GLAST Expression Research Articles

Exogenous leptin alleviates glutamate-excitotoxic injury caused by cerebral ischemia–reperfusion in mice by affecting the expression of glutamate transporters

Ischemic stroke is characterized by high morbidity and mortality and a lack of effective therapeutic interventions. Leptin plays an important role in regulating oxidative stress, angiogenesis, hematopoiesis, etc. Although recent studies have found a neuroprotective effect of leptin, little is known about its role in cerebral ischemia. This study explores the possible roles and potential preventative mechanisms of leptin in cerebral ischemia–reperfusion injury (CIRI). An in vivo middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was used to replicate the CIRI model, low (0.5 mg/kg), medium (1 mg/kg) and high (2 mg/kg) concentrations of leptin were injected intraperitoneally immediately after inserting the embolic line. After 1.5 h of ischemia and 24 h of reperfusion, we examined the neural function of the mice, collected brain tissue for histological examination, and screened for the optimal concentrations of leptin intervention. On this basis, we observed the changes of cortical apoptosis injury, intracellular calcium fluorescence intensity and astrocyte glial fibrillary acidic protein (GFAP) expression and morphological changes. In addition, we also tested the expression of transporters and metabolism-related enzymes (VGLUT-1, VGLUT-2, GLAST, GLT-1, GS, ATP1α1), the expression of inflammatory factors and the content of glutamate (Glu). Compared with the I/R group, we found that leptin improved neurological deficits, reduced the area of infarcts, maintained the normal morphology of astrocytes (AST), downregulated the expression of VGLUT-1, and upregulates the expression of GLT-1 and GLAST, thereby reducing the content of Glu in the synaptic cleft. Our studies suggest that leptin may have a neuroprotective effect by decreasing the excitotoxicity of glutamate.

Metabolic cross‐talk between astrocytes and neurons: implications for Alzheimer’s disease

AbstractBackgroundConverging evidence indicates that declining brain energetics contributes to the initiation and progression of neurodegenerative disorders, including Alzheimer’s disease (AD). Upstream events often characterizing AD include hypometabolism of glucose and oxygen in the brain, which may cause mitochondrial dysfunction, energetic failure, and oxidative stress (Moreira et al., 2010). Astrocytes, which have a main role in supporting neuronal function and metabolism, might contribute to the development of neurodegenerative diseases. By modulating the release of gliotransmitters (e.g. glutamate), they provide a fine control of neuronal metabolism and cell‐to‐cell communication, both of them involved in AD (Monterey et al., 2021). Impaired astrocytes metabolism may incite an inflammatory response that, in turn, negatively impacts neuronal viability.MethodPrimary rat cortical astrocytes were isolated from the cortex of Wistar rat pups. A metabolic stress was induced by using glyceraldehyde (GA) (Magi et al., 2021). We evaluated cell viability, the release of tumor necrosis factor alpha (TNFα) and the involvement of the nuclear factor‐kappa B (NF‐kB) pathway. In order to check the glutamate turnover, we analyzed the expression of GLAST and GLT1 and we evaluated the ATP content after glutamate exposure under physiological conditions. We also explored whether damaged astrocytes could elicit neuronal death in a co‐culture setting.ResultExposure to increasing concentrations of GA caused a concentration‐dependent cell injury in rat cortical astrocytes. GA (1mM) started to induce a significant cell damage after 24 h. After 48 h, GA exposure activated the NF‐kB pathway and the release of TNFα. GA treatment significantly increased GLAST and GLT1 levels after 48h. When injured astrocytes were co‐cultured with cortical neurons, neurons viability significantly decreased. Under physiological condition, glutamate significantly improved intracellular ATP levels after 4 and 8 hours of treatment, paving the way to the evaluation of its action in metabolic‐impaired setting, as induced by GA treatment.ConclusionOur results suggest that metabolically impaired astrocytes may damage neighboring neurons, suggesting astrocytes involvement in the pathophysiological development and progression of AD. Therefore, astrocytes can be considered a key therapeutic and neuroprotective target for future studies.

