The Expression And Regulation Of GSK-3β,CDK-5 And PP2A In Differentiated Neural Stem Cells Of Rats

Neural stem cells (NSCs) are cells that have self-renewal ability and multidifferentiation potential. NSCs are transformed into neurons as its immature forms changed to mature forms, which is coincident with the change of the tau protein's location and expression. Tau protein is an important microtubule-associated protein, and localizes primarily in the axon of neurons. Its major role is to promote the formation and increase the stability of microtubules, therefore it plays an important part in maintenance of the morphology and function of cells. The normal tau protein is a phosphoprotein, and it can not only influence the formation of microtubules when it is hyperphosphorylated, but also can cause various kinds of depositions to localize in the neurons, which will cause damage of the neurons and diseases of the nervous system.Tau protein's phosphorylation is regulated by the relative activity of protein kinases(which catalyze hyperphosphorylation)and protein phosphatases (which catalyze dephosphorylation). Glycogen synthakinase-3β(GSK-3β) and cyclin-dependent kinase-5 (CDK-5) belong to Ser/Thr kinase, which can promote tau protein hyperphosphorylation in vivo and vitro and play a very important role in the regulation of tau phosphorylation. The decreasing activity of protein phosphatases, especially the activity of protein phosphatase-2A (PP2A), also play a key part in the regulation of tau phosphorylation.It has been proved that the mature neurons in the brain express GSK-3β,CDK-5 and PP2A. Aβ_25～35 and ginsenoside Rb1 can also regulate the expression of GSK-3β,CDK-5 and PP2A .However, this hasn't been reported for the neurons which are from the differentiated NSCs. Thus, in this study we intend to investigate the expression of GSK-3β,CDK-5 and PP2A and the regulation of them by Aβ_25～35 and ginsenoside Rb1 after NSCs are transformed into neurons.MethodsNeural stem cells of 24h old rats were isolated and cultured from the dentate gyrus of the hippocampus. The third passage of the differentiated cells from neurospheres was induced into neurons by adding 10% fetal bovine serum and removing mitogens. Undifferentiated neural stem cells, neurons and astrocytes were identified separately with anti-nestin ,anti-NSE and anti-GFAP antibodies by using immunocytochemical staining, and the positive percentage of cultured cells was evaluated. The NSCs were divided into 3 experimental groups after they had been induced for one week. (1)The control group: cultured for another 36h without additional treatment. (2)Aβ_25～35 treatment group: cultured for another 24h ,and then Aβ_25～35(20μmol/L) was added for 12h. (3) Ginsenoside Rb1 pre-treatment group: Pre-treated with ginsenoside Rb1(10μmol/L) for 24h, and then Aβ_25～35(20μmol/L) was added for 12h. After total 36h, each group of cells was collectd. The expressions of GSK-3β,CDK-5 and PP2A were detected with RT-PCR. At the same time the cell cycles of each group were determined by means of flow cytometer. In addition, the expressions of PP2A and GSK-3β(pTyr279, 216) were tested by the immunofluorescence cytochemical staining.Results1. Freshly isolated single NSCs from the dentate gyrus of newborn rats hippocampus were smaller, round and contained more opaque particles. After 3 passages, the neurospheres grew significantly bigger and the particles mostly disappeared. The specific markers of the NSCs(Nestin) were expressed on primary culture and the passed cells. After 3 days induction, most floating neurospheres began to adhere to the bottom of the bottle and grew outwards like in the shape of a thorn. After 7 days induction, most cells of the neurospheres grew outwards and formed axons which were interlaced with one another. Immunocytochemistry found that differentiated cells showed the specific markers of neurons(NSE) and astrocytes(GFAP).2. The cell cycles were determined by means of flow cytometer. The Aβ_25～35 and the ginsenoside Rb1 treatment groups were restrained in G0/G1 phase of the cycle. Thus, the proliferation of the cells was inhibited. The proliferation ability of the Aβ_25～35 treatment group was reduced more significantly.3. The cells of the control group expressed GSK-3β,CDK-5 and PP2A . The expressions of GSK-3βand CDK-5 in the Aβ_25～35 and the ginsenoside Rb1 groups were more than those in the control group, moreover, the expressions of GSK-3βand CDK-5 were the greatest in the Aβ_25～35 treatment group. The difference was considered significant (p<0.01); However, the expression of PP2A was lower in the Aβ_25～35 and the ginsenoside Rbl teatment groups than the control group, and the expression of PP2A was the lowest in the Aβ_25～35 group. The difference was considered significant (p<0.01).4. Immunofluorescence cytochemisty showed that the expressions of PP2A in the Aβ_25～35 and the ginsenoside Rb1 treatment groups were all reduced more than that in the control group, especially in the Aβ_25～35 group. However, the expressions of GSK-3β(pTyr279,216) in the Aβ_25～35 and the ginsenoside Rb1 treatment groups were all increased more than that in the control group, and especially in the Aβ_25～35 group.Conclusions1. NSCs which were induced in vitro expressed GSK-3β,CDK-5 and PP2A;2. Aβ_25～35 and ginsenoside Rb1 can regulate the expressions of GSK-3β,CDK-5 and PP2A which are obtained from the differentated NSCs in vitro.