High-affinity Glutamate Transporters Research Articles

Abstract 6993: SLC1A4 is associated with metabolic reprogramming in triple-negative breast cancer

Abstract The solute carrier protein (SLC) family is transporters as integral membrane proteins that transport solutes in an ATP-independent manner. The SLC1 family consists of five high-affinity glutamate transporters and two neutral amino acid transporters, among which SLC1A4 transports the neutral amino acids and is known to participate in various biological processes, including metabolism and tumorigenesis. However, the relationship between SLC1A4 and breast cancer remains unclear. Here, we aimed to verify the role of SLC1A4 in triple-negative breast cancer (TNBC). To reveal the characteristics of SLC1A4 in TNBC cell lines, five cell lines (HCC38/OE, HCC1395/OE, HCC1937/OE, MDA-MB-468/KD, and Hs578T/KD) that regulate gene expression of SLC1A4 were established using a lentiviral system. The two major energy-producing pathways, glycolysis and oxidative phosphorylation, were measured according to SLC1A4 gene expression. In addition, cell proliferation, cell migration, and cell invasion ability were confirmed upon overexpression or knock-down of SLC1A4. To confirm cancer stem cell function according to SLC1A4 gene expression, ALDH activity and sphere formation ability were evaluated. To ensure transcript changes due to SLC1A4 expression, RNA sequencing was performed. Anti-cancer drug (cisplatin, carboplatin, paclitaxel, and doxorubicin) sensitivity was also tested. The result showed that energy production efficiency was significantly reduced in cells where SLC1A4 expression was suppressed. In addition, in cells overexpressing the SLC1A4 gene, the presence of SLC1A4 was confirmed to increase cell proliferation, migration, and invasion ability significantly. Transcriptome analysis results showed notable changes in genes related to signaling receptor binding. Regarding material transport, to investigate whether there was a change in amino acid uptake ability according to SLC1A4 gene expression, no difference was observed. In the drug responses, doxorubicin showed a significant sensitive response in the absence of SLC1A4 gene expression. Taken together, SLC1A4 is a potential oncogene that not only induces rapid proliferation, aggressive invasion, and migration through metabolic reprogramming but also contributes to increasing cancer stem cell capabilities. In addition, it shows the sensitive response to ADR when SLC1A4 is low or absent, so it can be used as a treatment strategy for TNBC. Citation Format: Soo Young Park, Da Sol Kim, Hayeon Kim, Min Ji Song, Han Suk Ryu. SLC1A4 is associated with metabolic reprogramming in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6993.

Increased Extrasynaptic Glutamate Escape in Stochastically Shaped Probabilistic Synaptic Environment.

Excitatory synapses in the brain are often surrounded by nanoscopic astroglial processes that express high-affinity glutamate transporters at a high surface density. This ensures that the bulk of glutamate leaving the synaptic cleft is taken up for its subsequent metabolic conversion and replenishment in neurons. Furthermore, variations in the astroglial coverage of synapses can thus determine to what extent glutamate released into the synaptic cleft could activate its receptors outside the cleft. The biophysical determinants of extrasynaptic glutamate actions are complex because they involve a competition between transporters and target receptors of glutamate in the tortuous space of synaptic environment. To understand key spatiotemporal relationships between the extrasynaptic landscapes of bound and free glutamate, we explored a detailed Monte Carlo model for its release, diffusion, and uptake. We implemented a novel representation of brain neuropil in silico as a space filled with randomly scattered, overlapping spheres (spheroids) of distributed size. The parameters of perisynaptic space, astroglial presence, and glutamate transport were constrained by the empirical data obtained for the ‘average’ environment of common cortical synapses. Our simulations provide a glimpse of the perisynaptic concentration landscapes of free and transporter-bound glutamate relationship, suggesting a significant tail of space-average free glutamate within 3 ms post-release.

