with each experiment including 4–6 replicates for each treatment. Reagents: 3% hydrogen peroxide solution (Sigma-Aldrich); recombinant mouse Wnt-5a (R&D Systems); anti-mouse Wnt-5a antibody (R&D Systems); anti-Fz5 antibody (against the extracel- lular cysteine-rich domain of human Fz5) (R&D Systems); Foxy5 peptide (Inbiolabs); Box5 peptide (Inbiolabs); recombinant mouse IL-10 (R&D Systems); and activated protein C (APC) (Sigma- Aldrich). Statistical analysis Data were expressed as the means 6 SEM. Statistical analysis was performed with the Prism software (GraphPad). We used the Student’s two-tailed t-test for statistical comparisons between any two groups, and one-way ANOVA analyses with a Bonferroni post-hoc test for comparisons between multiple groups. Results Increased expression of key proteins in the non-canonical Wnt signaling pathway in the hippocampus of AD mouse models Previous studies revealed down-regulation of canonical Wnt signaling in the brain of APPswe/PSEN1DE9 AD mouse models [27]. Consistent with these findings, we observed decreased expression of proteins in the canonical pathway, including Wnt3a, Fz1 and b-catenin (data not shown). In contrast, in preliminary studies, we also observed up-regulated expression of several proteins implicated in the non-canonical Wnt signaling pathway, including Wnt5a and its receptor Fz5, at various postnatal stage (3.5–9.5 months). At 3.5 months of age, APP transgene was clearly expressed (Fig. 1A). APP processing expected to generate Ab was evident in the transgenic hippocampi (Fig. 1B). In addition, immunohistochemistry staining with an antibody that recognized Ab revealed clustered signals in the cell body layer (Fig. 1C), which may include Ab that starts accumulating. Because memory deficits have not manifested at this stage [44], we reasoned that the up- regulation of non-canonical Wnt signaling is potentially an early molecular abnormality during AD pathogenesis. Therefore, we performed more detailed analysis on the non-canonical Wnt signaling in mice at 3.5 months of age. Western blotting analysis showed that, compared with wild-type controls, Wnt5a protein increased 1.5 fold in the hippocampus of the 26Tg mouse (p,0.01; Fig. 2A). Similarly, Fz5 protein was also significantly up- regulated in the 26Tg hippocampus (1.6 fold; p,0.01; Fig. 2B). Because activation of non-canonical Wnt signaling can cause aCaMKII phosphorylation [45], we next determined the protein level of phosphorylated aCaMKII (pT286-aCaMKII). pT286- aCaMKII was increased 2.6 fold (p,0.05; Fig. 2C). Fluorescent immunostaining showed that, in comparison with control, the increase of Wnt5a (Fig. 2D) and Fz5 (Fig. 2E) occurred in all hippocampal fields, including CA1, CA2/CA3 and dentate gyrus (DG), of the 26Tg mouse, which resembled the spatial pattern of Ab plaques (Fig. 1C). These data together suggest that non- canonical Wnt signaling is up-regulated in the 26Tg AD hippocampus. Rapid up-regulation of non-canonical Wnt signaling by Ab oligomers in primary neuron culture We next sought to understand the mechanism by which non- canonical Wnt signaling is activated in the AD mouse brain. Because Ab is a key pathological agent for AD, we hypothesized that it causes the up-regulated expression of Wnt5a and other relevant proteins measured in 26Tg mice. To test this hypothesis, Wnt5a in AD Figure 2. Wnt5a/CaMKII signaling is up-regulated in the hippocampus of the 26Tg AD mouse model. A, B, and C. Protein levels of Wnt5a (A), Fz5 (B) and pT286-aCaMKII (C) in the wild-type and 26Tg hippocampus. In summary graphs of quantitative data, the levels of target proteins were normalized with the b-actin loading control and expressed as relative units to the wild-type controls. Data presented in graphs are means 6 SEM from six pairs of mice. * p,0.05. D and E. Distribution of Wnt5a (D) and Fz5 (E) protein in the wild-type and 26Tg hippocampus. DAPI staining was performed to visualize the cell bodies. doi:10.1371/journal.pone.0022920.g002 PLoS ONE | www.plosone.org 3 August 2011 | Volume 6 | Issue 8 | e22920