Glutamine Synthetase Deficiency Research Articles

Glutamine synthetase deficiency in murine astrocytes results in neonatal death

Glutamine synthetase (GS) is a key enzyme in the "glutamine-glutamate cycle" between astrocytes and neurons, but its function in vivo was thus far tested only pharmacologically. Crossing GS(fl/lacZ) or GS(fl/fl) mice with hGFAP-Cre mice resulted in prenatal excision of the GS(fl) allele in astrocytes. "GS-KO/A" mice were born without malformations, did not suffer from seizures, had a suckling reflex, and did drink immediately after birth, but then gradually failed to feed and died on postnatal day 3. Artificial feeding relieved hypoglycemia and prolonged life, identifying starvation as the immediate cause of death. Neuronal morphology and brain energy levels did not differ from controls. Within control brains, amino acid concentrations varied in a coordinate way by postnatal day 2, implying an integrated metabolic network had developed. GS deficiency caused a 14-fold decline in cortical glutamine and a sevenfold decline in cortical alanine concentration, but the rising glutamate levels were unaffected and glycine was twofold increased. Only these amino acids were uncoupled from the metabolic network. Cortical ammonia levels increased only 1.6-fold, probably reflecting reduced glutaminolysis in neurons and detoxification of ammonia to glycine. These findings identify the dramatic decrease in (cortical) glutamine concentration as the primary cause of brain dysfunction in GS-KO/A mice. The temporal dissociation between GS(fl) elimination and death, and the reciprocal changes in the cortical concentration of glutamine and alanine in GS-deficient and control neonates indicate that the phenotype of GS deficiency in the brain emerges coincidentally with the neonatal activation of the glutamine-glutamate and the associated alanine-lactate cycles.

Epilepsy in glioblastoma multiforme: correlation with glutamine synthetase levels

The hypothesis addressed by this study is that a glutamine synthetase (GS) deficiency in neoplastic astrocytes is a possible molecular basis associated with seizure generation in glioblastoma multiforme (GBM). Quantitative Western blot analysis of GS was performed in 20 individuals operated for malignant glioma. The levels of GS in patients with GBM and epilepsy were significantly lower (range 0.04-1.15; mean 0.35 +/- 0.36; median 0.25) than in non-epileptic GBM individuals (range 0.78-3.97; mean 1.64 +/- 0.99; median 1.25; P = 0.002). No relationship has been found between histological features (i.e. necrosis, gliosis, stroma, inflammatory cells, giant cells, and haemosiderine) and GS expression or epilepsy. Even though the epileptogenesis in glioma is multifactorial, it is conceivable that a down-regulation of GS may have an important pro-epileptogenic role in GBM, through the slowing of glutamate-glutamine cycle. This study suggests that seizures in GBM are coupled with a highly localized enzyme deficiency. The manipulation of GS activity might constitute a novel principle for inhibiting seizures in patients with glioma epilepsy.

Recurrent seizures and brain pathology after inhibition of glutamine synthetase in the hippocampus in rats.

An excess of extracellular glutamate in the hippocampus has been linked to the generation of recurrent seizures and brain pathology in patients with medically intractable mesial temporal lobe epilepsy (MTLE). However, the mechanism which results in glutamate excess in MTLE remains unknown. We recently reported that the glutamate-metabolizing enzyme glutamine synthetase is deficient in the hippocampus in patients with MTLE, and we postulated that this deficiency is critically involved in the pathophysiology of the disease. To further explore the role of glutamine synthetase in MTLE we created a novel animal model of hippocampal glutamine synthetase deficiency by continuous (approximately 28 days) microinfusion of methionine sulfoximine (MSO: 0.625 to 2.5 microg/h) unilaterally into the hippocampus in rats. This treatment led to a deficiency in hippocampal glutamine synthetase activity by 82-97% versus saline. The majority (>95%) of the MSO-treated animals exhibited recurrent seizures that continued for several weeks. Some of the MSO-treated animals exhibited neuropathological features that were similar to mesial temporal sclerosis, such as hippocampal atrophy and patterned loss of hippocampal neurons. However, many MSO-treated animals displayed only minimal injury to the hippocampus, with no clear evidence of mesial temporal sclerosis. These findings support the hypothesis that a deficiency in hippocampal glutamine synthetase causes recurrent seizures, even in the absence of classical mesial temporal sclerosis, and that restoration of glutamine synthetase may represent a novel approach to therapeutic intervention in this disease.

