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.

Amino acids as biomarkers in the SOD1(G93A) mouse model of ALS.

The development of therapies for Amyotrophic Lateral Sclerosis (ALS) has been hindered by the lack of biomarkers for both identifying early disease and for monitoring the effectiveness of drugs. The identification of ALS biomarkers in presymptomatic individuals might also provide clues to the earliest biochemical correlates of the disease. Previous attempts to use plasma metabolites as biomarkers have led to contradictory results, presumably because of heterogeneity in both the underlying genetics and the disease stage in the clinical population. To eliminate these two sources of heterogeneity we have characterized plasma amino acids and other metabolites in the SOD1(G93A) transgenic mouse model for ALS. Presymptomatic SOD1(G93A) mice have significant differences in concentrations of several plasma metabolites compared to wild type animals, most notably in the concentrations of aspartate, cystine/cysteine, and phosphoethanolamine, and in changes indicative of methylation defects. There are significant changes in amino acid compositions between 50 and 70days of age in both the SOD1(G93A) and wild type mice, and several of the age-related and disease-related differences in metabolite concentration were also gender-specific. Many of the SOD1(G93A)-related differences could be altered by treatment of mice with methionine sulfoximine, which extends the lifespan of this mouse, inhibits glutamine synthetase, and modifies brain methylation reactions. These studies show that assaying plasma metabolites can effectively distinguish transgenic mice from wild type, suggesting that one or more plasma metabolites might be useful biomarkers for the disease in humans, especially if genetic and longitudinal analysis is used to reduce population heterogeneity.

A glutamine synthetase inhibitor increases survival and decreases cytokine response in a mouse model of acute liver failure.

BACKGROUND: Acute liver failure (ALF) can be induced in mice by administering Escherichia coli lipopolysaccharide (LPS) and D-galactosamine (D-GalN), which induce an inflammatory response involving tumour necrosis factor (TNF)-α production and a hepatocyte-specific transcriptional block. Under these conditions, binding of TNF-α to its cognate receptor on hepatocytes eventually leads to their apoptosis. AIMS: As part of an effort to identify drugs to treat this disease model, we have investigated whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) could play a protective role, given its effectiveness in the inhibition of brain swelling associated with hyperammonaemia. METHODS: Mouse survival, glutamine synthetase activity, hepatocyte apoptosis and induction of inflammatory cytokines were measured in mice treated with MSO before an intraperitoneal injection of LPS/D-GalN. The effect of MSO on viability and on TNF-α release was also assessed on inflammatory and liver cells. RESULTS: We have found that, in mice treated with LPS/D-GalN, MSO (i) drastically increases animal survival; (ii) sharply reduces glutamine synthetase activity, without inhibiting its other target, γ-glutamyl cysteine synthetase; (iii) inhibits death receptor-mediated apoptosis in hepatocytes upstream to cytokine binding; (iv) strongly reduces the overall inflammatory cytokine response, including a significant decrease in TNF-α induction in vivo and ex vivo, and in the interferon-γ level and signalling. CONCLUSIONS: These results demonstrate that the MSO target glutamine synthetase is required for the early steps of the cytokine response to endotoxins, and that its pharmacological inhibition may be exploited to treat inflammation.

In vitro suppression of inflammatory cytokine response by methionine sulfoximine.

BACKGROUND: The glutamine synthetase inhibitor methionine sulfoximine (MSO), shown previously to prevent death caused by an inflammatory liver response in mice, was tested on in vitro production of cytokines by mouse peritoneal macrophages triggered with lipopolysaccharide (LPS). RESULTS: MSO significantly reduced the production of Interleukin 6 (IL-6) and Tumor Necrosis Factor Alpha (TNFα) at 4 and 6 h after LPS-treatment. This reduction did not result from decreased transcription of IL-6 and TNFα genes, and therefore appeared to result from post-transcriptional inhibition of synthesis of these cytokines. MSO treatment did not inhibit total protein synthesis and did not reduce the production of a third LPS-triggered cytokine CXCL1, so the effect was not a toxic or global downregulation of the LPS response. The anti-inflammatory effects of a glutamine synthetase inhibitor were seen even though the medium contained abundant (2 mM) glutamine, suggesting that the target for this activity was not glutamine synthetase. In agreement with this hypothesis, the L,R isomer of MSO, which does not inhibit glutamine synthetase and was previously thought to be inert, both significantly reduced IL-6 secretion in isolated macrophages and increased survival in a mouse model for inflammatory liver failure. CONCLUSIONS: Our findings provide evidence for a novel target of MSO. Future attempts to identify the additional target would therefore also provide a target for therapies to treat diseases involving damaging cytokine responses.

