Mechanisms for Reversible Regulation Between G13 and Rho Exchange Factors

The Journal of Biological Chemistry. Jan, 2002  |  Pubmed ID: 11698392

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.

Focal Loss of the Glutamate Transporter EAAT2 in a Transgenic Rat Model of SOD1 Mutant-mediated Amyotrophic Lateral Sclerosis (ALS)

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2002  |  Pubmed ID: 11818550

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.

Oxidative Stress and Dopamine Deficiency in a Genetic Mouse Model of Lesch-Nyhan Disease

Brain Research. Developmental Brain Research. Feb, 2002  |  Pubmed ID: 11882343

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.

A Neuronal Glutamate Transporter Contributes to Neurotransmitter GABA Synthesis and Epilepsy

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2002  |  Pubmed ID: 12151515

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.

Paving New Pathways

Nature Medicine. Sep, 2002  |  Pubmed ID: 12205454

Evidence That Accumulation of Ceramides and Cholesterol Esters Mediates Oxidative Stress-induced Death of Motor Neurons in Amyotrophic Lateral Sclerosis

Annals of Neurology. Oct, 2002  |  Pubmed ID: 12325074

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.

Therapeutic Developments in the Treatment of Amyotrophic Lateral Sclerosis

Expert Opinion on Investigational Drugs. Oct, 2002  |  Pubmed ID: 12387699

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.

Cyclooxygenase 2 Inhibition Protects Motor Neurons and Prolongs Survival in a Transgenic Mouse Model of ALS

Annals of Neurology. Dec, 2002  |  Pubmed ID: 12447931

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.

Models of Amyotrophic Lateral Sclerosis

Current Protocols in Neuroscience / Editorial Board, Jacqueline N. Crawley ... [et Al.]. Nov, 2002  |  Pubmed ID: 18428572

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder caused by degeneration of the motor neurons in cortex, brainstem and spinal cord. Two experimental models of ALS are described in this unit: organotypic cultures of spinal cord, and transgenic mice expressing a human mutant superoxide dismutase 1 (SOD1) gene. Appropriate animal and cell culture models of ALS can be used to help unravel the sequence of events in motor neuronal degeneration and test potential therapies.

Aggregate Formation in Cu,Zn Superoxide Dismutase-related Proteins

The Journal of Biological Chemistry. Apr, 2003  |  Pubmed ID: 12551935

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.

Topiramate Protects Against Motor Neuron Degeneration in Organotypic Spinal Cord Cultures but Not in G93A SOD1 Transgenic Mice

Neuroscience Letters. Feb, 2003  |  Pubmed ID: 12566164

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.

Of Mice and Men: Reconciling Preclinical ALS Mouse Studies and Human Clinical Trials

Annals of Neurology. Apr, 2003  |  Pubmed ID: 12666108

NAALADase Inhibition Protects Motor Neurons Against Chronic Glutamate Toxicity

European Journal of Pharmacology. Jun, 2003  |  Pubmed ID: 12826236

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.

Human Embryonic Germ Cell Derivatives Facilitate Motor Recovery of Rats with Diffuse Motor Neuron Injury

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2003  |  Pubmed ID: 12832537

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.

Translocation of Glutamate Transporter Subtype Excitatory Amino Acid Carrier 1 Protein in Kainic Acid-induced Rat Epilepsy

The American Journal of Pathology. Aug, 2003  |  Pubmed ID: 12875997

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.

Reduced Expression of Glutamate Transporter EAAT2 and Impaired Glutamate Transport in Human Primary Astrocytes Exposed to HIV-1 or Gp120

Virology. Jul, 2003  |  Pubmed ID: 12890621

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.

Retrograde Viral Delivery of IGF-1 Prolongs Survival in a Mouse ALS Model

Science (New York, N.Y.). Aug, 2003  |  Pubmed ID: 12907804

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.

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

Journal of Neurochemistry. Sep, 2003  |  Pubmed ID: 12911626

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.

Climbing Fiber Activation of EAAT4 Transporters and Kainate Receptors in Cerebellar Purkinje Cells

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jan, 2004  |  Pubmed ID: 14715943

Cerebellar Purkinje cells (PCs) express two glutamate transporters, EAAC1 (EAAT3) and EAAT4; however, their relative contribution to the uptake of glutamate at synapses is not known. We found that glutamate transporter currents recorded at climbing fiber (CF)-PC synapses are absent in mice lacking EAAT4 but unchanged in mice lacking EAAC1, indicating that EAAT4 is preferentially involved in clearing glutamate from CF synapses. However, comparison of CF synaptic currents between wild-type and transporter knock-out mice revealed that ionotropic glutamate receptors are responsible for >40% of the current previously attributed to transporters, indicating that PCs remove <10% of the glutamate released by the CF. The receptors responsible for the nontransporter component accounted for 5% of the CF EPSC, had a slower time course and lower occupancy than AMPA receptors at CF synapses, and exhibited pharmacological properties consistent with kainate receptors. In GluR5 knock-out mice, this current was dramatically reduced, indicating that CF excitation of PCs involves two distinct classes of ionotropic glutamate receptors, AMPA receptors and GluR5-containing kainate receptors.

Glutamate Transporter Expression and Function in Human Glial Progenitors

Glia. Jan, 2004  |  Pubmed ID: 14730707

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

Reality and Immortality--neural Stem Cells for Therapies

Nature Biotechnology. Mar, 2004  |  Pubmed ID: 14990948

Altered Expression of the Glutamate Transporter EAAT2b in Neurological Disease

Annals of Neurology. Apr, 2004  |  Pubmed ID: 15048885

Functional studies suggest that up to 95% of all glutamate transport is handled by the glutamate transporter EAAT2. Amino and C-terminal antibodies demonstrate that under normal conditions EAAT2 is specific to astrocytes. A truncated splice variant of EAAT2, known as EAAT2b, also has been identified in astrocytes and some neurons. In vitro studies suggest EAAT2b transports glutamate similar to EAAT2, although the contribution of EAAT2b to normal clearance of extracellular glutamate is unknown. To investigate EAAT2b biology in pathological conditions, we examined the cellular and regional distribution of EAAT2b in amyotrophic lateral sclerosis. Using epitope-specific, affinity purified antibodies, we found that EAAT2b tissue levels were increased by more than twofold in amyotrophic lateral sclerosis motor cortex, whereas EAAT2 levels were decreased by up to 95%. EAAT2b distribution in normal human cortex was largely confined to the neuropil-like EAAT2, with occasional faint neuronal expression. In contrast, amyotrophic lateral sclerosis motor cortex had an obvious qualitative increase in neuropil EAAT2b staining and a drastic increase in neuronal soma and dendritic EAAT2b immunostaining. Despite these increases in EAAT2b immunostaining, functional transporter studies demonstrated a large loss of EAAT2 function. These studies clearly document altered regulation and splicing of the dominant glutamate transporter EAAT2 under conditions of neurological stress.

Glutamate Receptor Antagonists Protect from Virus-induced Neural Degeneration

Annals of Neurology. Apr, 2004  |  Pubmed ID: 15048893

Neuronal damage during acute viral encephalomyelitis can result directly from virus infection or indirectly from the host immune response to infection. In neurodegenerative diseases and stroke, neuronal death also can result from excess release of excitatory amino acid neurotransmitters, such as glutamate. To determine the role of glutamate excitotoxicity in fatal alphavirus-induced paralytic encephalomyelitis, we treated mice infected with neuroadapted Sindbis virus (NSV) with antagonists of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtypes of glutamate receptors. Both apoptotic and necrotic neurons in the hippocampus were decreased in animals treated with MK-801, an NMDA receptor antagonist, or GYKI-52466, an AMPA receptor antagonist. However, only AMPA receptor blockade prevented damage to spinal cord motor neurons and protected mice from paralysis and death due to NSV infection. Protection was not caused by altered virus replication because treatment did not affect virus distribution and actually delayed virus clearance. These results provide evidence that NSV infection activates neurotoxic pathways that result in aberrant glutamate receptor stimulation and neuronal damage. Furthermore, AMPA receptor-mediated motor neuron death is an important contributor to paralysis and mortality in acute alphavirus-induced encephalomyelitis.

Glutamate Transporters: Animal Models to Neurologic Disease

Neurobiology of Disease. Apr, 2004  |  Pubmed ID: 15056453

Glutamate is the primary excitatory amino acid neurotransmitter in the central nervous system and its activity is carefully modulated in the synaptic cleft by glutamate transporters. A number of glutamate transporters have been identified in the central nervous system and each has a unique physiologic property and distribution. Glutamate transporter dysfunction may either be an initiating event or part of a cascade leading to cellular dysfunction and ultimately cell death. Animal models of glutamate transporter dysfunction have revealed a significant role for these proteins in pathologic conditions such as neurodegenerative diseases, epilepsy, stroke, and central nervous system tumors. Recent work has focused on glutamate transporter biology in human diseases with an emphasis on how manipulation of these transporter proteins may lead to therapeutic interventions in neurologic disease.

Retinal Colocalization and in Vitro Interaction of the Glutamate Transporter EAAT3 and the Serum- and Glucocorticoid-inducible Kinase SGK1 [correction]

Investigative Ophthalmology & Visual Science. May, 2004  |  Pubmed ID: 15111600

The serum- and glucocorticoid-inducible kinase SGK1 regulates several epithelial channels and transporters, the related protein kinase B (PKB) regulates glucose transport. SGK1 is expressed in the brain and could thus regulate glial and/or neuronal transport processes. The present study explores whether SGK1 is expressed in the retina and whether it regulates EAAT3, a Na(+)-coupled glutamate transporter. EAAT3 is expressed in retinal ganglion cells and accomplishes the clearance of glutamate from synaptic clefts.

