Understanding the Role of GARP Proteins in Rod Outer Segment Disc Formation and Retinal Degeneration

项目摘要 人类的视觉开始于视网膜，在视网膜处，光捕获刺激外部视网膜内的电信号。 感光神经元的节细胞器。外节是一个修改的初级纤毛建成与 数以百计的扁平膜盘，最大限度地吸收光的表面。光毒性 损伤椎间盘膜，需要外节通过 在基础上增加新的光盘。外节的长度是通过吞噬旧的椎间盘来维持的 从远端被邻近的视网膜色素上皮细胞。独特的建筑外部 节隔对感光细胞的健康至关重要，而突变会破坏下面的光盘堆栈， 多种形式的人类视网膜退化。本提案的目的是调查 有助于形成和维持椎间盘，以便更好地了解 遗传性视网膜变性 视杆细胞光感受器表达三种蛋白质，包括富含谷氨酸的蛋白质（GARP） 结构域：环核苷酸门控通道（CNG）的β1亚基、GARP 1和GARP 2。在小鼠中， 所有三种GARP蛋白质的基因切除导致从外膜生长的外来膜 节段基底，椎间盘合成缺陷的标志。我有新的初步数据表明 关键的椎间盘形态发生蛋白原钙粘蛋白21（PCDH 21）在该模型中失调。这些数据 这导致我的假设，即GARP蛋白有助于招募PCHD 21到萌芽的前沿 新生椎间盘和GARP蛋白的丢失导致椎间盘生长速率降低， 视网膜变性我进一步确定了CNGβ1亚基对于稳定PCDH 21是必需的， 在外部部分的基础上，使其成为目标1的主要焦点。我将确认PCDH 21的丢失， 外节基底驱动椎间盘形态发生中的缺陷，并测试CNGβ1是否足以 调节PCDH 21和椎间盘形成。 最近对椎间盘形成的分子机制的深入了解揭示了 需要定量测量椎间盘合成率，但这样做的工具缺乏灵敏度， 多才多艺。作为一种解决方案，我开发了一种诱导型小鼠模型， 外段靶向，跨膜报告，我将使用它来量化新的光盘添加。在目标2中，我 将描述这种新的小鼠模型，然后定量比较椎间盘生长的速率， 野生型、GARPKO和CNGβ1-KO小鼠视杆细胞之间的差异，以了解椎间盘如何失调 形成有助于视网膜变性。这些目标的完成将提供机械的洞察力 GARP蛋白在椎间盘形成和外节生长动力学中的作用， 一个定量的工具，为未来的调查光盘更新的健康和疾病。

PROJECT SUMMARY Human vision begins in the retina where light capture stimulates an electrical signal within the outer segment organelle of photoreceptor neurons. The outer segment is a modified primary cilium built with hundreds of flattened membranous discs that maximize the light-absorbing surface. Phototoxicity damages the disc membranes requiring that the outer segment be continuously renewed through the addition of new discs at the base. Outer segment length is maintained by phagocytosis of old discs from the distal tip by the adjacent retinal pigment epithelial cells. The unique architecture of the outer segment compartment is critical to photoreceptor health and mutations that disrupt disc stacks underly many forms of human retinal degeneration. The purpose of this proposal is to investigate components that contribute to forming and maintaining discs in order to better understand the pathogenesis of inherited retinal degeneration. Rod photoreceptors express three proteins containing the glutamic acid rich protein (GARP) domain: the β1-subunit of the cyclic nucleotide-gated channel (CNG), GARP1, and GARP2. In mice, genetic ablation of all three GARP proteins results in extraneous membrane outgrowth from the outer segment base, a hallmark of defects in disc synthesis. I have new preliminary data indicating that the key disc morphogenesis protein, protocadherin 21 (PCDH21), is dysregulated in this model. These data led to my hypothesis that GARP proteins help to recruit PCHD21 to the leading edge of the budding nascent discs and the loss of GARP proteins results in reduced rates of disc growth that contributes to retinal degeneration. I have further identified that the CNGβ1 subunit is necessary to stabilize PCDH21 at the outer segment base, making it the primary focus of Aim 1. I will confirm that loss of PCDH21 from the outer segment base drives defects in disc morphogenesis and test whether CNGβ1 is sufficient to regulate PCDH21 and disc formation. Recent insights into the molecular mechanisms underlying disc formation have revealed the urgent need to quantitatively measure rates of disc synthesis, yet the tools to do so lack sensitivity and versatility. As a solution, I developed an inducible mouse model that expresses a photoconvertible, outer segment targeted, transmembrane reporter that I will use to quantify new disc addition. In Aim 2, I will characterize this novel mouse model followed by a quantitative comparison of rates of disc growth between wildtype, GARPKO, and CNGβ1-KO mouse rods to understand how dysregulated disc formation contributes to retinal degeneration. Completion of these aims will provide mechanistic insight into the role GARP proteins play in disc formation and outer segment growth kinetics as well as provide a quantitative tool for future investigation of disc renewal in health and disease.