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

Abstract

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 ( EC 50 = 2.2 μmol/L), a dose-dependent decrease in Complex II-linked respiration ( IC 50 = 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 ( EC 50 = 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 Q o 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.