| BackgroundRecent studies have shown that DNA N6-methyladenine(N6-m A)modification,an essential epigenetic regulatory modality,is abundant in the mammalian genome.The adenine methylation in genomic DNA can coordinate with histone methylation to alter chromatin accessibility.The methylation level of genomic DNA in organisms changes dynamically during growth,development,and aging.This dynamic change allows the expression of genes to change dynamically along the timeline,thus determining the cell’s fate.ALKBH1,a homolog of the Alk B dioxygenase family,has been shown to act as a DNA N6-m A demethylase in mammals.Genomic DNA N6-m A levels are regulated bidirectionally by the methyltransferase N6AMT1 and the demethylase ALKBH1.It has also been shown that in mouse brain cells or human glioblastoma cells,decreased N6-m A levels or elevated ALKBH1 expression can lead to upregulation of neuronal development-related gene levels,thus affecting neuronal development as well as proliferation,migration,and invasion of neural tumor cells.It is well known that mature mammalian neuronal cells lose the ability to divide and regenerate.After the injury,neuron axon regeneration and lengthening mechanisms must be initiated to generate new synaptic connections to restore normal neural function.Thus,the ability of neuronal axon regeneration is crucial in nerve injury repair.Neuronal cells in the embryonic period have a solid ability to divide and regenerate,which is related to their different gene expression levels.Through epigenetic regulation of genomic DNA and transcriptomic m RNA,transforming mature neurons,which have lost the intrinsic regenerative capacity,from the "mature state" to the "developmental state," thus having a robust axonal regeneration capacity,has become a popular research direction in neural injury and regeneration.ObjectivesThis research project explores whether DNA N6-m A is present in the genome of mature mammalian neurons and whether ALKBH1 regulates the N6-m A methylation level of mature neuronal genomic DNA.On this basis,we observed the effect of ALKBH1 on the axonal regeneration ability of mature neuronal cells through the regulation of ALKBH1 expression.Further,we analyzed the molecular mechanism of ALKBH1 affecting the axonal regeneration ability of neurons by altering the genomic DNA N6-m A methylation level,revealing a new direction of regulating axonal regeneration ability.MethodsIn this research project,we confirmed the existence of DNA N6-m A in mammalian neurons by dot blotting genomic DNA in different cells.Besides,the methylation level is regulated by the demethylase ALKBH1 and methyltransferase N6AMT1.With the RNAi approach,ALKBH1 and N6AMT1 on axon regeneration were observed by in vitro DRG sensory neuron culture and electroporation transfection and in vivo DRG electroporation transfection of the corresponding si RNAs.Furthermore,GO analysis was performed using the RNA-seq database of glioblastoma(GSE16089)in GEO to explore the difference in gene expression after ALKBH1 knockout.We selected some neural differentiation regulatory genes(GO: 0045664)as target genes.With quantitative real-time PCR,the expressions of these genes under different conditions(control group,ALKBH1 knockout group,N6AMT1 knockout group,and ALKBH1+N6AMT1 co-knockout group)were detected to investigate the effects of genomic DNA N6-m A levels changes on the expression of genes that related to neural development,revealing the mechanism of ALKBH1 regulating the axonal regeneration ability of mature neurons.Results(1)ALKBH1 regulates N6-m A DNA methylation in mammalian sensory neurons: Dot blotting of genomic DNA from different cells showed that DNA N6-m A exists in mammalian neurons and can be detected by N6-m A specific antibodies,and the intensity of its signal positively correlates with the concentration of N6-m A.Knockdown of the demethylase ALKBH1 caused a significant increase in N6-m A levels(p < 0.01),and knockdown of the methyltransferase N6AMT1 caused no significant change in N6-m A level compared to the control group.However,coknockdown of ALKBH1 and N6AMT1 reduced the N6-m A level more than the knockdown of ALKBH1 alone(p < 0.05),but no significant difference was observed in the control group.