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ALS研究进展2015英文

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DiscoveryToday󰀂Volume00,Number00󰀂February2016

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TeaserAreviewofthecurrentmodels,animalmodels,usedinALSand

newmodelstodrugAtheofPascalineClerc1,ScottLipnick2andCatherineWillett1TheHumaneSocietyoftheUnitedStates,700ProfessionalDrive,Gaithersburg,MD20879,USA

MassachusettsGeneralHospital,HarvardMedicalSchool,DepartmentofMedicine,55FruitStreet,Boston,MA02114,USA

12Althoughamyotrophiclateralsclerosis(ALS),alsoreferredas‘LouGehrig’sDisease,’wasfirstdescribedin1869andthefirstdisease-associatedgenewasdiscoveredalmost20yearsago,thediseaseetiologyisstillnotfullyunderstoodandtreatmentoptionsarelimitedtoonedrugapprovedbytheUSFoodandDrugAdministration(FDA).Theslowtranslationalprogresssuggeststhatcurrentresearchmodelsarenotidealtostudysuchacomplicateddiseaseandneedtobere-examined.ProgresswillrequiregreaterinsightintohumangenesandbiologyinvolvedinALS

susceptibility,aswellasadeeperunderstandingofdiseasephenotypeatthehistologicalandmolecularlevels.Improvinghumandiseaseoutcomewillrequiredirectingfocustowardimprovedassessmenttechnologiesandinnovativeapproaches.

PascalineClerchasdedicatedherresearchtostudyingthemetabolismoftheliverandbrain,mostimportantlytheroleofmitochondriaincelldeathpathways.Astheseniordirectorofpolicyandadvocacyforanimal

researchissuesatTheHumaneSocietyoftheUnitedStates,sheiscurrentlyworkingwithscientistsandpolicymakersinindustry,academia,andGovernmentintheUSAandinternationally,tofacilitatethedevelopmentandimplementationofmethodsthatreduceorreplaceanimalsincosmeticsassessmentprocessesandinbiomedicalresearch.

Introduction

SincethefirstgeneassociatedwithALSwasidentifiedalmost20yearsago,researchershavereliedprimarilyonanimalmodelstostudythemechanismsofdiseaseprogressionandtoidentifytherapies.Thesestudies,fundedmainlybytheALSAssociationandtheNationalInstituteofHealth(http://report.nih.gov/categorical_spending.aspx),havecostnearlyUS$700millionoverthepast10yearsintheUSAalone.Oneofthemainfocuseshasbeenonrecapitulatingthehumandiseaseinanimalmodels,whichhasresultedintheidentificationofasingledrug,riluzole,whichwasapprovedbytheFDAforALStreatmentin1995.However,thebenefitsofriluzolearelimitedtoextendingthelifespanortimetotracheostomybyanaverageof3months.Overthepastdecade,investigationalnewdrug(IND)applicationsbasedondatacollectedusinganimalmodelsofALShaveresultedin11humanclinicaltrials,allofwhichfailedtodemonstrateefficacy[1].Infact,somedrugs,whicheffectivelysloweddiseaseprogressioninmice,resultedinacceleratingtheprogressioninhumans[2].TherepeatedfailureofdrugtranslationfromanimalmodelstohumansseenwithALSisdisappointingintermsoffinancialand,moreimportantly,humancosts.Here,wereviewthecurrentmodelsusedinALSresearchandsuggestare-examinationofthefieldtofocustheresearchonmorehuman-basedapproaches.

ScottLipnickreceivedaBScinphysicsand

economicsfromBrandeisUniversity.HethenobtainedaPhDin

biomedicalphysicsattheUniversityofCalifornia,LosAngeles.After

graduating,hebecamean

AmericanAssociationfortheAdvancementof

Science(AAAS)ScienceandTechnologyPolicyFellowattheNationalInstitutesofHealthCentrefor

RegenerativeMedicine.HethenbecametheDirectorofScientificProgramsattheNewYorkStemCellFoundationResearchInstitute.DrLipnickcurrentlyholdspositionsattheMassachusettsGeneralHospitalandHarvardUniversity,withresearchfocusingonbigdataandtranslationalusesofstemcells.

CatherineWillettbeganhercareerasa

developmentalbiologistusingzebrafishtoelucidatetheimmunesystemandlaterasamodelfor

preclinicaldrugscreening.Since2006,shehasfocusedonthescience,

policy,andregulatoryaspectsofreplacinganimalsasthebasisofchemicalsafetyassessmentandis

currentlytheDirectorofRegulatoryToxicology,RiskAssessmentandAlternativesattheHumaneSocietyoftheUnitedStatesandcoordinatoroftheHumanToxicologyProjectConsortium.Shehasnumerouspublicationsonnon-animalapproachesandadvisesinternationalcompaniesandgovernmentsontheregulatoryuseofsuchapproaches.

Correspondingauthor:.Clerc,P.(pclerc@humanesociety.org)

1359-6446/ß2016ElsevierLtd.Allrightsreserved.http://dx.doi.org/10.1016/j.drudis.2016.02.002

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ALS

ALSisaprogressive,fatalneurodegenerativediseasecharacterizedbylossofmotorneuronsinthebrainandspinalcord.AccordingtotheNationalALSRegistry(http://www.cdc.gov/mmwr/preview/mmwrhtml/ss6307a1.htm),12,000peopleintheUSAhaveadefinitediagnosisofALS,foraprevalenceof3.9casesper100,000persons.Thediseaseismorecommonamongwhitemales,non-Hispanicsandpersonsaged60–69years.Between90%and95%oftheseindividualsdevelopasporadicformofALS(sALS),occurringwithoutknowngeneticriskfactors,andhavenofamilyhistoryofthedisease.Theremaining5–10%ofcasesareinheritedandclassifiedasfamilialALS(fALS),whichisassociatedwithmorethanadozengenes.BothrecessiveanddominantformsofALShavebeenidentified,butmostfALSispassedoninanautosomal-dominantmanner.

ALSisacomplexdisorderwithaspectrumofphenotypesthatcouldcompriseasinglediseaseorrepresentseveralcloselyrelateddisorderswithdifferentcausesbutsimilarclinicalmanifestations.Theclinicalprogressionisgenerallyrapid,beginningwithmusclelossfollowedbymuscledegeneration,paralysis,andrespiratoryproblems.Mostpatientssuccumbtothediseasewithin3–5yearsafterdiagnosisbecauseofrespiratoryorcardiacdeficits.Althoughmotorfunctiondeclines,sensory,cognitive,andemotionalcapa-bilitiesaregenerallyleftintact.AlthoughtheetiologyunderlyingALSprogressionisunknown,variouscellularpopulationsandprocessesareknowntobeinvolved.Keypathologicalfeaturesincludemotorneuronloss(lesions),retractionofmotorneuronaxonsfromneuromuscularjunctions,theappearanceofinclusionbodieswithinneuronsandastrocytes,ubiquitin-positiveproteinaggregatesinneurons,andblood–brainbarrierdisruption.Al-thoughmanygenemutationshavebeenshowntobeassociatedwiththedevelopmentoffALS,ageneticcontributiontosALSisstillunclear.RiskfactorsforsALSincludegender[3],exposuretotoxicchemicals[4,5],andtraumaexperiencedduringmilitaryservice[6].

