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Biomedical subjects

J D Rothstein

Publications and source records attributed to J D Rothstein.

At least 19 recordsLinked to original sources
PubMed2003

Modulation of spinal nociceptive processing through the glutamate transporter GLT-1.

GLT-1 is the predominant glutamate transporter in most brain regions and therefore plays a major role in terminating synaptic transmission and protecting neurons from glutamate neurotoxicity. In the present study we assessed (i) the regulation of GLT-1 expression in the spinal cord after peripheral nociceptive stimulation and (ii) the nociceptive behavior of rats following inhibition or transient knockdown of spinal GLT-1. Formalin injection into one hindpaw caused a rapid transient upregulation of GLT-1 protein expression in the spinal cord which did not occur when rats were pretreated with morphine (10 mg/kg, i.p.) suggesting that the nociceptive input specifically caused the increase of GLT-1 transcription. Inhibition of GLT-1 by the transportable inhibitor trans-pyrrolidine-2,4-dicarboxylic acid resulted in a significant reduction of nociceptive behavior in the rat formalin assay. Similar results were obtained with a transient reduction of GLT-1 protein expression by antisense oligonucleotides. These data suggest that inhibition of GLT-1 activity or expression reduces excitatory synaptic efficacy and thereby nociception. Mechanisms that might explain this phenomenon may include activation of inhibitory metabotropic glutamate receptors, postsynaptic desensitization or disturbance of glutamate recycling.

Analgesics, Opioid↗
PubMed2001

Altered expressions of glutamate transporter subtypes in rat model of neonatal cerebral hypoxia-ischemia.

Glutamate transporters are essential for maintaining the extracellular levels of glutamate at synaptic clefts and are regulated developmentally in a subtype-specific manner. We investigated chronological changes of immunoreactivities for glial glutamate transporters GLAST and GLT-1 and a neuronal glutamate transporter, EAAC1, in postnatal 7-day-old rat neocortices and hippocampi at 12, 24, 48 and 72 h after hypoxia-ischemia. Glutamate transporter subtypes are differentially expressed in the ischemic core and the boundary area of the neonatal rat brain with hypoxia-ischemia. Expressions of these glutamate transporters decreased in the ischemic core at 12 h, then immunoreactivities for GLAST and GLT-1 were recovered at the hippocampus. This was accompanied by a GFAP-positive gliosis at 72 h, whereas these immunoreactivities were reduced at the neocortex in the ischemic core. Glial glutamate transporters, especially GLAST, were noted in some astrocytes appearing as apoptosis as well as shrunken pyramidal neurons mainly in the boundary area of the neocortex. Increased perikaryal expression of EAAC1 was associated with that of MAP2 at the border of the boundary area. These temporal and regional expressions of glutamate transporters may contribute towards understanding the excitotoxic cell death mechanism in hypoxic-ischemic encephalopathy during the perinatal period.

Amino Acid Transport System X-AG↗
PubMed2001

Differential synaptic localization of GluR2 and EAAC1 in the macaque monkey entorhinal cortex: a postembedding immunogold study.

The synaptic distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit GluR2 and neuronal glutamate transporter subunit EAAC1 were studied using immunogold in layer II of the macaque monkey entorhinal cortex. Immunoreactivity for EAAC1 and GluR2 was frequent at asymmetric synapses and their associated membrane. The synaptic localization of EAAC1 differed considerably from that of GluR2, in that GluR2 immunolabelling was most commonly located within the postsynaptic density, but EAAC1 localization was more heterogeneous and was predominant at the edge of postsynaptic densities and perisynaptic zones. Since EAAC1 may play an important role in clearing glutamate from the synaptic cleft and intercellular spaces, the high perisynaptic expression of EAAC1 in these neurons could presumably offer a powerful mechanism through which high concentrations of glutamate could be efficiently removed from the synapses following release and interaction with glutamate receptors. The distribution of EAAC1 may also offer protection for these neurons against excessive glutamatergic stimuli that may occur under certain pathological conditions.

Amino Acid Transport System X-AG↗
PubMed2001

Antisense knockdown of the glial glutamate transporter GLT-1, but not the neuronal glutamate transporter EAAC1, exacerbates transient focal cerebral ischemia-induced neuronal damage in rat brain.

