Mechanisms and impacts of astrocyte inflammasome activation during viral encephalitis

PROJECT SUMMARY Viral infections of the central nervous system (CNS) pose a particularly difficult challenge to the host immune system. Virus must be cleared from the CNS without significant immune-mediated damage to neuronal tissues. In recent years, it has been increasingly recognized that survivors of viral encephalitides, such as West Nile virus neuroinvasive disease (WNND), suffer from longterm neurocognitive sequelae. Past research from our laboratory has modeled this phenomenon in mice by direct inoculation of attenuated West Nile virus into the third ventricle of the brain. In this model, mice recover from encephalitis, but immune- mediated damage causes adverse cognitive sequelae. Inflammatory astrocytes produce interleukin-1β after viral infection and interferon-γ-producing T cells drive microglia activation. Each of these pathways negatively impacts hippocampal synapse recovery, hippocampal neurogenesis, and visuospatial learning. Astrocytes are not directly infected by West Nile virus in vivo, but they nonetheless display NLRP3 inflammasome activation and downstream production of active caspase-1 and interleukin-1β. The signals that drive activation of the NLRP3 inflammasome activation in astrocytes during viral infection remain completely unknown. However, astrocytes express the P2 purinergic receptor P2RX7, which can mediate inflammasome activation in macrophages exposed to high concentrations of extracellular ATP. Here, I propose that neuronal synapse elimination during viral encephalitis releases supraphsyiologic levels of extracellular ATP, leading to astrocyte inflammasome activation. In Aim 1, I will seek to confirm this hypothesis by generating a novel mouse strain that specifically and inducibly lacks P2RX7 on astrocytes. In addition, I propose that heightened extracellular ATP levels signal to neuronal stem cells via P2RX7, driving their differentiation towards reactive astrogenesis rather than neurogenesis. I hypothesize that this pathway may contribute towards the lack of hippocampal neurogenesis in our model of WNND. In Aim 2, I will investigate this possibility by infecting animals with P2RX7-deficient neural stem cells with West Nile virus and monitoring for changes in hippocampal neurogenesis and visuospatial learning ability. Together, these Aims will characterize novel functions of the purinergic receptor P2RX7 during neuroinvasive viral infection, potentially identifying a target for pharmacologic therapies that reduce the severity of post-infectious neurocognitive sequelae.