The Effect Of Ginsenoside And Aβ On Tau Phosphorylation In Differentiated Neural Stem Cells Of Rats

Neural stem cells(NSCs) belong to the multipotential stem cells, which have a properity of proliferation and self-renewing, they were widely distributed in embryo, adult brain and peripheral nervous system, and that NSCs can be induced into neurons, astrocytes and oligodendrocytes by cytokines to compensate for the damaged nervous tissue. However, this compensation does not occured in some nervous disease, such like Alzheimer's disease (AD) disease, neural cells keep lossing, which may be associated with the microenvironment (like high level of Aβ) in brain of AD disease. We suppose that the high level of Aβmay affect the differentiation of NSCs,or may be the differentiated neural cells can not form normal structure and function.Tau protein is the highest content among microtubule associated protein (MAPs) in neural cells, which have a close relationship with neural cells function. Microtubule play an important role not only in maintaining neural cells configuration, but also in performing many cellular functions, for example the protein transport and signal transduction. Tau protein can promote microtubule stability and assembly, decrease tubulins decomposition.Our former experiment was found that in the process of mesenchymal stem cells (MSCs) differentiate into neural cells, the expression of tau protein was accord with cell configuration, when the MSCs gradually change shape into neural cells. The results indicate that during the process of MSCs differentiate to neural cells, a large number of tau protein are needed to promote formation of microtubule, in order to help the outgrow of axon and dendrites. other investigation shown that, in the process of NSCs differentiation, the expression of tau protein was accord with the maturities degree of neural cell .Tau protein takes participate in the development of axon ,makes sure the microtubule composite in the right way during the generate of dendrites, consequently, NSCs can transform into neural cells in morphology. Tau Protein is a pohosphorylation protein, the ability of association with microtubule is regulated by tau phosphorylation negatively. Normal level of phosphorylation is necessary for the function of tau protein ,however, high level of tau phosphorylation disrupt microtubule-based processes,format tau filament and handicap axonal transport ,owing to deceased microtubule binding.In the process of NSCs differentiate into neural cells, there is no reports about tau phpsphorylation. We choosed two representative tau phosphorylation residues Tau[pS396] Tau[ps262] and GSK-3p[pTyr279,216] in Our experiment, by immunocytochemistric and western-bloting methods, to observe the nomal expression of tau phosphorylation in NSCs differentiation. We also exposed differentiate cells by aggregated Aβ25-35 and ginsenoside Rb1,to investigate whether they could effect the tau phosphorylation in the process of NSCs differentiation, and explore the possible molecular mechanism.MethodsIsolate and culture rat's neural stem cells from the dentate gyrus of hippocampus, after 3-4 passages, differentiation of single cells from neurospheres was induced by adding 10% Fetal bovine serum, removal of mitogens. Undifferentiated neural stem cells, neurons and astrocytes were identified separately with anti-nestin ,anti-NSE and anti-GFAP antibodies by using immunocytochemical staining, Three groups are divided in experiment after differentiation for 7 days. 1. control group: cultured for another 36h without additional treatment. 2. Aβ_25-35 -treated group: cultured for another 24h,received AP25-35 (20μmol/L)for 12 hours. 3. Ginsenoside Rb1-pretreated group: given ginsenoside Rb1 (10μmol/L)24 hours,then receive AP25.35 (20μmol/L)for 12 hours at the same time with group 2. After total 36h, each group of cells was collectd. In order to detect the levels of Tau[pS396] Tau[ps262] and GSK-3β[pTyr279,216],western-blot and immunocytochemistry were performd. All data presented in the format of X|-±S .Deal data with SPSS12.0 software.Results1. The cells of the control group expressed Tau[pS396] and Tau[pS262].2. The positive rates of Tau[pS396] and Tau[pS262] were the highest in AP25-35 -treated group by immunocytochemistry, Ginsenoside Rb1-pretreated group and control group were decreased in turn, the differences between groups were significant(p<0.01). 3. The expression of Tau[pS396] Tau[pS262] and GSK-3β[pTyr279,216]were the highest in Aβ_25-35 -treated group by Western-blot experiments, Ginsenoside Rb1-pretreated group and control group were decreased in sequence. The differences between groups were significant(p<0.01).Conclusions1. There is a certain levels of phosphorylation in tau protein in neural stem cells differentiation.2. In neural stem cells differentiation, GSK-3βactivation by Aβ_25-35 may lead to txtensive tau phosphorylation. Ginsenoside Rb1 can attenuate Aβ_25-35-induced tau protein hyperosphorylation by inhibiting the activation of GSK-3β.