Figure 1

Dimethyl fumarate (DMF) protects from oxidative stress by enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) abundance and translocation to the nucleus in a time- and concentration-dependent manner. A) DMF protects from oxidative glutamate toxicity, a model of endogenous oxidative stress where extracellular glutamate blocks the cystine import ultimately causing glutathione (GSH) depletion and cell death. Primary cortical cultures or hippocampal HT22 cells were preincubated with 10 μM DMF, monomethylfumarate (MMF) or vehicle for 24 h and exposed to the indicated concentrations of glutamate for 24 h before cell viability was measured by the Cell Titer Blue (CTB) assay. B) DMF and MMF increase cellular GSH concentrations. HT22 cells were treated with 10 μM DMF, MMF or vehicle for 24 h and exposed to the indicated concentrations of glutamate for 8 h before intracellular glutathione was measured enzymatically. C-F) Dose and time-course of DMF and MMF effect on oxidative glutamate toxicity. HT22 cells were treated for the indicated times and concentrations with DMF, MMF or vehicle before addition of glutamate. Viability was quantitated 24 h later as described above. G) Time course of DMF effects on glutathione content. HT22 cells were incubated with 10 μM DMF for the indicated periods of time before intracellular glutathione was measured enzymatically. H) DMF enhances Nrf2 abundance quantitated by immunoblots done on nuclear fractions from HT22 cells treated with the indicated concentrations of DMF for 4 h. I) and J) DMF induces nuclear localization of Nrf2 but has no effect on the nuclear translocation of NF-κB as shown by high content imaging. I) HT22 cells were treated with vehicle (n = 9,561 cells), 10 μM DMF for 24 h (n = 8,170 cells) or with 25 μM TBHQ (n = 3,281 cells) as positive control for 4 h. J) HT22 cells were treated with vehicle or 10 ng/ml TNFα in the presence or absence of 10 μM DMF (vehicle n = 1,048 cells, DMF n = 943 cells, TNFα n = 1,410 cells, DMF + TNFα n = 1,085 cells). Cells were fixed, stained and nuclear localization analyzed by immunocytochemistry. K) DMF has no effect on GSH levels in fibroblasts derived from Nrf2-deficient mice. Cells were treated with 10 μM DMF (black bars) or vehicle (white bars) for 24 h before GSH was measured enzymatically. Graphs of all experiments represent the means ± standard error of the mean (SEM) of three independent experiments performed in triplicate. *P < 0.05, two-way ANOVA with Bonferroni post hoc test in A), B), G), I), J), and paired Student’s t-test in K).