiments in aim 2 will characterize the role of ezrin in the aged RPE microvilli. Ezrin is an actin-binding protein involved in processes such as cell senescence and signal transduction. In the RPE, we have shown that ezrin is key to the development of apical microvilli and basal infoldings. This research will therefore also pursue a comparison of ezrin binding-partners in young and aged RPE apical microvilli and -MV fraction to better understand senescence-related changes in this cell. We will also investigate whether a differential post-translational modification of ezrin in aged RPE microvilli is somehow correlated to the age-related decrease of RPE apical microvilli. The long-term goal of this research is to identify proteins specifically affected by normal and pathological aging processes. The decline in vision in elderly, a direct consequence of changes in the neuro-retina and RPE, is a well-documented phenomenon. This research project will characterize physiological RPE ageing and relate it to ocular age-related diseases.