Human adipose-derived stem cells enhance the glutamate uptake function of GLT1 in SOD1-bearing astrocytes

Impaired glutamate uptake function of astrocytes associated with accumulation of extracellular glutamate is a well-documented feature of amyotrophic lateral sclerosis (ALS). Enhancing the uptake function of astrocytic glutamate transport 1 (GLT1) may be a potential treatment for this disease. Human adipose-derived stem cells (hADSCs) are capable of secreting a large number of cytokines which exhibit diverse pharmacological effects. Therefore, we investigate the influence of the soluble factors released by hADSCs on the GLT1 in primary astrocytes cultured from SOD1^G93A mice, a widely studied mutant human SOD1 transgenic model of ALS. Our data indicate that soluble factors from hADSCs significantly upregulate the expression of GLT1 in SOD1^G93A-bearing astrocytes, which result in enhanced glutamate uptake function. The upregulation of GLT1 is accompanied by the inhibition of caspase-3 activation in mutant astrocytes. In addition, we find that hADSCs cocultured with SOD1^G93A-bearing astrocytes produce more VEGF, HGF and IGF-1, which are reported to have neuroprotective effects. Our results suggest that hADSCs may be a potential candidate in cellular therapy for ALS.     

A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain

We have identified a novel carboxyl-terminal splice-variant of the glutamate transporter GLT1, which we denote as GLT1c. Within the rat brain only low levels of protein and message were detected, protein expression being restricted to end feet of astrocytes apposed to blood vessels or some astrocytes adjacent to the ventricles. Conversely, within the retina, this variant was selectively and heavily expressed in the synaptic terminals of both rod- and cone-photoreceptors in both humans and rats. Double-immunolabelling with antibodies to the carboxyl region of GLT1b/GLT1v, which is strongly expressed in apical dendrites of bipolar cells and in cone photoreceptors revealed that in the rat GLT1c was co-localised with GLT1b/GLT1v in cone photoreceptors but not with GLT1b/GLT1v in bipolar cells. GLT1c expression was developmentally regulated, only appearing at around postnatal day 7 in the rat retina, when photoreceptors first exhibit a dark current. Since the glutamate transporter EAAT5 is also expressed in terminals of rod photoreceptor terminals these data indicate that rod photoreceptors express two glutamate transporters with distinct properties. Similarly, cone photoreceptors express two glutamate transporters. We suggest that differential usage of these transporters by rod and cone photoreceptors may influence the kinetics of glutamate transmission by these neurons.     

Cocaine-mediated downregulation of microglial miR-124 expression involves promoter DNA methylation

Neuroinflammation plays a critical role in the development of reward-related behavior in cocaine self-administration rodents. Cocaine, one of most commonly abused drugs, has been shown to activate microglia both in vitro and in vivo. Detailed molecular mechanisms underlying cocaine-mediated microglial activation remain poorly understood. microRNAs (miRs) belonging to a class of small noncoding RNA superfamily have been shown to modulate the activation status of microglia. miR-124, one of the microglia-enriched miRs, functions as an anti-inflammatory regulator that maintains microglia in a quiescent state. To date, the possible effects of cocaine on microglial miR-124 levels and the associated underlying mechanisms have not been explored. In the current study, we demonstrated that cocaine exposure decreased miR-124 levels in both BV-2 cells and rat primary microglia. These findings were further validated in vivo, wherein we demonstrated decreased abundance of miR-124 in purified microglia isolated from cocaine-administered mice brains compared with cells from saline administered animals. Molecular mechanisms underlying these effects involved cocaine-mediated increased mRNA and protein expression of DNMTs in microglia. Consistently, cocaine substantially increased promoter DNA methylation levels of miR-124 precursors (pri-miR-124-1 and ?2), but not that of pri-miR-124-3, both in vitro and in vivo. In summary, our findings demonstrated that cocaine exposure increased DNA methylation of miR-124 promoter resulting into its downregulation, which, in turn, led to microglial activation. Our results thus implicate that epigenetic modulation of miR-124 could be considered as a potential therapeutic approach to ameliorate microglial activation and, possibly, the development of cocaine addiction.     

