Methionine Sulfoximine Research Articles

Ammonium chloride reduces excitatory synaptic transmission onto CA1 pyramidal neurons of mouse organotypic slice cultures.

Acute liver dysfunction commonly leads to rapid increases in ammonia concentrations in both the serum and the cerebrospinal fluid. These elevations primarily affect brain astrocytes, causing modifications in their structure and function. However, its impact on neurons is not yet fully understood. In this study, we investigated the impact of elevated ammonium chloride levels (NH4Cl, 5 mM) on synaptic transmission onto CA1 pyramidal neurons in mouse organotypic entorhino-hippocampal tissue cultures. We found that acute exposure to NH4Cl reversibly reduced excitatory synaptic transmission and affected CA3-CA1 synapses. Notably, NH4Cl modified astrocytic, but not CA1 pyramidal neuron, passive intrinsic properties. To further explore the role of astrocytes in NH4Cl-induced attenuation of synaptic transmission, we used methionine sulfoximine to target glutamine synthetase, a key astrocytic enzyme for ammonia clearance in the central nervous system. Inhibition of glutamine synthetase effectively prevented the downregulation of excitatory synaptic activity, underscoring the significant role of astrocytes in adjusting excitatory synapses during acute ammonia elevation.

Multivariate data analysis of process parameters affecting the growth and productivity of stable Chinese hamster ovary cell pools expressing SARS-CoV-2 spike protein as vaccine antigen in early process development.

The recent COVID-19 pandemic revealed an urgent need to develop robust cell culture platforms which can react rapidly to respond to this kind of global health issue. Chinese hamster ovary (CHO) stable pools can be a vital alternative to quickly provide gram amounts of recombinant proteins required for early-phase clinical assays. In this study, we analyze early process development data of recombinant trimeric spike protein Cumate-inducible manufacturing platform utilizing CHO stable pool as a preferred production host across three different stirred-tank bioreactor scales (0.75, 1, and 10 L). The impact of cell passage number as an indicator of cell age, methionine sulfoximine (MSX) concentration as a selection pressure, and cell seeding density was investigated using stable pools expressing three variants of concern. Multivariate data analysis with principal component analysis and batch-wise unfolding technique was applied to evaluate the effect of critical process parameters on production variability and a random forest (RF) model was developed to forecast protein production. In order to further improve process understanding, the RF model was analyzed with Shapley value dependency plots so as to determine what ranges of variables were most associated with increased protein production. Increasing longevity, controlling lactate build-up, and altering pH deadband are considered promising approaches to improve overall culture outcomes. The results also demonstrated that these pools are in general stable expressing similar level of spike proteins up to cell passage 11 (~31 cell generations). This enables to expand enough cells required to seed large volume of 200-2000 L bioreactor.

Time-resolved fluoroimmunoassay for Aspergillus detection based on anti-galactomannan monoclonal antibody from stable cell line

Invasive Aspergillosis is a high-risk illness with a high death rate in immunocompromised people due to a lack of early detection and timely treatment. Based on immunology study, we achieved an efficient production of anti-galactomannan antibody by Chinese hamster ovary (CHO) cells and applied it to time-resolved fluoroimmunoassay for Aspergillus galactomannan detection. We first introduced dual promoter expression vector into CHO host cells, and then applied a two-step screening strategy to screen the stable cell line by methionine sulfoximine pressurization. After amplification and fermentation, antibody yield reached 4500 mg/L. Then we conjugated the antibodies with fluorescent microspheres to establish a double antibody sandwich time-resolved fluoroimmunoassay, which was compared with the commercial Platelia™ Aspergillus Ag by clinical serum samples. The preformed assay could obtain the results in less than 25 min, with a limit of detection for galactomannan of approximately 1 ng/mL. Clinical results of the two methods showed that the overall percent agreement was 97.7% (95% CI: 96.6%–98.4%) and Cohen's kappa coefficient was 0.94. Overall, the assay is highly consistent with commercial detection, providing a more sensitive and effective method for the rapid diagnosis of invasive aspergillosis.

