The Study Of The Relationship Between Mitochondrial DNA Methylation And Alzheimer's Disease

Part I Altered mitochondrial DNA methylation and mitochondrial DNA copy numbers in an APP/PS1 transgenic mouse model of Alzheimer disease[Background]Alzheimer's disease(AD)is a chronic neurodegenerative disease characterized by progressive loss of memory and other cognitive deterioration.It's the most prevalent cause of dementia.However,the etiology of AD is not well understood to date.Several studies have reported that mitochondrial dysfunction played an important role in the pathogenesis of AD.Recent studies have shown the existence of mitoepigenetics,which plays an important role in many diseases related to mitochondrial dysfunction.Therefore,some researchers suggested that mitoepigenetics may be a potential pathogenesis of AD.Epigenetics refers to the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence.The major epigenetic modifications include DNA methylation and hydroxymethylation,histone modifications and non-coding RNAs.The DNA methylation plays important roles in regulating gene expression without changing the primary DNA sequence.Over the pase decades,studies on DNA methylation in AD mostly focused on nuclear DNA.The mitochondrial DNA(mtDNA)methylation is a relatively new field compared to nuclear DNA.The presence of methylated nucleotides in the mtDNA has been a quite debatable and controversial in the past 40 years.Several studies showed that mtDNA methylation may play an important role in many pathophysiological processes by regulating mtDNA replication and expression.Recent studies showed that mtDNA methylation is associated with age,environmental factors and many diseases,but there are only two studies in AD.Both of the two studies focused on the mitochondrial D-loop,while other mitochondrial genes are rarely studied.The relationship between mtDNA methylation and AD hasn't been established.Therefore,further investigations are needed to identify the relationship between mtDNA methylation and AD,and to explore the possible mechanism of mtDNA methylation in the pathogenesis of AD.[Objectives]The aim of this study was to analyze the relationship between mtDNA methylation and AD using the amyloid precursor protein/presenilin 1(APP/PS1)transgenic mice,a model of familial AD.We examined mtDNA methylation changes in four specific mtDNA loci by bisulfite pyrosequencing,including the mitochondrial D-loop,12S rRNA gene,Cytochrome b(Cyt b)and Cytochrome oxidase ?(COX ?)genes.We also examined the mitochondrial structure,mtDNA copy numbers and mitochondrial gene expression levels to find the association with mtDNA methylation,add evidence to the functional significance of mtDNA methylation and explore the possible mechanism of mtDNA methylation in AD.[Methods]1.Animals:Male APP/PS1 transgenic mice and wild-type(C57BL/6J)mice were used at 9 months of age.2.The ultrastructures of the hippocampi CA1 of mice were observed by transmission electron microscopy.3.We used ten mice of each group for DNA extraction.MtDNA was extracted from the hippocampus of each mouse.MtDNA methylation changes were detected by bisulfite conversion and pyrosequencing.We choosed four specific mtDNA loci,including the D-loop,the 12S rRNA gene,Cyt b gene and COX ? gene.The methylation percentage at each CpG site was established by the ratio of cytosine-to-thymidine conversion.4.Relative mtDNA copy number of each mouse was measured by the quantitative real-time PCR(qRT-PCR)method,including the mitochondrial 12S rRNA gene,Cyt b gene and COX ? gene.The results were calculated as the ratio of mtDNA/18S rRNA gene copies.5.We used ten mice of each group for RNA extraction.RNA was extracted from the murine hippocampi and reverse transcription was done.We measured the mitochondrial gene expression levels by qRT-PCR.Gene expression was normalized as the amplification levels of three mitochondrial genes against the ?-actin amplification level using the 2-??Ct method[Results]1.Transmission electron microscopy was used to observe the ultrastructure of mitochondria in hippocampi CA1 zone of mice.In the APP/PS1 transgenic mice group,there were many damaged degeneration mitochondria,manifested as swelling of mitochondria,unclear structure and cristae fracture.In the age-matched C57BL/6J mice group,the morphology of mitochondria was normal and the cristae was clear.2.The results of bisulfite PCR combined with pyrosequencing showed that the average methylation levels of different CpG sites were all less than 7%.The percentage of mtDNA methylation represented only a small part of the total mtDNA and there was a differential mtDNA methylation profile in different regions of mtDNA.The mtDNA methylation reduced in the D-loop and increased in the 12S rRNA gene,Cyt b gene and COX II gene in the hippocampi of APP/PS1 transgenic mice compared to age-matched C57BL/6J mice.Until now,we presented the hypermethylation of the mitochondrial 12S rRNA gene,Cyt b gene and COX II gene in AD for the first time.This is also the first study so far to report the hypomethylation of the D-loop in APP/PS1 transgenic mice at 9 months of age.3.The relative mtDNA copy numbers were expressed as the ratios of the mitochondrial 12S rRNA gene,Cyt b gene and COX II gene copies to nuclear 18S rRNA gene copies.The mtDNA copy numbers were significantly decreased in the hippocampi of APP/PS1 transgenic mice compared to age-matched C57BL/6J mice.4.The mitochondrial gene expression levels of the 12S rRNA gene,Cyt b gene and COX II gene were reduced significantly in the hippocampi of APP/PS1 transgenic mice compared to age-matched C57BL/6J mice.