Functional Modulation of the Glutamate Transporter Variant GLT1b by the PDZ Domain Protein PICK1

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulatory effect on trafficking of AMPA-type glutamate receptors. The 11 extreme C-terminal residues specific for the GLT1b variant are essential for its specific interaction with the PICK1 PDZ domain, but a functional consequence of this interaction has remained unresolved. To identify a functional effect of PICK1 on GLT1a or GLT1b separately, we employed the Xenopus laevis expression system. GLT1a and GLT1b displayed similar electrophysiological properties and EC₅₀ for glutamate. Co-expressed PICK1 localized efficiently to the plasma membrane and resulted in a 5-fold enhancement of the leak current in GLT1b-expressing oocytes with only a minor effect on [³H]glutamate uptake. Three different GLT1 substrates all caused a slow TBOA-sensitive decay in the membrane current upon prolonged application, which provides support for the leak current being mediated by GLT1b itself. Leak and glutamate-evoked currents in GLT1a-expressing oocytes were unaffected by PICK1 co-expression. PKC activation down-regulated GLT1a and GLT1b activity to a similar extent, which was not affected by co-expression of PICK1. In conclusion, PICK1 may not only affect glutamatergic neurotransmission by its regulatory effect on glutamate receptors but may also affect neuronal excitability via an increased GLT1b-mediated leak current. This may be particularly relevant in pathological conditions such as amyotrophic lateral sclerosis and cerebral hypoxia, which are associated with neuronal GLT1b up-regulation.     

GLT1, glial glutamate transporter,is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high‐affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin‐like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin‐positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 515–526, 1999     

GLT1, glial glutamate transporter, is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain.

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high-affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin-like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin-positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development.     

Insulin increases glutamate transporter GLT1 in cultured astrocytes

The astroglial cell-specific glutamate transporter subtype 2 (excitatory amino acid transporter 2, GLT1) plays an important role in excitotoxicity that develops after damage to the central nervous system (CNS) is incurred. Both the protein kinase C signaling pathway and the epidermal growth factor (EGF) pathway have been suggested to participate in the modulation of GLT1, but the modulatory mechanisms of GLT1 expression are not fully understood. In the present study, we aimed to evaluate the effects of insulin on GLT1 expression. We found that short-term stimulation of insulin led to the upregulation of both total and surface expressions of GLT1. Akt phosphorylation increased after insulin treatment, and triciribine, the inhibitor of Akt phosphorylation, significantly inhibited the effects of insulin. We also found that the upregulation of GLT1 expression correlated with increased kappa B motif-binding phosphoprotein (KBBP) and GLT1 mRNA levels. Our results suggest that insulin may modulate the expression of astrocytic GLT1, which might play a role in reactive astrocytes after CNS injuries.     

Neuronal Soluble Factors Differentially Regulate the Expression of the GLT1 and GLAST Glutamate Transporters in Cultured Astroglia

Abstract: The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down-regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron-astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron-glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.     

Interleukin-17 (IL-17)-induced MicroRNA 873 (miR-873) Contributes to the Pathogenesis of Experimental Autoimmune Encephalomyelitis by Targeting A20 Ubiquitin-editing Enzyme

