RP2/视杆细胞光转导蛋白/视网膜色素变性致病机制

In the progression of retinitis pigmentosa (RP), rod photoreceptors are affected first (leading to night blindness), followed by the gradual loss of cone photoreceptors which results in tunnel vision and finally blindness. For a long time, psychologists and clinical researchers have been puzzled by the mechanism of why rods rather than cones are specifically impaired in the early stage of RP. In the preliminary studies, we knocked out the RP2 gene in zebrafish through the TALEN technology, and observed that the RP2 knockout zebrafish exhibit similar symptoms of typical RP patients. Furthermore, iTRAQ quantitative proteomics study revealed that in 2 mpf RP2 knockout zebrafish, the rod phototransduction proteins are down-regulated specifically. These results suggest the reduced protein levels of rod phototransduction proteins may be the main cause of rod dysfunction in early stage of RP. In this project, we are going to detect the pathologic changes of the photoreceptors at morphological and structrural level in RP2 deficient zebrafish, analyze the correlation between the disease progression and the expression of phototransduction proteins, and further explore the molecular pathways of how RP2 regulates the protein level of rod phototransduction proteins, reveal the pathogenesis of rod specific impairment in early stage caused by RP2 elimination. Our work will help to elucidate the disease-causing mechanism of retinitis pigmentosa 2, and for the first time explain why rods are affected first in RP progression, which has important academic significance and potential application value.

视网膜色素变性（RP）发病过程中，视杆细胞首先受累并表现出夜盲症状，进而引发视锥细胞逐渐死亡最后导致失明。长期以来，为何RP早期特异性地影响视杆细胞而非视锥细胞，一直困扰着生理学家和临床研究者。前期工作中我们应用TALEN技术在斑马鱼中敲除RP2基因，发现RP2敲除斑马鱼呈现与RP患者类似的表型；iTRAQ定量蛋白组学研究显示2个月时RP2敲除特异性下调视杆细胞的光转蛋白，提示视杆细胞光转导蛋白的下调可能是RP早期视杆受累的重要原因。本项目中，我们将观察RP2缺陷斑马鱼感光细胞在形态结构方面的病理改变，分析其与光转导蛋白表达的关系，并进一步探讨RP2调控视杆细胞光转导蛋白的分子途径，揭示RP2突变引发早期视杆细胞特异性损伤的机制。本项目将有助于阐明RP2发病的病理机制，并首次解释RP发病为何从视杆细胞受损开始的问题，具有重要的科学意义和潜在的应用价值。

视网膜色素变性（Retinitis pigmentosa, RP）发病过程中，视杆细胞首先受累并表现出夜盲症状，进而引发视锥细胞逐渐死亡最后导致失明。RP2是常见的X连锁RP致病基因，其突变导致RP的机制尚不明确。本项目应用TALEN技术首次构建了rp2敲除斑马鱼模型，发现其具有进行性视网膜变性表型，且表现出视杆细胞先于视锥细胞受损的特征，与典型的RP症状类似。通过iTRAQ定量蛋白组学分析和western blot验证，发现rp2敲除斑马鱼视网膜中视杆细胞光转导蛋白显著下调，而视锥细胞光转导蛋白则无明显变化。发现视杆细胞光转导蛋白的下调早于视网膜组织结构的变化，并进一步发现视杆细胞光转导蛋白Gnat1和Grk1的定位存在异常。利用anti-Farnesyl抗体，对斑马鱼视网膜中总的异戊二烯化蛋白的分布进行了检测，发现异戊二烯化蛋白的定位确实出现异常。我们还发现大多数人类RP2的致病突变可促进该蛋白降解并成功解析其分子机制。我们的研究首次通过体内实验证明了RP2可能通过ARL3和UNC119/PDE6D调控视杆细胞光转导蛋白的运输和蛋白水平，并揭示了RP2突变导致视网膜变性的分子机制，具有重要的科学意义和潜在的应用价值。

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