Mammalian ion-coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.     

Functions of glutamate transporters in cerebellar Purkinje cell synapses

Glutamate transporters play a critical role in the maintenance of low extracellular concentrations of glutamate, which prevents the overactivation of post‐synaptic glutamate receptors. Four distinct glutamate transporters, GLAST/EAAT1, GLT‐1/EAAT2, EAAC1/EAAT3 and EAAT4, are distributed in the molecular layer of the cerebellum, especially near glutamatergic synapses in Purkinje cells (PCs). This review summarizes the current knowledge about the differential roles of these transporters at excitatory synapses of PCs. Data come predominantly from electrophysiological experiments in mutant mice that are deficient in each of these transporter genes. GLAST expressed in Bergmann glia contributes to the clearing of the majority of glutamate that floods out of the synaptic cleft immediately after transmitter release from the climbing fibre (CF) and parallel fibre (PF) terminals. It is indispensable to maintain a one‐to‐one relationship in synaptic transmission at the CF synapses by preventing transcellular glutamate spillover. GLT‐1 plays a similar but minor role in the uptake of glutamate as GLAST. Although the loss of neither GLAST nor GLT‐1 affects cerebellar morphology, the deletion of both GLAST and GLT‐1 genes causes the death of the mutant animal and hinders the folium formation of the cerebellum. EAAT4 removes the low concentrations of glutamate that escape from uptake by glial transporters, preventing the transmitter from spilling over into neighbouring synapses. It also regulates the activation of metabotropic glutamate receptor 1 (mGluR1) in perisynaptic regions at PF synapses, which in turn affects mGluR1‐mediated events including slow EPSCs and long‐term depression. No change in synaptic function is detected in mice that are deficient in EAAC1.     

Detergent-insoluble EAAC1/EAAT3 aberrantly accumulates in hippocampal neurons of Alzheimer's disease patients

Disturbed glutamate homeostasis may contribute to the pathological processes involved in Alzheimer's disease (AD). Once glutamate is released from synapses or from other intracellular sources, it is rapidly cleared by glutamate transporters. EAAC1 (also called EAAT3 or SLC1A1) is the primary glutamate transporter in forebrain neurons. In addition to transporting glutamate, EAAC1 plays other roles in regulating GABA synthesis, reducing oxidative stress in neurons, and is important in supporting neuron viability. Currently, little is known about EAAC1 in AD. To address whether EAAC1 is disturbed in AD, immunohistochemistry was performed on tissue from hippocampus and frontal cortex of AD and normal control subjects matched for age and gender. While EAAC1 immunostaining in cortex appeared comparable to controls, in the hippocampus, EAAC1 aberrantly accumulated in the cell bodies and proximal neuritic processes of CA2-CA3 pyramidal neurons in AD patients. Biochemical analyses showed that Triton X-100-insoluble EAAC1 was significantly increased in the hippocampus of AD patients compared to both controls and Parkinson's disease patients. These findings suggest that aberrant glutamate transporter expression is associated with AD-related neuropathology and that intracellular accumulation of detergent-insoluble EAAC1 is a feature of the complex biochemical lesions in AD that include altered protein solubility.     

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer's disease

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na(+)-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V(max), without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.     

Facilitated activation of metabotropic glutamate receptors in cerebellar Purkinje cells in glutamate transporter EAAT4-deficient mice

Around excitatory synapses in cerebellar Purkinje cells (PCs), GLAST and EAAT4 are expressed as predominant glial and neuronal glutamate transporters, respectively. EAAC1, another subtype of neuronal glutamate transporter, is also expressed in PCs. EAAT4 is co-localized with metabotropic glutamate receptors (mGluRs) at perisynaptic sites in excitatory synapses in PCs, and this neuronal transporter was reported to be involved in the regulation of mGluR activation induced by the stimulation of parallel fibers (PFs). However, it remains to be elucidated whether only EAAT4 is specifically involved in mGluR activation among the glutamate transporters expressed near excitatory synapses in PCs. Here we examined mGluR-mediated excitatory postsynaptic currents (mGluR-EPSCs) evoked by PF stimulation in cerebellar slices of mice deficient in EAAT4, EAAC1, or GLAST. PF-evoked mGluR-EPSCs showed larger amplitude and faster rising kinetics in EAAT4-deficient mice than in the wild-type mice. In contrast, there was no significant difference in either the amplitude or the rising kinetics of mGluR-EPSCs in GLAST- or EAAC1-deficient mice compared to wild-type mice. We conclude that EAAT4 is most closely involved in mGluR activation in PCs among the glutamate transporters.     

