Wnt5a in AD Figure 6. Wnt5a activates molecular pathways critical for inflammatory response. A. The time course of NIK protein dynamics after Wnt5a (200 ng/ml) treatment. B. Heat-inactivated Wnt5a cannot induce NIK. C. The time course of IkB-a protein dynamics after Wnt5a (200 ng/ml) treatment. D. Heat-inactivated Wnt5a cannot induce of IkB-a protein. * p,0.05; *** p,0.001 (vs. 0 h in A and C, or vs. vehicle in B and D). doi:10.1371/journal.pone.0022920.g006 Anti-inflammation factors attenuate Ab neurotoxicity The results described above indicate that up-regulated Wnt5a may mediate Ab neurotoxicity. We then sought to understand the mechanism by which Wnt5a contributes to Ab neurotoxicity. Because recent studies revealed a critical role of Wnt5a in the regulation of inflammatory responses in peripheral systems, we were interested in determining if Wnt5a contributes to Ab neurotoxicity by regulating inflammation. To this end, we first investigated the contribution of neuroin- flammation to the Ab-induced sensitization of cortical cultures to H2O2 stress. We found that IL-10, a prototypic anti- inflammatory cytokine, caused concentration-dependent rescue effects on Ab toxicity (Fig. 5). IL-10 started to rescue Ab/H2O2- induced cell death at 0.25 ng/ml and reached a plateau effect at 1 ng/ml (Fig. 5A). Heat-inactivated IL-10 did not have any rescuing effect (Fig. 5A). To further confirm the involvement of inflammation in the Ab toxicity, we determined the effect of activated protein C (APC), another well established anti- inflammatory protein. The results showed that APC displayed a similar rescue effect as IL-10 (Fig. 5B). These results indicate that neuroinflammatory response likely contributes to Ab neurotoxicity. Wnt5a regulates proteins that involve in inflammatory response in cortical cultures Recent studies reveal an important role of Wnt5a in control of inflammatory response in non-neuronal peripheral tissues [56,57,58], but its involvement in regulation of inflammatory response in nervous systems has not been reported. To investigate the potential contribution Wnt5a signaling to neuroinflammation, we examined the effect of Wnt5a on the protein expression of NF- kB-inducing kinase (NIK), a key positive regulator of inflammation that controls NF-kB activity by promoting the processing of p100, the inactive NF-kB2 precursor, to produce the functional p52 subunit [59]. We observed that NIK protein began to increase 5 min after Wnt5a administration (200 ng/ml), and this increase continued gradually, peaked at 60 min. and stayed at a higher level afterward (Fig. 6A). Heat-inactivated Wnt5a did not induce a NIK increase (Fig. 6B), indicating that Wnt5a protein activates inflammatory pathways in cortical cultures. We also investigated the effect of Wnt5a on the expression of IkB-a protein, an inhibitory regulator of inflammation response by sequestering NF- kB in the cytosol [60]. Interestingly, Wnt5a also induced a gradual increase of IkB-a (Fig. 6C). In contrast to the NIK increase, the IkB-a up-regulation did not begin until 30–60 min after Wnt5a PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e22920