A cell-based model of alpha-synucleinopathy for screening compounds with therapeutic potential of Parkinson's disease.
OBJECTIVE
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.


CONCLUSIONS
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.
Zhao
Da-Long
DL
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
Zou
Li-Bo
LB
Zhou
Lan-Fang
LF
Zhu
Ping
P
Zhu
Hai-Bo
HB
D000818:Animals; D000978:Antiparkinson Agents / Q000737:chemistry* / Q000494:pharmacology / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D006801:Humans; D008954:Models, Biological; D015394:Molecular Structure; D009447:Neuroblastoma; D010300:Parkinson Disease / Q000188:drug therapy* / Q000473:pathology; D014158:Transcription, Genetic; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
D000978:Antiparkinson Agents; D051844:alpha-Synuclein