doi:10.1016/j.freeradbiomed.2004.08.021

Serial Review: The Powerhouse Takes Control of the Cell: The Role ofMitochondria in Signal TransductionSerial Review Editor: Victor J. Darley-Usmar

MITOCHONDRIAL DYSFUNCTION AND OXIDATIVE STRESS:

CAUSE AND CONSEQUENCE OF EPILEPTIC SEIZURES

Manisha Patel

Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA

(Received 13 July 2004; Revised 27 August 2004; Accepted 27 August 2004)

Available online 16 September 2004

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Abstract—Mitochondrial dysfunction has been implicated as a contributing factor in diverse acute and chronic

neurological disorders. However, its role in the epilepsies has only recently emerged. Animal studies show that epileptic

seizures result in free radical production and oxidative damage to cellular proteins, lipids, and DNA. Mitochondria

contribute to the majority of seizure-induced free radical production. Seizure-induced mitochondrial superoxide

production, consequent inactivation of susceptible iron–sulfur enzymes, e.g., aconitase, and resultant iron-mediated

toxicity may mediate seizure-induced neuronal death. Epileptic seizures are a common feature of mitochondrial

dysfunction associated with mitochondrial encephalopathies. Recent work suggests that chronic mitochondrial oxidative

stress and resultant dysfunction can render the brain more susceptible to epileptic seizures. This review focuses on the

emerging role of oxidative stress and mitochondrial dysfunction both as a consequence and as a cause of epileptic

seizures. D 2004 Elsevier Inc. All rights reserved.

Keywords—Free radicals

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Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1952

Role of cellular metabolism in epilepsy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1952

Oxidative stress and mitochondrial dysfunction: consequence of prolonged epileptic seizures . 1952

Seizure-induced oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1953

Seizure-induced mitochondrial dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . 1954

Pathological consequences of elevated mitochondrial O2S� . . . . . . . . . . . . . . . . . . 1955

Mitochondrial oxidative stress and dysfunction: cause of epileptic seizures . . . . . . . . . . 1957

Therapeutic approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1959

Free Radical Biology & Medicine, Vol. , No. , pp. 1951–1962, 2004Copyright D 2004 Elsevier Inc.

Printed in the USA. All rights reserved0891-5849/$-see front matter

1951

part of a series of reviews on bThe Powerhouse Takes Control of the Cell: The Role of Mitochondria in Signal Transduction.Q The fully be found on the home page of the journal.

atel received her Ph.D. in Pharmacology and Toxicology from Purdue University and postdoctoral fellowship in Neuroscience at Duke

s currently an Assistant Professor in the Department of Pharmaceutical Sciences at the University of Colorado Health Sciences Center.

uses on the role of reactive species in neurodegeneration.

spondence to: Manisha Patel, Department of Pharmaceutical Sciences, School of Pharmacy, 4200 East Ninth Avenue, Box C238,

62, USA; Fax: +1 303 315 0274; E-mail: manisha.patel@uchsc.edu.

M. Patel1952

INTRODUCTION

The epilepsies are a group of clinical syndromes that

affect more than 50 million people worldwide. Epileptic

seizures can be convulsive or nonconvulsive episodes

characterized by synchronized abnormal electrical activ-

ity arising from a group of cerebral neurons. The

incidence of epilepsy is high in children younger than

5 years of age, but precipitously rises in individuals

older than 65 years [1]. In contrast with genetic forms of

epilepsy, acquired epilepsy accounts for approximately

60% of all cases and is usually preceded by injury such

as an episode(s) of prolonged seizures or status

epilepticus (SE), childhood febrile seizures, hypoxia,

or trauma [2]. These initial insults are thought to set in

motion complex molecular, biochemical, and structural

changes that result over time in the development of

spontaneous recurring seizures. The process whereby an

initial insult or injury leads to the development of the

epileptic condition is referred to as epileptogenesis.

Among the events that occur in response to the initial

insult, neuronal death has received significant attention

as the propagating factor that links the initial insult with

the epileptic condition. However, to date the role of

neuronal death in the development of epilepsy remains

controversial. Accumulating evidence suggests that

neuronal cell death may be both a cause and a

consequence of epileptic seizures. The evidence that

seizures cause brain injury comes from the demonstra-

tion that intense seizure activity associated with SE can

cause hippocampal damage in part by excessive

activation of glutamate receptors and resultant excito-

toxicity [3–5]. The idea that neuronal death can cause

epilepsy is supported by the fact that surgical removal of

a damaged hippocampus improves the condition of

epilepsy patients [6]. Because there is evidence that

injury per se can set in motion molecular events that

trigger the epileptic condition, it is important to under-

stand the biochemical causes and consequences of

epileptic seizures. Identifying these biochemical mech-

anisms may lead to therapies that interrupt the process of

epileptogenesis.

