Basic Sciences
Critical Role of Flavin and Glutathione in Complex
I–Mediated Bioenergetic Failure in Brain Ischemia/
Reperfusion Injury
Anja Kahl, MD*; Anna Stepanova, MS*; Csaba Konrad, PhD; Corey Anderson, BS;
Giovanni Manfredi, MD, PhD; Ping Zhou, PhD; Costantino Iadecola, MD; Alexander Galkin, PhD
Background and Purpose—Ischemic brain injury is characterized by 2 temporally distinct but interrelated phases: ischemia
(primary energy failure) and reperfusion (secondary energy failure). Loss of cerebral blood flow leads to decreased oxygen
levels and energy crisis in the ischemic area, initiating a sequence of pathophysiological events that after reoxygenation
lead to ischemia/reperfusion (I/R) brain damage. Mitochondrial impairment and oxidative stress are known to be early
events in I/R injury. However, the biochemical mechanisms of mitochondria damage in I/R are not completely understood.
Methods—We used a mouse model of transient focal cerebral ischemia to investigate acute I/R-induced changes of
mitochondrial function, focusing on mechanisms of primary and secondary energy failure.
Results—Ischemia induced a reversible loss of flavin mononucleotide from mitochondrial complex I leading to a transient
decrease in its enzymatic activity, which is rapidly reversed on reoxygenation. Reestablishing blood flow led to a
reversible oxidative modification of mitochondrial complex I thiol residues and inhibition of the enzyme. Administration
of glutathione-ethyl ester at the onset of reperfusion prevented the decline of complex I activity and was associated with
smaller infarct size and improved neurological outcome, suggesting that decreased oxidation of complex I thiols during
I/R-induced oxidative stress may contribute to the neuroprotective effect of glutathione ester.
Conclusions—Our results unveil a key role of mitochondrial complex I in the development of I/R brain injury and provide
the mechanistic basis for the well-established mitochondrial dysfunction caused by I/R. Targeting the functional
integrity of complex I in the early phase of reperfusion may provide a novel therapeutic strategy to prevent tissue
injury after stroke.
Visual Overview—An online visual overview is available for this article. (Stroke. 2018;49:1223-1231. DOI: 10.1161/
STROKEAHA.117.019687.)
Key Words: flavin mononucleotide ◼ glutathione ◼ mitochondria ◼ oxidative stress ◼ reperfusion
S
to ischemia/reperfusion (I/R) damage.4 Mitochondria play a
key role in ischemic brain injury, both through impairment of
mitochondrial ATP production with bioenergetic dysfunction
and oxidative stress and by mediating cell death pathways.5,6
The lack of oxygen resulting from ischemia leads to impaired
mitochondrial ATP production (primary energy failure), collapse of the mitochondrial membrane potential, and, consequently, activation of intrinsic cell death pathways.7,8
After reperfusion, there is a transient restoration of bioenergetic state, which is followed by a second phase of energy
depletion (secondary energy failure) leading to delayed tissue
damage.9,10 This sequence of events has been confirmed by several laboratories, which have also ruled out microcirculatory
troke remains a leading cause of death and disability
worldwide.1 Despite decades of research, tissue-type
plasminogen activator and endovascular devices are the only
available treatment options.2 However, because of a narrow
therapeutic time window and potential contraindications, only
3% to 5% of stroke patients are able to benefit from these
interventions.3 This highlights the need for a broader understanding of tissue injury mechanisms to develop more effective treatments.
The loss of cerebral blood flow leads to decreased oxygen
levels, impairment of mitochondrial oxidative phosphorylation
and energy failure in the ischemic area, initiating a sequence
of pathophysiological events that after reoxygenation lead
Received September 11, 2017; final revision received February 1, 2018; accepted February 16, 2018.
From the Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New York, NY (A.K., A.S., C.K., C.A., G.M., P.Z., C.I., A.G.); and
School of Biological Sciences, Queen’s University Belfast, United Kingdom (A.S., A.G.)
