Emerging Roles for the Unfolded Protein Response in the Developing Nervous System

Review

Emerging

Roles

for

the

Unfolded

Protein

Response

in

the

Developing

Nervous

System

Juliette

D.

Godin,

*

Catherine

Creppe,

Sophie

Laguesse,

and

Laurent

Nguyen

*

Theunfoldedproteinresponse(UPR)is ahomeostaticsignalingpathway trig-geredbyproteinmisfoldingintheendoplasmicreticulum(ER).Beyondits pro-tectiverole,itplaysimportantfunctionsduringnormaldevelopmentinresponse toelevateddemand forproteinfolding. Several UPR effectors show dynamic temporalandspatialexpressionpatterns thatcorrelatewith milestonesof the central nervous system (CNS) development. Here, we discuss recent studies suggestingthatadynamicregulationof UPRsupportsgeneration, maturation, and maintenance of differentiated neurons in the CNS. We further highlight studiessupportingadevelopmentalvulnerabilityofCNStoUPRdysregulation, whichunderliesneurodevelopmentaldisorders.Webelievethatabetter under-standingofUPRfunctionsmayprovidenovelopportunitiesfortherapeutic strat-egiestofightER/UPR-associatedhumanneurologicaldisorders.

TheUPRIsaGuardianofCellularHomeostasisintheCentralNervous System

Thecentralnervoussystem(CNS)ofvertebratesincludestwomainstructures:thebrainandthe spinalcord.Theearlystagesofneuraldevelopmentaresimilaracrossallvertebratespeciesand startwiththeclosureoftheneuraltube(NT).Themorphogeneticeventsthatshapethebrain occurlaterwiththeswellingandfoldingoftheanteriorpartoftheNTintothreevesicles:the forebrain,midbrain,andhindbrain.TheposteriorpartoftheNTbecomesthespinalcord.The forebrainfurther splits intotwo additionalvesicles, one becomingthe telencephalonwhose dorsalpart generatesthe cerebral cortex. The cortexis an exquisite productof vertebrate evolution that computes higher cognitive functions and whose complex cytoarchitectonics reflectthegreatdiversity ofneurons andtheir migratorybehaviorsthattakeplaceduringits formation[1].Inmammals,thelaminarorganizationofthecortexarisesinside-outasprogenitors generatesuccessive wavesofpyramidal neurons inthe corticalwall[2]andinterneuronsin subpallialregions[themedialandcaudalganglioniceminences(MGEandCGE,respectively) andthepreopticarea(POA)][3,4].Projectionneuronstravelshortdistancesalongradialglial fibersandinterneuronsnavigatealongtangentialpathstosettleinthecorticalplate[5].While poorneuronsurvivalisthehallmarkofneurodegenerativedisorders,disruptingtheproduction, migration,ordifferentiationofcorticalneuronscanleadtocorticalmalformationsoften associ-atedwiththeetiologyofpsychiatricorneurologicaldisorders[6–8].

Duringneurogenesis,proteinsynthesisincreasesinprogenitorstomeetthecellulardemand imposedby theproliferationandmaturationofneurons. Activationofproteinqualitycontrol

Trends

Theunfoldedproteinresponse(UPR)is anevolutionaryconservedmechanism whosefunctiongoesbeyond regula-tionofcellularproteostasis.

SeveralUPReffectors areexpressed duringcentralnervous system(CNS) developmentandrecentstudieshave highlightedcriticalrolesforUPRduring neurodevelopmentaswellasneuronal maintenanceintheCNS.

Cellularandmolecularconditionsthat enhanced ER stress also deregulate UPRsignalingduringdevelopmentand can interfere with themyelination process aswellasleadtobrainmalformations.

DynamicallyregulatedUPRfinelytunes theneurogenesisprocessaswellas themorphologicalmaturationof neu-rons.However,itsupstreamregulators remaintobeidentified.

1

InstitutdeGénétiqueetdeBiologie MoléculaireetCellulaire(IGBMC), INSERMU964,CNRSUMR7104, UniversityofStrasbourg,Illkirch,France

2

GIGA-Neurosciences,Universityof Liège,C.H.U.SartTilman,Liège4000, Belgium

3

InterdisciplinaryClusterforApplied Genoproteomics(GIGA-R),University ofLiège,C.H.U.SartTilman,Liège 4000,Belgium

4

WalloonExcellenceinLifesciences andBiotechnology(WELBIO), UniversityofLiège,C.H.U.Sart Tilman,Liège4000,Belgium

