regulation of gene expression in experimental autoimmune encephalomyelitis indicates early neuronal...

Regulation of gene expression in experimentalautoimmune encephalomyelitis indicates earlyneuronal dysfunction

Arnaud Nicot,1,2 Pillarisetty V. Ratnakar,1 Yacov Ron,3 Chiann-Chyi Chen3 and Stella Elkabes1,2

1Department of Neuroscience, University of Medicine and

Dentistry of New Jersey, New Jersey Medical School,

Newark, 2Veterans Affairs, Neurology Service,

East Orange, 3Department of Molecular Genetics and

Microbiology, University of Medicine and Dentistry of

New Jersey, Robert W. Johnson Medical School,

Piscataway, NJ, USA

Correspondence to: Stella Elkabes, PhD, Department of

Neuroscience, MSB, H-506, New Jersey Medical School/

UMDNJ, 185 South Orange Avenue, Newark, NJ 07103,

USA

E-mail: [email protected]

SummaryMultiple sclerosis is an in¯ammatory, demyelinatingdisease of the CNS. Whereas oligodendrocytes havebeen considered the primary neural cell type mostaffected, recent evidence indicates that axonal andneuronal degeneration also occurs in both multiplesclerosis and experimental autoimmune encephalomyeli-tis (EAE), an animal model reproducing many featuresof multiple sclerosis. The molecular mechanisms under-lying neuronal de®cits in multiple sclerosis and EAEremain elusive. To address this issue, we have analysedthe expression of genes encoding proteins that playcritical roles in ion homeostasis, exocytosis, mitochon-drial function and impulse conduction in the Lewis ratlumbar spinal cord during the clinical course of acuteEAE. Transcript and protein levels of plasma mem-brane Ca2+ ATPase 2 (PMCA2), an essential ion pumpexpressed exclusively in grey matter and involved inCa2+ extrusion, synapsin IIa and syntaxin 1B, important

regulators of vesicular exocytosis, were dramaticallydecreased coincident with the onset of clinical symp-toms. In contrast, changes in the expression of severalother ion pumps, vesicular proteins, mitochondrialenzymes and sodium channels occurred at moreadvanced disease stages. Moreover, exposure of spinalcord slice cultures to kainic acid signi®cantly reducedPMCA2 mRNA levels. Taken together, our ®ndings sug-gest that glutamate, which recently has been implicatedin EAE pathogenesis, suppresses neuronal PMCA2expression leading to Ca2+ dyshomeostasis at initialclinical phases. Consequently, perturbations in Ca2+ bal-ance and neurotransmitter exocytosis may partiallyunderlie aberrant neuronal function and communi-cation at onset of symptoms. Altered mitochondrialfunction and impulse conduction may exacerbate neuro-logical de®cits at subsequent disease stages.

Keywords: EAE; exitotoxicity; synaptic protein; neuronal injury; ATP2B2

Abbreviations: DIG = digoxigenin; EAE = experimental autoimmune encephalomyelitis; PMCA2 = plasma membrane

Ca2+ ATPase 2; SERCA2 = sarcoplasmic and endoplasmic reticulum Ca2+-activated ATPase 2; SNAP-25 = synaptosome-

associated protein of 25 kDa

IntroductionMultiple sclerosis is a CNS disease leading to progressive

neurological de®cits and permanent disability (Steinman,

1996; Prineas and McDonald, 1997; Antel, 1999; Hickey,

1999). Experimental autoimmune encephalomyelitis (EAE),

the animal model of multiple sclerosis, is induced by

immunization of rodents with immunogenic myelin com-

ponents or by adoptive transfer with T cells reactive

against CNS myelin antigens (Wekerle et al., 1994;

Steinman, 1999; van der Goes and Dijkstra, 2001). The

clinical symptoms and histopathology of EAE may be diverse

depending on the antigen and the animal strain employed. It

has been hypothesized that distinct EAE models reproduce

different features of multiple sclerosis, a complex disease

with variable clinical course (Wekerle et al., 1994).

Immunization of adult Lewis rats with myelin basic protein

results in acute, monophasic EAE manifested by progressive

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ascending weakness leading to hindlimb paralysis and

quadriplegia.

Classically, demyelination of structurally intact axons and

the resulting impairment in saltatory nerve conduction have

been considered as the causes of functional de®cits in

multiple sclerosis and EAE (McFarlin and McFarland,

1982a, b; Raine, 1997). However, the lack of correlation

between myelin loss and symptom severity suggests that EAE

and multiple sclerosis are more complex than initially

believed (Rivera-Quinones et al., 1998; Antel, 1999).

Indeed, studies reporting axonal dysfunction and damage

(Ferguson et al., 1997; Trapp et al., 1998, 1999; De Stefano

et al., 1999; Bitsch et al., 2000; Bjartmar and Trapp, 2001;

Bjartmar et al., 2001), neuronal loss or apoptosis (Smith et al.,

2000; Peterson et al., 2001) indicate that multiple sclerosis

and EAE are not only demyelinating diseases but neuronal

disorders as well (Waxman, 2000a, b; Filippi, 2001).

The triggers that cause aberrant neuronal axonal function

and destruction of myelin are not well de®ned. A correlation

between in¯ammatory reaction and axonal injury has been

observed (Ferguson et al., 1997; Trapp et al., 1998).

Activated resident microglia and macrophages have also

been implicated in myelin and oligodendrocyte damage (for a

review see Sriram and Rodriguez, 1997). Recent studies

indicate that glutamate excitotoxicity may be a factor

contributing to axonal, neuronal and oligodendrocyte injury

during EAE (Pitt et al., 2000; Smith et al., 2000). Moreover, it

has been suggested that increased calcium in¯ux through

voltage-dependent calcium channels induces axonal degen-

eration and neuronal injury (Kornek et al., 2000, 2001).

Calcium dyshomeostasis, which has been implicated in many

pathological conditions, may also result from the abnormal

function of pumps that extrude Ca2+ from cells. This

possibility has not been investigated in EAE or multiple

sclerosis.

In summary, in contrast to the well-de®ned histopathology,

the molecular events underlying neuronal impairment con-

tributing to neurological de®cits and persistent disability in

EAE and multiple sclerosis remain elusive. To delineate

molecular mechanisms responsible for the onset and pro-

gression of symptoms in EAE, we de®ned the expression

pattern of genes encoding proteins that play essential roles in

neuronal function during the clinical course of the disease.

