Disease mechanism of Usher syndrome 2

项目摘要 视网膜色素变性是一种主要的遗传性视网膜退行性疾病，发病率为1：1。 全球4000人。RP合并听力损失的特征是Usher综合征(USH)，这是 遗传性聋盲的主要原因。目前，无论是RP还是USH都没有治愈方法。USH2是 最常见的USH临床型。USH2A(前列蛋白)、ADGRV1(黏附G蛋白偶联受体V1)，以及 WHRN(Whirlin)已被鉴定为三个USH2致病基因。其中，USH2A是主要的 USH和常染色体隐性遗传RP(Arrp)的致病基因，ADGRV1是第二常见的USH2 致病基因。在光感受器中，Uherin和ADGRV1蛋白与Whirlin相互作用并形成多蛋白 内、外段睫状膜复合体，但个体的功能 USH2蛋白以及整个USH2复合体还没有完全阐明。这一知识鸿沟阻碍了 发展以疾病机制为基础的治疗方法。由于许多患者携带USH2A突变， 针对USH2A的治疗药物的开发和评估是一个活跃的研究领域。然而， 这些研究进展缓慢，因为未知的早期视网膜分子缺陷和 USH2动物和/或体外视网膜模型中迟发性视网膜变性。在我们的初步研究中，我们 对USH2突变小鼠视网膜进行TMT标记的定量蛋白质组和磷酸蛋白质组研究 在出生后30天，远在视网膜变性之前。我们发现光感受器外段发生了变化 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。亲和纯化与质谱联用实验 确定了与USH2相互作用的候选蛋白，这些候选蛋白富含与USH2蛋白生物学相关的蛋白质 USH2突变视网膜早期受影响的分子和细胞过程。我们还发现蛋白质定位错误 在年轻的USH2突变体锥体中光反应减弱。在我们广泛的前期工作的基础上，我们 建议解决本申请中的以下具体目标：1)确定和调查迎宾-以及 视网膜中的ADGRV1相互作用蛋白；2)确定视网膜中的初级分子缺陷 不同的Ush2a和Adgrv1突变小鼠；3)揭示早发性视锥细胞功能障碍的分子原因 在USH2突变体视网膜中。如果成功，我们的研究将揭示USH2复合体在光感受器和 深入了解USH2基因导致视网膜变性的致病机制 突变。这项研究也将为USH和USH的基于机制的新疗法的发展提供信息。 ARRP，并发现有价值的生物标志物，可用于疗效评估。

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.