*Correspondence:godin@igbmc.fr

(J.D.Godin)andlnguyen@ulg.ac.be

pathwaysplaysacriticalroletomaintaincellularproteostasisduringthisprocess.Amongthese mechanisms,theevolutionaryconservedunfoldedproteinresponse(UPR)adjuststhe endo-plasmicreticulum(ER)environmentupondetectionofmisfolded/unfoldedproteinstoensure thatproteinfoldingcapacityisbalancedwithneeds.TheERisresponsibleforthesynthesisand folding of secretory proteins as well as others dedicated to either the cell surface or the membraneofotherintracellularorganelles.Differentcellstressconditions,including dysregu-lationofcalciumhomeostasisorredoxstatus,elevatedrateofsecretoryproteinsynthesisor theiralteredglycosylationcaninterferewiththeirproperfoldingleadingtoa‘collectivelynamed’ ERstress. This stress isfurthermanaged by activationof theUPR that either restores ER homeostasis(theUPRadaptivepathway)ortriggerscelldeath(theUPRapoptosispathway)[9]. TheUPRpathwayplaysacriticalroleinsynthesis,folding,andstructuralmaturationof approxi-matelyone-thirdofallproteinsproducedinthecell,mostofthembeingdedicatedtosecretionor membrane integration[10]. When theprotein folding capacity of cellsisoverwhelmed, they experienceERstressandactivateUPR.Thissituationoccurschronicallyindiseasecondition orin‘professionally’secreting cellssuch aspancreatic b cellswhereadaptiveUPR ensures homeostasis[11].Inhighereukaryotes,UPRtransductioninvolvestheactivationofERmembrane receptorsthatcontainanERlumenaldomainthatsensestheaccumulationofmisfoldedproteins, includingtheinositol-requiringenzyme1(Ire-1),theproteinkinase(PKR)-likeERkinase(Perk),and theactivatingtranscriptionfactor6(Atf6)(Box1).TheactivationoftheERstressresponseisa prosurvivalmechanism,whichexpandstheERandreducestranslationtolimitERproteinloading. Inaddition,itpromoteschaperoneexpressiontohelpclientproteinstorefoldproperly.TheUPR alsoclearssomemisfoldedproteinsthroughactivationoftheER-associateddegradation(ERAD) processthatpromotesubiquitylationanddegradationoftheseproteinsbythe26Sproteasome [12].However,whentheUPRcannotcopewiththeoverloadofmisfoldedproteins,then,thecells activatesa‘terminal’UPR,whichfinallyleadstoapoptosis[13](Box2).

Thisreview sumsupthecurrentknowledgeaboutthephysiologicalroles playedbyUPR in cellularhomeostasisduringCNSdevelopmentandhowdisruptingitsactivityunderlies pathol-ogy(Figure1,KeyFigure).

TheUPRContributestoCellFateAcquisition duringCNSDevelopment

UPRsignalingisactivatedduringneurogenesisinvariousanimalmodels[14–21](Table1).Its activationisindicativeofaphysiologicalfunctioninneuronalcommitmentandcellfate acquisi-tion.Thefirstlineofevidencecomesfrominvitrostudies.First,atleasttwoofthethreearmsof UPR(PerkandIre-1)areturnedonduringneuronaldifferentiationofmouseembryonicstem (mES) cells [22]. Second, ER stress induction by thapsigargin, tunicamycin, orbrefeldin A facilitatesneuronaldifferentiationandinhibits theglialdifferentiationofmES cells[22].Third, ERstressinducersalsofavorneurogenesisattheexpenseofgliogenesisafterdifferentiationof mouseembryoniccarcinomaP19cellsusingretinoicacid[23].Theseinitialstudiessuggestthat UPRisanactivefactorforneuronalcommitmentanddifferentiation.Thissectiondescribesin vivostudiessupportingnoncanonicalrolesforUPRsignalinginregulating:(i)thelateralinhibition and(ii)cellfateduringneurogenesis.

TheUPRFunctionsinLateralInhibition

Lateralinhibitionisamechanismthatgovernscellfatedecisionbetweenneighboringcells.The firstevidenceofUPRcontributiontolateralinhibitioncomesfromtheDrosophilaneurectoderm, whereitdictatesthecellfatechoicebetweenneuralandepidermalprogenitors.TheDrosophila ER-resident protein pecanex (pcx) controls the fate of neuroblasts [24]. Disruption of pcx function in neuroblasts impairs lateral inhibition and results in the increased generation of neuroblasts at theexpense ofepidermoblasts. This phenotype likelyresults fromdefective ERactivityasitissuppressedbyectopicexpressionoftwoUPRinducers,theactivatedXbp1or

Box1.TheUPRIsRequiredfortheMaintenanceofERHomeostasis

TheERisaspecializedorganelleinvolvedinthepost-translationalmodificationandfoldingofnewlysynthetizedsecretoryandmembraneproteins.Theoverloadingof misfoldedproteinsthatoccurswhentheERfoldingcapacityisoverwhelmedtriggersastressthatactivatesUPRsignalingtodealwiththealterationofERfunctions. ThethreemembranousERstresssensorsareIre-1,Perk,andAtf6(FigureI).TheiractivationengagestheUPR[51,52].ActivationofIre-1leadstoXbp1splicing,which generatesatranscriptionalfactor(Xbp1s)thatcontrolsUPRtargetgeneexpression[53].Moreover,Ire-1activationcanalsomediatemRNAdegradationbyaspecific processcalledRIDD(regulatedIre-1-dependentdecay)tolimittheERproteinload[54].Atf6activationinducesitsownintramembranecleavagefortranslocationtothe nucleus[Atf6(N)]whereitbindstotheregulatoryregionofUPRtargetgenes[55].Perkisactivatedbydimerizationandautophosphorylation.ItinduceseIF2/ phosphorylationatserine51thatpreventstherecyclingoftheinactiveGDPboundeIF2/toitsactiveGTPboundformbyeIF2B.Thisfurtherpreventstheformationof tRNAMet

ternaryinitiationcomplexesandresultsingeneraltranslationattenuation.However,somemRNAsthatharboraninternalribosomalentrysiteorasmallopen readingframeintheir50UTRcanbypassthetranslationinhibition[56].ThisisthecaseforAtf4,whoseupregulationuponPerkactivationpromotesUPRtargetgene induction[51].AnegativefeedbackloopcanfurtherbeactivateddownstreamAtf4topromotethedephosphorylationofeIF2/bytheGADD34/PP1Cphosphatase complex,whichrestoresproteinsynthesis(reviewedin[57]).

