Increasing evidence indicates that the Eph receptors and their ephrin ligands are involved in the regulation of interactions between neurons and astrocytes. Moreover, astrocytic ephrin‐A3 reverse signaling mediated by EphA4 receptors is necessary for controlling the abundance of glial glutamate transporters. However, the role of ephrin‐A3 reverse signaling in astrocytic function and neuronal death under ischemic conditions remains unclear. In the present study, we found that the EphA4 receptor and its ephrin‐A3 ligand, which were distributed in neurons and astrocytes, respectively, in the hippocampus showed a coincident up‐regulation of protein expression in the early stage of ischemia. Application of clustered EphA4 decreased the expressions of astrocytic glutamate transporters together with astrocytic glutamate uptake capacity through activating ephrin‐A3 reverse signaling. In consequence, neuronal loss was aggravated in the CA1 region of the hippocampus accompanied by impaired hippocampus‐dependent spatial memory when clustered EphA4 treatment was administered prior to transient global ischemia. These findings indicate that EphA4‐mediated ephrin‐A3 reverse signaling is a crucial mechanism for astrocytes to control glial glutamate transporters and prevent glutamate excitotoxicity under pathological conditions.
Recent evidence suggests that the predominant astrocyte glutamate transporter, GLT‐1/ Excitatory Amino Acid Transporter 2 (EAAT2) is associated with mitochondria. We used primary cultures of mouse astrocytes to assess co‐localization of GLT‐1 with mitochondria, and tested whether the interaction was dependent on neurons, actin polymerization or the kinesin adaptor, TRAK2. Mouse primary astrocytes were transfected with constructs expressing V5‐tagged GLT‐1, pDsRed1‐Mito with and without dominant negative TRAK2. Astrocytes were visualized using confocal microscopy and co‐localization was quantified using Volocity software. Image analysis of confocal z‐stacks revealed no co‐localization between mitochondria and GLT‐1 in pure astrocyte cultures. Co‐culture of astrocytes with primary mouse cortical neurons revealed more mitochondria in processes and a positive correlation between mitochondria and GLT‐1. This co‐localization was not further enhanced after neuronal depolarization induced by 1 h treatment with 15 mM K+. In pure astrocytes, a rho kinase inhibitor, Y27632 caused the distribution of mitochondria to astrocyte processes without enhancing GLT‐1/mitochondrial co‐localization, however, in co‐cultures, Y27632 abolished mitochondrial:GLT‐1 co‐localization. Disrupting potential mitochondrial: kinesin interactions using dominant negative TRAK2 did not alter GLT‐1 distribution or GLT‐1: mitochondrial co‐localization. We conclude that the association between GLT‐1 and mitochondria is modest, is driven by synaptic activity and dependent on polymerized actin filaments.
Glutamate transport is a critical process in the brain that maintains low extracellular levels of glutamate to allow for efficient neurotransmission and prevent excitotoxicity. Loss of glutamate transport function is implicated in epilepsy, traumatic brain injury, and amyotrophic lateral sclerosis. It remains unclear whether or not glutamate transport can be modulated in these disease conditions to improve outcome. Here, we show that sirtuin (SIRT)4, a mitochondrial sirtuin, is up‐regulated in response to treatment with the potent excitotoxin kainic acid. Loss of SIRT4 leads to a more severe reaction to kainic acid and decreased glutamate transporter expression and function in the brain. Together, these results indicate a critical and novel stress response role for SIRT4 in promoting proper glutamate transport capacity and protecting against excitotoxicity.
α1‐adrenoceptors (α1‐ARs) stimulation has been found to enhance excitatory processes in many brain regions. A recent study in our laboratory showed that α1‐ARs stimulation enhances glutamatergic transmission via both pre‐ and post‐synaptic mechanisms in layer V/VI pyramidal cells of the rat medial prefrontal cortex (mPFC). However, a number of pre‐synaptic mechanisms may contribute to α1‐ARs‐induced enhancement of glutamate release. In this study, we blocked the possible post‐synaptic action mediated by α1‐ARs to investigate how α1‐ARs activation regulates pre‐synaptic glutamate release in layer V/VI pyramidal neurons of mPFC. We found that the α1‐ARs agonist phenylephrine (Phe) induced a significant enhancement of glutamatergic transmission. The Phe‐induced potentiation was mediated by enhancing pre‐synaptic glutamate release probability and increasing the number of release vesicles via a protein kinase C‐dependent pathway. The mechanisms of Phe‐induced potentiation included interaction with both glutamate release machinery and N‐type Ca2+ channels, probably via a pre‐synaptic Gq/phospholipase C/protein kinase C pathway. Our results may provide a cellular and molecular mechanism that helps explain α1‐ARs‐mediated influence on PFC cognitive functions.
