Increased expression and function of glutamate transporters

in multiple sclerosis

Ainara Vallejo-Illarramendi,a Marıa Domercq,a Fernando Perez-Cerda,a

Rivka Ravid,b and Carlos Matutea,*

aDepartamento de Neurociencias, Universidad del Paıs Vasco, E-48940, Leioa, Vizcaya, SpainbNetherlands Brain Bank, Meibergdreef 33, 1105 AZ Amsterdam ZO, The Netherlands

Received 13 April 2005; revised 13 June 2005; accepted 28 June 2005

Available online 2 August 2005

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.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Multiple sclerosis; Glutamate; Oligodendrocytes; Astrocytes;

Excitotoxicity

Introduction

Multiple sclerosis (MS) is a chronic, degenerative disease of

the CNS, which is characterized by focal lesions with inflamma-

tion, 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). Auto-

immunity 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 patho-

logical 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

0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.nbd.2005.06.017

* Corresponding author. Fax: +34 94 601 3400.

E-mail address: onpmaalc@lg.ehu.es (C. Matute).

Available online on ScienceDirect (www.sciencedirect.com).

www.elsevier.com/locate/ynbdi

Neurobiology of Disease 21 (2006) 154 – 164

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.

Materials and methods

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%

formaldehyde-fixed tissue used for immunohistochemical studies.

Clinical characteristics for control and patient groups are

presented in Table 1. We used clinical data together with

macroscopic tissue analysis to classify MS samples as normal

appearing (NAON) or damaged optic nerves (DON), when

showing macroscopic plaques, atrophy and/or optic neuritis. For

Table 1

Clinical characteristics

Reference Code Gender Age PMD (hours) Duration of

MS (years)

MS type Optic nerve

alterations

MS samples

MS1 98-176 M 83 7:05 52 PP No

MS2 99-054 F 58 8:10 20 SP Yes

MS3 98-185 F 70 8:55 19 PP Yes

MS4 98-158 F 76 16:31 53 SP Yes

MS5 98-087 F 55 7:35 >11 SP No

MS6 98-179 F 60 8:50 36 PP Yes

MS7 99-062 F 79 10:00 44 PP No

MS8 99-025 F 64 7:45 35 SP Yes

MS9 98-066 F 75 4:50 >27 PP/SP No

MS10 01-018 F 48 8:10 8 SP No

MS11 01-003 M 69 10:00 34 PP Yes

MS12 99-119 F 38 5:15 10 RR No

MS13 00-024 F 52 8:25 16 PP/SP Yes

MS14a 99-109 M 70 6:25 22 SP Yes

MS15a 99-121 M 51 7:50 29 SP Yes

MS16a 99-066 M 69 16:45 46 PP No

Control samples

C1, 14, 16 98-101 M 72 6:45

C2, 5 98-125 F 58 6:15

C3 98-104 F 74 7:25

C4 98-051 F 94 16:50

C6 98-061 F 64 6:00

C7 98-042 F 79 9:45

C8 98-148 F 54 5:35

C9 98-112 F 84 9:20

C10, 13 00-050 F 52 6:50

C11 00-090 M 70 7:45

C12 00-025 F 41 13:30

C15 98-127 M 56 5:25

Optic nerve alterations consisted of macroscopic plaques, atrophy and/or optic neuritis.

Abbreviations: Reference, Netherlands Brain Bank bar-coded numbers; A, microarrays; qPCR, real-time quantitative PCR; IH, immunohistochemistry; C,

control; MS, multiple sclerosis; M, male; F, female; PMD, postmortem delay; PP, primary progressive; SP, secondary progressive; RR, relapsing remitting.a These samples have been used only for immunohistochemistry.

A. Vallejo-Illarramendi et al. / Neurobiology of Disease 21 (2006) 154–164 155

comparisons, MS samples were matched with control samples for

age, sex and postmortem delay.

RNA extraction and cDNA synthesis

Total RNA was extracted from optic nerve samples by the

guanidinium/phenol/chloroform method. Synthesis of cDNA was

carried out with a reverse transcription (RT) kit (Applied

Biosystems, Madrid, Spain) using random hexamers as primers.

The reaction mixture was incubated at 48-C for 30 min followed

by 5 min at 95-C to inactivate reverse transcriptase. Mock reverse

transcription (without enzyme) for each sample served as negative

controls.

