【作者】 蔡同建；

【导师】 陈景元； 骆文静；

【作者基本信息】 第四军医大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 【背景】锰是一种机体必需的微量元素,但过多的锰进入体内则会产生毒性作用。锰的毒性作用主要表现为神经系统功能异常,慢性锰中毒主要表现为锥体外系神经功能障碍,类似于帕金森病(Parkinson’s disease,PD)的症状。tau蛋白是微管相关蛋白家族的成员之一,在神经系统的发育和维持微管功能等方面具有重要作用,多种神经系统退行性疾病中都出现了与tau蛋白相关的特征性病理改变。tau蛋白过度磷酸化可影响其正常功能,引起一系列的病理损害。tau蛋白过度磷酸化后,其促微管聚合功能丧失,神经元微管解体,易于聚集成双螺旋丝(paired helical filaments,PHF)以及形成神经原纤维缠结(neurofibrillary tangle,NFT)结构,并转变成为毒性分子,可结合神经元中正常的微管相关蛋白,使微管结构崩解,神经元退变。多种蛋白激酶系统参与了tau蛋白的磷酸化过程,其中PI3K/Akt/GSK-3β以及MAPK信号通路是近年来研究tau蛋白磷酸化过程中涉及较多的两个信号通路。此外,氧化应激也被认为与神经元内tau蛋白的磷酸化过程密切相关,而我们此前的研究证实,锰可以通过诱导氧化应激水平的增高从而发挥其神经毒性效应。【目的】从细胞水平研究锰对神经元tau蛋白磷酸化水平的影响及其在锰诱导的神经毒性中的作用,并且从信号转导和氧化应激的角度探讨这个过程中的分子机制,从而为进一步阐明锰神经毒性的机制以及提出有效的锰神经毒性防护措施提供新的思路和理论依据。【方法】(1)利用培养的高分化型PC12细胞建立锰诱导多巴胺能神经元损伤的细胞模型。(2)MTT、LDH活性检测、流式细胞术、电镜等检测细胞毒性。(3)免疫荧光染色检测细胞骨架形态的变化。(4)Western Blot技术检测tau蛋白磷酸化水平以及相关信号通路的变化。(5)ROS水平检测了解细胞内氧化应激的水平。【结果】1.锰对PC12细胞损伤的诱导作用MTT结果显示,随着锰作用浓度的增高,PC12细胞活力逐渐下降。LDH活性检测结果显示,随着锰作用浓度的增高,PC12的损伤逐渐加重。流式细胞仪分析结果显示,锰可诱导PC12细胞凋亡率的显著增高。电镜检测可发现,锰染毒后PC12细胞可出现典型的凋亡超微结构特征。2.锰可诱导PC12细胞骨架的改变锰在诱导PC12细胞损伤的同时,也使细胞骨架形态出现了显著改变。免疫荧光染色结果显示,对照组细胞骨架形态清晰,排列规则、有序,细胞突起细长。锰作用后,PC12细胞骨架形态模糊、崩解,失去原有的规则排列方式。细胞胞体变大,突起缩短、或者消失。同时,细胞核出现固缩、碎裂等变化。此外,锰诱导细胞骨架改变的程度与其作用浓度呈正相关。3.锰诱导PC12细胞tau蛋白磷酸化水平的增高在不同浓度锰(0、100、300、500μmol/L)作用相同时间(6h)后以及300μmol/L锰作用不同时间(0、0.5、1、3、6h)后,PC12细胞的tau蛋白在Ser199、Ser202、Ser396和Ser404等位点均出现了磷酸化水平的增高。4.锰诱导PC12细胞tau蛋白磷酸化的信号转导机制研究结果显示,锰可诱导ERK MAPK、PI3K/Akt以及GSK-3β的激活,抑制这些信号通路的激活均能够抑制锰所诱导的tau蛋白磷酸化、细胞骨架的改变以及细胞损伤,而抑制ERK MAPK的激活还可抑制GSK-3β活性的增高,表明在这个过程中ERK MAPK可能在GSK-3β的上游。5.氧化应激在锰诱导的tau蛋白过度磷酸化过程中的作用锰可以诱导PC12细胞氧化应激水平的增高,表现为ROS水平的增高。而抗氧化剂NAC可抑制锰所诱导的PC12细胞凋亡和tau蛋白的过度磷酸化。同时,细胞骨架的形态也有所改善。【结论】1.在诱导PC12细胞增殖抑制以及损伤、凋亡的同时,锰可以诱导微管相关蛋白tau磷酸化水平的增高。tau的过度磷酸化可进一步诱导细胞骨架结构和功能的异常并进一步诱导PC12细胞的损伤。2.锰所诱导的tau蛋白磷酸化过程受到蛋白激酶的调控,其中锰可通过激活ERK MAPK进一步激活GSK-3β信号通路从而诱导tau蛋白的磷酸化,同时锰还可能通过诱导PI3K/Akt信号通路的激活诱导tau蛋白的过度磷酸化和相应的细胞毒性。3.锰可以通过诱导氧化应激水平的增高并进一步诱导tau蛋白的过度磷酸化。通过补充抗氧化剂有望成为抑制锰毒性的一个有效途径。综上所述,我们的研究初步阐明了锰对多巴胺能神经元tau蛋白磷酸化水平的影响并初步探讨了相关的分子机制,结果将为进一步阐明锰神经毒性的分子机制以及探寻有效的防护措施提供新的思路和理论依据。更多还原