Identification of the isomer of methionine sulfoximine that extends the lifespan of the SOD1 G93A mouse.

In previous studies methionine sulfoximine (MSO) significantly extended the lifespan of the SOD1 G93A mouse model for ALS. Those studies used commercially available MSO, which is a racemic mixture of the LS and LR diastereomers, leaving unanswered the question of which isomer was responsible for the therapeutic effects. In this study we tested both purified isomers and showed that the LS isomer, a well-characterized inhibitor of glutamine synthetase, extends the lifespan of these mice, but the LR isomer, which has no known activity, does not.

Therapeutic effects of methionine sulfoximine in multiple diseases include and extend beyond inhibition of glutamine synthetase.

INTRODUCTION: Methionine sulfoximine (MSO), a well-characterized inhibitor of glutamine synthetase, displays significant therapeutic benefits in animal models for several human diseases. This amino acid might therefore be a viable candidate for drug development to treat diseases for which there are few effective therapies. Areas covered: We describe the effects of MSO on brain swelling occurring in overt hepatic encephalopathy resulting from liver failure, the effects of MSO on excitotoxic damage involved in amyotrophic lateral sclerosis (ALS) or resulting from stroke, and the effects of MSO on a model for an inflammatory immune response involved in a range of diseases. We conclude that these results imply the existence of another therapeutic target for MSO in addition to glutamine synthetase. Expert opinion: We summarize the various diseases for which MSO treatment might be a candidate for drug development. We discuss why MSO has limited enthusiasm in the scientific and medical communities for use in humans, with a rebuttal to those negative opinions. And we conclude that MSO should be considered a candidate drug to treat brain swelling involved in overt hepatic encephalopathy and diseases involving an inflammatory immune response.

Is Huntington's a glutamine storage disease?

Huntington's disease is a neurological disorder caused by the expansion of a polyglutamine tract in the protein huntingtin. Several other neurological diseases also result from the expansion of polyglutamine regions in different proteins. Despite intense efforts, no definitive biochemical or physiological role for huntingtin has been described, nor has a function been assigned to the polyglutamine region in unaffected individuals. This article presents the hypothesis that polyglutamine expansions within huntingtin and other polyglutamine proteins provide a function in and of themselves. Incorporating multiple glutamine residues into a protein during synthesis, and releasing them during protein turnover, may represent a means of minimizing interruptions in brain levels of glutamine and glutamate during periods of malnutrition. The number and variety of different proteins containing polyglutamine expansions can be interpreted as a series of evolutionary "experiments" toward a nontoxic form for glutamine storage.

Effect of sex on lifespan, disease progression, and the response to methionine sulfoximine in the SOD1 G93A mouse model for ALS.

OBJECTIVE: To investigate the role of sex and the role of ammonia and amino acid metabolism, specifically the activity of glutamine synthetase, in survival and disease progression in amyotrophic lateral sclerosis. METHODS: We tested treatment with methionine sulfoximine (MSO) on the lifespan and neuromuscular ability of male and female SOD1 mice as measured by their ability to maintain their grip on an inverted wire grid. We also tested the effects of castration and ovariectomization on those measurements. RESULTS: MSO treatment improves the survival of both male and female mice, but the effects are significantly greater on female mice. Saline-treated (control) female mice have delayed neuromuscular degeneration compared with saline-treated male mice, and MSO further delays disease progression in females, to a greater extent than in males. Ovariectomization or castration completely eliminates the effect of the drug on either survival or neuromuscular deterioration. CONCLUSIONS: Sex is an important factor in disease progression and the response of SOD1 mice to a drug targeting a central enzyme in nitrogen metabolism, with female sex hormones playing a greater role than male sex hormones. Glutamine synthetase, or its reactants and products, therefore plays a role in this disease, and the sex specificity of treatments aimed at this or other metabolic targets may therefore be an important factor in the development of therapies to treat amyotrophic lateral sclerosis.

Methionine sulfoximine, an inhibitor of glutamine synthetase, lowers brain glutamine and glutamate in a mouse model of ALS.

In an effort to alter the levels of neurochemicals involved in excitotoxicity, we treated mice with methionine sulfoximine (MSO), an inhibitor of glutamine synthetase. Since glutamate toxicity has been proposed as a mechanism for the degeneration of motor neurons in a variety of neurodegenerative diseases, we tested the effects of MSO on the transgenic mouse that overexpresses the mutant human SOD1(G93A) gene, an animal model for the primary inherited form of the human neurodegenerative disease amyotrophic lateral sclerosis (ALS). This treatment in vivo reduced glutamine synthetase activity measured in vitro by 85%. Proton magnetic resonance spectroscopy, with magic angle spinning of intact samples of brain tissue, showed that MSO treatment reduced brain levels of glutamine by 60% and of glutamate by 30% in both the motor cortex and the anterior striatum, while also affecting levels of GABA and glutathione. Kaplan-Meyer survival analysis revealed that MSO treatment significantly extended the lifespan of these mice by 8% (p<0.01). These results show that in the SOD1(G93A) model of neurodegenerative diseases, the concentration of brain glutamate (determined with (1)H-MRS) can be lowered by inhibiting in vivo the synthesis of glutamine with non-toxic doses of MSO.

