Search PubMed⌕ Search

Biomedical subjects

J D Rothstein

Publications and source records attributed to J D Rothstein.

At least 73 records · Page 4Linked to original sources
PubMed1997

ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions.

High levels of familial Amyotrophic Lateral Sclerosis (ALS)-linked SOD1 mutants G93A and G37R were previously shown to mediate disease in mice through an acquired toxic property. We report here that even low levels of another mutant, G85R, cause motor neuron disease characterized by an extremely rapid clinical progression, without changes in SOD1 activity. Initial indicators of disease are astrocytic inclusions that stain intensely with SOD1 antibodies and ubiquitin and SOD1-containing aggregates in motor neurons, features common with some cases of SOD1 mutant-mediated ALS. Astrocytic inclusions escalate markedly as disease progresses, concomitant with a decrease in the glial glutamate transporter (GLT-1). Thus, the G85R SOD1 mutant mediates direct damage to astrocytes, which may promote the nearly synchronous degeneration of motor neurons.

ATP-Binding Cassette Transporters↗
PubMed1997

Endozepine stupor in children.

Recurring episodes of stupor in adults have been shown to be related to increased levels of endozepines, which are endogenous ligands for the GABAA receptors. We report here two children presenting with recurrent episodes of stupor associated with fast EEG activity who had increased levels of endozepine-4 in plasma. Mass spectroscopy did not reveal commercially available benzodiazepines. Interictal endozepine-4 levels were normal. In one of the patients, administration of flumazenil (0.25 mg i.v.), a benzodiazepine inverse agonist, induced improvement of consciousness and attenuation of EEG fast activity. In conclusion, children presenting with recurrent episodes of stupor and EEG fast activity should be evaluated for endozepine levels and can be effectively treated with i.v. flumazenil.

Carrier Proteins↗
PubMed1997

Alterations in glutamate transporter protein levels in kindling-induced epilepsy.

There is increasing evidence that levels of glutamate are elevated in certain brain regions immediately prior to and during induction and propagation of seizures. Modulation of high-affinity glutamate uptake is a potential mechanism responsible for the elevated levels observed with Seizures. To date, three distinct Na(+)-dependent glutamate transporters have been cloned from rat and rabbit: GLT-1, GLAST, and EAAC-1. We performed a series of experiments to determine whether levels of these transporters are altered in amygdala-kindled rats. Levels of GLT-1, GLAST, and EAAC-1 were examined in three brain regions (hippocampus, piriform cortex/amygdala, and limbic forebrain) by quantitative immunoblotting using subtype-specific antibodies. GLAST protein was down-regulated in the piriform cortex/amygdala region of kindled rats as early as 24 h after one stage 3 seizure and persisting through multiple stage 5 seizures. In contrast, kindling induced an increase in EAAC-1 levels in piriform cortex/amygdala and hippocampus once the animals had reached the stage 5 level. NO changes in GLT-1 were observed in any region examined. Changes in transporter levels could contribute to the changes in glutamate levels seen with kindling.

ATP-Binding Cassette Transporters↗
PubMed1997

Distribution of glutamate transporter subtypes during human brain development.

In the mature brain, removal of glutamate from the synaptic cleft plays an important role in the maintenance of subtoxic levels of glutamate. This requirement is handled by a family of glutamate transporters, EAAT1, EAAT2, EAAT3, and EAAT4. Due to the involvement of glutamate also in neuronal development, it is believed that glutamate transport plays a role in developmental processes as well. Therefore, we have used immunohistochemical and immunoblot analysis to determine the distribution of the four glutamate transporters during human brain development using human pre- and postnatal brain tissue. Regional analysis showed that each transporter subtype has a unique distribution during development. EAAT2 was the most prominent glutamate transporter subtype and was highly enriched in cortex, basal ganglia, cerebellum, and thalamus in all ages examined. EAAT1 immunoreactivity was lower than that of EAAT2, with predominant localization in cortex, basal ganglia, hippocampus, and periventricular region. EAAT3 was located mainly in cortex, basal ganglia, and hippocampus, and EAAT4 was found only in cortex, hippocampus, and cerebellar cortex. The distinct regional distribution of various EAAT subtypes and also the transient expression of specific EAAT subtypes during development suggest multiple functional roles for glutamate transporters in the developing brain.

ATP-Binding Cassette Transporters↗
PubMed1997

Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons.