Our ®ndings suggest that abnormal Ca2+ extrusion and

vesicular exocytosis may initiate neuronal dysfunction. At

later clinical stages, changes in the expression of other genes

that encode proteins involved in mitochondrial function

and impulse conduction may contribute further to disease

progression.

Material and methodsInduction of EAEFemale adult Lewis rats (200±250 g body weight) were

immunized in hindlimb footpads with 100 mg of guinea pig

myelin basic protein (Sigma, St Louis, MO, USA, or

generously provided by Dr A. Ben-Nun) in 50 ml of saline

emulsi®ed in 50 ml complete Freund's adjuvant (Difco,

Detroit, MI, USA). Control rats received only saline/

adjuvant. Animals were monitored daily for clinical symp-

toms. A very stereotyped clinical onset and disease course

was observed. Neurological impairment was scored as: 0, no

neurological symptoms; 1, limp tail [stage E1, observed on

day 10 post-injection (PI)]; 2, hindlimb weakness (stage E2,

day 11 PI); 3, hindlimb paralysis (stage E3, day 12±13 PI); 4,

quadriplegia (stage E4; day 14±15 PI); 5, moribund (past day

16 PI). During the observation period, rats were given food

and water ad libitum. Some animals were sacri®ced by

exposure to CO2/O2. Others were anaesthetized with a

mixture of ketamine/xylazine and perfused with saline

followed by 4% paraformaldehyde in 0.1 M phosphate buffer

pH 7.5. Lumbar spinal cords were dissected out and frozen

immediately on dry ice. The tissue was kept at ±80°C until

further use. All animal procedures were performed according

to IACUC and institutional guidelines.

Isolation of RNATotal RNA was isolated from 100±150 mg of tissue obtained

by pooling 2±3 lumbar spinal cords. The Trizol One Step

Isolation method was utilized according to the manufacturer's

instructions (Life Technologies, Grand Island, NY, USA). All

RNA samples were treated with DNase I to remove any trace

of contaminating genomic DNA and repuri®ed by phenol/

chloroform extraction. RNA was quanti®ed by measurement

of optical density. Samples were stored at ±20°C in the

presence of RNase inhibitor.

cDNA microarray analysisThis was performed utilizing the Atlas Rat 1.2 microarrays

according to the manufacturer's instructions (Clontech, Palo

Alto, CA, USA). The microarray includes 1176 rat cDNAs

immobilized on nylon membranes, nine housekeeping

cDNAs and negative controls. The list of the cDNAs can be

found at the Clontech web site (http://www.clontech.com/

atlas/genelists/7854-1_Ra12.txt). Total RNA was puri®ed

from lumbar spinal cords of control and EAE rats (three rats

per group; EAE clinical score 3, E3). A 25 mg aliquot of

pooled RNA was reverse transcribed in the presence of

MMLV reverse transcriptase, gene-speci®c primer mix,

0.5 mM each of dCTP, dGTP and dTTP, and 35 mCi of

[a-33P]dATP (2500 Ci/mmol; Amersham, Piscataway, NJ,

USA) in a 30 ml reaction volume at 50°C for 25 min using the

Atlas pure total RNA labelling system (Clontech). The arrays

were pre-hybridized in Express Hyb hybridization buffer

(Atlas hybridization kit) containing heat-denatured salmon

testes DNA (Sigma) for 30 min at 68°C. Subsequently, the

probe mixture (2±10 3 106 c.p.m.) and 5 ml of human

Cot-1DNA were added to the pre-hybridization buffer.

Hybridization was performed overnight at 68°C. The arrays

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were then washed four times in 23 SSC (standard saline

citrate), 1% SDS, and once in 0.13 SSC, 0.5% SDS for

30 min, each wash at 68°C, and exposed to phosphorimager

screens for 5 days. A Storm phosphorimager (Molecular

Dynamics, Sunnyvale, CA, USA) and ImageQuant software

were used to scan images. Gene expression was custom

analysed using Atlas Image 2.0 software by Clontech. The

intensity of each spot, quanti®ed after background correction,

re¯ected the level of expression of each gene. Experimental

variation in the overall intensity of the signal was corrected

by normalization of the gene signal with respect to the

housekeeping genes in the same array. For each gene, a ratio

was generated by dividing the intensity of the spot by the

average of the sum of housekeeping genes. Only those

housekeeping genes with signal intensity below saturation

were considered for normalization. Reproducibility of this

technique (80% concordance of signals) was veri®ed by

hybridizing probe mixtures generated from two aliquots of

the same RNA source to identical arrays. The signal levels of

genes selected for further analysis in our study were 30- to 40-

fold over the background. In addition, there was at least a

2-fold difference in the expression of the selected genes in

EAE versus control (Table 1).

RT±PCRThis was performed utilizing the Retroscript reverse tran-

scription kit and SuperTaq Polymerase (Ambion, Austin, TX,

USA) according to the manufacturer's instructions. A 1±2 mg

aliquot of DNase-treated total RNA was reverse transcribed

in a total volume of 20 ml. A 2±5 ml aliquot of RT mix was

then used for PCR, which was performed in a total volume of

50 ml by denaturation at 94°C for 30 s, annealing at 55°C for

45 s and polymerization at 68°C for 1 min. PCR was

completed by 10 min extension at 72°C. PCRs were

performed for 30±35 cycles, within the linear range.

Products were separated on a 1.5% agarose gel containing

ethidium bromide (0.5 mg/ml) and the image was captured

using an Alpha digital imaging system. The optical density in

each band was then quanti®ed utilizing the Un-Scan-It

software (Silk Scienti®c, Orem, UT, USA). Some PCR

products were analysed further by sequencing.

Preparation of riboprobes for in situhybridizationRT±PCR products generated by use of speci®c primers for

PMCA2 or a-tubulin were cloned into pCR II-TOPO vector

containing SP6 and T7 promoters (Invitrogen, Carlsbad, CA,

USA) and sequenced for further con®rmation. Antisense and

sense riboprobes were generated using the Digoxigenin (DIG)

RNA Labelling Kit according to the manufacturer's instruc-

tions (Boehringer Mannheim, Indianapolis, IN, USA). A 1 mg

aliquot of linearized plasmid was transcribed at 37°C for 2 h

in the presence of 1 mM ATP, CTP and GTP, 0.65 mM UTP,

0.35 mM DIG-UTP, 20 U of RNasin, 40 U of SP6 or T7 RNA

polymerase in 40 mM Tris buffer pH 8.0, 10 mM NaCl,

10 mM dithiothreitol and 6 mM MgCl2. The DNA template

was removed by treatment with DNase I and the probe was

precipitated with LiCl and ethanol. The concentration of

DIG-labelled riboprobe was determined by utilizing DIG

quanti®cation test strips according to the manufacturer's

instructions (Boehringer Mannheim).

