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

Norrin/Frizzled4 信号在视网膜血管生成中的作用：TSPAN12 的作用

基本信息

批准号：
8668328
负责人：
Harald Junge
金额：
$ 37.03万
依托单位：
UNIVERSITY OF COLORADO
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8668328
关键词：
Affect Binding Biological Assay Biological Models Biology Blindness Blood Blood Vessels Blood capillaries Bypass Cells Chimera organism Complex Cre-LoxP Defect Development Dimerization Disease Distant Endothelial Cells Exudative age-related macular degeneration Eye Development Family Genes Genetic Goals Homo Human Genetics Immunoprecipitation In Vitro Inherited Integral Membrane Protein Investigation Lead Ligands Ligation Lipoprotein Receptor Luciferases Mediating Mediator of activation protein Membrane Proteins Modeling Molecular Molecular Genetics Morphogenesis Mus Mutant Strains Mice Mutation Neurons Norrie&apos s disease Pathway interactions Patients Pattern Pericytes Phenocopy Phenotype Play Point Mutation Proteins Reporter Reporting Retina Retinal Retinal Diseases Role Signal Pathway Signal Transduction System Tacrolimus Binding Proteins Testing Therapeutic Intervention Vascular Diseases Vascular Endothelial Cell Vascularization Vision base capillary cell type human PHEMX protein in vivo loss of function member neovascular novel programs proliferative diabetic retinopathy public health relevance receptor receptor binding research study response retina blood vessel structure retinal angiogenesis retinal progenitor cell trafficking

项目摘要

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.

描述（由申请人提供）：我们的总体目标是使用小鼠作为模型系统，了解发育和眼部疾病背景下的视网膜血管生成。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性年龄相关性黄斑变性，是视力受损和失明的主要原因。视网膜中的中心血管生成信号传导途径之一是Norrin/Frizzled 4信号传导。Norrin通过其受体Frizzled 4和血管内皮细胞上的共受体LRP 5来激活β-连环蛋白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。受损的Norrin/Frizzled 4信号传导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。本申请的重点是膜蛋白TSPAN 12，这是Norrin/Frizzled 4信号传导的一种新的重要组分。Tspan 12基因破坏了小鼠norrin和frizzled 4突变小鼠的表型，并且TSPAN 12在基于细胞的测定中是Norrin/Frizzled 4信号传导的强促进剂。我们有三个目标：i）我们假设TSPAN 12在视网膜内皮细胞中是功能上所需的，并且限制的TSPAN 12表达调节途径激活的时空模式。该模型预测，内皮细胞中tspan 12基因的条件性破坏，而不是其他细胞类型中的条件性破坏，概括了功能丧失表型，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系统来测试这个模型。ii）TSPAN 12仅在Norrin诱导的而不是Wnt诱导的Frizzled 4信号传导中需要。这是Norrin/Frizzled 4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TSPAN 12一起起作用。相反，Wnt可以自主诱导所需的蛋白质相互作用（例如，Norrin和Wnt的不同之处在于它们同时结合受体和共受体的能力）。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，Wnt 3a和TSPAN 12促进的相互作用，以及通过确定在基于细胞的报告基因测定中消除或强制相关蛋白质相互作用的后果来测试这一假设。iii）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tspan 12的突变。我们将确定报告的突变是否损害了TSPAN 12在Norrin/Frizzled 4信号传导中的功能。基于我们对TSPAN 12蛋白相互作用的研究，我们将询问是否有一个或几个突变损害了TSPAN 12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TSPAN 12的折叠或运输。结论：Norrin/Frizzled 4信号转导作为治疗干预的靶点正在研究中。TSPAN 12在Norrin/Frizzled 4信号传导中功能的精细机制模型应该有助于在视网膜新生血管疾病中靶向该途径的努力。