Jaroslava Folbergrová, Dr.Sc. (1932 – 2024)

Jaroslava Folbergrová, an outstanding scientist, passed away on Sunday, January 28 at the age of 91. She was born in 1932 in Nový Bydžov, Czechoslovakia. She studied biochemistry in Charles University, School of Mathematics and Physics in Prague. She started her scientific carrier with studies of the metabolism of ammonia in the brain. She came to the Institute of Physiology, Czechoslovak Academy of Sciences, Prague, in 1958 and she worked there for incredible 65 years till the very end of her life. All the time she was interested in brain metabolism mostly during and after epileptic seizures – at first in Department of Cytochemistry of Nerve Cell, headed by Zdeněk Lodin, where she defended Ph.D. thesis on the „Effect of methionine sulfoximine on brain metabolism“. She defended her „Doctor of Science (Dr.Sc.)“ thesis in 1990 and continued working in this laboratory till 2000 when she entered Department of Developmental Epileptology of the Institute of Physiology of the Czech Academy of Sciences. Her research was further focused on brain metabolism in relation to epileptic seizures and also to brain hypoxia. Two international collaborations were important for her scientific life: Eighteen months spent in 1965-1967 in Washington University in St. Louis, MO where she worked under the leadership of prof. Oliver Lowry, and longlasting collaboration with prof. Siesjo in Lund University, Sweden. Her stage in prof. Lowry’s Institute resulted in four highly cited papers (including one with 255 citations). Repeated visits in Lund led to nearly 50 primary publications in prestigious international journals (some of these papers also reached more than 200 citations). She progressively extended her field of interest to developing brain and the role of oxidative stress in epileptic seizures and their metabolic and functional consequences. Jarka Folbergrová was fully devoted to her scientific work. It was common to see her leaving the institute in the late afternoon with her notebook and a lot of papers to continue in her work at home. She will be missed in the Institute because she very well represented the tradition both at our country and abroad. She was also very friendly person and an inspiring colleague.

Modulation of host glutamine anabolism enhances the sensitivity of small cell lung cancer to chemotherapy

Small cell lung cancer (SCLC) is one of the deadliest human cancers, with a 5-year survival rate of ∼7%. Here, we performed a targeted proteomics analysis of human SCLC samples and thereby identified hypoxanthine phosphoribosyltransferase 1 (HPRT1) in the salvage purine synthesis pathway as a factor that contributes to SCLC malignancy by promoting cell survival in a glutamine-starved environment. Inhibition of HPRT1 by 6-mercaptopurine (6-MP) in combination with methotrexate (MTX), which blocks the de novo purine synthesis pathway, attenuated the growth of SCLC in mouse xenograft models. Moreover, modulation of host glutamine anabolism with the glutamine synthetase inhibitor methionine sulfoximine (MSO) in combination with 6-MP and MTX treatment resulted in marked tumor suppression and prolongation of host survival. Our results thus suggest that modulation of host glutamine anabolism combined with simultaneous inhibition of the de novo and salvage purine synthesis pathways may be of therapeutic benefit for SCLC.

Increased NMDARs in neurons and glutamine synthetase in astrocytes underlying autistic-like behaviors of Gabrb1−/− mice

Mutations of the GABA-A receptor subunit β1 (GABRB1) gene are found in autism patients. However, it remains unclear how mutations in Gabrb1 may lead to autism. We generated Gabrb1-/- mouse model, which showed autistic-like behaviors. We carried out RNA-seq on the hippocampus and found glutamatergic pathway may be involved. We further carried out single-cell RNA sequencing on the whole brain followed by qRT-PCR, immunofluorescence, electrophysiology, and metabolite detection on specific cell types. We identified the up-regulated Glul/Slc38a3 in astrocytes, Grin1/Grin2b in neurons, glutamate, and the ratio of Glu/GABA in the hippocampus. Consistent with these results, increased NMDAR-currents and reduced GABAAR-currents in the CA1 neurons were detected in Gabrb1-/- mice. NMDAR antagonist memantine or Glul inhibitor methionine sulfoximine could rescue the abnormal behaviors in Gabrb1-/- mice. Our data reveal that upregulation of the glutamatergic synapse pathway, including NMDARs at neuronal synapses and glutamine exported by astrocytes, may lead to autistic-like behaviors.

