【作者】 尹君；

【导师】 王建枝；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 第一部分Tau蛋白通过Akt途径使细胞抵抗凋亡阿尔茨海默病(Alzheimer’s disease, AD)包括两种典型的病理学特征,细胞内过度磷酸化的微管相关蛋白tau组成的神经原纤维缠结(neurofibrillary tangles, NFTs)和细胞外由β-淀粉样肽(Aβ)形成的老年斑(senile plaques,SP)。尽管AD和tau相关疾病患者的神经元长期持续处在促凋亡的环境中,然而大多数含有NFTs的神经元不发生凋亡,而是慢性退行性变性,其中机制尚不清楚。本研究小组最近的研究发现:tau蛋白过度磷酸化使神经细胞逃逸凋亡。本文在小鼠成神经瘤(mouse Neuroblastoma 2a, N2a)细胞中进一步探讨神经细胞逃逸凋亡的机制。结果显示:(1)在细胞中过度表达tau蛋白或其空载体(对照)并给予血清饥饿刺激后,过度表达tau蛋白的实验组细胞凋亡率明显低于对照组;细胞中过度表达的tau蛋白与caspase-3活性片段和(或)浓缩/碎裂的细胞核不共定位。(2)tau蛋白对细胞的保护作用与其相对增高的磷酸化水平相关。(3)与对照组比,表达tau蛋白的细胞中Akt激活,其下游凋亡相关因子FOXO3磷酸化增强,p53和Bax水平降低。(4)用抑制剂阻断Akt途径后,tau蛋白的抗凋亡作用消失;(5)与不表达tau蛋白的细胞比,去血清饥饿处理组PI3K的催化亚基p110含量显著升高,而磷酸化的调节亚基p85(p-p85)的相对水平没有明显改变。此外,本研究还发现在细胞核组分中含有tau蛋白,去血清饥饿处理使细胞核总tau蛋白和Ser396位点磷酸化的tau蛋白含量均明显降低。上述研究结果提示:tau蛋白通过激活PI3K/Akt途径使细胞逃逸凋亡,而PI3K的激活可能与tau蛋白核内水平降低有关。本研究部分阐述了tau蛋白抗凋亡的分子机制。第二部分β-淀粉样肽通过Akt/FOXO3/Bim途径引起神经细胞凋亡阿尔茨海默病(Alzheimer’s disease, AD)患者的特征性脑病理学改变之一是由β-淀粉样肽(Aβ)组成的细胞间沉积的大量老年斑(senile plaques, SP)。大量研究报道,Aβ具有细胞毒性,可引起细胞凋亡。体内和体外实验都证明Aβ能引起机体生存通路中重要激酶Akt的活性降低。FOXO是最近发现的一个与细胞生存与分化密切相关的转录因子。FOXO为Akt的下游底物,当Akt激活时,使FOXO的Thr32位磷酸化,FOXO不发挥其转录因子的效应,避免了一些促凋亡蛋白或配体的表达,细胞凋亡减少。本研究的目的是观察Akt/FOXO是否介导Aβ引起的细胞凋亡。结果显示:在过量表达Aβ的APPswe+ PS1?9双转基因细胞中,Akt活性下降, FOXO3的Thr32位磷酸化下降,促凋亡蛋白Bim的水平升高,神经细胞凋亡增多。该研究结果提示:Aβ可能通过Akt/FOXO3/Bim途径促进细胞凋亡。本研究为阐明Aβ引起神经细胞凋亡的机制提出了一条新通路。第三部分褪黑素阻止过氧化亚硝酸盐引起的tau蛋白过度磷酸化在大鼠整体研究褪黑素对过氧化亚硝酸盐(peroxynitrite,PN)引起的tau蛋白过度磷酸化以及相关激酶,包括糖原合酶激酶-3β(GSK-3β)和丝裂原活化蛋白激酶(MAPK)家族的影响。大鼠在双侧海马注射PN的前体SIN-1造模之前1 h,侧脑室注射褪黑素进行保护。利用免疫印迹法检测tau蛋白磷酸化以及相关激酶的改变。结果发现:(1)注射SIN-1可引起tau蛋白在pS396位点发生过度磷酸化,同时,GSK-3β和MAPK家族中的p38被激活;(2)提前注射褪黑素则可阻止这些变化;(3)褪黑素可改善PN所引起的氧化应激。本研究结果提示:褪黑素可能通过清除氧自由基和抑制相关激酶,阻止PN引起的tau蛋白过度磷酸化。第四部分脂多糖侧脑室注射对大鼠海马tau蛋白磷酸化水平的影响目的研究脂多糖( lipopolysaccharide,LPS)对大鼠海马神经细胞tau蛋白磷酸化的影响。方法大鼠侧脑室注射LPS(5μg和50μg)后于2,4,8 h提取海马神经细胞蛋白,利用免疫印迹法检测tau蛋白磷酸化的情况。结果(1)大鼠海马神经细胞tau蛋白在LPS注射2 h时后出现过度磷酸化,之后出现去磷酸化。(2)注射LPS后出现了tau蛋白表达总量增加。结论LPS能引起大鼠海马神经细胞tau蛋白磷酸化水平。更多还原

【Abstract】 Part 1 Tau protein antagonizes cell apoptosis through Akt pathwayTwo major neuropathological features of Alzheimer’s disease (AD) are intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated microtubule-associated protein tau, and the extracellular senile plaques composed ofβ-amyloid (Aβ). Though exposing to a proapoptotic environment, most NFTs-bearing nourons degenerate chronically rather than go to apoptosis in AD patients and other taupathies, and the mechanisms are still elusive. Recently, we have found that tau hyperphosphorylation plays a role in anti-apoptosis of the cells. Here we investigated the underlying mechanisms involved in the protective effects of tau. We found that mouse Neuroblastoma 2a (N2a) cells overexpressing tau were more resistent to serum withdrawal-induced apoptosis than the wild type cells. The protective effect was positively correlated with the relatively increased phosphrylation level of tau. We also found that the Akt was activated in cells overexpressing tau. Simultaneously, the phosphorylation level of FOXO3 increased (representing decreased activity) and the expression of p53 and Bax decreased. The antiapoptotic effects of tau almost disappeared by inhibition of Akt. Upon serum starvation, the expression level of catalytic subunit of PI3K (p110) increased significantly in cells overexpressing tau compared with the control cells, whereas the relative level of the phosphorylated p85 was not changed. Additonally, we also detected tau proteins in the nuclear