Axonal Growth of Embryonic Stem Cell-derived Motoneurons in Vitro and in Motoneuron-injured Adult Rats

Proceedings of the National Academy of Sciences of the United States of America. May, 2004  |  Pubmed ID: 15118094

We generated spinal motoneurons from embryonic stem (ES) cells to determine the developmental potential of these cells in vitro and their capacity to replace motoneurons in the adult mammalian spinal cord. ES cell-derived motoneurons extended long axons, formed neuromuscular junctions, and induced muscle contraction when cocultured with myoblasts. We transplanted motoneuron-committed ES cells into the spinal cords of adult rats with motoneuron injury and found that approximately 3,000 ES cell-derived motoneurons (25% of input) survived for >1 month in the spinal cord of each animal. ES cell-derived axonal growth was inhibited by myelin, and this inhibition was overcome by administration of dibutyryl cAMP (dbcAMP) or a Rho kinase inhibitor in vitro and in vivo. In transplanted rats infused with dbcAMP, approximately 80 ES cell-derived motor axons were observed within the ventral roots of each animal, whereas none were observed in transplanted rats not treated with dbcAMP. Because these cells replicate many of the developmental and mature features of true motoneurons, they are an important biological tool to understand formation of motor units in vitro and a potential therapeutic tool to reconstitute neural circuits in vivo.

Astrocyte Glutamate Transporters Regulate Metabotropic Glutamate Receptor-mediated Excitation of Hippocampal Interneurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. May, 2004  |  Pubmed ID: 15140926

Clearance of extracellular glutamate is essential for limiting the activity of metabotropic glutamate receptors (mGluRs) at excitatory synapses; however, the relative contribution of transporters found in neuronal and glial membranes to this uptake is poorly understood. Hippocampal interneurons located at the oriens-alveus border express mGluR1alpha, a metabotropic glutamate receptor that regulates excitability and synaptic plasticity. To determine which glutamate transporters are essential for removing glutamate at these excitatory synapses, we recorded mGluR1-mediated EPSCs from oriens-lacunosum moleculare (O-LM) interneurons in acute hippocampal slices. Stimulation in stratum oriens reliably elicited a slow mGluR1-mediated current in O-LM interneurons if they were briefly depolarized to allow Ca2+ entry before stimulation. Selective inhibition of GLT-1 [for glutamate transporter; EAAT2 (for excitatory amino acid transporter)] with dihydrokainate increased the amplitude of these responses approximately threefold, indicating that these transporters compete with mGluRs for synaptically released glutamate. However, inhibition of all glutamate transporters with TBOA (DL-threo-b-benzyloxyaspartic acid) increased mGluR1 EPSCs >15-fold, indicating that additional transporters also shape activation of these receptors. To identify these transporters, we examined mGluR1 EPSCs in mice lacking GLAST (for glutamate-aspartate transporter; EAAT1) or EAAC1 (for excitatory amino acid carrier; EAAT3). A comparison of responses recorded from wild-type and transporter knock-out mice revealed that the astroglial glutamate transporters GLT-1 and GLAST, but not the neuronal transporter EAAC1, restrict activation of mGluRs in O-LM interneurons. Transporter-dependent potentiation of mGluR1 EPSCs led to a dramatic increase in interneuron firing and enhanced inhibition of CA1 pyramidal neurons, suggesting that acute or prolonged disruption of transporter activity could lead to changes in network activity as a result of enhanced interneuron excitability.

Thrombin and Lysophosphatidic Acid Receptors Utilize Distinct RhoGEFs in Prostate Cancer Cells

The Journal of Biological Chemistry. Jul, 2004  |  Pubmed ID: 15143072

Thrombin and lysophosphatidic acid (LPA) receptors play important roles in vascular biology, development, and cancer. These receptors activate rho via G(12/13) family heterotrimeric G proteins, which are known to directly activate three distinct rho guanine nucleotide exchange factors (rhoGEFs) that contain a regulator of G protein signaling (RGS) domain (RGS-rhoGEFs). However, it is not known which, if any, of these RGS-rhoGEFs (LARG (leukemia-associated rhoGEF), p115rhoGEF, or PDZrhoGEF) plays a role in G protein-coupled receptor-stimulated rho signaling. Using oligonucleotide small interfering RNAs that suppress specific RGS-rhoGEF expression, we show that thrombin receptor stimulation of rho is primarily mediated by LARG in HEK293T and PC-3 prostate cancer cell lines. In contrast, the LPA-stimulated rho response in PC-3 cells is dependent on PDZrhoGEF expression. Suppression of p115rhoGEF had no effect. Thus different rhoGEFs (LARG and PDZrhoGEF) mediate downstream rho signaling by the thrombin and LPA receptors.

Viral-induced Spinal Motor Neuron Death is Non-cell-autonomous and Involves Glutamate Excitotoxicity

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Aug, 2004  |  Pubmed ID: 15329404

Neuroadapted Sindbis virus (NSV) is a neurotropic virus capable of inducing the death of spinal motor neurons in mice and rats. In this study we investigated the mechanisms that underlie NSV-induced motor neuron death. We found that many degenerating spinal motor neurons were not infected directly with NSV, suggesting that bystander cell death occurs. An excitotoxic mechanism was confirmed when blockade of calcium-permeable AMPA receptors attenuated motor neuron death both in vitro and in vivo. Blockade of astroglial glutamate reuptake potentiated NSV-induced motor neuron loss in vivo, suggesting that astrocyte-mediated removal of perisynaptic glutamate is important in limiting NSV-induced excitotoxic injury. Astroglial glutamate transport was reduced markedly in the spinal cord during NSV infection, in advance of motor neuron injury in susceptible mice. In contrast, we found 5.6-fold elevated glutamate uptake in the spinal cords of mice resistant to NSV-induced paralysis. Likewise, minocycline markedly increased spinal cord glutamate transport and protected mice from NSV-induced motor neuron death. These studies suggest that NSV infection triggers a cascade of events in the spinal cord resulting in impaired astrocytic glutamate transport and excitotoxic injury of motor neurons mediated via calcium-permeable AMPA receptors. Similar changes may occur in other motor neuron disorders such as amyotrophic lateral sclerosis or West Nile Virus-induced poliomyelitis, suggesting a common tissue injury pathway.

Neural Stem Cells Protect Against Glutamate-induced Excitotoxicity and Promote Survival of Injured Motor Neurons Through the Secretion of Neurotrophic Factors

Molecular and Cellular Neurosciences. Nov, 2004  |  Pubmed ID: 15519246

Besides their capacity to give rise to neurons and/or glia, neural stem cells (NSCs) appear to inherently secrete neurotrophic factors beneficial to injured neurons. To test this potential, we have implanted NSCs onto or adjacent to spinal cord cultures. When NSCs were placed adjacent to the spinal cord sections, motor neuron axons grew toward the NSCs. Furthermore, conditioned medium from NSCs cultures was also able to induce similar axonal outgrowth, suggesting that these NSCs secrete soluble factors that have tropic and/or trophic properties. ELISA revealed that the NSCs secrete glial cell-line-derived factor (GDNF) and nerve growth factor (NGF). Interestingly, preincubation of the conditioned medium with GDNF-blocking antibodies abolished axonal outgrowth. We also showed that NSCs can protect spinal cord cultures from experimentally induced excitotoxic damage. The neuroprotective potential of NSCs was further confirmed in vivo by their ability to protect against motor neuron cell death.

Beta-lactam Antibiotics Offer Neuroprotection by Increasing Glutamate Transporter Expression

Nature. Jan, 2005  |  Pubmed ID: 15635412

Glutamate is the principal excitatory neurotransmitter in the nervous system. Inactivation of synaptic glutamate is handled by the glutamate transporter GLT1 (also known as EAAT2; refs 1, 2), the physiologically dominant astroglial protein. In spite of its critical importance in normal and abnormal synaptic activity, no practical pharmaceutical can positively modulate this protein. Animal studies show that the protein is important for normal excitatory synaptic transmission, while its dysfunction is implicated in acute and chronic neurological disorders, including amyotrophic lateral sclerosis (ALS), stroke, brain tumours and epilepsy. Using a blinded screen of 1,040 FDA-approved drugs and nutritionals, we discovered that many beta-lactam antibiotics are potent stimulators of GLT1 expression. Furthermore, this action appears to be mediated through increased transcription of the GLT1 gene. beta-Lactams and various semi-synthetic derivatives are potent antibiotics that act to inhibit bacterial synthetic pathways. When delivered to animals, the beta-lactam ceftriaxone increased both brain expression of GLT1 and its biochemical and functional activity. Glutamate transporters are important in preventing glutamate neurotoxicity. Ceftriaxone was neuroprotective in vitro when used in models of ischaemic injury and motor neuron degeneration, both based in part on glutamate toxicity. When used in an animal model of the fatal disease ALS, the drug delayed loss of neurons and muscle strength, and increased mouse survival. Thus these studies provide a class of potential neurotherapeutics that act to modulate the expression of glutamate neurotransmitter transporters via gene activation.