Distinct evolution of type I glutamine synthetase in Plasmodium and its species-specific requirement

Malaria parasite lacks canonical pathways for amino acid biosynthesis and depends primarily on hemoglobin degradation and extracellular resources for amino acids. Interestingly, a putative gene for glutamine synthetase (GS) is retained despite glutamine being an abundant amino acid in human and mosquito hosts. Here we show Plasmodium GS has evolved as a unique type I enzyme with distinct structural and regulatory properties to adapt to the asexual niche. Methionine sulfoximine (MSO) and phosphinothricin (PPT) inhibit parasite GS activity. GS is localized to the parasite cytosol and abundantly expressed in all the life cycle stages. Parasite GS displays species-specific requirement in Plasmodium falciparum (Pf) having asparagine-rich proteome. Targeting PfGS affects asparagine levels and inhibits protein synthesis through eIF2α phosphorylation leading to parasite death. Exposure of artemisinin-resistant Pf parasites to MSO and PPT inhibits the emergence of viable parasites upon artemisinin treatment.

1581-P: Systemic Alteration of Amino Acids Metabolism in Glutamate Dehydrogenase Gain-of-Function Mutation Knockin Mice

Patients with gain-of-function (GoF) mutations of glutamate dehydrogenase (GDH) have protein-sensitive hypoglycemia, hyperammonemia, epilepsy, and intellectual disability (HI/HA syndrome). Studies of mouse models with a beta-cell specific expression of H454Y hGDH suggest that the mechanisms of HI are caused by increased glutaminolysis induced by GDH GoF in islets. However, the mechanism of hyperammonemia is still unknown. In order to study the systemic effects of GDH GoF, H454Y GDH knockin mice (H454Y-GDH-KI) were created. Enzyme kinetic of GDH in the brain, kidney and liver confirmed that H454Y was successfully expressed with impaired GTP inhibition. H454Y-GDH-KI mice have a ~2.5-fold elevation of plasma ammonia and fasting hypoglycemia, similar to HI/HA patients. Tissues (brain, liver and kidney) and plasma amino acids profiles were determined by LC-MS. Compared to wildtype, the amino acids profile in the kidney was unaltered in KI mice. In contrast, KI liver had 60% lower glutamine, unchanged glutamate and aspartate, increased branch chain amino acids, etc. The glutamine of KI brain was 40% lower than the wildtype, glutamate and GABA was unchanged. Interestingly, plasma glutamine in KI mice was 30% higher compared to controls. A similar phenomenon has also been observed in HI/HA patients. We hypothesize that hyperammonemia is mainly caused by the increased glutaminolysis in KI liver. Higher plasma glutamine can be explained by the increased activity of tissue glutamine synthetase (GS). GS and glutamine serve as an ammonia sink, which is the main mechanism for the removal of increased ammonia in KI mice. We therefore applied the GS inhibitor, methionine sulfoximine (MSO) to KI mice. MSO significantly increased ammonia levels in KI mice after a single dose of injection. These studies in H454Y-GDH-KI mice suggest that GDH GoF in the liver is the main cause of hyperammonemia. GS plays an important role in ammonia removal. Disclosure C.Su: None. D.Han: Employee; Nanjing AscendRare Pharmaceutical Technology. S.Meng: Employee; Nanjing AscendRare Pharmaceutical Technology. Q.Zhu: Employee; Nanjing AscendRare Pharmaceutical Technology. X.Zhou: Employee; Nanjing AscendRare Pharmaceutical Technology. H.Jiang: None. J.Li: None. C.Gong: None. C.Li: Employee; Hua Medicine, Nanjing AscendRare Pharmaceutical Technology. Funding Beijing Natural Science Foundation (L212037)

Inhibition of nitrogen assimilation promotes carbon-based secondary metabolism in callus of Cyclocarya paliurus

