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TITLE:Defectsintheneuroendocrineaxiscauseglobaldevelopmentdelayina
DrosophilamodelofNGLY1Deficiency
AUTHORS:TamyPortilloRodriguez,JoshuaD.Mast,TomHartl,EthanO.Perlstein*
AFFILIATION:PerlaraPBC,6000ShorelineCourt,Suite204,SouthSanFrancisco,
California94080,UnitedStates
*correspondingauthor:(415)347-8493,ethan@perlara.com
MAJORCLASSIFICATION:BiologicalSciences
MINORCLASSIFICATION:Genetics
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ABSTRACT
N-glycanase1(NGLY1)Deficiencyisararemonogenicmulti-systemdisorderfirst
describedin2014.NGLY1isevolutionarilyconservedinmodelorganisms,including
theDrosophilamelanogasterNGLY1homolog,Pngl.Hereweconductedanatural
historystudyandchemical-modifierscreenonanewflymodelofNGLY1Deficiency
engineeredwithanonsensemutationinPnglatcodon420,resultingintruncation
oftheC-terminalcarbohydrate-bindingPAWdomain.Homozygousmutantanimals
exhibitglobaldevelopmentdelay,pupallethalityandsmallbodysizeasadults.We
developeda96-well-plate,image-based,quantitativeassayofDrosophilalarvalsize
foruseinascreenofthe2,650-memberMicrosourceSpectrumcompoundlibraryof
FDAapproveddrugs,bioactivetoolcompounds,andnaturalproducts.Wefound
thatthecholesterol-derivedecdysteroidmoltinghormone20-hydroxyecdysone
(20E)rescuedtheglobaldevelopmentaldelayinmutanthomozygotes.Targeted
expressionofahumanNGLY1transgenetotissuesinvolvedinecdysteroidogenesis,
e.g.,prothoracicgland,alsorescuesglobaldevelopmentaldelayinmutant
homozygotes.Finally,theproteasomeinhibitorbortezomibisapotentenhancerof
globaldevelopmentaldelayinourflymodel,evidenceofadefectiveproteasome
“bounce-back”responsethatisalsoobservedinnematodeandcellularmodelsof
NGLY1Deficiency.Together,theseresultsdemonstratethetherapeuticrelevanceof
anewflymodelofNGLY1Deficiencyfordrugdiscovery,biomarkerdiscovery,
pharmacodynamicsstudies,andgenemodifierscreens.
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INTRODUCTION
Recessiveloss-of-functionmutationsintheevolutionarilyconservedgeneNGLY1
resultinanultra-raregeneticdiseasecalledNGLY1Deficiency,whichis
characterizedbyglobaldevelopmentaldelay,seizures,smallheadandextremities,
chronicconstipation,lackoftears,andfloppybody(Ennsetal.,2014).NGLY1,short
forN-glycanase1,encodesadeglycosylatingenzymethathydrolyzesN-linked
glycansfromasparagineresiduesofglycoproteins,liberatingoligosaccharidesfor
degradationandrecycling(Suzukietal.,2016).Acomprehensiveclinicalsnapshot
byNationalInstitutesofHealth(NIH)establishedpotentialmeasurableclinical
endpointsandabaselineofdiseaseprogressioninacohortof12patients(Lamet
al.,2017).
ThepathophysiologyofNGLY1Deficiencyhasnotyetbeenfullyresolved.Before
2014,littlewasknownaboutNGLY1function.Itselucidationisthefocusof
numerousresearchgroupsemployingadiversityofdiseasemodels,includingmodel
organismsandhumancells.TwomodelsofthepathogenesisofNGLY1Deficiency
havebeenproposed.
Thefirstmodelisrootedinbiochemistryanddefectsincellularglycoprotein
homeostasis(Huangetal.,2015).NGLY1isanessentialcomponentoftheconserved
proteinqualitycontrolsystemknownasendoplasmic-reticulum-associated
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degradation(ERAD),bridgingp97/VCP-mediatedretrotranslocationofproteins
fromtheERtothecytoplasmforbulkdeglycosylationandsubsequentdegradation
bytheubiquitin-proteasomesystem(UPS)(Suzuki,2015).Theabsenceof
cytoplasmicN-glycanaseactivityhasbeenproposedtoresultininappropriate
cleavageofN-glycansfromglycoproteinsbythedownstreamcytoplasmicenzyme
endo-β-N-acetylglucosaminidase(ENGase),whosenormalsubstrateissoluble
oligosaccharideliberatedbyNGLY1.SuchglycoproteinsmisprocessedbyENGase
wouldretainasingleGlcNacresiduethatmaydestabilizeproteinsandpromote
theiraggregation.SuzukiandcolleaguesobservedevidenceofN-GlcNac
misprocessingandaccumulationinvitroinNGLY1-/-mouseembryonicfibroblasts
(Huangetal.,2015;Fujihiraetal.,2017).BasedonthecollectiveworkofSuzukiand
colleagues,inhibitionofENGasehasbeenproposedasatherapeuticthesisfor
NGLY1Deficiency(Fujihiraetal.,2017;Bietal.,2017).Indeed,aNGLY1-/-ENGase-/-
doublemutantmouseisviablewhileaNGLY1-/-singlemutantdisplaysvarying
degreesoflethalitydependingongeneticbackground(Fujihiraetal.,2017).