ActivationoftheUPRsensorsinducestheexpressionofvariousclassesofproteins.Amongthem,thechaperonesensureproperproteinfoldingtopreventprotein aggregationandapoptosis.BesideitsintrinsicrolesassuppressorofapoptosisandforthemaintenanceofERintegrity(reviewedin[58]),thechaperoneGrp78(also namedBip)controlstheactivationofthethreeERstresssensors:Ire-1,Perk,andAtf6[59].Undernormalconditions,Grp78bindstoERsensorstopreventtheir signalingcascades.But,uponERstress,Grp78isreleasedfromthemembraneandinducesthedimerizationofPerkandIre-1andthetranslocationofAtf6fromthe ERtotheGolgiwhereitiscleavedforitsfurthertranslocationtothenucleus.Meanwhile,thecellalsoprogressivelyactivatestheERAD,ubiquitin/proteasome,and autophagysignalingpathwaystogetridofthemisfoldedproteinsthataccumulateintheER[60].Nevertheless,aprolongedUPRactivationisoftenassociatedwiththe incapacityofthecelltocopewithERstressandultimatelyleadtocelldeathbyapoptosis[61].

ER Grp78 Misfolded protein A 6(N) A6 RIDD Splicing Degraded mRNA Translaon Proteasome ERAD mRNA Ire-1 Perk A4

P

P

_P

_P

Gene expression

Xbp1 ER chaperones ERAD ER chaperones

Oxidave response CHOP, apoptosis Autophagy

Amino acid metabolism

ER chaperones Lipid synthesis ERAD ER translaon Golgi Cleavage Nucleus A4 A 6(N)

P

adominantnegative formHsc70-3 (Heat-shockcognate 70-3), theDrosophila homolog of Grp78[24].HowactivationofUPRcompensatesforproperNotchsignalingintheabsenceof pcxremainstobeestablished.InterestinglyERstressinducersdonotaffectNotchsignaling duringtheretinoicacid(RA)-inducedneuronaldifferentiationofP19cells[23],suggestingthat

Box2.ImpairmentofUPRSignalingUnderliesCNSDisorders

ThemaintenanceandregulationofproteostasisisimportantandprolongedUPRactivationinducescelldeathandhas beenmorewidelyassociatedwithneurodegenerativediseases[62,63].Indeed,chronicERstressandsustainedUPR signalingareemergingaskeycontributorstoseveralhumanpathologiesrangingfromcancers,heartdiseases,and pulmonaryfibrosis,toneurologicaldisorders(reviewedin[11]).Oneofthepathologicalhallmarksofneurodegenerative diseasesisthetoxicaccumulationofmisfoldedproteinsandtheiraggregationthatinterferewithneuronviability[64]. AccumulationofproteinaggregatescorrelateswithUPRactivationandcelldeathinHuntington'sdisease,Parkinson's disease,andamyotrophiclateralsclerosis(ALS)[65–68].ThecausalroleofERstressandterminalUPRactivationin neurodegenerativedisordersisfurthersupportedbyrecentdataobtainedwithexperimentalmousemodels[69].In addition,neurodevelopmentaldisordersoffamilialorigin,suchasWolframsyndromeandautism-relatedCNTNP2,or inducedbyprenatalchronicalcoholexposure(fetalalcoholsyndrome)andcharacterizedbymicrocephalyalsoinvolve theinductionofERstressandUPRcomponentsasaputativetriggerofneuronalcelldeath[70–72].Recentmolecular dataalsoincriminateUPReffectorsasdownstreamtargetsofDISC1variantsinschizophrenia[73,74].

IntheCNS,theoligodendrocytesaretheglialcellsthatproducethegreateramountofplasmamembranetosupportthe myelinatingprocess,makingthemverysusceptibletoERstress.Alongthisline,recentobservationssuggestthatthe UPRanditsdownstreamtargetsareactivatedduringthedemyelinationprocessobservedinpatientsufferingfromeither multiplesclerosis[75]orPelizaeus–Merzbacher disease(PMD) [76].PMD isan X-linkeddysmyelinatingdisease characterizedbyERaccumulationofmisfoldedPLPproteinsthatfurthertriggersERstressandUPRinoligodendrocytes

[77].CorrelationbetweenERstressandpoormyelinationisalsoexemplifiedbytheautosomal-recessivehypomyelinating disordernamedvanishingwhitematterdisease,whichiscausedbyapointmutationintheproteinsynthesisfactoreIF2B thatimpairsitsresponsetophosphorylatedeIF2/incellstressconditionandtriggersUPRactivation[47,78].