It has been postulated for several years that the high affinity neuronal glutamate uptake system plays a role in clearing glutamate from the synaptic cleft. Four different glutamate transporter subtypes are now identified, the major neuronal one being EAAC1. To be a good candidate for the reuptake of glutamate at the synaptic cleft, EAAC1 should be properly located at synapses, either at pre- or postsynaptic sites. We have investigated the distribution of EAAC1 in primary cultures of hippocampal neurons, which represent an advantageous model for the study of synaptogenesis and synaptic specializations. We have demonstrated that EAAC1 immunoreactivity is segregated in the somatodendritic compartment of fully differentiated hippocampal neurons, where it is localized in the dendritic shaft and in the spine neck, outside the area facing the active zone. No co-localization of EAAC1 immunoreactivity with the stainings produced by typical presynaptic and postsynaptic markers was ever observed, indicating that EAAC1 is not to be considered a synaptic protein. Accordingly, the developmental pattern of expression of EAAC1 was found to be different from that of typical synaptic markers. Moreover, EAAC1 was expressed in the somatodendritic compartment of hippocampal neurons already at stages preceding the formation of synaptic contacts, and was also expressed in GABAergic interneurons with identical subcellular distribution. Taken together, these data rule against a possible role for EAAC1 in the clearance of glutamate from within the cleft and in the regulation of its time in the synapse. They suggest an unconventional non-synaptic function of this high-affinity glutamate carrier, not restricted to glutamatergic fibres.

Amino Acid Sequence↗
PubMed1997

Localization of the high-affinity glutamate transporter EAAC1 in rat kidney.

Most amino acids filtered by the glomerulus are reabsorbed in the kidney via specialized transport systems. Recently, the cDNA encoding a high-affinity glutamate transporter, EAAC1, has been isolated and shown to be expressed at high levels in the kidney. To determine the potential role of EAAC1 in renal acidic amino acid reabsorption, the distribution of EAAC1 mRNA and protein in rat kidney was examined. In situ hybridization revealed that EAAC1 mRNA is expressed predominantly in S2 and S3 segments of the proximal tubules and at low levels in the inner stripe of outer medulla and inner medulla. Polyclonal antibodies raised against the carboxy terminus of EAAC1 recognized a single band of approximately 70 kDa on Western blots of membrane protein from kidney cortex and medulla. Immunofluorescence microscopy revealed intense signals in the luminal membrane of S2 and S3 segments and weaker signals in S1 segments, descending thin limbs of long-loop nephrons, medullary thick ascending limbs, and distal convoluted tubules. These results are consistent with EAAC1 encoding the previously described apical high-affinity glutamate transporter in the kidney that mediates reabsorption of acidic amino acids in tubules beyond early proximal tubule S1 segments. Potential additional roles of EAAC1 in acid/base balance, cell volume regulation, and amino acid metabolism are discussed.

Amino Acid Transport System X-AG↗
PubMed1996

Tumor necrosis factor alpha inhibits glutamate uptake by primary human astrocytes. Implications for pathogenesis of HIV-1 dementia.

Human immunodeficiency virus (HIV) infection is commonly associated with neurological disease that occurs in the apparent absence of extensive infection of brain cells by HIV, suggesting that indirect mechanisms account for neuropathogenesis in the CNS, perhaps including changes in the normal neuroprotective functions of astrocytes. To test this hypothesis, we examined the effect of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNFalpha), produced by HIV-1-infected macrophages and microglia, on glutamate transport by primary human fetal astrocytes (PHFAs). A dose-dependent inhibition of high affinity glutamate uptake sites was observed 12-24 h after addition of exogenous recombinant human TNFalpha to PHFAs. This effect was specific since it was blocked by a neutralizing monoclonal antibody directed against TNFalpha. Furthermore, the inhibitory effect was reproduced by a monoclonal antibody that is an agonist at the 55-kDa TNF receptor. These results suggest that the neurotoxic effects of TNFalpha may be due in part to its ability to inhibit glutamate uptake by astrocytes, which in turn may result in excitotoxic concentrations of glutamate in synapses.

AIDS Dementia Complex↗
PubMed1996

Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex.

Glutamate transport is critical for synaptic inactivation of glutamate and prevention of excitotoxicity. The following four glutamate transporters have been identified in human brain: EAAT1, EAAT2, EAAT3, and EAAT4. Deficient glutamate transport has been identified in the motor cortex and the spinal cord of tissue from amyotrophic lateral sclerosis (ALS) patients. The defect appears to be due to a selective loss of the astroglial specific glutamate transporter protein EAAT2. In these studies we sought to extend our understanding of glutamate transporters in ALS by examining the mRNA for each transporter subtype in ALS motor cortex. All tissue was matched for age and postmortem delay. There was no quantitative change in mRNA for EAAT1, EAAT2, or EAAT3 in ALS motor cortex, even in patients with a large loss of EAAT2 protein (95% decrease compared with control) and decreased tissue glutamate transport (73% decrease compared with control). These studies suggest that the dramatic abnormalities in EAAT2 may be due to translational or post-translational processes.

