Figure 4

Dimethyl fumarate (DMF)-treated neuronal cells but not splenocytes secrete neuroprotective glutathione (GSH). A) HT22 cells and splenocytes show increased intracellular glutathione after 24 h pretreatment with 10 μM DMF, but only HT22 cells release glutathione into the extracellular space. GSH was quantitated enzymatically and normalized to cellular protein content and vehicle-treated cells, respectively. Released GSH in the medium was quantitated after 4 h incubation in cystine-free medium following a 24 h incubation in medium supplemented with 10 μM DMF (black bars) or vehicle (white bars). B) HT22 cells treated with conditioned medium (CM) from HT22 cells but not splenocytes take up released GSH and C) are protected from glutamate toxicity. HT22 cells were treated for 24 h with conditioned medium before addition of the indicated concentrations of glutamate for another 24 h. Viability was quantitated by the CTB assay. (D) Viability of unstimulated splenocytes quantitated by flow cytometry using Annexin V and 7AAD staining is unaffected by HT22 conditioned medium. Graphs of all experiments represent the means ± standard error of the mean (SEM) of three independent experiments performed in triplicate. *P < 0.05, paired Student’s t-test for all assays except two-way analysis of variance (ANOVA) with Bonferroni post hoc test for (C).