Glutamine synthetase is essential in early mouse embryogenesis

Glutamine synthetase (GS) is expressed in a tissue-specific and developmentally controlled manner, and functions to remove ammonia or glutamate. Furthermore, it is the only enzyme that can synthesize glutamine de novo. Since congenital deficiency of GS has not been reported, we investigated its role in early development. Because GS is expressed in embryonic stem (ES) cells, we generated a null mutant by replacing one GS allele in-frame with a beta-galactosidase-neomycine fusion gene. GS(+/LacZ) mice have no phenotype, but GS(LacZ/LacZ) mice die at ED3.5, demonstrating GS is essential in early embryogenesis. Although cells from ED2.5 GS(LacZ/LacZ) embryos and GS(GFP/LacZ) ES cells survive in vitro in glutamine-containing medium, these GS-deficient cells show a reduced fitness in chimera analysis and fail to survive in tetraploid-complementation assays. The survival of heavily (>90%) chimeric mice up to at least ED16.5 indicates that GS deficiency does not entail cell-autonomous effects and that, after implantation, GS activity is not essential until at least the fetal period. We hypothesize that GS-deficient embryos die when they move from the uterine tube to the harsher uterine environment, where the embryo has to catabolize amino acids to generate energy and, hence, has to detoxify ammonia, which requires GS activity.

Recent advances in amino acid and organic acid metabolism

This paper focuses on the three areas in this field in which there have been advances in amino acid and organic acid metabolism. These are the description of glutamine synthetase deficiency, the elucidation of the mechanism of pyridoxine-dependent convulsions, and a hypothesis to explain the neurological complications of some organic acidaemias.

Congenital Glutamine Deficiency with Glutamine Synthetase Mutations

Glutamine synthetase plays a major role in ammonia detoxification, interorgan nitrogen flux, acid-base homeostasis, and cell signaling. We report on two unrelated newborns who had congenital human glutamine synthetase deficiency with severe brain malformations resulting in multiorgan failure and neonatal death. Glutamine was largely absent from their serum, urine, and cerebrospinal fluid. Each infant had a homozygous mutation in the glutamine synthetase gene (R324C and R341C). Studies that used immortalized lymphocytes expressing R324C glutamine synthetase (R324C-GS) and COS7 cells expressing R341C-GS suggest that these mutations are associated with reduced glutamine synthetase activity.

Inborn error of amino acid synthesis: Human glutamine synthetase deficiency

Glutamine synthetase (GS) is ubiquitously expressed in human tissues, being involved in ammonia detoxification and interorgan nitrogen flux. Inherited systemic deficiency of glutamine based on a defect of glutamine synthetase was recently described in two newborns with an early fatal course of disease. Glutamine was largely absent in their serum, urine and cerebrospinal fluid. Each of the patients had a homozygous mutation in the glutamine synthetase gene and enzymatic investigations confirmed that these mutations lead to a severely reduced glutamine synthetase activity. From the observation in the first patients with congenital glutamine synthetase deficiency, brain malformation can be expected as one of the leading signs. In addition, other organ systems are probably involved as observed in one of the index patients who suffered from severe enteropathy and necrolytic erythema of the skin. Deficiency of GS has to be added to the list of inherited metabolic disorders as a rare example of a defect in the biosynthesis of an amino acid.

MARS treatment for a patient presenting with acquired hepatic glutamine synthetase deficiency after orthotopic liver transplantation

We report a 48-year-old man presenting with refractory hyperammonemia after orthotopic liver transplantation. On investigation he was found to have acquired hepatic glutamine synthetase deficiency, a rare condition that occurs after organ transplantations. The patient was started on continuous venovenous hemofiltration treatment, but the hyperammonemia did not respond. The patient was then subjected to molecular adsorption recirculation system (MARS) therapy and the ammonia level gradually improved with successive treatments. In conclusion, the response was unlikely due to the hemofiltration component of MARS alone but more probably due to the removal of putative albumin-bound toxin by the adsorption circuit that had ameliorated the internal milieu of the graft and reversed the enzyme deficiency.

