Aicardi-Goutières syndrome associated mutation at ADAR1 gene locus activates innate immune response in mouse brain
Authors: Guo Xinfeng , Wiley Clayton A. , Steinman Richard A. , Sheng Yi , Ji Beihong , Wang Junmei , Wang Tony , Zenatai Mazen , Billiar Timothy R. , Wang Qingde 
Published: April 2021

Abstract
BackgroundAicardi-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 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 IFN pathway and causes autoimmune pathogenesis in the brain is yet to be determined.MethodsMutations in the ADAR1 gene found in AGS patients were introduced into mouse genome via CRISPR/Case9 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. ResultsMice 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 mutant brain.Conclusions We demonstrated that an AGS-associated mutation in ADAR1 was sufficient to activate the IFN pathway in the brain. Neurons and microglia expressed different ISGs. The ADAR1 p.K999N mutant mouse replicated aspects of the brain interferonopathy of AGS. Other brain changes seen in AGS (gliosis, calcification, death) did not occur, indicating that clinical AGS mutations may be necessary but not sufficient for development of the full phenotype. This mutant mouse presents a robust tool for investigation of AGS and neuroinflammatory diseases including the modeling of potential “second hits” that enable severe phenotypes of clinically variable diseases.