Anisomycin过度激活丝裂原活化蛋白激酶使tau发生过度磷酸化

阿尔茨海默病(Alzheimer's disease,AD)的病理特征之一是神经元内存在神经原纤维缠结(neurofibrillary tangles,NFTs),后者是由过度磷酸化的微管相关蛋白tau形成的双股螺旋细丝(paired helical filaments,PHFs)构成.为了探讨丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)在微管相关蛋白tau磷酸化中的作用及机制,本实验用0.1 μg/mL、0.2 μg/mL和0.4μg/mL三种不同浓度的MAPK激动剂anisomycin处理小鼠成神经瘤细胞株(mouse neuroblastoma cells,N2a),检测MAPK活性的变化及其与tau蛋白多个AD相关位点过度磷酸化的关系,并检测糖原合酶激酶-3(glycogen synthase kinase-3,GSK-3)和蛋白激酶A(protein kinase A,PKA)的活性变化.结果显示,anisomycin以剂量依赖的方式激活MAPK活性,但免疫印迹结果显示tau蛋白的Ser-198/199/202位点和Ser-396/404位点的过度磷酸化只在anisomycin浓度为0.4 μg/mL时出现,三种浓度的anisomycin均未引起tau蛋白Ser-214位点磷酸化的改变;同时,GSK-3活性在anisomycin为0.1 μg/mL时没有明显变化,当anisomycin浓度升高到0.2 μg/mL和0.4 μg/mL时出现明显增高,而PKA的活性没有明显的改变.使用GSK-3的特异性抑制剂氯化锂(LiCl)则完全阻断MAPK被过度激活导致的tau蛋白磷酸化水平的增高,而同时MAPK活性不受影响.以上结果提示:过度激活MAPK可以导致tau蛋白Ser-198/199/202和Ser-396/404位点过度磷酸化,其机制可能涉及MAPK激活GSK-3的间接作用.

Overactivated mitogen-activated protein kinase by anisomycin induces tau hyperphosphorylation

One of the pathological feathers of Alzheimer's disease (AD) is neurofibrillary tangles (NFTs), which consist of paired helical filaments (PHFs) formed by hyperphosphorylated microtubule-associated protein tau. To study the role of mitogen-activated protein kinase (MAPK) in tau hyperphosphorylation and the underlying mechanism, wild type mouse neuroblastoma cells (N2a) were dealt with different concentrations (0.1 mug/mL, 0.2 mug/mL and 0.4 mug/mL) of anisomycin (an activator of MAPK) for 6 h. The relationship between MAPK activity and tau phosphorylation at some Alzheimer-sites was analyzed, and the activities of protein kinase A (PKA) and glycogen synthase kinase-3 (GSK-3) were detected. The results showed that anisomycin activated MAPK in a dose-dependent manner, but tau hyperphosphorylation at Ser-198/199/202 and Ser-396/404 sites was only observed when the concentration of anisomycin was at the level of 0.4 mug/mL, and the alteration of tau phosphorylation at Ser-214 showed no significant difference in different groups. 0.2 mug/mL and 0.4 mug/mL of anisomycin led to an increase in the activity of GSK-3, respectively, but had no effect on the activity of PKA. Lithium chloride, a specific inhibitor of GSK-3, completely abolished the anisomycin-induced elevation of tau phosphorylation without any effect on the activity of MAPK. In conclusion, overactivation of MAPK up to a certain degree induces tau hyperphosphorylation at Ser-198/199/202 and Ser-396/404 sites, and this is probably related to the effect of activated GSK-3 by MAPK.