| Alzheimer’s disease (AD) is a commonly neurodegenerative disease of the central nervous system (CNS) in the elderly. The traditional treatment of AD was drug treatment, including beta-amyloid protein drugs, anti-cholinesterase drugs (CHEI), anti-inflammatory agent, free radical scavenger, antioxidant, potassium channels blockers, improving cerebral metabolism drugs, and so on. However, the effect of the drug treatment is limited. These drug treatments can not stop the progress of this disease, but only relieve the symptoms relatively. Therefore, looking for methods of treatment and prevention of AD will become of increasing concern in the future.Nowadays, with the high-tech’s improving development, transplantation of the neural stem cells (NSCs) offers a promising method for remedying AD. Moreover, the NSCs can be used as a donor cells for the treatment of the neurodegenerative disease in the central nervous system (CNS). Nowadays, it has been found that stimulate the generation of endogenous nerve cells and transplant the exogenous stem cells make have a therapeutic effect on AD, though these techniques are not successful in the clinical practice yet. We suggest that one of the reasons might be the change of the microenvironment for the survival of the NSCs, caused by the deposition of beta-amlyoid protein (Aβ) in AD brain. There has been evidence that the cholinergic neurotransmitters released after peripheral vagus nerve being stimulated can interact with the a7-nAChR receptors in macrophages to inhibit the synthesis and secretion of inflammatory mediators of macrophages so that the peripheral inflammation is weakened. Meanwhile, it has demonstrated that the expression of a7-nAChR is identified on the microglial cells in vivo and in vitro. Therefore, it is necessary for us to investigate the mechanism why the inflammation mediated by Aβ could lead to the disability of the NSCs, and to prove the activation of a7-nAChR in microglia could improve microenvironment which can promote the proliferation, differentiation and apoptosis of NSCs.In this paper, we aim to investigate the neuroprotective effect of activation a7-nAChR in microglia against Aβ1-42-induced neurotoxicity on neural stem cells. Firstly, the critical process is to master the technique of culture neural stem cells and microglia from newborn rat hippocampus in vitro. Secondly, we would establish a co-culture model of neural stem cells and microglia with the cell culture transwell inserts in vitro, in order to observe the effect of Aβ1-42on proliferation, differentiation and apoptosis of NSCs. Further, we investigate the neuroprotective effect of activation a7-nAChR in microglia against Aβ1-42-induced neurotoxicity on neural stem cells in co-culture system. Finally, the expression of β-catenin and GSK-3β, two signaling proteins of the wnt/β-catenin signaling pathway, were examined by western blot assays on NSCs after co-cultivation, in order to determine the molecular mechanism of the wnt/β-catenin signaling pathway on the survival of NSCs. Further understanding of this study may enhance the development of clinical trals combining drugs improving the microvironment in brain with NSCs transplantation in the treatment of AD.Methods1. Cell culture of hippocampal neural stem cellsHippocampus were removed from newborn Sprague-Dawley rats (within24hours) and digested with0.125%trypsin-EDTA. Then these cells were suspended in DMEM/F12medium containing20ng/ml basic fibroblast growth factor (bFGF), epidermal growth factor (EGF),2%B27,2%L-glutamine and1%penicillin/streptomycin. The cells were then plated onto25cm2cultured flasks at a density of1×106. The primary neurospheres were grown for7days and then passaged. The third generation of neurospheres were identified on the basis of cell morphology and expression of specific marker, nestin. Also, the neuronspheres were digested into a single-cell suspension and incubated with10μM5-bromo-2’deoxyurine (5-BrdU) in order to assess the proliferation of the cells. The neurospheres were incubated in differentiation medium consisted of DMEM/F12(1:1) supplemented with10%fetal bovine serum (FBS) for7days, then immunofluorescence staining for glial fibrillary acidic protein (GFAP) and microtubule-associate protein (MAP-2) were used to investigate the potential multiple differentiation of the cells.2. Culture of microgliaHippocampus were removed from the newborn SD rats (within48hours) and digested with0.125%trypsin-EDTA. Cells were suspended in DMEM/F12medium containing10%FBS and then plated at5×105onto poly-L-lysine-coated75-cm2flasks. Cells were grown at37℃in5%CO2, and medium was changed at day2of the culture. After9-10days of culture, flasks were gently shaken and the supernatant containing the microglia was collected. The purified microglia cells were identified on the basis of cell morphology and expression of specific marker, CDllb/c. And then quantitative analysis was carried out by counting the number of immunoreactive cells per total number of