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Keywords:

  • Glutamate metabolism;
  • Glutamine synthetase;
  • Hippocampus;
  • Inflammation;
  • Mesial temporal sclerosis

Summary

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

Approximately one-third of all patients with epilepsy continue to suffer from seizures even after appropriate treatment with antiepileptic drugs. Medically refractory epilepsies are associated with considerable morbidity and mortality, and more efficacious therapies against these disorders are clearly needed. However, the discovery of better therapies has been lagging due to an incomplete understanding of the mechanisms underlying the development of epilepsy (epileptogensis) and seizures (ictogenesis) in humans. An increasing number of studies have suggested that an abnormal amplification of glutamatergic activity—often referred to as the “glutamate hypothesis”—is involved in the pathophysiology of seizures and certain types of medically refractory epilepsies. For example, elevated levels of extracellular glutamate in hyperexcitable areas of the brain, up-regulation of glutamate receptors, and loss of the glutamate-metabolizing enzyme, glutamine synthetase (GS), have all been reported in patients with mesial temporal lobe epilepsy (MTLE). Moreover, it appears that glial cells, particularly the astrocyte, may play a key role in the glutamate overflow in MTLE. Proliferation of astrocytes is a hallmark of the epileptogenic focus in MTLE, and the proliferated cells are characterized by several unique features that are permissive for the excessive accumulation and release of astrocytic glutamate. Here, we assess recent data regarding the glutamate excess in epilepsy, review the role of glutamine synthetase, and discuss the implications of astrocytes in the pathophysiology of MTLE.


Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

Mesial temporal lobe epilepsy (MTLE) is one of the most common types of medically refractory epilepsies, and anteromedial temporal lobectomy with removal of the hippocampus is used for seizure control in some of these patients (Spencer et al., 1984). Neuropathological studies of the resected epileptogenic tissue typically reveal mesial temporal sclerosis—a collective term for several distinct pathologies affecting the limbic structures of the brain (reviewed in Gloor, 1991). One of these pathologies is hippocampal sclerosis, which is characterized by atrophy, induration, glial proliferation, and preferential loss of neurons in CA1, CA3, and the dentate hilus of the hippocampus, while neurons in the subiculum, CA2, and dentate granule cell layer are relatively spared (Sommer, 1880).

Glial proliferation (gliosis) contributes to the epileptogenicity of the human hippocampus in MTLE. In a series of 62 patients whose intracranial EEGs recorded multiple spontaneous seizures from a subsequently resected hippocampus, the proportion of seizures with onsets preceded by periodic 2-Hz spiking was significantly and directly correlated with glial density in CA3. Electrical seizure duration was significantly and directly correlated with glial density in CA2 and CA3 (Spencer et al., 1999). Hippocampal neuronal counts did not correlate with any EEG variables. Consistent with this view of gliosis being an essential component of epileptic tissue, studies in epileptic rodent models have found that glial proliferation is a common feature, even in the absence of neuronal loss (Drage et al., 2002; Vessal et al., 2005). Furthermore, depth electrode recordings from patients with MTLE indicate that the seizures originate from the sclerotic (gliotic) hippocampus (Spencer, 1994). Surgical removal of the sclerotic hippocampus is associated with an excellent clinical outcome, resulting in cessation of the seizures in about 85% of the patients (Engel class I) (de Lanerolle et al., 2003). Taken together, these observations indicate that the sclerotic hippocampus is critically involved in the pathophysiology of MTLE (Mathern et al., 1997).

An excess of extracellular glutamate in the sclerotic hippocampus may be one of the key molecular causes of seizures and brain damage in MTLE. Firstly, a large number of studies in laboratory animals have shown that glutamate and glutamate analogues cause seizures and neuronal loss similar to MTLE, suggesting that glutamate is a crucial element in the pathophysiology of this disease (Olney et al., 1972; Olney et al., 1986). Secondly, interictal extracellular glutamate is elevated five-fold more in the epileptogenic vs. the nonepileptogenic human hippocampus, measured in vivo by simultaneous depth electrode EEG and microdialysis (Fig. 1A) (Cavus et al., 2005). Paradoxically, interictal extracellular glutamate concentrations are considerably higher in patients with hippocampal sclerosis (MTLE) than in patients without this pathology (non-MTLE), despite the 60–80% neuronal loss and doubling of glial density in the sclerotic hippocampus (Petroff et al., 2003; Kim et al., 2004; Petroff et al., 2004). Thirdly, in humans, extracellular hippocampal glutamate increases six-fold above the interictal level during the seizure (30-times higher than normal), and remains markedly elevated (12-times higher than normal) for at least 20 min after the cessation of seizure activity (Fig. 1B) (During & Spencer, 1993). Finally, isotopic tracer (13C) studies during epilepsy surgery suggest that the accumulation and impaired clearance of glutamate in MTLE is due to a slowing of the glutamate–glutamine cycle metabolism in the sclerotic hippocampus compared with the nongliotic epileptogenic hippocampus or the normal occipital neocortex (Fig. 1 C) (Petroff et al., 2002). A pertinent question therefore is: What causes the slowing of glutamate–glutamine cycling in MTLE, and how does this increase the levels of extracellular glutamate?

