Wnt5a in AD Wnt5a (200 ng/ml)+Ab/H2O2). F. Protein levels of pT286-aCaMKII in cortical cultures treated with Ab (500 nM), H2O2 (100 mM), or the combination of Ab and H2O2. G. Protein levels of pT286-aCaMKII in cortical cultures treated with anti-Wnt5a (4 mg/ml), anti-Fz5 (4 mg/ml) antibodies or Box5 (200 mM), in the presence of Ab (500 nM) and H2O2 (100 mM). * p,0.05; NS, not significant. doi:10.1371/journal.pone.0022920.g004 These results demonstrated that the non-canonical Wnt5a signaling is up-regulated by Ab peptide, especially the oligomers. Suppression of Wnt5a signaling alleviates Ab neurotoxicity The observations of the upregulation of the Wnt5a signaling in early AD mouse brains and by Ab in cortical cultures suggest a potential role of Wnt5a signaling in AD pathogenesis. We next sought to directly test this idea by determining the significance of Ab-mediated Wnt5a up-regulation on Ab cytotoxicity in cortical cultures. Previous work revealed concentration-dependent neuro- toxicity of Ab peptides [37,49,50], with high concentrations of Ab (.5.0 mM) causing neuron death in cultures and low concentra- tions of Ab (0.1,1.0 mM) sensitizing neurons to become vulnerable to further stresses [50]. To mimic the early AD brain condition when the non-canonical Wnt signaling proteins are aberrantly upregulated, in these experiments we used a relatively low concentration (500 nM) of Ab that can upregulate Wnt5a and Fz5 (Fig. 3). Ab oligomer at this concentration did not cause cell death, as measured by MTT assays (Fig. 4A). We also used trypan blue staining to confirm this observation (Fig. S1). This finding is consistent with the idea that in the early stages of AD pathogenesis Ab may not cause neuron death. Next, we wanted to investigate if Ab at this concentration causes the cells to become more vulnerable to detrimental stresses that are relevant to AD pathogenesis. To this end, we tested the effect of Ab on the sensitivity of the cultures to hydrogen peroxide (H2O2) stresses. Hydrogen peroxide was chosen because it is generated during the very early stages of aggregation of the amyloid peptides [51] and is critically involved in AD pathogenesis [49,52]. At low concentra- tions (25–100 mM), hydrogen peroxide did not cause cell death by itself; however, when co-administrated with Ab oligomers (500 nM) it caused marked cell death (Fig. 4A, Fig. S1). These results indicate that neurons exposed to 500 nM Ab are more vulnerable to other detrimental factors. We also showed that H2O2 neither stimulated aCaMKII phosphorylation by itself nor significantly enhanced the Ab-induced phsphorylation (Fig. 4F), indicating that H2O2 did not activate the non-canonical pathway. To determine the potential role of Wnt5a up-regulation in Ab- induced sensitization to H2O2 stress, we neutralized Wnt5a in media with specific anti-Wnt5a antibody. As shown in Fig. 4C, the antibody (0.5–8.0 mg/ml) displayed a rescue effect on cell death in a concentration-dependent manner, which reached statistical significance after 4.0 mg/ml. Recent studies identified a modified Wnt5a-derived hexapeptide (Box5) that can specifically antagonize Wnt5a in malignant melanoma cells [53]. Similar to the anti- Wnt5a antibody, Box5 also had a rescue effect on Ab/H2O2- induced cell death in a concentration-dependent manner (Fig. 4E). Because Fz-5 is a Wnt5a receptor [54], we further tested the effect of anti-Fz5 antibody (against the extracellular cystein-rich domain). Consistent with the findings with anti-Wnt5a antibody and Box5, the anti-Fz5 antibody also had a rescue effect (Fig. 4C). We further tested the effect of, anti-Wnt5a, anti-Fz5 antibodies and Box5 on the phosphorylation of aCaMKII, a molecular marker of the activation of the non-canonical Wnt/Ca2+ pathway. The results showed that all these reagents significantly blocked Ab/H2O2-induced up-regulation of p-aCaMKII (Fig. 4G), and thus provide an independent molecular confirmation of the activity of anti-Wnt5a, anti-Fz5 antibodies and Box5. These results together strongly suggest that Ab-induced Wnt5a up- regulation at least partially mediates Ab neurotoxicity. Exogenous Wnt5a potentiates Ab neurotoxicity Because Ab-induced Wnt5a up-regulation is critical for Ab neurotoxicity (Fig. 4C and E), we next investigated if exogenous Wnt5a can potentiate Ab/H2O2-induced cell injury. To this end, we tested the effect of recombinant Wnt5a on Ab/H2O2 neurotoxicity. As shown in Fig. 4B, compared with Ab/H2O2- treated cultures, addition of purified recombinant Wnt5a protein (50–800 ng/ml) increased cell death in a concentration-dependent manner. This potentiation activity of Wnt5a was completely abolished when the protein was heat-inactivated (Fig. 4B). This observation indicates that the biochemical activity of Wnt5a rather than the mere presence of Wnt5a peptide is essential for the potentiation. In addition, Foxy5, a Wnt5a-derived hexapeptide (the same peptide as Box5 but differently modified) that can mimic Wnt5a-induced activities in malignant melanoma and in breast cancer cells [55], was also able to potentiate the neurotoxicity of Ab/H2O2 (Fig. 4D). These results indicate that Wnt5a is sufficient to elicit neurotoxicity. Figure 5. Anti-inflammatory factors alleviate Ab neurotoxicity. A. Effects of IL-10 on cell viability of cortical cultures challenged with Ab/H2O2 for 24 hrs. B. Effects of APC. Data (means 6 SEM) are from three independent experiments. * p,0.05; ** p,0.01; # p,0.05 (vs. the group treated with active IL-10 (1 ng/ml) or APC (1 mg/ml) in the presence of Ab/H2O2). doi:10.1371/journal.pone.0022920.g005 PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e22920