However,aNGLY1-/-ENGase-/-doublemutantmouseisnothealthy.Additional
pathogenicmechanismsarerequiredtoexplainNGLY1Deficiency.
Thesecondmodelofpathogenesisisrootedingeneticsanddefectsinthe
deglycosylationofspecificglycoproteinclients,includingbutnotlimitedtothe
masterregulatoroftheconserved26Sproteasome“bounce-back”response,
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NFE2L1(thetranscriptionfactornuclearfactorerythroid2like1alsoknownas
Nrf1)(Radhakrishnan,2010).Nrf1,belongstotheancientbasicleucinezipper
familyoftranscriptionfactorsthatregulatemanydevelopmentalandstress
responsepathwaysinanimals(Kim,2016).TheflyNrf1homolog,cap-n-collar(cnc)
increasestheexpressionofproteasomesubunitgenes,aswellasoxidativeand
redoxstressresponsepathways(Grimberg,2011).Inanunbiasedscreenforgenetic
modifiersoftheproteasomebounce-backresponseinnematodes,Lehrbachand
RuvkundiscoveredthatthenematodehomologofNGLY1,PNG-1,deglycosylatesthe
ER-membraneboundisoformofthenematodehomologofNrf1,SKN-1A.They
furtherdemonstratedthatdeglycosylationofSKN-1AbyPNG-1isrequiredforSKN-
1Atranslocationtothenucleus,andtranscriptionalactivity(Lehrbach,2016).
Inacomplementarystudy,BertozziandcolleaguesrevealedthatNGLY1activityis
requiredforNrf1signalinginmouseembryonicfibroblastsmiceinthesame
mannerthatPNG-1activityisrequiredforSKN-1Afunctioninnematodes(Tomlin,
2017).Infact,theyshowedthatchemicalinhibitionofNGLY1functionpotentiates
cytotoxicitycausedbyproteasomeinhibitioninhumancancercelllines(Tomlin,
2017),whichmirrorstheobservationinnematodesthatpng-1loss-of-function
mutantsareextremelyhypersensitivetoproteasomeinhibitionbybortezomib
(Lehrbach,2016).Jafar-NejadandcolleaguesshowedthattheflyNGLY1/Pnglis
requiredduringembryonicandlarvaldevelopmentinDrosophilaforpost-
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translationalprocessingofDpp,theflyhomologoftheconservedbonemorphogen
protein(BMP)family(Galeone,2017),openingupthepossibilitythatNGLY1is
requiredforthefunctionofmultipleglycoproteinclients.
Herewecarriedoutphenotyping,high-throughputassaydevelopmentanda
chemical-modifierscreenonanewflymodelofNGLY1Deficiency,hereinreferred
toasngly1PL.Of~2,650bioactivecompounds,theecdysteroidinsectmolting
hormone20-hydroxyecdysone(20E)suppressedglobaldevelopmentaldelayin
mutanthomozygotes.ExpressionofahumanNGLY1transgeneintheprothoracic
gland(PG)andsitesofecdysteroidogenesisrescuedglobaldevelopmentaldelayin
mutanthomozygotes.Thesedataindicatethatcellautonomousdefectsinecdysone-
producingorgansandtissuesmayexplaintheglobaldevelopmentaldelayofthe
ngly1PLflies.nglyPLfliesarealsohypersensitivetotheproteasomeinhibitor
bortezomibandtheorganicsolventdimethylsulfoxide(DMSO).Together,these
observationscanbeparsimoniouslyaccommodatedbyamodelwherein
NGLY1/PnglisrequiredforNrf1/cncfunctionintheDrosophilaneuroendocrine
axis.
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METHODS
ngly1PLallelegeneration
Acassettecontainingastopcodonandmw+wasclonedintoamodifiedversionof
pUC57.Homologyarmstothengly1locuswereclonedupstreamanddownstream
ofthecassette.TheguideRNA(GCTGAGGAATAACTTTCGATCGG)wasclonedinto
pCDF3(Portetal.,2014).pCDF3andpUC57withngly1homologyarms,stopcodon,
andmw+wereinjectedintovas-Cas9(Bloomingtonstock#51323).Two
independentmw+F1strainswereestablishedandbalancedstockswerecreated.
Sequencingconfirmedtheintegrationofthestopcodonandmw+(Figure1A)
immediately3’tobp1906699(Release5.57)inchromosome2R(NT_033778.3).