ATP-Binding Cassette Transporters↗
PubMed1996

Differential abundance of glutamate transporter subtypes in amyotrophic lateral sclerosis (ALS)-vulnerable versus ALS-resistant brain stem motor cell groups.

Previous studies have suggested that defective high-affinity glutamate uptake, due mainly to a major loss of the astroglial-specific GLT-1 glutamate transporter, underlies the selective motoneuron degeneration observed in sporadic ALS (24, 28). If a defect in glutamate transport underlies the pathogenesis of sporadic ALS, the glutamate transporter subtype found to be lost in sporadic ALS should be present in abundance in the affected motor nuclei under normal conditions. To investigate this, we used immunohistochemical methods to analyze the localization of two subtypes of high-affinity glutamate transporters in the cranial motor nuclei of normal monkey brain stem: GLT-1, localized to astroglia; and EAAC1, localized to neurons. Our results indicated that all motor cell groups of monkey brain stem are rich in the GLT-1 glutamate transporter, which is localized to astroglial cells and processes that surround and envelop motoneuron cell bodies and dendrites. Image analysis indicated that the abundance of GLT-1 immunoreactive astroglial elements in ALS-vulnerable motor cell groups (i.e., the trigeminal, facial, and hypoglossal motor cell groups) is higher than in ALS-resistant motor cell groups (i.e., the oculomotor, trochlear, and abducens motor cell groups), and statistical analysis showed that this difference is significant. Our results also indicated that both ALS-vulnerable and ALS-resistant motor cell groups of monkey brain stem are relatively poor in EAAC1 immunoreactivity. Therefore, in the case of a loss in the GLT-1 glutamate transporter in sporadic ALS, glutamate may increase in the vicinity of motoneurons in all brain-stem motor cell groups, but especially in the ALS-vulnerable motor cell groups, which are normally richer in GLT-1. Increased extracellular glutamate could lead to excess entry of Ca2+ into motoneurons via glutamate-gated or voltage-activated Ca2+ channels and produce degeneration of those motoneurons unable to resist the insult. Since motoneurons in the ALS-resistant motor cell groups of the brain stem are enriched in the Ca2+ buffering protein parvalbumin, they should be better able to resist the damage than the majority of motoneurons in the ALS-vulnerable motor cell groups, which lack parvalbumin (20).

ATP-Binding Cassette Transporters↗
PubMed1996

Differential expression of three glutamate transporter subtypes in the rat retina.

The immunocytochemical distribution of the three excitatory amino acid transporter subtypes GLT-1, GLAST-1 and EAAC-1 was studied in the rat retina using antibodies raised against synthetic peptides corresponding to the C-terminus of each transporter subtype (Rothstein et al. 1994). A comparative immunoblot analysis of rat cortex, cerebellum and retina membrane proteins suggested the following rank order of glutamate transporter subtype expression in retina: GLAST-1>/=EAAC-1>GLT-1. GLAST-1 immunoreactivity was seen in Müller cells and astrocytes. EAAC-1 was found in horizontal cells, in amacrine and displaced amacrine cells, and in ganglion cells. A minority of bipolar cells also expressed EAAC-1. GLT-1 was preferentially expressed by different types of bipolar cells; however, it was also found in some amacrine cells. The functional role of this differential distribution of glutamate transporters in the retina is discussed.

ATP-Binding Cassette Transporters↗
PubMed1996

Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.

Three glutamate transporters have been identified in rat, including astroglial transporters GLAST and GLT-1 and a neuronal transporter EAAC1. Here we demonstrate that inhibition of the synthesis of each glutamate transporter subtype using chronic antisense oligonucleotide administration, in vitro and in vivo, selectively and specifically reduced the protein expression and function of glutamate transporters. The loss of glial glutamate transporters GLAST or GLT-1 produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis. The loss of the neuronal glutamate transporter EAAC1 did not elevate extracellular glutamate in the striatum but did produce mild neurotoxicity and resulted in epilepsy. These studies suggest that glial glutamate transporters provide the majority of functional glutamate transport and are essential for maintaining low extracellular glutamate and for preventing chronic glutamate neurotoxicity.

ATP-Binding Cassette Transporters↗
PubMed1996

Therapeutic horizons for amyotrophic lateral sclerosis.

