Amyloid β peptide (1–42; Human Aβ₄₂; Abcam plc., #ab120301), Hoechst 33258 (Sigma-Aldrich, #B2883), recombinant mouse Wnt5a protein (Bio-Techne China Co. Ltd. R&D Systems #645-WN), Roscovitine (ROS; Selleck.cn, #S1153), KN62 (Selleck.cn, #S7422), Calmodulin Kinase IINtide, Myristoylated (Ntide; Merck Millipore, #208921) and Z-VAD-FMK (Selleck.cn, #S7023) were added to the media at the indicated concentrations and time points. For transient gene transfection, DIC5 cortical neurons were transfected using the calcium phosphate transfection method described previously (Kingston et al., 2003). Lipofectamine 3000 (Thermo Fisher Scientific Inc.) was used for transient gene or siRNA transfection of HEK293 cells.

The following primary antibodies were used: Anti-Wnt7a antibody (Abcam plc., #ab100792), Anti-Wnt5a antibody (Abcam plc., #ab72583), Anti-beta Tubulin antibody (Abcam plc., #ab6046), Anti-Cyclin D1 antibody (Abcam plc., #ab16663), Anti-E2F1 antibody (Abcam plc., #ab94888), phospho-Rb (Ser795) antibody (Cell Signaling Technology, Inc., #9301), Anti-β-Catenin antibody (Abcam plc., #ab32572), Anti-Histone H1 antibody (Abcam plc., #ab71594), Anti-phospho-CaMKII alpha (Thr286)/beta (Thr287; Abcam plc., #ab32678), Flag (Sigma-Aldrich, #F1804).

Primary cortical neurons of either sex were established from cortices dissected from newborn C57/BL6 mice (<24 h) as described previously (Sciarretta and Minichiello, 2010). Animals were purchased from the Experimental Animal Center of Sun Yat-sen University. Ethical approval was obtained from the Animal Ethics Committee of Sun Yat-sen University. Briefly, the whole cerebral cortex was isolated and cells were dissociated in a trypsin solution (2.5 mg/mL in Hank’s buffer salt solution) for 10 min at 37°C. The cortex cell suspension was centrifuged and re-suspended, seeded at a density of 1.5 × 10⁶ cells/ml in plates pre-coated with poly-d-lysine (Sigma, St. Louis, MO, USA), and grown in Neurobasal™-A medium containing 2% B27 supplement (Thermo Fisher Scientific Inc., Waltham, MA, USA), L-glutamine (0.25 mM, Invitrogen), GlutaMax-I (0.25 mM, Thermo Fisher Scientific Inc.), 1% penicillin (100 U/ml, Invitrogen), 1% streptomycin (100 μg/ml, Invitrogen) in a humidified incubator with 5% CO₂ at 37°C. HEK293 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and were cultured at 37°C in 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 1% penicillin (100 U/ml, Invitrogen), 1% streptomycin (100 μg/ml, Invitrogen), L-glutamine (292 μg/ml, Invitrogen) and 10% fetal bovine serum (FBS; hyClone Laboratories).

Human cortical tissues obtained from surgical resection of patients with IV grade glioblastoma (glioblastoma adjacent normal cortical tissues) were fixed in 4% paraformaldehyde overnight at 4°C. Before sampling, routine processing, and paraffin embedding were performed. Written informed consent had been obtained from the family members of patients before the surgery. The human tissue study was approved by the Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University.

Male mice (8–12 week-old from Experimental Animal Center of Sun Yat-sen University, Guangzhou, China), maintained at an ambient temperature of 22–24°C under a 12:12 h light:dark cycle, were used in this experiment. Animals were divided into three groups (n = 10 each group): (1) sham-operated plus physiological saline treatment; (2) Human Aβ₄₂ (3 nmol/10 μl) i.c.v. (intracerebroventricularly) injection plus physiological saline treatment; and (3) Human Aβ₄₂ (3 nmol/10 μl) plus recombinant mouse Wnt5a protein treatment (6 nmol/10 μl) i.c.v. injection. To obtain the aggregated form of Aβ₄₂, the peptide solution was placed in an incubator at 37°C for 72 h. Seven days after injection, mice were perfused transcardially with 4% paraformaldehyde in phosphate buffered saline (PBS). The brains were post fixed for 24 h and were embedded in paraffin wax. Serial coronal sections (5 μm thickness) were cut from various sections of the brain.