Glial Restricted Precursors Protect Against Chronic Glutamate Neurotoxicity of Motor Neurons in Vitro

Glia. Apr, 2005  |  Pubmed ID: 15657939

We have examined the expression of glutamate transporters in primary and immortalized glial precursors (GRIPs). We subsequently transduced these cells with the GLT1 glutamate transporter and examined the ability of these cells to protect motor neurons in an organotypic spinal cord culture. We show that glial restricted precursors and GRIP-derived astrocytes predominantly express glutamate transporters GLAST and GLT1. Oligodendrocyte differentiation of GRIPs results in downregulation of all glutamate transporter subtypes. Having identified these precursor cells as potential vectors for delivering glutamate transporters to regions of interest, we engineered a line of GRIPS that overexpress the glutamate transporter GLT1. These cells (G3 cells) have a nearly fourfold increase in glutamate transporter expression and at least a twofold increase in the V(max) for glutamate transport. To assess whether G3 seeding can protect motor neurons from chronic glutamate neurotoxicity, G3s were seeded onto rat organotypic spinal cord cultures. These cultures have previously been used extensively to understand pathways involved in chronic glutamate neurotoxicity of motor neurons. After G3 seeding, cells integrated into the culture slice and resulted in levels of glutamate transport sufficient to enhance total glutamate uptake. To test whether neuroprotection was related to glutamate transporter overexpression, we isolated GRIPS from the GLT1 null mouse to serve as controls. The seeding of G3s resulted in a reduction of motor neuron cell death. Hence, we believe that these cells may potentially play a role in cell-based neuroprotection from glutamate excitotoxicity.

Expression of Glutamate Transporter Subtypes During Normal Human Corticogenesis and Type II Lissencephaly

Brain Research. Developmental Brain Research. Mar, 2005  |  Pubmed ID: 15804404

Glutamate transporters are thought to have an important role in central nervous system (CNS) development. We investigated the expression of the sodium-dependent high-affinity glutamate transporters EAAT1, EAAT2, and EAAT3 in 11 human autopsied cases without neurological disorders and in four cases with type II lissencephaly including Walker Warburg's syndrome (WWS) and Fukuyama-type congenital muscular dystrophy (FCMD), both of which are classified as migration disorders of the human brain. Expression of glutamate transporter subtypes was differentially regulated during normal human corticogenesis. Although EAAT1 and EAAT2 were mainly localized to the cortical astrocytes in the postnatal brain, EAAT1 was enriched in the proliferative zones and radial glia from 13 gestational weeks (GW) to 20 GW. EAAT2 was abundant in the intermediate zone until 23 GW, and transiently expressed in the radial fibers of the transitional form of radial glia into mature astrocytes as well as partly in the corticofugal axonal bundles. EAAT3 immunoreactivity was robust in the apical dendrites of the pyramidal neurons in the marginal zone and cortical plate during corticogenesis, and decreased postnatally. In the individuals with type II lissencephaly, glutamate transporters were expressed in the extrusion of neuroglial tissue. Bundles of EAAT2-immunoreactive radial fibers were prominent in the specimens at 20 GW. Thus, glutamate transporters are differentially regulated during normal and impaired corticogenesis. Altered glutamate transporter expression in type II lissencephaly suggests that glutamate metabolism is involved in the formation of the normal cortex and contributes to the disorganized cortex seen in migration disorders.

Signal-crosstalk Between Rho/ROCK and C-Jun NH2-terminal Kinase Mediates Migration of Vascular Smooth Muscle Cells Stimulated by Angiotensin II

Arteriosclerosis, Thrombosis, and Vascular Biology. Sep, 2005  |  Pubmed ID: 15994438

Rho and its effector Rho-kinase/ROCK mediate cytoskeletal reorganization as well as smooth muscle contraction. Recent studies indicate that Rho and ROCK are critically involved in vascular remodeling. Here, we tested the hypothesis that Rho/ROCK are critically involved in angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs) by mediating a specific signal cross-talk.

Aquaporin 4 is Increased in Association with Human Immunodeficiency Virus Dementia: Implications for Disease Pathogenesis

Journal of Neurovirology. Dec, 2005  |  Pubmed ID: 16338747

Changes in astrocyte shape and function are known to occur in association with human immunodeficiency virus (HIV) dementia (HIVD). However, the causes and consequences of such changes are not completely understood. In vitro data suggest that changes in the expression of aquaporin 4 (AQP4), the aquaporin subtype expressed by astrocytes, can significantly influence cell shape and physiology. In the present study, the authors therefore investigated the possibility that AQP4 levels may be altered in HIVD. Using Western blot, the authors show that immunoreactivity for AQP4 is elevated in brain homogenates from the mid frontal gyrus of patients who died with HIVD (P < .005 HIV seronegative versus HIVD). Of interest, a significant increase was also observed in homogenates from HIV-infected individuals without dementia (P < .05 HIV seronegative versus neurologically normal HIV seropositive). In the present study the authors also examined the stimulated expression of AQP4 in cultured cells. Previous in vitro studies have shown that AQP4 expression may be increased by stimuli that induce cytoskeletal changes and/or the activation of p38 mitogen-activated protein (MAP) kinase. The authors therefore focused on tumor necrosis factor (TNF)-alpha, which has been linked to p38 MAP kinase activation, and thrombin, which may also induce changes in the actin cytoskeleton. Both may be elevated with HIVD. Again using Western blot, the authors show an increase in both AQP4 and phosphorylated p38 MAP kinase in homogenates from TNF-alpha- and thrombin-stimulated organotypic cerebellar and spinal cord cultures. Together, these studies suggest that AQP4 expression may be altered in HIVD and/or in response to exogenous proteinases. Additional studies may be warranted to determine whether altered AQP4 expression represents a protective and/or maladaptive response to central nervous system (CNS) inflammation.

Spectrin Mutations Cause Spinocerebellar Ataxia Type 5

Nature Genetics. Feb, 2006  |  Pubmed ID: 16429157

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.

Interaction of PDZRhoGEF with Microtubule-associated Protein 1 Light Chains: Link Between Microtubules, Actin Cytoskeleton, and Neuronal Polarity

The Journal of Biological Chemistry. Apr, 2006  |  Pubmed ID: 16478718

Rat (r) PDZRhoGEF, initially identified as a glutamate transporter EAAT4-associated protein, is a member of a novel RhoGEF subfamily. The N terminus of the protein contains a PDZ and a proline-rich domain, two motifs known to be involved in protein-protein interactions. By using the yeast two-hybrid approach, we screened for proteins that interact with the N terminus of rPDZRhoGEF. The light chain 2 of microtubule-associated protein 1 (LC2) was the only protein identified from the screen that does not contain a type I PDZ-binding motif at its extreme C terminus (-(S/T)Xphi-COOH, where phi is a hydrophobic amino acid). However, the C terminus does conform to a type II-binding motif (-phiXphi). We report here that rPDZRhoGEF interacts with LC2 via the PDZ domain, and the interaction is abolished by mutations in the carboxylate-binding loop. The specificity of the interaction was confirmed using GST fusion protein pull-down assays and coimmunoprecipitations. Expression of rPDZRhoGEF mutants that are unable to interact with proteins via the carboxylate-binding loop induced changes in cell morphology and actin organization. These mutants alter the activation of RhoGTPases, and coexpression of dominant-negative RhoGTPases prevent the morphological changes. Furthermore, in cells expressing wild type rPDZRhoGEF, drug-induced microtubule depolymerization produces changes in cell morphology that are similar to those induced by rPDZRhoGEF mutants. These results indicate that modulation of the guanine nucleotide exchange activity of rPDZRhoGEF through interaction with microtubule-associated protein light chains may coordinate microtubule integrity and the reorganization of actin cytoskeleton. This coordinated action of the actin and microtubular cytoskeletons is essential for the development and maintenance of neuronal polarity.

Regulation of Astrocytic Glutamate Transporter Expression by Akt: Evidence for a Selective Transcriptional Effect on the GLT-1/EAAT2 Subtype

Journal of Neurochemistry. May, 2006  |  Pubmed ID: 16573655

In the nervous system, astrocytes express different ratios of the two glial glutamate transporters, glutamate transporter subtype 1 (GLT-1) and glutamate/aspartate transporter (GLAST), but little is known about the signaling pathways that independently regulate their expression. Treatment with dibutyryl-cAMP, epidermal growth factor (EGF) or other growth factors both induces expression of GLT-1 and increases expression of GLAST in astrocyte cultures. The induction of GLT-1 is correlated with morphological and biochemical changes that are consistent with astrocyte maturation. Pharmacological studies suggest that phosphatidylinositol 3-kinase (PI-3K) and the nuclear transcription factor-kappaB (NF-kappaB) may be involved in the induction of GLT-1 expression. In several signaling systems Akt, also known as protein kinase B (PKB), functions downstream of PI-3K. In these present studies we used lentiviral vectors engineered to express dominant-negative (DN), constitutively active (CA), or null variants of Akt to study the possible involvement of Akt in the regulation of GLT-1. Expression of DN-Akt attenuated the EGF-dependent induction of GLT-1. Expression of CA-Akt caused a dose- and time-dependent increase in GLT-1 protein, increased GLT-1 mRNA levels, increased dihydrokainate-sensitive (presumably GLT-1 mediated) transport activity, and caused a change in astrocyte morphology to a more stellate shape, but had no effect on GLAST protein levels. Finally, the expression of CA-Akt increased the expression of a reporter construct containing a putative promoter fragment from the human homolog of GLT-1, called EAAT2. From these studies, we conclude that Akt induces the expression of GLT-1 through increased transcription and that Akt can regulate GLT-1 expression without increasing GLAST expression in astrocytes.

