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Alternative Mitochondrial Electron Transfer as a Novel Strategy for Neuroprotection*

  • Yi Wen
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Wenjun Li
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Ethan C. Poteet
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Luokun Xie
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Cong Tan
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Liang-Jun Yan
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Xiaohua Ju
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Ran Liu
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Hai Qian
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Marian A. Marvin
    Affiliations
    the Departments of Neurology and
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  • Matthew S. Goldberg
    Affiliations
    the Departments of Neurology and

    Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and
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  • Hua She
    Affiliations
    the Departments of Pharmacology and

    Neurology, Emory University, Atlanta, Georgia 30322
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  • Zixu Mao
    Affiliations
    the Departments of Pharmacology and

    Neurology, Emory University, Atlanta, Georgia 30322
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  • James W. Simpkins
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Shao-Hua Yang
    Correspondence
    To whom correspondence should be addressed: Dept. of Pharmacology and Neuroscience University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107-2699. Tel.: 817-735-2250; Fax: 817-735-2091;
    Affiliations
    From the Department of Pharmacology and Neuroscience, Institute for Alzheimer's Disease and Aging Research, University of North Texas Health Science Center, Fort Worth, Texas 76107,
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01NS054687 (to S.-H. Y.), R01NS054651 (to S.-H. Y.), P01AG22550 (to J. W. S.), P01AG10485 (to J. W. S.), ES015317 (to Z. M.), AG023695 (to Z. M.), and NS048254 (to Z. M.). This work was also supported by Alzheimer's Association Grant NIRG57698 (to Y. W.) and Texas Garvey Foundation (to Y. W.).
Open AccessPublished:March 18, 2011DOI:https://doi.org/10.1074/jbc.M110.208447
      Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.

      Introduction

      Mitochondria are the powerhouses and the major source of free radicals in almost all cells. Mitochondrial dysfunction is implicated in numerous neuropathological diseases, including Parkinson disease (PD)
      The abbreviations used are: PD, Parkinson disease; AD, Alzheimer disease; MB, methylene blue; MBH2, leucomethylene blue; ETC, electron transport chain; ROS, reactive oxygen species; ICA, internal carotid artery; DCFH2, 2,7-dichlorofluorescin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; ECAR, extracellular acidification rate; TH, tyrosine hydroxylase; cyt c, cytochrome c; Veh/Sal, vehicle/saline; ROT/Sal, rotenone/saline; ROT/MB, rotenone/MB; ICA, internal carotid artery; ANOVA, analysis of variance; DOPAC, 3,4-dihydroxyphenylacetic acid; VTA, ventral tegmental area; SNC, substantia nigra pars compacta.
      (
      • Liu W.
      • Vives-Bauza C.
      • Acín-Peréz R.
      • Yamamoto A.
      • Tan Y.
      • Li Y.
      • Magrané J.
      • Stavarache M.A.
      • Shaffer S.
      • Chang S.
      • Kaplitt M.G.
      • Huang X.Y.
      • Beal M.F.
      • Manfredi G.
      • Li C.
      ,
      • Betarbet R.
      • Sherer T.B.
      • MacKenzie G.
      • Garcia-Osuna M.
      • Panov A.V.
      • Greenamyre J.T.
      ), Alzheimer disease (AD) (
      • Yao J.
      • Irwin R.W.
      • Zhao L.
      • Nilsen J.
      • Hamilton R.T.
      • Brinton R.D.
      ), and stroke (
      • Vosler P.S.
      • Graham S.H.
      • Wechsler L.R.
      • Chen J.
      ). Other than ATP production, mitochondria participate in diverse cell signaling events and are essential organelles for cell survival. The oxidative phosphorylation machinery is composed of five complexes (complexes I–V). From Krebs cycle intermediates (NADH and FADH2), electrons feed into complex I or II and are transferred to complex III and then to complex IV and finally to the oxygen molecules. Energy released during the electron transfer is utilized to actively pump out proton from the mitochondrial matrix to the intermembrane space, generating the electrochemical gradient of proton across the inner membrane, which is ultimately utilized by complex V to produce ATP (
      • Wallace D.C.
      ). However, a small portion of electrons leaking from the electron transport chain (ETC), mostly at complex I and complex III, react with the oxygen molecule and yield superoxide anion, which can be converted into other reactive oxygen species (ROS). When ROS production overwhelms the endogenous antioxidant systems, they can potentially damage various cellular components, including proteins, lipids, and nucleic acids. ROS is implicated in aging and various pathological processes and has been proposed as the key culprit for many neurodegenerative diseases. Thus, mitochondrial targeting strategies, such as free radical scavengers, mitochondrial signaling regulation, and ETC component supplementation, have been extensively studied (
      • Chaturvedi R.K.
      • Beal M.F.
      ,
      • Galluzzi L.
      • Blomgren K.
      • Kroemer G.
      ). Disappointingly, none of these neuroprotective strategies has been proven successful in any neurological diseases in clinical trials (
      • O'Collins V.E.
      • Macleod M.R.
      • Donnan G.A.
      • Horky L.L.
      • van der Worp B.H.
      • Howells D.W.
      ,
      • Olanow C.W.
      • Kieburtz K.
      • Schapira A.H.
      ).
      Methylene blue (MB) is a heterocyclic aromatic compound that has many biological and medical applications. It is an FDA-approved drug for methemoglobinemia and an antidote to cyanide poisoning. Previous publications have suggested that MB improves mitochondrial function (
      • Atamna H.
      • Nguyen A.
      • Schultz C.
      • Boyle K.
      • Newberry J.
      • Kato H.
      • Ames B.N.
      ,
      • Tokumitsu Y.
      • Ui M.
      ). In the present study, we demonstrate that MB functions as an alternative electron carrier that efficiently shuttles electrons between NADH and cytochrome c (cyt c). This process reroutes electron transfer upon complex I and III inhibition, reduces electron leakage, and attenuates ROS overproduction. MB is protective in a rotenone-induced animal model of Parkinsonism and an animal model of ischemic stroke induced by middle cerebral artery occlusion.