Glutamate transporter contribution to retinal ganglion cell vulnerability in a rat model of multiple sclerosis

Glial glutamate transporters actively participate in neurotransmission and have a fundamental role in determining the ambient glutamate concentration in the extracellular space. Their expression is dynamically regulated in many diseases, including experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In EAE, a downregulation has been reported which may render neurons more susceptible to glutamate excitotoxicity. In this study, we have investigated the expression of GLAST (EAAT1) and GLT-1 (EAAT2) in the retina of Brown Norway rats following induction of myelin oligodendrocyte glycoprotein (MOG)-EAE, which results in retinal ganglion cell (RGC) degeneration and dysfunction. In addition, we tested whether AAV-mediated overexpression of GLAST in the retina can protect RGCs from degeneration. To address the impact of glutamate transporter modulation on RGCs, we performed whole-cell recordings and measured tonic NMDA receptor-mediated currents in the absence and presence of a glutamate-uptake blocker. We report that αOFF-RGCs show larger tonic glutamate-induced currents than αON-RGCs, in line with their greater vulnerability under neuroinflammatory conditions. We further show that increased AAV-mediated expression of GLAST in the retina does indeed protect RGCs from degeneration during the inflammatory disease. Collectively, our study highlights the neuroprotective role of glutamate transporters in the EAE retina and provides a characterization of tonic glutamate-currents of αRGCs. The larger effects of increased extracellular glutamate concentration on the αOFF-subtype may underlie its enhanced vulnerability to degeneration.

N-Acetylcysteine normalizes brain oxidative stress and neuroinflammation observed after protracted ethanol abstinence: a preclinical study in long-term ethanol-experienced male rats

RationaleUsing a preclinical model based on the Alcohol Deprivation Effect (ADE), we have reported that N-Acetylcysteine (NAC) can prevent the relapse-like drinking behaviour in long-term ethanol-experienced male rats.ObjectivesTo investigate if chronic ethanol intake and protracted abstinence affect several glutamate transporters and whether NAC, administered during the withdrawal period, could restore the ethanol-induced brain potential dysfunctions. Furthermore, the antioxidant and anti-inflammatory effects of NAC during abstinence in rats under the ADE paradigm were also explored.MethodsThe expression of GLT1, GLAST and xCT in nucleus accumbens (Nacc) and dorsal striatum (DS) of male Wistar was analysed after water and chronic ethanol intake. We used the model based on the ADE within another cohort of male Wistar rats. During the fourth abstinence period, rats were treated for 9 days with vehicle or NAC (60, 100 mg/kg; s.c.). The effects of NAC treatment on (i) glutamate transporters expression in the Nacc and DS, (ii) the oxidative status in the hippocampus (Hip) and amygdala (AMG) and (iii) some neuroinflammatory markers in prefrontal cortex (PFC) were tested.ResultsNAC chronic administration during protracted abstinence restored oxidative stress markers (GSSG and GGSH/GSH) in the Hip. Furthermore, NAC was able to normalize some neuroinflammation markers in PFC without normalizing the observed downregulation of GLT1 and GLAST in Nacc.ConclusionsNAC restores brain oxidative stress and neuroinflammation that we previously observed after protracted ethanol abstinence in long-term ethanol-experienced male rats. This NAC effect could be a plausible mechanism for its anti-relapse effect. Also, brain oxidative stress and neuroinflammation could represent and identify plausible targets for searching new anti-relapse pharmacotherapies.

EphA4/ephrinA3 reverse signaling mediated downregulation of glutamate transporter GLAST in Müller cells in an experimental glaucoma model.

