The Disquisition Of Screening DNA Methylation Profiling At The Midterm Stage Of Neurodegeneration In An AD Model In Mouse With Presenilin-1/Presenilin-2 Conditional Double Knockout

Objective:Alzheimer’s diseases(AD)a kind of degenerative neurodegenerative disorder,which is mainly exhibited as the progressive cognitive impairment and memory deficit.Higher prevalence,is the most common type of dementia.One of the most pathological changes in the brain of Alzheimer`s disease(AD)is neuronal loss,cortical and hippocampal atrophy typical of senile plaques(senileplaques,SP),amyloid(β-amyloidprotein,Aβ)deposition,neurofibrillary tangles(neurofibrillary tangles,NFT)and hyperpho sphorylation of Tau protein and so on.The clinical symptoms include gradual memory loss appear,cognitive dysfunction,personality changes and late organism dysfunction,disease progress gradually deteriorate,severe cases can involve life.Unfortunately,however,In nervous system disorders,such as Alzheimer’s disease(AD),it has not been such a breakthrough,but there are also some important discoveries.Over the past more emphasis on the study of the DNA sequence itself,and the incidence of some diseases and the emergence of the phenotype and not just changes in DNA sequences.Researchers had three pairs of identical twins familial Alzheimer’s disease(FAD)patients,and found that even though the two twins have identical genomes,age of onset,clinical symptoms are not exactly the same.This shows that in the case of the nucleotide sequence of the corresponding gene has not changed,a number of organisms phenotype was changed.In recent years,a large number of studies have shown that DNA methylation changes are closely correlated with AD,and is closely related genes such as amyloid precursor protein gene(APP),apolipoprotein E(Apo E-4),PS-1gene(Psen-1)and PS-2 gene(Psen-2)have occurred in abnormal methylation changes.Therefore,DNA methylation is a core component of the epigenetic machinery that regulates gene transcription and plays a vital role in normal cellular function by changing chromatin structure,DNA conformation,DNA stability DNA-protein interactions and methods to control gene expression status.The preliminary study successfully keep and breed early DKO mice model of Alzheimer’s disease;obtaining 30 abnormal gene methylation.Well,it is not there more of aberrant methylation of genes involved in the pathogenesis of AD as well? In order to investigate whether there are some other abnormal methylation of genes involved in the pathogenesis of AD,we have reproduced and identified double knockout of Alzheimer’s presenilin-1 and presenilin-2Knockout Mice(DKO mice)that meet the requirement of the animal model of AD,exhibiting neurodegeneration like AD,such as atrophy of cortex,neuronal apoptosis,tauprotein hyperphosphorylation and cognitive disorders.Then,detect and analyze the DNA methylation changes in the hippocampus of mice at the midium disease stage,expecting to obtain the abnormal methylation of genes.Methods: The wild-type mice and DKO mice were bred and reproduced respectively.And the DKO mice were produced by crossing the heterozygous offspring.The genomic DNA was extracted from the tail tissue of each genotype mice and detected by using PCR method and Agarose Gel Electrophoresis.The experimental group selected d KO mice with 12 month age,the same age of wild-type mice served as control.Extract the genomic DNA from the hippocampus of mice,and use Reduced Representation Bisulphite Sequencing(RRBS)to detect the distribution and pattern of genomic DNA methylation,and then use related biological software to analyze the data in order to obtain the abnormal methylation of the genes.Results: In this study,we have succeeded in breeding and reproducing d KO mice of AD mouse model.By using Bismark(v O.7.4)software to compare with the reference genome sequence of mouse,and using DAVID online site to analyze genes GO and KEGG.We identified 127 genes with abnormal methylated regions(DMR),3025 aberrant methylated sites were screened,which involving 2758 genes,and they distribute in different chromosomes.Conclusion: The correct breeding and identification methods are to obtain PS1/PS2 double knockout mice’s effective way.In this study,we obtained the global informationof aberrant methylated genes of d KO mice by using RRBS,which provides important basis for establishing genomic DNA methylation map and getting aberrant methylated genes,as well as clues that may be used in the medth prevention,diagnosis and treatment of Alzheimer’s disease.