Biomedical subjects

J D Rothstein

Publications and source records attributed to J D Rothstein.

At least 55 records · Page 3Linked to original sources

PubMed1998

Multiple signaling pathways regulate cell surface expression and activity of the excitatory amino acid carrier 1 subtype of Glu transporter in C6 glioma.

Neuronal and glial sodium-dependent transporters are crucial for the control of extracellular glutamate levels in the CNS. The regulation of these transporters is relatively unexplored, but the activity of other transporters is regulated by protein kinase C (PKC)- and phosphatidylinositol 3-kinase (PI3K)-mediated trafficking to and from the cell surface. In the present study the C6 glioma cell line was used as a model system that endogenously expresses the excitatory amino acid carrier 1 (EAAC1) subtype of neuronal glutamate transporter. As previously observed, phorbol 12-myristate 13-acetate (PMA) caused an 80% increase in transporter activity within minutes that cannot be attributed to the synthesis of new transporters. This increase in activity correlated with an increase in cell surface expression of EAAC1 as measured by using a membrane-impermeant biotinylation reagent. Both effects of PMA were blocked by the PKC inhibitor bisindolylmaleimide II (Bis II). The putative PI3K inhibitor, wortmannin, decreased L-[3H]-glutamate uptake activity by >50% within minutes. Wortmannin decreased the Vmax of L-[3H]-glutamate and D-[3H]-aspartate transport, but it did not affect Na+-dependent [3H]-glycine transport. Wortmannin also decreased cell surface expression of EAAC1. Although wortmannin did not block the effects of PMA on activity, it prevented the PMA-induced increase in cell surface expression. This trafficking of EAAC1 also was examined with immunofluorescent confocal microscopy, which supported the biotinylation studies and also revealed a clustering of EAAC1 at cell surface after treatment with PMA. These studies suggest that the trafficking of the neuronal glutamate transporter EAAC1 is regulated by two independent signaling pathways and also may suggest a novel endogenous protective mechanism to limit glutamate-induced excitotoxicity.

Amino Acid Transport System X-AG↗

PubMed1998

Mutations in the glutamate transporter EAAT2 gene do not cause abnormal EAAT2 transcripts in amyotrophic lateral sclerosis.

Recently, variant mRNA transcripts for the astroglial glutamate transporter EAAT2 have been detected in brain tissues of 60% of patients with sporadic amyotrophic lateral sclerosis (SALS). We have tested the hypothesis that the gene for EAAT2 may be defective in some ALS cases. In 16 familial ALS (FALS) pedigrees without mutations in SOD1, we failed to detect genetic linkage to the EAAT2 locus. We next characterized the genomic organization of the EAAT2 gene and used single-strand conformation polymorphism analysis of genomic DNA to identify one novel mutation in a single SALS patient and two novel mutations in 2 affected FALS siblings. In the SALS patient, the mutation substitutes serine for an asparagine that might be involved in N-linked glycosylation of the EAAT2 protein. In the 2 affected individuals in the FALS family, we detected both a mutation in the 5' end of intron 7 and a silent G --> A transition at codon 234 in exon 5. It remains unclear whether this intron 7 mutation is related to the defective mRNA splicing. These studies indicate that germline mutations in the EAAT2 gene are infrequent and do not explain the presence of variant mRNA transcripts of EAAT2 in more than one-half of ALS cases.

Amyotrophic Lateral Sclerosis↗

PubMed1998

Use of antisense oligodeoxynucleotides to inhibit expression of glutamate transporter subtypes.

Antisense ODN are highly effective tools to selectively inhibit synthesis of glutamate transporter subtypes. They have been used to evaluate the biology of individual transporter subtypes in vivo and in vitro. Appropriate use of antisense ODN, however, requires a number of important controls to validate the specificity of their effects. Ultimately, the efficacy at inhibiting the synthesis of transporters proteins reflects several variables: the actual efficacy of the antisense to directly inhibit synthesis, the rate of degradation of the ODN, the rate of intracellular penetration of the ODN, the rate of new protein synthesis for the transporter subtype, and the actual penetration of the ODN into CNS tissue. The methods described in this chapter will help one to optimize each of these parameters.

