The Role And Mechanism Of NOX2 Mediated Complexin â…¡ On Glutamate Excitotoxicity In Experimental Stroke

Background and ObjectiveDespite considerable clinical and animal model studies have implicated that several mechanisms are involved in the pathogenesis of ischemic stroke, glutamate-induced excitotoxicity has been recognized to play an important role in the pathogenesis of ischemic stroke. As one of the excitatory neurotransmitters, physiologically, glutamate is released at presynaptic terminals by fusion of neurotransmitter-containing vesicles with the presynaptic plasma membrane, a process known as SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein-dependent synaptic vesicle exocytosis. Synaptic vesicle fusion is driven by assembly of trans-SNARE complexes from the membrane proteins, including soluble N-ethylmaleimide sensitive factor attachment protein-25 (SNAP25), vesicle-associated membrane protein (VAMP) and Syntaxin. In recent years, some specific proteins have been identified that are responsible for the regulation of SNARE-mediated fusion. Among them, complexins are regarded as key regulators of synaptic exocytosis for neurotransmitter release. However, whether complexins are involved in the pathogenesis of SNAREs mediated glutamate-induced excitotoxicity in ischemic stroke is no report till now.MethodIn this study, we used an in vivo transient middle cerebral artery occlusion (MCAO) model and in vitro primary neuronal cell cultures to test whether complexins, the regulators of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion, are contribute to glutamate-mediated excitotoxicity in ischemic stroke. In order to find the effective pathway to block the complexin mediated neuronal excitotoxicity, we further use NOX2 knockout mice and knock down neuronal cells to observe the regulation role of NADPH oxidase on the expression of complexins in experimental stroke.ResultsWe first identified the upregulation of complexin â…  and complexin â…¡ in the ischemic brain and evaluated the potential role in ischemic stroke showing that gene silencing of complexin â…¡ ameliorated cerebral injury as evidenced by reduced infarction volume,neurological deficit, neuron necrosis accompanied by decreased glutamate levels. We further demonstrated complexin â…¡ expression was mediated by NOX2 in both cerebral ischemia reperfusion injured animals subjected to MCAO and primary cultured neurons subjected to oxygen-glucose deprivation (OGD), which contributed to cerebral ischemia induced glutamate release and neuron necrosis via SNARE signaling. We then explored that whether the regulation of complexins is associated with NADPH oxidase-derived ROS and contributes to glutamate-mediated excitotoxicity in ischemic stroke by using both knockout mice and knoneurons.ConclusionThese findings for the first time provide evidence that complexins play important role in glutamate-mediated neuronal excitotoxicity in ischemic stroke. In addition, this study for the first time links NADPHoxidase-derived ROS to glutamate-mediated neuronal excitotoxicity by complexins in ischemic stroke. Pharmacological targeting of NOX2-mediated glutamate signalingpathways via complexin â…¡ at multiple levels may help design a new approach to develop therapeutic strategies for prevention and the treatment of ischemic stroke.