image

Figure 1. Extracellular glutamate and glutamate–glutamine cycling in the human epileptogenic hippocampus. (A) Interictal concentrations of extracellular glutamate measured by in vivo microdialysis are increased approximately five-fold in the epileptogenic hippocampus vs. the contralateral nonepileptogenic hippocampus (p < 0.0001; Redrawn from data in Cavus et al. (Cavus et al., 2005); with permission, John Wiley & Sons, Inc. © 2002). (B) Extracellular glutamate measured by in vivo microdialysis is increased six-fold in the epileptogenic hippocampus during a seizure vs. interictally (p < 0.05; Adapted and redrawn from During et al (During & Spencer, 1993); with permission, Elsevier B. V. © 1993; and Cavus et al. (Cavus et al., 2005); with permission, John Wiley & Sons, Inc. © 2002). Note the delayed clearance of extracellular glutamate in the epileptogenic hippocampus. (C) Magnetic resonance spectroscopy (MRS) of epilepsy patients prior to surgery reveal decreased cycling of glutamate to glutamine in the sclerotic (gliotic) hippocampus in MTLE. The glutamate–glutamine cycle remains directly proportional with energy metabolism over the entire range of cerebral electrical and metabolic activity. As a consequence of this linear relationship, the ratio of the glutamate–glutamine cycle to the tricarboxylic acid (TCA) cycle rates remains relatively constant (0.4–0.5) from deep anesthesia with marked slowing of the EEG through bicuculline-induced status epilepticus over a three-fold variation in the rate of mitochondrial glucose oxidation. This ratio is the same in animal models and human subjects. The low values measured in mesial temporal sclerosis suggest significant impairment of the glutamate-glutamine cycle involving dysfunction of glutamine synthetase. (Redrawn from data in Petroff et al. (Petroff et al., 2002); with permission, Blackwell Publishing, Inc. © 2002) hippocampus. (C) Magnetic resonance spectroscopy (MRS) of epilepsy patients prior to surgery reveal decreased cycling of glutamate to glutamine in the sclerotic (gliotic) hippocampus in MTLE. The glutamate–glutamine cycle remains directly proportional with energy metabolism over the entire range of cerebral electrical and metabolic activity. As a consequence of this linear relationship, the ratio of the glutamate–glutamine cycle to the tricarboxylic acid (TCA) cycle rates remains relatively constant (0.4–0.5) from deep anesthesia with marked slowing of the EEG through bicuculline-induced status epilepticus over a three-fold variation in the rate of mitochondrial glucose oxidation. This ratio is the same in animal models and human subjects. The low values measured in mesial temporal sclerosis suggest significant impairment of the glutamate-glutamine cycle involving dysfunction of glutamine synthetase. (Redrawn from data in Petroff et al. (Petroff et al., 2002); with permission, Blackwell Publishing, Inc. ©2002).

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Regulation of Brain Glutamate

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

The extracellular concentration of glutamate in the brain is influenced by a series of interactions involving multiple cellular compartments, transmembrane transporter molecules, enzymes, and metabolic intermediates (Fig. 2). The astrocyte has a central role in this regulatory environment due to its: (a) anatomical position between the blood vessels and neurons, (b) unique capacity for anaplerosis, (c) rapid uptake of extracellular glutamate, and (d) presence of glutamine synthetase (GS). Simply stated, the flux of glutamate in the brain can be separated into a series of distinct, yet interconnected pathways (Fig. 2) (Hyder et al., 2006):

