Alternative mitochondrial electron transfer as a novel strategy for neuroprotection

Abstract

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.

Figures

FIGURE 1.

MB improves mitochondrial respiration and reduces anaerobic glycolysis. A, effects of MB on cellular oxygen consumption in HT-22 cells. Cellular oxygen consumption was monitored with sequential injection of MB/vehicle, oligomycin, FCCP, and rotenone. The vehicle- and MB-treated groups had a significant difference in OCR at all time points (n = 5). B–D, OCR (mitochondrial respiration) and ECAR were monitored with similar sequential treatment in A after 4-h MB/vehicle incubation at the indicated concentration. MB induced a significant dose-dependent increase in OCR (B), a dose-dependent decrease in ECAR (C), and a dose-dependent attenuation of rotenone-induced OCR inhibition (D) (n = 5). E, effects of MB on cellular ATP levels. Shown is a representative assay of intracellular ATP level in HT22 cells treated with the indicated concentration of MB. *, p < 0.05; ***, p < 0.001. Error bars, S.E.

FIGURE 2.

Dose-dependent effects of MB in mitochondrial complex I-III and II-III activity in mitochondrial extracts. A, representative assay of complex I-III activity; B, complex I-III activity with increasing dose of MB or acetyl MB, which is redox-disabled; C, chemical structure of MB, MBH2, and acetyle MB. D, complex I-III activity with increasing dose of MB with/without the presence of complex I inhibitor (rotenone). E, complex I-III activity with increasing dose of MB with/without the presence of complex III inhibitor (antimycin A). F, complex II-III activity with increasing dose of MB with/without the presence of complex III inhibitor (antimycin A). *, **, and ***, significant difference from those without antimycin. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.E.

FIGURE 3.

MB forms a redox cycle between complex I and III. A and C, MB is a complex I substrate accepting electrons from NADH in a dose-dependent manner and is insensitive to rotenone inhibition. Activity with coenzyme Q1 was used as 100% control. **, significant difference between the indicated groups. B and D, reduced MB donates electrons to oxidized cyt c in a dose-dependent manner. ***, significant difference from the MB-only group. ROT, rotenone; CoQ1, coenzyme Q1. Top panels, representative assays (A and B). Bottom panels, quantitative analysis of the corresponding assay (C and D). Error bars, S.E.

FIGURE 4.

Effects of MB in rotenone- and antimycin A-induced mitochondrial dysfunction and cell death. A and B, neuroprotective effect of MB on rotenone-induced (A) and antimycin A-induced (B) cytotoxicity in HT22 cells. **, significant difference from vehicle. C, MB has no protection against H2O2 (released by glucose oxidase)-induced cytotoxicity. D and E, effects of MB on rotenone induced mitochondrial specific superoxide (D) and total cellular ROS (E). The y axis depicts the event (cell number in count × 103), and the x axis depicts fluorescent intensity. Shown is the representative result from three independent sets of experiments. Error bars, S.E.

FIGURE 5.

Effects of MB on rotenone-induced neurological and behavioral deficit in rats. A, MB prevents rat body weight loss induced by rotenone treatment. B, MB improved coordinated motor function deficiency induced by chronic rotenone treatment. **, significant difference between the indicated groups (p < 0.01). An asterisk in the individual sessions indicates that there is significant difference between ROT/Sal and both of the other two groups (p < 0.05). C, effects of MB on randomized blinded neurological assessments. ***, significant difference between the indicated groups (p < 0.001). D and E, effects of MB on catalepsy measurements in rotenone- and MB-treated rats with the bar test (D) and grid test (E). F, effects of MB on stride length in rotenone- and MB-treated rats in the gait test. ROT, rotenone-treated; Sal, saline (vehicle for MB); Veh, vehicle for rotenone. * and **, significant difference between the indicated groups in D–F. Error bars, S.E.

FIGURE 6.

Effects of MB and rotenone on dopamine and DOPAC levels, mitochondrial complex I-III activity, and total ROS in vivo. A and B, effects of rotenone and MB treatment on striatum dopamine (A) and DOPAC (B) levels. C and D, mitochondrial I-III activity (C) and total ROS (D) in rotenone- and MB-treated rats. All numbers were normalized to percentage of control groups. * and **, significant difference between the indicated groups in all panels. Error bars, S.E.

FIGURE 7.

Effects of MB on rotenone-induced nigrostriatal dopaminergic neurodegeneration. Coronal brain sections from control (Veh/Sal), rotenone-treated (ROT/Sal), and rotenone/MB-treated (ROT/MB) rats were immunostained for TH (left) and ubiquitin (right). TH immunohistochemistry was shown at low magnification and high magnification in the SNC region. Dopaminergic denervation, cell body shrinkage, and neuronal loss were clearly observed in the ROT/Sal group, and all were improved in the MB treatment group. Dopaminergic fibers were sparse in VTA in the ROT/Sal group and improved in the ROT/MB group. Neurons with positive ubiquitin immunostaining were observed only in the ROT/Sal group. Scale bars, 200 μm.

FIGURE 8.

MB attenuates rotenone induced fluoro-jade B-labeled neurodegeneration in TH-positive dopaminergic neurons. A, many degenerated cell bodies of nigrostriatal dopaminergic neurons were positively labeled by fluoro-jade B in the SNC area in ROT/Sal animals. B, nigral neurons were double labeled with fluoro-Jade B and TH, showing that these were degenerating dopaminergic neurons in SNC. The arrowheads indicate the presence of double labeled neurons with both TH and fluoro-jade B. Such fluoro-jade B-positive neurons were identified in half of the ROT/Sal group that had severe neuronal loss and behavioral deficiency. No fluoro-jade B-positive neurons were detected in Veh/Sal or ROT/MB groups. Scale bar, 100 μm (A) and 50 μm (B).

FIGURE 9.

MB reduces cerebral ischemia reperfusion damage induced by transient focal cerebral ischemia. A, ischemia/reperfusion injury, induced by 1 h of middle cerebral artery occlusion and 24 h of reperfusion, inhibits mitochondrial complex activities (I, III, I-III, and IV). B, ischemic lesion depicted by triphenyltetrazolium chloride staining (white area) in the representative brain sections from control-treated (top) and MB-treated (bottom) rats at 24 h after ischemic stroke. C, quantification of ischemic lesion volume with vehicle or MB treatment. All data are represented as mean ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 10.

Illustrations of the proposed mechanism by which MB facilitates electron transfers in the oxidative phosphorylation chain in the presence of rotenone and antimycin A inhibition.