The Integrin Ligand MFG-E8 and Photoreceptor Outer Segment Renewal.

描述（由申请人提供）：视网膜中每天都会发生老化的光感受器外段尖端（OS）脱落及其被视网膜色素上皮（RPE）吞噬。RPE细胞吞噬OS的失败导致啮齿动物和人类视网膜营养不良。此外，延迟的OS退化可能有助于年龄相关性黄斑变性的发生或进展。因此，了解外段更新的分子机制是很重要的。我们已经在完整的视网膜中发现了一个信号通路，它需要迅速清除脱落的OS。缺乏α -v- β 5整合素受体的小鼠缺乏同步吞噬导致年龄相关的感光功能障碍和脂褐素积累。整合素信号需要整合素受体的配体参与。因此，我们假设整合素配体/s的及时出现或募集有助于刺激视网膜中的节律信号。到目前为止还没有发现这样的整合素配体。我们的初步数据表明分泌的整合素配体蛋白MFG-E8是该功能的候选者。MFG-E8介导凋亡细胞的整合素依赖性吞噬，这一过程类似于OS吞噬。MFG-E8在体外由RPE表达并参与RPE吞噬。该探索性项目将实现两个特定目标：目的1将在缺乏MFG-E8的新小鼠模型中探索视觉功能、视网膜形态、体内和体外RPE吞噬和吞噬信号。这些实验将确定MFG-E8是否作为视网膜中av bbbb35整合素的必要配体。目的2将研究MFG-E8在视网膜下间隙的活性是否与avB5的激活、每日OS脱落和吞噬有关。我们将进一步探讨是否光感受器尖端的脱落暴露磷脂酰丝氨酸增加MFG-E8结合位点。我们将结合分子生物学、显微镜学和生物化学来检测小鼠视网膜中MFG-E8的转录、蛋白表达和蛋白定位与外段更新的时间过程的关系。我们将开发新的亲和试验，以量化完整视网膜OS脱落时磷脂酰丝氨酸暴露和MFG-E8结合。本探索性项目的结果为全面研究促进视网膜长期功能所必需的及时外节更新的光感受器尖端和分泌蛋白的变化提供了重要的前提。

DESCRIPTION (provided by applicant): Shedding of aged photoreceptor outer segment tips (OS) and their phagocytosis by the retinal pigment epithelium (RPE) take place daily in the retina. Failure of RPE cells to phagocytose OS causes retinal dystrophy in rodents and humans. Furthermore, delayed OS degradation may contribute to development or progression of age-related macular degeneration. It is thus important to understand the molecular mechanisms of outer segment renewal. We have identified a signaling pathway in intact retina that is required to clear shed OS promptly. Lack of synchronized phagocytosis in mice deficient in alpha-v-beta5 integrin receptors causes age-related photoreceptor dysfunction and lipofuscin accumulation. Integrin signaling requires ligand engagement of integrin receptors. We thus hypothesize that timely appearance or recruitment of integrin ligand/s serves to stimulate rhythmic signaling in the retina. No such integrin ligand has been identified to date. Our preliminary data suggest the secreted integrin ligand protein MFG-E8 as candidate for this function. MFG-E8 mediates integrin-dependent phagocytosis of apoptotic cells, a process similar to OS phagocytosis. MFG-E8 is expressed by the RPE and participates in RPE phagocytosis in vitro. This exploratory project will achieve two specific objectives: Aim 1 will explore visual function, retinal morphology, in vivo and in vitro RPE phagocytosis and phagocytic signaling in a new mouse model that lacks MFG-E8. These experiments will determine if MFG-E8 functions as a necessary ligand for av|35 integrin in the retina. Aim 2 will investigate if MFG-E8 activity in the sub-retinal space correlates with avB5 ihtegrin's activation, daily OS shedding and phagocytosis. It will further explore if photoreceptor tips destined for shedding expose phosphatidylserine to increase MFG-E8 binding sites. We will combine molecular biology, microscopy, and biochemistry to detect MFG-E8 transcription, protein expression, and protein localization in mouse retina in relation to the time course of outer segment renewal. We will develop novel affinity assays to quantify phosphatidylserine exposure and MFG-E8 binding at the time of OS shedding in intact retina. The results of this exploratory project are an important prerequisite for comprehensive studies of changes in photoreceptor tip and secreted proteins that promote the timely outer segment renewal that is essential for long-term retinal function.