ATP-Binding Cassette Transporters↗

PubMed1998

Regional and cellular expression of glial (GLT1) and neuronal (EAAC1) glutamate transporter proteins in ovine fetal brain.

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate which can have either neurotrophic or neurotoxic effects in the developing brain, depending on its concentration. Using immunoblotting and immunocytochemistry, we tested the hypotheses that expression of neuronal and glial glutamate transporter proteins was regionally and temporally regulated in the developing ovine brain and that expression of the glial isoform early in development was not cell-type specific. Immunoblots for the neuronal glutamate transporter EAAC1 revealed a major band of immunoreactivity at 69,000 nmol. wt, whereas glial glutamate transporter-1 (GLT1) immunoreactivity was observed as 73,000 and 146,000 mol. wt proteins. EAAC1 and GLT1 are regulated differently during development, with EAAC1 immunoreactivity being most abundant at 60 and 71 days completed gestation (term=145 days) and dissipating thereafter, while GLT1 immunoreactivity was most abundant at 136 days gestation. By immunocytochemistry EAAC1 expression is neuronal throughout gestation with intense labelling of dendrites within the telencephalon evident at 60 days. Neuropil, neuronal cell bodies and processes are EAAC1-immunoreactive throughout gestation with no evidence of astrocytic or oligodendroglial immunoreactivity. In contrast, GLT1 is expressed by neuronal and non-neuronal cell types during midgestation with astrocyte selectivity developing by 136 days. During midgestation, GLT1 is transiently expressed in neurons of the subplate, cranial nerve nuclei, basal ganglia, and cerebellar cortex. The major finding of this study, that GLT1 is transiently expressed in various neuronal populations at midgestation demonstrates that the cell-type specificity of the GLT1 phenotype is developmentally regulated and depends on brain maturity.

ATP-Binding Cassette Transporters↗

PubMed1998

Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is characterized by selective upper and lower motor neuron degeneration, the pathogenesis of which is unknown. About 60%-70% of sporadic ALS patients have a 30%-95% loss of the astroglial glutamate transporter EAAT2 (excitatory amino acid transporter 2) protein in motor cortex and spinal cord. Loss of EAAT2 leads to increased extracellular glutamate and excitotoxic neuronal degeneration. Multiple abnormal EAAT2 mRNAs, including intron-retention and exon-skipping, have now been identified from the affected areas of ALS patients. The aberrant mRNAs were highly abundant and were found only in neuropathologically affected areas of ALS patients but not in other brain regions. They were found in 65% of sporadic ALS patients but were not found in nonneurologic disease or other disease controls. They were also detectable in the cerebrospinal fluid (CSF) of living ALS patients, early in the disease. In vitro expression studies suggest that proteins translated from these aberrant mRNAs may undergo rapid degradation and/ or produce a dominant negative effect on normal EAAT2 resulting in loss of protein and activity. These findings suggest that the loss of EAAT2 in ALS is due to aberrant mRNA and that these aberrant mRNAs could result from RNA processing errors. Aberrant RNA processing could be important in the pathophysiology of neurodegenerative disease and in excitotoxicity. The presence of these mRNA species in ALS CSF may have diagnostic utility.

Amyotrophic Lateral Sclerosis↗

PubMed1998

The genetic and molecular mechanisms of motor neuron disease.

Significant progress has been made in the identification of genes and chromosomal loci associated with several types of motor neuron disease. Of particular interest is recent work on the pathogenic mechanisms underlying these diseases, especially studies in in vitro model systems and in transgenic and gene-targeted mice.

