title: A cell-based model of alpha-synucleinopathy for screening compounds with therapeutic potential of Parkinson's disease.
abstract: 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.
authors: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors: Da-Long
authors: DL
authors: Zhao
authors: Li-Bo
authors: LB
authors: Zou
authors: Lan-Fang
authors: LF
authors: Zhou
authors: Ping
authors: P
authors: Zhu
authors: Hai-Bo
authors: HB
authors: Zhu
mesh_terms: 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*
chemical_list: D000978:Antiparkinson Agents; D051844:alpha-Synuclein