KeyFigure

The

Unfolded

Protein

Response

(UPR)

Functions

in

the

Developing

Central

Nervous

System

(CNS)

Progenitor

Differenaon

Cell fate acquision Cell fate maintenance

Maturaon Neuritogenesis Secreon Clearing of extra neurons FASD Microcephaly WFS ASD Schizophrenia FASD SMA

UPR UPR UPR

Newborn

neuron Differenated neuron Cell death

Figure1.EmergingphysiologicalrolesofUPRsignalingatmilestonestagesofbraindevelopment:(i)generationanddifferentiationofneurons,(ii)maturationofnewborn neurons,and(iii)maintenanceofmatureneurons.EffectorsofthethreeUPRsignalingcascades(Box1)aredynamicallyexpressedduringCNSdevelopment[14–16,79]. ChemicalinductionofUPRinprogenitorsfavorsneuronaldifferentiation[18,22,23],whilegeneticablationofkeyUPReffectorsimpairsneurogenesisandcellfate commitment[18,30,33,35].Altogether,thesestudiessuggestthataprogressiveincreaseofUPRisrequiredfornormaldifferentiation.Neuronsfurtherrequirea permanentUPRsignalingtomature:bothreductionandelevationofUPRsignalleadtodefectinneuritogenesis[17,19,23,31].Finally,elevatedUPRsignalingduringCNS developmentislikelyrequiredtoeliminatetheexcessofneuronsbyapoptosis[30,79,80].Inthelowerpanel,neurologicaldisorderslinkedtoderegulationofUPRare indicated,supportingaparticularvulnerabilityoftheCNStoUPRdysfunction[18,63,70,72–74,81,82](Box2).Abbreviations:FASD,fetalalcoholspectrumdisorder; WFS,Wolframsyndrome;ASD,autismspectrumdisorder;SMA,spinalmuscularatrophy.

UPR ratherfunctionsinvivo tocontrol thelateral inhibition process.Notch-mediatedlateral inhibitionisalsorequiredformaintenanceofneuralprogenitorsinthedevelopingmammalian nervoussystem[25].ItisthustemptingtospeculatethattheUPRsignalingcouldalsomediate lateralinhibitioninthedevelopingcerebralcortexorspinalcord.

TheUPRPromotesNeurogenesisduringCerebralCortexDevelopment

Thecerebralcortexcontainslayersofneuronssequentiallygeneratedbydistinctlineage-related progenitors[26,27].Attheonsetofcorticogenesis,thefirst-bornprogenitors(apicalprogenitors, APs) divide asymmetrically to give birth directly to neurons. Later, they switch to indirect neurogenesisandgenerateintermediateprogenitors(IPs),which thengiverisetoprojection neuronsofallcorticallayers(Figure2)[26].DynamicregulationofUPRsignalsisproposedto governtheswitchfromdirecttoindirectneurogenesis(Figure2)[18].Laguesseandcolleagues presentmultiple lines ofevidence supporting this idea. First,there isan inverse correlation betweentheintensity ofUPR signalingandtherateofindirect neurogenesis [16,18],with a progressivereductionofAtf4signalinginAPsascorticogenesisproceeds[18].Second,the induction of ER stress with tunicamycin at midcorticogenesis (when indirect neurogenesis predominates)promotesdirectneurogenesis attheexpenseofIPs. Third, depletionofAtf4 attheonsetofcorticogenesis(whendirectneurogenesisisprominent)increasesthegeneration ofIPsattheexpense ofneurons [18].Thoseresults demonstratethataprogressive down-regulation of UPR in cortical progenitors acts as a physiological signal to amplify IPs and promotes indirect neurogenesis (Figure 2). The physiological relevance of this pathway is indicatedbyexperimentsshowingneurodevelopmentalphenotypesinmicewithexacerbated UPRsignals.Indeed,micedeficientfortheElongatorcomplexmaintainahighlevelofPerk– eIF2/–Atf4signalingthroughoutthecorticaldevelopment[18].ThiselevatedUPRresultsfrom defectiveproteintranslationand/orfoldingresultingfromlackofspecifictRNAmodifications [18,28].Thosemicedisplayaseveremicrocephalythatresultsfromadecreasedrateofindirect neurogenesis(Figure 2).Importantly, downregulationofAtf4levelinElongatordeficient pro-genitorsrescuesthebalancebetweendirectandindirectneurogenesis[18].Thissuggeststhat UPR dysregulation may underlie neurodevelopmental disorder (Box 2). Accordingly, Grp78 mutantmicearealsomicrocephalic[19].Assuch,itisworthtestingthelevelofUPRsignalingin thosemice.Interestingly,onlythePerk–eIF2/–Atf4branchofUPRisstrengthenedinElongator

Table1.DistributionofPrincipalUPREffectorsduringCNSDevelopment

UPREffector AnimalModel SpecialandTemporalDistribution Refs

Xbp1 Mousebrain Embryo>>adult [79]

Mouseretina Embryo>>adult [79]

Mouseforebrain E12>>E18 [17]

Drosophilaphotoreceptor Larvaeandearlypupalstage [33,36]

C.elegans Embryoandlarvae

Neurons>>non-neuronalcells

[20]

Atf4 Mousecortex E12>E14>E16

Progenitor>postmitoticneurons

[16,18]

MouseOSNs Lowlevel [30]

Drosophilaphotoreceptor Fromearlypupalstages [34]

Atf5 OSNs Highexpression(Atf5>>>Atf4) [30]

Mousecortex E12>E14>E16 [18]

Atf6 Mousebrain Embryo>>adult [79]

Mouseretina Embryo>>adult [79]

deficientmice,suggestingthatAtf6andIre-1pathwaysonlyplayminorrolesinthispathological context.However, one cannot excludeaphysiological roleofthetwo otherbranches.This remainstobetested.

