Increased glucose metabolism and impaired glutamate transport in human astrocytes are potential early triggers of abnormal extracellular glutamate accumulation in hiPSC-derived models of Alzheimer's disease.

Glutamate recycling between neurons and astrocytes is essential to maintain neurotransmitter homeostasis. Disturbances in glutamate homeostasis, resulting in excitotoxicity and neuronal death, have been described as a potential mechanism in Alzheimer's disease (AD) pathophysiology. However, glutamate neurotransmitter metabolism in different human brain cells, particularly astrocytes, has been poorly investigated at the early stages of AD. We sought to investigate glucose and glutamate metabolism in AD by employing human induced pluripotent stem cell (hiPSC)-derived astrocytes and neurons carrying mutations in the amyloid precursor protein (APP) or presenilin-1 (PSEN-1) gene as found in familial types of AD (fAD). Methods such as live-cell bioenergetics and metabolic mapping using [13 C]-enriched substrates were used to examine metabolism in the early stages of AD. Our results revealed greater glycolysis and glucose oxidative metabolism in astrocytes and neurons with APP or PSEN-1 mutations, accompanied by an elevated glutamate synthesis compared to control WT cells. Astrocytes with APP or PSEN-1 mutations exhibited reduced expression of the excitatory amino acid transporter 2 (EAAT2), and glutamine uptake increased in mutated neurons, with enhanced glutamate release specifically in neurons with a PSEN-1 mutation. These results demonstrate a hypermetabolic phenotype in astrocytes with fAD mutations possibly linked to toxic glutamate accumulation. Our findings further identify metabolic imbalances that may occur in the early phases of AD pathophysiology.

Glutamate transport system as a key constituent of glutamosome: Molecular pathology and pharmacological modulation in chronic pain.

Neural uptake of glutamate is executed by the structurally related members of the SLC1A family of solute transporters: GLAST/EAAT1, GLT-1/EAAT2, EAAC1/EAAT3, EAAT4, ASCT2. These plasma membrane proteins ensure supply of glutamate, aspartate and some neutral amino acids, including glutamine and cysteine, for synthetic, energetic and signaling purposes, whereas effective removal of glutamate from the synaptic cleft shapes excitatory neurotransmission and prevents glutamate toxicity. Glutamate transporters (GluTs) possess also receptor-like properties and can directly initiate signal transduction. GluTs are physically linked to other glutamate signaling-, transporting- and metabolizing molecules (e.g., glutamine transporters SNAT3 and ASCT2, glutamine synthetase, NMDA receptor, synaptic vesicles), as well as cellular machineries fueling the transmembrane transport of glutamate (e.g., ion gradient-generating Na/K-ATPase, glycolytic enzymes, mitochondrial membrane- and matrix proteins, glucose transporters). We designate this supramolecular functional assembly as 'glutamosome'. GluTs play important roles in the molecular pathology of chronic pain, due to the predominantly glutamatergic nature of nociceptive signaling in the spinal cord. Down-regulation of GluTs often precedes or occurs simultaneously with development of pain hypersensitivity. Pharmacological inhibition or gene knock-down of spinal GluTs can induce/aggravate pain, whereas enhancing expression of GluTs by viral gene transfer can mitigate chronic pain. Thus, functional up-regulation of GluTs is turning into a prospective pharmacotherapeutic approach for the management of chronic pain. A number of novel positive pharmacological regulators of GluTs, incl. pyridazine derivatives and ß-lactams, have recently been introduced. However, design and development of new analgesics based on this principle will require more precise knowledge of molecular mechanisms underlying physiological or aberrant functioning of the glutamate transport system in nociceptive circuits. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

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, ß-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.

High-affinity glutamate transporters in chronic pain: an emerging therapeutic target.

