title: Soluble amyloid triggers a myeloid differentiation factor 88 and interferon regulatory factor 7 dependent neuronal type-1 interferon response in vitro.
abstract: BACKGROUND
  Neuro-inflammation has long been implicated as a contributor to the progression of Alzheimer's disease in both humans and animal models. Type-1 interferons (IFNs) are pleiotropic cytokines critical in mediating the innate immune pro-inflammatory response. The production of type-1 IFNs following pathogen detection is, in part, through the activation of the toll-like receptors (TLRs) and subsequent signalling through myeloid differentiation factor-88 (Myd88) and interferon regulatory factors (IRFs). We have previously identified that neuronal type-1 IFN signalling, through the type-1 interferon alpha receptor-1 (IFNAR1), is detrimental in models of AD. Using an in vitro approach, this study investigated the TLR network as a potential production pathway for neuronal type-1 IFNs in response to Aβ.
  
  
  METHODS
  Wildtype and Myd88(-/-) primary cultured cortical and hippocampal neurons were treated with 2.5 μM Aβ1-42 for 24 to 72 h or 1 to 10 μM Aβ1-42 for 72 h. Human BE(2)M17 neuroblastoma cells stably expressing an IRF7 small hairpin RNA (shRNA) or negative control shRNA construct were subjected to 7.5 μM Aβ1-42/Aβ42-1 for 24 to 96 h, 2.5 to 15 μM Aβ1-42 for 96 h or 100 ng/ml LPS for 0.5 to 24 h. Q-PCR was used to analyse IFNα, IFNβ, IL-1β, IL-6 and TNFα mRNA transcript levels. Phosphorylation of STAT-3 was detected by Western blot analysis, and cell viability was assessed by MTS assay.
  
  
  RESULTS
  Reduced IFNα, IFNβ, IL-1β, IL-6 and TNFα expression was detected in Aβ1-42-treated Myd88(-/-) neurons compared to wildtype cells. This correlated with reduced phosphorylation of STAT-3, a downstream type-1 IFN signalling mediator. Significantly, Myd88(-/-) neuronal cultures were protected against Aβ1-42-induced neurotoxicity compared to wildtype as determined by MTS assay. Knockdown of IRF7 in M17 cells was sufficient in blocking IFNα, IFNβ and p-STAT-3 induction to both Aβ1-42 and the TLR4 agonist LPS. M17 IRF7 KD cells were also protected against Aβ1-42-induced cytotoxicity.
  
  
  CONCLUSIONS
  This study confirms that the neuronal type-1 IFN response to soluble amyloid is mediated primarily through TLRs. This production is dependent upon Myd88 and IRF7 signalling. This study suggests that targeting this pathway to modulate neuronal type-1 IFN levels may be beneficial in controlling Aβ-induced neurotoxicity.
authors: Department of Pharmacology and Therapeutics, University of Melbourne, 8th floor, Medical building, Grattan St, Parkville, Melbourne, 3010, VIC, Australia. mminter.student@unimelb.edu.au.
authors: Myles Robert
authors: MR
authors: Minter
authors: Department of Pharmacology and Therapeutics, University of Melbourne, 8th floor, Medical building, Grattan St, Parkville, Melbourne, 3010, VIC, Australia. bmain@student.unimelb.edu.au.
authors: Bevan Scott
authors: BS
authors: Main
authors: Department of Pharmacology and Therapeutics, University of Melbourne, 8th floor, Medical building, Grattan St, Parkville, Melbourne, 3010, VIC, Australia. kbrody@unimelb.edu.au.
authors: Kate Maree
authors: KM
authors: Brody
authors: Department of Pharmacology and Therapeutics, University of Melbourne, 8th floor, Medical building, Grattan St, Parkville, Melbourne, 3010, VIC, Australia. haiqingz@unimelb.edu.au.
authors: Moses
authors: M
authors: Zhang
authors: Department of Pharmacology and Therapeutics, University of Melbourne, 8th floor, Medical building, Grattan St, Parkville, Melbourne, 3010, VIC, Australia. juliett@unimelb.edu.au.
authors: Juliet Marie
authors: JM
authors: Taylor
authors: Department of Pharmacology and Therapeutics, University of Melbourne, 8th floor, Medical building, Grattan St, Parkville, Melbourne, 3010, VIC, Australia. pcrack@unimelb.edu.au.
authors: Peter John
authors: PJ
authors: Crack
mesh_terms: D016229:Amyloid beta-Peptides / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D050839:Interferon Regulatory Factor-7 / Q000378:metabolism*; D007370:Interferon Type I / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000473:pathology; D010446:Peptide Fragments / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
chemical_list: D016229:Amyloid beta-Peptides; D050839:Interferon Regulatory Factor-7; D007370:Interferon Type I; D008070:Lipopolysaccharides; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D010446:Peptide Fragments; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C075222:amyloid beta-protein (1-42)