The biosynthesis and accumulation of secondary metabolites are critical important to quality formation of medicinal plants, which are usually give way to primary processes and growth. Here, methionine sulfoximine (MSO) was used to inhibit the nitrogen assimilation in callus of Cyclocarya paliurus. The newly assimilated nitrogen characterized by 15N atom percentage excess, and the levels of amino acid and protein were reduced. The other primary processes such as carbohydrate metabolism and lipid metabolism were also repressed. In addition, the expression of the growth-related target of rapamycin (TOR) signaling was repressed, indicating nitrogen assimilation inhibition led to a systematic down-regulated primary metabolisms and resulted in a disruption of growth. In contrast, the biosynthesis of flavonoids and triterpenoids, antioxidase system, and the SnRK2-mediated abscisic acid (ABA) and jasmonic acid (JA) signaling were induced, which can improve plant stress resistance and defense. Nitrogen assimilation inhibition led to the carbon metabolic flux redirection from primary processes to secondary pathways, and facilitated the biosynthesis of flavonoids and triterpenoids in calluses of C. paliurus. Our results provide a comprehensive understanding of metabolic flux redirection between primary and secondary metabolic pathways and a potential means to improve the quality of medicinal plants.

Attenuation of initial pilocarpine-induced electrographic seizures by methionine sulfoximine pretreatment tightly correlates with the reduction of extracellular taurine in the hippocampus.

Initiation and development of early seizures by chemical stimuli is associated with brain cell swelling resulting in edema of seizure-vulnerable brain regions. We previously reported that pretreatment with a nonconvulsive dose of glutamine (Gln) synthetase inhibitor methionine sulfoximine (MSO) mitigates the intensity of initial pilocarpine (Pilo)-induced seizures in juvenile rats. We hypothesized that MSO exerts its protective effect by preventing the seizure-initiating and seizure-propagating increase of cell volume. Taurine (Tau) is an osmosensitive amino acid, whose release reflects increased cell volume. Therefore, we tested whether the poststimulus rise of amplitude of Pilo-induced electrographic seizures and their attenuation by MSO are correlated with the release of Tau from seizure-affected hippocampus. Lithium-pretreated animals were administered MSO (75 mg/kg ip) 2.5h before the induction of convulsions by Pilo (40 mg/kg ip). Electroencephalographic (EEG) power was analyzed during 60 min post-Pilo, at 5-min intervals. Extracellular accumulation of Tau (eTau) served as a marker of cell swelling. eTau, extracellular Gln (eGln), and extracellular glutamate (eGlu) were assayed in the microdialysates of the ventral hippocampal CA1 region collected at 15-min intervals during the whole 3.5-h observation period. The first EEG signal became apparent at ~10min post-Pilo. The EEG amplitude across most frequency bands peaked at ~40 min post-Pilo, and showed strong (r~ .72-.96) temporal correlation with eTau, but no correlation with eGln or eGlu. MSO pretreatment delayed the first EEG signal in Pilo-treated rats by ~10min, and depressed the EEG amplitude across most frequency bands, to values that remained strongly correlated with eTau (r> .92) and moderately correlated (r~ -.59) with eGln, but not with eGlu. Strong correlation between attenuation of Pilo-induced seizures and Tau release indicates that the beneficial effect of MSO is due to the prevention of cell volume increase concurrent with the onset of seizures.

Enhancing CHO cell productivity through a dual selection system using Aspgand Gsin glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexateor methionine sulfoximine,respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfectionof Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

Mechanism-Based Design of the First GlnA4-Specific Inhibitors.

γ-Glutamylamine synthetases are an important class of enzymes that play a key role in glutamate-based metabolism. Methionine sulfoximine (MSO) is a well-established inhibitor for the archetypal glutamine synthetase (GS) but inhibitors for most GS-like enzymes are unknown. Assuming a conserved catalytic mechanism for GS and GS-like enzymes, we explored if subtype-selective inhibitors can be obtained by merging MSO with the cognate substrates of the respective GS-like enzymes. Using GlnA4Sc from Streptomyces coelicolor, an enzyme recently shown to produce γ-glutamylethanolamine, we demonstrate that MSO can be reengineered in a straightforward fashion into potent and selective GlnA4Sc inhibitors. Linkage chemistry as well as linker length between the MSO moiety and the terminal hydroxyl group derived from ethanolamine were in agreement with the postulated phosphorylated catalytic intermediate. The best GlnA4 inhibitor 7 b potently blocked S. coelicolor growth in the presence of ethanolamine as the sole nitrogen source. Our results provide the first GlnA4Sc -specific inhibitors and suggest a general strategy to develop mechanism-based inhibitors for GS-like enzymes.

