IL-6 and Cognitive Changes Figure 1. Aging increases plasma and brain levels of IL-6, and induces Nox2 protein expression which is blocked by targeted deletion of IL-6. (a) Plasma IL-6 was measured by ELISA in young (4 month) and old (24 month) C57BL6 mice and was significantly increased in old mice; n = 4, mean6SEM (F(1,6) = 9.530, P = 0.021). (b) IL-6 mRNA expression was determined by real-time PCR in brain and was also increased significantly in old animals; n = 4, mean6SEM (F(1,6) = 10.288, P = 0.018). (c) Confocal imaging of brain slices prepared from young (4 months) and old (23 months) mice depicting Nox2 (green) and MAP2 (red) immunoreactivity in the CA1 hippocampal region. Nox2 expression was increased in old animals with respect to young, whereas this staining was absent in slices prepared from Nox2-deficient (gp91phox -/-, 12 months old) mice. Bar = 100 mm. For immunofluorescence detection AlexaFluor 488 and 568 conjugated secondary antibodies were used. Merged images show co- localization in yellow. (d, e) Western blot analysis of brain protein extracts obtained from young and old wild-type and IL-6-/- mice showed increased Nox2 expression with age which was significantly lower in old IL-6-/- mouse brain (n = 4, p,0.05, mean6SEM). Expression in young animals, either WT or IL-6-/-, was below detection levels. Lack of the Nox2 band in brain proteins extracted from an old gp91phox-/- mouse (far right lane) confirmed specificity of the anti-Nox2 antibody and location of Nox2- specific band. N = 4 animals per age per genotype (F(3,12) = 9.704, P = 0.002, *young versus old, P,0.05; # old versus old-IL6-/-, P,0.05 by Tukey’s). doi:10.1371/journal.pone.0005518.g001 resulted in substantially higher superoxide levels, as documented by DHE oxidation (Fig. 2d, bottom). Effects of IL-6 and IL-6 deficiency on Nox2 activity: oximetry and EPR spectroscopy in synaptosomes from IL- 6-treated and IL-6-/- mice To further study the effects of IL-6 on neuronal Nox2 activation in aging, we carried out a series of studies in synaptosomes prepared from young and old wild-type and IL-6-/- mice, and in old mice injected with IL-6. We have previously reported the use of synaptosomes to carry out concurrent measurement of oxygen consumption (oximetry) and superoxide radical production (by electron paramagnetic resonance EPR)[18] allowing assessment of both Nox-dependent oxygen utilization and superoxide produc- tion in the same sample. NADPH-stimulated O2 consumption was significantly higher in synaptosomes prepared from old animals compared to young (F(1,16) = 11.03, P,0.05), and this increase was completely prevented by co-incubation with the Nox inhibitor, apocynin (F(1,16) = 40.95, P,0.0001) (Fig. 3a). Superoxide pro- duction in the same samples, assessed by EPR, was also higher in old animals (F(1,4) = 24.92, P,0.001) (Figure 3b, c). The increase in synaptosomal superoxide production in old mice was signifi- cantly blocked by two different Nox inhibitors, aminoethyl- benzenesulfonylfluoride (AEBSF) and apocynin (Figure 3c). Sta- tistics for 200 mM apocynin were: young: 24.166.5% inhibition, P = 0.02; old: 33.563.3% inhibition, P = 0.01, and for 200 mM AEBSF were: young: 25.264.1% inhibition, P = 0.003; old: 32.4610.7% inhibition, P = 0.04. Control studies verified that neither apocynin nor AEBSF were acting as direct superoxide scavengers, and we confirmed that synaptic mitochondrial respiration and superoxide production was not altered by the conditions used to study Nox2 activity (Fig. S2). Previous studies have reported that Nox2 and Nox4 are the only NADPH oxidase isoforms expressed in the adult mouse nervous PLoS ONE | www.plosone.org 3 May 2009 | Volume 4 | Issue 5 | e5518