Current Literature In Basic Science
Taming the rAMPAnt Fire with Fat
Seizure Control by Decanoic Acid Through Direct AMPA Receptor Inhibition. Chang P, Augustin K, Boddum K, Williams S, Sun M, Terschak JA, Hardege JD, Chen PE, Walker MC, Williams RSB. Brain 2016:139;431–443. doi: 10.1093/brain/awv325.
The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones b-hydroxybutyrate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage- and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet.
Commentary A ketogenic diet (KD; long-chain triglycerides providing up to 90% of caloric intake) has been utilized as dietary treatment for epilepsy since the 1920s and represents an effective epilepsy therapy (1, 2). In its classic form, long-chain triglycerides provide the vast majority (see above) of daily caloric intake; there is severe carbohydrate restriction and fluid intake limit. Because of these impediments, the attrition rate is high (especially in adult patients) and, therefore, long-chain triglyceride (LCT) KD has been used mostly in medically intractable childhood epilepsies. More recently, a KD variant with medium chain triglycerides (MCT) was successfully implemented for treatment of refractory epilepsy (3). While on this type of diet, MCT provide 65 to 75 percent of daily calories and the restriction on carbohydrates is not so severe, resulting in greater diet variety. The attrition rate is still high as well as the occurrence of side effects (diarrhea, vomiting, cramps). This MCT KD has limited use in infants, however (4). Both diets have similar efficacy in childhood epilepsies, although there were more reports of vomiting and lack of energy in patients on the LCT KD (5). Despite these and other side effects, a ketogenic diet has a well supported role in controlling epilepsy, and some support for its use in cancer, neurodegenerative diseases, and migraine (6). Epilepsy Currents, Vol. 17, No. 1 (January/February) 2017 pp. 54–56 © American Epilepsy Society
54
Because of restrictions and side effects, research has been steered to identify an effective metabolite or component of the KD that could be used individually as a KD in a pill (7), that is, to provide a simple, effective therapy system with all the benefits of KD with limited side effects and, thus, good patient compliance. The KD, as per its name, leads to ketosis. Thus, initial studies sought to identify whether the ketone bodies forming because of the diet (acetone, acetoacetate, and bhydroxybutyrate) carry the positive features of the KD against seizures. These studies used in vivo models of seizures, but no effects of ketone bodies were observed on GABA- or glutamate-mediated synaptic transmission (8). Other proposed or validated mechanisms of action of the KD include carbohydrate reduction tested by 2-deoxyglucose administration (9), activation of ATP-sensitive potassium channels (KATP), inhibition of mammalian target of rapamycin (mTOR), inhibition of glutamatergic transmission, increase in GABA production, changes in bioenergetics, improvements in mitochondrial function, anti-inflammatory effects, affecting anti-oxidant and mitochondrial genes, and increases in poly-unsaturated fatty acids (with neuroprotective features) in patients (8, 10–12). Many of these mechanisms are quite complex, difficult to grasp, and even more difficult to be molded into a “pill”. Chang and colleagues selected a slightly different approach: in four sequential experiments, they investigated one of the major constituents of the MCT KD providing about 40% of the medium-chains, the ten-carbon decanoic acid. This fatty acid has anticonvulsant effects in in vivo models of seizures, including the 6-Hz threshold and maximal electroshock
Taming the rAMPAnt fire with fat
tests in mice (13). These in vivo effects are similar to another constituent of the MCT KD, octanoic acid (eight carbons). Interestingly, after ingestion, plasma levels of decanoic acid rise, suggesting that this compound is present in the circulation of patients with epilepsy on this type of KD. First, the authors determined efficacy of bath-applied decanoic acid in two simple models of interictal activity recorded in the area CA1 of hippocampal slices in vitro: 1) perfusion with 2 mM pentylenetetrazole (in 6 mM potassium) to block inhibitory GABA-A receptors and 2) low Mg2+ to activate excitatory Nmethyl-D-aspartate (NMDA) receptors. In both models, decanoic acid—but not the ketones acetoacetate or b-hydroxybutyrate—completely suppressed pharmacologically evoked