Loss of glutamine synthetase in the human epileptogenic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy

High extracellular glutamate concentrations have been identified as a likely trigger of epileptic seizures in mesial temporal lobe epilepsy (MTLE), but the underlying mechanism remains unclear. We investigated whether a deficiency in glutamine synthetase, a key enzyme in catabolism of extracellular glutamate in the brain, could explain the perturbed glutamate homoeostasis in MTLE. The anteromedial temporal lobe is the focus of the seizures in MTLE, and surgical resection of this structure, including the hippocampus, leads to resolution of seizures in many cases. By means of immunohistochemistry, western blotting, and functional enzyme assays, we assessed the distribution, quantity, and activity of glutamine synthetase in the MTLE hippocampus. In western blots, the expression of glutamine synthetase in the hippocampus was 40% lower in MTLE than in non-MTLE samples (median 44 [IQR 30-58] vs 69 [56-87]% of maximum concentration in standard curve; p=0.043; n=8 and n=6, respectively). The enzyme activity was lower by 38% in MTLE vs non-MTLE (mean 0.0060 [SD 0.0031] vs 0.0097 [0.0042] U/mg protein; p=0.045; n=6 and n=9, respectively). Loss of glutamine synthetase was particularly pronounced in areas of the MTLE hippocampus with astroglial proliferation, even though astrocytes normally have high content of the enzyme. Quantitative immunoblotting showed no significant change in the amount of EAAT2, the predominant glial glutamate transporter in the hippocampus. A deficiency in glutamine synthetase in astrocytes is a possible molecular basis for extracellular glutamate accumulation and seizure generation in MTLE. Further studies are needed to define the cause, but the loss of glutamine synthetase may provide a new focus for therapeutic interventions in MTLE.

Giant coronary aneurysms

THE LANCET • Vol 363 • January 3, 2004 • www.thelancet.com 37 30 Gorovits R, Avidan N, Avisar N, Shaked I, Vardimon L. Glutamine synthetase protects against neuronal degeneration in injured retinal tissue. Proc Natl Acad Sci USA 1997; 94: 7024–29. 31 Szegedy L. Enzyme histochemical studies in the rat hippocampus, cerebellar cortex and brainstem motor nuclei during methionine sulphoximine convulsions. Cell Mol Biol 1978; 23: 43–53. 32 Folbergrova J, Passonneau JV, Lowry OH, Schulz DW. Glycogen, ammonia and related metabolites in the brain during seizures evoked by methionine sulphoximine. J Neurochem 1969; 16: 191–203. 33 Laming PR, Cosby SL, O’Neill JK. Seizures in the Mongolian gerbil are related to a deficiency in cerebral glutamine synthetase. Comp Biochem Physiol C 1989; 94: 399–404. 34 Walz W, Lang MK. Immunocytochemical evidence for a distinct GFAP-negative subpopulation of astrocytes in the adult rat hippocampus. Neurosci Lett 1998; 257: 127–30. 35 Condorelli DF, Dell’Albani P, Kaczmarek L, et al. Glial fibrillary acidic protein messenger RNA and glutamine synthetase activity after nervous system injury. J Neurosci Res 1990; 26: 251–57. 36 Blondet B, Hantaz-Ambroise D, Ait-Ikhlef A, Cambier D, Murawsky M, Rieger F. Astrocytosis in wobbler mouse spinal cord involves a population of astrocytes which is glutamine synthetasenegative. Neurosci Lett 1995; 183: 179–82. 37 Derouiche A, Heimrich B, Frotscher M. Loss of layer-specific astrocytic glutamine synthetase immunoreactivity in slice cultures of hippocampus. Eur J Neurosci 1993; 5: 122–27. 38 Jackson MJ, Zielke HR, Max SR. Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture. Neurochem Res 1995; 20: 201–07. 39 Stanimirovic DB, Ball R, Small DL, Muruganandam A. Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro. Int J Dev Neurosci 1999; 17: 173–84. 40 Vardimon L, Ben-Dror I, Avisar N, Oren A, Shiftan L. Glucocorticoid control of glial gene expression. J Neurobiol 1999; 40: 513–27. 41 Beer J, Mielke K, Zipp M, Zimmermann M, Herdegen T. Expression of c-jun, junB, c-fos, fra-1 and fra-2 mRNA in the rat brain following seizure activity and axotomy. Brain Res 1998; 794: 255–66. 42 Huang TL, O’Banion MK. Interleukin-1 beta and tumor necrosis factor-alpha suppress dexamethasone induction of glutamine synthetase in primary mouse astrocytes. J Neurochem 1998; 71: 1436–42. 43 Kruchkova Y, Ben-Dror I, Herschkovitz A, David M, Yayon A, Vardimon L. Basic fibroblast growth factor: a potential inhibitor of glutamine synthetase expression in injured neural tissue. J Neurochem 2001; 77: 1641–49. 44 Liu Z, Holmes GL. Basic fibroblast growth factor-induced seizures in rats. Neurosci Lett 1997; 233: 85–88.

