毛喉萜对tau蛋白磷酸化及聚集的影响

The Effect of Forskolin on tau Phosphorylation and tau Inclusion Body Formation

目的:本实验旨在细胞水平研究毛喉萜(forskolin)对tau蛋白磷酸化、聚集体形成及tau蛋白与微管结合能力的影响。方法:利用基因转染技术建立过度表达人类最长tau(tau441)的人肾母细胞瘤细胞(HEK293),免疫印迹技术检测细胞内tau蛋白的磷酸化状况,tau与微管结合能力改变;免疫荧光技术和thioflavin染色检测细胞内tau蛋白聚集体形成情况。结果:在HEK293转tau细胞株,总tau蛋白的表达没有明显改变,tau蛋白在Ser214、Ser262和Ser396/404位点过度磷酸化。与此同时,随着tau蛋白的过度磷酸化,tau蛋白与微管的结合能力下降,细胞内有磷酸化的tau蛋白聚集增加。结论:蛋白激酶A的激活可以引起tau蛋白的过度磷酸化,磷酸化的位点包括蛋白激酶A和非蛋白激酶A的磷酸化位点,而过度磷酸化的tau蛋白又损害了tau与微管结合,形成了大量细胞内聚集体,提示了蛋白激酶A的激活可能是超早期阿尔茨海默病病理发展过程的关键因素之一。

Aim：To investigate the effect of forskolin on tau phosphorylation,tau inclusion body formation and tau function in cultured cells.Methods：Human embryonic kidney cells（HEK293） were stably transfected with the longest human isoform of tau（tau441）（HEK293/tau441）.Tau phosphorylation and microtubule binding activity of tau were detected by Western blotting.Tau aggregates formation was determined by immunofluorescence and thioflavin staining.Results：Western blotting results demonstrated that the level of total tau was not altered significantly,but the levels of phosphorylated tau at various sites detected by a panel of site-specific tau antibodies（PS214 PS262 and PHF-1） were significantly increased after forskolin incubation. The microtubule-binding activity of tau was decreased and tau accumulation was increased.Conclusion： These results indicate that in vitro activation of PKA can induce tau hyperphosphorylation not only at PKA site but also at some non-PKA sites.And tau phosphorylation induced by forskolin impaired tau function and increased tau aggregates formation which might be the underlining mechanism of Alzheimer＇s disease.These changes indicate the PKA activation may be a key process in early stage of Alzheimer＇s disease.