Study of the mechanism of abnormal phosphorylation of Alzheimer's disease.

在阿尔茨海默病（AD）中，微管相关蛋白tau变得异常磷酸化并聚集成成对的螺旋丝（PHF）。刺激tau蛋白磷酸化和tau蛋白聚集的生化机制尚不清楚。为了研究tau蛋白过度磷酸化的机制，我们使用过表达几种蛋白激酶的神经母细胞瘤细胞研究其磷酸化。本研究获得了以下结果：（1）制备了表达Ca^2+/钙调蛋白依赖性蛋白激酶II（CaM kinase II）的细胞系，CaM kinase II促进了神经突起的生长。(2)神经元细胞的分化受CaM激酶II和蛋白激酶C的调节。(3)在Thr-286处自磷酸化的激酶的Ca^<2+>非依赖性活性参与神经突生长。(4)在P19细胞神经分化过程中，CaM激酶Ⅱ δ亚型的表达被诱导，其剪接方式发生改变。(5)δ异构体的剪接模式不仅在培养的神经元细胞分化过程中，而且在大鼠前脑和小脑发育过程中也有明显的细胞类型变化。(6)获得了表达人tau蛋白cDNA的P19细胞株，并通过维甲酸诱导P19细胞异常神经分化，这些细胞被认为是AD的有用模型。(7)Tau蛋白由大肠杆菌产生，并将其用作各种蛋白激酶的底物。使用纯化的tau研究形成不溶性tau的条件。(8)在老年脑和分化的神经母细胞瘤细胞中研究了磷酸化AD tau特异位点的蛋白激酶。本研究为tau蛋白的磷酸化及其在神经元分化中的作用提供了新的认识。

In Alzheimer's disease (AD), microtubule-associated protein tau becomes abnormally phosphorylated and aggregates into paired helical filaments (PHFs). The biochemical mechanism of stimulation of tau phosphorylation and tau aggregation remaines unclear. To investigate the mechanism of hyperphosphorylation of tau, we studied its phosphorylation using neuroblastoma cells overexpressing several protein kinases. The following results were obtained in this study ; (1) Cell lines expressing Ca^<2+>/calmodulin-dependent protein kinase II (CaM kinase II) were prepared, and Cam kinase II promoted neurite outgrowth. (2) The differentiation of neuronal cells were regulated by CaM kinase II and protein kinase C.(3) Ca^<2+>-independent activity of the kinase autophosphorylated at Thr-286 involved for neurite outgrowth. (4) The expression of δ isoform of CaM kinase II was induced, and the splicing pattern of the isoform changed, during neural differentiation of P19 cells. (5) Cell type distinctive changes of splicing pattern of δ isoform were observed not only during differentiation of cultured neuronal cells, but also during development of rat forebrain and cerebellum. (6) Cell lines of P19 expressing human tau cDNA were obtained, and abnormal neural differentiation of P19 cell induced by retinoic acid, and these cells were thought to be as useful model of AD.(7) Tau protein was produced by E.coli and used it as substrate of various protein kinases. The condition forming insoluble tau was investigated using purified tau. (8) The protein kinase that phosphorylated specific sites of AD tau was investigated in the aged brain and differentiated neuroblastoma cells. Present study provide some insight of the phosphorylation of tau and role of tau on neuronal differentiation.