title: 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.
  
  
  CONCLUSIONS
  In spite of the complexity of the innate immune response, PKR inhibition could be an interesting anti-inflammatory strategy in AD.
authors.affiliation: Research Group on Brain Aging, GReViC EA 3808, 6 rue de la Milétrie BP 199, 86034 Poitiers Cedex, France.
authors.forename: J
authors.initials: J
authors.lastname: Couturier
authors.forename: M
authors.initials: M
authors.lastname: Paccalin
authors.forename: M
authors.initials: M
authors.lastname: Morel
authors.forename: F
authors.initials: F
authors.lastname: Terro
authors.forename: S
authors.initials: S
authors.lastname: Milin
authors.forename: R
authors.initials: R
authors.lastname: Pontcharraud
authors.forename: B
authors.initials: B
authors.lastname: Fauconneau
authors.forename: G
authors.initials: G
authors.lastname: Page
mesh_terms: D000544:Alzheimer Disease / Q000473:pathology / Q000503:physiopathology; D016229:Amyloid beta-Peptides / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007249:Inflammation / Q000139:chemically induced* / Q000503:physiopathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D016328:NF-kappa B / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D016207:Cytokines; D007093:Imidazoles; D007211:Indoles; D016328:NF-kappa B; D019892:eIF-2 Kinase; D051550:I-kappa B Kinase