ThefirststudyassociatingageneticassociationinALS,specifi-callyCu/Znsuperoxidedismutase(SOD1),waspublishedin1993[7].SOD1isaubiquitousproteinthoughttoberesponsibleforprotectingcellsfromoxidativestressbyneutralizingcytoplasmicfreeradicals.SOD1isasolubleproteinlocatedinthenucleus,cytosol,mitochondria,andperoxisomes,whereitconvertssuper-oxideradicalstooxygenandhydrogenperoxide.KnockoutandfunctionalstudiesindicatedthatmostmutationsinSOD1resultingainoftoxicfunctionbydestabilizingtheproteinandcausingittofoldintoanon-native,harmfulconformation[8].Currenttheoriessuggestthatthesemisfoldedproteinsproduceatoxicproteinaggregatebuildup[9],althoughthecompletemechanismsofdiseaseprogressionareunknown.MutationsinSOD1accountfor20%offALSand1–3%ofsALScases.DifferentmutationsinSOD1alsohavepenetrancethatcanvarydependingonfactorssuchasage,sex,orethnicity[10].

From2001to2011,mutationsassociatedwithfALSwerefoundinalmost20additionalgenes,mostofwhichwereassociatedwithonly1–5%offALScases,althoughC9ORF72,anewlyidentifiedgeneofunknownfunction,islinkedto30%offALS[11].ManyALS-associatedgeneswereidentifiedusinglinkageanalysisandcandidategenesequencingonDNAfromaffectedfamilies,al-thoughsomewerediscoveredthroughhistologicalassociations.

Forexample,theTDP-43proteinwasfoundtobepresentinmotorneuroninclusionbodiesinthebrainandspinalcordofmanypatientswithALS[12],suggestinggenedysregulation.MutationsintheTARDNA-BindingProtein(TARDBP)gene,responsibleforTDP-43proteinsynthesis,werelateridentifiedbygenesequencingofpatientDNA[13].Thewild-typeformofTDP-43interactswithanotherALS-associatedprotein,FUS,bothofwhichcanbindRNAandDNA,andareinvolvedintranscriptionalrepression,pre-mRNAsplicing,andtranslationregulation[14,15].However,thefunctionofFUSandTARDBPinpatientswithALScarryingthemutatedformofthegenesremainsunknown.

SoonafterthediscoveryofALS-associatedgenes,animalmodelsexpressingmutanthumanALS-associatedgenesweredevelopedandwidelyadoptedtostudythemechanismofactionbehindvariantsinvivo[16].GiventhatsALSandfALSareclinicallysimilarandbelievedtohavesimilarpathologicalmechanisms[17],mousemodelsoffALSwerethoughttorepresentgeneralALSpathologyintermsofidentificationofinterventionstosloworreversethediseaseprogression.Overthepast20years,althoughrodents,especiallymice,havebeenthemostcommonlyusedmodelsforALS[18],differentspecies,includingCaenorhabditiselegans[19],Drosophila[20],zebrafish[21,22],andevennonhumanprimates[23],havealsobeenusedasmodelorganisms.Here,wedescribethemostcommonnonhumananimalmodelsofALSanddiscussthecaveatsinfluencingtheirvalidityandtranslationalapplicabil-ity(Table1).Additionally,wesummarizecurrent,andsuggestpotentialadditional,alternativemethodsforstudyingALSpro-gressionandidentifyingtherapies,withafocusoninvitroandnon-animalapproachestoimprovetranslationalefficiencyandbenefitthepatients.

Currentuseofnonhumananimals

Variousspecies,fromvertebratestoinvertebrates,havebeenusedtodevelopedALSmodels.WehavebeenabletolearnaboutALSfromthosemodels,but,aswithanymodelsystem,therearelimitationsintheirabilitytorecapitulatethediseaseandintheknowledgegatheredfromtheiruse.The‘ideal’modelwouldreproducethehumandiseasesymptomsidenticallyandwiththesameprogression(‘face’validity)andneurobiologicalmecha-nismofaction(constructvalidity),whilealsoservingasaplatformfortheevaluationoftherapeuticinterventionsinhumans(pre-dictivevalidity).

Caninedegenerativemyelopathy

NospeciesotherthanhumansareknowntonaturallydevelopALS;however,dogscanexperienceasimilarneuromusculardiseasewithsomeALS-likeclinicalfeatures.Currently,twoSOD1mutations,E40KandT18S,havebeenidentifiedbygenome-wideassociationandsequencingstudieswithcaninedegenerativemyelopathy(DM)[24,25].DMandALSaresimilarinthatbothareage-related,fataldiseaseswithprogressivelossofbothupperandlowermotorneu-ronswithsubsequentmuscledegeneration.Thediseasesarealsosimilarhistologically:spinalcordsofdogswithDMandhumanswithALSexhibitlesions,ubiquitin-positiveinclusionbodies,signsofoxidativestress,andneuromusculardenervation[24,26].How-ever,therearesignificantdifferencesbetweenthetwodiseases.CanineDMbeginswithuppermotorneurondefects[24],whereashumanALSdysfunctionbeginsineitherupperorlowermotor

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TABLE1

AsummaryofALSresearchmethods

MethodsExamples

Features

Limitations

Refs

Invivo

CaenorhabditisCellularfocuswithoutsystemiccomplexityLacksspinalcordandlowermotorneurons[19,30,43]elegansaffectedbyALS

Drosophila

MorecomplexitythanC.elegans,butlessWild-typehumangeneexpressionistoxic;[20,35,44]thanvertebrates

relativelysimplisticcomparedwithhumanZebrafishCheap,quick,simplistic;vertebrate

Notmammalian;relativelynewmodels[21,31,36]Mouse

Manydifferentmodels;shortlifespanandOverexpressionartifacts;uniquebiological[18,37,38,80]

diseaselength

propertiesfromhumans(lifespan,metabolism,etc.)

