Elsevier

Neurobiology of Disease

Volume 75, March 2015, Pages 151-158
Neurobiology of Disease

Mitochondrial respiration deficits driven by reactive oxygen species in experimental temporal lobe epilepsy

https://doi.org/10.1016/j.nbd.2014.12.025Get rights and content

Highlights

  • Mitochondrial respiration deficits occur in experimental TLE.

  • Mitochondrial reserve capacity is decreased in experimental TLE.

  • Reactive oxygen species drive deficits in mitochondrial respiration.

Abstract

Metabolic alterations have been implicated in the etiology of temporal lobe epilepsy (TLE), but whether or not they have a functional impact on cellular energy producing pathways (glycolysis and/or oxidative phosphorylation) is unknown. The goal of this study was to determine if alterations in cellular bioenergetics occur using real-time analysis of mitochondrial oxygen consumption and glycolytic rates in an animal model of TLE. We hypothesized that increased steady-state levels of reactive oxygen species (ROS) initiated by epileptogenic injury result in impaired mitochondrial respiration. We established methodology for assessment of bioenergetic parameters in isolated synaptosomes from the hippocampus of Sprague–Dawley rats at various times in the kainate (KA) model of TLE. Deficits in indices of mitochondrial respiration were observed at time points corresponding with the acute and chronic phases of epileptogenesis. We asked if mitochondrial bioenergetic dysfunction occurred as a result of increased mitochondrial ROS and if it could be attenuated in the KA model by pharmacologically scavenging ROS. Increased steady-state ROS in mice with forebrain-specific conditional deletion of manganese superoxide dismutase (Sod2fl/flNEXCre/Cre) in mice resulted in profound deficits in mitochondrial oxygen consumption. Pharmacological scavenging of ROS with a catalytic antioxidant restored mitochondrial respiration deficits in the KA model of TLE. Together, these results demonstrate that mitochondrial respiration deficits occur in experimental TLE and ROS mechanistically contribute to these deficits. Furthermore, this study provides novel methodology for assessing cellular metabolism during the entire time course of disease development.

Introduction

Metabolic impairment underlies the etiology of various neurological disorders such as Alzheimer's disease, Parkinson's disease, traumatic brain injury and stroke (Beal, 2004, Rahman, 2012). Its role in the etiology of epilepsy is receiving increased attention (Kudin et al., 2002, Kovac et al., 2012, Rowley and Patel, 2013). Epilepsy is one of the most common neurological disorders with an incidence of ~ 1% in the population (Delgado-Escueta et al., 1999). Temporal lobe epilepsy (TLE), a form of acquired epilepsy is usually triggered by an insult such as brain injury and the development of chronic seizures after a silent period devoid of seizures. The process by which epilepsy develops i.e. epileptogenesis is known to involve multiple molecular and physiological changes that alter brain circuitry to promote excitability. In human TLE, two characteristic changes in metabolism are known to occur, the mechanistic basis of which is unclear. First, glucose utilization or increased metabolism occurs during seizures or ictal events. Secondly, the period between seizures or interictal period is typified by glucose hypometabolism (Chugani et al., 1994, Lee et al., 2012). Attempts to understand the role of glucose overutilization during seizures have led to studies demonstrating the anticonvulsant effects of limiting glycolysis with 2-deoxyglucose, fructose 1,6 bisphosphate or ketogenic diets (Bough et al., 2006, Lian et al., 2007, Stafstrom et al., 2009).

Multiple lines of evidence support a role of mitochondria in the development of epilepsy, which was first recognized by the occurrence of epilepsy in patients with inherited mitochondrial disorders (Wallace et al., 1988, Mecocci et al., 1993). Almost all known functions of mitochondria have a capacity to impact epilepsy, but ATP production and the generation of reactive oxygen species (ROS) are prime candidates due to the energy demands of seizures and sensitivity of mitochondrial processes to oxidative damage. In studies of patients with TLE, the role of mitochondria has been suggested by inhibition of complex I of the electron transport chain (ETC) and reduction in N-acetyl aspartate levels in the hippocampus (Kunz et al., 2000, Vielhaber et al., 2008). Recent work from our group has shown increases in production of mitochondrial ROS, reactive nitrogen species (RNS), oxidative damage to oxidant-sensitive mitochondrial proteins (aconitase and complex I) and glutathione depletion in animal models of TLE (Jarrett et al., 2008, Waldbaum et al., 2010, Ryan et al., 2012, Ryan et al., 2013). Increased mitochondrial ROS and oxidative damage to ETC enzymes suggest that mitochondrial respiration may be impaired in TLE. However, no studies to date have demonstrated if mitochondrial respiration is altered in experimental TLE and if so, determined its mechanistic basis. Here, we sought to determine if there are functional mitochondrial deficits through measurement of oxidative phosphorylation (OXPHOS) in experimental TLE. Assessment of mitochondrial respiration is a necessary step in asking if mitochondria are actually “dysfunctional” or have altered activities of individual enzymes (Brand and Nicholls, 2011). Furthermore, it is important to understand if chronic epilepsy renders brain mitochondria deficient in their reserve capacity, a measure of their ability to respond to an additional bioenergetic demand.

Section snippets

Reagents

KA was purchased from AG Scientific, Inc. product # K-1013. All other reagents were purchased from Sigma Aldrich or Fisher Scientific.

Animals

Animal studies were carried out according to the National Institute of Health Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Institute Animal Care and Committee (IACUC) at the University of Colorado Anschutz Medical Campus. Adult male Sprague–Dawley rats (300–350 g) were injected with KA (11 mg/kg, s.c.), or saline. 90 min

Mitochondrial respiration deficits and altered glycolysis in KA model of experimental TLE

Isolated brain synaptosomes allow simultaneous measurement of glycolysis and mitochondrial respiration, two major energy producing processes. Synaptosomal preparations are ideally suited for temporal metabolic assessment of brain tissue from animals as they contain synaptic machinery, including neuronal and astrocytic mitochondria. We adapted extracellular flux analysis to assess mitochondrial function by mitochondrial OCR in synaptosomes from animals subjected to epileptogenic injury. This

Discussion

Two primary lines of evidence support ROS-dependent deficits in mitochondrial oxygen consumption in experimental models of TLE. First, deficits in mitochondrial respiration occurred in acute and chronic phases of injury-induced TLE. The temporal profile of mitochondrial respiration deficits is reminiscent of increased steady-state ROS levels shown previously in these models (Jarrett et al., 2008, Waldbaum et al., 2010, Ryan et al., 2012). Secondly, a role for ROS in mitochondrial respiration

Conflict of interest

B.J.D. holds equity and serves as a consultant for Aeolus Pharmaceuticals which is developing metalloporphyrins as therapeutics.

Acknowledgments

This work is supported by NIH RO1NS039587(M.P.), NIH UO1NS083422 (M.P.), NIH 5 F31 NS077739-03 (S.R.). The authors wish to thank Drs. Yogendra Raol, Andrew White and the UCAMC In Vivo Neurophysiology and Pathology Core Facilities.

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