Protein kinase C–dependent trafficking of glutamate transporters excitatory amino acid carrier 1 and glutamate transporter 1b in cultured cerebellar granule cells

Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.     

Sustained metabolic inhibition induces an increase in the content and phosphorylation of the NR2B subunit of N-methyl-D-aspartate receptors and a decrease in glutamate transport in the rat hippocampus in vivo

The concentration of glutamate is regulated to ensure neurotransmission with a high temporal and local resolution. It is removed from the extracellular medium by high-affinity transporters, dependent on the maintenance of the Na(+) gradient through the activity of Na(+),K(+)-ATPases. Failure of glutamate clearance can lead to neuronal damage, named excitotoxic damage, due to the prolonged activation of glutamate receptors. Severe impairment of glycolytic metabolism during ischemia and hypoglycemia, leads to glutamate transport dysfunction inducing the elevation of extracellular glutamate and aspartate, and neuronal damage. Altered glucose metabolism has also been associated with some neurodegenerative diseases such as Alzheimer's and Huntington's, and a role of excitotoxicity in the neuropathology of these disorders has been raised. Alterations in glutamate transporters and N-methyl-D-aspartate (NMDA) receptors have been observed in these patients, suggesting altered glutamatergic neurotransmission. We hypothesize that inhibition of glucose metabolism might induce changes in glutamatergic neurotransmission rendering neurons more vulnerable to excitotoxicity. We have previously reported that sustained glycolysis impairment in vivo induced by inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), facilitates glutamate-mediated neuronal damage. We have now investigated whether this facilitating effect involves altered glutamate uptake, and/or NMDA receptors in the rat hippocampus in vivo. Results indicate that metabolic inhibition leads to the progressive elevation of extracellular glutamate and aspartate levels in the hippocampus, which correlates with decreased content of the GLT-1 glutamate transporter and diminished glutamate uptake. In addition, we observed increased Tyr(1472) phosphorylation and protein content of the NR2B subunit of the NMDA receptor. Results suggest that moderate sustained glycolysis inhibition alters glutamatergic neurotransmission.     

Modulation of Na(+),K(+) pumping and neurotransmitter uptake by beta-amyloid

Micromolar concentrations of beta-amyloid (Abeta), a 40/42-amino-acid-long proteolytic fragment (Abeta(1-40/42)) of the amyloid precursor protein, was shown previously to play a crucial role in pathogenesis of Alzheimer's disease. We used the Xenopus oocyte expression system to investigate specific effects of micromolar concentrations of Abeta(1-42) on the neurotransmitter transporters for gamma-aminobutyric acid (GABA), GAT1, and for the excitatory amino acid glutamate, EAAC1, which are driven by the transmembrane Na(+) gradient that is regulated by the Na(+),K(+)-ATPase. Brief treatment with Abeta(1-42), up to 80 min, leads to a significant inhibition of ion translocation by the Na(+),K(+)-ATPase (30-40%); also glutamate uptake is inhibited (20%) while GABA uptake is not affected. Since reduced glutamate uptake will result in elevated, neurotoxic concentrations of extracellular glutamate, we investigated the effects of Abeta(1-42) and the smaller fragments, Abeta(12-28) and Abeta(25-35), on EAAC1 in more detail. Prolonged incubation in 1 microM Abeta(1-42) leads to further, strong inhibition of glutamate uptake and EAAC1-mediated current (after 4 h inhibition amounts to more than 80%). Abeta(12-28) is less effective with 50% inhibition after 4 h of incubation at 20 microM. Abeta(1-42) and Abeta(12-28) affect EAAC1-mediated current to a similar extent as the rate of glutamate uptake. The effects on EAAC1-mediated current are irreversible if Abeta were applied for longer time periods. Peptides directly microinjected into the oocyte are ineffective suggesting that the observed effect were mediated by extracellular proteins. Abeta(25-35) hardly affects EAAC1-mediated current or glutamate uptake. The results demonstrate that Abeta specifically inhibits the Na(+),K(+) pump and EAAC1. The domain between amino acids 12 and 28 of Abeta seems to play a crucial role for inhibition of EAAC1. The inhibition of EAAC1 by neurotoxic, elevated extracellular glutamate levels may contribute to Alzheimer's pathogenesis.     