Loss of the Astrocyte Glutamate Transporter GLT1 Modifies Disease in SOD1(G93A) Mice

Experimental Neurology. Sep, 2006  |  Pubmed ID: 16753145

Recent studies have highlighted the role of astrocytes in the development of motor neuron disease in animal models. The astrocyte glutamate transporter GLT1 is responsible for a significant portion of glutamate transport from the synaptic cleft; regulating synaptic transmission and preventing glutamate excitotoxicity. While previous studies have demonstrated reductions in GLT1 with SOD1-mediated disease progression, it is not well established whether a reduction in this astrocyte-specific transporter alters the pathobiology of motor neuron degeneration in the SOD1(G93A) mouse. In order to address this possible astrocyte-specific influence, we crossed the SOD1(G93A) mouse line with a mouse heterozygous for GLT1 (GLT1+/-) exhibiting a significant reduction in transporter protein. Mice that carried both the SOD1 mutation and a reduced amount of GLT1 (SOD1(G93A)/GLT1+/-) exhibited an increase in the rate of motor decline accompanied by earlier motor neuron loss when compared with SOD1(G93A) mice. A modest reduction in survival was also noted in these mice. Dramatic losses of the GLT1 protein and reduced glutamate transport in the lumbar spinal cords of the SOD1(G93A)/GLT1+/- animals were also observed. GLT1 was not significantly changed in cortices from these animals suggesting that the effect of mutant SOD1 on GLT1 production/function was largely targeted to spinal cord rather than cortical astrocytes. This study suggests that astrocytes, and the astrocyte glutamate transporter GLT1, play a role in modifying disease progression and motor neuron loss in this model.

Trial of Celecoxib in Amyotrophic Lateral Sclerosis

Annals of Neurology. Jul, 2006  |  Pubmed ID: 16802291

To determine whether chronic treatment with celecoxib, a cyclooxygenase-2 inhibitor that has been shown to be beneficial in preclinical testing, is safe and effective in amyotrophic lateral sclerosis (ALS).

Recovery from Paralysis in Adult Rats Using Embryonic Stem Cells

Annals of Neurology. Jul, 2006  |  Pubmed ID: 16802299

We explored the potential of embryonic stem cell-derived motor neurons to functionally replace those cells destroyed in paralyzed adult rats.

The Glutamate-aspartate Transporter GLAST Mediates Glutamate Uptake at Inner Hair Cell Afferent Synapses in the Mammalian Cochlea

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2006  |  Pubmed ID: 16855093

Ribbon synapses formed between inner hair cells (IHCs) and afferent dendrites in the mammalian cochlea can sustain high rates of release, placing strong demands on glutamate clearance mechanisms. To investigate the role of transporters in glutamate removal at these synapses, we made whole-cell recordings from IHCs, afferent dendrites, and glial cells adjacent to IHCs [inner phalangeal cells (IPCs)] in whole-mount preparations of rat organ of Corti. Focal application of the transporter substrate D-aspartate elicited inward currents in IPCs, which were larger in the presence of anions that permeate the transporter-associated anion channel and blocked by the transporter antagonist D,L-threo-beta-benzyloxyaspartate. These currents were produced by glutamate-aspartate transporters (GLAST) (excitatory amino acid transporter 1) because they were weakly inhibited by dihydrokainate, an antagonist of glutamate transporter-1 (excitatory amino acid transporter 2) and were absent from IPCs in GLAST-/- cochleas. Furthermore, D-aspartate-induced currents in outside-out patches from IPCs exhibited larger steady-state currents than responses elicited by L-glutamate, a prominent feature of GLAST, and examination of cochlea from GLAST-Discosoma red (DsRed) promoter reporter mice revealed that DsRed expression was restricted to IPCs and other supporting cells surrounding IHCs. Saturation of transporters by photolysis of caged D-aspartate failed to elicit transporter currents in IHCs, as did local application of D-aspartate to afferent terminals, indicating that neither presynaptic nor postsynaptic membranes are major sites for glutamate removal. These data indicate that GLAST in supporting cells is responsible for transmitter uptake at IHC afferent synapses.

Glucocorticoid Regulation of GLT-1 Glutamate Transporter Isoform Expression in the Rat Hippocampus

Neuroendocrinology. 2006  |  Pubmed ID: 17028421

In the rat hippocampus, the predominate glutamate transporters are GLT-1 and its recently identified isoform, GLT-1b. Chronic restraint stress increases GLT-1b expression throughout the hippocampus while more selectively increasing GLT-1 expression in the CA3 region. These studies suggest that GLT-1b expression is regulated by stress levels of glucocorticoids (GCs) and GLT-1 expression is regulated by stress-induced increases in extracellular glutamate levels in the CA3 region.

Amyotrophic Lateral Sclerosis 2-deficiency Leads to Neuronal Degeneration in Amyotrophic Lateral Sclerosis Through Altered AMPA Receptor Trafficking

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2006  |  Pubmed ID: 17093100

Amyotrophic lateral sclerosis (ALS), the most common adult-onset motor neuron disease is caused by a selective loss of motor neurons. One form of juvenile onset autosomal recessive ALS (ALS2) has been linked to the loss of function of the ALS2 gene. The pathogenic mechanism of ALS2-deficiency, however, remains unclear. To further understand the function of alsin that is encoded by the full-length ALS2 gene, we screened proteins interacting with alsin. Here, we report that alsin interacted with glutamate receptor interacting protein 1 (GRIP1) both in vitro and in vivo, and colocalized with GRIP1 in neurons. In support of the physiological interaction between alsin and GRIP1, the subcellular distribution of GRIP1 was altered in ALS2(-/-) spinal motor neurons, which correlates with a significant reduction of AMPA-type glutamate receptor subunit 2 (GluR2) at the synaptic/cell surface of ALS2(-/-) neurons. The decrease of calcium-impermeable GluR2-containing AMPA receptors at the cell/synaptic surface rendered ALS2(-/-) neurons more susceptible to glutamate receptor-mediated neurotoxicity. Our findings reveal a novel function of alsin in AMPA receptor trafficking and provide a novel pathogenic link between ALS2-deficiency and motor neuron degeneration, suggesting a protective role of alsin in maintaining the survival of motor neurons.

Mechanisms of Disease: Astrocytes in Neurodegenerative Disease

Nature Clinical Practice. Neurology. Dec, 2006  |  Pubmed ID: 17117171

The term neurodegenerative disease refers to the principal pathology associated with disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease and Parkinson's disease, and it is presumed that neurodegeneration results in the clinical findings seen in patients with these diseases. Decades of pathological and physiological studies have focused on neuronal abnormalities in these disorders, but it is becoming increasingly evident that astrocytes are also important players in these and other neurological disorders. Our understanding of the normative biology of astrocytes has been aided by the development of animal models in which astrocyte-specific proteins and pathways have been manipulated, and mouse models of neurodegenerative diseases have also revealed astrocyte-specific pathologies that contribute to neurodegeneration. These models have led to the development of targeted therapies for pathways in which astrocytes participate, and this research should ultimately influence the clinical treatment of neurodegenerative disorders.

Analysis of IFT74 As a Candidate Gene for Chromosome 9p-linked ALS-FTD

BMC Neurology. 2006  |  Pubmed ID: 17166276

A new locus for amyotrophic lateral sclerosis--frontotemporal dementia (ALS-FTD) has recently been ascribed to chromosome 9p.

Degeneration of Respiratory Motor Neurons in the SOD1 G93A Transgenic Rat Model of ALS

Neurobiology of Disease. Jan, 2006  |  Pubmed ID: 16084734

The transgenic mutant superoxide dismutase (SOD1) mice and rats have been important tools in attempting to understand motor neuron pathology and degeneration but the mechanism behind death in this model has not been studied. We studied the electrophysiologic and pathologic properties of the cervical motor neurons and phrenic nerves in mutant SOD1 rats and demonstrated motor neuron loss, progressive reduction of phrenic nerve compound muscle action potential amplitudes, phrenic nerve fiber loss, and diaphragm atrophy suggesting respiratory insufficiency as a significant contributing factor leading to SOD1 rat death. Unlike previous observations suggesting that a dying-back process may be occurring in the mouse model of the disease, we did not observe differences between proximal and distal axon loss in phrenic nerves of SOD1 rats. This may reflect a unique feature of respiratory motor neuron biology or may be related to the relatively rapid course of decline in the rat model when compared with the mouse SOD1 model. Significant motor neuron loss was also noted in the lumbosacral spinal cord with relative sparing of motor neurons in the cranial nuclei. Taken together, these data suggest that respiratory motor neuron loss results in significant electrophysiologic changes and diaphragmatic atrophy. These changes may play a significant role resulting in death of these animals.

Selective Up-regulation of the Glial Na+-dependent Glutamate Transporter GLT1 by a Neuroimmunophilin Ligand Results in Neuroprotection

Neurobiology of Disease. Mar, 2006  |  Pubmed ID: 16274998

Excessive accumulation of extracellular glutamate results in neuronal death. Termination of synaptic glutamate transmission and the prevention of excitotoxicity depend on rapid removal of glutamate by high affinity Na+-dependent transporters. The astroglial transporter GLT1 is the predominant subtype, responsible for the bulk of extracellular clearance and for limiting excitotoxicity. This protein is crucial in the prevention of chronic glutamate neurotoxicity, and is markedly decreased in amyotrophic lateral sclerosis (ALS). Recent studies have shown that GLT1 expression can be induced in vitro and in vivo by various factors, but little is known about the signaling pathways mediating its regulation. The FK506-binding protein (FKBP) immunophilins are ubiquitous cytosolic proteins, concentrated in neural tissue (neuroimmunophilins). GPI-1046 is a synthetic, nonimmunosuppressive derivative of FK506 shown to exert neuroprotective and neuroregenerative actions in several systems. In the present study, we demonstrated that GPI-1046 induces selective expression of GLT1 in vitro and in vivo, associated with a marked increase in DHK-sensitive Na+-dependent glutamate transport. Furthermore, treatment with GPI-1046 was shown to protect motor neurons in an in vitro model of chronic excitotoxicity, and to prolong the survival of transgenic ALS mice. These studies suggest that neuroimmunophilins can regulate GLT1 and that their ligands could serve as therapies for neurodegenerative disorders.

