Figure 4

Knocking down Fzd-1 counteracts Wnt1- induced TH⁺ neuroprotection. Enriched neuronal cultures were processed for Real time PCR using specific primers for Fzd receptors. The effect of knocking down Fzd-1 in Wnt1 neuroprotection was studied with Fzd-1 sense (Fzd-1Ct) or antisense (Fzd-1AS) oligonucleotides. Differences were analyzed by ANOVA followed by Newman-Keuls test, and considered significant when p < 0.05. A: Differential expression and regulation of Fzd transcripts by SD, 6-OHDA and MPP⁺. B: Western blot (wb) analysis showing down-regulation of Fzd-1 levels in neuronal cultures exposed to the cytotoxic stimuli and the significant reversal induced by Wnt1. *p < 0.05 vs cultures without cytotoxic insult. Pre-treatment with Fzd-1^AS induced an almost 40-60% decrease of Fzd-1. C-H: Representative confocal images of dual staining with Fzd-1 (green) and TH (red) showing colocalization (orange to yellow) in PBS (C-D) controls. Note the marked loss of Fzd-1 in TH neurons exposed to 6-OHDA (E) or MPP⁺ (F), an effect efficiently counteracted by Wnt1 pre-treatment (G). I-J: Survival of TH⁺ neurons by cell counting (C), [³H]dopamine incorporation (D). K-L: Effect of Fzd-1 ^AS or Fzd-1^Ct, in β-catenin protein and Caspase3-like activity. Fzd-1^AS pre-treatment prevents Wnt1-induced increased β-catenin protein levels (K) and reverses Wnt1-induced Caspase3-inhibition (L) in 6-OHDA and MPP⁺-treated cultures. *p < 0.05 vs PBS. *p < 0.05 vs control siRNA. Note that Wnt1 efficiently reversed the dramatic decrease of neurite length caused by 6-OHDA or MPP⁺ in Fzd-1^Ct-treated (M, N), as opposed to Fzd-1 knocked down cultures (O, P). *p < 0.05 vs cultures without insult (within each each experimental group).