      DISCUSSION

      Neuroprotection is a therapeutic approach that aims to prevent or attenuate neuronal degeneration and loss of function in neurological diseases. Neuroprotective strategies, including but not limited to free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades (
      • Ginsberg M.D.
      ). Unfortunately, despite promising results from preclinical studies, outcomes of clinical neuroprotection trials have been repeatedly disappointing (
      • O'Collins V.E.
      • Macleod M.R.
      • Donnan G.A.
      • Horky L.L.
      • van der Worp B.H.
      • Howells D.W.
      ,
      • Löhle M.
      • Reichmann H.
      ). Increasing evidence has indicated that mitochondrial dysfunction is a common pathological mechanism that underlines many neuropathological conditions. Endogenous ETC component supplementation, such as with coenzyme Q10, was explored for their neuroprotective effects (
      • Storch A.
      • Jost W.H.
      • Vieregge P.
      • Spiegel J.
      • Greulich W.
      • Durner J.
      • Müller T.
      • Kupsch A.
      • Henningsen H.
      • Oertel W.H.
      • Fuchs G.
      • Kuhn W.
      • Niklowitz P.
      • Koch R.
      • Herting B.
      • Reichmann H.
      ,
      • Yang L.
      • Calingasan N.Y.
      • Wille E.J.
      • Cormier K.
      • Smith K.
      • Ferrante R.J.
      • Beal M.F.
      ). In addition, other identified neuroprotectants, such as estrogen, have been found to increase gene expression for many key components of the ETC complexes (
      • O'Lone R.
      • Knorr K.
      • Jaffe I.Z.
      • Schaffer M.E.
      • Martini P.G.
      • Karas R.H.
      • Bienkowska J.
      • Mendelsohn M.E.
      • Hansen U.
      ). However, the consistent failure of all of these approaches has cast doubt on the current neuroprotective strategies. In the present study, we identified alternative mitochondrial electron transfer as a novel neuroprotective strategy. The alternative electron transfer through MB redox cycle bypasses the inhibition of ETC complex I and III and avoids overproduction of free radical, hence providing neuroprotection in both chronic and acute neurological diseases.
      Current evidence suggests that it is not the mitochondrial energy defect per se but rather the overproduction of ROS induced by blockage of mitochondrial complexes that accounts for the neurodegeneration in many neuropathological conditions (
      • Fukui H.
      • Moraes C.T.
      ,
      • Cassarino D.S.
      • Fall C.P.
      • Swerdlow R.H.
      • Smith T.S.
      • Halvorsen E.M.
      • Miller S.W.
      • Parks J.P.
      • Parker Jr., W.D.
      • Bennett Jr., J.P.
      ,
      • Gandhi S.
      • Wood N.W.
      ). In fact, cells can survive via many alternative mechanisms other than mitochondrial oxidative phosphorylation, such as anaerobic glycolysis and autophagy. In our observation, complex IV (KCN) or complex V (oligomycin) inhibition reduces cell growth but does not convey direct cytotoxicity. Previous studies indicate that neither complex IV nor complex V is involved in electron leakage and ROS production (
      • Ladiges W.
      • Wanagat J.
      • Preston B.
      • Loeb L.
      • Rabinovitch P.
      ,
      • Ganguly A.
      • Basu S.
      • Chakraborty P.
      • Chatterjee S.
      • Sarkar A.
      • Chatterjee M.
      • Choudhuri S.K.
      ). We found that inhibition of complex IV or V induced anaerobic glycolytic activity and lactate production, which compensated for the decrease of ATP production (data not shown). On the other hand, complex I/III blockage directly cause the leakage of electrons, which react oxygen molecules, lead to ROS generation, and induce cytotoxicity. Thus, an alternative electron transfer pathway can bypass complex I/III blockage, avoid ROS production, and provide neuroprotection.
      MB has a very low redox potential of 11 mV and is very efficient cycling between oxidized and reduced forms (
