Upregulation Of Glutamate Transporter-1 Research Articles

Ceftriaxone Modulates Ubiquitination of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionic Acid Receptors to Improve Long-Term Potentiation Impairment Induced by Exogenous β-Amyloid in a Glutamate Transporter-1 Dependent Manner.

Α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are crucial for properties of synaptic plasticity, such as long-term potentiation (LTP). LTP impairment can occur early in the onset of Alzheimer's disease (AD). The downregulation or decreased abundance of AMPAR expression in the postsynaptic membrane is closely associated with LTP impairment. Ceftriaxone (Cef) can improve LTP impairment in the early stages of AD in a mouse model. The purpose of this study was to explore the mechanism underlying this process from the aspects of AMPAR expression and ubiquitination degree. In this study, we found that β-amyloid (Aβ) treatment induced hippocampal LTP impairment and AMPAR downregulation and ubiquitination. Cef pretreatment ameliorated Aβ-induced hippocampal LTP impairment, reduced AMPAR ubiquitination, and increased AMPAR expression, especially in the plasma membrane, in Aβ-treated mice. Administration of USP46 siRNA and DHK (a specific blocker of glutamate transporter-1) significantly inhibited the above effects of Cef, suggesting a role for anti-AMPAR ubiquitination and upregulation of glutamate transporter-1 (GLT-1) in the Cef-induced improvements mentioned above. The above findings demonstrate that pretreatment with Cef effectively mitigated Aβ-induced impairment of hippocampal LTP by suppressing the ubiquitination process of AMPARs in a GLT-1-dependent manner. These results provide novel insights into the underlying mechanisms elucidating the anti-AD by Cef.

Marrow Mesenchymal Stem Cell-Derived Exosomes Upregulate Astrocytic Glutamate Transporter-1 Expression via miR-124/mTOR Pathway against Oxygen-Glucose Deprivation/Reperfusion Injury.

Experimental investigations have reported the efficacy of marrow mesenchymal stem cell-derived exosomes (MSC-Exos) for the treatment of ischemic stroke. The therapeutic mechanism, however, is still unknown. The purpose of the study is to show whether MSC-Exos increases astrocytic glutamate transporter-1 (GLT-1) expression in response to ischemic stroke and to investigate further mechanisms. An in vitro ischemia model (oxygen-glucose deprivation/reperfusion, OGD/R) was used. MSC-Exos was identified by Western blot (WB) and transmission electron microscopy (TEM). To further investigate the mechanism, MSC-Exos, miR-124 inhibitor, and mimics, and a mTOR pathway inhibitor (rapamycin, Rap) were used. The interaction between GLT-1 and miR-124 was analyzed by luciferase reporter assay. The GLT-1 RNA expression and miR-124 was assessed by quantitative real-time polymerase chain reaction (qRTPCR). The protein expressions of GLT-1, S6, and pS6 were detected by WB. Results demonstrated that MSC-Exos successfully inhibited the decrease of GLT-1 and miR-124 expression and the increase of pS6 expression in astrocytes after OGD/R. miR-124 inhibitor suppressed the effect of MSC-Exos on GLT-1 upregulation after OGD/R. Rapamycin notably decreased pS6 expression with significantly higher GLT-1 expression in astrocytes injured by OGD/R. Luciferase activity of the reporter harboring the wild-type or mutant GLT-1 3'UTR was not inhibited by miR-124 mimics. Further results showed that the inhibiting effect of MSC-Exos on pS6 expression and promoting effect of MSC-Exos on GLT-1 expression could be reversed by miR-124 inhibitor after OGD/R; meanwhile, the above conditions could be reversed again by rapamycin. Results show that miR-124 and the mTOR pathway are involved in regulation of MSC-Exos on GLT-1 expression in astrocytes injured by OGD/R. miR-124 does not directly target GLT-1. MSC-Exos upregulates GLT-1 expression via the miR-124/mTOR pathway in astrocytes injured by OGD/R.

Liver Metabolomics and Inflammatory Profiles in Mouse Model of Fentanyl Overdose Treated with Beta-Lactams.