All experimental procedures were carried out in accordance with the guidelines of the Animal Care and Use Committee of Sun Yat-sen University. The mice weighing 20–24 g were anesthetized with sodium pentobarbital (Sigma-Aldrich Co., at a dose of 70 mg/kg) and placed in a stereotaxic apparatus (Stoelting, USA). According to the atlas of Paxinos, Franklin, and Franklin (Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates, Fourth Edition), 8 mm 26-gauge stainless-steel guide cannulas, closed by stylets, were implanted over the lateral ventricle (0.5 mm posterior to bregma, 1.0 mm lateral to midline, 2.0 mm ventral to skull surface). Body temperature of the mice was maintained at 37°C. The injection lasted 5 min and the needle with the syringe was left in place for 2 min after the injection for the completion of drug infusion. After surgery, mice were housed individually. All experiments were carried out between 9:00 a.m. and 6:00 p.m.

The overexpression plasmids of GFP, Wnt5a (mouse) and Cyclin D1 (mouse) were incorporated into the pcDNA-Flag expression vector.

Total RNA was extracted and isolated from cells using TRIzol reagent (Invitrogen) as described previously (Peirson and Butler, 2007). First strand cDNA was synthesized from 1 μg of mRNA using Superscript III reverse transcriptase (Invitrogen) and oligo (dT) as primers. Q-PCR was performed in triplicate on an ABI Prism 7000 sequence detection system using an ABI SYBR Green PCR mixture as described by the manufacturer. PCR cycling conditions were as follows: initial denaturation at 95°C for 5–10 min followed by 40 cycles of 95°C for 30 s 1 min of annealing, and 1 min of extension at 72°C. The annealing temperature was adapted for the specific primer set used. Fluorescence data were collected during the annealing stage of amplification and specificity of the amplification was verified by melting curve analysis. Cycle threshold (Ct) values were calculated using identical threshold values for all experiments. β-actin was used as a control and for normalization. Relative RNA expression was calculated using the formula ratio = 2 (Ctref−Cttarget). Data shown represent the mean and SE of three separate experiments. The following primer pairs were used: wnt1 forward (5′-ATG AAC CTT CAC AAC AAC GAG-3′) and reverse (5′-GGT TGC TGC CTC GGT TG-3′); wnt2 forward (5′-CTG GCT CTG GCT CCC TCT G-3′) and reverse (5′-GGA ACT GGT GTT GGC ACT CTG-3′); wnt2b forward (5′-CGT TCG TCT ATG CTA TCT CGT CAG-3′) and reverse (5′-ACA CCG TAA TGG ATG TTG TCA CTA C-3′); wnt3 forward (5′-CAA GCA CAA CAA TGA AGC AGG C-3′) and reverse (5′-TCG GGA CTC ACG GTG TTT CTC-3′); wnt3a forward (5′-CAC CAC CGT CAG CAA CAG CC-3′) and reverse (5′-AGG AGC GTG TCA CTG CGA AAG-3′); wnt4 forward (5′-GAG AAG TGT GGC TGT GAC CGG-3′) and reverse (5′-ATG TTG TCC GAG CAT CCT GAC C-3′); wnt5a forward (5′-CTC CTT CGC CCA GGT TGT TAT AG-3′) and reverse (5′-TGT CTT CGC ACC TTC TCC AAT G-3′); wnt5b forward (5′-ATG CCC GAG AGC GTG AGA AG-3′) and reverse (5′-ACA TTT GCA GGC GAC ATC AGC-3′); wnt6 forward (5′-TGC CCG AGG CGC AAG ACT G-3′) and reverse (5′-ATT GCA AAC ACG AAA GCT GTC TCT C-3′); wnt7a forward (5′-ATC TCC GGA TCG GTG ACT TC-3′) and reverse (5′-AGG CCT GGG ATC TTG TTA CAG-3′); wnt7b forward (5′-TCT CTG CTT TGG CGT CCT CTA C-3′) and reverse (5′-GCC AGG CCA GGA ATC TTG TTG-3′); wnt8a forward (5′-ACG GTG GAA TTG TCC TGA GCA TG-3′) and reverse (5′-GAT GGC AGC AGA GCG GAT GG-3′); wnt8b forward (5′-TTG GGA CCG TTG GAA TTG CC-3′) and reverse (5′-AGT CAT CAC AGC CAC AGT TGT C-3′); wnt9a forward (5′-GCA GCA AGT TTG TCA AGG AGT TCC-3′) and reverse (5′-GCA GGA GCC AGA CAC ACC ATG-3′); wnt9b forward (5′-AAG TAC AGC ACC AAG TTC CTC AGC-3′) and reverse (5′-GAA CAG CAC AGG AGC CTG ACA C-3′); wnt10a forward (5′-CCT GTT CTT CCT ACT GCT GCT GG-3′) and reverse (5′-CGA TCT GGA TGC CCT GGA TAG C-3′); wnt10b forward (5′-TTC TCT CGG GAT TTC TTG GAT TC-3′) and reverse (5′-TGC ACT TCC GCT TCA GGT TTT C-3′); wnt11 forward (5′-CTG AAT CAG ACG CAA CAC TGT AAA C-3′) and reverse (5′-CTC TCT CCA GGT CAA GCA GGT AG-3′); wnt16 forward (5′-AGT AGC GGC ACC AAG GAG AC-3′) and reverse (5′-GAA ACT TTC TGC TGA ACC ACA TGC-3′); β-actin forward (5′-CGT CTT CCC CTC CAT CG-3′) and reverse (5′-CTC GTT AAT GTC ACG CAC-3′).

