| Neural stem cells(NSCs)are a subset of precursor cells that possess the ability to self-renew and to produce neurons,astrocytes,and oligodendrocytes in the embryonic development or adult brain.Neurons differentiation started the earliest,followed by astrocytes,and finally oligodendrocytes.At the early stage of brain development,NSCs in the ventricle area were first divided symmetrically to increase the number of cells.Subsequently,..these cells differentiated into neurons through asymmetric division,which can directly generate one neuron and one NSC,or generate neurons indirectly by generating neuronal progenitor cells(NPCs).After neuronal differentiation,NSCs can differentiate to form astrocytes and oligodendrocytes.Proliferation and differentiation of NSCs,maturation and migration and the resulting tissue growth,stratification and other processes are the basis of the development of CNS.Therefore,the mechanism of self-renewal and differentiation of NSCs has become an important basic scientific issue in the field of NSCs.Melatonin is an indole hormone secreted mainly by pineal gland synthesis and is also synthesized in many peripheral tissues such as skin,retina,gastrointestinal and so on.Melatonin synthesis regulated by the photoperiod,firstly,the optical signal through the retinal hypothalamus fiber afferent,passed to the suprachiasmatic nucleus,after sympathetic nerve fibers through the superior cervical ganglion,dominate the pineal body to secrete melatonin,showing reduced secretion during the day,increased secretion of darkness in the circadian rhythm changes.Melatonin has a broad and important physiological role in living organisms,including the regulation of biological rhythms,improved sleep,anti-oxidation,immune regulation,inhibition of tumors and so on.Most importantly,melatonin is secreted by the brain tissue,and easy to cross the blood-brain barrier,therefore,there is a great deal of melatonin in the brain,melatonin has a protective effect on the pathological changes of the brain caused by stroke,Alzheimer’s disease,Parkinson’s disease and epilepsy.More and more studies have shown that the neuroprotective effect of melatonin may be related to the regulation of NSCs,especially the regulation of the fate of NSCs by melatonin.The differentiation of neural stem cells is highly regulated in time and space by the intracellular and extracellular environment.The mechanism of NSCs differentiation is mainly the regulation of the transcriptional level of certain determinants,whose transcriptional level is activated or inhibited during specific differentiation stages.The bHLH family is a type of transcription factor that plays a decisive role in the differentiation of neural stem cells,the expression of different bHLH factors has the characteristics of space-time and influence each other and regulate each other to form the regulatory network of the development of NSCs.Different bHLH factor expression has different characteristics,they affect each other and regulate each other,together form a regulatory network of the development of NSCs.BHLH transcription factors are divided into two types:one is the inhibitory type,such as Hes;the other is activated,including Neurogenin(Ngn),Mash1,NeuroD1.NSCs will maintain proliferative status when the inhibitory bHLH transcription factor is dominant.When the activated bHLH transcription factor is dominant,it can promote the differentiation of NSCs into neurons.Ngn1 and NeuroD1 play a decisive role in the differentiation of neurons.The mechanism is to form a heterodimer with the E protein,which binds to the E-box sequence(CANNTG)on the target gene sequence,while raising a large number of transcriptional activators to form a large transcriptional activation complex that activates the target gene transcription The Ngn1 plays an important role in the differentiation of neurons,which is a most important target gene,NSCs successfully complete the complex and fine process of differentiation into neurons.BHLH factor-mediated differentiation of neural stem cells is regulated by multiple aspects.More and more studies have shown that epigenetic regulation is a very important regulatory approach.Numerous studies have highlighted the active role of the dynamic structure of chromatin in the regulation of gene expression for the cell fate decisions of NSCs.Nucleosomes composed of a histone octamer and 147 base pairs of DNA are the primary structural units of chromatin.Posttranslational modifications of histones may be one of the most important ways to regulate gene expression.Among the many modifications that have been characterized,histone