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

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 beta-amyloid (A beta) peptides, the nuclear factor-kappa B (NF-kappa B) pathway could be an interesting target. In virus-infected cells, double-stranded RNA-dependent protein kinase (PKR) controls the NF-kappa B 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 A beta 42-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 A beta neurotoxicity (72 h), co-cultures were treated with compound C16, a specific inhibitor of PKR. Levels of tumor necrosis factor-alpha (TNF alpha), interleukin (IL)-1 beta, and IL-6 were assessed by ELISA. Levels of P-T451-PKR and activation of I kappa B, NF-kappa B and caspase-3 were assessed by western blotting. Apoptosis was also followed using annexin V-FITC immunostaining kit. Subcellular distribution of P-T451-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 A beta 42-induced activation of I kappa B and NF-kappa B, strongly decreased production and release of tumor necrosis factor (TNF alpha) and interleukin (IL)-1 beta, 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.