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Aicardi-Goutières syndrome-associated mutation at ADAR1 gene locus activates innate immune response in mouse brain
Journal of Neuroinflammation ( IF 9.3 ) Pub Date : 2021-07-31 , DOI: 10.1186/s12974-021-02217-9
Xinfeng Guo 1 , Clayton A Wiley 2 , Richard A Steinman 3 , Yi Sheng 4 , Beihong Ji 5 , Junmei Wang 5 , Liyong Zhang 1 , Tony Wang 6 , Mazen Zenatai 1 , Timothy R Billiar 1 , Qingde Wang 1, 7, 8
Affiliation  

Aicardi-Goutières syndrome (AGS) is a severe infant or juvenile-onset autoimmune disease characterized by inflammatory encephalopathy with an elevated type 1 interferon-stimulated gene (ISG) expression signature in the brain. Mutations in seven different protein-coding genes, all linked to DNA/RNA metabolism or sensing, have been identified in AGS patients, but none of them has been demonstrated to activate the IFN pathway in the brain of an animal. The molecular mechanism of inflammatory encephalopathy in AGS has not been well defined. Adenosine Deaminase Acting on RNA 1 (ADAR1) is one of the AGS-associated genes. It carries out A-to-I RNA editing that converts adenosine to inosine at double-stranded RNA regions. Whether an AGS-associated mutation in ADAR1 activates the IFN pathway and causes autoimmune pathogenesis in the brain is yet to be determined. Mutations in the ADAR1 gene found in AGS patients were introduced into the mouse genome via CRISPR/Cas9 technology. Molecular activities of the specific p.K999N mutation were investigated by measuring the RNA editing levels in brain mRNA substrates of ADAR1 through RNA sequencing analysis. IFN pathway activation in the brain was assessed by measuring ISG expression at the mRNA and protein level through real-time RT-PCR and Luminex assays, respectively. The locations in the brain and neural cell types that express ISGs were determined by RNA in situ hybridization (ISH). Potential AGS-related brain morphologic changes were assessed with immunohistological analysis. Von Kossa and Luxol Fast Blue staining was performed on brain tissue to assess calcification and myelin, respectively. Mice bearing the ADAR1 p.K999N were viable though smaller than wild type sibs. RNA sequencing analysis of neuron-specific RNA substrates revealed altered RNA editing activities of the mutant ADAR1 protein. Mutant mice exhibited dramatically elevated levels of multiple ISGs within the brain. RNA ISH of brain sections showed selective activation of ISG expression in neurons and microglia in a patchy pattern. ISG-15 mRNA was upregulated in ADAR1 mutant brain neurons whereas CXCL10 mRNA was elevated in adjacent astroglia. No calcification or gliosis was detected in the mutant brain. We demonstrated that an AGS-associated mutation in ADAR1, specifically the p.K999N mutation, activates the IFN pathway in the mouse brain. The ADAR1 p.K999N mutant mouse replicates aspects of the brain interferonopathy of AGS. Neurons and microglia express different ISGs. Basal ganglia calcification and leukodystrophy seen in AGS patients were not observed in K999N mutant mice, indicating that development of the full clinical phenotype may need an additional stimulus besides AGS mutations. This mutant mouse presents a robust tool for the investigation of AGS and neuroinflammatory diseases including the modeling of potential “second hits” that enable severe phenotypes of clinically variable diseases.

中文翻译:

ADAR1基因位点的Aicardi-Goutières综合征相关突变激活小鼠大脑的先天免疫反应

Aicardi-Goutières 综合征 (AGS) 是一种严重的婴儿或青少年发病的自身免疫性疾病,其特征是脑内 1 型干扰素刺激基因 (ISG) 表达特征升高的炎症性脑病。已经在 AGS 患者中发现了七种不同的蛋白质编码基因的突变,这些基因都与 DNA/RNA 代谢或传感有关,但它们都没有被证明可以激活动物大脑中的 IFN 通路。AGS 中炎症性脑病的分子机制尚未明确。作用于 RNA 1 的腺苷脱氨酶 (ADAR1) 是 AGS 相关基因之一。它进行 A-to-I RNA 编辑,将双链 RNA 区域的腺苷转化为肌苷。ADAR1 中的 AGS 相关突变是否会激活 IFN 通路并导致大脑中的自身免疫发病机制尚待确定。在 AGS 患者中发现的 ADAR1 基因突变通过 CRISPR/Cas9 技术被引入小鼠基因组。通过 RNA 测序分析测量 ADAR1 的脑 mRNA 底物中的 RNA 编辑水平,研究了特定 p.K999N 突变的分子活性。通过分别通过实时 RT-PCR 和 Luminex 测定法测量 mRNA 和蛋白质水平的 ISG 表达来评估大脑中的 IFN 通路激活。表达 ISG 的大脑和神经细胞类型中的位置由 RNA 原位杂交 (ISH) 确定。通过免疫组织学分析评估潜在的 AGS 相关脑形态学变化。对脑组织进行 Von Kossa 和 Luxol Fast Blue 染色以分别评估钙化和髓鞘。携带 ADAR1 p.K999N 的小鼠虽然比野生型同胞小,但仍能存活。神经元特异性 RNA 底物的 RNA 测序分析揭示了突变 ADAR1 蛋白的 RNA 编辑活性改变。突变小鼠大脑内的多种 ISG 水平显着升高。脑切片的 RNA ISH 显示神经元和小胶质细胞中 ISG 表达的选择性激活呈斑块状。ISG-15 mRNA 在 ADAR1 突变脑神经元中上调,而 CXCL10 mRNA 在相邻星形胶质细胞中升高。在突变脑中未检测到钙化或神经胶质增生。我们证明了 ADAR1 中的 AGS 相关突变,特别是 p.K999N 突变,激活了小鼠大脑中的 IFN 通路。ADAR1 页。K999N 突变小鼠复制 AGS 脑干扰素病的各个方面。神经元和小胶质细胞表达不同的 ISG。在 K999N 突变小鼠中未观察到在 AGS 患者中观察到的基底神经节钙化和白细胞营养不良,这表明除了 AGS 突变之外,完整临床表型的发展可能需要额外的刺激。这种突变小鼠为研究 AGS 和神经炎性疾病提供了一个强大的工具,包括对潜在的“二次打击”进行建模,从而使临床可变疾病的严重表型成为可能。表明除了 AGS 突变之外,完整临床表型的发展可能需要额外的刺激。这种突变小鼠为研究 AGS 和神经炎性疾病提供了一个强大的工具,包括对潜在的“二次打击”进行建模,从而使临床可变疾病的严重表型成为可能。表明除了 AGS 突变之外,完整临床表型的发展可能需要额外的刺激。这种突变小鼠为研究 AGS 和神经炎性疾病提供了一个强大的工具,包括对潜在的“二次打击”进行建模,从而使临床可变疾病的严重表型成为可能。
更新日期:2021-08-01
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