Equal amounts of protein (40–50 mg) were size-fractionated using 6%–15% SDS-PAGE gradient gels. The resolved proteins were electrophoretically transferred onto polyvinylidene difluoride membranes and analyzed by immunoblotting using an ECL chemiluminescence reagent and XAR film (Kodak, XBT-1) according to the manufacturer’s protocol. Primary antibodies were used at optimized dilutions along with the appropriate HRP-conjugated secondary antibodies. The data were collected from at least three independent experiments.

Brainstem neurons were grown on 13 mm round glass coverslips and processed according to the immunofluorescence protocol as described previously (Xie et al., 2011). Cells were placed on ice, washed twice with PBS and fixed with 0.37% PFA in PBS for 10 min followed by permeabilization with 0.1% Triton X-100 in TBS and blocking in 3% donkey serum. Cells were then washed twice in PBS and treated by cold blocking buffer for 1 h. After sequential treatment with NH₄Cl (50 mM in 20 mM glycine) for 10 min, the indicated antibody (1:200 in bovine serum albumin) was added and incubated overnight at 4°C. After an additional incubation for 1 h at room temperature with Hoechst 33258 and fluorescein isothiocyanate-conjugated secondary antibody (Invitrogen; 1:400 in bovine serum albumin), the slides were mounted in anti-fading solution (Permafluor, Beckman Coulter, Krefeld, Germany) and stored at 4°C, followed by confocal laser-scanning microscopy. A score of 0–3 was assigned to describe the level of Wnt5a protein based on the red fluorescence area in the cytoplasm of neurons (grade 0, <5%; grade 1, 5%–33%; grade 2, 33%–66%; and grade 3, >66%).

Approximately 10⁷ cells were harvested into 10 ml of isotonic fractionation buffer (250 mM sucrose, 0.5 mM EDTA, 20 mM Hepes, and 500 μM Na₃VO₄ at pH 7.2) supplemented with protease inhibitor cocktail complete (Roche Molecular Biochemicals) and centrifuged at 900 g for 5 min. The pellet was then resuspended in 200 μl fractionation buffer, homogenized with a ball-bearing homogenizer and centrifuged at 900 g for 5 min to enrich the nuclei. The post-nuclear supernatant was centrifuged at 20,000 g for 15 min to collect the heavy membrane fraction enriched in mitochondria, the supernatant was as cytoplasm without mitochondrion.

