P2X7-dependent constitutive Interleukin-1ß release from pyramidal neurons of the normal mouse hippocampus: Evidence for a role in maintenance of the innate seizure threshold.

Disruption of Interleukin-1ß (IL-1ß) signaling sensitized mice to convulsant stimuli, suggesting that this quintessential cytokine of the innate immune system contributes to maintenance of the innate seizure threshold (ST). However, much remains unknown about where and how IL-1ß secretion occurs in the normal brain. This study examined the possibility that neurons of the hippocampus are key sources of constitutive IL-1ß secretion and that the release from these cells is dependent on the purinoceptor, P2X7. It was posited that treatment with the P2X7 antagonist, JNJ-47965567 (JNJ), would cause IL-1ß to accumulate in cells that produce it, and consequently, lower the ST. No IL-1ß immunoreactivity was detected in any region of the hippocampal formation of mice treated with the JNJ vehicle, Sulfobutylether-ß-cyclodextrin. In contrast, prominent immunoreactivity was discovered in the pyramidal neurons of the CA3 region 60 min after treatment with the P2X7 antagonist. Lower levels were found in CA1 neurons, and no immunoreactivity was detected in granule cells of the dentate gyrus. JNJ also increased IL-1ß immunoreactivity in the cell bodies of hippocampal neurons in culture. Interestingly, JNJ potentiated bicuculline-induced Fos and COX-2 mRNA expression in the cultures and this was blocked by an NMDA receptor antagonist. Moreover, pentylenetetrazole-induced seizure severity and incidence of convulsions were increased in mice treated with JNJ and this resembled that observed with IL-1 signaling-deficient mice. Overall, the results from this study support the notion that constitutive P2X7-dependent IL-1ß release from hippocampal pyramidal neurons contributes to maintenance of the ST in the normal brain, perhaps by modulating neuronal excitability. These findings may have implications for epilepsy, a brain disorder in which the ST is compromised.

Hyperexcitability and brain morphological differences in mice lacking the cystine/glutamate antiporter, system xc.

System xc- (Sxc- ) is a heteromeric antiporter (L-cystine/L-glutamate exchanger) expressed predominately on astrocytes in the central nervous system. Its activity contributes importantly to the maintenance of the ambient extracellular glutamate levels, as well as, to cellular redox homeostasis. Since alterations in glutamate levels and redox modifications could cause structural changes, we analyzed gross regional morphology of thionin-stained brain sections and cellular and subcellular morphology of Golgi-Cox stained layer V pyramidal neurons in the primary motor cortex (PM1) of mice naturally null for SLC7A11 (SLC7A11sut/sut )-the gene that encodes the substrate specific light chain (xCT) for Sxc- . Intriguingly, in comparison to age- and sex-matched wild-type (SLC7A11+/+ ) littermate controls, we found morphologic changes-including increased dendritic complexity and mushroom spine area in males and reduced corpus callosum and soma size in females-that have previously been described, in each case, as morphological correlates of excitability. Consistent with this, we found that both male and female SLC7A11sut/sut mice had lower convulsive seizure thresholds and greater seizure severity than their sex-matched wild-type (SLC7A11+/+ ) littermates after acute challenge with two pharmacologically distinct chemoconvulsants: the Glu receptor agonist, kainic acid (KA), or the GABAA receptor antagonist, pentylenetetrazole (PTZ). These results suggest that the loss of Sxc- signaling in males and females perturbs excitatory/inhibitory (E/I) balance in vivo, potentially through its regulation of cellular and subcellular morphology.

Interleukin 1ß Regulation of the System xc- Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR.

System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1ß, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1ß increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1ß induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1ß-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1ß-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1ß-induced stabilization of astrocyte xCT mRNA.

Spontaneous Glutamatergic Synaptic Activity Regulates Constitutive COX-2 Expression in Neurons: OPPOSING ROLES FOR THE TRANSCRIPTION FACTORS CREB (cAMP RESPONSE ELEMENT BINDING) PROTEIN AND Sp1 (STIMULATORY PROTEIN-1).

