Disease mechanism of Usher syndrome 2

项目摘要 视网膜色素变性（RP）是一种主要的遗传性视网膜变性疾病，患病率为1 全球4,000家。RP合并听力损失的特征是Usher综合征（USH），这是一种 遗传性眼盲的主要原因目前，RP或USH都没有治愈方法。USH 2是 最常见的USH临床类型。USH 2A（usherin）、ADGRV 1（粘附G蛋白偶联受体V1）和 WHRN（whirlin）是USH 2的3个致病基因。其中，USH 2A是主要的 USH和常染色体隐性遗传RP（arRP）的致病基因，ADGRV 1是第二常见的USH 2 致病基因在光感受器中，usherin和ADGRV 1蛋白与whirlin相互作用并形成多蛋白 复合体位于内外节之间的睫状体周膜上，但个体的功能 USH 2蛋白以及整个USH 2复合物尚未完全阐明。这种知识差距阻碍了 发展基于疾病机制的治疗方法。由于许多患者携带USH 2A突变， 靶向USH 2A的治疗剂的开发和评价是一个活跃的研究领域。但 由于未知的早期视网膜分子缺陷， 在USH 2动物和/或体外视网膜模型中的迟发性视网膜变性。在初步研究中，我们 在USH 2突变小鼠视网膜中进行TMT标记定量蛋白质组学和磷酸化蛋白质组学研究 出生后30天，视网膜变性之前很久。我们发现光感受器外节 膜蛋白，脂化蛋白伴侣，BBSome及其货物，细胞外基质成分， 和蛋白激酶A信号通路。亲和纯化结合质谱实验 鉴定的USH 2蛋白相互作用候选物，其富含与蛋白质相互作用相关的蛋白质。 USH 2突变体视网膜中早期受影响的分子和细胞过程。我们还发现了蛋白质错误定位 和年轻的USH 2突变体视锥细胞中的光响应降低。在我们广泛的初步工作的基础上，我们 建议在本申请中解决以下具体目标：1）识别和研究usherin-以及 视网膜中的ADGRV 1相互作用蛋白; 2）确定三种视网膜中的主要分子缺陷 不同的Ush 2a和Adgrv 1突变小鼠;和3）揭示早发性视锥细胞功能障碍的分子原因 在USH 2突变视网膜中。如果成功，我们的研究将揭示USH 2复合物在光感受器中的作用， 深入了解USH 2基因引起的视网膜变性的致病机制 突变。这项研究还将为开发基于机制的USH新疗法提供信息， arRP和发现有价值的生物标志物，可应用于治疗评价。

Project Summary Retinitis pigmentosa (RP) is the predominant inherited retinal degenerative disease with a prevalence of 1 in 4,000 worldwide. RP combined with hearing loss is characterized as Usher syndrome (USH), which is the leading cause of inherited deaf-blindness. Currently, no cure is available for either RP or USH. USH2 is the most common USH clinical type. USH2A (usherin), ADGRV1 (adhesion G protein-coupled receptor V1), and WHRN (whirlin) have been identified as three USH2 causative genes. Among them, USH2A is the major causative gene for USH and autosomal recessive RP (arRP), and ADGRV1 is the second most frequent USH2 causative gene. In photoreceptors, usherin and ADGRV1 proteins interact with whirlin and form a multiprotein complex at the periciliary membrane between the inner and outer segment, but the functions of individual USH2 proteins as well as the entire USH2 complex have not been fully elucidated. This knowledge gap hinders the development of disease mechanism-based treatments. Since many patients carry USH2A mutations, the development and evaluation of therapeutics that target USH2A is an active area of investigation. However, the progress of these studies has been slowed down, because of the unknown early retinal molecular defects and late onset retinal degeneration in USH2 animal and/or in vitro retinal models. In our preliminary studies, we performed TMT-labeling quantitative proteomic and phosphoproteomic studies in USH2 mutant mouse retinas at postnatal day 30 long before retinal degeneration. We discovered alterations in photoreceptor outer segment membrane proteins, lipidated protein chaperones, BBSome and its cargos, extracellular matrix constituents, and protein kinase A signaling pathway. Affinity purification coupled with mass spectrometry experiments identified USH2 protein-interacting candidates that are enriched with proteins biologically associated with the early affected molecular and cellular processes in USH2 mutant retinas. We also found protein mislocalization and light response reduction in young USH2 mutant cones. Building on our extensive preliminary work, we propose to address the following specific aims in this application: 1) identify and investigate usherin- and ADGRV1-interacting proteins in the retina; 2) determine primary molecular defects in the retina of three different Ush2a and Adgrv1 mutant mice; and 3) uncover the molecular cause of early onset cone dysfunction in USH2 mutant retinas. If successful, our study will reveal the role of the USH2 complex in photoreceptors and provide insight into the pathogenic mechanisms underlying retinal degeneration caused by USH2 gene mutations. This study will also inform the development of mechanism-based new therapeutics for USH and arRP and discover valuable biomarkers that can be applied to therapeutic evaluation.