应用斑马鱼模型研究PROM1突变导致视网膜变性的分子机制

The clinical phenotype of retinal degeneration caused by PROM1 mutation is complicated. However, the molecular function of PROM1 in photoreceptors and exact mechanism of retinal degeneration cuased by PROM1 mutation remains unclear. In our previous work, we constructed the world’s first Prom1 knockout zebrafish model, and observed that the thickness of ONL was significantly thinner in the Prom1-deficient zebrafish, which consistent with the clinical symptoms of human retinal degeneration; in addition, we also observed the cone is more severely damaged than rod in the Prom1-deficient zebrafish, and the CP (Consisting mainly of F-actin) was abnormal, suggesting that structural changes of CP may be one of the main reason for photoreceptor apoptosis. In this project, by furter detecting the pathological changes of the retina, we will cleariry the different effects on cones and rods in Prom1-/- zebrafish, reveal the pathological mechanism of retinal degeneration cuased by PROM1 mutation, and provide an important theoretical basis for clinical diagnosis. Besides, by studying the molecular mechanism of PROM1 regulating actin polymerization and dissociation, we will understand the molecular function of PROM1 in photoreceptor, and provide relevant molecular targets for clinical treatment of retinal degeneration.

PROM1突变导致的视网膜变性临床症状比较复杂。目前，该基因的功能及其突变导致视网膜变性的具体病理机制尚不清楚。前期工作中我们构建了世界上首个Prom1敲除的斑马鱼模型，发现敲除型斑马鱼的外核层在早期明显变薄，视锥受累明显重于视杆，这与人类PROM1突变导致的CRD表型相似；此外，Prom1敲除斑马鱼的感光细胞CP (主要成分是F-actin) 形态出现异常，提示CP结构受损可能是导致感光细胞凋亡的重要原因。本项目将在前期工作基础上，通过深入观察与分析Prom1敲除斑马鱼发生视网膜变性的具体病理过程，并着重研究Prom1敲除斑马鱼中不同感光细胞的受损机制以及PROM1调控Actin聚合与解离的作用机制，从而揭示PROM1在感光细胞中的生理功能及其突变后导致视网膜变性的分子机制，为该病的临床诊断和治疗提供重要的理论依据以及相关的分子靶点。

人类prominin 1 (PROM1)基因突变后会导致视网膜色素变性、黄斑变性以及视锥-视杆营养不良等多种不同类型的视网膜变性疾病。有文献应用prom1敲除小鼠模型，发现PROM1对感光细胞外节膜盘的形态发生具有重要作用，但对于PROM1的具体分子功能及其突变后引起的复杂临床表型及病理机制仍不清楚。本研究应用斑马鱼模型探讨PROM1突变后导致视网膜变性的机制。斑马鱼中有两个预测的与PROM1功能同源的基因，分别是：prom1a和prom1b。我们采用TALEN技术分别构建了这两种基因敲除型斑马鱼品系，结果发现prom1b在视网膜感光细胞中具有重要的生理功能。Prom1b缺失后导致感光细胞外节形态异常，但视杆细胞和视锥细胞受损程度显著不同。观察prom1b敲除斑马鱼，发现7天的幼鱼视网膜视锥细胞的数量在便可见明显减少；视杆细胞的外节膜盘形态结构呈现异常，但其细胞数量至9个月龄时仍未见减少。免疫荧光结果显示，一种参与膜盘形成的关键蛋白Prph2在斑马鱼视锥细胞中的表达量明显高于视杆细胞；Prom1b缺失后导致Prph2蛋白出现错误定位，并造成Prph2蛋白二聚体与单体的比例显著下降，提示Prph2定位和多聚化的改变可能是造成Prom1b敲除后斑马鱼视网膜感光细胞变性的重要原因。本项目揭示了PROM1在视网膜中新的生理功能，阐明了该基因突变导致视网膜变性的新的病理机制。