Although NADPH oxidase (NOX)-mediated oxidative stress is considered one of the major mechanisms triggering the pathogenic actions of ischemic stroke and very recent studies have indicated that NADPH oxidase is a major source of reactive oxygen species (ROS) production controlling glutamate release, how neuronal NADPH oxidase activation is coupled to glutamate release is not well understood. Therefore, in this study, we used an in vivo transient middle cerebral artery occlusion model and in vitro primary cell cultures to test whether complexins, the regulators of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion, are associated with NOX2-derived ROS and contribute to glutamate-mediated excitotoxicity in ischemic stroke. In this study, we first identified the upregulation of complexin II in the ischemic brain and evaluated its potential role in ischemic stroke showing that gene silencing of complexin II ameliorated cerebral injury as evidenced by reduced infarction volume, neurological deficit, and neuron necrosis accompanied by decreased glutamate levels, consistent with the results from NOX2(-/-) mice with ischemic stroke. We further demonstrated that complexin II expression was mediated by NOX2 in primary cultured neurons subjected to oxygen-glucose deprivation (OGD) and contributed to OGD-induced glutamate release and neuron necrosis via SNARE signaling. Taken together, these findings for the first time provide evidence that complexin II is a central target molecule that links NADPH oxidase-derived ROS to glutamate-mediated neuronal excitotoxicity in ischemic stroke.
Keywords: 2,3,5-triphenyltetrazolium chloride; Cerebral ischemia injury; Complexin; Free radicals; HE; HPLC; Hematoxylin and eosin; High-performance liquid chromatography; LV; Lentivirus; MCAO; Middle cerebral artery occlusion; NADPH oxidase; NOX; O(2)(•−); OGD; Oxygen–glucose deprivation; ROS; Reactive oxygen species; SNAP25; SNARE; SNARE complex; Soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Soluble N-ethylmaleimide-sensitive factor attachment protein-25; Superoxide; TTC; WT; Wild type.
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