Proteomic Studies On The Mechanism Of Cytotoxicity Induced By Exogenous Dopamine In Differentiated SH-SY5Y Cells

Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of dopamine neurons in the substantia nigra,decreased striatal dopamine level, and the presence of eosinophilic inclusions called Lewy body in the cytoplasm of the spared neurons. The etiology and pathogenesis of PD have not been fully elucidated till now. Growing evidences suggest that the oxidative stress and the mitochondrial dysfunction are involved in the etiopathogenesis of PD.Dopamine (DA) is an essential neurotransmitter under physiological condition and is also an endogenous or exogenous neurotoxin. Reactive oxygen species (ROS) are produced in the course of normal and pathological metabolic DA oxidation, including auto-oxidation or enzyme-catalyzed reaction or both of them. Because of their high reactivity, accumulation of ROS beyond the immediate needs of the cell may affect cellular structure and functional integrity, by bringing about oxidative degradation of critical molecules, such as the DNA, proteins and lipids, resulting in oxidative stress. Recently, many evidences from experiments and postmortem of familial and sporadic PD display that the oxidative damage exists in the dopaminergic neurons of the substantia nigra pars compacta. The oxidative stress and mitochondrial dysfunction result in the accumulation of abnormal proteins in affected cells, the defects of cell function and even cell death. Oxidative stress and mitochondrial dysfunction may be the unifying pathway of various types of PD.Up to now, the toxic effects of DA on important protein molecules in PD patients and PD models have not been fully understood. To explore the mechanisms of DA toxicity at protein level, present study had performed the followed experiments: 1. applied RA and TPA in succession to induce the differentiation of SH-SY5Y cells; 2. applied exogenous DA to differentiated SH-SY5Y cells to evaluated the toxicity of DA on morphology, viability, and apoptosis; 3. applied DIGE and MALDI-TOF MS to identify differentially expressed proteins in differentiation course of SH-SY5Y; 4. applied DIGE and MALDI-TOF MS to identify differentially expressed proteins in exogenous DA-treated differentiated SH-SY5Y cells.In present study, administration of RA for 72h and TPA for additional 72h successfully induced the differentiation of SH-SY5Y cells. Compared to undifferentiated SH-SY5Y cells which have not or have shorter processes, marked extension of processes was observed in differentiated SH-SY5Y cells. The differentiated SH-SY5Y cells obtained typical neuronal phenotype.In present study, exogenous dopamine was added to differentiated SH-SY5Y cells. The viability of the cells was assayed by MTT and the apoptotic rate of the cells was measured by Hoechst33342 staining after incubation at different-concentration of dopamine for different period. In spite of its protective effect at low does (50μmol/L), DA was toxic at higher concentration (100μmol/L～500μmol/L). The viability of differentiated SH-SY5Y cells decreased with the prolonged treatment time and the increased dose of dopamine. The apoptotic rate of differentiated SH-SY5Y cells increased with the prolonged treatment time and the increased dose of dopamine.In present study, DIGE and MALDI-TOF MS were applied to identify differentially expressed proteins in differentiation course of SH-SY5Y. Three significantly up-regulated proteins were confidently identified including tubulin alpha1 (α-tubulin 1), laminin-binding protein (LBP), and GTP-binding regulatory alpha2 inhibitory subunit (Galphai2). The significantly up-regulation ofα-tubulin 1, LBP, and Galphai2 may involve in the differentiation of SH-SY5Y cells characterized by marked extension of neuronal-like processes.In present study, DIGE and MALDI-TOF MS were applied to identify differentially expressed proteins in differentiated SH-SY5Y treated by exogenous DA. A total of ten significantly changed proteins (of which, seven was increased and three was decreased) were unequivocally identified, including glucose-regulated protein 78 (GRP78), glucose-regulated protein 75 (GRP75), 14-3-3 zeta, prolyl-4-hydroxylase beta subunit (P4HB), protein disulfide isomerase-related protein (PDI),tubulin alpha- ubiquitous chain,β-actin (ACTB), keratin9 (KRT9), ATP synthase beta subunit (Fi- ATPase) and annexinA5. The significantly changed proteins involve in molecular chaperones, cytoskeletons, energy metabolism, and apoptosis.For the first time, present research analyzed and identified the changes at the protein level in the PD model induced by DA in differentiated SH-SY5Y cells. Among the identified differential proteins, tubulin alpha-ubiquitous chain,β-actin and keratin9, have not be reported in previous studies in the PD model induced by DA. These findings provide new valuable clues for future PD study and these proteins may become the new candidate targets of medicine treatment.