ROLE OF CELLULAR METABOLISM IN EPILEPSY

The most compelling evidence for mitochondrial

(dys)function in epilepsy is the recognition that dra-

matic metabolic and bioenergetic changes occur as a

consequence of both acute seizure episodes and chronic

epilepsy [7–9]. The acute consequence of seizures

associated with SE is an increase in cellular glucose

uptake and metabolism that is unparalleled by most

conditions [10–12]. Cerebral blood flow is increased to

match this hypermetabolism [10,13,14] and the

increased rate of glycolysis exceeds pyruvate utilization

by pyruvate dehydrogenase, resulting in increased

lactate buildup. Whereas hypermetabolism occurs in

human epileptic foci during ictal phases (seizure

episodes), hypometabolism is prevalent during interictal

phases (between seizure episodes) [11]. A bioenergetic

defect has been measured by 31P and the loss of

mitochondrial N-acetyl aspartate in human epileptic

tissue, implicating the involvement of mitochondria in

the altered neurotransmitter metabolism [7,15,16]. The

precise mechanism(s) of hypermetabolism in seizure-

induced brain damage and subsequent development of

epilepsy remains to be determined. These findings

provide a sound basis for the management of intractable

epilepsies with diet restriction or modification, e.g.,

ketogenic diet. Seizure-induced brain damage and

subsequent epilepsy can be inhibited by caloric restric-

tion or a ketogenic diet [17]. Interestingly these

paradigms are also known to limit free radical

formation.

OXIDATIVE STRESS AND MITOCHONDRIAL

DYSFUNCTION: CONSEQUENCE OF PROLONGED

EPILEPTIC SEIZURES

Because neuronal death may be an important factor

contributing to epileptogenesis, mechanisms that influ-

ence neuronal viability may also play a role in the

process of epileptogenesis. Such mechanisms (for exam-

ple, oxidative stress), could independently contribute to

the disease progression in addition to serving as

processes that underlie neuronal injury. Reactive oxygen

species (ROS) and reactive nitrogen species (RNS) are

thought to play important roles in diverse nervous system

disorders such as stroke, spinal cord injury, Parkinson

disease, Alzheimer disease, Huntington disease, Frei-

drich ataxia, and amyotrophic lateral sclerosis (ALS)

[18]. Whereas a precise role for ROS/RNS in the

epilepsies remains to be defined, a general role for

ROS in seizure-induced neuronal death is supported in

part by the observations that repeated seizures result in

increased oxidation of cellular macromolecules [19,20]

and that compounds with antioxidant properties (super-

oxide dismutase (SOD) mimetics, vitamin C, spin traps,

and melatonin) prevent seizure-induced pathology

[19,21–24]. Moreover, oxidative stress is thought to be

an important consequence of glutamate receptor activa-

tion and excitotoxicity [25–28], which play a critical role

in epileptic brain damage [4]. Finally, seizure-induced

neuronal death involves overlapping processes of

necrosis and apoptosis [29–33], which are strongly

influenced by mitochondrial function and oxidative

stress. Seizure activity initiates the mitochondrial path-

way of apoptosis via involvement of proapoptotic

Mitochondria and epilepsy 1953

factors, cytochrome c translocation, and caspase-3/9

activation [34,35].

SEIZURE-INDUCED OXIDATIVE STRESS

Studies in this laboratory are directed toward deter-

mining whether prolonged seizure activity in animals

results in the increased production of ROS and if

oxidative injury contributes to seizure-induced brain

damage. Two general approaches are taken to address

this. Because reactive species are transient, unstable, and

localized to cellular compartments, their measurement in

biological systems, particularly in vivo, is challenging.

The selective oxidation of certain cellular macromole-

cules to oxidative attack renders them suitable as

surrogate markers in vivo. In the first approach,

seizure-induced oxidative damage to susceptible targets

of oxidative damage (protein, lipids, and DNA) is

assessed after kainate-induced SE. Systemic or intra-

hippocampal or intra-amygdaloid injection of kainate, an

excitotoxic analog of glutamate, results in SE and a

rather selective pattern of neuronal loss in limbic areas of

the brain [36].

The presence of a labile iron motif in the iron–sulfur

(Fe-S) center of cytosolic and mitochondrial aconitase(s)

renders them sensitive byO2S� (Fig. 1) and related species

[37–39], allowing the measurement of aconitase activity

to serve as an index of steady-state O2S� levels. Measure-

ment of O2S� production using endogenous aconitase

inactivation as a surrogate marker has several advantages

in that it is highly sensitive to inactivation by O2S�, is

relatively specific for O2S�, and allows estimation of

compartmentalized (mitochondrial or cytosolic) O2S�

production [19,40]. This is evident by the demonstration

that brain and heart mitochondrial aconitase activity is

drastically diminished in Sod2 homozygous knockout

(Sod2�/�) mice [41,42]. In rats, kainate-induced seizures

inactivate O2S�-susceptible mitochondrial aconitase but

not oxidatively stable fumarase [41]. This inactivation is

Fig. 1. The Fe-S cluster of aconitase. Conversion of the active [4Fe-4Saccompanied by the release of an unligated iron atom and formation o