Burgeoning evidence supports a role for cyclooxygenase metabolites in regulating membrane excitability in various forms of synaptic plasticity. Two cyclooxygenases, COX-1 and COX-2, catalyze the initial step in the metabolism of arachidonic acid to prostaglandins. COX-2 is generally considered inducible, but in glutamatergic neurons in some brain regions, including the cerebral cortex, it is constitutively expressed. However, the transcriptional mechanisms by which this occurs have not been elucidated. Here, we used quantitative PCR and also analyzed reporter gene expression in a mouse line carrying a construct consisting of a portion of the proximal promoter region of the mouse COX-2 gene upstream of luciferase cDNA to characterize COX-2 basal transcriptional regulation in cortical neurons. Extracts from the whole brain and from the cerebral cortex, hippocampus, and olfactory bulbs exhibited high luciferase activity. Moreover, constitutive COX-2 expression and luciferase activity were detected in cortical neurons, but not in cortical astrocytes, cultured from wild-type and transgenic mice, respectively. Constitutive COX-2 expression depended on spontaneous but not evoked excitatory synaptic activity and was shown to be N-methyl-d-aspartate receptor-dependent. Constitutive promoter activity was reduced in neurons transfected with a dominant-negative cAMP response element binding protein (CREB) and was eliminated by mutating the CRE-binding site on the COX-2 promoter. However, mutation of the stimulatory protein-1 (Sp1)-binding site resulted in an N-methyl-d-aspartate receptor-dependent enhancement of COX-2 promoter activity. Basal binding of the transcription factors CREB and Sp1 to the native neuronal COX-2 promoter was confirmed. In toto, our data suggest that spontaneous glutamatergic synaptic activity regulates constitutive neuronal COX-2 expression via Sp1 and CREB protein-dependent transcriptional mechanisms.

Inhibition of System Xc(-) Transporter Attenuates Autoimmune Inflammatory Demyelination.

T cell infiltration into the CNS is a significant underlying pathogenesis in autoimmune inflammatory demyelinating diseases. Several lines of evidence suggest that glutamate dysregulation in the CNS is an important consequence of immune cell infiltration in neuroinflammatory demyelinating diseases; yet, the causal link between inflammation and glutamate dysregulation is not well understood. A major source of glutamate release during oxidative stress is the system Xc(-) transporter; however, this mechanism has not been tested in animal models of autoimmune inflammatory demyelination. We find that pharmacological and genetic inhibition of system Xc(-) attenuates chronic and relapsing-remitting experimental autoimmune encephalomyelitis (EAE). Remarkably, pharmacological blockade of system Xc(-) 7 d after induction of EAE attenuated T cell infiltration into the CNS, but not T cell activation in the periphery. Mice harboring a Slc7a11 (xCT) mutation that inactivated system Xc(-) were resistant to EAE, corroborating a central role for system Xc(-) in mediating immune cell infiltration. We next examined the role of the system Xc(-) transporter in the CNS after immune cell infiltration. Pharmacological inhibitors of the system Xc(-) transporter administered during the first relapse in a SJL animal model of relapsing-remitting EAE abrogated clinical disease, inflammation, and myelin loss. Primary coculture studies demonstrate that myelin-specific CD4(+) Th1 cells provoke microglia to release glutamate via the system Xc(-) transporter, causing excitotoxic death to mature myelin-producing oligodendrocytes. Taken together, these studies support a novel role for the system Xc(-) transporter in mediating T cell infiltration into the CNS as well as promoting myelin destruction after immune cell infiltration in EAE.

Interleukin-1ß protects astrocytes against oxidant-induced injury via an NF-κB-dependent upregulation of glutathione synthesis.

Astrocytes produce and export the antioxidant glutathione (GSH). Previously, we found that interleukin-1ß (IL-1ß) enhanced the expression of astrocyte system xc (-) , the transporter that delivers the rate-limiting substrate for GSH synthesis-cyst(e)ine. Herein, we demonstrate directly that IL-1ß mediates a time-dependent increase in extracellular GSH levels in cortical astrocyte cultures, suggesting both enhanced synthesis and export. This increased GSH production was blocked by inhibition of nuclear factor-κB (NF-κB) activity but not by inhibition of p38 MAPK. To determine whether this increase could provide protection against oxidative stress, the oxidants tert-butyl hydroperoxide (tBOOH) and ferrous sulfate (FeSO4 ) were employed. IL-1ß treatment prevented the increase in reactive oxygen species produced in astrocytes following tBOOH exposure. Additionally, the toxicity induced by tBOOH or FeSO4 exposure was significantly attenuated following treatment with IL-1ß, an effect reversed by concomitant exposure to l-buthionine-S,R-sulfoximine (BSO), which prevented the IL-1ß-mediated rise in GSH production. IL-1ß failed to increase GSH or to provide protection against t-BOOH toxicity in astrocyte cultures derived from IL-1R1 null mutant mice. Overall, our data indicate that under certain conditions IL-1ß may be an important stimulus for increasing astrocyte GSH production, and potentially, total antioxidant capacity in brain, via an NF-κB-dependent process.