Gamma-aminobutyric acid enhances hypoxia tolerance of juvenile Chinese mitten crab (Eriocheir sinensis) by regulating respiratory metabolism and alleviating neural excitotoxicity

With climate change and intensive aquaculture development, environmental hypoxia in aquaculture water has become a common challenge for many aquatic species. Therefore, it is crucial to improve the hypoxic tolerance of animals through nutritional strategies. This study explored the positive role of dietary gamma-aminobutyric acid (GABA) supplementation in enhancing hypoxia tolerance of juvenile Eriocheir sinensis through respiratory regulation and alleviation of hypoxia-induced neural excitotoxicity. Acute hypoxia stress significantly up-regulated the mRNA expression level of hypoxia-inducible factor 1α, oxygen consumption rate and anaerobic respiratory metabolism-related enzyme activities. On the other hand, aerobic respiratory metabolism-related enzyme activities were significantly decreased. However, dietary GABA supplementation remodeled the respiratory metabolism pattern of juvenile crabs exposed to hypoxia stress. In addition, acute hypoxic stress significantly increased the contents of free glutamate and GABA in the nervous tissue. The expression levels of N-Methyl-d-aspartate-related receptor genes and calcium-dependent degradation enzyme-related genes were significantly up-regulated. Similarly, neuronal apoptosis rates, expression levels of apoptosis-related genes, and vesicular glutamate transporter genes were also significantly increased. The high-affinity neuronal glutamate transporter decreased significantly in the crabs exposed to hypoxia stress. However, dietary GABA supplementation could effectively prevent acute hypoxia stress-induced neural excitotoxicity. Furthermore, dietary GABA could significantly improve the redox status in vivo exposed to hypoxia stress. In conclusion, acute hypoxia stress can affect respiratory metabolism and redox state and induce neural excitotoxicity in juvenile E. sinensis. GABA supplementation could improve hypoxia tolerance through multiple physiological regulation pathways.

Neural excitotoxicity and the toxic mechanism induced by acute hypoxia in Chinese mitten crab (Eriocheir sinensis)

Hypoxia can induce neural excitotoxicity in mammals, but this adverse effect has not been investigated in aquatic animals to date, especially in crustaceans. This study explored the induction effect and toxic mechanism of acute hypoxia stress (1.0±0.1mg dissolved oxygen /L) for 24h on neural excitotoxicity in juvenile Chinese mitten crab, Eriocheir sinensis. The results showed that hemolymph glucose and serum lactic acid content were significantly increased, and the mRNA expression of crustacean hyperglycemic hormone and hypoxia-inducible factor 1α were significantly up-regulated in the hypoxia group compared with control. RNA-Seq results confirmed that acute hypoxia stress had a more significant impact on carbohydrate metabolism than lipid and protein metabolism. In addition, the TUNEL assay showed that the apoptosis rate of nerve cells was significantly higher in the hypoxia group than in the control, and similar trends were observed in the expression of apoptosis-related genes. RNA-Seq results also showed that acute hypoxia stress-induced neuronal apoptosis by regulating multiple apoptosis-related pathways. Moreover, free glutamate and GABA contents in the nerve tissue of thoracic ganglia were significantly higher in the hypoxia group than in the control group. Furthermore, the mRNA expression of NMDA related receptors was significantly up-regulated in the hypoxia group compared with the control. Similar trends were observed in the expression of calcium-dependent degrading enzymes and endogenous antioxidant-related proteins or enzymes. Meanwhile, the mRNA expression level of high-affinity neuronal glutamate transporter in the hypoxia group was significantly up-regulated compared with the control, whereas the vesicular glutamate transporter was significantly down-regulated. Furthermore, NMDA-R antagonists (MK-801 and Ro25-6981) injection showed that NMDA-R served as the bridge and core position of glutamate-induced neural neurotoxicity. This study provides a new perspective and theoretical guidance for exploring the regulation of hypoxic tolerance in E. sinensis.