Guest Editor for this article was Miguel A Perez-Pinzon, PhD.
*Dr Kahl and A. Stepanova contributed equally.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
117.019687/-/DC1.
Correspondence to Alexander Galkin, PhD, Division of Neonatology, Department of Pediatrics, Columbia University, 3959 Broadway, CHN 1201, New
York, NY 10032. E-mail ag4003@cumc.columbia.edu
© 2018 The Authors. Stroke is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article
under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original
work is properly cited.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.117.019687
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failure or changes in substrate availability as the cause of the
secondary energy depletion and cell death.11,12 In fact, data
indicate that secondary energy failure after transient ischemia might be the result of delayed mitochondrial damage,
likely because of oxidative stress.11,12 Mitochondrial electron
transport chain (ETC) enzymes are known to become rapidly
over-reduced in the absence of oxygen and to be damaged by
subsequent reoxygenation.11,13,14 However, despite intensive
research, the molecular mechanisms of mitochondria damage
in I/R remain to be elucidated.
Here, we used a mouse model of middle cerebral artery
occlusion (MCAO) to investigate acute I/R-induced changes
of mitochondrial function, focusing on the molecular and
biochemical mechanisms of primary and secondary energy
failure. Our results suggest a central role of mitochondrial
complex I (C-I) impairment in the development of bioenergetic
failure after acute I/R brain injury. Protection of C-I enzymatic
function during ischemia and the initial stages of reperfusion
could be an effective approach to prevent subsequent detrimental events in the I/R cascade, ultimately preserving neuronal
integrity and reducing brain damage after stroke.
pH 7.4) with 80 strokes of a Dounce homogenizer. The homogenate
was centrifuged at 1000g for 5 minutes at 4°C and the supernatant
was collected and used for respiration analysis. Respiration was measured using Oxygraph-2k (Oroboros Instruments).
For isolation of mitochondria, brain homogenates were centrifuged for 15 minutes at 20 000g. The obtained membrane pellet was
rinsed twice with (in mmol/L): 250 sucrose, 50 Tris-HCl (pH 7.5), 0.2
EDTA medium, and subsequently resuspended in the same medium.
Frozen aliquots were stored at −80°C until use. Protein content was
determined by bicinchoninic acid assay (Sigma) with 0.1% deoxycholate for solubilization of mitochondrial membranes.
Mitochondria and Respiratory Chain Analysis
Activities of respiratory chain enzyme and citrate synthase were measured spectrophotometrically as described.22 Flavin mononucleotide
(FMN) was determined fluorometrically.23 Immunoblot analyses were
performed using OXPHOS antibody cocktail (ab110413; Abcam).22
Experimental Design and Statistical Analysis
Mice were randomly assigned to the experimental groups, and
analyses were performed by an investigator blinded to the treatment
protocol. Data are expressed as mean±SEM. Differences were considered statistically significant when *P<0.05. Details of statistical
analyses are indicated in the Figure legends and online-only Data
Supplement.
Materials and Methods
All data and materials have been made publicly available at the
https://pure.qub.ac.uk/portal/ repository, and a detailed Methods section is available in the online-only Data Supplement.
