Elsevier

Neurobiology of Disease

Volume 45, Issue 3, March 2012, Pages 1068-1076
Neurobiology of Disease

Mitochondrial oxidative stress and epilepsy in SOD2 deficient mice: Attenuation by a lipophilic metalloporphyrin

https://doi.org/10.1016/j.nbd.2011.12.025Get rights and content

Abstract

Epileptic seizures are a common feature associated with inherited mitochondrial diseases. This study investigated the role of mitochondrial oxidative stress in epilepsy resulting from mitochondrial dysfunction using cross-bred mutant mice lacking mitochondrial manganese superoxide dismutase (MnSOD or SOD2) and a lipophilic metalloporphyrin catalytic antioxidant. Video-EEG monitoring revealed that in the second to third week of postnatal life (P14-P21) B6D2F2 Sod2−/− mice exhibited frequent spontaneous motor seizures providing evidence that oxidative stress-induced mitochondrial dysfunction may contribute to epileptic seizures. To confirm the role of mitochondrial oxidative stress in epilepsy a newly developed lipophilic metalloporphyrin, AEOL 11207, with high potency for catalytic removal of endogenously generated reactive oxygen species was utilized. AEOL 11207-treated Sod2−/− mice showed a significant decrease in both the frequency and duration of spontaneous seizures but no effect on seizure severity. A significant increase in the average lifespan of AEOL 11207-treated Sod2−/− mice compared to vehicle-treated Sod2−/− mice was also observed. Indices of mitochondrial oxidative stress and damage (aconitase inactivation, 3-nitrotyrosine formation, and depletion of reduced coenzyme A) and ATP levels affecting neuronal excitability were significantly attenuated in the brains of AEOL 11207-treated Sod2−/− mice compared to vehicle-treated Sod2−/− mice. The occurrence of epileptic seizures in Sod2−/− mice and the ability of catalytic antioxidant therapy to attenuate seizure activity, mitochondrial dysfunction, and ATP levels suggest that ongoing mitochondrial oxidative stress can contribute to epilepsy associated with mitochondrial dysfunction and disease.

Highlights

►The role of mitochondrial oxidative stress in epilepsy was investigated. ►Mice deficient in mitochondrial antioxidant (Sod2−/−) showed spontaneous electrographic and motor seizures. ►Seizures, oxidative stress and bioenergetic parameters in Sod2−/− mice were attenuated by a catalytic antioxidant. ►Mitochondrial oxidative stress is a contributing mechanism to epilepsy in Sod2-/- mice.

Introduction

Epileptic seizures commonly occur in patients with inherited mitochondrial disease (Mecocci et al., 1993, Wallace et al., 1988) suggesting that mitochondrial dysfunction can contribute to seizures (Kunz, 2002, Patel, 2004). General and partial seizures with mitochondrial encephalopathy can be caused by mitochondrial dysfunction arising from mitochondrial mtDNA mutations (Shoffner et al., 1990, Wallace et al., 1988). Mitochondrial dysfunction is a consequence of many neurological insults such as neonatal or adult hypoxia, trauma and infections which are known risk factors for epilepsy development (Beal, 1998, Douglas et al., 2010, Jensen et al., 1991, Jensen et al., 1992, Mustafa et al., 2010). These data strongly suggest that mitochondrial dysfunction per se may be a common pathway contributing to epilepsy development. Mitochondria have important functions that include cellular ATP production, control of apoptotic/necrotic cell death, reactive oxygen species (ROS) formation and calcium homeostasis. Which of these critical mitochondrial functions contributes to increased seizure susceptibility associated with inherited or acquired epilepsies remains unknown. Results from this and other laboratories suggest that mitochondrial oxidative stress and resultant dysfunction are not only a consequence of seizure activity but also render the brain more susceptible to age-related epileptic seizures (Jarrett et al., 2008, Kudin et al., 2002, Liang et al., 2000, Liang and Patel, 2004, Waldbaum et al., 2010).

