Neuroprotective Actions of Methylene Blue and Its Derivatives

Abstract

Methylene blue (MB), the first lead chemical structure of phenothiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia. We have previously demonstrated that MB could function as an alternative mitochondrial electron transfer carrier, enhance cellular oxygen consumption, and provide protection in vitro and in rodent models of Parkinson’s disease and stroke. In the present study, we investigated the structure-activity relationships of MB in vitro using MB and six structurally related compounds. MB reduces mitochondrial superoxide production via alternative electron transfer that bypasses mitochondrial complexes I-III. MB mitigates reactive free radical production and provides neuroprotection in HT-22 cells against glutamate, IAA and rotenone toxicity. Distinctly, MB provides no protection against direct oxidative stress induced by glucose oxidase. Substitution of a side chain at MB’s 10-nitrogen rendered a 1000-fold reduction of the protective potency against glutamate neurototoxicity. Compounds without side chains at positions 3 and 7, chlorophenothiazine and phenothiazine, have distinct redox potentials compared to MB and are incapable of enhancing mitochondrial electron transfer, while obtaining direct antioxidant actions against glutamate, IAA, and rotenone insults. Chlorophenothiazine exhibited direct antioxidant actions in mitochondria lysate assay compared to MB, which required reduction by NADH and mitochondria. MB increased complex IV expression and activity, while 2-chlorphenothiazine had no effect. Our study indicated that MB could attenuate superoxide production by functioning as an alternative mitochondrial electron transfer carrier and as a regenerable anti-oxidant in mitochondria.

Methylene Blue Reduces Acute Cerebral Ischemic Injury via the Induction of Mitophagy

“…MB is a lipophilic compound that has been used for the treatment of multiple disorders, including methemoglobinemia, malaria and cyanide poisoning (9,10,49). Recently, MB has been shown to play neuroprotective roles in a variety of mitochondria-associated cytotoxicity paradigms, such as stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease and optic neuropathy (30,47). In this study, we demonstrated for the first time that MB attenuates ACI injury by increasing the induction of mitophagy.…”

“…We discovered that the dramatic decline in the MMP under OGD conditions was greatly hindered by MB, and unexpectedly, MB in the absence of OGD induced a decrease in the MMP. Based on MB's redox properties that low MB concentrations favor dimerization and reduction, whereas high concentrations promote oxidation (7), we propose the following hypothesis for this phenomenon: in functional mitochondria, MB in its reduced form donates H + (47), which tends to cause an elevation of MMP; MB in its oxidized form accepts H + (53), which tends to cause a decline of MMP. However, in dysfunctional mitochondria, these effects may be just opposite.…”

“…Because mitophagy is directly induced by the loss of MMP (33,47), and because the reduction in MMP can be attenuated by MB (47), we analyzed the MMP via flow cytometry and measured mitophagy via Western blot in the OGD model. The MMP sharply fell after the cells were exposed to OGD for 2 h and MB significantly attenuated this decline, even though MB in the absence of OGD induced a decrease in the MMP (Figures 7A-B).…”

Rotenone-induced nigrostriatal toxicity is reduced by methylene blue

“…22,23 Methylene blue is a reduction-oxidation agent with potent antioxidant properties, that prevents formation of mitochondrial oxygen free radicals. [24][25][26] The dye also acts as an enhancer of the electron transport chain, thereby promoting oxygen consumption. 27,28 Methylene blue readily crosses the blood-brain barrier, resulting in high concentrations in the central nervous system.…”

“…Several in vitro and in vivo studies indicate an antioxidant and neuroprotective effect for methylene blue. 25,26,59 When infused into the rat striatum along with rotenone, methylene blue (8.8 µg) prevented a decrease in cytochrome oxidase activity and any perilesional increase in oxidative stress (dihydroethidium fluorescence). 60 In rats treated subcutaneously with rotenone 5 mg/kg for 8 days, methylene blue given at 500 µg/kg improved locomotor deficits and prevented striatal dopamine depletion.…”