Deficiency of glutamate transporter GLAST in Müller cells may be culpable for excessive extracellular glutamate, which involves in retinal ganglion cell (RGC) damage in glaucoma. We elucidated how GLAST was regulated in rat chronic ocular hypertension (COH) model. Western blot and whole-cell patch-clamp recordings showed that GLAST proteins and GLAST-mediated current densities in Müller cells were downregulated at the early stages of COH. In normal rats, intravitreal injection of the ephrinA3 activator EphA4-Fc mimicked the changes of GLAST in COH retinas. In purified cultured Müller cells, EphA4-Fc treatment reduced GLAST expression at mRNA and protein levels, which was reversed by the tyrosine kinase inhibitor PP2 or transfection with ephrinA3-siRNA (Si-EFNA3), suggesting that EphA4/ephrinA3 reverse signaling mediated GLAST downregulation. EphA4/ephrinA3 reverse signaling-induced GLAST downregulation was mediated by inhibiting PI3K/Akt/NF-κB pathways since EphA4-Fc treatment of cultured Müller cells reduced the levels of p-Akt/Akt and NF-κB p65, which were reversed by transfecting Si-EFNA3. In Müller cells with ephrinA3 knockdown, the PI3K inhibitor LY294002 still decreased the protein levels of NF-κB p65 in the presence of EphA4-Fc, and the mRNA levels of GLAST were reduced by LY294002 and the NF-κB inhibitor SN50, respectively. Pre-injection of the PI3K/Akt pathway activator 740 Y-P reversed the GLAST downregulation in COH retinas. Western blot and TUNEL staining showed that transfecting of Si-EFNA3 reduced Müller cell gliosis and RGC apoptosis in COH retinas. Our results suggest that activated EphA4/ephrinA3 reverse signaling induces GLAST downregulation in Müller cells via inhibiting PI3K/Akt/NF-κB pathways, thus contributing to RGC damage in glaucoma.

Disturbance of glutamate metabolism and inhibition of CaM-CaMKII-CREB signaling pathway in the hippocampus of mice induced by 1,2-dichloroethane exposure

1,2-Dichloroethane (1,2-DCE) is a highly toxic neurotoxicity, and the brain tissue is the main target organ. At present, long-term exposure to 1,2-DCE has been shown to cause cognitive dysfunction in some studies, but the mechanism is not clear. The results of this study showed that long-term 1,2-DCE exposure decreased learning and memory abilities in mice and impaired the structure and morphology of neurons in the hippocampal region. Moreover, except for the mRNA level of PAG, the enzymatic activities and protein levels of GS and PAG, as well as the mRNA level of GS were inhibited. With increasing dose of exposure, the protein and mRNA expression of GLAST and GLT-1 also decreased. Contrarily, there were protein and mRNA expression upregulation of GluN1, GluN2A and GluN2B in the hippocampus, as well as increased levels of extracellular Glu and intracellular Ca2+. In addition, 1,2-DCE exposure also downregulated the protein expression levels of CaM, CaMKII and CREB. Taken together, our results suggest that long-term 1,2-DCE exposure impairs the learning and memory capacity in mice, which may be attributed to the disruption of Glu metabolism and the inhibition of CaM- CaMKII-CREB signaling pathway in the hippocampus.

Glutamate-aspartate transporter 1 attenuates oxygen-glucose deprivation-induced injury by promoting glutamate metabolism in primary cortical neurons.

Ischemic stroke is a common cerebral disease. However, the treatment for the disease is limited. Daurian ground squirrel (GS;Spermophilus dauricus), a hibernating mammalian species, is highly tolerant to ischemia. In the present study, GS neurons in a non-hibernating state were found to be more resistant to oxygen-glucose deprivation (OGD), an ischemic model in vitro. We leveraged the differences in the endurance capacity of GS and rats to investigate the mechanisms of resistance to ischemia in GSneurons. We first identified glutamate-aspartate transporter 1 (GLAST) as a cytoprotective factor that contributed to tolerance against OGD injury of GS neurons. The expression of GLAST in GS neurons was much higher than that in rat neurons. Overexpression of GLAST rescued viability in rat neurons, and GS neurons exhibited decreased viability following GLAST knockdown under OGD conditions. Mechanistically, more glutamate was transported into neurons after GLAST overexpression and served as substrates for ATP production. Furthermore, eukaryotic transcription initiation factor 4E binding protein 1 was downregulated by GLAST to rescue neuronal viability. Our findings not only revealed an important molecular mechanism underlying the survival of hibernating mammals but also suggested that neuronal GLAST may be a potential target for ischemic stroke therapy.

