Ciliary pcoket matrix in photoreceptor health

视网膜色素变性25(RP25)是一种常见的常染色体隐性遗传病，由异常引起 闭眼同源蛋白(EYS)。然而，人们对突变的机制知之甚少。 导致RP25，目前还没有有效的治疗方法。发展机械化联合国的挑战之一是- 对EYS功能的理解是，小鼠没有EYS基因。为了克服这一重大意义- 无法跨越，我们为RP25开发了一个斑马鱼模型。斑马鱼表达眼睛基因。眼亏虚 斑马鱼表现出与RP25患者类似的年龄依赖性光感受器退化。因此，目标是 这个项目的目的是破译由眼睛突变引起的视网膜退化的机制 斑马鱼模型系统。EYS是一种分泌蛋白，含有多种表皮生长因子(EGF)和 层粘连蛋白球状(LG)结构域。后者是一种保守的结构域结构，能够与O-结合 α的甘露糖-营养不良多糖，细胞表面细胞外基质的受体。初步数据显示 EYS蛋白与O-甘露糖基多聚糖结合，并与边界附近的构造复合体共存。 光感受器细胞的纤毛/过渡带。O-甘露糖糖基化异常是综合征的基础 先天性肌营养不良合并脑畸形的视网膜萎缩。有趣的是，亚象 在视网膜色素变性76中发现参与O-甘露糖糖基化的一种酶的突变 (RP76)。结构复合体蛋白与严重的纤毛疾病有关，如Joubert综合征。我们的中心- Tral假说认为，EYS是与O-甘露糖基相互作用的睫状囊基质的关键成员 连接纤毛/过渡带的构造杂岩。 目的1：确定EYS在维持光感受器健康中的作用。 目的2：确定O-甘露糖聚糖在光感受器存活中的作用。 目的3：描述EYS与连接纤毛/过渡带的构造杂岩的相互作用。 多学科的方法，包括免疫-EM，冷冻-EM，免疫组织学染色，遗传操作 斑马鱼胚胎中的离子，以及免疫共沉淀、邻近连接等生化实验 实验，并将使用蛋白质组学方法来实现这些目标。总之，拟议的研究将 为该病的分子致病机制提供见解，揭示EYS是如何调节睫状体功能的。 并揭示了与睫状袋中的眼睛相互作用的蛋白质。它有潜力 将几种形式的视网膜退行性变连接到一条共同的生物途径，其中细胞-细胞外基质 EYS介导的相互作用有助于维持睫状囊结构的完整性，以维持光感受器 生死存亡。因此，我们将更好地理解光感受器退化引起的机制。 通过异常眼球，这将为发展治疗干预提供新的机会。

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.