Regulation of RPE phagocytosis via avb5 integrin

描述（由申请人提供）：本研究项目的长期目标是确定哺乳动物眼睛中视网膜色素上皮（RPE）吞噬以昼夜节律脱落的光感受器外段片段（POS）的分子机制，并了解由于衰老和突变导致这些机制的具体变化如何导致视网膜变性。有丝分裂后的RPE细胞一生中每天消化数千个POS盘，是已知最活跃的吞噬细胞。RPE吞噬异常直接导致无法治愈的人类致盲疾病，影响数百万人，包括年龄相关性黄斑变性和视网膜色素变性。因此，确定RPE对视网膜外节更新的基本过程的贡献与我们对人类视网膜疾病的理解直接相关。最近，我们开发并探索了新的实验方法，首次允许直接定量地确定基因突变和年龄相关变化如何通过RPE在体内和培养中改变吞噬蛋白的活性。Shed POS需要通过其视网膜配体mgf - e8连接RPE的aV - 5受体，通过局灶黏附激酶刺激RPE细胞内的细胞质信号通路，该通路指向酪氨酸激酶受体MerTK，这对于有效的POS吞噬是必不可少的。通过aV¿5受体同步清除POS对于长期视网膜健康至关重要：衰老的aV¿5缺陷小鼠缺乏吞噬节律导致促氧化剂RPE脂褐素过度积累和视力丧失。在目的1中，我们提出确定aV¿5和MerTK表面受体之间缺失的分子联系，并揭示Src激酶在RPE吞噬中的作用。一种新的底物筛选方法将识别出直接针对POS吞噬的Src底物，并确定Src活性是否足以促进MerTK激活和POS吞噬。我们的初步数据表明，在POS结合初始爆发后，RPE细胞保留了充足的表面aV¿5和MerTK受体，但不能促进长时间的POS结合或吞噬信号。因此，在目标2和3中，我们提出确定RPE细胞在完成每日POS清除后主动特异性抑制aV¿5和MerTK功能的分子机制。我们将验证RPE细胞通过特异性蛋白水解整合素相关蛋白来终止POS吞噬的假设，整合素相关蛋白切断了aV¿5受体在肌动蛋白细胞骨架上的锚定，并通过刺激特异性蛋白酪氨酸磷酸酶来灭活MerTK。防止过早或过量RPE吞噬活性的机制一直被认为存在，但据我们所知从未被研究过。我们认为这种抑制机制可以确保视网膜中POS脱落和吞噬的健康平衡。我们已经获得了适当的体内模型，特异性活性测定和检测试剂进行鉴定

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