Besidesitscontributiontotheregulationofcorticalneurogenesis,Atf4alsocontrolscellcycle progressionoftheearliestprogenitors,theneuroepithelialprogenitors.Thoseprogenitorsdivide symmetricallytoself-renewandasmallpercentagedividesasymmetricallytogivebirthtothe earlybornneurons.StabilizationofAtf4(byoverexpressingaformthatcannotbedegraded)in thoseprogenitorsleads tocell cyclearrest inG1 andpositioningdefects [16].Interestingly, CyclinDexpressionrescuestheAtf4-dependentproliferationphenotypebutnotthemigration phenotype.ThisimpliesthatAtf4playsadualroleinearlycorticogenesis[16]:(i)itcontrolscell proliferationthroughregulationofCyclinDpromoteractivityand(ii)itcontrolsmigrationofearliest bornneuronsbyunknownmechanisms.ItisnotclearwhetherthisfunctionofAtf4requires upstreamactivationoftheUPR.However,theabilityofAtf4topotentlysuppressproliferation andelicitG1arrestmayrequirePerkactivationasithasbeenshowntotriggerG1arrestthrough repressionofCyclinDtranslationinfibroblast[29].Altogether,thosestudiesshowthatneuronal progenitorsareacutelysensitivetoAtf4dosageandthatproperlevelofAtf4isrequiredfor efficientneurogenesisinthedevelopingmousebrain.

UPRandOlfactoryReceptorChoiceinMammalianOlfactorySensoryNeurons

Mammalianolfactorysensoryneurons(OSNs)fatechoiceisguidedbytheexpressionofasingle olfactoryreceptor(OR)alleleandafeedbacksignalthatlockstheORchoice.Recentfindings suggest that UPR components participate to the OR feedback process by detecting OR proteinsintheER[30].PerkandAtf5knockout(KO)mice,aswellaseIF2/phosphorylation mutants exhibit unstable OR expression, suggesting that the Perk/eIF2//Atf5 pathway is

+ –

Neuron generated via direct neurogenesis APs

UPR level

Key:

UPR _UPR

Neuron generated via indirect neurogenesis IPs Time Pre-neurogenesis Indirect neurogenesis Direct neurogenesis Time Pre-neurogenesis Indirect neurogenesis Direct neurogenesis

Wild-type cortex Elp3-deficient cortex

Figure2.TheUnfoldedProteinResponseRegulatestheBalancebetweenDirectandIndirectNeurogenesisintheDevelopingCortex.Wild-type corticogenesis(leftpanel)ischaracterizedbyaprogressivedecreaseofUPRsignalinglevelsinapicalprogenitors(APs–depictedinblue–darkblueandlightblueshowing intenseandlowUPRsignals,respectively).Attheearlystepsofcorticogenesis,APsgenerateneuronsbydirectneurogenesis:theydividetogiverisetooneAPandone neuron(magenta).Asdevelopmentprogresses,APsproduceneurons(purple)throughthegenerationofproliferativeintermediateprogenitors(IPs–orangecells).Indirect neurogenesiscontributestotheincreasedrateofneuronproductionascorticogenesisproceeds.Uponelp3deletion(rightpanel),themaintenanceofhighUPRlevel throughoutdevelopmentfavorsdirectneurogenesisattheexpenseofindirectneurogenesisandthereforeleadstoareductionoftheneuronaloutputandmicrocephaly (Adaptedfrom[83]).

requiredtostabilizeORexpressionandultimatelymaintainthefateofOSNs[30].Ire-1andAtf6 branches are unlikely to beinvolved inthis feedback process as nophenotype has been observedinXbp1mutantsandasAtf6isonlyslightlyexpressedinOSNs.Mechanistically,the authorsnicelyshowedthatincreasedeIF2/-dependenttranslation(translationinitiation func-tion,Box1)ofnuclearAtf5afterORexpressioninitiatestranscriptionofAdenylatecyclase3, whichthensignalstolockORexpression[30].Noteworthy,Atf4KOmicedonotexhibitOR instabilityphenotype,indicatingtheuseofanoncanonicalPerkpathwayindevelopingOSNs.

TheUPRFacilitatesVariousNeuronalDifferentiationProcesses

TheUPRplaysanimportantphysiologicalfunctiontocopewiththeconsiderabledemandfor proteinfoldingthataccompaniesneuronalbranchingandtheproductionofsecretoryfactors duringcelldifferentiation.WhilethefullspectrumofdownstreamtargetsoftheUPRpathway remainstobediscoveredindifferentiatingneurons,theyconvergetowardmembrane produc-tionandvesiculartrafficking(Box3).

