纤毛内Hedgehog通路异常在剪切缺陷致视网膜色素变性中的作用机制研究

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy. It is characterized by progressive loss of photoreceptor cells（especially the rod）. Hedgehog pathway plays an important role in mammalian photoreceptor cell development. We have shown that mutation of SNRNP200 and possible other pre-mRNA splicing genes is an important cause of autosomal dominant RP (adRP). Our preliminary studies showed that certain cilium genes (the structure containing the key components of hedgehog pathway) and the hedgehog-targeted genes (i.e. Ptch1 and Gli1) were dramatically downregulated in pre-mRNA mutation-induced defect of splicing model. On the basis of our previous population studies, in the current proposal, we will first establish in vitro and in vivo splicing defect models-caused by pre-mRNA mutation. Experiments will be performed to prove that splicing defect-induced with pre-mRNA mutation will affect the structure and function of cilia via downregulating cilia genes, therefore leading to hedgehog signaling pathway in-activation, which then results in photoreceptor cell dysplasia and apoptosis. This project aims to explore the core pathogenesis of adRP associated with splicing defect, and to clarify the function role of cilia/hedgehog pathway abnormality in the pre-mRNA mutation-induced defect of splicing.

视网膜色素变性(retinitis pigmentosa, RP)是一种重大遗传性致盲眼病，其核心病理改变是光感受器细胞中视杆细胞进行性退行性变、凋亡。大量研究证明Hedgehog信号通路在光感受器细胞发育和存活过程中发挥着重要作用。我们在前期收集RP家系的研究中发现SNRNP200等剪切基因突变是常染色体显性遗传RP（adRP）的重要致病病因。进一步预实验分析剪切基因突变所致基因表达改变时，发现Hedgehog通路下游靶基因Ptch1，Gli1以及纤毛（Hedgehog核心元件所在结构）相关基因表达下调。因此，本项目将在上述研究基础上，拟通过构建剪切缺陷小鼠模型和光感受器细胞模型，证实剪切缺陷影响的纤毛结构和功能，进而下调纤毛内Hedgehog信号通路的活化，最终导致视杆细胞发育异常、凋亡。本项目旨在探究剪切缺陷致adRP的核心病理机制，明确纤毛内Hedgehog通路异常在其中的作用。

视网膜色素变性(retinitis pigmentosa, RP)是其中常见的一种遗传性视网膜疾病。我们在对前期临床收集的adRP患者样本进行研究时分别发现了SNRNP200和PRPF4这两个重要的adRP致病基因，其突变均会影响剪切复合体解旋并进而造成多个剪接基因表达水平的改变，以及剪切子复合体再循环障碍。为了进一步研究剪切突变特异性引起视网膜色素变性的机制，我们用全基因组深度测序技术发现一组纤毛表达基因和Hedgehog信号通路下游靶基因Gli1表达水平明显下调。并且原代培养hPrp4Pro315Leu和hSnrnp200P1087L的成纤维细胞，发现在hPrp4Pro315Leu和hSnrnp200P1087L纤毛长度和生长率明显减少 。Gli1(Gli1是Hedgehog信号通路重要的下游靶点)区别于对照组的顶端表达，在hPrp4Pro315Leu和hSnrnp200P1087L突变中，并未观察到Gli1的表达。构建剪切缺陷的小鼠模型，HE染色结果显示WT和突变小鼠眼球大小基本一致，但是光感受其结构和IS/OS在SNRNP200 R1090L knock in小鼠视网膜明显紊乱，用原位杂交的方法检测Shh（sonic hedgehog，hedgehog信号通路的配体）在剪切缺陷小鼠中的表达。SNRNP200 R1090L knock in小鼠中shh表达明显减弱。本研究解释Pre-mRNA剪切缺陷导致adRP的核心病理机制，明确纤毛内Hedgehog信号通路异常在其中的作用，并且提出Hedgehog可作为治疗视网膜色素变性的新靶点，从而为临床治疗光感受器细胞损伤后的再生以及视觉功能的修复提供重要思路