Effects of co-exposure to lead and manganese on learning and memory deficits

Lead (Pb) and manganese (Mn) are common neurotoxins. However, individuals are subject to co-exposures in real life, and it is therefore important to study these metals in combination. Weaning Sprague-Dawley rats were given ad libitum access to drinking water solutions containing Pb (100 mg/L), Mn (2.5 mg/mL) or a mixture, and each treatment had its own minocycline (50 mg/(kg•day)) supplement group. The results showed a significant difference in spatial memory and induction levels of hippocampal long-term potentiation (LTP) in all exposure groups when compared with controls. The combined-exposure group exhibited the most pronounced effect when compared with each of the single-metal exposure groups. Microglia displayed activation at day 3 after exposure alone or in combination, while astrocytes showed activation at day 5, accompanied by decreased expression levels of GLAST, GLT-1, and GS. Furthermore, the levels of glutamate in the synaptic cleft increased significantly. When microglial activation was inhibited by minocycline, the activation of astrocytes and the expression of GLAST, GLT-1, and GS were both reversed. In addition, upon minocycline treatment, hippocampal LTP impairment and cognitive injury were significantly alleviated in each of the exposure groups. These results suggest that combined exposure to Pb and Mn can cause greater effects on cognition and synaptic plasticity when compared to single-metal exposure groups. The reason may involve abnormal activation of microglia leading to excessive regulation of astrocytes, resulting in glutamate reuptake dysfunction in astrocytes and leading to perturbed cognition and synaptic plasticity.

Repeated cocaine or methamphetamine treatment alters astrocytic CRF2 and GLAST expression in the ventral midbrain.

Dopamine neurons in the substantia nigra (SN) and ventral tegmental area (VTA) play a central role in the reinforcing properties of abused drugs including methamphetamine and cocaine. Chronic effects of psychostimulants in the SN/VTA also involve non-dopaminergic transmitters, including glutamate and the stress-related peptide corticotropin-releasing factor (CRF). In the SN/VTA, astrocytes express a variety of membrane-bound neurotransmitter receptors and transporters that influence neurotransmission. CRF receptor type 2 (CRF2) activity in the VTA is important for stress-induced relapse and drug-seeking behaviour, but the localization of its effects is incompletely understood. Here, we first identified CRF2 transcript in astrocytes of the SN/VTA using RNA-Seq in Aldh1l1;NuTRAP mice and confirmed it using in situ hybridization (RNAscope) in wild-type mice. We then used immunofluorescence to quantify the astrocytic marker protein S100β, glial-specific glutamate/aspartate transporter GLAST, and CRF2 in the SN/VTA following 12 days of treatment (i.p.) with methamphetamine (3 mg/kg), cocaine (10mg/kg), or saline. We observed a significant decrease in GLAST immunofluorescence in brains of psychostimulant treated mice compared with saline controls. In addition, we observed increased labelling of CRF2 in drug treated groups, a decrease in the number of S100β positive cells, and an increase of co-staining of CRF2 with both S100β and tyrosine hydroxylase (dopamine neurons). Our results suggest a significant interaction between CRF2, GLAST, and astrocytes in the midbrain that emerges with repeated exposure to psychostimulants. These findings provide rationale for future investigation of astrocyte-based strategies for altering cellular and circuit function in response to stress and drug exposure.

Differential effects of inhibitors of PTZ-induced kindling on glutamate transporters and enzyme expression.