Transient focal cerebral ischemia leads to extensive neuronal damage in cerebral cortex and striatum. Normal functioning of glutamate transporters clears the synaptically released glutamate to prevent excitotoxic neuronal death. This study evaluated the functional role of the glial (GLT-1) and neuronal (EAAC1) glutamate transporters in mediating ischemic neuronal damage after transient middle cerebral artery occlusion (MCAO). Transient MCAO in rats infused with GLT-1 antisense oligodeoxynucleotides (ODNs) led to increased infarct volume (45 +/- 8%; p < 0.05), worsened neurological status, and increased mortality rate, compared with GLT-1 sense/random ODN-infused controls. Transient MCAO in rats infused with EAAC1 antisense ODNs had no significant effect on any of these parameters. This study suggests that GLT-1, but not EAAC1, knockdown exacerbates the neuronal death and thus neurological deficit after stroke.

ATP-Binding Cassette Transporters↗
PubMed2001

Modulation of the neuronal glutamate transporter EAAC1 by the interacting protein GTRAP3-18.

Excitatory amino-acid carrier 1 (EAAC1) is a high-affinity Na+-dependent L-glutamate/D,L-aspartate cell-membrane transport protein. It is expressed in brain as well as several non-nervous tissues. In brain, EAAC1 is the primary neuronal glutamate transporter. It has a polarized distribution in cells and mainly functions perisynaptically to transport glutamate from the extracellular environment. In the kidney it is involved in renal acidic amino-acid re-absorption and amino-acid metabolism. Here we describe the identification and characterization of an EAAC1-associated protein, GTRAP3-18. Like EAAC1, GTRAP3-18 is expressed in numerous tissues. It localizes to the cell membrane and cytoplasm, and specifically interacts with carboxy-terminal intracellular domain of EAAC1. Increasing the expression of GTRAP3-18 in cells reduces EAAC1-mediated glutamate transport by lowering substrate affinity. The expression of GTRAP3-18 can be upregulated by retinoic acid, which results in a specific reduction of EAAC1-mediated glutamate transport. These studies show that glutamate transport proteins can be regulated potently and that GTRAP can modulate the transport functions ascribed to EAAC1. GTRAP3-18 may be important in regulating the metabolic function of EAAC1.

ATP-Binding Cassette Transporters↗
PubMed2001

Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins.

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters. To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1-5). GLAST and GLT-1 are localized primarily in astrocytes, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9-11) and EAAT5 (ref 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites. This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4--the glutamate transporter expressed predominately in the cerebellum--or in targeting and/or anchoring or clustering the transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.

Amino Acid Sequence↗
PubMed2001

Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues.

The mechanisms leading to neurodegeneration in ALS (amyotrophic lateral sclerosis) are not well understood, but cytosolic protein aggregates appear to be common in sporadic and familial ALS as well as transgenic mouse models expressing mutant Cu/Zn superoxide dismutase (SOD1). In this study, we systematically evaluated the presence of these aggregates in three different mouse models (G93A, G85R, and G37R SOD1) and compared these aggregates to those seen in cases of sporadic and familial ALS. Inclusions and loss of motor neurons were observed in spinal cords of all of these three mutant transgenic lines. Since a copper-mediated toxicity hypothesis has been proposed to explain the cytotoxic gain-of-function of mutant SOD1, we sought to determine the involvement of the copper chaperone for SOD1 (CCS) in the formation of protein aggregates. Although all aggregates contained CCS, SOD1 was not uniformly found in the inclusions. Similarly, CCS-positive skein-like inclusions were rarely seen in ALS neurons. These studies do not provide strong evidence for a causal role of CCS in aggregate formation, but they do suggest that protein aggregation is a common event in all animal models of the disease. Selected proteins, such as the glutamate transporter GLT-1, were not typically observed within the inclusions. Most inclusions were positively stained with antibodies recognizing ubiquitin, proteasome, Hsc70 in transgenic lines, and some Hsc70-positive inclusions were detected in sporadic ALS cases. Overall, these observations suggest that inclusions might be sequestered into ubiquitin-proteasome pathway and some chaperone proteins such as Hsc70 may be involved in formation and/or degradation of these inclusions.