Amyotrophic Lateral Sclerosis↗

PubMed1998

Traumatic brain injury down-regulates glial glutamate transporter (GLT-1 and GLAST) proteins in rat brain.

Excess activation of NMDA receptors is felt to participate in secondary neuronal damage after traumatic brain injury (TBI). Increased extracellular glutamate is active in this process and may result from either increased release or decreased reuptake. The two high-affinity sodium-dependent glial transporters [glutamate transporter 1 (GLT-1) and glutamate aspartate transporter (GLAST)] mediate the bulk of glutamate transport. We studied the protein levels of GLT-1 and GLAST in the brains of rats after controlled cortical impact-induced TBI. With use of subtype-specific antibodies, GLT-1 and GLAST proteins were quantitated by immunoblotting in the ipsilateral and contralateral cortex at 2, 6, 24, 72, and 168 h after the injury. Sham-operated rats served as control. TBI resulted in a significant decrease in GLT-1 (by 20-45%; p < 0.05) and GLAST (by 30-50%; p < 0.05) protein levels between 6 and 72 h after the injury. D-[3H]Aspartate binding also decreased significantly (by 30-50%; p < 0.05) between 6 and 72 h after the injury. Decreased glial glutamate transporter function may contribute to the increased extracellular glutamate that may mediate the excitotoxic neuronal damage after TBI. This is a first report showing altered levels of glutamate transporter proteins after TBI.

ATP-Binding Cassette Transporters↗

PubMed1998

Endozepine stupor. Recurring stupor linked to endozepine-4 accumulation.

Recurring stupor can be caused by repeated metabolic, toxic or structural brain disturbances. Recently, cases of recurring stupor, with fast EEG activity were shown to display increased endogenous benzodiazepine-like activity during the episodes of stupor. Patients with recurring stupor underwent extensive metabolic and toxicologic screening, EEG and brain imaging. Endozepines and exogenously administered benzodiazepines were assayed in plasma and CSF by means of mass spectrometry. Flumazenil, a benzodiazepine antagonist was administered and the behavioural and EEG responses monitored. Treatment with oral flumazenil was attempted in selected cases. Twenty patients were found with recurring stupor. Episodes had begun between ages 18 and 67 years, and in nine patients, had disappeared spontaneously after 4-6 years with symptoms. Stupor lasted hours or days. Onset of the episodes and frequency were unpredictable. Patients were normal between attacks. Stupor was characterized by initial drowsiness, staggering and behavioural changes, followed by deep sleep and spontaneous recovery with post-ictal amnesia. Biochemical screening and brain imaging were always normal. Ictal EEG showed fast background activity, and flumazenil transiently awoke the patients and normalized the EEG. In the nine cases examined, endozepine-4 levels were increased during the stupor. Oral flumazenil reduced the frequency of the attacks in three of these nine patients. Recurring episodes of stupor may be due to increased endozepine-4. We propose the term 'endozepine stupor' for such episodes. Endozepine-4 is an endogenous ligand for the benzodiazepine recognition site at the GABAA receptor, with unknown molecular structure.

Adult↗

PubMed1998

EAAC1, a high-affinity glutamate tranporter, is localized to astrocytes and gabaergic neurons besides pyramidal cells in the rat cerebral cortex.

High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil. EAAC1-positive neurons were more numerous in layers II-III and V-VI, i.e. throughout all projection layers. Most EAAC1-positive neurons were pyramidal, although nonpyramidal cells were also observed. Some EAAC1-positive non-pyramidal neurons stained positively with an antiserum to GAD, thus demonstrating that EAAC1 is not confined to glutamatergic neurons. Non-neuronal EAAC1-positive cells were also observed in the white matter, and some of them stained positively with an antiserum to GFAP. Ultrastructural studies showed that EAAC1-ir was in neuronal cell bodies, dendrites and dendritic spines, but not in axon terminals, i.e. exclusively postsynaptic. Analysis of the type of axon terminals synapsing on EAAC1-ir profiles showed that 97% of them formed asymmetric contacts, thus indicating that EAAC1 is located at the very sites of excitatory amino acid release. Unexpectedly, EAAC1-ir was also found in a few astrocytic processes located in both the gray and the white matter. The localization of EAAC1 may explain the pathological symptoms that follow EAAC knockout (seizures and mild toxicity), as seizures could be due to the loss of EAAC1-mediated fine regulation of neuronal excitability at axodendritic and axospinous synapses, whereas the mild toxicity may be related to the functional inactivation of astrocytic EAAC1.

