基于GJA8-Atg 蛋白调控的晶状体上皮细胞自噬异常及其分子机制研究

Gap junction protein alpha 8(GJA8), which mediates the intercellular communication and substance exchange in lens cells, plays an important role in lens’ proliferation, differentiation, osmotic balance and metabolism homeostasis.GJA8 is one of the cataract disease-associated genes .By cloning and functional analysis, we have found that GJA8 can regulate the autophagy process in lens epithelial cells (LECs). Based on this, we plan to explore the molecular pathway between GJA8 and autophagy in LECs by electron microscopy, Western blotting, immunofluorescence, immune co-precipitation, related genes knockdown cell lines and animal models. . To be detailed: We will investigate the interaction between GJA8 and pre-autophagosome-related proteins (autophagy-related proteins(Atg) family, Beclin1, Vps34(phosphoinositide-3-kinase class 3 ,PIK3C3),Vps15(phosphoinositide 3-kinase regulatory subunit 4,PIK3R4),etc.),and detect the expression of the autophagy-related protein( microtubule-associated protein 1 light chain (LC3), nucleoporin p62 (P62), gamma-aminobutyric aci receptor-associated protein (GABARAP), etc.).Zebra fish models will also be established by CRISPR/Cas9 (the clustered, regularly interspaced, short palindromic repeats-associated protein systems)to confirm our results. In conclusion, we plan to explore the potential regulatory networks connecting GJA8-Atg proteins to autophagy process in LECs, and uncover the mechanism for GJA8 in embryonic lens development and cataract formation. Our research will contribute to the new targeting points for the prevention and treatment of lens disease, and lead new directions in clinical work.

缝隙连接蛋白α8(gap junction protein alpha 8，GJA8)参与细胞间信息传递与物质交换，对晶状体的生长发育、渗透平衡及代谢稳态具有重要作用。GJA8是白内障疾病相关候选基因。课题组在前期对GJA8克隆和功能研究中，首次发现其具有调控晶状体上皮细胞自噬的功能。本项目拟采用电镜观察、蛋白印迹、免疫荧光、免疫共沉淀、构建多种自噬相关基因敲减细胞系和动物模型等方法研究GJA8与自噬之间的分子通路，探索GJA8与前自噬体相关蛋白（Atg家族、Beclin1等）间的相互作用，检测自噬标记蛋白LC3、P62、GABARAP等的表达，并经CRISPR/Cas9技术构建斑马鱼动物模型验证，建立GJA8-Atg蛋白对晶状体自噬的调控反馈网络，以期从自噬的角度探索并阐述GJA8在晶状体胚胎发育异常和白内障发生发展中的作用机制，寻找晶状体疾病防治的新靶点，为临床工作提供新思路。

本项目拟在前期白内障家系中发现的GJA8基因突变与该基因主要的缝隙连接通道功能无关，提示GJA8基因存在其他的重要细胞功能，并发现与细胞自噬相关，课题组由此展开对GJA8基因在自噬相关调控机制中的研究，采用CRISPR/Cas9构建GJA8基因敲除斑马鱼动物模型，电镜观察、蛋白印迹、免疫荧光、免疫共沉淀等方法研究GJA8与自噬之间的分子通路，探索GJA8与前自噬体相关蛋白（Atg家族）间的相互作用，检测自噬标记蛋白LC3等的表达，并在斑马鱼动物模型中验证，建立GJA8-Atg蛋白对晶状体自噬的调控反馈网络，完成了从自噬的角度探索并阐述GJA8基因在晶状体发育和白内障发生发展中的作用机制，发现了晶状体疾病防治的新靶点，为临床工作提供新思路。