Amyotrophic Lateral Sclerosis↗
PubMed2001

Microglial response to the neurotoxicity of 6-hydroxydopamine in neonatal rat cerebellum.

Depletion of noradrenaline in newborn rats by 6-hydroxydopamine (6-OHDA) affects the postnatal development and reduces the granular cell area in the neocerebellum (lobules V-VII). During the first postnatal month, Bergmann glial fibers guide the migration of immature granule cells to the internal granule cell layer. Microglia and Bergmann glia may play an important role in this process, but the exact mechanism behind this phenomenon is not known. We studied the effect of systemic administration of 6-OHDA on the expression and localization on microglia and Bergmann glia in the neonatal cerebellum by immunohistochemistry. In the neocerebellum, 6-OHDA treatment caused a significant increase in the number of activated microglia. The increase was observed mainly in the granule cell layer and the cerebellar medulla. Bergmann glial cells in treated brains were abnormally located, did not form intimate associations with Purkinje cells, and the glial fibers were structurally different. Our findings indicate that a noradrenergic influence may be necessary for the normal maturation and migration of granule cells, and abnormal migration may be the result of Bergmann glia destruction and the activation of microglia. Activated microglia in the granule cell layer may be used as a marker for an injured cerebellar area.

Age Factors↗
PubMed2001

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine.

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25--35 mg kg(-1) day(-1) orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9 +/- 4.5%) than in the posterior (53.2 +/- 15.4%) cortex. L-[(3)H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6 +/- 8.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3 +/- 10.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1 +/- 0.7 microM) and the KCl-evoked (28.7 +/- 7.7 microM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.

ATP-Binding Cassette Transporters↗
PubMed2000

Differential synaptic localization of the glutamate transporter EAAC1 and glutamate receptor subunit GluR2 in the rat hippocampus.

EAAC1, a neuron-specific glutamate transporter, is likely to play an important role in the regulation of glutamate levels in the synaptic cleft. Ultrastructural studies have demonstrated that the glutamate receptor subunit proteins (e.g., GluR2) are frequently preferentially located at the postsynaptic density of asymmetric synapses. While the glutamate/glutamate receptor interaction is likely to be influenced by the activity and location of the transporter molecules, the spatial localization of the transporter molecules relative to the receptor molecules is not well delineated. Thus, we analyzed the cellular, ultrastructural, and synaptic distribution of EAAC1 in the context of the distribution of the AMPA receptor subunit GluR2 in the hippocampus. While GluR2 and EAAC1 are both present in hippocampal projection neurons, their intracellular distribution patterns differ. Both GluR2 and EAAC1 are present in the dendritic membranes and cytoplasm; however EAAC1 has a distinctive punctate distribution in the dendrite compared to the more diffuse labeling reflected by GluR2. Pre-embedding ultrastructural studies also revealed cytoplasmic and membrane-associated pools of EAAC1 within dendritic shafts and spines, as well as in a subset of axonal profiles and terminals. Postembedding double label immunogold localization demonstrated a similar intraneuronal distribution, but in addition showed that membrane-associated EAAC1 is not intermingled with GluR2 within the synaptic complex, but in contrast is primarily located perisynaptically, often immediately outside the synaptic specialization. In addition, there is a significant presynaptic pool of EAAC1, whereas GluR2 is essentially absent from the pre-synaptic profile. Thus, membrane-associated EAAC1 within the synaptic region is ideally situated to restrict the site of action of glutamate with respect to ionotropic receptors to the synaptic cleft, as well as regulate glutamate levels in the perisynaptic and presynaptic domains, the ultrastructural sites that have been associated with metabotropic receptor localization.

Amino Acid Transport System X-AG↗
PubMed2000

Calcium channel agonists and dystonia in the mouse.