RatLargersize;shortlifespananddiseaselengthDiseasephenotypedifferencesfrommouse[22,46,80,107]Dog

Naturallyoccurringdisease(DM)

DMsimilarto,butnotsameasALS[24,26,29]NonhumanBetterdiseasemimicthanotherspecies

Expensive,longlived,ethicalissues

[23]primates

Invitrocultures

Yeast

Simple,modelsproteinaggregationwellNotsystemic,neural,orevenhuman[36,86,87]

PatientdissociatedNaturaldiseasegeneexpression;easy,directFinitelifespan;difficulttotransfect

motorneuronsdrugapplication

NSC34

Immortalmotorneuron-likeline;human;Induceddiseasephenotype;nottruemotormanipulable

neuronline

Stemcells

Cantransplantordifferentiateforstudy/Notsystemic,transplantationstilltransplant

experimental

IPSCs

TissuesliceEasilymanipulated;moresystemicthancellSamerecapitulationissuesaswholeanimal;culture(animal)

cultures

distantconnectionssevered

Insilico

ComputermodelingHighthroughput,hypothesisdrivenRequiresaprioriknowledge;underdevelopedVirtualmice

Quick,cheap

Sameissuesasmousemodel

GeneticNGS

Identifiessequencemutations

Requirespreviouslinkage;requiresseveral[11,82,83]

affectedindividualswithsamemutationGWAS

IdentifiesSNPslinkedtodisease;canidentifyNeedlargesamplesize;genetic

severalSNPsatonce

heterogeneityinALSpopulation;remainingmutationscanberiskrelated

MolecularIdentifiesclinicaltargets;givesinsightsintoExpensiveathighthroughputlevels;directphenotyping

diseasepathogenesis

versusindirecttargetsofdiseaseunclear

Availabledata

Patientdata

Cheap,quick,easilyaccessible;largesampleReliesonpersonalreporting;limiteddataset[84,85]

sizes

breadth

Medicalrecords

Easytrendanalysis;lesssubjectivethanLimiteddatasetbreadth

personalreporting

neurons[27].MosthumanSOD1mutationsaredominant[27],withhumans,buthavesignificantdisadvantages,includingtime,whereasDMappearstoberecessivewithincompletepenetrancespace,funding,andethicalconcerns[23].

[24],suggestingthatSOD1functionsorrequirementsareuniqueinThezebrafishisauniqueorganisminthatithasaconserved,humans.Additionally,menaremoresusceptibletoALSthanwom-simplifiedvertebratenervoussystem,ashortlifespan,andisame-enandmostALSissporadic[3,28],whereasDMindogsisequalnabletogeneticmanipulationandtherapeuticscreening.Further-betweenthesexes,althoughthereisbreedsusceptibility,andgen-more,70%ofhumangeneshaveatleastonezebrafishortholog,anderallyoccursinafamilialpattern[29].

arecentstudyshowedthat82%ofgenesassociatedwithhumanThesimilaritiestoALSindicatethatinformationregardingthediseaseshaveazebrafishortholog.ResearchersstudyingALShavemechanismandprogressionofDMmayyieldinsightintoALS.foundthattheuseofzebrafishmodelsprovidesuniqueinsightsintoMosthistologicalresearchonALSisdoneontissuefromindivid-systemic,cellular,andmolecularpathwaysassociatedwithdifferentualswhodiedatlatestagesofdiseaseprogressionandlittleisgeneticvariants.Theflexibilityofthismodelandsimilaritytotheknownaboutthemanifestationsofearly-stageALS.However,dogshumangenomehaveinfluenceditsrisingpopularityinneurode-withDMareofteneuthanizedearlyindiseaseprogressionbecausegenerativeresearchandhasincreasedourunderstandingoftheofdeteriorationintheirqualityoflife[24]and,therefore,early-biologicalactivityofgenesassociatedwithhumandiseases.How-stageDMdiseasetissuesareavailable.

ever,despiteevolutionaryrelationsoranatomicalsimilaritiesbe-tweenzebrafishandhuman,biochemicalmechanismsorTransgenicanimals

physiologicalresponsesaredifferent,anddataaccumulatedusingRodentshavebeenpreferredtootherspeciesbecauseoftheirmorezebrafishareoftennotrelevanttohumans.Invertebratemodels,complexcentralnervoussystem(CNS),andbecausetheyareeasiersuchasC.elegansandDrosophila,havealsobeenusedtodescribethetohandleandhaveashorttimetomanifestdisease-likepheno-diseasephenotypeandprogressionatthecellularandmoleculartypes.Nonhumanprimatemodelshavealsobeendevelopedbasedlevelsinarelativelycell-autonomousmanner,despitetheirlackofontheirpresumedrelevancebecauseofphylogeneticsimilarity

lowermotorneurons.

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SOD1transgenicanimals.SincethediscoveryofSOD1asanALS-associatedgenein1994,avarietyoftransgenicSOD1mutantanimalmodelshavebeencreated.Morethan150mutationsofSOD1havebeenidentifiedinfALSandover20rodentmodelshavebeengenerated,mostofwhichoverexpressamutantvariantofhumanSOD1.TheoldestandmostwidelyusemodelistheG93AhSOD1overexpressionmouse,whichatonepointwasusedinupto97%ofALSdrugstudies[18].Transgenicmutanthuman(h)SOD1overexpressionmodelsalsoexistinC.elegans(G93A,G85R,G37R,andA4V)[30],Drosophila(G85RandA4V)[20],andzebrafish(G93R,G37R,andA4V)[21,31].

TheSOD1modelswereinitiallydevelopedtobeageneralmodelforALS(SOD1-relatedandnon-SOD1-relatedfALS,aswellassALS).AlthoughSOD1mutationsarenotpresentinsALS,therearephenotypicsimilaritieswithfALS,andithasbeenassumedthatfamilialandsporadicformssharedthesameneuronaldegenera-tionpathwayandstudyingthefamilialformcouldprovideinsightintothesporadicform.However,thevalidityoftheseassumptionshasbeenquestioned.IthasbeenshownthattissuesfrompatientswithsALSandnon-SOD1fALSexhibitaggregatesofTDP-43inmotorneuronsandglia[32],yetpatientswithALSandSOD1mutationsdisplayTDP-43-negativeneuralaggregates[32].TheG93AhSOD1mousemimicsthehumanSOD1phenotypewithTDP-43-negativeaggregatestaining[33],indicatingthatmouseSOD1modelsmaybetterrepresentthephenotypeofpatientswithALSandhSOD1mutations,butnotthosewithsALSornon-SOD1fALSmutations,whichaccountfor98%ofALScases.

TARDBPtransgenicanimals.In2008,TARDBPwasidentifiedasanassociatedgenein3–5%offALScasesand2%ofsALScases[34].Sincethen,mutanthumanTARDBPhasbeenoverexpressedinbothC.elegansandDrosophila,whichexhibitsomehumanALScharacteristics,includinglossofmotilityandreducedlifespan[35].ThezebrafishTARDBPmodelisnotyetwellcharacterized,butdoesexhibitmotordefectsandaxondeficiencies[36].MouseandratoverexpressionmodelsappeartorecapitulateoverarchinghumanALSphenotypesinthattheyexhibitneuronalubiquitin-positiveinclusions,motorneurondegeneration,axondegenera-tion,motorloss,paralysis,anddeath[37,38].Givenconcernsaboutthelackofrelevanceofexpressingmodelsthatexhibitearlyparalysisanddeath,arecentmodelwasdevelopedexpressinglowerlevelsofTDP-43tomimicthehumanproteinexpressionlevel[39].Todate,onenonhumanprimateTDP-43modelhasbeendevelopedbyoverexpressingthehumanwild-typefromofTDP-43inthespinalcordsofcynomolgusmonkeytorecapitulatetheredistributionofTDP-43fromthenucleustothecytoplasm,whichwasnotobservedinratmodels[23].

