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Jeffrey D Rothstein

Publications and source records attributed to Jeffrey D Rothstein.

17 recordsLinked to original sources
PubMed2003

Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neuromuscular disease that is associated with the degeneration of spinal and brainstem motor neurons, leading to atrophy of limb, axial, and respiratory muscles. The cause of ALS is unknown, and there is no effective therapy. Neurotrophic factors are candidates for therapeutic evaluation in ALS. Although chronic delivery of molecules to the central nervous system has proven difficult, we recently discovered that adeno-associated virus can be retrogradely transported efficiently from muscle to motor neurons of the spinal cord. We report that insulin-like growth factor 1 prolongs life and delays disease progression, even when delivered at the time of overt disease symptoms.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120.

L-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V(max) for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40-70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-alpha (TNF-alpha) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-alpha production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease.

Astrocytes↗
PubMed2003

NAALADase inhibition protects motor neurons against chronic glutamate toxicity.

Glutamate toxicity is implicated in the pathogenesis of amyotrophic lateral sclerosis. The neuropeptide N-acetyl-aspartyl glutamate (NAAG) appears to function both as a storage form for glutamate and as a neuromodulator at glutamatergic synapses. N-acetylated-alpha-linked acidic dipeptidase (NAALADase; also termed glutamate carboxypeptidase II) yields N-acetyl aspartate (NAA) and glutamate. Prior studies have demonstrated NAALADase upregulation in motor cortex and increased NAAG, NAA and glutamate in cerebrospinal fluid from amyotrophic lateral sclerosis patients. The potent NAALADase inhibitor, 2-(phosphonomethyl)-pentanedioic acid (2-PMPA), was tested in an in vitro model of chronic glutamate-mediated motor neuron degeneration. Neuroprotection was determined (1) biochemically, by measuring choline acetyltransferase activity, (2) immunohistochemically, by counting neurofilament-H-positive motor neurons and (3) morphologically, with phase contrast microscopy. 2-PMPA (10 microM) had significant neuroprotective effects on motor neurons as evidenced by increased choline acetyltransferase activity, decreased motor neuron loss and improved gross morphology. Results suggest that NAALADase inhibitors protect against chronic glutamate-mediated motor neuron degeneration and may prove therapeutic towards amyotrophic lateral sclerosis.

Animals↗
PubMed2003

Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury.

We have investigated the potential of human pluripotent cells to restore function in rats paralyzed with a virus-induced motor neuronopathy. Cells derived from embryonic germ cells, termed embryoid body-derived (EBD) cells, introduced into the CSF were distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchyma in paralyzed, but not uninjured, animals. Some of the transplanted human cells expressed the neuroglial progenitor marker nestin, whereas others expressed immunohistochemical markers characteristic of astrocytes or mature neurons. Rare transplanted cells developed immunoreactivity to choline acetyltransferase (ChAT) and sent axons into the sciatic nerve as detected by retrograde labeling. Paralyzed animals transplanted with EBD cells partially recovered motor function 12 and 24 weeks after transplantation, whereas control animals remained paralyzed. Semi-quantitative analysis revealed that the efficiency of neuronal differentiation and extension of neurites could not account for the functional recovery. Rather, transplanted EBD cells protected host neurons from death and facilitated reafferentation of motor neuron cell bodies. In vitro, EBD cells secrete transforming growth factor-alpha (TGF-alpha) and brain-derived neurotrophic factor (BDNF). Neutralizing antibodies to TGF-alpha and to BDNF abrogated the ability of EBD-conditioned media to sustain motor neuron survival in culture, whereas neutralizing antibodies to BDNF eliminated the axonal outgrowth from spinal organotypics observed with direct coculture of EBD cells. We conclude that cells derived from human pluripotent stem cells have the capacity to restore neurologic function in animals with diffuse motor neuron disease via enhancement of host neuron survival and function.

Alphavirus Infections↗
PubMed2003

Topiramate protects against motor neuron degeneration in organotypic spinal cord cultures but not in G93A SOD1 transgenic mice.

