The Mechanism Of Homocysteine-mediated DNA 6mA Methylation On Bdnf Transcription In Mice And Its Role In Stress Cognitive Impairment

As a systemic range of nonspecific responses in the body,stress occurs when stimulated by various internal and external environmental factors.Long-term or high-intensity stress has a clear negative effect on cognitive function in animals.Brain derived neurotrophic factor(BDNF)is an important molecule in the maintenance of synaptic structure and cognitive function development,and its inhibition by stress may be an important factor in the development of cognitive impairment due to stress,but its mechanism is not fully understood.Homocysteine(Hcy)is a small amino acid,previous studies have found that Hcy levels increase during stress.In addition to the recognized role of Hcy in inducing cardiovascular damage,recent studies have found that it is also involved in the development of cognitive impairment.However,the mechanism is not fully understood.In this study,we systematically observed the inhibitory effect of Hcy on hippocampal Bdnf during the occurrence of stress-related cognitive impairment.In addition,from the perspective of DNA 6mA methylation modification,we revealed the transcriptional regulation mechanism of Hcy on Bdnf,thus providing a new theoretical basis for elucidating the molecular mechanism of stress-induced cognitive impairment occurrence.This study also provides important ideas for controlling medical measures and intervention targets for the prevention and treatment of stress-related cognitive impairment.Ⅰ.Study of the role of homocysteine in the cognitive impairment of mice caused by chronic stressIn order to investigate the effect of stress on cognitive function in mice,male C57BL/6 mice from 15 to 25 g were selected for stress intervention using chronic unpredictable mild stress(CUMS)model from 0 to 8 weeks.The behavioral and biochemical indexes of CUMS mice and control group(Ctrl)were examined at the time points of 0,2,4,6 and 8 week.The behavioral and biochemical parameters were measured at the end of0,2,4,6 and 8 week for CUMS mice and control group(Ctrl).The animal models were evaluated by sucrose preference test.Spatial learning memory and emotional and autonomous activities were elevated by Morris water maze test and open-field test.Hippocampal Bdnf,Psd95 and Syp transcription and protein expression were measured by real-time fluorescence PCR(q-PCR)and western blotting(WB).Hippocampal BDNF distribution and neuronal dendritic spine density were measured by immunofluorescence(IF)and Golgi staining.Plasma Hcy level were measured by enzyme cycling method.The sugarwater preference experiment and body weight showed that the CUMS model was successfully established.Results showed that the plasma Hcy level increased significantly after 6 weeks of stress,meanwhile,cognitive function,Bdnf transcription and expression in the hippocampus,transcription and expression of synaptic function genes such as Psd95 and Syp and dendritic spine density decreased significantly.Moerover,Hcy was significantly associated with cognitive function and hippocampal Hcy and negatively correlated with cognitive function and hippocampal Bdnf level.To investigate the role of Hcy in the stress process,male C57BL/6 mice from 15 to25 g were selected for the stress mouse model intervention using 8-week CUMS.The down-and up-regulation of Hcy was achieved by B-complex vitamins gavage based on CUMS(CUMS+VBco)and given high methionine diet(Met diet).The results showed that Hcy was well regulated by the method above,and the hippocampal Bdnf level was significantly increased after down-regulation of Hcy based on CUMS,the transcription and protein levels of synaptic function genes increased,the dendritic spine density increased and the cognitive impairment caused by CUMS significantly improved in mice.In contrast,upregulation of Hcy decreased hippocampal Bdnf levels,significantly decreased synaptic function gene and protein levels,and significantly decreased dendritic spine density,inducing the appearance of cognitive impairment.HT22 cells were intervened using concentrations of 0,50,150,200,and 250 μM Hcy.Biochemical indexes were detected using the above method 72 hours after the intervention.Results showed that the level of Bdnf,which is widely present in the cell cytoplasm,decreased and the level of synaptic function genes and proteins decreased as the concentration of Hcy increased during the intervention of HT22 cells from 0 to 150 μM Hcy.Ⅱ .Effect of homocysteine on DNA 6mA modification during chronic stressTo clarify the alteration pattern of DNA 6mA modification in the hippocampal genome of mice during stress,animals mentioned above were selected for stress intervention using CUMS from 0 to 8 weeks,and biochemical indexes were examined in CUMS mice and their Ctrl groups at the time points of 0,2,4,6 and 8 week,respectively.The distribution and level of DNA 6mA modification in hippocampal genome were detected by dot blot assay(DB),DpnⅠ enzyme digestion assay and 6m A immunofluorescence staining.Results showed that DNA 6mA was widely present in the nuclei of CA1,CA3 and the granular cell layer in the dentate gyrus of the hippocampus,and the degree of DNA 6mA modification decreased with the increase of stress duration.To further investigate whether Hcy is involved in DNA 6mA modification during stress,mice in the 8-week CUMS intervention group,CUMS+VBco