Glycogen Synthase Kinase-3 and TBI Figure 6. SB-216763 inhibits the GSK-3 pathway. A) Represen- tative images of western blots for phospho-GSK-3ß, phospho-LRP6, phospho-ß-Catenin, phospho-Akt, and phospho-ERK in response to i.p. injection of SB-216763 (2.5 and 5.0 mg/kg) or vehicle (0 mg/kg) (n = 3/ group). B) SB-216763 significantly increased the phosphorylation of GSK-3ß in a dose-dependent manner. The phosphorylation of the GSK-3 substrates C) LRP-6 and D) ß-Catenin, were found to be decreased following SB-216763 administration. The phosphorylation levels of E) Akt and F) ERK were unaffected by SB-216763 administration. Data are presented as the mean 6 SEM. *, P,0.05 by one-way ANOVA. doi:10.1371/journal.pone.0024648.g006 examination did not reveal any overt differences in ipsilateral hippocampal neuronal loss (as indicated using NeuN immunore- activity) between injured animals treated with vehicle and those given SB-216763 (Figure 8E), with both groups having demon- strable neuron loss in the dentate gyrus (DG) and CA3 subfields. Neither ipsilateral (vehicle: 28.3062.98 mm3 vs. SB-216763: 3 28.2962.98 mm ; Student’s t-test p = 0.99) nor contralateral (vehicle: 14.4862.81 mm3 vs. SB-216763: 12.3962.61 mm3; Student’s t-test p = 0.59) cortical tissue loss was different between the two groups. Discussion Using a rodent model of TBI, we examined if post-injury inhibition of GSK-3 activity is neuroprotective, and if this neuroprotection is associated with improved motor and cognitive outcomes. Our findings show that daily, post-injury administration of the GSK-3 inhibitor lithium for five days significantly improved hippocampal-dependent learning and memory and reduced Figure 7. Post-injury administration of SB-216763 improves motor function. A) Schematic of the drug injection and behavioral testing paradigm. Rats (n = 10/group) were injected (i.p.) for the first 5 days post-injury with either 5.0 mg/kg SB-216763 or vehicle. SB-216763- treated rats had B) comparable vestibulomotor deficits, but made significantly fewer C) ipsilateral and D) contralateral foot faults. E) Body weight was unaffected by SB-216763 administration. Data are presented as the mean 6 SEM. {, P,0.05 by two-way repeated measures ANOVA. doi:10.1371/journal.pone.0024648.g007 hippocampal CA3 neuron loss. In contrast, the GSK-3-selective inhibitor SB-216763 improved motor function, but did not provide overt neuroprotection and only modestly improved learning and memory. One of the most studied mechanisms of GSK-3 regulation is the canonical Wnt pathway. In the absence of Wnt, GSK-3 exists as a part of a destruction complex consisting of the scaffold protein Axin, the tumor suppressor APC, and casein kinase Ia (CKIa) that binds to, and regulates, ß-Catenin-mediated gene expression [9] (see Figure 1A). Phosphorylation of ß-Catenin by CKIa and GSK- 3 marks it for proteosomal degradation. Upon Wnt binding to the Frizzled receptor, GSK-3 is recruited to the plasma membrane where it can phosphorylate the Frizzled co-receptor LRP6 on five PPPSP motifs. These phosphorylation events prime the receptor for subsequent phosphorylation by CKIc [30]. Several studies have indicated that the phosphorylation of LRP6 is crucial for reducing ß-Catenin phosphorylation and degradation [14]. When the phosphorylation of LRP6 was examined in hippocampal extracts following TBI, we observed a transient, but significant increase in Ser1490 phosphorylation, suggesting GSK-3 transloca- tion. However, as other kinases have also been shown to phosphorylate LRP6 on Ser1490, we cannot rule out their involvement in the increased phosphorylation we observed [14]. Regardless of the mechanism underlying its phosphorylation, it appears that phosphorylated LRP6 acts, at least in part, to inhibit GSK-3. Using phosphorylated peptide mimetic of LRP6, it has been shown that peptides containing the PPPSPxS motif of LRP6 PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24648