Fumaric acid esters have proven to be effective medications in relapsing-remitting multiple sclerosis with neuroprotective effects. In this study, we investigated the impact of fumaric acid esters on primary murine microglia in vitro compared to DMSO vehicle control. Monomethyl fumarate (MMF) increased MTT reduction in a dose-dependent manner, whereas dimethyl fumarate (DMF) exhibited a biphasic response with low concentrations enhancing MTT reduction and higher concentrations inducing toxicity. Notably, complementary analyses of cell number and cell death did not reveal differences between MMF-treated and control conditions, indicating that the increased MTT reduction reflects enhanced cellular metabolic activity rather than increased viability. Consistent with this interpretation, MMF-treated cells exhibited higher basal and maximal oxygen consumption, spare respiratory capacity, and ATP production in the Seahorse XF Cell Mito Stress Test. Proteomic analysis did not indicate an upregulation of mitochondrial respiratory chain proteins, but instead suggested a qualitative shift in mitochondrial homeostasis, including increased expression of mitophagy-associated proteins. MMF-treated Nrf2-deficient microglia showed a blunted increase in MTT reduction, suggesting an involvement of Nrf2 in mediating MMF-induced metabolic effects. Additionally, MMF modulated the microglial iron metabolism and reduced the uptake of non-transferrin-bound iron and altered the gene expression of iron transport proteins, promoting a shift toward the uptake of less toxic, transferrin-bound iron. MMF mitigated iron-induced toxicity and was associated with upregulation of the ferroptosis suppressor protein, indicating a protective response to iron overload. Together, these findings suggest that MMF enhances microglial metabolic activity and mitochondrial function while reducing iron-mediated toxicity, thereby contributing to its neuroprotective effects.