Neurons responsible for sensing noxious stimuli and conducting pain signals from periphery to the spinal cord are predominantly glutamatergic. Members of the SLC1A family of high-affinity glutamate transporters (GluTs) are differentially expressed in sensory neurons and surrounding glial cells. These plasma membrane proteins along with glutamate/cystine exchanger, light chain of cystine/glutamate exchanger, are responsible for fine tuning of extracellular glutamate concentrations and, thus, for modulation of excitatory signalling in the spinal cord. Emerging data point at key roles of GluTs in molecular mechanisms of chronic pain and analgesia, incl. development of opioid tolerance. Pharmacological inhibition or antisense down-regulation of spinal GluTs can induce/aggravate pain behaviours, whereas increasing of expression of GluTs by viral gene transfer or positive pharmacological modulators can mitigate chronic pain. Furthermore, some drugs, originally introduced for targeting different pathological conditions, but in parallel exhibiting analgesic properties (e.g. anti-convulsants valproate and riluzole, ß-lactam- and tetracycline antibiotics, tricyclic anti-depressants), can enhance glutamate transport in the spinal cord. Thus, molecular modulation of GluTs may turn into prospective therapeutic approach for the management of chronic pain. However, precise pharmacological targeting of this transport system requires in-depth elucidation of molecular factors and signalling pathways underlying expression and activity of individual GluT subtypes, including their splice variants. Neurons conducting pain signals from periphery to the spinal cord are predominantly glutamatergic. High-affinity glutamate transporters (GluTs) regulate extracellular glutamate concentrations and, thus, modulate excitatory signalling in pain circuits. The present review critically analyses accumulated data on the roles of GluTs in molecular mechanisms of chronic pain, as well as perspectives for targeting this transport system in pain therapies.

Limited energy supply in Müller cells alters glutamate uptake.

The viability of retinal ganglion cells (RGC) is essential for the maintenance of visual function. RGC homeostasis is maintained by the surrounding retinal glial cells, the Müller cells, which buffer the extracellular concentration of neurotransmitters and provide the RGCs with energy. This study evaluates if glucose-deprivation of Müller cells interferes with their ability to remove glutamate from the extracellular space. The human Müller glial cell line, Moorfields/Institute of Ophthalmology-Müller 1, was used to study changes in glutamate uptake. Excitatory amino acid transporter (EAAT) proteins were up-regulated in glucose-deprived Müller cells and glutamate uptake was significantly increased in the absence of glucose. The present findings revealed an up-regulation of EAAT1 and EAAT2 in glucose-deprived Müller cells as well as an increased ability to take up glutamate. Hence, glucose deprivation may result in an increased ability to protect RGCs from glutamate-induced excitotoxicity, whereas malfunction of glutamate uptake in Müller cells may contribute to retinal neurodegeneration.

Tonic activation of group I mGluRs modulates inhibitory synaptic strength by regulating KCC2 activity.

The furosemide-sensitive potassium-chloride cotransporter (KCC2) plays an important role in establishing the intracellular chloride concentration in many neurons within the central nervous system. Consequently, modulation of KCC2 function will regulate the reversal potential for synaptic GABAergic inputs, thus setting the strength of inhibitory transmission. We show that tonic activation of group I metabotropic glutamate receptors (mGluR1s) regulates inhibitory synaptic strength via modulation of KCC2 function in pyramidal neurons of the hippocampal CA3 area. Specifically, group I mGluRs signal via activation of a protein kinase C-dependent pathway to alter KCC2 activity, thereby altering the intracellular chloride concentration, and thus inhibitory synaptic input. This interaction between the glutamatergic and chloride transport systems highlights a novel homeostatic mechanism whereby ambient glutamate levels directly regulate the inhibitory synaptic tone by setting the activity level of KCC2. Thus, mGluRs are poised to play a pivotal role in providing a direct interplay between the excitatory and inhibitory systems in the hippocampus.

Glutamate transporter GLAST/EAAT1 directs cell surface expression of FXYD2/gamma subunit of Na, K-ATPase in human fetal astrocytes.