Sex- and age-dependent contribution of System xc- to cognitive, sensory, and social behaviors revealed by comprehensive behavioral analyses of System xc- null mice.

Background: System xc- (Sxc-) is an important heteromeric amino acid cystine/glutamate exchanger that plays a pivotal role in the CNS by importing cystine into cells while exporting glutamate. Although certain behaviors have been identified as altered in Sxc- null mutant mice, our understanding of the comprehensive impact of Sxc- on behavior remains incomplete. Methods: To address this gap, we compared motor, sensory and social behaviors of male and female mice in mice null for Sxc- (SLC7A11sut/sut) with wildtype littermates (SLC7A11+/+) in a comprehensive and systematic manner to determine effects of genotype, sex, age, and their potential interactions. Results: Motor performance was not affected by loss of Sxc- in both males and females, although it was impacted negatively by age. Motor learning was specifically disrupted in female mice lacking Sxc- at both 2 and 6 months of age. Further, female SLC7A11sut/sut mice at both ages exhibited impaired sociability, but normal spatial and recognition memory, as well as sensorimotor gating. Finally, pronounced open-space anxiety was displayed by female SLC7A11sut/sut when they were young. In contrast, young SLC7A11sut/sut male mice demonstrated normal sociability, delayed spatial learning, increased open-space anxiety and heightened sensitivity to noise. As they aged, anxiety and noise sensitivity abated but hyperactivity emerged. Discussion: We find that the behavioral phenotypes of female SLC7A11sut/sut are similar to those observed in mouse models of autism spectrum disorder, while behaviors of male SLC7A11sut/sut resemble those seen in mouse models of attention deficit hyperactivity disorder. These results underscore the need for further investigation of SLC7A11 in neurodevelopment. By expanding our understanding of the potential involvement of Sxc-, we may gain additional insights into the mechanisms underlying complex neurodevelopmental conditions.

Neuromodulatory role of endogenous interleukin-1ß in acute seizures: possible contribution of cyclooxygenase-2.

The function of endogenous interleukin-1ß (IL-1ß) signaling in acute seizure activity was examined using transgenic mice harboring targeted deletions in the genes for either IL-1ß (Il1b) or its signaling receptor (Il1r1). Acute epileptic seizure activity was modeled using two mechanistically distinct chemoconvulsants, kainic acid (KA) and pentylenetetrazole (PTZ). KA-induced seizure activity was more severe in homozygous null (-/-) Il1b mice compared to their wild-type (+/+) littermate controls, as indicated by an increase in the incidence of sustained generalized convulsive seizure activity. In the PTZ seizure model, the incidence of acute convulsive seizures was increased in both Il1b and Il1r1-/- mice compared to their respective +/+ littermate controls. Interestingly, the selective cyclooxygenase (COX)-2 inhibitor, rofecoxib, mimicked the effect of IL-1ß deficiency on PTZ-induced convulsions in Il1r1+/+ but not -/- mice. Together, these results suggest that endogenous IL-1ß possesses anticonvulsive properties that may be mediated by arachidonic acid metabolites derived from the catalytic action of COX-2.

The Cystine/Glutamate Antiporter, System xc -, Contributes to Cortical Infarction After Moderate but Not Severe Focal Cerebral Ischemia in Mice.

