Could Glutamine Feed Epileptogenesis?

Abstract

Mitochondrial Glutamine Metabolism Drives Epileptogenesis in Primary Hippocampal Neurons

Helmut Kubista, Francesco Gentile, Klaus Schicker, Thomas Köcher, Stefan Boehm, and Matej Hotka, J Neurosci, May 21, 2025;45(21):e0110252025. https://doi.org/10.1523/JNEUROSCI.0110-25.2025.

All available antiseizure medications aim at symptomatic control of epilepsy, but there is no strategy to stop the development of the disease. The main reason is the lack of understanding of the epileptogenic mechanisms. Closing this knowledge gap is an essential prerequisite for developing disease-modifying therapies that can prevent the onset of epilepsy. Using primary cocultures of hippocampal neurons and glial cells derived from rat pups of either sex, we show that epileptiform paroxysmal depolarization shifts (PDS) induce neuronal glucose hypometabolism which is compensated for by increased glutaminolysis. Glutaminolysis not only provides sufficient ATP to support electrical activity but also leads to decreased vesicular glutamate release, thereby promoting neuronal hypersynchrony. Moreover, prolonged promotion of PDS increased neuronal arborization and synaptic density, which in combination with spontaneous recovery of neuronal glucose metabolism led to seizure-like discharge activity. Since inhibition of glutaminolysis did not prevent the PDS-induced morphogenesis but eliminated seizure-like activity, we propose that glutaminolysis is a causative process linking neuronal metabolism with electrical activity thereby driving epileptogenesis.

Commentary

A cellular model of epileptogenesis was established to investigate metabolic alterations underlying the in vitro development of spontaneous seizure-like events. ¹ The model employed bicuculline (Bic), a GABA_A receptor blocker, and Bay K86444 (BayK), an L-type calcium opener, which were added to primary rat hippocampal neurons grown in the presence of glia. This drug combination immediately induced paroxysmal depolarization shifts (PDS) in single neurons, which resemble pre- and interictal spiking. PDS continued at 1 h and increased in duration at 24 and 72 h by 3–7-fold. The over 10 s lasting events at 3 days were classified as seizure-like events, indicating that this drug combination in hippocampal cell cultures models “epileptogenesis.” The control condition consisted of hippocampal cells exposed to Bic and isradipine (Isra), an L-type calcium channel blocker, where neurons showed increased firing of action potentials, although not PDS nor development of ictal-like behavior at 3 days. Using calcium imaging, prolonged incubation Bic with BayK, but not Isra, also led to synchronization and augmentation in amplitude of intracellular [calcium] increases over 3 days. This ultimately led to spontaneous synchronized long-lasting [calcium] _i increases in neurons, again indicating the development of epileptiform events. Using an intracellular sensor, reduced neuronal ATP/ADP ratios were recorded during PDS, which could be attenuated when physiological amounts of glutamine were added, or perfusion was stopped to allow transfer of metabolites between glia and neurons. In the absence of glia, the metabolism of glucose via glycolysis and the tricarboxylic acid (TCA) cycle decreased during PDS, similar to interictal glucose hypometabolism seen in vivo. Instead, in vitro oxidation of glutamine increased, which correlated with improved intracellular ATP/ADP ratios in the presence of glutamine.

The development of epileptiform discharges over the next days in this epileptogenesis model was inhibited by blocking the TCA cycle enzyme α-ketoglutarate dehydrogenase or knockout of glutamate dehydrogenase (GDH), which converts glutamate to α-ketoglutarate and vice versa. Applying a pharmacological GDH inhibitor also prevented seizure-like activity in a similar cellular epileptogenesis model induced by 10 min exposure to glutamate. The accompanying increases in dendritic arborizations and synapse densities alone were not sufficient to lead to “epileptogenesis.” During “epileptogenesis” at 1 and 2 days, synaptic glutamate release was reduced, which recovered at 3 days concomitant with a doubling of glucose uptake and occurrence of seizure-like events. Thus, the authors postulated that glutaminolysis drives epileptogenesis.

The paper demonstrates that glucose (2 mM) and glutamine (0.1 mM) supplied in concentrations found in brain extracellular fluid^2,3 were oxidized in purified rat hippocampal neurons in vitro contributing to energy production. ¹ Glutamine was converted to glutamate, which was then oxidized to α-ketoglutarate and further metabolized in the TCA cycle. When neuronal activity was high (with Bic + Isra), the metabolism of glutamine into TCA metabolites (glutaminolysis) amounted to about ∼15% relative to glucose. During PDS, glutaminolysis about doubled, while glucose oxidation dropped by ∼20%-50%, indicating that glutamine may supply similar amounts of energy as glucose in extreme conditions in vitro. Glutamine oxidation in the TCA cycle has been observed in other studies in vitro, namely in synaptosomes, ⁴ acutely prepared rodent brain slices and neurons derived from human induced pluripotent stem cells ⁵ but information about neuronal activity in these preparations is lacking. There is indirect evidence from animal models with mitochondrial disorders that glutaminolysis occurs in vivo in animals. ⁶ Only one research group showed using in vivo microdialysis that radiolabeled glutamine infused into the awake rat brain is oxidized in brain. ⁷ Among the amino acids, glutamine levels are the highest in blood and extracellular fluid in brain. Glutamine can cross the blood–brain barrier, ³ and is also released from astrocytes, which synthetize glutamine from glutamate after uptake from the extracellular fluid (glutamate/glutamine cycle) or from glucose (Figure 1). ⁸ Interestingly, many studies have also shown that glutamate, but not glutamine, provides energy via oxidation to α-ketoglutarate and within the TCA cycle specifically in astrocytes.^7,9,10 Thus, although glucose is the main external brain fuel, once inside the brain, glucose produces sizeable amounts of glutamate and glutamine, which can supply energy in a cell-specific manner to glia and neurons, respectively (Figure 1).