TheUPRSignalsinNeuritogenesis

Duringdevelopment, dendritesacquire theirmorphology byconsiderable branchsprouting, whichcomes withan increasedneedofproteinproduction. Interestingly, inCaenorhabditis elegans,lossofire-1impairsdendriticmorphogenesisofthehighlybranchedneuronsbuthas noimpactonneuronswithfewerdendriticoraxonalbranches[31],suggestingapreferentialrole forIre-1inlargeandcomplicateddendriticarbordevelopment.Inaccordance,increasedUPR activity in low-branched neurons efficiently induces ectopic branches. Noteworthy, Ire-1 mutantsdonotshowanyaxonaldefect.Thereisanapparentdiscrepancywithresultsobtained inculturedmousecorticalneurons,showingareductionofaxonalbranchesinXbp1–/–neurons [17].TheroleofXbp1onaxogenesismaydependonthephysiologicalcontextandneedsfurther investigation.BothXbp1mRNAsplicingandRIDD(Ire-1-dependentdecayofmRNA,Box1) pathwayregulatedendriticbranchingofwormsensoryneurons,showingthattheentireIre-1 armoftheUPRpathwayisinvolvedindendriticmorphogenesis.Remarkably,thetwoothers armsoftheUPRpathway(Atf6andPerk)onlycontributemarginallytodendritogenesis.The authorsnicelyshowedthatUPRactivitywasspecificallyinducedduringthetimeofdendritic branching.ThephysiologicalincreaseoftheIre-1/Xbp1pathwayupregulatesspecific chaper-ones,suchasHSP-4,thathelpstofoldDMA-1,whoseoverexpressionisrequiredfordendritic branching.ThephysiologicalroleoftheRIDDpathwayandthenatureofitstargetsindendritic morphogenesisofwormsensoryneuronsarelessclear.

TheroleofUPRsignalingindendritogenesisisfurthersupportedbyexperimentswithcultured mousehippocampalneurons,demonstratingthatbothXbp1andEif2/areactivatedinneurites inresponsetoBDNF(brain-derivedneurotrophicfactor)[17].Indeed,BDNF-inducedneurite

Box3.UPRSignalingConvergestowardMembraneSynthesisinDifferentiatingNeurons

WhilethedownstreamtargetsoftheUPRpathwayarenotalwaysknownindifferentiatingneurons,mechanismsoften convergetowardmembranerequirementandvesiculartrafficking.WediscussedthatIre-1contributestoRhodopsin deliveryviadegradationoffatpmRNA[33].Howdoeselevatedfatplevelleadtorhabdomeremorphogenesisdefectsin Ire-1mutants?Fatpmediatestheuptakeoffattyacidsintocellsandfattyacidsareprecursorsforthebiosynthesisof phosphatidicacids,amajorcomponentoftheplasmamembrane.Increasedlevelsofphosphatidicacidwereshownto impairphotoreceptorapicalmembranetransportanddisruptrhabdomeremorphogenesis[84],causingaphenotype similartothatofIre-1mutantphotoreceptor.Itisthereforeproposedthatincreasedfatpelevatesphosphatidicacidand thuspreventsmembrane-associatedRhodopsindeliverytotherhabdomere.Inlinewiththis,theimpairmentof BDNF-inducedneuriteoutgrowthinXbp1–/–neuronsmayalsorelyondefectivemembranesynthesis[17].Indeed,Xbp1is reportedtopromotelipidbiosynthesisinfibroblastcells[85]andsecretoryorgans[86].OnecanenvisagethatUPRis usedtocopewithincreaseddemandbytheexocytosispathway.FurtherstudiesarerequiredtodeterminewhetherUPR maycontrolexocytosisindevelopingneurons;thiscouldhavemanyconsequencesonvesiculartransport,an indis-pensableprocessforproperneuronaldifferentiationandmaturation.

outgrowthisstronglyimpairedinXbp1–/–neurons.BDNF-inducedproteinsynthesisinneurites couldinitiateXbp1splicing.SplicedXbp1isthentransportedtothenucleuswhereitservesasa signaltransducerforneuriteoutgrowth[17].However,moleculartargetsdownstreamofXbp1 signalingforneuriteoutgrowthremaintobeelucidated(Box3).Whiletherearenoinvivostudies supportingthishypothesis,theERADpathwaymayalsopromoteneuronalmaturation.Indeed, downregulationofHrd-1level,onewell-knownERAD-associatedE3ubiquitinligase, counter-actsthedeleteriouseffectofmildERstressondendriteextensionduringRA-inducedneuronal differentiationofP19cells[23].

Altogether,thesestudiessuggestthatUPRsignalsneedtobetemporallyandspatially fine-tuned during neuronal differentiation. Its levels are indeed critical for neuronal maturation, neuritesbeingvulnerableto bothlowandhighsignals.UPRmay thereforebeahomeostat regulatingneuronaldifferentiation.