Table 1 Differentially expressed genes in the lumbar spinal cord during EAE

Gene Expressionchanges

Ratio of spotintensity (E/C)

GeneBankaccession No.

Function

Plasma membrane Ca2+ ATPase 2 Decrease 0.10 J03754 Ion homeostasisNa+/K+ transporting ATPase b2 subunit Decrease <0.10 J04629 Ion homeostasisSERCA2 Decrease 0.50 J04022 Ion homeostasisSynapsin IIa Decrease 0.15 M27925 Vesicular functionSyntaxin 1B Decrease 0.40 M95735 Vesicular functionSNAP-25 Decrease 0.50 AB003991 Vesicular functionRab12 Increase 6.30 M83676 Vesicular functionRab14 Increase 8.30 M83680 Vesicular functionRab16 Increase 3.30 M83681 Vesicular functionCytochrome c oxidase subunit Vb Increase 13.0 D10952 Mitochondrial functionMitochondrial cytochrome c subunit IV Increase 4.80 X14209 Mitochondrial functionNa+ channel b1 subunit Decrease <0.10 M91808 Impulse conductionNa+ channel b2 subunit Decrease 0.30 U37026 Impulse conductionNa+ channel II Decrease 0.30 X03639 Impulse conduction

cDNA microarray analysis identi®ed differentially expressed genes in the spinal cord during EAE, including those listed above whichshowed robust changes in transcript levels (at least 2-fold difference between EAE and control) and which were highly expressed (at least30- to 40-fold over background). The genes are classi®ed according to their best characterized function. The results obtained with all thelisted genes were corroborated subsequently by RT±PCR in 2±3 independent experiments. The ratio of spot intensity re¯ects the relativetranscript levels in EAE over control.

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In situ hybridizationControls and EAE (E3) rats (six animals/group) were

sacri®ced by cardiac perfusion of saline followed by 4%

paraformaldehyde in 0.1 M phosphate buffer pH 7.5. Lumbar

spinal cords were dissected out, post-®xed in phosphate

buffer for 4 h, cryoprotected in 10 and 15% sucrose and cryo-

sectioned at 10 mm. Sections obtained from control and EAE

lumbar spinal cords were mounted on the same slides to

minimize experimental variations, ®xed in 4% paraform-

aldehyde/phosphate buffer for 15 min at room temperature,

and treated with 6% H2O2 in phosphate-buffered saline (PBS)

containing 0.1% Tween (PBST). Sections were then digested

with proteinase K (1 mg/ml) for 15 min at 37°C, followed by

incubations in glycine (2 mg/ml, 10 min) and 4% para-

formaldehyde/0.2% gluteraldehyde/PBST for 15 min. Pre-

hybridization was performed in buffer containing 50%

deionized formamide, 53 SSC pH 4.5, 1% SDS, 50 mg/ml

yeast tRNA and 50 mg/ml heparin at 65°C for 1 h.

Hybridization was performed overnight at 65°C in the same

buffer containing 1 mg/ml DIG-labelled sense or antisense

probe. The sections were then washed in 53 SSC/50%

formamide/1% SDS at 70°C, followed by 43 SSC/50%

formamide at 65°C, blocked in 10% heat-inactivated goat

serum/PBS for 1 h at room temperature and incubated in anti-

DIG±alkaline phosphatase-conjugated antibody (1 : 1000) in

PBS at 4°C overnight. Endogenous alkaline phosphatase

activity was blocked by pre-treatment with 10 mM levami-

sole. The colour reaction was developed overnight in the

presence of 250 mg/ml NBT (4-nitro blue tetrazolium

chloride) and 130 mg/ml BCIP (5-bromo-4-chloro-3-indolyl-

phosphate) as substrate.

ImmunocytochemistryLumbar spinal cord sections, prepared as described above,

were ®rst treated with 10% goat serum/0.1% Triton X-100/

PBS for 1 h at room temperature. They were then incubated in

ED-1 (1 : 500; Serotec, Oxford, UK)/0.1% Triton X-100/PBS

overnight at 4°C. The rest of the procedure was performed at

room temperature. The sections were washed in PBS,

incubated in anti-mouse IgG [1/500 (v/v)] followed by

avidin±biotin complex (Vectastain ABC kit, Vector

Laboratories, Burlingame, CA, USA). Immunopositive cells

were visualized by 3,3¢¢-diaminobenzidine reaction product.

Western blot analysisLumbar spinal cords of controls or EAE (E1±E3) rats (three

animals/group) were homogenized on ice with a motorized

pestle in 1 ml of buffer A (10 mM HEPES pH 7.0, 10%

sucrose, 0.4 mM phenylmethylsulfonyl ¯uoride, 5 mM

EDTA, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 2 mg/ml

pepstatin). The homogenate was then passed ®ve times

through a syringe with a 22-gauge needle and centrifuged at

800 g for 5 min. The supernatant was removed and

centrifuged at 20 000 g for 45 min. The crude plasma

membrane preparation was suspended in buffer A and

aliquots were kept at ±70°C until use. Total protein concen-

trations were determined utilizing the CBQCA protein

quanti®cation kit according to the manufacturer's instructions

(Molecular Probes, Eugene, OR, USA). A 5 mg aliquot of

total protein was loaded on each lane of an 8% SDS±

polyacrylamide Novex, Tris-glycine gel (Invitrogen).

Electrophoresis was performed for 90 min at 125 V. The

protein was then electrotransferred onto a polyvinylidene

di¯uoride (PVDF) membrane for 45 min at 50 V, and stained

with BLOT-FastStain (Chemicon, Temecula, CA, USA)

according to the manufacturer's instructions. Immuno-

detection was performed employing a Western Breeze kit

(Invitrogen). Primary antibodies were utilized as follows:

anti-PMCA2 (1 : 5000, polyclonal; Research Diagnostic,

Flanders, NJ, USA), anti-synapsin IIa (1 : 5000, monoclonal;

BD Sciences, Palo Alto, CA, USA) and anti-a-tubulin

(1 : 20 000, monoclonal; Sigma). Signal was visualized by

exposure of blots to Hyper®lm ECL (Amersham Pharmacia

Biotech, Piscataway, NJ) for 30±120 s. Between each

immunoblot, membranes were stripped with Re-Blot

recycling kit (Chemicon). Bands were quanti®ed using the

Un-scan-it software (Silk Scienti®c).

