Ž .Journal of Neuroimmunology 89 1998 131–141

Role of the target organ in determining susceptibility to experimentalautoimmune myasthenia gravis

A. Hoedemaekers a, J.-L. Bessereau b, Y. Graus c, T. Guyon d, J.-P. Changeux b,S. Berrih-Aknin d, P. van Breda Vriesman a, Marc H. De Baets e,)

a Maastricht UniÕersity, Department of Immunology, P.O. Box 616, 6200 MD Maastricht, Netherlandsb CNRS-UAD-1284, Institut Pasteur, Unite de Neurobiologie Moleculaire, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France´

c The Netherlands Cancer Institute, DiÕision of Immunology, Plesmanlaan 121, 1066 CX Amsterdam, Netherlandsd CNRS-URA-1159, Hopital Marie Lannelongue, 133 AÕenue de la Resistance, 92350 Le Plessis-Robinson, Franceˆ ´

e Maastricht UniÕersity, Department of Neurology, PO Box 5800, 6202 AZ Maastricht, Netherlands

Received 6 February 1998; revised 22 April 1998; accepted 22 April 1998

Abstract

Ž .Injection of anti-AChR antibodies in passive transfer experimental autoimmune myasthenia gravis EAMG results in increasedŽ .degradation of acetylcholine receptor AChR and increased synthesis of AChR a-subunit mRNA. Passive transfer of anti-Main

Ž .Immunogenic Region MIR mAb 35 in aged rats does not induce clinical signs of disease nor AChR loss. The expression of the AChRsubunit genes was analyzed in susceptible and resistant rats. In aged EAMG resistant rats, no increase in the amount of AChR a-subunitmRNA was measured. In vivo AChR degradation experiments did not show any increase in AChR degradation rates in aged resistant rats,in contrast to young susceptible rats. Taken together, these data demonstrate that resistance of the AChR protein to antibody-mediateddegradation is the primary mechanism that accounts for the resistance to passive transfer EAMG in aged rats. q 1998 Elsevier ScienceB.V. All rights reserved.

Keywords: Experimental autoimmune myasthenia gravis; Acetylcholine receptor; Autoimmunity

1. Introduction

Ž .Myasthenia gravis MG is an autoimmune diseasecharacterized by weakness of voluntary muscles and exces-

Ž .sive fatigue reviewed in Drachman, 1994 . Signs andsymptoms of MG are caused by an antibody-mediatedattack against AChR in the postsynaptic membrane, result-ing in impairment of neuromuscular transmission. TheAChR is a transmembrane glycoprotein composed of fourdifferent subunits in a stoichiometry a bgd in embryonic2

type AChR. In the adult form, the g subunit is replaced byŽan ´ subunit reviewed in Hall and Sanes, 1993; Duclert

.and Changeux, 1995 . The immunopathogenic mechanismsresponsible for loss of functional AChR include: cross-lin-king of AChR by antibodies, resulting in increased inter-nalization; activation of the complement system causingfocal lysis of the postsynaptic membrane; direct interfer-

) Corresponding author. Tel.: q31 43 387058; fax: q31 43 3877055;e-mail: mdba@ms-azm-3.azm.nl

ence of a small subset of anti-AChR antibodies withbinding of acetylcholine to the AChR or with ionchannelfunction.

Although the pathogenesis of MG is entirely antibody-mediated, anti-AChR antibody titers or the amount ofAChR complexed with antibody correlate poorly with the

Žseverity of neuromuscular dysfunction Lindstrom et al.,.1976a,b; Roses et al., 1981; Verschuuren et al., 1992 .

However, it has been shown previously that certain AChRa-subunit gene haplotypes are associated with a higher

Ž .susceptibility for MG Garchon et al., 1994 . Furthermore,a higher frequency of microsatellite variants was found inMG patients compared to controls. In addition, analysis ofAChR a-subunit mRNA concentrations in muscle biopsiesfrom MG patients revealed increased levels in the amountsof a-subunit mRNA in severely affected MG patients,whereas no increase was found in moderately ill MGpatients, independently of the anti-AChR antibody titersŽ .Guyon et al., 1994 . It seems therefore likely that theautoantigen itself influences susceptibility and clinicalcourse of MG.

0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0165-5728 98 00126-X

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141132

Passive transfer experimental autoimmune myastheniaŽ .gravis EAMG is a good model to analyse the response of

the target organ to the antibody mediated immune attack inMG. It is induced by injection of monoclonal or polyclonalanti-AChR antibodies. In susceptible rats, clinical signs of

Ždisease and AChR loss develop within 24–48 h Toyka et.al., 1975; Tzartos et al., 1987 . Impairment of neuromuscu-

lar transmission in these rats triggers a cascade of events,resulting in increased levels of mRNA coding for the

Ždifferent subunits of the AChR Asher et al., 1988; Asher.et al., 1990 . The increase in AChR MRNA is accompa-

nied by elevated levels of the mRNAs encoding the myo-Žgenic transcription factors of the MyoD family Asher et

.al., 1993 .Aged BN rats, in contrast to young BN rats, are rela-

Žtively resistant to induction of EAMG Graus et al., 1993;.Hoedemaekers et al., 1997a,b,c . Passive transfer of mono-

clonal anti-AChR antibodies does not induce muscularweakness nor clinical signs of disease in aged rats. Thisresistance could not be attributed to deficient antibodyuptake, increased antibody clearance, inaccessibility of theAChR for antibody, absence of infiltrating macrophages or

Ždeficient complement activation Graus et al., 1993;.Hoedemaekers et al., 1997a,b,c . Therefore, it is most

likely that resistance to EAMG in aged rats resides at thelevel of the target organ. The mechanisms that couldaccount for this age-related resistance include a moreefficient AChR neosynthesis in aged rats or resistance ofthe AChR protein to degradation. In order to test thesehypotheses, we analyzed the AChR gene expression inyoung susceptible and aged resistant rats upon induction ofpassive transfer EAMG. Furthermore, we determined thein vivo AChR degradation rates and analyzed possibledifferences in the composition of the postsynaptic mem-brane in young and aged rats. The results of this study mayelucidate possible factors determining susceptibility andseverity of disease in MG patients at the level of the targetorgan.