The target sequences for Wnt5a-specific siRNAs were 5′-GAC CUG GUC UAC AUC GAC CTT-3′ and 5′-AGU GCA AUG UCU UCC AAG UTT-3′; Cyclin D1 specific siRNAs were 5′-CCA AUA GGU GUA GGA AAU AGC GCT G-3′ and 5′-AAC ACC AGC TCC TGT GCT GCG-3′ all of which and the negative control siRNA (no silencing small RNA fragment) were synthesized by GenChem Co. (Shanghai, China).

Cells were stained with Hoechst 33258 (5 μg/ml) to visualize nuclear morphology. Apoptosis was quantified by scoring the number of cells with pyknotic nuclei (chromatin condensation or nuclear fragmentation) relative to the total number of Hoechst 33258-positive cells in the same visual field. Cells were counted in an unbiased manner (at least 1000 cells for each group) and were scored blindly without previous knowledge of their treatment.

Homogenates of treated cortical neurons were quantified and used to detect CaMKII activity, using the SignaTECT^® Calcium/Calmodulin-Dependent Protein Kinase Assay System (Promega, #V8161) as described by the manufacturer. In brief, the assay quantifies [γ-³²P]-labeling of a proprietary CaMKII-specific, biotinylated peptide substrate that is based on the T286 autophosphorylation site, followed by streptavidin capture; this recognition sequence is conserved in mouse. Controls include calmodulin and substrate omission. The substrate is specific for CaMKII and does not detect CaMKIV, PKC or calcineurin (Hanson et al., 1989; Goueli et al., 1995). Samples were assayed in triplicate and protein concentration was determined using the bicinchoninic acid (BCA) and Western blotting method. Results were expressed as relative CaMKII activity.

Human cortical tissues obtained from surgical resection of patients with IV grade glioblastoma (glioblastoma adjacent normal cortical tissues) or mouse brains were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.6 overnight at 4°C. Dehydration of tissue was through a series of 80%, 95% (v/v) ethanol 1 h each followed by 100% ethanol overnight. Two 100% (v/v) xylene washes were done for 1 h each and then 1 h in 60°C Paraplast Plus (Tyco/Healthcare). After a change of Paraplast Plus, tissue was incubated in a 60°C vacuum oven for 2 h prior to placing in molds to cool and solidify. Sections, 5 μm thick, were cut and mounted. Sections were deparaffinized by drying on superfrost plus slides (Fisher), heating at 56°C overnight, and then washing through mixed xylenes, 100% ethanol, 95% ethanol, ddH₂O. Slides were immersed in 10 mm citrate buffer, pH 6.0, dry heated for 10 min each to unmask antigen sites, and then cooled and washed in PBS. Endogenous peroxidase activity was inhibited by rinsing the slides in 3% hydrogen peroxide for 5 min. Non-specific binding was blocked by 5 min incubation with the Super Block Solution (ScyTek Laboratories). After washing in PBS, sections were incubated for 30 min at room temperature with indicated antibodies. Sections were washed extensively with PBS and subsequently treated with the Ultra Tek Anti-Polyvalent kit (ScyTek Laboratories). Finally, sections were treated with 3,3′-diaminobenzidine as chromogenic and mounted.

All experiments were repeated at least three times using independent culture preparations. All measurements were performed blindly. The significance of difference between means was analyzed by the ANOVA and post hoc Bonferroni/Dunn tests (for multiple comparisons) and by the Student’s t test (for single comparisons). The results are represented as means ± SEM. Statistical significance was determined by value of p < 0.05 or p < 0.01 for all analyses.