Rateofeclosionandrescuebyhumanngly1transgenestudies
ngly1PL/CyO;actin-switch-gal4;UAS-human-ngly1,w+/+maleswerecrossedto
ngly1PL/CyO,Act-GFPvirginfemales.Thephenotypesofeclosingflieswere
recordedondays9-14afterparentsweremated.Wefoundthattheactin-switch-
gal4transgenicstrainexpressedGal4,despitetheabsenceofRU486.
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Flystrains
Actin5C-switch-gal4(stock#9431)wasobtainedfromtheBloomingtonstock
center.PnglexcisionallelesandUAS-human-ngly1wereobtainedfromDr.Hamed
Jafar-Nejad.2_286,phmandspokGAL4driverswereobtainedfromDr.Michael
O’Connor.
Larvalsizemeasurements
0-4houroldlarvaewereplacedintopetridisheswithstandardflyfoodmediafor3
daysat25°C.Then,larvaewereremovedfromthefood,rinsedinPBS,andplacedin
PBStobeimaged.Theareasofnineteenheterozygousandtwentyhomozygous
larvaeweremeasuredinImageJ.
Platepreparationforchemicalmodifierscreening
100nLcompoundorDMSO(vehicle)wasdispensedfrommotherplatesintowellsof
96welldaughterplatesusingtheEchoacousticdispenser(LabCyte).Then,100uL
ofmoltenstandardflyfoodmedia(molasses,agar,yeast,propionicacid)lacking
cornmeal,butcarrying0.025% bromophenolblue,wasdispensedusingaMultiFlo
(Bio-TekInstruments).Bromophenolbluedyeinthegutsofthelarvaincreases
theirsignaloverbackgroundlaterduringimaging.Plateswereplacedonaplate
shakerandshakenfor1minute,duringwhich,themoltenagarsolidifiedwith
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thoroughlymixedcompound/DMSOineachwell.Plateswerethencoveredwith
adhesivealuminumsealsandstoredat4°Cforuptotwoweeks.
Culturingflylarvaein96-wellplates
Ngly1PL/CyO,Act-GFPflieswereculturedinalargepopulationcage(Genessee)for
uptotwoweeks,wheretheylaideggsongrapejuiceagartrays(Genessee)coated
withathinstripofactiveyeastpaste.Eggcollectionswererestrictedto~6hours
duringmorninghoursandwereplacedinto25°Cfor~24hrs.~0-6hrold1stinstar
larvaewererinsedoffofthetrayswithroomtemperaturewaterandcollectedin
funnel-attached40micronsievestypicallyusedforcellstraining.Toremove
embryocontamination,thelarvae/embryomixturewasincubatedtwotimesin
10.3%inoculationsolution(sodiumchloride,sucrose,10%TritonX-100)forthree
minutes.Mostembryosfloattothetopofthissolutionafterthreeminutes,while
larvaeremaininsuspension.Embryosfromthemixture’ssolutioncanthereforebe
removedwitha10mlpipette.Embryoswerethenaddedtosortingsolution
(Polyethyleneglycol,COPAS200xGPsheathreagent,Tween20)anddrawnintoa
BioSorter(UnionBiometrica)forsortinganddispensinginto96well
compound/mediabearingplates.ThreeGFPnegativehomozygotesweregated
fromheterozygotesanddispensedintothe11leftmostcolumnsoftheplate,and
wellsG12andH12.ThreeGFPpositiveheterozygotesweredispensedintowells
A12,B12,C12,D12,E12,andF12.Plateswerethencoveredwithpermeable
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adhesivesealsandincubatedat25°Cforthreedays.Approximately18plateswere
dispensedintoperday,andlarvalviabilitywasnotaffectedbytheirtime
submergedindispensingsolution(<4hours).
Preparationofplatesforimaging
Ontheterminaldayoftheassay(day3),gaspermeablesealswereremovedand
plateswerefilledwith20%sucrosesolutionmadeacidicwithhydrochloricacid(pH
2)andcarryingdefoamer(FiveStarDefoamer105-2oz).Plateswerethenvortexed
andmoresolutionwasaddedtosuspendlarvaetoafixedfocalplane.Plateswere
thenimaged.
Imageanddataanalysis
TheFlyImagerusesaSonya7riicamera,controlledoverUSBbythegphoto
softwaretogeneratefullplateimagesthatarethenrunthroughalarvaldetection
algorithm.Thealgorithmfirstbuildsanimageofwhatthewellwouldlooklikewhen
itisempty.Todothisitexcludesareasnearedges(sincethoseareprobablylarvae)
andinterpolatesacrossthegaps.Itthenlooksatthedifferencebetweentheimage
andestimatedemptybackground,selectingareaswithahighdifferencetobelarvae.
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20Efeedingandeffectondevelopmentaltiming
20E(Enzo)wasdissolvedin100%ethanolandaddedtomoltenstandardflyfood
mediaat200µMandaddedtovials.Anequivalentsetofvialswithanequalamount
ofethanolwasaddedfornegativecontrols.0-6hourlarvaeweredispensedwith
theBioSorterintothevialsandincubatedat25°Cfor12days.Thenumberoflarvae
thatpupariatedwererecordedonkeydaysat8am,12pm,and4pm,andthenumber
ofadultsthateclosedwererecordedondays9–12atasingletime.
