1. Department of pediatrics, First Hospital of Jilin University, Changchun, Jilin Province, China;
2. Department of Pharmacology, College of Basic Medical Sciences, Jilin University, Chang Chun, Jilin Province, China;
3. Departments of Neurology and Neuroscience Center, First Hospital of Jilin University, Changchun, Jilin Province, China;
4. Key Laboratory of Medical Cell Biology, Institute of Translational Medicine, China Medical University, Shenyang, Liaoning Province, China.
# signify an equal contribution to the study.
* signify an equal contribution to the study.
Parkinson's disease (PD) is one of the most common nervous system degenerative diseases. However, the etiology of this disease remains elusive. Here, a proteasome inhibitor (PSI)-induced undifferentiated SH-SY5Y PD model was established to analyze protein alterations through proteomic study. Methods: Cultured undifferentiated SH-SY5Y cells were divided into a control group and a group treated with 2.5 µM PSI (PSI-treated group). An methyl thiazolyl tetrazolium (MTT) assay was applied to detect cell viability. Acridine orange/ethidium bromide (AO/EB), α-synuclein immunofluorescence and hematoxylin and eosin (H&E) staining were applied to evaluate apoptosis and cytoplasmic inclusions, respectively. The protein spots that were significantly changed were separated, analyzed by 2D gel electrophoresis and DIGE De Cyder software, and subsequently identified by MALDI-TOF mass spectrometry and database searching. Results: The results of the MTT assay showed that there was a time and dose dependent change in cell viability following incubation with PSI. After 24 h incubation, PSI resulted in early apoptosis, and cytoplasmic inclusions were found in the PSI-treated group through H&E staining and α-synuclein immunofluorescence. Thus, undifferentiated SH-SY5Y cells could be used as PD model following PSI-induced inhibition of proteasomal function. In total, 18 proteins were differentially expressed between the groups, 7 of which were up-regulated and 11 of which were down-regulated. Among them, 5 protein spots were identified as being involved in the ubiquitin proteasome pathway-induced PD process. Conclusions: Mitochondrial heat shock protein 75 (MTHSP75), phosphoglycerate dehydrogenase (PHGDH), laminin binding protein (LBP), tyrosine 3/tryptophan 5-monooxygenase activation protein (14-3-3ε) and YWHAZ protein (14-3-3ζ) are involved in mitochondrial dysfunction, serine synthesis, amyloid clearance, apoptosis process and neuroprotection. These findings may provide new clues to deepen our understanding of PD pathogenesis.
Keywords: Parkinson's disease, ubiquitin proteasome system, SH-SY5Y cells, ptoteasomes inhibitor, proteomics