Microarray procedure

Total RNA extracted from the optic nerves of control and MS

patients was used to synthesize cDNA with a primer containing

poly T and T7 RNA polymerase promoter sequences. Double-

strand cDNA was synthesized by means of the Superscript Choice

System (Invitrogen, Barcelona, Spain), and it was used as a

template for in vitro transcription to generate biotinylated cRNA

(Enzo Diagnostics, Farmingdale, NY). Labeled RNA was purified

with the Qiagen RNeasy Mini Kit spin columns (Qiagen, Hilden,

Germany) and quantified by spectrophotometry. Test3 arrays

(Affymetrix, Santa Clara, CA) were hybridized to check cRNA

integrity before running the expression arrays. In all cases, the

GADPH 3V/5V ratio, indicative of RNA sample quality, was below

2. Labeled cRNAwas fragmented and added to Affymetrix human

genome U133 arrays. After sample hybridization, microarrays

were extensively washed and scanned using a confocal scanner.

Fluorescence intensities were analyzed by Microarray Suite 5.0

software (Affymetrix). The expression level, ‘‘mean intensity’’, for

each gene was determined by calculating the mean of the average

differences of all replicates (perfect match–mistmach oligonucleo-

tides) for each probe set. The data filtering criteria applied were

based on the statistical significance (unpaired t test) of expression

changes, fixed at P < 0.0025.

Real-time quantitative PCR (qPCR)

The levels of expression of the glutamate transporters EAAT1,

EAAT2, EAAT3 and several splice variants as well as the levels of

different MS markers were analyzed using qPCR. Glial fibrillary

acidic protein (GFAP), CD8, tumor necrosis factor-a (TNFa) and2V,3V-cyclic nucleotide 3V-phosphodiesterase (CNPase) were used

as markers of astroglial reactivity, infiltration, inflammation and

oligodendrocyte viability, respectively. Primers (Supplementary

Table 1) were designed with PrimerExpress software (Applied

Biosystems). qPCR reactions were carried out with 20–80 ng of

reverse transcribed RNA and 50–300 nM of forward and reverse

primers (Genotek, Sabadell, Spain) diluted in SYBRGreen PCR

universal master mix reagent (Applied Biosystems) in an ABI

PRISM 7000 Sequence Detection System instrument (Applied

Biosystems). We verified that generated fluorescence was not

overestimated by contamination resulting from residual genomic

DNA amplification (using RT negative controls) and from primer

dimer formation or external DNA contamination (no template

controls). qPCR products were also subjected to a dissociation

protocol to ensure that a single amplicon of the expected melting

temperature was indeed obtained.

The amount of cDNA was calculated from the appropriate

standard curve of a stock cDNA obtained from human optic

nerve. Correlation values were 0.995–0.99996. Duplicate qPCR

reactions were performed for each cDNA sample, and the

standard deviations of the Ct values considered were �0.38.

Several constitutive genes (Supplementary Table 1) were used as

endogenous references to compensate for different RT efficiencies

and normalize the variability in the initial quantities of total RNA

so that accurate comparison of gene expression levels could be

made between the different samples. Target genes were normal-

ized by means of a normalization factor (FN5), based on the

geometric mean of multiple internal control genes and calculated

by recently developed software (Vandesompele et al., 2002). The

best and worst scoring housekeeping genes of the six genes

analyzed were ubiquitin C and cyclophilin A, respectively.

Consequently, we eliminated data from the cyclophilin A gene

and calculated a normalization factor based on the expression

levels of the remaining 5 housekeeping genes (FN5). Expression

of endogenous genes used to normalize is showed in Fig. 2D.

Data are expressed as fold change in gene expression compared

to the matched controls. The relative abundance of the different

transporters or splicing variants was determined by using the DCtmethod (see User Bulletin 2; Applied Biosystems). Normalized

data from qPCR in the control and MS groups were compared by

one tail paired Student’s t test. The Spearman non-parametric test

was used for correlation analysis. All results are expressed as

mean T SEM.