2. Materials and methods

2.1. Animals

Ž . Ž .Inbred young 10 weeks old and aged 2 years oldfemale BN rats were obtained from the breeding coloniesof the Department of Experimental Animal Services of the

ŽMaastricht University or from TNO Leiden, The Nether-.lands . All animals were maintained under specified

pathogen free conditions.

2.2. Induction of passiÕe transfer EAMG

Rats were injected intraperitoneally with 20 pmol AChRbinding capacityr100 g b.wt. of concentrated culture su-pernatant containing rat anti-AChR mAb 35 as previously

Ž .described Graus et al., 1993 . mAb 35 is a rat IgG1 mAbdirected against the MIR on the a-subunit of the AChRŽ .Tzartos et al., 1987; Loutrari et al., 1992 . Control ratswere injected intraperitoneally with an equal volume ofPBS. All rats were sacrificed at 48 h after induction ofEAMG. The severity of clinical illness in EAMG wasassessed by measuring weight loss and signs of muscular

Ž .weakness Lennon et al., 1975; Verschuuren et al., 1990 .

2.3. Determination of AChR concentration

The concentration of AChR in carcass or hind limb wasŽdetermined as described previously Lindstrom et al.,

.1976a,b; Verschuuren et al., 1992 . Briefly, frozen tissuewas homogenized and AChR was extracted with 2% Tri-

Žton X-100 Sigma, Brunschwig Chemie, Amsterdam,.Netherlands . An aliquot of 250 ml of each extract was

y9 125 Ž125 .labelled with 2=10 M I-a-Bungarotoxin I-a-BT ,incubated overnight with excess rat anti-AChR antibodiesand precipitated by goat anti-rat antibodies. The AChRconcentration was expressed as pmoles 125I-a-BT precipi-tated.

2.4. Quantification of a-subunit AChR mRNA by RT-PCR

The amount of rat a-subunit mRNA was determined ina competitive RT-PCR. Internal control RNA, transcribedfrom a mutant fragment of human a-subunit AChR DNA,containing the P3Aqexon, was used as an internal control

Ž .as described previously Guyon et al., 1994 . Total RNAŽ .was isolated from Extensor Digitorum Longus EDL mus-

Žcle by guanidine isothiocyanate extraction Chomczynski.and Sacchi, 1987 . The EDL muscle used for quantifica-

tion of AChR a-subunit mRNA levels was removed intoto, to ensure that the amount of RNA extracted derivedfrom comparable numbers of neuromuscular junctions inall samples.

Sense and anti-sense primers were chosen to be comple-mentary to the rat a-subunit cDNA sequence spanning theregion from AA 20 to 230.

5X Primer QPCRy1R :Ž .5X-GGC-TCC-GAA-CAT-GAG-ACT-CG-3X

3X Primer QPCRy2R :Ž .5X-GCA-GAC-GCT-GCA-TGA-CGA-AG-3X

RNA was reverse transcribed into first strand cDNA ina 20 ml reaction volume containing 1 mg of total rat RNAtogether with serial dilutions of internal control RNA,using 25 pmol QPCR-2R and expand reverse transcriptaseŽ .Boehringer, Mannheim, Germany . Subsequent amplifica-tion was carried out in the presence of trace amounts of32 Ž 5 .P labelled QPCR-1R approximately 10 cpmrreaction .PCR products were electrophoresed in an 8% acrylamidegel, dried and subsequently analyzed in a phosphor imager.The amount of radioactivity in the two amplified PCR

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141 133

products was determined and graphed as a function of theamount of internal control RNA. The initial concentrationof target RNA was deduced from the graph at the pointwhere the targetrinternal control ratios1, and was ex-pressed as the number of molecules of rat AChR a-subunitmRNArmg total RNA. The amplification efficiencies oftarget and internal control RNA were verified in a kinetic

Ž .PCR and found to be comparable data not shown .

2.5. In situ hybridization

Extensor carpi radialis muscle was frozen in isopentaneand kept at y808C until use. Cryostat sections were fixedin 4% paraformaldehyde in PBS. cRNA probes from AChR´-and a-subunit were transcribed using T7 or SP6 RNA

Ž .polymerase Promega, Madison, WI, USA and labelledX Ž 35 . Ž .with uridine 5 - a- S thiotriphosphate 1000 Cirmmol .

The ´-subunit probe was derived from a rat cDNA tem-Žplate, subcloned in pSP72 a kind gift of Dr. V. Witze-.mann, Heidelberg, Germany , while the a-subunit probe

Žwas derived from a mouse cDNA template Klarsfeld et.al., 1991 . Hybridization was performed as previouslyŽ .described Fontaine et al., 1988 . To identify muscle end-

plate regions, serial sections were stained for acetylcholin-esterase activity using a modified Karnovsky–Roots stain-

Ž .ing Liu and Salpeter, 1994 . Sections were examined bylight- or dark-field microscopy and recorded with a Hama-matsu C4880 CCD camera. Semi-quantitative analysis wasperformed using the Khoros software package under the

Ž .Unix operating system Konstatinides and Rasure, 1994 .Levels of extrasynaptic transcription were analyzed bydark-field microscopy and expressed as number of grainsper mm2. Synaptic expression of AChR mRNAs wasanalysed on light field images. A fixed sized circularregion of 12 mm radius was superimposed on the synapticregions. The fraction of the area occupied by grains wastaken as an index of mRNA abundance and expressed asarbitrary units.

2.6. DenerÕation of hind limb muscle

Denervation of the left hind limb muscle was performedunder ether anaesthesia by excision of 5–10 mm of the left

sciatic nerve. At 10 days post denervation rats were sacri-ficed and left and right hind limb muscles were separatelyfrozen in liquid nitrogen. The AChR concentration wasmeasured in each hind limb as described.