image

Figure 2. Diagram of glutamate metabolism in the central nervous system. The three main pathways of glutamate metabolism (see text for details) are identified by roman numerals and separate colors. Only the most important metabolic intermediates, enzymes, and transmembrane transporter molecules are indicated. The glutamine–glutamate-GABA pathway is not shown; however, the site of entry into the TCA cycle of GABA is indicated. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; BCAA, branched chain amino acids; BCAT, branched chain aminotransferases; BC α-ketoacid, branched chain α-ketoacid; CS, citrate synthase; EAATs, excitatory amino acid transporters; GDH, glutamate dehydrogenase; GLUTs, glucose transporters; GS, glutamine synthetase; HEX, hexokinase; LDH, lactate dehydrogenase; ME, cytosolic malic enzyme; MCTs, monocarboxylate transporters; PAG, phosphate activated glutaminase; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase complex; SA, system A transporters; SN, system N transporters; VCYCLE, glutamate–glutamine cycling measured by MRS; VGLUTs, vesicular glutamate transporters; VTCA astro, TCA cycling in astrocytes measured by MRS; VTCA neuron, TCA cycling in neurons measured by MRS.

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(1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

Because blood glutamate does not readily enter the brain, the bulk of brain glutamate is synthesized from blood-derived glucose (Lajtha et al., 1959). The importance of glucose as the main metabolic substrate of the brain is reflected by the avid uptake of blood glucose via glucose transporter molecules present on endothelial cells, astrocytes, neurons, and possibly also other cell types in the brain (Qutub & Hunt, 2005). The microvessels of the brain are almost completely surrounded by astrocytic processes (perivascular end-feet), so blood glucose is believed to enter the astrocytic compartment before it reaches the neuron. Once in the astrocyte, glucose may take one of several routes: (a) synthesis of glycogen, (b) glycolysis with synthesis of lactate, which may be transported to the neurons and used as a fuel in the latter cells (Magistretti et al., 1993; Pellerin et al., 1998), (c) glycolysis with synthesis of pyruvate and oxaloacetate via pyruvate carboxylase (i.e., anaplerosis; discussed under 2) below), and (d) glycolysis with synthesis of pyruvate and acetyl coenzyme A, followed by entry of the tricarboxylic acid (TCA) cycle and synthesis of α-ketoglutarate. The latter intermediate may be converted to glutamate via one of several enzymes, including glutamate dehydrogenase (GDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and branched chain aminotransferases (BCAT).

It should be noted that even though astrocytes may be the first target of blood-derived glucose (after the endothelial cells), neurons also contain glucose transporters and use glucose as metabolic fuel. Moreover, the enzymes necessary for synthesizing glutamate from α-ketoglutarate are present in neurons, indicating that these cells too, can produce glutamate from α-ketoglutarate.

(2) Anaplerosis: Replenishment of TCA cycle intermediates

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

Because the synthesis of glutamate from α-ketoglutarate leads to a net loss of TCA cycle intermediates, replenishment of the cycle (anaplerosis) is necessary. Astrocytes are the predominant and, at least in vivo, probably the only compartment capable of anaplerosis in the brain. This is due to the unique presence of pyruvate carboxylase (PC), which catalyzes the formation of oxaloacetate from pyruvate (Shank et al., 1985), in astrocytes.

(3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

In the astrocyte, glutamate is converted to glutamine via the astrocyte- specific enzyme, GS (Martinez-Hernandez et al., 1977). Glutamine is then transferred to neurons via glutamine transporters on astrocytes and neurons. In the neuron, glutamine is converted to glutamate via phosphate-activated glutaminase (PAG) (Svenneby, 1970; Kvamme et al., 2001), packaged in synaptic vesicles, and released into the extracellular space. In the extracellular space, glutamate is taken up by astrocytes via glutamate transporters (Danbolt, 2001) and reconverted to glutamine via GS. Glutamine is subsequently ready for transfer to neurons, and the cycle is complete (Hyder et al., 2006).

Although the regulation of brain glutamate is more complex than described here, the pathways above provide a useful and testable foundation for exploration of glutamate homeostasis in health and disease.

Loss of Glutamine Synthetase in MTLE

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

GS is an integral element of the glutamate-glutamine cycle, and patients with MTLE are severely deficient in this enzyme (Eid et al., 2004). Based on investigations of surgically resected brain tissue from patients with MTLE, we recently demonstrated a considerable (35–40%) loss of GS protein (Fig. 3A) and activity (Fig. 3B) in astrocytes (Fig. 4) of the hippocampus (Eid et al., 2004). A similar loss of enzyme was not present in surgically resected hippocampi from patients with other types of temporal lobe epilepsy or in hippocampi from autopsy control subjects, suggesting that this phenomenon may be unique to MTLE. A subsequent study has independently confirmed this finding (van der Hel et al., 2005). Moreover, GS activity is reduced in the amygdala of MTLE patients (Steffens et al., 2005).

