EPAC2下调通过激活CDK5诱导Tau蛋白过度磷酸化

发布时间：2018-06-23 22:26

本文选题：EPAC2 + Tau磷酸化　；参考：《华中科技大学》2015年博士论文

【摘要】：[背景] 阿尔茨海默病(Alzheimer's Disease, AD)是一种常见的、进行性发展的致死性神经退行性疾病,其主要病理学特征是脑内神经元细胞内的神经原纤维缠结(NFTs)和细胞外的老年斑(SPs)的形成。NFTs的主要成分是过度磷酸化的Tau。异常磷酸化的Tau丧失促微管组装和稳定微管的功能,并聚集成为成对螺旋丝(PHF),最终导致NFTs的形成、突触的退变和神经元的丢失,在AD的病程发生、发展中起了关键作用。鸟嘌呤核苷酸交换蛋白(EPAC)作为cAMP直接激活的下游蛋白,在生物进程调节中发挥着重要作用。EPAC1/2敲除小鼠表现出学习记忆和社会交往的障碍,且EPAC2亚型在AD患者脑中]nRNA水平表达下降,提示EPAC2下调参与AD的病程调节。然而EPAC2信号如何调节体内相关通路,从而影响学习记忆尚不清楚。 [目的] 探讨EPAC2调节的信号通路对Tau蛋白异常过度磷酸化的调控机制；探究如何逆转EPAC2下调后带来的学习记忆障碍。 [方法] 在探讨EPAC2信号通路对Tau蛋白异常过度磷酸化调控机制的研究中,我们应用了EPAC2-/-转基因小鼠和si-EPAC2质粒,应用免疫印迹实验在整体水平和细胞水平分别检测Tau蛋白磷酸化相关抗体的表达,并分离出可溶性和不可溶蛋白组分对Tau蛋白的溶解性进行探讨。为了验证是否有Tau蛋白过度磷酸化导致的神经纤维缠结,我们进行了Bielschowsky银染。为了探究造成上述结果的机制,我们应用免疫印迹反应筛选了一系列与Tau蛋白磷酸化相关的激酶酯酶,发现调节Tau蛋白磷酸化关键的激酶CDK5被激活,再根据文献研究找到CDK5上游因子Calpain并进行活性检测。为了解释EPAC2下调与Calpain的关联性,应用ELISA从多个免疫因子中最终筛选出TGFβ1这个EPAC2和Calpain的中间连接因子。为了逆转EPAC2下调带来的Tau病变,我们应用了Calpain抑制剂Calpain inhibitorⅣ和CDK5抑制剂Roscovitine脑立体定位注射到EPAC2-/-小鼠侧脑室,用免疫印迹检测是否对EPAC2下调导致的Tau过度磷酸化有修复作用。 [结果] 1、EPAC2下调导致Tau蛋白过度磷酸化,不可溶Tau蛋白增加、神经纤维缠结形成等Tau病变。为了探讨EPAC2下调对Tau蛋白的影响,我们首先应用EPAC2-/-转基因小鼠和si-EPAC2质粒在动物和细胞水平造成EPAC2下调的环境,再取样分析Tau蛋白的磷酸化程度。结果显示从6月龄开始,EPAC2下调后Tau蛋白磷酸化水平增加,pT205、 pT231、pS396、pS4O4位点的磷酸化程度也增加。随后我们把6月龄EPAC2+/+和EPAC2-/-小鼠脑组织样品分为可溶组分和不可溶组分,发现EPAC2-/-、鼠的Tau蛋白在不可溶组分中明显增加。Bielschowsky银染显示,EPAC2-/-小鼠脑内9月龄时即出现明显Tau过度磷酸化形成的神经纤维缠结。 2、EPAC2下调通过激活CDK5影响Tau蛋白磷酸化调节。 Tau蛋白磷酸化是相关激酶酯酶调节失衡的结果,为了找出在EPAC2下调体系中影响Tau蛋白磷酸化的关键因子,我们对相关激酶酯酶进行了筛查,最后发现CDK5在EPAC2下调后活性成分表达明显增加,下调CDK5可逆转EPAC2下调引起的Tau蛋白过度磷酸化。 3. EPAC2下调通过激活Calpain活化CDK5。 Calpain是调节CDK5活性主要因子,检测显示EPAC2-/-小鼠Calpain活性明显高于EPAC2+/+小鼠。在EPAC2-/-小鼠体内下调Calpain后CDK5活性降低,Tau磷酸化病变得到逆转。 4、EPAC2下调激活TGF-β1/Calpain/CDK5通路影响Tau蛋白磷酸化调节。经验证,EPAC2对Calpain. CDK5的激活不是通过传统的Ca2+活化造成的,为了找到EPAC2对CDK5的调节作用,我们筛选出TGF-β1这个炎症因子,进一步通过TGF-β1/Calpain/CDK5通路进行研究。 [结论] 1、EPAC2下调导致Tau蛋白过度磷酸化,不可溶Tau蛋白增加、神经纤维缠结形成等Tau病变。 2、EPAC2下调通过激活CDK5影响Tau蛋白磷酸化调节。 3、EPAC2下调通过激活Calpain活化CDK5。 4、EPAC2下调激活TGF-β1/Calpain/CDK5通路影响Tau蛋白磷酸化调节。
[Abstract]:[background]
Alzheimer's Disease (AD) is a common, progressive, fatal neurodegenerative disease. Its main pathological feature is the formation of neurogenic fibrous tangles (NFTs) and extracellular senile plaques (SPs) in the neurons of the brain. The main component of the.NFTs is over phosphorylated Tau bereavement of the abnormal phosphorylation of Tau.. The function of microtubule assembly and stabilization of microtubules and the aggregation of PHF, which eventually lead to the formation of NFTs, the degeneration of synapses and the loss of neurons, play a key role in the development of AD.
Guanine nucleotide exchange protein (EPAC) is a downstream protein directly activated by cAMP, which plays an important role in the regulation of biological processes..EPAC1/2 knockout mice show learning memory and social interaction, and the EPAC2 subtype decreases in the]nRNA level in the brain of AD patients, suggesting that the EPAC2 downregulation is involved in the course regulation of AD. However, EPAC2 signal It is not clear how to regulate the related pathways in vivo and thus affect learning and memory.
[Objective]
Objective to investigate the regulatory mechanism of EPAC2 regulated signaling pathway on abnormal overphosphorylation of Tau protein, and explore how to reverse learning and memory impairment induced by EPAC2 downregulation.
[method]
In the study of the regulation mechanism of abnormal overphosphorylation of Tau protein by EPAC2 signaling pathway, we applied EPAC2-/- transgenic mice and si-EPAC2 plasmids. The expression of Tau protein phosphorylation related antibodies was detected by immunoblot test at the overall level and cell level, and the soluble and insoluble protein components were separated to Tau. In order to verify whether there is a neurofibrillary tangles caused by excessive phosphorylation of Tau protein, we have carried out Bielschowsky silver staining.