Interleukin 17 (IL-17), produced mainly by T helper 17 (Th17) cells, is increasingly recognized as a key regulator in various autoimmune diseases, including human multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Although several microRNAs (miRNAs) with aberrant expression have been shown to contribute to the pathogenesis of MS and EAE, the mechanisms underlying the regulation of abnormal miRNA expression in astrocytes upon IL-17 stimulation remain unclear. In the present study, we detected the changes of miRNA expression profiles both in the brain tissue of EAE mice and in cultured mouse primary astrocytes stimulated with IL-17 and identified miR-873 as one of the co-up-regulated miRNAs in vivo and in vitro. The overexpression of miR-873, demonstrated by targeting A20 (TNFα-induced protein 3, TNFAIP3), remarkably reduced the A20 level and promoted NF-κB activation in vivo and in vitro as well as increasing the production of inflammatory cytokines and chemokines (i.e. IL-6, TNF-α, MIP-2, and MCP-1/5). More importantly, silencing the endogenous miR-873 or A20 gene with lentiviral vector of miR-873 sponge (LV-miR-873 sponge) or short hairpin RNA (shRNA) of A20 (LV-A20 shRNA) in vivo significantly lessened or aggravated inflammation and demyelination in the central nervous system (CNS) of EAE mice, respectively. Taken together, these findings indicate that miR-873 induced by IL-17 stimulation promotes the production of inflammatory cytokines and aggravates the pathological process of EAE mice through the A20/NF-κB pathway, which provides a new insight into the mechanism of inflammatory damage in MS.     

The GLT‐1 (EAAT2; slc1a2) glutamate transporter is essential for glutamate homeostasis in the neocortex of the mouse

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.

     

Ceftriaxone attenuates hypoxic-ischemic brain injury in neonatal rats

Background  

Perinatal brain injury is the leading cause of subsequent neurological disability in both term and preterm baby. Glutamate excitotoxicity is one of the major factors involved in perinatal hypoxic-ischemic encephalopathy (HIE). Glutamate transporter GLT1, expressed mainly in mature astrocytes, is the major glutamate transporter in the brain. HIE induced excessive glutamate release which is not reuptaked by immature astrocytes may induce neuronal damage. Compounds, such as ceftriaxone, that enhance the expression of GLT1 may exert neuroprotective effect in HIE.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Astrocytes repress the neuronal expression of GLAST and GLT glutamate transporters in cultured hippocampal neurons from embryonic rats

Glutamate extracellular levels are regulated by specific transporters. Five subtypes have been identified. The two major ones, GLAST and GLT (glutamate transporters 1 and 2, respectively), are localized in astroglia in normal mature brain. However, in neuron-enriched hippocampal cultures, these proteins are expressed in neurons during the early in vitro development (Plachez et al., 2000). Here, we show that, in these cultures, GLAST and GLT neuronal expression is transient and no longer observed after 7 days in vitro, a stage at which the few astrocytes present in the culture are maturing. Moreover, we demonstrate that these few astrocytes are responsible for the repression of this neuronal expression. Indeed, addition of conditioned medium prepared from primary cultures of hippocampal astrocytes, to cultured hippocampal neurons, rapidly leads to the suppression of neuronal GLAST expression, without affecting neuronal GLT expression. However, when neurons are seeded and co-cultured on a layer of hippocampal astrocytes, they do not develop any immunoreactivity towards GLAST or GLT antibodies. Altogether, these results indicate that glia modulate the expression of GLAST and GLT glutamate transporters in neurons, via at least two distinct mechanisms. Neuronal GLAST expression is likely repressed via the release or the uptake of soluble factors by glia. The repression of neuronal GLT expression probably results from glia-neuron interactions. This further reinforces the fundamental role of direct or indirect neuron-glia interactions in the development of the central nervous system.     

Dopamine denervation of the prefrontal cortex increases expression of the astrocytic glutamate transporter GLT‐1

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.

     

MicroRNA-1 Downregulation Increases Connexin 43 Displacement and Induces Ventricular Tachyarrhythmias in Rodent Hypertrophic Hearts