Recent theories on the pathogenesis of motor neuron disease and research on motor neuron injury have resulted in new putative therapies, which include treatment with various neurotrophic factors, antioxidants and anti-excitotoxicity agents. Clinical and preclinical studies have now provided the first agents that reproducibly alter the course of amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis↗
PubMed1996

Fimbria-fornix transections selectively down-regulate subtypes of glutamate transporter and glutamate receptor proteins in septum and hippocampus.

The effects of CNS axotomy on glutamate transporter and glutamate receptor expression were evaluated in adult rats following unilateral fimbria-fornix transections. The septum and hippocampus were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted by using antibodies directed against glutamate transporters (GLT-1, GLAST, and EAAC1) and glutamate receptors (GluR1, GluR2/3, GluR6/7, and NMDAR1), and they were assayed for glutamate transport by D-[3H]aspartate binding. GLT-1 was decreased at 7 and 14 days postlesion within the ipsilateral septum and at 7 days postlesion in the hippocampus. GLAST was decreased within the ipsilateral septum and hippocampus at 7 and 14 days postlesion. No postlesion alterations in EAAC1 immunoreactivity were observed. D-[3H]Aspartate binding was decreased at 7, 14, and 30 days postlesion within the ipsilateral septum and 14 days postlesion in the hippocampus. GluR2/3 expression was down-regulated at 30 days postlesion within the ipsilateral septum, whereas GluR1, GluR6/7, and NMDAR1 immunoreactivity was unchanged. In addition, no alterations in glutamate receptor expression were detected within hippocampal homogenates. This study demonstrates a selective down-regulation of primarily glial, and not neuronal, glutamate transporters and a delayed, subtype-specific down-regulation of septal GluR2/3 receptor expression after regional deafferentation within the CNS.

ATP-Binding Cassette Transporters↗
PubMed1996

Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC1).

Several subtypes of sodium-dependent high affinity (SDHA) glutamate transporters have been pharmacologically differentiated in brain tissue. Recently, four distinct cDNAs (EAAC1, GLT1, GLAST, and EAAT4) encoding Na+-dependent glutamate transporters have been isolated, but the properties of some of these transporters do not fully match the properties of transport observed in brain tissue or astrocyte-enriched cultures. The purpose of the current investigation was to determine whether the pharmacological properties of EAAC1 parallel those observed in cortical or cerebellar synaptosomes, C6 glioma, or primary astrocyte-enriched cultures. EAAC1 cRNA was expressed in Xenopus oocytes, an expression system with no detectable endogenous Na+-dependent glutamate transport activity. EAAC1-mediated glutamate transport was >98% Na+ dependent, and the transport was saturable and consistent with a single site. Glutamate transport activates in EAAC1-injected oocytes and C6 glioma have similar Km values for glutamate (Km = 15-24 microM) and Na+ (apparent Km = 35-50mM), and these values markedly differ from those observed in rat synaptosomes (glutamate, Km = 1-5 microM; Na+, Km = 13-20 mM). Several excitatory amino acid analogues were tested as inhibitors of L-[3H] glutamate transport in oocytes expressing EAAC1 cRNA. The potencies of several compounds for inhibition of EAAC1-mediated transport differed from those previously observed in cerebellar synaptosomes and astrocyte-enriched cultures. Although EAAC1-mediated transport and cortical synaptosomal transport have similar pharmacological profiles, five excitatory amino acid analogues were > or= 3-fold more potent as inhibitors of transport into cortical synaptosomes than of transport into EAAC1-injected oocytes. For example, L-trans-pyrrolidine-2,4-dicarboxylate was approximately 5-fold more potent in cortical synaptosomes, and dihydrokainate was approximately 10-fold more potent in cortical synaptosomes than in EAAC1-injected oocytes. In contrast, all of the compounds examined inhibit transport observed in C6 glioma wtih potencies similar to that observed in oocytes injected with EAAC1 cRNA. Consistent with these data, C6 glioma expressed EAAC1- but not GLT1- and GLAST-like immunoreactivity. Although this immunoreactivity migrated as proteins of slightly different molecular masses in each system, treatment with N-glycosidase F shifted all proteins to a molecular mass consistent with that predicted from the cDNA sequence. In cortical synaptosomes, EAAC1-, GLT1-, and GLAST-like immunoreactives were apparent. These results indicate that (i) EAAC1 but not GLAST or GLT1 transporters are expressed in C6 glioma, (ii) synaptosomes contain a heterogeneous population of transporters, (iii) EAAC1 does not account for the pharmacology previously observed in cortical synaptosomes, and (iv) based on the pharmacology and tissue distribution of EAAC1, GLT1, GLAST, and EAAT4, it appears that there are additional glutamate transporter subtypes.

