CRISPR/dCas9-Dnmt3a-mediated targeted DNA methylation of APP rescues brain pathology in a mouse model of Alzheimer’s disease

Background:Aberrant DNA methylation patterns have been observed in neurodegenerative diseases,including Alz-heimer’s disease(AD),and dynamic changes in DNA methylation are closely associated with the onset and progres-sion of these diseases.Particularly,hypomethylation of the amyloid precursor protein gene(APP)has been reported in patients with AD.Methods:In this study,we used catalytically inactivated Cas9(dCas9)fused with Dnmt3a for targeted DNA methyla-tion of APP,and showed that the CRISPR/dCas9-Dnmt3a-mediated DNA methylation system could efficiently induce targeted DNA methylation of APP both in vivo and in vitro.Results:We hypothesized that the targeted methylation of the APP promoter might rescue AD-related neuronal cell death by reducing APP mRNA expression.The cultured APP-KI mouse primary neurons exhibited an altered DNA-methylation pattern on the APP promoter after dCas9-Dnmt3a treatment.Likewise,the APP mRNA level was significantly reduced in the dCas9-Dnmt3a-treated wild-type and APP-KI mouse primary neurons.We also observed decreased amyloid-beta(Aβ)peptide level and Aβ42/40 ratio in the dCas9-Dnmt3a-treated APP-KI mouse neurons compared to the control APP-KI mouse neurons.In addition,neuronal cell death was significantly decreased in the dCas9-Dnmt3a-treated APP-KI mouse neurons.Furthermore,the in vivo methylation of APP in the brain via dCas9-Dnmt3a treatment altered Aβplaques and attenuated cognitive and behavioral impairments in the APP-KI mouse model.Conclusions:These results suggest that the targeted methylation of APP via dCas9-Dnmt3a treatment can be a potential therapeutic strategy for AD.

APOE ε4-dependent effects on the early amyloid pathology in induced neurons of patients with Alzheimer’s disease

Background: The ε4 allele of apolipoprotein E (APOE ε4) is the strongest known genetic risk factor for late-onset Alzheimer’s disease (AD), associated with amyloid pathogenesis. However, it is not clear how APOE ε4 accelerates amyloid-beta (Aβ) deposition during the seeding stage of amyloid development in AD patient neurons. Methods: AD patient induced neurons (iNs) with an APOE ε4 inducible system were prepared from skin fibroblasts of AD patients. Transcriptome analysis was performed using RNA isolated from the AD patient iNs expressing APOE ε4 at amyloid-seeding and amyloid-aggregation stages. Knockdown of IGFBP3 was applied in the iNs to investigate the role of IGFBP3 in the APOE ε4-mediated amyloidosis. Results: We optimized amyloid seeding stage in the iNs of AD patients that transiently expressed APOE ε4. Remarka-bly, we demonstrated that Aβ pathology was aggravated by the induction of APOE ε4 gene expression at the amyloid early-seeding stage in the iNs of AD patients. Moreover, transcriptome analysis in the early-seeding stage revealed that IGFBP3 was functionally important in the molecular pathology of APOE ε4-associated AD. Conclusions: Our findings suggest that the presence of APOE ε4 at the early Aβ-seeding stage in patient iNs is critical for aggravation of sporadic AD pathology. These results provide insights into the importance of APOE ε4 expression for the progression and pathogenesis of sporadic AD.