Immunohistochemical analysis

Optic nerve samples for histology (Table 1) were fixed with 4%

formaldehyde in phosphate buffered saline (PBS, pH 7.4) for 4–8

days. Subsequently, they were cryoprotected in 20% sucrose in

PBS and stored at �80-C until cryostat sectioning. Tissue was

permeabilized with 0.1% Triton X-100 in incubation solutions or

with absolute ethanol for 10 min previously to immunohisto-

chemistry procedure.

Conventional immunoperoxidase histochemistry was carried

out as previously described (Domercq et al., 1999). Some

sections were hematoxylin–eosin or luxol fast blue stained,

and others were immunoreacted with antibodies to glial fibril-

lary acid protein (GFAP, 0.1 Ag/ml, Chemicon, Temecula, CA)

or myelin basic protein (MBP, 4 Ag/ml, Sternberger Mono-

clonals, Lutherville, MD) to assess the presence of inflamma-

tion, cellular infiltration, demyelination and astroglial reactivity

in optic nerve samples from MS patients (see Supplementary

Fig. 1).

To evaluate the glutamate transporters expression in human

optic nerve, double immunofluorescence staining was carried out

on normal human optic nerve sections. Longitudinal sections were

incubated for 48 h at 4-C with antibodies to EAAT1 (0.4–1.0 Ag/ml for EAAT1) or EAAT2 (0.05–0.2 Ag/ml for EAAT2) (Rothstein

et al., 1994). Sections were subsequently incubated with antibodies

to adenomatus polyposis coli protein (APC, 2.0 Ag/ml, Calbio-

chem, Darmstadt, Germany), marker for oligodendrocytes (Bhat et

al., 1996) or to GFAP (0.1 Ag/ml, Chemicon), marker for astrocyte-

cytoskeleton. Appropriate fluorescent secondary antibodies were

used for each combination. Control staining was done in the

absence of each one of the primary antibodies. Labeling was

examined with a Zeiss Axioplan photomicroscope. Selected

fluorescence digital microphotographies were deconvoluted using

A. Vallejo-Illarramendi et al. / Neurobiology of Disease 21 (2006) 154–164156

Aquacosmos* software (Hamamatsu, Photonics Iberica, Barce-

lona, Spain) to improve labeling.

Western blot

The level of glutamate transporter expression was determined in

total homogenates of control and MS optic nerves. Samples (10 Agof protein per lane) were loaded and size separated in 10% SDS-

PAGE. Proteins were blotted onto nitrocellulose filters (Amersham

Biosciences, Barcelona, Spain) and incubated with antibodies

directed against EAAT1 (1 Ag/ml, Santa Cruz Biotechnology, Santa

Cruz, CA), EAAT2 (1 Ag/ml) and h-actin (1:1000; Sigma, Madrid,

Spain). Immunoblots were visualized using enhanced chemilumi-

nescence (Pierce, Rockford, IL). Protein bands densitometry was

performed using Scion Image for Windows (Scion, Frederick,

MD), and actin immunoreactivity was used to normalize glutamate

transporters signal.

Glutamate uptake in glial plasmalemmal vesicles

Glial plasmalemmal vesicles (GPV) were obtained from

postmortem human optic nerves according to a procedure

described earlier (Nakamura et al., 1993). Briefly, optic nerves

were homogenized in 0.32 M sucrose, 1 mM EDTA using a

Teflon-glass homogenizer and centrifuged at 1000 � g for 10

min. The supernatant was layered (33,500 � g, 5 min) on a

discontinuous four-step 2, 6, 10 and 20% Percoll (Amersham

Biosciences) gradient, and the GPVs collected at the interface

between 2 and 6% Percoll. GPVs were further centrifuged at

33,500 � g for 20 min.

Glutamate uptake in GPVs was determined at 37-C in saline

solution containing (in mM): NaCl, 140; KCl, 5; MgCl2, 2; CaCl2,

2; HEPES, 10; glucose, 10 (pH 7.4). GPVs were incubated in the

presence of 1 AM of 3H-glutamate, and uptake was stopped by

filtration through glass fiber filter papers (Whatman, Brentford,

UK) in a vacuum filtering manifold. Filters were washed 5 times

with 2 ml of ice-cold PBS within 15 s, and their radioactivity was

counted with a scintillation cocktail. Sodium-dependent uptake

was calculated as the difference between the amount of radio-

activity in the presence of sodium and the amount observed in the

choline-containing buffer.

