Zhao Da-Long, Zou Li-Bo, Zhou Lan-Fang, Zhu Ping, Zhu Hai-Bo
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Acta Pharmacol Sin. 2007 May;28(5):616-26. doi: 10.1111/j.1745-7254.2007.00539.x.
To develop a cell-based model by stable transfection of SH-SY5Y with mutant A53T human alpha-synuclein, recapitulating neurotoxicity of alpha -synuclein overexpression.
The overexpression of mutant alpha -synuclein was analyzed by Western blotting, immunocytochemistry, and RT-PCR. Cell viability was processed when treated with different concentrations of 1-methyl-4-phenylpyridinium (MPP+) and exogenous dopamine (DA) for 24, 48, and 72 h by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Early apoptosis and late apoptosis/necrosis were analyzed by flow cytometry using Annexin V and propidium iodide double staining, respectively. DNA was isolated and applied to agarose gel for electrophoresis; the typical DNA "ladder"represented severe apoptosis. We also used this model to screen 99 compounds with therapeutic potential by MTT assay.
One of the stably-transfected clones overexpressed exogenous genes on both the protein level and the transcriptive level. Significant differences in cytotoxicity were found between the pcDNA3.1(+) group and the pcDNA3.1(+)-hm alpha-synuclein group in the presence of the same concentration of MPP+ and DA within the same incubation time. The level of either early apoptosis or late apoptosis/necrosis was remarkably increased in transfected cells compared with the control after treatment with 100 micromol/L MPP+ for 24 h. In addition, the presence of the typical DNA "ladder" was observed in the pcDNA3.1(+)-hm alpha-synuclein group when treated with 200 micromol/L MPP+ for 48 h. After the screening experiment, 12 of the 99 compounds were found to decrease DA-induced cytotoxicity on cell viability.
We established a cell-based model which is useful for studying the function of alpha-synuclein and screening compounds with therapeutic potential. In addition, it was identified that cells overexpressing A53T mutant alpha-synuclein were significantly vulnerable against MPP+ or dopamine exposures.
通过用突变型A53T人α-突触核蛋白稳定转染SH-SY5Y细胞，建立一种细胞模型，以重现α-突触核蛋白过表达的神经毒性。
通过蛋白质印迹法、免疫细胞化学和逆转录-聚合酶链反应分析突变型α-突触核蛋白的过表达情况。用不同浓度的1-甲基-4-苯基吡啶鎓（MPP+）和外源性多巴胺（DA）处理细胞24、48和72小时后，采用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法检测细胞活力。分别使用膜联蛋白V和碘化丙啶双重染色，通过流式细胞术分析早期凋亡和晚期凋亡/坏死情况。提取DNA并应用于琼脂糖凝胶进行电泳；典型的DNA“梯形条带”代表严重凋亡。我们还使用该模型通过MTT法筛选了99种具有治疗潜力的化合物。
其中一个稳定转染的克隆在蛋白质水平和转录水平上均过表达外源基因。在相同孵育时间内，相同浓度的MPP+和DA存在下，pcDNA3.1(+)组和pcDNA3.1(+)-hmα-突触核蛋白组之间的细胞毒性存在显著差异。用100μmol/L MPP+处理24小时后，与对照组相比，转染细胞中的早期凋亡或晚期凋亡/坏死水平均显著升高。此外，用200μmol/L MPP+处理48小时后，在pcDNA3.1(+)-hmα-突触核蛋白组中观察到典型的DNA“梯形条带”。筛选实验后，发现99种化合物中有12种可降低DA诱导的细胞活力毒性。
我们建立了一种细胞模型，可用于研究α-突触核蛋白的功能并筛选具有治疗潜力的化合物。此外，已确定过表达A53T突变型α-突触核蛋白的细胞对MPP+或多巴胺暴露显著敏感。
