Role Of P21 Waf1/cip1 In Manganese-induced Neurotoxicity

Manganese is a common environmental and occupational pollutant. However, excessive intake of mnganese by respiratory and digestive tract can cause toxicity named as manganism. Therefore,more attention has been paid to the manganese-induced effect on the human health. Chronic manganese exposure can cause some extracorticospinal tract symptoms, which may result from the selective accumulation of manganese in the brain. Some recent researches showed that abnormal cerebral metabolism and lesion of DAergic neuron caused by manganese are the main factors of shaking palsy. But the exact mechanisms of manganese neurotoxicity are still being unraveled.Our recent study found that in addition to dopaminergic cell injury, manganese can also upregulate the p21 levels in nigrostriatal regions and DAergic PC12 cells. p21, as a cyclin dependent kinase inhibitor, is a member of CIP family, which can inhibit the cell growth by bind to CDK-cyclin complexes. It is not only a negative regulator of cell cycle, but also a mediator of cell death through both p53-dependent and p53-independent mechanisms. Recent studies found that the abnormal cell growth and cell proliferation are closely related with neurodegenerative diseases and the cell cycle-related proteins may play an important role in the pathological process of these diseases. In addition, the research on nerve cells also confirmed that the cell cycle proteins contributed to the neuronal apoptosis.Objective:In this study, with the establishment of the animal and cell model of manganism, the change in p21 expression was observed to investigate the effect of p21 in manganese-induced neurotoxicity and the underlying regulation mechanisms in this process. Our work may further some new theoretical bases and ways for the knowledge of manganese-induced neurotoxicity.Method:1.The animal model of manganism was established by stereotaxic injection into the rat striatum;Immunofluorescence was conducted for dopaminergic neuron injury, and western blot and RT-PCR were used for the expression of p21;2.PC12 cell line was used to establish the in vitro model of manganism, and the cytotoxic effect of manganese was analyzed through MTT assay, trypan blue staining and flow cytometry assay;3.The role of p21 in manganese-induced cytotoxicity was determined by western blot, immunofluorescence staining and the p21siRNA transfection;4.The regulation mechanisms of manganese on p21 expression were analyzed by western blot, RT-PCR and luciferase assay. Results:1.The effect of manganese on dopaminegic neuron injury and p21 levelsWe created the animal model of manganism by intrastriatal microinjections. The results of immunofluorescence staining showed that after manganese administration, there was a marked loss of TH-ir neurons in the substantia nigra compared with the control group, and the mRNA and protein levels of p21 were increased simultaneously.2.The role of p21 in manganese-induce dopaminergic PC12 cell injuryThe results of MTT assay and trypan blue staining showed that manganese inhibited the cell viability of PC12 cells in a dose-and time-dependent fashion. The inhibition of cell growth and cell cycle progression were observed by flow cyometry assay after manganese treatment. As shown by RT-PCR and western blot, manganese upregulated the mRNA and protein levels of p21, but it did not have the same effect on the other related factors. The results of immunofluorescence staining showed that the p21 protein was translocated from cytoplasm to nucleus in PC12 cells after manganese administrition. And silencing of p21 by siRNA can overcome the cell growth inhibition and G2 cell cycle arrest induced by manganese in PC12. Thus these results indicated an important role of p21 in manganese-induce cytotoxicity in PC12 cells.3.The regulation mechanisms of manganese-induced p21 expression in PC12 cellsWe found that p21 is mainly regulated at transcriptional level by manganese. The luciferase assay analysis showed that treatment of PC12 cells with manganese resulted in an increase in p21 promoter activity. And manganese did not stabilize the p21 mRNA and protein. Our results also showed that manganese can cause in vitro and in vivo DNA damage and p53 activation, but whether enhanced p21 expression occurs through p53-dependent mechanisms needs to be studied further.Conclusion:Our results showed that,for the in vivo study,manganese resulted in the selective dopaminergic neuron losses and the increased levels of p21 mRNA and protein. And manganese had a cytotoxic effect on dopaminergic PC12 in vitro, possibly by induction of p21. Manganese enhanced the p21 promoter activity, but it did not stabilize the p21 mRNA and protein. In vivo and in vitro studies also showed that manganese exposure result in DNA damage and p53 activation, and they may contribute to the induction of p21. But whether manganese activates p21 transcription through a p53 dependent pathway remains to be proved further.In conclusion, our research has been focused on the p21 induced by manganese in SD rats and PC12 cells and its underlying regulation mechanisms. Our results may further our understanding of manganese-induced neurotoxicity and provide some new theoretical bases and ways for the prevention of mangnism.