Preparation of rat optic nerves, drug perfusion and quantification

of glutamate transporter transcripts

Adult male Sprague–Dawley rats were deeply anesthetized

with chloroform and then decapitated. Optic nerves were freed

from their meninges in artificial cerebrospinal fluid (aCSF: 126

mM NaCl, 3 mM KCl, 2 mM MgSO4, 26 mM NaHCO3, 1.25

mM NaH2PO4 and 2 mM CaCl2I2H2O) supplemented with 10

mM glucose. Subsequently, the nerves were placed into a

chamber and perfused for 3 h with oxygen-saturated aCSF, to

which glutamate (1 mM) alone or in the presence of CNQX (30

AM, Sigma) was applied. After perfusion, total RNA was

extracted, and synthesis of cDNA was carried out as described

above. Rat glutamate transporter transcripts GLAST, GLT-1 and

EAAC1, the murine counterparts of human EAAT1, 2 and 3

respectively, were quantified by qPCR as described above, using

the corresponding rat primer pairs (Supplementary Table 1).

Gene expression levels were normalized using three constitutive

genes (see Supplementary Table 1). Normalized data were

compared by a one tail non-paired Student’s t test, and data

expressed as mean T SEM.

Cloning, transfection and transport studies

cDNA amplicons encoding EAAT2 and the EAAT2 exon 7

skipping splice variant were obtained by RT-PCR from control

human optic nerve RNA using the following primer pair:

forward: 5V-CCT GAC ATG GCA TCT ACG GAA G-3V; reverse:5V-ATT TCT CAC GTT TCC AAG G-3V. PCR was carried out

with the Taq Platinum Master Mix (Invitrogen) employing an

annealing temperature of 55-C and an elongation time of 2 min

for 35 cycles. The PCR reaction was purified using the GenElute

PCR DNA Purification kit (Sigma) and ligated into the

pTARGET vector (Promega Corporation, Madison, WI). Tran-

sient transfections into HEK-293 cells were carried out using

LipofectAMINE Plus reagent (Invitrogen). Sodium-dependent

glutamate uptake was performed 3–4 days after transfection, as

described previously (Domercq et al., 1999). Uptake was

measured using 100 nM l-[3H]-glutamic acid in the presence

of increasing concentrations of unlabeled substrate, at 37-C for 5

min. Data are expressed as a mean T SEM of at least 3

independent experiments performed in triplicate. Uptake data

were plotted by nonlinear regression using Prism software

(GraphPAD Software, San Diego, CA).

Results

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 microarrays

employed, we found that the expression levels of the glutamate

transporters EAAT1 and EAAT2 were altered in optic nerve MS

samples (average fold increase 1.5 and 1.7, respectively). To

validate these results, we used qPCR and extended this study to

other members of the glutamate transporter family, as described

below.

Molecular characterization of postmortem MS and control tissue

All samples were characterized by studying the expression

levels of GFAP, TNFa, CD8 and CNPase which are commonly

used to analyze MS lesions (Lassmann et al., 1998; Lucchinetti et

al., 1999). This analysis revealed an average increase in the levels

of mRNA encoding GFAP (2.0-fold, P < 0.05), TNFa (5.5-fold,

P < 0.05) and CD8 (8.9-fold, P < 0.05) in MS samples compared

to matched controls (Fig. 1A). In contrast, CNPase expression

showed a significant reduction (0.7-fold, P < 0.05) (Fig. 1A).

Moreover, MBP transcripts were decreased in 8 cases but

increased in the remaining 5 samples. Of these latter samples,

three (MS1, 5 and 8) presented a strong upregulation of MBP, a

feature which is likely indicative of intense remyelinating activity

which has been reported in some MS lesions (Graumann et al.,

A. Vallejo-Illarramendi et al. / Neurobiology of Disease 21 (2006) 154–164 157

2003). These results indicate that the MS samples studied are in

general characterized by astroglial reactivity, inflammation and

oligodendrocyte death.

