AIM: To develop a cell-based model by stable transfection of SH-SY5Y with mutant A53T human alpha-synuclein, recapitulating neurotoxicity of alpha -synuclein overexpression. METHODS: 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. 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(+)-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. CONCLUSION: 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.
AIM: To develop a cell-based model by stable transfection of SH-SY5Y with mutant A53Thumanalpha-synuclein, recapitulating neurotoxicity of alpha -synuclein overexpression. METHODS: 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. 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(+)-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. CONCLUSION: 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.
Authors: Olga Maria Lage; María C Ramos; Rita Calisto; Eduarda Almeida; Vitor Vasconcelos; Francisca Vicente Journal: Mar Drugs Date: 2018-08-14 Impact factor: 5.118