viable cells as determined by DAPI staining.3. Transwell co-culture assayWe established a co-culturre model of neural stem cells (NSCs) and microglia in the cell culture transwell inserts in vitro.A. NSCs Control group:the chamber contains of NSCs, but the well contains of medium.B. Aβ1-42+NSCs group:the chamber contains of NSCs, but the well contains of a final concentration of10μM of Ap1-42.C. NSCs and Microglia Co-culture group:NSCs and microglia were placed into the chamber and the well at a ratio of1:1, respectively.D. NSCs and Aβ1-42+Microglia Co-culture group:the chamber contains of NSCs, but the well contains of microglia treated by Aβ1-42.E. NSCs and Nicotine+Aβ1-42+Microglia Co-culture group:the chambert contains of NSCs, but the well contains of microglia pretreated by a final concentration of10μM of Nicotine before treated by Aβ1-42.F. Sulindac+NSCs and Microglia Co-culture group:the chamber contains of NSCs treated by a final concentration of200nM of Sulindac, but the well contains of microglia.G. Sulindac+NSCs and Aβ1-42+Microglia Co-culture group:the chamber contains of NSCs treated by Sulindac, but the well contains of microglia treated by Aβ1-42.H. Sulindac+NSCs and Nicotine+Aβ1-42+Microglia Co-culture group:the chamber contains of NSCs treated by Sulindac, but the well contains of microglia pretreated by Nicotine before treated by Aβ1-42.4. NSCs proliferation examined by BrdU incorporation assays The neuronspheres were detached into single cells and then incubated with a final concentration of10μM of BrdU for24hours. Then, cells were treated according to the experimental protocols. After96h, the proliferations of NSCs were examined by BrdU immunofluorescence assays.5. NSCs differentiation examined by immunocytochemistryThe neurospheres were incubated in differentiation medium for7days and then treated according to the experimental protocols. After96h, immunofluorescences staining for MAP-2and choline acetyltranferase (CHAT) were used to investigate the potential multiple differentiation of NSCs.6. NSCs apoptosis examined by FACSThe neuronspheres were detached into single cells for suspension culture, and then treated according to the experimental protocols. After96h, the apoptosis of NSCs were determined using the Annexin V/PI Apoptosis Detection kit according to the manufacturer’s protocol. Cell counts were taken in a FACSCalibu and analyzed with Cell Quest Pro software.7. Western Blot assaysThe neuronspheres were cultured in the proliferative medium and differentiated medium, respectively. And then, the expression of β-catenin and GSK-3β protein were examined by western blot assays on NSCs after co-cultivation.8. Statistical analysisAll the data were expressed as mean±standard deviation and analyzed using SPSS16.0statistical software. Statistical analysis of the data was done by One-Way ANOVA using LSD post hoc tests in multiple comparisons of means. A P value of less than0.05is considered statistically significant.Results1. NSCs Culture and identificationThe cultured NSCs proliferated, forming neurospheres. The rat NSCs after the culture and passage were proved by positive both Nestin staining and BrdU staining. The differentiated cells in the sphere could express specific antigens of neurons (positive MAP-2) and astrocytes (positive GFAP).2. Microglia Culture and identificationThe original generation of mixed-cultured cells mainly contains astrocytes and a small number of microglia, neurons and oligodendrocytes. After9-10days of culture, it had found stratification in the mixed-cultured cells. Then the flasks were gently shaken and the supernatant containing the microglia was collected. Morphologically, the microglia has platode and orbicular-ovate body and long processes. About95%of the cells were determined to be microglia based on the ration of CDllb/c positive cells to total cells counted by DAPI nuclear staining.3. Transwell co-culture systemWe successfully established a co-culture model of neural stem cells (NSCs) and microglia in a cell co-culture system in vitro.4. The proliferation of the NSCsThe proliferative rate of the Aβ1-42+NSCS group were significantly lower than that of the NSCs Control group (P<0.001). But compared the NSCs and Microglia Co-culture group to the NSCs Control group, the difference of the rate has not statistically significant (P>0.05). Compared with the NSCs and Microglia Co-culture group and the Aβ1-42+NSCS group, the rate were decreased in the NSCs and Aβ1-42+Microglia Co-culture group (all of P<0.01). When microglia’s a7-nAChR were activated against Aβ1-42toxicity, the rate were higher than that of the NSCs and AP1-42+Microglia Co-culture group (P<0.01), but still lower than that of the NSCs Control group and the NSCs and Microglia Co-culture group (all of P<0.01). The proliferative rate of the Sulindac+NSCs and Microglia Co-culture decreased significantly, comparing with the NSCs and Microglia Co-culture group (P<0.01). When microglia’s a7-nAChR were activated against Aβ1-42toxicity in the