Rescue of developmental delay with ring and prothoracic gland expression of human
ngly1
Ringglandexpressionofhumanngly1wasdrivenusingtheGAL4/UASsystem
(BrandandPerrison,1993)andringglanddrivers286-GAL4,spok-GAL4,andphm-
GAL4inngly1PL/ex20compoundheterozygotes.Adultswereallowedtolayeggsfor8-
16hours,andthegenotypesforalleclosingflieswererecorded.Thenumberofring
glandrescuedflieseclosedwasnormalizedtothenumberofeclosedsibling
homozygotesnotcarryingthedriver.
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RESULTS
Generationofthengly1PLflyandcharacterizationofitsphenotypes
WeusedCRISPR/Cas9tocreateanewalleleofPnglwithaprematurestopcodon
andaselectablemarkerinsertedupstreamofthePAWdomain,hereinreferredto
simplyas“ngly1PL“(Fig.1A).Webenchmarkedngly1PLagainstthepreviously
describedPnglgeneticnullmutantgeneratedbyP-elementexcision(Pnglex20),
whichcausesdevelopmentaldefectsandsemi-lethalitywithfewadultescapers
(Funakoshi,2010).ngly1PLhomozygotesaredelayedinthelarval-to-pupal
transitionbyoneday,anddelayedtoeclosionby2days(Fig.1B).Asthree-day-old
larvae,ngly1PLhomozygotesare~75%thesizeoftheirheterozygotesiblings(P<
1X10-10,student’st-test)(Fig.1D).Consistentwiththesemi-lethalityofPnglexcision
alleles,wealsoobservedthatthengly1PLhomozygotemutantexhibitscold-sensitive
pupallethality,meaningnosurvivalbelow18˚C(datanotshown).Timetoeclosion
inngly1PLhomozygotesiscompletelyrescuedbyubiquitousexpressionofahuman
NGLY1(hNGLY1)transgene(Fig.1B).Thesmallbodysizephenotypeofngly1PL
homozygoteadultsisalsorescuedbyubiquitousexpressionofhNGLY1(Fig.1C).
Theseresultswhenfliesarerearedinstandardvialssuggestedphenotypessuitable
forhigh-throughput,whole-organismphenotypicscreeningateachstageoffly
developmentfrom1stinstarlarvaeonwards.
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Wedecidedtooptimizeahigh-throughputlarvalsizeassayin96-wellplatesfor
severalreasons.Assayminiaturizationfrom30mLvialsto96-wellplatesinvolves
reducingthenumberofanimalstestedbyafactorof10,e.g.,30animalspervial
versus3animalsperwell.Thelarvalsizedifferencebetweenngly1PLheterozygote
larvaeversusngly1PLhomozygotelarvaewasmorestatisticallyrobustin96-well
platesthanthetimingtopupationdifferenceorthetimingtoeclosiondifference.A
3-dayassayversusa7-11dayassayallowedforfasteroptimizationcycletimes.
Timetopupationandtimetoeclosionwouldbesecondaryassaysinvialformatto
ensureprimaryscreeninghitsrescueglobaldevelopmentaldelay,notjust
developmentaldelayinlarvae.
Asapreludetodrugscreening,weestablishedthetolerabilityofngly1PL
homozygotelarvaetodimethylsulfoxide(DMSO),theorganicsolventforalmost
everycompoundlibrary,includingtheMicrosourceSpectrumcollection.The
maximumtolerateddoseofDMSO,therefore,setsaceilingonthefinalassay
screeningconcentration.Unexpectedly,weobservedthatngly1PLhomozygotesare
extremelyhypersensitivetoDMSOcomparedtotheheterozygotecontrol(Figure
2).Weestimatedamaximumtolerateddoseinthengly1PLhomozygoteof0.1%v/v
DMSO(14mM)(Fig.2A,C),whichisactuallyseveralfoldmoresensitivethanPngl
excisionallele20(Fig.2B).Incomparison,wild-typeDrosophilaexhibitadult
lethalitystartingat1%v/vDMSO,larvallethalityrangingfrom2%v/vto3%v/v
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DMSO,anda“noobservedadverseeffectlevel”(NOAEL)doseof0.3%v/vDMSO
(Nazir,2003).DMSOhypersensitivityappearstobespecifictothengly1PL
homozygoteinalimitedcomparisontoothermutants(datanotshown).Forthe
purposesofdrugscreening,weexploitedDMSOasasensitizerinthelarvalsize
assay.Exposureofmutantlarvaeto0.1%v/vDMSO,whichwouldentailafinal
assayscreeningconcentrationof10µMforeachlibrarycompound,waspotent
enoughtoincreasethedynamicrangeandimprovetherobustnessoftheassay
whilesparinglarvallethality.
