Fibrillar beta-amyloid peptide Aβ1–40 activates microglial proliferation via stimulating TNF-α release and H2O2 derived from NADPH oxidase: a cell culture study

Aiste Jekabsone,Anna Tickler,Palwinder K Mander,Guy C Brown,Martyn Sharpe

doi:10.1186/1742-2094-3-24

Aiste Jekabsone, Anna Tickler + Show 3 more

Open Access

https://doi.org/10.1186/1742-2094-3-24

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Abstract

BackgroundAlzheimer'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.MethodsPrimary 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.ResultsWe found that 1 μM fibrillar (but not soluble) Aβ1–40 peptide induced microglial 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.ConclusionThese 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.

Highlights

Alzheimer's disease is characterized by the accumulation of neuritic plaques, containing activated microglia and β-amyloid peptides (Aβ)
Materials Apocynin was purchased from Calbiochem; EUK-8 and EUK-134 were synthesized as previously described in [18]; Amplex Red, Dulbecco's Modified Eagle Medium (DMEM), Earl's Balanced Salt Solution (EBSS), Isolectin GS-IB4 from Griffonia simplicifolia conjugated with AlexaFluor488, Phosphate-Buffered Saline (PBS), readyto-use Streptavidin-horse radish peroxidase (HRP) conjugate were purchased from Invitrogen; Anti-rat TNF-α monoclonal antibody was purchased from R&D Systems; soluble TNF receptor inhibitor/Fc chimera was purchased from GenScript Corporation; Biotin anti-rat TNF-α polyclonal antibody was purchased from Insight Biotechnology Ltd.; all other chemicals were purchased from Sigma
When microglia were incubated with fibrillar Aβ1–40 for 48 hours, cells continued to proliferate to 372 ± 53% of the initial number, whereas in the untreated control the number of cells increased to 223 ± 28% of the initial number (Fig. 2)

Summary

Introduction

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. In this study, we investigated whether Aβ can stimulate microglial proliferation and cytokine production via activation of NADPH oxidase to produce hydrogen peroxide. Alzheimer's disease is characterised by neuritic plaques that contain dead and dying neurons and their processes, inflammatory-activated microglia and β-amyloid peptides Aβ1–40 and Aβ1–42 [1,2]. Β-Amyloid and cytokines cause inflammatory activation of glia, and inflammatoryactivated microglia are consistently found in the neuritic plaques of Alzheimer's patients [1,2]. We and others believe that NADPH oxidase activation is the key event converting resting microglia to activated, proliferating, cytotoxic microglia; and, that blocking oxidase activation may block inflammatory neurodegeneration [3,6,8,9,10,11,12]

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