2.7. In ÕiÕo AChR degradation

In vivo AChR degradation was measured using 125I-a-Ž .BT as described previously Wilson et al., 1983 , with

modifications. Briefly, rats were given a subcutaneoussublethal injection of 2.5 mg 125I-a-BTr100 g b.wt., 24 hprior to induction of EAMG. The dose injected had beendetermined in preliminary experiments and was chosen tolabel approximately 30% of the available a-BT bindingsites. At 24 h after the 125I-a-BT administration, animalsreceived an intraperitoneal injection of mAb 35 or PBS asdescribed above. Rats were sacrificed 48 h after the initial125I-a-BT injection. To determine the amount of AChRlabelled in vivo, excess rat anti-AChR antibodies were

Ž .added to aliquots of in vivo labelled AChR extracts andprecipitated using goat-anti-rat antibodies. Total AChRconcentration was measured by adding 2=10y9 M 125I-a-BT in a radioimmunoassay as described above. Degrada-tion rates at later points in time were not obtained becauseof limited availability of aged rats and high mortality ratesin young mAb 35 injected rats.

2.8. Immunohistochemistry

Muscle biopsy cryosections of young and aged ratswere acetone-fixed for 10 min at 48C and air-dried for 5min. After washing three times in PBS, the sections werepre-incubated in PBS containing 2% bovine serum albu-min and subsequently incubated with the respective mAbor an irrelevant isotype-matched mAb, together with rho-

Ždamine-labelled a-Bungarotoxin Molecular Probes, Eu-.gene, OR, US for 1 h at room temperature, in order to

co-localize AChR in the same section. Mouse anti-s-laminin IgG1 mAb was purchased from the Developmental

Ž .Studies Hybridoma Bank University of Iowa, IA, US .Goat polyclonal anti-agrin antibodies were kindly donated

Žby Dr. J. van den Born University Hospital Nijmegen,. Ž .The Netherlands Raats et al., 1998 . Mouse anti-43K

IgG1 mAb were kindly donated by Prof. Dr. S. Froehner

Table 1Aged BN rats are clinically resistant to passive transfer EAMG

Ž . Ž .Age weeks mAb 35rPBS Ratsrgroup Weight change % Clinical EAMG score

y q qq qqq

10 mAb 35 ns5 y9.7"2.8 y y 3 210 PBS ns5 q1.3"1.6 5 y y y

130 mAb 35 ns7 y0.2"0.9 7 y y y130 PBS ns6 y0.8"0.6 6 y y y

Passive transfer EAMG was induced in young and aged BN rats. Aged rats were clinically resistant, whereas young rats developed significant weight lossand muscular weakness. Weight change is expressed as mean"SD of at least five rats.

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141134

Fig. 1. Resistance to EAMG in aged rats is not a result of increasedŽ .AChR a-subunit mRNA synthesis. Total AChR concentration A and

Ž .number of AChR a-subunit mRNA molecules B were measured inyoung and aged rats at 48 h after induction of passive transfer EAMG.Ž .A EAMG resistant aged rats had similar concentrations of AChRcompared to aged control rats, whereas EAMG susceptible young rats

Ž .showed significant AChR loss P -0.05 . AChR concentration is ex-pressed as pmol AChRrcarcass. Bars represent the mean"SD of at least

Ž .five rats. B In EAMG resistant aged rats no increase in the number ofa-subunit mRNA molecules was found compared to age-matched controlrats. In young rats, however, a significant increase in the mean number of

Ž .a-subunit mRNA molecules was measured P -0.05 . The amount ofa-subunit mRNA is expressed as number of molecules per mg total RNAextracted. Points represent individual rats.

Ž . ŽUniversity of North Carolina, Chapel Hill, NC Froehner,.1984 . Mouse anti-utrophin mAb MANCH0 7 is specific

Ž .for the C-terminal domain of utrophin James et al., 1996Žand was kindly donated by Prof. G. Morris North East

.Wales Institute, Wrexham, UK . After washing with PBS,the slides were incubated for 1 h with FITC labelled

Žanti-mouse or anti-goat Ig Cappel, Organon Technika,.Boxtel, Netherlands . A minimum of 10–15 endplates per

group were examined semi-quantitatively under confocallaser optics with appropriate filters. Results were correctedfor differences in endplate size and expressed as ratiomean fluorescence intensity of FITC to rhodamine stain-ing.

2.9. Statistical analysis

The Wilcoxon rank and Student’s t-test were used forstatistical analysis.

3. Results

3.1. Aged rats are resistant to induction of passiÕe transferEAMG

Upon injection of the mAb 35 aged rats developed noŽ .weight loss nor muscular weakness Table 1 . Young rats

showed severe signs of muscular weakness and lost 9.6"Ž . Ž .1.3% of their initial weight mean"SD P-0.05 . No

Fig. 2. Denervation results in upregulation of AChR a-subunit mRNAand protein synthesis in EAMG resistant aged rats. The amount of

Ž . Ž .a-subunit mRNA A and total AChR concentration B was measured inŽ .young and aged rats 10 days after denervation. A A similar increase in

the number of AChR a-subunit mRNA molecules was found in youngand aged rats. The amount of a-subunit mRNA is expressed as number ofmolecules per mg total RNA extracted. Points represent individual rats.Ž .B This increased AChR a-subunit mRNA synthesis resulted in simi-larly increased AChR concentrations in young and aged rats. AChRconcentration is expressed as pmol AChR per leg. Bars represent themean"SD of at least five rats.

