[1]冯利杰,张 瑾,丁 倩,等.自噬参与神经细胞中过表达tau和异常磷酸化tau蛋白的降解[J].中国药理学通报,2015,(03):356-362.[doi:10.3969/j.issn.1001-1978.2015.03.013]
 FENG Li-jie,ZHANG Jin,DING Qian,et al.Autophagy involved in overexpressed tau and okadaic acid-induced hyperphosphorylated tau degradation[J].Chinese Pharmacological Bulletin,2015,(03):356-362.[doi:10.3969/j.issn.1001-1978.2015.03.013]
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自噬参与神经细胞中过表达tau和异常磷酸化tau蛋白的降解()
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《中国药理学通报》[ISSN:/CN:]

卷:
期数:
2015年03期
页码:
356-362
栏目:
论著
出版日期:
2015-03-25

文章信息/Info

Title:
Autophagy involved in overexpressed tau and okadaic acid-induced hyperphosphorylated tau degradation
作者:
冯利杰13张 瑾23丁 倩13朱 娜23王 鹏23沈玉君3沈玉先13
安徽医科大学 1.基础医学院,2.药学院,3.生物药物研究所,安徽 合肥 230032
Author(s):
FENG Li-jie13ZHANG Jin23DING Qian13ZHU Na23WANG Peng23SHEN Yu-jun3SHEN Yu-xian13
1.School of Basic Medical Sciences, 2.School of Pharmacy, 3.Institute of Biopharmaceutical Research, Anhui Medical University,Hefei 230001
关键词:
阿尔茨海默病 tau 磷酸化 自噬 蛋白降解 细胞毒性
Keywords:
tau phosphorylation autophagy protein degradation cytotoxicity Alzheimer's disease
分类号:
R-332; R329.24; R341; R745.7
DOI:
10.3969/j.issn.1001-1978.2015.03.013
文献标志码:
A
摘要:
目的 观察自噬对神经细胞内源性tau、过表达tau和异常磷酸化tau蛋白水平的影响,探讨不同形式tau蛋白降解的可能机制。方法 体外培养小鼠神经瘤母细胞株N2a和敲除Atg5基因的小鼠胚胎成纤维细胞株MEF,分别转染GFP-tau质粒,并用蛋白磷酸酯酶抑制剂冈田酸(okadaic acid, OA)诱导tau蛋白过度磷酸化,自噬诱导剂雷帕霉素(rapamycin)和溶酶体抑制剂氯化铵(NH4Cl)处理细胞,Western blot法检测tau、磷酸化tau以及自噬相关蛋白LC3和p62的表达; 放线菌酮(cycloheximide, CHX)示踪法观察tau和磷酸化tau的降解; GFP-tau和RFP-Lamp1质粒共转染N2a细胞,观察tau和溶酶体的定位; 免疫荧光染色和DAB染色观察自噬抑制对表达tau细胞形态的影响。结果 与溶媒对照组相比,NH4Cl和rapamycin处理组细胞内源性tau蛋白水平无明显变化; 过表达tau的细胞中,NH4Cl能增加tau和OA诱导的磷酸化tau蛋白水平,而rapamycin处理组细胞中tau和磷酸化tau水平降低,尤其是高分子量的磷酸化tau寡聚体明显减少; CHX实验证明自噬抑制能减缓tau和磷酸化tau的降解; tau和溶酶体在细胞内存在共定位; 过 表达tau的N2a细胞经NH4Cl处理后,胞质中出现大量tau聚集体,细胞核变小、固缩甚至消失。结论 自噬参与神经细胞中过表达tau和异常磷酸化tau蛋白的降解,抑制自噬能增加tau的细胞毒作用。
Abstract:
Aim To observe the effect of autophagy on the levels of endogenous tau, overexpressed tau and hyperphosphorylated tau induced by okadaic acid(OA)in Neuro2A cells. On this basis we are supposed to explore the role of autophagy in tau degradation. Methods Neuro2A and Atg5 knockdown(Atg5-/-)MEFs cells were cultured and transfected with GFP-tau plasmid, and equal amount of empty vector was used as control. Twenty-four hours after transfection, cells were incubated with or without OA for 12 h, followed by treated with a lysosome inhibitor NH4Cl or an autophagy inducer rapamycin for 6 h, and the levels of tau, phosphorylated tau, LC3 and p62 were detected by Western blot. The cycloheximide(CHX)chase analysis was employed to determine tau or phosphorylated tau degradation. Meanwhile, expression and intracellular localization of tau and lamp1 in Neuro2A cells were observed under confocal microscopy. Immunohistochemistry was used to observe the morphology of tau-positive cells. Results Our study showed that autophagy activation or inhibition had no influence on the levels of endogenous tau. On the contrary, the levels of tau and phosphorylated tau were increased in tau-transfected Neuro2A cells treated with NH4Cl, which was reversed on induction of autophagy by rapamycin, especially high molecular weight polymer tau. CHX chase results showed that the clearance of tau and phosphorylated tau were delayed in Atg5-/- MEF cells. GFP-tau and RFP-Lamp1 were colocalized in Neuro2A cells. Furthermore, NH4Cl treatment significantly facilitated tau aggregation and increased tau cytotoxicity.Conclusion Autophagy is involved in overexpressed tau and OA-induced hyperphosphorylated tau degradation, whereas it is not involved in endogenous tau degradation.Inhibition of autophagy enhances tau aggregation and cytotoxicity.

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备注/Memo

备注/Memo:
收稿日期:2014-12-04,修回日期:2015-01-04 基金项目:国家自然科学基金(No 81302755); 安徽省教育厅重点项目(No KJ2013A159); 高等学校博士学科点专项科研基金(No 20123420120002) 作者简介:冯利杰(1980-),女,博士,讲师,E-mail:fenglijie1128@sina.com; 沈玉先(1965- ),女,博士,教授,博士生导师,研究方向:功能性蛋白与药物作用靶点,Tel:0551-65113750,E-mail:shenyx@ustc.edu.cn
更新日期/Last Update: 2015-03-25