imageimage

Figure 3. Glutamine synthetase protein and activity in the human epileptogenic hippocampus. (A) Quantitative Western blotting reveals that glutamine synthetase protein is reduced by 40% (p < 0.05) in the sclerotic (MTLE) vs. the nonsclerotic (non-MTLE) hippocampus (Redrawn from data in Eid et al (Eid et al., 2004); with permission, Elsevier B. V. © 2004). (B) The activity of glutamine synthetase is decreased by 38% (p < 0.05) in tissue homogenates of the sclerotic (MTLE) vs. the nonsclerotic (non-MTLE) hippocampus (Redrawn from data in Eid et al (Eid et al., 2004); with permission, Elsevier B. V. © 2004).

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Figure 4. Glutamine synthetase in astrocytes of the sclerotic hippocampus in MTLE. Immunohistochemistry for glutamine synthetase reveals labeling of numerous astrocytes in a control hippocampus obtained at autopsy (A–C). Specifically, astrocytes in the subiculum (B) and CA1 (C) are strongly labeled. In contrast, in a sclerotic MTLE hippocampus (D-F), labeling of astrocytes is observed in the subiculum (D, E) whereas CA1 is devoid of immunoreactive cells (C, E). Scale bars, A = 0.5 mm (same magnification for D); B = 100 μm (same magnification for C, E, F). Abbreviations: alv, alveus; dg, dentate gyrus; sub, subiculum. (Eid, et al., 2004); reprinted with permission, Elsevier B. V., (c) 2004).

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We postulate that the deficiency in GS will slow the conversion of glutamate to glutamine and cause accumulation of extracellular and astrocytic glutamate. This conclusion is based on two key findings. First, the known stoichiometry of glutamate transport across the glial plasma membrane suggests that rapid metabolism of intracellular glutamate is a prerequisite for efficient glutamate clearance from the extracellular space (Otis & Jahr, 1998); Second, studies of organotypic hippocampal cultures show that glutamate accumulates and glutamine decreases in astrocytes after experimental inhibition of GS by methionine sulfoximine (MSO) (Laake et al., 1995).

Because it is possible that the higher than normal amounts of extracellular glutamate in MTLE could be caused not only by a deficiency in GS, but by a loss of astrocytic glutamate transporters, we also assessed the presence of two of the most abundant of these transporters in the hippocampus, EAAT1 (GLAST) and EAAT2 (GLT), by immunohistochemistry and quantitative western blots (Fig. 5) (Eid et al., 2004; Bjørnsen et al., 2007). We detected no difference in EAAT1 or EAAT2 protein expression between whole hippocampi from patients with MTLE and control hippocampi. Furthermore, GS-deficient astrocytes in the MTLE hippocampi expressed EAAT 1 and EAAT2. Thus, glutamate appears to have access to the cytosol of the GS-deficient astrocytes in MTLE. Taken together, these data suggest that the loss of GS is likely to cause the observed slowing in glutamate–glutamine cycling (Petroff et al., 2002) and accumulation of extracellular glutamate in MTLE (During & Spencer, 1993; Cavus et al., 2005).

image

Figure 5. Levels of glutamate transporters EAAT1 and EAAT2 in the human epileptogenic hippocampus. Quantitative Western blotting of tissue homogenates from sclerotic (MTLE) and nonsclerotic (non-MTLE) hippocampi reveal no change in protein concentrations of glutamate transporters EAAT1 and EAAT2 between the two patient categories (Bjornsen et al., 2007); reprinted with permission, Elsevier B. V., © 2007.

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However, the reduction in GS activity in MTLE does not necessarily imply causality, that is the putative link between enzyme dysfunction and the emergence of epilepsy requires experimental validation. One approach is to explore the effects of GS deficiency in laboratory animals. This can be accomplished by systemic administration of MSO to rodents, and such studies have indeed demonstrated that GS inhibition results in seizure activity (Folbergrova et al., 1969; Szegedy, 1978). However, general inhibition of GS is not representative of human MTLE where the enzyme is impaired locally in the hippocampus and, possibly, in the amygdala (see above). Moreover, high doses of MSO also inhibit gamma-glutamylcysteine synthetase, thus reducing the level of intracellular glutathione, an important antioxidant molecule (Shaw & Bains, 2002). A better way would be to develop an animal model of chronic, localized hippocampal GS deficiency, similar to the situation in human MTLE, followed by careful characterization of this model by continuous video-EEG monitoring and pertinent neuropathological studies.