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141 135

Fig. 3. Induction of passive transfer EAMG does not affect synaptic nor extrasynaptic expression of AChR a- and ´-subunit mRNA in aged rats.Ž .Dark-field photographs from muscle biopsies of young and aged EAMG and control rats showing autoradiographic grains. A–D Longitudinal muscle

Ž . Ž . Ž . Ž . Ž .section of aged control A , aged EAMG B , young control C and young EAMG D rat hybridized with AChR a-subunit probe. E–H LongitudinalŽ . Ž . Ž . Ž .muscle section of aged control A , aged EAMG B , young control C and young EAMG D rat hybridized with AChR ´-subunit probe. Bar is 100 mm.

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141136

weight loss nor muscular weakness was observed in PBSŽ .treated control rats. Determination of AChR concentra-

tion revealed no AChR loss in mAb 35 injected aged ratsŽ .compared to age matched control rats Fig. 1A . In con-

Ž .trast, 52"7% mean"SD AChR loss was measured inŽ .young female rats P-0.05 compared to age-matched

control rats. The clinical resistance in aged rats was inaccordance with the absence of AChR loss.

Upon induction of passive transfer EAMG, serum mAb35 levels were comparable in young and aged rats asdetermined by ELISA using Torpedo AChR as antigenŽ .Graus et al., 1993 . Furthermore, immunohistochemicalanalysis of muscle biopsies revealed similar depositions of

Žmembrane attack complex in young and aged rats Graus.et al., 1993 . These results confirm previous experiments

showing that the interaction of the mAb 35 with the AChRis similar in young and aged rats.

3.2. AChR a-subunit mRNA leÕels remain stable in EAMGresistant aged rats

Induction of EAMG in susceptible rats has been shownŽto result in an increase in AChR mRNA levels Asher et

.al., 1993 . We measured the amount of AChR a-subunitmRNA in young and aged rats, before and after injectionof mAb 35. a-subunit mRNA was quantified by competi-tive RT-PCR. The number of a-subunit mRNA moleculesmeasured in aged control rats was 4.6"0.7=108rmg

Ž .RNA mean"SEM and found to be comparable to that inŽ .young control rats Fig. 1B . Injection of mAb 35 did not

result in an increase in the number of a-subunit mRNAŽ 8 .molecules 3.4"0.5=10 rmg RNA in resistant aged

rats. In contrast, the mean number of a-subunit mRNAmolecules increased significantly in young rats, from 4.1"0.5=108rmg RNA in young control rats to 12.1"1.6

Fig. 4. Synaptic expression of AChR a- and ´-subunit mRNA remains unchanged in resistant aged rats, but is strongly reduced in young susceptible rats.Ž . Ž . Ž .ArB Semi-quantitative analysis of synaptic AChR a-subunit A and ´-subunit B transcripts in young and aged EAMG and control rats. In aged rats,induction of EAMG induced no apparent changes in size and density of synaptic clusters, whereas in young rats a strong decrease was measured. Levels of

Ž .synaptic mRNA transcripts are expressed as arbitrary units. Bars represent the mean"SEM of at least 40 analyses. CrD Semiquantitative analysis ofŽ . Ž .extrasynaptic AChR a-subunit C and ´-subunit D transcripts in young and aged EAMG and control rats. Extrasynaptic distribution of AChR a- and

´-subunit mRNA did not exceed background levels as determined by hybridization with the respective sense probes. Extrasynaptic expression is expressedas number of autoradiographic grainsrmm2. Bars represent mean"SEM of at least 18 analyses.

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141 137

=108rmg RNA in susceptible young rats at 48 h afterŽ .injection of mAb 35 P-0.05 .

3.3. DenerÕation results in upregulation of a-subunitmRNA synthesis in EAMG resistant aged rats

To establish whether aged rats are still capable ofincreasing their AChR a-subunit mRNA and protein syn-thesis, hind legs of young and aged female rats weredenervated. Motor nerve section is known to cause adramatic increase in AChR a-subunit mRNA and protein

Ž .synthesis reviewed in Duclert and Changeux, 1995 . Inaged rats, the number of a-subunit mRNA moleculesrmgRNA increased from 7.6"2.5 to 146"27.5=108rmg

Ž . ŽRNA mean"SEM at 10 days after denervation P-. Ž .0.05 Fig. 2A . A comparable increase was found in

young rats, viz. from 9.1"4.4 to 154"24.2=108rmgŽ .RNA P-0.05 . To verify whether the a-subunit mRNA

was also translated into protein, AChR concentration wasmeasured in individual hind limbs. In aged rats, the AChRconcentration increased from 4.9 " 1.1 to 13.4 " 1.8

Ž .pmolrleg P-0.05 . Similar increases were found inŽ .young rats 4.7"1.2 to 10.7"2.3 pmolrleg, P-0.05

Ž .Fig. 2B .

3.4. Synaptic expression of a- and ´-subunit mRNAs is notaffected in aged EAMG rats

In normal innervated muscle, AChR genes are preferen-tially expressed in the subjunctional regions of the musclefiber. Yet, synaptic domains represent less than one per-

Ž .cent of the fibers Hall and Sanes, 1993 . Variations inAChR mRNA levels quantified in total muscle RNA bymass techniques, mainly represent variations in extrasynap-tic regions of the myofiber. This implies that synapticcompensatory mechanisms would have remained unno-ticed in the RT-PCR experiments described above. There-fore, a- and ´-subunit mRNA expression was analysed bymeans of in situ hybridisation experiments.

In aged control rats, both a- and ´-subunit transcriptsŽ .were densely accumulated at the endplate regions Fig. 3 .