Validation of a System xc– Functional Assay in Cultured Astrocytes and Nervous Tissue Samples

Disruption of the glutamatergic homeostasis is commonly observed in neurological diseases and has been frequently correlated with the altered expression and/or function of astrocytic high-affinity glutamate transporters. There is, however, a growing interest for the role of the cystine-glutamate exchanger system xc– in controlling glutamate transmission. This exchanger is predominantly expressed in glial cells, especially in microglia and astrocytes, and its dysregulation has been documented in diverse neurological conditions. While most studies have focused on measuring the expression of its specific subunit xCT by RT-qPCR or by Western blotting, the activity of this exchanger in tissue samples remains poorly examined. Indeed, the reported use of sulfur- and carbon-radiolabeled cystine in uptake assays shows several drawbacks related to its short radioactive half-life and its relatively high cost. We here report on the elaborate validation of a method using tritiated glutamate as a substrate for the reversed transport mediated by system xc–. The uptake assay was validated in primary cultured astrocytes, in transfected cells as well as in crude synaptosomes obtained from fresh nervous tissue samples. Working in buffers containing defined concentrations of Na+, allowed us to differentiate the glutamate uptake supported by system xc– or by high-affinity glutamate transporters, as confirmed by using selective pharmacological inhibitors. The specificity was further demonstrated in primary astrocyte cultures from transgenic mice lacking xCT or in cell lines where xCT expression was genetically induced or reduced. As such, this assay appears to be a robust and cost-efficient solution to investigate the activity of this exchanger in physiological and pathological conditions. It also provides a reliable tool for the screening and characterization of new system xc– inhibitors which have been frequently cited as valuable drugs for nervous disorders and cancer.

Buffering by Transporters Can Spare Geometric Hindrance in Controlling Glutamate Escape.

The surface of astrocyte processes that often surround excitatory synapses is packed with high-affinity glutamate transporters, largely preventing extrasynaptic glutamate escape. The shape and prevalence of perisynaptic astroglia vary among brain regions, in some cases providing a complete isolation of synaptic connections from the surrounding tissue. The perception has been that the geometry of perisynaptic environment is therefore essential to preventing extrasynaptic glutamate escape. To understand to what degree this notion holds, we modelled brain neuropil as a space filled with a scatter of randomly sized, overlapping spheres representing randomly shaped cellular elements and intercellular lumen. Simulating release and diffusion of glutamate molecules inside the interstitial gaps in this medium showed that high-affinity transporters would efficiently constrain extrasynaptic spread of glutamate even when diffusion passages are relatively open. We thus estimate that, in the hippocampal or cerebellar neuropil, the bulk of glutamate released by a synaptic vesicle is rapidly bound by transporters (or high-affinity target receptors) mainly in close proximity of the synaptic cleft, whether or not certain physiological or pathological events change local tissue geometry.

ASCT1 and ASCT2: Brother and Sister?

The SLC1 family includes seven members divided into two groups, namely, EAATs and ASCTs, that share similar 3D architecture; the first one includes high-affinity glutamate transporters, and the second one includes SLC1A4 and SLC1A5, known as ASCT1 and ASCT2, respectively, responsible for the traffic of neutral amino acids across the cell plasma membrane. The physiological role of ASCT1 and ASCT2 has been investigated over the years, revealing different properties in terms of substrate specificities, affinities, and regulation by physiological effectors and posttranslational modifications. Furthermore, ASCT1 and ASCT2 are involved in pathological conditions, such as neurodegenerative disorders and cancer. This has driven research in the pharmaceutical field aimed to find drugs able to target the two proteins.This review focuses on structural, functional, and regulatory aspects of ASCT1 and ASCT2, highlighting similarities and differences.