MCAO Model
All procedures were approved by the Institutional Animal Care
and Use Committee of Weill Cornell Medicine and performed in
accordance with the ARRIVE guidelines (Animals in Research:
Reporting In Vivo Experiment).15 Transient MCAO was induced
using an intraluminal filament as described.16 In brief, 7- to 9-weekold, male mice were anesthetized with 1.5% to 2.0% isoflurane and
rectal temperature was maintained at 37.3±0.3°C. Cerebral blood
flow was measured with laser-Doppler flowmetry (Periflux System
5010; Perimed) in the ischemic center (2 mm posterior, 5 mm lateral
to bregma). After 35 minutes, the filament was retracted and cerebral blood flow reestablished. This duration of cerebral ischemia
has been used extensively by us17,18 and others19 and leads to reproducible infarct volumes of 50 to 60 mm3 and measurable neurological deficits. Only animals that exhibited a reduction in cerebral
blood flow 85% during MCAO and in which cerebral blood flow
recovered by 80% after 10 minutes of reperfusion were included in
the study.20,21 Three days after, MCAO functional impairment was
assessed and infarct volume was quantified in cresyl violet–stained
sections and corrected for swelling, as previously described.16
Administration of Glutathione-Ester and
Glutathione Content Measurement
Reduced glutathione-ethyl ester (G1404; Sigma Aldrich) was administered immediately after the initiation of reperfusion via jugular
vein (400 mg/kg). Saline injections served as control. Total glutathione content was determined using Glutathione Assay Kit (703002;
Cayman).
Results
Multiphasic Impairment of
Mitochondrial Respiration in I/R
We studied I/R-induced changes of mitochondrial function
in a mouse model of focal ischemia after transient MCAO.
Figure 1A shows representative traces of malate/glutamate-supported respiration of brain homogenates of sham
and after 35 minutes ischemia. ADP-stimulated mitochondrial respiration showed multiphasic impairment after I/R
(Figure 1B). A decline (59.0±5.9% of sham control; P<0.05;
n=4 per group) was observed during ischemia, followed by
a partial recovery (79.6±5.4% of control; P>0.05; n=5 per
group) at 10 minutes of reperfusion, and by a subsequent
profound decline in respiration (50.7±6.2% of control;
P<0.05; n=5 per group) at 30 minutes of reperfusion. These
early changes in mitochondrial function were followed by a
recovery of respiration at 1 hour of reperfusion (84.7±2.3%
of control; P>0.05; n=5 per group) and then by a progressive
decline in respiration, occurring 2 to 24 hours (55±7.8% of
control; P<0.05; n=5 per group, at 24 hours) after reperfusion (Figure 1B).
Citrate synthase activity, an indicator of mitochondrial content, did not significantly differ from control at any time point
(P>0.05; n=3–7 per group; Figure 1C). Further, we did not
detect significant changes in the protein levels of ETC complexes I to V and mitochondrial respiratory control ratio, during the ischemic phase or within 24 hours after reperfusion
(Figure I in the online-only Data Supplement).
Mitochondrial Measurements
After MCAO alone or MCAO with a period of recirculation as indicated, mice were decapitated. Brains were removed and a standardized
4 mm MCA area tissue sample dissected using a mouse brain matrix
(Zivic Instruments). The brain sample was homogenized in ice-cold
isolation buffer (in mmol/L: 210 mannitol, 70 sucrose, 1 ethylene
glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 5 HEPES,
Activities of Individual Mitochondrial Membrane
Complexes Are Differently Affected in I/R
We note that brain homogenates included nonsynaptic mitochondria but also synaptosomes containing synaptic mitochondria. The synaptic mitochondria, however, do not contribute
Kahl et al
Mitochondrial Complex I Impairment in Stroke
1225
Figure 1. Multiphasic pattern of mitochondrial respiratory decline after ischemia/reperfusion (I/R). A, Representative traces of mitochondrial oxygen consumption from sham (red) and 35 minute ischemia (black) in whole tissue homogenates. Addition of 1 mM cyanide (KCN)
almost fully inhibited respiration in homogenates. B, Effect of I/R injury on ADP-stimulated oxygen consumption (sham: n=19; different
time points n as indicated by the dots; Kruskal–Wallis test with Dunn multiple comparisons test; C) citrate synthase (C) and NADH oxidase (D) activity in the same preparations.
to ADP-stimulated respiration, because of restricted ADP
access to synaptosomes. Respiration measured in whole tissue homogenates is a product of several processes including
transport of substrates, activities of NAD-dependent dehydrogenases, and ETC.