We have now developed such a model using continuous (∼28 days) infusion of MSO (0.5 μg/h) into the hippocampus of rats (Wang et al., 2006). Treated animals showed long-term inhibition of hippocampal GS activity, but had normal brain tissue glutathione concentrations. Spontaneous recurrent seizures commenced after an initial seizure-free period, and neuropathological analysis revealed changes that closely resembled human MTLE, including preferential neuronal loss in area CA1 of the hippocampus. These findings suggest that inhibition (or loss) of GS may be critically involved in the pathophysiology of MTLE. Furthermore, intrahippocampal infusion of MSO may prove to be a useful model for further experimental studies of epilepsy.

Mechanisms of Glutamine Synthetase Deficiency in MTLE

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

What causes the GS deficit in astrocytes of the MTLE hippocampus? Even though the enzyme has been investigated extensively in animals and in vitro for decades, there is a remarkable lack of information on its regulation in human neurological diseases, particularly MTLE. However, a careful review of the literature reveals pieces of information that could be of particular relevance for the fate of GS in epilepsy.

One possibility is that GS is down-regulated due to the loss of neurons in the MLTE hippocampus (Sommer, 1880). Using hippocampal slice cultures, Derouiche and colleagues showed that the expression of GS protein in astrocytes is markedly reduced in an environment absent of neurons, particularly glutamatergic axon terminals (Derouiche et al., 1993). These authors concluded that a neuron-derived factor, possibly glutamate, may be necessary for full expression of the protein. However, glutamate is unlikely to be the regulator of GS in MTLE, because the levels of extracellular glutamate are more than five-fold increased in the MTLE hippocampus (During & Spencer, 1993; Petroff, et al. 2004; Cavus et al., 2005).

Is it possible that the lack of another neuron-derived factor causes the loss of GS in MTLE? In fact, studies of retinal cell cultures have shown that direct glial-neuron contact is required for induction of GS in Müller cells (Linser & Moscona, 1983). Such contact leads to cytoskeletal changes, a decrease in the levels of transcription factor c-jun, and up-regulation of mRNA for GS (Vardimon et al., 2006). Conversely, high levels of c-jun, which repress the GS gene are seen after loss of cell–cell interactions (Vardimon et al., 2006), and, interestingly, also after epileptic seizures (Beer et al., 1998). Thus, elevations in c-jun may be a potential cause of the GS deficiency in MTLE.

GS is also regulated by glucocorticoids and proinflammatory cytokines. Dexamethasone increases transcription of the GS gene in astrocytes in culture (Hallermayer et al., 1981; Patel & Hunt, 1985; Jackson et al., 1995) and in C6 glioma cells (Pishak & Phillips, 1980). The inductive effect of glucocorticoids is mediated by binding of the glucocorticoid receptor to a glucocorticoid response element in the regulatory region of the GS gene, and this effect is blocked by the proinflammatory cytokines interleukin 1β (IL-1β) and tumor necrosis factor-α (Huang & O'Banion, 1998). Moreover, lipopolysaccharide and interferon-γ inhibit the induction of GS caused by N-methyl-d-aspartate (NMDA) receptor stimulation in cultured astrocytes (Muscoli et al., 2005). Interestingly, microglial activation (Beach et al., 1995), which is associated with alterations of proinflammatory cytokines and chemokines, is indeed a prominent feature of the MTLE hippocampus (Crespel et al., 2002; Kanemoto et al., 2003). The possibility that inflammation plays a role in the regulation of GS in MTLE therefore clearly deserves further attention.

The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

The metabolism of glutamate and glutamine in epileptic human tissue has been studied during surgery with stable nonradioactive isotopes (Petroff et al., 2004), and in vitro using standard enzyme assays (Eid et al., 2004). We have extended the findings by measuring 13C glutamine synthesis in incubated brain slices prepared from resected human tissue. To control for variables unique to each epilepsy patient, for example medications and seizure frequency, brain slices prepared from the epileptogenic human hippocampus were compared to brain slices from the middle temporal gyrus of the same patient (Fig. 6). Electrophysiology was studied in adjacent slices along with quantitative histology. The methodological rationale is that 13C glucose is converted sequentially to 13C-labeled intermediates, notably 13C glutamate and 13C glutamine (Fig. 2). The concentration of 13C-labeled isotopes relative to unlabeled isotopes (isotopic enrichment, IR) can then be determined in timed tissue samples using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This analytical approach has the advantage of requiring very small tissue amounts (10–15 mg wet weight). The combination of using brain tissue slices incubated in 13C-labeled metabolic precursors with LC/MS/MS allows for an assessment of metabolic activity in valuable tissue samples, for example human surgical specimens or rats with distinct genetic or behavioral characteristics. Moreover, because small quantities of tissue are needed, pharmacological manipulations can be used to examine the regulation of the different enzymatic pathways involved. For example, the tissue can be exposed to an ammonia challenge to examine its capacity of to detoxify the ammonia and glutamate released by neurons during glutamatergic and GABAergic neurotransmission. In other words, addition of low doses of ammonium chloride (NH4Cl) to the bath can probe the extent by which GS activity can be enhanced by one of its substrates (glutamate and ATP).

