DAPK1磷酸化Tau对抗自身诱导的细胞凋亡

发布时间：2018-07-08 16:47

本文选题：死亡相关蛋白激酶1 + 阿尔茨海默病　；参考：《华中科技大学》2013年博士论文

【摘要】：[背景] 阿尔茨海默病(Alzheimer disease, AD)的两大特征性病理改变为：过度磷酸化的Tau蛋白在细胞内的聚集形成神经原纤维缠结(neurofibrillary tangles, NFTs),和p-淀粉样多肽(β-amyloid, Aβ)在细胞外的沉淀形成老年斑(senile plaques, SPs),导致神经元的退化和功能缺失,出现渐进性记忆障碍、认知功能障碍、语言障碍及人格改变等痴呆症状。死亡相关蛋白激酶1(death-associated protein kinase1, DAPK1)是由钙／钙调蛋白调节的、可引起其底物在丝、苏氨酸位点发生磷酸化修饰的蛋白激酶,它在肿瘤细胞表达减少,而在神经退行性病变中表达增多,其作用是诱导细胞凋亡、自噬和坏死,并在信号传导、肿瘤的发生发展和神经退行性病变中起重要作用。用全基因组关联研究(genome-wide association study, GWAS)和单核苷酸多态分析(gene-centric single nucleotide polymorphisms, SNP)研究证明DAPK1的表达与迟发型阿尔茨海默病密切相关。本课题组研究发现,Tau蛋白磷酸化能够对抗外源性的凋亡诱导剂诱导的凋亡,从而最终导致神经元发生退行性变。但Tau蛋白磷酸化能否对抗脑内内源性凋亡诱导因子如DAPK1诱导的细胞凋亡,目前尚不清楚。 [目的] 探讨DAPK1对Tau蛋白磷酸化的作用及Tau蛋白磷酸化在DAPK1诱导的细胞凋亡中的作用。 [方法] 实验选取10月龄C57小鼠、hTau小鼠及Tg2576小鼠,分离皮层和海马等脑组织,用Western Bolt方法检测DAPK1和Tau蛋白在各组织中的表达；用免疫组化观察DAPK1及AD相关磷酸化位点在皮层和海马中的分布。分别用HEK293/wt、N2a和HEK293/tau细胞转染wtDAPK、siDAPK、mutant DAPK1、siMARK2质粒48小时,或24小时后采用无血清诱导细胞凋亡,采用免疫组织化学或免疫印迹检测Tau蛋白磷酸化、磷酸酯酶或蛋白激酶的活性及凋亡蛋白的表达水平；采用CCK8检测细胞活性,Hochest和TUENL染色检测细胞凋亡情况；采用免疫共沉淀检测DAPK1和Tau蛋白的相互作用；采用细胞免疫荧光的方法检测DAPK1和Tau蛋白共定位情况。 [结果] 结果发现hTau转基因小鼠脑片DAPK1的表达量和正常C57小鼠相比明显升高,并且表达部位也由突起和细胞膜转入胞浆。通过无血清诱导凋亡的方式使DAPK1激活,发现无论细胞的活性还是凋亡因子的表达以及细胞核固缩等指标,Tau蛋白的过表达可明显改善细胞凋亡。HEK293/Tau细胞内转染wtDAPK1质粒或siDAPK1质粒后,可调节Tau蛋白的Thr231, Ser262和Ser396位点磷酸化水平。与Thr231, Ser262和Ser396位点磷酸化有关的蛋白激酶和磷酸酯酶：PP2A, GSK-3β、PKA、CaMKⅡ、Cdc2、Cdk5和MARK2,不涉及DAPK1诱导的Tau蛋白磷酸化的机制中。采用免疫共沉淀方法,发现Tau和DAPK1可以形成复合体；免疫荧光染色发现DAPK1与总Tau(Tau-5或R134d)和pT231在细胞膜上有共定位。用kinase区缺失的突变DAPK1质粒转染HEN293/Tau细胞后,发现pT231,pS262和pS396水平没有改变。 [结论] DAPK1可以通过磷酸化Tau蛋白的Thr231,Ser262和Ser396位点,而对抗其诱导的细胞凋亡。
[Abstract]:Background background

The expression of protein kinase 1 ( DAPK1 ) , which is regulated by calcium / calcium modulated protein , can induce apoptosis , autophagy and necrosis of neurons . It also plays an important role in the development of apoptosis , cognitive dysfunction , language disorder and personality change . The death - associated protein kinase 1 ( death - associated protein kinase1 , DAPK1 ) is a protein kinase which can induce apoptosis , autophagy and necrosis .

Purpose of the project

To investigate the role of DAPK1 on Tau protein phosphorylation and the role of Tau protein phosphorylation in DAPK1 - induced apoptosis .

Methodology

The expression of DAPK1 and Tau protein in various tissues was determined by Western Bolt method in the brain tissues of 10 - month - old C57 mice , hTau mice and Tg2576 mice , isolated cortex and hippocampus .
The distribution of DAPK1 and AD - related phosphorylation sites in cortex and hippocampus was observed by immunohistochemistry .

The activity of Tau protein phosphorylation , phosphatase or protein kinase activity and the level of apoptosis protein were detected by immunohistochemistry or Western blot after transfection of pAPK , siDAPK , mutant DAPK1 , siMARK2 plasmid for 48 hours , or 24 hours respectively .
The apoptosis of the cells was detected by the cell activity , Hochest and TUENL staining .
The interaction of DAPK1 and Tau protein was detected by immunoconjugate precipitation .
Cell immunofluorescence was used to detect the co - localization of DAPK1 and Tau protein .

The result is not valid .

The results showed that the expression of DAPK1 was significantly higher in hTau transgenic mice than in normal C57 mice .
Immunofluorescence staining showed that DAPK1 was co - located with Tau - 5 or R134d and pT231 on the cell membrane . After transfection of HEN293 / Tau cells with the mutant DAPK1 plasmid , the level of pT231 , pS262 and pS396 was not changed .

Conclusion

DAPK1 can counter its induced apoptosis by phosphorylating the Thr 231 , Ser262 and Ser396 sites of Tau protein .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.16

【共引文献】