discharges. Second, the authors measured stimulus-evoked, pharmacologically isolated excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs), recorded again in CA1 pyramidal cells of hippocampal slices. Here, they found that addition of decanoic acid to the perfusate suppressed pharmacologically isolated AMPA EPSCs without affecting GABA-A receptormediated IPSCs. It should be emphasized that the concentration of decanoic acid needed for 40% suppression of AMPA EPSCs (300 µM) corresponded to peak concentrations found in plasma of children on the MCT KD. Even steady state concentrations found in children (about 100 µM) were capable of 20% suppression of AMPA EPSCs. These concentrations did not affect AMPA receptor-mediated paired-pulse currents, suggesting that decanoic acid has a true postsynaptic effect, likely mediated by the AMPA receptor. In the third experiment, to further the analysis, the authors expressed AMPA receptors in Xenopus laevis oocytes either as homomers (GluA1 subunits) or heteromers (GluA1/GluA2 or GluA2/GluA3) and made recordings from the oocytes. Glutamate, the natural AMPA receptor agonist, was applied to the bath to evoke AMPA receptor-mediated currents. Stepwise increase in concentration of decanoic acid was eventually able to suppress glutamate-evoked currents and provided data for calculation of 50% inhibitory concentration (IC50). In contrast, if octanoic acid was applied in ascending concentrations, its effect on AMPA currents was much weaker (about sevenfold). If valproic acid—a branched-chain fatty acid isomer of octanoic acid and frequently used anticonvulsant drug—was used, there was no suppression of AMPA currents. Addition of potent AMPA receptor antagonist GYKI 52466 confirmed specificity of AMPA current blockade by decanoic acid. Expanded recordings determined that decanoic acid had a different potency on different subunit compositions of AMPA receptors (lowest IC50 = 0.52 mM at GluA2/GluA3 subunit combination and highest IC50 = 2.09 mM at homomeric GluA1). There was no shift in the glutamate dose-response curve after addition of decanoic acid, suggesting that the binding site for decanoic acid is different from that for glutamate (i.e., decanoic acid provides noncompetitive antagonism). The effect of decanoic acid was also membrane voltage dependent; the efficacy was higher if the membrane was more hyperpolarized (–80 mV) and less potent if the membrane was less hyperpolarized (–40 mV). The combination of these findings suggests that the binding site for decanoic acid sits within the pore (ionophore) of the AMPA receptor. To explore this possibility, the authors’ fourth experiment used an in silico approach investigat-
ing interactions of the decanoic acid molecule with the rat homotetrameric GluA2 receptor. Twenty-five conformations with the lowest binding energy identified receptor residues most frequently participating in binding. These residues were in the pore of the AMPA receptor but did not correspond to the binding site for perampanel, an AMPA receptor antagonist recently introduced for drug-resistant epilepsy (14). In conclusion, the authors convincingly showed that decanoic acid, a constituent of the MCT KD that is increased in plasma of patients using the diet, utilizes antagonism at excitatory AMPA receptor as the likely mechanism of its anticonvulsant action in concentrations relevant to human use. Decanoic acid noncompetitively antagonizes the endogenous ligand glutamate within the pore of the AMPA receptor at a site different from perampanel. Unfortunately, the authors did not attempt to use a combination of perampanel and decanoic acid to explore a cooperative effect of both compounds that may have significant clinical implications. Despite this clearly delineated mechanism, it seems that there are additional benefits of decanoic acid, such as antioxidant effects and improvement of mitochondrial function (15), that may further promote its use in the treatment of refractory epilepsy. by Libor Velíšek, MD, PhD References 1. Levy RG, Cooper PN, Giri P. Ketogenic diet and other dietary treatments for epilepsy. Cochrane Database Syst Rev 2012:CD001903. 2. Lambrechts DA, de Kinderen RJ, Vles JS, de Louw AJ, Aldenkamp AP, Majoie HJ. A randomized controlled trial of the ketogenic diet in refractory childhood epilepsy. Acta Neurol Scand 2016. doi: 10.1111/ ane.12592. 3. Huttenlocher PR, Wilbourn AJ, Signore JM. Medium-chain triglycerides as a therapy for intractable childhood epilepsy. Neurology 1971;21:1097–1103. 