MEKK3-MKK3-P38信号模块与RAGE作用位点的筛选及其在高糖诱导海马神经元损伤中的作用机制

Diabetes induced cognitive deficit is closely related to hippocampal neurons injury. NF-κB nuclear translocation plays a key role in hippocampal neurons injury induced by high glucose. Phosphorylated P38 can promote NF-κB nuclear translocation. The preliminary experiments showed that high sugar accelerated the combination of RAGE and MEKK3-MKK3-P38 signal module, increase the P38 phosphorylation and NF-κB nuclear translocation. However, which proteins of MEKK3, MKK3, P38 or OSM can directly bind to RAGE, and interacting sites between them, have not been reported. .In this project, mammalian two-hybrid system will be performed to select the "target protein" which can combine with RAGE directly from MEKK3-MKK3-P38 signal module. Furthermore, we will use GST pull-down, confocal laser scanning (CLS) and immunoprecipitation methods to validate the above experimental results. These above technologies will be used to screen and validate the interacting sites between RAGE and “target protein” following sites mutation respectively. Moreover, we will mutate interacting sites and explore the effects and mechanisms of them in hippocampal neurons injury induced by high glucose. This project will provide new experimental evidence for searching new drug target to prevent hippocampal neurons damage induced by high glucose.

糖尿病引起的认知障碍与海马神经元损伤密切相关，而NF-κB核转位是高糖诱导海马神经元损伤的关键因素，并且P38磷酸化可促进NF-κB核转位。预实验发现：高糖刺激可使RAGE与MEKK3-MKK3-P38信号模块结合，并促进该模块组装，促使P38磷酸化和NF-κB核转位。然而，RAGE是与该模块中MEKK3、MKK3或P38直接作用，还是与其支架蛋白OSM作用，及具体作用位点，尚不明确。.本项目拟用哺乳动物双杂交技术，从MEKK3-MKK3-P38信号模块中筛选出与RAGE直接作用的“靶蛋白”，用GST pull-down、激光共聚焦和免疫共沉淀等技术进行验证；分别构建RAGE和“靶蛋白”的位点突变体，再以上述技术筛选并验证它们之间的作用位点；分别突变RAGE和“靶蛋白”上的作用位点，研究其在高糖诱导海马神经元损伤中的作用机制，为高糖诱导海马神经元损伤的防治提供新的药物靶点和实验依据。

糖基化终末产物受体（RAGE）及其相关的信号通路与糖尿病认知功能障碍的病理进程紧密相关。但是高糖环境下，RAGE促进海马神经元损伤及认知功能障碍的分子机制尚未完全阐明。.通过细胞实验和动物实验证明，高糖刺激下RAGE可以与MEKK3-MKK3-p38信号模块及其支架蛋白OSM结合，并且促进该模块的组装，进而活化其下游p38MAPK/NF-κB信号通路活化，导致海马神经元损伤，促进糖尿病认知功能障碍。通过蛋白的对接模拟，GST pull-down，免疫共沉淀和激光共聚焦等实验证明RAGE胞内端氨基酸2-5可与信号模块中的MKK3的329位氨基酸直接结合。突变RAGE胞内端氨基酸2-5，能够抑制RAGE与MKK3结合，减轻高糖刺激引起的细胞损伤。此外，RAGE突变体能够减少db/db小鼠海马区RAGE与MKK3结合，减少神经元丢失，提高糖尿病小鼠的认知功能。此部分内容依据整理成文，投稿在PNAS杂志，目前正在审稿阶段。.在开展上述研究的实验中，我们发现：RAGE的高表达，能够促进糖尿病小鼠海马区炎症因子的表达，导致小鼠焦虑抑郁样行为。抑制海马区RAGE的表达，通过减少海马区炎症因子的表达，改善糖尿病小鼠的焦虑和抑郁症状。.此外，我们还发现抑制p38MAPK信号通路，能够减轻小鼠的神经炎症。. 实验的开展按照研究计划进行，并且全部完成：此项研究中，我们阐明了高糖环境下RAGE表达增加，引起海马神经元丢失和动物认知功能障碍的分子机制。为阐明糖尿病脑病的病理机制提供理论依据，同时也为临床上糖尿病脑病的治疗提供有效的药物靶点。