Elsevier

Neurobiology of Disease

Volume 21, Issue 1, January 2006, Pages 154-164
Neurobiology of Disease

Increased expression and function of glutamate transporters in multiple sclerosis

https://doi.org/10.1016/j.nbd.2005.06.017Get rights and content

Abstract

Recent studies have shown that glutamate excitotoxicity may be a component in the etiology of multiple sclerosis (MS). Glutamate transporters determine the levels of extracellular glutamate and are essential to prevent excitotoxicity. We have analyzed here the expression of the glutamate transporters EAAT1, EAAT2 and EAAT3 in control and in MS optic nerve samples. We observed an overall increase in the level of the glutamate transporters EAAT1 and EAAT2 mRNA and protein. In turn, functional assays showed that glutamate uptake was also increased in MS samples. Furthermore, glutamate transporter increases were mimicked in rat optic nerves treated with excitotoxic levels of glutamate. Together, these results indicate that enhanced expression of glutamate transporters in MS constitutes a regulatory response of glial cells to toxic levels of glutamate in the CNS during inflammation and neurodegeneration.

Introduction

Multiple sclerosis (MS) is a chronic, degenerative disease of the CNS, which is characterized by focal lesions with inflammation, demyelination, infiltration of immune cells, oligodendroglial death and axonal damage (Trapp et al., 1998, Prineas et al., 2002). These cellular alterations are accompanied by neurological deficits, such as sensory disturbances, lack of motor coordination and visual impairment (Steinman, 2001). MS usually begins with an autoimmune inflammatory reaction to myelin components and progresses later to a chronic phase in which oligodendrocytes, myelin and axons degenerate (Steinman et al., 2002). Autoimmunity may be triggered by microbial protein sequences which share homologies with components of the myelin sheath and which in turn are recognized by T cells (Wucherpfennig et al., 1997). Activated lymphocytes penetrate into the CNS and open the blood–brain barrier to plasma constituents which otherwise cannot reach the nervous tissue and are toxic to neurons and glia (Weiner and Selkoe, 2002). Although the precise cause of MS remains unclear, several lines of evidence support the hypothesis that glutamate excitotoxicity may be involved in this pathology (Matute et al., 1997, McDonald et al., 1998, Pitt et al., 2000, Smith et al., 2000).

Oligodendrocytes are highly vulnerable to excitotoxic signals mediated by AMPA and kainate receptors (Matute et al., 1997, McDonald et al., 1998, Sanchez-Gomez and Matute, 1999, Sanchez-Gomez et al., 2003), and activation of these receptors in vivo can induce inflammation, demyelination and other pathological features which are typical of MS lesions (Matute, 1998). Strong evidence supporting an association between glutamatergic hyperfunction and MS has come from pharmacological studies showing that amantadine decreases the rate of relapse of MS patients (Plaut, 1987) and that memantine, as well as AMPA antagonists, ameliorates the neurological sequelae of experimental autoimmune encephalomyelitis (EAE) (Wallstrom et al., 1996, Pitt et al., 2000, Smith et al., 2000).

Moreover, alterations in glutamate homeostasis in MS have given further credence to the potential involvement of the glutamatergic system in this pathology. Thus, the levels of glutamate are increased in the cerebrospinal fluid of patients with acute MS (Stover et al., 1997) and secondary progressive MS (Sarchielli et al., 2003), as well as in serum prior to the onset of clinical relapse (Westall et al., 1980). In addition, glutamine synthase and glutamate dehydrogenase, enzymes responsible for glutamate degradation, are downregulated in EAE and MS white matter (Hardin-Pouzet et al., 1997, Werner et al., 2001), whereas the glutamate producing enzyme, glutaminase, shows increased immunoreactivity in macrophages and microglia in active MS lesions (Werner et al., 2001). Together, these data strongly suggest that the levels of glutamate are likely to be increased in EAE as well as in MS.

High affinity glutamate transport is the major mechanism by means of which the CNS maintains low levels of extracellular glutamate (Danbolt, 2001). To date, five human excitatory amino acid transporters (EAAT1–5) have been cloned (Arriza et al., 1994, Danbolt, 2001). It is conceivable that the altered expression or function of these glutamate transporters may be responsible for increased extracellular glutamate in the diseased CNS. Truncated splicing variants of human EAAT2 (Lin et al., 1998, Meyer et al., 1998) and EAAT3 (Matsumoto et al., 1999), which regulate the function of normal EAAT proteins (Lin et al., 1998), could be additionally involved in the pathogenesis of neurodegenerative disorders, including MS. In the present study, we determined the levels of expression of EAAT1–3 and their splicing variants in optic nerves from MS and controls. The results indicate that the expression and function of glutamate transporters are indeed increased in optic nerves of MS patients and that high levels of glutamate can drive upregulation of the expression of these transporters in the isolated rat optic nerve. On the basis of the present results, enhanced expression of glutamate transporters in MS represents an adaptive response to increased levels of glutamate in the CNS during inflammation and neurodegeneration.

Section snippets

Human tissue samples

Postmortem optic nerve samples from 16 long-standing MS patients and 12 control subjects (who died from non-neurological diseases) were obtained at autopsy under the management of the Netherlands Brain Bank. All patients and controls had previously given written approval for the use of their tissue, according to the guidelines of the Netherlands Brain Bank. Samples obtained included frozen tissue (−80°C) used for microarray, real-time PCR analysis, Western blot and functional studies and 4%

Analysis of EAAT expression by DNA microarrays

Since glutamate homeostasis is altered in MS (Matute et al., 2001), we analyzed if the expression of glutamate-related enzymes as well as glutamate receptors and transporters were altered in this disease using DNA microarrays. To this end, we used 4 human optic nerve samples (MS 10, 11, 12, 13; Table 1) obtained at autopsy from MS patients and matched controls from subjects who did not suffer any neurological or psychiatric disorder. Of the 193 glutamate-related genes annotated in the DNA

Discussion

The results reported here indicate that the expression of the glutamate transporters EAAT1 and EAAT2 is increased in postmortem optic nerves from MS patients. EAAT1 is the most abundant glutamate transporter transcript in human optic nerves, and its expression is consistently higher in the MS samples examined. In contrast, upregulation of EAAT2 expression in optic nerves from MS patients correlates with the presence of damage. In addition, we also observed increases in the expression of EAAT1

Acknowledgments

Supported by Fundación “La Caixa”, Ministerio de Salud y Consumo, Gobierno Vasco and the Universidad del País Vasco. We thank Dr. M.M. Panicker for advice in cloning and transfection experiments and Dr. D.J. Fogarty for having corrected the English of the manuscript. AV-I and MD hold fellowships from the Gobierno Vasco.

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