Background: Alzheimer's disease is characterized by the accumulation of neuritic plaques,containing activated microglia and β-amyloid peptides (Aβ). Fibrillar Aβ can activate microglia,resulting in production of toxic and inflammatory mediators like hydrogen peroxide, nitric oxide,and cytokines. We have recently found that microglial proliferation is regulated by hydrogen peroxide derived from NADPH oxidase. Thus, in this study, we investigated whether Aβ can stimulate microglial proliferation and cytokine production via activation of NADPH oxidase to produce hydrogen peroxide.Methods: Primary mixed glial cultures were prepared from the cerebral cortices of 7-day-old Wistar rats. At con... More
Background: Alzheimer's disease is characterized by the accumulation of neuritic plaques,containing activated microglia and β-amyloid peptides (Aβ). Fibrillar Aβ can activate microglia,resulting in production of toxic and inflammatory mediators like hydrogen peroxide, nitric oxide,and cytokines. We have recently found that microglial proliferation is regulated by hydrogen peroxide derived from NADPH oxidase. Thus, in this study, we investigated whether Aβ can stimulate microglial proliferation and cytokine production via activation of NADPH oxidase to produce hydrogen peroxide.Methods: Primary mixed glial cultures were prepared from the cerebral cortices of 7-day-old Wistar rats. At confluency, microglial cells were isolated by tapping, replated, and treated either with or without Aβ. Hydrogen peroxide production by cells was measured with Amplex Red and peroxidase. Microglial proliferation was assessed under a microscope 0, 24 and 48 hours after plating. TNF-α and IL-1β levels in the culture medium were assessed by ELISA. Results: We found that 1 μM fibrillar (but not soluble) Aβ1− 40 peptide induced microglia proliferation and caused release of hydrogen peroxide, TNF-α and IL-1β from microglial cells.Proliferation was prevented by the NADPH oxidase inhibitor apocynin (10 μM), by the hydrogen peroxide-degrading enzyme catalase (60 U/ml), and by its mimetics EUK-8 and EUK-134 (20 μM);as well as by an antibody against TNF-α and by a soluble TNF receptor inhibitor. Production of TNF-α and IL-1β, measured after 24 hours of Aβ treatment, was also prevented by apocynin,catalase and EUKs, but the early release (measured after 1 hour of Aβ treatment) of TNF-α was
insensitive to apocynin or catalase.Conclusion: These results indicate that Aβ1− 40-induced microglial proliferation is mediated both by microglial release of TNF-α and production of hydrogen peroxide from NADPH oxidase. This
suggests that TNF-α and NADPH oxidase, and its products, are potential targets to prevent Aβ-induced inflammatory neurodegeneration.