Downregulation of the muscle-specific microRNA-1 (miR-1) mediates the induction of pathologic cardiac hypertrophy. Dysfunction of the gap junction protein connexin 43 (Cx43), an established miR-1 target, during cardiac hypertrophy leads to ventricular tachyarrhythmias (VT). However, it is still unknown whether miR-1 and Cx43 are interconnected in the pro-arrhythmic context of hypertrophy. Thus, in this study we investigated whether a reduction in the extent of cardiac hypertrophy could limit the pathological electrical remodeling of Cx43 and the onset of VT by modulating miR-1 levels. Wistar male rats underwent mechanical constriction of the ascending aorta to induce pathologic left ventricular hypertrophy (LVH) and afterwards were randomly assigned to receive 10mg/kg valsartan, VAL (LVH+VAL) delivered in the drinking water or placebo (LVH) for 12 weeks. Sham surgery was performed for control groups. Programmed ventricular stimulation reproducibly induced VT in LVH compared to LVH+VAL group. When compared to sham controls, rats from LVH group showed a significant decrease of miR-1 and an increase of Cx43 expression and its ERK1/2-dependent phosphorylation, which displaces Cx43 from the gap junction. Interestingly, VAL administration to rats with aortic banding significantly reduced cardiac hypertrophy and prevented miR-1 down-regulation and Cx43 up-regulation and phosphorylation. Gain- and loss-of-function experiments in neonatal cardiomyocytes (NCMs) in vitro confirmed that Cx43 is a direct target of miR-1. Accordingly, in vitro angiotensin II stimulation reduced miR-1 levels and increased Cx43 expression and phosphorylation compared to un-stimulated NCMs. Finally, in vivo miR-1 cardiac overexpression by an adenoviral vector intra-myocardial injection reduced Cx43 expression and phosphorylation in mice with isoproterenol-induced LVH. In conclusion, miR-1 regulates Cx43 expression and activity in hypertrophic cardiomyocytes in vitro and in vivo. Treatment of pressure overload-induced myocyte hypertrophy reduces the risk of life-threatening VT by normalizing miR-1 expression levels with the consequent stabilization of Cx43 expression and activity within the gap junction.     

PI3K/Akt/mTOR signaling regulates glutamate transporter 1 in astrocytes

Reduction in or dysfunction of glutamate transporter 1 (GLT1) is linked to several neuronal disorders such as stroke, Alzheimer’s disease, and amyotrophic lateral sclerosis. However, the detailed mechanism underlying GLT1 regulation has not been fully elucidated. In the present study, we first demonstrated the effects of mammalian target of rapamycin (mTOR) signaling on GLT1 regulation. We prepared astrocytes cultured in astrocyte-defined medium (ADM), which contains several growth factors including epidermal growth factor (EGF) and insulin. The levels of phosphorylated Akt (Ser473) and mTOR (Ser2448) increased, and GLT1 levels were increased in ADM-cultured astrocytes. Treatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor or an Akt inhibitor suppressed the phosphorylation of Akt (Ser473) and mTOR (Ser2448) as well as decreased ADM-induced GLT1 upregulation. Treatment with the mTOR inhibitor rapamycin decreased GLT1 protein and mRNA levels. In contrast, rapamycin did not affect Akt (Ser473) phosphorylation. Our results suggest that mTOR is a downstream target of the PI3K/Akt pathway regulating GLT1 expression.     

MIR-206 regulates connexin43 expression during skeletal muscle development

Skeletal myoblast fusion in vitro requires the expression of connexin43 (Cx43) gap junction channels. However, gap junctions are rapidly downregulated after the initiation of myoblast fusion in vitro and in vivo. In this study we show that this downregulation is accomplished by two related microRNAs, miR-206 and miR-1, that inhibit the expression of Cx43 protein during myoblast differentiation without altering Cx43 mRNA levels. Cx43 mRNA contains two binding sites for miR-206/miR-1 in its 3′-untranslated region, both of which are required for efficient downregulation. While it has been demonstrated before that miR-1 is involved in myogenesis, in this work we show that miR-206 is also upregulated during perinatal skeletal muscle development in mice in vivo and that both miR-1 and miR-206 downregulate Cx43 expression during myoblast fusion in vitro. Proper development of singly innervated muscle fibers requires muscle contraction and NMJ terminal selection and it is hypothesized that prolonged electrical coupling via gap junctions may be detrimental to this process. This work details the mechanism by which initial downregulation of Cx43 occurs during myogenesis and highlights the tight control mechanisms that are utilized for the regulation of gap junctions during differentiation and development.