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Figure 6. Glutamate–glutamine cycling in human hippocampal slices measured by LC/MS/MS. The diagram depicts the ratio of isotopically enriched (IR, see text for details) 13C-glutamine to IR 13C-glutamate in slices of the nonepileptogenic neocortex (middle temporal gyrus) and epileptogenic MTLE hippocampus after incubation for 4 h in 13C glucose. This ratio reflects the activity of astrocytic glutamine synthetase. The two left bars indicate that the synthesis of glutamine by glutamine synthetase is comparable between the neocortex and hippocampus under “standard” conditions of 10 mM 13C glucose. However, when 1 mM ammonium chloride is added to the bath, glutamine synthesis in the neocortex, but not in the hippocampus, is markedly increased (two right bars). Notably, ammonia increases glutamate synthesis in both the neocortex and the hippocampus (data not shown). This implies that glucose metabolism, primarily glial anaplerosis, is increased by the ammonia challenge as expected from the known stoichiometry of GS. These findings suggest that there is limited capacity for glutamine synthesis in the hippocampus compared with the neocortex of patients with MTLE. The error bars show the standard error (n = 4, paired samples).

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For this set of experiments, we exposed 400 μm hippocampal and neocortical slices from resected temporal lobes to 10 mM 13C glucose (the normal glucose concentration used in brain slice studies) for 4 h. (See (Errante et al., 2002) for details of the human slice preparation.) Prior experiments in rat brain slices had indicated that glutamate and glutamine are in equilibrium within this period (unpublished observations). Although true steady-state labeling is not achieved in slices until 6–7 h (Duarte et al., 2007), these data showed that, under physiological conditions in rats, GABA and glutamate are in equilibrium at the time point when the human studies were undertaken.

Data obtained from a total of four patients with MTLE (Fig. 6) demonstrated that under physiological conditions (i.e., 10 mM glucose), the degree of glutamate–glutamine cycling, as determined by (IR 13C glutamine/IR 13C glutamate), is comparable between the anterior hippocampus and the neocortex. However, when GS activity was increased by adding 1 mM NH4Cl to the tissue, a marked increase in the degree of glutamate–glutamine cycling occurred in the neocortex, but not in the hippocampus (Fig. 6). These data suggest that, in the sclerotic hippocampus, there is sufficient GS to maintain the glutamate and glutamine needed for both glutamatergic and GABAergic synaptic transmission under physiological conditions. However, during periods of increased activity, for example when ammonia is synthesized by neuronal phosphate activated glutaminase (PAG; Fig. 2), GS may not be able to increase both glutamate clearance and glutamine synthesis. This scenario is relevant, because even though the majority of neurons are lost in the MTLE hippocampus, a subpopulation of neurons with particularly high levels of PAG is still present (Eid, et al. 2007). Given the sensitivity of GABAergic neurotransmission to glutamine availability (Liang et al., 2006), both excitatory and inhibitory networks will be impaired by the “sluggish” GS activity demonstrated in these studies.

Can Astrocytes Release Glutamate in MTLE?

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

An increasing number of studies have shown that astrocytes can be significant sources of extracellular glutamate (Volterra & Meldolesi, 2005), and there are a variety of pathways by which release of glutamate from astrocytes can take place. One mechanism is due to astrocyte swelling (Kimelberg et al., 1995); this is certainly a possibility in MTLE, as we were able to demonstrate an increase in mRNA expression of the astrocyte-specific protein aquaporin-4 (AQP4) in the sclerotic hippocampus of patients with this disease (Lee et al., 2004). Using quantitative immunogold electron microscopy, we found that the astrocytic end-feet had lower than normal expression of the protein, indicating a loss of the normal polar distribution of AQP4 on astrocytes (Eid, et al. 2005). Such a change in AQP4 distribution may decrease the elimination of metabolic water from the astrocyte into the blood and lead to astrocyte swelling and high extracellular K+, which can further facilitate swelling (Amiry-Moghaddam et al., 2003; Lee et al., 2004; Eid et al., 2005). Astrocyte swelling can be compensated for by release of glutamate, along with chloride and other anions, through volume sensitive organic anion channels, thus increasing the extracellular concentration of glutamate (Anderson & Swanson, 2000).

