The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms

doi:10.1016/S0197-0186(00)00019-X

Neurochemistry International

Volume 37, Issues 2–3, 1 August 2000, Pages 163-170

https://doi.org/10.1016/S0197-0186(00)00019-X Get rights and content

Abstract

High-affinity glutamate transporters ensure termination of glutamatergic neurotransmission and keep the synaptic concentration of this amino acid below excitotoxic levels. However, neuronal glutamate transporters, EAAC1 and EAAT4, are located outside the synaptic cleft and contribute less significantly to the glutamate uptake in the brain than two astroglial transporters, GLAST and GLT1. Aberrant functioning of the glutamate uptake system seems to be linked to some neurodegenerative disorders (eg amyotrophic lateral sclerosis, ALS). Expression of glutamate transporters is differentially regulated via distinct cellular mechanisms. GLT1, which is expressed at very low levels in cultured astrocytes, is strongly induced in the presence of neurons. The present immunocytochemical data provide further evidence that neuronal soluble factors, rather than physical contact between neurons and glia, determine the induction of GLT1 in astrocytes. This effect is apparently mediated by yet undefined growth factor(s) via the tyrphostin-sensitive receptor tyrosine kinase (RTK) signalling, that in turn, supports the downstream activation of p42/44 MAP kinases and the CREM and ATF-1 transcription factors. RTK-independent simultaneous activation of the CREB transcription factor suggests a possible involvement of complementary pathway(s). Neuronal soluble factors do not affect expression of GLAST, but induce supporting machinery for differential regulation of GLAST via the astroglial metabotropic glutamate receptors, mGluR3 and mGluR5. Thus, long-term treatment with the group I mGluR agonist, DHPG, causes down-regulation of GLAST, whereas the group II agonist, DCG-IV, has an opposite effect on the expression of GLAST in astrocytes. However, in BT4C glioma cells glutamate or other transportable substrates (D-aspartate and L-2,4-trans-PDC) induced cell-surface expression of EAAT4 in a receptor-independent manner. The activity-dependent trafficking of this transporter which also exhibits properties of a glutamate-gated chloride channel may play functional roles not only in neuronal excitability, but in glioma cell biology as well.

Introduction

L-Glutamic acid is the major amino acid transmitter of excitatory signals in the mammalian CNS (Fonnum, 1984). However, prolonged activation of glutamate receptors can be highly toxic for post-synaptic neurons and, thus, cause their death (Choi, 1994). The elevated concentration of glutamate in the synaptic cleft is rapidly inactivated by diffusion and by the astroglial high-affinity glutamate uptake system (Schousboe, 1981), which is so far represented by two carrier proteins, GLT1 and GLAST. Other known glutamate transporters, EAAC1, EAAT4 and EAAT5 are expressed in neurons, predominantly at extra-synaptic locations. It is noteworthy that EAAT4 and EAAT5 possess properties of glutamate-gated chloride channels with yet unclear functions (Kanai and Hediger, 1992; Pines et al., 1992, Storck et al., 1992, Fairman et al., 1995, Arriza et al., 1997; for more references see, Gegelashvili and Schousboe, 1997, Gegelashvili and Schousboe, 1998, Robinson and Dowd, 1997, Danbolt et al., 1998a).

Aberrant expression and functioning of glutamate transporters may lead to accumulation of toxic concentrations of glutamate and thus promote neuronal degeneration (Tanaka et al., 1997), a likely scenario in amyotrophic lateral sclerosis, ALS (Rothstein et al., 1995, Trotti et al., 1999). However, very little is known about the factors regulating expression and activity of high-affinity glutamate transporters. Recent studies indicate that they might be regulated via different signalling pathways and cellular mechanisms. cAMP or activation of the astroglial ionotropic glutamate receptors can up-regulate GLAST (Gegelashvili et al., 1996b), whereas other neuronal factors induce expression of astroglial GLT1 (Gegelashvili et al., 1997, Swanson et al., 1997, Schlag et al., 1998). Furthermore, trafficking of EAAC1 to the cell surface is apparently regulated via protein kinase C and IP3 kinase pathways (Davis et al., 1998).

The present study demonstrates new mechanisms of regulation of high-affinity glutamate transporters: involvement of growth factor signalling in neuron-dependent expression of GLT1, differential regulation of GLAST via metabotropic glutamate receptors, and, finally, the glutamate- or other substrate-dependent trafficking of EAAT4 to the cell surface.

Section snippets

Materials

(±)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD); trans-4-carboxy-L-proline/L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC); (RS)-α-methyl-4-carboxyphenylglycine (MCPG); (RS)-3,5-dihydroxyphenylglycine (DHPG); (2S, 1′R, 2′R, 3′R)-2-(2′,3′-dicarboxycyclopropyl)-glycine (DCG-IV) were from Tocris Cookson, UK. Biotinylation kit, including sulfo-NHS-biotin and Immunopure immobilized monomeric avidin was purchased from Pierce (Rockford, IL, USA); Tyrphostin A23 (AG18) was

Soluble factors determine predominantly astroglial induction of GLT1 in mixed cultures

GLT1, the major forebrain astroglial glutamate transporter (Lehre and Danbolt, 1998), is hardly detectable in neonatal astrocytic cultures (Kondo et al., 1995, Gegelashvili et al., 1996a, Gegelashvili et al., 1996b).

However, in mixed cortical neuron–astroglial cultures a dramatic up-regulation of GLT1 was observed and this effect could be mimicked by application of neuron-conditioned media (NCM) in pure astroglial cultures (Gegelashvili et al., 1997, Swanson et al., 1997, Schlag et al., 1998).

Acknowledgments

The authors would like to thank Prof. Jan Albrecht, The Polish Academy of Sciences, and Elsevier Science for organizing and supporting an excellent meeting on Glutamate and Glutamine Homeostasis in the CNS (Wierzba, Poland, June 19–23, 1999). The expert secretarial assistance of Ms. Hanne Danø and Ms. Tea Gobronidze, as well as the expert technical assistance of Ms. Inge Damgaard is cordially acknowledged. Authors would like to thank Prof. Klaus Edvardsen. The work has been supported by grants

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The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms

Abstract

Introduction

Section snippets

Materials

Soluble factors determine predominantly astroglial induction of GLT1 in mixed cultures

Acknowledgments

J. Biol. Chem.

Neurochem. Int.

Prog. Brain Res.

Prog. Brain Res.

Methods Enzymol.

Brain Res. Bull.

J. Biol. Chem.

Neuron

Neurosci. Lett.

Int. Rev. Neurobiol.

CREB-mediated transcriptional control

Crit. Rev. Eukaryot. Gene Exp.

Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance

Proc. Natl. Acad. Sci. USA

Metabotropic glutamate receptor mGluR5 in astrocytes: pharmacological properties and agonist regulation

J. Neurochem.

Glioma cell interactions with fetal rat brain aggregates in vitro and with brain tissue in vivo

Cancer Res.

Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta

J. Neurosci.

Opposite influence of the metabotropic glutamate receptor subtypes mGluR-3 and -5 on astrocyte proliferation in culture

Glia

Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas

Neurochem. Res.

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

J. Neurosci.

The glutamate transporter EAAT4 in rat cerebellar Purkinje cells: A glutamate-gated chloride channel concentrated near the synapse in parts of the dendritic membrane facing astroglia

J. Neurosci.

An excitatory amino-acid transporter with properties of a ligand-gated chloride channel

Nature

Glutamate: a neurotransmitter in mammalian brain

J. Neurochem.

Glutamate receptor agonists up-regulate glutamate transporter GLAST in astrocytes

Neuroreport