In order to explore the role of Wnt in Aβ₄₂ induced neuronal cell death, primary cultured mouse cortical neurons were used in this study (Figure 1A). In the current study, after 5 days culture in vitro, cortical neurons were treated with Aβ₄₂. We found Aβ caused a concentration-dependent increase of cortical neuron apoptosis (Figure 1B). A significant of cortical neuron apoptosis was observed at concentrations from 0.5 μM to 4 μM (Figure 1B), these results were consistent with previous studies.

Earlier reports have shown that Aβ₄₂ may promote neuronal apoptosis by regulating the expression of Wnt family members (Bozyczko-Coyne et al., 2001; Majd et al., 2008). To study which Wnt isoforms take part in accommodation of Aβ₄₂ induced cytotoxicity, the mRNA expression levels of all 19 Wnt gene family members in cortical neurons were analyzed by RT-PCR. As illustrated in Table 1, most Wnt members were expressed only at very low levels in both control and Aβ₄₂ treated group, Aβ₄₂ mainly altered the expression of Wnt5a, Wnt7a, Wnt9b and Wnt11. We next confirmed the mRNA and protein expression levels of these genes. We found the mRNA and protein expression levels of Wnt9b and Wnt11 were undetectable (data not shown); the mRNA expression level of Wnt7a was upregulated after Aβ₄₂ treatment, but the protein level of Wnt7a was not changed (Figures 1C,D); Aβ₄₂ significantly suppressed the expression level of Wnt5a (Figures 1C,D). These results indicate that Aβ₄₂ suppresses the expression of Wnt5a on both mRNA and protein levels.

We next determined the expression level of Wnt5a protein in human brains. Unfortunately, we were unable to obtain postmortem brains derived from AD patients. As an alternative, we collected the adjacent normal brain tissues of malignant glioma from 32- to 86-year-old patients. Immunohistochemical staining of the adjacent normal brain tissues of malignant glioma revealed that Wnt5a levels were consistently expressed in the neurons, this result was consistent with previous study (Howng et al., 2002); however, the Wnt5a levels were decreased with the increase of age (Figure 2A). These results indicate a role of Wnt5a in the aging of human brain.

To examine the effect of Aβ₄₂ on the expression of Wnt5a in vivo, a solution of Aβ₄₂ was injected into the lateral ventricles of mice. Seven days after the injection, tissues from the cerebral cortex were collected to assess the expression of Wnt5a. We found that the protein level of Wnt5a was decreased in cortical neurons after Aβ₄₂-treatment (Figure 2B). This observation was further supported by quantitative PCR analysis (Figure 2C). Therefore, Aβ₄₂ suppresses the expression of Wnt5a on both mRNA and protein levels in vivo.

To investigate the function of Wnt5a in cortical neuron survival and apoptosis, two siRNAs were designed to reduce mouse Wnt5a protein levels. These siRNAs completely abolished the expression of a transfected mouse Flag-Wnt5a construct in HEK293 cells (Figure 3A). In cortical neurons, Wnt5a siRNAs were co-transfected with the marker plasmid pCMV-GFP, immunofluorescence analysis using anti-Wnt5a antibody revealed that the expression of Wnt5a was abolished by the two Wnt5a siRNAs (Figure 3B), demonstrating the efficacy of the siRNAs in cortical neurons. Furthermore, siRNAs specifically targeted against Wnt5a induced cortical neuron apoptosis, and the Wnt5a siRNAs induced apoptosis was inhibited by Caspase inhibitor Z-VAD (Figure 3C). These results indicate that Wnt5a is required for the survival of cortical neuron.

To further confirm the effect of Wnt5a on cortical neuron survival, transfection of neurons with wild-type Wnt5a also significantly increased neuron survival during Aβ₄₂ treatment, and the effect induced by Wnt5a overexpression was abolished by Wnt5a siRNAs (Figure 3D). Moreover, we added various concentration (0, 50, 100, 250, 500, 1000 ng/ml) of recombinant Wnt5a protein to the culture medium of Aβ₄₂ treated neurons. The suppressive effect of recombinant Wnt5a protein on Aβ₄₂ induced neuronal apoptosis was dose-dependent (Figure 3E). Taken together, Wnt5a acts as a mediator of cortical neuron survival, and Aβ₄₂ promotes cortical neuron apoptosis by downregulating the expression of Wnt5a.