Fentanyl is a highly potent opioid analgesic that is approved medically to treat acute and chronic pain. There is a high potential for overdose-induced organ toxicities, including liver toxicity, and this might be due to the increase of recreational use of opioids. Several preclinical studies have demonstrated the efficacy of beta-lactams in modulating the expression of glutamate transporter-1 (GLT-1) in different body organs, including the liver. The upregulation of GLT-1 by beta-lactams is associated with the attenuation of hyperglutamatergic state, which is a characteristic feature of opioid use disorders. A novel experimental beta-lactam compound with no antimicrobial properties, MC-100093, has been developed to attenuate dysregulation of glutamate transport, in part by normalizing GLT-1 expression. A previous study showed that MC-100093 modulated hepatic GLT-1 expression with subsequent attenuation of alcohol-increased fat droplet content in the liver. In this study, we investigated the effects of fentanyl overdose on liver metabolites, and determined the effects of MC-100093 and ceftriaxone in the liver of a fentanyl overdose mouse model. Liver samples from control, fentanyl overdose, and fentanyl overdose ceftriaxone- or MC-100093-treated mice were analyzed for metabolomics using gas chromatography-mass spectrometry. Heatmap analysis revealed that both MC-100093 and ceftriaxone attenuated the effects of fentanyl overdose on several metabolites, and MC-100093 showed superior effects. Statistical analysis showed that MC-100093 reversed the effects of fentanyl overdose in some metabolites. Moreover, enrichment analysis revealed that the altered metabolites were strongly linked to the glucose-alanine cycle, the Warburg effect, gluconeogenesis, glutamate metabolism, lactose degradation, and ketone body metabolism. The changes in liver metabolites induced by fentanyl overdose were associated with liver inflammation, an effect attenuated with ceftriaxone pre-treatments. Ceftriaxone normalized fentanyl-overdose-induced changes in liver interleukin-6 and cytochrome CYP3A11 (mouse homolog of human CYP3A4) expression. Our data indicate that fentanyl overdose impaired liver metabolites, and MC-100093 restored certain metabolites.

Bone marrow stromal cells-derived exosomes reduce neurological damage in traumatic brain injury through the miR-124-3p/p38 MAPK/GLT-1 axis

BackgroundMounting evidence indicates that stem cell-derived exosomal miRNAs have therapeutic effects on traumatic brain injury (TBI). This research is focused on exploring the molecular processes of miR-124-3p obtained from bone marrow stromal cells-derived exosomes (BMSCs-Exos) in attenuating posttraumatic glutamate-mediated excitotoxicity. MethodsWe created a TBI rat model and analyzed the expression profile of miRNA through miRNA microarray. The miR-124-3p and p38 MAPK levels were analyzed utilizing RT-qPCR and western blotting. Dual-luciferase reporter (DLR) assay showed the targeting relationship between miR-124-3p and p38 MAPK. We subsequently conducted a TUNEL assay and flow cytometry to evaluate the neuronal apoptotic rate in an in vitro glutamate-mediated excitotoxicity model treated with BMSCs-Exos enriched with miR-124-3p (BMSCs-ExosmiR-124-3p). Moreover, the levels of p38 MAPK and glutamate transporter-1 (GLT-1) were measured by western blotting. Furthermore, BMSCs-ExosmiR-124-3p were administered to the TBI rats, and their neuroprotective effects were observed using western blotting, immunohistochemistry, histological staining, magnetic resonance imaging (MRI), and Morris water maze (MWM). ResultsThe results revealed that the brains of TBI rats exhibited lowered miR-124-3p and enhanced p38 MAPK levels. DLR assay demonstrated miR-124-3p's role in targeting p38 MAPK and negatively regulating its expression. In vitro and in vivo studies confirmed that BMSCs-ExosmiR-124-3p attenuated glutamate-mediated excitotoxicity by downregulating p38 MAPK and upregulating GLT-1 expressions via transferring exosomal miR-124-3p. Moreover, histopathological evaluation and MRI results showed that BMSCs-ExosmiR-124-3p remarkably alleviated neuronal cell death and minimized the lesion volumes post-TBI. MWM outcomes illustrated that BMSCs-ExosmiR-124-3p treatment could substantially improve neurological function post-TBI. Furthermore, the effects of treatment with p38 MAPK inhibitor SB203580 were similar to BMSCs-ExosmiR-124-3p. ConclusionOverall, the outcomes of the current report highlighted that BMSCs-ExosmiR-124-3p can lead to the upregulation of GLT-1 in TBI rat models by inhibiting the p38 MAPK signaling pathway, hence alleviating glutamate-mediated excitotoxicity and attenuating neurological damage post-TBI.

Repeated sevoflurane exposures inhibit neurogenesis by inducing the upregulation of glutamate transporter 1 in astrocytes.