specific for the mitochondrial aconitase and not its

cytosolic counterpart that is also sensitive to O2S�,

implicating mitochondria as the major site of seizure-

induced O2S� production. Maximal inactivation of

mitochondrial aconitase occurs several hours after the

cessation of SE (16 h post-kainate injection) and at times

preceding the death of susceptible hippocampal neurons

[19]. Kainate-induced mitochondrial aconitase inactiva-

tion and hippocampal neuronal loss are attenuated in

transgenic mice overexpressing Sod2 [19] and exacer-

bated in mice partially deficient in Sod2 [43]. Mangane-

se(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP), a

broad-spectrum antioxidant, protects rats against seizure-

induced mitochondrial aconitase inactivation and hippo-

campal damage without decreasing behavioral seizure

intensity or frequency [19]. These findings support a role

for mitochondrial O2S� in hippocampal pathology pro-

duced by kainate-induced seizures. Inactivation of mito-

chondrial aconitase and a second oxidant-sensitive

enzyme, a-ketoglutarate dehydrogenase, has been sub-

sequently shown to occur after pilocarpine-induced

seizures [44]. Furthermore, seizure-induced O2S� pro-

duction has also been demonstrated by hydroethidium

staining in the rat model of lithium/pilocarpine-induced

SE [45].

Seizure-induced lipid peroxidation has been observed

by measuring products such as thiobarbituric acid reactive

substances [46] and F2-isoprostanes (F2-IsoP’s) [47]. F2-

IsoP’s are a novel class of prostaglandin F2-like com-

pounds, produced in vivo by a noncyclooxygenase and

free radical-catalyzed mechanism involving the perox-

idation of arachidonic acid [48]. F2-IsoP’s serve as a

sensitive, stable, and reliable marker of free radical-

induced lipid peroxidation in vivo. Prolonged seizures

produce a large increase in prostaglandin derivatives,

including prostaglandin-F2a, a precursor of F2-IsoP

[49,50]. In adult rats, kainate-induced SE produces a large

subregion-specific increase in F2-IsoP levels in the highly

vulnerable CA3 region before cell damage. Interestingly,

]2+ cluster of aconitase to the inactive [3Fe-S]+ form by O2

S� isf H2O2.

M. Patel1954

the dentate gyrus, a region that contains granule neurons

which are resistant to kainate-induced neuronal death, also

shows marked increases in F2-IsoP levels [20]. It remains

to be determined whether these potentially bioactive lipid

derivatives exert a cytotoxic or signaling role in response

to seizure activity and if mitochondrial oxidative stress

contributes to their formation.

Seizure-induced oxidative DNA damage has been

demonstrated by using the measurement of 8-hydroxy-2-

deoxyguanosine (8-OHdG), an oxidatively modified

guanine adduct, as an index of oxidative DNA damage

[19,51]. The ratio of steady-state levels of 8-OHdG to 2-

deoxyguanine (2-dG) reflects oxidative DNA damage

[52]. Kainate administration in adult rats produces a large

increase in the ratio of oxidized:nonoxidized bases (8-

OHdG:2-dG) at times preceding overt cell death,

suggesting a potential role for oxidative DNA damage

in the sequence of events leading to seizure-induced

neuronal death. More recent studies confirm that

mitochondrial, not nuclear, DNA is the source of this

seizure-induced increased 8-OHdG level (L. P. Liang and

M. Patel, unpublished observations), as would be

expected by the close proximity of mitochondrial DNA

to the major source of O2S� production, i.e., the electron

transport chain (ETC), which accounts for the severalfold

more oxidized DNA bases in mitochondrial DNA in

comparison to nuclear DNA [53–55].

Two lines of evidence suggest that seizure-induced

oxidative damage correlates with neuronal vulnerability.

First, oxidative damage occurs to a greater extent in brain

areas that are vulnerable to kainate-induced brain damage

(hippocampal areas CA3 and CA1 and piriform cortex)

compared to resistant areas (dentate gyrus and cerebel-

lum). Second, seizure-induced mitochondrial oxidative

stress shows a strong age dependence [56,57], which

may explain the age-related vulnerability of the brain to

seizure-induced brain damage [58,59]. The higher

expression of uncoupling protein-2 (UCP-2) in the

neonatal, but not the adult, brain was shown to be an

underlying factor that renders the neonatal brain more

resistant to seizure-induced free radical production and

neuronal injury [57]. The mechanism by which UCP-2

inhibits seizure-induced ROS production in the neonatal

brain may be related to dissipation of the mitochondrial

membrane potential by UCP-2-mediated proton leak and

mitochondrial calcium overload [57].