acetylation is reported to be the one most strongly correlated with transcriptional activation,and it occurs mainly on the N-terminal tails of histones H3 and H4.Acetylation of histone proteins allows for easy access to genomic DNA by relaxing the chromatin architecture and providing docking sites for additional transcriptional regulation factors.This dynamic balance between acetylation/deacetylation status of histones and controls the initiation or closure of gene transcription.We used mouse embryonic NSCs to explore the effect of melatonin on the fate of NSCs and its regulation on the transcriptional level of decisive factor bHLH.It was proved that melatonin activates ERK1/2 signaling pathway through receptor MT1 in epigenetic mechanism,and improves the activity of acetyltransferase(HAT)of CBP/p300,which increases the activity of Ngn1 and NeuroD1 Subunit histone H3lysine 14 acetylation levels,enhanced both transcription,thus promoting the differentiation of neurons.Our study confirms the important role of epigenetic regulation in the fate of NSCs cells,and helps to understand the role and mechanism of melatonin in the nervous system and promote its clinical application.Part 1 The effect of melatonin on the differentiation of neural stem cellsIn order to investigate the role of melatonin in the differentiation of NSCs,the pluripotency,cell cycle and terminal differentiation ratio of NSCs were detected in multiple stages of NSCs differentiation.The results showed that the numbers of nestin-positive cells of pluripotency marker gene in melatonin-treated group were significantly decreased compared with the control group when cells were cultured for 3,5,7 days in differentiation medium,indicating that the number of neural stem cells with multiple differentiation potential were decreased,melatonin may promote the process of NSCs differentiation.The differentiation of neural stem cells,accompanied by cell cycle withdrawal,PHH3 can specifically marker in the M phase of cells,melatonin-treated NSCs PHH3 positive rate decreased significantly,indicating that more cells out of the cell cycle.The RNA level of cyclin-dependent kinase inhibitor p21,p27,were increased significantly,also illustrates this point.The effect of melatonin on the differentiation of NSCs can be further determined by the marker gene gene Tuj1 and the marker gene GFAP of astrocytes.The results showed that the number of neurons increased significantly after melatonin treatment.While the number of astrocytes did not change significantly.Therefore,it was suggested that melatonin could significantly promote the differentiation of NSCs into neurons at the early stage of differentiation,and did not affect the differentiation of NSCs into astrocytes.For a detailed analysis of the molecular mechanisms of melatonin regulation of NSCs differentiation,we examined the transcriptional level of the differentiation fate determinants,the Ngn1,NeuroD1,Hes1 and Hes5 in the bHLH factor.The results showed that the expression of Ngn1 and NeuroD1 was significantly increased.Indicating that melatonin affects the expression of both,and this is likely to be melatonin to promote the differentiation of NSCs the root causes.Hes1 and hes5 are two kinds of inhibitory Bhlh factors,which are highly expressed in the dry state of NSCs.The decrease of both expression is the increase of NSCs differentiation.Mammalian nervous system has two kinds of melatonin receptor MT1,MT2.There was no significant change in the expression of MT1 and MT2 during differentiation.Luzindole is an inhibitor of MT1 and MT2,and 4P-PDOT is a specific inhibitor of MT2.The results showed that MT1 and MT2 were inhibited,melatonin lost the regulation of NSCs differentiation,and the specific inhibition of MT2,melatonin can still promote the differentiation of NSCs to neurons.Thus melatonin regulates NSCs differentiation by receptor MT1.The results of this study show that melatonin can affect the differentiation process of neural stem cells by receptor MT1.After treatment with melatonin,the larger proportion of NSCs is selected to differentiate into neurons,and the pluripotency of cells is decreased,Withdrawal of the cell cycle,Tuj1 and MAP2 and other neuronal marker gene expression increased.But does not affect the progress of glioblast differentiation of NSCs.Melatonin regulates the transcriptional level of bHLH by the above-mentioned biological effects.Melatonin can promote the transcription of Ngn1 and NeuroD1,inhibit the transcription of Hes1 and Hes5.This study first reveals the effect of melatonin on the regulation of bHLH factor in the differentiation of