Systemic administration of the L-type calcium channel agonists +/-Bay K 8644 or FPL 64176 causes a characteristic pattern of motor dysfunction in normal C57BL/6J mice that resembles generalized dystonia. There is no associated change in the electroencephalogram, confirming that the motor disorder does not reflect epileptic seizures. However, the electromyogram reveals an increase in baseline motor unit activity with prolonged phasic discharges consistent with dystonia. The duration and severity of dystonia is dependent on the dose administered and the age of the animal at testing. The effects are transient, with the return of normal motor behavior 1-4 hours after treatment. Similar effects can be provoked by intracerebral administration of small amounts of the drugs, indicating a centrally mediated response. Dystonia can be attenuated by co-administration of dihydropyridine L-type calcium channel antagonists (nifedipine, nimodipine, and nitrendipine) but not by non-dihydropyridine antagonists (diltiazem, verapamil, and flunarizine). These results implicate abnormal function of L-type calcium channels in the expression of dystonia in this model.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2000

TGFbeta trophic factors differentially modulate motor axon outgrowth and protection from excitotoxicity.

Transforming growth factor (TGF) beta-like trophic factors have been shown to be protective in acute neuronal injury paradigms. In the current study, we analyzed and compared members of this growing family, including glial cell line-derived neurotrophic factor (GDNF), neurturin, nodal, persephin, and TGFbeta1, for protection against chronic glutamate toxicity. In parallel, we developed a organotypic spinal cord culture system to study the ability of these factors to promote motor axon outgrowth across white matter. Using these systems, we were able to differentiate the neuroprotective effect of the TGFbeta-like factors from their motor axon outgrowth-promoting activity. GDNF, neurturin, persephin, nodal, and TGFbeta1 all protected against excitotoxic motor neuron degeneration. Low amounts of GDNF (1 ng/ml) and high concentrations of neurturin induced vigorous motor axon outgrowth. In contrast, nodal, persephin, and TGFbeta1 did not induce motor axon outgrowth. Both GDNF and neurturin bind to Ret receptor complexes and were capable of activating the MAP kinase pathway. A specific inhibitor of MAP kinase kinase, PD98059, inhibited the motor axon outgrowth-promoting activity of the GDNF but not the neuroprotective activity. Similarly, the specific PI3K inhibitors, LY294002 and wortmannin, were able to inhibit the promotion of motor axon outgrowth by GDNF, but did not affect neuroprotective activity. Our results suggest that the neurite outgrowth-promoting effect of GDNF is mediated through the PI3K and MAP kinase pathways. The neuroprotective effect of GDNF appears to be through a separate pathway.

Androstadienes↗
PubMed2000

Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia.

Glial (GLT-1 and GLAST) and neuronal (EAAC1) high-affinity transporters mediate the sodium dependent glutamate reuptake in mammalian brain. Their dysfunction leads to neuronal damage by allowing glutamate to remain in the synaptic cleft for a longer duration. The purpose of the present study is to understand their contribution to the ischemic delayed neuronal death seen in gerbil hippocampus following transient global cerebral ischemia. The protein levels of these three transporters were studied by immunoblotting as a function of reperfusion time (6 h to 7 days) following a 10 min occlusion of bilateral common carotid arteries in gerbils. In the vulnerable hippocampus, there was a significant decrease in the protein levels of GLT-1 (by 36-46%, P < 0.05; between 1 and 3 days of reperfusion) and EAAC1 (by 42-68%, P < 0.05; between 1 and 7 days of reperfusion). Histopathological evaluation showed no neuronal loss up to 2 days of reperfusion but an extensive neuronal loss (by approximately 84%, P < 0.01) at 7 days of reperfusion in the hippocampal CA1 region. The time frame of GLT-1 dysfunction (1-3 days of reperfusion) precedes the initiation of delayed neuronal death (2-3 days of reperfusion). This suggests GLT-1 dysfunction as a contributing factor for the hippocampal neuronal death following transient global cerebral ischemia. Furthermore, decreased EAAC1 levels may contribute to GABAergic dysfunction and excitatory/inhibitory imbalance following transient global ischemia.

ATP-Binding Cassette Transporters↗
PubMed2000

Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB.

The glial glutamate transporter GLT-1 may be the predominant Na(+)-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) for Na(+)-dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kappaB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron-conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kappaB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury.

ATP-Binding Cassette Transporters↗