Sevoflurane is a widely used general anaesthetic in paediatric patients. Although repeated sevoflurane exposure is known to cause neurodevelopmental disorders in children, the mechanism of this neurotoxicity remains largely unknown. Herein, we investigated the role of glutamate transporter 1 (GLT1) in sevoflurane-induced decreased neurogenesis. Neonatal rat pups (postnatal Day 7, PN7) were exposed to 3% sevoflurane for 2 h for three consecutive days. Neuron loss and decreased neurogenesis have been observed in the neonatal rat brain, along with decreased number of astrocytes. Apoptotic astrocytes were observed after repeated sevoflurane exposure in vitro, resulting in decreased levels of brain-derived neurotrophic factor (BDNF). Calcium overload was observed in astrocytes after repeated sevoflurane exposure, in addition to upregulation of GLT1. Inhibition of GLT1 activity ameliorates repeated sevoflurane exposure-induced cognitive deficits in adult rats. Mechanically, the upregulation of GLT1 was caused by the activation of mRNA translation. RNA-sequencing analysis further confirmed that translation-related genes were activated by repeated sevoflurane exposure. These results indicate that cognitive deficits caused by repeated sevoflurane exposure during PN7-9 are triggered decreased neurogenesis. The proposed underlying mechanism involves upregulation of apoptosis in astrocytes induced by GLT1; therefore, we propose GLT1 as a potential pharmacological target for brain injury in paediatric practice.

Exosomes derived from bone marrow mesenchymal stem cells attenuate neurological damage in traumatic brain injury by alleviating glutamate-mediated excitotoxicity

BackgroundTraumatic brain injury (TBI) is one of the major contributors to disability and death worldwide. Glutamate-mediated excitotoxicity, one of the secondary injuries occurring after TBI, leads to extreme neuronal apoptosis, and can be a potential target for intervention. Bone marrow mesenchymal stem cells-derived exosomes (BMSCs-Exos) have demonstrated neuroprotective effects on TBI. However, their precise role and the underlying mechanism by which they regulate glutamate-mediated excitotoxicity have not yet been determined. Therefore, this study aimed to determine whether BMSCs-Exos alleviate glutamate excitotoxicity post-TBI and their associated mechanism. MethodsBMSCs-Exos were extracted from the BMSCs incubation medium and identified by transmission electron microscopy, nanoparticle trafficking analysis, and western blotting. The neuroprotective effects of BMSCs-Exos on glutamate excitotoxicity were investigated in the glutamate-mediated excitotoxicity neuronal cell model and the TBI rat model (TBI induced by controlled cortical impact) using western blotting and TUNEL assay. Cortical lesion samples were collected post-TBI on day-1 and day-14 to study histology. In addition, cortical lesion volume on days 1, 3 and 7 following TBI was determined using T2-weighted magnetic resonance imaging (MRI), and cognitive function was assessed at 4 weeks following TBI using the Morris water maze (MWM) test. ResultsBMSCs-Exos were observed to be spherical with a mean diameter of 109.9 nm, and expressed exosomal markers CD9, CD81 and TSG101. BMSCs-Exos were efficiently endocytosed by astrocytes after co-incubation for 24 h. In vitro studies revealed that 125 μM of glutamate significantly induced neuronal apoptosis, which was attenuated by BMSCs-Exos in astrocyte–neuron co-cultures. This attenuation was mediated by the upregulation of glutamate transporter-1 (GLT-1) level and the downregulation of p-p38 MAPK level in astrocytes. Similar results were obtained in vivo, wherein we verified that PKH67-labeled BMSCs-Exos administered intravenously could reach the perilesional cortex crossing the blood-brain barrier and significantly reduce glutamate levels in the perilesional cortex of the TBI rat, accompanied by increased GLT-1 level and downregulation in p-p38 MAPK level. Additionally, western blotting and TUNEL staining also revealed that BMSCs-Exos significantly downregulated the expression of pro-apoptosis markers, including cleaved caspase-3 and cleaved caspase-9, and attenuated neuronal apoptosis following TBI. Immunohistochemical analysis and Nissl staining showed that BMSCs-Exos significantly increased GLT-1-positive cells, and the number of apoptotic neurons decreased in the perilesional cortex. Moreover, MRI and MWM results revealed that BMSCs-Exos significantly minimized cortical lesion volume and ameliorated cognitive function after TBI. The underlying neuroprotective mechanism of BMSCs-Exos may be due to an increase in GLT-1 level in astrocytes by blocking the p38 MAPK signaling pathway. ConclusionTaken together, our findings demonstrate that the implementation of BMSCs-Exos may be an effective prospective therapy for attenuating post-TBI neurological damage.

Ceftriaxone as a Novel Therapeutic Agent for Hyperglutamatergic States: Bridging the Gap Between Preclinical Results and Clinical Translation.