Epilepsy is a neurological disorder resulting from abnormal neuronal firing in the brain. Glutamate transporters and the glutamate-glutamine cycle play crucial roles in the development of seizures. In the present study, the correlation of epilepsy with glutamate transporters and enzymes was investigated. Herein, male Wistar rats were randomly allocated into four groups (six animals/group); 35mg/kg pentylenetetrazole (PTZ) was used to induce a kindling model of epilepsy. Once the kindling model was established, animals were treated for 15days with either valproic acid (VPA, 350mg/kg) or ceftriaxone (CEF, 200mg/kg) in addition to the control group receiving saline. After treatment, electrocorticography (ECoG) was performed to record the electrical activity of the cerebral cortex. The glutamate reuptake time (T80 ) was also determined in situ using an in vivo voltammetry. The expression levels of glutamate transporters and enzymes in the M1 and CA3 areas of the brain were determined using a real-time polymerase chain reaction (RT-PCR). ECoG measurements showed that the mean spike number of the PTZ+VPA and PTZ+CEF groups was significantly lower (p<0.05) than that of the PTZ group. Compared with the PTZ group, VPA or CEF treatment decreased the glutamate reuptake time (T80 ). The expression levels of EAAC1, GLT-1, GLAST, glutamine synthetase (GS), and glutaminase were increased in the PTZ group. Treatment with VPA or CEF enhanced the expression levels of GLT-1, GLAST, EAAC1, and GS, whereas the glutaminase expression level was reduced. The current results suggest that VPA or CEF decreases seizure activity by increasing glutamate reuptake by upregulating GLT-1 and GLAST expression, implying a possible mechanism for treating epilepsy. Also, we have suggested a novel mechanism for the antiepileptic activity of VPA via decreasing glutaminase expression levels. To our knowledge, this is the first study to measure the glutamate reuptake time in situ during the seizure (i.e., real-time measurement).

Deficiency in MT5-MMP Supports Branching of Human iPSCs-Derived Neurons and Reduces Expression of GLAST/S100 in iPSCs-Derived Astrocytes.

For some time, it has been accepted that the β-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the β-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer’s disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100β expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.

Inhibiting spinal secretory phospholipase A2 after painful nerve root injury attenuates established pain and spinal neuronal hyperexcitability by altering spinal glutamatergic signaling.

Neuropathic injury is accompanied by chronic inflammation contributing to the onset and maintenance of pain after an initial insult. In addition to their roles in promoting immune cell activation, inflammatory mediators like secretory phospholipase A2 (sPLA2) modulate nociceptive and excitatory neuronal signaling during the initiation of pain through hydrolytic activity. Despite having a known role in glial activation and cytokine release, it is unknown if sPLA2 contributes to the maintenance of painful neuropathy and spinal hyperexcitability later after neural injury. Using a well-established model of painful nerve root compression, this study investigated if inhibiting spinal sPLA2 7 days after painful injury modulates the behavioral sensitivity and/or spinal dorsal horn excitability that is typically evident. The effects of sPLA2 inhibition on altered spinal glutamatergic signaling was also probed by measuring spinal intracellular glutamate levels and spinal glutamate transporter (GLAST and GLT1) and receptor (mGluR5, GluR1, and NR1) expression. Spinal sPLA2 inhibition at day 7 abolishes behavioral sensitivity, reduces both evoked and spontaneous neuronal firing in the spinal cord, and restores the distribution of neuronal phenotypes to those of control conditions. Inhibiting spinal sPLA2 also increases intracellular glutamate concentrations and restores spinal expression of GLAST, GLT1, mGluR5, and GluR1 to uninjured expression with no effect on NR1. These findings establish a role for spinal sPLA2 in maintaining pain and central sensitization after neural injury and suggest this may be via exacerbating glutamate excitotoxicity in the spinal cord.