MutantmodelsofTARDBPexpressionarestillrelativelynew,andlackcharacterizationinthecontextofthehumanTARDBPALSphenotype,butrecentpublicationshavereportedthat50TARDBPmutationsmightbeassociatedwithALS,whereasothersreportthatthosemutationsarestillrareinALS[40,41].FUStransgenicanimals.In2009,FUSwasidentifiedasanassoci-atedgenein5%offALScasesand1%ofsALScases[42].InclusionsofFUSinthecytoplasmhavebeenshowntoberecurrentinpatientswithALSandFUSmutations.Sincethen,ahandfulofmodelsoverexpressingthemutanthumanFUS(hFUS)havebeencreatedinC.elegans,Drosophila,zebrafish,andrat.BoththemutanthFUSoverexpressionDrosophilaandC.elegansmodels

exhibitadecreasednumberofneuromuscularjunctions,motorneurondamage,cytoplasmicFUS-positiveproteinaggregation,locomotorimpairment,andprematuredeath[43,44].Overexpres-sionofmutanthFUSinzebrafishisnotfullycharacterized,butdoesresultinmotorneurondefectsandmotordeficits[45].Inrodents,itlooksliketheoverexpressionofmutatedorwild-typeFUSinducesdifferentphenotypesdependingonthespecies.Themu-tanthFUSoverexpressionratmodelexhibitsproteinaggregates,neurodegeneration,andmuscleatrophy[46],whilerecentstudieshaveshownthatoverexpressionofwild-typeFUSinratsinducescognitivedefectsinagedanimalswithoutmotorphenotypeorspinalcordpathology.However,inmice,overexpressionofwild-typeFUSinducesmotorneurondegenerationwhensignificantamountsofproteinaccumulateinthecytoplasm[47].

ConcernsregardingtherelevanceofnonhumananimalstoALS

GiventheirabilitytorecapitulatesomekeyALSfeatures,animalmodelshavebeenusedtostudyALSforalmosttwodecades;however,thevalidityofanimalsasmodelsforhumandiseaseshasbeenchallengedandisgenerallydebated[18,48–51](http://dana.org/News/Details.aspx?id=42802).Often,theunderlyingmechanismsofactionandresultingclinicalmanifestationfoundinanimalmodelsnotonlydonotcorrelatewiththatinhumans,butalsoguideresearchersandresourcesalongfruitlessavenues.StudieshaveidentifiedtroublingdisparitiesbetweenhistologicalphenotypesofpopularALSanimalmodelsandhumanALS[32,33].Disparitiesalsoexistwithinresearchresultsinthesamemodelsatthesameordifferentinstitutions,indicatingalackofreproducibility[18].Additionally,someconcernshavebeenraisedregardingthedifferentformsofcelldeaththatmayexistbetweenhumansandmicemodelsofALS[52].Althoughanimalmodelshaveyieldedsignificantinformationregardingbiology,theiruseinmodelingcomplexhumandisordersislimited.

Overexpressionissues

Althoughmostbroaddiseaseclinicalcharacteristics,suchasmotorneurondegeneration,motorloss,anddeath,arereplicatedinsomeanimalALSmodels,thevalidityofoverexpressionmodelshasbeencalledintoquestion.ItisunknownwhetherALS-associ-atedgenesareupregulatedinpatientswithALS,yetmostanimalmodelsofALSutilizeoverexpressionofamutanthumanALS-associatedgene.Asdescribedabove,theoverexpressionofFUSusingdifferentpromotersandonsetofexpressionindifferentspecies(ratsandmice)andstrainsresultindifferentphenotypes[46,47].Modelsresultingfromgeneoverexpressionhaveseveraltohundredsofcopiesofthehumantransgeneandexpressionofthemutanthumangeneisseveral-foldhigherthanendogenousani-malgeneexpression[53].Inthemostpopularmousemodel(G93ASOD1),25copiesofthehumantransgenehavebeeninsertedandhSod1isexpressedatlevels13-foldhigherthanendogenousmSod1[53].Interestingly,overexpressionofwild-typegenesinmicecausessomeneuronaldefectsandvacuoleaccumulation,whicharenotobservedinhumans[47,54],likelyindicativeofmitochon-drialswellingfromtoxicproteinoverload.Consistentwithanoverloadhypothesis,theexogenousgenecopynumbersofeithermutantorwild-typegenesinmiceisstronglycorrelatedwithdiseasephenotypeseverity[55,56].Overexpressioneffectsarealso

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seeninDrosophila,wherewild-typehTARDBPoverexpressiontohumansbutnotanimals,asisthecasewiththalidomide.Expo-causesdose-dependentmotordefectsandseverereductionofsureofpregnantwomentothalidomideduringthe1970sresultedinlifespan[57].However,dependingontheleveloverexpressionseverefetaldevelopmentaldefects;however,thalidomidetestinginofproteinslinkedtoALS,non-ALS-relevantphenotypescanbeanimalshadshowninconsistentandspecies-and/orstrain-specificobserved,rangingfromphotoreceptordegenerationtodisruptionresponses[64,65].Speciesvariationsindrugtoxicologyresponsesofeyearchitectureandlarvallethality,highlightingthetoxicarelikelyduetodifferencesintheuptakeand/orexcretion,metab-effectsandlackofspecificityofthismodel.

olism,anddistributionofdrugs,aswellasimmuneresponsesOverall,thesestudiessuggestthatgeneoverexpressioncauses[66,67].Thesecriticaldifferencesindicatethatanimalsandanimaltoxiceffects,someofwhicharesimilartoanALS-likephenotypemodelsmaynotbeidealcandidatesforthetoxicologytestingthatisbutarenotrelatedtoALSdiseaseprogression,therebycomplicat-necessaryforestablishingthesafetyofnewdrugs.

ingdiseaserecapitulationandunderstanding.Animalmodelsarecreatedtounderstanddiseaseprogressionandpathogenesis,sup-Aging,environment,gender,andCNSphysiology

portthedevelopmentoftreatments,andcollectpreclinicaldata;ALSisstronglyageassociated,butthelackofage-relatedALS-likeartifactsofgeneoverexpressioncanconfoundresearchresults,diseasesoutsideofhumans(withthepossibleexceptionofdogs)misleadfuturestudies,and,therefore,delaytheavailabilityofnewsuggeststheneedtoappreciatethebiologicaldifferencesbetweendrugstopatients.