Na+-dependent uptake of excitatory neurotransmitter glutamate in astrocytes increases cell energy demands primarily due to the elevated ATP consumption by glutamine synthetase and Na+, K+-ATPase. The major pool of GLAST/EAAT1, the only glutamate transporter subtype expressed by human fetal astrocytes in undifferentiated cultures, was restricted to the cytoplasmic compartment. Elevated glutamate concentrations (up to 50 microM) stimulated both glutamate uptake and Na+, K+-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/gamma subunit of Na+, K+-ATPase. Intracellular accumulation of glutamate or its metabolites per se was not responsible for these changes since metabolically inert transport substrate, D-aspartate, exerted the same effect. Nanomolar concentrations of TFB-TBOA, a novel nontransportable inhibitor of glutamate carriers, almost completely reversed the action of glutamate or D-aspartate. In the same conditions (i.e. block of glutamate transport) monensin, a potent Na+ ionophore, had no significant effect neither on the activation of Na+, K+-ATPase nor on the cell surface expression of gamma subunit or GLAST. In order to elucidate the roles of gamma subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures gamma subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of gamma subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of gamma subunit effectively abolished the glutamate uptake-dependent activation of Na+, K+-ATPase. Following withdrawal of siRNA from cultures, the expression levels of gamma subunit and the sensitivity of Na+, K+-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/gamma subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of gamma subunit on the kinetic parameters of catalytic alpha subunit(s) of Na+, K+-ATPase.

High-affinity glutamate transporter GLAST/EAAT1 regulates cell surface expression of glutamine/neutral amino acid transporter ASCT2 in human fetal astrocytes.

Neutral amino acid transporter ASCT2, together with high-affinity glutamate transporters, belongs to the SLC1 gene family of Na(+)-dependent solute carriers and is one of the major transporters of glutamine in cultured astrocytes. Besides glutamine and other high-affinity substrates--alanine, serine, cysteine or threonine, ASCT2 can also translocate protonated glutamate. The present study elucidated substrate-dependent trafficking of ASCT2 in differentiated primary cultures of human fetal astrocytes. The differentiation induced by 8-bromo-cAMP caused dramatic up-regulation of two co-localized and functionally linked astroglial proteins--glutamate transporter GLAST, that is the only high-affinity router of glutamate into cultured astrocytes, and glutamine synthetase (GS), a cytosolic enzyme that converts at least a part of the arriving glutamate into glutamine. In order to distinguish individual intracellular effects of these two substrates on ASCT2, in some cultures glutamine synthetase was effectively knocked down using siRNA silencing technique. In control conditions, regardless of GS levels, almost the entire ASCT2 immunoreactivity was restricted to the cytosol. Both glutamine and alanine, though to different extents, induced partial redistribution of ASCT2 from the cytosolic compartment to the plasma membrane. However, in cultures with high GS expression, micromolar concentrations of glutamate exhibited more pronounced effect on ASCT2 trafficking than the preferred substrates of this carrier. In contrast, glutamate had no effect on ASCT2 distribution in cultures devoid of GS. D-Aspartate, a metabolically inert substrate effectively transported by GLAST, had no effect in any cell culture utilized. It seems that intracellular glutamine produced by GS from glutamate that, in turn, is supplied by GLAST, is a more potent inducer of ASCT2 trafficking to the cell surface than the ASCT2-mediated translocation of extracellular substrates. At lower pH values (6.2-6.7), the cell surface pool of ASCT2 was significantly larger than at physiological pH. In addition, high concentrations of glutamate, independently from GLAST or glutamate receptor activation, induced further arrival of ASCT2 to the plasma membrane. The pH-dependent functional activation of ASCT2 and the ASCT2-mediated glutamate uptake may play important roles during ischemic acidosis or synaptic activity-induced local acidification.

Expression of glutamate transporter subtypes in cultured retinal pigment epithelial and retinoblastoma cells.

