Altered expression of glutamate transporters under hypoxic conditions in vitro

Regulation of extracellular excitotoxins by glial and neuronal glutamate transporters is critical to maintain synaptic terminal integrity. Factors interfering with the normal functioning of these transporters might be involved in neurodegeneration. Among them, recent studies have shown that hypoxia alters glutamate transporter function; however, it is unclear if hypoxia has an effect on the expression of glutamate transporters and which intracellular signaling pathways are involved. The C6 rat glial and GT1--7 mouse neuronal cell lines were exposed to hypoxic conditions (5% CO(2), 95% N(2)) and levels of glutamate transporter mRNA were determined by ribonuclease protection assay. After 21 hr, there was a 100% increase in levels of rat excitatory amino acid transporter 3 (EAAT3) mRNA in C6 cells and a 600% increase in levels of murine EAAT2 mRNA in GT1--7 cells. There was a similar increase in mRNA levels after hypoxia in C6 cells transfected with human EAAT2, whereas reoxygenation normalized the expression levels of glutamate transporters. Although the expression of EAATs was associated with increased immunoreactivity by Western blot, functioning of the transporters was decreased as evidenced by D-aspartate uptake. Finally, although the protein kinase C stimulator phorbol-12-myristate-13-acetate enhanced EAAT2 mRNA levels after hypoxia, protein kinase C inhibitor bisindolylmaleimide I had the opposite effect. Taken together, this study suggests that the hypoxia is capable of upregulating levels of EAATs via a protein kinase C-dependent compensatory mechanism. This increased expression is not sufficient to overcome the decreased functioning of the EAATs associated with decreased ATP production and mitochondrial dysfunction.     

Increased expression of the neuronal glutamate transporter (EAAT3/EAAC1) in hippocampal and neocortical epilepsy

PURPOSE: To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy. METHODS: The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis. RESULTS: The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with non-dysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue. CONCLUSIONS: Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types.     

Nonsynaptic localization of the excitatory amino acid transporter 4 in photoreceptors

Excitatory amino acid transporters (EAATs) are involved in regulating extracellular glutamate levels at synaptic regions in the CNS. EAAT1, 2, 3, and 5 have been found in the mammalian retina, but the presence of EAAT4 has remained controversial. Recently, we found a high level of EAAT4 mRNA in the human retina, and this observation lead us to examine whether EAAT4 was expressed in the mammalian retina. Immunoblotting studies showed the presence of EAAT4-immunoreactive proteins in human and mouse retinas, corresponding to EAAT4 monomers and dimers. Immunohistochemistry revealed that EAAT4 was localized in rod and cone photoreceptor outer segments in the human retina, and in the outer and inner segments of mouse and ground squirrel retinas. In no case was EAAT4 found in the outer plexiform layer or in any other layer in the retina. EAAT4 expression by photoreceptors was confirmed by immunoblotting a purified rod outer segment preparation, which showed the presence of a 50-kDa EAAT4-immunoreactive protein. In addition, the EAAT4-associated protein, GTRAP41, was found in the human, mouse, and squirrel retinas as well as in the rod outer segment preparation. Further immunocytochemical and co-immunoprecipitation experiments demonstrated that GTRAP41 was colocalized and interacted in vivo with EAAT4. Importantly, glutamate uptake and drug inhibition experiments showed that an EAAT4-like glutamate uptake system is present in the rod outer segments. Finally, we examined whether glutamate signaling mediated by EAAT4 can modulate rod outer segment phagocytosis by the retinal pigment epithelium. Results of the present study show that EAAT4 is present in the outer segments, a nonsynaptic region of photoreceptors, where it might provide a feedback mechanism for sensing extracellular glutamate or serve as an outer barrier to prevent glutamate from escaping from the retina.     