The ventrolateral thalamus (VL) is a primary relay point between the basal ganglia and the primary motor cortex (M1). Using dual probe microdialysis and locomotor behavior monitoring, we investigated the contribution of VL input into M1 during amphetamine (AMPH)‐stimulated monoamine release and hyperlocomotion in rats. Tetrodotoxin (10 μM) perfusion into the VL significantly lowered hyperactivity induced by AMPH (1 mg/kg i.p.). This behavioral response corresponded to reduced cortical glutamate and monoamine release. To determine which glutamate receptors the thalamocortical projections acted upon, we perfused either the α‐amino‐3‐(3‐hydroxy‐5‐methyl‐isoxazol‐4‐yl)propanoic acid (AMPA)/kainate receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo[f]quinoxaline‐2,3‐dione (NBQX) (10 μM) or the N‐methyl‐D‐aspartic acid (NMDA) receptor antagonist (MK‐801) intracortically followed by systemic AMPH. The results show that AMPA/kainate, and to a lesser extent NMDA receptors, mediated the observed effects. As glutamate–monoamine interactions could possibly occur through local or circuit‐based mechanisms, we isolated and perfused M1 tissue ex vivo to determine the extent of local glutamate–dopamine interactions. Taken together, these results demonstrate that AMPH generates hyperlocomotive states via thalamocortical signaling and that cortical AMPA receptors are an important mediator of these effects.
Prion protein (PrP) plays crucial roles in regulating antioxidant systems to improve cell defenses against cellular stress. Here, we show that the interactions of PrP with the excitatory amino acid transporter 3 (EAAT3), γ‐glutamyl transpeptidase (γ‐GT), and multi‐drug resistance protein 1 (MRP1) in astrocytes and the interaction between PrP and EAAT3 in neurons regulate the astroglial and neuronal metabolism of the antioxidant glutathione. Ablation of PrP in astrocytes and cerebellar neurons leads to dysregulation of EAAT3‐mediated uptake of glutamate and cysteine, which are precursors for the synthesis of glutathione. In PrP‐deficient astrocytes, levels of intracellular glutathione are increased, and under oxidative stress, levels of extracellular glutathione are increased, due to (i) increased glutathione release via MRP1 and (ii) reduced activity of the glutathione‐degrading enzyme γ‐GT. In PrP‐deficient cerebellar neurons, cell death is enhanced under oxidative stress and glutamate excitotoxicity, when compared to wild‐type cerebellar neurons. These results indicate a functional interplay of PrP with EAAT3, MRP1 and γ‐GT in astrocytes and of PrP and EAAT3 in neurons, suggesting that these interactions play an important role in the metabolic cross‐talk between astrocytes and neurons and in protection of neurons by astrocytes from oxidative and glutamate‐induced cytotoxicity.
Both dopamine and glutamate are critically involved in cognitive processes such as working memory. Astrocytes, which express dopamine receptors, are essential elements in the termination of glutamatergic signaling: the astrocytic glutamate transporter GLT‐1 is responsible for > 90% of cortical glutamate uptake. The effect of dopamine depletion on glutamate transporters in the prefrontal cortex (PFC) remains unknown. In an effort to determine if astrocytes are a locus of cortical dopamine–glutamate interactions, we examined the effects of chronic dopamine denervation on PFC protein and mRNA levels of glutamate transporters. PFC dopamine denervation elicited a marked increase in GLT‐1 protein levels, but had no effect on levels of other glutamate transporters; high‐affinity glutamate transport was positively correlated with the extent of dopamine depletion. GLT‐1 gene expression was not altered. Our data suggest that dopamine depletion may lead to post‐translational modifications that result in increased expression and activity of GLT‐1 in PFC astrocytes.
Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT‐1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT‐1 and GLAST, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we injected GLT‐1 knockout (KO) mice and wild‐type littermates with [1‐13C]glucose and [1,2‐13C]acetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and 13C labeling in neocortex. Whereas the cerebellum in GLT‐1‐deficient mice had normal levels of glutamate, glutamine, and 13C labeling of metabolites, glutamate level was decreased but labeling from [1‐13C] glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from [1,2‐13C] acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the cortex.
