Effect of Wnt1-induced TH+ neuroprotection on active GSK-3β and Caspase3-like activity. Purified neuronal cultures were exposed to SD, 6-OHDA or MPP+ insults as described, and DA neuroprotection examined as above. Active GSK-3β (GSK-3β phospho-Tyr216) protein levels were determined by western blotting (wb). Part of the cultures were pre-treated with Wnt1 (100 ng/ml) or the the specific GSK-3β antagonist, AR-14418 (5 μM), alone or in combination, while other cell cultures were exposed to the specific antagonist of Wnt/β-catenin pathway, Dkk1 (100 ng/ml), 1 hr before Wnt1 (100 ng/ml) treatment. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B: Active GSK-3β (GSK-3β phospho-Tyr216) protein levels as determined by wb. A sharp up-regulation is observed after SD, 6-OHDA or MPP+ insults, an effect reversed by Wnt1 and AR pre-treatment. B: Survival of TH+ neurons exposed to the described insults in the absence or the presence of the different treatments. * p < 0.05 when compared to control cultures without cytotoxic insults. Note that both Wnt1 and AR increase TH+ neuron survival, and the combined Wnt1+AR treatment further increase TH+ neuron survival. By contrast, exposure of Dkk1-pretreated cultures to SD, 6-OHDA or MPP+ reduced Wnt1 protective effect on cell survival. C: Caspase-3 activity shows a sharp inhibition of DEVD-like activity in neurons pre-treated with Wnt1 or AR and exposed to the different insults, whereas previous exposure to Dkk1, counteracts Wnt1-induced Casapse3 inhibition. * p < 0.05 when compared to control cultures without cytotoxic insults.