Topiramate is a novel anti-convulsant, structurally distinct from other known anti-convulsants. A number of independent studies suggest that topiramate has anti-excitotoxic properties. It has been found to diminish release of glutamate from neurons and block (-amino-3-hydoxy-5-methylisoxazole-4-proprionic acid glutamate receptor evoked currents. Since activation of non-N-methyl-D-aspartate glutamate receptors is thought to play a role in the selective loss of motor neurons in amyotrophic lateral sclerosis (ALS), we determined whether topiramate could protect against chronic glutamate-mediated motor neuron degeneration. An organotypic spinal cord culture system was used in which glutamate transport is inhibited by pharmacological blockade. After 3 weeks of treatment, topiramate was found to significantly prevent motor neuron degeneration in this culture model. However, the drug did not increase survival in G93A SOD1 transgenic mice, an animal model of ALS. These studies suggest that topiramate could be useful as a neuroprotectant, but were not effective in more complex motor injury paradigms such as the mouse model of ALS.

Amyotrophic Lateral Sclerosis↗
PubMed2003

Aggregate formation in Cu,Zn superoxide dismutase-related proteins.

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.

Amino Acid Motifs↗
PubMed2003

Translocation of glutamate transporter subtype excitatory amino acid carrier 1 protein in kainic acid-induced rat epilepsy.

Glutamate excitotoxicity has been implicated in the pathophysiology of epilepsy. Systemic injection of kainic acid (KA) in the rat produces an animal model of human temporal lobe epilepsy. We examined the temporal expression of the sodium-dependent neuronal glutamate transporter, excitatory amino acid carrier 1 (EAAC1), in KA-induced rat epilepsy. As an early alteration, perinuclear deposits of EAAC1 protein were found mainly in the large pyramidal neurons at the hippocampus, neocortex, piriform cortex, and amygdala with the reduction of neuropil staining 6 hours after KA injection. Immunoelectron microscopic study revealed that the perinuclear EAAC1 immunoreactivity corresponded to the translocation to the Golgi complex. At this time point, EAAC1 mRNA was down-regulated. The intracellular aggregation of EAAC1 primarily disappeared by 24 hours. In vitro studies indicated that internalization of EAAC1 from the plasma membrane to the intracellular compartment by KA treatment was associated with the reduction of electrogenic transporter currents. Our results suggest that the transient EAAC1 internalization participates in the modulation of the transporter function preventing excessive glutamate uptake to pyramidal neurons during the early stage of epilepsy.

Amino Acid Transport System X-AG↗
PubMed2003

Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B.

Surface expression of the glial glutamate transporter EAAT1 is stimulated by insulin-like growth factor 1 through activation of phosphatidylinositol-3-kinase. Downstream targets include serum and glucocorticoid-sensitive kinase isoforms SGK1, SGK2 and SGK3, and protein kinase B. SGK1 regulates Nedd4-2, a ubiquitin ligase that prepares cell membrane proteins for degradation. To test whether Nedd4-2, SGK1, SGK3 and protein kinase B regulate EAAT1, cRNA encoding EAAT1 was injected into Xenopus oocytes with or without additional injection of wild-type Nedd4-2, constitutively active S422DSGK1, inactive K127NSGK1, wild-type SGK3 and/or constitutively active T308D,S473DPKB. Glutamate induces a current in Xenopus oocytes expressing EAAT1, but not in water-injected oocytes, which is decreased by co-expression of Nedd4-2, an effect reversed by additional co-expression of S422DSGK1, SGK3 and T308D,S473DPKB, but not K127NSGK1. Site-directed mutagenesis of the SGK1 phosphorylation sites in the Nedd4-2 protein (S382A,S468ANedd4-2) and in the EAAT1 protein (T482AEAAT1, T482DEAAT1) significantly blunts the effect of S422DSGK1. Moreover, the current is significantly larger in T482DEAAT1- than in T482AEAAT1-expressing oocytes, indicating that a negative charge mimicking phosphorylation at T482 increases transport. The experiments reveal a powerful novel mechanism that regulates the activity of EAAT1. This mechanism might participate in the regulation of neuronal excitability and glutamate transport in other tissues.

Animals↗
PubMed2002

A neuronal glutamate transporter contributes to neurotransmitter GABA synthesis and epilepsy.