Fatal hyperammonemia after orthotopic lung transplantation.

A case of fatal hyperammonemia complicating orthotopic lung transplantation was previously reported. To describe the incidence, clinical features, and treatment of hyperammonemia associated with orthotopic lung transplantation. Retrospective cohort analysis. Academic medical center and lung transplantation center in Philadelphia, Pennsylvania. 145 sequential adult patients who underwent orthotopic lung transplantation. Plasma ammonium levels. Six of the 145 patients who had had orthotopic lung transplantation developed hyperammonemia, all within the first 26 days after transplantation. The 30-day post-transplantation mortality rate was 67% for patients with hyperammonemia compared with 17% for those without hyperammonemia (P = 0.01). Development of major gastrointestinal complications (P = 0.03), use of total parenteral nutrition (P < 0.001), and lung transplantation for primary pulmonary hypertension (P = 0.045) were associated with hyperammonemia. Hyperammonemia is a potentially fatal event occurring after orthotopic lung transplantation. It is associated with high nitrogen load, concurrent medical stressors, primary pulmonary hypertension, and hepatic glutamine synthetase deficiency.

Seizures in the mongolian gerbil are related to a deficiency in cerebral glutamine synthetase

1. Seizure prone (SP)-gerbils ( Meriones unguiculatus) tested repeatedly in an open field exhibited habituation of seizures after one or two trials and subsequently showed more ambulatory activity than non-seizure prone (NSP) individuals. 2. A subset of 5 SP and 5 NSP animals were killed and portions of each cerebral hemisphere, the cerebellum and the brainstem medulla were analysed for glutamine synthetase (GS). 3. GFAP immunohistochemistry was used on forebrain sections to assay astrocyte density. 4. It was found by MANOVA, PCA and regression analyses that seizures and ambulatory activity were related to a deficiency in cerebral GS. 5. Rearing behaviour was related to medullary brainstem and cerebellar GS concentrations. 6. The decreased GS of the seizure-prone gerbils was not apparently associated with a deficiency of astrocytes, perhaps the reverse. 7. The results are discussed in relation to glial-neuronal interactions modulating arousal and the propensity for seizures.

Glutamine synthetase activity in Huntington's disease

Glutamine synthetase activity was measured in seven brain areas post-mortem from control patients, and those with Hungtington's disease. The activity of the enzyme was reduced in the frontal and temporal cortex, putamen and cerebellum, but not in the hippocampus, thalamus or olivary nucleaus. The results do not suggest a generalised deficiency of glutamine synthetase in Huntington's disease. However, as this enzyme is localised to astrocytic cells, the reduction in activity in areas of neuronal devastation, where the ratio of astrocytes to neurones is increased, may reflect a greater functional deficit. The enzyme plays a crucial role in cerebral ammonia assimilation and its inhibition in laboratory animals is known to produce neuronal toxicity. A reduction in its activity in Huntington's disease may well contribute to the neuronal pathology in certain areas.

Free amino acids of normal and abnormal human placenta

The free amino acid content of human placenta has not previously been reported, although such levels are known for many normal and malignant tissues. Free amino acids in the placenta increase during pregnancy although they are already quite high by 20 weeks. Compared to studies of uterus and endometrium, placental aspartic acid is high and glutamic acid is normal, while asparagineglutamine is uniformly low, suggesting a deficiency of asparagine and glutamine synthetase. In hydatidiform mole, aspartic and glutamic acid levels are low while asparagine and glutamine levels are comparable to those of normal placenta, suggesting that transamination is not a major metabolic activity of trophoblast. Placental arginine is high compared to levels in normal uterus. All other essential amino acids show little variation. Free amino acid levels are low in infarcted placenta and high in toxemic placenta. All tissue samples exhibited low levels of valine and proline, suggesting they are active, rather than degenerating.