      • Atamna H.
      • Nguyen A.
      • Schultz C.
      • Boyle K.
      • Newberry J.
      • Kato H.
      • Ames B.N.
      ). In the current study, we determined the action of MB in ETC complex activities. Our results demonstrated that MB functions as an alternative electron carrier similar to the endogenous coenzyme Qs in mitochondrial ETC. On the other hand, MB-mediated electron transfer is insensitive to either rotenone or antimycin A inhibition, suggesting that MB provides an alternative route for electron transfer. Upon the completion of the redox cycle (MB → MBH2 → MB), electrons from NADH are delivered to cyt c in an alternate route that is insensitive to complex I and III blockage. Such a mechanism is further confirmed by a de novo synthesized MB derivative with the disabled redox center by N-acetylation. Acetyl-MB completely lost its ability to enhance electron transfer between complex I and III. The alternative electron transfer through MB prevents the “electron leakage” induced by complex I/III inhibition and avoids the massive ROS production during complex I/III inhibition (Fig. 10).
      Figure thumbnail gr10
      FIGURE 10Illustrations of the proposed mechanism by which MB facilitates electron transfers in the oxidative phosphorylation chain in the presence of rotenone and antimycin A inhibition.
      In cell cultures, MB provides protection against mitochondrial inhibition-induced ROS overproduction, mitochondrial dysfunction, and cytotoxicity. Given the high enzymatic efficacy and potency, the alternative electron transfer strategy requires much lower concentrations for neuroprotection as compared with the traditional free radical scavengers. Consistently, the neuroprotective effects of MB occur at very low concentrations with an EC50 of 0.1762 nm against glutamate toxicity in HT22 cells. Therefore, the alternative electron transfer strategy is fundamentally different from traditional free radical scavenger approach. Rather, it avoids the production of ROS by rerouting electron transfer and bypasses complex I/III inhibition. Indeed, MB failed to provide protection against direct hydrogen peroxide insult even with much higher concentrations.
      We further determined the effects of this novel neuroprotective strategy in animal models of PD and ischemic stroke. From both epidemiological (
      • Dhillon A.S.
      • Tarbutton G.L.
      • Levin J.L.
      • Plotkin G.M.
      • Lowry L.K.
      • Nalbone J.T.
      • Shepherd S.
      ) and basic research (
      • Betarbet R.
      • Sherer T.B.
      • MacKenzie G.
      • Garcia-Osuna M.
      • Panov A.V.
      • Greenamyre J.T.
      ,
      • Nicklas W.J.
      • Vyas I.
      • Heikkila R.E.
      ), growing evidence has indicated that complex I dysfunction is the primary factor in PD pathogenesis. It has been known that complex I activity is deficient in the brain as well as peripheral tissues in PD patients (reviewed in Refs.
      • Fukui H.
      • Moraes C.T.
      ,
      • Gandhi S.
      • Wood N.W.
      ,
      • Mizuno Y.
      • Ohta S.
      • Tanaka M.
      • Takamiya S.
      • Suzuki K.
      • Sato T.
      • Oya H.
      • Ozawa T.
      • Kagawa Y.
      , and
      • Haas R.H.
      • Nasirian F.
      • Nakano K.
      • Ward D.
      • Pay M.
      • Hill R.
      • Shults C.W.
      ). Such concepts are further proved by the recently developed rotenone PD model (
      • Betarbet R.
      • Sherer T.B.
      • MacKenzie G.
      • Garcia-Osuna M.
      • Panov A.V.
      • Greenamyre J.T.
      ,
      • Greenamyre J.T.
      • MacKenzie G.
      • Peng T.I.
      • Stephans S.E.
      ). Many features of PD have been observed in the systemic rotenone model, including complex I impairment (
      • Betarbet R.
      • Sherer T.B.
      • MacKenzie G.
      • Garcia-Osuna M.
      • Panov A.V.
      • Greenamyre J.T.
