Abstract
Ketone Bodies Mediate Antiseizure Effects through Mitochondrial Permeability Transition
Kim do Y, Simeone KA, Simeone TA, Pandya JD, Wilke JC, Ahn Y, Geddes JW, Sullivan PG, Rho JM. Ann Neurol 2015;78:77–87.
OBJECTIVE: Ketone bodies (KB) are products of fatty acid oxidation and serve as essential fuels during fasting or treatment with the high-fat antiseizure ketogenic diet (KD). Despite growing evidence that KB exert broad neuroprotective effects, their role in seizure control has not been firmly demonstrated. The major goal of this study was to demonstrate the direct antiseizure effects of KB and to identify an underlying target mechanism. METHODS: We studied the effects of both the KD and KB in spontaneously epileptic Kcna1-null mice using a combination of behavioral, planar multi-electrode, and standard cellular electrophysiological techniques. Thresholds for mitochondrial permeability transition (mPT) were determined in acutely isolated brain mitochondria. RESULTS: KB alone were sufficient to: (1) exert antiseizure effects in Kcna1-null mice, (2) restore intrinsic impairment of hippocampal long-term potentiation and spatial learning-memory defects in Kcna1-null mutants, and (3) raise the threshold for calcium-induced mPT in acutely prepared mitochondria from hippocampi of Kcna1-null animals. Targeted deletion of the cyclophilin D subunit of the mPT complex abrogated the effects of KB on mPT, and in vivo pharmacological inhibition and activation of mPT were found to mirror and reverse, respectively, the antiseizure effects of the KD in Kcna1-null mice. INTERPRETATION: The present data reveal the first direct link between mPT and seizure control, and provide a potential mechanistic explanation for the KD. Given that mPT is increasingly being implicated in diverse neurological disorders, our results suggest that metabolism-based treatments and/or metabolic substrates might represent a worthy paradigm for therapeutic development.
Commentary
Epilepsy has been treated for nearly a century with diets intended to manipulate energy metabolism. In particular, the high-fat ketogenic diet has garnered increased interest since the mid-1990s as a therapy for drug-resistant epilepsy, especially in children (1). Caloric restriction and the ketogenic diet induce ketosis, which is characterized by increased hepatic production of ketone bodies. Hypothetically, ketone bodies may be used as alternate fuels by the brain when glucose levels are low. Not unexpectedly, compliance with the restrictions of the ketogenic diet is problematic for some patients, and it is necessary to have a mechanistic understanding of its seizure-suppressing effects in order to develop effective epilepsy therapies based on ketone body activity. Several hypotheses have been promoted to explain the antiseizure and neuroprotective effects of the ketogenic diet—and of ketone bodies in particular—but clear mechanisms have not been forthcoming. Ketone bodies increase cellular ATP levels and decrease reactive oxygen species (ROS), two indexes of mitochondria-based cellular energy metabolism (2), but how this occurs and how these actions might translate to anti-seizure effects are unclear. Kim and colleagues describe the effects of both the ketogenic diet and ketone bodies on the mitochondrial permeability transition (mPT) pore to develop a mechanism explaining how the ketogenic diet and ketone bodies suppress seizures in a potassium channel-deficient transgenic mouse model of epilepsy, the Kcna1-null mutant (3). Like ketone bodies, the mPT regulates cellular ATP and ROS levels (4), implicating cellular bioenergetics as a critical regulator of seizure expression. Effects of ketone bodies on mPT activity and cognition were also differentiated from those of phenobarbital, a prototypical antiseizure drug. As a cellular surrogate for learning and memory, long-term potentiation (LTP) of hippocampal synapses can be negatively affected by seizures or anti-seizure medications. Thus, the investigation by Kim and colleagues not only aimed to definitively identify a cellular mechanism for the effects of ketone bodies on seizures but also to differentiate effects on cognition from those of an antiseizure drug. The authors concluded that effects of the ketogenic diet are mediated by direct actions of ketone bodies operating through mitochondrial—and, therefore, cellular—energy metabolism. These results provide a plausible explanation for the effects of the ketogenic diet on seizures and should prompt more detailed investigation of how modulation of cellular bioenergetics affects seizures and a multitude of other neurological disorders that appear to be influenced by cellular metabolism (5, 6).
