GSK-3在阿尔茨海默病样细胞骨架蛋白过度磷酸化中的作用(英)

神经原纤维缠结是阿尔茨海默病（Alzheimer disease, AD）的特征性病理改变.蛋白激酶和蛋白磷酸酯酶失衡可导致骨架蛋白的异常过度磷酸化，而异常过度磷酸化的tau 和神经丝 (neurofilament, NF) 是神经原纤维缠结的组成部分.在众多激酶中，糖原合酶激酶-3（glycogen synthase kinase-3，GSK-3）可能是AD神经退行性变起重要作用.为深入探讨GSK-3在AD样神经退行性变中的作用，以磷酯酰肌醇三磷酸激酶（phosphatidylinositol 3-kinase，PI3K）的特异性抑制剂渥曼青霉素（wortmannin，WT）处理野生型鼠成神经瘤细胞株（wild type mouse neuroblastoma cell lines, N2a wt），系统观察WT处理N2a wt不同时间点（1 h、3 h、6 h）细胞代谢率、细胞形态、细胞骨架蛋白tau和NF的磷酸化状态改变以及细胞的命运，并分析了GSK-3活性与上述参数改变之间的相关性.结果发现：1 μmol/L WT处理细胞1 h，GSK-3活性与未经WT处理的对照组相比明显增高，并伴有Ser9磷酸化的GSK-3水平的降低； NF磷酸化程度增强，tau在Ser198/Ser199/Ser202位点的磷酸化增强. 1 μmol/L WT处理细胞3 h，GSK-3活性与对照组和处理1 h 组相比明显下降，NF磷酸化程度较1 h降低，但仍高于正常水平.1 μmol/L WT处理细胞6 h，细胞形态、GSK-3活性、Ser9磷酸化形式的GSK-3β的表达、NF磷酸化程度与对照组相比均无明显改变.WT呈剂量依赖性降低细胞代谢率.1 μmol/L WT处理细胞1 h和3 h导致细胞变圆，突起变短甚至消失.1 μmol/L WT处理细胞1 h，用TUNEL法和电子显微镜技术未观察到细胞凋亡.研究结果提示：在N2a细胞中过度激活GSK-3可导致神经细丝和tau蛋白的AD样过度磷酸化，从而引起神经细胞的AD样退行性变.

Activation of Glycogen Synthase Kinase 3 Induces Alzheimer-like Hyperphosphorylation of Cytoskeleton Protein and Cell Damage

Neurofibrillary tangles are the neuropathological hallmarks of Alzheimer disease (AD). Abnormally hyperphosphorylated tau and neurofilament (NF) are the components of neurofibrillary tangles. Hyperphosphorylation may be the result of an imbalanced regulation between protein kinases and protein phosphatases. Among the many kinases,glycogen synthase kinase-3(GSK-3) might be a key participator in neurodegeneration of AD. To investigate the role of GSK-3 on Alzheimer-like neurofibrillary degeneration, the wild type mouse neuroblastoma cell lines (N2awt) were treated with wortmannin (WT), an inhibitor of phosphatidylinositol 3-kinase (PI3K), and the effect of WT on cell metabolism, cell morphology, cell apoptosis, phosphorylation of NF and tau were detected, as well as the relationship between the alternations of these parameters and GSK-3 activity. It was found (1) that treatment of the cell with 1 μmol/L WT led to a transient (at 1h) activation of GSK-3 with a concurrent increase in phosphorylation of NF and tau. At 3h, the activity of GSK-3 was decreased and the hyperphosphorylation of NF was partially restored. (2) that WT decreased the cell metabolism detected by MTT assay in a dose dependent manner. (3) that treatment of the cell with 1 μmol/L WT for 1 h or for 3h induced retraction of cell processes. (4) that no typical apoptotic damage was seen by transient stimulation of GSK-3 activity. It is suggested that transit overactivation of GSK-3 led to Alzheimer-like hyperphosphorylation of cytoskeleton protein and impairment in cell viability.