Amino Acid Transport System X-AG↗

PubMed1998

Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels approximately 5-fold, increased GLAST protein approximately 2-fold, and increased GLT-1 protein >/=8-20-fold. These increases in protein expression were accompanied by 2-fold increases in the Vmax and Km values for Na+-dependent L-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase approximately 2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of either N-methyl-D-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.

ATP-Binding Cassette Transporters↗

PubMed1998

Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta.

Concentrative absorption of glutamate by the developing placenta is critical for proper fetal development. The expression of GLAST1, GLT1, EAAC1, and EAAT4, known to be capable of D-aspartate-inhibitable and Na(+)-coupled glutamate transport (system X-AG), was evaluated in day 14 vs. day 20 rat chorioallantoic placenta. Steady-state mRNA levels were greater at day 20 for all transporters. Immunohistochemistry determined that the expression of GLAST1, GLT1, and EAAC1 was greater throughout the day 20 placenta and was asymmetric with respect to cellular localization. EAAT4 protein was not detected. System X-AG activity was responsible for most of the Na(+)-dependent glutamate uptake and was greater in day 20 than in day 14 apical and basal membrane subdomains of the labyrinth syncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 protein were identified on day 20, and quantities were greater in basal than in apical membranes. GLT1 expression, unchanged in apical membranes, was decreased in basal membranes. These data correlate transporter mRNA and protein content with transport activity and demonstrate an increasing capacity for glutamate absorption by the developing placenta.

ATP-Binding Cassette Transporters↗

PubMed1997

Glutamate transporter protein subtypes are expressed differentially during rat CNS development.

Extracellular glutamate concentrations are regulated by glial and neuronal transporter proteins. Four glutamate transporter subtypes have been identified in rat brain; GLAST and GLT-1 are primarily astrocytic, whereas EAAC1 and EAAT4 are neuronal. Using immunoblotting and immunohistochemistry with subtype-specific antipeptide antibodies, we examined the protein expression and regional and cellular localization of each glutamate transporter subtype in embryonic and postnatal rat CNS. Each transporter had a specific pattern of expression. GLAST immunoreactivity was low prenatally but became enriched in cerebellar Bergmann glia early postnatally and then was also present in forebrain later postnatally. The post-translational modification of GLAST was unique among the subtypes; glycosylated GLAST increased with maturation, whereas nonglycosylated protein decreased in abundance postnatally. GLT-1 was present in fetal brain and spinal cord, with expression progressively increasing to adult levels throughout the neuraxis by postnatal day 26. Transient expression of GLT-1 immunoreactivity along axonal pathways was observed prenatally, in contrast to the exclusive localization of GLT-1 to astrocytes in the adult CNS. EAAC1, localized to neurons, was enriched in forebrain, diencephalon, and hindbrain during prenatal and postnatal development. EAAC1 expression was greater in newborn brain compared with adult brain. EAAT4 had a region-specific distribution; EAAT4 was mainly in cerebellum, localized to Purkinje cells, with much lower levels in forebrain. EAAT4 levels increased in cerebellum with age. We conclude that during CNS development the expression of glutamate transporter subtypes is differentially regulated, regionally segregated, and coordinated.

ATP-Binding Cassette Transporters↗

PubMed1997

The development of excitatory synapses in cultured spinal neurons.

