Sandra J. Hewett

Mechanisms of the antioxidant effects of nitric oxide

The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.

The Cystine/Glutamate Antiporter System xc− in Health and Disease: From Molecular Mechanisms to Novel Therapeutic Opportunities

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

Chemotherapy for the Brain: The Antitumor Antibiotic Mithramycin Prolongs Survival in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) is a fully penetrant autosomal-dominant inherited neurological disorder caused by expanded CAG repeats in the Huntingtin gene. Transcriptional dysfunction, excitotoxicity, and oxidative stress have all been proposed to play important roles in the pathogenesis of HD. This study was designed to explore the therapeutic potential of mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic. Pharmacological treatment of a transgenic mouse model of HD (R6/2) with mithramycin extended survival by 29.1%, greater than any single agent reported to date. Increased survival was accompanied by improved motor performance and markedly delayed neuropathological sequelae. To identify the functional mechanism for the salubrious effects of mithramycin, we examined transcriptional dysfunction in R6/2 mice. Consistent with transcriptional repression playing a role in the pathogenesis of HD, we found increased methylation of lysine 9 in histone H3, a well established mechanism of gene silencing. Mithramycin treatment prevented the increase in H3 methylation observed in R6/2 mice, suggesting that the enhanced survival and neuroprotection might be attributable to the alleviation of repressed gene expression vital to neuronal function and survival. Because it is Food and Drug Administration-approved, mithramycin is a promising drug for the treatment of HD.

A microtiter trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture

An automated method for the determination of neuronal cell death using trypan blue is described. Following various excitotoxic insults, murine mixed cortical cell cultures are stained with trypan blue (0.05%; 15 min), followed by SDS (1%) lysis. The absorbance of the dye is measured spectrophotometrically at 590 nm using a microtiter plate reader. When compared to the biochemical lactate dehydrogenase assay, no statistical difference in the calculated levels of excitotoxic neuronal cell death was noted between the assays in any given paradigm. This method is fast and reliable. It eliminates the need for cell counting, thus allowing for high volume sample analysis with a minimum of sample error. Utility of this trypan blue absorbance spectrophotometric assay is likely to extend beyond the study of excitotoxic neuronal injury and should complement existing methods for measuring neuronal viability and cytotoxicity in cell culture.

Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes

Cultures of astrocytes can be readily established and are widely used to study the biological functions of these glial cells in isolation. Unfortunately, contamination by microglia can confound results from such studies. Herein, a simple and highly effective modification of a common procedure to remove microglia from astrocyte cultures is described. After becoming confluent, astrocytes were exposed to a mitotic inhibitor for 5-6 days then treated with 50-75 mM l-leucine methyl ester (LME) for 60-90 min. Unlike previous protocols that employed lower LME concentrations on subconfluent cultures or during passage of astrocytes, this protocol effectively depleted microglia from high-density astrocyte monolayers. This was evidenced by the selective depletion of microglial-specific markers. Purified monolayers appeared morphologically normal 24h after LME treatment and expressed nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) proteins upon stimulation with LPS plus IFNgamma, albeit to a lower level than unpurified monolayers. This difference could be attributed to removal of contaminating microglia from monolayers and not to astrocyte dysfunction, since LME treatment did not alter global protein synthesis and a reactive phenotype could be induced in the purified monolayers. Thus, this modified protocol selectively depletes microglia from high-density primary astrocyte monolayers without compromising their functional integrity.

Interleukin-1β: a bridge between inflammation and excitotoxicity?

Interleukin‐1 (IL‐1) is a proinflammatory cytokine released by many cell types that acts in both an autocrine and/or paracrine fashion. While IL‐1 is best described as an important mediator of the peripheral immune response during infection and inflammation, increasing evidence implicates IL‐1 signaling in the pathogenesis of several neurological disorders. The biochemical pathway(s) by which this cytokine contributes to brain injury remain(s) largely unidentified. Herein, we review the evidence that demonstrates the contribution of IL‐1β to the pathogenesis of both acute and chronic neurological disorders. Further, we highlight data that leads us to propose IL‐1β as the missing mechanistic link between a potential beneficial inflammatory response and detrimental glutamate excitotoxicity.

System xc− Activity and Astrocytes Are Necessary for Interleukin-1β-Mediated Hypoxic Neuronal Injury

The purpose of this study was to elucidate the cellular/biochemical pathway(s) by which interleukin-1β (IL-1β) contributes to the pathogenesis of hypoxic–ischemic brain damage. In vivo, IL-1 receptor type I (IL-1RI)-deficient mice showed smaller infarcts and less neurological deficits than wild-type animals after a 90 min reversible middle cerebral artery occlusion. In vitro, IL-1β mediated an enhancement of hypoxic neuronal injury in murine cortical cultures that was lacking in cultures derived from IL-1RI null mutant animals and was blocked by the IL-1 receptor antagonist or an IL-1RI blocking antibody. This IL-1β-mediated potentiation of hypoxic neuronal injury was associated with an increase in both cellular cystine uptake ([cystine]i) and extracellular glutamate levels ([glutamate]e) and was prevented by either ionotropic glutamate receptor antagonism or removal of l-cystine, suggesting a role for the cystine/glutamate antiporter (System xc−). Indeed, dual System xc−/metabotropic glutamate receptor subunit 1 (mGluR1) antagonism but not selective mGluR1 antagonism prevented neuronal injury. Additionally, cultures derived from mGluR1-deficient mice exhibited the same potentiation in injury after treatment with IL-1β as wild-type cultures, an effect prevented by System xc−/mGluR1 antagonism. Finally, assessment of System xc− function and kinetics in IL-1β-treated cultures revealed an increase in velocity of cystine transport (Vmax), in the absence of a change in affinity (Km). Neither the enhancement in [cystine]i, [glutamate]e, or neuronal injury were observed in chimeric cultures consisting of IL-1RI+/+ neurons plated on top of IL-1RI−/− astrocytes, highlighting the importance of astrocyte-mediated alterations in System xc− as a novel contributor to the development and progression of hypoxic neuronal injury.

SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death.

3‐Morpholinosyndnomine (SIN‐1) has been reported to be a peroxynitrite (OONO−) donor because it produces both nitric oxide (NO) and superoxide ( O2−·) upon decomposition in aqueous solution. However, SIN‐1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN‐1 caused a concentration‐dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3‐nitrotyrosine into the culture medium. The increase in 3‐nitrotyrosine immunoreactivity, a footprint of OONO− production, was specific for SIN‐1 as exposure to neurotoxic concentrations of an NO donor (Z)‐1‐[2‐aminoethyl)‐N‐(2‐ammonioethyl) aminodiazen‐1‐ium‐1,2‐diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN‐1‐induced injury and 3‐nitrotyrosine staining were prevented by the addition of either 5,10,15,20‐Tetrakis (4‐sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO− decomposition catalyst, or uric acid, an OONO− scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO− from SIN‐1 as well as its cytotoxicity. Removal of O2−· and the subsequently formed H2O2 by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein‐bound tyrosine but actually enhanced the cytotoxicity of SIN‐1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN‐1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non‐NMDA receptors contributed to the SIN‐1‐mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN‐1 initiates neural cell death in an OONO−‐dependent or ‐independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO‐related species generated.

Analysis of the Neuroprotective Effects of Various Nitric Oxide Donor Compounds in Murine Mixed Cortical Cell Culture

Abstract: Nitric oxide (NO) has been implicated in both the pathogenesis of and protection from NMDA receptor‐mediated neuronal injury. This apparent paradox has been attributed to alternate redox states of nitrogen monoxide, whereby, depending on the redox milieu, nitrogen monoxide can be neuroprotective via nitrosation chemistry or react with superoxide to form secondary toxic species. In our murine mixed cortical cell culture system, the NONOate‐type NO donors diethylamine/NO complex sodium (Dea/NO), (Z)‐[N‐(3‐ammoniopropyl)‐N‐(n‐propyl)amino]diazen‐1‐ium‐1,2‐diolate (Papa/NO), and spermine/NO complex sodium (Sper/NO), as well as the S‐nitrosothiols S‐nitroso‐L‐glutathione (GSNO) and S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP) (NO+ equivalents), decreased NMDA‐induced neuronal injury in a concentration‐dependent manner. 8‐Bromo‐cyclic GMP did not mimic the inhibitory effects of the donors, suggesting that the neuroprotection was not the result of NO‐stimulated neuronal cyclic GMP production. Furthermore, neuronal injury induced by exposure of cultures to H2O2 was not altered by the presence of Dea/NO, indicating the absence of a direct antioxidant effect. NONO‐ates did, however, reduce NMDA‐stimulated uptake of 45Ca2+, whereas high potassium‐induced 45Ca2+ accumulation, a measurement of entry via voltage‐gated calcium channels, was unaffected. The parallel reduction of 45Ca2+ accumulation and NMDA neurotoxicity by NONOates mimicked that seen with an NMDA receptor antagonist. Electrochemical measurements of NO via an NO‐sensitive electrode demonstrated that neuroprotective concentrations of all donors produced appreciable amounts of NO over the 5‐min time frame. Determination of the formation of NO+ equivalents, as assessed by N‐nitrosation of 2,3‐diaminonaphthylene, revealed little or no observable N‐nitrosation by Sper/NO, GSNO, and SNAP with significant N‐nitrosation observed by Papa/NO and Dea/NO. However, addition of ascorbate (400 μM) effectively prevented the nitrosation of 2,3‐diaminonaphthylene produced by Dea/NO and Papa/NO without altering their neuroprotective properties or their effects on 45Ca2+ accumulation. Present results indicate that the intrinsic NO/NO+ characteristics of NO donor compounds may not be a good predictor of their ability to inhibit NMDA receptor‐mediated neurotoxicity at the cellular level.

Regulation of system xc− activity and expression in astrocytes by interleukin-1β: implications for hypoxic neuronal injury

We recently demonstrated that interleukin-1β (IL-1β) increases system x(c)(-) (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes, and microglia all express system x(c)(-) subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL-1β stimulation increases mRNA for xCT--the light chain that confers substrate specificity--in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL-1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild-type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL-1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL-1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild-type neurons plated on top of sut astrocytes. Nor was it observed in wild-type cultures treated with a system x(c)(-) inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL-1β selectively regulates system x(c)(-) activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1β found under hypoxic conditions.We recently demonstrated that interleukin‐1β (IL‐1β) increases system xc− (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes, and microglia all express system xc− subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL‐1β stimulation increases mRNA for xCT—the light chain that confers substrate specificity—in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL‐1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL‐1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild‐type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL‐1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL‐1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild‐type neurons plated on top of sut astrocytes. Nor was it observed in wild‐type cultures treated with a system xc− inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL‐1β selectively regulates system xc− activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL‐1β found under hypoxic conditions.