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

描述（由申请人提供）：视网膜中每天发生老化感光细胞外节尖端（OS）的脱落及其被视网膜色素上皮（RPE）的吞噬作用。RPE细胞吞噬OS的失败导致啮齿动物和人类的视网膜营养不良。此外，延迟的OS退化可能有助于年龄相关性黄斑变性的发展或进展。因此，重要的是要了解外节更新的分子机制。我们已经确定了一个信号通路，在完整的视网膜，需要清除脱落OS迅速。缺乏α-v-β 5整联蛋白受体的小鼠缺乏同步吞噬作用导致年龄相关的光感受器功能障碍和脂褐素积累。整合素信号传导需要整合素受体的配体接合。因此，我们假设整合素配体的及时出现或募集用于刺激视网膜中的节律信号。迄今为止尚未鉴定出这样的整联蛋白配体。我们的初步数据表明，分泌的整合素配体蛋白MFG-E8作为该功能的候选者。MFG-E8介导凋亡细胞的整联蛋白依赖性吞噬作用，这是一个类似于OS吞噬作用的过程。MFG-E8由RPE表达并参与体外RPE吞噬作用。该探索性项目将实现两个具体目标：目标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吞噬作用和吞噬信号传导。这些实验将确定MFG-E8是否作为av的必需配体起作用。|视网膜中的35整合素。目的2将研究视网膜下腔中的MFG-E8活性是否与avB 5整合蛋白的活化、每日OS脱落和吞噬作用相关。它将进一步探索用于脱落的光感受器尖端是否暴露磷脂酰丝氨酸以增加MFG-E8结合位点。我们将结合联合收割机分子生物学、显微镜和生物化学来检测MFG-E8在小鼠视网膜中的转录、蛋白表达和蛋白定位与外节更新的时间过程的关系。我们将开发新的亲和力测定来定量完整视网膜中OS脱落时的磷脂酰丝氨酸暴露和MFG-E8结合。这个探索性项目的结果是全面研究感光体尖端和分泌蛋白变化的重要前提，这些蛋白促进了对长期视网膜功能至关重要的及时外节更新。

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.