Figure 1.

Simplified proposed brain metabolism of glutamine and glutamine as discussed here. Glucose is the main external fuel provided by the blood. Glucose produces significant amounts of glutamate in neurons and some glutamine in astrocytes, which both may be used as internal brain fuels (see discussion above). Note that in addition to glucose, glutamine can be oxidized in the TCA cycle of neurons, ¹ while in astrocytes glutamate can provide energy.^4,10 More research is needed regarding in vivo oxidation of these internal fuels as well as the conditions and extent to which this occurs and plays a role in disorders of the brain, such as epilepsy. Based on results in neuronal cultures, ¹ glucose-derived metabolites in neurons are depicted in blue, while glutamine-derived metabolites are shown in red and underlined. Transporters and enzymes, namely Glut1, PC and GS, largely localized in astrocytes are green. The plasma and brain extracellular concentrations of glucose, glutamine, and glutamate are shown. Abbreviations: AAT = aspartate aminotransferase, αKG = α-ketoglutarate, ETC = electron transport chain, GDH = glutamine dehydrogenase, Glu = glutamate, Glut1 = glucose transporter, Gln = glutamine, GS = glutamine synthetase, OAA = oxaloacetate, PAG = phosphate-dependent glutaminase, PC = pyruvate carboxylase, TCA = tricarboxylic acid. Please see the color version in BioRender for best viewing. Created in BioRender. Borges, K. (2025) https://BioRender.com/bu896ps.

Is glutaminolysis a contributor to epileptogenesis? The development of epileptiform discharges in two in vitro epileptogenesis models was inhibited by genetic deletion of GDH and a GDH inhibitor. ¹ In vivo mitochondrially localized GDH produces glutamate in the reversible reaction (NH₃ + α-ketoglutarate + NAD(P)H ↔ glutamate + NAD(P)+), but it can also oxidize glutamate to α-ketoglutarate.^2,9 Traditionally, aspartate aminotransferase has been thought to be the main enzyme to convert glutamate to α-ketoglutarate (Glutamate + Oxaloacetate ↔ Aspartate + α-ketoglutarate), but little effect was seen in vitro, when this enzyme was blocked with aminooxyacetic acid.^1,4 However, most in vivo studies rely on healthy anesthetized rodents and anesthesia reduces brain activity and concomitantly energy needs and metabolism. Therefore, we still know little about in vivo metabolism of glutamine and glutamate and to which extent glutaminolysis may play a role in epilepsy. However, blocking glutamine entry into the TCA cycle as a means to potentially inhibit epileptogenesis in vivo is expected to have serious ramifications for neuronal energy and carbon metabolism, as glutamine provides needed carbons for the TCA cycle in neurons (anaplerosis). ⁵ On another note, providing high amounts of glutamine to rats increased seizure severity in an animal model where seizures were induced by blocking glutamine synthetase. ¹¹ Although there was no information on alteration in brain glutamine levels or glutaminolysis, this indicates that glutamine can promote seizures.

Glucose hypometabolism and increased glutaminolysis coincided with reduced glutamate release during development of synchronized neuronal activity. ¹ The authors explain that reductions in synaptic fidelity can be expected to reduce the transmission of smaller neuronal events, thereby promoting the propagation of more intense or prolonged activity across the network, which ultimately enhances synchronicity. ¹ At first it is surprising that vesicular glutamate levels were affected, as brain contains 3–10 times more glutamate than glucose and only small amounts of glutamate are released as neurotransmitter under normal conditions. ¹⁰ However, the reduction in glutamate release described by Kubista and colleagues ¹ becomes conceivable when taking into consideration (i) excessive glutamate release (where some may be lost through perfusion or diluted into medium), (ii) glutamine is largely consumed as fuel and maybe less used as a source of glutamate, while at the same time (iii) glutamate production from glucose is also reduced.

Taken together, this ground-breaking paper raises important questions on the use of neuronal and glial fuels during neuronal stimulation and epileptogenesis. Future work is needed to investigate brain glutamate and glutamine metabolism in vivo in awake subjects with and without epilepsy to reveal to which extent they may play a role as energy substrates under different conditions as well as the generation of seizures and epilepsy.