早老蛋白-1过度表达对神经母细胞瘤NG-108的tau蛋白磷酸化的影响

摘要

目的 观察早老蛋白-1(Presenilin-1,PS1)的过度表达对tau蛋白磷酸化的影响.方法 在NG-108细胞上转染不同的PS1质粒, 利用免疫双标,免疫印记, 免疫细胞化学,MTT,Western blot 等方法, 观察早老蛋白-1的过度表达对tau蛋白磷酸化的影响.结果 免疫双标显示,在散发性AD(sporadic Alzheimer disease)患者脑内, PS1主要分布于神经元,并与磷酸化tau蛋白部分共存.免疫印迹显示, 与空质粒组相比,转染野生型PS1组和转染一种家族性AD(familial Alzheimer disease,FAD)突变型早老蛋白-1(mPS1)质粒组的NG-108细胞中磷酸化的tau蛋白均增加.MTT方法未检测到这两组的转染细胞与对照组有显著差异.用Confocal显微镜观察PS1-EGFP质粒转染组, 发现在转染后早期12 h,PS1-EGFP主要分布在细胞膜表面或细胞器上,随后PS1-EGFP主要在细胞浆中弥散分布.同时,免疫细胞化学检测显示,部分PS1-EGFP 转染细胞中有明显的tau蛋白磷酸化,并且这些磷酸化tau蛋白能与PS1-EGFP蛋白一起形成聚集体.在给予磷酸酯酶抑制剂岗田酸后, PS1-EGFP 转染细胞比未转染细胞含有更多的磷酸化的tau蛋白.结论 野生型PS1可能参与了散发性阿茨海默氏病中的tau蛋白的病理改变.%Purpose To study the effect of overexpressing either wild type or a familial Alzheimer disease mutant presenilin 1 (mPS1) on tau phosphorylation in neuroblastoma NG-108 cells. Methods Three different plasmids transfected NG-108 cells respectively. Immunostaining and confocal microscopic technique were used to study the distribution of presenilin 1 and phosphorylated tau. Immunoblot test was applied to investigate the change of tau phosphorylation. Results Immunostaining showed that in brain of sporadic Alzheimer disease, PS1 mainly distributed in neuron and partially colocalized with the phosphorylated tau. Immunoblot tests showed that the cells transected either wild type PS1 or mPS1 contained more phorphorylated tau than the control cells. However, MTT test showed no significant difference between mock transfected cells and the wPS1 or mPS1 transfected cells. In addition, after transfection of the constructed PS1-EGFP vector, overexpressed EGFP-PS1 was located at cell surface membrane and subcellular organelles at earlier time at 12 hr, then EGFP-PS1 diffused in cytosol. Immunocytochemical observations demonstrated that some of the PS1-EGFP transfected cells contained more phosphorylated tau protein, which formed aggresome with PS-1-EGFP. When treated with phosphotase inhibitor okadaic acid, in the PS1-EGFP transfected cells accumulated more phosphorylated tau than the un-transfected cells. Conclusions Wild type PS1 is possibly involved in tauopathy in sporadic Alzheimer's disease.