Yet, the density and size of the synaptic a- and ´-subunitclusters were lower than those observed in young control

Ž . Ž .rats P-0.001 , Fig. 4 . Injection of mAb 35 in aged ratsdid not result in any apparent changes in the distribution ordensity of the clusters of grains at the level of the end-

Ž .plates Figs. 3 and 4 . Extrasynaptic distribution of both a-and ´-subunit transcripts did not exceed background levelsas determined by hybridization with a- and ´-subunit

Ž .sense RNA probes Figs. 3 and 4 . In contrast, injection ofmAb 35 in young rats elicited a dramatic reduction of thesize and density of the synaptic clusters of grains for both

Ž . Žthe a- and ´-subunit mRNA transcripts P-0.001 Figs..3 and 4 . The overall extrasynaptic distribution of both a-

and ´-subunit transcripts in mAb 35 injected susceptibleyoung rats remained unaffected and did not exceed back-

Ž .ground levels Figs. 3 and 4 , though in some sections, we

Fig. 5. Aged rats are resistant to antibody mediated AChR degradation. Invivo AChR degradation rates were measured in young and aged rats afterinduction of passive transfer EAMG. AChR was labelled with 125I-a-BT24 h prior to induction of EAMG. At 24 h after induction of EAMG nosignificant decrease in amount of 125I-a-BT labelled AChR was found in

Ž .aged rats 12.8"8.6% decrease compared to age matched control rats ,whereas young rats showed a significant decrease of 59.5"4.2% in theamount of 125I-a-BT labelled AChR. Results are expressed as % loss of125I-a-BT labelled AChR. The fraction of AChR labelled at the time

Žpoint of EAMG induction was considered 100% actual fraction of125I-a-BT labelled AChR was 29.6"1.8% and 28.7"1.0% in young and

.aged rats, respectively . Points represent mean"SD of at least five ratsper group.

observed erratic fibers with high levels of a-subunit mRNAŽ .in extrasynaptic regions not shown .

3.5. Aged rats are resistant to antibody mediated AChRprotein degradation

The AChR protein degradation rates were measured innormal and mAb 35 treated young and aged rats. AChRwas labelled in vivo with 125I-a-BT, 24 h prior to induc-tion of passive transfer EAMG. The fraction of AChRlabelled at the moment of induction of EAMG was re-

Ž 125garded as 100% the actual fraction of I-a-BT labelledŽ .AChR was 28.7"1.0% and 29.6"1.8% mean"SD in

.young and aged rats, respectively . Twenty-four hoursafter injection of mAb 35, aged rats showed no significantdecrease in the amount of 125I-a-BT labelled AChR com-

Žpared to age matched PBS treated rats 12.8"8.6 and. Ž .0.2"13.0% decrease, respectively Fig. 5 . MAb 35 in-

jected susceptible young rats, however, showed a signifi-cant decrease in 125I-a-BT labelled AChR of 59.5"4.2%Ž . 125P-0.05 . No significant decrease in the amount of I-a-BT labelled AChR was measured in young PBS treated

Ž .rats 9.9"5.6% .

3.6. Protein composition of the postsynaptic membranechanges with aging

Proteins of the basal lamina and the postsynaptic cyto-skeleton are known to play an important role in clusteringand anchoring of AChR in the postsynaptic membraneŽ .Sanes, 1995 . Possible changes in the amounts of s-

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141138

Ž . Ž . Ž .Fig. 6. Protein composition of the postsynaptic membrane changes with aging. The relative amounts of s-laminin A , agrin B , rapsyn C and utrophinŽ .D were analyzed by confocal laser microscopy. Muscle biopsy cryosections from young and aged rats were stained by immunofluorescence doublestaining using rhodamine labelled a-BT to identify endplate regions together with the respective mAb followed by FITC labelled 2nd step antibodies tostain for postsynaptic membrane proteins. Results are expressed as ratio mean fluorescence intensity FITC to rhodamine staining. A minimum of 10–15endplates per group was analyzed.

laminin, agrin, 43K and utrophin were analyzed semiquan-titatively in muscle biopsies of young and aged rats byconfocal laser microscopy. The amount of s-laminin wassignificantly increased in normal old compared to normal

Žyoung rats mean intensity ratio 0.66"0.06 vs. 0.46". Ž .0.06, P-0.001 , Fig. 6A . The amount of agrin signifi-

cantly decreased with aging from 0.98"0.22 in normalŽ .young rats to 0.64"0.10 in normal aged rats P-0.001 ,

Ž .Fig. 6B . Similar to s-laminin, the amount of 43K proteinalso increased with aging from 1.22"0.15 to 1.52"0.15,

Ž .respectively in normal young and aged rats P-0.001 ,Ž .Fig. 6C . The amount of utrophin was comparable in

Ž .young and aged rats Fig. 6D .

4. Discussion

In this study we examined the role of AChR in deter-mining susceptibility to EAMG using susceptible young

and resistant aged rats. No increase in AChR a-subunitmRNA was found upon induction of EAMG in aged rats.In contrast, a threefold increase in AChR a-subunit mRNAwas measured in EAMG susceptible young rats by compet-itive RT-PCR. Nevertheless, upon denervation aged ratswere found to be potentially capable of increasing theAChR a-subunit mRNA synthesis to the same level asyoung rats. In situ hybridization showed that the distribu-tion of a- and ´-subunit transcripts was confined to thesynapse and was not increased in aged rats after injectionof mAb 35. These results suggest that injection of mAb 35in aged rats does not induce AChR neosynthesis to com-pensate for AChR loss. Measurements of in vivo AChRdegradation rates confirmed that in aged rats the AChRprotein in the postsynaptic membrane is resistant to anti-body mediated degradation. Semiquantitative analysis ofseveral proteins of the postsynaptic membrane revealedthat the protein composition of the basal lamina andcytoskeleton changes with aging.