An additional approach to assessing the role of

oxidative stress in seizure-induced brain damage is the

use of SOD mutant mice. Whereas oxidized proteins,

lipids, and DNA are useful surrogate markers for detecting

the presence of oxidative stress, it is difficult to identify the

reactive species that is important in the oxidative insult

and its cellular source. Mice that are transgenic/knockout

for the three endogenous SODs in the cytoplasm

(CuZnSOD or Sod1) [60], mitochondria (MnSOD or

Sod2) [61], and extracellular compartment (EC-SOD or

Sod3) [62] allow verification of the role of O2S� in the

injury process as well as testing which cellular compart-

ment contributes to the injury-induced O2S� production.

Hippocampal damage produced by local application of

kainate has been studied in Sod1, Sod2, and Sod3

transgenic mice and knockout mice. These studies show

that overexpression of Sod2 [19] and, to a lesser extent,

Sod3 [63], but not Sod1 (L.-P. Liang and M. Patel,

unpublished observations), protects against kainate-

induced hippocampal damage. Moreover, mice over-

expressing Sod2 were protected against kainate-induced

mitochondrial aconitase inactivation and hippocampal cell

death [19], whereas mice with partial Sod2 deficiency

(Sod2�/+) showed exacerbation of these effects [43].

Together with the demonstration that seizures inactivate

mitochondrial, not cytosolic, aconitase, these studies

suggest that mitochondrial O2S� may play a dispropor-

tionately greater role in seizure-induced O2S� production.

SEIZURE-INDUCED MITOCHONDRIAL DYSFUNCTION

These studies demonstrate that prolonged seizures

acutely result in oxidative damage to lipids, DNA, and

susceptible proteins. However, whether oxidative stress

and/or mitochondrial dysfunction occurs several weeks

after SE is unknown. Kudin et al. [64] addressed this

issue by assessing mitochondrial dysfunction several

weeks after pilocarpine-induced SE. They show that

the activities of complexes I and IV of the ETC

decrease and complex II increases 1 month after

piloparpine SE. This is accompanied by lowered

mitochondrial membrane potential measured by rhod-

amine-123 fluorescence in the CA1 and CA3 areas.

These changes may be attributed to decreased mito-

chondrial DNA copy number that results in down-

regulation of oxidative phosphorylation (OXPHOS)

enzymes encoded by mtDNA. Together, these studies

suggest that the acute effect of SE, i.e., increased

mitochondrial oxidative stress, may result over time in

oxidative damage to mtDNA (as suggested by

increased levels of 8-OHdG) and decreased expression

of mitochondrially encoded proteins required for the

functioning of the ETC. Seizure-induced accumulation

of oxidative mitochondrial DNA lesions and resultant

somatic mtDNA mutations could over a period of time

render the brain more susceptible to subsequent

epileptic seizures, particularly in the context of

advancing age. A link between mitochondrial dysfunc-

tion and epilepsy is strengthened by the finding that

some patients with temporal lobe epilepsy show

mitochondrial complex I deficiency in the seizure foci

[65], which is the leading cause of increased O2S�

Mitochondria and epilepsy 1955

production and mitochondrial dysfunction in patients

with Parkinson disease [66].

Mechanistic studies assessing the role of mitochon-

drial functions on neuronal excitability have been

attempted by measuring seizure-like events (SLE) in

hippocampal explant cultures [67–69]. The SLEs, usu-

ally generated by a combination of electrical stimulation

of the axons of the granule neurons (mossy fibers) and

lowering the Mg2+ concentration in the bathing medium

(0 Mg2+) to relieve the voltage-dependent Mg2+ blockade

of the NMDA receptors, result in increased mitochon-

drial calcium accumulation, mitochondrial depolariza-

tion, decreased nicotinamide adenine dinucleotide

(NAD(P)H) autofluoresence, and O2S� generation [68].

The O2S� production measured by hydroethidium pro-

gressively increases during and with each consecutive

SLE along with increases in both cytosolic and mito-

chondrial calcium, thus providing a link between

mitochondrial calcium, free radical production, and

neuronal death.

PATHOLOGICAL CONSEQUENCES OF ELEVATED

MITOCHONDRIAL O2

S�

The studies above demonstrate that mitochondrial

oxidative stress is an early consequence of SE and this

may contribute to the persistent mitochondrial dysfunc-

tion observed in the epileptic brain. Based on the

measurement of mitochondrial aconitase inactivation,

seizure-induced mitochondrial O2S� production occurs

several hours after the cessation of acute SE [70]. The

source of seizure-induced O2S� production which results

in mitochondrial aconitase inactivation remains to be

identified, but may involve inhibition of an ETC complex.