PURPOSE: Glutamate is the major excitatory neurotransmitter in the retina and glutamate uptake is essential for normal glutamate signalling. Retinal diseases may induce neurochemical changes which affect retinal cells including retinal pigment epithelium (RPE). The aim of the study was to investigate the expression of glutamate transporter subtypes in RPE and retinoblastoma cells and to clarify the effect of proliferation modulators on the levels of the expressed transporter in the RPE cell line. METHODS: Cultured pig RPE cells and two human RPE cell lines, D407 and ARPE-19, as well as the human retinoblastoma cell line Y79 were used. Glutamate transporter expression was evaluated with Western blot analysis and immunocytochemistry. RESULTS: The study revealed unexpected expression of neuronal glutamate transporter/chloride channel EAAT4 in these three cell lines, but not in cultured pig RPE cells, whereas another glutamate carrier, EAAC1, was present in all cell types utilized. Other transporter subtypes, GLT1, GLAST and EAAT5 were not found. Neither tamoxifen, known to inhibit both proliferation and glutamate uptake in RPE cells, nor retinoic acid nor insulin, also known to affect cell proliferation rates, were capable of changing the total levels of EAAT4 in APRE-19 cells. CONCLUSIONS: Neuronal glutamate transporter EAAC1 is expressed in RPE cells. The robust expression of EAAT4 in cell lines may reflect a role of EAAT4 in cell proliferation and migration. Unaltered steady-state expression of this carrier and chloride-channel protein hints at posttranslational mechanisms of regulation of EAAT4.

Beta-amyloid and brain-derived neurotrophic factor, BDNF, up-regulate the expression of glutamate transporter GLT-1/EAAT2 via different signaling pathways utilizing transcription factor NF-kappaB.

Malfunctioning of high-affinity glutamate transporters is believed to contribute to the accumulation of toxic concentrations of glutamate and, thus, trigger the cellular mechanisms of neurodegeneration. Emerging data point to the presence of excitotoxic component in Alzheimer's disease (AD) and aberrant expression of glutamate transporters in this neurodegenerative malady. Neuronal soluble factors are essential for differential expression and fine tuning of the astroglial glutamate transporters, GLT-1/EAAT2 and GLAST/EAAT1. However, the nature of factors specifically affecting glutamate uptake in AD is largely unknown. The overproduction of neurotoxic beta-amyloid peptide (Abeta), a major constituent of amyloid plaques, and marked down-regulation of BDNF, a neuroprotective factor, are hallmarks of AD pathophysiology. None of these typically neuronal factors was capable of changing the pattern of glutamate transporter expression in undifferentiated rat astrocytes that predominantly expressed GLAST. In differentiated astrocytes, BDNF and, to a lesser extent, subtoxic concentrations of Abeta 1-42 (1-5 microM) induced the expression of GLT-1 and increased glutamate uptake, whereas the GLAST levels were unaltered by these factors. The BDNF-dependent up-regulation of GLT-1 in differentiated astrocytes was partially antagonized by the activation of metabotropic glutamate receptor 4 (mGluR4), but not by group I or II mGluRs. Activation of transcription factor NF-kappaB appeared to be a shared essential, but not a sufficient molecular event in the BDNF- or Abeta-dependent induction of GLT-1. The BDNF-dependent activation of NF-kappaB and up-regulation of GLT-1 was critically dependent on the upstream activation of p42/p44 MAP kinase signaling, whereas the inhibition of these MAP kinases dramatically increased the Abeta-dependent activation of NF-kappaB and production of GLT-1. The capacity to up-regulate astroglial glutamate uptake system, that apparently represents a novel element in the neuroprotective repertoire of BDNF, can, however, provide adverse effect under certain insults when glutamate transporters start operating in reverse direction. The Abeta-dependent up-regulation of GLT-1/EAAT2, more pronounced under the deficit of MAP kinase signaling, may attenuate synaptic efficacy and, thus contribute to the impairment of neuroplasticity in AD.