In this paper, we report an important role of Wnt5a signaling in the regulation of Ab-evoked neurotoxicity and neuroinflamma- tion. We observed that (1) Wnt5a/CaMKII signaling is up- regulated at the early stages of AD development in an APPswe/ PSEN1DE9 transgenic mouse model, (2) Ab activates Wnt5a signaling in primary cortical cultures, (3) Ab-induced Wnt5a up- regulation is a critical molecular step leading to the development of Ab neurotoxicity in cultures, (4) Wnt5a stimulates inflammatory processes, and (5) Wnt5a is critical for Ab-induced inflammatory response. Our results suggest that abnormally up-regulated non- canonical Wnt5a signaling may regulate chronic neuroinflamma- tion in AD brains. Materials and Methods Preparation of Ab42 peptides Recombinant human Ab (1–42) (Chemicon) was used to prepare monomers (Ab-mon), oligomers (Ab-olig) and fibrils (Ab-fib), as described [37]. For monomer preparation, 5 mM Ab42 in DMSO was diluted directly with cell culture media. For oligomer preparation, 5 mM Ab42 in DMSO was diluted to 100 mM in ice-cold D-MEM/F-12 (Invitrogen), vortexed for 300, centrifugated (10,000 g; room temperature) for 19, and incubated at 4uC for 24 h. For fibril preparation, 10 mM HCl was added to the Ab42 solution (5 mM in DMSO) to bring Ab to a final concentration of 100 mM, vortexed for 300, centrifugated (10,000 g, room temperature) for 19, and incubated with shaking at 37uC for 24 h. Products from such preparations are mixtures that contain Ab monomers and the intended aggregates [37]. The quality of the preparations was confirmed by Western blotting analysis with the 6E10 (Covance; 1:5000), and the molecular mass was estimated by rainbow pre-stained protein markers (GE Healthcare). Animals and hippocampus dissection All procedures were approved by Animal Care and Use Committees of the University of Texas Medical Branch. Male APP (amyloid precursor protein)/presenilin-1 double transgenic (APPswe/PSEN1DE9, 26Tg) mice [38] and wild-type littermates at 3.5 months of age were anesthetized and then decapitated. The hippocampi were rapidly collected on ice for Western blotting, immunohistochemistry, or fluorescent immunostaining.. Wnt5a in AD coated dishes at a density of 1.56105 cells/cm2. Immunostaining with cell-type markers indicate that the cultures are mixtures of neuron (,60%) and glial cells (,40%). Cultures at 12–14 DIV were used in this study. All experimental treatments were carried out by adding the administrated agents into the freshly changed culture media. Only morphologically healthy neuronal cultures were used for drug treatments. Western blotting and antibodies Western blotting was performed as described [40]. The intensity of non-saturated bands on Western blots was quantified by densitometry analysis with NIH ImageJ. GAPDH or b-actin were used as loading controls. Rabbit-anti-APP antibody was from Sigma-Aldrich (1:3000); rabbit-anti-Wnt5a (1:1000) and rabbit- anti-Fz5 (1:1000) antibodies were from Abcam; rabbit-anti-p- aCaMKII(Thr286) (1:1000), rabbit-anti-NIK (1:1000), and rabbit- anti-IkB-a (1:1000) antibodies were from Cell Signaling Technol- ogy; and mouse-anti-GAPDH (1:1000) and rabbit-anti-b-actin (1:1000) antibodies were from Santa Cruz Biotechnology. Immunohistochemistry and immunofluorescence Immunohistochemistry was performed on 5 mm-thick paraffin- embedded sections. The sections were deparaffined, dehydrated and treated with 1% H2O2 as decribed [41] Antigen retrieval was carried out using citrate buffer (0.01 M; pH 6.0). The sections were then incubated with the primary antibody recognizing Ab (1–40/42) (Millipore; 1:100) overnight at 4uC followed by incubation with biotinylated goat anti-rabbit IgG. After treatment with streptavidin-HRP and staining with DAB, the sections were stained with hematoxylin and mounted. Indirect fluorescence immunostaining was performed, as described [42].with rabbit anti-Wnt5a (abcam; 5 mg/ml) and rabbit anti-Fz5 (abcam; 1 mg/ ml) primary antibodies and Cy3-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch; 1:200). Measurement of secreted cytokines Cortical cultures (12–14 DIV) were treated with Wnt5a (200 ng/ml), and media were collected at 0.5, 1, 2, 6, 12 and 24 h after Wnt5a application. Tumor necrosis factor alpha (TNF- a) and interleukin 1 beta (IL-1b) in collected media were quantified using Mouse TNF-a or IL-1b Quantikine ELISA Kits (R&D Systems), respectively. Neuron cultures C57BL E18 cortical cultures were prepared, as described [39]. The cells were plated on poly-D-lysine (30,000–70,000; Sigma) - MTT assay and trypan blue staining The MTT assay and trypan blue staining were performed as described in [43]. Three independent experiments were performed Figure 1. Ab increases significantly in the hippocampus of the APPswe/PSEN1DE9 (26Tg) mouse at the age of 3.5 months. A, APP in the wild-type and 26Tg hippocampus (3.5-months). B. APP processing in the hippocampus revealed by immunoblotting with the monoclonal antibody 6E10 (recognizing residues 1–16 of Ab). C. Immunohistochemical staining of the hippocampus with 6E10. Note the clustered Ab signals (arrows) in the cell body layer of the 26Tg hippocampus. Similar results were obtained from 6 pairs of wild-type and 26Tg mice. doi:10.1371/journal.pone.0022920.g001 PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e22920