Project Summary/Abstract The putative nucleotide sensor ZBP1 can trigger necroptotic cell death or transcriptional responses, and genetic studies indicate that this pathway is required for host defense against an array of viral pathogens. Furthermore, recent studies show that in some circumstances ZBP1 can become activated under sterile conditions, implicating endogenous cellular products as potential ZBP1 ligands. However, despite long study, the ligand responsible for activating ZBP1 and its interplay with other components of the nucleotide sensing machinery remain controversial, with double-stranded RNA, ribonucleoprotein, and viral Z-form nucleic acid species all suggested as ligand. ZBP1 shares its key nucleotide sensing domain with only one other mammalian protein, ADAR1. ADAR1 inactivates endogenous dsRNA species to limit autoinflammatory pathology, and mutations in ADAR1 in human patients lead to the severe autoimmune disease Acardi- Goutieres syndrome (AGS). We hypothesize that ADAR1 and ZBP1 compete for a common endogenous ligand, whose inactivation by ADAR1 is required to limit ZBP1 activation. In support of this idea, the pathology observed in a newly-developed mouse model of human ADAR1 mutation was fully rescued by ablation of ZBP1. This finding supports our hypothesis, and also implies that autoimmune pathology associated with loss of ADAR1 function is caused by activation of ZBP1-dependent inflammation and cell death. Using these observations as a starting point, the work proposed here will investigate ZBP1 activation and function by pursuing three Aims: First, we will use ADAR1 mutation and additional new mouse models to facilitate isolation and identification of an endogenous ZBP1 ligand. Second, we will assess the contribution of necroptosis as well as ZBP1-mediated inflammatory signaling to the pathology of an animal model of human AGS triggered by ADAR1 mutation, and test the ability of necroptosis inhibitors to ameliorate this pathology. Third, we will investigate the role of other dsRNA sensors, including MDA5 and PKR, to ZBP1 ligand formation and necroptotic pathway activation. Together this work will both reveal key aspects of ZBP1 function, and identify ZBP1-dependent necroptosis as a potentially treatable target to ameliorate AGS.