Wu Fengxia, Poon Wai Sang, Lu Gang, Wang Ancong, Meng Haiwei, Feng Lei, Li Zhenping, Liu Shuwei
Department of Anatomy, Shandong University School of Medicine, 44 Wen-hua Xi Road, Jinan, Shandong Province 250012, PR China.
Brain Res. 2009 Oct 6;1292:173-9. doi: 10.1016/j.brainres.2009.07.067. Epub 2009 Jul 29.
Alpha-synuclein is one of the main constituents of Lewy bodies and plays an important role in the pathology of Parkinson's disease. Mutation or overexpression of alpha-synuclein causes Parkinson's disease, and downregulation of alpha-synuclein resists MPP(+)-induced cell death, but the mechanism remains elusive. In this study, we attempted to explore the effect of alpha-synuclein knockdown on mitochondrial function in MPP(+)-treated SH-SY5Y cells. We reconstructed the short hairpin RNA expression vector, pGenesil-2, specially targeting alpha-synuclein mRNA, and it was stably transfected into SH-SY5Y cells. Cell viability, nuclear morphology, and mitochondrial membrane potential were then detected, and the expression of alpha-synuclein, cytochrome c, Bcl-2 and Bax were analyzed by Western blotting. The results showed that after exposure to 500 microM MPP(+) for 24 h, about 41.0+/-1.5% control cells showed low mitochondrial membrane potential. However, the percentage was 13.6+/-1.2% in MPP(+) treated alpha-synuclein knockdown cells. MPP(+) induced cytochrome c release significantly, which was about 3.1-fold compared with that of control. However, in alpha-synuclein knockdown cells, the release of cytochrome c was blocked, which was about 1.4-fold compared with that of control. The Bcl-2/Bax ratio of SH-SY5Y cells reduced to 35.5+/-3.8% after MPP(+) treatment, and this ratio was 85.2+/-3.0% in MPP(+) treated alpha-synuclein knockdown cells. These data suggest that knockdown of alpha- synuclein might be an effective means in rescuing MPP(+)-induced mitochondrial dysfunction of SH-SY5Y cells.
α-突触核蛋白是路易小体的主要成分之一，在帕金森病的病理过程中起重要作用。α-突触核蛋白的突变或过表达会导致帕金森病，而α-突触核蛋白的下调可抵抗1-甲基-4-苯基吡啶离子（MPP(+)）诱导的细胞死亡，但其机制仍不清楚。在本研究中，我们试图探讨α-突触核蛋白敲低对MPP(+)处理的SH-SY5Y细胞线粒体功能的影响。我们构建了专门靶向α-突触核蛋白mRNA的短发夹RNA表达载体pGenesil-2，并将其稳定转染到SH-SY5Y细胞中。然后检测细胞活力、细胞核形态和线粒体膜电位，并通过蛋白质免疫印迹法分析α-突触核蛋白、细胞色素c、Bcl-2和Bax的表达。结果显示，在500微摩尔MPP(+)处理24小时后，约41.0±1.5%的对照细胞显示出线粒体膜电位降低。然而，在MPP(+)处理的α-突触核蛋白敲低细胞中，这一比例为13.6±1.2%。MPP(+)显著诱导细胞色素c释放，与对照相比约为3.1倍。然而，在α-突触核蛋白敲低细胞中，细胞色素c的释放被阻断，与对照相比约为1.4倍。MPP(+)处理后，SH-SY5Y细胞的Bcl-2/Bax比值降至35.5±3.8%而在MPP(+)处理的α-突触核蛋白敲低细胞中，该比值为85.2±3.0%。这些数据表明，敲低α-突触核蛋白可能是挽救MPP(+)诱导的SH-SY5Y细胞线粒体功能障碍的有效手段。