Dysregulation of glutamate homeostasis is a well-established core feature of neuropsychiatric disorders. Extracellular glutamate concentration is regulated by glutamate transporter 1 (GLT-1). The discovery of a beta-lactam antibiotic, ceftriaxone (CEF), as a safe compound with unique ability to upregulate GLT-1 sparked the interest in testing its efficacy as a novel therapeutic agent in animal models of neuropsychiatric disorders with hyperglutamatergic states. Indeed, more than 100 preclinical studies have shown the efficacy of CEF in attenuating the behavioral manifestations of various hyperglutamatergic brain disorders such as ischemic stroke, amyotrophic lateral sclerosis (ALS), seizure, Huntington’s disease, and various aspects of drug use disorders. However, despite rich and promising preclinical data, only one large-scale clinical trial testing the efficacy of CEF in patients with ALS is reported. Unfortunately, in that study, there was no significant difference in survival between placebo- and CEF-treated patients. In this review, we discussed the translational potential of preclinical efficacy of CEF based on four different parameters: (1) initiation of CEF treatment in relation to induction of the hyperglutamatergic state, (2) onset of response in preclinical models in relation to onset of GLT-1 upregulation, (3) mechanisms of action of CEF on GLT-1 expression and function, and (4) non-GLT-1-mediated mechanisms for CEF. Our detailed review of the literature brings new insights into underlying molecular mechanisms correlating the preclinical efficacy of CEF. We concluded here that CEF may be clinically effective in selected cases in acute and transient hyperglutamatergic states such as early drug withdrawal conditions.

Intrathecal Administration of the α1 Adrenergic Antagonist Phentolamine Upregulates Spinal GLT-1 and Improves Mirror Image Pain in SNI Model Rats.

Mirror image pain (MIP) is a type of extraterritorial pain that results in contralateral pain or allodynia. Glutamate transporter-1 (GLT-1) is expressed in astrocytes and plays a role in maintaining low glutamate levels in the synaptic cleft. Previous studies have shown that GLT-1 dysfunction induces neuropathic pain. Our previous study revealed bilateral GLT-1 downregulation in the spinal cord of a spared nerve injury (SNI) rat. We hypothesized that spinal GLT-1 is involved in the mechanism of MIP. We also previously demonstrated noradrenergic GLT-1 regulation. Therefore, this study aimed to investigate the effect of an α1 adrenergic antagonist on the development of MIP. Rats were subjected to SNI. Changes in pain behavior and GLT-1 protein levels in the SNI rat spinal cords were then examined by intrathecal administration of the α1 adrenergic antagonist phentolamine, followed by von Frey test and western blotting. SNI resulted in the development of MIP and bilateral downregulation of GLT-1 protein in the rat spinal cord. Intrathecal phentolamine increased contralateral GLT-1 protein levels and partially ameliorated the 50% paw withdrawal threshold in the contralateral hind paw. Spinal GLT-1 upregulation by intrathecal phentolamine ameliorates MIP. GLT-1 plays a role in the development of MIPs.

Role of suppressing GLT-1 and xCT in ceftriaxone-induced attenuation of relapse-like alcohol drinking in alcohol-preferring rats.

Alcohol dependence results in long-lasting neuroadaptive changes in meso-corticolimbic system, especially in the nucleus accumbens (NAc), which drives relapse-like ethanol drinking upon abstinence or withdrawal. Within NAc, altered glutamate homeostasis is one of the neuroadaptive changes caused by alcohol dependence. Accumbal glutamate homeostasis is tightly maintained through glutamate transporter 1 (GLT-1) and cystine-glutamate antiporter (xCT). But the role of GLT-1 and xCT in relapse-like ethanol drinking is poorly understood. Here, we used alcohol-preferring (P) rats in relapse-like ethanol drinking paradigm to (a) determine the effect of relapse-like ethanol drinking on gene and protein expression of GLT-1 and xCT in NAc, measured by quantitative polymerase chain reaction (qPCR) and Western blot, respectively; (b) examine if glutamate uptake is affected by relapse-like ethanol drinking in NAc, measured by radioactive glutamate uptake assay; (c) elucidate if upregulation of either/both GLT-1 or/and xCT through ceftriaxone is/are required to attenuate relapse-like ethanol drinking. The GLT-1 or xCT protein expression was suppressed during ceftriaxone treatments through microinjection of GLT-1/xCT anti-sense vivo-morpholinos. We found that relapse-like ethanol drinking did not affect the gene and protein expression of GLT-1 and xCT in NAc. The glutamate uptake was also unaltered. Ceftriaxone (200 mg/kg body weight, i.p.) treatments during the last 5 days of abstinence attenuated relapse-like ethanol drinking. The suppression of GLT-1 or xCT expression prevented the ceftriaxone-induced attenuation of relapse-like ethanol drinking. These findings confirm that upregulation of both GLT-1 and xCT within NAc is crucial for ceftriaxone-mediated attenuation of relapse-like ethanol drinking.

