Regulation of RPE phagocytosis via avb5 integrin

描述(申请人提供)：本研究项目的长期目标是确定视网膜色素上皮(RPE)吞噬哺乳动物眼睛昼夜节律中脱落的光感受器外片段(POS)的分子机制，并了解这些机制中因衰老和突变而发生的具体变化如何导致视网膜退化。有丝分裂后的RPE细胞终生每天消化数千个POS盘，是已知最活跃的吞噬细胞。RPE吞噬功能的异常直接导致了影响数百万人的不可治愈的人类致盲疾病，包括老年性黄斑变性和视网膜色素变性。因此，确定RPE在视网膜外段更新的基本过程中的作用直接关系到我们对人类视网膜疾病的理解。最近，我们开发和探索了新的实验方法，首次允许直接和定量地确定基因突变和与年龄相关的变化如何改变体内和培养中RPE吞噬蛋白的活性。去除POS需要通过视网膜配体MFG-E8连接RPE的AV？5受体，以刺激RPE细胞中的细胞质信号通路，通过粘着斑激酶到达酪氨酸激酶受体MerTK，而MerTK是有效吞噬POS所必需的。通过AV？5受体同步清除POS对于长期的视网膜健康是必不可少的：老化的AV？5缺陷小鼠缺乏吞噬节律会导致促氧化性RPE脂褐素过度积累和视力丧失。在目标1中，我们建议鉴定AV？5和MerTK表面受体之间缺失的分子连接，并揭示Src激酶在RPE吞噬中的作用。一种新的底物筛选将识别POS吞噬作用的直接Src底物，并确定Src活性是否足以促进MerTK激活和POS吞噬。我们的初步数据显示，在最初的POS结合爆发后，RPE细胞保留了丰富的表面AV？5和MerTK受体，但未能促进延长的POS结合或吞噬信号。因此，在目标2和目标3中，我们建议确定RPE细胞在完成日常POS清除后主动和特异性抑制AV？5和MerTK功能的分子机制。我们将测试RPE细胞终止POS吞噬功能的假设，这是通过整合素相关蛋白的特定蛋白分解，切断AV？5受体与肌动蛋白细胞骨架的锚定，以及通过刺激特定的蛋白酪氨酸磷酸酶使MerTK失活来实现的。防止过早或过度的RPE吞噬活性的机制长期以来一直被认为是存在的，但据我们所知，从未被研究过。我们认为，这种抑制机制可能确保POS在视网膜内的脱落和吞噬的健康平衡。我们已经采购了合适的活体模型、特定活性分析和检测试剂来鉴定它们

DESCRIPTION (provided by applicant): It is the long-term goal of this research project to identify molecular mechanisms used by the retinal pigment epithelium (RPE) to phagocytose photoreceptor outer segment fragments (POS) shed in a circadian rhythm in the mammalian eye and to understand how specific changes in these mechanisms due to aging and mutations cause retinal degeneration. Digesting several thousand POS disks every day for life, post-mitotic RPE cells are the most active phagocytes known. Abnormalities in RPE phagocytosis directly contribute to incurable human blinding diseases affecting millions, including age-related macular degeneration and retinitis pigmentosa. Identifying the contributions of the RPE to the fundamental retinal process of outer segment renewal is thus directly relevant to our understanding of human retinal disease. Recently, we developed and explored novel experimental approaches that allow for the first time to directly and quantitatively determine how genetic mutations and age-related changes alter activity of phagocytic proteins by the RPE in vivo and in culture. Shed POS require ligation of aV¿5 receptors of the RPE by their retinal ligand MFG-E8 to stimulate a cytoplasmic signaling pathway in RPE cells via focal adhesion kinase towards the tyrosine kinase receptor MerTK that is indispensable for efficient POS engulfment. Synchronized POS clearance via aV¿5 receptors is essential for long-term retinal health: lack of phagocytic rhythm in aging aV¿5 - deficient mice causes excessive accumulation of pro-oxidant RPE lipofuscin and loss of vision. In aim 1 we propose to identify the missing molecular links between aV¿5 and MerTK surface receptors and unravel the role of Src kinase in RPE phagocytosis. A novel substrate screen will identify direct Src substrates specific to POS phagocytosis and determine if Src activity is sufficient to promote MerTK activation and POS engulfment. Our preliminary data show that RPE cells following an initial burst of POS binding retain ample surface aV¿5 and MerTK receptors but fail to promote prolonged POS binding or engulfment signaling. In aims 2 and 3 we therefore propose to identify molecular mechanisms used by RPE cells to actively and specifically inhibit aV¿5 and MerTK functions following completion of diurnal POS clearance. We will test the hypothesis that RPE cells terminate POS phagocytosis by specific proteolysis of integrin associated proteins that severs anchorage of aV¿5 receptors to the actin cytoskeleton, and by stimulating specific protein tyrosine phosphatases that inactivate MerTK. Mechanisms that prevent untimely or excess RPE phagocytic activity have long been assumed to exist but to our knowledge never been studied. We propose that such inhibitory mechanisms may ensure the healthy balance of POS shedding and engulfment in the retina. We have procured appropriate in vivo models, specific activity assays and detection reagents for their identification