Our recent studies have shown that endogenous zinc, co‐released with glutamate from the synaptic terminals of vertebrate retinal photoreceptors, provides a feedback mechanism that reduces calcium entry and the concomitant vesicular release of glutamate. We hypothesized that zinc feedback may serve to protect the retina from glutamate excitotoxicity, and conducted in vivo experiments on the retina of the skate (Raja erinacea) to determine the effects of removing endogenous zinc by chelation. These studies showed that removal of zinc by injecting the zinc chelator histidine results in inner retinal damage similar to that induced by the glutamate receptor agonist kainic acid. In contrast, when an equimolar quantity of zinc followed the injection of histidine, the retinal cells were unaffected. Our results are a good indication that zinc, co‐released with glutamate by photoreceptors, provides an auto‐feedback system that plays an important cytoprotective role in the retina.
Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium‐evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4‐ and 7‐fold increase in potassium‐evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal‐dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability.
Zinc has been implicated in neurodegeneration following ischemia. In analogy with calcium, zinc has been proposed to induce toxicity via mitochondrial dysfunction, but the relative role of each cation in mitochondrial damage remains unclear. Here, we report that under conditions mimicking ischemia in hippocampal neurons – normal (2 mM) calcium plus elevated (> 100 μM) exogenous zinc – mitochondrial dysfunction evoked by glutamate, kainate or direct depolarization is, despite significant zinc uptake, primarily governed by calcium. Thus, robust mitochondrial ion accumulation, swelling, depolarization, and reactive oxygen species generation were only observed after toxic stimulation in calcium‐containing media. This contrasts with the lack of any mitochondrial response in zinc‐containing but calcium‐free medium, even though zinc uptake and toxicity were strong under these conditions. Indeed, abnormally high, ionophore‐induced zinc uptake was necessary to elicit any mitochondrial depolarization. In calcium‐ and zinc‐containing media, depolarization‐induced zinc uptake facilitated cell death and enhanced accumulation of mitochondrial calcium, which localized to characteristic matrix precipitates. Some of these contained detectable amounts of zinc. Together these data indicate that zinc uptake is generally insufficient to trigger mitochondrial dysfunction, so that mechanism(s) of zinc toxicity must be different from that of calcium.
Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4‐dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca2+ levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP‐treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin – PI3K – MAPK pathways, and up‐regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up‐regulated in response to DNP. CREB (cAMP‐response element‐binding protein) signaling, Arc and brain‐derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up‐regulated in response to DNP, and DNP‐treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP‐induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up‐regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity.
The exact effect of glycine pre‐treatment on brain ischemic tolerance (IT) remains quite controversial. The objective of this study was to investigate the potential effects of glycine on IT. We used rat models of both in vitro ischemia (oxygen and glucose deprivation) and in vivo ischemia (transient middle cerebral artery occlusion). Low doses of glycine (L‐Gly) significantly decreased hippocampal ischemic LTP (i‐LTP), infarct volume, and neurological deficit scores which were administered before ischemia was induced in rats, whereas high doses of glycine exerted deteriorative effects under the same condition. These findings suggested that exogenous glycine may induce IT in a dose‐dependent manner. Furthermore, L‐Gly‐dependent neuronal protection was inversed by L689, a selective NMDAR glycine site antagonist both in vitro (abolished i‐LTP depression) and in vivo (increased infarct size reduction), but not glycine receptor (GlyR) inhibitor strychnine. Importantly, L‐Gly‐induced IT was achieved by NR2A‐dependent cAMP‐response element binding protein phosphorylation. These data imply that glycine pre‐treatment may represent a novel strategy for inducing IT based on synaptic NMDAR‐dependent neuronal transmission.
Treatments to inhibit or repair neuronal cell damage sustained during focal ischemia/reperfusion injury in stroke are largely unavailable. We demonstrate that dietary supplementation with the antioxidant di‐tert‐butyl‐bisphenol (BP) before injury decreases infarction and vascular complications in experimental stroke in an animal model. We confirm that BP, a synthetic polyphenol with superior radical‐scavenging activity than vitamin E, crosses the blood–brain barrier and accumulates in rat brain. Supplementation with BP did not affect blood pressure or endogenous vitamin E levels in plasma or cerebral tissue. Pre‐treatment with BP significantly lowered lipid, protein and thiol oxidation and decreased infarct size in animals subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. This neuroprotective action was accompanied by down‐regulation of hypoxia inducible factor‐1α and glucose transporter‐1 mRNA levels, maintenance of neuronal tissue ATP concentration and inhibition of pro‐apoptotic factors that together enhanced cerebral tissue viability after injury. That pre‐treatment with BP ameliorates oxidative damage and preserves cerebral tissue during focal ischemic insult indicates that oxidative stress plays at least some causal role in promoting tissue damage in experimental stroke. The data strongly suggest that inhibition of oxidative stress through BP scavenging free radicals in vivo contributes significantly to neuroprotection.
Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose‐6‐phosphate dehydrogenase, the rate‐limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time‐ and dose‐dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes.
Exposure to atmospheric particulate matter PM2.5 (aerodynamic diameter ≤ 2.5 μm) has been epidemiologically associated with respiratory illnesses. However, recent data have suggested that PM2.5 is able to infiltrate into circulation and elicit a systemic inflammatory response. Potential adverse effects of air pollutants to the central nervous system (CNS) have raised concerns, but whether PM2.5 causes neurotoxicity remains unclear. In this study, we have demonstrated that PM2.5 impairs the tight junction of endothelial cells and increases permeability and monocyte transmigration across endothelial monolayer in vitro, indicating that PM2.5 is able to disrupt blood–brain barrier integrity and gain access to the CNS. Exposure of primary neuronal cultures to PM2.5 resulted in decrease in cell viability and loss of neuronal antigens. Furthermore, supernatants collected from PM2.5‐treated macrophages and microglia were also neurotoxic. These macrophages and microglia significantly increased extracellular levels of glutamate following PM2.5 exposure, which were negatively correlated with neuronal viability. Pre‐treatment with NMDA receptor antagonist MK801 alleviated neuron loss, suggesting that PM2.5 neurotoxicity is mediated by glutamate. To determine the potential source of excess glutamate production, we investigated glutaminase, the main enzyme for glutamate generation. Glutaminase was reduced in PM2.5‐treated macrophages and increased in extracellular vesicles, suggesting that PM2.5 induces glutaminase release through extracellular vesicles. In conclusion, these findings indicate PM2.5 as a potential neurotoxic factor, crucial to understanding the effects of air pollution on the CNS.
Dysregulation of prefrontal cortical glutamatergic signalling via NMDA receptor hypofunction has been implicated in cognitive dysfunction and impaired inhibitory control in such neuropsychiatric disorders as schizophrenia, attention‐deficit hyperactivity disorder and drug addiction. Although NMDA receptors functionally interact with metabotropic glutamate receptor 5 (mGluR5), the consequence of this interaction for glutamate release in the prefrontal cortex (PFC) remains unknown. We therefore investigated the effects of positive and negative allosteric mGluR5 modulation on changes in extracellular glutamate efflux in the medial PFC (mPFC) induced by systemic administration of the non‐competitive NMDA receptor antagonist dizocilpine (or MK801) in rats. Extracellular glutamate efflux was measured following systemic administration of the positive allosteric mGluR5 modulator [S‐(4‐Fluoro‐phenyl)‐{3‐[3‐(4‐fluoro‐phenyl)‐[1,2,4]‐oxadiazol‐5‐yl]‐piperidin‐1‐yl}‐methanone] (ADX47273; 100 mg/kg, p.o.) and negative allosteric mGluR5 modulator [2‐chloro‐4‐{[1‐(4‐fluorophenyl)‐2,5‐dimethyl‐1H‐imidazol‐4‐yl]ethynyl}pyridine] (RO4917523; 0.3 mg/kg, p.o.), using a wireless glutamate biosensor in awake, freely moving rats. The effect of MK801 (0.03–0.06 mg/kg, s.c.) on mPFC glutamate efflux was also investigated in addition to the effects of MK801 (0.03 mg/kg, s.c.) following ADX47273 (100 mg/kg, p.o.) pre‐treatment. ADX47273 produced a sustained increase in glutamate efflux and increased the effect of NMDA receptor antagonism on glutamate efflux in the mPFC. In contrast, negative allosteric mGluR5 modulation with RO4917523 decreased glutamate efflux in the mPFC. These findings indicate that positive and negative allosteric mGluR5 modulators produce long lasting and opposing actions on extracellular glutamate efflux in the mPFC. Positive and negative allosteric modulators of mGluR5 may therefore be viable therapeutic agents to correct abnormalities in glutamatergic signalling present in a range of neuropsychiatric disorders.
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