Because O2S� has limited reactivity, its role in patho-

physiological processes has been attributed to more

potent oxidants such as peroxynitrite (ONOO�) and

HOS. However, experimental evidence over the past

several decades continues to support a role for O2S� in

toxic injury. A central mechanism of O2S� toxicity is

direct oxidation and consequent inactivation of Fe-S

proteins such as aconitases. Direct oxidative inactivation

of susceptible Fe-S enzymes is thought to be the basis of

O2S� toxicity in bacteria and yeast [71]. Evidence for this

comes from the following studies: (1) increasing O2S�

concentrations by redox cycling agents, e.g., paraquat, or

SOD deficiency inactivates dehydratases containing Fe-S

centers such as aconitases, dihydroxy acid dehydratase, 6-

phosphogluconate dehydratase, and fumarases A and B

[38]; (2) an increase in free iron concentrations as

measured by electron paramagnetic spin resonance

studies can be observed concomitant with O2S�-mediated

inactivation of Fe-S enzymes, suggesting that Fe-S

enzymes can be a source of free iron [72]; (3) oxidative

inactivation of Fe-S enzymes and the resultant increase in

free iron can lead to DNA damage, mutagenesis,

decreased growth, and nutritional auxotrophies in Escher-

ichia coli [73]; and (4) these deficits can be overcome by

complementation with functional SOD genes or catalytic

antioxidants such as the metalloporphyrins [74,75].

Whether O2S� mediates toxicity via oxidative inacti-

vation of Fe-S enzymes depends on the abundance of the

Fe-S enzymes, competing targets for O2S� within its

close vicinity, the rate of reaction of O2S� with these

targets, and the availability of substrates that may protect

the Fe-S enzymes from inactivation. The fate of

mitochondrial O2S� is controlled by its reactivity with

several important targets that include Sod2, nitric oxide

(NO), and susceptible Fe-S centers. The most important

of these targets is Sod2. It is estimated that the majority

of mitochondrial O2S� is consumed by Sod2 based on

the estimation of steady-state [O2S�] in the picomolar

range [76] and SOD’s extremely high rate of reaction

(1.8 � 109 M�1 s�1) coupled with its micromolar

subcellular concentrations. The remainder of O2S� is

available to react with the nanomolar levels of NO and

micromolar levels of Fe-S targets, most notably aconi-

tase, at estimated rate constants of 6.9 � 1010 [77] and

~107 M�1 s�1 [37], respectively. Based on these

assumptions, it has been estimated that in E. coli, 75%

of O2S� reacts with labile Fe-S centers and 25% with NO

[78]. Therefore, the labile Fe-S enzymes such as

mitochondrial aconitase, which are abundant in the brain

and offer an important target for O2S�, particularly of

steady-state [O2S�], increase and endogenous Sod2 is

compromised. The preferential attack of the Fe-S center

of aconitase by O2S� is attributed in large part to the

electrophilic nature of a single unligated, solvent-

exposed iron moiety (Fea; for review see [40]). Cluster

oxidation results in instability and promotes the loss of

Fea from the [4Fe-4S]2+ cluster with concomitant loss of

enzyme activity (Fig. 1). Additionally, substrate-free

aconitase is inactivated with a much higher second-order

rate constant by O2S� compared with O2, H2O2, and

ONOO� [37,79,80]. Oxidative inactivation of aconitase

in this manner may have at least two major consequen-

ces. First, the formation of an inactive [3Fe-4S]+ cluster

results in the concomitant release of Fe2+ and H2O2. In

fact, O2S�-mediated inactivation of Fe-S-containing

enzymes may pose a significant oxidative burden

because it provides equimolar amounts of H2O2 per

mole of O2S� [78]. The release of Fe2+ and H2O2,

ingredients of the Fenton reaction, can result in gen-

eration of the potent HOSradicals, which can oxidize

mitochondrial proteins, DNA, and lipids, thereby ampli-

fying O2S�-initiated oxidative damage (Fig. 2). Second,

even partial inhibition of mitochondrial aconitase could

result in tricarboxylic acid (TCA) cycle dysfunction that

Fig. 2. Proposed scheme of events that result in seizure-induced mitochondrial O2

S� toxicity via oxidative aconitaseinactivation. Under normal conditions, the fate of mitochondrial O2

S� is controlled by its reactivity with several targets thatinclude Sod2, NO, and Fe-S centers. Elevated steady-state mitochondrial O2

S� production resulting from sustained seizureactivity favors oxidative inactivation of the [4Fe-4S]2+-containing aconitase and simultaneous release of Fe2+ and H2O2.Generation of HO

Svia Fenton chemistry can damage mitochondrial proteins, DNA, and lipids, thereby amplifying O2

S�-initiated oxidative damage. The release of Fe may affect cellular iron homeostasis, and decreased aconitase activity couldinterrupt its TCA cycle functions.

M. Patel1956

could impact neuronal excitability and seizure threshold

by altering neurotransmitter levels via astrocytic–neuro-

nal glutamate–glutamine cycling and/or a mere decline in

ion exchange by declining ATP levels.

