NE_II_L: Neurosciences II. Lectures
Gergely Sváb1, Márton Kokas1, Ildikó Sipos2, Attila Ambrus1, László Tretter1
1 Department of Biochemistry, Laboratory of Neurobiochemisrty, Institute of Biochemistry and Molecular Biology, Semmelweis University
2 Department of Neurology, Semmelweis University
Introduction: Methylene blue (MB) has been used in human therapy in various pathological conditions. Its effects in neurodegenerative disease models are promising. MB acts on multiple cellular targets and mechanisms, but many of its potential beneficial effects are ascribed to be mitochondrial. MB can ameliorate deleterious effects of the inhibition of respiratory complex I (CI) because MB is able to accept electrons from NADH and can donate electrons to the distal components of electron transport chain. Bypassing the inhibition of CI can improve bioenergetic parameters in mitochondria treated with CI inhibitors. There are controversial reports about the effects of MB in complex III (CIII) inhibited mitochondria.
Aims: Our aim was to (re)investigate, whether inhibition of the mitochondrial respiratory CIII could be bypassed by MB. It has been studied in rodent brain mitochondria, whether MB could donate electrons directly to cytochrome c (cyt c). Besides corroborating our earlier results, unusual bioenergetic reactions to ADP and to the adenine nucleotide translocase inhibition were described and analyzed in the presence of CIII inhibitors and MB. The oxidation-reduction dependent shuttling of MB between the mitochondria and the extramitochondrial space was described.
Method: Mitochondria were isolated from rodent’s brain. Oxygen consumption assays were performed using high-resolution respirometry system. ΔΨm was determined with two different methods: safranin O and TMRM fluorescence. NAD(P)H autofluorescence was monitored using a fluorescence spectrophotometer. Oxidoreduction state and localization of MB was detected by the absorbance changes at 660 nm.
Results: MB induced a significant increase in oxygen consumption and was able to partially restore the Δψm. The partial rescue of oxygen consumption as well as Δψm indicated that MB could indeed be beneficial under those pathological conditions that affect CIII.
Conclusion: MB is indeed capable of transferring electrons from the respiratory chain and/or reducing equivalents directly to cyt c, bypassing the CIII. Unexpected bioenergetic phenomena are presented in terms of mitochondrial bioenergetics parameters in the presence of MB and CIII inhibitors. These phenomena might be accounted for by the dynamic charge- and membrane potential-dependent changes of MB’s extra and intramitochondrial localization.
Semmelweis University, János Szentágothai Doctoral School of Neurosciences