组蛋白去乙酰化酶2激活糖原合酶激酶3β磷酸化tau蛋白

发布时间：2018-04-10 20:33

本文选题：阿尔茨海默病 + 组蛋白去乙酰化酶-2　；参考：《华中科技大学》2013年硕士论文

【摘要】：背景：阿尔茨海默病(Alzheimer’s disease,AD)是一种进行性发展的致死性神经退行性疾病，临床表现为认知和记忆功能不断恶化，日常生活能力进行性减退，并有各种神经精神症状（抑郁、焦虑不安、幻觉、妄想和失眠等）和行为障碍（踱步、攻击行为、坐立不安等）；主要病理特征是胞内神经原纤维缠结(neurofibrillary tangles, NFT)、胞外老年斑(Senile Plaques, SP)的聚集及神经元进行性丢失等。过度磷酸化的tau是胞内神经原纤维缠结的最主要的成分。研究发现组蛋白去乙酰化酶2（Histone deacetyltransferase-2，HDAC2）在AD病人脑中、AD转基因小鼠模型CK-p25和5xFAD及体外AD相关的神经毒性损伤模型中表达量均显著增加，并且其含量增加与部分学习记忆及突触可塑性相关蛋白表达量的下降密切相关，敲除HDAC2可以有效逆转AD转基因小鼠的神经退行性病变。但老年痴呆患者中增加的HDAC2与磷酸化的tau之间的关系尚不清楚。目的：离体水平探讨HDAC2的水平升高与AD样tau蛋白过度磷酸化之间的关系及可能的机制。方法：蛋白免疫印迹技术检测瞬时转染pEGFP-HDAC2于HEK293/tau细胞(人胚肾293细胞稳定转染人tau全长蛋白)内后微管相关蛋白tau的磷酸化水平的改变情况及tau磷酸化相关激酶和酯酶的蛋白含量和活性水平；免疫共沉淀检测HDAC2与GSK-3β之间是否有直接作用；细胞免疫荧光技术检测HDAC2和GSK-3β在细胞内的分布和定位情况。结果： 1. HDAC2导致293/tau细胞中tau蛋白pT231、pS404位点过度磷酸化：过表达HDAC2明显增加了293/tau细胞中pT231、pS404位点磷酸化水平，分别至140.2%(p=0.017)和150.7%(p=0.0058)，总tau含量未发生改变； 2.同家族的HDAC1、HDAC3不改变tau磷酸化水平：过表达I型HDACs的其它两个成员HDAC1、HDAC3后293/tau细胞内pT231、pS396、 pS404、R134d含量均未变化（p0.05）； 3. HDAC2激活GSK-3β使tau过度磷酸化：过表达HDAC2后检测t-GSK-3β、pS9-GSK-3β、pY216-GSK-3β后发现GSK-3β的抑制性位点磷酸化的s9蛋白水平降低而t-GSK-3β并无变化，此外，pS21-GSK-3α，t-GSK-3α的含量也并未发生改变，表明HDAC2过表达后激活GSK-3β，而非GSK-3α；同时检测其他tau磷酸化相关的激酶PKA、PKC、ERK及酯酶PP2Ac发现他们的蛋白水平和活性并没有随着HDAC2的过表达而变化，这些结果提示，HDAC2过表达引起的tau过度磷酸化可能是通过激活GSK-3β而不是PKA、PKC、ERK或PP2A起作用。pEGFP-N1-dnGSK与HDAC2共转后tau磷酸化水平没有上升（p0.05）及转染HDAC2后给予GSK-3β抑制剂SB216763均不改变tau磷酸化水平进一步证明GSK-3β激活介导了HDAC2过表达引起的tau过度磷酸化。 4. HDAC2与GSK-3β并非直接结合在一起发挥致tau过度磷酸化的作用：pEGFP-HDAC2和HA-GSK-3β共转染293/tau后免疫共沉淀检测HA及GFP发现二者并没有直接的结合，，这提示HDAC2对GSK-3β的激活并非直接作用。结论：I型HDACs中，只有HDAC2过表达可以诱导tau蛋白pT231、pS404位点发生过度磷酸化，并且这一过程是通过激活GSK-3β实现的。
[Abstract]:Background : Alzheimer ' s disease ( AD ) is a progressive progressive fatal neurodegenerative disease characterized by deteriorating cognitive and memory functions , progressive deterioration in daily life , and various neuropsychiatric symptoms ( depression , anxiety , illusion , delusion , insomnia , etc . ) and behavioral disorders ( pacing , aggression , restlessness , etc . ) ;
The main pathological characteristics were the accumulation of neurotrophin ( NFT ) , extracellular senile plaque ( SP ) and progressive loss of neurons .

Objective : To investigate the relationship between level elevation of HDAC2 and hyperphosphorylation of AD - like tau protein in vitro .

Methods : The level of phosphorylation of tau and the protein content and activity level of tau phosphorylation related kinase and esterase were detected by Western blot in human embryonic kidney 293 cells ( human embryonic kidney 293 cells stably transfected with human tau full - length protein ) .
immune codeposition detect whether there is a direct effect between HD2 2 and gsk - 3 beta ;
Cell immunofluorescence assay was used to detect the distribution and localization of HDAC2 and gsk - 3.beta . in cells .

Results :

1 . The phosphorylation level of pT231 and pS404 in 293 / tau cells was significantly increased in 293 / tau cells by HDAC2 , and the phosphorylation level of pT231 and pS404 in 293 / tau cells was significantly increased , and the phosphorylation level of pT231 and pS404 in 293 / tau cells was increased to 140.2 % ( p = 0 . 017 ) and 150.7 % ( p = 0.0058 ) , respectively , and the total tau content did not change ;

2 . HDAC1 and HDAC3 of the same family did not change the level of tau phosphorylation : the levels of pT231 , pS396 , pS404 , R134d in 293 / tau cells after overexpression of HDAC1 and HDAC3 did not change ( p . 05 ) ;

3 . The phosphorylation of tau hyperphosphorylation was observed after overexpression of HDAC3 , and the level of phosphorylation of pS9 - 3 - 尾 , pS9 - gsk - 3.beta . , pS9 - gsk - 3.beta . was decreased and the levels of t - gsk - 3beta were not changed . Furthermore , the levels of pS21 - gsk - 3伪 , t - gsk - 3伪 did not change .
At the same time , it was found that the level and activity of PKC , ERK and PP2Ac associated with phosphorylation of other tau phosphorylation were not changed with the overexpression of HD2 . These results suggested that the hyperphosphorylation of tau caused by overexpression of HDAC2 could be mediated by the activation of gsk - 3beta rather than the phosphorylation of protein kinase , PKC , ERK or PP2A .

Conclusion : It is concluded that only the overexpression of HDAC2 can induce hyperphosphorylation of tau protein pT231 and pS404 , and this process is achieved by activation of gsk - 3beta . Conclusion : In type I HDAC , only HDAC2 overexpression can induce tau protein pT231 and pS404 site to be hyperphosphorylated .

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

【共引文献】