Abstract
2-Deoxy-d-Glucose Enhances Tonic Inhibition through the Neurosteroid-Mediated Activation of Extrasynaptic GABAa Receptors
Forte N, Medrihan L, Cappetti B, Baldelli P, Benfenati F. Epilepsia 2016;57(12):1987–2000. doi:10.1111/epi.13578.
OBJECTIVE: The inhibition of glycolysis exerts potent antiseizure effects, as demonstrated by the efficacy of ketogenic and low-glucose/nonketogenic diets in the treatment of drug-resistant epilepsy. ATP-sensitive potassium (KATP) channels have been initially identified as the main determinant of the reduction of neuronal hyperexcitability. However, a plethora of other mechanisms have been proposed. Herein, we report the ability of 2-deoxy-D-glucose (2-DG), a glucose analog that inhibits glycolytic enzymes, of potentiating γ-aminobutyric acid (GABA)ergic tonic inhibition via neurosteroid-mediated activation of extrasynaptic GABAA receptors. METHODS: Acute effects of 2-DG on the ATP-sensitive potassium currents, GABAergic tonic inhibition, firing activity, and interictal events were assessed in hippocampal slices by whole-cell patch-clamp and local field potential recordings of dentate gyrus granule cells. RESULTS: Acute application of 2-DG activates two distinct outward conductances: a KATP channel–mediated current and a bicuculline-sensitive tonic current. The effect of 2-DG on such GABAergic tonic currents was fully prevented by either finasteride or PK11195, which are specific inhibitors of the neurosteroidogenesis pathway acting via different mechanisms. Moreover, the oxidized form of vitamin C, dehydroascorbic acid, known for its ability to induce neurosteroidogenesis, also activated a bicuculline-sensitive tonic current in a manner indistinguishable from that of 2-DG. Finally, we found that the enhancement of KATP current by 2-DG primarily regulates intrinsic firing rate of granule cells, whereas the increase of the GABAergic tonic current plays a key role in reducing the frequency of interictal events evoked by treatment of hippocampal slices with the convulsive agent 4-aminopyridine. SIGNIFICANCE: We demonstrated, for the first time, that 2-DG potentiates the extrasynaptic tonic GABAergic current through activation of neurosteroidogenesis. Such tonic inhibition represents the main conductance responsible for the antiseizure action of this glycolytic inhibitor.
Commentary
The publication outlining the modern version of the ketogenic diet for epilepsy is nearing its 100-year anniversary in 2021 (1). As this milestone nears, we have the opportunity to document both clinical and basic science progress in metabolism-based therapy for epilepsy. There has been an explosion in novel types of diets and our understanding of their impact on seizures. Mechanism-based studies have been less forthcoming but have been increasingly published in recent years (2, 3). Novel and undefined mechanisms have been invoked as the explanation for some of these treatments, but the recent work by Forte et al. shows that indeed, the mechanism for glycolysis inhibition might have been in closer reach than any of us appreciated previously.
The ketogenic diet, the most widely recognized form of metabolism-based therapy for medically intractable epilepsy, involves significant restriction of calorie intake from carbohydrate, modest restriction of protein (which is necessary for induction of systemic ketosis [4]), and compensatory supplementation of fat (5). The classical debate of the relative importance of carbohydrate restriction versus fat supplementation is not likely to be settled anytime soon, but a reductionist approach (i.e., examining each diet component in isolation) can provide interesting answers. Forte et al. studied the electrophysiological properties of one aspect of the ketogenic diet, glycolysis inhibition, with the small molecule 2-deoxy-D-glucose (2-DG). 2-DG has been shown previously to protect against induced seizures in animals and in vitro overexcitation, mediated at least in part to changes in ATP-sensitive potassium (KATP) channels (6–8). However, this new work provides another key to the puzzle in the form of neurosteroids and their binding target, extrasynaptic GABAA receptors, which have not received wide attention in epilepsy.
For a brief review, neurosteroids are endogenous progesterone metabolites that have unique binding sites on GABAA receptors, particularly those with δ-subunits. Examples include allopregnanolone and allotetrahydrodeoxycorticosterone, which act at both synaptic and extrasynaptic GABAA receptors; the latter mediates tonic inhibition (9).