Differential astrocyte and oligodendrocyte vulnerability in murine Creutzfeldt-Jakob disease

ABSTRACT Glial vulnerability to prions is assessed in murine Creutzfeldt-Jakob disease (CJD) using the tg340 mouse line expressing four-fold human PrP M129 levels on a mouse PrP null background at different days following intracerebral inoculation of sCJD MM1 brain tissues homogenates. The mRNA expression of several astrocyte markers, including glial fibrillary acidic protein (gfap), aquaporin-4 (aqp4), solute carrier family 16, member 4 (mct4), mitochondrial pyruvate carrier 1 (mpc1) and solute carrier family 1, member 2 (glial high-affinity glutamate transporter, slc1a2) increases at 120 and 180 dpi. In contrast, the mRNA expression of oligodendrocyte and myelin markers oligodendrocyte transcription factor 1 (olig1), olig2, neural/glial antigen 2 (cspg), solute carrier family 16, member 1 (mct1), myelin basic protein (mbp), myelin oligodendrocyte glycoprotein (mog) and proteolipid protein 1 (plp1) is preserved. Yet, myelin regulatory factor (myrf) mRNA is increased at 180 dpi. In the striatum, a non-significant increase in the number of GFAP-positive astrocytes and Iba1-immunoreactive microglia occurs at 160 dpi; a significant increase in the number of astrocytes and microglia, and a significant reduction in the number of Olig2-immunoreactive oligodendrocytes occur at 180 dpi. A decrease of MBP, but not PLP1, immunoreactivity is also observed in the striatal fascicles. These observations confirm the vulnerability and the reactive responses of astrocytes, together with the microgliosis at middle stages of prion diseases. More importantly, these findings show oligodendrocyte vulnerability and myelin alterations at advanced stages of murine CJD. They confirm oligodendrocyte involvement in the pathogenesis of CJD.

Effect of High-Oleic Peanut Intake on Aging and Its Hippocampal Markers in Senescence-Accelerated Mice (SAMP8).

In many previous studies, the preventive effects of peanut against aging and cognitive impairment have often been unclear, so to clarify the effects we first investigated effective markers for evaluating its effects in the hippocampus of senescence-accelerated mouse prone/8 (SAMP8) mice, mainly using proteomics. The effects of dietary high-oleic peanuts on the hair appearance of SAMP8, the expression of effective markers in the hippocampus, and the TBARS and amino acid contents of the hippocampus were examined. Hippocampus solute carrier family 1 (glial high-affinity glutamate transporter), calcium/calmodulin-dependent protein kinase type II, and sodium- and chloride-dependent GABA transporter, which all are considered to be closely related to glutamic acid concentration were decreased by feeding of the samples, and the GABA/glutamic acid ratio in the hippocampus was increased by feeding with the samples. The formation of glial fibrillary acidic protein and synapsin-2, which showed higher levels in the SAMP8 than in SAMR1, and the protein expression of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein and dihydropteridine reductase, which are considered to be related to the formation of adrenergic neuron transmitters, were reduced by the feeding of peanuts and their germ-rich fraction. Ferulic acid, as an ester and minor component in peanuts, could be partly connected to the effect of peanuts. These results indicate that high-oleic peanuts and their germ-rich fraction can protect against aging and cognitive impairment by regulating protein expression, which could be measured by the proteomics of the above hippocampus proteins of SAMP8 and the hippocampal GABA/glutamic acid ratio.

Propranolol decreases DRD3 and SLC1A2 gene expression in patients with essential tremor

BackgroundEssential tremor (ET) is the most common disease among movement disorders. Genes such as essential tremor 1-4 (ETM 1-4), HS1-binding protein-3 (HS1BP3), dopamine receptor D3 (DRD3), leucine-rich repeat and Ig domain containing 1 (LINGO1), glial high affinity glutamate transporter member 2 (SLC1A2), FUS, high temperature requirement A2 (HTRA2) and TENM4 had been shown to be responsible for the genetic inheritance of the disease. The aim of the present study was to investigate the effect of propranolol on the expression of DRD3, SLC1A2, and HTRA2 genes in patients with ET.MethodsA study of non-randomized experimental design was conducted involving 76 subjects. They were divided into two groups: 38 patients with ET in the patient group (Group 1) and 38 healthy people in the control group (Group 2). DRD3, SLC1A2 and HTRA2 gene expressions were assessed before and after 8 weeks of propranolol treatment. Fahn-Tolosa-Marin tremor scale results were compared before and after propranolol administration. Kruskal Wallis test was used to determine differences in gene expressions between the groups.ResultsD3 dopamine receptor and SLC1A2 gene expression in the patient group appeared to be lower than in the control group (p<0.001). However, the HTRA2 gene expression level was significantly higher in the patient group (p<0.001). Conclusion D3 dopamine receptor and SLC1A2 gene expressions were decreased in ET patients which at first glance can be explained in relation to etiology, but after treatment it was not increased as expected but decreased even more.

