The possibility that P2X7 receptor (P2X7R) expression in microglia would mediate neuronal damage via reactive oxygen species (ROS) production was examined in the APPswe/PS1dE9 mouse model of Alzheimer's disease (AD). P2X 7 R was predominantly expressed in CD11b-immunopositive microglia from 3 months of age before Aβ plaque formation. In addition, gp91 phox , a catalytic subunit of NADPH oxidase, and ethidium fluorescence were detected in P2X 7 R-positive microglial cells of animals at 6 months of age, indicating that P2X 7 R-positive microglia could produce ROS. Postsynaptic density 95-positive dendrites showed significant damage in regions positive for P2X 7 R in the cerebral cortex of 6 month-old mice. Taken together, up-regulation of P2X 7 R activation and ROS production in microglia are parallel with Aβ increase and correlate with synaptotoxicity in AD.

Present study demonstrated that fibrillar β-amyloid peptide (fAβ 1-42 ) induced ATP release, which in turn activated NADPH oxidase via the P2X 7 receptor (P2X 7 R). Reactive oxygen species (ROS) production in fAβ1-42-treated microglia appeared to require Ca 2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca 2+. Considering previous observation of superoxide generation by Ca 2+ influx through P2X 7 R in microglia, we hypothesized that ROS production in fAβ-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAβ1-42-induced Ca 2+ influx was mediated through P2X 7 R activation. In serial experiments, we found that microglial pretreatment with the P2X 7 R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (100 µM) or oxidized ATP (100 µM) inhibited fAβ-induced Ca 2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAβ1-42-stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAβ-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.

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A degree of brain inflammation is required for repair of damaged tissue, but excessive inflammation causes neuronal cell death. Here, we observe that IL-10 is expressed in LPS-injected rat cerebral cortex, contributing to neuronal survival. Cells immunopositive for IL-10 were detected as early as 8 h post-injection and persisted for up to 3 d, in parallel with the expression of IL-1β, TNF-α , and iNOS. Double immunofluorescence staining showed that IL-10 expression was localized mainly in activated microglia. Next, we examined the neuroprotective effects of IL-10 using IL-10 neutralizing antibody (IL-10NA). Blockade of IL-10 action caused a significant loss of neurons both 3 d and 7 d after LPS injection. Further, the induction of mRNA species encoding IL-1β, TNF-α , and iNOS, reactive oxygen species (ROS) production, and NADPH oxidase activation, increased after co-injection of LPS and IL-10NA, compared to the levels seen after injection of LPS alone. Taken together, these results clearly suggest that LPS-induced endogenous expression of IL-10 in microglia contributes to neuronal survival by inhibiting brain inflammation.

Recent studies have reported that the "cholinergic anti-inflammatory pathway" regulates peripheral inflammatory responses via α 7 nicotinic acetylcholine receptors (α 7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-α and prostaglandin E 2 in microglial cultures. In a previous study we showed that ATP released by β -amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X7 receptor (P2X7R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar β amyloid peptide (1-42) (fAβ 1-42)-induced ROS production by modulating ATP efflux-mediated Ca 2+ influx through P2X 7 R. Nicotine inhibited ROS generation in fAβ 1-42-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and α -bungarotoxin, a selective α 7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca 2+ influx in fAβ 1-42 -stimulated microglia. Moreover, ATP release from fAβ 1-42-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca 2+ influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X7R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fAβ 1-42 -stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X 7 R.