Proteomics Study Of The Early Parkinson's Disease Of Cells Model Treated With Proteasomal Inhibitor

Backgroud: Parkinson's disease(PD) is a kind of common slowly neurodegenerative disorder whose primary pathologic features are the degeneration of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy body in the cytoplasm of the spared neurons, the loss of dopaminergic neurons in SNc result in a reduction in striatal dopamine content. After people's exploration for so many years, the etiology and etiopathogenesis of PD can not be elucidated till now. Neuropatholgenesis in PD is associated with oxidative stress, mitochondrial dysfunction, and excitotoxicity but it is not completely clear how these events contribute to the neurodegenerative process. Recent evidence suggests that failure of the ubiquitin proteasome system(UPS), leading to protein accumulation, contributes to the degeneration of dopaminergic neurons and Lewy body formation in the SNc in both familial and sporadic forms of PD. As the major degradation system of cellular unwanted proteins, the UPS achieves its function with the conjugation of a chain of ubiquitin to the targeted proteins under the action of different enzymes, may be a common feature in the etiopathogenesis of both familial and sporadic PD.Objective: To investigate the early changed protens in a model of Parkinson's disease(PD) induced by ubiquitin-proteasome dysfunction, which can provide the theoretical basis for PD.Method: PC12 cells, derived from a rat adrenal pheochromocytoma, were cultured and proteasomal inhibitor PSI was added to the cells, then establishment of PD cell model induced by proteasomal inhibitor. Proteins of untreated(DMSO) and PSI-treated PC12 cells were extracted after incubation, and then the maps of the changed proteins were established by 2D-DIGE system.The altered protein spots were identified with MALDI-TOF Pro MS. Method of indendified proteins were verified by immunological technique and Western blot technique.Results: 1. In this experiment, PC12 cells, derived from a rat adrenal pheochromocytoma, were cultured and proteasomal inhibitor 10μM PSI was added to the cells. The viability of the cells was assayed by MTT and trypan blue staining after incubation in different-concentration of PSI for different period. The viability of PC12 cells decreased with the prolonged treatment time and the increased dose of PSI. According to AO&EB and Hoechst33342 stain observation, PSI administration resulted in cell apoptosis. Eosinophilic cytoplasmic inclusions were detected in PC12 cells treated with PSI after H&E staining andα-synuclein immunostaining. The two most important pathologic features of PD can be induced in PC12 cells by treatment of proteasomal inhibitor PSI. So the cells after short-time inhibition of proteasomal function can be used as a model of PD. 2. A proteomic approach with 2-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry was used to determine proteins in PC12 cells that were differentially expressed following treatment by 10μM PSI. 14 were indendified, of which 8 increased and 4 decreased. In addition to, p47 were identified as two isoform, one was up-regulated and anther one was down-regulated in PSI treated groups, compared with control groups. For the first time, eIF3εand microtubule-associated protein RP/EB family member 1 protein were detected differentlly expressed respectively in this PD model. 3. ERp29 was validated by immunostaining method and Western blot, which can futher prove method of indendified proteins.Conclusion: At this stage of investigation, we found early changed protens in a model of Parkinson's disease (PD) induced by ubiquitin-proteasome dysfunction. These findings suggest that, these proteins participate in the formation of PSI-induced inclusions and apoptosis, which can provide the theoretical basis for PD.