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Skeletal Muscle in Motor Neuron Diseases: TherapeuticTarget and Delivery Route for Potential Treatments.
Luc Dupuis, Andoni Echaniz-Laguna
To cite this version:Luc Dupuis, Andoni Echaniz-Laguna. Skeletal Muscle in Motor Neuron Diseases: Therapeutic Targetand Delivery Route for Potential Treatments.. Current Drug Targets, Bentham Science Publishers,2010, pp.1250-61. <inserm-00497537>
Dupuis,L&Echaniz‐Laguna,A 1/1
Skeletalmuscleinmotorneurondiseases:therapeutictarget
anddeliveryrouteforpotentialtreatments
LucDUPUIS1,2*&AndoniECHANIZLAGUNA1,2,3
(1) Inserm, U692, Strasbourg, F-67085 France (2) Université de Strasbourg, Faculté de Médecine, UMRS692, Strasbourg, F-67085
France (3) Département de Neurologie, Hôpital Civil de Strasbourg, 1 Place de l'Hôpital, BP426,
67091 Strasbourg,France
* To whom correspondance should be addressed: Luc DUPUIS, INSERM U692, Faculté de Médecine, bat 3, 8e étage, 11 rue Humann, STRASBOURG, F-67085, France ; Telephone: (+33) 3 68 85 30 91; Fax: (+33) 3 68 85 30 65; e-mail : [email protected]
Dupuis,L&Echaniz‐Laguna,A 2/2
Abstract(237words):
Lowermotorneuron(LMN)degenerationoccursinseveraldiseasesthataffectpatients
fromneonatestoelderlyandcaneitherbegeneticallytransmittedoroccursporadically.
AmongdiseasesinvolvingLMNdegeneration,spinalmuscularatrophy(SMA)andspinal
bulbarmuscularatrophy(Kennedy’sdisease,SBMA)arepuregeneticdiseaseslinkedto
loss of the SMN gene (SMA) or expansion of a polyglutamine tract in the androgen
receptorgene(SBMA)whileamyotrophiclateralsclerosis(ALS)caneitherbeofgenetic
originoroccursporadically.Inthisreview,ouraimistoputforwardthehypothesisthat
muscle fiber atrophy andweaknessmight not be a simple collateral damage of LMN
degeneration,butinsteadthatmusclefibersmaybethesiteofcrucialpathogenicevents
inthesediseases.InSMA,theSMNgenewasshowntoberequiredformusclestructure
and strength as well as for neuromuscular junction formation, and a subset of SMA
patients develop myopathic pathology. In SBMA, the occurence of myopathic
histopathologyinpatientsandanimalmodels,alongwithneuromuscularphenotypeof
animalmodelsexpressingtheandrogenreceptorinmuscleonlyhasleadtotheproposal
thatSBMAmayindeedbeamuscledisease.Lastly,inALS,atleastpartofthephenotype
might be explained by pathogenic events occuring in skeletalmuscle. Apart from its
potential pathogenic role, skeletalmusclepathophysiological eventsmight be a target
fortreatmentsand/orbeapreferentialroutefortargetingmotorneurons.
Dupuis,L&Echaniz‐Laguna,A 3/3
1) Introduction: lower motor neuron degeneration affects first
neuromuscularjunctions
Voluntary locomotion is governed bymotor units, composed of lowermotor neurons
(LMNs) (alpha‐motor neurons) that innervate skeletalmuscle fibers and control their
contraction.MyelinatedaxonsoftheLMNscontactmusclefibersonadiscreteregionof
the fiber, highly speciallized, called the neuromuscular junction (NMJ). A number of
pathologiestargetthiscriticalregion.First,myasthenicsyndromesaffecttheefficacyof
synaptic transmission through either immunological or genetic processes. In these
pathologies,theabsolutenumberofNMJsremainroughlythesamebuttheirefficacyto
triggeramuscleactionpotentialinresponsetomotorneuronstimulationisdecreased
leadingtofatigablemuscleweakness.NMJsarealsoaffectedduringlowermotorneuron
degeneration.Inthisreview,wewillfocusonpathologiesinvolvingLMNdegeneration,
butsparingsensoryinnervation.Thisgroupofdiseasesaffectspatientsfromneonatesto
elderly people and can either be genetically transmitted or occur sporadically in a
family.Thetable1summarizestheclinical featuresofthediseaseswewill focusonin
thisreview,namelyspinalmuscularatrophy(SMA)[1],spinalbulbarmuscularatrophy
(Kennedy’sdisease,SBMA)[2]andamyotrophiclateralsclerosis(ALS)[3].
During LMN degeneration, NMJs are progressively destroyed, leading to fully
denervated muscle fibers and neurogenic figures in histopathology. The figure 1
presents typicalhistopathologicalandelectrophysiological resultsobtained inpatients
afflictedwith a LMN degeneration. One is able to observe thatmuscle fibers of these
patients display spontaneous denervation activities as judged in electromyography
(figure 1A). Histopathology shows the occurence of small grouped, angulated fibers
(figure 1B‐D). It is thought that reinnervation of muscle fibers by the same motor
neuron leads to these grouped fibers. NMJ destruction during LMN degeneration is
associatedwiththedegenerationof lowermotorneuroncellbodies,hencetheirname
andawidelyacceptedparadigmwasthatLMNdeathwasthecauseofNMJdestruction
inthesepathologies.Indeed,muscledenervationbynervetransectionleadstoroughly
similarhistopathological figures,suggestingthat inLMNdegeneration, thesamecause
(disruption of the nerve to muscle communication) leads to the same consequence
(muscle denervation). However, recent histopathological evidences suggest that
Dupuis,L&Echaniz‐Laguna,A 4/4
grouped atrophic fibers are highly homogenous in type in polyneuropathies, but
heterogenousinLMNdegeneration[4].Thereasonforthisdyscrepancyisunknown,but
might indeed reflect profound differences in the mechanisms of
denervation/reinnervationbetweenpurelydenervatingdiseasesanddiseaseswithLMN
degeneration.