For ETC activity measurements, substrate delivery into all
mitochondrial populations was ensured by addition of the membrane-permeabilizing agent alamethicin. To specifically assay
for I/R-induced changes in ETC complexes, we assessed the
overall activity of the respiratory chain by measuring NADH
oxidase (complexes I+III+IV; Figure 1D). The temporal profile
of NADH oxidase activity changes strongly corresponded to
the multiphasic pattern observed for the mitochondrial respiration (Figure 1B), suggesting that the observed mitochondrial
dysfunction is a result of I/R-induced ETC impairment.
Next, we measured the activities of individual ETC complexes: succinate dehydrogenase (C-II), ferrocytochrome c
oxidase (C-IV), and NADH:ubiquinone oxidoreductase (C-I),
as well as succinate:cytochrome c reductase (C-II+C-III).
C-II–linked activities were not affected at any time point after
I/R, indicating that C-II and C-III were not responsible for the
I/R-induced mitochondrial dysfunction (P>0.05; n=4–7 per
group; Figure 2A; C-II+III data not shown).
C-IV activity was lower at all time points compared with
sham (63.7±9.1% at 24 hours; P<0.05; n=4; Figure 2B).
However, the decline of C-IV did not follow the multiphasic
pattern observed in ADP-stimulated respiration and NADH
oxidase activity, suggesting that the mechanism of C-IV and
NADH oxidase impairment is different and that C-IV is not
responsible for the I/R-induced changes.
Complex I Impairment Is Associated With the
Multiphasic Pattern of Respiratory Decline in I/R
To elucidate the mechanisms of C-I impairment, we measured
the physiological activity and the relative amount of C-I using
two different approaches. The physiological activity of C-I
was assessed as NADH:Q1 reductase. The relative content of
C-I (proportional to flavin [FMN] content in the enzyme) was
determined as oxidation of NADH by hexaammineruthenium
(HAR). The NADH:HAR reaction occurs only at the head of
the enzyme, where HAR accepts electrons from the FMN, the
first redox center of C-I.24
The physiological activity of C-I (Figure 2C) followed
the same pattern as ADP-stimulated respiration (Figure 1B)
and NADH oxidase activity (Figure 1D), indicating
that the impairment of oxidative phosphorylation in the
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Figure 2. Enzymatic activities of
respiratory chain complexes are differently affected after ischemia/reperfusion (I/R). A, Complex II (C-II), (B)
C-IV, and (C) C-I NADH:Q1 and (D) C-I
NADH:hexaammineruthenium (HAR)
reductase activities were measured in
whole tissue homogenates; n=4 to 12 per
group; Kruskal–Wallis test.
ischemic tissue was because of a specific dysfunction of
C-I. Interestingly, NADH:HAR reductase showed an apparent decrease in the relative content of C-I after 35 minutes of
ischemia (68.7±1.3%; P=0.0001; n=6 per group), followed
by a rapid recovery after reoxygenation (97.0±3.9%;
P=0.99; n=6 per group) and a slow gradual decline at subsequent time points after I/R injury (78.7±4.3%; P=0.0003;
n=8 per group; Figure 2D).
Figure 3. Complex I (C-I) impairment is associated with the multiphasic pattern of respiratory decline observed in ischemia/reperfusion
(I/R). A, Overall activity of C-I at critical time points after I/R in mitochondrial membranes. B, In vitro time course of the reductive inactivation of NADH:hexaammineruthenium (HAR) reductase activity in mitochondrial membranes. Ischemic over-reduction of the ETC resulted
in a decrease of the relative amount of C-I (red line) compared with control (black line). Addition of reduced FMN restored NADH:HAR
reductase activity (arrow). C, Decrease of FMN in mitochondrial membranes obtained from the ischemic area after 35 minutes of middle
cerebral artery occlusion (MCAO) compared with sham controls (n=6–12 per group; P=0.0048; ANOVA). n.s. indicates not signficant.
