NMDA receptor blockade attenuates Japanese encephalitis virus infection-induced microglia activation

Abstract

Neurodegeneration and neuroinflammation are key components in the pathogenesis of Japanese Encephalitis caused by Japanese Encephalitis Virus (JEV) infection. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotoxic and pro-inflammatory properties in a cell context-dependent manner. Herein, potential roles of the NMDA receptor in excitatory neurotoxicity and neuroinflammation and effects of NMDA receptor blockade against JEV pathogenesis were investigated in rat microglia, neuron/glia, neuron cultures, and C57BL/6 mice. In microglia, JEV infection induced glutamate release and activated post-receptor NMDA signaling, leading to activation of Ca2+ mobilization and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), accompanied by pro-inflammatory NF-κB and AP-1 activation and cytokine expression. Additionally, increased Dynamin-Related Protein-1 protein phosphorylation, NAPDH Oxidase-2/4 expression, free radical generation, and Endoplasmic Reticulum stress paralleled with the reactive changes of microglia after JEV infection. JEV infection-induced biochemical and molecular changes contributed to microglia reactivity and pro-inflammatory cytokine expression. NMDA receptor antagonists MK801 and memantine alleviated intracellular signaling and pro-inflammatory cytokine expression in JEV-infected microglia. JEV infection induced neuronal cell death in neuron/glia culture associated with the concurrent production of pro-inflammatory cytokines. Conditioned media of JEV-infected microglia compromised neuron viability in neuron culture. JEV infection-associated neuronal cell death was alleviated by MK801 and memantine. Activation of NMDA receptor-related inflammatory changes, microglia activation, and neurodegeneration as well as reversal effects of memantine were revealed in the brains of JEV-infected mice. The current findings highlight a crucial role of the glutamate/NMDA receptor axis in linking excitotoxicity and neuroinflammation during the course of JEV pathogenesis, and proposes the anti-inflammatory and neuroprotective potential of NMDA receptor blockade.