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

描述(由申请人提供)：我们的总体目标是以小鼠为模型系统，在发育和眼部疾病的背景下了解视网膜血管生成。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老年性黄斑变性，是导致视力减退和失明的主要原因。视网膜中的中心血管生成信号通路之一是Norrin/Frizzled4信号通路。Norrin通过其受体Frizzled4和血管内皮细胞上的辅助受体LRP5信号来激活？连环蛋白信号和转录程序。该通路在指导视网膜血管形态发生和血-视网膜屏障的形成中起着关键作用。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。这一应用的重点是膜蛋白TSPAN12，它是Norrin/Frizzled4信号的一个新的和重要的组成部分。Tspan12基因扰乱了小鼠的表型Norrin和Frizzled4突变小鼠，TSPAN12在基于细胞的分析中是Norrin/Frizzled4信号的强大促进器。我们有三个目标：i)我们假设TSPAN12在功能上是视网膜内皮细胞所必需的，并且受限的TSPAN12表达调节通路激活的时空模式。该模型预测，内皮细胞中tspan12基因的条件性破坏，但在其他细胞类型中不是，重述了功能丧失的表型，例如，视网膜内毛细血管缺乏和血-视网膜屏障完整性受损。我们将使用CRE-Lox系统来测试该模型。Ii)TSPAN12只有在Norrin诱导的信号中才需要，而在Wnt诱导的Frizzled4信号中不需要。这是Norrin/Frizzled4信号的一个有趣而又无法解释的特征。我们假设去甲肾上腺素不能有效地诱导去甲肾上腺素受体复合体中的蛋白质相互作用，因此与TSPAN12一起作用。相反，Wnts可以自主地诱导所需的蛋白质相互作用(例如，Norrin和Wnts在同时结合受体和辅助受体的能力上不同)。我们将通过表征Norrin受体复合体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，以及通过确定在基于细胞的报告分析中取消或强制相关蛋白质相互作用的后果来检验这一假设。最近的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的tspan12基因突变。我们将确定已报道的突变是否损害了TSPAN12在Norrin/Frizzled4信号中的功能。在我们对TSPAN12蛋白质相互作用的研究基础上，我们将询问是否一个或几个突变损害了TSPAN12的关键蛋白质相互作用。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信号通路作为治疗干预的靶点正在研究中。TSPAN12在Norrin/Frizzled4信号中作用的精细机制模型应该有助于在视网膜新生血管疾病中针对这一途径的努力。

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.