Abstract
Aim:
To develop a cell-based model by stable transfection of SH-SY5Y with mutant A53T human α-synuclein, recapitulating neurotoxicity of α-synuclein overexpression.
Methods:
The overexpression of mutant α-synuclein was analyzed by Western blotting, immunocytochemistry, and RT-PCR. Cell viability was processed when treated with different concentrations of 1-methyl-4-phenyl-pyridinium (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.
Results:
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(+)-hmα-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 μmol/L MPP+ for 24 h. In addition, the presence of the typical DNA “ladder” was observed in the pcDNA3.1(+)-hmα-synuclein group when treated with 200 μmol/L MPP+ for 48 h. After the screening experiment, 12 of the 99 compounds were found to decrease DA-induced cytotoxicity on cell viability.
Conclusion:
We established a cell-based model which is useful for studying the function of α-synuclein and screening compounds with therapeutic potential. In addition, it was identified that cells overexpressing A53T mutant α-synuclein were significantly vulnerable against MPP+ or dopamine exposures.
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Project supported by the National “973” program (No 2004CB518906), The Chang-jiang Scholars and innovative researech team in University (PCSIRJ, No IRT0514), the National Natural Sciences Foudation of China (No 30370720 and No 30572343).
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Zhao, Dl., Zou, Lb., Zhou, Lf. et al. A cell-based model of α-synucleinopathy for screening compounds with therapeutic potential of Parkinson's disease. Acta Pharmacol Sin 28, 616–626 (2007). https://doi.org/10.1111/j.1745-7254.2007.00539.x
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DOI: https://doi.org/10.1111/j.1745-7254.2007.00539.x
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