Establishment Of APOE4 Genotype-based Induced Pluripotent Stem Cell Model And Related Clinical Research In Alzheimer’s Disease

Alzheimer’s disease(AD)is one of the most common neurodegenerative diseases,which belongs to the familiar type of dementia,characterized by progressive cognitive decline,aphasia,apraxia,executive dysfunction,and behavioral changes.Because of its increasing incidence yearly and irreversibility,it poses a significant burden on the affected society,economy and families.AD is a chronic disease with long incubation period and prodromal period,with an average clinical course of 8-10 years.Because of the early insidiousness of the disease and no effective treatments,early detection and intervention may be helpful for delay the progression of disease and improve symptoms.Early identification and intervention are especially important for patients with mild cognitive impairment(MCI).However,current clinical examination methods are limited,and many clinical features related to the onset of AD have not been fully identified.In terms of basic research,the pathogenesis of AD is unclear,and many classical cell and animal models could not entirely simulate the environment in which AD occurs,causing certain limitations to related mechanisms and drug exploration.Therefore,this study sets about building ADrelated individualized cell models and exploring the characteristics of single nucleotide polymorphisms and resting state EEG related to the pathogenesis of AD in the basic and clinical aspects.Part Ⅰ: The establishment,identification and differentiation of induced pluripotent stem cells based on APOE4 genotype in Alzheimer’s diseaseObjectives: Apolipoprotein E(APOE)genotype is considered to be one of the largest genetic factors of sporadic AD.Many studies have shown that APOE4 is associated with β-amyloid(Aβ)and neurofibrillary tangles(NFTs).Therefore,we plan to establish two induced pluripotent stem cell(iPSC)models with the background of APOE4 genotype for the study of AD pathogenesis and drug screening.Methods: Peripheral blood mononuclear cells(PBMC)were collected from a 66-year-old MCI woman and her healthy daughter,and the genotypes of APOE are both ε4/ε4.PBMC was reprogrammed using nonintegrative Sendai viral vectors containing reprogramming factors OCT4,KLF4,SOX2 and C-MYC.The pluripotency of transgene-free iPSCs was confirmed by immunocytochemistry and ability of differentiation spontaneously into 3 germ layers in vitro.Moreover,the iPSC line displayed a normal karyotype.Conclusions: Our study has successfully established the individual iPSC models based on the APOE4 genotype.iPSCs can be obtained by transducing four genes into PBMC using Sendai virus.The iPSCs model has the characteristics of normal cell morphology and pluripotent differentiation,and might offer a good platform to study the pathological mechanisms and drug testing studies in AD and MCI.Part Ⅱ: Genetic Association of FERMT2,HLA-DRB1,CD2 AP,and PTK2 B Polymorphisms with Alzheimer’s Disease Risk in the Southern Chinese PopulationObjectives: This study aimed to explore the relationship between 18 single nucleotide polymorphisms(SNPs)and AD within the southern Chinese population.Methods: A total of 420 participants,consisting of 215 AD patients and 205 sex-and agematched controls,were recruited.The SNa Pshot technique and polymer chain reaction(PCR)were used to detect the 18 SNPs.Combined with the apolipoprotein E(APOE)ε4 allele and age at onset,we performed an association analysis between these SNPs and AD susceptibility.Furthermore,we analyzed SNP-associated gene expression using the expression quantitative trait loci analysis.Conclusions: Our study identifies the G-allele at rs17125924 as a risk factor for developing AD,especially in APOE ε4 carriers.In addition,we found that rs9271058 of HLA-DRB1,rs9473117 of CD2 AP and rs73223431 of PTK2 B were associated with EOAD.Using the genotype-tissue expression(GTEx)and Braineac database,we found a significant association between rs9271058 genotypes and HLA-DRB1 expression levels,while the CC genotype at rs9473117 and the TT genotype of rs73223431 increased CD2 AP and PTK2 B gene expression,respectively.Further studies with larger sample sizes are needed to confirm our results.Part Ⅲ: Exploring changes of power spectral density and functional connectivity in patients with Alzheimer’s disease and mild cognitive impairmentObjectives: Mild cognitive impairment(MCI)is considered as a prodromal stage of Alzheimer’s disease(AD),but not all patients with MCI will convert to AD.The power spectrum analysis and functional connection changes of the electroencephalogram(EEG)are still controversial in relevant AD researches.We explored changes in the power spectrum and functional connectivity of MCI and AD patients in the Southern Chinese Population,and analyzed whether they have the ability to distinguish the development of MCI.Methods: We respectively enrolled 26 patients in each of the AD,MCI and cognitively normal groups.The 20 scalp electrodes resting state EEG was used to detect the brain waveform of the subjects.The power spectrum value of each band and the weighted phase delay index(w PLI)were used to analyze the differences in EEG changes among the groups.At the same time,the MCI group was divided into two groups according to the follow-up results,and the difference in the characterizations of EEG between the converted MCI(cMCI)and the stable(s MCI)at baseline was identified.Conclusions: In the AD group,the power spectral density of theta band is significantly higher than that in the MCI group and the control group.Compared with the s MCI group,the power spectral density of the cMCI group in the theta frequency band also has similar tendency,indicating that the increase of theta frequency band is altered at the early phase of disease.In addition,the differences in functional connectivity among the three groups are mainly in the lower frequency bands.The phase synchronization of AD patients in delta frequency band is the lowest among three groups,while the phase synchronization of AD patients in theta frequency band is significantly stronger than that of MCI group and control group.In s MCI and cMCI groups,w PLI-related functional connectivity analysis contributes to identifying the progression of AD.