Photoreceptor Renewal by Retinal Pigmented Epithelium Phagocytosis

描述(申请人提供)：脊椎动物视网膜有两个主要组成部分，神经视网膜，它使用被称为杆和锥的特殊光感受器细胞来收集光线并将信息传输到大脑，以及视网膜色素上皮(RPE)，它是血液的一部分：视网膜屏障，发挥几个重要的功能。RPE通过从血流中提供代谢物，参与视觉生色团的再生，并通过吞噬作用定期清除最老的感光细胞部分，来维持视网膜的健康。光感受器细胞通过不断地从其底部产生新的外节段，同时以昼夜节律的方式在顶端释放成熟的外节段来维持几乎恒定的长度。因此，有丝分裂后的RPE细胞在一生中吞噬大量的物质，处理光感受器生存和更新所需的细胞废物。这一功能是维持视力所必需的，因为参与RPE吞噬功能的基因突变可能导致进行性视网膜退化。在这里，我们提出了一套定量分析来剖析RPE吞噬光感受器的遗传因素。首先，我们将使用下一代大规模并行RNA测序(RNA-Seq)来绘制整个昼夜节律时间线上的小鼠眼睛转录组。此外，我们将使用定量蛋白质组学方法来更全面地了解导致脱落光感受器外部片段被吞噬的信号转导途径。最后，我们将在培养中培养原代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。这些方法将提供对负责RPE吞噬的复杂信号网络的洞察，并潜在地提高我们对吞噬过程的总体理解。 公共卫生相关性：视网膜的健康和维护需要神经视网膜和视网膜色素上皮(RPE)之间的密切互动。这种相互作用为神经视网膜提供营养物质和底物，以及一个连续的过程，在这个过程中，RPE吞噬光感受器的最老部分，以允许它们更新。这项研究将增加我们对RPE吞噬如何发生的理解，并可能为与其他吞噬过程有关的机制问题提供答案。

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.