humansandotherspecies.Eachspecieshasauniquelifespan,tissue-specificcellularturnoverrates,oxidativestressresponses,Speciesbiologicaldifferencesimpacttranslationalpotential

andgene-expressionprofiles,allofwhichcontributetoaging,andAsmentionedabove,modelsofALSinC.elegansandDrosophilaarearelikelytocontributetodifferencesinaging-relateddiseasesimilartohumanALSinthatbothexhibitinclusionbodies,defectsprogression.

inneuromuscularjunctionsignaling,glialabnormalities,progres-AgingandALSarealsolinkedtolocalfactors,suchasenviron-sivemotordefects,andevendeath[20,30,35,43].However,thosement,diet,andstress[68,69];however,differentanimal-housingmodelsdonotexhibitmotorneurondeath[20,30],asobservedinfacilitiesandresearchgroupshavevaryinganimalcolonyman-humanswithALS,wholosebothupperandlowermotorneurons.agementmethods,whichaffectanimalstressandbehaviorThisdifferencecouldbeduetotheshortlifespanofthesespeciesor[70–72].Thesameanimalmodelstudiedattwodifferentfacilitiestothedifferencesbetweentheneuralcellphysiologyofvertebratesorbytwodifferentinvestigatorscanexhibitdifferentdiseasefea-andinvertebrates.ZebrafishG93RmutantsresembleALSinthattures,thusconfoundingresultsthroughoutthefield.Additionally,theyexhibitthemaindiseasecharacteristics,includingspinalgenderisariskfactorforALS,becausemenareslightlymorelikelytomotorneuronloss,muscledegeneration,motordeficits,neuro-developthediseasethanwomenandfemaleALS-modelmicetendmuscularjunctiondefects,paralysis,andearlydeath[31].Howev-tolivelongerthanmales[28,73];however,genderisnotalwayser,thetransgeniczebrafishmodelisrelativelynewandnotyettakenintoaccountinanimalstudies[18],whichcancontributetofullyunderstood,anddevelopingnewinterventionsforhumanpoorreproducibility.Additionally,althoughthehumangenomeisALSbasedonstudiesconductedonzebrafishisnotaguaranteeofapproximately97.5%similartothemousegenome,differencesinsuccess,despiteitsresemblancetothediseaseinhumans.

DNAmethylationorhistonemodificationcanbeverydifferentSimilartozebrafish,somerodentmodelsofALS,includingSOD1amongspecies,leadingtovariabilityingeneexpression.

models,alsoappeartorecapitulatemanyofthecharacteristicALSTheanatomyandphysiologyoftheorgansystemsaffectedbyphenotypes,includingneuromuscularjunctiondisruption,upperALS(CNSandskeletalmuscle)alsodifferbetweenspecies,whichandlowermotorneuronloss,muscledegeneration,respiratorylikelyinfluencesdifferencesindiseasephenotypeanddrugproblems,immuneabnormalities,blood–brainbarrierdisruptions,responses.Importantanatomicbraindifferencesexistbetweenandproteinaggregation[16,58–60].Transgenicmodels,suchasspecies[74,75],andevenbetweenstrainswithinasinglespeciesSOD1andTARDBPmodels,alsodemonstrateprogressivemotor[76].AccordingtoEisen,ALSandotherneurodegenerativediseasesdeclinethateventuallyleadstoparalysisanddeath.However,somearehuman-specificdiseases.Indeed,theevolutionofHomosapiensstrainsofG93ASOD1transgenicratshavemoreaggressivediseaseledtoneocorticalchanges,withthedevelopmentofnewcorticalphenotypesanddifferentmotorneurondegenerationpatternsareasandincreasedinterconnectionsthatarenotonlyresponsiblecomparedwiththeirmousecounterparts[22],indicatinguniquefortheacquisitionofbipedalismandopposablethumbs,butalsodiseasemanifestationsamongspecies.Giventhattransgenicani-thebrainregionstargetedbyALS[77].Spinalcordanatomymals,suchasG93ASOD1rodentsexhibitingsomesimilaritieswithbetweenspeciesisalsounique;thelengthofthehumanspinalALS,displayspecies-specificdiseasephenotypes,thissuggeststhatcordisseveraltimesthesizeofthecordinmostanimals.Specieseffectsobservedinanimalmodelscannotfullybetranslatedintoalsodifferatthemolecularlevel,becauserodentsandhumanshumansbecauseofcriticalbiologicaldifferences.

exhibituniquegeneexpressionpatternsinthebrain[78].TheseThesebiologicaldifferencescaninfluencediseasemanifesta-differencesinphysiology,complexity,andexpressionundoubt-tions,treatmentresponses,andtoxicologicalthresholds.Foredlyaffecttheabilityofanimalmodelstomimichumanneuralexample,administrationofcertainchemicals,suchasmethyldiseasephenotypes.

tert-butylether(MTBE),afueladditive,inmaleratsinducedendogenousa2u-globulinnephropathyandledtokidneytumors;Concernsaboutthemethodologiesusedtocreatehowever,humansdonotproducea2u-globulinand,therefore,animalmodelsofALS

responddifferently[61,62].Similarly,penicillinissafeinhumans,Giventhattransgenicanimalsaredevelopedusinginbredstrainsbuttoxictoguineapigs[63].Inotherinstances,drugsareharmful

thatarerelativelygeneticallyhomogenous,resultsusingthesame

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transgeniclineshouldbereproduciblebydifferentresearchgroups.However,thishasoftennotbeenthecaseforALSanimalmodelsforseveralreasons.Asmentionedabove,somedifferencesresultfromanimalcolonymanagementprotocols.Inthesemod-els,diseaseprogressionisoftenassessedbasedonqualitativemeasures,suchasgripstrength,wirehanging,gaitanalysis,andneurologicalscoring[79].Thelackofstandardquantitativemea-suresmakesitdifficulttocompareresultsbetweenstudies.Inaddition,drugstudiesontransgenicmiceareoftenunderpowered,lackrandomizationofgroups,andoutcomeevaluationsarenotperformedblind[18,80].Infact,thelackofstandardizedmeth-odologyledtheEuropeanALS/MNDgrouptodevelopandpublishtheStandardOperatingProceduresforALSpreclinicalanimalresearch[79].However,itisstilltooearlytotellwhethertheseguidelineswillbeusedorimpactthereproducibilityofresults.