Glutamine synthetase plays an important role in ammonium tolerance of Myriophyllum aquaticum

High-strength ammonium (NH4+), the main characteristic of swine wastewater, poses a significant threat to the rural ecological environment. As a novel phytoremediation technology, Myriophyllum aquaticum wetlands have high tolerance and removal rate of NH4+. Glutamine synthetase (GS), a pivotal enzyme in nitrogen (N) metabolism, is hypothesized to play an important role in the tolerance of M. aquaticum to high NH4+. Herein, the responses of M. aquaticum to GS inhibition by 0.1 mM methionine sulfoximine (MSX) under 15 mM NH4+ were investigated. After 5 days, visible NH4+ toxicity symptoms were observed in MSX-treated plants. Compared with the control, the NH4+ accumulation in the leaves increased by 20.99 times, while that of stems and roots increased by 3.27 times and 47.76 %, suggesting that GS inhibition had a greater impact on the leaves. GS inhibition decreased pigments in the leaves by 8.64 %–41.06 %, triggered oxidative stress, and affected ions concentrations in M. aquaticum. The concentrations of glutamine (Gln) and asparagine decreased by 63.46 %–97.43 % and 12.37 %–76.41 %, respectively, while the concentrations of most other amino acids increased after 5 days of MSX treatment, showing that GS inhibition reprogrammed the amino acids synthesis. A decrease in Gln explains the regulations of N-related genes, including increased expression of AMT in roots and decreased expression of GS, GOGAT, GDH, and AS, which would cause further NH4+ accumulation via promoting NH4+ uptake and decreasing NH4+ assimilation in M. aquaticum. This study revealed for the first time that GS inhibition under high NH4+ condition can lead to phytotoxicity in M. aquaticum due to NH4+ accumulation. The physiological and molecular responses of the leaves, stems, and roots confirmed the importance of GS in the high NH4+ tolerance of M. aquaticum. These findings provide new insights into NH4+ tolerance mechanisms in M. aquaticum and a theoretical foundation for the phytoremediation of high NH4+-loaded swine wastewater.

Ammonium-dependent regulation of ammonium transporter ZmAMT1s expression conferred by glutamine levels in roots of maize

In maize, two root epidermis-expressed ammonium transporters ZmAMT1;1a and ZmAMT1;3 play major roles in high-affinity ammonium uptake. However, the transcriptional regulation of ZmAMT1s in roots for ensuring optimal ammonium acquisition remains largely unknown. Here, using a split root system we showed that ZmAMT1;1a and ZmAMT1;3 transcript levels were induced by localized ammonium supply to nitrogen-deficient roots. This enhanced expression of ZmAMT1s correlated with increases in 15NH4+ influx rates and tissue glutamine concentrations in roots. When ammonium was supplied together with methionine sulfoximine, an inhibitor of glutamine synthase, ammonium-induced expression of ZmAMT1s disappeared, suggesting that glutamine rather than ammonium itself regulated ZmAMT1s expression. When glutamine was supplied to nitrogen-deficient roots, expression levels of ZmAMT1s were enhanced, and negative feedback regulation could subsequently occur by supply of glutamine at a high level. Thus, our results indicated an ammonium-dependent regulation of ZmAMT1s at transcript levels, and a dual role of glutamine was suggested in the regulation of ammonium uptake in maize roots.

Endogenous glutamine is rate-limiting for anti-CD3 and anti-CD28 induced CD4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1–4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

Direct formation and site-selective elaboration of methionine sulfoximine in polypeptides.

Sulfoximines are emerging moieties for medicinal and biological chemistry, due in part to their efficacy in selective inhibition of amide-forming enzymes such as γ-glutamylcysteine synthetase. While small-molecule sulfoximines such as methionine sulfoximine (MSO) and its derivatives are well studied, structures with methionine sulfoximine residues within complex polypeptides have been generally inaccessible. This paper describes a straightforward means of late-stage one-step oxidation of methionine residues within polypeptides to afford NH-sulfoximines. We also present chemoselective subsequent elaboration, most notably by copper(ii)-mediated N-H cross-coupling at methionine sulfoximine residues with arylboronic acid reagents. This development serves as a strategy to incorporate diverse sulfoximine structures within natural polypeptides, and also identifies the methionine sulfoximine residue as a new site for bioorthogonal, chemoselective bioconjugation.

