β-Amyloid peptide (Aβ) is a major protein in the brain associated with Alzheimer's and Parkinson's diseases. The purpose of this study was to investigate the role of macrophage antigen-1 (MAC1) receptor, an integrin scavenger receptor in microglia, and subsequent signaling events in mediating Aβ-induced neurotoxicity. We have previously reported that NADPH oxidase (PHOX) on microglia and superoxide produced by PHOX are critical for Aβ-induced loss of dopaminergic neurons. However, the upstream signaling pathway of superoxide production remains unclear.

Background: Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions.Methods: Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (³H-naloxone) binding assays were performed in wild type and gp91^phox-/-neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47^phoxto plasma membrane was assessed by western blot.Results: Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 μM, respectively. Competitive binding of³H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively.³H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91^phox; in contrast, reduced³H-naloxone binding was found in neutrophils deficient in gp91^phoxor in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of³H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91^phoxantibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47^phoxto the membrane, leading to NOX2 inactivation.Conclusions: Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.