Keyer and Imlay first demonstrated the release of iron

from E. coli aconitase and its involvement in O2S�-

mediated toxicity [73]. Vasques-Vivar et al. [81] tested

the idea that O2S�-mediated inactivation of mitochon-

drial aconitase can be a source of HOSradical formation

by Fenton chemistry initiated by the coreleased Fe2+ and

H2O2. This study demonstrates the formation of HOS

radicals originating from mitochondrial aconitase upon

oxidative inactivation by electron paramagnetic reso-

nance. These studies suggest that purified aconitase is

capable of generating HOSradicals in a cell-free system.

Whether aconitase inactivation results in free radical

formation and toxicity in vivo remains unknown. We

recently addressed this question in the 1-methyl-4-

phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of

experimental parkinsonism. MPTP administration mobi-

lizes an early pool of chelatable iron in mitochondrial

fractions that temporally coincides with the inactivation

of mitochondrial aconitase [82]. Both mitochondrial

chelatable iron and aconitase inactivation are inhibited

in mice overexpressing Sod2, which are resistant to

MPTP neurotoxicity. This study suggests that iron-

dependent mitochondrial O2S� toxicity can contribute

to neurodegeneration in vivo.

In addition to catalyzing the formation of HOS

radicals, both Fe2+ and H2O2 released by aconitase

Mitochondria and epilepsy 1957

inactivation can play independent roles in gene expres-

sion and cell signaling, respectively. The short-term fate

of the iron released from Fe-S clusters most likely

involves complexation with low-molecular-weight che-

lators such as citrate and ADP. However, the long-term

consequences of this iron on cellular iron homeostasis

remain to be determined. Cellular iron status is main-

tained by iron-dependent regulation of the synthesis of

iron storage (ferritin) and uptake (transferrin receptor;

Tfr) proteins via the binding of iron-responsive proteins

(IRPs) to stem–loop structures in the 5Vand 3Vuntranslatedregions (UTRs) of ferritin and Tfr mRNAs, respectively

[83]. Cytosolic aconitase regulates cellular iron via its

dual function as IRP1 [84]. An increase in cellular iron

results in decreased RNA binding activity of IRPs,

increased translation of iron-storage proteins, e.g.,

ferritin, and simultaneous stabilization and decreased

synthesis of Tfr mRNA [85]. The presence of an iron-

responsive element in the 5VUTR of mitochondrial

aconitase mRNA [86] has been shown to alter mitochon-

drial aconitase synthesis in response to cellular iron

availability. Increased cellular iron levels result in

enhanced synthesis of mitochondrial aconitase and

ferritin [87], whereas iron deficiency decreases the

mitochondrial aconitase synthesis [88]. The impact of

O2S�-mediated iron release from mitochondrial aconitase

on cellular iron homeostasis remains to be fully

determined. Recent work shows that increased expres-

sion of Tfr mRNA occurs in conjunction with MPP+-

induced aconitase inactivation in cerebellar granule

neurons in vitro [89]. This suggests increased efforts

made by the cell to accumulate iron in the face of

mitochondrial oxidative stress, perhaps due to the

localized changes in subcellular iron changes.

Posttranslational inactivation of Fe-S-containing mito-

chondrial aconitase by O2S� may pose a significant

oxidative burden because it can provide equimolar

amounts of H2O2 per mole of O2S�. H2O2 can oxidize

the mitochondrial GSH pool leading to decreased GSH/

GSSG ratios. Diminished GSH/GSSG impacts the

mitochondrial redox status, resulting in a more oxidized

environment. Additionally, GSH and other cellular

reductants (e.g., NADH) are important for reductive

repair of proteins and DNA. For example, aconitase can

be repaired by reinsertion of iron in the presence of

cellular reductants such as GSH. Finally, mitochondrial

GSH/GSSH ratios may function in regulating the thiol/

disulfide status of proteins involved in neuronal viability

(e.g., the mitochondrial permeability transition pore)

[90]. Using the glutathione redox couple as an indicator

of oxidative stress, seizure-induced changes in mitochon-

drial and tissue redox status were recently assessed.

Whereas tissue GSH/GSSH levels are minimally

changed after kainate-induced seizures in rat hippo-

campus, mitochondrial GSH/GSSG levels show an early

and persistent decline predominantly due to increased

GSSG levels [91]. This is accompanied by a mild

increase in tissue glutathione peroxidase activity and

decrease in glutathione reductase activity.