Inthisreview,wewouldliketoputforwardthehypothesisthatmusclefibersmightnot
bethecollateraldamagesofLMNdegeneration,butrather,oneoftheirmurderers.For
this,wewillfirstdescribehowmuscleisinvolvedinthepatterninganddevelopmentof
fully functionalNMJsandhowmuscleparticipates in thepathologyofALS, SBMAand
SMA.Wewillalsogiveinsightsintohowdrugsmighttargetmusclepathophysiological
events or target motor neurons but be delivered through muscles to treat LMN
degeneration.
2) Muscleandmotorneuron:apeertopeerdialogtoestablishneuromuscular
junctions
The establishment of neuromuscular synapses is a highly controlled developmental
event.Motor axons, emerging from the embryonic spinal cord, contact newly formed
myofibers around E13. It was long known that motor neurons and muscles are
interdependantfortheirdevelopment.Indeed,duringdevelopment,motorneuronsdie
if deprivedof their targetmuscle[5‐8]. Conversely,muscledevelopment is arrested in
the absence ofmotor neurons. A neurocentric view of the development of NMJs long
prevailed andpostulated that thisprocesswasmainlydrivenbymotorneurons,with
musclefibersbeingpassiveplayersofNMJdevelopment.Indeed,motorneuronssecrete
averyactiveisoformoftheglycoproteinagrin(neuralagrin),thatissufficienttocluster
nicotinic acetylcholine receptors on themyofiber [9, 10] through a complex receptor
pathway involving the tyrosine kinaseMusk and a co‐receptor LRP4 [11, 12]. In this
model,thepresenceofagrinsecretedbythenerveissufficienttoclusterAchRandform
NMJs.However, in theabsenceof nerves, clustersofnicotinicacetylcholine receptors
still form in the correct , central, regionof thedeveloppingmuscle[13,14].Moreover,
diaphragmmusclesfromE14embryosculturedinthepresenceofrecombinantagrinas
wellasmusclesfromtransgenicmiceoverexpressingaminiaturisedformofagrininthe
Dupuis,L&Echaniz‐Laguna,A 5/5
whole muscle fiber retain the formation of AchR clusters in their central region[15].
Conversely,thetransientmuscleoverexpressionofaconstitutivelyactiveformofErbB2
widened the zone of AchR transcription and of the region where NMJs formed[16].
Altogether, these data show that the sitewhere NMJs form in skeletalmuscle is pre‐
patterned bymuscle cells, independantly of nerve derived cues such as agrin. In the
currentmodel,NMJs thus form through a dialog betweenmuscle andmotor neurons,
muscledriving the zonewhere NMJswill formandneurons strengthening thenewly
formedcontactsbythesecretionofagrin.
ThispathwayofNMJdevelopmenthasbeenlargelystudiedinthecontextofcongenital
myasthenicsyndromes inwhichmutations inMusk[17],ormorerecently inagrin[18]
werefound.Uptonow,thereisnodocumentedassociationbetweenthispathwayand
LMN degeneration. However, it is striking to note that a mutation in Musk, when
introduced in knockin mice, not only leads to the full phenotypic spectrum of
myasthenic syndrome, but also to denervation of endplates andmolecular features of
denervation[19].Inlinewiththis,therecentlydescribedmutationinagrininapatient
withcongenitalmyastheniav[18] leads tonotonly topost‐synapticdefectsbutalso to
pre‐synapticpathologysuggestingthattheAgrin/Muskpathwaymightalsobeinvolved
inthemaintenanceofNMJsinadultsand,thuspotentiallyinLMNdegeneration.
3) Musclepathogenicroleinspinalmuscularatrophy
3‐1) spinalmuscularatrophy
Childhoodspinalmuscularatrophy(hereaftercalledspinalmuscularatrophy,SMA)isa
child‐onsetmotorneurondisease involvingmutations in thesurvivalofmotorneuron
gene[20].SMApatientsaredividedinthreeclinicalgroupsaccordingtotheseverityof
their disease[1]. Type I SMA is characterizedby severe, generalizedmuscleweakness
andhypotoniaatbirthorwithinthefirst6months,withdeathusuallyoccuringwithin
the first 2 years. Type II children are able to sit, although they cannot stand orwalk
unaided,andtheysurvivebeyond2years.IntypeIIISMApatientshaveproximalmuscle
weakness,startingaftertheageof18months.
Dupuis,L&Echaniz‐Laguna,A 6/6
Inthehumangenome,theSMNgeneisduplicatedwithahighlyhomologouscopycalled
SMN2.BothSMNandSMN2genesareexpressedandonlyfivenucleotidesaredifferent
between both genes.We showed that even the promoter sequences and activities of
these two genes were strikingly similar[21]. Importantly, only SMN1 deletions cause
SMA,whileupto5%ofindividualsarelackingtheSMN2gene.Thesubtledifferencein
nucleotide sequencebetweenSMN1 and2hasno effect on the encodedopen reading
framebutprofoundeffectsonSMN2 splicing. Indeed,oneof thenucleotidesdivergent
betweenSMN1andSMN2createsanewsplicingsiteinSMN2mRNAbyskippingexon7.
Thus,theSMN1geneproducesexclusivelyfull lengthfullyfunctionaltranscripts,while
thetranscriptsderivedfromSMN2lackexon7.Mostimportantly,theamountofSMN2
protein are strikingly invertly correlatedwith the clinical severity of disease[22, 23],
suggesting that SMN2 is a modifying gene in SMA. Thus, SMA is caused by SMN1
mutation and the severity of the disease is linked to the potential compensation by
SMN2proteinproducts.ThatSMA is causedby the lackof full‐lengthSMNproteins is
largely substantiated by the evidence that ablating specifically the exon 7 in motor
neuronsleadtomotorneurondegenerationinmice[24‐26].
3‐2) ahousekeepingfunctionforSMNprotein?
WhatisthefunctionofSMN,lostinSMA,thatmightleadtomotorneurondegeneration?