【Abstract】 【Background】Manganese is an essential trace element and it is required for many ubiquitous enzymatic reactions. But too much absorption of manganese can cause toxicity named as manganism. The central nerve system is particularly susceptible to this excess manganese. Chronic manganese exposure can cause some extracorticospinal tract symptoms, which are similar to those of Parkinson’s disease.Tau protein is a member of microtubule-associated protein family, which plays an important role in the development of nervous system and the normal function of microtubule. The tau-associated pathological changes can be found in several neurodegenerative diseases. The hyperphosphorylation of tau can lead to the disturbance of its function and a series of pathological lesions. A lot of articles show that the hyperphosphorylation of tau is a common phenomenon in several kinds of neurodegenerative diseases. After the hyperphosphorylation, tau protein will lose its normal microtubule-binding ability, which will lead to the disassembly of microtubule structures in neurons and their degeneration. So, the hyperphosphorylation of tau protein has been regarded as a critical event in Alzheimer’s disease (AD), Parkinson’s disease (PD) and other neurodegenerative diseases.A large number of research results show that various protein kinases are involved in the phosphorylation of tau protein. Of them, PI3K/Akt/GSK-3βand mitogen-activated protein kinases (MAPK) are rather important. However, oxidative stress is also important in the phosphorylation of tau protein in neurons. Our previous work has showed that the treatment of manganese can lead to neurotoxicity through the elevation of oxidative stress level.【Aims】In the present study, we have investigated the effect of manganese on tau phosphorylation in neurons, and the mechanism of tau phosphorylation from signal transduction and oxidative stress. Our work may further our knowledge of manganese-induced neurotoxicity and provide some new theoretical bases and ways for the inhibition of manganese-induced neurotoxicity.【Methods】(1) High differentiated rat pheochromocytoma PC12 cell line was used to the model building of manganese-induced neurotoxicity in vitro. (2)The cytotoxicity was analyzed through MTT assay, LDH activity analysis, flow cytometry and electron microscopy. (3)The change of cytoskeleton was detected by an immunofluorescence method. (4)Westen blot was used to determine the phosphorylation of tau and the alteration of relative signal transduction pathways. (5)For investigating the oxidative stress level in PC12 cells, ROS contents were determined.【Results】 1.The cytotoxicity of manganese on PC12 cellsThe results of MTT showed that manganese inhibited the cell viability of PC12 cells in a concentration-dependent manner. The activity of LDH indicated that the injuries of PC12 cell induced by manganese were enhanced with the increase of manganese concentration. Treated by manganese, the apoptosis index of the cells increased as indicated by flow cytometry. Electron microscopy results showed that PC12 cells treated with manganese presented typical features of apoptosis.2.The change of cytoskeleton in PC12 cells induced by manganeseAs shown by immunofluorescence, the cytoskeleton in control group showed normal characteristics such as clear structure, regular arrangement, and long lengthy neurites. However, after the treatment by manganese, the cytoskeleton lost regular arrangement and the morphology of cytoskeleton turned to be fuzzy. Cell bodies enlarged while neurites shortened or disappeared. At the same time, cell nucleus showed typical apoptotic features such as pyknosis and fragmentation.3.The increased level of hyperphosphorylation of tau protein in PC12 cells caused by manganese treatmentAt the same concentration of MnCl(2300μmol/L), the phosphorylation level of tau protein at Ser199, Ser202, Ser396, and Ser404 increased with the time prolonged (0, 0.5, 1, 3, 6h) . Treated for the same period ( 6h ) , the phosphorylation level of tau at same sites increased with the increase of manganese concentrations (0, 100, 300, 500μmol/L).4.The signal transduction mechanism of manganese-induced tau hyperphosphorylation in PC12 cellsIn the present research, manganese induced the hyperphosphorylation of tau protein in PC12 cells with the activation of some protein kinases. Manganese also induced the activation of ERK MAPK, PI3K/Akt, and GSK-3βpathway, while the inhibitor of these kinases inhibited the hyperphosphorylation of tau protein, the alteration of cytoskeleton, and the cell injuries induced by manganese. However, the inhibition of ERK MAPK also inhibited the activation of GSK-3β, which suggesting that ERK MAPK may be the upstream of GSK- 3βduring the process.5.The role of oxidative stress in tau hyperphosphorylation induced by manganeseWith the treatment of manganese, the content of ROS increased. Antioxidant NAC inhibited the apopotis and tau hyperphosphorylation in PC12 cells induced by manganese. At the same time, the morphological changes of cytoskeleton were also prevented.【Conclusions】1.Our reasearch shows that manganese can induce tau hyperphosphorylation in PC12 cells, as well as the proliferation arrest and apoptosis. Such hyperphosphorylation can lead to the disfunction of cytoskeleton, which may further manganese-induced cytotoxicity in PC12 cells.2.The phosphorylation of tau induced by manganese is regulated by several protein kinases. In these protein kinases, ERK MAPK can lead to the hyperphosphorylation of tau through the activation of GSK-3βpathway. Besides, manganese can also lead to the activation of PI3K/Akt, which may be involved in the phosphorylation of tau and the cytotoxicity.3.Manganese can induce the elevation of oxidative stress, which may lead to the hyperphosphorylation of tau in PC12 cells. The supplement of antioxidant may be effective in the inhibition of manganese-induced toxicity. In conclusion, our research has been focused on the tau phosphorylation induced by manganese in PC12 cells and its mechanism. The results may further our understanding of manganese-induced neurotoxicity for an effective strategy to prevent manganese-induced neurotoxicity.更多还原