fraction of the cells, and starvation led to a significantly decreased level of nuclear tau proteins. Our findings suggest that tau leads the cells to escape apoptosis by activating PI3K/Akt pathway and the activation of PI3K involves the decreased level of nuclear tau proteins. Part 2β-?amyloid induces neuronal apoptosis with Akt/FOXO3/Bim-involved pathwayOne of the major neuropathological features of Alzheimer’s disease (AD) is the formation of extracellular senile plaques, which are composed of beta-amyloid (Aβ). Aβi?s neurotoxic and it causes neuron apoptosis. Aβcan also decrease the activity of Akt, a well-known survival factor, in vivo and in vitro. FOXO is a down-stream substrate of Akt. Phosphorylation of FOXO at Thr32 by Akt restrains the transcription activity of FOXO and thus attenuates cell apoptosis through down-regulating the expression of proapoptotic factors. Here we investigated the involvement of Akt/FOXO in Aβ-induced apoptosis by using N2a/APPswe+PS1?9 cells, which overproduce Aβ. We found that the activity of Akt was reduced in N2a/APPswe+PS1?9 compared with the wild type control cells. Simultaneously, the FOXO3 was activated and the level of Bim, a proapoptotic transcript, was elevated. Moreover, the level of the apoptosis was increased in N2a/APPswe+PS1?9 cells. These data suggest that Aβmay induce neuron apoptosis through Akt/FOXO3/Bim-involved pathway. Part 3 Melatonin arrests peroxynitrite-induced tau hyperphosphorylationand the overactivation of protein kinases in rat brainAbstract: The purpose of this study was to examine the in vivo effect of melatonin (MEL) on peroxynitrite-induced tau hyperphosphorylation and the involvement of glycogen synthase kinase-3β(GSK-3β) and mitogenactivated protein kinase (MAPK) families. Melatonin was injected into the right cerebroventricle of the rats 1 hr before the bilateral hippocampal injection of 3-morpholino-sydnonimine chloride (SIN-1), the recognized donor of peroxynitrite. Thereafter, the phosphorylation level of tau and the activity of the kinases were analyzed. The injection of SIN-1 induced hyperphosphorylation of tau at pS396 epitope with a concomitant activation of GSK-3βand selective MAPK isoforms including p38α, p38β, and p38δbut not p38γ. The effect of peroxynitrite was confirmed using uric acid, a recognized scavenger of peroxynitrite. Preinjection of MEL significantly arrested the peroxynitrite-induced hyperphosphorylation of tau and the activation of GSK-3βand MAPKs. Melatonin also ameliorated peroxynitrite-induced oxidative stress. We conclude that MEL can efficiently arrest peroxynitrite-induced tau hyperphosphorylation, and the underlying mechanism may involve scavenging the reactive species and suppressing the activated GSK-3βand p38 MAPK family. Part 4 Effect of Intracerebroventricular Injection of LPS on tau Hyperhosphorylation in Rat HippocampusAbstract Objective: To investigate the effect of lipopolysaccharide (LPS) intracerebroventricular injection on hyperphosphorylation of tau. Methods: Western blot was used to analyze the hyperphosphorylation of tau from different rats killed at 2, 4 and 8 h respectively after LPS injection. Results: (1) Hyperphosphorylation of tau protein was seen after 2 h of LPS injection, then, the tau protein was dephosphorylated. (2) Intraperitoneal injection of LPS resulted in a significant increase in total level of tau. Conclusion: LPS affects tau expression and phosphorylation.更多还原