MITOCHONDRIAL OXIDATIVE STRESS AND

DYSFUNCTION: CAUSE OF EPILEPTIC SEIZURES

As described above, oxidative stress and mitochon-

drial dysfunction occur as a consequence of prolonged

epileptic seizures and may contribute to seizure-induced

brain damage. However, as with each of the diverse

signaling events activated by seizures, the crucial ques-

tion is whether acute changes in seizure-induced free

radical production and mitochondrial dysfunction are

epileptogenic, i.e., resulting in chronic redox alterations

that increase seizure susceptibility and result in the

development of subsequent epilepsy. The most prominent

example of mitochondrial dysfunction causing epilepsy is

the occurrence of epilepsy in mitochondrial disorders

arising due to mutations in mtDNA or nuclear DNA. For

example, myoclonic epilepsy with ragged red fibers

(MERRF) is a syndrome in which a single mutation of

the mitochondrially encoded tRNALys results in a disorder

consisting of myoclonic epilepsy and a characteristic

myopathy with ragged red fibers [92]. Defects in complex

I and complex IV of mitochondrial OXPHOS are the

leading mechanism by which this mitochondrial gene

mutation produces MERRF [92,93]. Several normal

functions of mitochondria, ranging from bioenergetic

functions to metabolic functions, can impact neuronal

excitability. These include cellular ATP production, ROS

formation, synthesis and metabolism of neurotransmit-

ters, fatty acid oxidation, calcium homeostasis, and

control of apoptotic/necrotic cell death. Furthermore,

these vital functions are closely intertwined and which

of these factors contributes to the seizures associated with

mitochondrial dysfunction remains unclear.

Several common neurological insults such as hypoxia,

trauma, and aging and neuronal diseases such as stroke

and Alzheimer disease render the brain susceptible to

epileptic seizures [1,94,95]. In fact, although epilepsy

occurs in all age groups, the incidence of epilepsy is

markedly increased in the elderly [1]. The ability to

produce oxidative stress and mitochondrial dysfunction

is common to each of these neuronal conditions. This

raises an intriguing possibility that mitochondrial dys-

function initiated by free radical production could

increase seizure susceptibility. In support of this idea,

seizure activity can be induced by paradigms which are

capable of increasing mitochondrial free radicals, for

example, increased oxygen tension (hyperbaric hyper-

oxia) [1,96] and local infusion of redox-active iron salts

M. Patel1958

[97] or mitochondrial toxins [98]. Mice partially deficient

in Sod2 (Sod2�/+) are particularly useful because they

provide a model of sublethal chronic elevation of

mitochondrial O2S� in which to test this hypothesis.

Work from this laboratory provides experimental evi-

dence linking mitochondrial oxidative stress with

increased seizure susceptibility induced by aging, envi-

ronmental stimulation, or kainate administration.

Whereas Sod2�/� mice show extensive mitochondrial

dysfunction and behavioral phenotypes such as ataxia

and seizures before neonatal death [41,99], Sod2�/+ mice

appear both biochemically and phenotypically normal at

birth but develop age-related deficits consistent with

chronic mitochondrial oxidative stress [100,101]. A

subset of Sod2�/+ mice developed spontaneous and

handling-induced seizures as a function of advancing

age [43]. The age-related onset of seizures in Sod2�/+

mice correlated with increased mitochondrial oxidative

stress (mitochondrial aconitase inactivation and mito-

chondrial, but not nuclear, 8-OHdG formation) and

mitochondrial dysfunction as measured by O2 utilization.

Before the age at which spontaneous and handling-

induced seizures occurred, Sod2�/+ mice showed

increased susceptibility to kainate-induced seizures and

hippocampal cell loss [43].

Although several factors associated with mitochon-

drial dysfunction have the ability to enhance seizure

Fig. 3. Proposed events leading to increased seizure susceptibility in Sosteady-state mitochondrial O2

S� levels and oxidative inactivationPosttranslational inactivation of aconitase by O2

S� can lead to the rradical formation via the Fenton reaction. Chronic free radical productiosensitive glutamate transporters and a failure to sequester glutamateextracellular glutamate and activation of excitatory amino acid receptseizures in Sod2�/+ mice. In addition to bioenergetic treatments, potentneuroprotective antiepileptic drugs.

susceptibility in Sod2�/+ mice, the most likely suspects

include targets sensitive to oxidative insults that raise

plasma membrane excitability. The high-affinity astro-

glial and neuronal glutamate transporters were examined

based on their known sensitivity to oxidative damage

[102] and their crucial role in maintaining low levels of

synaptic glutamate from reaching neurotoxic levels.

Sod2�/+ mice show an age-dependent decrease in the

hippocampal expression of glial glutamate transporters

(GLT-1 and GLAST), which may explain their increased

vulnerability to epileptic seizures [43]. Chronic O2S�

production in the mitochondrial matrix can be rapidly

converted to H2O2 by dismutation and Fe-S inactivation.