Optical monitoring of glutamate release at multiple synapses in situ detects changes following LTP induction

Information processing and memory formation in the brain relies on release of the main excitatory neurotransmitter glutamate from presynaptic axonal specialisations. The classical Hebbian paradigm of synaptic memory, long-term potentiation (LTP) of transmission, has been widely associated with an increase in the postsynaptic receptor current. Whether and to what degree LTP induction also enhances presynaptic glutamate release has been the subject of debate. Here, we took advantage of the recently developed genetically encoded optical sensors of glutamate (iGluSnFR) to monitor its release at CA3-CA1 synapses in acute hippocampal slices, before and after the induction of LTP. We attempted to trace release events at multiple synapses simultaneously, by using two-photon excitation imaging in fast frame-scanning mode. We thus detected a significant increase in the average iGluSnFR signal during potentiation, which lasted for up to 90 min. This increase may reflect an increased amount of released glutamate or, alternatively, reduced glutamate binding to high-affinity glutamate transporters that compete with iGluSnFR.

Potassium and glutamate transport is impaired in scar-forming tumor-associated astrocytes

Unprovoked recurrent seizures are a serious comorbidity affecting most patients who suffer from glioma, a primary brain tumor composed of malignant glial cells. Cellular mechanisms contributing to the development of recurrent spontaneous seizures include the release of the excitatory neurotransmitter glutamate from glioma into extracellular space. Under physiological conditions, astrocytes express two high affinity glutamate transporters, Glt-1 and Glast, which are responsible for the removal of excess extracellular glutamate. In the context of neurological disease or brain injury, astrocytes become reactive which can negatively affect neuronal function, causing hyperexcitability and/or death. Using electrophysiology, immunohistochemistry, fluorescent in situ hybridization, and Western blot analysis in different orthotopic xenograft and allograft models of human and mouse gliomas, we find that peritumoral astrocytes exhibit astrocyte scar formation characterized by proliferation, cellular hypertrophy, process elongation, and increased GFAP and pSTAT3. Overall, peritumoral reactive astrocytes show a significant reduction in glutamate and potassium uptake, as well as decreased glutamine synthetase activity. A subset of peritumoral astrocytes displayed a depolarized resting membrane potential, further contributing to reduced potassium and glutamate homeostasis. These changes may contribute to the propagation of peritumoral neuronal hyperexcitability and excitotoxic death.

Role of Glutamatergic Excitotoxicity in Neuromyelitis Optica Spectrum Disorders.

Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory disorder mediated by immune-humoral responses directed against central nervous system (CNS) antigens. Most patients are positive for specific immunoglobulin G (IgG) auto-antibodies for aquaporin-4 (AQP4), a water channel present in astrocytes. Antigen-antibody binding promotes complement system cascade activation, immune system cell infiltration, IgG deposition, loss of AQP4 and excitatory amino acid transporter 2 (EAAT2) expression on the astrocytic plasma membrane, triggering necrotic destruction of spinal cord tissue and optic nerves. Astrocytes are very important cells in the CNS and, in addition to supporting other nerve cells, they also regulate cerebral homeostasis and control glutamatergic synapses by modulating neurotransmission in the cleft through the high-affinity glutamate transporters present in their cell membrane. Specific IgG binding to AQP4 in astrocytes blocks protein functions and reduces EAAT2 activity. Once compromised, EAAT2 cannot take up free glutamate from the extracellular space, triggering excitotoxicity in the cells, which is characterized by overactivation of glutamate receptors in postsynaptic neurons. Therefore, the longitudinally extensive myelitis and optic neuritis lesions observed in patients with NMOSD may be the result of primary astrocytic damage triggered by IgG binding to AQP4, which can activate the immune-system cascade and, in addition, downregulate EAAT2. All these processes may explain the destructive lesions in NMOSD secondary to neuroinflammation and glutamatergic excitotoxicity. New or repurposed existing drugs capable of controlling glutamatergic excitotoxicity may provide new therapeutic options to reduce tissue damage and permanent disability after NMOSD attacks.