Recent studies in animals have provided evidence that Ca2+ elevations in astrocytes induce excitotoxic release of glutamate from these cells (Bezzi et al., 1998). In studies of hippocampal slice models of epilepsy, astrocytic glutamate generated paroxysmal depolarization shifts in neurons suggesting that astrocytes may play an important role in interictal events related to seizures (Tian et al., 2005). Fellin et al. (Fellin et al., 2006) confirmed the results,and showed that astrocytes are activated during a period of epileptiform activity and that activation of these cells results in increased glutamate release. However, these authors concluded that astrocyte-derived glutamate is not necessary for the initiation of epileptiform activity, although it may modulate and enhance the duration of seizure-like events.

There are several pathways by which Ca2+-dependent glutamate release may occur. One mechanism involves glutamate-mediated coactivation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate and metabotropic glutamate receptors (mGluRs) on astrocytes. Binding of glutamate to these receptors leads to high intracellular levels of prostaglandin E2 (PGE2), an increase in intracellular Ca2+ oscillations, and release of glutamate (Bezzi et al., 1998) in a wave-like fashion (Innocenti et al., 2000). PGE2 activation is mediated through the inducible prostaglandin G/H synthetase (PGHS-2), also known as cyclooxygenase-2 (O'Banion et al., 1996). Notably, the PGE2 pathway can also be directly or indirectly activated by the transcription factor NFκB.

Several lines of evidence suggest that Ca2+-dependent glutamate release from astrocytes may also take place in the epileptogenic human brain. First, astrocytes in primary cultures derived from human MTLE hippocampi exhibit increased frequency of intracellular Ca2+ waves compared to cultures from non-MTLE hippocampi (Cornell-Bell, unpublished; Cornell-Bell et al., 1991). Astrocytes from neocortical epileptic foci in childhood epilepsy (Rasmussen's encephalitis) (Manning & Sontheimer, 1997) and epileptogenic mass lesions associated with temporal lobe epilepsy (Cornell-Bell & Williamson, 1993) likewise exhibit elevated Ca2+ oscillation frequency. Secondly, a recent gene expression profiling study of MTLE hippocampi (Lee et al., 2007), and earlier biochemical and immunohistochemical studies (Crespel et al., 2002; Perosa et al., 2002) point to a number of up-regulated molecules in sclerotic areas of the brain. These molecules are associated with mechanisms that may activate the PGE2/Ca2+ dependent glutamate release pathway via an increase in NFκB. The proinflammatory cytokine IL-1β), and the chemokine stromal derived factor 1α (SDF-1α) are both involved in these processes. Interestingly, there is a strong association of a polymorphism in the IL-1β gene in patients with hippocampal sclerosis compared to patients without sclerosis and nonepileptic controls (Berkovic & Jackson, 2000; Kanemoto et al., 2000). The biallelic polymorphism, which is in the promoter region of the IL-1β gene, predisposes to high production of IL-1β in patients with hippocampal sclerosis

IL-1β, acting through interleukin receptors on astrocytes and the transcription factor NFκB, can induce a large number of other molecules of relevance for glutamate release (John et al., 2005). These molecules include members of the tumor necrosis factor superfamily, chemokines and chemokine receptors, growth factors, extracellular matrix and matrix metalloproteinases, and gap junction proteins (John et al., 2005) as well as genes regulating the morphology and migration of astrocytes (John et al., 2004). Microarray data from our laboratory show that several of the genes that can be induced by IL-1β are up-regulated in the sclerotic hippocampus (Lee et al., 2007). Among these are proteins of the ezrin-radixin-moesin (ERM) family, S100β (Barger & Van Eldik, 1992), hyaluronan (hyaluronic acid, HA), CD44 (Pure & Cuff, 2001), CXCR4, and several other chemokines. IL-1β, the ERM proteins, CD44, S100β, and HA are directly or indirectly involved in intracellular Ca2+ release. SDF-1α can bind to the CXCR4 receptor, which is up-regulated on astrocytes and microglia in MTLE, and lead to an increase in intracellular Ca2+ through a complex signaling pathway that involves PGE2 (Bezzi et al., 2001).

