Aicardi-Goutières syndrome-associated mutation at ADAR1 gene locus activates innate immune response in mouse brain.
Aicardi-Goutières syndrome-associated mutation at ADAR1 gene locus activates innate immune response in mouse brain.
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DOI：
10.1186/s12974-021-02217-9
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发表时间：
2021-07-31
影响因子：
9.3
通讯作者：
Wang Q
中科院分区：
文献类型：
--
作者：
Guo X;Wiley CA;Steinman RA;Sheng Y;Ji B;Wang J;Zhang L;Wang T;Zenatai M;Billiar TR;Wang Q
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，其特征是炎性脑病，脑中1型干扰素刺激基因（ISG）表达标志升高。已经在AGS患者中鉴定了7种不同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，但没有一种基因被证明可以激活动物大脑中的IFN途径。AGS中炎性脑病的分子机制尚未明确。腺苷脱氨酶1（Adenosine Deaminase Acting on RNA 1，ADAR 1）是一种与AGS相关的基因。它进行A-to-I RNA编辑，在双链RNA区域将腺苷转化为肌苷。ADAR 1中的AGS相关突变是否激活IFN途径并导致大脑中的自身免疫发病机制尚未确定。在AGS患者中发现的ADAR 1基因突变通过CRISPR/Cas9技术引入小鼠基因组。通过RNA测序分析测量ADAR 1脑mRNA底物中的RNA编辑水平，研究特异性p.K999N突变的分子活性。通过分别通过实时RT-PCR和Luminex测定在mRNA和蛋白质水平上测量ISG表达来评估脑中的IFN途径活化。通过RNA原位杂交（ISH）确定表达ISGs的脑和神经细胞类型的位置。潜在的AGS相关的脑形态学变化进行了评估与免疫组织化学分析。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ADAR 1 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ADAR 1蛋白的RNA编辑活性改变。突变小鼠在脑内表现出显著升高的多种ISG水平。脑切片的RNA原位杂交显示ISG在神经元和小胶质细胞中以斑片状模式表达的选择性激活。ISG-15 mRNA在ADAR 1突变的脑神经元中上调，而CXCL 10 mRNA在相邻的星形胶质细胞中升高。在突变体脑中未检测到钙化或神经胶质增生。我们证明了ADAR 1中的AGS相关突变，特别是p.K999N突变，激活了小鼠大脑中的IFN途径。ADAR 1 p.K999N突变小鼠复制了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K999 N突变小鼠中未观察到AGS患者中观察到的基底神经节钙化和脑白质营养不良，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AGS和神经炎性疾病的研究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。在线版本包含补充材料，可通过10.1186/s12974-021-02217-9获得。
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. The online version contains supplementary material available at 10.1186/s12974-021-02217-9.
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影响因子：
7.3
作者：
Bartsch K;Damme M;Regen T;Becker L;Garrett L;Hölter SM;Knittler K;Borowski C;Waisman A;Glatzel M;Fuchs H;Gailus-Durner V;Hrabe de Angelis M;Rabe B
通讯作者：
Rabe B
影响因子：
1.4
作者：
Gavazzi F;Cross ZM;Woidill S;McMann JM;Rand EB;Takanohashi A;Ulrick N;Shults J;Vanderver AL;Adang L
通讯作者：
Adang L
影响因子：
32.4
作者：
Gall A;Treuting P;Elkon KB;Loo YM;Gale M Jr;Barber GN;Stetson DB
通讯作者：
Stetson DB
影响因子：
6.4
作者：
Izzotti, Alberto;Pulliero, Alessandra;Fazzi, Elisa
通讯作者：
Fazzi, Elisa
影响因子：
8.8
作者：
Mannion NM;Greenwood SM;Young R;Cox S;Brindle J;Read D;Nellåker C;Vesely C;Ponting CP;McLaughlin PJ;Jantsch MF;Dorin J;Adams IR;Scadden AD;Ohman M;Keegan LP;O'Connell MA
通讯作者：
O'Connell MA