Expression and distribution of ‘high affinity’ glutamate transporters GLT1, GLAST,EAAC1 and of GCPII in the rat peripheral nervous system

l ‐Glutamate is one of the major excitatory neurotransmitters in the mammalian central nervous system, but recently it has been shown to have a role also in the transduction of sensory input at the periphery, and in particular in the nociceptive pathway. An excess of glutamate is implicated in cases of peripheral neuropathies as well. Conventional therapeutic approaches for treating these diseases have focused on blocking glutamate receptors with small molecules or on reducing its synthesis of the receptors through the inhibition of glutamate carboxypeptidase II (GCPII), the enzyme that generates glutamate. In vivo studies have demonstrated that the pharmacological inhibition of GCPII can either prevent or treat the peripheral nerve changes in both BB/Wor and chemically induced diabetes in rats. In this study, we characterized the expression and distribution of glutamate transporters GLT1, GLAST, EAAC1 and of the enzyme GCPII in the peripheral nervous system of female Wistar rats. Immunoblotting results demonstrated that all glutamate transporters and GCPII are present in dorsal root ganglia (DRG) and the sciatic nerve. Immunofluorescence localization studies revealed that both DRG and sciatic nerves were immunopositive for all glutamate transporters and for GCPII. In DRG, satellite cells were positive for GLT1 and GCPII, whereas sensory neurons were positive for EAAC1. GLAST was localized in both neurons and satellite cells. In the sciatic nerve, GLT1 and GCPII were expressed in the cytoplasm of Schwann cells, whereas GLAST and EAAC1 stained the myelin layer. Our results give for the first time a complete characterization of the glutamate transporter system in the peripheral nervous system. Therefore, they are important both for understanding glutamatergic signalling in the PNS and for establishing new strategies to treat peripheral neuropathies.     

H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine

The H(+)-electrochemical gradient was originally considered as a driving force for solute transport only across cellular membranes of bacteria, plants and yeast. However, in the mammalian small intestine, a H(+)-electrochemical gradient is present at the epithelial brush-border membrane in the form of an acid microclimate. Over recent years, a large number of H(+)-coupled cotransport mechanisms have been identified at the luminal membrane of the mammalian small intestine. These transporters are responsible for the initial stage in absorption of a remarkable variety of essential and non-essential nutrients and micronutrients, including protein digestion products (di/tripeptides and amino acids), vitamins, short-chain fatty acids and divalent metal ions. Proton-coupled cotransporters expressed at the mammalian small intestinal brush-border membrane include: the di/tripeptide transporter PepT1 (SLC15A1); the proton-coupled amino-acid transporter PAT1 (SLC36A1); the divalent metal transporter DMT1 (SLC11A2); the organic anion transporting polypeptide OATP2B1 (SLC02B1); the monocarboxylate transporter MCT1 (SLC16A1); the proton-coupled folate transporter PCFT (SLC46A1); the sodium-glucose linked cotransporter SGLT1 (SLC5A1); and the excitatory amino acid carrier EAAC1 (SLC1A1). Emerging research demonstrates that the optimal intestinal absorptive capacity of certain H(+)-coupled cotransporters (PepT1 and PAT1) is dependent upon function of the brush-border Na(+)-H(+) exchanger NHE3 (SLC9A3). The high oral bioavailability of a large number of pharmaceutical compounds results, in part, from absorptive transport via the same H(+)-coupled cotransporters. Drugs undergoing H(+)-coupled cotransport across the intestinal brush-border membrane include those used to treat bacterial infections, hypercholesterolaemia, hypertension, hyperglycaemia, viral infections, allergies, epilepsy, schizophrenia, rheumatoid arthritis and cancer.     