Astatisticallyrobusthigh-throughputimage-basedlarvalsizechemicalmodifierpilot
screenyieldedonevalidatedhit,20-hydroxyecdysone(20E)
Wepositedthatthesmalllarvalsizephenotypeofngly1PLhomozygotemutants
couldbeusedtodiscoversmall-moleculesuppressorsthatprovideinsightintothe
pathophysiologyofNGLY1/Pngldeficiencyinthefly.Tothatend,wedevelopeda
novelimage-basedassaytocultureDrosophilalarvaeinclear-bottom96-wellplates
whereeachwelleithercontainsauniquesmallmoleculeorvehicle.Usinga96-well-
plateformatallowedustotakeadvantageofexistinglabautomationformanaging
multi-wellplatesinhigh-throughputscreening(HTS)applications,includinga
whole-organismsorteranddispenser.Ourmethodincludesstepstodissolveand
dilutepre-existingflyfoodsothatlarvaecanbefloatedtoafixedfocalplaneand
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thenimagedwithcustomplateimager.Softwarewaswrittentomeasuretheoverall
areaoffloatedlarvaetoenabletheidentificationofsmallmoleculesthat
significantlyincreasethesizeofngly1PLhomozygotelarvaevsvehicle-fedlarvae.
WeselectedtheMicrosourceSpectrumcollectionforapilotscreen.Thelibrary
contains2,532uniquecompoundsincluding~600FDAapproveddrugs,~800
compoundsthathavereachedclinicaltrialstagesintheUSA,~400drugsthathave
beenmarketedinEuropeorJapanbutnottheUSA,~600bioactivetoolcompounds,
and~800naturalproducts.Threelarvaeperwellwereculturedin96-wellplates
withcontrolwellscomprisingthetwooutermostcolumns.Weusedngly1PL
homozygouslarvaefedvehicle(0.1%v/vDMSO)asanegativecontrol.Weinclude
twopositivecontrolgroups:thefirstgroupconsistofngly1PLheterozygouslarvae
fedvehicle,andthesecondgroupconsistsofngly1PLhomozygouslarvaecultured
withoutDMSO.Theremaining80wellscontainedngly1PLhomozygouslarvaefeda
uniquelibrarycompoundat10µMplus0.1%v/vDMSO.Animageofa
representativedrugscreeningplatewithzoomed-inreferencewellsisshownin
Figure3.
Weperformedthescreenintriplicate,meaningthreeindependentbiological
replicates.Therewasarobuststatisticalseparationbetweenpositiveandnegative
controls(Fig.4A).Onaverage,ngly1PLhomozygoteswerehalfthesizeofngly1PL
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heterozygotes,althoughsomevariationinsizewasobservedineachgenotype.75%
ofallscreeningplates(73of96)hadaZ’factor>0;mostofthescreeningplates
withhighvariabilitybelongedtothesecondreplicate(Fig.4B).Aweakpositive
correlationexistedinpairwisecomparisonsofeachbiologicalreplicatewhenall
datapointsareincluded(Fig.5A).WhenweonlyincludedwellswithZscoresless
than-2orgreaterthan2,thepositivecorrelationincreasedsignificantlyonaverage
(Fig.5B).Forexample,inthepairwisecomparisonofreplicate1versusreplicate2,
thecorrelationimprovesfromR2=0.05977inthefulldatasettoR2=0.48171inthe
reduceddataset(withpositiveandnegativecontrolsremovedaswell).
Weinitiallyconsidered162pre-hitswithaZscoreof>2intwoofthreebiological
replicatesasprimaryscreeningpositives(Table1).Overtwo-thirdsofthosepre-
hitsprovedtobefalsepositivesforoneoftwoofthefollowingreasons.First,
uneatenflyfoodinthewelloccasionallyincreasedtheimagebackgroundartificially
inflatingthecalculatedareaofthelarvae.Second,becausePnglmutantsare
hypersensitivetoDMSO,anyfailureincompounddispensingorvariabilityinDMSO
levelsduetohydrationresultedinlargerlarvae.Forty-fivecompoundswithaZ-
scoreof>2intwoofthreereplicateswereconsideredfurtherbecausetheirwells
didnothaveobvioushighbackgroundorlow/noDMSO.Therawimagesofthewells
containingthose45compoundsweremanuallyinspected,and22hadaclear
differencefromcontrols.Ultimately,22compoundswerefoundtohaveaZof>2in
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twoofthreereplicatesanduponmanualinspection,thewellswiththose
compoundsappearedtohavelarvaelargerthanthenegativecontrolsinthesame
plate.Twoofthose22compoundshadaZof>2inthreeofthreereplicates,were
countedtwiceinthiscomparison,andthusthefinalsetofpre-hitswetestedfurther
consistsof18uniquecompounds.
