Forced expression of Nurr1 and Foxa2 protects mDA neurons from toxic insults in vitro.Figure 3A Representative image for TH+ (mDA neuron), GFAP+ (astrocyte), and Iba-1+ (microglia) cells in mDA neuron-glia cultures used in the gain-of-function experiments. B mDA neurons in the cultures transduced with Nurr1 and/or Foxa2 are resistant to toxic stimuli induced by H2O2 (50-500 μM), 6-OHDA (50-1,000 μM), or MPP+ (50-500 μM). The mDA neuron-glia cultures were transduced with lentiviruses expressing Nurr1 (N), Foxa2 (F), Nurr1 + Foxa2 (NF), or control (C) and treated with the toxin for 8 h. Viable TH+ cells were counted on the following day. Shown in the graphs are percent TH+ cells relative to the respective toxin-untreated cultures. TH+ cells of C, N, F, and NF were compared at the same concentrations of the toxins. Significantly different from the control (C)*, from N#, and from F‡ at P < 0.05, n = 10 cultures each; one-way ANOVA followed by Bonferroni post hoc test. C, D Forced expression of Nurr1 and Foxa2 in glia exerts neuroprotective roles on mDA neurons in a paracrine mode. Experimental scheme (C) to test the effects of paracrine factors released from Nurr1-/Foxa2-expressing glia. Mixed astrocytes + microglia cultures (derived from VM tissue of mousepups on postnatal day 1) were transduced with N, F, NF, or C, and conditioned media (CM) were prepared from the transduced glia and added to mDA neuron cultures. Two days after the CM treatment, H2O2 (500 μM, 8 h)-mediated cell death was measured by counting viable TH+ cells (D). Significantly different from the control (C-CM)*, N-CM#, F-CM‡ at P < 0.05. P-values: 0.031 (N-CM*), 0.021 (F-CM*), 0.017 (NF-CM*), 0.022 (NF-CM#), and 0.027 (NF-CM‡); one-way ANOVA followed by Bonferroni post hoc test. E-G NF-expressing glia secrete factors that reduce oxidative stress by inducing Nrf2-mediated antioxidant gene expressions. Oxidative stress measured by DCF (oxidative stress indicator) staining (E). Primary cultured mDA neurons were pre-treated with C-CM (upper) or NF-CM (lower) for 2 days and then exposed to H2O2 (250 μM) in the presence of the CM. Four hours later, DCF staining was carried out. E-G NF-expressing glia secrete factors that reduce oxidative stress by inducing Nrf2-mediated antioxidant gene expressions. Oxidative stress measured by DCF (oxidative stress indicator) staining. (F) Expression array for antioxidant genes. mDA neuron cultures were pre-treated with C-CM, N-CM, F-CM, or NF-CM for 2 days before exposure to 500 μM H2O2 for 8 h, and mRNA expression levels of 39 antioxidant genes were estimated using an RT2 PCR Profiler ArrayR. Volcano plot (left) demonstrating a tendency for increased antioxidant gene expression in mDA neuron cultures with NF-CM treatment. The pink lines indicate the threshold of 1.5-fold changes in gene expression. The 23 antioxidant genes up-regulated 1.5-fold (circled) by NF-CM treatment relative to C-CM treatment are listed on the bar graph (right). E-G NF-expressing glia secrete factors that reduce oxidative stress by inducing Nrf2-mediated antioxidant gene expressions. Oxidative stress measured by DCF (oxidative stress indicator) staining. (G) NF-CM treatment of mDA neurons activates cytosolic Nrf2proteins (0 min) by inducing nuclear translocation (30 min). Shown in the graph are cytosolic and nuclear Nrf2protein levels (MFI) 30 min after C-CM, N-CM, F-CM, and NF-CM treatments. *P < 0.05, n = 50-60 cells in each group. P-values: 0.032 (N-CM*), 0.029 (F-CM*), 0.019 (NF-CM*) for the Nrf2 intensity in nucleus, 0.029 (F-CM*), 0.039 (NF-CM*) for the Nrf2 intensity in cytosol; one-way ANOVA followed by Bonferroni post hoc test.