Immunohistochemical studies of synapses in the CNS have demonstrated that glutamate receptors (GluRs) are concentrated at postsynaptic sites in vivo and in vitro (Baude et al., 1995). The mechanisms leading to receptor clustering at excitatory synapses are far less understood than those governing acetylcholine receptor accumulation at the neuromuscular junction () or glycine receptor aggregation at central inhibitory synapses (). Using cultured rat spinal cord neurons, we demonstrate that clustering of the AMPA receptor subunit GluR1 is among the earliest events in excitatory synapse formation in vitro, coincident with the onset of miniature EPSCs and in many cases preceding presynaptic vesicle accumulation. Postsynaptic receptor clustering is induced in a highly specific and reiterative pattern, independent of receptor activation, by contact with a subset of axons capable of inducing receptor clusters. The subunit composition of AMPA receptor clusters varied significantly between neurons but was invariant within a given neuron. The presence of either GluR2 or GluR3 was common to all receptor clusters. Neither high-affinity glutamate transporters nor NMDA receptors appeared to be concentrated with AMPA receptor subunits at these excitatory synapses.

ATP-Binding Cassette Transporters↗

PubMed1997

Decreased glutamate transporter (GLT-1) expression in frontal cortex of rats with acute liver failure.

It has been suggested that reduced astrocytic uptake of neuronally released glutamate contributes to the pathogenesis of hepatic encephalopathy in acute liver failure. In order to further address this issue, the recently cloned and sequenced astrocytic glutamate transporter GLT-1 was studied in brain preparations from rats with ischemic liver failure induced by portacaval anastomosis followed 24 h later by hepatic artery ligation and from appropriate sham-operated controls. GLT-1 expression was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of GLT-1 transcript was significantly decreased in frontal cortex at coma stages of acute liver failure. Western blotting using a polyclonal antibody to GLT-1 revealed a concomitant decrease in expression of transporter protein in the brains of rats with acute liver failure. Reduced capacity of astrocytes to reuptake neuronally released glutamate, resulting from a GLT-1 transporter deficit and the consequently compromised neuron-astrocytic trafficking of glutamate could contribute to the pathogenesis of hepatic encephalopathy and brain edema, two major complications of acute liver failure.

ATP-Binding Cassette Transporters↗

PubMed1997

Glutamatergic enteric neurons.

We tested the hypothesis that glutamate, the major excitatory neurotransmitter of the CNS, is also an excitatory neurotransmitter in the enteric nervous system (ENS). Glutamate immunoreactivity was found in cholinergic enteric neurons, many of which were identified as sensory by their co-storage of substance P and/or calbindin. Glutamate immunoreactivity was concentrated in terminal varicosities with a majority of small clear synaptic vesicles. The immunoreactivities of both AMPA and NMDA receptor subunits were also detected on neurons in both submucosal and myenteric plexuses. The immunoreactivity of the EAAC1 neuronal glutamate transporter was widespread in both plexuses. Glutamate evoked depolarizing responses in myenteric neurons that had fast and slow components. The fast component was mimicked by AMPA, and the slow component was mimicked by NMDA. The fast component and the response to AMPA mimicked fast EPSPs evoked in 2/AH neurons; moreover, fast EPSPs as well as fast glutamate and AMPA responses were blocked by selective AMPA antagonists and potentiated by the glutamate uptake inhibitor L-(-)-threo-3-hydroxyaspartic acid. These observations demonstrate, for the first time, the presence of glutamatergic neurons and glutamate-mediated neurotransmission in the ENS.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1997

Neuronal regulation of glutamate transporter subtype expression in astrocytes.