SMNformsalargemultiproteincomplexwithat least7otherproteins(calledgemins)
both inthecytoplasmandinthenucleuswhere it isconcentrated inastructurecalled
gems (for "gemini of coiled bodies"), associated with Cajal bodies[27]. This complex
including SMN and gemins appears crucial for the biogenesis of small nuclear
ribonucleoprotein particles (snRNPs) [28‐30] that are involved in the splicing of pre‐
mRNA.ConsistentwithakeyroleinRNAmetabolism,theablationofexon7ofSMNin
eithermuscleorneuronleadstostrongup‐regulationofanumberofgenesinvolvedin
pre‐mRNA splicing, ribosomal RNA processing, or RNA decay [31]. Furthermore, a
recent study used exon microarray in SMA mice and observed widely distributed
splicing defects in numerous mRNAs[32]. Interestingly, while splicing defects were
prominentlyobservedinallthetissuesstudied,thegenesthatwereabnormalyspliced
were different between tissues, suggesting that the selectivity of SMA for the
neuromuscularsystemisexplainedbytissuespecificalterationsinmRNAsplicing.
Dupuis,L&Echaniz‐Laguna,A 7/7
3‐3) SMNisrequiredformusclefunction
TheSMNproteinisubiquitouslyexpressedandthetissuespecificablationofSMNleads
to drastic alterations in the physiology of the targeted tisue[24‐26, 33]. A number of
studieshaveconvincinglyshownthatSMNexpressionwasrequiredformusclestructure
and function. Invitro,Shafeyandcollaboratorsdemonstrated thatSMNknockdown in
C2C12 myoblasts lead to decreased myoblast proliferation and impaired myotube
fusion[34]. Consistently, in vivo, exon 7 ablation of the SMN gene in mouse skeletal
muscle leads toamassivemuscledystrophyanddeathof theanimals [24] andRNAi
knockdownofSMN indrosophilamusclesis letal indrosophila inamuchmoresevere
waythanneuronalknockdown[35].Thesestudieswereallbasedontheknock‐downor
knock‐out of the SMN gene, a situation much more drastic of what occurs in real
pathological situations. Their relevance for the human pathology is however
strengthenedbytheworkofArnoldandcollaborators[36]thathadpreviouslyobserved
decreased proliferation and impaired fusion of type I SMAmyoblasts. More recently,
Martínez‐Hernández and collaborators showed thatmyotubesof fetuses affectedwith
type I SMA were smaller and with abnormal arrangements suggesting delayed
developmentalmaturation[37].Importantly,aboutaquarterofSMAtype3arereported
to have a dystrophic phenotype with high serum creatine kinase (CK) levels and
“myopathic”histopathology[38].Thus,SMNisrequiredfornormalmusclestructureand
developmenteitherinexperimentalsituationsorinSMApatients.
The function of SMN in muscle is likely to be related to sarcomeric structure. In
drosophila bearing an hypomorphic allele of SMN, Rajendra and collaborators in
2007observedaseveredisorganizationofmusclefilamentsassociatedwithdecreased
actinexpression[39].Indeed,SMNhypomorphdisplayedasimilarphenotypethanflies
withablationofonemuscleisoformofactin.TheseauthorssuggestedafunctionforSMN
in sarcomere formation since both endogenous and transgenic SMN localized to the
sarcomericregionof flymuscle.Sucha functionwas furthersuggestedbytheworkof
Walker and collaborators[40]. Indeed, not only SMN, but also its associated proteins
localize to the sarcomeric Z‐disc in both cardiac and skeletalmyofibrils of themouse.
This localization of the SMN complex appeared independent of its role in pre‐mRNA
splicingsincesnRNPsthemselveswerenotfoundinthesarcomere.SMAmutantmuscles
exhibitnumerousmorphologicaldefects, includingvacuolesandalteredZ‐discspacing
Dupuis,L&Echaniz‐Laguna,A 8/8
andanoverall lossofsarcomericuniformityandalignment.Whetherthisphenotypeis
primarilymyopathicorifitisasecondaryconsequenceofdenervationisnotknownbut
these results establish that myofibrils from SMA type I mice display defects that are
consistentwith those observed in othermyopathies. Furthermore, these observations
are consistentwith theoccurenceof congenital heartdefects in SMA.This functionof
SMNinsarcomereformationmightberelatedtothepostulatedroleofSMNinbeta‐actin
mRNAtranslation[41].Inall,thedataavailablepointtoakeyfunctionforSMNinmuscle
developmentandfunction.
3‐4) SMAisadevelopmentalpathologyofNMJs
Recent evidence suggest that the SMA pathology begins at NMJs. Indeed, Kariya and
collaboratorsusedsevere‐andmild‐SMN2expressingmousemodelsofSMAaswellas
material fromhumanpatients tounderstandthe initialstagesofneurodegeneration in
thehumandisease[42].Inthesestudies,theearliestdefectsappearattheNMJ.Indeed,
lack of SMN protein lead to the failure of post‐natal development of the NMJ. In
particular, NMJs of SMA mice showed impaired maturation of acetylcholine receptor
(AChR) clusters into ‘pretzels’ that were reflected in functional deficits at the NMJ
characterized by intermittent neurotransmission failures. Similar results were also
reportedbyKongandcollaboratorswhichobservedimmaturityofNMJsinSMAmiceas
assessed for instance by persistance of embryonic gene expression, reduced post‐
synaptic apparatus and electrophysiological abnormalities[43]. Importantly, the pre‐
synapticpathologyofsevereSMAmiceisdissociatedfromthepost‐synapticpathology,
suggestingthatbotheventsmightbeindependent[44].Thesedataareindeedconsistent
withearlier studies showing reducedexpressionofAchRgenes in SMAmyocytes[36].