Gene Network Dysregulation in the Trigeminal Ganglia and Nucleus Accumbens of a Model of Chronic Migraine-Associated Hyperalgesia.

The pharmacological agent nitroglycerin (NTG) elicits hyperalgesia and allodynia in mice. This model has been used to study the neurological disorder of trigeminovascular pain or migraine, a debilitating form of hyperalgesia. The present study validates hyperalgesia in an established mouse model of chronic migraine triggered by NTG and advances the understanding of the associated molecular mechanisms. The RNA-seq profiles of two nervous system regions associated with pain, the trigeminal ganglia (TG) and the nucleus accumbens (NAc), were compared in mice receiving chronic NTG treatment relative to control (CON) mice. Among the 109 genes that exhibited an NTG treatment-by-region interaction, solute carrier family 32 (GABA vesicular transporter) member 1 (Slc32a1) and preproenkephalin (Penk) exhibited reversal of expression patterns between the NTG and CON groups. Erb-b2 receptor tyrosine kinase 4 (Erbb4) and solute carrier family 1 (glial high affinity glutamate transporter) member 2 (Slc1a2) exhibited consistent differential expression between treatments across regions albeit at different magnitude. Period circadian clock 1 (Per1) was among the 165 genes that exhibited significant NTG treatment effect. Biological processes disrupted by NTG in a region-specific manner included adaptive and innate immune responses; whereas glutamatergic and dopaminergic synapses and rhythmic process were disrupted in both regions. Regulatory network reconstruction highlighted the widespread role of several transcription factors (including Snrnp70, Smad1, Pax6, Cebpa, and Smpx) among the NTG-disrupted target genes. These results advance the understanding of the molecular mechanisms of hyperalgesia that can be applied to therapies to ameliorate chronic pain and migraine.

Riluzole Impairs Cocaine Reinstatement and Restores Adaptations in Intrinsic Excitability and GLT-1 Expression

Adaptations in glutamate signaling within the brain's reward circuitry are observed following withdrawal from several abused drugs, including cocaine. These include changes in intrinsic cellular excitability, glutamate release, and glutamate uptake. Pharmacological or optogenetic reversal of these adaptations have been shown to reduce measures of cocaine craving and seeking, raising the hypothesis that regulation of glutamatergic signaling represents a viable target for the treatment of substance use disorders. Here, we tested the hypothesis that administration of the compound riluzole, which regulates glutamate dynamics in several ways, would reduce cocaine seeking in the rat self-administration and reinstatement model of addiction. Riluzole dose-dependently inhibited cue- and cocaine-primed reinstatement to cocaine, but did not affect locomotor activity or reinstatement to sucrose seeking. Moreover, riluzole reversed bidirectional cocaine-induced adaptations in intrinsic excitability of prelimbic (PL) and infralimbic (IL) pyramidal neurons; a cocaine-induced increase in PL excitability was decreased by riluzole, and a cocaine-induced decrease in IL excitability was increased to normal levels. Riluzole also reversed the cocaine-induced suppression of the high-affinity glutamate transporter 1 (EAAT2/GLT-1) in the nucleus accumbens (NAc). GLT-1 is responsible for the majority of glutamate uptake in the brain, and has been previously reported to be downregulated by cocaine. These results demonstrate that riluzole impairs cocaine reinstatement while rectifying several cellular adaptations in glutamatergic signaling within the brain's reward circuitry, and support the hypothesis that regulators of glutamate homeostasis represent viable candidates for pharmacotherapeutic treatment of psychostimulant relapse.

