Neurotoxic Activation of Microglia Is Promoted by a Nox1-Dependent NADPH Oxidase

Abstract

Reactive oxygen species (ROS) modulate intracellular signaling but are also responsible for neuronal damage in pathological states. Microglia, the resident CNS macrophages, are prominent sources of ROS through expression of the phagocyte oxidase which catalytic subunit Nox2 generates superoxide ion (O 2 ⅐Ϫ ). Here we show that microglia also express Nox1 and other components of nonphagocyte NADPH oxidases, including p22 phox , NOXO1, NOXA1, and Rac1/2. The subcellular distribution and functions of Nox1 were determined by blocking Nox activity with diphenylene iodonium or apocynin, and by silencing the Nox1 gene in microglia purified from wild-type (WT) or Nox2-KO mice. [Nox1-p22 phox ] dimers localized in intracellular compartments are recruited to phagosome membranes during microglial phagocytosis of zymosan, and Nox1 produces O 2 ⅐Ϫ in zymosan-loaded phagosomes. In microglia activated with lipopolysaccharide (LPS), Nox1 produces O 2 ⅐Ϫ , which enhances cell expression of inducible nitric oxide synthase and secretion of interleukin-1␤. Comparisons of microglia purified from WT, Nox2-KO, or Nox1-KO mice indicate that both Nox1 and Nox2 are required to optimize microglial production of nitric oxide. By injecting LPS in the striatum of WT and Nox1-KO mice, we show that Nox1 also enhances microglial production of cytotoxic nitrite species and promotes loss of presynaptic proteins in striatal neurons. These results demonstrate the functional expression of Nox1 in resident CNS phagocytes, which can promote production of neurotoxic compounds during neuroinflammation. Our study also shows that Nox1-and Nox2-dependent oxidases play distinct roles in microglial activation and that Nox1 is a possible target for the treatment of neuroinflammatory states.

TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ

“…Activation of IFN-γ receptors triggers various responses in macrophages and microglia (37,51). Moreover, there is cross-talk between TLR4 and the IFN-γ receptor via autocrine and paracrine loops at various intracellular downstream cascades (37,52). The promoter of iNOS gene (Inos) is operated by both STAT1 and NFκΒ transcription factors, activated by IFN-γ receptor and TLR4, respectively (37,53); thus, coincident STAT1/NFκB signaling up-regulates iNOS transcription and subsequent NO release to the critical neurotoxic level (11,32,34).…”

“…The promoter of iNOS gene (Inos) is operated by both STAT1 and NFκΒ transcription factors, activated by IFN-γ receptor and TLR4, respectively (37,53); thus, coincident STAT1/NFκB signaling up-regulates iNOS transcription and subsequent NO release to the critical neurotoxic level (11,32,34). TLR4 and/or IFN-γ receptor activation also up-regulate NADPH oxidase, which generates superoxide anions (20,52). Superoxide anions can rapidly oxidize NO, resulting in highly toxic peroxynitrite (27).…”

“…Superoxide anions can rapidly oxidize NO, resulting in highly toxic peroxynitrite (27). Thus, iNOS and NADPH oxidase in microglia are strong candidates for the mechanism that induces inflammatory neurodegeneration (14,34,44,52).…”

Novel Neuroinflammatory Targets in the Chronically Injured Spinal Cord

“…Alternative forms include NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2, and differ in their requirements for cytosolic and membrane subunits. In addition to NOX2, microglia have been shown to express functional NOX1 [12] and NOX4 [13]. Furthermore, NOX1, NOX2, and NOX4 are expressed in neurons, astrocytes, endothelial cells, pericytes and fibroblasts, among other cells [14,15].…”

Binding of EBP50 to Nox organizing subunit p47 phox is pivotal to cellular reactive species generation and altered vascular phenotype

“…Ex vivo EPR studies on aortic rings also showed absence of an O 2 •-signal in response to AngII in EBP50 KO, correlating with findings in Nox1 KO. Moreover, in vivo data demonstrated that in response to another robust inducer of Nox-derived ROS, LPS (27)(28)(29)(30)(31)(32)(33), vascular medial smooth muscle oxidation was absent in EBP50 KO mice. Because Nox1 is a major source of O 2…”