Study On The Role Of N6-methyladenosine In The Pathogenesis Of SCA3/MJD

Background:Spinocerebellar ataxia type 3(SCA3/MJD)is a kind of hereditary neurodegenerative disease caused by abnormal expansion of CAG repeat in the exon 10 of the ATXN3 gene.The core pathogenic process of the disease is the aggregation of mutant ataxin-3 protein containing abnormally expanded polyglutamine(poly Q)fragment,which forms an intranuclear inclusion body in the cell and causes selective injury and death of neurons.At present,the pathogenesis of SCA3/MJD is not clear.The recognized hypotheses include aggregation of toxic protein fragments,disorder of autophagy pathway,and so on.Epigenetic modification also participates in the pathogenesis of SCA3/MJD.N6-methyladenosine(m6A)is the most common RNA modification in mammals.The abundance of m6 A is high in the nervous system.The epigenetic modification regulation mechanism of m6 A has been proven to play an important role in a variety of neurodegenerative diseases,including Parkinson’s disease(PD),Alzheimer’s disease(AD)and Huntington’s disease(HD).However,there is still a lack of research on the role and mechanism of m6 A methylation modification in the pathogenesis of SCA3/MJD.Objective:1.To explore the change of m6 A methylation level and the m RNA expression of m6 A methylation related molecules in SCA3/MJD patients and its relationship with disease severity;2.To explore the possible role and mechanism of m6 A methylation modification in SCA3/MJD.Methods:1.Overall m6 A level in the peripheral blood were detected in 44 healthy control(HC),13 SCA3/MJD pre-ataxic stage patients and 134 ataxic stage patients,and the m RNA expression of m6 A methylation related molecules were detected in 24 HC,11 pre-ataxic stage patients and 24 ataxic stage patients.Differentia analysis among the three group and correlation analysis between the overall m6 A level together with m RNA expression of m6 A methylation related molecules and clinical characteristics were conducted.2.We constructed two kinds of SCA3/MJD disease cell models,293T-c DNA-84 Q and 293T-CDS-84 Q,by constructing and transfecting plasmids containing c DNA and CDS sequence of the ATXN3 gene into HEK-293 T cells.The overall m6 A level together with the expression of m6 A methylation related molecules was detected in the cell model.We further inhibited the m6 A methyltransferase METTL3 and the m6 A demethylase ALKBH5,and observed the effect of the overall m6 A level on the gene expression of ATXN3 change in the cell model.3.Three 6-month-old and three 18-month-old MJD84.2 transgenic model mice together with three wild-type(wt)mice of the same ages were selected.M6 A immunoprecipitation sequencing(Me RIP-seq)and RNA sequencing(RNA-seq)were conducted on the cerebellum of selected mice,and the functional enrichment analysis of differential methylation genes and differential expression genes were performed respectively.Weighted gene co-expression network analysis(WGCNA)was used for combined analysis of Me RIP-seq data and RNA-seq data,to explore the potential target genes with changes of both m6 A level and expression level.Functional enrichment and primary validation were carried out to explore the possible mechanism of changes in the m6 A level participating in the pathogenesis of SCA3/MJD.Results:1.In the overall m6 A detection,the difference analysis found that the overall m6 A level in the peripheral blood of SCA3/MJD ataxic stage patients was significantly higher than that of HC(HC:0.0137±0.0060%,pre-ataxic:0.0134±0.0048%,ataxic:0.0160 ±0.0050%;p=0.038)(p=0.042).Correlation analysis found that the overall m6 A level of SCA3/MJD ataxic patients was negatively correlated with disease duration and Scale for Assessment and Rating of Ataxia(SARA)(disease duration,r=-0.243,p=0.006;SARA,r=-0.256,p=0.004).After adjustment for gender,age and expanded CAG repeats of ATXN3,the overall m6 A level was significantly related to the age of onset(AO),disease duration,SARA and the scores of International Cooperative Ataxia Rating Scale(ICARS)(AO,r=0.194,p=0.036;disease duration,r=-0.198,p=0.032;SARA,r=-0.351,p=0.000;ICARS,r=-0.254,p=0.006).In the m RNA expression level of m6 A methylation related molecules detection,the