ApoE4通过calpain-CDK5通路介导tau蛋白过度磷酸化加剧5xFAD转基因小鼠认知功能的损害

发布时间：2018-03-04 21:04

  本文选题：阿尔茨海默病　切入点：ApoE4　出处：《福建医科大学》2015年博士论文　论文类型：学位论文

【摘要】：【目的】过度磷酸化的tau蛋白是阿尔茨海默病(Alzheimer’s disease,AD)的病理特征神经元纤维缠结的主要组成成分。而目前普遍认为,ApoE中的ε4等位基因已经成为了AD最为主要的危险相关基因,且参与了AD的各种病理过程。到目前为止,学者们对apoE4与Aβ的关系已经进行了广泛的研究,然而apoE4与tau蛋白磷酸化的关系仍不是很清楚。故本文拟从在体和体外两个方面进行探讨。在体,用人源的APOE基因(ε3和ε4)置换小鼠的APOE基因,然后与apoE-/-/5xFAD转基因小鼠进行交配,从而培育出ApoE/5xFAD(简称EFAD)转基因小鼠。通过对EFAD转基因小鼠进行研究,拟在AD的遗传背景下观察内源性apoE的不同基因型(apoE3和apoE4)对5xFAD转基因小鼠的认知功能、tau蛋白磷酸化水平以及神经纤维形态的影响,并探讨引起tau蛋白磷酸化改变的可能上游机制。体外,通过对表达Aβ的N2a/APP695细胞株给予外源性apoE肽段及相关抑制剂干预,进一步证实在体所观察到的tau蛋白磷酸化及机制。最终为AD的治疗及新药的开发提供一定的理论依据。【方法】1、在体部分,本实验采用SPF级3月龄及7月龄雄性E3FAD转基因小鼠和E4FAD转基因小鼠,每组9-12只。首先,通过Morris水迷宫检测不同月龄不同APOE基因型转基因小鼠学习记忆能力是否存在差异。然后,通过改良的Marsland氏银染方法对大脑皮层神经纤维进行形态学染色,观察不同月龄APOE的不同基因型对小鼠神经纤维形态的影响;其次,通过Western blot和免疫组化的方法探索EFAD转基因小鼠大脑皮层的taupT205、taupS396、taupS404和taupT231的磷酸化水平及总tau的水平。接下来,通过Western blot的方法对tau蛋白磷酸化的相关激酶GSK3β和CDK5及其调节亚单位P35、P25的表达水平进行检测。最后,通过Western blot的方法对calpain-CDK5信号通路中calpain的表达情况进行了检测。2、体外部分,对稳定表达突变型APP695的小鼠神经母细胞瘤细胞株(N2a/APP695)予以200nM的外源性重组人apoE3和apoE4肽段干预24小时,因此实验分为apoe3干预组、apoe4干预组和溶媒对照组。首先,采用westernblot的方法对不同组taupt205、taups396、taups404和taupt231的磷酸化水平进行检测,验证动物实验中所观察到的现象是否能在体外进行模拟。然后,对tau蛋白激酶cdk5及其调节亚单位p35、p25的表达水平进行检测,同时进一步应用cdk5抑制剂进行干预,进一步在体外确认是否cdk5影响tau蛋白磷酸化。最后,对calpain的表达水平进行检测,从而证实其中所包含的机制。【结果】1、在体实验结果显示:a.行为学结果表明:在3月龄时,e3fad转基因小鼠与e4fad转基因小鼠之间的学习记忆能力无明显统计学差异;而在7月龄时,与e3fad转基因小鼠相比,e4fad转基因小鼠学习记忆能力明显下降,表现在前5天训练时寻找平台的潜伏期明显延长;第6天平台探索实验中,在平台所在象限停留时间明显缩短、穿越原平台所在位置的次数明显减少。b.marsland氏银染结果显示:在3月龄时,e3fad转基因小鼠与e4fad转基因小鼠的皮层神经纤维均比较完整,且走行很规律,但e4fad转基因小鼠的神经纤维稍增粗、深染,提示e4fad转基因小鼠的神经纤维已有轻微损害。然而,在7月龄时,e3fad转基因小鼠的神经纤维仍然完整且走行规律,但e4fad转基因小鼠的神经纤维已明显破坏,表现为神经纤维的断裂且排列的杂乱无序。c.westernblot和组化结果显示:与e3fad转基因小鼠相比,早在3月龄时,e4fad转基因小鼠大脑皮层taupt205的磷酸化水平明显增加;到7月龄时,e4fad转基因小鼠taupt205、taups396、taups404和taupt231的磷酸化水平均明显增加。且e4fad转基因小鼠脑内cdk5、p35、p25及calpian明显增高,但gsk3β活性在两组之间无明显的统计学差异。2、体外实验结果表明:a.给予n2a/app695细胞株200nm外源性重组人apoe3和apoe4肽段干预后,apoe4组taupt205、taups396、taups404和taupt231的磷酸化水平明显增加;cdk5、p35、p25及calpian的表达量也明显增加。b.在给予cdk5抑制剂后,taupt205、taups396、taups404和taupt231的磷酸化水平明显下降。【结论】1、apoe4所导致的tau蛋白磷酸化水平的增加早于学习记忆能力的下降。2、在5xfad转基因小鼠中,tau蛋白磷酸化水平的增加是由于cdk5活性升高所引起,而与GSK3β无关。3、ApoE4能够通过calpain-CDK5信号通路介导tau蛋白的过度磷酸化,从而破坏神经元的骨架结构,最终导致5xFAD转基因小鼠的认知功能损害。
[Abstract]:[Objective] hyperphosphorylation of tau protein of Alzheimer's disease (Alzheimer 's disease, AD) a major component of the pathological characteristics of neurofibrillary tangles. But it is generally believed that the ApoE 4 allele AD has become the most dangerous gene related major, and participate in a variety of pathological processes AD so far, the scholars on the relationship between the apoE4 and A beta have been widely studied, but the relationship between apoE4 and tau phosphorylation is not very clear. Therefore, this paper discusses from two aspects in vivo and in vitro. In vivo, APOE gene of human origin (E 3 and E 4 APOE) gene replacement in mice, and apoE-/-/5xFAD transgenic mice were mated, to develop a ApoE/5xFAD (EFAD) transgenic mice. Through the study of EFAD transgenic mice to different genotypes observed endogenous apoE in the genetic background of AD (under APO E3 and apoE4) on the cognitive function of 5xFAD transgenic mice, tau protein phosphorylation and morphology of nerve fibers, and to explore the causes may change the phosphorylation of tau upstream mechanism. In vitro by N2a/APP695 cells on the expression of A beta exogenous apoE peptide inhibitors and related intervention, further confirmed that tau protein phosphorylation and mechanism of the observed in vivo. Finally developed for treatment and new drug AD to provide a theoretical basis. [Methods] 1, in part, this experiment adopts 3 month old grade SPF and 7 month old male E3FAD transgenic mice and E4FAD transgenic mice, 9-12 rats in each group. First, the ability of learning and memory by Morris water maze test in different months in different genotypes of APOE transgenic mice and whether there are differences. Then, the morphology of nerve fibers in the cerebral cortex stained by Marsland's modified silver staining to observe the different Effect of