Genetic Modifiers of Enhanced S-cone Syndrome –Role of the External Limiting Membrane

项目概要/摘要 增强型 S 锥综合征 (ESCS) 是一种遗传性视网膜变性，其特征是视网膜色素变性增加 S 锥体数量、视网膜发育不良和进行性感光器变性导致早睡 失明和视力丧失。该疾病是由决定杆命运的突变引起的 人类和 rd7 小鼠模型中的转录因子 NR2E3。疾病的表现差异很大 人类并依赖于小鼠的遗传背景，证明了遗传修饰剂的存在。 小鼠模型表明视网膜发育不良与该病的发生密切相关 外部限制膜 (ELM) 的断裂，这是穆勒细胞之间的粘附/紧密连接网络 顶突和光感受器内节。 ELM 的碎片也出现在其他 与发育不良相关的视网膜疾病，例如由于基因突变导致的莱伯先天性黑蒙（LCA） crumbs1 (CRB1) 和 RP27，以及糖尿病视网膜病变。 我们已经鉴定出一种遗传修饰剂，可以防止 rd7 小鼠中的 ELM 断裂 和 Nrl ko 小鼠 (RP27) 型号。这一发现强烈表明 ELM 的致病作用 光感受器发育不良发展过程中的碎片化，并提供了建立光感受器发育不良的工具 ELM 断裂发生的机制、它们如何导致视网膜发育不良以及这如何影响疾病 进展。 为了确定这些机制，我们将： 1) 确定 ELM 连接的自然历史 使用细胞连接标记分析在 wt 和 rd7 模型中形成和光感受器分化 蛋白质。 2) 通过单细胞 RNAseq 分析鉴定 ELM 片段化涉及的细胞类型。 3） 确定修饰蛋白如何改变连接蛋白向细胞膜的募集。 4）识别 我们生成的其他遗传修饰菌株的分子基础，以进一步了解 NR2E3 疾病途径。

PROJECT SUMMARY/ABSTRACT Enhanced S-cone syndrome (ESCS) is an inherited retinal degeneration characterized by an increased number of S-cones, retinal dysplasia, and progressive photoreceptor degeneration leading to early night blindness and loss of visual acuity. The disease is caused by mutations in the rod-fate determining transcription factor NR2E3 in humans and the rd7 mouse model. The disease presentation is highly variable in human and dependent on genetic background in the mouse, demonstrating the existence of genetic modifiers. The mouse model demonstrates that the retinal dysplasia is intimately associated with the occurrence of breaks in the external limiting membrane (ELM), a network of adherens/tight junctions between Müller cell apical processes and photoreceptor inner segments. Fragmentation of the ELM is also observed in other retinal diseases associated with dysplasia, such as Leber's Congenital Amaurosis (LCA) due to mutations in crumbs1 (CRB1) and RP27, and in diabetic retinopathy. We have identified a genetic modifier that prevents the fragmentation of the ELM in both the rd7 mouse and the Nrl ko mouse (RP27) models. This discovery strongly suggests a causative role for the ELM fragmentation in the development of photoreceptor dysplasia and provides a tool with which to establish the mechanisms by which ELM breaks occur, how they lead to retinal dysplasia, and how this affects disease progression. In order to identify these mechanisms we will: 1) Determine the natural history of ELM junction formation and photoreceptor differentiation in wt and the rd7 model using marker analysis of cell junction proteins. 2) Identify the cell types involved in the ELM fragmentation by single cell RNAseq analysis. 3) Determine how the modifier protein alters recruitment of junctional proteins to the cell membrane. 4) Identify the molecular basis of additional genetic modifier strains that we have generated to gain further insight into the NR2E3 disease pathways.