GSK3β在GRK5基因缺陷致AD轴突转运缺陷中的作用机制研究

Axonal transport defects is one of the early common pathologic features for Alzheimer's Desease (AD) and neurodegenerative diseases, and it also might be the contributing factor of AD. In our previous study, it has been showed that aged G-protein coupled receptor kinase-5 (GRK5) knockout mice (GRK5KO) displayed mild cognitive impairment associated with increased swollen axonal clusters (SACs) in hippocampus. However, the ultrastructural features and the pathogenesis mechanism of hippocampal SACs have yet to be elucidated. Our recent study indicated that the activity of glycogen synthase kinase-3(GSK-3) β and the level of phosphorylated kinesin light chain (KLC) were increased in the hippocampus of aged GRK-/- mice. It was reported that the elevation of GSK3 β activity could lead to hyperphosphorylation of tau, and GSK3β could also phosphorylate KLC and trigger the chaperone-dependent dissociation of kinesin-1 from its cargo, eventually leading to axonal transport defects. Based on these evidences, we hypothesize that elevation of GSK3β induced by GRK5 deficiency can cause AD-like axonal transport deficits. In this proposal, the relationship between the development as well as build-up of hippocampal SACs and the age of GRK5-/- mice will be investigate, and the ultrastructural features of SACs will be examined by immune electron miscroscopy. The role of activation of GSK-3β in the abberant phosphorylation of tau and KLC will be evaluated. And the mechanism that GRK5 deficiency induced the elevation of GSK3β activity will be determined. If our hypothesis is correct, it will help us to reveal the intimate relationship with GRK5 deficiency and early AD-like pathological changes, and provide a new drug target for the prevention and treatment of early AD.

前期研究发现老龄GRK5基因缺陷（GRK5KO）小鼠出现了AD样早期的病理改变-轴突转运缺陷，形态学表现为海马内出现了肿胀轴突丛（SACs），但其超微结构本质及其形成机制尚不清楚。我们还发现GSK-3β活性及磷酸化的KLC水平在老龄GRK5-/-小鼠海马内升高。而GSK-3β活性增高据认为可致tau过度磷酸化，并可磷酸化KLC使其与它转运的货物分离而致轴突转运障碍。据此我们提出了"GRK5基因缺陷致GSK-3β活性增高介导AD轴突转运缺陷"的假说。本项目拟用免疫电镜、激光共聚焦、体内受体阻断等方法，研究GRK5-/-小鼠海马内SACs出现和积累与年龄的相关性及其超微结构本质；观察GSK-3β激活在GRK5缺陷导致的海马内tau和KLC畸变磷酸化中的作用；探讨GRK5缺陷引起GSK-3β活性升高的机制。可望阐明GRK5缺陷引起AD样早期病理改变的分子机制，为早期AD防治提供新的药物作用靶点。

目前关于GRK5在AD中发挥的功能的研究并不深入，课题组较早开展GRK5在AD中研究，并取得了一系列结果：功能性GRK5缺陷可增加AD病理进程，导致小鼠海马内出现轴突肿胀（SACs）及短时记忆受损，其机制涉及Aβ聚集、胆碱能神经元功能障碍以及反应性炎性增生等病程机制，课题组随着研究深入首次提出了GRK5对AD中TAU异常磷酸化相关的假设并深入研究。本项目建立GRK5敲除的转基因小鼠，通过细胞水平以及动物水平的系统研究，结合对GRK5基因SNPs的病例对照研究，获得如下结果：1）AD小鼠模型中随着年龄的增加功能性GRK5减少，p-GSK3β活性减低，p-Tau增加；SH-SY5Y细胞中随着Aβ42浓度的增加，也得到同样支持结果；2）在GRK5KO 的小鼠模型中随着年龄的增加，p-GSK3β活性减低，p-tau增加；在GRK5RNAi的细胞模型中也可得到相应结论，反过来说明GRK5缺陷可显著加速AD病理进程；3）GRK5-OE细胞中p-GSK3β活性增加，p-tau减低，说明功能性GRK5增多可作为AD的一种保护因素。4）给予AD小鼠腹腔注射GSK3β抑制剂后，p-GSK3β活性增加，p-tau减低，而GRK5未见明显变化，说明GSK3β可能是一个关键调节位点；5）当GSK3β抑制剂应用于两种神经细胞系后，GSK3β表达降低，p-tau减弱，但GRK5表达基本不变。说明GRK5可能是通过GSK3β调节tau蛋白磷酸化；6）通过分析筛选出两个单核苷酸突变位点：Arg304His（rs2230345）和Gln41Leu（rs2230345），分别构建慢病毒载体构建突变细胞模型，发现Gln41Leu突变可以抑制GRK5由胞膜转入胞浆及Tau过度磷酸化的作用；7）在病例对照分析中，GRK5基因Gln41Leu与晚发型AD相关，对AD有有保护作用。综合来说本课题结果明确GRK5在AD的病理进程中的早中期开始出现膜转位效应，并通过GRK5-GSK3beta-Tau通路最中对AD的病理进程有巨大贡献，而GRK5与膜转位有关的SNP位点也与AD的易感性相关，这些结果为我们更加明确了GRK5在AD中的重要作用，也提示我们GRK5是AD早中期病理进程中的一个潜在的重要标志物和靶点，研究结果同时将β淀粉样学说、tau蛋白学说及胆碱能学说通过GRK5联系起来，为我们理解AD的作用机制提供参考。