β-catenin depletion abolishes Wnt1- induced TH+ neuroprotection. Enriched neuronal cultures were transiently transfected with β-catenin small interference RNA (β-cateninsiRNA) (sc-29210) or control siRNA (β-cateninCt, see text for details), before being exposed to either Wnt1 or PBS, and DA neuron survival assessed by counting TH+ neurons, by assessing [3H]dopamine incorporation and Caspase3 activity. Differences analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A-B: Effect of SD, 6-OHDA and MPP+ treatments in β-catenin mRNA (A) and protein levels (B) showing a significant decrease of β-catenin. Note that preventive application of Wnt1 increases β-catenin both at a mRNA (A) and protein levels (B). *p < 0.05 vs PBS. Depletion of β-catenin via the introduction of β-catenin siRNA shows an almost 40-60% reduction in β-catenin mRNA by Real time PCR (A) and western blotting (B). *p < 0.05 vs control siRNA C-D: Survival of TH+ neurons by cell counting (C), [3H]dopamine incorporation (D). Note that in β-catenin siRNA pre-treated cultures, the application of Wnt1 failed to protect TH+ neurons against 6-OHDA or MPP+, whereas in cultures pre-treated with a control siRNA, Wnt1 treatment increased TH+ neuron survival (C) and [3H]dopamine incorporation (D). E-L: Representative immunocytochemical images show the ability of Wnt1 to efficiently counteracts TH neuron death and neurite loss, an effect abolished by β-catenin silencing *p < 0.05 vs cultures without cytotoxic insult; ** p < 0.05 compared to siRNACt+ cytotoxic insult (within each each experimental group); ° p < 0.05 compared to Wnt1 treated cultures in the presence of siRNACt.