Kahl et al
On the basis of the results above, we identified 35 minutes
ischemia and 30 minutes, 1 hour, and 24 hours of reperfusion
as critical time points for the development of mitochondrial
dysfunction in I/R injury. To further elucidate the mechanism
of C-I impairment, we assayed NADH:HAR and NADH:Q1
reductase activity in preparations of mitochondrial membranes isolated at these time points.
As shown in Figure 3A, 35 minutes ischemia resulted in
a robust decline of NADH:Q1 reductase and NADH:HAR
activity (74.2±4.2% and 80.5±2.7%, n=5 per group, respectively). A significant decrease in NADH:Q1 activity at 30
minutes (71.2±2.3%; n=4 per group), recovery at 1 hour
(92.7±2.1%; n=6 per group), and another activity decline
at 24 hours (58.9±3.3%; n=4 per group) of reperfusion was
observed, confirming the results in whole tissue homogenates.
Conversely, NADH:HAR activity showed a transient recovery at 30 minutes and 1 hour (92.9±2.9%; n=4 per group;
90.9±3.5%, n=6 per group, respectively) followed by a gradual decline at 24 hours after reoxygenation (77.2±2.0%; n=4
per group; Figure 3A).
The drop in physiological NADH:Q1 reductase activity
could be explained by two fundamentally different mechanisms: a decline in C-I content or a decrease in the catalytic
efficiency of C-I (number of NADH molecules oxidized by 1
enzyme molecule per minute). To estimate the relative catalytic efficiency of C-I, the ratio of NADH:Q1/NADH:HAR
reductase (Q1/HAR) was calculated as previously described.25
No significant reduction in the catalytic efficiency of C-I after
35 minutes of ischemia (92.1±4.2%; P>0.05; n=5 per group)
was observed. After reperfusion, a substantial decline in the
efficiency of the enzyme was found at 30 minutes (76.8±2.8%;
P<0.05; n=4 per group), followed by a complete recovery at
1 hour (102.6±4.0%; P>0.05; n=6 per group), and another
decline at 24 hours (79.4±6.0%; P>0.05; n=4 per group) after
I/R (Figure 3A). Note that although there was a significant
decrease in NADH:Q1 and NADH:HAR reductase activities
at 35 minutes ischemia, the Q1/HAR ratio did not change.
This could be interpreted as decrease in the number of functional C-I molecules in the membrane with no change in the
individual C-I enzyme catalytic efficiency (Q1/HAR).
Mitochondrial Complex I Impairment in Stroke
1227
the decrease in NADH:HAR reductase activity in the same
samples (Figure 3A, red bar) indicating ischemia-induced loss
of FMN from the enzyme without a decrease in C-I content.
Glutathione Improves C-I Dysfunction
and Neurological Outcome After I/R
Reperfusion-induced oxidative stress is one of the main contributors to tissue injury in I/R.28,29 Intracellular glutathionedependent enzymatic systems regulate the thiol-based redox
homeostasis and play a major role in the protection against
oxidative stress. As shown in Figure 4A, I/R resulted in a
significant decline of total glutathione content in the affected
area in comparison to the contralateral hemisphere or sham.
To test if reduced glutathione is able to confer a C-I–linked
neuroprotection in vivo, we administered membrane-permeable glutathione-ethyl ester at the onset of reperfusion.
Glutathione-ethyl ester restored total glutathione content in
the ipsilateral hemisphere to control values (Figure 4A), indicating that it is able to penetrate into the brain tissue and interact with cellular glutathione pool. Furthermore, administration
Functional Impairment C-I in Ischemia
Is Because of a Loss of FMN
To further explore the transient decrease of NADH:HAR
reductase after 35 minutes of ischemia, we performed in vitro
experiments. Brain mitochondrial membranes from naive
animals were incubated in conditions of metabolic reductive
hypoxia,26 mimicking the over-reduction of the ETC in ischemia (Figure 3B). The first redox center of C-I, noncovalently
bound FMN, is capable of dissociating from the enzyme.27 We
found that incubation of mitochondrial membranes in reductive conditions resulted in a rapid decline of the HAR reductase
activity over time, which seemed as a decrease of C-I content.
