Gp91phox (NOX2) in classically activated microglia exacerbates traumatic brain injury. J Neuroinflammation 7:41

Department of Emergency and Critical Care Medicine, Showa University School of Medicine, Shinagawa-Ku, Tokyo 142-8555, Japan.
Journal of Neuroinflammation (Impact Factor: 5.41). 07/2010; 7(1):41. DOI: 10.1186/1742-2094-7-41
Source: PubMed


We hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.
Unilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNgamma or IL-4.
Gp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNgamma, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.
Classical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

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    • "Upregulation of the NADPH oxidase subunit gp91phox and of the high throughput NOS, iNOS, is a distinctive hallmark of glial activation [66]. Gp91phox deficient mice do not mount a robust ROS response following traumatic brain injury, and hence are relatively protected from cerebral damage [67]. iNOS, on the other hand, seems to have a dichotomous role in the brain. "
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    ABSTRACT: Background A20 (TNFAIP3) is a pleiotropic NFκB-dependent gene that terminates NFκB activation in response to inflammatory stimuli. The potent anti-inflammatory properties of A20 are well characterized in several organs. However, little is known about its role in the brain. In this study, we investigated the brain phenotype of A20 heterozygous (HT) and knockout (KO) mice. Methods The inflammatory status of A20 wild type (WT), HT and KO brain was determined by immunostaining, quantitative PCR, and Western blot analysis. Cytokines secretion was evaluated by ELISA. Quantitative results were statistically analyzed by ANOVA followed by a post-hoc test. Results Total loss of A20 caused remarkable reactive microgliosis and astrogliosis, as determined by F4/80 and GFAP immunostaining. Glial activation correlated with significantly higher mRNA and protein levels of the pro-inflammatory molecules TNF, IL-6, and MCP-1 in cerebral cortex and hippocampus of A20 KO, as compared to WT. Basal and TNF/LPS-induced cytokine production was significantly higher in A20 deficient mouse primary astrocytes and in a mouse microglia cell line. Brain endothelium of A20 KO mice demonstrated baseline activation as shown by increased vascular immunostaining for ICAM-1 and VCAM-1, and mRNA levels of E-selectin. In addition, total loss of A20 increased basal brain oxidative/nitrosative stress, as indicated by higher iNOS and NADPH oxidase subunit gp91phox levels, correlating with increased protein nitration, gauged by nitrotyrosine immunostaining. Notably, we also observed lower neurofilaments immunostaining in A20 KO brains, suggesting higher susceptibility to axonal injury. Importantly, A20 HT brains showed an intermediate phenotype, exhibiting considerable, albeit not statistically significant, increase in markers of basal inflammation when compared to WT. Conclusions This is the first characterization of spontaneous neuroinflammation caused by total or partial loss of A20, suggesting its key role in maintenance of nervous tissue homeostasis, particularly control of inflammation. Remarkably, mere partial loss of A20 was sufficient to cause chronic, spontaneous low-grade cerebral inflammation, which could sensitize these animals to neurodegenerative diseases. These findings carry strong clinical relevance in that they question implication of identified A20 SNPs that lower A20 expression/function (phenocopying A20 HT mice) in the pathophysiology of neuroinflammatory diseases.
    Journal of Neuroinflammation 07/2014; 11(1):122. DOI:10.1186/1742-2094-11-122 · 5.41 Impact Factor
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    • "NOX expression has previously been reported to increase in several CNS injury models, including brain [17,27] and spinal cord injury [18,20,36]. In the spinal cord, NOX expression is up-regulated through 6 months after injury in rodents [19], and ROS production has been reported from 6 hours to several months post-injury in humans [35,36]. "