The Regulation of Glutamate Transporter 1 in the Rapid Antidepressant-Like Effect of Ketamine in Mice.

Accumulating evidence suggests that glutamate clearance plays a critical role in the pathophysiology and treatment of depression. Preclinical and clinical studies have demonstrated that ketamine provides an immediate and sustained antidepressant effect. However, the precise mechanism of its action remains to be elucidated. Glutamate transporter 1 (GLT1) participates in glutamate clearance; therefore, we hypothesized that GLT1 may play an important role in the antidepressant effect of ketamine. In this study, we determined that GLT1 inhibition blocks the antidepressant-like properties of ketamine and alters the phosphorylation of the mammalian target of rapamycin (mTOR) in the prefrontal cortex (PFC). Our results show that pretreatment with dihydrokainic acid (DHK), a GLT1 inhibitor, alleviated the antidepressant-like effect of ketamine, and decreased the level of phosphorylated mTOR (pmTOR) in mice (which is normally upregulated by ketamine). In addition, inhibition of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor and L-type voltage-dependent calcium channel (L-VDCC) significantly abolished the antidepressant-like effect of ketamine. Moreover, inhibition of L-VDCC significantly blocked the upregulation of GLT1 and BDNF in the PFC of mice. The inhibition of the AMPA receptor only significantly alleviated BDNF. Our results provide insight into the role of GLT1 as the critical presynaptic molecule participating in the pathophysiological mechanism of depression and contributing to the antidepressant-like effect of ketamine. In addition, our study confirms that both AMPA receptor and L-VDCC are crucial factors in the immediate antidepressant-like effect of ketamine.

Sulbactam improves binding property and uptake capacity of glutamate transporter-1 and decreases glutamate concentration in the CA1 region of hippocampus of global brain ischemic rats.

Glutamate transporter-1 (GLT-1) removes most glutamate in the synaptic cleft. Sulbactam confers neuronal protection against ischemic insults in the hippocampal CA1 region accompanied by the upregulation of GLT-1 expression in rats. The present study further investigates the effect of sulbactam on the binding property and uptake capacity of GLT-1 for glutamate, and the change in extracellular glutamate concentration in the hippocampal CA1 region of rats with global brain ischemia. The binding property and uptake capacity of GLT-1 were measured using a radioligand binding and uptake assay, respectively, with L-3H-glutamate. The extracellular glutamate concentration was detected using microdialysis and high-performance liquid chromatography-mass spectrometry. Neuropathological evaluation was performed based on thionin staining. It was shown that sulbactam pre-treatment changed GLT-1 binding property, including increased Bmax and decreased Kd values, increased GLT-1 uptake capacity for glutamate, and inhibited the elevation of extracellular glutamate concentration in rats with global cerebral ischemia. These effects of sulbactam were accompanied by its neuronal protection on the hippocampal CA1 neurons against delayed neuronal death resulted from ischemic insult. Furthermore, administration of GLT-1 antisense oligodeoxynucleotides, which inhibited the expression of GLT-1, blocked the aforementioned sulbactam-related effects, which suggested that GLT-1 upregulation mediated the above effect although other mechanisms independent of the upregulation of GLT-1 expression could not be excluded. It could be concluded that sulbactam improves the binding property and uptake capacity of GLT-1 for glutamate and then reduces the glutamate concentration and excitotoxicity during global cerebral ischemia, which contributes to the neuroprotection of sulbactam against brain ischemia.

Targeted overexpression of glutamate transporter-1 reduces seizures and attenuates pathological changes in a mouse model of epilepsy

Astrocytic glutamate transporters are crucial for glutamate homeostasis in the brain, and dysregulation of these transporters can contribute to the development of epilepsy. Glutamate transporter-1 (GLT-1) is responsible for the majority of glutamate uptake in the dorsal forebrain and has been shown to be reduced at epileptic foci in patients and preclinical models of temporal lobe epilepsy (TLE). Current antiepileptic drugs (AEDs) work primarily by targeting neurons directly through suppression of excitatory neurotransmission or enhancement of inhibitory neurotransmission, which can lead to both behavioral and psychiatric side effects. This study investigates the therapeutic capacity of astrocyte-specific AAV-mediated GLT-1 expression in the intrahippocampal kainic acid (IHKA) model of TLE. In this study, we used Western blot analysis, immunohistochemistry, and long-term-video EEG monitoring to demonstrate that cell-type-specific upregulation of GLT-1 in astrocytes is neuroprotective at early time points during epileptogenesis, reduces seizure frequency and total time spent in seizures, and eliminates large behavioral seizures in the IHKA model of epilepsy. Our findings suggest that targeting glutamate uptake is a promising therapeutic strategy for the treatment of epilepsy.

