Mitochondrial oxidative stress and increased seizure susceptibility in Sod2−/+ mice☆
Introduction
Mitochondrial oxidative stress is thought to play an important mechanistic role in numerous acute and chronic neuronal disorders [1], [2]. However, its role in the epilepsies is incompletely understood. It has been previously demonstrated that oxidative stress and mitochondrial dysfunction occur as a consequence of prolonged epileptic seizures and may play an important role in seizure-induced brain damage [3], [4]. The converse is unclear, i.e., whether mitochondrial oxidative stress and resultant dysfunction can contribute to epileptic seizures. Generalized and partial seizures are associated with myoclonic epilepsy and mitochondrial encephalopathies arising from mitochondrial DNA (mtDNA) mutations. Myoclonic epilepsy with ragged red fibers (MERRF) is the first epilepsy in which a molecular defect was identified and linked with an epilepsy syndrome [5], [6]. The molecular error in MERRF arises from a single mutation of the tRNALys resulting in a disorder consisting of myoclonic epilepsy and a characteristic myopathy with ragged red fibers [5]. Defects in complex I and complex IV of mitochondrial oxidative phosphorylation (OXPHOS) are the leading mechanism by which this mitochondrial gene mutation produces MERRF [6]. However, the biochemical basis of epileptic seizures in mitochondrial disorders remains unclear.
Mitochondria have several important functions that include cellular ATP production, reactive oxygen species formation, synthesis and metabolism of neurotransmitters, fatty acid oxidation, and control of apoptotic/necrotic cell death. The generation of mitochondrial ATP is a concerted effort of the tricarboxylic acid (TCA) cycle and OXPHOS. Each of these vital interrelated mitochondrial functions is crucial for normal brain function, and a defect in one or more of these can be a likely cause of seizures. Mitochondrial dysfunction can therefore have a major impact on certain epilepsies, particularly those associated with mtDNA mutations [4], [7]. However, which of these factors contributes to seizures associated with mitochondrial dysfunction remains unclear. Whereas epilepsies arising from mtDNA mutations are rare, epilepsies arising from pathological insults, e.g., hypoxia–ischemia or trauma that can increase oxidative stress and mitochondrial dysfunction, are common and underscore the importance of understanding mitochondrial dysfunction in seizure disorders.
An animal model with chronically increased steady-state mitochondrial O2− levels provides a useful tool to ask whether mitochondrial oxidative stress plays a role in seizure disorders. Mutant mice deficient in manganese superoxide dismutase (MnSOD or SOD2), a critical mitochondrial antioxidant, provide such a model. Generation of mice lacking each of the three compartmentalized SODs reveals that complete lack of SOD2 is lethal, whereas mice lacking CuZnSOD or ECSOD are viable and do not exhibit remarkable phenotypes [8], [9], [10]. The Sod2 gene was initially inactivated in mice from two genetic backgrounds, an outbred CD1 and an inbred C57BL/6 mixed background [8], [11]. SOD2 homozygous knockout (Sod2−/− tm1CJE) mice exhibit dilated cardiomyopathy, fatty liver, and neurodegenerative disease [8]. Mitochondrial disease in these mice is characterized by biochemical defects that include a dramatic inactivation of brain mitochondrial aconitase, a marker of steady-state O2− levels [12], and exhibit frequent seizures before neonatal death [13]. The Sod2−/− mouse model underscores the importance of acute mitochondrial O2− toxicity. However, because the Sod2−/− mice are neonatal lethal, Sod2 heterozygous knockout (Sod2−/+) mice are better suited to study chronic mitochondrial O2− toxicity that may mimic physiological insults. The Sod2−/+ mice are normal at birth, but develop biochemical abnormalities associated with chronically increased mitochondrial O2− production as they age [14], [15]. Using the Sod2−/+ model of sublethal chronic mitochondrial O2− stress, we asked whether mitochondrial oxidative stress increases susceptibility to seizures induced by aging, environmental stimulation, or excitotoxin administration.
Section snippets
Genotyping of Sod2−/+ mice
Animal studies were conducted in accordance with National Jewish Medical and Research Center (NJC) institutional policies. Age-matched Sod2−/+ mice and their littermate controls (Sod2+/+) were evaluated for seizures and indices of mitochondria oxidative stress. Sod2 knockout mice (congenic in the C57BL/6 background), generously provided by Dr. Brian Day, were originally purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Genotyping of Sod2−/+ mice was performed by polymerase chain
Decreased SOD2 activity in Sod2−/+ mice
To determine if partial inactivation of the Sod2 gene resulted in diminished SOD2 enzyme activity, we measured SOD2 activity of brain homogenates from 3–4 month old Sod2−/+ and Sod2+/+ mice. Brain homogenates of Sod2−/+ mice showed ∼50% SOD2 activity (3.03 ± 0.13 units/mg protein, n = 8) compared to Sod2+/+ mice (5.83 ± 0.36 units/mg protein, n = 8).
