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

Abstract

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.

Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking

“…Our data now reveal interesting similarities between SN1 regulation and mechanisms involved in the regulation of the glial glutamate transporter GLT and glutamate-aspartate transporter GLAST: PMA-induced activation of PKC also stimulates internalization of the glial glutamate transporters GLT and GLAST (Kalandadze et al, 2002;Vanoni et al, 2004;Guillet et al, 2005). Furthermore, coimmunoprecipitation of GLT and PKC␣ has been demonstrated (Gonzalez et al, 2005).…”

Internalization and degradation of the glutamate transporter GLT-1 in response to phorbol ester

“…These studies suggest that at least a portion of internalized GLT-1 is targeted for degradation after internalization. Others have shown that GLT-1 can be internalized in to co-expression with the variant of superoxide dismutase associated with amyotrophic lateral sclerosis (Vanoni et al, 2004). It will be interesting in future studies to determine if the cellular machinery contributing to phorbol esterdependent internalization of GLT-1 also mediate internalization caused by this or other stimuli.…”

Syntaxin 1A promotes the endocytic sorting of EAAC1 leading to inhibition of glutamate transport

“…Increasing evidence show that the dysfunctions in endocytosis are common themes in many neurological disorders, especially neurodegenerative diseases (Cataldo et al, 2000;Nixon, 2005;Smith et al, 2005;Vanoni et al, 2004), thus interference of abnormal endocytosis may be potential therapeutic strategies in neurological disease. Our study demonstrates that syntaxin 1A is an intrinsic regulator of KAinduced EAAC1 endocytic sorting.…”