Elevations in extracellular glutamate in the brain are implicated in the pathogenesis of several neurological conditions, including mesial temporal lobe epilepsy. The underlying mechanisms of this elevation are not completely understood, however, and there are no effective methods that reduce the elevation or limit its neurotoxic effects. Glutamate is normally cleared from the extracellular space and replenished in axon terminals by a series of compartmentalized processes collectively known as the glutamate-glutamine cycle. A critical step of the cycle is the conversion of glutamate to glutamine by the astrocyte-specific enzyme glutamine synthetase. In several neurological conditions including mesial temporal lobe epilepsy, studies have demonstrated that glutamine synthetase activity is pathologically low. Because glutamine synthetase is thought to be critical for glutamate metabolism, its deficiency has been postulated as a possible mechanism for the increased glutamate observed in the extracellular fluid of the epileptogenic areas of the brain.
The branched-chain amino acids valine, leucine, and isoleucine are thought to contribute to de novo synthesis of glutamate in the brain by transferring an amino nitrogen to the tricarboxylic acid intermediate alpha-ketoglutarate. The branched-chain amino acids have gained increasing attention in recent years for the important roles they play in cell signaling, immune modulation, protein metabolism, and glutamate synthesis. It was previously unknown, however, if increasing peripheral branched-chain amino acids concentrations can increase extracellular glutamate concentrations in the brain during physiological or in pathological conditions, particularly when glutamate metabolism is perturbed (i.e. in glutamine synthetase deficiency). Moreover, the effects of branched-chain amino acids on seizures and neuronal viability were unknown.
The objective of this thesis was to use state of-the-art methods in microdialysis, isotope tracing, mass spectrometry and video-intracranial electroencephalogram recordings to study the metabolism and functional effects of branched-chain amino acids and glutamate in naïve and glutamine synthetase-inhibited, epileptic rats. The central hypothesis was that increased extracellular concentrations of branched-chain amino acids in the brain, in combination with alterations in enzymatic processes of glutamate metabolism, are key pathogenic features that result in brain glutamate excess and seizures in mesial temporal lobe epilepsy. To achieve the objective of this thesis, we pursued 3 specific aims.
In Aim 1, we determined the effects of intravenous branched-chain amino acid administration on brain extracellular fluid concentrations of glutamate and glutamine in naïve rats. We found that the administration of a high-dose bolus of branched-chain amino acids significantly increased the concentrations of branched-chain amino acids and glutamine in the extracellular compartment of the brain. Glutamate concentrations transiently increased, but the elevation was not statistically significant. In Aim 2, we determined the effects of intravenous isotope-labeled leucine administration on brain extracellular fluid concentrations of glutamate and glutamine in glutamine synthetase-inhibited rats. We found that glutamine synthetase-inhibited rats, like normal rats, were remarkably efficient in handling glutamate. Moreover, we demonstrated that leucine influx across the blood brain barrier is highly dependent on glutamine levels in the extracellular fluid of the brain. In Aim 3, we investigated the effects of chronic oral branched-chain amino acid supplementation on spontaneous and induced seizures, and neuron loss in glutamine synthetase-inhibited epileptic rats. We found that the branched-chain amino acid supplementation was ineffective in reducing the frequency and severity of spontaneous seizures, but increased the threshold to pentylenetetrazole-induced seizures. Furthermore, chronic branched-chain amino acid supplementation resulted in increased loss of hippocampal hilar neurons. Future studies will explore the impact of glutamine and leucine dysregulation in the brain on cell signaling and immune modulation, which may play an important role in epilepsy as well as other disorders. We will also further explore the mechanisms underlying the branched-chain amino acid-induced neuron loss.
|School Location:||United States -- Connecticut|
|Source:||DAI-B 79/05(E), Dissertation Abstracts International|
|Subjects:||Neurosciences, Biochemistry, Physiology|
|Keywords:||Branched-Chain Amino Acids, Glutamate, Glutamine, Leucine, Neurometabolism, Temporal Lobe Epilepsy|
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