The authors reasoned that 2-DG-mediated increases in pentose phosphate pathway (PPP) activity and resultant increases in NADPH levels lead to increased synthesis of neurosteroids, and subsequently, decreased neuronal excitability via extrasynaptic GABAA receptors. The possibility that the inhibitory effects of 2-DG involved more than just KATP-receptor modulation was raised by the finding that pretreatment of cultured hippocampal granule cell neurons with the KATP inhibitor glibenclamide led to some residual tonic outward current activation in field potential recordings. Extrasynaptic GABAA receptors mediate tonic inhibitory current (owing to the outward flow of chloride); and following up on the prior finding, when neurons were pretreated with the GABAA inhibitor bicuculline, 2-DG still produced some outward current that was then blocked by glibenclamide. Further, the 2-DG-mediated change was eliminated by the PPP inhibitor 6-amino nicotinamide. Together, these results indicate that there were two components to the 2-DG-evoked tonic outward current (i.e., KATP- and GABAA-sensitive). 2-DG did not affect spontaneous GABAergic postsynaptic currents. The next question was whether this GABAergic current was the result of increased neurosteroid levels (recall that 2-DG-induced decreases in glycolysis lead to increased PPP activity and thus increased NADPH, which is a cofactor for 5α-reductase, the rate-limiting enzyme in neurosteroid synthesis). Two small molecules were used to stop neurosteroid synthesis: the 5α-reductase inhibitor, finasteride, and an inhibitor of cholesterol transport across the mitochondrial inner membrane, PK11195. Both inhibitors decreased 2-DG-induced tonic outward current, demonstrating a dependence of this inhibitory current on neurosteroid synthesis.
Effects on neuronal firing at the single cell level (hippocampal dentate granule cells) were explored using current clamp recordings. Pretreatment with 2-DG led to a reduction of firing rate in response to current injection but glibenclamide pretreatment abolished the effect of 2-DG on neuron firing. However, pretreatment with bicuculline had no effect. Thus, the effect of 2-DG on single neurons appeared to be primarily due to enhancement of KATP, not GABAA, conductance. To further explore the mechanism of antiseizure effects noted in prior work at the network level (6), interictal events were provoked by the potassium channel (KCNA) blocker 4-aminopyridine (4-AP). There was a modest but incomplete reduction in interictal events when neurons were pretreated with 4-AP and glibenclamide followed by 2-DG, indicating that the mechanism was not entirely attributable to KATP activity. In contrast, pretreatment with finasteride or PK 11195 (neurosteroid synthesis inhibitors) followed by 2-DG application nearly eliminated this response, consistent with a neurosteroid-induced effect. Furthermore, when GABAA receptors were inactivated with bicuculline, 2-DG had no effect on interictal events, further implicating GABAA receptors. The discrepancy between single cell and network level effects was explained by the differences between the localization of KATP channels (including hippocampal granule cells) and neurosteroid synthesis-containing glutamatergic cells (10,11). Single cell recordings of hippocampal CA1 and CA2 neurons, which have higher levels of 5-alpha reductase mRNA than granule cells (10), may have shown different results.
The authors provide evidence for a novel and previously unanticipated mechanism for glycolysis inhibition in seizure control. There are a few remaining questions about this elegant work. The authors used both sexes of mice in their work, which is now becoming a priority in both preclinical and clinical research. Of interest, an examination of progesterone metabolites (neurosteroids) might be one area where a separate analysis of the sexes might provide richer information than pooled data. It is unclear which sexes of mice provided tissue in different experiments and whether the variability noted in the experimental groups (evidenced by large error bars) could be attributed to sex and/or different phases of the estrus cycle. To further strengthen the link between glycolysis inhibition and seizure control, it also would be useful to know the concentrations of anticonvulsant neurosteroids in mice treated with 2-DG (12). Because many forms of pediatric epilepsy, where metabolism-based treatments are most frequently utilized, involve regions outside the hippocampus (the only type of neurons studied here), it would be interesting to know if these in vitro effects also occur in cortical glutamatergic neurons. Finally, testing these hypotheses in models other than 4-AP and also in mammalian seizure/epilepsy models seems to be a logical next step.
The data shown here provide direct evidence that multiple mechanisms may be in play for metabolism-based antiseizure treatments. They also may indicate a similarity between 2-DG and fructose-1,6-bisphosphate, which also favors metabolism via the PPP over glycolysis and has been shown to have antiseizure properties in some animal models (13). In summary, the authors revealed a link between metabolism-based therapies, which are clinically underutilized and mechanistically underexplored (relative to most other drugs) and known receptors (in this case, extrasynaptic GABAA receptors). These data have implications for all metabolism-based seizure and epilepsy therapies and suggest that we have only started to explore the tip of this iceberg. What a great way to celebrate a 100th birthday!