Early Postnatal Exposure to a Low Dose of Nanoparticulate Silver Induces Alterations in Glutamate Transporters in Brain of Immature Rats.

Due to strong antimicrobial properties, silver nanoparticles (AgNPs) are used in a wide range of medical and consumer products, including those dedicated for infants and children. While AgNPs are known to exert neurotoxic effects, current knowledge concerning their impact on the developing brain is scarce. During investigations of mechanisms of neurotoxicity in immature rats, we studied the influence of AgNPs on glutamate transporter systems which are involved in regulation of extracellular concentration of glutamate, an excitotoxic amino acid, and compared it with positive control—Ag citrate. We identified significant deposition of AgNPs in brain tissue of exposed rats over the post-exposure time. Ultrastructural alterations in endoplasmic reticulum (ER) and Golgi complexes were observed in neurons of AgNP-exposed rats, which are characteristics of ER stress. These changes presumably underlie substantial long-lasting downregulation of neuronal glutamate transporter EAAC1, which was noted in AgNP-exposed rats. Conversely, the expression of astroglial glutamate transporters GLT-1 and GLAST was not affected by exposure to AgNPs, but the activity of the transporters was diminished. These results indicate that even low doses of AgNPs administered during an early stage of life create a substantial risk for health of immature organisms. Hence, the safety of AgNP-containing products for infants and children should be carefully considered.

Astrocytes from cortex and striatum show differential responses to mitochondrial toxin and BDNF: implications for protection of striatal neurons expressing mutant huntingtin

BackgroundEvidence shows significant heterogeneity in astrocyte gene expression and function. We previously demonstrated that brain-derived neurotrophic factor (BDNF) exerts protective effects on whole brain primary cultured rat astrocytes treated with 3-nitropropionic acid (3NP), a mitochondrial toxin widely used as an in vitro model of Huntington’s disease (HD). Therefore, we now investigated 3NP and BDNF effects on astrocytes from two areas involved in HD: the striatum and the entire cortex, and their involvement in neuron survival.MethodsWe prepared primary cultured rat cortical or striatal astrocytes and treated them with BDNF and/or 3NP for 24 h. In these cells, we assessed expression of astrocyte markers, BDNF receptor, and glutamate transporters, and cytokine release. We prepared astrocyte-conditioned medium (ACM) from cortical and striatal astrocytes and tested its effect on a cellular model of HD.ResultsBDNF protected astrocytes from 3NP-induced death, increased expression of its own receptor, and activation of ERK in both cortical and striatal astrocytes. However, BDNF modulated glutamate transporter expression differently by increasing GLT1 and GLAST expression in cortical astrocytes but only GLT1 expression in striatal astrocytes. Striatal astrocytes released higher amounts of tumor necrosis factor-α than cortical astrocytes in response to 3NP but BDNF decreased this effect in both populations. 3NP decreased transforming growth factor-β release only in cortical astrocytes, whereas BDNF treatment increased its release only in striatal astrocytes. Finally, we evaluated ACM effect on a cellular model of HD: the rat striatal neuron cell line ST14A expressing mutant human huntingtin (Q120) or in ST14A cells expressing normal human huntingtin (Q15). Neither striatal nor cortical ACM modified the viability of Q15 cells. Only ACM from striatal astrocytes treated with BDNF and ACM from 3NP + BDNF-treated striatal astrocytes protected Q120 cells, whereas ACM from cortical astrocytes did not.ConclusionsData suggest that cortical and striatal astrocytes respond differently to mitochondrial toxin 3NP and BDNF. Moreover, striatal astrocytes secrete soluble neuroprotective factors in response to BDNF that selectively protect neurons expressing mutant huntingtin implicating that BDNF modulation of striatal astrocyte function has therapeutic potential against neurodegeneration.Graphical abstract

Reciprocal communication between astrocytes and endothelial cells is required for astrocytic glutamate transporter 1 (GLT-1) expression