[Conclusions]1.The present work showed the presence of mtDNA methylation,which representing only a small part of the total mtDNA.There were aberrant mtDNA methylation changes in APP/PS1 transgenic mice and different methylation profiles in different regions of mtDNA.We conclude that mtDNA methylation may play a key role in AD pathology.2.The APP/PS1 transgenic mice exhibited decreased mtDNA copy numbers and lower gene expression levels compared to age-matched C57BL/6J mice,strengthening the phenomenon of decreased mitochondrial biogenesis and mitochondrial dysfunction.3.We conclude that mtDNA methylation may play an important role in AD pathology by regulating mtDNA copy numbers and the expression levels of mitochondrial genes,which warrants larger further investigations.Part II Mitochondrial DNA methylation in the peripheral blood of patients with Alzheimer disease[Background]In recent years,there are many studies on the relationship between AD and DNA methylation,at either global or gene specific level.But some of the results are conflicting.Several researchers have also detected the DNA methylation in the peripheral blood of AD patients.Those studies have found abnormal DNA methylation changes in the peripheral blood of AD patients,which could serve as biomarkers for the diagnosis of AD.In addition,aberrant DNA methylation of multiple genes in peripheral blood could also be used as potential epigenetic biomarkers for predicting the transformation from amnesia mild cognitive impairment(aMCI)to AD.However,most of these studies are focused on nuclear DNA methylation.Compared with nuclear DNA,mtDNA methylation is a relatively new field.There is growing evidence that mtDNA can be methylated.Since mitochondrial impairment is a key feature of AD,some researchers have proposed that the mitochondrial dysfunction of AD can be partially explained by mtDNA methylation.But,there are only two studies performed so far to address this issue.One of the two studies observed a significant reduction of mitochondrial D-loop methylation in the peripheral blood of late-onset AD(LOAD)patients,suggesting that mtDNA epimutations deserve further investigations in AD pathogenesis.However,the methylation levels of other mtDNA regions haven't been investigated in the peripheral blood of AD patients.Therefore,we designed the second part of the experiment.[Objectives]The aim of this study was to analyze the mtDNA methylation changes in the peripheral blood of AD patients.We measured mtDNA methylation levels of the mitochondrial D-loop and MTRNR1(mitochondrially encoded 12S rRNA)gene by bisulfite pyrosequencing to find evidence for mtDNA methylation as a biomarker of AD diagnosis.[Methods]1.Study population:a total of 24 AD patients and 28 matched controls were collected at the Second Hospital of Shandong University between September 2017 and September 2019.2.Venous blood samples were withdrawn and the DNA from each sample was extracted from the peripheral blood.The DNA were treated with sodium bisulfite in order to convert all unmethylated cytosines into uracil.We utilized bisulfite pyrosequencing to investigate the mtDNA methylation levels,including the mitochondrial D-loop and MTRNR1 gene.3.To determine the predictive probability of mtDNA methylation as an AD biomarker,we performed receiver operating characteristic(ROC)curve analysis.[Results]1.No significant differences were observed in the sex,age and education between AD patients and healthy controls(all P>0.05).There was a significant difference in MMSE score between the two groups(P<0.01).2.The results of bisulfite pyrosequencing showed that the average methylation levels of different CpG sites were no more than 8%.The mtDNA methylation reduced in the D-loop and increased in the MTRNR1 gene in the peripheral blood of AD patients compared to the healthy controls.We presented the hypermethylation of the MTRNR1 gene in the peripheral blood of AD patients for the first time.3.The Spearman correlation analysis results showed that methylation level of CpG site 1(at 61 position of the mtDNA)in the D-loop was positively associated with MMSE score in AD patients(r=0.947,P<0.01).However,no significant correlations were observed in other CpG sites.4.The methylation levels of D-loop at CpG sites 1 and 2 were less accurate for AD diagnosis,while the methylation levels of both D-loop at CpG sites 3,4 and the MTRNR1 gene at CpG sites 1,5 showed moderate prediction accuracy.Among them,the 1st CpG site within the MTRNR1 gene at 886 position of the mtDNA showed the best performance,and achieved a high area under the curve(AUC)of 0.869(95%CI:0.770-0.968)with 83.3%sensitivity and 78.6%specificity at a cut-off of 2.50%.[Conclusions]1.Our data showed that mtDNA methylation represented only a small part of the total mtDNA.The mtDNA showed hypomethylation of the D-loop and hypermethylation of the MTRNR1 gene,which suggested that mtDNA methylation may be implicated in the etiology of AD.2.The methylation level of CpG site 1 in the D-loop at 61 position of mtDNA was positively associated with MMSE score in AD patients.3.The mtDNA methylation,especially the 1st CpG site within the MTRNR1 gene at 886 position of the mtDNA,could serve as non-invasive and potential biomarkers.