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    ABSTRACT: Brain injury results in an increase in the activity of the reactive oxygen species generating NADPH oxidase (NOX) enzymes. Preliminary studies have shown that NOX2, NOX3, and NOX4 are the most prominently expressed NOX isotypes in the brain. However, the cellular and temporal expression profile of these isotypes in the injured and non-injured brain is currently unclear. Double immunofluorescence for NOX isotypes and brain cell types was performed at acute (24 hours), sub-acute (7 days), and chronic (28 days) time points after controlled cortical impact-induced brain injury or sham-injury in rats. NOX2, NOX3, and NOX4 isotypes were found to be expressed in neurons, astrocytes, and microglia, and this expression was dependent on both cellular source and post-injury time. NOX4 was found in all cell types assessed, while NOX3 was positively identified in neurons only, and NOX2 was identified in microglia and neurons. NOX2 was the most responsive to injury, increasing primarily in microglia in response to injury. Quantitation of this isotype showed a significant increase in NOX2 expression at 24 hours, with reduced expression at 7 days and 28 days post-injury, although expression remained above sham levels at later time points. Cellular confirmation using purified primary or cell line culture demonstrated similar patterns in microglia, astrocytes, and neurons. Further, inhibition of NOX, and more specifically NOX2, reduced pro-inflammatory activity in microglia, demonstrating that NOX is not only up-regulated after stimulation, but may also play a significant role in post-injury neuroinflammation. This study illustrates the expression profiles of NOX isotypes in the brain after injury, and demonstrates that NOX2, and to a lesser extent, NOX4, may be responsible for the majority of oxidative stress observed acutely after traumatic brain injury. These data may provide insight into the design of future therapeutic approaches.
    Journal of Neuroinflammation 12/2013; 10(1):155. DOI:10.1186/1742-2094-10-155 · 5.41 Impact Factor
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    • "NADPH oxidase is a multiunit enzyme composed of several subunits that include several isoforms of NOX (NOX 1–5) (Bedard and Krause, 2007). Although few studies have evaluated the role of gp91 phox (catalytic subunit of the enzyme, NOX 2 ) in TBI, experimental evidences suggest that use of specific NADPH oxidase inhibitors may have significant efficacy in the treatment of TBI (Dohi et al., 2010; Lo et al., 2007). A candidate for this is Apocynin, a natural organic compound isolated from the Himalayan medicinal herb Picrohiza kurroa used as an efficient inhibitor of the NADPH–oxidase complex (Hayashi et al., 2005; Stolk et al., 1994; Van den Worm et al., 2001). "
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    ABSTRACT: Traumatic brain injury (TBI) is a devastating disease that commonly causes persistent mental disturbances and cognitive deficits. Although studies have indicated that overproduction of free radicals, especially superoxide (O2(-)) derived from Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a common underlying mechanism of pathophysiology of TBI, little information is available regarding the role of apocynin, an NADPH oxidase inhibitor, in neurological consequences of TBI. Therefore, the present study evaluated the therapeutic potential of apocynin for treatment of inflammatory and oxidative damage, in addition to determining its action on neuromotor and memory impairments caused by moderate fluid percussion injury in mice (mLFPI). Statistical analysis revealed that apocynin (5 mg/kg), when injected subcutaneously (s.c.) 30 min and 24 h after injury, had no effect on neuromotor deficit and brain edema, however it provided protection against mLFPI-induced object recognition memory impairment 7 days after neuronal injury. The same treatment protected against mLFPI-induced IL-1β, TNF-α, nitric oxide metabolite content (NOx) 3 and 24 hours after neuronal injury. Moreover, apocynin treatment reduced oxidative damage (protein carbonyl, lipoperoxidation) and was effective against mLFPI-induced Na(+)K(+)-ATPase activity inhibition. The present results were accompanied by effective reduction in lesion volume when analyzed 7 days after neuronal injury. These data suggest that superoxide (O2(-)) derived from NADPH oxidase can contribute significantly to cognitive impairment, and that the post injury treatment with specific NADPH oxidase inhibitors, such as apocynin, may provide a new therapeutic approach to the control of neurological disabilities induced by TBI.
    Neurochemistry International 09/2013; 63(6). DOI:10.1016/j.neuint.2013.09.012 · 3.09 Impact Factor
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