All18ofthepre-hitsfromthescreenwereordered,dissolved,andretestedin
attempttoreproducerescueinvialformatwithlargernumbersofanimals(Fig.5C,
Table2).Onecompound,20-hydroxyecdysone(20E),rescuedthedevelopmental
delayofngly1PLhomozygotelarvaedevelopmenttopupae(Fig.6B),buthadno
effectondevelopmentaltimingofngly1PLheterozygotelarvae(Fig.6A).Moreover,
thesuppressiveeffectof20Epersistedtoadulthood,resultinginastatistically
significant4-foldincreaseineclosionpercentage(Fig.6E).Asacontroltoruleouta
simpleecdysonebiosynthesisdefect,weshowedthatthe20Eprecursor7-
hydroxycholesterol(7D)failedtorescuengly1PLhomozygotelarvaedevelopmentto
pupae(Fig.6D).Ifsynthesisof20Eisfaulty,itislikelyatapointdownstreamof7D.
Wecouldnotreproduciblyvalidateanyoftheother17pre-hits,sowefocusedour
effortsonunderstandingthemechanism-of-action(MoA)of20E.Therefore,this
pilotscreenhadanextremelylowhitrateof0.04%(1/2532).
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20Eimplicatestheflyneuroendocrineaxisasparticularlysensitivetolossof
NGLY1/Pnglfunction
20EdrivesmetamorphosisinDrosophilaandarthropodsgenerally(Faunes,2016).
Dietarycholesterolformsthebasisof20E,andall20Eprecursorsaresynthesizedin
theprothoracicgland,anorganof<50cellsthatcomprisespartofthelargerring
gland.Theimmediate20Eprecursor,ecdysoneor“E”,ispackagedintosecretory
vesicles,secreted,anddistributedbythehemolymphthroughouttheanimals.Eis
convertedto20Eintheseperipheraltissues,andinitiatessignalingcascadesand
geneexpressioninducingphysiological,morphological,andbehavioralchangeswith
eachmolt,ordevelopmentaltransition.
Totestwhetherthe20EinsufficiencyinPngl-deficientmutantsisautonomousto
theringgland,weexpressedaUAS-drivenhNGLY1transgenethatcanrescueglobal
developmentaldelaywhenexpressedubiquitously(Fig.1B,C),intheringgland
withthe2-286-GAL4driver.Tocontrolforoff-targetmutationsduetostrain
backgroundconfoundingourresults,wetestedthetrans-heterozygouscombination
ofPnglalleles,ngly1PL/ex20.Mostngly1PL/ex20larvaenotexpressingthehuman
ngly1transgeneeclosedonDay10.Incontrast,mostngly1PL/ex20larvaeexpressing
humanNGLY1intheringglandpupatedonDay9(Fig.7A).Asidefromthering
gland,2-286-GAL4alsodrivesexpressioninthesalivarygland,fatbody,andcuticle
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inlarvae(Timmonsetal.,1997).Weobservedasimilarrescueeffectwhenhuman
ngly1transgeneexpressionisdrivenbytworing-gland-specificdrivers,phantom-
GAL4(phm)(Fig.7B)andspookier-GAL4(spok)(Fig.7C).phmandspokencode
cytochromeP450monooxygenasesintheecdysonebiosyntheticpathway(Rewitz,
2007).Together,thesedataindicatethatNGLY1/Pnglisnecessaryfornormal
functionoftheringglandtoenableproperlevelsof20Etocirculatethroughoutthe
developinganimalanddrivedevelopmentaltransitions.
NGLY1/Pngldeficientfliesarehypersensitiveproteasomeinhibition
Asmentionedabove,lossofNGLY1sensitizesnematodesandhumancancercell
linestoproteasomeinhibition.Wepredictedthatngly1PLhomozygotemutants
wouldexhibithypersensitivitytobortezomib.Indeed,bortezomibcaused100%
lethalityofngly1PLhomozygouslarvaeat5µM,whilelethalitywasnotobservedin
ngly1PLheterozygouslarvaeuntiladoseof25µM(Figure8.).Inaddition,thesizeof
ngly1PLhomozygousmutantswasreducedby<50%whentreatedwith1µM
bortezomib,andto~50%ngly1PLheterozygouslarvaeat~10µMbortezomib.
Thesedataindicatethatthehalf-maximalinhibitoryconcentration(IC50)of
bortezomibtoreducelarvalgrowthis~10Xlessinngly1PLhomozygote.
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DISCUSSION
WesuccessfullygeneratedanewDrosophilamodelofNGLY1Deficiency,optimized
ahigh-throughputwhole-animalphenotypicassayoflarvalsize,andthen
demonstratedtherapeuticrelevanceinaproof-of-conceptdrugrepurposingscreen.