The Effect of GLT-1 Upregulation on Extracellular Glutamate Dynamics.

Pharmacological upregulation of glutamate transporter-1 (GLT-1), commonly achieved using the beta-lactam antibiotic ceftriaxone, represents a promising therapeutic strategy to accelerate glutamate uptake and prevent excitotoxic damage in neurological conditions. While excitotoxicity is indeed implicated in numerous brain diseases, it is typically restricted to select vulnerable brain regions, particularly in early disease stages. In healthy brain tissue, the speed of glutamate uptake is not constant and rather varies in both an activity- and region-dependent manner. Despite the widespread use of ceftriaxone in disease models, very little is known about how such treatments impact functional measures of glutamate uptake in healthy tissue, and whether GLT-1 upregulation can mask the naturally occurring activity-dependent and regional heterogeneities in uptake. Here, we used two different compounds, ceftriaxone and LDN/OSU-0212320 (LDN), to upregulate GLT-1 in healthy wild-type mice. We then used real-time imaging of the glutamate biosensor iGluSnFR to investigate functional consequences of GLT-1 upregulation on activity- and regional-dependent variations in glutamate uptake capacity. We found that while both ceftriaxone and LDN increased GLT-1 expression in multiple brain regions, they did not prevent activity-dependent slowing of glutamate clearance nor did they speed basal clearance rates, even in areas characterized by slow uptake (e.g., striatum). Unexpectedly, ceftriaxone but not LDN decreased glutamate release in the cortex, suggesting that ceftriaxone may alter release properties independent of its effects on GLT-1 expression. In sum, our data demonstrate the complexities of glutamate uptake by showing that GLT-1 expression does not necessarily translate to accelerated uptake. Furthermore, these data suggest that the mechanisms underlying activity- and regional-dependent differences in glutamate dynamics are independent of GLT-1 expression levels.

Pharmacological upregulation of GLT-1 alleviates the cognitive impairments in the animal model of temporal lobe epilepsy.

It is known that hippocampal epileptogenesis is accompanied by hyperexcitability, glutamate-related neuronal dysfunctions and consequently cognitive deficits. However, the neuroprotective role of astrocytic glutamate uptake through the Glutamate Transporter-1 (GLT-1) remains to be unknown in these processes. Therefore, to assess the effect of glutamate uptake, pharmacological upregulation of GLT-1 using ceftriaxone administration (200 mg/kg/day, i.p, 5 days) was utilized in Li-PIL animal models of temporal lobe epilepsy (TLE). Glutamate concentration and glutamine synthetase activity were analyzed using biochemical assays. In addition, GLT-1 gene expression was assessed by RT-qPCR. Finally, cognitive function was studied using Morris water maze (MWM) test and novel object recognition task (NORT). Our results demonstrated that the acute phase of epileptogenesis (first 72 hours after Status Epilepticus) was accompanied by an increase in the hippocampal glutamate and downregulation of GLT-1 mRNA expression compared to controls. Ceftriaxone administration in epileptic animals led to a reduction of glutamate along with elevation of the level of glutamine synthetase activity and GLT-1 expression in the acute phase. In the chronic phase of epileptogenesis (4 weeks after Status Epilepticus), glutamate levels and GLT-1 expression were decreased compared to controls. Ceftriaxone treatment increased the levels of GLT-1 expression. Furthermore, impaired learning and memory ability in the chronic phase of epileptogenesis was rescued by Ceftriaxone administration. This study shows that astrocytic glutamate uptake can profoundly impact the processes of hippocampal epileptogenesis through the reduction of glutamate-induced excitotoxicity and consequently rescuing of cognitive deficits caused by epilepsy.

Photobiomodulation Therapy Ameliorates Glutamatergic Dysfunction in Mice with Chronic Unpredictable Mild Stress-Induced Depression.