In these studies, young Kcna1 knock-out mice were used to first show efficacy of the ketogenic diet and of a chronically infused stable ketone body—beta-hydroxy butyrate (BHB)—on spontaneous seizure frequency. The ketogenic diet increased blood ketone body concentrations, and both the ketogenic diet and BHB treatment attenuated seizures. In addition, ketone body application in vitro diminished cellular hyperexcitability in slice cultures, even in the presence of normalized glucose concentration. The effects of ketone bodies in vitro (and, by implication, the antiseizure effects in vivo) therefore occurred independent of glycolytic restriction. Neurons from the brains of Kcna1 knock-out mice displayed a lower threshold for calcium-induced mPT pore transition; both the ketogenic diet and ketone bodies reversed this lowered mPT threshold in a manner that required the CypD subunit. This is relevant because cyclosporine A is an established, but non-specific, mPT inhibitor that binds the CypD subunit of the pore, offering a potentially translational option for investigation of this mechanism in patients. Representing an important advance toward a mechanism, the authors found that the effects of ketone bodies on mitochondrial function were energy dependent, rather than affecting mitochondrial membrane potential, ROS status, or redox status. The effects of ketone bodies on mPT in mitochondria from Kcna1 knock-out mice were prevented when mPT threshold was elevated pharmacologically, and a specific CypD antagonist (NIM811) had antiseizure effects in the epileptic mice. Thus, although a direct effect of ketone bodies on the CypD subunit was not shown conclusively, these results strongly imply that the antiseizure effects of the ketogenic diet and ketone bodies are mediated by a metabolism-dependent interaction with a specific subunit of the mPT complex. If a direct effect on the drugable CypD subunit can be confirmed, this discovery offers a new potential therapeutic avenue for ameliorating seizures that are otherwise pharmacoresistant.
Negative implications of uncontrolled seizures include effects on cognition, and continual use of some conventional antiseizure medications may also negatively affect cognitive function. As expected, Kcna1 knock-out mice performed worse than wild-type mice in a spatial learning task, and the ketogenic diet reversed this trend. In conjunction with the spatial learning deficit, hippocampal LTP deficiency was observed in the epilepsy model, and this deficiency was abrogated in mice fed the ketogenic diet or treated systemically with ketone bodies. In addition to seizure-suppressing effects, ketone body treatment was therefore also deemed to be nootropic (i.e., cognition-enhancing). Conversely, treatment with phenobarbital at a concentration that was effective against seizures failed to improve the hippocampal LTP deficit observed in the Kcna1 knock-out mice and did not alter mPT activation. Thus, although effects of conventional and ketone body treatments are convergent with respect to antiseizure therapy, the divergent mechanisms of action of the two treatments resulted in significant differences in cognitive outcomes.
The findings of Kim et al. show, for the first time, that modulation of the mPT pore provides a mechanistic explanation for the antiseizure effects of the ketogenic diet and ketone bodies. Ketone bodies were also effective against both seizures and intrinsic impairment of hippocampal LTP in this developmental epilepsy model. Identification of the mPT pore as a target for the seizure-controlling effects of the ketogenic diet may offer a new avenue for antiseizure therapy. Critically, ketone bodies may not directly target the mPT pore; rather, the effect appears to be modulated by changes in mitochondrial bioenergetics, perhaps by offering an alternative biofuel for mitochondrial respiration. The effectiveness of the ketogenic diet has been mainly established for seizures in the young, and there is less known about its efficacy in adults. Although ketone body-mediated mPT pore modulation was demonstrated to have antiseizure effects in the developmental epilepsy model used here, the relevance of the mechanism to other types of epilepsy—especially acquired epilepsy in adults—is unknown. Ketone bodies may also have broad neuroprotective effects, in addition to the antiseizure and nootropic actions described by Kim and colleagues (6). Investigation into the efficacy of ketone bodies, mPT pore inhibition, or modulation of cellular bioenergetics on other types of epilepsy as well as epileptogenesis are critical next steps in translating cellular bioenergetics into therapies for epilepsy.