Spinal cord slice culturesAdult rats were sacri®ced by exposure to CO2/O2. Lumbar

spinal cords were excised, sliced into 1 mm sections and

immediately placed in 1 ml of de®ned medium consisting of

L-15: neurobasal medium (2 : 1; Gibco/BRL, Rockville, MD,

USA) containing insulin (5 mg/ml), transferrin (100 mg/ml),

selenium (40 ng/ml), putrescine (16 ng/ml), progesterone

(60 ng/ml) and 1 U of penicillin/streptomycin. Kainic acid

was then introduced at the concentrations indicated in the

Results. Controls were maintained in medium containing

vehicle. The slices were incubated for 6 h at 37°C. At the end

of the incubation period, the slices were homogenized in

Trizol reagent and RNA was prepared as described above.

Some slices were ®xed in 4% paraformaldehyde, cryopro-

tected in 10 and 20% sucrose and frozen on dry ice. Cryostat

sections (10 mm) were mounted on poly-L-lysine-coated

slides and utilized for assessment of DNA fragmentation by

TUNEL assay, a hallmark of cell death.

TUNEL assay was performed utilizing the ApopTag kit

(Intergen, Purchase, NY, USA) according to the manufac-

turer's instructions. Eight control and eight kainic acid-

treated slices were analysed. At least ®ve sections from each

slice were used for TUNEL assay. Control and experimental

samples were mounted on the same slide, side by side. The

experiment was repeated twice and yielded similar results.

ResultsTo identify differentially expressed genes in the EAE lumbar

spinal cord, we ®rst performed cDNA microarray analysis at



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disease stage E3. We selected a number of differentially

expressed genes for further studies because they are

expressed exclusively or mainly by neurons, they play critical

roles in important cellular functions including ion home-

ostasis, neurotransmitter exocytosis, energy metabolism or

impulse conduction, and their transcript level was altered

greatly. The genes were classi®ed according to the best

characterized function of the protein which they encode

(Table 1). Thus, cDNA microarray pro®ling provided an

initial and global insight into potential cellular mechanisms

that may be affected during a de®ned stage of the disease. We

further corroborated these ®ndings by semi-quantitative RT±

PCR utilizing the same source of RNA as that employed for

cDNA microarray analysis (stage E3). Subsequently, we

further validated our ®ndings by performing at least 2±3

additional, independent experiments employing distinct

groups of rats (2±3 rats/group) during the course of the

disease (stages E1±E3), as reported below.

PMCA2 mRNA and protein levels are decreasedat onset of symptomsOne of our observations by cDNA microarray analysis was a

pronounced change in the expression of several pumps which

modulate ion, and in particular Ca2+, homeostasis including

plasma membrane Ca2+ ATPase 2 (PMCA2), sarcoplasmic

and endoplasmic reticulum Ca2+-activated ATPase 2

(SERCA2) and Na+/K+ transporting ATPase b2 subunit

(Table 1). As Ca2+ balance plays a pivotal role in the function

and survival of cells, including neurons, we further analysed

these genes and corroborated our microarray ®ndings by

semi-quantitative RT±PCR utilizing speci®c primers for each

pump (Table 2). We examined the expression of the

aforementioned ion pumps during the course of the disease

starting at E1 (tail weakness only; clinical score 1) in order to

determine the clinical stage when the ®rst signi®cant changes

in expression occur. Transcript levels of PMCA2, a major

pump mediating Ca2+ extrusion from cells (Carafoli, 1987;

Miller, 1991; Garcia and Strehler, 1999), were signi®cantly

decreased at E1 [Fig. 1, 88% decrease, P < 0.001 by analysis

of variance (ANOVA); Scheffe's post hoc test] and con-

sistently remained low until E3, the latest clinical stage

examined. Transcript levels of SERCA2, which mediates the

sequestration of intracellular Ca2+ into the endoplasmic

reticulum, and Na+/K+ transporting ATPase b2 subunit,

which regulates Na+ homeostasis, were not signi®cantly

altered at E1 or E2 but dramatically decreased at E3 (Fig. 1;

100 and 90% decrease for SERCA2 and Na+/K+ ATPase,

respectively, P < 0.001 by ANOVA, Scheffe's post hoc test).

To ascertain that the changes in PMCA2 mRNA were also

re¯ected at the protein level, we performed western blot

analysis utilizing an antibody which previously has been

reported to be speci®c for the PMCA isoform 2 (Stauffer et al.,

1995). Indeed, we did not detect any signal when crude

membranes from kidney, a tissue which does not express

PMCA2, were tested, indicating speci®city of the antibody

(data not shown). In contrast, in spinal cord, one large band of

mol. wt ~135±140 kDa and another one of 130 kDa were

visualized (Fig. 2), in accordance with the 130, 135 and

138 kDa PMCA2 isoforms previously described (Stauffer

et al., 1995). The intensity of both bands decreased by 50% at

E1, and by 80% at subsequent stages (Fig. 2). The results by

western analysis con®rm that the reduction in PMCA2

mRNA is accompanied by a decrease in protein level. Our

®ndings, taken together, raise the possibility of ion dys-

homeostasis as one of the mechanisms leading to neural

dysfunction or injury during EAE.

Table 2 List of primers utilized in RT±PCR

Gene 5¢ primer 3¢ primer Predictedsize (bp)

Plasma membrane Ca2+ ATPase 2 667±690 1659±1681 1014Na+/K+ transporting ATPase b2 subunit 499±522 1039±1062 563SERCA2 548±570 1357±1379 823Synapsin IIa 1884±1909 2531±2556 672Syntaxin 1B 211±235 918±942 731SNAP-25 1±22 588±609 609Rab12 43±66 518±542 499Rab14 201±222 629±651 450Rab16 24±45 468±490 466Cytochrome c oxidase subunit Vb 17±38 382±403 386Mitochondrial cytochrome c subunit IV 91±122 551±572 481Na+ channel b1 subunit 333±356 858±834 525Na+ channel b2 subunit 319±339 735±755 436Na+ channel II 213±233 772±793 580a-Tubulin 68±90 687±708 640

The region selected for the design of primers and the predicted size for each RT±PCR product arepresented. GenBank accession Nos are given in Table 1.