Sulindac+NSCs and Nicotine+Aβ1-42+Microglia Co-culture group, the difference of the rate has not statistically significant (P>0.05) comparing with that of the Sulindac+NSCs and Aβ1-42+Microglia Co-culture group, but has statistically significant with that of the NSCs and Nicotine+Aβ1-42+Microglia Co-culture group (P<0.01).5. The differentiation of the NSCsIt is resulted that there was significant difference of the positive MAP-2and CHAT between the NSCs Control group and the Aβ1-42+NSCs group (all of P<0.01), but not difference between the NSCs Control group and the NSCs and Microglia Co-culture group (all of P>0.05). In the co-cultured system, the positive rate in the NSCs and Aβ1-42+Microglia Co-culture group decreased significantly. However, the activation of the microglia’s a7-nAChR make the positive rate increased significantly (all of P<0.05). When the NSCs were treated by sulindac, the Aβ1-42protein can significantly reduce the ratio of the neuron differentiated from NSCs (all of P<0.05). With the help of the microglia’s a7-nAChR, these inhibition of the differentiation can not release (all of P>0.05).6. The apoptosis of the NSCsComparing with the NSCs Control group, the apoptosis rate of NSCs significantly decreased in the Aβ1-42+NSCs group (P<0.01). But the difference of the rate has not statistically significant in the the NSCs and Microglia Co-culture group (P>0.05). Compared with the Aβ1-42+NSCs group, the apoptosis rate of NSCs decreased in the NSCs and Aβ1-42+Microglia Co-culture group (P<0.01). When microglia’s a7-nAChR were activated by Nicotine, the apoptosis rate of NSCs were lower than that of the NSCs and Aβ1-42+Microglia Co-culture group (P<0.01), but still higher than that of the NSCs Control group and the Aβ1-42+NSCs group (all of P<0.01). The apoptosis rate in the Sulindac+NSCs and Microglia Co-culture group were higher than that of the NSCs and Microglia Co-culture group (P<0.01), but lower than these of the Sulindac+NSCs and Aβ1-42+Microglia Co-culture group and the NSCs and Nicotine+Aβ1-42+Microglia Co-culture group (all of P<0.01). Compare with the NSCs and Aβ1-42+Microglia Co-culture group, the apoptosis rate increased significantly in the Sulindac+NSCs and Aβ1-42+Microglia Co-culture group (P<0.01). The rate in the Sulindac+NSCs and Nicotine+Aβ1-42+Microglia Co-culture group were higher than that of the NSCs and Nicotine+Aβ1-42+Microglia Co-culture grou (P<0.01).7. Effect of the Wnt/β-Catenin signaling on the proliferation of the NSCsWestern blot analysis of total levels of β-catenin and GSK-3β from the proliferation NSCs showed that the level of expression of β-catenin was lower in the Sulindac+NSCs and Microglia Co-culture group than that of in the NSCs and Microglia Co-culture (P<0.05). But the difference of the up-regulation of GSK-3(3was statistically significant (P<0.01). P-catenin protein level tend to be less in the Sulindac+NSCs and Aβ1-42+Microglia Co-culture group compared to that of in the NSCs and Aβ1-42+Microglia Co-culture group, but the GSK-3β protein level was up-regulation(all of P<0.05). When microglia’s a7-nAChR were activated against Aβ1-42toxicity, the up-regulation of the β-catenin protein levels have no difference (P>0.05). But the up-regulation of GSK-3β protein levels was statistically significant (P<0.01).8. Effect of the Wnt/β-Catenin signaling on the differentiation of the NSCsThe level of expression of β-catenin in the Sulindac+NSCs and Microglia Co-culture group was lower than that of in the NSCs and Microglia Co-culture (P<0.05). The up-regulation of GSK-3P protein levels was also statistically significant (P<0.01). Compare to the NSCs and Aβ1-42+Microglia Co-culture group, both of the the β-catenin and the GSK-3β protein levels were down-regulation in the Sulindac+NSCs and Aβ1-42+Microglia Co-culture group (all of P<0.01). When microglia’s a7-nAChR were activated against Aβ1-42toxicity, the up-regulation of the β-catenin and the GSK-3β protein levels were statistically significant comparing to that of in the NSCs and Nicotine+Aβ1-42+Microglia Co-culture group (all of P<0.01).Conclusions1. We successfully established a co-culture model of neural stem cells (NSCs) and microglia in a Transwell co-culture system in vitro.2. The Aβ1-42protein could inhibit the survival of neural stem cells and significantly reduce the ratio of the neuron differentiated by neural stem cells, especially that of the cholinergic neuron.3. The inhibition of the survival of neural stem cells induced by Aβ1-42may be strengthening in presence of microglia.4. In the co-culture system, the proliferation and differentiation of neural stem cells can be enhanced by activating the α7-nAChR in microglia.5. Activation of a7-nAChR in microglia could improve the growth microenvironment of neural stem cells. In the co-culture system, the Wnt/β-catenin signaling pathway can play a different role on the proliferation, differentiation and apoptosis of neural stem cells. |
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