Ciliary pcoket matrix in photoreceptor health

视网膜色素变性25（RetinitisPigmentosa 25，RP 25）是一种常见的常染色体隐性遗传病， 闭眼同源蛋白（EYS）。然而，人们对突变的机制知之甚少， EYS引起RP 25，目前尚无有效的治疗方法。开发一个机械化的联合国系统的挑战之一是， 对EYS功能的理解是小鼠没有EYS基因的事实。为了克服这一重大... 我们已经为RP 25开发了一个斑马鱼模型。斑马鱼表达EYS基因。眼缺陷 斑马鱼表现出与RP 25患者相似的年龄依赖性光感受器退化。因此，目标 该项目的主要目的是利用EYS基因的突变来破译由EYS突变引起的视网膜变性的机制。 斑马鱼模型系统。EYS是一种含有多种表皮生长因子（EGF）的分泌蛋白， 层粘连蛋白球状（LG）结构域。后者是一个保守的结构域结构，能够结合O- α-肌营养不良聚糖的甘露糖基聚糖，细胞外基质的细胞表面受体。初步数据显示 EYS蛋白与O-甘露糖聚糖结合，并与构造复合物共定位在连接点附近， 感光细胞中的连接纤毛/过渡区。异常O-甘露糖基化是综合征的基础 先天性肌营养不良伴脑畸形的视网膜萎缩。有趣的是， 在色素性视网膜炎中发现了参与O-甘露糖基化的酶之一的突变76 （RP76）。结构复合物蛋白与严重的纤毛病变如Joubert综合征有关。我们的中心- tral假说认为EYS是与O-甘露糖基聚糖相互作用的睫状囊基质的关键成员 以及纤毛/过渡带连接处的构造复合体。 目的1：确定EYS在维持感光细胞健康中的作用。 目的2：确定O-甘露聚糖在感光细胞存活中的作用。 目的3：表征EYS与连接纤毛/过渡带的构造复合体的相互作用。 多学科的方法，包括免疫电镜，冷冻电镜，免疫组织学染色，遗传操作， 斑马鱼胚胎中的作用，以及生物化学实验，如免疫共沉淀，邻近连接， 分析和蛋白质组学方法将被用来实现这些目标。总之，拟议的研究将 提供对疾病分子致病机制的见解，揭示EYS如何调节睫状功能， 在光感受器的作用，并揭示了蛋白质的相互作用与EYS在睫状袋。它有可能 将几种形式的视网膜变性连接到一个共同的生物途径中， 由EYS介导的相互作用有助于维持睫状囊结构的完整性， 生存因此，我们将有一个更好的了解光感受器变性的机制， 这将为开发治疗干预措施提供新的机会。

Retinitis pigmentosa 25 (RP25) is a common autosomal recessive genetic disease caused by abnormal eyes shut homolog protein (EYS). There is, however, little understanding of the mechanism by which mutations to EYS cause RP25 and there is no effective therapy. One of the challenges in developing a mechanistic un- derstanding of EYS function is the fact that the mouse does not have the EYS gene. To overcome this signifi- cant hurdle, we have developed a zebrafish model for RP25. Zebrafish express the EYS gene. EYS-deficient zebrafish show age-dependent photoreceptor degeneration similar to patients with RP25. Therefore, the goal of this project is to decipher the mechanisms of retinal degeneration caused by mutations in EYS using the zebrafish model system. EYS is a secreted protein containing multiple epidermal growth factor (EGF) and laminin globular (LG) domains. The latter is a conserved domain structure that is capable of binding to O- mannosyl glycans of α-dystroglycan, a cell surface receptor for extracellular matrix. Preliminary data indicate that the EYS protein binds to O-mannosyl glycans and is co-localized with the tectonic complex near the con- necting cilium/transition zone in photoreceptor cells. Abnormal O-mannosyl glycosylation underlie syndromic retinal atrophy found in congenital muscular dystrophies with brain malformations. Interestingly, hypomorphic mutations in one of the enzymes involved in O-mannosyl glycosylation are found in retinitis pigmentosa 76 (RP76). Tectonic complex proteins are implicated in severe ciliopathies such as Joubert syndrome. Our cen- tral hypothesis is that EYS is a key member of the ciliary pocket matrix interacting with O-mannosyl glycans and the tectonic complex at the connecting cilium/transition zone. Aim 1: Determine the roles of EYS in maintaining photoreceptor health. Aim 2: Determine the roles of O-mannosyl glycans in photoreceptor survival. Aim 3: Characterize EYS interactions with the tectonic complex at connecting cilium/transition zone. Multidisciplinary approaches including immuno-EM, cryo-EM, immunohistological staining, genetic manipu- lations in zebrafish embryos, and biochemical experiments such as co-immunoprecipitation, proximity ligation assay, and proteomic methods will be employed to accomplish the aims. Together, the proposed research will provide insights on molecular pathogenic mechanisms of the disease, uncover how EYS regulates ciliary func- tions in photoreceptors, and reveal the proteins that interact with EYS at the ciliary pocket. It has the potential to link several forms of retinal degeneration into a common biological pathway where cell-extracellular matrix interactions mediated by EYS help maintain the ciliary pocket structural integrity to maintain photoreceptor survival. We, thus, will have a better understanding of the mechanisms of photoreceptor degeneration caused by abnormal EYS, which will provide new opportunities to develop therapeutic interventions.