The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring-freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.

Action Potentials↗
PubMed2002

Oxidative stress and dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease.

Lesch-Nyhan disease, a neurogenetic disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase, is associated with a prominent loss of striatal dopamine. The current studies address the hypothesis that oxidant stress causes damage or dysfunction of nigrostriatal dopamine neurons in a knockout mouse model of the disease, by assessing several markers of oxidative damage and free radical scavenging systems. Some of these measures provided evidence for an increase in oxidative stress in the mutant mice (aconitase activity, oxidized glutathione, and lipid peroxides), but others did not (superoxide dismutase, protein thiol content, carbonyl protein content, total glutathione, glutathione peroxidase, catalase, and thiobarbituric reducing substances). Immunolocalization of heme-oxygenase 1 provided no evidence for oxidative stress restricted to specific elements of the striatum or midbrain in the mutants. Striatal dopamine systems of the mutant mice were more vulnerable to a challenge with the neurotoxin 6-hydroxydopamine, but they were not protected by cross-breeding the mutants with transgenic mice over-expressing superoxide dismutase. Overall, these data provide evidence for increased oxidative stress, but the failure to protect the knockout mice by over-expressing SOD1 argues that oxidative stress is not the sole process responsible for the loss of striatal dopamine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2002

Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS).

Transgenic overexpression of Cu(+2)/Zn(+2) superoxide dismutase 1 (SOD1) harboring an amyotrophic lateral sclerosis (ALS)-linked familial genetic mutation (SOD1(G93A)) in a Sprague-Dawley rat results in ALS-like motor neuron disease. Motor neuron disease in these rats depended on high levels of mutant SOD1 expression, increasing from 8-fold over endogenous SOD1 in the spinal cord of young presymptomatic rats to 16-fold in end-stage animals. Disease onset in these rats was early, approximately 115 days, and disease progression was very rapid thereafter with affected rats reaching end stage on average within 11 days. Pathological abnormalities included vacuoles initially in the lumbar spinal cord and subsequently in more cervical areas, along with inclusion bodies that stained for SOD1, Hsp70, neurofilaments, and ubiquitin. Vacuolization and gliosis were evident before clinical onset of disease and before motor neuron death in the spinal cord and brainstem. Focal loss of the EAAT2 glutamate transporter in the ventral horn of the spinal cord coincided with gliosis, but appeared before motor neuron/axon degeneration. At end-stage disease, gliosis increased and EAAT2 loss in the ventral horn exceeded 90%, suggesting a role for this protein in the events leading to cell death in ALS. These transgenic rats provide a valuable resource to pursue experimentation and therapeutic development, currently difficult or impossible to perform with existing ALS transgenic mice.

Amino Acid Substitution↗
PubMed2002

Evidence that accumulation of ceramides and cholesterol esters mediates oxidative stress-induced death of motor neurons in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the spinal cord resulting in progressive paralysis and death. The pathogenic mechanism of ALS is unknown but may involve increased oxidative stress, overactivation of glutamate receptors, and apoptosis. We report abnormalities in sphingolipid and cholesterol metabolism in the spinal cords of ALS patients and in a transgenic mouse model (Cu/ZnSOD mutant mice), which manifest increased levels of sphingomyelin, ceramides, and cholesterol esters; in the Cu/ZnSOD mutant mice, these abnormalities precede the clinical phenotype. In ALS patients and Cu/Zn-SOD mutant mice, increased oxidative stress occurs in association with the lipid alterations, and exposure of cultured motor neurons to oxidative stress increases the accumulation of sphingomyelin, ceramides, and cholesterol esters. Pharmacological inhibition of sphingolipid synthesis prevents accumulation of ceramides, sphingomyelin, and cholesterol esters and protects motor neurons against death induced by oxidative and excitotoxic insults. These findings suggest a pivotal role for altered sphingolipid metabolism in the pathogenesis of ALS.

Aged↗
PubMed2002

Cyclooxygenase 2 inhibition protects motor neurons and prolongs survival in a transgenic mouse model of ALS.

