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An Experimental Study on the Effect of Dopaminergic Neurotoxins on Cell Cycle Reentry

Author: ZhangZhenTao
Tutor: CaoXueBing；SunShengGang
School: Huazhong University of Science and Technology
Course: Neurology
Keywords: Cell cycle apoptosis PC12 cells Parkinson’s disease Neurotoxins Cyclin Cyclin dependent kinase cell cycle ERK1/2 pathway RB
CLC: R741
Type: Master's thesis
Year: 2007
Downloads: 44
Quote: 0
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Abstract


PartⅠThe effect of dopaminergic neurotoxins on cell viability and cell cycle Objective To investigate the effect of dopaminergic neurotoxins on cell viability and cell cycle. Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor, were treated by dopaminergic neurotoxins (Lactacystin, MPP+, 6-OHDA and rotenone). Cell viability was measured by MTT, flow cytometry was used to detect cell apoptosis and the changes of cell cycle. Results The majority of NGF treated PC12 cells stayed in the G0/G1 phase of cell cycle. Dopaminergic neurotoxins markedly inhibited the viability of neurodifferentiated PC12 cells and increased the apoptosis rate in a time and concentration dependent manner (P < 001). As shown by flow cytometric, the cell percentage in G0/G1 phase decreased and that in the G2/M phase increased (P < 001). Conclusion Cell cycle reentry promote the cell apoptosis induced by dopaminergic neurotoxins. Part IIThe effect of dopaminergic neurotoxins on cell cycle regulator Objective To investigate the effect of dopaminergic neurotoxins on cell cycle regulator. Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor, were treated by dopaminergic neurotoxins (Lactacystin, MPP+, 6-OHDA and rotenone). The expression of Cyclins (CyclinA, CyclinD1, CyclinB1 and CyclinE) and Cyclin Dependent Kinases (CDK2, CDK4 and CDC2) mRNA was assayed by RT-PCR.Results After treated with dopaminergic neurotoxins, the expression of CyclinA, CyclinD1 and CDC2 mRNA increased (P < 001) and the expression of CyclinB1, CyclinE, CDK2 and CDK4 mRNA did not change (P > 005).Conclusion Dopaminergic neurotoxins can induce cell cycle reentry in dopaminergic neurons thought the high expression of CyclinA, CyclinD1 and CDC2. Part IIIThe role of ERK1/2 pathway activation and RB phosphorylation in the cell cycle reentry of dopaminergic neuronsObjective To investigate the role of ERK1/2 pathway activation and RB phosphorylation in the cell cycle reentry of dopaminergic neurons.Methods PC12 cells as dopaminergic neurons, differentiated by nerve growth factor, were treated by dopaminergic neurotoxins (Lactacystin, MPP+, 6-OHDA and rotenone). Immunocytochemistry and western blot were used to study the activation state of ERK1/2 pathway and the phosphorylation of RB.Results After treated with dopaminergic neurotoxins, the protein level of ERK1/2 did not change (P > 005), but the protein level of phosphorylatated ERK1/2 increased dramatically 4 h after treated by the dopaminergic neurotoxins and lasted for at lease 24 h (P < 001). Protein level of tumor suppressor retinoblastoma (RB) protein decreased while the protein level of phosphorylated RB increased (P < 001).Conclusion ERK1/2 pathway activation and RB phosphorylation promote the cell cycle reentry and cell apoptosis induced by dopaminergic neurotoxins.

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