Climbing Fiber-Mediated Spillover Transmission to Interneurons Is Regulated by EAAT4

Neurotransmitter spillover is a form of communication not readily predicted by anatomic structure. In the cerebellum, glutamate spillover from climbing fibers recruits molecular layer interneurons in the absence of conventional synaptic connections. Spillover-mediated signaling is typically limited by transporters that bind and reuptake glutamate. Here, we show that patterned expression of the excitatory amino acid transporter 4 (EAAT4) in Purkinje cells regulates glutamate spillover to molecular layer interneurons. Using male and female Aldolase C-Venus knock-in mice to visualize zebrin microzones, we find larger climbing fiber-evoked spillover EPSCs in regions with low levels of EAAT4 compared with regions with high EAAT4. This difference is not explained by presynaptic glutamate release properties or postsynaptic receptor density but rather by differences in the glutamate concentration reaching receptors on interneurons. Inhibiting glutamate transport normalizes the differences between microzones, suggesting that heterogeneity in EAAT4 expression is a primary determinant of differential spillover. These results show that neuronal glutamate transporters limit extrasynaptic transmission in a non–cell-autonomous manner and provide new insight into the functional specialization of cerebellar microzones.SIGNIFICANCE STATEMENT Excitatory amino acid transporters (EAATs) help maintain the fidelity and independence of point-to-point synaptic transmission. Whereas glial transporters are critical to maintain low ambient levels of extracellular glutamate to prevent excitotoxicity, neuronal transporters have more subtle roles in shaping excitatory synaptic transmission. Here we show that the patterned expression of neuronal EAAT4 in cerebellar microzones controls glutamate spillover from cerebellar climbing fibers to nearby interneurons. These results contribute to fundamental understanding of neuronal transporter functions and specialization of cerebellar microzones.     

Neuronal glutamate transporters regulate glial excitatory transmission

In the CNS, excitatory amino acid transporters (EAATs) localized to neurons and glia terminate the actions of synaptically released glutamate. Whereas glial transporters are primarily responsible for maintaining low ambient levels of extracellular glutamate, neuronal transporters have additional roles in shaping excitatory synaptic transmission. Here we test the hypothesis that the expression level of the Purkinje cell (PC)-specific transporter, EAAT4, near parallel fiber (PF) release sites controls the extrasynaptic glutamate concentration transient following synaptic stimulation. Expression of EAAT4 follows a parasagittal banding pattern that allows us to compare regions of high and low EAAT4-expressing PCs. Using EAAT4 promoter-driven eGFP reporter mice together with pharmacology and genetic deletion, we show that the level of neuronal transporter expression influences extrasynaptic transmission from PFs to adjacent Bergmann glia (BG). Surprisingly, a twofold difference in functional EAAT4 levels is sufficient to alter signaling to BG, although EAAT4 may only be responsible for removing a fraction of released glutamate. These results demonstrate that physiological regulation of neuronal transporter expression can alter extrasynaptic neuroglial signaling.     

共聚焦激光扫描显微镜研究大鼠脑缺血谷氨酸载体GLAST mRNA和EAAT1的表达与其细胞分布

目的通过应用共聚焦激光扫描显微镜技术(confocal laser scanning microscope, CLSM)观察脑缺血后谷氨酸载体GLAST mRNA和EAAT1蛋白表达的细胞定位,探讨CLSM技术中三维重建和三维显示在观察基因和蛋白在神经细胞上空间定位的应用.方法对脑缺血后采用原位杂交和免疫组织化学相结合的荧光双标技术标记的脑片进行双通道断层扫描以及三维数据重组.结果脑缺血后,荧光免疫组化双标显示大脑皮质缺血半暗区内的谷氨酸载体EAAT1蛋白与星形胶质细胞和神经元均有共表达;原位杂交结合免疫组化的荧光双标显示缺血周边区谷氨酸载体GLAST mRNA与星形胶质细胞和神经元有明显的共表达.结论共聚焦激光扫描显微镜观察脑缺血后谷氨酸载体GLAST mRNA和EAAT1蛋白在神经胶质细胞和神经元上的空间定位,为进一步研究脑缺血后谷氨酸载体系统作用机制提供了更为准确的形态依据.     

Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity

The expression of excitatory amino acid transporters (EAATs) in rat hippocampus was studied following kainic acid-induced seizure activity in vivo and in hippocampal slice cultures. Protein and mRNA levels of the glial (EAAT2) and neuronal (EAAT3) transporters were determined with affinity-purified antibodies and oligonucleotide probes, respectively. Kainate treatment decreased EAAT3 immunoreactivity in stratum lacunosum moleculare within 4 h of seizure onset. Upon pyramidal cell death (5 days after kainate treatment), EAAT3 immunoreactivity in stratum pyramidale of CA1 and in stratum lacunosum moleculare was almost completely eliminated. The rapid effect of kainate on EAAT3 expression was confirmed by in situ hybridization; EAAT3 mRNA levels were decreased in CA1 and CA3 regions within 4-8 h of seizure onset. Kainate treatment had an opposite effect on levels and expression of EAAT2. Developmental studies indicated that the rapid regulation of transporter expression was not observed in rats younger than 21 days, an observation congruent with previous reports regarding the resistance of young rats to kainate. In hippocampal organotypic cultures, which lack a major excitatory input from the entorhinal cortex, kainate produced a slow decrease in [3H]d-aspartate uptake. This study indicates that an early effect of kainate treatment consists of down-regulation of the neuronal transporter EAAT3 in restricted hippocampal regions, together with a modest increase in the expression of the glial transporter EAAT2. Differential regulation of neuronal and glial glutamate transporters may thus play a role in kainate-induced seizure, neurotoxicity and neuronal plasticity.     

Role of neuronal glutamate transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical neurons

Summary. Cysteine uptake is the rate-limiting process in glutathione synthesis. Previously we have shown that the inhibitors of excitatory amino acid transporters (EAATs) significantly enhance glutamate toxicity via depletion of intracellular glutathione. In this study we show evidence that the neuronal glutamate transporter EAAT3 is directly enrolled in cysteine uptake in cultured neurons. Neuronal cysteine uptake was dependent on the extracellular sodium, and was suppressed by EAAT inhibitors. Cysteine uptake was suppressed by extracellular glutamate and aspartate, substrates of EAATs, and not by substrates of cysteine transporters. Intracellular glutathione levels were reduced by EAAT inhibitors, and not by inhibitors of cysteine transporters. Knock down of EAAT3 expression using antisense oligonucleotide significantly reduced cysteine uptake, intracellular glutathione level, and neuronal viability against oxidative stress. These facts indicate that EAAT3 functions as a cysteine transporter, and this function seems to be unique and distinct from cysteine transporters that have been reported.     

Increased numbers of coassembled PSD-95 to NMDA-receptor subunits NR2B and NR1 in human epileptic cortical dysplasia

PURPOSE: Glutamatergic transmission between neurons occurs at chemical synapses. The N-methyl-d-aspartate (NMDA)-receptor subclass of ionotropic glutamate receptors has been implicated in the epileptogenic mechanisms in human cortical dysplasia (CD). NMDA receptors are clustered at the postsynaptic membrane by anchoring to the postsynaptic density protein PSD-95, a putative ion channel-clustering protein. In this study, we quantitatively investigated the coassembly of PSD-95 to NR2B and NR1 in human epileptogenic cortex as compared with nonepileptic cortex. METHODS: We used coimmunoprecipitation and immunoblotting techniques to quantify and compare the numbers of coassembled PSD-95 with NR2B, PSD-95 with NR1, and NR2B with NR1 in the membrane proteins of brain tissues resected from four patients (aged 3.5, 6, 14, and 18 years) with medically intractable neocortical epilepsy associated with CD. The resected cortical tissues were grouped into epileptic and nonepileptic, as determined by prolonged subdural electrode recordings in three patients and direct intraoperative electrocorticographic recording in one patient. RESULTS: In all patients, the amounts of immunoprecipitated complexes, which reflect the numbers of coassembled PSD-95 proteins to NR2B subunits, were increased in epileptic cortex as compared with nonepileptic cortex. CONCLUSIONS: These results suggest that increased coassembly of NR2B and NR1 with PSD-95 may underlie one of the cellular mechanisms that contribute to the in situ increased hyperexcitability, leading to seizure generation in focal CD.     

Altered glutamate reuptake in relapsing-remitting and secondary progressive multiple sclerosis cortex: correlation with microglia infiltration, demyelination, and neuronal and synaptic damage