4. van der Louw E, van den Hurk D, Neal E, Leiendecker B, Fitzsimmon G, Dority L, Thompson L, Marchio M, Dudzinska M, Dressler A, Klepper J, Auvin S, Cross JH. Ketogenic diet guidelines for infants with refractory epilepsy. Eur J Paed Neurol 2016;20:798–809. 5. Neal EG, Chaffe H, Schwartz RH, Lawson MS, Edwards N, Fitzsimmons G, Whitney A, Cross JH. A randomized trial of classical and mediumchain triglyceride ketogenic diets in the treatment of childhood epilepsy. Epilepsia 2009;50:1109–1117. 6. Stafstrom CE, Rho JM. The ketogenic diet as a treatment paradigm for diverse neurological disorders. Front Pharmacol 2012;3:59. 7. Rho JM, Sankar R. The ketogenic diet in a pill: Is this possible? Epilepsia 2008;49(suppl 8):127–133. 8. Rho JM. How does the ketogenic diet induce anti-seizure effects? Neurosci Lett 2015. doi: 10.1016/j.neulet.2015.07.034. 9. Garriga-Canut M, Schoenike B, Qazi R, Bergendahl K, Daley TJ, Pfender RM, Morrison JF, Ockuly J, Stafstrom C, Sutula T, Roopra A. 2-Deoxy-D-glucose reduces epilepsy progression by NRSF-CtBPdependent metabolic regulation of chromatin structure. Nat Neurosci 2006;9:1382–1387. 10. Danial NN, Hartman AL, Stafstrom CE, Thio LL. How does the ketogenic diet work? Four potential mechanisms. J Child Neurol 2013;28:1027–1033. 11. Kim DY, Abdelwahab MG, Lee SH, O’Neill D, Thompson RJ, Duff HJ, Sullivan PG, Rho JM. Ketones prevent oxidative impairment of
55
Taming the rAMPAnt fire with fat
hippocampal synaptic integrity through KATP channels. PLoS One 2015;10:e0119316. 12. Simeone TA, Matthews SA, Samson KK, Simeone KA. Regulation of brain PPARgamma2 contributes to ketogenic diet anti-seizure efficacy. Exp Neurol 2016. doi: 10.1016/j.expneurol.2016.08.006. 13. Wlaz P, Socala K, Nieoczym D, Zarnowski T, Zarnowska I, Czuczwar SJ, Gasior M. Acute anticonvulsant effects of capric acid in seizure tests in mice. Prog Neuropsychopharmacol Biol Psychiatry 2015;57:110–116.
56
14. Rogawski MA, Hanada T. Preclinical pharmacology of perampanel, a selective non-competitive AMPA receptor antagonist. Acta Neurol Scand Suppl 2013;127:19–24. 15. Tan KN, Carrasco-Pozo C, McDonald TS, Puchowicz M, Borges K. Tridecanoin is anticonvulsant, antioxidant, and improves mitochondrial function. J Cereb Blood Flow Metab 2016. DOI: 10.1177/0271678X16659498.
Taming the rAMPAnt Fire with Fat
Seizure Control by Decanoic Acid Through Direct AMPA Receptor Inhibition. Chang P, Augustin K, Boddum K, Williams S, Sun M, Terschak JA, Hardege JD, Chen PE, Walker MC, Williams RSB. Brain 2016:139;431–443. doi: 10.1093/brain/awv325.
The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones b-hydroxybutyrate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage- and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet.
Commentary A ketogenic diet (KD; long-chain triglycerides providing up to 90% of caloric intake) has been utilized as dietary treatment for epilepsy since the 1920s and represents an effective epilepsy therapy (1, 2). In its classic form, long-chain triglycerides provide the vast majority (see above) of daily caloric intake; there is severe carbohydrate restriction and fluid intake limit. Because of these impediments, the attrition rate is high (especially in adult patients) and, therefore, long-chain triglyceride (LCT) KD has been used mostly in medically intractable childhood epilepsies. More recently, a KD variant with medium chain triglycerides (MCT) was successfully implemented for treatment of refractory epilepsy (3). While on this type of diet, MCT provide 65 to 75 percent of daily calories and the restriction on carbohydrates is not so severe, resulting in greater diet variety. The attrition rate is still high as well as the occurrence of side effects (diarrhea, vomiting, cramps). This MCT KD has limited use in infants, however (4). Both diets have similar efficacy in childhood epilepsies, although there were more reports of vomiting and lack of energy in patients on the LCT KD (5). Despite these and other side effects, a ketogenic diet has a well supported role in controlling epilepsy, and some support for its use in cancer, neurodegenerative diseases, and migraine (6). Epilepsy Currents, Vol. 17, No. 1 (January/February) 2017 pp. 54–56 © American Epilepsy Society
54