Targeting an Old Mechanism in a New Disease-protection of Glutamatergic Dysfunction in Depression

Biological Psychiatry. Jan, 2007  |  Pubmed ID: 17223439

Therapeutic Immunization with a Glatiramer Acetate Derivative Does Not Alter Survival in G93A and G37R SOD1 Mouse Models of Familial ALS

Neurobiology of Disease. Apr, 2007  |  Pubmed ID: 17276077

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. The cause of motor neuron degeneration remains largely unknown, and there is no potent treatment. Overexpression of various human mutant superoxide dismutase-1 (SOD1) genes in mice and rats recapitulates some of the clinical and pathological characteristics of sporadic and familial ALS. Glatiramer acetate (GA) is an approved drug for the treatment of multiple sclerosis and neuroprotective properties in some neurodegenerative conditions. A recent report suggested that GA immunization could delay disease progression in some, but not all, G93A SOD1 transgenic mouse models of amyotrophic lateral sclerosis (ALS). Moreover, it has been theorized that derivatives of GA could enhance immunogenicity and positively affect disease outcomes. The purpose of our study was to assess the neuroprotective efficacy of TV-5010, a high molecular weight GA, in three different SOD1 mutant mouse models. We used large numbers of two SOD1 transgenic mouse strains overexpressing the G93A mutation, B6SJL-TgN[SOD1-G93A]1Gur and B6.Cg-Tg(SOD1-G93A)1Gur/J, and the SOD1 mutant mouse overexpressing G37R (line 29). Regardless of the frequency of injections and the dose, treatment with TV-5010 was ineffective at altering either disease onset or survival in both SOD1 G93A mutants used and in the SOD1 G37R transgenic mice; in multiple studies, disease was accelerated. These studies suggest that, at a range of dosing regimens and carrier used, TV-5010 immunization was ineffective in delaying disease in multiple preclinical therapeutic models for ALS. The biological response in animals, and ultimate clinical translation, will ultimately be dependent on careful and appropriate dose, route and carrier paradigms.

TDP-43 in Amyotrophic Lateral Sclerosis: Pathophysiology or Patho-babel?

Annals of Neurology. May, 2007  |  Pubmed ID: 17469124

Variations in Promoter Activity Reveal a Differential Expression and Physiology of Glutamate Transporters by Glia in the Developing and Mature CNS

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2007  |  Pubmed ID: 17581948

Glutamate transporters regulate excitatory neurotransmission and prevent glutamate-mediated excitotoxicity in the CNS. To better study the cellular and temporal dynamics of the expression of these transporters, we generated bacterial artificial chromosome promoter Discosoma red [glutamate-aspartate transporter (GLAST)] and green fluorescent protein [glutamate transporter-1 (GLT-1)] reporter transgenic mice. Analysis of these mice revealed a differential activation of the transporter promoters not previously appreciated. GLT-1 promoter activity in the adult CNS is almost completely restricted to astrocytes, often and unexpectedly in a nonoverlapping pattern with GLAST. Spinal cord GLT-1 promoter reporter, protein density, and physiology were 10-fold lower than in brain, suggesting a possible mechanism for regional sensitivity seen in disease. The GLAST promoter is active in both radial glia and many astrocytes in the developing CNS but is downregulated in most astrocytes as the mice mature. In the adult CNS, the highest GLAST promoter activity was observed in radial glia, such as those located in the subgranular layer of the dentate gyrus. The continued expression of GLAST by these neural progenitors raises the possibility that GLAST may have an unanticipated role in regulating their behavior. In addition, GLAST promoter activation was observed in oligodendrocytes in white matter throughout many (e.g., spinal cord and corpus callosum), but not all (e.g., cerebellum), CNS fiber tracts. Overall, these studies of GLT-1 and GLAST promoter activity, protein expression, and glutamate uptake revealed a close correlation between transgenic reporter signals and uptake capacity, indicating that these mice provide the means to monitor the expression and regulation of glutamate transporters in situ.

Intraparenchymal Spinal Cord Delivery of Adeno-associated Virus IGF-1 is Protective in the SOD1G93A Model of ALS

Brain Research. Dec, 2007  |  Pubmed ID: 17963733

The potent neuroprotective activities of neurotrophic factors, including insulin-like growth factor 1 (IGF-1), make them promising candidates for treatment of amyotrophic lateral sclerosis (ALS). In an effort to maximize rate of motor neuron transduction, achieve high levels of spinal IGF-1 and thus enhance therapeutic benefit, we injected an adeno-associated virus 2 (AAV2)-based vector encoding human IGF-1 (CERE-130) into lumbar spinal cord parenchyma of SOD1(G93A) mice. We observed robust and long-term intraspinal IGF-1 expression and partial rescue of lumbar spinal cord motor neurons, as well as sex-specific delayed disease onset, weight loss, decline in hindlimb grip strength and increased animal survival.

Analysis of Cerebellar Purkinje Cells Using EAAT4 Glutamate Transporter Promoter Reporter in Mice Generated Via Bacterial Artificial Chromosome-mediated Transgenesis

Experimental Neurology. Jan, 2007  |  Pubmed ID: 17022974

The EAAT4 glutamate transporter helps regulate excitatory neurotransmission and prevents glutamate-mediated excitotoxicity in the cerebellum. Immunohistochemistry and in situ hybridization have previously defined a cerebellar cell population expressing this protein. These methods, however, are not well suited for evaluating the dynamic regulation of the transporter and its gene-especially in living tissues. To better study EAAT4 expression and regulation, we generated bacterial artificial chromosome (BAC) promoter eGFP reporter transgenic mice. Histological analysis of the transgenic mice revealed that the EAAT4 promoter is active predominantly in Purkinje cells, but can also be modestly detected in other neurons early postnatally. EAAT4 promoter activity was not present in non-neuronal cells. Cerebellar organotypic slice cultures prepared from BAC transgenic mice provided a unique reagent to study transporter and Purkinje cell expression and regulation in living tissue. The correlation of promoter activity to protein expression makes the EAAT4 BAC promoter reporter a valuable tool to study regulation of EAAT4 expression.

Reticulon RTN2B Regulates Trafficking and Function of Neuronal Glutamate Transporter EAAC1

The Journal of Biological Chemistry. Mar, 2008  |  Pubmed ID: 18096700

Excitatory amino acid transporters (EAATs) are the primary regulators of extracellular glutamate concentrations in the central nervous system. Their dysfunction may contribute to several neurological diseases. To date, five distinct mammalian glutamate transporters have been cloned. In brain, EAAC1 (excitatory amino acid carrier 1) is the primary neuronal glutamate transporter, localized on the perisynaptic membranes that are near release sites. Despite its potential importance in synaptic actions, little is known concerning the regulation of EAAC1 trafficking from the endoplasmic reticulum (ER) to the cell surface. Previously, we identified an EAAC1-associated protein, GTRAP3-18, an ER protein that prevents ER exit of EAAC1 when induced. Here we show that RTN2B, a member of the reticulon protein family that mainly localizes in the ER and ER exit sites interacts with EAAC1 and GTRAP3-18. EAAC1 and GTRAP3-18 bind to different regions of RTN2B. Each protein can separately and independently form complexes with EAAC1. RTN2B enhances ER exit and the cell surface composition of EAAC1 in heterologous cells. Expression of short interfering RNA-mediated knockdown of RTN2B decreases the EAAC1 protein level in neurons. Overall, our results suggest that RTN2B functions as a positive regulator in the delivery of EAAC1 from the ER to the cell surface. These studies indicate that transporter exit from the ER controlled by the interaction with its ER binding partner represents a critical regulatory step in glutamate transporter trafficking to the cell surface.

The Endoplasmic Reticulum Exit of Glutamate Transporter is Regulated by the Inducible Mammalian Yip6b/GTRAP3-18 Protein

The Journal of Biological Chemistry. Mar, 2008  |  Pubmed ID: 18167356

GTRAP3-18 interacts with and reduces the activity of the neuronal specific Na(+)/K(+) glutamate transporter, EAAC1 both in vitro and in vivo. GTRAP3-18 and the related isoform, JM4, are distant relatives of the Rab GTPase-interacting factor PRA1, and share a topology of four transmembrane domains and cytosolic termini. GTRAP3-18 and JM4 are resident endoplasmic reticulum (ER) proteins. The physiological role of GTRAP3-18 is poorly understood. We demonstrate for the first time that GTRAP3-18 is a regulator of ER protein trafficking. Expression of GTRAP3-18 delays the ER exit of EAAC1, as well as other members of the excitatory amino acid transporter family. GTRAP3-18 uses hydrophobic domain interactions in the ER membrane to self-associate and cytoplasmic interactions at the C terminus to regulate trafficking. The features of GTRAP3-18 activity are consistent with recent phylogenic sequence analyses suggesting GTRAP3-18 and JM4 be reclassified as mammalian isoforms of the yeast protein family Yip, Yip6b, and Yip6a, respectively.

Motor Neuron Disease Occurring in a Mutant Dynactin Mouse Model is Characterized by Defects in Vesicular Trafficking

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Feb, 2008  |  Pubmed ID: 18305234

Amyotrophic lateral sclerosis (ALS), a fatal and progressive neurodegenerative disorder characterized by weakness, muscle atrophy, and spasticity, is the most common adult-onset motor neuron disease. Although the majority of ALS cases are sporadic, approximately 5-10% are familial, including those linked to mutations in SOD1 (Cu/Zn superoxide dismutase). Missense mutations in a dynactin gene (DCTN1) encoding the p150(Glued) subunit of dynactin have been linked to both familial and sporadic ALS. To determine the molecular mechanism whereby mutant dynactin p150(Glued) causes selective degeneration of motor neurons, we generated and characterized mice expressing either wild-type or mutant human dynactin p150(Glued). Neuronal expression of mutant, but not wild type, dynactin p150(Glued) causes motor neuron disease in these animals that are characterized by defects in vesicular transport in cell bodies of motor neurons, axonal swelling and axo-terminal degeneration. Importantly, we provide evidence that autophagic cell death is implicated in the pathogenesis of mutant p150(Glued) mice. This novel mouse model will be instrumental for not only clarifying disease mechanisms in ALS, but also for testing therapeutic strategies to ameliorate this devastating disease.