Cortical involvement in multiple sclerosis (MS) is emerging as an important determinant of disease progression. The mechanisms responsible for MS cortical pathology are not fully characterized. The objective of this study was to assess the role of excitotoxicity in MS cortex, evaluating excitatory amino acid transporter (EAAT) expression and its relationship with demyelination, inflammation, gliosis, and neuronal and synaptic pathology. EAATs are essential in maintaining low extracellular glutamate concentrations and preventing excitotoxicity. Ten MS brains (3 relapsing-remitting MS cases and 7 secondary progressive MS cases) were evaluated by immunohistochemistry for myelin basic protein, CD68, HLA-DR, EAAT1, EAAT2, glial fibrillary acidic protein, phosphorylated c-Jun N-terminal kinase (pJNK), synaptophysin, and neurofilaments. Cortical lesions were frequently observed in MS brains in variable numbers and extensions. In cortical lesions, activated microglia infiltration correlated with focal loss of EAAT1, EAAT2, and synaptophysin immunostaining, and with neuronal immunostaining for pJNK, a protein involved in response to excitotoxic injury. No reduction of EAATs or synaptophysin immunostaining was observed in demyelinated cortex in the absence of activated microglia. Alterations of the mechanisms of glutamate reuptake are found in cortical MS lesions in the presence of activated microglia and are associated with signs of neuronal and synaptic damage suggestive of excitotoxicity. Excitotoxicity may be involved in the pathogenesis of demyelination and of neuronal and synaptic damage in MS cortex.     

Alternative splicing of glutamate transporter EAAT2 RNA in neocortex and hippocampus of temporal lobe epilepsy patients

RATIONALE: Altered expression of glutamate transporter EAAT2 protein has been reported in the hippocampus of patients with temporal lobe epilepsy (TLE). Two alternative EAAT2 mRNA splice forms, one resulting from a partial retention of intron 7 (I7R), the other from a deletion of exon 9 (E9S), were previously implicated in the loss of EAAT2 protein in patients with amyotrophic lateral sclerosis. METHODS: By RT-PCR we studied the occurrence of I7R and E9S in neocortical and hippocampal specimens from TLE patients and non-neurological controls. RESULTS: Both splice forms were found in all neocortical specimens from TLE patients (100% I7R, 100% E9S). This was significantly more than in controls (67% I7R, 60% E9S; P < 0.05). We also detected I7R and E9S in all seven motor cortex post-mortem samples from patients with amyotrophic lateral sclerosis. Within the TLE patient group, both splice variants appeared significantly more in non-sclerotic (100%), than in sclerotic hippocampi (69%, P < 0.05). CONCLUSION: These data indicate that the epileptic brain, especially that of TLE patients without hippocampal sclerosis, is highly prone to alternative EAAT2 mRNA splicing. Our data confirm that the presence of alternative EAAT2 splice forms is not disease specific.     

Over-expression of the human EAAT2 glutamate transporter within neurons of mouse organotypic hippocampal slice cultures leads to increased vulnerability of CA1 pyramidal cells

Excitatory amino acid transporters (EAATs) maintain the balance between pathological and physiological conditions by limiting the extracellular concentration of glutamate within the CNS and thus preventing excitotoxic injury. The loss of EAAT2 has been associated with the development of neurological diseases such as amyotrophic lateral sclerosis. It has therefore been suggested that the over-expression of specific EAATs may provide some degree of neuroprotection. However, the inability to isolate and study the function of the different EAAT isoforms in a cell type-specific manner has made it difficult to determine the exact contribution of individual EAATs toward neuroprotection or neurodegeneration in the context of excitotoxic injury. To address this question, we transduced hippocampal slice cultures from 1-week-old C57B/6 mice with recombinant adeno-associated virus carrying an EAAT2 gene expression cassette. EAAT2 gene expression was driven in neurons with the neuron-specific enolase promoter. Using this model system, we were able to induce a significant increase in the expression of functional EAAT2. Consequently, a significant increase in CA1 neuronal damage was observed in slices over-expressing EAAT2 in neurons following an acute exposure to exogenous glutamate. These data suggest that the increased expression of EAAT2 within neurons may contribute to neurodegeneration.     

Parasynaptic NMDA receptor signaling couples neuronal glutamate transporter function to AMPA receptor synaptic distribution and stability

At synapses, two major processes occur concomitantly after the release of glutamate: activation of AMPA receptors (AMPARs) to conduct synaptic transmission and activation of excitatory amino acid transporters (EAATs) for transmitter removal. Although crosstalk between the receptors and EAATs is conceivable, whether and how the transporter activity affects AMPAR synaptic localization remain unknown. Using cultured hippocampal and cortical rat neurons, we show that inhibition of glutamate transporters leads to rapid reduction in AMPAR synaptic accumulation and total AMPAR abundance. EAAT inactivity also results in elevated internalization and reduced surface expression of AMPARs. The reduction in AMPAR amount is accompanied by receptor ubiquitination and can be blocked by suppression of proteasome activity, indicating the involvement of proteasome-mediated receptor degradation. Consistent with glutamate spillover, effect of EAAT inhibition on AMPAR distribution and stability is dependent on the activation of parasynaptically localized NR2B-containing NMDA receptors (NMDARs). Moreover, we show that neuronal glutamate transporters, especially those localized at the postsynaptic sites, are responsible for the observed effect during EAAT suppression. These results indicate a role for neuron-specific glutamate transporters in AMPAR synaptic localization and stability.     