Accumulating evidence indicates that dysfunction of the glutamatergic neurotransmission has been widely involved in the pathophysiology and treatment of depression. Photobiomodulation therapy (PBMT) has been demonstrated to regulate neuronal function both in vitro and in vivo. Herein, we aim to investigate whether the antidepressant phenotype of PBMT is associated with the improvement of glutamatergic dysfunction and to explore the mechanisms involved. Results showed that PBMT decreased extracellular glutamate levels via upregulation of glutamate transporter-1 (GLT-1) and rescued astrocyte loss in the cerebral cortex and hippocampus, which also alleviated dendritic atrophy and upregulated the expression of AMPA receptors on the postsynaptic membrane, ultimately exhibiting behaviorally significant antidepressant effects in mice exposed to chronic unpredictable mild stress (CUMS). Notably, PBMT also obtained similar antidepressant effects in a depressive mouse model subcutaneously injected with corticosterone (CORT). Evidence from in vitro mechanistic experiments demonstrated that PBMT treatment significantly increased both the GLT-1 mRNA and protein levels via the Akt/NF-κB signaling pathway. NF-κB-regulated transcription was in an Akt-dependent manner, while inhibition of Akt attenuated the DNA-binding efficiency of NF-κB to the GLT-1 promoter. Importantly, in vitro, we further found that PKA activation was responsible for phosphorylation and surface levels of AMPA receptors induced by PBMT, which is likely to rescue excitatory synaptic transmission. Taken together, our research suggests that PBMT as a feasible therapeutic approach has great potential value to control the progression of depression.

Activation of p38 MAPK participates in the sulbactam-induced cerebral ischemic tolerance mediated by glial glutamate transporter-1 upregulation in rats

Our previous studies have shown that sulbactam can play a neuroprotection role in hippocampal neurons by upregulating the expression and function of glial glutamate transporter-1 (GLT-1) during ischemic insult. Here, using rat global cerebral ischemia model, we studied in vivo the role of p38 mitogen-activated protein kinases (MAPK) in the sulbactam-induced GLT-1 upregulation and neuroprotection against ischemia. The hippocampal CA1 field was selected as observing target. The expressions of phosphorylated-p38 MAPK and GLT-1 were assayed with western blot analysis and immunohistochemistry. The condition of delayed neuronal death (DND) was assayed with neuropathological evaluation under thionin staining. It was shown that administration of sulbactam protected CA1 hippocampal neurons against ischemic insult accompanied with significantly upregulation in the expressions of phosphorylated-p38 MAPK and GLT-1. The time course analysis showed that sulbactam activated p38 MAPK before the GLT-1 upregulation in either normal or global cerebral ischemic rats. Furthermore, inhibiting p38 MAPK activation by SB203580 blocked the GLT-1 upregulation and neuroprotection induced by sulbactam. The above results suggested that p38 MAPK, at least partly, participated in the sulbactam-induced brain tolerance to ischemia mediated by GLT-1 upregulation in rats.

Effect of Modulation of the Astrocytic Glutamate Transporters' Expression on Cocaine-Induced Reinstatement in Male P Rats Exposed to Ethanol.

Reinforcing properties of ethanol and cocaine are mediated in part through the glutamatergic system. Extracellular glutamate concentration is strictly maintained through several glutamate transporters, such as glutamate transporter 1 (GLT-1), cystine/glutamate transporter (xCT) and glutamate aspartate transporter (GLAST). Previous findings revealed that cocaine and ethanol exposure downregulated GLT-1 and xCT, and that β-lactam antibiotics restored their expression. In this study, we investigated the effect of ampicillin/sulbactam (AMP/SUL) (200mg/kg, i.p.), a β-lactam antibiotic, on cocaine-induced reinstatement and locomotor activity in male alcohol preferring (P) rats using free choice ethanol (15 and 30%, v/v) and water. We also investigated the effect of co-exposure to ethanol and cocaine (20mg/kg, i.p.) on GLT-1, xCT and GLAST expression in the nucleus accumbens (NAc) core, NAc shell and dorsomedial prefrontal cortex (dmPFC). Cocaine exposure decreased ethanol intake and preference. Cocaine and ethanol co-exposure acquired place preference and increased locomotor activity compared to ethanol-exposed rats. GLT-1 and xCT expression were downregulated after cocaine and ethanol co-exposure in the NAc core and shell, but not in dmPFC. AMP/SUL attenuated reinstatement to cocaine as well attenuated the decrease in locomotor activity and ethanol intake and preference. These effects were associated with upregulation of GLT-1 and xCT expression in the NAc core/shell and dmPFC. GLAST expression was not affected after ethanol and cocaine co-exposure or AMP/SUL treatment. Our findings demonstrate that astrocytic glutamate transporters within the mesocorticolimbic area are critical targets in modulating cocaine-seeking behavior while being consuming ethanol.

Hippocampal Synaptic Dysfunction in a Mouse Model of Huntington Disease Is Not Alleviated by Ceftriaxone Treatment.