Altogether,SMAshouldnowbeviewedasadevelopmentalNMJsynaptopathy
3‐5) SMAasaglobalneuromuscularpathology
GiventhatSMNhasacrucialfunctioninmuscle,andthatSMApathologyisinitiatedat
theneuromuscularsynapse,onecouldhypothesizethatSMAisindeedamuscledisease
spreading to the motor neuron. Indeed, muscle knockdown of SMN leads to a more
drastic phenotype than neuronal knockdown in Drosophila [35]. This is however not
true inall animalmodels, sinceSMN ablation ispartially rescuedbyneuronalbutnot
Dupuis,L&Echaniz‐Laguna,A 9/9
muscle transgenic expression in the nematode[45]. Furthermore, overexpression of
SMNinskeletalmusclesdidnotallowtherescueofthephenotypeofsevereSMAmice,
whileamorewidespreadoverexpressionincludinghighlevelsinneuronsandlowlevels
inmuscle stronglyalleviated thephenotypeof thesemice [46].However, it shouldbe
kept in mind that the transgenic overexpression of SMN2 in these animals might be
sufficientonitsowntofullyrescuethemusclephenotype.Inthissituation,oneshould
notexpectanevenmoreincreasedsurvivalofthemiceafterincreasingSMNexpression.
Inanycase,thebunchoflitteraturecurrentlyavailablestronglysuggeststhatSMAisa
globalneuromuscularpathology, involvingpathologicalevents inmuscleandneurons,
andpotentiallyanumberofothercelltypes(figure2).
4) MusclepathogenicroleinKennedy’sdisease
4‐1) Kennedy’sdisease
Spinalandbulbarmuscularatrophy (SBMA),orKennedy'sdisease, isapurelygenetic
LMNdegenerationcausedbyexpansionofaCAGrepeatinthefirstexonoftheandrogen
receptor(AR)gene, leadingtoanARproteinwithanexpandedpolyglutamine(polyQ)
tract [47]. Like in other triplet repeat disorder, including Huntington’s disease, the
disease develops only in individuals bearing an expansion of the polyQ tract over a
certainthreshold(>36Gln)andthelengthofthepolyQiscorrelatedwithanumberof
clinicalparameters[48,49].SBMAisanX‐linked,genderspecificdiseasesinceonlymale
carriers of a pathogenic mutation are affected. The phenotype of females carrying a
pathogenicalleleisabsentorverymildevenifhomozygousforanexpandedCAGrepeat
[50,51].SBMApatientsdevelopproximalmuscleweakness,fasciculations,andatrophy,
alongwithlowermotorneurondegenerationinthebrainstemandspinalcord[2,52].A
subsetofpatientsalsodisplayadditionalfeatures,associatedwithandrogendysfunction
such as androgen insensitivity, oligozoospermia or azoospermia, testicular atrophy,
feminizedskinchanges,andgynecomastia[53,54].
DifferentanimalmodelsofSBMAhavebeengeneratedintherecentyears.Inparticular,
expansions of CAG repeats have been introduced in knock‐in mice[55‐57] or in
transgenicmice[58‐61].For instance,malemicewitha113polyglutamine tract in the
Dupuis,L&Echaniz‐Laguna,A 10/10
endogenousARdeveloppedanSBMA‐likeneuromuscularpathology,withearlymuscle
pathology but late spinal cord disease and relatively spared motor neurons. In this
mousemodel, therewasprominentearlydeathofmales causedbyobstructionof the
urinary tract [55]. Interestingly, affected males display several signs of androgen
insensitivity,ascommonlyobservedinSBMA.
ThephysiologicalfunctionofthemutantproteininSBMAisextremelywelldocumented.
The AR is a transcription factor, whose transcriptional activity is activated by
testosterone or dihydrotestosterone[62]. After ligand activation, AR drives the
expression of its target genes, including key factors for muscle growth, male
reproductivefunctionandmalesecondarysexualphenotypes[63].Thelackoffunctional
AR protein leads to testicular feminization and complete infertility in both mice and
humans[64, 65]. In skeletal muscle, AR is especially required for the maintenance in
musclemassandfibertype[66]aswellasmusclestrengthinmales[67].
Studiesinanimalmodelsindicatethathormonalligandsarecrucialforthedevelopment
of SBMA. First, only male transgenic mice develop an SBMA‐like phenotype. Second,
castration of transgenic males alleviates the phenotype while testosterone‐treated
females develop the pathology [58, 60, 61]. This is reminiscent of the absence of
phenotype of women carriers of SBMA mutations [50, 51]. Consistently, a phase 2
clinical trial has recently suggested that androgen deprivation by leuprorelin acetate
maybebeneficialtopatients[68]Thus,SBMAisduetoatoxicgainoffunctioninmutant
ARunmaskedbythepresenceoftheligand.
4‐2) MyopathicpathologyinSBMA
Studies in both patients and mice have shown that, apart from lower motor neuron
degeneration,SBMApatientsdisplaymyopathic features. Inparticular,SBMApatients
showedCKelevationinmusclebiopsies[69].Recently,wedescribedafamilywithearly
onset and rapidly progressive SBMAmimicking muscle dystrophy [70]. Three out of
sevenpatients in this familydisplayed increasedCK levels indicating rhabdomyolysis,
suggesting that theoccurenceofsuchmuscleabnormalities is indeedverycommon in
SBMA. It should however be noted that CK elevation is a poor indicator ofmyopathy
since it is subject to large variations and increased in a number of conditions. Apart
Dupuis,L&Echaniz‐Laguna,A 11/11
from plasma CK levels more elevated than expected in denervative diseases, SBMA
patients also often show myopathic abnormalities in muscle biopsies with centrally
nucleatedfibersorothermyopathicfeatures[69]andsuchabnormalitiesdonotappear
tobethesoleconsequenceofdenervation.Inthesameline,Yuandcollaboratorshave
shownthatmusclepathologylongpreceedsspinalcordpathologyinananimalmodelof
SBMA.Furthermore,theseanimalsdisplaymixedfeaturesofmyopathyanddenervation
and showed prominent and lethal myotonic discharges [55]. Thus, while SBMA was
considered as a puremotor neuron disorder, recent research reevaluated this notion
andshowedthatSBMAisamixedconditioninvolvingnotonlyneurogenicdenervation,
butalsomyopathicfeatures.
4‐3) AmyogenicoriginforKDrelatedmotorneurondegeneration?
