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

了解 GARP 蛋白在视杆外节盘形成和视网膜变性中的作用

• 批准号: 10748725
• 负责人: Molly Naylor
• 金额: $ 7.45万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-26 至 2026-08-25
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10748725
• 关键词: Ablation; Acceleration; Actins; Address; Affect; Alternative Splicing; Architecture; Automobile Driving; Blindness; Cell Death; Cell membrane; Cell physiology; Cellular biology; Cilia; Data; Defect; Diameter; Disease; Distal; Electric Stimulation; Event; Exons; Future; Genes; Genetic; Glutamic Acid; Goals; Growth; Health; Human; Impairment; Inherited; Investigation; Kinetics; Knock-out; Knockout Mice; Knowledge; Lead; Length; Life; Light; Maintenance; Measures; Membrane; Modeling; Molecular; Morphogenesis; Mus; Mutation; Neurons; Opsin; Organelles; Pathogenesis; Phagocytosis; Photoreceptors; Phototoxicity; Physiologic pulse; Play; Process; Proteins; Radiolabeled; Regulation; Reporter; Research; Retina; Retinal Degeneration; Retinal Diseases; Rod; Role; Seminal; Shapes; Signal Transduction; Structure of retinal pigment epithelium; Surface; Tertiary Protein Structure; Testing; Vision; Visualization; Wild Type Mouse; absorption; base; cyclic-nucleotide gated ion channels; depolymerization; experimental study; in vivo; inherited retinal degeneration; insight; mouse model; novel; programs; recruit; retinal rods; rod outer segment disc; tool; trafficking; vision science

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.

项目摘要 人类视觉始于视网膜，光捕获刺激外部的电信号 光感受器神经元的细节细胞器。外部细分市场是一个修改的主要纤毛 数百张扁平的膜盘，可最大程度地吸收光线表面。光毒性 损坏椎间盘膜，要求外部段通过 在基础上添加新光盘。外部段长度通过旧椎间盘的吞噬作用来维持 从邻近的视网膜色素上皮细胞的远端尖端。外部的独特架构 细分区室对于受到光感受器的健康和破坏椎间盘堆积的突变至关重要 许多形式的人类残留变性。该建议的目的是调查组件 这有助于形成和维护椎间盘，以便更好地了解 继承的残余变性。 杆感光体表达含有富含谷氨酸蛋白（GARP）的三种蛋白 结构域：环状核苷酸门控通道（CNG），GARP1和GARP2的β1亚基。在老鼠中， 所有三种GARP蛋白的遗传消融导致外部膜出生 片段基础，椎间盘合成中缺陷的标志。我有新的初步数据，表明 在该模型中，关键的椎间盘形态发生蛋白，蛋白质粘蛋白21（PCDH21）失调。这些数据 导致我的假设是GARP蛋白有助于将PCHD21招募到萌芽的前沿 新生的椎间盘和GARP蛋白的损失导致椎间盘生长速率降低，这有助于 视网膜变性。我进一步确定CNGβ1亚基是稳定PCDH21所必需的 在外部段基础上，使其成为目标1的主要重点。我将确认从 外部段底座驱动椎间盘形态发生中的缺陷，并测试CNGβ1是否足以使 调节PCDH21和圆盘形成。 最近对椎间盘形成的分子机制的见解表明了紧急 需要定量测量光盘合成的速率，但是要做的工具缺乏灵敏度和 多功能性。作为解决方案，我开发了一种诱导小鼠模型，该模型表达了光转换， 外部段针对的，跨膜记者，我将用来量化新的光盘添加。在AIM 2中，我 将表征这种新型的小鼠模型，然后对圆盘生长速率进行定量比较 在WildType，Garpko和CNGβ1-KO小鼠杆之间，以了解椎间盘的失调 形成有助于视网膜变性。这些目标的完成将提供机械洞察力 GARP蛋白在椎间盘形成和外部片段生长动力学中扮演的角色以及提供 一种定量工具，用于未来研究健康和疾病中的椎间盘恢复。