Age-related Changes in Epithelial Microvilli

描述(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段之间建立亲密的相互作用对视觉至关重要。RPE具有高度专门化、独特的功能，对神经视网膜的动态平衡至关重要。这些包括光感受器脱落外节的吞噬作用，营养物质向光感受器细胞内的定向运输和从光感受器细胞中的废物的去除，以及视觉色素的运输和再生。RPE的顶端微绒毛在调节这些活动中起着关键作用。这一建议的假设是，对年轻的和老年的视网膜色素上皮(RPE)顶端微绒毛进行蛋白质组学比较，将有助于深入了解RPE的基本功能，特别是受衰老影响。这项建议探讨了可以研究RPE顶端微绒毛的概念，以了解RPE中发生的与年龄相关的关键变化。目标1中的实验将使用一种基本的方案来从年轻和老年大鼠身上获得完整的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与每个年龄段的样本相关的蛋白质组成和重大的翻译后修饰。共聚焦和免疫电子显微镜分析将进一步量化相关蛋白质表达的变化。目标2中的实验将表征Ezrin在老年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，我们已经证明Ezrin是顶端微绒毛和基底部折叠发育的关键。因此，本研究还将对年轻和老年RPE顶端微绒毛和-MV组分中的Ezrin结合伙伴进行比较，以更好地了解该细胞中与衰老相关的变化。我们还将调查老年RPE微绒毛中Ezrin的差异翻译后修饰是否与RPE顶端微绒毛的年龄相关。这项研究的长期目标是确定受正常和病理性衰老过程特别影响的蛋白质。老年人视力下降是神经视网膜和RPE变化的直接后果，这是一个有据可查的现象。这项研究项目将描述生理性RPE老化的特征，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.