To confirm these in vitro findings, we determined the content
of noncovalently bound FMN in mitochondrial membranes
isolated at the critical time points after I/R. We found a significant decline of FMN content in the samples obtained after
35 minutes of ischemia (Figure 3C). This drop correlated with
Figure 4. Restoration of total glutathione (GSH) tissue content
attenuates ischemic injury and improves neurological outcome
72 hours after middle cerebral artery occlusion (MCAO). A, GSH
ester treatment at the onset of reperfusion restored total GSH
content in the ischemic brain area 30 minutes after reperfusion (n=3–4 per group; ANOVA). B, Reduced infarct volume in
GSH-treated mice compared with controls 72 hours after MCAO
(n=8–9 per group; P=0.0002; t test). C, Representative images
of corresponding Nissl-stained brain sections of a GSH-treated
mouse compared with control 3 days after MCAO. The red
dashed line indicates the infarct area. D, GSH-treated mice show
a significantly reduced motor impairment indicated by hanging
wire test (6–8 per group; P<0.0001; t test).
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of glutathione-ethyl ester led to a 61% reduction in infarct
volume (glutathione: 25.9±4.4 mm3 versus control: 66.8±7.0
mm3; P=0.0002; n=8–9 per group; Figure 4B and 4C), which
correlated with decreased body weight loss (glutathione:
7.0±2.4% versus control: 19.8±2.9%; P=0.0037; n=8–9
per group; Figure IIA in the online-only Data Supplement).
Overall functional outcome, assessed by the hanging wire test
(Figure 4D) and modified Bederson score (Figure IIB in the
online-only Data Supplement), was also improved compared
with saline-treated controls.
Glutathione Prevents Mitochondrial Dysfunction
and C-I Activity Decline Early After I/R
To test the effect of glutathione-ethyl ester administration on
mitochondrial function in vivo, we measured mitochondrial
respiration 30 minutes after the onset of reperfusion comparing glutathione-treated mice and saline-treated controls.
A significant increase in respiration was observed in tissue
homogenates prepared from the ischemic area of glutathionetreated mice compared with saline-treated animals subjected
to MCAO (P=0.03; n=5–6 per group; Figure 5A). These findings were associated with an increase in NADH:Q1 activity
(P=0.0002; n=6 per group; Figure 5B). NADH:HAR activity
was not affected (P>0.05; n=6 per group; Figure 5C) at 30
minutes of reperfusion.
Mitochondrial membranes isolated from the ischemic area
of untreated animals at the critical time points after MCAO
were preincubated ex vivo with thiol-reducing agent glutathione. NADH:Q1 and NADH:HAR activity were measured before and after glutathione incubation (Figure 5D).
Pre-incubation with glutathione was able to recover NADH:Q1
activity in membranes obtained at 30 minutes of reperfusion
(Figure 5D), indicating that reversible oxidation of C-I thiols
is the underlying post-translational modification early after
reperfusion. In contrast, glutathione treatment did not affect
NADH:Q1 activity at 24 hours of reperfusion, pointing to an
irreversible decline of C-I catalytic efficiency at later time
points.