These findingsprompted to test thehypothesis thatLMNpathologyof SBMApatients
indeed resulted from a myogenic pathology. This idea is further supported by the
crucialroleofARintheskeletalmuscle[63‐65]andbytheintriguingobservationthat
AR accumulates at NMJs[71]. Indeed, the pathology of animal models of SBMA is
primarilymuscular,withbothneurogenicandmyogenicpathologyoccuringlongbefore
motor neuron degeneration is observed [55]. Indeed, in most SBMA animal models,
there is no detectablemotor neuron degeneration [55, 58, 59].Most importantly, the
musclespecificoverexpressionofanon‐expandedARleadstoaSBMA‐likephenotype.
In this model, the phenotype was androgen dependent, witha pathology strictly
affecting males but alleviated by castration[72]. Importantly, mice present with a
pathology not restricted to muscle (muscle necrosis) but also show axonal loss and
denervationrelatedchangesingeneexpressionbutnotlossofMNcellbodies[72].Last,
the pathology did not affect females except when treated with testosterone, and the
cessation of testosterone treatment in females allows complete recovery of the
phenotype [73, 74]. These studies thus provide the convincing proof of principle that
SBMAmight be a LMNdegeneration ofmuscle origin [74] (figure 3). Furtherwork is
neededtodelineatethemechanismslinkingmuscleARandNMJdenervation,aswellas
thepathogenicroleofpolyglutamineexpansion.
Dupuis,L&Echaniz‐Laguna,A 12/12
5) Musclepathogenicroleinamyotrophiclateralsclerosis
5‐1) Amyotrophiclateralsclerosis
Amyotrophic lateral sclerosis (ALS) is the most frequent LMN degeneration of adult
onset. ALS worldwide incidence is estimated to be of 1–3 new cases per 100000
individuals, which ranks ALS as the most frequent neurodegenerative disease after
Alzheimer's and Parkinson's diseases[3]. Like SMA and SBMA, ALS presents with a
progressive paralysis affecting first either limb muscles (spinal onset) or cranial
muscles (bulbar onset). A difference between ALS and other diseases with LMN
degenerationisthatLMNsarenottheonlyneuronalcelltypetobeaffected.Indeed,in
paralleltoLMNinvolvement,uppermotorneurons(corticospinalmotorneurons)also
degenerate.MostoftheALSpatientsdiewithintwotofiveyearsafterthediagnosisbut
the disease is heterogeneous in its duration and clinical presentation. No current
treatment isabletostopthediseaseprocess.RiluzoleremainstheonlyFDAapproved
drugandincreasesthesurvivalofthepatientsbyafewmonths.
MostALScasesarenotassociatedwithafamilyhistoryandare,hence,termedsporadic;
the remainders (20%) are of genetic origin, generally transmittedwith an autosomal
dominantinheritance.SporadicandfamilialALSareclinicallyindistinguishable.Indeed,
even in a single affected family, the clinical presentation of the patients may vary,
strongly suggesting that genetic and/or environmental cues are of pathological
importance[75‐77].Severalgenes includingangiogenin[78],vapb[79],dynactin[80,81],
andmorerecentlytdp43[8286],fus[87,88]andfig4[89]havebeengeneticallylinkedto
familial forms of ALS, but how these specific mutations lead to ALS is currently
unknown[90].TherecentdescriptionofamousemodeloverexpressingmutantTDP‐43
will hopefully lead to new insights in the field[91]. On the contrary, themechanisms
underlyingALSlinkedtomutationsinthesod1gene,thefirstandmajorgenelinkedto
familialALSin1993,havebeenextensivelystudied[75‐77].Itisinterestingtonotethat
variations in the SMN1 and2 genes involved in SMAhave been reported to be a risk
factorinALSalso[92‐97].
Dupuis,L&Echaniz‐Laguna,A 13/13
5‐2) MyopathicfeaturesinALS
Myopathic features have been rarely described in ALS and are certainly much less
frequent than in SMAor SBMA.However, itwas reported that a largenumberofALS
muscle biopsies displayed myopathic features[98, 99] and moderately elevated CK
levels[100].Theextentofthesefindingsishoweververylimitedsincesimilarchanges
in magnitude are observed upon denervation. Most interestingly, a number of case
reportstudiessuggest thateven typicalALSpatientsmight initiallydisplaymyopathic
features.Forinstance,apatientbearingaSOD1mutationdisplayedCKelevationbefore
EMG abnormalities [101]. Moreover, ALS patients might present ragged‐red muscle
fibers,indicativeofmitochondrialpathologyintheirmuscles[102].Itisnoteworthythat
mitochondrial alterations in skeletal muscle might contribute to the pathology[103].
However, this interpretation is complicated by the fact that detectablemitochondrial
dysfunctioninmuscleisaratherlateevent[104,105]thatcouldindeedbetheresultof
muscle denervation. Thus, ALS is also associated with myopathic changes, although
milderthaninSMAorSBMAandmitochondrialdefectsinmusclemightbeassociatedin
atleastasubsetofALScases.
5‐3) NMJdestructionisthecriticalpathogeniceventinSOD1‐linkedALS
StudiesinALSduringthelasttenyearshavebeenfocusedontheelucidationofSOD1‐
linkedALS, through the use of transgenicmice overexpressingmutant SOD1 (mSOD1
mice) isoforms. Between 3 and 12 months of age, mSOD1 mice develop muscle
weakness linked to muscle denervation and both upper and lower motor neuron
degeneration.ThefirsteventinthisdiseaseprocessisthedestructionoftheNMJ,more
specifically of the postsynaptic apparatus, followed by axonal degeneration and late‐
onset degeneration ofmotor neuron cell body[106, 107]. In years 2000, the accepted
paradigmwas that neuronal expression ofmSOD1was responsible formotor neuron
degeneration,leadingtomuscledenervationandparalysis.Thus,mostresearchefforts
sought tounderstandthemechanisms leading frommutantSOD1expression inmotor
neuronstocelldeath.Indeed,apoptosiswasshowntobethemainmechanismofmotor
neurondeath inALSandmultiplesignalingpathwaysarenowknowntocontribute in
vivo to the degeneration of motor neuron cell body [108]. However, none of these
Dupuis,L&Echaniz‐Laguna,A 14/14
pathwaysdidseemtocontribute tooverallsurvivalofmSOD1miceandallpreclinical
trialsbasedon interferencewithcelldeathpathwaysonlymarginallyaffectedmSOD1
micelifespanMostimportantly,clinicaltrialsbasedonthoseresults,includingtherecent
minocyclinetrial[109],notonlydidnotimprovepatientoutcome,butinsomeinstances
evenworsenit.
