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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 aL‐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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