不同亚细胞定位的alpha-突触核蛋白对SK-N-SH细胞的毒性影响

Effects of Subcellular Localized alpha-Synuclein on the Viability of SK-N-SH Cells

在帕金森病中,alpha-突触核蛋白(α-synuclein)累积与聚集所产生的细胞毒性是发病的重要原因。本文目的是探求不同亚细胞定位的α-突触核蛋白对于细胞的毒性影响。分别在α-突触核蛋白前插入核输出序列(nuclear export sequence,NES)或核定位序列(nuclear localization sequence,NLS),使其特定地表达在细胞质或细胞核中。构建成功的质粒分别在神经母细胞瘤SK-N-SH细胞中表达,通过免疫荧光与Western印迹法检测蛋白质的表达情况。结果显示,NES-α-synuclein特异性地在细胞质中表达,NLS-α-synuclein特异性地在细胞核中表达。乳酸脱氢酶法检测结果表明,相较于WT-α-synuclein组,NES-α-synuclein组的乳酸脱氢酶的释放量减少26. 54%,NLS-α-synuclein组的乳酸脱氢酶释放量增加12. 85%。CCK8法检测细胞活性结果表明,相较于WT-α-synuclein组,NES-α-synuclein组的细胞活力提高35. 51%,NLS-α-synuclein组的细胞活力减少7. 93%。上述结果提示,胞质内表达α-synuclein对于细胞的毒性更小,而细胞核内表达的α-synuclein对细胞有更强的毒性作用。这些发现为研究帕金森病的分子机制提供了新的研究思路。

The cytotoxicity caused by the accumulation and aggregation of alpha-synuclein is an important cause in the pathogenesis of Parkinson’s disease. In this study,we analyzed the impact of α-synuclein expressed in different subcellular localization on the cell viability. Firstly, we created constructions of α-synuclein tagged with either a nuclear export sequence( NES) or a nuclear localization sequence( NLS) to localize the protein in the nucleus or cytoplasm,respectively. Subsequently,the plasmids were separately expressed in SK-N-SH cells,and the Western blotting data showed that NES-α-synuclein protein was majorly constrained in the cytoplasm,and NLS-α-synuclein was mainly expressed in the nucleus. Next,we employed CCK8 and LDH assays to detect the effects of α-synuclein on cytotoxicity when it was expressed in different cell compartments. The results of LDH assay showed that release of LDH in the NES-α-synuclein group was decreased by 26. 54% as compared with the WT-α-synuclein group,and release of LDH in the NLS-α-synuclein group was increased by 12. 85%. At the same time,the results of CCK8 assay showed that compared with the WT-α-synuclein group,the cell viability of the NES-α-synuclein group increased by 35. 51%,whereas the cell viability of the NLS-α-synuclein group decreased by 7. 93%. The results showed that expression of α-synuclein in the cytoplasm partially rescued its toxic effects,and α-synuclein expressed in the nucleus had stronger toxic effects on cells. Our findings showed the differential effects of subcellular localization of α-synuclein on the cells,providing a potential strategy for studying the molecular mechanism of Parkinson’s disease.