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PMCA2 expression is restricted to grey matterTo determine the cell type-speci®c expression and modula-

tion of PMCA2, we performed in situ hybridization on lumbar

spinal cord sections utilizing DIG-labelled riboprobes spe-

ci®c for this isoform. PMCA2 mRNA was localized exclu-

sively to grey matter (Fig. 3A±D). We did not detect any

signal in white matter even when the substrate reaction was

continued for 48 h. Many cells in the grey matter, including

those in the ventral horn exhibiting motor neuron-like

morphology, expressed PMCA2 (Fig. 3C and D). The

exclusive expression of PMCA2 mRNA in spinal cord grey

matter is consistent with the ®ndings of Stauffer et al. (1997)

reporting PMCA2 immunoreactivity primarily associated

with neurons in the brain.

In situ hydridization studies further con®rmed the results

obtained by microarray analysis or RT±PCR, and indicated a

Fig. 1 Modulation of ion pump expression during the clinical course of EAE as assessed by RT±PCR.RNA obtained from rats exhibiting tail weakness only (clinical score 1, E1), hindlimb weakness (clinicalscore 2, E2), hindlimb paralysis (clinical score 3, E3) and unaffected controls (C) was subjected to RT±PCR. Each lane shows the results obtained with pooled RNA with distinct groups of rats (2±3 rats/group).The expression of a-tubulin, a housekeeping gene not modulated in EAE, was utilized as control forexperimental variations. PCRs for the various ion pumps and a-tubulin were performed in parallelutilizing the same reverse transcription mix for comparison. RNA subjected to PCR without reversetranscription con®rmed the lack of contaminating genomic DNA (negative control). PCR was performedfor 35 cycles. The ®gure shows a representative gel and the graphs present the mean 6 SEM of threeindependent experiments (n = 5±6 with 2±3 rats per group, as indicated above). ***P < 0.001,signi®cantly different from control by ANOVA, Scheffe's post hoc test.



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striking reduction in PMCA2 mRNA levels in EAE spinal

cord (Fig. 3E and F) when compared with controls. The

magnitude of the change observed was variable in individual

rats (n = 6). In some animals, PMCA2 expression was

decreased dramatically to almost undetectable levels, as

illustrated in Fig. 3, whereas in others a less pronounced but

clearly discernible diminution was observed.

To ensure that the decrease in PMCA2 expression was not

due solely to a generic reduction in mRNA, we performed

in situ hybridization utilizing DIG-labelled riboprobes for

a-tubulin, a housekeeping gene not modulated during EAE.

a-Tubulin expression in experimental samples was compar-

able with that of controls (Fig. 3H and I). Adjacent sections

from control and experimental spinal cords were also stained

with cresyl violet, which con®rmed the lack of extensive cell

loss in grey matter (Fig. 3J and K). To ascertain further that

the decrease in PMCA2 mRNA levels was not due to grey

matter cell death, we investigated the distribution of TUNEL-

positive cells in the spinal cord during EAE. TUNEL-positive

cells were scarce in the grey matter even at clinical stage E3,

did not exhibit neuronal morphology and localized mostly to

white matter (results not shown). Taken together, these

®ndings indicate that cell death alone cannot account for the

88% decrease in PMCA2 mRNA levels at stages E1±E3,

although its contribution at more advanced clinical phases

cannot be ruled out. Thus, the reduction in PMCA2 mRNA

levels during EAE may be attributed to decreased gene

expression rather than loss of PMCA2-expressing cells,

especially because it occurs at the earliest clinical times,

before extensive neuronal death is evident (Smith et al.,

2000). It is noteworthy that at this stage of the disease, the

in¯ammatory reaction in the lumbar spinal cord is quite

extensive, as indicated by the distribution of ED-1-immuno-

positive activated microglia/macrophages in the grey and

white matter (Fig. 3L±N).

PMCA2 expression is modulated by kainic acidGlutamate excitotoxicity has been implicated recently in

EAE as a trigger that induces neuronal and oligodendrocyte

loss and axonal injury (Pitt et al., 2000; Smith et al., 2000). It

has also been reported that kainic acid rapidly suppresses

hippocampal PMCA expression, in vivo. Reductions in

PMCA2 levels in the dentate gyrus were already observed

4 h following the administration of kainic acid, in vivo

(Garcia et al., 1997). Taken together, these ®ndings suggested

that glutamate might be a possible modulator of PMCA2

expression in the spinal cord during EAE. To begin assessing

this possibility, we exposed lumbar spinal cord slices to

de®ned medium containing kainic acid (4±100 mM) or

medium alone for 6 h at 37°C. Kainic acid (4 mM) decreased

PMCA2 transcript levels by 86% as compared with controls

(Fig. 4A and B; P < 0.001 by t test). Exposure to 20 or 100 mM

kainic acid completely suppressed PMCA2 expression (not

shown). In contrast, a-tubulin mRNA levels were unaltered,

indicating speci®city in the changes observed (Fig. 4A).

Previous reports have shown that prolonged exposure

of neurons to kainic acid induces cell death, as indicated

by DNA fragmentation and TUNEL immunoreactivity

(Simonian et al., 1996; Cheung et al., 1998; Venero et al.,

1999; Fujikawa et al., 2000; Giardina and Beart, 2001).

Although our cultures were treated with kainic acid only for

6 h, we ascertained that the decrease in PMCA2 expression

was not due to death of grey matter cells, by performing

TUNEL assay on sections obtained from sister cultures

maintained under the same experimental conditions. In

controls, TUNEL-positive cells were infrequent (Fig. 4C)

and were found only occasionally in the white matter and in

the immediate vicinity of the dorsal root entry zone.

Following exposure to kainate, TUNEL-positive cells were

sparse in the ventral horn or intermediate zone grey matter,

regions rich in PMCA2-expressing cells (Fig. 4D), whereas

they increased in the dorsal root entry zone and white matter

(Fig. 4E). We conclude that the decrease in PMCA2 mRNA

levels in response to kainic acid is due to alterations in

expression rather than to cell death.

