GSK-3β的苏木化（SUMO）修饰在阿尔兹海默病突触病变中的作用及机制研究

GSK-3β plays an key role in Aβ generation, tau protein phosphorylation, to occur nutrition neuritis and injure neuron. However, in the present study, it is largely unknowed whether increasing GSK-3β activity results in synaptic damage and possible mechanism in Alzheimer’s disease (AD). Our preliminary study discovered that the SUMO-1 levels is increased to detect in cerebral cortex and hippocampus of AD. Aβ enhances SUMO-1, leads to GSK-3β SUMOylation and an increase of GSK-3β level. Therefore, we hypothesized that GSK-3β SUMOylation rose GSK-3β activity and inhibited GSK-3β ubiquitination and degeneration. It will cause synaptic damage in AD by altering expression of synaptic proteins and protein complexes and synapse plasticity. Thus, we intends to construct GSK-3β-SUMO-1 plasmid, specific SUMOylation mutant sites GSK-3β K292R plasmid and wild GSK-3β plasmid and its adeno-associated virus. Then, the plasmids and virus were transfectd and infected in AD transgenic mices, SD rats and primary cultured neurons. In the end, to use those technology of gene transfect, western blot, ELISA, laser confocal, LTP, patch clamp, electron microscope and cell calcium imaging would verificate the hypothesis at cellular and animal levels. The results of the project will might be a new clues of AD pathogenesis.

GSK-3β介导的Aβ生成、tau蛋白磷酸化、营养性神经炎发生、神经元受损是引起阿尔兹海默病（AD）的关键事件，但其活性上调是否导致AD患者突触损伤及其可能机制不明。申请者前期研究发现AD脑内SUMO-1表达增加；Aβ上调SUMO-1，进一步促进GSK-3β的苏木（SUMO）化，且观察到GSK-3β水平增加。因此，我们推测苏木化上调GSK-3β活性、拮抗GSK-3β泛素化抑制其降解，进而通过对突触相关蛋白及突触可塑性进行异常调控，导致AD突触病变。为此，本项目拟通过构建GSK-3β-SUMO-1、特异性苏木化位点GSK-3β K292R -SUMO-1突变体及野生型GSK-3β质粒及腺相关病毒，在AD 转基因鼠及SD大鼠、培养的原代细胞或细胞株，运用基因转染、免疫印迹、ELISA、激光共聚焦、LTP、膜片钳、电镜和钙成像等技术，从细胞和整体水平验证此假说，为AD发病机制研究提供新线索。

GSK-3β介导的Aβ生成、tau蛋白磷酸化、营养性神经炎发生、神经元受损是引起阿尔兹海默病（AD）的关键事件，但其活性上调是否导致AD患者突触损伤及其可能机制不明。申请者前期研究发现AD脑内SUMO-1表达增加；Aβ上调SUMO-1，进一步促进GSK-3β的苏木（SUMO）化，且观察到GSK-3β水平增加。本项目通过构建GSK-3β-SUMO-1、特异性苏木化位点GSK-3β K292R -SUMO-1突变体及野生型GSK-3β质粒及腺相关病毒，在AD 转基因鼠及SD大鼠、培养的原代细胞或细胞株，运用基因转染、免疫印迹、ELISA、激光共聚焦、LTP、膜片钳等技术，从细胞和整体水平验证了GSK-3β能发生苏木化修饰并上调GSK-3β活性，进而通过对突触相关蛋白及突触可塑性进行异常调控，同时还促进了tau蛋白过度磷酸化，进而导致AD突触病变这一假说，为AD发病机制研究提供了新线索。同期还开展了土家医道地药材板桥党参、头顶一颗珠通过GSK-3β信号通路途径治疗阿尔兹海默病机制及疗效研究并已完成。综合上述结果，本结果为GSK-3 β与苏木化（SUMO化）修饰之间的关系以及促进突触病变和tau蛋白过度磷酸化提供了重要的启示性线索。项目资助发表论文十六篇，待发表四篇。根据实验结果撰写的论文获第六届全国大学生基础医学创新研究暨实验设计论坛优秀成果奖二等奖。培养已获得硕士学历学位研究生11名，在读研究生5名。项目投入经费38万元，支出22.48845万元，各项支出基本与预算相符，剩余经费15.511550万元，剩余经费计划用于本项目研究后续支出。