      ), oxidative damage (
      • Testa C.M.
      • Sherer T.B.
      • Greenamyre J.T.
      ), accumulation and aggregation of α-synuclein in select dopaminergic neurons, and impairment of nigral ubiquitin-proteasome system function with accumulation of polyubiquitinated proteins (
      • Betarbet R.
      • Sherer T.B.
      • MacKenzie G.
      • Garcia-Osuna M.
      • Panov A.V.
      • Greenamyre J.T.
      ). Moreover, two genetic causes of PD, mutations in Parkin and PINK1, have been associated with defective complex I activity (
      • Liu W.
      • Vives-Bauza C.
      • Acín-Peréz R.
      • Yamamoto A.
      • Tan Y.
      • Li Y.
      • Magrané J.
      • Stavarache M.A.
      • Shaffer S.
      • Chang S.
      • Kaplitt M.G.
      • Huang X.Y.
      • Beal M.F.
      • Manfredi G.
      • Li C.
      ,
      • Müftüoglu M.
      • Elibol B.
      • Dalmizrak O.
      • Ercan A.
      • Kulaksiz G.
      • Ogüs H.
      • Dalkara T.
      • Ozer N.
      ). Consistently, overexpression of NDI1, a yeast gene encoding a single subunit rotenone-insensitive NADH-quinone oxidoreductase, in rodents could prevent respiratory deficiencies and attenuate Parkinson-like symptoms in animals treated with rotenone or MPTP (
      • Seo B.B.
      • Nakamaru-Ogiso E.
      • Flotte T.R.
      • Matsuno-Yagi A.
      • Yagi T.
      ). Our in vivo studies indicated that MB attenuates complex I inhibition-induced neurodegeneration in dopaminergic neurons and provides protection against rotenone-induced Parkinson-like behavioral, neurochemical, and neuropathological features.
      The role of ROS in the pathogenesis of cerebral ischemia reperfusion injury is well known. Reperfusion produces a burst in ROS formation after cerebral ischemia and has been known as one of the major mechanisms by which reperfusion worsens ischemic damage (
      • Schaller B.
      • Graf R.
      ). Antioxidant has been viewed as one of the most promising neuroprotective strategies for the treatment of ischemic stroke. However, the failure of the SAINT II trial has raised concerns regarding the traditional free radical trapping strategy (
      • Diener H.C.
      • Lees K.R.
      • Lyden P.
      • Grotta J.
      • Davalos A.
      • Davis S.M.
      • Shuaib A.
      • Ashwood T.
      • Wasiewski W.
      • Alderfer V.
      • Hårdemark H.G.
      • Rodichok L.
      ). Instead of neutralizing the free radical, the alternative electron transfer strategy blocked the overproduction of ROS generated by the inhibition of ETC complex I and III. In addition, our study demonstrated that MB increases the oxygen consumption rate and decreases the extracellular acidification rate, which could potentially prevent the superoxide production derived from the excessive oxygen supply during the reperfusion. Consistently, our in vivo study demonstrated that MB, as an alternative electron carrier, significantly decreased the cerebral ischemia reperfusion damage induced by transient focal cerebral ischemia.
      There are other considerations for the role of alternative electron transfer in mitochondrial functions. The mitochondrial ETC removes electrons from an electron donor (NADH or FADH2) and passes them to a terminal electron acceptor (O2) via a series of redox reactions. These reactions are coupled to the creation of a proton gradient across the mitochondrial inner membrane, which functions as the direct driving force for ATP synthesis. In ETC, complexes I, III, and IV function as the proton pumps. An alternate route to ETC could bypass the proton pump function in these complexes and might reduce the efficiency of ATP production. However, our study demonstrated that MB could enhance ATP production, suggesting that MB does not comprise the function of complexes I, III, and IV as proton pumps.
