An Experimental Study On The Role Of Cell Cycle Aberrant In The Pathogenesis Of Parkinson's Disease

Objective To investigate the role of cell cycle aberrant in neuronal death induced by dopaminergic neurotoxins.Methods Neuronal PC12 cells were exposed to dopaminergic neurotoxins (lactacystin, MPP+, and 6-OHDA). Cell viability was measured by MTT. Flow cytometry was used to detect cell apoptosis and the distribution of cell cycle. The role of cell cycle inhibitors was assessed by MTT and flow cytometry.Results After differentiated by NGF, PC12 cells acquire a neuronal phenotype similar to that of sympathetic neurons. The cell viability of neuronal PC12 cells decreased in a time-and concentration-dependent manner. After exposure to lactacystin, MPP+, and 6-OHDA, the apoptotic rate increased in a time-dependent manner. At the same time, the percentage of cells in G0/G1 phase of cell cycle decreased and that in G2/M phase increased. Flavopiridol, a cell cycle inhibitor attenuated the cell cycle re-entry and cell injury induced by neurotoxins.Conclusion Cell cycle aberrant mediates cell apoptosis induced by dopaminergic neurotoxins.Objective To investigate effect of dopaminergic neurotoxins on the expression of cell cycle regulators in neuronal PC 12 cells.Methods Neuronal PC12 cells were exposed to dopaminergic neurotoxins (lactacystin, MPP+, and 6-OHDA). The mRNA levels of cyclin (cyclin A, cyclin D1, cyclin B1 and cyclin E) and cyclin dependent kinases (CDK2, CDK4 and CDK1) was assayed by RT-PCR. The protein levels of cell cycle regulators (pRB, p-pRB, cyclin B1, CDK4 and CDK1) were assessed by Western blot and immunocytochemistry.Results After exposure to dopaminergic neurotoxins, the mRNA levels of cyclin A, cyclin Dl and CDK1 increased, while the mRNA levels of cyclin B1, cyclin E, CDK2 and CDK4 did not change. The protein levels of p-pRB, cyclin B1, CDK4 and CDK1 increased after exposure lactacystin.Conclusion Neurotoxins induce cell cycle re-entry via influencing the expression and degradation cell cycle regulators.Objective To investigate the role of DNA polymerases in DNA replication and apoptotic death in neurons after re-entering the cell cycleMethods Cerebellar granule cells were exposed to MPP+. Cell viability was measured by MTT. Cell cycle distribution of neurons was measured by flow cytometry, and cell apoptosis was detected by flow cytometry and Hoechst staining. BrdU incorporation was used to detect the cells in S phase of cell cycle. Western blot was used to detect the expression of cyclin A, cyclin B, DNA primase, DNA pol-P, DNA pol-8 and pol-s. The effect of cell cycle inhibitors and DNA pols inhibitors on cell cycle, cell viability and cell apoptosis was assessed as well.Results After exposure to MPP+, some of the postmitotic neurons re-entered S phase of the cell cycle. The neuronal cell cycle re-entry and apoptosis were attenuated by flavopiridol, which is a broad inhibitor of cyclin-dependent kinases (CDKs). MPP+ exposure significantly increased the expression of DNA pol-βand primase but did not affect the expression of the canonical replicative DNA pols, including DNA pol-δand pol-ε. Dideoxycytidine, which is a pharmacological inhibitor of DNA pol-β, attenuated the neuronal apoptosis mediated by MPP+. In a similar manner, the expression of a dominant negative form of DNA pol-P was also neuroprotective.Conclusion DNA pol-βmay have a causal role in MPP+-induced neuronal apoptosis.Objective To investigate the role of MAPK signaling pathway in cell cycle re-entry and cell injury.Methods Neuronal PC12 cells were exposed to MPP+. Cell viability was measured by MTT. Flow cytometry was used to detect the distribution of cell cycle. Western blot was used to detect the expression of ERK, p-ERK, JNK, p-JNK, P38 and p-P38. The specific inhibitors of ERK, JNK, and P38 pathway was used to determine the role of different MAPK signaling pathway in cell cycle re-entry and cell viability.Results After exposure to MPP+, the expression of p-ERK, p-JNK, and p-P38 increased. PD98059, an inhibitor of ERK1/2 pathway inhibited the cell cycle re-entry and cell viability lose induced by MPP+. SP600125, an inhibitor of JNK pathway attenuated cell viability loss of PC 12 cells, but did not affected the cell cycle re-entry. SB202190, an inhibitor of P38 pathway did not affect the cell viability and cell cycle re-entry.Conclusion ERK1/2 signaling pathway mediates cell cycle re-entry induced by MPP+. Objective To investigate the role of oxidative stress in the activation of ERK1/2 pathway in and cell cycle re-entry.Methods Neuronal PC12 cells were exposed to MPP+. Cell viability was measured by MTT. Flow cytometry was used to detect the distribution of cell cycle. Intracellular reactive oxygen species (ROS) was measured using the fluorescent probe 2,7-dichlorofluorescein diacetate (H2DCFDA). Western blot was used to detect the expression of ERK1/2 and p-ERK. Catalase, an antioxidant was used to determine the role of oxidative stress in the activation of ERK1/2 pathway and cell cycle re-entry.Results After exposure MPP+, the level of intracellular ROS increased, and the cells re-entered the cell cycle. Pretreatment with catalase attenuated the ROS formation, cell cycle re-entry and cell viability loss induced by MPP+.Conclusion Oxidative stress mediates the activation of ERK1/2 pathway in and neuronal cell cycle re-entry.Aim:To investigate the neuroprotective effects of morin on 1-methyl-4-phenylpyridinium ion (MPP+)-induced apoptosis in neuronal differentiated PC12 cells as well as in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD.Methods:Neuronal differentiated PC 12 cells were challenged with MPP+ in the presence or absence of morin. Cell viability was determined by the MTT method. Cell apoptosis and cell cycle distribution were measured by flow cytometry. Intracellular ROS formation was assayed by fluorescence assay. In the MPTP mouse model of PD, the striatal dopamine content was detected by HPLC. The survival of dopaminergic neurons were measured by immunohistochemistry.Results:MPP+ induced loss of cell viability and apoptosis in neuronal PC12 cells. Concomitant treatment with morin significantly attenuated cell viability loss, apoptosis, and oxidative stress induced by MPP+. Furthermore, morin attenuated cell cycle re-entry induced by MPP+. In mice, MPTP induced significant nigrostriatal lesions. When administered prior to MPTP, morin attenuated behavioral deficits, dopaminergic neuronal death and striatal dopamine depletion in the MPTP mouse model.Conclusion:Morin is neuroprotective both in vitro and in vivo. The neuroprotective effect of morin may be attributed to its antioxidant and antiproliferative properties.