GLT-1, GLAST, and EAAC1 are high-affinity, Na(+)-dependent glutamate transporters identified in rat forebrain. The expression of these transporter subtypes was characterized in three preparations: undifferentiated rat cortical astrocyte cultures, astrocytes cocultured with cortical neurons, and astrocyte cultures differentiated with dibutyryl cyclic AMP (dBcAMP). The undifferentiated astrocyte monocultures expressed only the GLAST subtype. Astrocytes cocultured with neurons developed a stellate morphology and expressed both GLAST and GLT-1; neurons expressed only the EAAC1 transporter, and rare microglia in these cultures expressed GLT-1. Treatment of astrocyte cultures with dBcAMP induced expression of GLT-1 and increased expression of GLAST. These effects of dBcAMP on transporter expression were qualitatively similar to those resulting from coculture with neurons, but immunocytochemistry showed the pattern of transporter expression to be more complex in the coculture preparations. Compared with astrocytes expressing only GLAST, the dBcAMP-treated cultures expressing both GLAST and GLT-1 showed an increase in glutamate uptake Vmax, but no change in the glutamate K(m) and no increased sensitivity to inhibition by dihydrokainate. Pyrrolidine-2,4-dicarboxylic acid and threo-beta-hydroxyaspartic acid caused relatively less inhibition of transport in cultures expressing both GLAST and GLT-1, suggesting a weaker effect at GLT-1 than at GLAST. These studies show that astrocyte expression of glutamate transporter subtypes is influenced by neurons, and that dBcAMP can partially mimic this influence. Manipulation of transporter expression in astrocyte cultures may permit identification of factors regulating the expression and function of GLAST and GLT-1 in their native cell type.

ATP-Binding Cassette Transporters↗

PubMed1997

4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4-hydroxynonenal potentiates oxidative cascades. Collectively, these findings suggest that 4-hydroxynonenal plays important roles in oxidative impairment of synaptic functions that would be expected to promote excitotoxic cascades.

ATP-Binding Cassette Transporters↗

PubMed1997

Cellular and synaptic localization of the neuronal glutamate transporters excitatory amino acid transporter 3 and 4.

Glutamate transport is a primary mechanism for the synaptic inactivation of glutamate. Excitatory amino acid transporter 4 (EAAT4) is a novel glutamate transporter with properties of a ligand-gated chloride channel that was recently cloned from human brain. The present study was an investigation of the protein expression and cellular localization of EAAT4 in human and rat brain, and comparison with another neuronal glutamate transporter, EAAT3 (rabbit excitatory amino acid carrier 1; EAAC1). Regional immunoblot analysis of EAAT4, using a monospecific oligopeptide (carboxy-terminal) affinity-purified polyclonal antibody, revealed that the protein was restricted to the central nervous system. The EAAT4 protein was largely expressed in cerebellum, with a much lower expression in hippocampus, neocortex, striatum, brain stem and thalamus. Immunohistochemical studies showed intense EAAT4 immunoreactivity in the human and rat cerebellar Purkinje cells with a somatodendritic localization. Other brain regions including neocortex, hippocampus, striatum showed faint neuropil staining of EAAT4. Immunogold localization identified EAAT4 protein at plasma membranes of Purkinje cell dendrites and spines. In the hippocampus and neocortex, EAAT4 immunoreactivity was found mainly at small calibre dendrites. Rarely, EAAT4 immunoreactivity was found in astrocytic cell processes of forebrain. In the cerebellum, EAAT4 localization partly overlapped with the neuronal localization of EAAT3 (EAAC1). Immunoreactivity for EAAT3 was enriched in the somatodendritic compartment of the Purkinje cells like EAAT4, but EAAT3 was also found in Purkinje cell axons and in boutons in deep cerebellar nuclei, as well as in granular cells and stellate cells. Our results indicate that EAAT4 protein is largely localized to cerebellar cortex and lower levels of EAAT4 protein are present in forebrain by immunoblot and immunohistochemistry. Both neuronal glutamate transporter EAAT3 (EAAC1) and EAAT4 are located at somatodendritic compartment of Purkinje cells, and probably contribute to glutamate re-uptake mechanisms at Purkinje cell synapses.

Aged↗