(2)ALKBH1 is necessary for sensory neuron axon regeneration in vitro: By isolating and culturing sensory neurons from adult mouse DRG in vitro and transfecting them with electroporation,combined with re-plating,we observed that knockdown of ALKBH1 significantly impaired the ability of axon regeneration(p < 0.0001),whereas knockdown of N6AMT1 showed no noticeable change in the axon regeneration,but after knocking down both ALKBH1 and N6AMT1,the axon regeneration ability was restored and showed no difference from the control group.Moreover,the number of neurons with axons showed no difference among the four conditions(si Non-Target,si ALKBH1,si N6AMT1,si ALKBH1 + si N6AMT1).(3)ALKBH1 is necessary for rapid axon regeneration in sensory neurons in vivo: By using quantitative real-time PCR,we detected that after peripheral nerve injury,the level of ALKBH1 m RNA in neurons was significantly increased at 1 day and returned to normal by 3 days.Based on this result,we further used in vivo electroporation transfection of mouse DRG and RNAi technology to knock down ALKBH1 and observed the axonal regeneration ability at different time points after sciatic nerve injury.The results showed that the knockdown of ALKBH1 did not affect axon regeneration ability before the axons entered a period of rapid regeneration(at 2days).However,the knockdown of ALKBH1 caused a significant effect(p < 0.0001)when axons entered a period of rapid regeneration(at 3 days),and this effect persisted until the m RNA of ALKBH1 returned to normal(at 4 days)(p < 0.05).At the same time,the knockdown of N6AMT1 did not affect the axon regeneration ability either before or during the fast regeneration phase.However,when both N6AMT1 and ALKBH1 were knocked down,the axon regeneration ability at the fast regeneration phase could be found to be rescued(p < 0.0001).(4)ALKBH1 downregulation leads to altered expression of neurodevelopmentrelated genes: it can be concluded by GO analysis of RNA SEQ data(GSE117632)of human glioblastoma in the public database GEO those genes with 2-fold and above downregulation after knockdown of ALKBH1,predominantly neuronal differentiation regulatory genes(GO: 0045664).Further,we selected some of these genes for quantitative real-time PCR in mouse DRG neurons.We observed that five of the m RNA levels(EFNA1,Id1,Nrn1,ATG9 B,and C1QL4)were significantly reduced.Moreover,none gene was detected to be statistically significantly upregulated.Several of these 5genes(Nrn1,Id1)have been shown to regulate axon growth or regeneration.However,when both N6AMT1 and ALKBH1 were knocked down,the levels of four genes were significantly affected based on one-way ANOVA analysis,in which the expressions of C1QL4 and Id1 were significantly reversed.Conclusions(1)The DNA N6-m A modification is present in the mammalian peripheral nerve system,and the demethylase ALKBH1 and the methyltransferase N6AMT1 regulate its level.The Knockdown of ALKBH1 increased the DNA N6-m A level,whereas the knockdown of N6AMT1 did not reduce the effect of the DNA N6-m A level.Simultaneous knockdown of N6AMT1 and ALKBH1 normalizes DNA N6-m A levels.(2)Knockdown of ALKBH1 in primary sensory neurons extracted from mouse DRGs inhibited axon regeneration ability,whereas knockdown of the methyltransferase N6AMT1 did not promote axon regeneration.However,the simultaneous knockdown of N6AMT1 and ALKBH1 restores regenerating ability.(3)Direct knockdown of ALKBH1 in mouse DRGs allows the observation that the rapid regenerative capacity of axons in the sciatic nerve is inhibited.Likewise,direct knockdown of N6AMT1 did not promote the ability of axons to regenerate rapidly in the sciatic nerve.However,the simultaneous knockdown of N6AMT1 and ALKBH1 restores the ability of axons to regenerate rapidly in the sciatic nerve.(4)Knockdown of ALKBH1 resulted in significant downregulation of multiple neurodevelopment-related genes(e.g.,Id1,Nrn1).It suggests that ALKBH1 may support sensory neuron axon regeneration by reducing the DNA N6-m A levels and subsequently upregulating these neuro-developmental associated genes.SignificanceThis study confirms that N6-m A is present in adult mouse DRG neurons and is dynamically regulated by the demethylase ALKBH1 and the methyltransferase N6AMT1.Elevated levels of genomic DNA methylation due to ALKBH1 knockdown further reduce the expression of downstream genes involved in promoting neural development and regeneration,thereby affecting the ability of neuronal axons to regenerate.This study provides a new direction to study the ability of neuronal axon regeneration. |
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