Astrocytes have diverse functions that are supported by their anatomic localization between neurons and blood vessels. One of these functions is the clearance of extracellular glutamate. Astrocytes clear glutamate using two Na+-dependent glutamate transporters, GLT-1 (also called EAAT2) and GLAST (also called EAAT1). GLT-1 expression increases during synaptogenesis and is a marker of astrocyte maturation. Over 20 years ago, several groups demonstrated that astrocytes in culture express little or no GLT-1 and that neurons induce expression. We recently demonstrated that co-culturing endothelia with mouse astrocytes also induced expression of GLT-1 and GLAST. These increases were blocked by an inhibitor of γ-secretase. This and other observations are consistent with the hypothesis that Notch signaling is required, but the ligands involved were not identified. In the present study, we used rat astrocyte cultures to further define the mechanisms by which endothelia induce expression of GLT-1 and GLAST. We found that co-cultures of astrocytes and endothelia express higher levels of GLT-1 and GLAST protein and mRNA. That endothelia activate Hes5, a transcription factor target of Notch, in astrocytes. Using recombinant Notch ligands, anti-Notch ligand neutralizing antibodies, and shRNAs, we provide evidence that both Dll1 and Dll4 contribute to endothelia-dependent regulation of GLT-1. We also provide evidence that astrocytes secrete a factor(s) that induces expression of Dll4 in endothelia and that this effect is required for Notch-dependent induction of GLT-1. Together these studies indicate that reciprocal communication between astrocytes and endothelia is required for appropriate astrocyte maturation and that endothelia likely deploy additional non-Notch signals to induce GLT-1.

The Expression of GLAST and GLT1 in a Transient Cerebral Ischemia Mongolian Gerbil Model.

PurposeExcitatory amino acid transporters (EAATs) have an indispensable function in the reuptake of extracellular glutamate. To investigate the relationship and the expression of neuronal and astrocytic markers after brain ischemia, the temporal profile of glial EAATs in both peripheral and core regions of the cortex was examined.MethodsTransient common carotid artery occlusion was used to induce unilateral transient forebrain ischemia of Mongolian gerbils, and post-ischemic brains (6 h to 2 w) were collected and prepared for immunohistochemical and Western blotting analysis of glutamine synthetase (GS), GLT-1, GLAST, S100β, and NeuN, and for Alizarin red staining of calcium deposits.ResultsThe expression of GLAST and GLT-1 were significantly escalated at 6 h both in the core and periphery regions, while reduced from 12 h to 2 w in the core region post-ischemia. GS-positive cells increased at 6 h both in the core and periphery regions, while the density of Alizarin red-positive cells increased and peaked at 12 h in the ischemic cortex. The density of S100β-positive cells decreased in the ischemic core and increased in the periphery region. Immunofluorescence staining showed that S100β and TUNEL double-positive cells increased at 12 h in the core region.ConclusionThe results of GLT-1 and GLAST expression in the cortex indicate that their up-regulation was time-dependent and occurred in the acute post-ischemia period, whereas their down-regulation was region-dependent and it is involved in the pathological progress of nerve cell and glial cell death, and has a series of cascade reactions.

Amyloid-ß peptides inhibit the expression of AQP4 and glutamate transporter EAAC1 in insulin-treated C6 glioma cells