We further checked whether there was any correlation between

the markers analyzed and the neuropathological characterization

of the tissue. This analysis showed larger increases in CD8 and

TNFa expression, which are markers of infiltration and inflam-

mation respectively, in damaged optic nerves (DON) comparing

to the normal appearing optic nerves (NAON; Fig. 1B). These

data are consistent with previous clinical and pathological

observations indicating that inflammation is the hallmark of

lesion activity (Lassmann et al., 1998; Graumann et al., 2003). In

contrast, we did not observe differences in GFAP or CNPase

expression between NAON or DON samples, suggesting that

astroglial reactivity and oligodendroglial loss may also occur in

normal appearing white matter in MS. DON specimens showed

areas of demyelination and astrogliosis as well as lymphocyte

infiltrates (Supplementary Figs. 1A–G), whereas NAON were

histologically indistinguishable from control samples but for the

occasional presence of an increased astroglial reactivity (Supple-

mentary Figs. 1H and I).

Quantification of EAAT1, EAAT2 and EAAT3 RNA transcripts in

normal and MS optic nerves

We analyzed the expression of EAAT1, EAAT2 and EAAT3

since their murine counterparts GLAST, GLT-1 and EAAC1

respectively are present in the rat optic nerve (Domercq et al.,

1999). By using qPCR, we verified that all three glutamate

transporters were also expressed in the human optic nerve. In

control samples (Fig. 2A), EAAT1 and EAAT2 showed the

highest levels (62 T 3% and 36 T 4% respectively), whereas the

expression of EAAT3 was very low compared to the other two

transporters (2 T 1%; Fig. 2A). The levels of EAAT1 expression

were higher in 11 out of 13 MS samples (1.7 average fold

increase; P < 0.005; Fig. 2B). In addition, EAAT2 mRNA

expression in MS samples was increased in 8 cases, similar in 4

cases and decreased in 1 case, and it was overall significantly

higher in MS compared to controls (1.8 average fold increase; P <

0.05; Fig. 2B). Finally, the mean level of expression of the

EAAT3 transporter was unaltered in MS (Fig. 2B).

We next analyzed whether the levels of the EAAT correlated

with the pathological and clinical data of the MS patients. No

significant differences were observed in the EAAT expression

levels between samples from patients with primary or secondary

MS (data not shown). However, we found that EAAT2 mRNA

levels were significantly higher in the DON samples as compared

to the NAON samples (¨2.5-fold; P < 0.05), while EAAT1 levels

increased independently of the presence of lesions in the MS optic

nerves (Fig. 2C). Expression levels of housekeeping genes used as

reference were not altered in these samples, as compared to

matched controls (Fig. 2D).

In addition to the principal forms of the EAAT2 transcripts

measured above, we analyzed the expression of EAAT2 alter-

native splice variants in control and MS optic nerves (Lin et al.,

1998; Meyer et al., 1998; Matsumoto et al., 1999) (see

Supplementary Table 1 and Supplementary Fig. 2). The quantity

of mRNAs encoding EAAT2 alternative splice variants was found

to be increased in the majority of MS samples assayed as

compared to controls (Supplementary Fig. 2). The overall increase

raised the proportion of alternative splice transcripts—which are

aberrant non-functional isoforms—from 4.37% in control nerves

to 7.32% in MS nerves. However, functional glutamate uptake

studies in transfected HEK-293 cells demonstrated that such

increase did not have any effect on overall EAAT2 activity (see

Supplementary Fig. 2).

Changes in the levels of EAAT1 and EAAT2 protein expression and

function in MS optic nerves

To examine whether changes in EAAT transcription involve

similar changes in the protein expression, we carried out

immunohistochemistry and Western blot analysis. The distribution

of the glutamate transporters EAAT1 and EAAT2 in control human

optic nerves (Fig. 3) was similar to that previously described in

white matter tracts (Domercq and Matute, 1999; Domercq et al.,

1999; Li et al., 1999). Thus, EAAT1 was located in APC+-

oligodendrocytes, and EAAT2 was mainly distributed throughout

Fig. 1. Characterization of MS samples by qPCR. (A) Expression of GFAP,

CD8, TNFa and CNPase in each individual MS sample compared to

controls which are matched for the same gender and similar age. Values,

indicated by bars, are 1.97 T 0.30, 8.89 T 4.42, 5.55 T 2.39 and 0.67 T 0.09

for GFAP, CD8, TNFa and CNPase, respectively. P < 0.05 in all instances.