The SLC36 family: proton-coupled transporters for the absorption of selected amino acids from extracellular and intracellular proteolysis

Whilst Na(+) has replaced H(+) as a major transport driving force at the plasma membrane of animal cells, the evolutionarily older H(+)-driven systems persist on endomembranes and at the plasma membrane of specialized cells. The first member of the SLC36 family, present in both intracellular and plasma membranes, was identified independently as a lysosomal amino acid transporter (LYAAT1) responsible for the export of lysosomal proteolysis products into the cytosol and as a proton/amino acid transporter (PAT1) responsible for the absorption of amino acids in the gut. In addition to LYAAT1/PAT1, the family comprises another characterized member, PAT2, and two orphan transporters. Both PAT1 and PAT2 mediate 1:1 symport of protons and small neutral amino acids such as glycine, alanine, and proline. Their mRNAs are broadly and differentially expressed in mammalian tissues. The PAT1 protein localizes to lysosomes in brain neurons, but is also found in the apical membrane of intestinal epithelial cells with a role in the absorption of amino acids from luminal protein digestion. In both cases, protons supplied by the lysosomal H(+)-ATPase or by the acidic microclimate of the brush border membrane drive transport of the amino acids into the cytosol. The subcellular localization and physiological role of PAT2 have still to be determined. SLC36 transporters are related distantly to other proton-coupled amino acid transporters, such as the vesicular neurotransmitter transporter VIAAT/VGAT (SLC32) and system N transporters (SLC38 family).     

Comparative analysis of glutamate transporter expression in rat brain using differential double in situ hybridization

 This study compares the mRNA expression pattern for the three glutamate transporters EAAC1, GLT1 and GLAST in rat brain, using a sensitive non-radioactive in situ hybridization technique. The results confirm the predominantly neuronal localization of EAAC1 mRNA, the astroglial and ependymal localization of GLAST mRNA and the astroglial and neuronal localization of GLT1 mRNA. Further, we demonstrate, using a novel differential double hybridization protocol, that the presence of GLT1 mRNA in neurons is more widespread than previously thought, and that it encompasses the majority of neurons in the neocortex, neurons in the external plexiform layer in the olfactory bulb, neurons in dorsal and ventral parts of the anterior olfactory nucleus, the majority of neurons in the anteromedial thalamic nuclei, the CA3 pyramidal neurons in the hippocampus and neurons in the inferior olive. In addition, we demonstrate marked variations in the expression levels of GLT1 and GLAST mRNAs in different brain areas, suggesting that their mRNA levels are regulated by different mechanisms. Finally, for EAAC1 we demonstrate also a widespread distribution and a marked heterogeneity in the expression levels. EAAC1 is strongly expressed by a heretofore unrecognized group of cells in white matter tracts such as the corpus callosum, fimbria-fornix or anterior commissure. Also, strong EAAC1 expression is present in groups of scattered cells in grey matter areas of much of the forebrain and the cerebellum. These results provide more detailed information about the precise cellular localization of these three glutamate transporters and their regulation at the mRNA level. Accepted: 29 January 1998     

Glutamate transporters GLAST and EAAT4 regulate postischemic Purkinje cell death: an in vivo study using a cardiac arrest model in mice lacking GLAST or EAAT4

Cerebellar Purkinje cells represent a group of neurons highly vulnerable to ischemia. Excitotoxicity is thought to be an important pathophysiological mechanism in Purkinje cell death following ischemia. The glutamate transporter is the only mechanism for the removal of glutamate from the extracellular fluid in the brain. Therefore, glutamate transporters are believed to play a critical role in protecting Purkinje cells from ischemia-induced damage. Two distinct glutamate transporters, GLAST and EAAT4, are expressed most abundantly in the cerebellar cortex. GLAST is expressed in Bergmann glia, whereas EAAT4 is concentrated in the perisynaptic regions of Purkinje cell spines. However, the in vivo functional significance of these glial and neuronal glutamate transporters in postischemic Purkinje cell death is largely unknown. To clarify the role of these glutamate transporters in the protection of Purkinje cells after global brain ischemia, we evaluated Purkinje cell loss after cardiac arrest in mice lacking GLAST or EAAT4. We found that Purkinje cells with low EAAT4 expression were selectively lost after cardiac arrest in GLAST mutant mice. This result demonstrates that GLAST plays a role in preventing excitotoxic cerebellar damage after ischemia in concert with EAAT4.     

