Proteomic Studies Of Proteasomal Inhibitor Treated Undifferentiation And Differentiation SH-SY5Y Cells

Parkinson's disease(PD) is one of the most common progressive neurodegenerative diseases with a terminal phase marked by resistance to therapy. Pathological hallmarks of the disease include loss of dopaminergic neurons in the substantia nigra (SN), as well as the presence of eosinophilic cytoplasmic inclusions (Lewy bodies) in residual neurons. The pathogenesis of PD are involved in many factors, including oxidative stress,mitochondrial dysfunction,cell apoptosis,ubiquitin-proteasome system (UPS) dysfunction, and so on. Recent studies suggest that defects in the capacity of the UPS to degrade unwanted proteins may be a common feature in the etiopathogenesis of both familial and sporadic PD.The cell line SH-SY5Y is a thrice-cloned neuroblastoma, originally from SK-N-SH. SH-SY5Y cells are known to be dopamine beta hydroxylase active, acetylcholinergic, glutamatergic and adenosinergic. The cells have very different growth phases, outlined in the surrounding pictures. The cells both propagate via mitosis and differentiate by extending neurites to the surrounding area. While induced by TPA, the differentiated cells look so close to nerve cell, so that the differentiated SH-SY5Y cells treated by proteasomal inhibitor PSI have been applied as a PD model.Objective To establish the PD model of undifferentiated and differentiated SH-SY5Y cells treated by PSI and to analyze the PSI-treated protein alterations in undifferentiated and differentiated SH-SY5Y cells. Methods In this experiment, undifferentiated and differentiated SH-SY5Y cells were cultured and proteasomal inhibitor PSI was added to the cells. Differentiated SH-SY5Y cells were previously cultured by TPA for 6 days. The cultured undifferentiated and differentiated SH-SY5Y cells were respectively divided into control group and 2.5μmol/L PSI-treated group. MTT assay was applied to detect cell viability, AO/EB,α-synuclein immunofluor- escence technology and HE stains were applied to show apoptosis and cytoplasmic inclusions, respectively. The specifically changed protein spots were separated and analyzed by 2D gel electrophoresis and DIGE DeCyder software, subsequently identified by MALDI-TOF mass spectrometry(MS) and database searching.Results1. In this experiment, SH-SY5Y cells were cultured together with 2.5umol/L proteasomal inhibitor PSI. The differentiated cells look so close to nerve cell. The viability of the cells was assayed by MTT after incubation in different-concentration of PSI for different period. The viability of SH-SY5Y cells decreased with the prolonged treatment time and the increased dose of PSI. According to fluorescent DNA stain observation, PSI administration resulted in cell apoptosis. Eosinophilic cytoplasmic inclusions were detected in SH-SY5Y cells treated with PSI after H&E staining andα-synuclein immunofluorescence technology. The two most distinctive pathologic features of PD can be induced in undifferentiated and differentiated SH-SY5Y 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. In treated group of undifferentiated SH-SY5Y cells, compared with that in control, the expressional levels of 18 protein spots were significantly changed, of which 7 were up-regulated and 11 were down-regulated. 5 protein spots were identified by mass spectrometry. The up-regulated proteins include MTHSP75. The down-regulated proteins include tyrosine3/tryptophan 5-monooxygenase activation protein, epsilon polypeptide (14-3-3 epsilon), phosphoglycerate dehydrogenase, YWHAZ protein, laminin-binding protein (LBP).3. In treated group of differentiated SH-SY5Y cells, compared with that in control, the expressional levels of 36 protein spots were significantly changed, of which 21 were up-regulated and 15 were down-regulated. 16 protein spots were identified by mass spectrometry, some of which were the same protein, finally 10 protein were determined. The up-regulated proteins include tumor rejection antigen (gp96), MTHSP75, chaperonin (HSP60), beta-actin. The down-regulated proteins include phosphoglycerate dehydrogenase, RuvB-like2, YWHAZ protein, proliferating cell nuclear antigen(PCNA), annexin V (annexin A5), ephrin a2.Conclusions1. Treatment of the undifferentiated and differentiated SH-SY5Y cells with 2.5μmol/L PSI for 24 h successfully established reliable experimental cell models of PD.2. We adopted advanced 2D-DIGE and MALDI-TOF MS and successfully identified significantly changed difference proteins in the undifferentiated and differentiated SH-SY5Y cells treated with PSI.3. In present study, it is the first time to have found the changed proteins of phosphoglycerate dehydrogenase, laminin-binding protein, RuvB-like2, ephrin a2 in PD model of undifferentiated and differentiated SH-SY5Y cells treated by PSI. These results provide valuable new clues for the further exploration of the pathogenetic mechanism of PD and novel targets for developing new drugs of PD therapy.