Discussion
In the present study, we established a spatiotemporal profile
of biochemical mechanisms contributing to the evolution of
mitochondrial bioenergetic failure in I/R using a mouse model
of transient MCAO. Using brain homogenates, we demonstrate an I/R-induced, multiphasic pattern of mitochondrial
respiratory dysfunction in the brain, which to our knowledge has not been described before (Figure 6). We observed
an initial decline in respiration after 35 minutes of ischemia,
which is in agreement with previously published studies.28,30
The rapid partial recovery of mitochondrial respiration after
10 minutes of reperfusion followed by a first reflow-induced
respiratory decline at 30 minutes of reoxygenation has never
been reported. This decline in tissue respiration was followed
by an almost full recovery at 1 hour with a slow decrease at
later reperfusion time points (4–24 hours). In the samples
from all time points, citrate synthase activity was similar to
the sham controls, indicating preservation of mitochondrial
mass, for 24 hours post-ischemia.
The rate of mitochondrial respiration can be used as a predictor of tissue survival after I/R.31 Our tissue preparations
Figure 5. Glutathione (GSH) ester treatment improves complex I (C-I)–mediated bioenergetic failure early after reperfusion. A, GSH ester
treatment ameliorates mitochondrial respiratory decline at 30 minutes of reperfusion (n=5–6 per group; P=0.03; Mann–Whitney U test). B,
C-I activity is significantly improved in GSH-treated mice compared with controls (n=5–6 per group; P<0.05; Mann–Whitney U test). C, No
change in the relative amount of C-I was observed. D, In vitro pre-incubation of whole tissue homogenates with GSH was able to partially
recover ischemia/reperfusion (I/R)-induced C-I activity decline 30 minutes after reperfusion (n=4 per group; P=0.0009; t tests). GSH treatment did not affect C-I activity in sham, 1 or 24 hours after reperfusion. MCAO indicates middle cerebral artery occlusion.
Kahl et al
Mitochondrial Complex I Impairment in Stroke
1229
Figure 6. Proposed biochemical mechanisms contributing to complex I (C-I)–mediated energy failure in brain ischemia/reperfusion (I/R).
A, Electron transfer within C-I during normoxia. B, Ischemic over-reduction of the ETC results in a reduction of C-I FMN. Reduced flavin
(FMNH2) loses the affinity for its binding site and dissociates from the enzyme. C, Reflow-induced reoxidation of FMNH2 by molecular
oxygen is associated with the generation of reactive oxygen species (ROS) and likely contributes to oxidative stress in the mitochondrial
matrix. D, Recovery of C-I function at the early stage of reperfusion is followed by oxidation of critical C-I thiol residues (-SH) at later
stages. MCAO indicates middle cerebral artery occlusion.
from the MCA area include a mixture of different brain cells
so the observed changes in mitochondrial respiration cannot
be exclusively attributed to only one cell type. Several publications have described a glia/neuron ratio of 0.4 to 0.35 in
mouse brain,32,33 suggesting that neuronal mitochondria may
contribute the major fraction of respiratory activity in brain
homogenates.
We identified C-I as the key respiratory enzyme responsible
for the multiphasic pattern of mitochondrial dysfunction in
I/R. C-I has a high degree of flux control over oxidative phosphorylation and is considered to be the rate-limiting component of NADH oxidase activity within the ETC.34 Supporting
our results, a comparable pattern of rotenone-sensitive C-I
activity decline within 4 hours after reperfusion has been
reported previously.35
The observed progressive decline in the enzymatic activity
of C-IV after I/R injury is likely because of a different mechanism.11 C-II and C-III were not significantly affected in I/R,
which is in agreement with previous in vivo studies investigating mitochondrial membrane complexes in stroke.7,28,34,35
Inhibition of NAD+-dependent respiration after ischemia
has been observed in many stroke studies,7,13,28,34,35 but the
mechanism was never established. Our results strongly suggest that ischemia induces a reversible release of FMN from
C-I that caused the robust decrease of enzyme activity, which
was rapidly restored within 10 minutes of reflow. C-I contains
1 molecule of noncovalently bound FMN per molecule of the
enzyme,36 and it is the main source of membrane-associated
flavin in mitochondria.37 FMN release is likely to occur in
ischemia because of complex I over-reduction via reverse
electron transfer.38 Reductive dissociation of C-I FMN has
been reported in vitro27,38 but has not been shown in physiological settings. The release of a significant amount of reduced
FMN (30–40 µmol/L) to the mitochondrial matrix is potentially harmful for the cell. On reperfusion, reduced FMN can
be quickly reoxidized by oxygen,39 generating an equimolar
amount of H2O2 in the matrix and significantly contributing to
I/R-induced oxidative stress and tissue injury.
