GLP-1受体介导的GSK-3β/PP2A平衡在糖尿病合并AD治疗中的作用及其机制研究

Diabetes mellitus is considered to be a risk factor for Alzheimer's disease (AD), but the pathogenesis is still unclear. Diabetes-related AD is lack of effective drugs. In our previous study, we have found the amelioration effect of neurodegenerative changes in cellular and rat models of diabetes-related AD by glucagon-like peptide-1 (GLP-1) receptor agonist through reversal abnormal activation of glycogen synthase kinase 3β (GSK-3β), but the effect and mechanism of GLP-1 on protein phosphatase 2A (PP2A) in diabetes-related AD especially the mechanism in regulation of the equilibrium between GSK3β and PP2A is unclear. Based on the unbalance in GSK3β/PP2A is a key risk factor for AD, in this project, first we will investigate the mechanism of deregulation of the equilibrium between GSK3β and PP2A in diabetes-related AD and the effect of GLP-1 receptor agonist; then we will intervene GSK3β/PP2A by using the transfection, RNA interference (RNAi) technologies and so on to explore the roles of GSK3β and PP2A activation/inactivation in AD and the regulation effect of GLP-1; finally we will clarify the roles of GSK-3β/cAMP response element binding protein (GSK-3β/CREB), insulin/phosphoinositide 3-kinase (insulin/PI3K) and mammalian target of rapamycin (mTOR) pathways in the regulation of the equilibrium between GSK3β and PP2A by GLP-1 receptor agonist. The aim of this project is to clarify the mechanism of GSK3β/PP2A equilibrium mediated by GLP-1 receptor and provide new experimental evidence for the potential targets of GLP-1 receptor agonist treatment of diabetes-related AD.

糖尿病增加阿尔茨海默病（AD）发生风险，但糖尿病合并AD发生机制不明确且缺乏有效药物。本课题组已证实GLP-1受体激动剂抑制糖原合成酶激酶-3β（GSK-3β）异常活化改善糖尿病相关AD动物、细胞模型病变，但糖尿病合并AD中GLP-1对蛋白磷酸酯酶2A（PP2A）的作用，特别是稳定GSK3β/PP2A平衡的机制尚不清楚，基于GSK3β/PP2A失衡是AD发生的关键因素，本项目首先观察糖尿病合并AD致GSK-3β/PP2A失衡及GLP-1干预作用；然后采用转染、RNAi等技术考察GSK-3β和PP2A激活或抑制在AD发病中的作用及GLP-1的调节；最后分析GSK-3β/核转录因子CREB、胰岛素/PI3K、mTOR途径在GLP-1调节GSK-3β/PP2A中的作用，旨在阐明GLP-1受体介导GSK-3β/PP2A平衡的分子机制，为GLP-1受体激动剂治疗糖尿病合并AD及新靶点提供实验依据。

近年，由糖尿病引起的神经系统损伤倍受关注，研究发现糖尿病增加AD发生风险，目前糖尿病相关AD的发生机制尚不明确，缺乏有效药物。研究表明GLP-1受体激动剂（GLP-1RA）具有神经保护作用，但其具体作用机理不明确。本项目基于AGEs建立了糖尿病相关AD细胞和动物模型，研究 GLP-1RA对AGEs致tau病变和与之相关的GSK-3β/PP2A的作用及机理。明确了GLP-1RA显著改善AGEs导致的神经元损伤以及神经元tau蛋白过度磷酸化；exendin-4显著缓解模型小鼠海马CA1区病理改变，并减少模型小鼠海马tau异常磷酸化；发现GLP-1RA通过抑制GSK-3β活性改善AGEs引起的tau蛋白异常磷酸化水平，而并不影响 PP2A 活性变化；且GSK-3β特异性抑制剂能显著改善AGEs导致的神经元tau蛋白病变，而用GSK-3β激活剂则明显阻断GLP-1RA 的神经保护作用，同时RNAi确证GSK-3β在其中靶点的地位。进一步，深入探讨GSK-3β介导GLP-1发挥作用的机理。发现GLP-1RA 能够显著降低AGEs引起的神经元胞内ROS异常升高，修复线粒体形态损伤，并提高线粒体膜电位水平；exendin-4 能够显著抑制AGEs导致的小鼠海马神经元胞内ROS生成水平，并降低氧化应激标志物8-羟基脱氧鸟苷的生成；同时通过对参与线粒体生物发生调控的主要因子的考察，发现GLP-1RA能够通过提高PGC-1α、NRF-1、Tfam 活性改善AGEs引起的氧化应激水平异常，并可提高小鼠模型海马线粒体内抗氧化酶活性；免疫共沉淀检测GSK-3β与线粒体生物发生调控因子的相互作用，同时探讨其在GLP-1信号系统和RAGE受体途径对话中的作用，发现GLP-1可能通过缓解AGEs对GSK-3β与PGC-1α相互作用的抑制而发挥相关活性。本项目从动物、细胞水平探讨了GLP-1RA防治糖尿病相关AD的分子机制，为治疗糖尿病相关AD的潜在靶点提供实验依据。通过本项目，已发表论文10篇（SCI论文9篇），参加国内外学术交流3次，培养研究生6名，其中博士研究生3名，硕士研究生3名。