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141 139

In both MG and EAMG, the antibody mediated AChRŽloss results in neuromuscular transmission defects Elmq-

vist et al., 1964; Stalberg et al., 1974; Verschuuren et al.,.1990; Hoedemaekers et al., 1997a,b,c . This impairment of

neuromuscular transmission has been reported to causeincreased transcription of the AChR a-, b-, ´-,and d-sub-

Žunit genes in both MG and EAMG Asher et al., 1988,.1990; Guyon et al., 1994 . Concomitantly, an increase in

MRF4 and myogenin mRNA was observed in EAMG,Žwhereas the levels of MyoD remained unchanged Asher

.et al., 1992; 1993 . In our study, however, induction ofEAMG in resistant aged rats did not result in an increasein the amount of AChR a-subunit mRNA. In addition, nochanges in the levels of MRF4, myogenin or MyoD were

Ž .detected by Northern blotting data not shown . Denerva-tion experiments, however, indicated that aged rats retainthe potential to increase AChR neosynthesis to a levelcomparable to that in young rats. Denervation as well asinduction of EAMG or injection of a-BT are known toincrease the expression of AChR a-subunit and myogeningenes through the impairment of nerve evoked electrical

Ž .activity Asher et al., 1991, 1993; Duclert et al., 1991 .This increase in the levels of AChR a-subunit and myo-genin mRNA is, however, quantitatively stronger in dener-vation experiments compared to EAMG or treatment witha-BT. This quantitative difference may be related to dis-ruption of the contact between nerve and muscle upondenervation andror to blocking of the release of neuralfactors, whereas these processes remain unaffected inEAMG. In situ hybridization experiments indicated thatthe expression of a- and ´-subunit transcripts remainsrestricted to synaptic areas in aged rats. Furthermore, noincrease in synaptic expression of a- and ´-subunit geneswas observed in EAMG resistant aged rats, excluding thepossibility of local compensatory AChR neosynthesis bysubsynaptic nuclei. Altogether, these results suggest that inEAMG resistant aged rats, neuromuscular transmission isfunctional and electrical activity-dependent repression ofextrasynaptic AChR gene expression is maintained. Thesefindings are supported by data from stimulated single fiberelectromyography experiments, showing that neuromuscu-lar transmission is not disturbed in EAMG resistant aged

Žrats compared to age-matched control rats Hoedemaekers.et al., 1997a,b,c .

Interestingly, we observed a strong synaptic decrease ina- and ´-subunit mRNA transcripts in EAMG susceptibleyoung rats. These results do not match with those ofprevious experiments in Lewis rats, which revealed a

Žsynaptic increase in a-subunit gene expression Asher et.al., 1993 . This difference may have resulted from a lower

Ždose of mAb 35 injected, resulting in less AChR loss 44%AChR loss in Asher et al., vs. 52% AChR loss in our

.experiments and less destruction of the neuromuscularjunction. Under normal conditions, motor innervation elic-its a compartmentalisation of AChR gene expression insynaptic domains by two distinct mechanisms. In extrasy-

naptic regions the electrical activity that spreads along thefiber is mainly responsible for the repression of AChRgene transcription, while nerve-derived factors and thespecialized basal lamina sustain high levels of AChR

Žbiosynthesis in the synaptic regions reviewed in Duclert.and Changeux, 1995; Sanes, 1995 . The maintenance of

high numbers of AChR at the endplate thus requirespositive signals. Recently it has been shown that positivesignalling by neuregulin is required for maintenance of

Ž .high AChR density in adult rats Sandrock et al., 1997 .Destruction of the endplates in EAMG may interfere withthis positive signalling and subsequently decrease theAChR mRNA transcription. Furthermore, the immune at-tack may interfere with the accessibility of the subsynapticnuclei for neurotrophic factors such as Neu differentiationfactorrARIA or CGRP. Our experiments suggest that inEAMG susceptible rats the increase in a-subunit mRNA,as measured in total muscle by RT-PCR, is mainly extrasy-naptic. Since the increase was only approximately three-fold, it is very likely that the expression levels remainedbelow the detection threshold of the in situ hybridisationexperiments. Alternatively, such an extrasynaptic increasemay not be homogeneously distributed over the musclefibers, as we detected in a few fibers showing a denerva-tion-like morphology. In addition, it has been shown thatcomplement component C5b-9 decreases the amount of

Ž .AChR a-subunit mRNA on myotubes Lang et al., 1997 .Complement activation to the final step of membraneattack complex was demonstrated in muscle biopsy sec-

Žtions of resistant and susceptible rats Graus et al., 1993;.Hoedemaekers et al., 1997a,b,c . However, small quantita-

tive differences between susceptible and resistant rats can-not be excluded, implying that decreased synaptic AChRmRNA transcription might result from a complement me-diated effect

In vivo AChR degradation experiments showed no lossof in vivo labelled AChR in EAMG resistant aged rats,whereas significant loss was measured in EAMG suscepti-ble young rats. Loss of 125I-a-BT in vivo is not due todisplacement by antibody, since the anti-MIR mAb 35

Ždoes not interfere with a-BT binding Tzartos et al.,.1991 . Since the loss of radioactivity with time is an

Žapproximation of the rate of AChR degradation Chang.and Huang, 1975 , these results indicate that in aged rats

the AChR protein in the postsynaptic membrane is resis-tant against antibody mediated AChR degradation.

The postsynaptic membrane is known to change mor-Žphologically in aged rats and humans Courtney and Stein-

.bach, 1981; Wokke et al., 1990 . Length and degree ofbranching of the postsynaptic membrane increase withenlargement of the postsynaptic area, and degeneration ofjunctional folds. Furthermore, it has been shown that mem-

Žbrane rigidity increases with aging Shinitzky and Baren-.holz, 1978 . Several components of the basal lamina and

the postsynaptic cytoskeleton are involved in AChR clus-tering and anchoring. Agrin and the synapse specific s-

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141140

laminin are components of the basal lamina. Agrin hasbeen shown to induce AChR clustering in cultured my-

Ž .otubes Reist et al., 1992 , whereas s-laminin has beenshown to affect synapse formation both in vitro and in

Ž .vivo Noakes et al., 1995; Porter and Sanes, 1995 . 43KŽ .protein and utrophin dystrophin-related protein are com-

ponents of the post-synaptic cytoskeleton and play animportant role in AChR anchoring by the formation of acrosslinking network. Our data indicate that the concentra-tion of these proteins change with aging. Changes in theconcentrations of these or other proteins may increase therigidity of the postsynaptic membrane, which may result inresistance to antigenic modulation and subsequent internal-ization.