Understanding the mechanisms underlying ischemic brain injury is of importance to the goal of devising novel therapeutics for protection and/or recovery. Previous work in our laboratory and in others has shown that activation of cystine/glutamate antiporter, system xc - (Sxc -), facilitates neuronal injury in several in vitro models of energy deprivation. However, studies on the contribution of this antiporter to ischemic brain damage in vivo are more limited. Since embolic or thrombotic transient or permanent occlusion of a cerebral blood vessel eventually leads to brain infarction in most stroke cases, we evaluated the contribution of Sxc - to cerebral ischemic damage by comparing brain infarction between mice naturally null for SLC7a11 (SLC7a11sut/sut mice) - the gene the encodes for the substrate specific light chain for system xc - - with their wild type (SLC7a11 + ⁣/ +) littermates following photothrombotic ischemic stroke of the middle cerebral artery (PTI) and permanent middle cerebral artery occlusion (pMCAo) rendered by cauterization. In the PTI model, we found a time-dependent reduction in cerebral blood flow that reached 50% from baseline in both genotypes 47-48 h post-illumination. Despite this, a remarkable reduction in incidence and total infarct volume of SLC7a11sut/sut mice was revealed 48 h following PTI as compared to SLC7a11+/+ mice. No difference in injury markers and/or infarct volume was measured between genotypes when occlusion of the MCA was permanent, however. Present data demonstrate a model-dependent differential role for Sxc - in focal cerebral ischemic damage, further highlighting that ischemic severity activates heterogeneous biochemical events that lead to damage engendered by stroke.

Prophylactic, prandial rofecoxib treatment lacks efficacy against acute PTZ-induced seizure generation and kindling acquisition.

PURPOSE: The goal of this study was to determine whether prophylactic prandial administration of rofecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, could alter seizure generation, kindling acquisition, and/or kindling maintenance in the mouse pentylenetetrazole (PTZ) epilepsy model. METHODS: Male CD-1 mice were fed ad libitum with control chow or chow formulated to deliver 30 mg/kg/day rofecoxib. After 5 days, mice were treated with a single dose of 40 or 55 mg/kg PTZ (acute paradigm) or 40 mg/kg PTZ delivered daily (kindling paradigm). Seizure severity was scored on a four-point behavioral scale and COX-2 expression was assessed in brain slices from a subset of mice 3 h or 72 h after acute PTZ or following establishment of kindling. KEY FINDINGS: Hippocampal COX-2 expression was transiently upregulated 3 h after an acute PTZ-induced convulsion and returned to baseline levels within 72 h, whereas it remained elevated for at least 72 h after the final seizure in the kindling paradigm. Despite this increase, chronic rofecoxib treatment did not attenuate the severity of acute PTZ-induced seizures and failed to alter kindling development or maintenance. SIGNIFICANCE: The present study demonstrates that prophylactic, prandial rofecoxib treatment lacks efficacy against acute PTZ-induced seizure generation and kindling acquisition, and does not reverse the kindled state once established.

Influence of glutamate and GABA transport on brain excitatory/inhibitory balance.

An optimally functional brain requires both excitatory and inhibitory inputs that are regulated and balanced. A perturbation in the excitatory/inhibitory balance-as is the case in some neurological disorders/diseases (e.g. traumatic brain injury Alzheimer's disease, stroke, epilepsy and substance abuse) and disorders of development (e.g. schizophrenia, Rhett syndrome and autism spectrum disorder)-leads to dysfunctional signaling, which can result in impaired cognitive and motor function, if not frank neuronal injury. At the cellular level, transmission of glutamate and GABA, the principle excitatory and inhibitory neurotransmitters in the central nervous system control excitatory/inhibitory balance. Herein, we review the synthesis, release, and signaling of GABA and glutamate followed by a focused discussion on the importance of their transport systems to the maintenance of excitatory/inhibitory balance.

The brain in flux: Genetic, physiologic, and therapeutic perspectives on transporters in the CNS.

Active and passive transporters constitute a gene family of approximately 2000 members. These proteins are required for import and export across the blood brain barrier, clearance of neurotransmitters, inter-cellular solute transfer, and transport across the membranes of subcellular organelles. Neurologic, neurodevelopmental, and psychiatric diseases have been linked to alterations in function and/or mutations in every one of these types of transporters, and many of the transporters are targeted by therapeutics. This is the 4th biennial special edition of Neurochemistry International that originates from a scientific meeting devoted to studies of transporters and their relationship to brain function and to neurodevelopmental, neurologic, and psychiatric disorders. This meeting provides the only international forum for the presentation and discussion of cutting-edge research on brain transporters covering fundamental aspects of transporter structure, function, and trafficking. Scientists describe the novel approaches being used to link this information to physiology/circuit function and behavior. The meeting also addresses translational topics surrounding mouse models of brain transporter disorders, novel human brain disorders arising from transporter mutations, and innovative therapeutic approaches centered on modification of transporter function. This special issue includes a sampling of review articles that address timely questions of the field and several primary research articles.