The pathogenesis of cell death in amyotrophic lateral sclerosis (ALS) may involve glutamate-mediated excitotoxicity, oxidative damage, and apoptosis. We used a transgenic mouse model of ALS to determine the effect of inhibition of cyclooxygenase-2 in treating the disease. Cyclooxygenase-2, present in spinal neurons and astrocytes, catalyzes the synthesis of prostaglandin E2. Prostaglandin E2 stimulates glutamate release from astrocytes, whereas cyclooxygenase-2 also plays a key role in the production of proinflammatory cytokines, reactive oxygen species, and free radicals. Treatment with a selective cyclooxygenase-2 inhibitor, celecoxib, markedly inhibited production of prostaglandin E2 in the spinal cords of ALS mice. Celecoxib treatment significantly delayed the onset of weakness and weight loss and prolonged survival by 25%. Spinal cords of treated ALS mice showed significant preservation of spinal neurons and diminished astrogliosis and microglial activation. Our results suggest that cyclooxygenase-2 inhibition may benefit ALS patients.

Amyotrophic Lateral Sclerosis↗
PubMed2002

Paving new pathways.

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Antineoplastic Agents↗
PubMed2002

Therapeutic developments in the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease characterised by the selective death of motor neurones. The mechanisms and processes responsible for the selective loss of motor neurones are still unknown, however several hypotheses have been put forward, including oxidative damage and/or toxicity from intracellular aggregates due to mutant superoxide dismutase-1 activity, axonal strangulation from cytoskeletal abnormalities, loss of trophic factor support and glutamate-mediated excitotoxicity. These theories are based on a better understanding of the genetics of amyotrophic lateral sclerosis and on biochemical and pathological analysis of post-mortem tissue. They have led to the development of appropriate animal and cell culture models, allowing the sequence of events in motor neuronal degeneration to be unravelled and potential therapeutic agents to be screened. Unfortunately, the majority of therapeutics found to be efficacious in the animal and cell culture models have failed in human trials. Riluzole is still the only proven therapy in humans, shown to extend survival of amyotrophic lateral sclerosis patients by approximately 3 months, but it has no effect on muscle strength. Other potential therapeutic approaches are being identified, including inhibition of caspase-mediated cell death, maintenance of mitochondrial integrity and energy production, regulation of glutamate homeostasis, reduction of inflammation and control of neurofilament synthesis. Hopefully, in the near future some new agents will be found that can alter the course of this devastating and fatal disease.

Amyotrophic Lateral Sclerosis↗
PubMed2001

Mechanisms for reversible regulation between G13 and Rho exchange factors.

The heterotrimeric G proteins, G(12) and G(13), mediate signaling between G protein-coupled receptors and the monomeric GTPase, RhoA. One pathway for this modulation is direct stimulation by Galpha(13) of p115 RhoGEF, an exchange factor for RhoA. The GTPase activity of both Galpha(12) and Galpha(13) is increased by the N terminus of p115 Rho guanine nucleotide exchange factor (GEF). This region has weak homology to the RGS box sequence of the classic regulators of G protein signaling (RGS), which act as GTPase-activating proteins (GAP) for G(i) and G(q). Here, the RGS region of p115 RhoGEF is shown to be distinctly different in that sequences flanking the predicted "RGS box" region are required for both stable expression and GAP activity. Deletions in the N terminus of the protein eliminate GAP activity but retain substantial binding to Galpha(13) and activation of RhoA exchange activity by Galpha(13). In contrast, GTRAP48, a homolog of p115 RhoGEF, bound to Galpha(13) but was not stimulated by the alpha subunit and had very poor GAP activity. Besides binding to the N-terminal RGS region, Galpha(13) also bound to a truncated protein consisting only of the Dbl homology (DH) and pleckstrin homology (PH) domains. However, Galpha(13) did not stimulate the exchange activity of this truncated protein. A chimeric protein, which contained the RGS region of GTRAP48 in place of the endogenous N terminus of p115 RhoGEF, was activated by Galpha(13). These results suggest a mechanism for activation of the nucleotide exchange activity of p115 RhoGEF that involves direct and coordinate interaction of Galpha(13) to both its RGS and DH domains.

Animals↗