While20EitselfshouldnotbeconsideredadrugcandidateforNGLY1Deficiencyin
humans,thefactthatitisachemicalsuppressorimplicatestheneuroendocrineaxis
inthepathophysiologyofPngldeficiencyinflies.Inotherwords,eventhough20Eis
aninsect-specificdevelopmentalhormone,theneuroendocrineaxisandsteroid-
deriveddevelopmentalhormonesareconservedinmammalsandmayplayarolein
NGLY1Deficiencyinhumans.Collectively,ourfindings–moststrikingly,
hypersensitivityofthengly1PLmutantbothtobortezomibandtoDMSO–alignwith
resultsobservedinnematodes(LehrbackandRuvkun,2017)andhumancells
(Tomlin,2017)thatpointtotheessentialandconservedroleofNGLY1inregulating
thefunctionofglycoproteinclients,specificallyNrf1andtheproteasomebounce-
backresponse.
Infact,wecanalreadyproposeamechanismtolinkNrf1functionto
ecdysteroidogenesisandtheneuroendocrineaxisinflies.TheflyhomologofNrf1is
thelongestisoformofcnc,CncC,whichcontainsaconservedN-terminalleucinerich
transmembraneregiontargetingCncCtotheER(Grimberg,2011).Specificlossof
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CncCintheprothoracicglandreducestheexpressionofecdysonebiosyntheticgenes
andresultsindelayedtimingtopupation(DengandKerppola,2013).RNA
interferenceoftheColoradopotatobeetlehomologofCncCalsoreduced
ecdysteroidogenesispathwaygeneexpressionanddelayedtimingpupation,which
couldberescuedby20Esupplementation(Sunetal.,2017).Ourdatamakeastrong
testableprediction:CncCisthefunctionalequivalentofSKN-1Ainnematodesand
Nrf1inmammals,andthereforeisasubstratefordeglycosylationbyPnglinflies.A
secondtestablepredictionisthattheflyhomologofnematodeDDI-1andhuman
DDI2,ringslost(ringo),actsdownstreamofPngltoproteolyzeCncC,generatinga
mature,nuclear-activespecies.
Thereareotherexplanationsforhow20Emightrescueglobaldevelopmentaldelay
inthengly1PLmutantthatdonotdirectlyinvolveNrf1/CncC,orthatmayactin
concertwithlossofNrf1/CncCactivity.First,ngly1PLmutantsmaynotpackage
ecdysoneintosecretoryvesiclesproperly,ormaybedefectiveinsecreting
ecdysone.Second,signaltransductionmediatedbytheinteractionbetween20Eand
theecdysonereceptor(EcR)mightnotbefullyoperational,andsoextra20Eboosts
thisflawedsignaling.Third,ecdysonesecretionbytheringglandisinducedbytwo
neuronsthatsynapseontotheprothoracicglandandsecretetheneuropeptide
prothoracicotropichormone,orPTTH.PTTHcontactsthereceptortyrosinekinase
Torsotoinitiatesignalingleadingtoecdysonesecretion.Perhapsthereisaflawin
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PTTHsecretionorTorsosignaling.Fourth,damagetodevelopinglarvaltissues,or
starvation,mayimpingeon20Etofinetuneorganismdevelopmentsothata
properlyproportionedandnourishedanimalcandevelopfullytoadulthood.The
ngly1PLmutantmayhavedamagedtissues,forexamplethroughprotein-aggregate
toxicityorproteasomestress;oritmayhavesomedegreeofstarvation,forexample
ifthegutcannotattainnutrientsproperly.20Efeedingmaybypassdelaysinduced
bythishypotheticaltissuedamage/starvation.
ACKNOWLEDGMENTS
WethanktheGraceScienceFoundationforfundingthiswork.WethankDr.Kevin
Leeforplanningandexperimentplanning.Drs.HamedJafar-NejadandTadashi
Suzukikindlyprovidedreagents.PeterSanddesignedandbuiltourcustomfly
imagerandanalysissoftware.Dr.TomLeeprovidedassistanceduringngly1PL
mutantstrainconstruction.Dr.RebeccaChoyprovidedearlyguidanceonhowto
scaleupflyhusbandry/culturing.
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Figure1.Genotypingandphenotypingngly1PLallele.A)WeusedCRISPRtocreateanalleleofngly1withastopcodonandwhite+transgeneinsertedupstreamofthePAWdomain.Sequencingconfirmedtheintegrationofthestopcodonandmw+immediately3’tobp1906699(Release5.57)inchromosome2R(NT_033778.3).B)Ubiquitousexpressionofahumanngly1transgenerescuedthe2-daydevelopmentdelaytoeclosionphenotypeobservedonngly1PLhomozygotes.C)ngly1PLhomozygousadultfliesaresmallerthantheirhomozygoussiblingsexpressingthehumanngly1transgene.D)ngly1PLhomozygousaredevelopmentallydelayed,at3daysold,thelarvaeare25%smallerthantheirheterozygoussiblings(p<0.01).