Smad3-dependent signaling underlies the TGF-ß1-mediated enhancement in astrocytic iNOS expression.

We previously demonstrated that transforming growth factor-beta1 (TGF-beta1), while having no effect alone, enhances nitric oxide (NO) production in primary, purified mouse astrocytes induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma), by recruiting a latent population of astrocytes to respond, thereby enhancing the total number of cells that express Nos2. In this investigation, we evaluated the molecular signaling pathway by which this occurs. We found that purified murine primary astrocytes express mRNA for TGFbetaRII as well as the TGFbetaRI subunit activin-like kinase 5 (ALK5), but not ALK1. Immunofluorescence microscopy confirmed the expression of TGFbetaRII and ALK5 protein in astrocytes. Consistent with ALK5 signaling, Smad3 accumulated in the nucleus of astrocytes as early as 30 min after TGF-beta1 (3 ng/mL) treatment and persisted upto 32 hr after TGF-beta1 administration. Addition of ALK5 inhibitors prevented TGF-beta1-mediated Smad3 nuclear accumulation and NO production when given prior to the Nos2 induction stimuli, but not after. Finally, astrocyte cultures derived from Smad3 null mutant mice did not exhibit a TGF-beta1-mediated increase in iNOS expression. Overall, this data suggests that ALK5 signaling and Smad3 nuclear accumulation is required for optimal enhancement of LPS plus IFNgamma-induced NO production in astrocytes by TGF-beta1.

Regulation of system x(c)(-)activity and expression in astrocytes by interleukin-1ß: implications for hypoxic neuronal injury.

We recently demonstrated that interleukin-1ß (IL-1ß) 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-1ß 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-1ß exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1ß 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-1ß 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-1ß 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-1ß selectively regulates system x(c)(-) activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1ß found under hypoxic conditions.

Sexually dimorphic and brain region-specific transporter adaptations in system xc- null mice.

System xc- is a heterodimeric amino acid antiporter that, in the central nervous system, is best known for linking the import of L-cystine (CySS) with the export of L-glutamate for the production and maintenance of cellular glutathione (GSH) and extracellular glutamate levels, respectively. Yet, mice that are null for system xc- are healthy, fertile, and, morphologically, their brains are grossly normal. This suggests other glutamate and/or cyst(e)ine transport mechanisms may be upregulated in compensation. To test this, we measured the plasma membrane expression of Excitatory Amino Acid Transporters (EAATs) 1-3, the Alanine-Serine-Cysteine-Transporter (ASCT) 1, the sodium-coupled neutral amino acid transporter (SNAT) 3 and the L Amino Acid Transporter (LAT) 2 in striatum, hippocampus and cortex of male and female mice using Western Blot analysis. Present results demonstrate brain region and transporter-specific changes occurs in female system xc- null mice with increased expression of EAAT1 and ASCT1 occurring in the striatum and cortex, respectively, and decreased SNAT 3 expression in cortex. In male system xc- null brain, only SNAT3 was altered significantly - increasing in the cortex, but decreasing in the striatum. Total levels of GSH and CyS were similar to that found in age and sex-matched littermate control mice, however, reductions in the ratio of reduced to oxidized GSH (GSH/GSSG) - a hallmark of oxidative stress - were found in all three brain regions in female system xc- null mice, whereas this occurred exclusively in the striatum of males. Protein levels of Superoxide dismutase (SOD) 1 were reduced, whereas SOD2 was enhanced in the hippocampus of male xc- null mice only. Finally, striatal vulnerability to 3-nitropropionic acid (3-NP)-mediated oxidative stress in either sex showed no genotype difference, although 3-NP was more toxic to female mice of either genotype, as evidenced by an increase in moribundity as compared to males.

Nitroxyl exacerbates ischemic cerebral injury and oxidative neurotoxicity.

