帕金森病基因PINK1和DJ-1的转录水平在神经毒损伤的MN9D细胞中的不同变化

doi:10.3969/j.issn.1006-7795.2015.05.015

首都医科大学学报 ›› 2015, Vol. 36 ›› Issue (5): 740-746.doi: 10.3969/j.issn.1006-7795.2015.05.015

帕金森病基因PINK1和DJ-1的转录水平在神经毒损伤的MN9D细胞中的不同变化

杨俭^1,2, 王勇^2,3, 汪璇^2,3, 杨兆菲^1,2, 宫晓丽^2,3, 王晓民^1,2

1. 首都医科大学基础医学院神经生物学系教育部神经变性病重点实验室, 北京 100069;
2. 北京脑重大疾病研究院, 北京 100069;
3. 首都医科大学基础医学院生理学与病理生理学系教育部神经变性病重点实验室, 北京 100069

收稿日期:2015-09-14 出版日期:2015-10-21 发布日期:2015-10-20
基金资助:
国家重点基础研究发展计划("973"计划)(2011CB504100),北京市自然科学基金(7082008),北京市属高等学校创新团队建设与教师职业发展计划项目(IDHT20140514)。

Parkinson's disease related gene PINK1 but not DJ-1 was down-regulated in neurotoxicity of 6-hydroxydopamine and 1-methyl-4-phenylpyridinium

Yang Jian^1,2, Wang Yong^2,3, Wang Xuan^2,3, Yang Zhaofei^1,2, Gong Xiaoli^2,3, Wang Xiaomin^1,2

1. Key Laboratory for Neurodegenerative Disorders of the Ministry of Education, Department of Neurobiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China;
2. Beijing Institute for Brain Disorders, Beijing 100069, China;
3. Key Laboratory for Neurodegenerative Disorders of the Ministry of Education, Department of Physiology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China

Received:2015-09-14 Online:2015-10-21 Published:2015-10-20
Contact: 王晓民 E-mail:xmwang@ccmu.edu.cn
Supported by:
This study was supported by Major State Basic Research Development Program of the China (973 Program) (2011CB504100), Natural Science Foundation of Beijing (7082008), Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges under Beijing Municipal Education Commission (IDHT20140514).

摘要/Abstract

摘要： 目的在细胞水平上探究神经毒素作用下,关键的帕金森(Parkinson's disease)致病基因 PINK1和DJ-1 的转录情况,并从表观遗传学角度对其机制进行初步探索。方法本研究使用MN9D细胞系作为研究对象。通过台盼蓝活细胞计数的方法筛选神经毒素6-羟基多巴胺(6-hydroxydopamine,6-OHDA)和1-甲基-4-苯基嘧啶离子(1-methyl-4-phenylpyridinium,MPP⁺)的半数致死剂量。通过半定量RT-PCR方法检测 DJ-1基因和PINK1 基因转录情况。通过RT-PCR以及免疫细胞荧光染色方法检测DNA去甲基化酶TET1的转录表达情况。结果 1)MN9D活细胞数在6-OHDA和MPP⁺ 作用下均发生了时间、剂量依赖性降低,6-OHDA和MPP⁺的半数致死剂量分别约为10 μmol/L和100 μmol/L。2)DJ-1的mRNA水平在6-OHDA或MPP⁺ 作用下未发生改变,而PINK1的mRNA水平则发生了明显的时间依赖下降。3)DNA去甲基化酶TET1的转录表达水平也发生显著下降。结论神经毒素作用下,帕金森病致病基因 PINK1 的转录发生下调,并可能是由DNA去甲基化酶TET1表达下调所介导的。

关键词: 帕金森病, 神经毒性, PINK1, DJ-1, DNA去甲基化酶TET1

Abstract: Objective To explore the transcriptional levels and molecular mechanism of key Parkinson's disease (PD)-related genes DJ-1 and PINK1 in neurotoxicity. Methods MN9D cells were used as dopaminergic neurons and treated with neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP⁺). Lethal doses 50 (LD 50) was determined by cell counting with Typan Blue staining. Transcriptional levels of DJ-1, PINK1 and demethylation enzyme TET1 were detected by semi-quantitative RT-PCR. Expressional levels of demethylation enzyme TET1 were detected by immune-cytofluorescent staining. Results LD50 at 10 μmol/L of 6-OHDA and 100 μmol/L of MPP⁺ were used to treat MN9D cells. In neurotoxicity of 6-OHDA and MPP⁺, transcriptional levels of PINK1 were significantly down-regulated, but no change in DJ-1 transcriptional levels was found. In addition, transcriptional and expressional levels of TET1 were down-regulated in neurotoxicity of 6-OHDA and MPP⁺. Conclusion These data suggest that down-regulated TET1 might contribute to the down-regulation of PINK1 in neurotoxicity of PD.

Key words: Parkinson's disease, neurotoxicity, PINK1, DJ-1, Ten-eleven translocation 1

中图分类号:

杨俭, 王勇, 汪璇, 杨兆菲, 宫晓丽, 王晓民. 帕金森病基因PINK1和DJ-1的转录水平在神经毒损伤的MN9D细胞中的不同变化[J]. 首都医科大学学报, 2015, 36(5): 740-746.

Yang Jian, Wang Yong, Wang Xuan, Yang Zhaofei, Gong Xiaoli, Wang Xiaomin. Parkinson's disease related gene PINK1 but not DJ-1 was down-regulated in neurotoxicity of 6-hydroxydopamine and 1-methyl-4-phenylpyridinium[J]. Journal of Capital Medical University, 2015, 36(5): 740-746.

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帕金森病基因PINK1和DJ-1的转录水平在神经毒损伤的MN9D细胞中的不同变化

Parkinson's disease related gene PINK1 but not DJ-1 was down-regulated in neurotoxicity of 6-hydroxydopamine and 1-methyl-4-phenylpyridinium

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