embryonic NSCs,suggesting that melatonin can determine the fate of NSCs by promoting the transcriptional level of Ngn1 and NeuroD1.Part 2 Epigenetic mechanism of regulating neural stem cells by melatoninWith the further research of stem cell,more and more evidence demonstrated that new epigenetic mechanisms were involved in the regulation of stem cell differentiation.Histone acetylation modification is the earliest and most thorough post-translational modification of epigenetic regulation,which is closely related to gene activation.Some studies reported that histone acetylation plays an important role in neurodevelopment,such as the proliferation and differentiation of NSCs,and the process of regulating neurogenesis.Melatonin has been shown to induce significant increases in histone H3 and histone H4 acetylation in mouse hippocampus.Firstly,we examined the changes in the histone acetylation sites associated with gene transcriptional activation,for example,H3K9,H3K14,H4K8,H4K16.Western Blot and immunofluorescence experiments have shown that the level of H3K14 was significantly increased with the treatment of melatonin.No obvious abnormalities were detected in other sites.The changes of H3K14 in Ngn1 and Neurod1 promoter regions were detected by ChIP experiment.The results showed that H3K14 promoter region was significantly increased with the treatment of melatonin,which suggests that melatonin regulates both transcripts by influencing the levels of Ngn1 and Neurod1 promoter regions H3K14.Histone modification is inseparable from the regulation of modified enzymes,histone acetylation modification is regulated by histone acetyltransferase and deacetylase.CBP/p300 is one of the HATs,their amino acid sequence and function were similar,which not noly play a role in histone acetylation,but also can raise the transcriptional activator to a specific gene,regulate gene expression.Both of which play an important role in the development of the nervous system.Although CBP/p300 can catalyze the acetylation of multiple loci,but the highest specificity was H3K14.Firstly,We detected its expression level by WB,and found no significant changes.Then we used the activity test kit of HAT,the results showed that both of their HAT enzyme activity were increased significantly with the treatment of melatonin.To further prove the above conclusions,We used C646 to inhibit HAT activity and found that the acetylation level of H3K14 was significantly decreased with the treatment of C646.It is worth noting that the transcriptional activation of Ngn1 and Neurod1 also disappeared,and the up-regulation of MAP2 and Tuj1 in the downstream also disappeared.This suggests that the histone modification was regulated by melatonin with the CBAT/p300 HAT activity,which further affects NSCs differentiation.We have already shown that melatonin regulates NSCs differentiation by MT1,whereas MT1 activates multiple intracellular signaling pathways,in which ERK1/2 and AKT are two more closely related to cell differentiation,so we first examined these two access path.Respectively,the two antisense inhibitors,PD98059 can inhibit ERK.,LY294002 can inhibit AKT.The results showed that the promotion of the neuronal differentiation by melatonin can be effectively inhibited with the treatment of PD98059,and there was no effect with the treatment of LY294002.It has been reported that ERK1/2 can promote the HAT enzyme activity of CBPp300,so we further detected the changes of CBP/p300 HAT enzyme activity.We found that melatonin treatment can not cause the increase of HAT activity after ERK1/2 pathway inhibition.The results of WB also show that H3K14 acetylation levels can not be affected by melatonin.This suggests that melatonin activates CBP,p300 enzyme activity through the ERK1/2 pathway,which produce a subsequent biological effects.Our study,for the first time,shows that melatonin can specifically enhance histone H3K14 acetylation during NSCs differentiation and further confirms the important role of epigenetic regulation in determining the fate of NSCs.Our study revealed for the first time that melatonin can regulate the chromatin structure in the promoter region and promote the transcription of Ngn1 and NeuroD1,which are the fate determinants of NSCs’ differentiation,and construct a regulatory network in which exogenous signals cooperate with internal transcription factors to regulate the fate of NSCs.Our study will deepen the understanding of the role of melatonin in determining the fate of NSCs and their role in the development of the central nervous system and promote their clinical application. |
PDF Full Text Request