JAK2/STAT3 Signaling Mediates IL-6-inhibited Neurogenesis Of Hippocampal Neural Stem Cells Through DNA Demethylation/Methylation

BackgroundNeural stem cells(neural stem cells,NSCs)is present in the peripheral and central nervous system with self-renewal and multiple differentiation potential,which can differentiated into neurons and oligodendrocytes,astrocyte.Adult neurogenesis in the brain mainly exist in the ventricular zone(SVZ)and hippocampal dentate gyrus granular zone(SGZ).These areas of NSCs in vitro could be isolated and cloned,meanwhile,have ability to respond to illness and injury,In the SVZ and SGZ,it would be occurred NSCs proliferation,differentiation and migration throughout the life of the individual,therefore,It is great significance of adult neurogenesis in the brain research to the clinical treatment of neurodegenerative diseases,brain damage repair,etc.NSCs instead the brain damage and degeneration is expected to become the new treatment of alzheimer’s disease(AD),Parkinson’s disease(PD)and other neurodegenerative diseases.In the occurrence and development of the neurodegenerative disease,the brain always exists glial cell activation as the main characteristics of the inflammatory response.Nerve inflammation is a double-edged sword,on the one hand,it induces or aggravates the degenerative diseases of the nervous system;On the other hand,it is conducive to the repair of the nervous system damage in certain circumstances.A lot of research shows that nerve inflammation is thought to be important neurobiological characteristics of AD,actively participate in the occurrence and development process of AD,meanwhile exists a large number of features of inflammation in the AD brain,the hippocampus excessive expression of IL-6 results in a lot of glial proliferation and nerve inflammation.IL-6 is mainly synthetic by microglia and astrocytes,through free and membrane combination of IL-6 receptor to induce polymerization,common homologous gp130 signalling receptor and then activate JAK-STAT signaling pathways.Studies have reported that JAK/STAT signaling pathway mediates the neurological development process.The pathway includes two protein family: Janus tyrosine kinase(JAKs)and Signal transduction and transcriptional activation factor(STAT).The STAT family has seven members,they can be activated by JAKs selectively.STAT3 is widely expressed in different types of cells,it plays an important role in the cell growth,differentiation,proliferation and survival.Specific gene expression,including a variety of epigenetic changes,such as DNA methylation/demethylation.DNA demethylation can occur in the passive and active way.DNA methylation refers to the DNA methyltransferase(DNA methytransferase Dnmt)under the catalysis of using S-adenosine methionine with methyl,will be the fifth cytosine methylation,carbon atoms make it into 5-methyl cytosine(5-mC),the process is often found in the gene sequence of CG.DNA methyltransferase,a kind of preventing DNA passive demethylation of enzyme,mainly include: Dnmt1,namely type maintain DNA methyltransferase.Apply to only one chain double-stranded DNA methylation,make its full methylation,it can participate in DNA replication in the double chain methylation of new synthetic chain;Dnmt3a and Dnmt3 b,from a synthetic methyl transferase.It can make the CpG island methylation,CpG island methylation suppresses gene expression,DNA methylation is extremely important to embryonic development and neural stem cells.TET proteins(TET1,2,and 3)drive DNA active demethylation,5 mC can be catalyzed to 5-hydroxy methyl cytosine(5-hmC),5-formyl cytosine(5-fC)and 5-carboxyl cytosine(5-caC).Mutual adjustment between DNMTs and TETs plays a vital role in the brain’s normal development and function.(Neurogenic differentiation 1,NeuroD1),as a kind of containing the bHLH structure domain E-box of neural differentiation factor,which plays an important role in nervous system development and neuronal differentiation.NeuroD1 has been highly expressed in the process of peripheral and central nervous system development,but its expression level will be reduced with the mature neurons.Aims:In the present study,we attempted to investigate the direct connection between JAK2/STAT3 signaling and DNA methylation/demethylation and their roles in aberrant neurogenesis induced by IL-6 insult.Methods:Part one:Cultured cells were performed to determine the role of IL-6 in differentiation of NSCs by immunofluorescence(IF)and EdU.The expression of p-JAK2,JAK2,p-STAT3,STAT3 protein were detected at different concentration and time.immunofluorescence(IF)were employed to determine the AG490 whether can weaken the effect of IL-6 on NSCs.Part two:To study the effect of AG490 on Tet1,Tet2 and Tet3 protein expression of NSCs treated by IL-6,Western Blot,Dot Blot method and IF were used to detect the whole genome 5-mC.After DNMT1 protein was knocked down,5-hmC was observed by Dot Blot method and IF methods.Finally,the expression of NeuN and GFAP was tested by IF.Part three:To study the