Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures.

Abstract : BACKGROUND: Inflammation may be involved in the pathogenesis of Alzheimer's disease (AD). There has been little success with anti-inflammatory drugs in AD, while the promise of anti-inflammatory treatment is more evident in experimental models. A new anti-inflammatory strategy requires a better understanding of molecular mechanisms. Among the plethora of signaling pathways activated by β-amyloid (Aβ) peptides, the nuclear factor-kappa B (NF-κB) pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR) controls the NF-κB signaling pathway. It is well-known that PKR is activated in AD. This led us to study the effect of a specific inhibitor of PKR on the Aβ42-induced inflammatory response in primary mixed murine co-cultures, allowing interactions between neurons, astrocytes and microglia. METHODS: Primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days, then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. Before exposure to Aβ neurotoxicity (72 h), co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, and IL-6 were assessed by ELISA. Levels of PT451-PKR and activation of IκB, NF-κB and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of PT451-PKR was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. Data were analysed using one-way ANOVA followed by a Newman-Keuls' post hoc test RESULTS: In these co-cultures, PKR inhibition prevented Aβ42-induced activation of IκB and NF-κB, strongly decreased production and release of tumor necrosis factor (TNFα) and interleukin (IL)-1β, and limited apoptosis. CONCLUSION: In spite of the complexity of the innate immune response, PKR inhibition could be an interesting anti-inflammatory strategy in AD.
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Journal of Neuroinflammation, BioMed Central, 2011, 8 (1), pp.72. 〈10.1186/1742-2094-8-72〉
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Julien Couturier, Marc Paccalin, Milena Morel, Faraj Terro, Serge Milin, et al.. Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures.. Journal of Neuroinflammation, BioMed Central, 2011, 8 (1), pp.72. 〈10.1186/1742-2094-8-72〉. 〈inserm-00663637〉

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