Messenger RNA Oxidation Occurs Early in Disease Pathogenesis and Promotes Motor Neuron Degeneration in ALS

PloS One. 2008  |  Pubmed ID: 18682740

Accumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration.

Focal Transplantation-based Astrocyte Replacement is Neuroprotective in a Model of Motor Neuron Disease

Nature Neuroscience. Nov, 2008  |  Pubmed ID: 18931666

Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction also occurs in human ALS and transgenic rodents expressing mutant human SOD1 protein (SOD1(G93A)). Here we investigated focal enrichment of normal astrocytes using transplantation of lineage-restricted astrocyte precursors, called glial-restricted precursors (GRPs). We transplanted GRPs around cervical spinal cord respiratory motor neuron pools, the principal cells whose dysfunction precipitates death in ALS. GRPs survived in diseased tissue, differentiated efficiently into astrocytes and reduced microgliosis in the cervical spinal cords of SOD1(G93A) rats. GRPs also extended survival and disease duration, attenuated motor neuron loss and slowed declines in forelimb motor and respiratory physiological functions. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter GLT1. These findings indicate the feasibility and efficacy of transplantation-based astrocyte replacement and show that targeted multisegmental cell delivery to the cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.

Current Hypotheses for the Underlying Biology of Amyotrophic Lateral Sclerosis

Annals of Neurology. Jan, 2009  |  Pubmed ID: 19191304

The mechanisms involved in selective motor neuron degeneration in amyotrophic lateral sclerosis remain unknown more than 135 years after the disease was first described. Although most cases have no known cause, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) have been implicated in a fraction of familial cases of the disease. Transgenic mouse models with mutations in the SOD1 gene and other ALS genes develop pathology reminiscent of the disorder, including progressive death of motor neurons, and have provided insight into the pathogenesis of the disease but have consistently failed to predict therapeutic efficacy in humans. However, emerging research has demonstrated that mutations and pathology associated with the TDP-43 gene and protein may be more common than SOD1 mutations in familial and sporadic ALS. Putative mechanisms of toxicity targeting motor neurons include oxidative damage, accumulation of intracellular aggregates, mitochondrial dysfunction, defects in axonal transport, growth factor deficiency, aberrant RNA metabolism, glial cell pathology, and glutamate excitotoxicity. Convergence of these pathways is likely to mediate disease onset and progression.

Presynaptic Regulation of Astroglial Excitatory Neurotransmitter Transporter GLT1

Neuron. Mar, 2009  |  Pubmed ID: 19323997

The neuron-astrocyte synaptic complex is a fundamental operational unit of the nervous system. Astroglia regulate synaptic glutamate, via neurotransmitter transport by GLT1/EAAT2. Astroglial mechanisms underlying this essential neuron-glial communication are not known. We now show that presynaptic terminals regulate astroglial synaptic functions, GLT1/EAAT2, via kappa B-motif binding phosphoprotein (KBBP), the mouse homolog of human heterogeneous nuclear ribonucleoprotein K (hnRNP K), which binds the GLT1/EAAT2 promoter. Neuron-stimulated KBBP is required for GLT1/EAAT2 transcriptional activation and is responsible for astroglial alterations in neural injury. Denervation of neuron-astrocyte signaling by corticospinal tract transection, ricin-induced motor neuron death, or neurodegeneration in amyotrophic lateral sclerosis all result in reduced astroglial KBBP expression and transcriptional dysfunction of astroglial transporter expression. Presynaptic elements dynamically coordinate normal astroglial function and also provide a fundamental signaling mechanism by which altered neuronal function and injury leads to dysregulated astroglia in CNS disease.

Tumor Necrosis Factor-alpha Modulates Glutamate Transport in the CNS and is a Critical Determinant of Outcome from Viral Encephalomyelitis

Brain Research. Mar, 2009  |  Pubmed ID: 19368827

Neuroadapted Sindbis virus (NSV) is a neuronotropic virus that causes a fulminant encephalomyelitis in susceptible mice due to death of motor neurons in the brain and spinal cord. We and others have found that uninfected motor neurons die in response to NSV infection, at least in part due to disrupted astrocytic glutamate transport, resulting in excitotoxic motor neuron death. Here, we examined the mechanisms of astrocyte dysregulation associated with NSV infection. Treatment of organotypic slice cultures with NSV results in viral replication, cell death, altered astrocyte morphology, and the downregulation of the astrocytic glutamate transporter, GLT-1. We have found that TNF-alpha can mediate GLT-1 downregulation. Furthermore, TNF-alpha deficient mice infected with NSV exhibit neither GLT-1 downregulation nor neuronal death of brainstem and cervical spinal cord motor neurons and have markedly reduced mortality. These findings have implications for disease intervention and therapeutic development for the prevention of CNS damage associated with inflammatory responses.

Regulation of the Na(+)-coupled Glutamate Transporter EAAT3 by PIKfyve

Neurochemistry International. May-Jun, 2009  |  Pubmed ID: 19418632

The Na(+), glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end,EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I(glu)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I(glu). Coexpression of either, SGK1 orPIKfyve, significantly enhanced I(glu) in EAAT3 expressing oocytes. The increased I(glu) was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I(glu) and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve coexpression on I(glu). The stimulating effect of PIKfyve on I(glu) was abolished by replacement of the serine by alanine in the SGK consensus sequence ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.

Epigenetic Regulation of Neuron-dependent Induction of Astroglial Synaptic Protein GLT1

Glia. Feb, 2010  |  Pubmed ID: 19672971

Astroglial glutamate transporter EAAT2/GLT1 prevents glutamate-induced excitotoxicity in the central nervous system. Expression of EAAT2/GLT1 is dynamically regulated by neurons. The pathogenesis of amyotrophic lateral sclerosis (ALS) involves astroglial dysfunction, including dramatic loss of EAAT2/GLT1. DNA methylation of gene promoters represents one of the most important epigenetic mechanisms in regulating gene expression. The involvement of DNA methylation in the regulation of astroglial EAAT2/GLT1 expression in different conditions, especially in ALS has not been explored. In this study, we established a procedure to selectively isolate a pure astrocyte population in vitro and in vivo from BAC GLT1 eGFP mice using an eGFP-based fluorescence-activated cell sorting approach. Astrocytes isolated from this procedure are GFAP+ and GLT1+ and respond to neuronal stimulation, enabling direct methylation analysis of GLT1 promoter in these astrocytes. To investigate the role of DNA methylation in physiological and pathological EAAT2/GLT1 expression, methylation status of the EAAT2/GLT1 promoter was analyzed in astrocytes from in vitro and in vivo paradigms or postmortem ALS motor cortex by bisulfite sequencing method. DNA demethylation on selective CpG sites of the GLT1 promoter was highly correlated to increased GLT1 mRNA levels in astrocytes in response to neuronal stimulation; however, low level of methylation was found on CpG sites of EAAT2 promoter from postmortem motor cortex of human amyotrophic lateral sclerosis patients. In summary, hypermethylation on selective CpG sites of the GLT1 promoter is involved in repression of GLT1 promoter activation, but this regulation does not play a role in astroglial dysfunction of EAAT2 expression in patients with ALS.

A Phase II Trial of Talampanel in Subjects with Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis : Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases. May, 2010  |  Pubmed ID: 19961264

Our objective was to determine if chronic treatment with the non-competitive AMPA antagonist talampanel is efficacious and safe in subjects with ALS. A double-blind, placebo-controlled, multicenter, randomized clinical trial of nine months treatment duration was conducted in 59 subjects with ALS, with 40 subjects receiving talampanel 50 mg p.o. t.i.d, and 19 subjects receiving placebo. Primary outcome measure was rate of decline in isometric arm strength (as measured by change in arm strength megaslope of the Tufts Quantitative Neuromuscular Exam (TQNE)). Other efficacy endpoints included rate of decline in respiratory function, isometric leg strength, bulbar function, fine motor function, the ALS Functional Rating Scale (ALSFRS), and survival. Secondary safety outcome measures were frequency of adverse events, neurological status, plasma concentration of talampanel, vital signs, routine laboratory tests, and electrocardiograms. Decline in muscle strength was 15% less in talampanel treated subjects, and decline in ALSFRS was 30% slower in talampanel treated subjects. Talampanel was safe in subjects with ALS. Mortality rates (8% talampanel, 5% placebo) and drug discontinuation rates (25% talampanel, 16% placebo) were similar in active treatment and placebo groups. Dizziness and somnolence occurred significantly more often in talampanel treated subjects. Although no efficacy measure reached statistical significance, there was a repeated trend toward slower decline in ALSFRS and isometric muscle strength in talampanel treated subjects. Talampanel was well tolerated in subjects with ALS. Although certain adverse events occurred more frequently in the active treatment group, the rate of subject drop-out after nine months did not exceed that seen in other trials. These findings provide strong support for a phase III trial to determine the efficacy of talampanel in subjects with ALS.