Finally, it has been proposed that the Ca2+ dependent glutamate release from astrocytes is a SNARE protein-dependent process that requires functional vesicle-associated proteins and release of glutamate by exocytosis (Araque et al., 2000). Interestingly, gene expression studies reveal that the vesicle-associated proteins SNAP23 and syntaxin-16 are increased in the MTLE hippocampus. SNAP23 is an analogue of SNAP25 and is expressed in astrocytes (Hepp et al., 1999). These findings points to the possibility of vesicular release of glutamate from astrocytes.

Conclusions and Future Directions

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

Loss of neurons and proliferation of glial cells are two characteristic features of the epileptogenic hippocampus in MTLE. Because neurons normally are the primary source of extracellular glutamate, it remains somewhat puzzling how high-excitotoxic levels of glutamate build up in a neuron-sparse area, such as the MTLE hippocampus, unless glutamate is inefficiently cleared by and/or released from astrocytes. The observation that astrocytes in the epileptogenic hippocampus in MTLE lack the glutamate metabolizing enzyme GS suggests that these cells accumulate glutamate, and possibly impair (by mass action) the clearance of glutamate from the extracellular space. Moreover, an increasing number of studies indicate that astrocytes are capable of releasing glutamate through a Ca2+-dependent mechanism, and that this capacity is enhanced in astrocytes of the MTLE hippocampus. An intriguing question is whether lack of GS and increased Ca2+-dependent glutamate release are properties of all “reactive” astrocytes, and if this may be a mechanism common to all seizure foci that contain “reactive” astrocytes, such as in mass lesions, traumatic brain injury, and tubers in tuberous sclerosis.

Even though these findings suggest a key role of glutamate, GS, and astrocytes in the pathophysiology of epilepsy, they do not necessarily imply causality, and further studies are needed to resolve this issue. For example, does a deficiency in GS in the hippocampus of laboratory animals lead to a clinical phenotype similar to MTLE, and would restoration of GS reverse the signs of the disease? Moreover, can the entire spectrum of astrocyte abnormalities seen in the sclerotic human hippocampus be reproduced in laboratory animals, and will the presence of such abnormal cells lead to epilepsy? If abnormal astrocytes are indeed causally involved in the pathogenesis of epilepsy, a large number of molecules deserve further exploration as potential therapeutic targets. This includes, not only GS, but also aquaporins, cytokines and other molecules proposed to influence the control of extracellular glutamate levels by astrocytes.

Acknowledgments

  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References

This work was supported by National Institute of Health (NIH-NINDS) Grant NS054801. We are most grateful to Drs. Dennis D. Spencer and Jung H. Kim for providing human epilepsy and autopsy control tissue, respectively, and Drs. Michael E. Hodsdon and Brian R. Smith for providing access to the mass spectrometry facility at the Department of Laboratory Medicine at Yale. We thank the following colleagues for their help with the studies presented here: Drs. Henning Beckstrøm, Lars P. Bjørnsen, Idil Cavus, Niels C. Danbolt, Svend Davanger, Arko Ghosh, Janniche Hammer, Petter Laake, James C.K. Lai, Gauri H. Malthankar-Phatak, Mahmood Amiry-Moghaddam, Kirsten Osen, Ole P. Ottersen, Elise Runden-Pran, Bjørg A. Roberg, Marion J. Thomas, Ingeborg A. Torgner, Yue Wang, and Hitten Zaveri. We also thank the following publishers and authors for their permission to use previously published data: John Wiley & Sons, Inc (Figs. 1A, 1B: Dr. Idil Cavus); Blackwell Publishing, Inc (Fig. 1C); Elsevier B. V. (Fig. 1A: Dr. Matthew During; Figs. 3, 4, 5).

Conflict of interest:  The contributing authors to this article have no conflicts of interest.

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  1. Top of page
  2. Mesial Temporal Lobe Epilepsy—A Disorder of Gliosis and Glutamate Excess
  3. Regulation of Brain Glutamate
  4. (1) Glucose as the source of glutamate conversion of blood-derived glucose to α-ketoglutarate and glutamate
  5. (2) Anaplerosis: Replenishment of TCA cycle intermediates
  6. (3) The glutamate-glutamine cycle: Trafficking of glutamate and glutamine between astrocytes and neurons
  7. Loss of Glutamine Synthetase in MTLE
  8. Mechanisms of Glutamine Synthetase Deficiency in MTLE
  9. The Activity of Glutamine Synthetase Can Be Studied Experimentally in Human Hippocampal Slices
  10. Can Astrocytes Release Glutamate in MTLE?
  11. Conclusions and Future Directions
  12. Acknowledgments
  13. References
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