Amino Acid Transport System X-AG↗
PubMed1995

Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis.

The pathogenesis of sporadic amyotrophic lateral sclerosis (ALS) is unknown, but defects in synaptosomal high-affinity glutamate transport have been observed. In experimental models, chronic loss of glutamate transport can produce a loss of motor neurons and, therefore, could contribute to the disease. With the recent cloning of three glutamate transporters, i.e., EAAC1, GLT-1, and GLAST, it has become possible to determine if the loss of glutamate transport in ALS is subtype specific. We developed C-terminal, antioligopeptide antibodies that were specific for each glutamate transporter. EAAC1 is selective for neurons, while GLT-1 and GLAST are selective for astroglia. Tissue from various brain regions of ALS patients and controls were examined by immunoblot or immunocytochemical methods for each transporter subtype. All tissue was matched for age and postmortem delay. GLT-1 immunoreactive protein was severely decreased in ALS, both in motor cortex (71% decrease compared with control) and in spinal cord. In approximately a quarter of the ALS motor cortex specimens, the loss of GLT-1 protein (90% decrease from control) was dramatic. By contrast, there was only a modest loss (20% decrease from control) of immunoreactive protein EAAC1 in ALS motor cortex, and there was no appreciable change in GLAST. The minor loss of EAAC1 could be secondary to loss of cortical motor neurons. As a comparison, glial fibrillary acidic protein, which is selectively localized to astroglia, was not changed in ALS motor cortex. Because there is no loss of astroglia in ALS, the dramatic abnormalities in GLT-1 could reflect a primary defect in GLT-1 protein, a secondary loss due to down regulation, or other toxic processes.

ATP-Binding Cassette Transporters↗
PubMed1995

Neuroprotective strategies in a model of chronic glutamate-mediated motor neuron toxicity.

A dramatic loss of glutamate transport has been observed in sporadic amyotrophic lateral sclerosis and has been postulated to contribute to the disease. Experimentally, this hypothesis was corroborated by mimicking the chronic loss of glutamate transport in postnatal rat spinal cord organotypic cultures through the use of glutamate transport inhibitors. This system is characterized by a relatively selective slow loss of ventral horn motor neurons resulting from glutamate transport inhibition. In this study, spinal cord organotypic cultures were used to test various drugs to evaluate their neuroprotective properties against this slow glutamate-mediated neurotoxicity The most potent neuroprotectants were drugs that altered glutamate neurotransmission, including non-NMDA receptor antagonists (GYKI-52466, PD144216, and PD13997) and drugs that could block presynaptic release or synthesis (riluzole and gabapentin). In addition, some antioxidants (U83836E and N-t-butyl-alpha-phenylnitrone) and inhibitors of nitric oxide synthesis (NG-monomethyl-L-arginine acetate) were modestly neuroprotective. The calcium endonuclease inhibitor aurintricarboxylic acid and the calcium release inhibitor dantrolene also provided partial motor neuron protection. However, several antioxidants and calcium channel antagonists had no excitotoxic neuroprotectant activity. This system provides a preclinical screening method for the burgeoning number of drugs postulated for clinical trials in motor neuron disease and a model to evaluate the mechanisms of chronic glutamate toxicity.

Animals↗
PubMed1995

Regional deafferentation down-regulates subtypes of glutamate transporter proteins.

Low extracellular glutamate content is maintained primarily by high-affinity sodium-dependent glutamate transport. Three glutamate transporter proteins have been cloned: GLT-1 and GLAST are astroglial, whereas EAAC1 is neuronal. The effects of axotomy on glutamate transporter expression was evaluated in adult rats following unilateral fimbria-fornix and corticostriatal lesions. The hippocampus and striatum were collected at 3, 7, 14, and 30 days postlesion. Homogenates were immunoblotted using antibodies directed against GLT-1, GLAST, EAAC1, and glial fibrillary acidic protein and assayed for glutamate transport by D-[3H]aspartate binding. GLT-1 immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 14 days postlesion. GLAST immunoreactivity was decreased within the ipsilateral hippocampus and striatum at 7 and 14 days postlesion. No alterations in EAAC1 immunoreactivity were observed. D-[3H]Aspartate binding was decreased at 14 days postlesion within the ipsilateral hippocampus and at 7 and 14 days postlesion within the ipsilateral striatum. By 30 days postlesion, glutamate transporters and D-[3H]aspartate binding returned to control levels. This study demonstrates the down-regulation of primarily glial, and not neuronal, glutamate transporters following regional disconnection.

ATP-Binding Cassette Transporters↗