Wnt5a in AD Figure 3. Up-regulation of proteins in the non-canonical Wnt signaling pathway by Ab peptide in cortical neuron cultures. A. Western blot analysis of the preparations of Ab (1–42) oligomers and fibrils. Ab preparations were, separated on SDS–PAGE (4–12% NuPAGE bis-Tris gel), and probed with the monoclonal antibody 6E10.. B, C, and D. Protein levels of Wnt5a (B), Fz5 (C) and pT286-aCaMKII (D) in cortical cultures treated with 500 nM of monomeric, oligomeric, and fibrillar Ab peptide for 24 hrs. E, F, and G. Concentration-dependent effects of Ab oligomer on the protein levels of Wnt5a (E), Fz5 (F) and pT286-aCaMKII (G). Although the dose effect of Ab on Wnt5a was most clearly observed at 24 hours post Ab administration, the effect on Fz5 could be observed at 30 min. H. Time course of the Ab oligomer effects (500 nM) on Wnt5a. In summary graphs of quantitative data, the levels of target proteins were normalized against GAPDH or b-actin and expressed as relative units to the vehicle-treated controls. Data in summary graphs are means 6 SEM from three independent experiments. * p,0.05; ** p,0.01. doi:10.1371/journal.pone.0022920.g003 we determined the effect of Ab on the expression of these proteins in mouse cortical cultures (12–14 DIV). A wide range of Ab concentrations (100 nM - 15 mM) have been used in previous in vitro studies. We chose to use a relatively low Ab concentration (500 nM) to better reflect the fact that the proteins are up- regulated at early AD stages when Ab begins to accumulate (Fig. 1C). Prior works have shown that 500 nM Ab is efficient to initiate some of the earliest and reversible AD-relevant pathologies [46,47]. More recent studies have indicated that different forms of Ab (monomer, oligomer and fibril) vary in their AD-related toxicity, with the oligomer being considered the most toxic form [37,48]. We prepared the oligomers and fibrils of Ab (1–42) (Fig. 3A) and compared the effect of Ab monomers, oligomers and fibrils on Wnt5a, Fz5 and pT286-aCaMKII protein expression in cortical cultures. As shown in Fig. 3B–D, Ab oligomer treatment for 24 h caused significant increases in Wnt5a (1.4 fold; p,0.05), Fz5 (1.4 fold; p,0.01) and pT286-aCaMKII (1.7 fold; p,0.01). Under the same experimental conditions, preparations of monomers and fibrils caused changes with lower magnitudes (Fig. 3B–D). Next, we investigated the dose effects of the oligomer. As shown in Fig. 3E–G, the oligomer increased expression of Wnt5a, Fz5, and pT286-aCaMKII proteins in a concentration- dependent manner (50 nM–5 mM). Although Wnt5a and p- CaMKII displayed a clear dose-effect at 24 hours after treatment, Fz5 showed this effect at 30 min. In addition, 500 nM of Ab oligomer caused a time-dependent increase in Wnt5a (Fig. 3H). PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e22920