Humanstudiesandnon-animalapproachesGeneticstudiesinhumans

ALSgeneticsubpopulationscanhaveuniquediseaseprogressionmechanisms

Previously,geneticstudieshavebeeninstrumentalinidentifyingassociatedmutationsinALS.TheidentificationofC9ORF72throughnext-generationsequencing(NGS),whichhadpreviouslybeenlinkedtofALSusingagenome-wideassociationstudy(GWAS)[11,82],wasahugebreakthroughintheALSfield,becauseC9ORF72isanassociatedmutationresponsibleforupto24%offALSand4%sALScases[82].GiventhatthegeneticcausesofmostsALScasesarestillunknown,furtherGWASandNGSstudieswouldbeusefultoidentifyotherALS-associatedgenes.Further-more,geneticstudiescanimproveourunderstandingofALSprogressionandmechanisms.MolecularphenotypingstudieshaveidentifieduniquegeneexpressionprofilesofspinalcordneuronalpopulationsincontrolsversuspatientswithALS[83],implicatingcertainpathwaysinALSprogression.Futuremolecularphenotypingstudiescouldidentifyearlyversuslatemarkers,therebypotentiallyinformingcausalrelations.

Asmentionedabove,transgenicanimals,especiallyrodents,areestablishedininbredand,therefore,geneticallyhomogenouslines[18].However,patientswithALSareclinicallyandgeneti-callydiverse.Asaresult,drugstudiesinALSmousemodelsshowingsmallbutpositiveeffectshavenottranslatedtopositivefindingsinhumanclinicaltrials,likelybecausetheeffectsofthedrugsarebothsmallandspecifictouniquerodentgeneticbackgrounds.

AccordingtotheguidelinesforpreclinicaltestingandcolonymanagementwhenworkingwithALSmice,establishedbyTheJacksonLaboratoryandPrize4Life,mixedstrainsofALSanimalmodelshaveanadvantagebecausetheybettermimicthehetero-geneoushumanpopulation.However,mixingstrainsbetweenexperimentalgroupscouldrequireanincreasednumberofani-malstoreachstatisticalsignificancefromaheterogeneoussubjectpool.Theuseofinadequatenumbersofanimalscannegativelyimpactreproducibilityandresultinmisleadingdataandwastedresources.Geneticissuescanevenoccurwithinasinglestrain;G93AhSOD1mousecoloniescanspontaneouslylosecopiesofG93AhSOD1,resultinginmisleadingdatafromdelayeddiseaseonsetanddeath[56].ThevariablegeneticsofALSanimalmodelcoloniesisacomplicatingfactorthatcaneasilyaffecttheaccuracyandreproducibilityofresults,andlikelyhascontributedtotheconfusionwithintheALSfield.

Humandataandpopulationmonitoring

Foryears,researchershavecombedavailablepatientdataforcausativelinkstoALSfromseveralpatientregistries,includingALSregistries[NationalALSRegistry,DepartmentofVeteran’sAffairs(VA)ALSRegistry,andfALSConnect],whichcontaindatafromthousandsofpatientsaswellasmedicalrecords.Thesedatabasesareeasilyandquicklyaccessible,free,containdataonalargepatientpopulation,and,therefore,havebenefittedresearchers.RegistrydatacanbeusedtoidentifygeneticandenvironmentalALSriskfactors,predictdiseaseprogression,orevenimprovetreatment[84,85].

Additionally,monitoringpopulationssusceptibletothediseasecouldhelpidentifybiologicalorphysicalmarkers,whichwouldgiveusabetterunderstandingoftheprogressionofthediseaseand,therefore,identifyearlydiagnostics.

Cellandtissuemodels

Clinicaltrialdesign

Inmoststudies,potentialdrugsorcompoundsaretestedonanimalmodelsbeforetheonsetofsymptoms,whichisnotrelevantwhenitcomestoclinicaltrialsbecauseitisimpossibletostarttreatingpatientspresymptomatically.Additionally,Vin-santetal.proposedthatthediseaseonsetinSOD1mutantmicestarts2monthsbeforeitwasinitiallythought(postnatalday30insteadof90);therefore,theauthorsuggestedthattreatmentshouldbetestedatpostnatalday30[81].AccordingtotheGuidelinesforpreclinicaltestingandcolonymanagement,itisrecommendedtoconductpreclinicaltrialspre-onsetand,ifthedrugsshowsomeeffect,toconducttheexperimentsatorpost-onset;therefore,thereisnovalidreasontoconducttheexperimentspre-onsetonanimalsiftheywilleventuallybeconductedatorpost-onset.

Yeastshavebeenusedtostudymolecularinteractionsassociatedwithneurodegenerativediseases,suchasALS.Yeastsareamenabletogeneticmanipulation,growquickly,areeasytostudyatasingle-celllevel,andhavemanyofthebasiccellularmechanismsinvolvedinneurodegeneration,suchasmitochondrialdefects,proteinmisfolding,andproteintrafficking/degradationimpair-ments.Assuch,yeastcanbeusedtostudycellularresponsestoknownALSgeneticmutationstodeterminetheunderlyingmech-anismsofALSdiseasepathology.Forexample,yeaststudiesshowedthatTDP-43isnormallylocalizedtothenucleus,butTDP-43overexpressionresultedincytoplasmic,toxicproteinaggregates[86].OtherALSmutationsincreasedcellularstressandalsoresultedinaggregates[87].Ofcourse,ALSisacomplex,multicellulardiseaseand,thus,asasingle-celledorganism,yeastisnotcapableofrecapitulatingALSinitssystemicentirety.Itisalsoimpossiblewithyeasttointerrogatethecomplexinteractionswithotherproteinsorpathwayssolelyinhumancellsorinthepartic-ularcellofinterest,themotorneuron.However,yeastasamodelhasprovenusefulindelineatingtheALSdiseasepathwayatthemolecularandcellularlevels,whichisacriticalfoundationuponwhichtobuildunderstandingofthediseaseatthetissueandmultiorganlevels.

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Otherculturedcellsaresimilarlyusefulindelineatingthequalitativeandquantitativemethodtocomparetheresponseoffundamentalmoleculardiseasepathway.CulturescancomprisetissuesfromdifferentALSgenotypes.Additionally,useofmarkersprimaryanimaland/orhumancellsfromanytissue,immortalcouldhelpstandardizeresultsbetweenlaboratories.

nonstemcelllines,andevenembryonic,fetal,oradultstemcells.Therecentdiscoveryofinducedpluripotentstemcells(iPSC)holdsInvitromodelsusingiPSCs

greatpromisebecausetheycanbegeneratedfromthoseaffectedIPSCsrepresenttheopportunitytointegratethecomplexgenomicbyALS,maintainingthedonorgenotype,andprovideanunlim-landscapeseeninhumansaffectedbybothfALSandsALS.Humanitedsourceofcellsformodelingthedisease.IncaseofprimaryiPSCsarederivedfromadultsomaticcellsthroughepigeneticanimalcells,neuralsubtypesfromdifferentspeciescanbeused,modificationtoyieldpluripotentcellsthatproliferateindefinitelytherebyimprovingspeciesandcellularrelevancyforbothpatho-intheundifferentiatedstate,yetretaintheabilitytodifferentiategenesisstudiesanddrugscreenings[88].Forexample,motorintoorgan-specificcelltypes.Motorneuronsandotherrelevantneuronandmotorneuron-likecultures(suchasNSC34cells)celltypes,suchasastrocytes,arerecreatedfromiPSCsobtaineddisplaycellstress,aggregation,andcelldeathinresponsetofromhumanswithALSandhealthycontrolscanbegeneratedtoALSmutations[36,87].Additionally,cellculturescangenerateevaluategeneticfactorsrelatedtothediseaseaswellasexposuretocellpopulationsfortransplantationtherapies;everythingfromvarioustoxins.Thedifferentiatedcellsarecharacterizedandcom-embryonicstemcellstoadultneuroblastscanbeleftundifferenti-paredinthehopeofidentifyingcellularphenotypesthatrecapit-ated,inducedtodifferentiate,oralteredandthenimplanted[89].ulatekeyaspectsofthedisease.