The results of this study show that the age-relatedresistance to EAMG is determined by resistance of theAChR to degradation, which may be related to differencesin composition of the postsynaptic membrane in aged rats.Furthermore, these results suggest that susceptibility andclinical course of MG is not only determined by theimmune attack towards the neuromuscular junction, butalso by the target organ.

Acknowledgements

We are greatly indebted to Mr. S. Garbai for his help inCCD microscopy and software usage. We wish to thankDr. B. Schutte for help and advice in confocal lasermicroscopy. We also thank Mrs. M. Vroomen for excellenttechnical assistance, Dr. V. Witzemann for supplying therat AChR ´-subunit cDNA probe and Dr. S. Tzartos forsupplying mAb 35. We thank Prof. S. Fuchs for criticalreview of the manuscript and helpful discussions. Thiswork was supported by grants from ‘Het Prinses BeatrixFonds’, ‘l’Association Francaise contre les Myopathies’,College de France direction de recherches et etudes tech-` ´

Ž . Ž .niques 87r211 and EC Biomed BMHI-CT93-1100 .

References

Asher, O., Fuchs, S., Zuk, D., Rapaport, D., Buonanno, A., 1992.Changes in the expression of mRNAs for myogenic factors and othermuscle-specific proteins in experimental autoimmune myastheniagravis. FEBS Lett. 299, 15–18.

Asher, O., Kues, W., Witzemann, V., Tzartos, S., Fuchs, S., Souroujon,M., 1993. Increased gene expression of acetylcholine receptor andmyogenic factors in passively transferred experimental autoimmunemyasthenia gravis. J. Immunol. 151, 6442–6450.

Asher, O., Neumann, D., Fuchs, S., 1988. Increased levels of acetyl-choline receptor a-subunit mRNA in experimental autoimmune myas-thenia gravis. FEBS Lett. 233, 277–281.

Asher, O., Neumann, D., Witzemenn, V., Fuchs, S., 1990. Acetylcholinereceptor gene expression in experimental autoimmune myastheniagravis. FEBS Lett. 267, 231–235.

Asher, O., Provenzano, C., Fuchs, S., 1991. Regulation of acetylcholine

receptor gene expression in rats treated with a-bungarotoxin. FEBSLett. 282, 242–246.

Chang, C.C., Huang, M.C., 1975. Turnover of junctional and extrajunc-tional acetylcholine receptors of the rat diaphragm. Nature 253,643–644.

Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolationby acid guanidinium thiocyanate–phenol–chloroform extraction. Anal.Biochem. 162, 156–159.

Courtney, J., Steinbach, J., 1981. Age changes in neuromuscular junctionmorphology and acetylcholine receptor distribution on rat skeletalmuscle fibres. J. Physiol. 320, 435–447.

Drachman, B., 1994. Myasthenia gravis. New Engl. J. Med. 330, 1797–1810.

Duclert, A., Changeux, J.-P., 1995. Acetylcholine receptor gene expres-sion at the developing neuromuscular junction. Physiol. Rev. 75,339–368.

Duclert, A., Piette, J., Changeux, J.-P., 1991. Influence of innervation onmyogenic factors and acetylcholine receptor a-subunit mRNA’s. Neu-roReport 2, 25–28.

Elmqvist, D., Hofman, W., Kugelberg, J., Quastel, D., 1964. An electro-physiological investigation of neuromuscular transmission in myasthe-nia gravis. J. Physiol. 174, 417–434.

Fontaine, B., Sassoon, D., Buckingham, M., Changeux, J.-P., 1988.Detection of the nicotinic acetylcholine receptor a-subunit mRNA byin situ hybridization at neuromuscular junctions of 15-day-old chickstraited muscles. EMBO J. 7, 603–609.

Froehner, S., 1984. Peripheral proteins of postsynaptic membranes fromTorpedo electric organ identified with monoclonal antibodies. J. CellBiol. 99, 88–96.

Garchon, H.-J., Djabiri, F., Viard, J.-P., Gajdos, P., Bach, J.-F., 1994.Involvement of human muscle acetylcholine receptor a-subunit geneŽ .CHRNA in susceptibility to myasthenia gravis. Proc. Natl. Acad.Sci. U.S.A. 91, 4668–4672.

Graus, Y.M.F., Verschuuren, J.J.G.M., Spaans, F., Jennekens, F., vanBreda Vriesman, P.J.C., De Baets, M.H., 1993. Age-related resistanceto experimental autoimmune Myasthenia Gravis in rats. J. Immunol.150, 4093–4103.

Guyon, T., Levasseur, P., Truffault, F., Cottin, C., Gaud, C., Berrih-Aknin,S., 1994. Regulation of acetylcholine receptor a-subunit variantsRNA in human myasthenia gravis. Quantification of steady-statelevels of messenger RNA in muscle biopsy using the polymerasechain reaction. J. Clin. Invest. 94, 16–24.

Hall, Z., Sanes, J., 1993. Synaptic structure and development: the neuro-muscular junction. Cell 72, 99–121.

Hoedemaekers, A., Graus, Y., Beijleveld, L., van Breda Vriesman, P., DeBaets, M., 1997a. Macrophage infiltration at the neuromuscular junc-tion does not contribute to AChR loss and age-related resistance toEAMG. J. Neuroimmunol. 75, 147–155.

Hoedemaekers, A., Graus, Y., van Breda Vriesman, P., De Baets, M.,1997b. Age- and sex-related resistance to experimental autoimmunemyasthenia gravis in Brown Norway rats. Clin. Exp. Immunol. 107,189–197.

Hoedemaekers, A., Verschuuren, J., Spaans, F., Graus, Y., Riemersma,S., Van Breda Vriesman, P., De Baets, M., 1997c. Age-relatedsusceptibility to experimental autoimmune myasthenia gravis: im-munological and electrophysiological aspects. Muscle Nerve 20,1091–1101.