Ontopofthis,weandothershaveshownthatevenacompleterescueofmotorneuron
cellbodiesdoesnotcuremSOD1mice.Forinstance,geneablationofbax,akeyplayerin
motor neuron apoptosis, completely rescued mSOD1 mice motor neurons from
apoptosis, while onlymodestly delayingmuscle denervation and animal death [110].
Along the same line, sodium valproate, a drug inhibiting epigenetic chromatin
remodelingduringapoptosis,oraninhibitorofp38MAPK,aproteinkinaseinvolvedin
initiating cell death, are able to rescue the cell bodies but have no effects onmuscle
denervationandanimallifespan[111,112].Last,ablatingmSOD1frommotorneurons,
while delaying onset modestly, did not cure the pathology[113]. Thus, the primary
pathogenic event, determining the survival of the animal, is not motor neuron death
itself, but rather the loss of motor neuron/muscle contacts. Hence, preservingmotor
neuroncellbodiesistherapeuticallynotsufficientsincetherescuedmotorneuronsare
unable to recreate destroyed NMJs. In this model, motor neuron degeneration
representsalate,secondaryconsequenceofsynapticdestruction
5‐4) IsmusclemutantSOD1expressionsufficienttotriggerALS?
InSBMA,recentworksuggestedthatthemuscleoverexpressionofAR,evennotbearing
an expanded polyglutamine tract could lead to the pathology[72]. Could mSOD1
expression in muscle also lead to such a concept in ALS? Indeed, the transgenic
overexpression of mSOD1 was sufficient to induce severe muscle atrophy associated
with significant reduction in muscle strength, sarcomere disorganization, significant
changes in mitochondria morphology and disposition, and disorganization of the
sarcotubular system. The authors involved several signalling pathways, including
autophagyandoxidativestress inthedeleteriouseffectsofmusclemSOD1expression.
However,whilemuscle‐restrictedmSOD1expressionpromotespinalcordastrocytosis
and inflammation, no motor neuron loss was observed. Thus, in this study, muscle
mSOD1 expressionwas not sufficient to support full blown ALS [114]. A very recent
Dupuis,L&Echaniz‐Laguna,A 15/15
studyconfirmedandsignificantlyextendedthis initialwork.WongandMartincreated
transgenicmice overexpressing eitherwild type or twomutations in the SOD1 cDNA
under the control of the skeletal muscle actin promoter[115]. These authors, in a
manner consistentwithDobrowolnyand collaborators, found important clues for this
overexpression triggering localized oxidative stress and subsequent muscle atrophy.
Most interestingly, they found NMJ denervation and animals developped paresis and
motorneurondegeneration.Inthisstudy,muscleSOD1expression,eitherwildtypeor
mutantwassufficienttotriggerthefullALSphenotype.Itisintriguingtonotethatthere
wasnodifferencebetweenwildtypeandmutantSOD1overexpression,reminiscentof
theeffectsofwildtypeARoverexpressioninmuscle[72].
Intriguingly,theknock‐downofmSOD1inmuscledidnotappeartobesufficienttoslow
downthepathology [116,117].Thissuggestseither thatmusclemSOD1expression is
not key in the pathology, or that even the small remaining amounts of mSOD1 in
knockeddownmuscleswere sufficient to lead to their toxic effects, or that theknock
downoccuredtoolateinanimallifespantoyielditsprotectivepotential.Inall,muscle
appears tobeoneof the sitesofmSOD1 toxicity, although it remainselusivewhether
NMJdestructionisprimarilydrivenbymusclemSOD1expression.
5‐5) MuscleenergymetabolismabnormalitiesasacauseofNMJdestruction
WhatcouldbethemechanismsunderlyingmuscletoxicitytomotorneuronsinALS?We
haveshownthattheneuriteoutgrowthinhibitorNogo‐Aismassivelyexpressedinthe
skeletal muscle of mSOD1 mice and ALS patients [118‐120]. Importantly, Nogo‐A
ablationincreasedmSOD1micelifespanwhile itsoverexpressioninmusclefibers lead
toNMJshrinkage[119].Thesefindingsprovidetheproofofprinciplethatalterationsin
musclegeneexpressionareabletomodulatethediseaseprocessinmSOD1mice.
From a more general point of view, our work suggests that abnormalities in muscle
energymetabolismmightthedirectcause.Weandothershaveobservedthatmusclesof
mSOD1mice display decreased cellular levels of ATP[121, 122], alongwith increased
expression of mitochondrial uncoupling proteins and of markers of both lipid and
carbohydrate use[123, 124]. Indeed, as previously mentioned, mitochondrial
dysfunctionoccuredinmusclesofmSOD1mice,and,toalesserextent,inmusclesofALS
patients[103].These findingssuggestedthatmusclesofALSpatientsandmSOD1mice
Dupuis,L&Echaniz‐Laguna,A 16/16
show an exacerbation of their energy metabolism. To determine whether this was
sufficienttoleadtomotorneurondegeneration,westudiedtheneuromuscularsystemof
mice overexpressing the mitochondrial uncoupling protein 1 in their muscles, as a
model ofmuscle restricted hypermetabolism. These animals displayed age‐dependent
deterioration of the NMJ that correlatedwith progressive signs of denervation and a
mild late‐onset motor neuron pathology[125]. Furthermore, NMJ regeneration and
functional recoverywereprofoundlydelayed following injuryof the sciatic nerve and
crossing these mice with mSOD1 mice exacerbated ALS‐like pathology[125]. Thus, a
muscle restricted mitochondrial defect is sufficient to generate motor neuron
degeneration.