Cellculturemodelsareevenbeginningtosupplement,andatAlthoughthesecellsarenotfreeofcaveats,suchasthedifficultytimesreplace,animaltestingfortoxicologicalstudiesbecausetheytoaccuratelydefinethestateofthecells,maturethecells,orarequicker,cheaper,moreeffective,andmoreethical[90].

obtaintherightcells,theyholdgreatpromiseforALSresearch,butSimilartoyeast,cellculturesarelimitedwithrespecttounder-effectiveintegrationiscontingentuponaccesstolargenumbersofstandingsystemicfunctionsanddefects;however,culturemodelsaffectedcelllinesandreproduciblepanelsoftheirdifferentiatedareusefulforidentifyingcell-specificdefectsandmechanisms.progeny.ThefirststudydemonstratingtheabilitytogenerateWhilegroups,suchastheorgan-on-a-chipprogramfundedbyiPSCsfroman82-year-oldwomanwithALSanddifferentiatethemDARPA,NIH,andFDA,arebeginningtodevelopcomplexengi-intomotorneuronswaspublishedin2008[94].Morerecentneeringplatformscapableofmodelinginteractionsbetweenmul-studieshaveevaluatedvariouscellularphenotypesrelatedtotipleorgansystems,theyarestillimmatureandhavenotshowntheprogressionofALS[95,96].Ifcellularphenotypesaresuccess-muchutility(http://www.ncats.nih.gov/research/reengineering/fullyidentified,theiPSC-basedmodelcanthenbeusedtoscreentissue-chip/tissue-chip.html).However,inthefuture,itispossibledrugsthatcorrectthephenotype,therebygeneratingamodelofthattheseplatformswillenablecellculturesystemstobetterhumanefficacyusinghumancells.Recently,researcherscon-modelcomplexinvivoenvironments.

ductedascreenofsmallmoleculesonmouseembryonicstemTissuesliceculturesaddcomplexityand,therefore,relevancecellsdifferentiatedintomotorneuronsandidentifiedonemole-relativetosinglecell-typecultures.Culturedtissuesmaintainlocalcule,kenpaullone,whichwasfoundtoprolongthesurvivalofenvironmentsandconnectivity,butallowrelativelyeasyaccesstobothwild-typeandSOD1mutantmotorneurons.Theauthorsalsomolecularandenvironmentalmanipulations.Assuch,slicecul-found,bytestingkenpaulloneonhumanmotoneuronsderivedturesareidealforunderstandingcellularandlocalsystempath-fromhumanembryonicstemcellsoriPSCs,thatthecompoundwaysunderlyingALSdiseaseprogressionandmechanisms.showedpromisingpreclinicalresults.Theirstudyalsodemonstrat-Althoughtheystillrequireanimalsasasourceofthetissue,edthatdexpramipexoleandolesoxime,twocompoundsthatareexperimentswithculturedtissuesusefeweranimalsperexperi-activeinALSmousemodelsandthatfailedinPhaseIIIclinicalment,allowmultipletestingperanimal,andarealsoquicker,trials,hadnoortoosmallaneffectonhuman-derivedmotorcheaper,andmoreamenabletotreatmentstargetinggeneexpres-neurons[97].Inamorerecentstudy,researcherswereabletosion.Forexample,tissuesliceculturescanbetransfectedusingrecapitulateaclinicalphenotype,specificallyincreasedexcitabil-virusesorparticle-mediateddelivery[91,92],whichcanbeusedtoityofmotorneurons,measuredfrompatientsusingiPSC-derivedeitherincreaseorknockdowntargetgeneexpression.Further-motorneuronsinadish.Throughthescreeningofdrugsknowntomore,thistechniquehasrecentlyshownpromisingdatainreduceneuronalexcitabilitypreviouslyapprovedfortreatmentofSOD1micemodels[93].Unfortunately,sliceculturesalsohaveepilepsy,theyshowedthatonedrug,retigabine,reversedtheseveraldisadvantages.Theyrequirelivingtissues,whicharenotcellularphenotype[98].Thisworkisnowthebasisforaclinicalwidelyavailablefromhumans.ALSmousemodeltissueshavetrialsupportedbyGlaxo-SmithKlineandrepresentsthefirstdrugquestionablevalidity,aspreviouslynotedfortransgenicanimals.identifiedusingiPSC-derivedtissueasadiseasemodel.

Additionally,ALSisanage-relateddisease,butsliceculturesarenotrecommendedfortissuesfromolderanimalsbecauseofpoorInsilicomodels

survivalresponsestomechanicaltraumainducedduringtheAscomputermodelingbecomesmoresophisticated,softwarecanpreparation.

beusedtomakepredictions,whichcouldinfluencethedirectionForallofthesetissuecultureapproaches,drug-screeningstudiesofinvivoandinvitroALSstudies.Insilicomodelingprogramscanwouldstronglybenefitfromtheidentificationofcellularmarkersbeusedtomakeavarietyofpredictions,whichcansavetime,ofdiseaseprogressionandhealth.Markerlevelscouldbeusedmoney,andlivesofbothhumansandanimals.Forexample,toquicklydeterminetreatmentefficacyandeventoxicityinasoftwarecangaugethefunctionalvariabilitybetweennormalspecies-specificmanner.Thiswouldalsoprovideanimportant

andmutantproteinsandcansuggestwhethermutationsresult

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inagainorlossoffunction.MostknownALSgenemutationsresultinproteinswithatoxicgaininfunction;however,recentsoftwarepredictionsindicatedthatmutationsinthenovelALSgene,SQSTM1,resultinalossoffunction[99].SoftwareprogramsalsopredictedmechanismsofdiseasedevelopmentinSOD1mutants[100,101].Additionally,virtualmicearebeingcreatedfromwell-characterizedmousemodels[102,103],andcanbetestedfordrugefficacyand,perhapsmoreimportantly,studiedforspecies-specificdifferencesindiseasemanifestation[102,104].Thesepredictionprogramswillbecriticaltounderstandingdiseasepathogenesisandtargetsfortreatment,bothinthegeneralALSpopulationandinindividualswithuniquemutations.However,predictionsoftwarerequiresaprioriknowledgeofassociativegenesandtheirmutations.