GLT-1 Promoter Activity in Astrocytes and Neurons of Mouse Hippocampus and Somatic Sensory Cortex

Frontiers in Neuroanatomy. 2010  |  Pubmed ID: 20161698

GLT-1 eGFP BAC reporter transgenic adult mice were used to detect GLT-1 gene expression in individual cells of CA1, CA3 and SI, and eGFP fluorescence was measured to analyze quantitatively GLT-1 promoter activity in different cells of neocortex and hippocampus. Virtually all GFAP+ astrocytes were eGFP+; we also found that about 80% of neurons in CA3 pyramidal layer, 10-70% of neurons in I-VI layers of SI and rare neurons in all strata of CA1 and in strata oriens and radiatum of CA3 were eGFP+. Analysis of eGFP intensity showed that astrocytes had a higher GLT-1 promoter activity in SI than in CA1 and CA3, and that neurons had the highest levels of GLT-1 promoter activity in CA3 stratum pyramidale and in layer VI of SI. Finally, we observed that the intensity of GLT-1 promoter activity in neurons is 1-20% of that measured in astrocytes. These results showed that in the hippocampus and neocortex GLT-1 promoter activity is observed in astrocytes and neurons, detailed the distribution of GLT-1 expressing neurons, and indicated that GLT-1 promoter activity in both astrocytes and neurons varies in different brain regions.

Quantitative Analysis of EAAT4 Promoter Activity in Neurons and Astrocytes of Mouse Somatic Sensory Cortex

Neuroscience Letters. Apr, 2010  |  Pubmed ID: 20211693

EAAT4-eGFP BAC reporter transgenic adult mice were used to detect EAAT4 gene expression in individual cells of cerebral cortex, and eGFP fluorescence was measured to compare EAAT4 promoter activity in different cells. Most eGFP+ cells were neurons; only rare GFAP+ profiles were eGFP+. About 10% of NeuN+ cells was eGFP+, and the percentage of NeuN/eGFP co-localization varied from 2 to 20% of NeuN+ cells throughout cortical layers: layers I and II-III showed the highest values of co-localization, layer IV the lowest. The intensity of eGFP fluorescence did not exhibit laminar variations. Finally, we observed that EAAT4 promoter activity in cortical neurons was 10% of that measured in cerebellar Purkinje cells, i.e., the cells displaying the highest intensity in the CNS. These results extend our knowledge on EAAT4 expression in the cerebral cortex of adult mice, and suggest that the role of EAAT4 in cortical glutamatergic transmission may be more important than previously thought.

Loss of Beta-III Spectrin Leads to Purkinje Cell Dysfunction Recapitulating the Behavior and Neuropathology of Spinocerebellar Ataxia Type 5 in Humans

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2010  |  Pubmed ID: 20371805

Mutations in SPTBN2, the gene encoding beta-III spectrin, cause spinocerebellar ataxia type 5 in humans (SCA5), a neurodegenerative disorder resulting in loss of motor coordination. How these mutations give rise to progressive ataxia and what the precise role beta-III spectrin plays in normal cerebellar physiology are unknown. We developed a mouse lacking full-length beta-III spectrin and found that homozygous mice reproduced features of SCA5 including gait abnormalities, tremor, deteriorating motor coordination, Purkinje cell loss, and cerebellar atrophy (molecular layer thinning). In vivo analysis reveals an age-related reduction in simple spike firing rate in surviving beta-III(-/-) Purkinje cells, whereas in vitro studies show these neurons to have reduced spontaneous firing, smaller sodium currents, and dysregulation of glutamatergic neurotransmission. Our data suggest an early loss of EAAT4- (protein interactor of beta-III spectrin) and a subsequent loss of GLAST-mediated uptake may play a role in neuronal pathology. These findings implicate a loss of beta-III spectrin function in SCA5 pathogenesis and indicate that there are at least two physiological effects of beta-III spectrin loss that underpin a progressive loss of inhibitory cerebellar output, namely an intrinsic Purkinje cell membrane defect due to reduced sodium currents and alterations in glutamate signaling.

Zones of Enhanced Glutamate Release from Climbing Fibers in the Mammalian Cerebellum

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. May, 2010  |  Pubmed ID: 20505095

Purkinje cells in the mammalian cerebellum are remarkably homogeneous in shape and orientation, yet they exhibit regional differences in gene expression. Purkinje cells that express high levels of zebrin II (aldolase C) and the glutamate transporter EAAT4 cluster in parasagittal zones that receive input from distinct groups of climbing fibers (CFs); however, the physiological properties of CFs that target these molecularly distinct Purkinje cells have not been determined. Here we report that CFs that innervate Purkinje cells in zebrin II-immunoreactive (Z(+)) zones release more glutamate per action potential than CFs in Z(-) zones. CF terminals in Z(+) zones had larger pools of release-ready vesicles, exhibited enhanced multivesicular release, and produced larger synaptic glutamate transients. As a result, CF-mediated EPSCs in Purkinje cells decayed more slowly in Z(+) zones, which triggered longer-duration complex spikes containing a greater number of spikelets. The differences in the duration of CF EPSCs between Z(+) and Z(-) zones persisted in EAAT4 knock-out mice, indicating that EAAT4 is not required for maintaining this aspect of CF function. These results indicate that the organization of the cerebellum into discrete longitudinal zones is defined not only by molecular phenotype of Purkinje cells within zones, but also by the physiological properties of CFs that project to these distinct regions. The enhanced release of glutamate from CFs in Z(+) zones may alter the threshold for synaptic plasticity and prolong inhibition of cerebellar output neurons in deep cerebellar nuclei.

NG2+ CNS Glial Progenitors Remain Committed to the Oligodendrocyte Lineage in Postnatal Life and Following Neurodegeneration

Neuron. Nov, 2010  |  Pubmed ID: 21092857

The mammalian CNS contains a ubiquitous population of glial progenitors known as NG2+ cells that have the ability to develop into oligodendrocytes and undergo dramatic changes in response to injury and demyelination. Although it has been reported that NG2+ cells are multipotent, their fate in health and disease remains controversial. Here, we generated PDGFαR-CreER transgenic mice and followed their fate in vivo in the developing and adult CNS. These studies revealed that NG2+ cells in the postnatal CNS generate myelinating oligodendrocytes, but not astrocytes or neurons. In regions of neurodegeneration in the spinal cord of ALS mice, NG2+ cells exhibited enhanced proliferation and accelerated differentiation into oligodendrocytes but remained committed to the oligodendrocyte lineage. These results indicate that NG2+ cells in the normal CNS are oligodendrocyte precursors with restricted lineage potential and that cell loss and gliosis are not sufficient to alter the lineage potential of these progenitors.

New Treatments in Amyotrophic Lateral Sclerosis

Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology. Jan, 2011  |  Pubmed ID: 21116264

Spatial and Temporal Changes in Promoter Activity of the Astrocyte Glutamate Transporter GLT1 Following Traumatic Spinal Cord Injury

Journal of Neuroscience Research. Jul, 2011  |  Pubmed ID: 21488085

After traumatic spinal cord injury (SCI), there is an opportunity for preserving function by attenuating secondary cell loss. Astrocytes play crucial roles in the adult CNS and are responsible for the vast majority of glutamate buffering, potentially preventing excitotoxic loss of neurons and oligodendrocytes. We examined spatial and temporal changes in gene expression of the major astrocyte glutamate transporter GLT1 following moderate thoracic contusion SCI using transgenic BAC-GLT1-eGFP promoter reporter mice. In dorsal column white matter, total intensity of GLT1-eGFP expression per region was significantly reduced following SCI at both lesion epicenter and at rostral and caudal areas where no tissue loss occurred. This regional decrease in GLT1 expression was due to significant loss of GLT1-eGFP(+) cells, partially accounted for by apoptosis of eGFP(+) /GFAP(+) astrocytes in both white and gray matter. There were also decreased numbers of GLT1-eGFP-expressing cells in multiple gray matter regions following injury; nevertheless, there was sustained or even increased regional GLT1-eGFP expression in gray matter as a result of up-regulation in astrocytes that continued to express GLT1-eGFP. Although there were increased numbers of GFAP(+) cells both at the lesion site and in surrounding intact spinal cord following SCI, the majority of proliferating Ki67(+) /GFAP(+) astrocytes did not express GLT1-eGFP. These findings demonstrate that spatial and temporal alterations in GLT1 expression observed after SCI result from both astrocyte death and gene expression changes in surviving astrocytes. Results also suggest that following SCI a significant portion of astrocytes lacks GLT1 expression, possibly compromising the important role of astrocytes in glutamate homeostasis.

John W. "Jack" Griffin, MD

Annals of Neurology. Jun, 2011  |  Pubmed ID: 21681788

Nuclear Factor-κB Contributes to Neuron-dependent Induction of Glutamate Transporter-1 Expression in Astrocytes

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2011  |  Pubmed ID: 21697367

The glutamate transporter-1 [GLT-1 (excitatory amino acid transporter 2)] subtype of glutamate transporter ensures crisp excitatory signaling and limits excitotoxicity in the CNS. Astrocytic expression of GLT-1 is regulated during development, by neuronal activity, and in neurodegenerative diseases. Although neurons activate astrocytic expression of GLT-1, the mechanisms involved have not been identified. In the present study, astrocytes from transgenic mice that express enhanced green fluorescent protein (eGFP) under the control of a bacterial artificial chromosome (BAC) containing a very large region of DNA surrounding the GLT-1 gene (BAC GLT-1 eGFP mice) were used to assess the role of nuclear factor-κB (NF-κB) in neuron-dependent activation of the GLT-1 promoter. We provide evidence that neurons activate NF-κB signaling in astrocytes. Transduction of astrocytes from the BAC GLT-1 eGFP mice with dominant-negative inhibitors of NF-κB signaling completely blocked neuron-dependent activation of a NF-κB reporter construct and attenuated induction of eGFP. Exogenous expression of p65 and/or p50 NF-κB subunits induced expression of eGFP or GLT-1 and increased GLT-1-mediated transport activity. Using wild-type and mutant GLT-1 promoter reporter constructs, we found that NF-κB sites at -583 or -251 relative to the transcription start site were required for neuron-dependent reporter activation. Electrophoretic mobility shift and supershift assays reveal that p65 and p50 interact with these same sites ex vivo. Finally, chromatin immunoprecipitation showed that p65 and p50 interact with these sites in adult cortex, but not in kidney (a tissue that expresses no detectable GLT-1). Together, these studies strongly suggest that NF-κB contributes to neuron-dependent regulation of astrocytic GLT-1 transcription.