Increased expression of glutamate transporters in subcortical white matter after transient focal cerebral ischemia

Transient focal cerebral ischemia leads to extensive excitotoxic glial damage in the subcortical white matter. Efficient reuptake of released glutamate is essential for preventing glutamate receptor overstimulation and neuronal and glial death. The present study evaluates the expression of the main glutamate transporters (EAAT1, EAAT2, and EAAT3) in subcortical white matter of the rat after transient middle cerebral artery occlusion. Western blot analysis and immunohistochemistry show an increase in the expression of EAAT1 and EAAT2 in subcortical white matter early after ischemia which subsequently decreases at longer reperfusion periods. However, expression of both EAAT1 and EAAT2 remains higher in astrocytes forming the gliotic scar and in microglial/macrophage cells at the border of or within the infarct area, respectively. Taken together, these results indicate that there is a transient enhanced expression of EAATs in the subcortical white matter early after ischemia. Our findings reveal an adaptive response of subcortical white matter to increased levels of glutamate during focal cerebral ischemia which may limit excitotoxic damage.     

Human brain neurons express a novel splice variant of excitatory amino acid transporter 5 (hEAAT5v)

Excitatory amino acid transporter 5 (EAAT5) is a protein that is known to be alternately spliced and to be abundantly expressed in the retina by populations of neurons including photoreceptors and bipolar cells. EAAT5 acts as a slow glutamate transporter and also as glutamate-gated chloride channel, the chloride conductance being large enough for EAAT5 to serve functionally as an “inhibitory” glutamate receptor. However, there has been a long-standing view that the classically spliced form of EAAT5 is not abundant or widespread in the brain and so it has not been extensively investigated in the literature. We recently identified a human-specific splicing form of EAAT5 that was not expressed by rodents but was shown to be a functional glutamate transporter. We have examined the expression of this form of EAAT5, hEAAT5v at the mRNA, and protein level in human brain, and show that populations of human cortical pyramidal neurons and cerebellar Purkinje cells show significant expression of hEAAT5v. Accordingly, we infer that EAAT5 may well be a player in modulating neuronal function in the human brain and propose that its localization in both glutamatergic and GABAergic neurons could be compatible with a role in influencing intracellular chloride and thereby neuronal parameters such as membrane potential rather than acting as a presynaptic glutamate transporter.     

Glutamate transporter expression and function in human glial progenitors

Glutamate is the major neurotransmitter of the brain, whose extracellular levels are tightly controlled by glutamate transporters. Five glutamate transporters in the human brain (EAAT1-5) are present on both astroglia and neurons. We characterize the profile of three different human astroglial progenitors in vitro: human glial restricted precursors (HGRP), human astrocyte precursors (HAPC), and early-differentiated astrocytes. EAAT 1, EAAT3, and EAAT4 are all expressed in GRPs with a subsequent upregulation of EAAT1 following differentiation of GRPs into GRP-derived astrocytes in the presence of bone morphogenic protein (BMP-4). This corresponds to a significant increase in the glutamate transport capacity of these cells. EAAT2, the transporter responsible for the bulk of glutamate transport in the adult brain, is not expressed as a full-length protein, nor does it appear to have functional significance (as determined by the EAAT2 inhibitor dihydrokainate) in these precursors. A splice variant of EAAT2, termed EAAT2b, does appear to be present in low levels, however. EAAT3 and EAAT4 expression is reduced as glial maturation progresses both in astrocyte precursors and early-differentiated astrocytes and is consistent with their role in adult tissues as primarily neuronal glutamate transporters. These human glial precursors offer several advantages as tools for understanding glial biology because they can be passaged extensively in the presence of mitogens, afford the potential to study the temporal changes in glutamate transporter expression in a tightly controlled fashion, and are cultured in the absence of neuronal coculture, allowing for the independent study of astroglial biology.