Norrin/Frizzled4 Signaling in Retinal Angiogenesis: The Role of TSPAN12

描述（由申请人提供）：我们的总体目标是在发育和眼部疾病的背景下了解视网膜血管生成，使用小鼠作为模型系统。视网膜血管疾病，包括增殖性糖尿病视网膜病变和新生血管性年龄相关性黄斑变性，是视力受损和失明的主要原因。Norrin/Frizzled4信号通路是视网膜血管生成的主要信号通路之一。Norrin通过其受体Frizzled4和辅助受体LRP5在血管内皮细胞上发出信号，激活ss-catenin信号传导和转录程序。该通路在指导视网膜血管形态发生和血视网膜屏障形成中起关键作用。Norrin/Frizzled4信号受损会导致家族性渗出性玻璃体视网膜病变，这是一种可导致失明的遗传性疾病。本应用的重点是膜蛋白TSPAN12，这是Norrin/Frizzled4信号传导的一个新的重要组成部分。Tspan12基因破坏了norrin和frzzled4突变小鼠的表型，并且在基于细胞的检测中，Tspan12是norrin / frzzled4信号传导的强促进剂。我们有三个目标：i)我们假设TSPAN12在视网膜内皮细胞中是功能性必需的，并且限制TSPAN12的表达调节了通路激活的时空模式。该模型预测，内皮细胞（而非其他细胞类型）的tspan12基因的条件性破坏概括了功能缺失的表型，例如，缺乏视网膜内毛细血管和血液-视网膜屏障完整性受损。我们将使用Cre-Lox系统来测试这个模型。ii) TSPAN12仅在norrin诱导的信号中是必需的，而在wnt诱导的Frizzled4信号中则不需要。这是Norrin/Frizzled4信号的一个有趣且无法解释的特征。我们假设Norrin不能有效地诱导Norrin受体复合物中的蛋白质相互作用，因此与TSPAN12一起作用。相反，Wnts可以自主诱导所需的蛋白质相互作用（例如，Norrin和Wnts同时结合受体和共受体的能力不同）。我们将通过表征Norrin受体复合物中的蛋白质相互作用，通过确定Norrin、Wnt3a和TSPAN12促进哪些相互作用，以及通过在基于细胞的报告基因分析中确定取消或强制相关蛋白质相互作用的后果来验证这一假设。最近几项人类遗传学研究独立报道了家族性渗出性玻璃体视网膜病变患者中tspan12的突变。我们将确定报道的突变是否会损害TSPAN12在Norrin/Frizzled4信号传导中的功能。基于我们对TSPAN12蛋白相互作用的研究，我们将询问是否一个或几个突变损害了TSPAN12的关键蛋白相互作用。我们还将确定一个或几个突变是否会损害TSPAN12的折叠或运输。结论：Norrin/Frizzled4信号作为治疗干预的靶点正在研究中。一个完善的TSPAN12在Norrin/Frizzled4信号中功能的机制模型应该有助于在视网膜新生血管疾病中靶向这一途径。

DESCRIPTION (provided by applicant): Our overall objective is to understand retinal angiogenesis in a context of development and ocular disease, using mice as a model system. Retinal vascular diseases, including proliferative diabetic retinopathy and neovascular age related macular degeneration, are major causes of impaired vision and blindness. One of the central angiogenic signaling pathways in the retina is Norrin/Frizzled4 signaling. Norrin signals via its receptor Frizzled4 and the co-receptor LRP5 on vascular endothelial cells to activate ss-catenin signaling and transcriptional programs. The pathway plays a critical role in instructing retinal vascular morphogenesis and blood-retina barrier formation. Impaired Norrin/Frizzled4 signaling causes familial exudative vitreoretinopathy, an inherited disease that can cause blindness. The focus of this application is on the membrane protein TSPAN12, a novel and essential component of Norrin/Frizzled4 signaling. Tspan12 gene disrupted mice phenocopy norrin and frizzled4 mutant mice, and TSPAN12 is a strong facilitator of Norrin/Frizzled4 signaling in cell-based assays. We have three objectives: i) We hypothesize that TSPAN12 is functionally required in retinal endothelial cells and that restricted TSPAN12 expression modulates the spatio-temporal pattern of pathway activation. This model predicts that conditional disruption of the tspan12 gene in endothelial cells, but not in other cell types, recapitulates the loss-of-function phenotypes, e.g., lack of intraretinal capillaries and impaired blood-retina barrier integrity. We will use the Cre-Lox system to test this model. ii) TSPAN12 is required only in Norrin-induced but not in Wnt-induced Frizzled4 signaling. This is an intriguing and unexplained feature of Norrin/Frizzled4 signaling. We hypothesize that Norrin cannot efficiently induce protein interactions in the Norrin receptor complex and therefore operates together with TSPAN12. In contrast, Wnts may induce the required protein interactions autonomously (e.g., Norrin and Wnts differ in their ability to bind receptor and co-receptor simultaneously). We will test this hypothesis by characterizing protein interactions in the Norrin receptor complex, by determining which interactions are promoted by Norrin, Wnt3a, and TSPAN12, and by determining the consequences of abolishing or forcing relevant protein interactions in cell-based reporter assays. iii) Several recent human genetic studies independently reported mutations in tspan12 in patients with familial exudative vitreoretinopathy. We will determine if the reported mutations impair the function of TSPAN12 in Norrin/Frizzled4 signaling. Building on our investigation of TSPAN12 protein interactions, we will ask if one or several of the mutations impair critical protein interactions of TSPAN12. We will also determine if one or several of the mutations impair the folding or trafficking of TSPAN12. Conclusion: Norrin/Frizzled4 signaling is under investigation as a target for therapeutic intervention. A refined mechanistic model of TSPAN12 function in Norrin/Frizzled4 signaling should aid efforts to target this pathway in neovascular diseases of the retina.