effect of AG490 on DNMT1 and DNMT3 a,DNMT3b protein expression of NSCs treated by IL-6,Western Blot,Dot Blot method and IF were used to detect the whole genome 5-mC.After DNMT1 protein was knocked down,5-mC was observed by Dot Blot method and IF methods.Finally,the expression of NeuN and GFAP was tested by IF.Part four:Western Blot and cell immunofluorescence(IF)were used to detect whether AG490 weakened the protein expression of IL-6-induced NeuroD1,Using the IF to observe the expression of DCX,GFAP after NeuroD1 protein was knocked down.Bisulfite sequencing(BSP)of NeuroD1 promotor area test 5-mC pattern.Using 5-hmC analysis kit quantify 5-hmC of NeuroD1 gene promoter region.Results:Part one:IF results show that IL-6 group compared with Control group,NeuN expression decreased and GFAP expression gradually increased in concentration dependence manner.EdU results showed that IL-6 group compared with Control group,the number of positive cells gradually decreased in concentration dependence manner.And Western Blot experiments results show that IL-6 concentration and time gradient group compared with the Control group,p-JAK2 and p-STAT3 expression gradually increased and the JAK2 and STAT3 expression has no obvious change.To prove JAK2 / STAT3 signaling pathways involved in the IL-6-inducted differentiation of NSCs,we used the JAK2 inhibitors AG490,the IF result showed: the NeuN expression in IL-6 groups decreased significantly compared with control group,IL-6+AG490 group compared with IL-6 group,NeuN expression is significantly enhanced.IL-6 group compared with the control group,GFAP expression obviously increased,the NeuN expression in IL-6+AG490 and IL-6 groups significantly reduced.Part two:WB results showed that IL-6 group compared with Control group,Tet3 protein obviously decreased,and increased in IL-6+AG490 group,Tets proteins were reduced by AG490.There are no significant difference between Control group and AG490 group.The expresses of Tet1 and Tet2 remain unchanged.TET3 also showed the concentration and time dependence.In order to further determine Tet3 in the role of methylation,Dot Blot analysis showed that 5-hmC in IL-6 group droped compared with control group,and IL-6+AG490 group compared with IL-6 group,5-hmC expression enhanced.IF results for 5-hmC were consistent with Dot Blot.In order to detect whether Tet3 protein activation participated in IL-6-induced differentiation,we knocked down Tet3 expression,the IF and Dot Blot results showed that shRNA Tet3 group compared with IL-6 groups,5-hmC expression decreased.Besides,we also found that knockdown Tet3 could obviously reduce the NeuN expression and improve the GFAP expression.Part three:WB results showed that IL-6 group compared with Control group,DNMT1 protein expression obviously increased,and decreased in IL-6+AG490 group,IL-6 induced increase in DNMT1 protein may be inhibited by AG490.There were no significant difference between Control group and AG490 group.DNMT3 a and DNMT3 b groups remained unchanged.DNMT1 also was in concentration and time dependent manner.In order to further determine the DNMT1 protein in the role of DNA methylation,Dot Blot analysis showed that IL-6 group compared with control group,5-mC enhanced.The results of IF for 5-mC were consistent with Dot Blot.In order to detect DNMT1 protein activation is involved in IL-6 differentiation,we knocked down DNMT1 expression,the results of IF and Dot Blot showed that shRNA DNMT1 group compared with IL-6 groups,5-mC expression decreased.At the same time,we also found that knockdown DNMT1 increased greatly NeuN expression,reduced GFAP expression.Part four:WB results showed that NeuroD1 protein in IL-6 group showed a marked decline compared with Control group,and increased in IL-6+AG490 group.AG490 reversed the decrease of IL-6-induced NeuroD1 protein.In line with WB,IF for NeuroD1 had similar results.In order to detect whether the activation of NeuroD1 protein takes part in IL-6 differentiation,we knocked down NeuroD1 expression,the results of IF showed that shRNA NeuroD1 group compared with IL-6 group,we also found that knockown of NeuroD1 could obviously reduce the DCX expression and improve the GFAP expression.BSP results showed that between NeuroD1 promoter region from 1599 to 2019,the expression of 5mC in IL-6 group increased 12% compared with control group,and reduced by 6% in IL-6+AG490 group.In order to investigate whether the 5-hmC participates in the regulation of neural differentiation,we employed 5-hmC analysis to test 5-hmC in NeuroD1 promoter area,the results showed that IL-6 group compared with control group,5-hmC reduced significantly,and increased significantly in IL-6+AG490 group.Conclusion:1)Blocking JAK2/STAT3 Signaling Attenuates IL-6-inhibited Neurogenesis of NSCs.2)JAK2/STAT3 Signaling Regulates TET3 Expression in IL-6-induced Differentiation of NSCs.3)JAK2/STAT3 Signaling Mediates IL-6-induced DNMT1 Activity in the Differentiation of NSCs.4)bHLH Transcription Factor NeuroD1 Participates in IL-6-induced Differentiation of NSCs.