Soluble amyloid triggers a myeloid differentiation factor 88 and interferon regulatory factor 7 dependent neuronal type-1 interferon response in vitro

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 A beta. Methods: Wildtype and Myd88(-/-) primary cultured cortical and hippocampal neurons were treated with 2.5 mu M A beta 1-42 for 24 to 72 h or 1 to 10 mu M A beta 1-42 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 mu M A beta 1-42/A beta 42-1 for 24 to 96 h, 2.5 to 15 mu M A beta 1-42 for 96 h or 100 ng/ml LPS for 0.5 to 24 h. Q-PCR was used to analyse IFN alpha, IFN beta, IL-1 beta, IL-6 and TNF alpha mRNA transcript levels. Phosphorylation of STAT-3 was detected by Western blot analysis, and cell viability was assessed by MTS assay. Results: Reduced IFN alpha, IFN beta, IL-1 beta, IL-6 and TNF alpha expression was detected in A beta 1-42-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 A beta 1-42-induced neurotoxicity compared to wildtype as determined by MTS assay. Knockdown of IRF7 in M17 cells was sufficient in blocking IFNa, IFN beta and p-STAT-3 induction to both A beta 1-42 and the TLR4 agonist LPS. M17 IRF7 KD cells were also protected against A beta 1-42-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 A beta-induced neurotoxicity.