title: Glutamate released by Japanese encephalitis virus-infected microglia involves TNF-α signaling and contributes to neuronal death.
abstract: The substantial activation of microglia in Japanese encephalitis virus (JEV)-induced Japanese encephalitis found in numerous studies demonstrates that the disease pathogenesis involves bystander damage caused by microglia-released mediators. Previously, we reported that microglia synthesized and secreted bioactive mediators with neurotoxic potential into the cultured supernatants in response to JEV infection. In this study, we found that the supernatants of JEV-infected microglia caused MK801-inhibitable neuronal damage in cultured neurons, indicating a potential excitotoxic mechanism. Infection with JEV was found to elicit the extracellular glutamate accumulation from microglia but not from neuron and astrocyte cultures. The glutaminase inhibitor 6-diazo-5-oxo-L-norleucine, cystine/glutamate antiporter inhibitor α-aminoadipic acid, and the gap junction inhibitor carbenoxolone reduced JEV infection-induced microglial glutamate release and neurotoxicity. We further demonstrated that tumor necrosis factor-alpha (TNF-α) was a key cytokine which stimulated extensive microglial glutamate release by up-regulating glutaminase expression via signals involving protein kinase C, cAMP responsive element-binding protein, and CAAT-enhancer-binding protein-beta. Although the elevated expression of excitatory amino acid transporter 1 and 2 was observed in JEV-infected cells, the glutamate uptake activity was significantly inhibited by TNF-α. The JEV infection-induced alterations, such as the extracellular glutamate release and glutamate-mediated excitoneurotoxicity, also occurred in neuron/glia cultures. Our findings support a potential link between neuroinflammation and the development of excitotoxic neuronal injury in Japanese encephalitis. The link between neuroinflammation and excitotoxic death may involve a mechanism in which TNF-α released by microglia plays a facilitory role in glutamate excitoneurotoxicity via up-regulation of glutamate synthesis and down-regulation of glutamate uptake.
authors.affiliation: Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan.
authors.forename: Chun-Jung
authors.initials: CJ
authors.lastname: Chen
authors.forename: Yen-Chuan
authors.initials: YC
authors.lastname: Ou
authors.forename: Cheng-Yi
authors.initials: CY
authors.lastname: Chang
authors.forename: Hung-Chuan
authors.initials: HC
authors.lastname: Pan
authors.forename: Su-Lan
authors.initials: SL
authors.lastname: Liao
authors.forename: Shih-Yun
authors.initials: SY
authors.lastname: Chen
authors.forename: Shue-Ling
authors.initials: SL
authors.lastname: Raung
authors.forename: Ching-Yi
authors.initials: CY
authors.lastname: Lai
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D017077:Culture Media, Conditioned / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D016291:Dizocilpine Maleate / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D004664:Encephalitis Virus, Japanese / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D018698:Glutamic Acid / Q000378:metabolism*; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism* / Q000821:virology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D016207:Cytokines; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D018698:Glutamic Acid; D016291:Dizocilpine Maleate; D002118:Calcium