Figure 7

Antagonizing Wnt signaling or Silencing Wnt1 in VM astrocytes fail to protect TH⁺ neurons. Wnt antagonism studies were performed with the extracellular-rich domain (CRD) of Fzd-1 (Fzd-1-CRD, 1 μ/ml) and the CRD of Fzd-2 (Fzd-2-CRD,1 μ/ml). Depletion of Wnt1 in VM astrocytes was achieved by introducing a small interference RNA targeting Wnt1, Astro ^siWnt1 (46) in indirect astrocyte-neuron co-cultures (42). A-C:Indirect astrocyte-neuron co-culture efficiently mitigated SD, 6-OHDA- and MPP⁺-induced decreased TH neuron survival (A), DA uptake (B), and Caspase3 activation (C), whereas these protective effects are reversed by Fzd-1-CRD pre-treatment, but not Fzd-2-CRD. * p < 0.05 when compared to control cultures without cytotoxic insults; ° p < 0.05 compared to neurotoxic exposure within each experimental group. D-F: Glial inserts transiently transfected with siRNA^wnt1 or siRNA^control were added on the top of the purified neurons at 9 DIV and exposed to cytotoxic stimuli. RT-qPCR analysis (D) and immunocytochemistry (E-F) show an almost 40-60% Wnt1 depletion by 72 h. G-H: In neurons co-cultured with Astro ^siWnt1, irrespective of the cytotoxic stimulus, a sharp reduction of TH⁺ neuronal count (G) and increased Caspase3-like activity (H) were observed, compared to neuron co-cultured with Astro^Ct (p < 0.05). In β-catenin siRNA- and Fzd-1^AS-knocked down neurons, astrocyte inserts failed to promote neuroprotection. I-S : Confocal images of TH staining (green) in the indirect cocultures showing the protective effect of astrocyte inserts + siRNA^Ct (L,O,R) as compared with siRNA^Wnt1 (M,P,S). The effect of indirect astrocyte coculture is emphasized in the PBS control cultures (I, N).