Astrocytic aquaporin 4 (AQP4) facilitates glutamate clearance via regulation of the glutamate transporter function, involved in the modulation of brain plasticity and cognitive function to prevent neurodegenerative disorders such as Alzheimer’s disease (AD). In in vitro studies, the C6 rat glioma cell line is a widely applied aging model system to investigate changes in glial cells associated with aging or AD. However, the neurotoxicity mechanism whether AQP4 mediate glutamate uptake in Aβ-stimulated C6 cell remain uncertain. In this study, we examined the effects of Aβ on the expression of AQP4, Glu transporters, Glu uptake, and cell viability in insulin-treated C6 cells. Our results showed that the expression of AQP4 mRNA and protein was significantly enhanced by insulin in older cultures (passage 45), and the expression was inhibited by Aβ at 10 μM. In addition, the cell viability and glutamate uptake in Aβ-treated C6 cells were decreased in dose-dependent manners. GFAP showed similar changes in gene and protein expression patterns as AQP4, but no significant alterations were seen in GLAST expression. In C6 cells, the glutamate transport was found to be EAAC1, not GLT-1. EAAC1 expression was decreased by the treatment of Aβ. Taken together, our findings suggest that C6 cells may have astrocytic characteristics, and the astrocytic cytotoxicity induced by Aβ was mediated by reduction of glutamate uptake through AQP4/EAAC1 pathway in C6 cells. This indicates that C6 glioma cells could be used to study the roles of AQP4 on astrocyte function in AD.

Regionally selective knockdown of astroglial glutamate transporters in infralimbic cortex induces a depressive phenotype in mice.

Elevation of energy metabolism and disturbance of astrocyte number/function in the ventral anterior cingulate cortex (vACC) contributes to the pathophysiology of major depressive disorder (MDD). Functional hyperactivity of vACC may result from reduced astrocytic glutamate uptake and increased neuronal excitation. Here we tested this hypothesis by knocking-down astrocytic glutamate transporter GLAST/GLT-1 expression in mouse infralimbic (IL, rodent equivalent of vACC) or prelimbic (PrL) cortices using RNAi strategies. Unilateral siRNA (small interfering RNA) microinfusion targeting GLAST or GLT-1 in mouse IL induced a moderate (20-30%) and long-lasting (7 days) decrease in their expression. Intra-IL GLAST-/GLT-1 siRNA microinfusion reduced the number of glial fibrillary acidic protein (GFAP)-positive and glutamine synthetase (GS)-positive astrocytes and evoked a depressive-like phenotype reversed by citalopram and ketamine. Intra-IL GLAST or GLT-1 knockdown markedly reduced serotonin (5-HT) release in the dorsal raphe nucleus (DR) and induced an overall reduction of brain-derived neurotrophic factor (BDNF) expression in ipsilateral and contralateral hemispheres. Egr-1 (early growth response protein-1) labeling suggests that both siRNAs enhance the GABAergic tone onto DR 5-HT neurons, leading to an overall decrease of 5-HT function, likely related to the widespread reduction on BDNF expression. Conversely, similar reductions of GLAST and GLT-1 expression in PrL did not induce a depressive-like phenotype. These results suggest that a focal glial change in IL translates into global change of brain activity by virtue of the descending projections from IL to DR and the subsequent attenuation of serotonergic function in forebrain, an effect perhaps related to the varied symptomatology of MDD.

Altered function of the glutamate-aspartate transporter GLAST, a potential therapeutic target in glioblastoma.

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration compared to GLAST-depleted glioma. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than taking up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.

Effect of Adenosine and Adenosine Receptor Antagonists on Retinal Müller Cell Inwardly Rectifying Potassium Channels under Exogenous Glutamate Stimulation.

The vitreousness of glaucoma subjects contains elevated glutamate, and excessive extracellular glutamate is toxic to retinal neurons. Therefore, glutamate clearance is potentially impaired in the retina of glaucoma subjects. Müller cells play an important role in maintaining low extracellular levels of neurotransmitters, such as glutamate. A better understanding of the cross-talk between adenosine and glutamate may provide a better characterization of the regulatory network in Müller cells. Here, Müller cells were purified from the rat retina on postnatal day 5 using the papain digestion method. Application of increasing concentrations of glutamate (0-20 mmol/L) caused a dose-dependent decrease in the expression levels of Kir4.1, Kir2.1, GLAST, and GS. Exogenous adenosine regulated Kir channels and subsequently promoted GLAST and GS expression levels in Müller cells under exogenous glutamate stimulation. These effects were partly dependent on adenosine receptors.