different genotypes of APOE month old mice on the fiber morphology of nerve; secondly, by Western blot and immunohistochemistry of EFAD transgenic mouse brain cortex taupT205, taupS396, taupS404 and taupT231 phosphorylation level and total tau level. Then, based on tau phosphorylation by Western blot correlation GSK3 and CDK5 kinase beta and its regulatory subunit P35, detect the expression level of P25. Finally, through the method of Western blot expression of calpain in calpain-CDK5 signaling pathway were detected by.2, in part, to the stable expression of murine neuroblastoma cell lines with mutant APP695 (N2a/APP695) 200nM to be exogenous recombinant human apoE3 and apoE4 peptide for 24 hours, so the experiment was divided into apoE3 treatment group, apoE4 intervention group and control group. Firstly, using the Westernblot method to the different groups of taupt20 5, taups396, taups404 were detected and the phosphorylation level of taupt231, whether the observed animal verification experimental phenomena can be simulated in vitro. Then, the tau protein kinase Cdk5 and its regulatory subunit p35, detect the expression level of p25, and further application of Cdk5 inhibitor intervention, further confirm whether Cdk5 in vitro effect of Tau protein phosphorylation. Finally, detect the expression level of calpain, thus confirming mechanism contained. [results] 1, the in vivo results showed that A. behavioral results showed that: in the 3 month old, the ability of learning and memory between e3fad transgenic mice and e4fad transgenic mice had no significant difference; while in 7 month old, compared with e3fad transgenic mice, e4fad transgenic mice significantly decreased learning and memory ability, performance in the first 5 days of training significantly prolonged the latency to find the platform balance; Sixth Taiwan exploration experiment, shorten the residence time in the platform quadrant and frequency of crossing the original platform location significantly reduced.B.marsland's silver staining results showed that: in 3 month old, cortical nerve fibers in e3fad transgenic mice and e4fad transgenic mice were relatively complete, and walking regularly, but the e4fad transgenic mice nerve fibers increased slightly coarse, dark stained, suggesting that e4fad transgenic mice nerve fibers have been slight damage. However, in 7 month old, e3fad transgenic mice nerve fibers still intact and running rules, but the e4fad transgenic mice nerve fiber has obvious damage, is broken and arranged out of order.C.westernblot and the immunohistochemical results showed: compared to the nerve fiber with e3fad transgenic mice, as early as 3 month old, e4fad transgenic mouse brain cortex taupt205 phosphorylation significantly increased to 7 month old, e4fad transgenic; Mouse taupt205, taups396, taups404 and taupt231 phosphorylation levels were significantly increased. And in the brain of e4fad transgenic mice Cdk5, p35, P25 and calpian were increased, but the GSK3 activity in the two groups had no statistically significant difference between the.2 and the experimental results show that: a. n2a/ APP695 cells 200nm exogenous recombinant human apoE3 and apoE4 peptide intervention group apoE4, taupt205, taups396, taups404 and taupt231 phosphorylation level was significantly increased; Cdk5, p35, P25 and calpian expression was significantly increased in the.B. inhibitor of Cdk5, taupt205, taups396, taups404 and taupt231 phosphorylation levels were significantly decreased. [Conclusion] 1, increase of tau the phosphorylation level of apoE4 in the early in the learning and memory ability decreased.2 in 5xfad transgenic mice, tau protein phosphorylation level increase is due to the increase of Cdk5 activity caused by.3, and has nothing to do with the GSK3 beta, ApoE4 It can mediate the excessive phosphorylation of tau protein through calpain-CDK5 signaling pathway, thereby disrupting the skeleton structure of neurons, and ultimately leading to cognitive impairment in 5xFAD transgenic mice.

【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R749.16

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