Wnt5a in AD Figure 7. Wnt5a evokes cytokine release. A. The temporal profiles of IL-1b in media of Wnt5a-treated cortical cultures measured by ELISA. B. Heat-inactivation abolished the activity of Wnt5a in eliciting IL-1b release. C. The temporal profiles of TNF-a in media after Wnt5a treatment. D. Effects of heat-inactivation on the Wnt5a activity in eliciting TNF-a release. * p,0.05 (vs. 0 h in A and C, or vs. vehicle in B and D). doi:10.1371/journal.pone.0022920.g007 treatment but continued for the rest of the time that measurements were recorded (Fig. 6C). It is possible that the lag in up-regulation of IkB-a functions to resolve the Wnt5a-initiated inflammatory response. Inflammatory response culminates with the secretion of an array of inflammatory cytokines. If Wnt5a indeed induces the inflam- matory response, specific inflammatory cytokines must be secreted after Wnt5a administration. Thus, we used ELISA to measure IL- 1b and TNF-a in culture media after Wnt5a treatment for 0.5, 1, 2, 6, 12, or 24 hrs. We observed that Wnt5a stimulation caused a 2.7-fold increase in IL-1b at 12 h, while no change was detected at other time points (Fig. 7A). On the other hand, TNF-a increased significantly at 6 h (2.3 fold), 12 h (4.3 fold) and 24 h (3.1 fold) (Fig. 7C). However, this effect of Wnt5a was completely abolished when the protein was heat-inactivated (Fig. 7B and D). These results together strongly suggest that Wnt5a can elicit the inflammatory response in neuron cultures. Blockage of Wnt5a signaling impairs Ab-induced up-regulation of proinflammatory cytokines Because Ab evoked inflammation-mediated neurotoxicity, we next investigated the effect of Wnt5a signaling inhibition on the Ab-elicited inflammatory response. As shown in Fig. 8A, Ab oligomers (500 nM) significantly increased IL-1b release (3.4 fold; p,0.05). Box5 peptide (200 mM), anti-Wnt5a (2 mg/ml) or anti-Fz5 (2 ug/ml) antibody suppressed the increase of IL-1b induced by Ab, by 58%, 59% and 55%, respectively (all 3 with p,0.05). In contrast, Foxy5 (200 mM), an agonist of Wnt5a, increased IL-1b release (1.5 fold, compared with Ab alone; p,0.05; Fig. 8A). Box5, anti-Wnt5a and anti-Fz5 antibodies caused similar effects on Ab-induced TNF-a release (Fig. 8B). These results together demonstrate that the suppression of Wnt5a signaling inhibits Ab-evoked inflammatory responses in cortical neuron cultures. Figure 8. Wnt5a is critical for Ab-elicited cytokine release. A. Inhibition of Wnt5a/Fz5 signaling impaired Ab-evoked IL-1b increase in media. B. Inhibition of Wnt5a/Fz5 signaling impaired Ab-evoked TNF-a increase in media. ELISA assay of IL-1b (A) and TNF-a (B) secreted in cortical cultures treated for 12 hrs with Ab (500 nM) or Ab and Box5 (200 mM), anti-Wnt5a (2 mg/ml) or anti-Fz5 (2 mg/ml) antibody. Foxy5, a Wnt5a agonist, potentiates the cytokine release. * p,0.05; ** p,0.01 (vs. vehicle). C. A working model for the role of Wnt5a signaling in AD pathogenesis. doi:10.1371/journal.pone.0022920.g008 Discussion In this study, we found that non-canonical Wnt5a signaling is up-regulated in mouse brains prior to AD phenotypes and by Ab peptide in cortical neuron cultures. The up-regulated Wnt5a signaling contributes to the inflammation-dependent Ab neuro- toxicity in cultures. We also found that Wnt5a up-regulates inflammation regulatory proteins and proinflammatory cytokines and that Wnt5a is required for the Ab-induced proinflammatory cytokines. These observations collectively suggest the following working model (Fig. 8C): accumulation of Ab in the brain aberrantly up-regulates Wnt5a signaling, which in turn evokes an inflammatory response that causes neurodegeneration or cell death in AD brains. The observed up-regulation of Wnt5a signaling is probably an early etiologically relevant event during AD development. Both Wnt5a and Fz5 proteins significantly increase in the APPswe/ PSEN1DE9 hippocampus at the age of 3.5 months (Fig. 2). Previous studies showed that this AD mouse model started to accumulate Ab plaques after 4 months of age [61] and did not develop cognitive impairments until 5–7 months of age [44]. Thus, PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e22920