detected immediately after addition of the substrate, NADPH, and in contrast to a number of other studies which have required agents, such as phorbol esters, to induce assembly of the Nox2 complex, required no other additions to observe O2 consumption and superoxide generation. These results suggest that in neurons and at the synapse, Nox2 is in the assembled, superoxide-capable form, and is responsible for a sustained increase in neuronal and synaptic superoxide production in the aged brain. Thus, we believe this to be the first report that Nox2 is the major source of neuronal and synaptic superoxide production in normal aging brain. There has been debate over whether apocynin inhibits the p47phox-dependent Nox isoforms (Nox1, Nox2, Nox4, and Nox5), with a recent paper suggesting that in vascular smooth muscle cells, apocynin may be acting as an antioxidant rather than a NADPH oxidase inhibitor, and that standard chemiluminescence- based assays for Nox activity may produce artifacts in the presence of apocynin [40]. However, there are also several recent studies supporting efficacy of apocynin as a Nox2 inhibitor[30,41], reviewed in [42], with the use of appropriate controls. We have previously shown, using EPR and oximetry as measures of Nox2 activity, that apocynin is not acting as an antioxidant in our system ([15,18]), that it lacks activity when Nox2 is not induced, and that it does not affect mitochondrial superoxide generation in the same sample [15,18]. Of note, myeloperoxidase, which is required to convert apocynin to its active dimer, is expressed in neurons and astrocytes in brain[43-45]. The role of IL-6 in mediating age-related induction of neuronal Nox2 was then examined in old IL-6-/- mice, and in wild-type mice treated directly with IL-6. IL-6-/- mice lack the age-related increase in Nox2 expression and superoxide production observed in old wild- type mice. Nox-dependent superoxide production in synaptosomes was also significantly lower in old IL-6-/- mice compared to wild- type mice, and was not statistically different than superoxide production in young synaptosomes. Consistent with a direct effect of IL-6 on Nox2 expression, direct systemic injection of IL-6 into mice increased Nox2 protein and activity, confirming the ability of peripheral IL-6 to mediate increased Nox2 expression and superoxide production in brain. Live-animal imaging of in vivo ox- DHE fluorescence followed by confocal microfluorimetry of cell- specific DHE oxidation in slices prepared from live-imaged animals provided additional support for increased superoxide production in aging that was increased even further by IL-6 injection. Factors responsible for peripheral IL-6 induction in aging humans include diet, hormone status, activity, and genet- ics[2,3,5,46,47], and while mechanisms which increase IL-6 in the nervous system are less well understood, direct entry of plasma IL-6 into brain has been shown[12]. Stress effects on the hypopituitary axis can also lead to local induction of IL-6 in brain and secondary release into blood, a potential additional source of IL-6 in both brain and plasma[3] (and references therein). Our ability to induce Nox2 in brain by direct peripheral IL-6 administration suggests that the former process may be operative in our experiments. Of note, these studies were carried out in male mice only, and given the fact that have previously reported higher CNS superoxide levels in aged female versus male C57BL6 mice[48], we speculate that gender-specific differences in inflammatory signaling in brain might contribute to this difference. Induction of Nox2 by IL-6 in neurons was dependent on NFkB signaling. The decreased expression of Nox2 we observed in old IL-6-/- mice compared to wild-type mice was accompanied by a significant reduction in nuclear NFkB activity. Although IL-1b and TNFa are strong inducers of NFkB activation, expression of these two cytokines, measured by RT-qPCR, was not statistically IL-6 and Cognitive Changes different between old wild-type and old IL-6-/- mice (data not shown) suggesting that the specific loss of IL-6 expression, not changes in these other cytokines, was responsible for decreased nuclear NFkB activity in aged IL-6-/- mouse brain. More importantly, direct application of IL-6 to cultured neurons resulted in a significant increase in Nox-2 expression and superoxide production which was fully blocked by the NFkB translocation inhibitor, SN50. It has recently been shown in epithelial tumor cells that IL-6 activates NFkB signaling through binding of unphosphorylated STAT3 (U-STAT3) to NFkB, promoting STAT3-NFkB translocation to the nucleus via nuclear targeting sequences in STAT3, resulting in subsequent transcription of a subset of NFkB-dependent genes[36]. However, it has become increasingly clear that NFkB signaling is highly complex, and is dependent on the stimulus, tissue, cell type, and context. For example, NFkB activated by TNFa induces transcription of a panel of genes, including IL-6 and RANTES, whereas NFkB activation by notch fails to induce these two genes. Therefore, although both Nox2 and p22phox are classic NFkB target genes which are induced by TNFa and IL-1b, this is to our knowledge, the first report that IL-6 acting through non-canonical NFkB signaling can directly induce Nox2 expression in neurons. Recent work has suggested that the brain maintains a physiological range of superoxide production that is required for normal nervous system function. Perturbations of superoxide levels in either direction are associated with impaired LTP, altered glutamatergic neurotransmission, and poorer cognitive perfor- mance. Elevated levels of superoxide associated with aging have specifically been shown to mediate deficits in learning and memory[27,49,50]. Thus, superoxide in brain has been the focus of several recent studies on cognitive deficits in aging. Increased neuronal superoxide production at the synapse, specifically the glutamatergic synapse, can affect a number of redox-regulated proteins important to synaptic activity and plasticity [18,51]. Dysregulation of the activity of these proteins would in turn affect normal glutamatergic transmission, and thus cognition. We recently reported that acute (days) elevation of superoxide production in brain by ketamine caused the loss of phenotype of PV-interneurons, as demonstrated by reduced PV and GAD67 immunoreactivity, which recovered over a few days. In contrast, here we show that sustained superoxide production by Nox2 during aging produces a significant decrease in these same neurons throughout hippocampus and prefrontal cortex. There was loss of 45%, 20%, 33%, and 17% of these neurons in hippocampal CA3, CA1, DG, and prefrontal cortex, respectively, in 24 month-old mice. This decline was completely rescued by treatment of mice with the small molecule SOD mimetic, C3, from middle age. Systemic treatment with C3 has been previously shown to reduce brain superoxide levels significantly. These results strongly suggest that sustained Nox-dependent superoxide pro- duction during aging may be responsible for the decline in PV- interneuron numbers. In addition, as predicted by our results showing that injected IL-6 induces brain Nox2, and that IL-6-/- mice lack induction of Nox2, there was preservation of these interneurons in aged IL-6-/- mice which was highly significant for CA3, and approached significance for CA1. While our data suggest that neurons are the primary source of Nox2-derived synaptic superoxide, and that in this model of non-pathological aging, very few astrocytes have detectable Nox2 protein or superoxide production, there is no way to rule out local Nox- dependent superoxide release by astrocytes at the synapse as contributing to the observed effects. PV-interneurons subserve an important role in cognitive processes, and are critically positioned to modulate hippocampal PLoS ONE | www.plosone.org 9 May 2009 | Volume 4 | Issue 5 | e5518