A Hexanucleotide Repeat Expansion in C9ORF72 is the Cause of Chromosome 9p21-linked ALS-FTD

Neuron. Oct, 2011  |  Pubmed ID: 21944779

The chromosome 9p21 amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD) locus contains one of the last major unidentified autosomal-dominant genes underlying these common neurodegenerative diseases. We have previously shown that a founder haplotype, covering the MOBKL2b, IFNK, and C9ORF72 genes, is present in the majority of cases linked to this region. Here we show that there is a large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72 on the affected haplotype. This repeat expansion segregates perfectly with disease in the Finnish population, underlying 46.0% of familial ALS and 21.1% of sporadic ALS in that population. Taken together with the D90A SOD1 mutation, 87% of familial ALS in Finland is now explained by a simple monogenic cause. The repeat expansion is also present in one-third of familial ALS cases of outbred European descent, making it the most common genetic cause of these fatal neurodegenerative diseases identified to date.

Human Nasal Olfactory Epithelium As a Dynamic Marker for CNS Therapy Development

Experimental Neurology. Dec, 2011  |  Pubmed ID: 21945230

Discovery of new central nervous system (CNS) acting therapeutics has been slowed down by the lack of useful applicable biomarkers of disease or drug action often due to inaccessibility of relevant human CNS tissue and cell types. In recent years, non-neuronal cells, such as astrocytes, have been reported to play a highly significant role in neurodegenerative diseases, CNS trauma, as well as psychiatric disease and have become a target for small molecule and biologic therapies. We report the development of a method for measuring pharmacodynamic changes induced by potential CNS therapeutics using nasal olfactory neural tissue biopsy. We validated this approach using a potential astrocyte-targeted therapeutic, thiamphenicol, in a pre-clinical rodent study as well as a phase 1 human trial. In both settings, analysis of the olfactory epithelial tissue revealed biological activity of thiamphenicol at the drug target, the excitatory amino acid transporter 2 (EAAT2). Therefore, this biomarker approach may provide a reliable evaluation of CNS glial-directed therapies and hopefully improve throughput for nervous system drug discovery.

Plasticity of Astroglial Networks in Olfactory Glomeruli

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2011  |  Pubmed ID: 21997206

Several recent findings have shown that neurons as well as astrocytes are organized into networks. Indeed, astrocytes are interconnected through connexin-formed gap junction channels allowing exchanges of ions and signaling molecules. The aim of this study is to characterize astrocyte network properties in mouse olfactory glomeruli where neuronal connectivity is highly ordered. Dye-coupling experiments performed in olfactory bulb acute slices (P16-P22) highlight a preferential communication between astrocytes within glomeruli and not between astrocytes in adjacent glomeruli. Such organization relies on the oriented morphology of glomerular astrocytes to the glomerulus center and the enriched expression of two astroglial connexins (Cx43 and Cx30) within the glomeruli. Glomerular astrocytes detect neuronal activity showing membrane potential fluctuations correlated with glomerular local field potentials. Accordingly, gap junctional coupling of glomerular networks is reduced when neuronal activity is silenced by TTX treatment or after early sensory deprivation. Such modulation is lost in Cx30 but not in Cx43 KO mice, indicating that Cx30-formed channels are the molecular targets of this activity-dependent modulation. Extracellular potassium is a key player in this neuroglial interaction, because (i) the inhibition of dye coupling observed in the presence of TTX or after sensory deprivation is restored by increasing [K(+)](e) and (ii) treatment with a K(ir) channel blocker inhibits dye spread between glomerular astrocytes. Together, these results demonstrate that extracellular potassium generated by neuronal activity modulates Cx30-mediated gap junctional communication between glomerular astrocytes, indicating that strong neuroglial interactions take place at this first relay of olfactory information processing.

Harmine, a Natural Beta-carboline Alkaloid, Upregulates Astroglial Glutamate Transporter Expression

Neuropharmacology. Jun, 2011  |  Pubmed ID: 21034752

Glutamate is the predominant excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Glutamate transporter EAAT2/GLT-1 is the physiologically dominant astroglial protein that inactivates synaptic glutamate. Previous studies have shown that EAAT2 dysfunction leads to excessive extracellular glutamate and may contribute to various neurological disorders including amyotrophic lateral sclerosis (ALS). The recent discovery of the neuroprotective properties of ceftriaxone, a beta lactam antibiotic, suggested that increasing EAAT2/GLT-1 gene expression might be beneficial in ALS and other neurological/psychiatric disorders by augmenting astrocytic glutamate uptake. Here we report our efforts to develop a new screening assay for identifying compounds that activate EAAT2 gene expression. We generated fetal derived-human immortalized astroglial cells that are stably expressing a firefly luciferase reporter under the control of the human EAAT2 promoter. When screening a library of 1040 FDA approved compounds and natural products, we identified harmine, a naturally occurring beta-carboline alkaloid, as one of the top hits for activating the EAAT2 promoter. We further tested harmine in our in vitro cell culture systems and confirmed its ability to increase EAAT2/GLT1 gene expression and functional glutamate uptake activity. We next tested its efficacy in both wild type animals and in an ALS animal model of disease and demonstrated that harmine effectively increased GLT-1 protein and glutamate transporter activity in vivo. Our studies provide potential novel neurotherapeutics by modulating the activity of glutamate transporters via gene activation. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Molecular Comparison of GLT1+ and ALDH1L1+ Astrocytes in Vivo in Astroglial Reporter Mice

Glia. Feb, 2011  |  Pubmed ID: 21046559

Astrocyte heterogeneity remains largely unknown in the CNS due to lack of specific astroglial markers. In this study, molecular identity of in vivo astrocytes was characterized in BAC ALDH1L1 and BAC GLT1 eGFP promoter reporter transgenic mice. ALDH1L1 promoter is selectively activated in adult cortical and spinal cord astrocytes, indicated by the overlap of eGFP expression with ALDH1L1 and GFAP, but not with NeuN, APC, Olig2, IbaI, PDGFRα immunoreactivity in BAC ALDH1L1 eGFP reporter mice. Interestingly, ALDH1L1 expression levels (protein, mRNA, and promoter activity) in spinal cord were selectively decreased during postnatal maturation. In contrast, its expression was up-regulated in reactive astrocytes in both acute neural injury and chronic neurodegenerative (G93A mutant SOD1) conditions, similar to GFAP, but opposite of GLT1. ALDH1L1(+) and GLT1(+) cells isolated through fluorescence activated cell sorting (FACS) from BAC ALDH1L1 and BAC GLT1 eGFP mice share a highly similar gene expression profile, suggesting ALDH1L1 and GLT1 are co-expressed in the same population of astrocytes. This observation was further supported by overlap of the eGFP driven by the ALDH1L1 genomic promoter and the tdTomato driven by a 8.3kb EAAT2 promoter fragment in astrocytes of BAC ALDH1L1 eGFP X EAAT2-tdTomato mice. These studies support ALDH1L1 as a general CNS astroglial marker and investigated astrocyte heterogeneity in the CNS by comparing the molecular identity of the ALDH1L1(+) and GLT1(+) astrocytes from astroglial reporter mice. These astroglial reporter mice provide useful in vivo tools for the molecular analysis of astrocytes in physiological and pathological conditions.

β-III Spectrin is Critical for Development of Purkinje Cell Dendritic Tree and Spine Morphogenesis

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2011  |  Pubmed ID: 22090485

Mutations in the gene encoding β-III spectrin give rise to spinocerebellar ataxia type 5, a neurodegenerative disease characterized by progressive thinning of the molecular layer, loss of Purkinje cells and increasing motor deficits. A mouse lacking full-length β-III spectrin (β-III⁻/⁻) displays a similar phenotype. In vitro and in vivo analyses of Purkinje cells lacking β-III spectrin, reveal a critical role for β-III spectrin in Purkinje cell morphological development. Disruption of the normally well ordered dendritic arborization occurs in Purkinje cells from β-III⁻/⁻ mice, specifically showing a loss of monoplanar organization, smaller average dendritic diameter and reduced densities of Purkinje cell spines and synapses. Early morphological defects appear to affect distribution of dendritic, but not axonal, proteins. This study confirms that thinning of the molecular layer associated with disease pathogenesis is a consequence of Purkinje cell dendritic degeneration, as Purkinje cells from 8-month-old β-III⁻/⁻ mice have drastically reduced dendritic volumes, surface areas and total dendritic lengths compared with 5- to 6-week-old β-III⁻/⁻ mice. These findings highlight a critical role of β-III spectrin in dendritic biology and are consistent with an early developmental defect in β-III⁻/⁻ mice, with abnormal Purkinje cell dendritic morphology potentially underlying disease pathogenesis.

Neuronal Activity Regulates Glutamate Transporter Dynamics in Developing Astrocytes

Glia. Feb, 2012  |  Pubmed ID: 22052455

Glutamate transporters (GluTs) maintain a low ambient level of glutamate in the central nervous system (CNS) and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here, we examined the subcellular distribution and dynamic remodeling of the predominant GluT GLT-1 (excitatory amino acid transporter 2, EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. Green fluorescent protein (GFP)-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse three-dimensional confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of GluTs in developing astrocyte membranes and their position relative to synapses.