Glutamate transporters, particularly glutamate transporter 1 (GLT-1), help to prevent the adverse effects associated with glutamate toxicity by rapidly clearing glutamate from the extracellular space. Since GLT-1 expression and/or function are reduced in many neurodegenerative diseases, upregulation of GLT-1 is a favorable approach to treat the symptoms of these diseases. Ceftriaxone, a β-lactam antibiotic reported to increase GLT-1 expression, can exert neuroprotective effects in a variety of neurodegenerative diseases; however, many of these diseases do not exhibit uniform brain pathology. In contrast, as a drug that readily crosses the blood–brain barrier, ceftriaxone administration is likely to increase GLT-1 levels globally throughout the neuroaxis. In Huntington disease (HD), low GLT-1 expression is observed in the striatum in postmortem tissue and animal models. While ceftriaxone was reported to increase striatal GLT-1 and ameliorate the motor symptoms in a mouse model of HD, the extrastriatal effects of ceftriaxone in HD are unknown. Using electrophysiology and high-speed imaging of the glutamate biosensor iGluSnFR, we quantified real-time glutamate dynamics and synaptic plasticity in the hippocampus of the Q175FDN mouse model of HD, following intraperitoneal injections of either saline or ceftriaxone. We observed an activity-dependent increase in extracellular glutamate accumulation within the HD hippocampus, which was not the result of reduced GLT-1 expression. Surprisingly, ceftriaxone had little effect on glutamate clearance rates and negatively impacted synaptic plasticity. These data provide evidence for glutamate dysregulation in the HD hippocampus but also caution the use of ceftriaxone as a treatment for HD.

Aquaporin 4 knockout increases complete freund's adjuvant-induced spinal central sensitization

Growing evidence suggests a critical role of astrocytes for pain regulation. The water channel protein aquaporin 4 (AQP4), a functional regulator of astrocytes, is involved in various neurological disorders. However, the pathophysiological roles of AQP4 in pain conditions remain unclear. In the present study, we investigated the effect of AQP4 gene knockout in central sensitization induced by complete Freund's adjuvant (CFA). The behavioral analysis revealed that mechanical allodynia and thermal hyperalgesia were more severe in AQP4 null mice than those of wild-type controls over the course of 11 days following CFA intraplantar injection. CFA caused activation of astrocytes with upregulated expression levels of AQP4 and glutamate transporter 1 (GLT1) in the dorsal horn of the spinal cord. AQP4 deficiency reduced GLT1 up-regulation, causing persistent expression of the neuronal activation marker Fos within superficial dorsal horn neurons, including glutamatergic neurons. However, AQP4 deletion did not affect CFA-evoked proinflammatory cytokine expression in the spinal cord. Together, these results have shown that AQP4 absence intensifies CFA-induced spinal central sensitization, which is associated with reduced compensatory up-regulation of GLT1, subsequently increasing glutamatergic overexcitation. Therefore, targeting spinal cord AQP4 may serve as a potential strategy for treatment of peripheral inflammation-evoked hyperalgesia.

Upregulation of GLT-1 via PI3K/Akt Pathway Contributes to Neuroprotection Induced by Dexmedetomidine.

Perioperative ischemic stroke usually leads to neurological dysfunction caused by neuron death. During the ischemic condition, excitotoxity due to extracellular glutamate accumulation is a main mechanism of neuron damage. The clearance of glutamate mainly depends on glutamate transporter-1 (GLT-1) which is expressed in astrocytes. Dexmedetomidine, an α2 adrenergic receptor agonist, is proved to induce neuroprotection. This study was set out to investigate the glutamate-related mechanism involved in the neuroprotective effect of dexmedetomidine. Middle cerebral artery occlusion (MCAO) was used as a model of ischemic stroke in our study. We determined Neurological deficit scores (NDS) and Magnetic resonance imaging (MRI) at three points (2, 6, and 24 h) after middle cerebral artery occlusion (MCAO) to evaluate the neuroprotective effect of dexmedetomidine. Besides, we performed western blot (6 and 24 h after MACO) and immunofluorescent staining (24 h after MCAO) to observe the expression of GLT-1. The effect and mechanism of dexmedetomidine on GLT-1 in primary cultured astrocytes were investigated using western blot and RT-PCR. Our results showed that pretreatment with dexmedetomidine improved NDS and reduced infarct volume as well as upregulating GLT-1 expression. Furthermore, using Atipamezole and LY294002, we found that dexmedetomidine significantly increased GLT-1 levels in astrocytes via activating α2 adrenergic receptor and PI3K/AKT pathway both in vitro and in vivo study. Overall, our present study indicated that dexmedetomidine had neuroprotective effects on ischemia stroke and upregulation of GLT-1 levels by PI3K/AKT dependent pathway might be the potential mechanism.