Aicardi-Goutières syndrome-associated mutation at ADAR1 gene locus activates innate immune response in mouse brain

Abstract

Background 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. Methods 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. Results 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. Conclusions 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.

Deciphering the Biological Significance of ADAR1–Z-RNA Interactions

“…To elucidate the biological significance of Z-RNA-binding to ADAR1 p150 in vivo, various studies, analyzing mutant mice harboring single or double point mutation(s) in Zα, have been reported [ 77 , 78 , 79 , 80 , 81 ]. Adar1 knock-in (KI) mice that harbor a P195A point mutation, corresponding to human AGS-causative P193A mutation ( Figure 2 ), show no overt phenotypes [ 80 , 81 ].…”

“…To elucidate the biological significance of Z-RNA-binding to ADAR1 p150 in vivo, various studies, analyzing mutant mice harboring single or double point mutation(s) in Zα, have been reported [ 77 , 78 , 79 , 80 , 81 ]. Adar1 knock-in (KI) mice that harbor a P195A point mutation, corresponding to human AGS-causative P193A mutation ( Figure 2 ), show no overt phenotypes [ 80 , 81 ]. However, Adar1 P195A/- and Adar1 P195A/p150− mice cannot survive beyond 3 and 4 months, respectively, with abnormalities in the spleen, kidney, and liver, and increased expression of ISGs, which is in contrast to the normal phenotypes of Adar1 +/− and Adar1 p150 +/− mice [ 80 ].…”

Case Report: Aicardi-Goutières Syndrome Type 6 and Dyschromatosis Symmetrica Hereditaria With Congenital Heart Disease and Mitral Valve Calcification – Phenotypic Variants Caused by Adenosine Deaminase Acting on the RNA 1 Gene Homozygous Mutations

“…Inserting, deleting, converting, or modifying ribonucleotides are the essential processes of enzymecatalyzed transformations. It is well-known that the adenosine deamination to inosine (A-to-I) is one of the most abundant RNA modifications and is catalyzed by ADAR in metazoans (8)(9)(10). If a mutated form of ADAR1 encounters amino acids, it cannot form an effective dimer structure and play its normal function, thus leading to the occurrence of disease (11).…”

“…Previous studies have shown that defects in ADAR1 activity caused by AGS mutations could lead to elevated levels of cytoplasmic double-stranded RNA, and finally result in interferon induction. Normal ADAR1 activity could prevent these RNAs from binding to the cytoplasmic dsRNA sensor IFIH1 through producing multiple IU mismatches ( 10 ). ADAR is widely expressed in mammals and considered as one of the housekeeping genes, but the molecular pathogenesis of DSH has not been well-demonstrated until now.…”

The most common human ADAR1p150 Zα domain mutation P193A is well tolerated in mice and does not activate the integrated stress response pathway

“…Similar to ADAR mutant AGS, BSN patients with ADAR mutation have a characteristic “interferon signature”. Recent knock-in mouse models have begun to address how distinct ADAR1 mutations, including those reported in AGS and BSN, impact the function of ADAR1 in vivo (de Reuver et al, 2021; Guo et al, 2021; Inoue et al, 2021; Maurano et al, 2021; Nakahama et al, 2021; Tang et al, 2021).…”