Role Of Tau Phosphorylation In Neurotoxicity Induced By Manganese

【Background】Manganese is an essential trace element and it is required for many ubiquitous enzymatic reactions. But too much absorption of manganese can cause toxicity named as manganism. The central nerve system is particularly susceptible to this excess manganese. Chronic manganese exposure can cause some extracorticospinal tract symptoms, which are similar to those of Parkinson's disease.Tau protein is a member of microtubule-associated protein family, which plays an important role in the development of nervous system and the normal function of microtubule. The tau-associated pathological changes can be found in several neurodegenerative diseases. The hyperphosphorylation of tau can lead to the disturbance of its function and a series of pathological lesions. A lot of articles show that the hyperphosphorylation of tau is a common phenomenon in several kinds of neurodegenerative diseases. After the hyperphosphorylation, tau protein will lose its normal microtubule-binding ability, which will lead to the disassembly of microtubule structures in neurons and their degeneration. So, the hyperphosphorylation of tau protein has been regarded as a critical event in Alzheimer's disease (AD), Parkinson's disease (PD) and other neurodegenerative diseases.A large number of research results show that various protein kinases are involved in the phosphorylation of tau protein. Of them, PI3K/Akt/GSK-3βand mitogen-activated protein kinases (MAPK) are rather important. However, oxidative stress is also important in the phosphorylation of tau protein in neurons. Our previous work has showed that the treatment of manganese can lead to neurotoxicity through the elevation of oxidative stress level.【Aims】In the present study, we have investigated the effect of manganese on tau phosphorylation in neurons, and the mechanism of tau phosphorylation from signal transduction and oxidative stress. Our work may further our knowledge of manganese-induced neurotoxicity and provide some new theoretical bases and ways for the inhibition of manganese-induced neurotoxicity.【Methods】(1) High differentiated rat pheochromocytoma PC12 cell line was used to the model building of manganese-induced neurotoxicity in vitro. (2)The cytotoxicity was analyzed through MTT assay, LDH activity analysis, flow cytometry and electron microscopy. (3)The change of cytoskeleton was detected by an immunofluorescence method. (4)Westen blot was used to determine the phosphorylation of tau and the alteration of relative signal transduction pathways. (5)For investigating the oxidative stress level in PC12 cells, ROS contents were determined.【Results】 1.The cytotoxicity of manganese on PC12 cellsThe results of MTT showed that manganese inhibited the cell viability of PC12 cells in a concentration-dependent manner. The activity of LDH indicated that the injuries of PC12 cell induced by manganese were enhanced with the increase of manganese concentration. Treated by manganese, the apoptosis index of the cells increased as indicated by flow cytometry. Electron microscopy results showed that PC12 cells treated with manganese presented typical features of apoptosis.2.The change of cytoskeleton in PC12 cells induced by manganeseAs shown by immunofluorescence, the cytoskeleton in control group showed normal characteristics such as clear structure, regular arrangement, and long lengthy neurites. However, after the treatment by manganese, the cytoskeleton lost regular arrangement and the morphology of cytoskeleton turned to be fuzzy. Cell bodies enlarged while neurites shortened or disappeared. At the same time, cell nucleus showed typical apoptotic features such as pyknosis and fragmentation.3.The increased level of hyperphosphorylation of tau protein in PC12 cells caused by manganese treatmentAt the same concentration of MnCl(2300μmol/L), the phosphorylation level of tau protein at Ser199, Ser202, Ser396, and Ser404 increased with the time prolonged (0, 0.5, 1, 3, 6h) . Treated for the same period ( 6h ) , the phosphorylation level of tau at same sites increased with the increase of manganese concentrations (0, 100, 300, 500μmol/L).4.The signal transduction mechanism of manganese-induced tau hyperphosphorylation in PC12 cellsIn the present research, manganese induced the hyperphosphorylation of tau protein in PC12 cells with the activation of some protein kinases. Manganese also induced the activation of ERK MAPK, PI3K/Akt, and GSK-3βpathway, while the inhibitor of these kinases inhibited the hyperphosphorylation of tau protein, the alteration of cytoskeleton, and the cell injuries induced by manganese. However, the inhibition of ERK MAPK also inhibited the activation of GSK-3β, which suggesting that ERK MAPK may be the upstream of GSK- 3βduring the process.5.The role of oxidative stress in tau hyperphosphorylation induced by manganeseWith the treatment of manganese, the content of ROS increased. Antioxidant NAC inhibited the apopotis and tau hyperphosphorylation in PC12 cells induced by manganese. At the same time, the morphological changes of cytoskeleton were also prevented.【Conclusions】1.Our reasearch shows that manganese can induce tau hyperphosphorylation in PC12 cells, as well as the proliferation arrest and apoptosis. Such hyperphosphorylation can lead to the disfunction of cytoskeleton, which may further manganese-induced cytotoxicity in PC12 cells.2.The phosphorylation of tau induced by manganese is regulated by several protein kinases. In these protein kinases, ERK MAPK can lead to the hyperphosphorylation of tau through the activation of GSK-3βpathway. Besides, manganese can also lead to the activation of PI3K/Akt, which may be involved in the phosphorylation of tau and the cytotoxicity.3.Manganese can induce the elevation of oxidative stress, which may lead to the hyperphosphorylation of tau in PC12 cells. The supplement of antioxidant may be effective in the inhibition of manganese-induced toxicity. In conclusion, our research has been focused on the tau phosphorylation induced by manganese in PC12 cells and its mechanism. The results may further our understanding of manganese-induced neurotoxicity for an effective strategy to prevent manganese-induced neurotoxicity.