Neurons in culture exposed to Aβ₄₂ could re-enter the cell cycle, cross the G1-S-phase transition and begin de novo DNA synthesis before apoptotic death occurs (Caricasole et al., 2003; Majd et al., 2008). And cell-cycle activation is causally related to neuronal death (Busser et al., 1998; Giovanni et al., 1999; Liu and Greene, 2001; Yang et al., 2003; Becker and Bonni, 2004). Meanwhile, Wnt pathway is the key pathway in activation of cell cycle (Davidson and Niehrs, 2010). Interestingly, Wnt5a can act as a cell proliferation inhibitor in many cancer cells and mouse B cells (Liang et al., 2003; Kremenevskaja et al., 2005; Bitler et al., 2011), suggesting a cell-cycle suppressor role for Wnt5a in some cell types. We speculate that Aβ may activate the cell-cycle pathway by reducing the expression of Wnt5a.

To determine the effect of Aβ₄₂ in inducing cortical neuron to re-enter the cell cycle, neurons were treated with Aβ₄₂ combined with or without CDKs inhibitor ROS (Bach et al., 2005), results showed that Aβ₄₂ induced neuronal apoptosis was significantly decreased by CDKs inhibitor (Figure 4A). Furthermore, CDKs inhibitor also inhibited Wnt5a siRNAs induced cortical neuron apoptosis (Figure 4B). These findings demonstrate that Aβ₄₂ could promote neurons re-enter the cell cycle by reducing the expression of Wnt5a.

Aberrations in cell cycle control are always controlled by cell cycle regulators, such as cyclins, Myc and E2F1 (van den Heuvel, 2005). To study which cell cycle regulators mediated the Aβ₄₂ activated cell cycle, the mRNA expression levels of cyclins, Myc and E2F1 in cortical neurons were analyzed by RT-PCR. As illustrated in Table 2, most cyclin members and Myc were expressed only at very low levels in both control and treatment group, Aβ₄₂ mainly altered the expression of Cyclin D1 and E2F1. We next confirmed that Aβ₄₂ significantly increased the expression level of Cyclin D1, and the upregulation of Cyclin D1 was eliminated by CDKs inhibitor ROS (inhibited the CDK mediated phosphorylation of Rb Ser795; Figures 4C,D). Accordingly, neurons treated with recombinant Wnt5a protein also abolished Aβ₄₂ induced Cyclin D1 expression (Figures 4E,F). However, the protein level of E2F1 was not changed after Aβ₄₂, CDKs inhibitor and Wnt5a treatment (Figures 4C,D). Taken together, these results suggest that Wnt5a prevents cell cycle activation by inhibiting the expression of Cyclin D1 in cortical neurons.

We next confirmed the expression level of Cyclin D1 protein in the adjacent normal brain tissues of malignant glioma from 32- to 86-year-old patients. As observed in previous studies (Hoozemans et al., 2002), trace amounts of Cyclin D1 was detected in the brain cortex of 32, 35 and 43 years old people, but the protein levels were upregulated with the increase of age (Figure 5A). These results also indicated a relationship between the neuronal aging and Cyclin D1.

To examine the effect of Wnt5a on Cyclin D1 in vivo, a solution of Aβ₄₂ or Aβ₄₂ combined with recombinant Wnt5a protein was injected into the lateral ventricles of mice. Seven days after the injection, tissues from the cerebral cortex were collected to assess the expression of Cyclin D1. We found that the protein level of Cyclin D1 was increased in cortical neurons after Aβ₄₂-treatment, and the increase of Cyclin D1 was inhibited by recombinant Wnt5a protein (Figure 5B). This observation was further supported by quantitative PCR analysis (Figure 5C). These in vivo results support the conclusion that Wnt5a could prevent Aβ₄₂ induced Cyclin D1 expression.