(B) Expression of GFAP, CD8, TNFa and CNPase in normal appearing

optic nerves (NAON; n = 6) and in damaged optic nerves (DON; n = 7)

from MS patients. CD8 and TNFa expression was more markedly

increased in DON samples (14.0 T 7.9 and 9.4 T 4.0, respectively) than

in NAON samples (2.9 T 0.9 and 1.1 T 0.3). *P < 0.05.

A. Vallejo-Illarramendi et al. / Neurobiology of Disease 21 (2006) 154–164158

GFAP+-astroglial processes (Fig. 3). The cell types expressing both

glutamate transporters were identical in MS and in control optic

nerves. However, immunoreactivity was more intense for EAAT1

and EAAT2 in MS optic nerves (data not shown). In agreement

with immunohistochemistry data, protein levels of EAAT1 and

EAAT2 showed about a two-fold increase in MS optic nerves

homogenates, as revealed by immunoblot analysis (n = 13; P <

0.05; Fig. 4A).

Finally, we analyzed whether the increase in EAAT1 and

EAAT2 transporters in MS samples was relevant to glutamate

uptake in these samples. To this end, we used a preparation of

glial plasmalemmal vesicles (GPVs) which shows a high

activity of glutamate uptake (Nakamura et al., 1993). GPVs

derived from human optic nerve showed low levels of glutamate

uptake (12.4 pmol/mg prot/min) as compared to GPV from

other brain regions (see Nakamura et al., 1993). However, we

detected a significant increase, about two-fold, in Na+-dependent

glutamate uptake in GPVs from MS optic nerves compared to

control GPVs (n = 6; P < 0.05; Fig. 4B), indicating that the

function of EAAT transporters is substantially enhanced in MS

optic nerves.

Glutamate induces glutamate transporter upregulation in rat optic

nerves

Upregulation of glutamate transporters in MS optic nerves

could be an adaptive and protective response to altered levels of

glutamate observed in this demyelinating disease (Sarchielli et al.,

2003). To test this possibility, we evaluated whether elevated

concentrations of glutamate are sufficient to induce an upregulation

of EAATs in optic nerves. To this end, isolated adult rat optic

nerves were perfused with oxygen-saturated aCSF, and expression

of glutamate transporters was assayed by qPCR. We found that the

proportion of the glutamate transporters GLAST, GLT-1 and

EAAC1, which are the rat homologs of EAAT1, 2 and 3 in rat

optic nerves, was 50 T 4%, 49% T 4% and virtually absent,

respectively (Fig. 5A). Addition of glutamate (1 mM, 3 h) to the

perfusate resulted in a significant increase of about 1.4-fold in the

expression of GLAST and GLT1 compared with controls (n = 5;

Fig. 5B). This effect was partially blocked by adding to the

perfusate the AMPA/kainate receptor antagonist CNQX. This

result indicates that glutamate upregulates the glutamate trans-

porters GLAST and GLT1 in rat optic nerves, a mechanism that

Fig. 2. Expression of EAAT1, EAAT2 and EAAT3 in control and multiple sclerosis (MS) samples. (A) Relative expression of EAAT1, EAAT2 and EAAT3

in control human optic nerves are 61.65 T 3.46%, 36.04 T 3.51% and 2.30 T 0.57% respectively. (B) Summary of EAAT1, EAAT2 and EAAT3 expression

in MS samples. Statistical analysis showed significant increases in EAAT1 (175 T 21%) and EAAT2 expression (187.5 T 41.6%) in MS samples as

compared to matched controls (n = 13). *P < 0.05; **P < 0.005. (C) Changes in EAAT expression in MS samples of optic nerves with or without

apparent damage. Increased EAAT1 expression in optic nerves from MS patients is independent of the presence of tissue damage and clinical symptoms of

lesion. Instead, EAAT2 expression is significantly increased in damaged optic nerves (DON, 2.5 T 0.7-fold increase) compared to normal appearing optic

nerves (NAON, 1.1 T 0.3). *P < 0.05. (D) Expression of endogenous genes used to calculate normalization factor in control and MS samples.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), h-2-microglobulin (B2M), ubiquitin C (UbC) and hypoxanthine guanine phosphoribosyl transferase

1 (HPRT1).

