Figure 4.

CDDO^TFEA drug treatment is neuroprotective by a GSH-dependent, astrocyte-mediated mechanism. (a) CDDO^TFEA-induced neuroprotection requires the presence of astrocytes. Mixed mouse cortical cultures (90% NeuN+ neurons, 10% GFAP+ astrocytes) or astrocyte-free mouse neuronal cultures (>98% NeuN+, <0.2% GFAP+) were treated with CDDO^TFEA (100 nM) for 24 h, before treatment with H₂O₂ (100 μM) for a further 24 h, after which cultures were fixed and levels of cell death assessed. ^*P<0.05 paired t-test (n=4). (b) CDDO^TFEA-induced neuroprotection is Nrf2-dependent. CDDO^TFEA-induced neuroprotection in mixed Nrf2^+/+ or Nrf2^−/− mouse cortical cultures was studied using the same protocols as (a). ^*P<0.05 paired t-test (n=4). (c) Enriched human astrocyte cultures were treated with CDDO^TFEA (250 nM)±BSO (200 μM) for 6 h, the drug washed out, and fresh minimal medium replaced for conditioning over 24 h. ACM was derived as previously and supplemented with H₂O₂. (d) Human neuronal viability was assessed upon treatment with H₂O₂ (50 μM) for 6 h, in ACM derived from untreated and CDDO^TFEA-treated enriched astrocytes. BSO co-treatment was used to block GCL activity, demonstrating GCL-dependent and -independent neuroprotective effects of ACM. (e–g) Representative TuJ1 and activated caspase-3 co-staining (scale bar, 50 μm). ^*P<0.05 ANOVA, Newman–Keuls post-test (n=3). (h) Human neuronal expression of NFE2L2 and GCLC mRNA with 6 h CDDO^TFEA (250 nM) treatment were examined by quantitative real-time PCR. (i) The effect of treatment with CDDO^TFEA (250 nM) for 24 h on human neuronal viability was determined over a range of H₂O₂ concentrations (0–200 μM, 6 h). ^*P<0.05 paired t-test (n=3)