Predictiveoutcomepathways

Inthechemicalfield,theOrganizationforEconomicCooperationandDevelopment(OECD),theUSEnvironmentalProtectionAgency,andtheEuropeanUnionJointResearchCentrearecol-laboratingtodevelopthenecessaryinfrastructuretohostaunified‘knowledgebase’ofadverseoutcomepathways(AOPs)thatcoversthebroadspectrumofbiologicalpathwaysthatarelikelytobeinvolvedinhumanhealthandecologicalriskassessment:theAdverseOutcomePathwayKnowledgeBase(AOPKB)

(http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing-safety-assessment-chemicals/improved_safety_assessment_chemicals/first-release-of-aop-wiki;http://www.epa.gov/research/priorities/docs/aop-wiki.pdf;http://aopkb.org/aopwiki/index.php/Main_Page;http://www.epa.gov/ncct/download_files/chemical_prioritization/AOPWikiTutorial%20v2.pdf).Buildingontheexist-ingAOPKB,wecoulddevelopanewpathwayknowledgebasefordifferentfordiseasemodelstounderstandthediseaseandidentifynewtherapeuticstargetsinamoreefficientmanner.

ALSisacomplexdiseasewithaspectrumofphenotypesthatcouldrepresentseveralcloselyrelateddisorderswithdifferentcausesbutsimilarphenotypes.Predictiveoutcomeofdiseasemodelswouldbenefitfromtheuseofdiseasepathwayapproaches,similartoAOPsusedintoxicology[105].ScientistsandclinicianshavealreadygatheredmuchinformationonALSpathways,someofwhichareintegratedinaKEGGpathwaymap(http://www.genome.jp/dbget-bin/www_bget?pathway:map05014).However,weneedtofocusonintegratingthisinformationinacommonandaccessibledatabasewheresequencesofmolecularchangeswithinthecellsleadingtothedevelopmentoftheclinicalcon-ditionscouldbelisted,asdescribedinFig.1.ThisapproachwouldallowfornewinformationtobeaddedasitisdiscoveredtogetabroaderunderstandingofadiseasesuchasALS,orofspecificpopulations,andtoidentifynewtherapeutictargets[106].

(a) ToxicantMolecularinteractionCellular responseOrgan responseOrganism responsePopulation responseChemical propertyReceptor/ligandDNA bindingProtein oxidationGene activationProtein productionAltered stagingAltered physiologyDisrupted homeostasis Altered development/functionLethalityImpaired developmentImpaired reproductionAltered sex ratioExtinctionMolecular initiating eventMechanism of action, toxicity pathwayAdverse outcomeAdverse outcome pathway(b) Initiating eventMolecularinitiating eventCellular responseOrgan responseBrain and spinal cordOrganism responseGene mutations:SOD1, TARDBP, FUS,…SOD1 protein destabilization Inhibition of TDP43/FUS binding to DNA and RNAPrevents transcriptional repression, pre-mRNA splicing, and translation regulationUbiquitin-positive protein aggregates in neuronesOxidative stressLoss of motor neurones in brain and spinal cordBlood–brain barrier disruptionMuscleMuscle degeneration, paralysis, and respiratory and cardiac deficitsDeathDrug Discovery Today FIGURE1

Adverseoutcomepathway.(a)Theadverseoutcomepathwayisabiologicalmapfromthemolecularinitiatingeventthroughtotheresultingadverseoutcomethatencompassesbothmechanismandmodeofaction.(b)Adaptationofthepathway-basedapproachtotheamyotrophiclateralsclerosis(ALS)modelsdescribingthekeyeventsleadingtotheadverseoutcomeattheorganismlevel.Adaptedfrom[105](a).

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Similarly,itwouldbepossibletomapandunderstandtheuniquediseasephenotypes,therapyresponses,andtoxicitydiseaseprocessofeachofthedifferentformsofALSbyusingthresholds.Advancesinmolecularbiology,humandiseasebio-computertechnology,genetic,andepidemiologydatabases.Thismonitoring,andcomputersciencecouldbeleveragedtodrivewouldenablescientiststodeveloptargetedandeffectivemanage-investmentsindevelopingnew,potentiallymorerelevantandmentdrugsandcures.Lessonscanalsobelearnedfromdataeffectiveresearchmethods,includinginsilico,diseasepathwaygeneratedduringclinicaltrials.Understandingwhyaclinicaltrialapproach-andhuman-basedstudies.Redirectingfundingtowardfailedisinthebestinterestofpatientsandrepresentsanimportantthesemodelswouldconsiderablyimprovetheirefficacy,validity,pieceofthediseasepathwaysmap.

andubiquity,aswellasaddingtoourunderstandingofhumanbiology,diseaseprogression,anddrugfunctions.Importantly,Concludingremarks

usingavarietyofalternativeswillimprovedatavalidityandThelackofclinicaltranslationfromanimalstohumansafter30deepenourunderstandingofbasicscience.Alternativetechni-yearsofresearchsuggeststhatanimalmodelsarenotanidealquesarenotonlyethicallysuperior,butalsooffermoredirect,systemforstudyingALSorfordevelopingdrugtherapies.Therobustinsightsintohumanbiologyanddisease,therebyacceler-diseasemechanismdatafromanimalmodelstudiesarenotco-atingthetransitionfrombenchtobedside.GiventhattheUShesiveandthepreclinicaldrugstudieshavenottranslatedwellCongressisre-examiningthewayinwhichdrugsarediscovered,intosuccessfulhumanclinicaltrials.Giventhatanimalsdonotdeveloped,anddeliveredthroughthe21stCenturyCuresAct,andnaturallydevelopALS,thecurrentmodelsareanimalssubjectedastheALSassociationhasreceivedmorethanUS$100milliontoartificialgeneexpressionanddiseaseconstruction.However,throughthe‘IceBucketChallenge,’alargeinvestmentisbeingtheseconstructedmodelshavephenotypesdistinctfromhumanmadedirectlytohelpthefamiliesofpatientswithALS.ThetimeisALS,therebylimitingmodelvalidityandmuddyingtheanalysesripeforre-examiningourapproachtoALSintervention;itisclearofanalreadycomplexdisease.Inaddition,therelevanceofthatweneedtoadoptanewapproachtobiomedicalresearchanimalsasmodelsingeneralisbeingquestioned.Criticalbiolog-basedonhumanbiologyandtheuseofnewandadvancedicaldifferencesexistbetweenanimalsandhumans,resultingin

technologies.

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