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First published online 5 October 2004
doi: 10.1242/jcs.01411

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Increased internalisation and degradation of GLT-1 glial glutamate transporter in a cell model for familial amyotrophic lateral sclerosis (ALS)

¹ Department of Pharmacology, School of Medicine, C.E.N.D. Center of Excellence on Neurodegenerative Diseases, University of Milan, IN-CNR Cellular and Molecular Pharmacology Section, Via Vanvitelli 32, 20129 Milano, Italy
² Laboratory of Molecular Neurobiology, Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Via Eritrea, 62 20157 Milano, Italy

^* Author for correspondence (e-mail: grazia.pietrini{at}unimi.it)

Accepted 15 July 2004

It has been suggested that glutamate-induced excitotoxicity plays a central role in the development of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The GLT-1 isoform of the glutamate transporter gene family is the most important transporter involved in keeping extracellular glutamate concentration below neurotoxic levels. Its loss and an increase in extracellular glutamate has been documented in cases of sporadic and familial ALS, as well as in animal models expressing ALS-linked Cu²⁺-Zn²⁺ superoxide dismutase (SOD1) mutations, but the underlying molecular mechanisms are still unclear. We developed and characterised a cell model consisting of polarised epithelial Madin-Darby Canine Kidney (MDCK) cell lines stably expressing wild-type SOD1 or the ALS-linked SOD1 G93A mutant, and analysed the expression of glutamate transporters after transient transfection of the corresponding cDNAs. Like ALS patients and animal models of ALS, the G93A-expressing MDCK cell system showed reduced total glial GLT-1 expression, with no change in the expression of the neuronal EAAC1 glutamate transporter isoform. Morphological analysis revealed the intracellular redistribution of GLT-1 to acidic compartments, whereas the surface distribution of other glutamate transporters (neuronal EAAC1 and glial GLAST) was not affected. Moreover, mutant SOD1 affected the cytosolic tail of GLT-1 because reduced protein expression of EAAC-GLT but not GLT-EAAC chimeras was found in G93A-expressing cell lines. GLT-1 downregulation was greatly induced by inhibition of protein synthesis, and prevented by treatment with chloroquine aimed at inhibiting the activity of acidic degradative compartments. Negligible effect on the protein level or distribution of GLT-1 was observed in cells overexpressing wild-type SOD1. The specific decrease in the GLT-1 isoform of glutamate transporters is therefore recapitulated in G93A-expressing MDCK cell lines, thus suggesting an autonomous cell mechanism underlying the loss of GLT-1 in ALS. Our data indicate that the continuous expression of mutant SOD1 causes the downregulation of GLT-1 by increasing the internalisation and degradation of the surface transporter, and suggest that the cytosolic tail of GLT-1 is required to target the transporter to degradation.

Key words: Endocytosis, Excitotoxicity, ALS-linked SOD1 G93A, Glutamate transporters, Oxidative stress, SOD1 mutations

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