High-affinity glutamate transporters and chronic pain

Glutamate serves as a major excitatory neurotransmitter in the mammalian central nervous system and is stored in synaptic cleft by an uptake system that is dependent on the high-affinity glutamate transporters (ETTAs), which locate in the plasma membrane of glial cells and neurons. ETTAs can rapidly terminate the action of glutamate and maintain its normal physiological functions. If the content or function of glutamate transporters is abnormal, it can result in many physiological dysfunctions. Studies have demonstrated that high-affinity glutamate transporters play an important role in the development of chronic pain, which might be a new therapeutic target for the pain.

Glutamate Transport System as a Novel Therapeutic Target in Chronic Pain: Molecular Mechanisms and Pharmacology.

The vast majority of peripheral neurons sensing noxious stimuli and conducting pain signals to the dorsal horn of the spinal cord utilize glutamate as a chemical transmitter of excitation. High-affinity glutamate transporter subtypes GLAST/EAAT1, GLT1/EAAT2, EAAC1/EAAT3, and EAAT4, differentially expressed on sensory neurons, postsynaptic spinal interneurons, and neighboring glia, ensure fine modulation of glutamate neurotransmission in the spinal cord. The glutamate transport system seems to play important roles in molecular mechanisms underlying chronic pain and analgesia. Downregulation of glutamate transporters (GluTs) often precedes or occurs simultaneously with development of hypersensitivity to thermal or tactile stimuli in various models of chronic pain. Moreover, antisense knockdown or pharmacological inhibition of these membrane proteins can induce or aggravate pain. In contrast, upregulation of GluTs by positive pharmacological modulators or by viral gene transfer to the spinal cord can reverse the development of such pathological hypersensitivity. Furthermore, some multi-target drugs displaying analgesic properties (e.g., tricyclic antidepressant amitriptyline, riluzole, anticonvulsant valproate, tetracycline antibiotic minocycline, β-lactam antibiotic ceftriaxone and its structural analog devoid of antibacterial activity, clavulanic acid) can significantly increase the spinal glutamate uptake. Thus, mounting evidence points at GluTs as prospective therapeutic target for chronic pain treatment. However, design and development of new analgesics based on the modulation of glutamate uptake will require more precise knowledge of molecular mechanisms underlying physiological or aberrant functioning of this transport system in the spinal cord.

Glutamate oxidation in astrocytes: Roles of glutamate dehydrogenase and aminotransferases.

The cellular distribution of transporters and enzymes related to glutamate metabolism led to the concept of the glutamate-glutamine cycle. Glutamate is released as a neurotransmitter and taken up primarily by astrocytes ensheathing the synapses. The glutamate carbon skeleton is transferred back to the presynaptic neurons as the nonexcitatory amino acid glutamine. The cycle was initially thought to function with a 1:1 ratio between glutamate released and glutamine taken up by neurons. However, studies of glutamate metabolism in astrocytes have shown that a considerable proportion of glutamate undergoes oxidative degradation; thus, quantitative formation of glutamine from the glutamate taken up is not possible. Oxidation of glutamate is initiated by transamination catalyzed by an aminotransferase, or oxidative deamination catalyzed by glutamate dehydrogenase (GDH). We discuss methods available to elucidate the enzymes that mediate this conversion. Methods include pharmacological tools such as the transaminase inhibitor aminooxyacetic acid, studies using GDH knockout mice, and siRNA-mediated knockdown of GDH in astrocytes. Studies in brain slices incubated with [15 N]glutamate demonstrated activity of GDH in astrocytes in situ. These results, in conjunction with reports in the literature, support the conclusion that GDH is active in astrocytes both in culture and in vivo and that this enzyme plays a significant role in glutamate oxidation. Oxidative metabolism of glutamate, primarily mediated by GDH, but also by transamination by aspartate aminotransferase, provides considerably more energy than is required to maintain the activity of the high-affinity glutamate transporters needed for efficient removal of glutamate from the synaptic cleft. © 2016 Wiley Periodicals, Inc.

