Figure 6

Modulation of astroglial Wnt1/β-catenin signaling directs towards TH neuron survival/death. Astrocyte-neuron co-cultures where shifted to serum deprived medium (SD, A), or received 6-OHDA or MPP⁺ (25 μM), with or without Dkk1 (100 ng/ml), a Wnt1-Ab, or AR-14418 (5 μM) as described. A-B: TH⁺ neuron counts (A), and [³H]dopamine uptake (B) 48 h after SD or 24 h after 6-OHDA or MPP⁺(25 μM) with or without Dkk1, Wnt1-Ab, or AR. * p < 0.05 compared to controls. C: Western blot analysis showing β-catenin levels in neurons exposed to astrocyte insert upon cytotoxic challenge with or without Wnt1-Ab or Dkk1. In this experimental condition, the glial inserts were added on the top of the purified neurons at 9 DIV. D: immunoblotting with Wnt1-Ab (50) using protein extracts from embryonic VM and the NIH/3T3 cell line. 50 ng of recombinant Wnt1 was used as a positive control. E: Dual staining with TH (red) and DAPI depicting TH⁺ neurons in a control (PBS) astrocyte-neuron co-culture. D-P: Confocal images showing dual staining with TH (green) and GFAP (red) in a typical astrocyte-neuron control co-culture at 9 DIV. Note the lenght and branching of TH⁺ processes. Astrocyte coculture induced a significant protection against SD (E), 6-OHDA (H), and the reversal induced by Dkk1 antagonism of Wnt/β-catenin signaling (F,I). By contrast, pharmacological activation of Wnt/β-catenin signaling with AR magnified TH neuroprotection (G,J,K). Astrocyte coculture increases Fzd-1 signal in the long and branched TH⁺ neurites and growth cones (L-N and insert).