α-突触核蛋白磷酸化参与小鼠多巴胺能神经元的保护作用

α-Synuclein phosphorylation takes part in mouse dopaminergic neuron protection

目的研究α-突触核蛋白(α-SYN)129位点磷酸化修饰是否具有神经元保护作用。方法应用反转录病毒感染小鼠多巴胺能神经元细胞MN9D,通过实时定量PCR检测各组细胞内α-SYN过表达情况,并通过免疫细胞化学方法检测野生型α-SYN(WT/α-SYN)及129位点磷酸化α-SYN(S129D/α-SYN)过表达后α-SYN在细胞内聚集情况,应用CCK-8检测细胞活力,观察WT/α-SYN及S129D/α-SYN对MN9D的细胞毒性,从而明确S129D/α-SYN对多巴胺能神经元的影响。结果实时定量PCR结果显示,WT/α-SYN及S129D/α-SYN在MN9D细胞内均过表达(P<0.01)。WT/α-SYN过表达组细胞活力明显下降(P<0.01),共聚焦显微镜观察未见明显的α-SYN聚集;S129D/α-SYN过表达组细胞内可观察到明显的α-SYN聚集体,同WT/α-SYN组相比细胞活力有明显提高(P<0.01)。结论129位点丝氨酸的磷酸化修饰在一定程度上减轻了WT/α-SYN过表达所引起的细胞毒性。

Objective To study the role of phosphorylation at Serine 129 in regulating the neurotoxicity of α-synuclein. Methods Wild type and phosphomimic mutant α-synuclein genes were over-expressed in mouse dopaminergic cells MN9D using retrovirus. The cell viability was examined using CCK-8 assay and cell morphology was observed by immunofluorescenee microscopy. Results The result of real time PCR showed that WT/α-SYN and S129D/α-SYN genes were overexpressed in MN9D as compared to uninfected MN9D and vector control group （P 〈 0. 01 ）. The cell viability was obviously reduced as a result of over-expressing both WT and phosphorylated α-synuclein genes. The viability of cells over-expressing wild type α-synuelein was much lower than that of the cells over-expressing phosphorylated α-synuclein（ P 〈 0. 05 ,P 〈 0. 01 ,P 〈 0. 01 ）. Insoluble α-synuclein aggregates were observed with confocal microscope in the MN 9 D cells over - expressing phosphorylated α - synuclein . Conclusion Over-expressing phosphorylated α-synuclein may lead to α-synuclein aggregated in MN9D cells. Phosphorylation of 129 Ser may protect the cells from toxicity induced by over expressing α-synuclein.