Decreased cysteine uptake by EAAC1 gene deletion exacerbates neuronal oxidative stress and neuronal death after traumatic brain injury.

Excitatory amino acid carrier type 1 (EAAC1), a high-affinity glutamate transporter, can expend energy to move glutamate into neurons. However, under normal physiological conditions, EAAC1 does not have a great effect on glutamate clearance but rather participates in the neuronal uptake of cysteine. This process is critical to maintaining neuronal antioxidant function by providing cysteine for glutathione synthesis. Previous study showed that mice lacking EAAC1 show increased neuronal oxidative stress following transient cerebral ischemia. In the present study, we sought to characterize the role of EAAC1 in neuronal resistance after traumatic brain injury (TBI). Young adult C57BL/6 wild-type or EAAC1 (-/-) mice were subjected to a controlled cortical impact model for TBI. Neuronal death after TBI showed more than double the number of degenerating neurons in the hippocampus in EAAC1 (-/-) mice compared with wild-type mice. Superoxide production, zinc translocation and microglia activation similarly showed a marked increase in the EAAC1 (-/-) mice. Pretreatment with N-acetyl cysteine (NAC) reduced TBI-induced neuronal death, superoxide production and zinc translocation. These findings indicate that cysteine uptake by EAAC1 is important for neuronal antioxidant function and survival following TBI. This study also suggests that administration of NAC has therapeutic potential in preventing TBI-induced neuronal death.

High-affinity glutamate transporter and glutamine synthetase content in longissimus dorsi and adipose tissues of growing Angus steers differs among suckling, weanling, backgrounding, and finishing production stages.

Skeletal muscle and adipose tissues play important roles in maintaining whole-body Glu and N homeostasis by the uptake of Glu and release of Gln. To test the hypothesis that expression of high-affinity Glu transporters (GLAST1, EAAT4, EAAC1, GLT-1) and glutamine synthetase (GS) would increase in longissimus dorsi and adipose tissue of newborn Angus steers randomly assigned ( = 6) to develop through suckling (S; 32 d) and/or weanling (W; 184 d), backgrounding (B; 248 d), and finishing (F; 423 d) production stages. Carcass quality was determined at slaughter to verify shifts in adipose and lean deposition with development. Expression of mRNA (RT-PCR/Southern) and relative protein abundance (Western analysis) were determined in tissue homogenates isolated from longissimus dorsi, and kidney and subcutaneous adipose. The effect of production stage or tissue type on carcass and protein abundance was assessed by 1-way ANOVA using the GLM procedure of SAS, and Fisher's protected LSD procedure was used to separate data means. Neither GLAST1 nor EAAT4 mRNA or protein was detected. EAAC1, GLT-1, and GS mRNA were identified in all tissues, but GLT-1 and GS protein were not detected in kidney or subcutaneous adipose, and GS protein was not detected in longissimus dorsi. The EAAC1 content of subcutaneous ( = 0.06) and kidney ( = 0.02) adipose was 2 times greater in B and F than W steers, whereas GS was 5 times greater ( < 0.07) in B than F steers (B = W > F). For longissimus dorsi, EAAC1 ( < 0.01) and GLT-1 ( < 0.04) content decreased with development (S > W > B = F, S = W > B = F, respectively). Within F steers, EAAC1 and GLT-1 mRNA was expressed by subcutaneous, kidney, omental, mesenchymal, and intramuscular adipose tissues, whereas GS mRNA was expressed by all except for intramuscular. Only EAAC1 protein was detected in any adipose tissue, with EAAC1 content being 104% and 112% greater ( < 0.01) in intramuscular than in kidney or subcutaneous adipose, respectively, and not differing ( > 0.45) from omental or mesenchymal adipose. These data demonstrate (1) longissimus dorsi and adipose tissues of steers developing through typical production stages have different capacities for Glu uptake and Gln synthesis, (2) the importance of EAAC1 and GS in adipose metabolism, and (3) the differential metabolic fate of Glu by adipose tissues as steers developed, as evidenced by the marked decrease of GS content in subcutaneous and kidney adipose of F steers.