Nitroxyl (HNO) donor compounds function as potent vasorelaxants, improve myocardial contractility and reduce ischemia-reperfusion injury in the cardiovascular system. With respect to the nervous system, HNO donors have been shown to attenuate NMDA receptor activity and neuronal injury, suggesting that its production may be protective against cerebral ischemic damage. Hence, we studied the effect of the classical HNO-donor, Angeli's salt (AS), on a cerebral ischemia/reperfusion injury in a mouse model of experimental stroke and on related in vitro paradigms of neurotoxicity. I.p. injection of AS (40 mumol/kg) in mice prior to middle cerebral artery occlusion exacerbated cortical infarct size and worsened the persistent neurological deficit. AS not only decreased systolic blood pressure, but also induced systemic oxidative stress in vivo indicated by increased isoprostane levels in urine and serum. In vitro, neuronal damage induced by oxygen-glucose-deprivation of mature neuronal cultures was exacerbated by AS, although there was no direct effect on glutamate excitotoxicity. Finally, AS exacerbated oxidative glutamate toxicity - that is, cell death propagated via oxidative stress in immature neurons devoid of ionotropic glutamate receptors. Taken together, our data indicate that HNO might worsen cerebral ischemia-reperfusion injury by increasing oxidative stress and decreasing brain perfusion at concentrations shown to be cardioprotective in vivo.

Decreased epileptogenesis in mice lacking the System xc - transporter occurs in association with a reduction in AMPA receptor subunit GluA1.

OBJECTIVE: Although the cystine/glutamate antiporter System xc - (Sxc -) plays a permissive role in glioma-associated seizures, its contribution to other acquired epilepsies has not been determined. As such, the present study investigates whether and how Sxc - contributes to the pentylenetetrazole (PTZ) chemical kindling model of epileptogenesis. METHODS: Male Sxc - null (sut/sut) mice and their wild-type littermates were administered PTZ (i.p.) daily for up to 21 days (kindling paradigm). Seizure severity was scored on a 5-point behavioral scale. Mossy fiber sprouting, cellular degeneration, and Sxc - light chain (xCT) messenger RNA (mRNA) were explored using Timm staining, thionin staining, and real-time quantitative polymerase chain reaction (qPCR), respectively. Levels of reduced and oxidized glutathione and cysteine were determined via high-performance liquid chromatography (HPLC). Plasma membrane protein levels of glutamate and γ-aminobutyric acid (GABA) receptor subunits as well as the K+/Cl- co-transporter KCC2 were quantified via western blot analysis. RESULTS: Repeated administration of PTZ produced chemical kindling in only 50% of Sxc - null mice as compared to 82% of wild-type littermate control mice. Kindling did not result in any changes in xCT mRNA levels assessed in wild-type mice. No cellular degeneration or mossy fiber sprouting was discernible in either genotype. Except for a small, but significant, decrease in oxidized cysteine in the hippocampus, no other change in measured redox couples was determined in Sxc - null mice. Cortical levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA1 were decreased in Sxc - null mice as compared to wild-type littermates, whereas all other proteins tested showed no difference between genotypes. SIGNIFICANCE: This study provides the first evidence that Sxc - signaling contributes to epileptogenesis in the PTZ kindling model of acquired epilepsy. Further data indicate that a reduction in AMPA receptor signaling could underlie the resistance to PTZ kindling uncovered in Sxc - null mice.

System x(c)- activity and astrocytes are necessary for interleukin-1 beta-mediated hypoxic neuronal injury.

The purpose of this study was to elucidate the cellular/biochemical pathway(s) by which interleukin-1beta (IL-1beta) 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-1beta 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-1beta-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 x(c)-). Indeed, dual System x(c)-/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-1beta as wild-type cultures, an effect prevented by System x(c)-/mGluR1 antagonism. Finally, assessment of System x(c)- function and kinetics in IL-1beta-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 x(c)- as a novel contributor to the development and progression of hypoxic neuronal injury.

Interleukin-1beta: 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-1beta to the pathogenesis of both acute and chronic neurological disorders. Further, we highlight data that leads us to propose IL-1beta as the missing mechanistic link between a potential beneficial inflammatory response and detrimental glutamate excitotoxicity.

Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death.

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.