5‐5) ALSasasystemicpathology
Muscle hypermetabolism of mSOD1 mice has broad consequences on the overall
energetic physiology of these animals, and provides clues for a potential therapeutic
strategy.Indeed,mSOD1miceshowbodyweightdeficitascomparedtowildtypesdue
toanincreaseinthebasalmetabolicrate,asareflectofmusclehypermetabolism[123].
Furthermore,energymetabolism,especially lipidmetabolism,wasstrikinglyalteredin
these animals[126]. We sought to determine whether correcting energy deficit of
mSOD1mice could delay their pathology and fed these mice with a diet enriched in
saturated fats. High fat fed mSOD1 mice lived longer and showed reduced muscle
denervation associated with improved motor neuron survival, a finding recently
confirmedbyMattson'sgroupinanothermSOD1strain[123,127].
ALSpatientsalsoshowabnormalitiesintheirsystemicenergyhomeostasis.Couratier’s
group has found an increased energy expenditure in these patients, similar to what
observed in mSOD1 mice[128‐132]. Most importantly, ALS patients show increased
blood lipid levels and hyperlipemia was associated with increased survival [133]and
betterrespiratorycapacityinthesepatients.However,thetranslationfrommSOD1mice
toALSpatientsisnotstraightforwardsincepatientsshowanimportanttrendtobecome
insulinresistant[134].Intheseconditions,theincreaseinenergyintake,intheformof
an high fat diet, might precipitate insulin resistance and worsen patient outcome.
Potential therapeutic strategies based on nutrition should take this trend to insulin
resistanceintoaccount.
Dupuis,L&Echaniz‐Laguna,A 17/17
In all, skeletal muscle is involved in ALS through an increase in its metabolic rate,
precipitating systemic defects in energy homeostasis, such as leanness. Musclemight
also directly influence NMJ stability through energy metabolism defects and yet
unknownsignallingpathways(figure4).
6) Muscleasadrugtargetinlowermotorneurondiseases
The previous results in either SMA, SBMA or ALS point to muscle being an active
contributor to these pathologies. Based on these studies, a number of therapeutic
strategieshavebeenproposedtotargetmusclepathology(figure5).
In SMAmice, treatmentwith follistatin, an inhibitorofmyostatin [135] that increases
musclemass,wasreportedtolessendiseaseseverityinSMAmice[136]butnotinALS
mice[116].InSMAmice,butnotinALSmice,follistatin‐treatedmiceperformedbetter
thantheirvehicle‐treatedlittermates.Themostwidelydocumentedfactorthatcouldbe
beneficial in LMN degeneration through a muscle action is IGF1. In 2002, Antonio
Musaroandcolleaguesgeneratedatransgenicmouselineoverexpressinganisoformof
IGF1 (termed mIGF1) that is retained locally in skeletal muscle and does not
systematically diffuse [137]. Crossing these mice with either mSOD1 mice [138] or
SBMAmice[139]potentlyincreasedthelifespanofbothmodels.InmSOD1mice,mIGF1
expressiondelayedthedisease,enhancedsurvival,stabilizedNMJsandhadalsodistant
protectiveeffectsbydecreasingastrocytosis[138]. InSBMAmice,mIGF1 increasedAR
phosphorylation, promoted the degradation of aggregated AR, rescued the muscle
phenotype,increasedmotorneuroncountsandpotentlyincreasedthelifespanofSBMA
mice[139].Thesedatathussuggestthatapharmacologicalinterventionsolelytargeted
atmuscles is able toprovideglobalprotection tomotorneurons. Indeed, it shouldbe
noted that the protectionmediated by exercise [140‐142], high fat feeding[123, 127],
creatine[143]orcarnitine[144]inanimalmodelsofLMNdegenerationmightwellbe
duetoanactionofthesetreatmentsonskeletalmuscleratherthanonmotorneurons.
Suchanindirecteffectissuggestedbytheprofoundeffectsofexerciseongenesinvolved
inNMJmaintenance[145].
Dupuis,L&Echaniz‐Laguna,A 18/18
7) Muscle a a delivery route to curemotor neurons in lowermotor neuron
diseases
WhileskeletalmuscleisinvolvedinatleastsomeofthekeypathogeniceventsinLMN
degeneration,onecouldalsoconsidertousemuscleasawaytotargetmotorneurons
(figure6).Forinstance,localproductionofneurotrophicfactorsbytheskeletalmuscle
might sustain neuronal survival and enhancemuscle reinnervation. Such an example
wasprovidedbythestudyofLiandcollaborators[146]showingthatmuscleproduction
of GDNF, but not astrocyte derived‐GDNF delayed disease onset and slowed down
diseaseprogressionofmSOD1mice.
Inthesameline,retrogradetransportofatherapeuticgenethroughmuscleinjectionsof
aviruscouldprovideprotectiontomotorneurons.Twodifferentexamplesprovidethe
proof of concept for such a strategy. First, delivery of IGF1 through AAV vectors in
skeletalmuscles leads toprotectionofmotorneuronsanddelayeddiseaseonset.This
protective effect might be due to retrograde transport of viral particles in the axon,
leading to IGF1 production inmotor neurons and autocrine protection of these cells.
Alternatively, and as suggested byA.Musaro and colleagues, IGF1 has profound local
effects on skeletalmuscle thatmight account for its potential for therapeutics in ALS
[138].Asecondexampleofa therapeutic strategyaimingatdeliveringaneurotrophic
factor though skeletal muscle is VEGF. M. Azzouz and collaborators used a lentiviral
vectortoprovideVEGFdirectlytomotorneurons throughretrogradetransport.They
observed that a single injection of a VEGF‐expressing lentiviral vector into various
musclesdelayedonsetandslowedprogressionof inmSOD1mice[147]. Interestingly,
eitherviraldeliveryofVEGForIGF1sloweddiseaseprogressionevenwhentreatment
wasinitiatedattheonsetofdisease,suggestingthatthesestrategiesmightberelevant
inALSpatients.