Unlike O2S�, H2O2 is freely permeable across cellular

membranes and can be generated in an equimolar amount

as O2S� by oxidative inactivation of mitochondrial

aconitase, which also showed parallel age-related decline

in Sod2�/+ mice. H2O2 generated in this manner could

oxidize astroglial glutamate transporters and lead to their

decreased expression (Fig. 3). This suggests that mito-

chondrial oxidative stress and resultant dysfunction are

sufficient to increase seizure susceptibility. Depletion of

ATP may be another important independent factor

contributing to increased seizure susceptibility associated

with mitochondrial dysfunction in part due to the

dependence of neurotransmitter and ion transport sys-

tems on ATP.

d2�/+ mice. Decreased Sod2 levels result in chronic elevation ofof vulnerable Fe-S-containing proteins such as aconitase.

elease of redox-active iron and H2O2 and generation of HOS

n in this manner can lead to oxidation and dysfunction of redox-. The process of aging uncovers this effect. Accumulation ofors could serve as the biochemical signal that initiates epilepticial therapeutic strategies include antioxidants, iron chelators, and

Mitochondria and epilepsy 1959

THERAPEUTIC APPROACHES

Therapies for the epilepsies are largely aimed at

decreasing neuronal excitability and thereby controlling

the occurrence of epileptic seizures. A recent therapeutic

approach is to search for antiepileptogenic rather than

antiepileptic drugs that would target underlying pro-

cesses that lead to the development of epilepsy [103].

Antiepileptogenic therapies aimed at mitochondrial bio-

energetics and oxidative stress pathways are in their

infancy and have been largely limited to animal studies.

Clinical trials of vitamin E as an add-on therapy for

refractory epilepsy have been controversial, with largely

failed attempts to influence the occurrence of epileptic

seizures in pediatric patients. Creatine, an endogenous

guanidine, functions with phosphocreatine and a mito-

chondrial form of creatine kinase as a spatial energy

buffer between the cytosolic and the mitochondrial

compartments. Administration of creatine has been

shown to be protective in animal models of CNS injury,

including trauma, ischemia, 3-nitropropionic acid,

MPTP-induced parkinsonism, and ALS [104–106].

Creatine supplementation has been effective in reducing

hypoxia-induced seizures in both rats and rabbits

[107,108]. Diet modification by ketogenic diet or caloric

restriction represents a nonpharmacologic strategy that

decreases seizure frequency in the epileptic EL mouse

[109,110]. The finding that newer antiepileptic drugs

such as zonisamide possess antioxidant properties [111]

raises the possibility that free radical scavenging may in

part underlie their antiepileptic actions.

Oxidative stress and neuronal damage but not behav-

ioral seizures induced by kainate can be ameliorated by at

least two types of SOD mimetics, the manganese

porphyrin MnTBAP [19] and the salen compound

EUK134 [22]. Other compounds with antioxidant proper-

ties that inhibit seizure-induced brain injury include the

hormone melatonin, a nitrone spin trap, and vitamin C

[21,24,112]. Whether chronic administration of antiox-

idant compounds has an effect on epileptogenesis remains

to be determined. Development of therapies influencing

mitochondrial bioenergetics and oxidative stress for the

epilepsies will ultimately depend on unraveling their role

in the disease process.

CONCLUSION

Oxidative stress and mitochondrial dysfunction occur

as a consequence of prolonged epileptic seizures and

influence seizure-induced brain injury. Mitochondria

contribute to a disproportionately greater extent to

seizure-induced O2S� production. Accumulating evi-

dence suggests that mitochondrial oxidative stress and

dysfunction initiated by O2S� radicals may contribute to

seizure-induced brain damage. Conversely, mitochondrial

oxidative stress and/or dysfunction can render the brain

more susceptible to epileptic seizures. Therefore, oxida-

tive stress and mitochondrial dysfunction may be both an

important cause and a consequence of prolonged seizures.

Insight into the mechanisms by which seizures initiate

oxidative stress and mitochondrial dysfunction and vice

versa may provide novel therapeutic approaches for the

treatment of epilepsies.

Acknowledgments—The author thanks the Epilepsy Foundationof America’s Partnership for Pediatric Epilepsy and NINDS(RO1NS39587 and RO1 NS45748) for their support.

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ABBREVIATIONS

ETC—electron transport chain

Fe-S— iron sulfur

F2-IsoP—F2-isoprostane

GSH—reduced glutathione

GSSG—glutathione disulfide

HOS

—hydroxyl radical

H2O2—hydrogen peroxide

IRP—iron-responsive protein

MnTBAP—manganese III tetrakis(4-benzoic acid)

porphyrin

mtDNA—mitochondrial DNA

NADH—nicotinamide adenine dinucleotide

NMDA—N-methyl-d-aspartate

NO—nitric oxide

ONOO�—peroxynitrite

OXPHOS—oxidative phosphorylation

O2S�—superoxide

RNS—reactive nitrogen species

ROS—reactive oxygen species

SE—status epilepticus

SLE—seizure-like event

SOD—superoxide dismutase

TCA—tricarboxylic acid

Tfr— transferrin receptor

UCP-2—uncoupling protein-2

UTR—untranslated region

2-dG—2-deoxyguanosine

8-OHdG—8-hydroxy-2-deoxyguanosine