      In addition to the capacity as an alternative electron carrier, many other functions of MB have previously been described. It was shown that MB can delay senescence and extend the life span of human IMR90 fibroblasts in tissue culture (
      • Atamna H.
      • Nguyen A.
      • Schultz C.
      • Boyle K.
      • Newberry J.
      • Kato H.
      • Ames B.N.
      ). It has also been indicated that MB can penetrate the blood brain barrier, increase cyt c oxidase (complex IV) activity, improve cognitive function in rats (
      • Callaway N.L.
      • Riha P.D.
      • Bruchey A.K.
      • Munshi Z.
      • Gonzalez-Lima F.
      ), and protect against methylmalonate-induced seizures (
      • Furian A.F.
      • Fighera M.R.
      • Oliveira M.S.
      • Ferreira A.P.
      • Fiorenza N.G.
      • de Carvalho Myskiw J.
      • Petry J.C.
      • Coelho R.C.
      • Mello C.F.
      • Royes L.F.
      ). As a century-old drug, MB has been in medical use for many years in various pathological conditions (
      • Naylor G.J.
      • Martin B.
      • Hopwood S.E.
      • Watson Y.
      ,
      • Peer G.
      • Itzhakov E.
      • Wollman Y.
      • Chernihovsky T.
      • Grosskopf I.
      • Segev D.
      • Silverberg D.
      • Blum M.
      • Schwartz D.
      • Iaina A.
      ). The therapeutic potential of MB has been demonstrated clinically against many diseases, including endotoxin-induced lung injury, bacterial lipopolysaccharide-induced fever (
      • Galili Y.
      • Kluger Y.
      • Mianski Z.
      • Iaina A.
      • Wollman Y.
      • Marmur S.
      • Soffer D.
      • Chernikovsky T.
      • Klausner J.P.
      • Robau M.Y.
      ,
      • Demirbilek S.
      • Sizanli E.
      • Karadag N.
      • Karaman A.
      • Bayraktar N.
      • Turkmen E.
      • Ersoy M.O.
      ,
      • Riedel W.
      • Lang U.
      • Oetjen U.
      • Schlapp U.
      • Shibata M.
      ), cyclosporin-induced kidney injury (
      • Rezzani R.
      • Rodella L.
      • Corsetti G.
      • Bianchi R.
      ), doxorubicin-induced heart injury (
      • Hrushesky W.J.
      • Olshefski R.
      • Wood P.
      • Meshnick S.
      • Eaton J.W.
      ), and pancreas injury induced by streptozotocin (
      • Haluzik M.
      • Nedvídková J.
      • Skrha J.
      ). Recently, MB was reported to attenuate Tau aggregation, and it is currently in phase II clinical trials for treatment of Alzheimer disease. Many other activities have also been demonstrated with MB, including inhibition of nitric-oxide synthase (
      • Marczin N.
      • Ryan U.S.
      • Catravas J.D.
      ,
      • Mayer B.
      • Brunner F.
      • Schmidt K.
      ) and monoamine oxidase A (
      • Ramsay R.R.
      • Dunford C.
      • Gillman P.K.
      ), which may also contribute to the illustrated neuroprotective action. However, some of these functions might be secondary to the currently described mitochondrial mechanism and warrant further investigation.
      In summary, we have identified a novel neuroprotective strategy represented by MB in the present study. We have demonstrated that MB could function as an electron carrier and provide an alternative electron transfer along ETC, avoid ROS overproduction induced by ETC blockage, and maintain mitochondrial function. We have tested this novel neuroprotective strategy in two animal models of neurological diseases. In an animal model of Parkinsonism, MB was able to attenuate rotenone-induced motor deficits and nigral-dopaminergic neuronal degeneration. In an ischemic stroke model, MB significantly reduced cerebral ischemia reperfusion damage. Considering that MB has been in clinical use for over a century with few known side effects, the identified novel neuroprotective strategy of alternative electron transfer is now ready for testing in clinical settings and might lead to the discovery of promising treatments for the mitochondria dysfunction-related neurological diseases, such as PD and stroke.

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