James, M., Man, Nt., Wise, C., Jones, G., Morris, G., 1996. Utrophin-dystroglycan complex in membranes of adherent cultured cells. CellMotil. Cytoskeleton 33, 163–174.

Klarsfeld, A., Bessereau, J.-L., Salmon, A.-M., Triller, A., Babinet, C.,Changeux, J.-P., 1991. An acetylcholine receptor a-subunit promotorconferring preferential synaptic expression in muscle of transgenicmice. EMBO J. 10, 625–632.

Konstatinides, K., Rasure, J., 1994. The Khoros software developmentenvironment for image and signal processing. IEEE Trans. ImageProc. 3, 243–252.

( )A. Hoedemaekers et al.rJournal of Neuroimmunology 89 1998 131–141 141

Lang, T., Badea, T., Wade, R., Shin, M., 1997. Sublytic terminalcomplement attack on myotubes decreases the expression of mRNAsencoding muscle specific proteins. J. Neurochem. 68, 1581–1589.

Lennon, V., Lindstrom, J., Seybold, M., 1975. Experimental autoimmunemyasthenia: a model of myasthenia gravis in rats and guinea pigs. J.Exp. Med. 141, 1365–1375.

Lindstrom, J., Seybold, M., Lennon, V., Whittingham, S., Duane, D.,1976a. Antibody to acetylcholine receptor in myasthenia gravis.Prevalence, clinical correlates and diagnostic value. Neurology 26,1054–1059.

Lindstrom, J.M., Lennon, V.A., Seybold, M.E., Whittingham, S., 1976b.Experimental autoimmune Myasthenia Gravis and Myasthenia Gravis:Biochemical and immunochemical aspects. Ann. NY Acad. Sci. 274,254–274.

Liu, E., Salpeter, M., 1994. In situ hybridization and cytochemistry:localization of mRNA at stained neuromuscular junctions with 33 P-labeled probes. J. Histochem. Cytochem. 42, 1407–1411.

Loutrari, L., Kokla, A., Tzartos, S.J., 1992. Passive transfer of experi-mental myasthenia gravis via antigenic modulation of acetylcholinereceptor. Eur. J. Immunol. 22, 2449–2452.

Noakes, P., Gautam, M., Mudd, J., Sanes, J., Merlie, J., 1995. Aberrantdifferentiation of neuromuscular junctions in mice lacking s-lamininrlaminin b 2. Nature 374, 258–262.

Porter, B., Sanes, J., 1995. Gated migration: neurons migrate on but notonto substrates containing s-laminin. Dev. Biol. 167, 609–616.

Raats, C.J.I., Bakker, M.A.H., Hoch, W., Tamboer, W.P.M., Groffen,A.J.A., van den Heuvel, L.P.W.J., Berden, J.H.M., van den Born, J.,1998. Differential expression of agrin in renal basement membranesas revealed by domain-specific antibodies. J. Biol. Chem., in press.

Reist, N., Werle, M., McMahan, U., 1992. Agrin released by motorneurons induces the aggregation of acetylcholine receptors at neuro-muscular junctions. Neuron 8, 865–868.

Roses, A., Olanow, W., McAdams, M., Lane, R., 1981. No directcorrelation between serum antiacetylcholine receptor antibody levelsand clinical state of individual patients with myasthenia gravis. Neu-rology 31, 220–224.

Sandrock, A., Dryer, S., Rosen, K., Gozani, S., Kramer, R., Theill, L.,Fischbach, G., 1997. Maintenance of acetylcholine receptor numberby neuregulins at the neuromuscular junction in vivo. Science 276,599–603.

Sanes, J., 1995. The synaptic cleft of the neuromuscular junction. Dev.Biol. 6, 163–173.

Shinitzky, M., Barenholz, Y., 1978. Fluidity parameters of lipid regionsdetermined by fluorescence polarization. Biochim. Biophys. Acta 515,367.

Stalberg, E., Ekstedt, J., Broman, A., 1974. Neuromuscular transmissionin myasthenia gravis studied with single fiber electromyography. J.Neurol. Neurosurg. Psychiatry 37, 540–547.

Toyka, K.V., Drachman, D.B., Pestronk, A., Kao, I., 1975. MyastheniaGravis: passive transfer from man to mouse. Science 190, 397–399.

Tzartos, S., Hochschwender, S., Vasquez, P., Lindstrom, J., 1987. Passivetransfer of experimental autoimmune myasthenia gravis by mono-clonal antibodies to the main immunogenic region of the acetyl-choline receptor. J. Neuroimmunol. 15, 185–194.

Tzartos, S.J., Barkas, T., Cung, M.T., Kordossi, A., Loutrari, H., Mar-raud, M., Papadouli, I., Sakarellos, C., Sophianos, D., Tsikaris, V.,1991. The main immunogenic region of the acetylcholine receptor.Structure and role in Myasthenia Gravis. Autoimmunity 8, 259–270.

Verschuuren, J., Spaans, F., De Baets, M., 1990. Single-fiber electromyo-graphy in experimental autoimmune Myasthenia Gravis. Muscle Nerve13, 485–492.

Verschuuren, J.J.G.M., Graus, Y.M.F., Theunissen, R.O.M., Yamamoto,T., Vincent, A., van Breda Vriesman, P.J.C., De Baets, M.H., 1992.Role of acetylcholine receptor antibody complexes in muscle inexperimental autoimmune Myasthenia Gravis. J. Neuroimmunol. 36,117–125.

Wilson, S., Vincent, A., Newsom-Davis, J., 1983. Acetylcholine receptorturnover in mice with passively transferred myasthenia gravis: I.Receptor degradation. J. Neurol. Neurosurg. Psychiatry 46, 377–382.

Wokke, J., Jennekens, F., van den Oord, C., Veldman, H., Smit, L.,Leppink, G., 1990. Morphological changes in the human end platewith age. J. Neurol. Sci. 95, 291–310.