Rotenone-induced neurotoxicity in PC 12 cells has been widely used to study the pathogenesis of Parkinson's disease. However, the precise mechanisms underlying rotenone-induced dopaminergic neuronal degeneration in Parkinson's disease remains unclear.OBJECTIVE:
To establish rotenone-induced neurotoxicity in PC 12 cells, and to investigate the possible action pathways to rotenone-induced neural cell injury at the protein level.DESIGN, TIME AND SETTING:
A controlled proteomics study was performed at the Department of Neurology, First Hospital, Jilin University between March 2006 and March 2007.MATERIALS:
PC 12 cells were obtained from Shanghai Cell Bank of Chinese Academy of Sciences, China. Rotenone was provided by Sigma, USA.METHODS:
PC 12 cells in logarithmic growth phase were treated under experimental and control conditions, respectively. A total of 0.5 μ mol/L rotenone, or the same amount of Dulbecco's modified eagle's medium (DMEM), was added in the experimental and control conditions, respectively.MAIN OUTCOME MEASURES:
Following 72 hours of rotenone treatment, cellular survival rate was determined by methyl thiazolyl tetrazolium assay, and apoptotic changes were detected by Hoechst 33342 staining. Total cellular protein was extracted to acquire differential protein expression data utilizing two-dimensional differential in-gel electrophoresis. To identify differential protein spots, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used.RESULTS:
In the MTT assay, the experimental condition induced significantly less cell survival compared to the control condition (P < 0.01). Hoechst 33342 staining revealed a larger number of apoptotic cells under the experimental condition compared to the control condition (P < 0.01), as determined by the presence of nuclear condensation, pyknosis, and nuclear fragmentation. Two-dimensional electrophoresis results showed that the differential expression of protein spots 1069 and 1538 was increased by 144% and 124%, respectively, while that of protein spot 1094 was decreased by 123% in the experimental condition compared to the control condition (P < 0.01). By MALDI-TOF-MS analysis and database retrieval, γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A were confirmed to be involved in rotenone-induced neural cell injury.CONCLUSION:
γ-enolase, triosephosphate isomerase 1, and eukaryotic translation initiation factor 4A might participate in rotenone-induced neurotoxicity in PC 12 cells.