Photoreceptor Renewal by Retinal Pigmented Epithelium Phagocytosis

描述（由申请人提供）：脊椎动物视网膜有两个主要组成部分，一个是神经视网膜，它使用称为视杆细胞和视锥细胞的特殊感光细胞来收集光线并将信息传递给大脑，另一个是视网膜色素上皮（RPE），它是血液视网膜屏障的一部分，并执行几个重要功能。RPE通过提供血液中的代谢物、参与视觉发色团的再生以及通过吞噬作用周期性地清除感光细胞中最老的部分来维持视网膜健康。光感受器细胞通过不断地从其基部产生新的外节，同时在尖端以昼夜节律的方式释放其成熟的外节，从而保持几乎恒定的长度。因此，有丝分裂后的RPE细胞在一生中吞噬大量的物质，处理光感受器存活和更新所需的细胞废物。这种功能是维持视力所必需的，因为参与RPE吞噬的基因突变可导致进行性视网膜变性。在这里，我们提出了一套定量分析，剖析遗传因素背后的光受体吞噬作用的RPE。首先，我们将采用下一代大规模平行rna测序（RNA-Seq）来绘制整个昼夜节律时间表中小鼠眼睛转录组的图谱。此外，我们将使用定量蛋白质组学方法来获得导致脱落光感受器外段吞噬的信号转导途径的更完整图像。最后，我们将培养原代RPE细胞，并使用新开发的技术来识别RPE吞噬过程中的第二信使信号。这些方法将提供对RPE吞噬的复杂信号网络的见解，并有可能提高我们对吞噬过程的理解。

DESCRIPTION (provided by applicant): The vertebrate retina has two main components, the neural retina, that employs specialized photoreceptor cells called rods and cones to collect light and transmit this information to the brain, and the retinal pigmented epithelium (RPE), which serves as part of the blood: retina barrier and performs several important functions. The RPE maintains retinal health by providing metabolites from the bloodstream, participating in the regeneration of the visual chromophore, and periodically removing the oldest portions of photoreceptor cells by phagocytosis. Photoreceptor cells maintain a virtually constant length by continuously generating new outer segments from their base while simultaneously releasing their mature outer segments at the tip in a circadian manner. Thus, post-mitotic RPE cells phagocytose an immense amount of material over a lifetime, disposing of cell waste required for photoreceptor survival and renewal. This function is required to maintain vision, as mutations in genes involved in RPE phagocytosis can lead to progressive retinal degeneration. Here, we propose a set of quantitative analyses to dissect the genetic factors underlying photoreceptor phagocytosis by the RPE. First, we will employ next generation massively parallel RNA-sequencing (RNA-Seq) to map the mouse eye transcriptome throughout the circadian timeline. Additionally, we will use quantitative proteomic approaches to gain a more complete picture of the signal transduction pathway leading to engulfment of shed photoreceptor outer segments. Lastly, we will grow primary RPE cells in culture and use newly developed techniques to discern second messenger signaling during RPE phagocytosis. These approaches will provide insights into the complex signaling networks responsible for RPE phagocytosis and potentially improve our understanding of phagocytic processes in general. PUBLIC HEALTH RELEVANCE: Retinal health and maintenance requires an intimate interaction between the neural retina and the retinal pigmented epithelium (RPE). This interaction provides nutrients and substrates to the neural retina, as well as a continuous process whereby the RPE engulfs the oldest portions of photoreceptors to permit their renewal. This study will increase our understanding of how RPE phagocytosis occurs and potentially provide answers to mechanistic questions pertaining to other phagocytic processes.