group,Met diet group and HT22 cells with Hcy intervention were used to detect genomic DNA 6mA modification using methods mentioned above.Results showed that the DNA 6mA methylation level in the hippocampal genome was significantly increased after down-regulation of Hcy,while the DNA6 m A modification level was significantly decreased after up-regulation of Hcy.The cellular genome also increased after Hcy intervention in HT22 cells.Further,to investigate the alteration pattern of DNA 6mA methylation in the Bdnf promoter during stress and the role of Hcy in this process,animal and cellular models mentioned above were selected,and DNA 6mA levels in the Bdnf promoter were detected by methylation dependent restriction endonucleases(MDRE)and 6m A-Methylation immunoprecipitation(6m A-Me DIP).Results showed that there was a ’GATC’ site in the Bdnf promoter where 6m A modification potentially occurred.The CUMS and Met diet groups with higher Hcy levels had significantly lower Bdnf promoter IV 6m A methylation modification,while the CUMS+VBco group had significantly lower methylation modification and was close to the level of the Ctrl group.In order to clarify the reasons for the alteration of DNA 6mA methylation modification caused by Hcy during stress,transcription and protein expression of methyltransferase N6amt1 and transmethylase Alkbh1 in hippocampus and cells were detected by qPCR and WB.Results showed that N6amt1 was not significantly altered in any group.The level of Alkbh1 increased significantly with the increase of stress time to 6 weeks,in addition,the level of Alkbh1 increased significantly in the Met diet group,and the level of Alkbh1 decreased significantly in the CUMS+VBco group compared with CUMS,which was close to the level of Ctrl group.Alkbh1 level decreased with the increase of Hcy concentration during the intervention of 0~150 μM Hcy in HT22 cells.Morever,this study also focused on the altered DNA 5m C methylation modification in the hippocampal genome under stress conditions.Results showed that stress led to a decrease in DNA 5m C methylation modification in the hippocampal genome,and this alteration trend was the same as the altered DNA 6mA modification.Ⅲ.Mechanistic study of DNA 6mA regulation of Bdnf transcriptionPrevious study found that elevated Hcy during cognitive impairment caused by CUMS leads to lower Bdnf levels,while Alkbh1 is elevated and genomic and Bdnf promoter DNA 6mA methylation modifications are reduced.To further investigate whether DNA 6mA affects Bdnf transcription and expression,HT22 cells and 15~25g male C57BL/6 mice were used to detect the effect of interference after infection with viral vectors interfering with Alkbh1 in the hippocampus,respectively,after administration of150μM Hcy and 8 weeks of CUMS intervention by the above method.Cellular results showed that interfering with Alkbh1 significantly increased the genomic and Bdnf promoter 6m A hypomethylation status caused by Hcy,animal results showed that interfering with Alkbh1 significantly increased the hippocampal genomic and Bdnf promoter 6m A hypomethylation status caused by CUMS without affecting plasma Hcy conditions,which is close to that of the Ctrl group.To further reveal the effect of DNA 6mA intervention by interfering with Alkbh1 on Bdnf transcript levels in CUMS mice and Hcy-interfered HT22 cell,WB was used to detect transcription factors YY1 and TFIIβ levels in hippocampus and HT22 cells,and Ch IP was used to detect the binding of Bdnf promoter to transcription factors YY1 and TFIIβ ability.Results showed that DNA 6mA had no significant effect on the expression of YY1 and TFⅡβ in the hippocampus of CUMS mice and Hcy-intervened cells,but significantly increased the binding level of Bdnf promoter to the above two transcription factors.Meanwhile,Bdnf level significantly increased in the hippocampus of CUMS mice and HT22 cells of Hcy-intervened cells,and the expression levels of synaptic function genes and proteins were significantly increased.CUMS mice showed a significant increase in dendritic spine density and improved cognitive function.In addition,animal and cellular level experiments showed that Bdnf levels in hippocampus and HT22 cells were significantly reduced after interfering with Yy1 and Tf IIβ.Since the regulation of DNA 6mA modification by interfering with Alkbh1 was nomspecific,the normal and the Bdnf promoter sequence with DNA 6mA modification were further constructed.DNA-immunoprecipitation(DNA-IP),6m A-electrophoretic mobility shift assay(6m A-EMSA)and in vitro transcription assay were used to evaluate the combination of promoter and the transcription factors.Results showed that the promoter DNA 6mA modification had a significant impact on the binding ability,binding efficiency and in vitro transcription ability of transcription factors.In conclusion,elevated Hcy during chronic stress may inhibit hippocampal Bdnf promoter DNA 6mA modification through high levels of Alkbh1,and further lead to a decrease in its binding capacity and efficiency to important transcription factors such as YY1 and TFIIβ,and in turn inhibits Bdnf transcription and protein expression,ultimately leading to a decrease in synaptic function protein levels and dendritic spine density and inducing cognitive impairment.This study provides new epigenetic evidence to uncover the mechanism of stress-induced cognitive impairment and provides a new idea to prevent and treat stress injury.