Synaptic protein expression is decreased atonset of symptomscDNA microarray analysis also indicated changes in the

expression of several neuronal proteins which mediate

exocytosis. The most prominent decrease occurred in

synapsin IIa, syntaxin 1B and SNAP-25 (synaptosome-

associated protein of 25 kDa) transcript levels, whereas the

expression of Rab12, 14 and 16 was increased (Table 1).

Some members of the Rab family GTPases previously have

Fig. 2 Modulation of PMCA2 and synapsin IIa protein levels atdifferent phases of EAE (E1, E2, E3) and control (C) as assessedby western blot analysis. The ®gure shows a representativewestern blot. a-Tubulin was used as control for experimentalvariations. Quanti®cation of PMCA2 (135/138 and 130 kDa) bandintensities revealed a 50% decrease at E1, and 80% at subsequentstages. As expected, a single band of 78 or 50 kDa was visualizedwith the synapsin IIa and a-tubulin antibodies, respectively.Quanti®cation of synapsin IIa band intensities indicated a 30, 60and 80% decrease at E1, E2 and E3, respectively. The experimentwas repeated twice and yielded similar results.

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Fig. 3 Cellular localization and modulation of PMCA2 mRNA assessed by in situ hybridization. Bright ®eld photomicrographs of lumbarspinal cord (L4±L5) sections obtained from controls (A±D) and diseased rats (E and F) hybridized to DIG-labelled antisense riboprobe.(G) A control section hybridized to a sense riboprobe. Labelled cells were found exclusively in grey matter of control sections (A).(B) A higher magni®cation photomicrograph of the section shown in A, demonstrating labelling in cells exhibiting motor neuron-likemorphology in the ventral horn (examples shown within the square) and in smaller cells of the intermediate zone (arrows). (C and D)Higher magni®cation photomicrographs of the cells in B (indicated by arrows and enclosed within the square, respectively). Expression ofPMCA2 dramatically decreased in the spinal cord during EAE (E), con®rming results obtained by cDNA microarray analysis and RT±PCR. Note that the diseased spinal cord is usually enlarged as compared with controls. (F) A higher magni®cation picture of the sectionshown in E. Arrows point at some weakly labelled cells. To ensure that the reduction in PMCA2 expression was not due to a genericdecrease in mRNA levels, adjacent sections obtained from control (H) and experimental (I) spinal cord were hybridized to an antisense a-tubulin riboprobe. (H and I) A group of motor neuron-like cells in the ventral horn expressing the housekeeping gene at comparablelevels. The presence of cresyl violet-stained cells in sections obtained from spinal cord of control (J) and experimental (K) rats indicatedthat the decrease in PMCA2 was not due to non-speci®c cell loss. To con®rm the in¯ammatory reaction in the lumbar spinal cord,consecutive sections were stained with haematoxylin±eosin (not shown) and immunolabelled with ED-1, a marker for activated microglia/macrophages. ED-1-immunopositive cells are rare in control (L), and very abundant in EAE (M) spinal cord grey and white matter. Thegrey matter in the control section is delineated by dashed lines. (N) A higher magni®cation photomicrograph of a region in the sectionshown in M. Arrows point at examples of immunopositive cells. Sections obtained from six controls and six EAE rats (clinical score 3,E3) were analysed. The reduction in PMCA2 expression was observed in all diseased rats, although the magnitude of the change wasvariable and ranged from dramatic decreases as illustrated in this ®gure to more moderate but distinctly discernable reductions. Scalebar = 400 mm (A, E, G, L and M); 200 mm (B and F); 100 mm (C and D); 40 mm (N).



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been implicated in vesicular function (for a review see

Geppert and Sudhof, 1998), although the exact role of the

aforementioned Rab isoforms has yet to be determined.

As synapsins, syntaxins and SNAP-25 are essential for

vesicular function and neurotransmitter exocytosis, we fur-

ther analysed the temporal pattern of their expression during

the course of EAE. At E1, transcript levels of synapsin IIa and

syntaxin 1B were decreased by 93 and 84%, respectively

(P < 0.001 by ANOVA, Scheffe's post hoc test) and remained

low thereafter (Fig. 5). In addition, western blot analysis

indicated 30, 60 and 80% reductions in synapsin IIa protein

levels at E1, E2 and E3, respectively (Fig. 2). SNAP-25

expression showed a tendency to decrease at E1 and E2, but

the results were more variable and did not reach statistical

signi®cance (Fig. 5). By E3, however, SNAP-25 levels had

decreased by 96% (P < 0.001 by ANOVA, Scheffe's post hoc

test), in agreement with a recent study on chronic EAE in

mice (Ibrahim et al., 2001). In contrast to synapsins, syntaxin

and SNAP-25, the transcript levels of Rab12, Rab14 and

Rab16 were 2.9-, 4.3- and 2.6-fold increased, respectively

(Fig. 6; P < 0.001 for Rab12 and 14, and P < 0.01 for Rab16,

by t test). These results raise the possibility of disturbances in

neurotransmission at onset of EAE symptoms.

Increased cytochrome c oxidase expressionsuggests deregulation of mitochondrialmetabolic activity at E2cDNA microarray analysis indicated a strong increase in

expression of cytochrome c oxidase subunits IV and Vb,

suggesting altered mitochondrial metabolic activity (Table 1).

We corroborated this ®nding by RT±PCR and found that the

most robust increases in cytochrome c oxidase IV and Vb

subunit expression (~11- and 15-fold, respectively) occur at

E2 and E3 (Fig. 7). Mitochondrial dysfunction has often been

associated with neuronal injury in CNS diseases and espe-

cially in response to intracellular Ca2+ overload or oxidative

stress (Fiskum, 2000; Hirai et al., 2001). It is conceivable that

changes in mitochondrial activity contribute to EAE path-

ology in a fashion similar to that observed in other CNS

disorders such as ischaemia.

Reduced sodium channel expression occurs atadvanced clinical phasesSodium channels are critical mediators of impulse conduction

along the axon. Therefore, the reduced expression of Na+

channel b1 and 2 subunits and brain sodium channel II, as

indicated by cDNA microarray analysis, was analysed further

by RT±PCR. We found that the expression of all the

aforementioned channels substantially decreased at E3

(Fig. 8; 80±90% decrease), con®rming the results obtained

by microarray analysis. In contrast, no changes were observed

at E1 and E2. Thus, potential abnormalities in Na+ channel

repertoires in nerve cells leading to impairment in impulse

activity may contribute to the progression of symptoms at

more advanced disease phases.