Because of restrictions and side effects, research has been steered to identify an effective metabolite or component of the KD that could be used individually as a KD in a pill (7), that is, to provide a simple, effective therapy system with all the benefits of KD with limited side effects and, thus, good patient compliance. The KD, as per its name, leads to ketosis. Thus, initial studies sought to identify whether the ketone bodies forming because of the diet (acetone, acetoacetate, and bhydroxybutyrate) carry the positive features of the KD against seizures. These studies used in vivo models of seizures, but no effects of ketone bodies were observed on GABA- or glutamate-mediated synaptic transmission (8). Other proposed or validated mechanisms of action of the KD include carbohydrate reduction tested by 2-deoxyglucose administration (9), activation of ATP-sensitive potassium channels (KATP), inhibition of mammalian target of rapamycin (mTOR), inhibition of glutamatergic transmission, increase in GABA production, changes in bioenergetics, improvements in mitochondrial function, anti-inflammatory effects, affecting anti-oxidant and mitochondrial genes, and increases in poly-unsaturated fatty acids (with neuroprotective features) in patients (8, 10–12). Many of these mechanisms are quite complex, difficult to grasp, and even more difficult to be molded into a “pill”. Chang and colleagues selected a slightly different approach: in four sequential experiments, they investigated one of the major constituents of the MCT KD providing about 40% of the medium-chains, the ten-carbon decanoic acid. This fatty acid has anticonvulsant effects in in vivo models of seizures, including the 6-Hz threshold and maximal electroshock
Taming the rAMPAnt fire with fat
tests in mice (13). These in vivo effects are similar to another constituent of the MCT KD, octanoic acid (eight carbons). Interestingly, after ingestion, plasma levels of decanoic acid rise, suggesting that this compound is present in the circulation of patients with epilepsy on this type of KD. First, the authors determined efficacy of bath-applied decanoic acid in two simple models of interictal activity recorded in the area CA1 of hippocampal slices in vitro: 1) perfusion with 2 mM pentylenetetrazole (in 6 mM potassium) to block inhibitory GABA-A receptors and 2) low Mg2+ to activate excitatory Nmethyl-D-aspartate (NMDA) receptors. In both models, decanoic acid—but not the ketones acetoacetate or b-hydroxybutyrate—completely suppressed pharmacologically evoked discharges. Second, the authors measured stimulus-evoked, pharmacologically isolated excitatory or inhibitory postsynaptic currents (EPSCs or IPSCs), recorded again in CA1 pyramidal cells of hippocampal slices. Here, they found that addition of decanoic acid to the perfusate suppressed pharmacologically isolated AMPA EPSCs without affecting GABA-A receptormediated IPSCs. It should be emphasized that the concentration of decanoic acid needed for 40% suppression of AMPA EPSCs (300 µM) corresponded to peak concentrations found in plasma of children on the MCT KD. Even steady state concentrations found in children (about 100 µM) were capable of 20% suppression of AMPA EPSCs. These concentrations did not affect AMPA receptor-mediated paired-pulse currents, suggesting that decanoic acid has a true postsynaptic effect, likely mediated by the AMPA receptor. In the third experiment, to further the analysis, the authors expressed AMPA receptors in Xenopus laevis oocytes either as homomers (GluA1 subunits) or heteromers (GluA1/GluA2 or GluA2/GluA3) and made recordings from the oocytes. Glutamate, the natural AMPA receptor agonist, was applied to the bath to evoke AMPA receptor-mediated currents. Stepwise increase in concentration of decanoic acid was eventually able to suppress glutamate-evoked currents and provided data for calculation of 50% inhibitory concentration (IC50). In contrast, if octanoic acid was applied in ascending concentrations, its effect on AMPA currents was much weaker (about sevenfold). If valproic acid—a branched-chain fatty acid isomer of octanoic acid and frequently used anticonvulsant drug—was used, there was no suppression of AMPA currents. Addition of potent AMPA receptor antagonist GYKI 52466 confirmed specificity of AMPA current blockade by decanoic acid. Expanded recordings determined that decanoic acid had a different potency on different subunit compositions of AMPA receptors (lowest IC50 = 0.52 mM at GluA2/GluA3 subunit combination and highest IC50 = 2.09 mM at homomeric GluA1). There was no shift in the glutamate dose-response curve after addition of decanoic acid, suggesting that the binding site for decanoic acid is different from that for glutamate (i.e., decanoic acid provides noncompetitive antagonism). The effect of decanoic acid was also membrane voltage dependent; the efficacy was higher if the membrane was more hyperpolarized (–80 mV) and less potent if the membrane was less hyperpolarized (–40 mV). The combination of these findings suggests that the binding site for decanoic acid sits within the pore (ionophore) of the AMPA receptor. To explore this possibility, the authors’ fourth experiment used an in silico approach investigat-
