The Effect Of LiCl And Aβ_25-35 On Tau Protein Phosphorylation During The Differentiation Of Mice Neural Stem Cells

BACKGROUNDNeural stem cells are stem cells which distribute in the nervous system, they can self-update and have various differentiated potentiality. Their main functions are to participate in the nervous system injury-repair or normal cell death-updating as back-up reserves. Recent studies have found that there are stem cells in the nervous system of adult mammals and primates central. Neural stem cells can be induced to differentiate into neurons or gliocytes.Tau protein is an important microtubule-associated protein, distributed widely in the central nervous system and peripheral nervous system. Normal Tau protein can promote microtubule assembly, maintain microtubule stability and connect with other cytoskeletal proteins. In some central nervous system diseases, Tau protein has been abnormally modified, such as over-phosphorylation. The abnormal Tau protein losses physiological functions, with the result of microtubule depolymerization, the destruction of cytoskeleton, normal axonal transport system impairment, and the loss and degeneration of synapsis. The research in vivo has showed that the transplanted neural stem cells display proliferation, migration and the formation of specific types of nerve cells. But in some central nervous system diseases, with sustained neuron loss, the added "save" mechanism has not occurred. This may be related to some internal and external factors which hinder the neural stem cells to normally differentiate and differentiate into the corresponding function of neurons. Thus in differentiated process of neural stem cell, the study on Tau protein phosphorylation and the mechanisms of it has become one of the key issues. So our purpose are the following. We investigate the phosphorylation of Tau protein during the differentiation of neural stem cells and the effect of LiCl and Aβ_25-35 on Tau protein phosphorylation during the differentiation of neural stem cells. We also try to find out the mechanism of Tau protein phosphorylation.METHODSNeural stem cells were isolated from the neonatal rat hippocampus dentate gyrus under aseptic conditions, then were cultured with D/F serum (included 20ng/mL bFGF, 20ng/mL EGF, 2%B27), under the condition of 37℃, 5%CO₂. When neural stem cells were cultured to the third generation, remove bFGF and EGF, added 10% FBS into it. The differentiated neural stem cells were separated into three groups: normal group, LiCl pre-treated group, Aβgroup.we observed the growth of cells and collected the cells. Immuocytochemistry and Western-blot was used to detect the expression of Tau[pS396], Tau[pS262] GSK-3β[pTry^279,216] in three groups. Statistic analysis: the experimental results were analyzed by SPSS 10.0. The results of immuocytochemistry were analyzed by chi-square test, the results of Western-blot were analyzed by one-way analysis of variance.RESULTS1. We observed morphous of three groups cells after cultivating 36 hours. The living cells of LiCl pre-treated group and Aβgroup are less than normal group, the cells of Aβgroup reduced obviously. In some fields, the adherent cells of normal group exceeded other groups. In the Aβgroup and LiCl pre-treated group, the refraction of adherent cells descended.2. During normal neural stem cell differentiation process there were expressions of Tau[pS396], Tau[pS262], GSK-3β[pTry^279,216], displayed positive staining in the endochylema and axon.3. The results of immuocytochemistry and Western-blot both indicated: the levels of Tau protein's phosphorylation in Aβgroup and normal group were higher than LiCl pre-treated group on Tau[pS396] Tau[pS262](P<0.01). The expressed levels of GSK-3β[pTyr^279,216] in Aβgroup and normal group were higher than LiCl pre-treated group.CONCLUSIONS1. During neural stem cell differentiation process, Tau protein has a certain degree of phosphorylation levels.2. Lithium chloride (lithium chloride, LiCl) can inhibit the activity of GSK-3β, reduce tau protein phosphorylation levels.3. Aβ_25-35 can raise Tau protein phosphorylation levels with LiCl-pretreated by upregulating the expression of activated GSK-3β.