DiscussionThe present study reports alterations in the expression of

speci®c genes in the lumbar spinal cord during the clinical

course of EAE, with special emphasis on the initial phase of

symptomatic disease. In particular, we analysed the temporal

expression pattern of genes which encode proteins playing

critical roles primarily in neuronal function. Our results,

taken together, indicate that changes in neuronal gene

expression coincide with onset of symptoms. Thus, our

®ndings provide novel insights into the potential molecular

mechanisms that may underlie the recently described

neuronal dysfunction or injury during EAE (Ferguson et al.,

1997; Trapp et al., 1998, 1999; De Stefano et al., 1999; Bitsch

et al., 2000; Smith et al., 2000; Bjartmar and Trapp, 2001;

Bjartmar et al., 2001; Peterson et al., 2001). We propose that

ion dyshomeostasis due to aberrant Ca2+ extrusion and

abnormal neurotransmitter release resulting from anomalies

in the expression of vesicular proteins may promote the onset

of neural de®cits. As the disease progresses, perturbed

mitochondrial function and reduced impulse conduction due

to decreases in Na+ channel expression may exacerbate

clinical symptoms.

Glutamate-mediated changes in PMCA2expression may induce neuronal dysfunction atonset of EAE symptomsIncreases in intracellular Ca2+ often have been associated

with cell dysfunction, injury and death in many pathological

conditions of the CNS, including ischaemia, Alzheimer's and

Parkinson's disease (for reviews see Paschen, 2000; O'Neill

et al., 2001; Paschen and Frandsen, 2001). Yet, the

contribution of Ca2+ dyshomeostasis to EAE and the affected

mechanisms have not been de®ned. A loss in Ca2+ balance

may result from defects in mechanisms that regulate Ca2+

entry, extrusion or intracellular sequestration. A role for

voltage-dependent channel-mediated Ca2+ in¯ux in axonal

and neuronal degeneration during multiple sclerosis and EAE

has been reported (Kornek et al., 2000, 2001). To our

knowledge, our study is the ®rst report implicating a pivotal

role for distinct ion pumps involved in calcium extrusion and

sequestration in EAE pathogenesis.

Whereas PMCAs are major pumps modulating Ca2+

extrusion, SERCA isoforms play critical roles in sequestra-

tion of Ca2+ into the endoplasmic reticulum. Our results

indicate a profound decrease in PMCA2 expression at the

earliest manifestation of symptoms, which is then followed

by a signi®cant reduction in SERCA2 transcript levels at

subsequent clinical phases. Such temporal changes in ion

pump expression may promote accumulation of Ca2+ in the

cytoplasm, increasing the vulnerability of neurons to cal-

406 A. Nicot et al.


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cium-mediated injury. This may be one possible candidate

mechanism underlying neuronal decline, and implicates

PMCA2 and SERCA2 as potential targets for therapeutic

interventions. Importantly, our results may be relevant to the

human disease, as a recent study found a decrease in PMCA2

trancript levels by microarray analysis of three multiple

sclerosis brains (Lock et al, 2002, online supplementary

table B).

The PMCA gene family encodes four isoforms which

initially were considered to be necessary only for the

maintenance of basal Ca2+ levels. However, emerging

evidence indicates that they may play important roles in

pathological conditions (Garcia and Strehler, 1999) espe-

cially after overstimulation of glutamate receptors which

causes intracellular Ca2+ overload (Choi, 1992; Mody and

MacDonald, 1995). PMCA2 is expressed primarily in brain

and heart, in a region- and cell-type speci®c manner (Garcia

and Strehler, 1999). The highly restricted tissue distribution

of PMCA2 suggests that this isoform plays unique,

specialized and non-redundant roles in the CNS. Indeed,

PMCA2-null mice exhibit deafness, and unsteady gait and

balance, indicating that the presence of other isoforms does

not compensate for the absence of PMCA2 (Kozel et al.,

1998).

The triggers that induce changes in the expression of Ca2+

pumps during EAE have not been de®ned. Glutamate is an

appealing candidate because of its well-known effects on

Ca2+ homeostasis (Choi, 1992) and because of the recently

reported contribution of excitotoxicity to EAE pathogenesis

(Pitt et al., 2000; Smith et al., 2000). Administration of

AMPA/kainate antagonists ameliorates EAE symptoms in

mice and rats, and prevents neuronal and oligodendrocyte

loss and axonal damage. Moreover, kainate-induced decrea-

ses in PMCA1 and PMCA2 expression in hippocampal

pyramidal cells are followed by neuronal death, in vivo

(Garcia et al., 1997). These ®ndings raise the possibility of

kainate receptor-mediated alterations in PMCA2 expression

in the spinal cord during EAE. In fact, we found that exposure

of spinal cord slice cultures to relatively low concentrations

of kainate (4 mM) for 6 h dramatically decreased PMCA2

transcript levels. Thus, even low glutamate levels may be

suf®cient to affect PMCA2 expression when cells are exposed

to the neurotransmitter continuously. We propose that, at

onset of EAE, persistent stimulation of kainate receptors

following moderate but sustained elevations in glutamate

concentrations may lead to downregulation of Ca2+ pump

expression, promote Ca2+ overload and initiate the previously

described injury cascade (Shields et al., 1998).

Fig. 4 (A) Modulation of PMCA2 expression by kainic acid in spinal cord slice cultures, assessed by RT±PCR analysis. C = control, i.e. slices maintained only in de®ned medium and vehicle; KA = slicesmaintained in medium containing 4 mM kainic acid; (PMCA2) negative control, i.e. RNA subjected toPCR without reverse transcription to ascertain the lack of contaminating genomic DNA. The graph in Bshows the densitometric quanti®cation of the bands in the gel and re¯ects the mean 6 SEM of threeindependent experiments (n = 6, ***P < 0.001 by t test). To rule out the possibility that the decrease inPMCA2 expression was due to cell death, slices in sister cultures were sectioned for assessment ofTUNEL-positive cells. (C and D) Photomicrographs of a representative region in the ventral horn incontrols and kainic acid-treated slices, respectively. Note that TUNEL-positive cells are absent in controls(C) and very few in experimental samples (D, arrows). In kainic acid-treated sections, small, TUNEL-positive cells were con®ned primarily to the edge of the dorsal root entry zone (E, arrows). Scalebar = 100 mm.