Mitochondrial function depends strongly on the maintenance of a cellular redox balance. Reperfusion triggers a burst
of reactive oxygen species formation directly damaging cells
via several different mechanisms.40 A critical component in
the mitochondrial antioxidant defense system is endogenous
glutathione. Reduced glutathione prevents or repairs oxidative
damage generated by reactive oxygen species. Glutathione
homeostasis is severely affected after I/R, therefore, making protein thiols a major target of oxidative damage.41–43
Mitochondrial respiratory enzymes are particularly susceptible to reactive oxygen species-mediated modulation of the
thiol redox systems.44,45
We demonstrate that restoring total glutathione levels in the
ischemic area at the onset of reperfusion is associated with
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protection of mitochondrial C-I activity and a robust neuroprotective effect. This is in agreement with previous studies showing a
cytoprotective action of membrane-permeable thiol antioxidants
against I/R-induced brain injury,46–48 but the mechanisms were
not completely understood. Here, we presented evidence suggesting a reversible oxidation of critical thiols of C-I early after
reperfusion, which is associated with a significant decrease in the
enzymatic activity. Reconstitution of glutathione levels in vivo
prevents mitochondrial bioenergetic dysfunction and C-I activity
decline at 30 minutes of reperfusion. It should be noted that, in
addition to protecting mitochondrial C-I, the antioxidant action of
glutathione could also have beneficial impact through other cellular pathways, including inhibition of apoptosis48 and prevention
of cytokine release.46 The ex vivo treatment of post-I/R mitochondrial membranes with glutathione recovered C-I activity at early,
but not at late time points after reperfusion. Our data suggest
that early reversible post-translational modifications of C-I are
followed by an irreversible enzyme damage. On the basis of the
neuroprotection of glutathione-ethyl ester and its positive effect
on mitochondrial bioenergetics at 30 minutes of reperfusion, it is
fair to speculate that this time point is particularly critical for the
evolution of tissue infarction in our I/R model.
Conclusions
We provide the first evidence that focal cerebral ischemia
induces a C-I–mediated pattern of mitochondrial respiratory
decline early after I/R. The ischemia-induced impairment
of C-I activity is because of the reversible dissociation of
reduced flavin from the enzyme (Figure 6). Because FMNH2
is a strong reactive oxygen species generator, this might be an
important mechanism for the development of transient oxidative stress after reintroduction of oxygen on reperfusion.
Administration of ethyl ester of glutathione at the onset of
reperfusion reduces infarction volume by 61% and improves
neurological outcomes. This neuroprotective effect is associated with an increase of mitochondrial respiration and
C-I activity. Thus, we conclude that reperfusion-induced
C-I decline at 30 minutes after reperfusion is the result of a
reversible modification of critical thiols of the enzyme. These
findings indicate that preventing oxidative thiol modification
of ETC early after the onset of reperfusion may be a viable
approach to ameliorate mitochondrial dysfunction after I/R
injury, ultimately reducing brain damage after stroke.
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17.
18.
19.
Sources of Funding
This study was supported by Medical Research Council UK grant
MR/L007339/1 (Dr Galkin) and National Institutes of Health grants
R01NS34179 (Dr Iadecola) and R01NS095692 (Drs Manfredi and
Iadecola). Dr Kahl was recipient of a postdoctoral research grant
from the Deutsche Forschungsgemeinschaft (KA3810/1-1).
20.
21.
Disclosures
None.
22.
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