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