The Effect Of Nicotine On Proliferation And Differentiation Of Mice Hippocampal Neural Stem Cells

Objective:Nicotine is able to regulate neural stem cells(NSCs)development through nAChR-a7 mediated signaling,and participate in neurogenesis,while the exchange protein directly activated by cAMP,PKA and its downstream signaling molecules are also involved in NSCs proliferation,differentiation,as well as neuron axon targeted growth.Therefore,we investigated whether nicotine induced EPAC and related signaling molecules alteration through nAChR-?7,which finally affects NSCs proliferation,differentiation,as well as neuron morphology plasticity.Methods:1.Isolated and cultured of primary mouse hippocampal NSCs from hippcampus of 1-day-old C57/BL6 mice.The cells were passaged to the 3rd generation expanding to 5th day.Then,the cells were performed with immunocytochemical staining.Nestin was used to identify neural stem cells,SOX2 was used to identify neural progenitor cells,and Ki-67 was used to detect the proliferation of NSCs.Further more,NSCs were cultured and induced to differentiate for 5 days.DCX,Tuj1,MAP2,GFAP and S100? antibodies were used for identification of NSCs differentiation ability by immunocytochemical staining.2.Nicotine cytotoxicity to NSCs was first detected by LDH toxicity assay when different concentrations of nicotine(1,10,100,200 and 400?M)were administrated to NSCs.Cell viability after nicotine exposure were then tested using CCK-8 method.Moreover,cell cycle of NSCs was determined using flow cytometry with ropidium iodide staining after different concentrations of nicotine was exposured to NSCs.Finally,western blotting was performed to detect p-p38 MAPK,p38 MAPK,pERK,ERK,pAKT,AKT,nAChR-a7,EPAC1,EPAC2,PKA,Rap1,p-CREB and CREB protein expression when NSCs was cultured for proliferation.3.NSCs were induced to differentiate with nicotine continuous treatment for 7 days to detect the influence of nicotine on NSCs.Immunocytochemical staining was used to detect DCX and GFAP positive cells.Western blotting was peformed to reveal the protein expression of DCX and GFAP after nicotine exposure to NSCs.Nicotine,8-CPT,ESI-09,6-BNZ,H-89,ESI-09 combined with nicotine and H-89 combined with nicotine were treated to NSCs for 7 days.Then immunocytochemical staining was used to analyze the dendritic branches of DCX-positive neurons,as well as the length of axons.In addition,qRT-PCR was used to detect the mRNA expression of nAChR-?7,EPC1,EPAC2,PKA and Rap1.4.NSCs were cultured and induced to differentiate with 10?M nicotine.Nicotine induced change of the signal were analyzed by western blotting.nAChR-a7 specific inhibitor ML A 10?M and MLA combined with nicotine were added to the culture for 6 hours to confirm nAChR-?7 mediated signaling by nicotine induction.nAChR-?7,EPC1,EPAC2,PKA and Rap1 were selected to measure protein expression.Results:1.NSCs were isolated and cultured from mouse hippocampus successfully.They can be subcultured and expanded every 3 days for further experiment The third passage of NSCs were positive for Nestin and SOX2,and the percentage of Ki-67 positive cells was 49%.NSCs have the ability to differentiate into neurons and astrocytes.DCX,Tuj 1,MAP2,GFAP and S100? staining were all positive.2.Different concentrations of nicotine(1,10,100,200,300 and 400?M)had no significant cytotoxic effects on NSCs.There is a dose dependent effect on the proliferation between 10 to 400?M.100 and 200?M nicotine significantly increase the cell viability of NSCs,and promote NSCs proliferation.We found p-p38 MAPK/p38 MAPK,pERK/ERK,pAKT/AKT,nAchR-a7 and pCREB/CREB were significantly up-regulated when NSCs were exposed to nicotine.3.Nicotine increased the number of neurons.However nicotine,EPAC,and PKA agonists reduced the number of dendritic branches of neurons,and shorten the length of axons.On the other hand,inhibitors of EPAC and PKA inhibitor played opposite roles,which increased the number of dendritic branches of neurons and augment the length of axons.Inhibition of EPAC and PKA signals improved the impairment of nicotine induced neuronal plasticity when ESI-09 or H89 was combined with nicotine,respectively.4.When NSCs were induced to differentiation,nAChR-a7 specific inhibitor(MLA)down-regulated nAChR-a7 protein expression,and nicotine-mediated downstream EPAC/Rapl signal,but had no significant effect on PKA protein expression.Conclusion:This study first demonstrated that nicotine activated P38-MAPK,ERK,and AKT signaling pathways,mediated changes in EPAC,PKA,and downstream signaling,and promoted proliferation in NSCs.Secondly,we found that nicotine has two sides effect on differentiated NPSCs.One side was to increase the number of DCX+immature neurons.The other side was to decrease plasticity of early differentiated immature neurons.Based our in vitro study,it will provide a novel insight into molecular mechanism for nicotine treatment of neurodegenerative diseases.Moreover,it may give a new molecular target for protecting NSCs from nicotine-induced neurodevelopmental toxicity.Our study provided a reasonable explanation for the neurodevelopmental toxicity caused by nicotine exposure,and suggested nAChR-a7 mediated Epac/Rapl signaling alternation may be a novel target for nicotine toxicity.Animal study is futher needed to verify our finding here.Figure[12]table[0]reference[52]...