To understand the role of oxidative stress in epilepsy associated with mitochondrial disease it is useful to utilize an animal model in which spontaneous epileptic seizures arise due to increased steady-state mitochondrial ROS. Mutant mice lacking manganese superoxide dismutase (MnSOD or SOD2), a critical mitochondrial antioxidant, provide such a model. Mitochondrial disease has been characterized in SOD2 deficient mice generated in several background strains with phenotypes characteristic of increased steady-state mitochondrial ROS. Sod2−/− mice bred from a C57B6 background (B6 Sod2−/−) are neonatal lethal (Lebovitz et al., 1996), whereas CD-1 Sod2−/− mice develop and live approximately 8–10 days postnatal (Melov et al., 1999). Recently, Sod2−/− mutant mice from a mixed background (C57BL/6J X DBA/2J, B6D2) have been generated, which live approximately 3 weeks without pharmacological intervention (Huang et al., 2001). Behavioral tonic–clonic seizures have been anecdotally reported starting the second to third week of postnatal life in B6D2F1 Sod2−/− mice (Lynn et al., 2005). Therefore, increased life-span of the cross-bred B6D2 Sod2−/− mice provides a model in which the underlying role of oxidative stress in mitochondrial disease epilepsy can be investigated.

Metalloporphyrin catalytic antioxidants are small molecule mimics of superoxide dismutase (SOD) and/or catalase (CAT), and are also potent detoxifiers of lipid peroxides and peroxynitrite (ONOO¯) (reviewed in (Day, 2004)). Because they are catalytic, and not merely bulk scavengers, these compounds are much more potent antioxidants than dietary additives such as vitamin E that act stoichiometrically. The manganese meso-porphyrin catalytic antioxidants combine the broad spectrum detoxification of reactive species like the stoichiometric antioxidants with the catalytic efficiency of the endogenous antioxidant enzymes. Additionally, these synthetic compounds can be chemically modified to increase their ability to cross the blood brain barrier (BBB), as well as their targeting to various subcellular compartments. Treatment of short-lived Sod2−/− mice in the CD-1 background with manganese tetrakis 5, 10, 15, 20- porphyrin (MnTBAP) ameliorated cardiomyopathy but not neurodegeneration (Melov et al., 1998) whereas EUK8 or EUK134 ameliorated spongiform encephalopathy and neurodegeneration (Melov et al., 2001). A major advancement in the field of catalytic antioxidants was the demonstration that AEOL 11207, a lipophilic metalloporphyrin, protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo following oral administration (Liang et al., 2007). This compound belongs to a series of metalloporphyrins which were designed to have greater lipid solubility, oral bioavailability, and cross the BBB. The objective of this study was to determine the role of mitochondrial oxidative stress in the development of epileptic seizures using B6D2 Sod2−/− mice. Seizure activity was monitored by video-EEG methods in conjunction with indices of mitochondrial oxidative stress. To confirm the role of mitochondrial oxidative stress in epilepsy in Sod2−/− mice we asked whether treatment with a lipophilic metalloporphyrin antioxidant could attenuate seizure activity.

Section snippets

Animals

Animal studies were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). All procedures were approved by the Institute Animal Care and Use Committee (IACUC) of the University of Colorado Denver (UCD), which is fully accredited by the American Association for the Accreditation of Laboratory Animal Care. Heterozygous MnSOD (Sod2−/+) mutant mice on a C57BL/6J (B6) background were crossed with DBA/2J (D2) wild

Seizure activity in Sod2−/− mice

Forty-six Sod2/ mice were initially digitally recorded for a minimum of 8 h/day beginning on P16 to establish seizure parameters and any age-related changes in behavioral seizure activity. A significant increase in seizure duration and numbers from P17 to P20 in vehicle or AEOL11207 treated Sod2/ mice was observed (Fig. 1), suggesting that elevated mitochondrial ROS over time may contribute to epilepsy progression. In order to obtain a higher degree of quantification and accuracy than

Discussion

The present study used SOD2 deficient mice and a novel lipophilic metalloporphyrin catalytic antioxidant, AEOL 11207, to establish the role of mitochondrial oxidative stress as a contributing mechanism in epilepsy resulting from mitochondrial dysfunction. Specifically, the results demonstrated that: (1) Sod2−/− mice from a mixed genetic background (B6D2F2) lived approximately 3 weeks, exhibited frequent spontaneous seizures by the second to third week postnatal, and possessed significant brain

Disclosure

B.J.D. holds equity and serves as a consultant for Aeolus Pharmaceuticals which is developing metalloporphyrins as therapeutics. M.P. has been a recipient of research grants from Aeolus Pharmaceuticals. M.P. and L.L. have a patent pending for therapeutic use of metalloporphyrins in epilepsy.

The following are the supplementary materials related to this article.

Supplementary materials.

Acknowledgments

This work is supported by grants from the NIH RO1NS039587 and R21NS53548 (MP), RO1AG24400 (TTH), CURE Innovator Award (MP), EFA (SW) and CCTSI (SW). The authors wish to thank Dr. Yogendra Raol and the UCD In Vivo Neurophysiology Core and Dr. Andrew White for help analyzing and interpreting EEG data.

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