The mechanism of Wnt5a-mediated inhibition of the Cyclin D1expression is unclear. Wnt5a has been proposed to activate both the canonical Wnt/β-catenin pathway (He et al., 1997; Toyofuku et al., 2000) and Wnt/Ca²⁺ pathway (Liang et al., 2003). Activation of the Wnt/Ca²⁺ pathway has been suggested to block the Wnt/β-catenin signaling cascade (Torres et al., 1996; Kühl et al., 2001; Ishitani et al., 2003). To dissect the mechanistic pathway utilized by Wnt5a in regulating cortical neuron cell-cycle activation, we analyzed β-catenin levels in Aβ₄₂ treated and untreated neurons. No difference was detected in β-catenin levels in whole-cell or in nuclear extracts between Aβ₄₂ treated and untreated neurons, suggesting that Wnt5a was neither activating nor inhibiting the canonical Wnt/β-catenin pathway in cortical neuron (Figures 6A,B).

Therefore, we examined the Wnt/Ca²⁺ pathway. CamKII activity assays showed that Aβ₄₂ treated cells possess 63.51% less activity than Aβ₄₂ untreated cells and the decrease of CamKII activity was recovered by Wnt5a recombinant protein stimulation (Figure 6C), even though Western blotting analysis revealed equal amounts of total CamKII in both samples (Figure 6C). Furthermore, we examined the level of phospho-CaMKII alpha (Thr286)/beta (Thr287) by Western blotting. CaMKII phosphorylation was decreased at as early as 0.5 h after Aβ₄₂ treatment and was also recovered by Wnt5a recombinant protein stimulation (Figure 6D). These results indicate that Wnt5a is signaling via the Wnt/Ca²⁺ pathway to activate CamKII.

To further confirm the ability of Wnt/Ca²⁺/CaMKII pathway to downregulate Cyclin D1 and inhibit cell-cycle activation, KN62 [a pan-inhibitor of CaMKs (Tokumitsu et al., 1990)] and CaMKII-Ntide [an endogenous selective inhibitor of CaMKII (Chang et al., 1998)], was used to treat cortical neurons. Western blotting analysis revealed that these two inhibitors elevated Cyclin D1 expression of both Aβ₄₂ untreated and Aβ₄₂ plus Wnt5a recombinant protein treated neurons (Figure 6E). Furthermore, we found CamKII inhibitors were sufficient to cause cortical neuron apoptosis in a Cyclin D1 dependent way (Figures 6F,G). Collectively, Wnt5a/CaMKII pathway down-regulates the expression of Cyclin D1.

Previous studies have established that overexpression of Cyclin D1 may lead to neuronal apoptosis (Kranenburg et al., 1996). To assess whether Cyclin Dl overexpression would induce this phenomenon, two siRNAs were designed to reduce mouse Cyclin D1 protein levels (Figure 6F). siRNAs specifically targeted against Cyclin D1 inhibited Aβ₄₂ induced cortical neuron apoptosis (Figure 7A). Furthermore, we confirmed that overexpression of Cyclin D1 could lead to cortical neuron apoptosis, and Cyclin D1 induced apoptosis was also abolished by Cyclin D1 siRNAs (Figure 7B). These results indicate that Cyclin D1 is required for Aβ₄₂ induced cortical neuron apoptosis.

Based on the obvious importance of Wnt5a and Cyclin D1 in cortical neuron survival and apoptosis, we hypothesized that Wnt5a might mediate the cortical neuron survival by inhibiting Cyclin D1 expression. Manipulations of Wnt5a and Cyclin D1, using knockdown, overexpression or recombinant protein approaches, were combined to investigate their functional relationship. The Wnt5a recombinant protein significantly blocked cortical neuron apoptosis in Aβ media (Figure 7C). In contrast, overexpression of wild-type Cyclin D1 removed the protection conferred by Wnt5a recombinant protein (Figure 7C). Overexpression of wild-type Cyclin D1 induced apoptosis could not prevent by adding Wnt5a recombinant protein, and the knockdown of endogenous Wnt5a also could not enhance the apoptosis rate (Figure 7D). These findings demonstrated that Cyclin D1 lies downstream of the Wnt5a-dependent pro-survival signaling cascade.