Immunosuppressant-like effects of phenylbutyrate on growth inhibition of Saccharomyces cerevisiae.

Phenylbutyrate (4-phenylbutyric acid; PB) and its metabolite, phenylacetate, are effective anti-neoplastic agents in tissue culture and have shown promise in clinical trials for a variety of neoplasms. PB is a drug of remarkably low toxicity that acts in vitro as a differentiating agent, causing reversion of the transformed phenotype by an unknown mechanism. We attempted to identify the cellular target(s) for PB using Saccharomyces as a model. PB inhibits growth of yeast on rich medium at concentrations of 0.1-1.0 mM, concentrations similar to plasma concentrations observed in human trials. Yeast cells treated with 1 mM PB remain over 90% viable for 24 h. PB inhibits tryptophan uptake, and resistance to PB can be conferred by tryptophan prototrophy, by supplementing tryptophan auxotrophs with the high levels of tryptophan, by overexpression of the aromatic amino acid permeases Tat1p or Tat2p, and by disruption of TAT1. Since tryptophan auxotrophy and transport influences resistance to PB, phytosphingosine, and the immunosuppressant FK506, these drugs might affect the same pathway. We isolated and characterized a mutant resistant to 1 mM PB and identified the mutant as bul1. A chromosomal BUL1 deletion displayed all phenotypes shown by the PB-resistant mutant.

Activity of the yeast Tat2p tryptophan permease is sensitive to the anti-tumor agent 4-phenylbutyrate.

4-Phenylbutyrate (PB) induces differentiation and is being intensively studied as a treatment for brain, prostate, breast, and hematopoietic cancer. While many different primary targets for PB have been proposed, the mechanism by which it causes cellular differentiation remains unknown. To identify the primary cellular target, we investigated its effects on Saccharomyces cerevisiae and showed that it inhibits tryptophan transport. We show here that PB and sorbic acid induce an ubiquitin-dependent turnover of the tryptophan permease Tat2p. However, the inhibition of transport is not a consequence of the loss of Tat2p, since it also occurs when turnover is prevented by deleting the Tat2p ubiquitination sites. When we tested the effects of PB and other growth inhibitory agents on the growth of amino acid auxotrophs, we found that several auxotrophs are hypersensitive to a number of chemically unrelated agents, including PB and some, but not all, weak acids; and this sensitivity is due to the inhibition of amino acid transport. For the inhibitory weak acids, inhibition is not confined to aromatic amino acid auxotrophs, nor is it a general weak acid stress response, since the degree of inhibition is independent of weak acid hydrophobicity and p Ka. Our results show that diverse agents affect the activity of the Tat2p permease rather than its stability and suggest the hypothesis that the anti-neoplastic action of PB is due to a decrease in the activity of surface receptors or other membrane proteins needed to maintain the transformed state.

The Escherichia coli F1F0 ATP synthase displays biphasic synthesis kinetics.

The F1F0 proton-translocating ATPase/synthase is the primary generator of ATP in most organisms growing aerobically. Kinetic assays of ATP synthesis have been conducted using enzymes from mitochondria and chloroplasts. However, limited data on ATP synthesis by the model Escherichia coli enzyme are available, mostly because of the lack of an efficient and reproducible assay. We have developed an optimized assay and have collected synthase kinetic data over a substrate concentration range of 2 orders of magnitude for both ADP and Pi from the synthase enzyme of E. coli. Negative and positive cooperativity of substrate binding and positive catalytic cooperativity were all observed. ATP synthesis displayed biphasic kinetics for ADP indicating that 1) the enzyme is capable of catalyzing efficient ATP synthesis when only two of three catalytic sites are occupied by ADP; and 2) occupation of the third site further activates the rate of catalysis.

Determination of proton flux and conductance at pH 6.8 through single FO sectors from Escherichia coli.

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single FO of the F1FO ATP synthase. Lipid vesicles reconstituted with just a few functional FO from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single FO sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type FO has a proton flux of 3100 +/- 500 H+/s/FO at a transmembrane potential of 106 mV (25 degrees C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.