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

阿尔茨海默病异常磷酸化机制的研究。

基本信息

批准号：
11557184
负责人：
YAMAUCHI Takashi
金额：
$ 8.64万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11557184/
关键词：
abnormal phosphorylation Alzheimer's disease calmodulin CaM kinase II neural differentiation protein kinase tau protein タウ CaM kinaseII P19細胞リン酸化

项目摘要

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.

在阿尔茨海默病(AD)中，微管相关蛋白tau异常磷酸化并聚集成成对的螺旋细丝(PHF)。促进tau磷酸化和tau聚集的生化机制尚不清楚。为了探讨tau蛋白过度磷酸化的机制，我们用过表达几种蛋白激酶的神经母细胞瘤细胞研究了tau蛋白的磷酸化。本研究获得了以下结果：(1)制备了表达钙调素依赖的蛋白激酶II(CaM KK II)的细胞系，并且CaM KK II促进神经突起生长。(2)神经细胞的分化受CaM激酶II和蛋白激酶C的调节。(3)Thr-286处参与突起生长的自磷酸化的激酶的活性不依赖于钙离子。(4)在P19细胞神经分化过程中，诱导了δ亚型CaM激酶II的表达，并改变了该亚型的剪接模式。(5)在培养的神经细胞分化过程中，以及大鼠前脑和小脑发育过程中，δ异构体剪接模式的细胞类型发生了明显的变化。(6)获得了表达人tau基因的P19细胞系，维甲酸诱导的P19细胞神经分化异常，可作为AD的动物模型。(7)在大肠杆菌中表达Tau蛋白，并将其作为多种蛋白激酶的底物。以纯化的牛磺酸为原料，考察了不溶性牛磺酸的形成条件。(8)在老年脑和分化的神经母细胞瘤细胞中，研究了使AD tau蛋白的特定位点磷酸化的蛋白激酶。目前的研究为tau的磷酸化及其在神经元分化中的作用提供了一些见解。