Methylene blue modulates mitochondrial electron flow via multiple regulatory sites in isolated brain mitochondria: Implications for neurodegeneration

Methylene blue (MB) is a redox-active compound capable of modulating the efficiency of the mitochondrial electron transport system (ETS). We investigated the acute dose-dependent effects of MB on oxygen consumption and H₂O₂ production in isolated brain mitochondria. High-resolution respirometry with fluorescence-based H₂O₂ detection enabled quantification of H₂O₂/O₂ flux ratios. At 5 μmol/L, MB increased Complex I activity (+11.7%), decreased Complex IV activity (-19.2%), and left Complex II + III activity unchanged, as determined spectrophotometrically. Functionally, MB caused a saturable increase in Complex I-linked respiration (EC50 = 2.2 μmol/L), a dose-dependent decrease in Complex II-linked respiration (IC50 = 0.57 μmol/L), unchanged Complex I + II-linked respiration, logarithmic inhibition of Complex IV-linked respiration, and saturable increase in rotenone inhibited Complex I-linked respiration (EC50 = 7.1 μmol/L). MB induced a biphasic response in H₂O₂ production and the H₂O₂/O₂ flux ratio, increasing up to 10 μmol/L and decreasing thereafter. Low-dose MB (1 μmol/L) did not alter oxygen flux or flux control ratios across SUIT-defined respiratory states but significantly increased H₂O₂ production and the H₂O₂/O₂ flux ratio prior to inhibitor addition. This increase required a functional ETS and originated from accelerated electron leak at Complex I and Complex III. MB established a rotenone-insensitive redox bypass from NADH-dependent dehydrogenases to cytochrome c and Complex IV. Dose-dependent decrease in Complex II-linked respiration, suggests effects of MB on Complex II, the Qo site of Complex III, or ubiquinol oxidation. MB redistributes electron flow and alters electron leakage at multiple sites of the ETS, which underlies its effects on mitochondrial dysfunction.