In order to explore the mechanism that causes the results, we screened a series of kinase esterases associated with the phosphorylation of Tau protein by immunoblotting. We found that the key kinase CDK5 regulating the phosphorylation of Tau protein was activated. Then, the CDK5 upstream factor Calpain was found and the activity detection was found according to the literature study. In order to explain the downregulation of EPAC2 and Calpain In association, ELISA was used to screen out the TGF and beta 1 intermediate linkage factor of EPAC2 and Calpain from multiple immune factors.
In order to reverse the Tau lesion caused by EPAC2 downregulation, we injected the Calpain inhibitor Calpain inhibitor IV and CDK5 inhibitor Roscovitine stereotaxic into the lateral ventricle of EPAC2-/- mice, and the immunoblotting was used to detect the repair of Tau overphosphorylation caused by EPAC2 downregulation.
[results]
1, down regulation of EPAC2 results in excessive phosphorylation of Tau protein, increased insoluble Tau protein, and formation of Tau lesions such as nerve fiber entanglement.
In order to investigate the effect of EPAC2 down regulation on Tau protein, we first used EPAC2-/- transgenic mice and si-EPAC2 plasmids to cause EPAC2 downregulation in animal and cell levels, and then sample and analyze the phosphorylation of Tau protein. The results showed that the level of Tau protein phosphorylation increased after EPAC2 downregulation, pT205, pT231, pS396, pS4O4 sites. The degree of phosphorylation was also increased. Then we divided the brain tissue samples of 6 month old EPAC2+/+ and EPAC2-/- mice into soluble components and insoluble components. It was found that EPAC2-/-, the Tau protein in the insoluble components increased obviously in the insoluble components, and the.Bielschowsky silver staining was found in the insoluble components of the EPAC2-/- mice. The neurons of Tau overphosphorylation appeared in the brain of EPAC2-/- mice. Fiber tangles.
2, EPAC2 downregulates the phosphorylation of Tau protein through activation of CDK5.
The phosphorylation of Tau protein is the result of the imbalance of the related kinase esterase regulation. In order to find out the key factor affecting the phosphorylation of Tau protein in the down regulation of EPAC2, we screened the related kinase esterase. Finally, it was found that the expression of CDK5 was obviously increased after the downregulation of EPAC2, and the lower modulation of CDK5 could reverse the Tau protein overregulation caused by the downregulation of EPAC2. Phosphorylation.
3. EPAC2 down regulated by activating Calpain to activate CDK5.
Calpain was the main factor regulating the activity of CDK5. The detection showed that the activity of Calpain in EPAC2-/- mice was significantly higher than that of EPAC2+/+ mice. The activity of CDK5 was reduced and the Tau phosphorylation lesion was reversed in the EPAC2-/- mice.
4, EPAC2 downregulates the activation of TGF- beta 1/Calpain/CDK5 pathway and regulates the phosphorylation of Tau protein.
It was verified that the activation of Calpain. CDK5 by EPAC2 was not caused by traditional Ca2+ activation. In order to find the regulatory role of EPAC2 to CDK5, we screened the inflammatory factor of TGF- beta 1 and further studied the TGF- beta 1/Calpain/CDK5 pathway.
[Conclusion]
1, down regulation of EPAC2 results in excessive phosphorylation of Tau protein, increased insoluble Tau protein, and formation of Tau lesions such as nerve fiber entanglement.
2, EPAC2 downregulates the phosphorylation of Tau protein through activation of CDK5.
3, EPAC2 downregulation activates Calpain by activating CDK5.
4, EPAC2 downregulates the activation of TGF- beta 1/Calpain/CDK5 pathway and regulates the phosphorylation 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】：2015
【分类号】：R749.16

【相似文献】