A. Vallejo-Illarramendi et al. / Neurobiology of Disease 21 (2006) 154–164 159

constitutes a possible adaptive response to elevated concentrations

of glutamate.

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

and EAAT2 protein. Moreover, we present evidence indicating that

the increase in EAAT1 and EAAT2 transporter expression is

relevant to glutamate uptake.

EAAT1 and EAAT2 glutamate transporter expression and

function are positively regulated by the presence of extracellular

glutamate (Thorlin et al., 1998; Duan et al., 1999). In addition,

enhanced expression of glutamate transporters has also been

detected in experimental paradigms involving increased glutamate

levels and neurodegeneration. Thus, upregulation of glutamate

transporters occurs in the retina after optic nerve crush and in

traumatic lesions to the spinal cord (Vera-Portocarrero et al.,

2002; Mawrin et al., 2003). Inflammation following facial nerve

axotomy, cortical injury or chronic infection with the simian

immunodeficiency virus results in an increased expression of

GLAST and GLT-1 in microglial cells (Lopez-Redondo et al.,

2000; Van Landeghem et al., 2001; Chretien et al., 2002).

Moreover, restraint stress leads to higher glutamate levels in the

hippocampus which renders neurons more vulnerable to neuro-

toxic insults (Lowy et al., 1995), despite a concomitant

upregulation of the GLT-1/EAAT2 transporter (Reagan et al.,

2004). Consistent with these findings, we also observed that

glutamate increases the expression of EAAT1 and EAAT2

glutamate transporters in the optic nerve. Therefore, the enhanced

expression of EAAT1 and EAAT2 in optic nerves from MS

patients reported in this study may be reflecting an adaptive

molecular change to limit glutamate excitotoxicity and ensuing

neurodegeneration. Indeed, a recent DNA microarray study in MS

samples showed upregulation of genes characteristic of neuro-

protective mechanisms against hypoxia (Graumann et al., 2003),

a condition which causes oligodendrocyte death (Lyons and

Kettenmann, 1998) and which can be prevented by glutamate

receptor antagonists (Matute et al., 1997; Tekkok and Goldberg,

2001).

In contrast to our findings, a previous immunohistochemical

study described decreased EAAT2 expression in oligodendrocytes

of spinal cord white matter in subjects with MS (Werner et al.,

2001). However, we did not detect EAAT2 expression in

oligodendrocytes in human optic nerve samples, in agreement

with previous findings in other species or regions (Rothstein et al.,

1994; Milton et al., 1997; Domercq et al., 1999; Li et al., 1999). In

addition, we observed that the increase in EAAT2 protein

correlates with the change observed in EAAT2 transcripts. The

apparent discrepancy with the previous study (Werner et al., 2001)

could be explained by differences in the regions analyzed, in the

antibodies used, the procedures to fix tissues and/or the disease

stages examined.

Fig. 3. Glutamate transporters in control human optic nerves. Microphotographs depict same fields in each row with immunofluorescence labeling of EAAT1

and EAAT2 transporters (left column) and of APC+ oligodendrocytes and GFAP+ astrocytes (middle column). Overlays (right column) show that EAAT1

colocalizes with the oligodendrocyte marker APC (top) but not with GFAP+ cells (middle). In turn, EAAT2 (bottom) is located in GFAP immunoreactive

processes (arrows) and somata indicating their astrocytic nature. Scale bar = 30 Am.

A. Vallejo-Illarramendi et al. / Neurobiology of Disease 21 (2006) 154–164160

Several lines of evidence support the idea that the extracellular

concentration of glutamate is increased in MS. Thus, the

concentration of glutamate in cerebrospinal fluid is higher in

acute than in silent MS and controls (Stover et al., 1997), and it

is associated with the severity and course of the disease

(Barkhatova et al., 1998). Similarly, glutamate levels are

increased in the cerebrospinal fluid of secondary progressive

MS patients (Sarchielli et al., 2003). In serum, glutamate rises

gradually a month or two prior to the onset of clinical relapse to

reach a peak during relapse and then slowly declines (Westall et

al., 1980). Finally, a rise in glutamate and glutamine content has

been detected in MS white matter by proton magnetic resonance

spectroscopic imaging (Tourbah et al., 1999; Srinivasan et al.,

2005). Collectively, these findings indicate that glutamate homeo-

stasis is altered in MS and that high glutamate levels are

associated with this disease.

