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

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Translational Neurodegeneration 2022年第1期11卷 331-342页

作者： Hanseul Park Jaein Shin Yunkyung Kim Takashi Saito takaomi C.saido Jongpil Kim Laboratory of Stem Cells and Gene Editing Department of Chemistry&Biomedical EngineeringDongguk UniversitySeoul 100‐715Republic of Korea Laboratory for Proteolytic Neuroscience RIKEN Center for Brain ScienceWako-shiSaitamaJapan Department of Neurocognitive Science Institute of Brain ScienceNagoya City University Graduate School of Medical SciencesNagoyaAichiJapan Institute for Cellular Rebooting Dongguk UniversitySeoulKorea

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.

关键词： Alzheimer’s disease APP dCas9 Dnmt3a Methylation editing

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Impairment of autophagic signaling in murine forebrain disrupts GABAA receptor trafficking leading to excitatoryinhibitory imbalance and ASD-like behaviours

Impairment of autophagic signaling in murine forebrain disru...

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中国神经科学学会第十二届全国学术会议

作者： Kelvin Hui Noriko Takashima Hiroshi Matsukawa Akiko Watanabe Per Nilsson Ryo Endo takaomi saido Shigeyoshi Itohara Takeo Yoshikawa Motomasa Tanaka Laboratory for Protein Conformation Diseases RIKEN Brain Science Institute Laboratory for Behavioral Genetics RIKEN Brain Science Institute Laboratory for Molecular Psychiatry RIKEN Brain Science Institute Laboroatory for Proteolytic Neuroscience RIKEN Brain Science Institute Department of Neurobiology Care Sciences and Society Karolinska Institutet

Many syndromic forms of autism spectrum disorder(ASD) are caused by mutations in proteins such as PTEN, TSC1, TSC2, and FMRP, which lie within the m TOR signaling pathway. While recent work has primarily focused on the dysregulation of protein translation via the deletion of these genes, few studies have examined how disruption of autophagy, another major downstream branch of m TOR signaling,contributes to neuronal dysfunction and ASD pathogenesis. To this end, we and others have previously examined mice deficient for autophagy in forebrain excitatory neurons via the deletion of Atg7 by Ca MKII-cre and observed ASD-like behavioural abnormalities in addition to neuronal dysfunctions from the molecular to the network level. As ASD is believed to be caused by an imbalance between excitatory and inhibitory signals in the brain, we have further extended our analysis to examine disruption of autophagy in forebrain GABAergic interneurons. Similar to disrupted autophagy in other neuronal and non-neuronal cell types, Atg7 deletion by Dlx5-cre results in time-dependent ubiquitin+ and p62+ aggregate formation in affected neurons. Notably, Dlx5-cre Atg7 c KO mice exhibit an overlapping set of ASD-like behavioural abnormalities previously observed in Ca MKII-cre Atg7 c KO animals. Furthermore, we have detected a disruption of the excitatory-inhibitory balance in both Ca MKII-cre and Dlx5-cre Atg7 c KO mice. Using primary cultured neurons, a specific reduction of surface GABAA receptors was observed in Atg7 c KO neurons. Unbiased mass spectrometric and subsequent antibody-based analyses demonstrated that members of the GABARAP(GABA receptor-associated protein) protein family accumulate and mislocalize to p62+ aggregates formed in affected neurons of both Ca MKII-cre and Dlx5-cre Atg7 c KO brains. Genetic manipulation of GABARAP family proteins and p62 in wild-type or Atg7 c KO neurons further revealed that GABAA receptor trafficking are influenced by p62 aggregates as they interact with and sequester GABARAP family proteins. F

关键词： autophagy autism protein aggregation GABAA receptors GABARAP

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