Spontaneous and handling-induced seizures in Sod2−/+ mice
Spontaneous seizures in the Sod2 knockout mouse cohorts (C57BL/6 and CD-1 backgrounds) were initially noticed and reported by the NJC Bioresource
Discussion
Four principal findings arise from this work. First, a subset of Sod2−/+ mice exhibits age-dependent spontaneous and handling-induced seizures. Second, the seizure disorder in this subset of Sod2−/+ mice correlates with age-related mitochondrial oxidative stress (mitochondrial aconitase inactivation and 8-OHdG formation) and mitochondrial dysfunction (diminished oxygen utilization) in the Sod2−/+ mice. Third, Sod2−/+ mice show increased susceptibility to kainate-induced seizures and hippocampal
Acknowledgements
This work was supported by NIH RO1NS39587 and Partnership for Pediatric Epilepsy, which includes the American Epilepsy Society, the Epilepsy Foundation, Anna and Jim Fantaci, Fight against Childhood Epilepsy and Seizures (f.a.c.e.s.), Neurotherapy Ventures Charitable Research Fund, and P.A.C.E. to M.P. The authors are grateful to Dr. Brian Day and the staff of the NJC Bioresource Center for their help with this project.
References (57)
Mitochondrial dysfunction in neurodegenerative diseases
Biochim. Biophys. Acta
(1998)Mitochondrial involvement in Parkinson's disease, Huntington's disease, hereditary spastic paraplegia and Friedrich's ataxia
Biochim. Biophys. Acta
(1999)- et al.
Mitochondrial superoxide production in kainate-induced hippocampal damage
Neuroscience
(2000) - et al.
Familial mitochondrial encephalomyopathy (MERRF): genetic, pathophysiological and biochemical characterization of a mitochondrial DNA disease
Cell
(1988) - et al.
Myoclonic epilepsy and red-ragged fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation
Cell
(1990) - et al.
Genetic modification of prenatal lethality and dilated cardiomyopathy in Mn superoxide dismutase mutant mice
Free Radic. Biol. Med
(2001) - et al.
Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice
J. Biol. Chem
(1998) - et al.
The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase
J. Biol. Chem
(1972) - et al.
Neuronal stress and injury in C57/BL mice after systemic kainic acid administration
Brain Res
(1998) - et al.
Cyclooxygenase-2 selective inhibitors aggravate kainic acid induced seizure and neuronal cell death in the hippocampus
Brain Res
(1999)
Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples
Brain Res. Brain Res. Protocols
Requirement for superoxide in excitotoxic cell death
Neuron
In vivo DNA damage: measurement of 8-hydroxy-2′-deoxyguanosine in DNA and urine by high-performance liquid chromatography with electrochemical detection
Methods Enzymol
Hydroxyl radical generation during mitochondrial electron transfer and formation of 8-hydroxydeoxyguanosine in mitochondrial DNA
Arch. Biochem. Biophys
Differences in ionotropic glutamate receptor subunit expression are not responsible for strain-dependent susceptibility to excitotoxin-induced injury
Brain Res. Mol. Brain Res
Localization of neuronal and glial glutamate transporters
Neuron
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate
Neuron
Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration?
Trends Pharmacol. Sci
Mitochondrial dysfunction associated with neuronal death following status epilepticus in rat
Epilepsy Res
Ascorbate attenuates the systemic kainate-induced neurotoxicity in the rat hippocampus
Brain Res
Parallel strain-dependent susceptibility to environmentally-induced stereotypies and stress-induced behavioral sensitization in mice
Physiol. Behav
Protective effect of adenosine receptor agonists in a new model of epilepsy—seizures evoked by mitochondrial toxin, 3-nitropropionic acid, in mice
Neurosci. Lett
Seizure-dependent modulation of mitochondrial oxidative phosphorylation in rat hippocampus
Eur. J. Neurosci
The role of mitochondria in epileptogenesis
Curr. Opin. Neurol
Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase
Nat. Genet
Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury
Nat. Genet
Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia
Proc. Natl. Acad. Sci. USA
Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice
Proc. Natl. Acad. Sci. USA
Cited by (150)
RGS14 limits seizure-induced mitochondrial oxidative stress and pathology in hippocampus
2023, Neurobiology of DiseaseDanhong injection alleviates cerebral ischemia/reperfusion injury by improving intracellular energy metabolism coupling in the ischemic penumbra
2021, Biomedicine and PharmacotherapyUpregulated Nmnat2 causes neuronal death and increases seizure susceptibility in temporal lobe epilepsy
2021, Brain Research BulletinSOD2 deficiency-induced oxidative stress attenuates steroidogenesis in mouse ovarian granulosa cells
2021, Molecular and Cellular EndocrinologyDevelopment of eugenol-loaded submicron emulsion and its antiepileptic effect through regulating the oxidative stress
2020, International Journal of Pharmaceutics
- ☆
Supplementary data for this article may be found on ScienceDirect.