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Aberrant neurotransmitter exocytosis may affectneuronal communication during the earlycourse of the diseaseIn addition to PMCA2, the expression of synapsin IIa and

syntaxin 1B, vesicular proteins that are crucial for exocytosis

in presynaptic terminals, was decreased dramatically at onset

of clinical symptoms. Synapsins are a family of synaptic

vesicle-associated phosphoproteins that are required for the

tethering of vesicles to each other and to cytoskeletal proteins

such as actin. Phosphorylation of synapsins liberates vesicles

from the cytoskeleton, enabling their traf®cking in the

presynaptic terminal. Synapsins are essential for the forma-

tion, maintenance and regulation of the reserve synaptic pool

in the vicinity of the active zone. Decreases in synapsin

Fig. 5 Modulation of genes encoding proteins involved in vesicular function during the course of EAE asassessed by RT±PCR. Each lane shows the results obtained with distinct groups of rats (pooled RNAfrom 2±3 rats/group). The expression of a-tubulin, a housekeeping gene not modulated in EAE, wasutilized as control for experimental variations. PCRs for the various vesicular proteins and a-tubulin wereperformed in parallel utilizing the same reverse transcription mix. RNA subjected to PCR without reversetranscription ascertained the lack of contaminating genomic DNA (not shown). PCR was performed for35 cycles. The ®gure shows representative gels, and graphs present the mean 6 SEM of threeindependent experiments (n = 5±6 with 2±3 rats per group, as indicated above). ***P < 0.001,signi®cantly different from control by ANOVA, Scheffe's post hoc test.

408 A. Nicot et al.


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expression reduce the number of synaptic vesicles distal to

the active zone, leading to synaptic fatigue (Pieribone et al.,

1995; Rosahl et al., 1995). Moreover, perturbations in

synapsin function inhibit or slow down neurotransmitter

release.

Syntaxins belong to a class of proteins called SNAREs

(soluble N-ethylmaleimide-sensitive factor attachment

protein receptor) hypothesized to be essential for the docking

and fusion of synaptic vesicles. Vesicle-associated SNAREs,

such as synaptobrevin, and target membrane-associated

SNAREs, such as syntaxins and SNAP-25, form a high

af®nity complex. Formation of the SNARE complex draws

vesicles to the target membrane and induces membrane

fusion. In our experimental paradigm, we observed a decrease

in both syntaxin 1B and SNAP-25. However, the dramatic

reduction in syntaxin 1B occurred at onset of symptoms,

whereas SNAP-25 showed a tendency to decrease, which was

statistically signi®cant only at a later clinical stage (E3).

Hence, changes in the expression of some synapsin and

SNARE proteins can interfere with normal neuronal com-

munication during EAE.

The notion of synapse pathology is suggested further by

changes observed in Rab12, 14 and 16 expression. Rab

isoforms belong to the Ras superfamily of small GTP-binding

proteins, and have been implicated in the attachment of

vesicles to target membranes. A member of this family, Rab3,

is localized to synaptic vesicles and limits the amount of

neurotransmitter released in response to Ca2+ signal.

However, a similar function for Rab12, 14 and 16 has not

yet been de®ned. Rab12 and 14 are also expressed in

oligodendrocytes and may play additional roles (Burcelin

et al., 1997). Therefore, the exact contribution of the

aforementioned Rab isoforms to EAE pathogenesis remains

to be determined.

Perturbations in mitochondrial function maycontribute to cellular injury at advanced EAEstagesCellular injury often has been associated with disturbances in

mitochondrial function. Interestingly, our microarray analysis

indicated a pronounced increase in the expression of

mitochondrial cytochrome c oxidase subunits IV and Vb, a

®nding which was validated further by RT±PCR. Cytochrome

c oxidase has been used frequently as a marker for neuronal

metabolic activity, especially in pathological conditions

involving oxidative stress. Neurons subject to oxidative

Fig. 6 Modulation of Rab isoform expression during EAE as assessed by RT±PCR. Each lane representsresults obtained with distinct groups of control and EAE rats in three independent experiments (2±3 rats/group). The graphs show the mean 6 SEM of the densitometric quanti®cation of bands (n = 3).***P < 0.001 and **P < 0.01, signi®cantly different from control by t test.



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damage show abnormalities in mitochondrial dynamics even

in the absence of any apparent indication of degeneration

(Sayre et al., 1997; Smith et al., 1997). Elevations in

cytochrome c oxidase expression or function in vulnerable

neuronal subpopulations in Alzheimer's disease, following

traumatic brain injury and preceding apoptotic death of spinal

cord motor neurons after sciatic nerve avulsion, have been

reported (Martin et al., 1999; Harris et al., 2001; Hirai et al.,

2001). Thus, an increase in cytochrome c oxidase expression

may re¯ect aberrant energy metabolism and oxidative

damage in the spinal cord during EAE.

Alterations in sodium channel expression mayexacerbate symptoms by affecting impulseconductionNeurological symptoms in multiple sclerosis and EAE

models associated with extensive myelin loss have been

attributed largely to a failure in impulse conduction in

demyelinated axons. However, our results indicating a

dramatic decrease in the expression of important Na+

channels at advanced EAE stages (E3) suggest that impaired

nerve conduction may also be due to alterations in Na+

channel levels. Indeed, the expression and function of various

other sodium channels are affected after nerve injury,

in¯ammation and demyelination (Dib-Hajj et al., 1996;

Cummins and Waxman, 1997; Schild et al., 1997; Gould

et al., 1998; Black et al., 2000). A change in the expression of

sensory neuron-speci®c sodium channels in Purkinje cells

during EAE has been reported (Black et al., 2000).

In conclusion, our results suggest that the onset of EAE

symptoms may be due partially to disruption of neuronal

function and communication as a consequence of changes in

mechanisms regulating calcium extrusion and neurotransmit-

ter exocytosis. Exacerbation of symptoms at later stages may

result from abnormalities in additional processes including

impulse conduction and energy metabolism. Future studies

will determine the role of the differentially expressed genes in

the onset and progression of EAE and their precise contri-

bution to neuronal dysfunction during the different stages of

the disease.

AcknowledgementsWe wish to thank Dr Peter Dowling for helpful discussions,

and Joseph Menonna for technical advice. This work was

supported by a grant from the F. B. Kirby foundation to S.E.

and by NIH- NS38272 to Y.R.

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Received July 6, 2002. Revised September 10, 2002.

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