Subcellular localization of the glutamate transporters GLAST and GLT at the neuromuscular junction in rodents

The vertebrate neuromuscular junction (NMJ) is known to be a cholinergic synapse at which acetylcholine (ACh) is released from the presynaptic terminal to act on postsynaptic nicotinic ACh receptors. There is now growing evidence that glutamate, which is the main excitatory transmitter in the CNS and at invertebrate NMJs, may have a signaling function together with ACh also at the vertebrate NMJ. In the CNS, the extracellular concentration of glutamate is kept at a subtoxic level by Na(+)-driven high-affinity glutamate transporters located in plasma membranes of astrocytes and neurons. The glutamate transporters are also pivotal for shaping glutamate receptor responses at synapses. In order to throw further light on the potential role of glutamate as a cotransmitter at the NMJ we used high-resolution immunocytochemical methods to investigate the localization of the plasma membrane glutamate transporters GLAST (glutamate aspartate transporter) and GLT (glutamate transporter 1) in rat and mice NMJ regions. Confocal laser-scanning immunocytochemistry showed that GLT is restricted to the NMJ in rat and mouse skeletal muscle. Lack of labeling signal in knock-out mice confirmed that the immunoreactivity observed at the NMJ was specific for GLT. GLAST was also localized at the NMJ in rat but not detected in mouse NMJ (while abundant in mouse brain). Post-embedding electron microscopic immunocytochemistry and quantitative analyses in rat showed that GLAST and GLT are enriched in the junctional folds of the postsynaptic membrane at the NMJ. GLT was relatively higher in the slow-twitch muscle soleus than in the fast-twitch muscle extensor digitorum longus, whereas GLAST was relatively higher in extensor digitorum longus than in soleus. The findings show--together with previous demonstration of vesicular glutamate, a vesicular glutamate transporter and glutamate receptors--that mammalian NMJs contain the machinery required for synaptic release and action of glutamate. This indicates a signaling role for glutamate at the normal NMJ and provides a basis for the ability of denervated muscle to be reinnervated by glutamatergic axons from the CNS.     

Increased expression of the glial glutamate transporter EAAT2 modulates excitotoxicity and delays the onset but not the outcome of ALS in mice

The glial glutamate transporter EAAT2 is primarily responsible for clearance of glutamate from the synaptic cleft and loss of EAAT2 has been previously reported in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. The loss of functional EAAT2 could lead to the accumulation of extracellular glutamate, resulting in cell death known as excitotoxicity. However, it is still unknown whether it is a primary cause in the cascade leading to neuron degeneration or a secondary event to cell death. The goals of this study were to generate transgenic mice overexpressing EAAT2 and then to cross these mice with the ALS-associated mutant SOD1(G93A) mice to investigate whether supplementation of the loss of EAAT2 would delay or rescue the disease progression. We show that the amount of EAAT2 protein and the associated Na+-dependent glutamate uptake was increased about 2-fold in our EAAT2 transgenic mice. The transgenic EAAT2 protein was properly localized to the cell surface on the plasma membrane. Increased EAAT2 expression protects neurons from L-glutamate induced cytotoxicity and cell death in vitro. Furthermore, our EAAT2/G93A double transgenic mice showed a statistically significant (14 days) delay in grip strength decline but not in the onset of paralysis, body weight decline or life span when compared with G93A littermates. Moreover, a delay in the loss of motor neurons and their axonal morphologies as well as other events including caspase-3 activation and SOD1 aggregation were also observed. These results suggest that the loss of EAAT2 may contribute to, but does not cause, motor neuron degeneration in ALS.     

The SLC32 transporter,a key protein for the synaptic release of inhibitory amino acids

The SLC32 family comprises a single member: the vesicular inhibitory amino acid transporter (VIAAT) or vesicular GABA transporter (VGAT). It belongs to a eukaryotic-specific superfamily of H(+)-coupled amino acid transporters, which also comprises the mammalian SLC36 and SLC38 transporters. VIAAT exchanges GABA or glycine for protons. It is present on synaptic vesicles of GABAergic and glycinergic neurons, and in some endocrine cells, where it ensures the H(+)-ATPase-driven uptake, and subsequent exocytotic release, of inhibitory amino acids. Despite a similar function in vesicular neurotransmitter loading, VIAAT is not related to the vesicular glutamate transporter (VGLUT, SLC17) or the vesicular monoamine transporter/vesicular acetylcholine transporter (VMAT/VACHT, SLC18) proteins.     