ing interactions of the decanoic acid molecule with the rat homotetrameric GluA2 receptor. Twenty-five conformations with the lowest binding energy identified receptor residues most frequently participating in binding. These residues were in the pore of the AMPA receptor but did not correspond to the binding site for perampanel, an AMPA receptor antagonist recently introduced for drug-resistant epilepsy (14). In conclusion, the authors convincingly showed that decanoic acid, a constituent of the MCT KD that is increased in plasma of patients using the diet, utilizes antagonism at excitatory AMPA receptor as the likely mechanism of its anticonvulsant action in concentrations relevant to human use. Decanoic acid noncompetitively antagonizes the endogenous ligand glutamate within the pore of the AMPA receptor at a site different from perampanel. Unfortunately, the authors did not attempt to use a combination of perampanel and decanoic acid to explore a cooperative effect of both compounds that may have significant clinical implications. Despite this clearly delineated mechanism, it seems that there are additional benefits of decanoic acid, such as antioxidant effects and improvement of mitochondrial function (15), that may further promote its use in the treatment of refractory epilepsy. by Libor Velíšek, MD, PhD References 1. Levy RG, Cooper PN, Giri P. Ketogenic diet and other dietary treatments for epilepsy. Cochrane Database Syst Rev 2012:CD001903. 2. Lambrechts DA, de Kinderen RJ, Vles JS, de Louw AJ, Aldenkamp AP, Majoie HJ. A randomized controlled trial of the ketogenic diet in refractory childhood epilepsy. Acta Neurol Scand 2016. doi: 10.1111/ ane.12592. 3. Huttenlocher PR, Wilbourn AJ, Signore JM. Medium-chain triglycerides as a therapy for intractable childhood epilepsy. Neurology 1971;21:1097–1103. 4. van der Louw E, van den Hurk D, Neal E, Leiendecker B, Fitzsimmon G, Dority L, Thompson L, Marchio M, Dudzinska M, Dressler A, Klepper J, Auvin S, Cross JH. Ketogenic diet guidelines for infants with refractory epilepsy. Eur J Paed Neurol 2016;20:798–809. 5. Neal EG, Chaffe H, Schwartz RH, Lawson MS, Edwards N, Fitzsimmons G, Whitney A, Cross JH. A randomized trial of classical and mediumchain triglyceride ketogenic diets in the treatment of childhood epilepsy. Epilepsia 2009;50:1109–1117. 6. Stafstrom CE, Rho JM. The ketogenic diet as a treatment paradigm for diverse neurological disorders. Front Pharmacol 2012;3:59. 7. Rho JM, Sankar R. The ketogenic diet in a pill: Is this possible? Epilepsia 2008;49(suppl 8):127–133. 8. Rho JM. How does the ketogenic diet induce anti-seizure effects? Neurosci Lett 2015. doi: 10.1016/j.neulet.2015.07.034. 9. Garriga-Canut M, Schoenike B, Qazi R, Bergendahl K, Daley TJ, Pfender RM, Morrison JF, Ockuly J, Stafstrom C, Sutula T, Roopra A. 2-Deoxy-D-glucose reduces epilepsy progression by NRSF-CtBPdependent metabolic regulation of chromatin structure. Nat Neurosci 2006;9:1382–1387. 10. Danial NN, Hartman AL, Stafstrom CE, Thio LL. How does the ketogenic diet work? Four potential mechanisms. J Child Neurol 2013;28:1027–1033. 11. Kim DY, Abdelwahab MG, Lee SH, O’Neill D, Thompson RJ, Duff HJ, Sullivan PG, Rho JM. Ketones prevent oxidative impairment of
55
Taming the rAMPAnt fire with fat
hippocampal synaptic integrity through KATP channels. PLoS One 2015;10:e0119316. 12. Simeone TA, Matthews SA, Samson KK, Simeone KA. Regulation of brain PPARgamma2 contributes to ketogenic diet anti-seizure efficacy. Exp Neurol 2016. doi: 10.1016/j.expneurol.2016.08.006. 13. Wlaz P, Socala K, Nieoczym D, Zarnowski T, Zarnowska I, Czuczwar SJ, Gasior M. Acute anticonvulsant effects of capric acid in seizure tests in mice. Prog Neuropsychopharmacol Biol Psychiatry 2015;57:110–116.
56
14. Rogawski MA, Hanada T. Preclinical pharmacology of perampanel, a selective non-competitive AMPA receptor antagonist. Acta Neurol Scand Suppl 2013;127:19–24. 15. Tan KN, Carrasco-Pozo C, McDonald TS, Puchowicz M, Borges K. Tridecanoin is anticonvulsant, antioxidant, and improves mitochondrial function. J Cereb Blood Flow Metab 2016. DOI: 10.1177/0271678X16659498.