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Figure2.ngly1PLhomozygotesarehypersensitivetodimethylsulfoxide(DMSO).A)Animageofngly1PLhomozygouslarvaegrownin96-wellplateformatonfooddosedwithdifferentconcentrations(0%-0.4%)ofDMSO.ngly1PLhomozygouslarvaearehypersensitivetoDMSO,adecreaseinlarvalsizeisobservedstartingat0.025%DMSOandlarvalsizeisdecreaseasthedoseofDMSOisincreased.ThetimetopupationofB)ngly1Ex20orC)ngly1PLhomozygouslarvaerearedonfoodtreatedwithDMSO.HomozygousmutantlarvaeexhibithypersensitivitytoDMSOshowingadelayedtimetopupariateandincreasedlethality.
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Figure3.Layoutofanngly1assayplatewithexamplesofcontrolsandpre-hitcompound.Larvaewereculturedin96-wellplateswithnegativecontrolsontheleftmostwells(column1)whichcarriedngly1PLhomozygouswithvehicleDMSOat0.1%.80testingwellswereinthemiddle(columns2-11)whichcarriedngly1PLhomozygouslarvaewithacompoundat10uM,andDMSOat0.1%.Rightmosttopwells(12A-12F)containpositivecontrolgroup1withngly1PLheterozygouslarvaeand0.1%DMSO.Rightmostbottomwells(12G-12H)containpositivecontrolgroup2withngly1PLhomozygouslarvaewithoutDMSO.ThecompoundinwellG4wasconsideredapre-hitcompound.
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Figure4.Theassayhadaconsistentdifferencebetweenpositiveandnegativecontrols.A)AconsistentseparationbetweenpositiveandnegativecontrolswithZ’Factor>0,indicatedarobustassaythatcouldidentifychemicalsuppressors.B)Threereplicatesofa32platelibrarywereanalyzedand73of96plateshadaZ’Factor>0whichfurthersuggestarobustassaytoidentifychemicalsuppressors.
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Figure5.Positivecorrelationbetween3Xreplicates.A)ThethreepairwisecomparisonsofZ-scoresshowpositivecorrelationsindicatingthatasetofsmallmoleculescouldmodifythesmalllarvalsizephenotypeB)ThepositivecorrelationsbetweenreplicatesismoreevidentwhenonlyplottingZ-scoresof<-2or>2.C)Larvalsizeisrescuedwhen20Eisfedtongly1PLhomozygouslarvae.
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Figure6.20-hydroxyecdysone(20E),butnotanearlierecdysonepathwayprecursor(7-dehydrocholesterol),partiallyrescuesdevelopmentaldelayandlethalityinngly1PLmutants.ThetimetopupationofA)ngly1PLheterozygousorB)ngly1PLhomozygouslarvaerearedonfoodtreatedwithdifferentconcentrationof20E.20Edidnotimpactdevelopmentalrateofheterozygousngly1PLlarvaetopupation,butrescueddevelopmentdelayofhomozygousngly1PLlarvaetopupationat200µM.ThetimetopupationofC)ngly1PLheterozygousorD)ngly1PLhomozygouslarvaerearedonfoodtreatedwithdifferentconcentrationof7-d.7-ddidnotimpactdevelopmentalrateofheterozygousorhomozygousngly1PLlarvaetopupation.E)Thefractionofanimalssurvivingtoeclosion.20E,butnot7-d,rescuedlarvallethalityofngly1PLhomozygousat200uM.
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Figure7:ngly1isnecessaryfornormalfunctionoftheringgland.Thefractionofngly1PL/ngly1Ex20compoundheterozygoteswithhumanngly1drivenbytheringglanddriver(blue)A)2_286-GAL4B)spokier-GAL4,orC)phantam-GAL4survivingtoeclosioncomparedtosiblingcontrolslackingadriver(red).A)Compoundheterozygousngly1PL/ngly1Ex20larvaeexpressing2_286>ngly1eclosedearlierandhadlowerlethalitythancontrolflies.Compoundheterozygousngly1PL/ngly1Ex20larvaeexpressingspokier>ngly1orphantam>ngly1eclosedlowerlethalitythancontrolflies.
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Figure8.NGLY1larvaeare≥10Xmoresensitivetotheproteasomeinhibitorbortezomib.Imagesof3dayngly1PL+/-heterozygousoragematchedngly1PL-/-mutantlarvaeraisedonfoodtreatedwith0,1,5,10or25µMbortezomib.Bortezomibdelayslarvaldevelopmentalprogressioninngly1PLhomozygouslarvaemoreseverelythanngly1PLheterozygouslarvaeleadingtosmallersizedlarvae,andislethalforhomozygotesatconcentrationsequaltoorgreaterthan5µM.
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Table 1. Whittling screen pre-hits to a set of promising compounds to test in validation studies. Ultimately, 22 compounds were found to have a Z of > 2 in 2 of 3 replicates and upon manual inspection, the wells with those cpds appeared to have larvae larger then the negative controls. Two of those 22 compounds had a Z of > 2 in 3 of 3 replicates, were counted twice in this comparison, and thus the final set to consider further were 18 unique compounds.
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