DrosophilaPhotoreceptorDifferentiation

InthedevelopingDrosophilaphotoreceptorcell,thegrowthoftherhabdomeresrequiresthe deliveryoflargeamountsofmembraneandproteins,includingRhodopsin-1(Rh1),intothis structure,imposingahighdemandforproteinfoldingandmembraneproductionintheER.Ire-1 accommodatesproperrhodopsin-1deliverytotherhabdomeresthroughtheregulationofFatp (fattyacidtransporterprotein)mRNA,apreviouslydescribedregulatorofRh1levels[32],by RIDD[33].AnotherarmoftheUPRmightalsobeinvolvedinnormalphotoreceptor develop-ment,asAtf4reporteractivityhasbeendetectedinDrosophilaphotoreceptorfromearlypupal stages[34].Furtherinvestigationsarerequiredtodefinetheprecisecontributionofthispathway toDrosophilaphotoreceptordifferentiation.Inaddition,thePerk/Atf4/CHOPpathwaymayhave aroleinmammalianretinas[35].Indeed,usingCHOP–/–mouseembryonicfibroblasts,Behrman andcolleaguesidentifiedrhodopsinasatargetofmiR-708,amicroRNAwhoseexpressionis controlledby CHOP[35].The physiological relevanceofthis mechanismstill remainsto be validatedinvivoinretinaswhereitcouldrepresentanadditionalmechanismtocopewiththe excessiveloadofrhodopsinandfacilitatephotoreceptordifferentiation.InDrosophila,theroleof Ire-1 in rhabdomere morphogenesis is independent of Xbp1but rather involves the RIDD mechanism[33,36].However,owingtoseveralXbp1–EGFPreporters,ithasbeenshownthat theactivationofXbp1startswellbefore rhabdomeregrowththatoccursatmidpupalstage, suggestingthatIre-1/Xbp1signalingmightberequiredatearlydevelopmentalstages[33,36]. Accordingly,ERdifferentiationdefectshavebeenobservedinIre-1mutantphotoreceptorat early pupal stage before Rhodopsin expression and the massive secretion that buildsthe rhabdomere[36].WhilenormalphotoreceptorshowsERamplificationwithroughmorphology, Ire-1mutantphotoreceptorshowsanabnormalERproliferationwithtangledtubularshape[36]. Thistubularshapeisunlikelytobereminiscentofaccumulationofmisfoldedproteins[37]orof rhodopsinaccumulation[38].Remarkably,Xbp1hypomorphicphotoreceptorsshownormalER morphologyandexpansion,evidencinganXbp1-independentroleofIre-1inthiscontext[36]. The mechanism by which Ire-1 supports photoreceptor ER differentiation remains to be determined(Box3).Altogether,one canspeculate thatIre-1 activityisrequiredto shape a dynamicERto anticipate apeakof proteinsynthesis.AsIre-1 mutants donot displayany defectsinspecificationorpolarityatthird-instarlarvalstage[33],onecanspeculatethatifIre-1/ Xbp1participatesinearlystepsofphotoreceptordevelopment,redundantmechanismsmay exist,throughotherbranchesoftheUPR,forinstance.Inaccordance,Atf4upregulationhas beenobserved inIre-1 mutants [33].In the Drosophila ommatidia, Ire-1 also mediatesthe secretionofSpacemaker/eyesshutintotheinter-rhabdomeralspace(IRS)byan Xbp1-inde-pendentmechanism[33,36].WhethertheRIDDpathwayalsoregulatesformationofIRShasnot beenanalyzedyet.Inconclusion,Ire-1-dependentpathwaysarecriticaltofacilitatethefolding andprocessingofthehighload ofsecretedproteinsbytheERas theDrosophilareceptor differentiates.

ReelinSecretoryCellsinDevelopingMammalianBrain

Duringbraindevelopment,Cajal–Retzius(CR)cells,foundinthemarginalzoneofthecortexandin thedentategyrus(DG)ofthehippocampus, secretereelin thatdiffusesandensurescorrect neuronalpolarizationandpositioning.Lossofreelinexpressionresultsincorticallayerinversion [39].MimuraandcolleaguesshowedthatGrp78mutantmicedisplayanoutside-inpatternoflayer formationinthecerebralcortexandmigrationdefectsincerebellumwherereelinisalsosecreted [19].Culturedcorticalmutantneuronsfailtosecretereelinbutrespondcorrectlytoreelinstimuli, suggestingthattheimpairedsecretionofreelinbyCRcellsratherthandefectiveresponsivenessis responsibleforthecorticalmalformationofGrp78mutantmice[19].Severalstudiespointtowarda physiologicalroleofGrp78inreelinfolding:(i)transcriptionofreelinisnormalinmutantCRcells;(ii) reelinproteinlevelsaredecreasedinmutantbrains;(iii)Grp78colocalizeswithreelin;and (iv) overexpressionofGrp78greatlyenhancesexpressionofreelinprotein.Grp78mutantmicedonot displaymigrationphenotypeinthehippocampus[19]suggestingthat:(i)Grp78doesnotfacilitate reelinfoldingintheDG;or(ii)compensatorymechanismsovercomethelossofGrp78;or/and(iii) otherERmolecularchaperonesarerequiredforproperreelinsecretion.Reelinalsoensuresthe correctpositioningofseveraltypesofneuronsinthedevelopingspinalcord[40,41].Itwouldbe interestingtotestwhetherUPRfunctioncouldbeextendedtomostofthereelin-sensitiveneurons. ItisnoteworthythatGrp78mutantmicedisplayadditionalphenotypecomparedwiththereeler mice[19].TheseincludemicrocephalyandscatteringofCRcellsthroughoutthecortex.Mutant Grp78maythereforeinterferewithothercrucialfactorsforbraindevelopment.Althoughprecise activationofUPRsignalinghasnotbeenassessedinthisbiologicalcontext,itisconceivablethat UPRisrequiredinCRcellstoenhanceERproteinfoldingcapacitytocopewithspecificprotein demandduringdevelopment.