m RNA expression of YTHDF2 was significantly increased in ataxic stage patients(HC: 1.00±0.40,pre-ataxic stage patients: 1.43±1.71,ataxic stage patients:1.89±1.29,p=0.003).Correlation analysis showed that the m RNA expression of YTHDF2 was significantly related to disease duration(r=0.562,p=0.004).After adjustment for gender,age and expanded CAG repeats of ATXN3,the m RNA expression of YTHDF2 was significantly correlated with AO,disease duration,SARA and ICARS scores(AO: r=-0.458,p=0.042,disease duration: r=0.458,p=0.042,SARA: r=0.476,p=0.034,ICARS: r=0.595,p=0.006).2.The m RNA expression of IGF2BP2 and IGF2BP3 was increased(p<0.05),and the protein expression of YTHDF2 was increased(p<0.01)in the 293T-c DAN-84 Q cell.After the intervention on METTL3,the overall m6 A level of 293T-c DNA-84 Q cells decreased,the expression of mutant ataxin-3 protein increased,and the cell viability decreased.After the intervention on ALKBH5,the overall m6 A level of 293T-c DNA-84 Q cells increased,the expression of mutant ataxin-3 protein decreased,and the cell viability increased.After knocking down YTHDF2,it was found that there was no significant change in the m6 A level but the expression of mutant ataxin-3 protein increased.However,there was no significant change in the expression of normal ataxin-3 protein in 293T-c DNA-20 Q cells.After knocking down YTHDF2 together with METTL3 or ALKBH5,it was found that the knock-down of YTHDF2 did not affect the changes in m6 A level mediated by the knock-down of METTL3 or ALKBH5,but could offset the changes in the expression of mutant ataxin-3 protein in the 293T-c DNA-84 Q cell model.When interfering with 293T-CDS-84 Q cells by inhibiting METTL3,the overall m6 A level of the cells decreased,but no change in the expression of mutant ataxin-3 protein was observed.3.In the MJD84.2 mouse model,the differentially methylated genes in both 6-month-old and 18-month-old mice were enriched in the biological processes such as Positive regulation of transcription,Signal transduction,Nervous system development,Multicellular organism development,and Cell differentiation,as well as the signal pathway of Neuroactive ligand-receptor interaction.The differentially expressed genes in 6-month-old mice were mainly enriched in the Cholinergic synapse signal transduction pathway through KEGG analysis,while differentially expressed genes in 18-month-old mice were mainly enriched in the Neuroactive ligand-receptor interactive pathway.In the combined analysis,compared with wt mice at the same age,MJD84.2mice at the age of 6 months had a total of 71 significantly differential genes,and MJD84.2 mice at the age of 18 months had a total of 171 significantly differential genes.The functional enrichment analysis found that the differential genes of MJD84.2 mice at the age of 6 months and 18 months enriched in the Neuroactive ligand-receptor interaction pathway and the TGF-β signal pathway.After screening and validation by mouse and cell models,it was found that the m RNA expressions of ADRB1 and GABRA4 genes were downregulated in SCA3/MJD,while the m RNA expression of BMP2 was upregulated.The m RNA expression of ADRB1 and BMP2 genes changed under different m6 A levels.Conclusions:1.The overall m6 A level in the peripheral blood of SCA3/MJD ataxic stage patients was higher than that of the healthy control,but it was significantly negatively correlated with the severity of the disease,suggesting that the overall m6 A level can reflect the severity of the disease to a certain extent.2.The m RNA expression of YTHDF2 in the peripheral blood of SCA3/MJD ataxic stage patients increased significantly compared to the healthy control,and positively correlated with the severity of the disease,suggesting that the m RNA expression of YTHDF2 is a potential biomarker to reflect the severity of the disease.3.In the SCA3/MJD cell model,m6 A methylation acted on the regulatory region and specifically negatively regulated the expression of ATXN3 gene with abnormal CAG repeat expansion via a YTHDF2-dependent manner.4.The change of m6 A methylation of related genes in Neuroactive ligand-receptor interaction pathway(ADRB1,GABRA4)and TGF-βsignal pathway(BMP2)may participate in the pathogenesis of SCA3/MJD.