The excitatory amino acid carrier 1 (EAAC1) in the rat nucleus of the solitary tract: subcellular localization suggests no major role in glutamate clearance

The excitatory amino acid carrier 1 (EAAC1) is a sodium-dependent glutamate transporter widely found in the mammalian brain and mainly localized in the somatodendritic compartment of neurons. The present study was performed to determine whether EAAC1 is present in the rat nucleus of the solitary tract (NST, a sensory brainstem nucleus involved in visceroception) and to document its subcellular localization. Using fluorescent immunolabeling, peroxidase immunostaining and quantitative immunogold labeling, we showed that both intracellular and plasma membrane-associated pools of EAAC1 transporters existed in dendrites of NST neurons. Although plasma membrane-associated transporters were more concentrated in the vicinity of synapses, no labeling was found at the axon–dendrite interface, suggesting that EAAC1 was not (or barely) expressed in this portion of dendritic membrane. Using computer simulation, we next showed that the ability of EAAC1 to efficiently take up synaptically released glutamate was very low outside the axon–dendrite interface. These data suggest that EAAC1 transporters present on NST dendrites may play a minor role if any in glutamate clearance.     

Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis.

BACKGROUND. Amyotrophic lateral sclerosis (ALS) is a chronic degenerative neurologic disorder characterized by the death of motor neurons in the cerebral cortex and spinal cord. Recent studies have suggested that the metabolism of glutamate, a potentially neurotoxic amino acid, is abnormal in patients with ALS. We hypothesized that the high-affinity glutamate transporter is the site of the defect. METHODS. We measured high-affinity, sodium-dependent glutamate transport in synaptosomes from neural tissue obtained from 13 patients with ALS, 17 patients with no neurologic disease, and 27 patients with other neuro-degenerative diseases (Alzheimer's disease in 15 patients and Huntington's disease in 12 patients). The groups were comparable with respect to age and the interval between death and autopsy. Synaptosomes were prepared from spinal cord, motor cortex, sensory cortex, visual cortex, striatum, and hippocampus. We also measured sodium-dependent transport of gamma-aminobutyric acid and phenylalanine in the synaptosomal preparations. RESULTS. In patients with ALS, there was a marked decrease in the maximal velocity of transport for high-affinity glutamate uptake in synaptosomes from spinal cord (-59 percent, P less than 0.001), motor cortex (-70 percent, P less than 0.001), and somatosensory cortex (-39 percent, P less than 0.05), but not in those from visual cortex, striatum, or hippocampus. The affinity of the transporter for glutamate was not altered. No abnormalities in glutamate transport were found in synaptosomes from patients with other chronic neurodegenerative disorders. The transport of gamma-aminobutyric acid and phenylalanine was normal in patients with ALS. CONCLUSIONS. ALS is associated with a defect in high-affinity glutamate transport that has disease, region, and chemical specificity. Defects in the clearance of extracellular glutamate because of a faulty transporter could lead to neurotoxic levels of extracellular glutamate and thus be pathogenic in ALS.     

TNFR1 mediates increased neuronal membrane EAAT3 expression after in vivo cerebral ischemic preconditioning

A short ischemic event (ischemic preconditioning) can result in subsequent resistance to severe ischemic injury (ischemic tolerance). Glutamate is released after ischemia and produces cell death. It has been described that after ischemic preconditioning, the release of glutamate is reduced. We have shown that an in vitro model of ischemic preconditioning produces upregulation of glutamate transporters which mediates brain tolerance. We have now decided to investigate whether ischemic preconditioning-induced glutamate transporter upregulation takes also place in vivo, its cellular localization and the mechanisms by which this upregulation is controlled. A period of 10 min of temporary middle cerebral artery occlusion was used as a model of ischemic preconditioning in rat. EAAT1, EAAT2 and EAAT3 glutamate transporters were found in brain from control animals. Ischemic preconditioning produced an up-regulation of EAAT2 and EAAT3 but not of EAAT1 expression. Ischemic preconditioning-induced increase in EAAT3 expression was reduced by the TNF-alpha converting enzyme inhibitor BB1101. Intracerebral administration of either anti-TNF-alpha antibody or of a TNFR1 antisense oligodeoxynucleotide also inhibited ischemic preconditioning-induced EAAT3 up-regulation. Immunohistochemical studies suggest that, whereas the expression of EAAT3 is located in both neuronal cytoplasm and plasma membrane, ischemic preconditioning-induced up-regulation of EAAT3 is mainly localized at the plasma membrane level. In summary, these results demonstrate that in vivo ischemic preconditioning increases the expression of EAAT2 and EAAT3 glutamate transporters the upregulation of the latter being at least partly mediated by TNF-alpha converting enzyme/TNF-alpha/TNFR1 pathway.