Viral deliverymight prove useful not only for providing a neuroprotective factor, but
also to restore the causative deficient gene. Indeed,multiplemuscular injections of a
lentiviral vector expressing SMN restored SMN expression in motor neurons and
delayed the pathology of severe SMA mice [148]. Conversely, similar viral strategies
mightbeusedtosilencethetoxicgenewhengainof functionof themutantprotein is
showntoleadtothepathogeniceffects.Inthisline,targetingofSOD1mutationsthrough
siRNA has been achieved by Ralph and collaborators in mSOD1 mice [149] through
Dupuis,L&Echaniz‐Laguna,A 19/19
muscleinjectionsofviralvectorsretrogradelytransportedtomotorneurons.Thismode
of delivery achieved what remains probably the most impressive protective effect in
mSOD1 mice. Thus, muscle might not only be a tissue to target, but also a route to
deliverdrugsortreatments.
8) Conclusion
As a conclusion,when considering all the data discussed above, onemayhypothesize
thatLMNdegenerationmaybe“synaptopathies”ratherthan«motorneurondiseases».
As such, one should focus on designing treatments to strengthen and stabilize the
remaining NMJs and/or to stimulate the generation of newly formed NMJs. To our
knowledge,notreatmenthasbeenspecificallydesignedtotargetNMJsinmotorneuron
diseases until now. It may worth to consider that a drug or treatment targeting the
commonfinalpathwayofdiseaseswithLMNdegenerationi.e.theNMJ,wouldbeequally
beneficialforALS,SMAandSBMApatients.
Lastly, it shouldbekept inmind thatwhileskeletalmuscle isamuchmore important
actor in LMN degeneration than previously anticipated, these affections targetmotor
neurons and involve other cell types, i.e. astrocytes, schwann cells and microglia[75,
150‐157].Anefficienttherapyshouldtakeallthesecellularactorsintoaccount.
Dupuis,L&Echaniz‐Laguna,A 20/20
Figureandlegendstofigures:
Figure1:electrophysiologicalandmusclepathologicalfeaturesofLMNdegenerationin
skeletalmuscle
A.Electrophysiological(EMG)recordingofapatientwithALSdemonstratingfibrillation
potentialsatrest,i.e.spontaneousdenervation‐relatedmuscleactivity.
B‐D.Muscle biopsy of a patientwithALS. All the features associatedwith neurogenic
disorders, i.e. groupingofatrophic fibers (B,C),predominanceofone typeof fiber (D),
andpresenceofangulatedfibers(D),areobserved.
(B:semi‐thinsection,X250;C:H‐Estaining,X200;D:NADH‐TRstaining,X100).
Figure2:potentialmechanismsinvolvingskeletalmuscleinSMA.
Loss of SMN1 leads to two consequences in skeletalmuscle. First, SMN1 loss leads to
abnormalities in sarcomere structure, which are a likely cause of muscle weakness.
Second,SMN1lossdecreasesthepotentialofmuscletoproducematureAchRsubunits,
leading to an abnormal development of NMJs. SMN1 loss in motor neurons has also
profoundeffectsonNMJdevelopment.Seetext(section3)forfurtherdetails.
Figure3:potentialmechanismsinvolvingskeletalmuscleinSBMA.
ThemutantARtoxicityisunmaskedbytestosterone.Thisleadstomyopathicfeatures,
suchasmyotonicdischargesandelevationofbloodCK,contributingtomuscleweakness
and letality. On the other hand,mutant AR toxicity (orwild type AR overexpression)
dismantlesNMJsthroughyetunknownmechanisms.ApathogenicfunctionofmutantAR
inmotorneuronshasalsobeendocumented.Seetext(section4)forfurtherdetails.
Figure4:potentialmechanismsinvolvingskeletalmuscleinSOD1‐linkedALS
Mutant SOD1 expression in skeletalmuscle leads to oxidative stress ,muscle atrophy
and weakness. mSOD1 mice skeletal muscles are also hypermetabolic but whether
mSOD1expression inmuscleor inother cell types is responsibleof thisphenotype is
unknown.MusclehypermetabolismissufficienttodriveNMJdestructionandsystemic
energydeficit.mSOD1expressioninbothmotorneuronsandglialcellsisalsoinvolved
intheoverallALSphenotypeofmSOD1mice.Seetext(section5)forfurtherdetails.
Dupuis,L&Echaniz‐Laguna,A 21/21
Figure5:targetingskeletalmuscleinLMNdegeneration
A potential therapeutic treatment for LMN degeneration might target deleterious
processesoccuringinmuscleitself.
Figure6:skeletalmuscleasadeliveryrouteinLMNdegeneration.
Skeletal muscle is also a privilegied route to deliver drugs targeting motor neurons
throughretrogradetransport.
Dupuis,L&Echaniz‐Laguna,A 22/22
Dupuis,L&Echaniz‐Laguna,A 23/23
Dupuis,L&Echaniz‐Laguna,A 24/24
Dupuis,L&Echaniz‐Laguna,A 25/25
Dupuis,L&Echaniz‐Laguna,A 26/26
Dupuis,L&Echaniz‐Laguna,A 27/27
Table1:clinicalpresentationofdiseaseswithLMNdegeneration
disease onset Cause LMN
involvement
UMN
involvement
Myopathic
features
SMA
typeI birth LossofSMN1 + ‐ +
typeII Before
18mo
LossofSMN1 + ‐ +
typeIII After
18mo
LossofSMN1 + ‐ ++
SBMA
SBMA Adult‐
onset
Expansionofthe
polyglutaminetractof
AR
+ ‐ +++
ALS
sALS Adult‐
onset
Unknown(sporadic) + + Occasionally
reported
fALS Adult
onset
Genetic(multipleloci:
sod1,tdp‐43,fus,vapb...)
+ + Occasionally
reported
Dupuis,L&Echaniz‐Laguna,A 28/28
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