Saul Brusilow: Understanding and treating diseases of ammonia toxicity

This article reviews the scientific career and accomplishments of the late Dr. Saul Brusilow, Professor of Pediatrics at Johns Hopkins. Dr. Brusilow's career was focused on diseases involving hyperammonemia. He and his colleagues developed a set of drugs that could lower ammonia levels in patients with genetic disorders of the urea cycle by providing alternative pathways for the synthesis of excretable nitrogenous molecules. Those drugs and their derivatives represent one of the earliest and most successful drug therapies for genetic diseases. Turning their attention to brain swelling caused by liver disease, Dr. Brusilow and colleagues developed the Osmotic Gliopathy Hypothesis to help explain the mechanism of ammonia toxicity, postulating that high ammonia drives glutamine synthetase in astrocytes to produce elevated levels of glutamine that act as a potent osmolyte, drawing water into the cell and causing cerebral edema. This hypothesis suggests that inhibiting glutamine synthetase with its well-characterized inhibitor, methionine sulfoximine, might prove therapeutic in cases of hepatic encephalopathy, a conclusion supported by their subsequent studies in animals. But although the drugs developed to treat hyperammonemia resulting from urea cycle disorders were successfully developed and approved by the FDA, the compound suggested as a treatment for hepatic encephalopathy was unable to attract sufficient interest and investment to be tested for use in humans.

Inhibition of Glutamate Release, but Not of Glutamine Recycling to Glutamate, Is Involved in Delaying the Onset of Initial Lithium-Pilocarpine-Induced Seizures in Young Rats by a Non-Convulsive MSO Dose.

Initial seizures observed in young rats during the 60 min after administration of pilocarpine (Pilo) were delayed and attenuated by pretreatment with a non-convulsive dose of methionine sulfoximine (MSO). We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was administered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-13C]acetate and [U-13C]glucose were i.p.-injected either together with Pilo (short period) or 15 min before sacrifice (long period). Their conversion to Glu and Gln in the hippocampus and entorhinal cortex was followed using [13C] gas chromatography-mass spectrometry. Release of in vitro loaded Glu surrogate, [3H]d-Asp from ex vivo brain slices was monitored in continuously collected superfusates. [3H]d-Asp uptake was tested in freshly isolated brain slices. At no time point nor brain region did MSO modify incorporation of [13C] to Glu or Gln in Pilo-treated rats. MSO pretreatment decreased by ~37% high potassium-induced [3H]d-Asp release, but did not affect [3H]d-Asp uptake. The results indicate that MSO at a non-convulsive dose delays the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.

Network-Related Changes in Neurotransmitters and Seizure Propagation During Rodent Epileptogenesis.

To test the hypothesis that glutamate and GABA are linked to the formation of epilepsy networks and the triggering of spontaneous seizures, we examined seizure initiation/propagation characteristics and neurotransmitter levels during epileptogenesis in a translationally relevant rodent model of mesial temporal lobe epilepsy. The glutamine synthetase (GS) inhibitor methionine sulfoximine was infused into one of the hippocampi in laboratory rats to create a seizure focus. Long-term video-intracranial EEG recordings and brain microdialysis combined with mass spectrometry were used to examine seizure initiation, seizure propagation, and extracellular brain levels of glutamate and GABA. All seizures (n = 78 seizures, n = 3 rats) appeared first in the GS-inhibited hippocampus of all animals, followed by propagation to the contralateral hippocampus. Propagation time decreased significantly from 11.65 seconds early in epileptogenesis (weeks 1-2) to 6.82 seconds late in epileptogenesis (weeks 3-4, paired t test, p = 0.025). Baseline extracellular glutamate levels were 11.6-fold higher in the hippocampus of seizure propagation (7.3 µM) vs the hippocampus of seizure onset (0.63 µM, analysis of variance/Fisher least significant difference, p = 0.01), even though the concentrations of the major glutamate transporter proteins excitatory amino acid transporter subtypes 1 and 2 and xCT were unchanged between the brain regions. Finally, extracellular GABA in the seizure focus decreased significantly from baseline several hours before a spontaneous seizure (paired t test/false discovery rate). The changes in glutamate and GABA suggest novel and potentially important roles of the amino acids in epilepsy network formation and in the initiation and propagation of spontaneous seizures.