DoesGlialDifferentiationRequireUPRSignaling?

Besidesitsactivityinneuronalcells,UPRmayalsoregulatethebiologyofglialcells.Indeed, canonicalUPRtransducersAtf6,Ire-1,andPerkareexpressedinglialcellsinvariousanimalor cellularmodels[23,42–45].However,severalstudiespointtowardalowormildcontributionof UPRtogliogenesis:(i)ERstressinducersinhibitglialdifferentiationinvitro[22,23];(ii)whilethe threeUPRbranchesareslightlyactivateduponinvitroastrocytesdifferentiation,thisinductionis notnecessaryfor astrogenesis[43];and(iii)Perkisdispensable foroligodendrocytenormal development[45].TogetherwithmanystudiesshowingastronginductionandneedofUPR signaling in pathological conditions [45–48], one can suggest that canonical UPR mainly protects glial cells frominjury rather than contributing to their development.Interestingly, a noncanonicalUPRpathwayhasbeenproposedtopromoteastrocytedifferentiation[43].OASIS isanalternativeUPRinducer,whichisexclusivelyactivated inastrocytes[49,50].Similarlyto Atf6,OASIS iscleavedbyS1PandS2PinresponsetoERstress(Box1).OASIS fragment translocatesinthenucleustoactivatetranscriptionofwell-knownUPRtargetgenessuchas Grp78[50].OASISknockoutmiceexhibitimpairedastrocytedifferentiationwithalargedecrease ofastrocytenumber,indicatingaroleforanoncanonicalUPRsignalingindevelopingastrocytes [43].Atthemolecularlevel,OASISpromotesthetranscriptionofgcm1(gliacellmissing1),a criticalgeneforastrocytedifferentiation[43].WhileOASIS-dependenttranscriptionofgcm1is increaseduponERstress[43],itnotclearifGrp78orotherUPRtargetgenesareactivatedupon astrocytedifferentiation.

ConcludingRemarksandFutureDirections

Whileinitiallyidentifiedasaregulatoryelementofhomeostaticprocesses,recentdatasupport that the UPR functions beyond ER proteostasis. By driving some critical aspects of the developmentandthemaintenanceofthenervoussystem,UPR signalingmustbeseenasa toolboxratherthanjustahomeostaticregulator.Moreover,accumulatingdataindicatethatthe activationprofileofUPRsignalingisuniqueineverysinglecellandemploysdifferentbranches andthusarraysofeffectorstocontrolspecificprocessessuchasneuriteoutgrowthorcellfate

OutstandingQuestions

Whyaresomebranchesof theUPR selectivelyactivated,dependingonthe physiologicalcontext?Istheactivation oftheUPRsignalingduringCNS devel-opmentexclusivelydependenton ele-vatedERstress?Canweenvisagethat otherstresscouldtriggerUPRinthe developingCNS?CanUPReffectors be activated independently of ER stressinthatcontext?

Could deregulationof UPRsignaling induce microcephaly phenotype in mouseafterdepletionof othertRNA modificationgenes?WouldUPR sig-nals be elevated in those models? Wouldthesamemechanism,thatis, an impaired balancebetween direct and indirect neurogenesis, be involved? Could we envisage UPR effectorlevelasbiomarkersfor neuro-developmental disorders? Would selectiveUPRinhibitorsbepromising therapeutically compounds to treat suchdiseases?

What isthephysiologicalfunctionof UPRsignalinginneuronalmigration? Towhat extent and how does any alterationofthisfunctioncontributeto neurodevelopmentalpathologies char-acterizedbyimpairedmigration (lissen-cephaly,polymicrogyria,pachygyria)?

CouldUPRsignalingbeasimportantin adultneurogenesisthanitisin embry-onicneurogenesis? Wouldthesame branchesoftheUPRbeinvolved?

IsanyUPR effectorexpressedinthe other type of progenitors, exclusively foundinprimates,theouterradialglial cells (oRGs)?Whatwouldbe thefunction ofUPRinhumanoRGs?Whatwouldbe theconsequenceofalteredUPRsignals forhumanneurodevelopment?

specification.DespitelackofapparentERstress,theUPRcanbeactivebutinmostcasesits upstreamregulatorsremaintobeidentified(seeOutstandingQuestions).Thus,itisanexciting timeforresearcherstobetterunderstandtheroleandtheregulationofUPRpathwaysthatare notonlyatplayduringCNSdevelopmentbutthatarealsoincriminatedinneurodegenerative disorders,makingthemattractivetargetsfortherapeuticdevelopment.

Acknowledgments

TheauthorsthankSandraBourandtheIGBMCcommunicationservice.L.N.andC.C.are,respectively,researchassociate andpostdoctoralresearcherfromtheFRS-FNRS(NationalScientificResearchFund).J.D.G.isfundedbytheATIP-Avenir jointCNRS-INSERMprogram,theFrenchNationalResearchAgency[ANR-10-LABX-0030-INRT(programme Investisse-mentsd’AvenirANR-10-IDEX-0002-02)]andtheFondation Fyssen.L.N.isfundedbyFRS-FNRS,theFondsLéon Fredericq,theFondationMédicaleReineElisabeth,theFondationSimoneetPierreClerdent,theBelgianSciencePolicy (IAP-VIInetworkP7/20),andtheARC(ARC11/16-01).

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