BackgroundInflammation 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.MethodsPrimary 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 testResultsIn 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.ConclusionIn spite of the complexity of the innate immune response, PKR inhibition could be an interesting anti-inflammatory strategy in AD.
Alzheimer Disease (AD)2 is the leading cause of dementia in the elderly, affecting 25 million people in the world with about 5% of genetic origin. The biological features of AD are neuronal loss, senile plaques predominantly composed of amyloid  peptide (A), and the presence of neurofibrillary tangles, leading to a progressive deterioration of cognitive function with loss of memory. Unfortunately, no definitive therapy for AD exists.The involvement of A is clearly established, but mechanisms that underlie neuronal death are currently under investigation. For 10 years, research has focused on pathways controlling translation (1-5). They revealed that activated double-stranded RNA-dependent protein kinase (PKR), which phosphorylates the ␣-subunit of eIF2, was associated with degenerating neurons in AD brains (6 -8). Furthermore, the levels of phosphorylated forms of PKR and eIF2␣ were significantly increased in AD cellular models exposed to A42 (2, 5) in the brain of AD transgenic mice (1, 9) and in lymphocytes of AD patients (10). These modifications were negatively correlated with cognitive and memory test scores performed in AD patients (3, 10, 11). Besides its important role as a translational regulator, PKR is a key mediator in different signaling pathways. Indeed, among its downstream targets, the Fas-associated protein with a death domain (FADD) and subsequent caspase-8 are responsible for PKR-induced apoptosis in virus-infected cells (12). Furthermore, the overexpression of a dominant-negative FADD construct rescued SH-SY5Y cells from either tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or A-induced neurotoxicity (13). FADD could be involved in apoptosis through its nuclear localization by interacting with the methylCpG binding domain protein 4 (MDB4), a protein known to be implicated in DNA repair, but that could promote apoptosis when associated with FADD (14). Furthermore, many postmortem studies have reported the role of the FADD-linked death receptor signaling pathway in the pathobiology of AD (15-17).However, control of FADD under the PKR activation in AD remains unknown. The present study performed a detailed analysis of PKR and FADD interaction in A-treated cells and in APP SL PS1 KI transgenic mice. The purpose is to determine if inhibition of PKR could reduce apoptosis through the decrease in this death adaptor activation. Here, we showed a physical interaction between PKR and FADD in nuclei of A42-treated SH-SY5Y and in APP SL PS1 KI mice. Furthermore, PKR inhibitory treatments prevented not only the nuclear translocation of FADD but also significantly decreased caspase-3 and -8 activities induced by A neurotoxicity.
EXPERIMENTAL PROCEDURES
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