The ADAR G1007R mutation in Aicardi-Goutières Syndrome causes neuroinflammation through RNA sensing signaling pathways in mice

The ADARG1007R mutation is one of the most frequent mutations found in type six Aicardi-Goutières Syndrome (AGS), a progressive inflammatory encephalopathy in pediatric patients, typically begins to manifest in newborns or infants. Due to the rarity of this disease and the limited accessibility of the human brain tissues, the pathologic features and the underlying mechanism of this mutation has not been well understood. In this study, we generated a genetic mutant mouse model which successfully recapitulated the genetic and pathologic features of human ADARG1007R AGS, including the early-onset brain inflammatory reaction in heterozygous mutant mice and the deep gray matter area ISG expression. Through analysis of the homozygous ADARG1007R/G1007 mice, we found that the G1007R mutation interrupts the RNA splicing donor site in the RNA transcripts that alters ADAR1 RNA splicing, leading to depletion of ADAR1 protein and embryonic death. Furthermore, we demonstrated that the brain inflammation could be reversed by deletion of the cellular RNA receptor MDA5 which blocks the cellular RNA sensing signaling pathway. Collectively, this study generated a clinically relevant AGS animal model, revealed the molecular mechanism of the ADARG1007R mutation in AGS, and demonstrated that the MDA5-dependent RNA sensing signaling pathway plays a critical role in neuroinflammation. Overall design: All mice used in this study were kept in a specific pathogen-free animal facility and all studies were carried out following protocols approved by our Institutional Animal Care and Use Committee. The G1007R mutation was introduced into the mouse genome using our CRISPR/Cas9 protocol as described previously. Considering the genetic diversity of human diseases of ADAR1 mutations, mice used in this study, including AdarG1007R/G1007R and AdarG1007R/G1007R; Ifih1-/- mice, were purposely maintained in C57/BL6J and DBA2 mixed background. Three repeats for wild-type (WT), three repeats for G1007R mutant, and three repeats for G1007R mutatant + MDA5 retreat

ADARG1007R突变是6型艾卡迪-古蒂耶综合征（Aicardi-Goutières Syndrome, AGS）中最常见的突变类型之一。艾卡迪-古蒂耶综合征是一种儿童进行性炎症性脑病，通常于新生儿或婴幼儿阶段发病。由于该疾病发病率极低且人类脑组织样本获取途径有限，目前学界对该突变的病理特征与潜在致病机制尚未形成充分认知。
本研究构建了一款基因工程突变小鼠模型，该模型成功复现了人类ADARG1007R型AGS的遗传与病理特征，包括杂合突变小鼠的早发性脑部炎症反应，以及深部灰质区域的干扰素刺激基因（Interferon Stimulated Gene, ISG）表达。通过对纯合子ADARG1007R/G1007R小鼠的分析，本研究发现G1007R突变会破坏RNA转录本中的RNA剪接供体位点，改变ADAR1的RNA剪接过程，进而导致ADAR1蛋白耗竭并引发胚胎致死。此外，本研究证实，通过敲除细胞RNA受体MDA5以阻断细胞RNA感知信号通路，可逆转脑部炎症反应。综上，本研究构建了具有临床相关性的AGS动物模型，揭示了ADARG1007R突变导致AGS的分子机制，并证明MDA5依赖的RNA感知信号通路在神经炎症中发挥关键作用。
总体实验设计：本研究中所有小鼠均饲养于无特定病原体（Specific Pathogen Free, SPF）级动物实验设施内，所有实验操作均严格遵循本机构动物实验伦理审查委员会批准的实验方案。参照此前报道的实验方案，本研究通过CRISPR/Cas9技术将G1007R突变引入小鼠基因组。考虑到人类ADAR1突变相关疾病存在遗传异质性，本研究使用的小鼠（包括AdarG1007R/G1007R纯合子小鼠，以及AdarG1007R/G1007R; Ifih1-/-双基因修饰小鼠）均特意维持在C57/BL6J与DBA2的混合遗传背景中。野生型（Wild Type, WT）、G1007R突变型以及G1007R突变型+MDA5干预组每组均设置3次生物学重复。