Several features may account for the increased levels of

glutamate observed in the CNS of MS patients. The rate of

glutamate entry across the blood–brain barrier under physiological

conditions is very low (Oldendorf and Szabo, 1976; Betz et al.,

1994). This creates a steep gradient in glutamate concentration

between the bloodstream (Divino Filho et al., 1998) and the

extracellular space in the CNS (Danbolt, 2001). Indeed, the

concentration of glutamate and other excitatory amino acids in

the CNS parenchyma significantly increases following experimen-

tal opening of the blood–brain barrier (Westergren et al., 1994).

Thus, blood–brain barrier distortion due to MS-associated

inflammation results in glutamate flooding of the CNS and

Fig. 4. Quantitative analysis of EAAT protein levels and function in optic nerves from MS patients and matched controls. (A) Western blot analysis of

EAAT1 and EAAT2 protein levels in control and MS optic nerves. Representative samples from each group and histograms showing the average density in

EAAT1 and EAAT2 transporters, normalized to h-actin, in MS optic nerves and matched controls (n = 13; *P < 0.05, **P < 0.01). (B) Na+-dependent

glutamate uptake determined in glial plasmalemmal vesicles from control and MS optic nerves. A significant increase in uptake in MS optic nerves was

detected (n = 6; *P < 0.05).

A. Vallejo-Illarramendi et al. / Neurobiology of Disease 21 (2006) 154–164 161

subsequent increase in the concentration of this amino acid

(Oldendorf and Szabo, 1976; Betz et al., 1994) which can

jeopardize the viability of oligodendrocytes which are highly

vulnerable to excitotoxicity (Matute et al., 1997; McDonald et al.,

1998). Furthermore, alterations in the glutamate producing enzyme

glutaminase in close proximity to dystrophic axons in MS have

been detected in activated macrophages/microglia (Werner et al.,

2001), a cell lineage which has the potential to produce and release

glutamate (Piani et al., 1991). Therefore, in addition to direct

glutamate leakage from the bloodstream, activated macrophages/

microglia in MS may also contribute to increases in the

concentration of local glutamate. Finally, glutamate transporter

activity is inhibited by reactive oxygen species (Trotti et al., 1998),

which are known players of MS neuropathology (Smith et al.,

1999) and thus impair further glutamate homeostasis.

EAAT1 and EAAT2 transporters are differently modulated in

the MS samples studied. This is probably because they are

expressed in different cell types, astrocytes vs. oligodendrocytes,

which respond differently to environmental changes. Indeed,

EAAT1 promoter activity is cell-type dependent and varies greatly

among the cells tested (Kim et al., 2003). Intriguingly, EAAT1

expression is consistently augmented in MS samples including

normally appearing optic nerves. This suggests that oligodendro-

cytes react more readily to subtle changes in glutamate concen-

tration, probably as a mechanism to limit excitotoxicity. Indeed,

inhibition of expression and functioning of glutamate transporters

in isolated optic nerves, which induces a three-fold increase in

extracellular glutamate, damages oligodendrocytes and induces

demyelination (Domercq et al., in press). The expression of

glutamate receptors by oligodendrocytes (Garcia-Barcina and

Matute, 1996, 1998) may provide a sensor by means of which

these cells regulate EAAT1. Determination of these early molecular

alterations in normal appearing white matter in MS can be useful to

elucidate the mechanisms of lesion formation and to develop new

treatments (Graumann et al., 2003).

In conclusion, the present study provides evidence for an

increased expression of EAAT1 and EAAT2 glutamate transporters

in the optic nerves of MS subjects that correlates with an increase

in glutamate uptake in these patients. Enhanced expression of

glutamate transporters may reflect a mechanism to limit oligoden-

drocyte excitotoxicity caused by high glutamate levels in MS.

Potentiation of this adaptive response with positive transcriptional

regulators of glutamate transporter expression such as EGF or h-lactams may prove to be of therapeutical value in the treatment of

MS.

Acknowledgments

Supported by Fundacion ‘‘La Caixa’’, Ministerio de Salud y

Consumo, Gobierno Vasco and the Universidad del Paıs 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.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.nbd.2005.06.017.

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