Notch signaling in the brain: In good and bad times

Notch

signaling

in

the

brain:

In

good

and

bad

times

Lavinia

Alberi

_,

_Sarah

_Hoey

_,

_Emanuele

_Brai

_,

_Alessandra

_Scotti

_,

_Swananda

_Marathe

a_{UnitofAnatomy,DepartmentofMedicine,UniversityofFribourg,Switzerland}

b_{DivisionofPsychiatryResearchandPsychogeriatricMedicine,UniversityofZurich,Switzerland}

c_{InstituteofAnatomy,UniversityofBern,Switzerland}

Notchsignalingisanevolutionarilyconservedpathway,whichisfundamentalforneuronal develop-mentandspecification.Inthelastdecade,increasingevidencehaspointedoutanimportantroleofthis pathwaybeyondembryonicdevelopment,indicatingthatNotchalsodisplaysacriticalfunctioninthe maturebrainofvertebratesandinvertebrates.Thispathwayappearstobeinvolvedinneuralprogenitor regulation,neuronalconnectivity,synapticplasticityandlearning/memory.Inaddition,Notchappears tobeaberrantlyregulatedinneurodegenerativediseases,includingAlzheimer’sdiseaseandischemic injury.ThemolecularmechanismsbywhichNotchdisplaysthesefunctionsinthematurebrainarenot fullyunderstood,butarecurrentlythesubjectofintenseresearch.Inthisreview,wewilldiscussoldand novelNotchtargetsandmolecularmediatorsthatcontributetoNotchfunctioninthematurebrainand willsummarizerecentfindingsthatexplorethetwofacetsofNotchsignalinginbrainphysiologyand pathology.



Published in "$JHLQJ5HVHDUFK5HYLHZVGRLMDUU"

which should be cited to refer to this work.

1. Introduction

Inthenameofnature’sconservationistcharacter,themature brain employsanarrayof developmentalpathwaystoregulate higher cognitive functions (Herz and Chen, 2006; Meffert and

Baltimore,2005;SpeeseandBudnik,2007).Notchsignalingisone

ofthebestexamples.Thispathwayisexpressedthroughoutthe lifespanofananimalfromdevelopment(delaPompaetal.,1997) toadulthood(Berezovskaetal.,1998),andcrosstalkswithother signalingcascadesinacontextdependentmanner.

Based on its temporally ubiquitous function, alteration to this cascade resultfromgrowtharrest (Swiatek et al.,1994)to brain diseases such as cerebralautosomal dominant arteriopa-thywithsubcorticalinfarctsandleukoencephalopathy(CADASIL)

(Arboleda-Velasquez et al., 2011), Alzheimer’s disease (AD)

(Berezovskaet al., 1998; Steineret al., 1999), Down syndrome

(Fischeretal.,2005), stroke(Arumugam etal.,2006)andbrain

tumors(Dangetal.,2006;Pierfeliceetal.,2011).ThefactthatNotch hassuchpleiotropiceffectsinthebrainandotherorganshasposed acriticalquestioninunravelingitsroleinthedifferentcelltypes. Studiestakingadvantageofcre/loxPmousegeneticshavebeenable toovercomethedevelopmentalrequirementofNotchin embry-onicgrowthandfocusonthespecificroleofNotchinthemature brainandinatime-dependentmanner(Table1).Inaddition,Notch reportermouselineshaveprovidedspatialandtemporalresolution ofNotchsignalingintheembryonicandmaturebrain,overcoming thepaucityofgoodantibodiesforNotchtargets(Table1).Taken together,mousegeneticstoolshavegreatlyimprovedour knowl-edgeontheroleofthissignalingpathwayinthepostnatalbrain. Followingthesestrongadvancements,remarkableworkis ongo-ingtountanglethecomplexityoftheNotchcascadeinthemature brain.Foralongtime,itwasthoughtthatNotchactivityinneurons wasrestrictedtoitstranscriptionalpotential(Costaetal.,2003;

Sestan etal., 1999).However, thereare nownumerous reports

indicatingthat Notchcrosstalkswithotherconservedpathways tocomplementitsarrayoffunctionsbothinphysiological(Alberi

etal.,2011;Giniger,1998;Hashimoto-Toriietal.,2008;Lugertetal.,

2010;Wangetal.,2004)andpathologicalconditions(Arumugam

etal.,2006,2011;Lealetal.,2012;Yangetal.,2004)(Fig.1).

Thisreviewaimstosummarizethemostrecentstudiesthathave revealedsomeofthemolecularmechanismsunderlyingNotch sig-nalinginthematuremammalianbrain.Wewilltrytoexplainsome oftheunsolvedmechanismsbylookingintootherbiological set-tings,suchascancerandDrosophilabiology,tofindinspirationand supportforfutureinvestigations.

Advancesinunderstandingthiscascadearehighlyrelevant,not onlyforbasicscience,butoffersaconcretepotentialfordeveloping therapeuticstrategiestocounteractalterationsinNotchsignaling. 2. TheNotchpathway:anoverview

2.1. TheNotchpathwayanditshallmarks

TheNotchpathwayishighlyconservedinmetazoans(Gazave et al., 2009).It occurs early onin development,when cell-to-cellcontactdeterminesmorphogenesis(Artavanis-Tsakonasetal., 1999). Notch receptors are membrane-tethered proteins with characteristicepidermal growthfactor(EGF)-like repeatsonthe extracellular portion (Rebay et al., 1991) and an intracellular transcriptionalactivesite(FortiniandArtavanis-Tsakonas,1994;

Schroeter etal., 1998).In mammals,nativeNotch is cleaved at

theendoplasmicreticulum(ER)byfurin(S1cleavage)thatsplits theextracellularandtransmembraneportionintoamature recep-tordimerheldbyacalcium(Ca2+_{)bond.Invertebrates,thereare}

fourNotchreceptorparalogues:Notch1,2,3and4.Thereceptors areactivatedbyspecificligandsoftheJaggedand Deltaserrate memberfamilies,whichareexpressedonthemembraneof adja-centcellsandcontainaDSL(Delta-serrate-lag-2)motif,essential forbindingtoNotch(Lindselletal.,1996;NyeandKopan,1995). WhenthejuxtaposedligandbindstoNotch,dimerizationofthe receptoroccurs(Mummetal.,2000).Theextracellularportionis endocytosedwiththeligandintheadjacentsignalingcellandthe membrane boundportionof thereceptor undergoessequential processing(Parksetal.,2000)(Fig.2).Thesecondcleavage(S2)

Table1

.

4

Mousemodel Phenotype Paper

Notch1LOFmodels

Notch1+/− Learningandmemorydeficit Costaetal.(2003)

Notch1as Synapticplasticitydeficit Wangetal.(2004)

RBPJK+/− Learningandmemorydeficit Costaetal.(2003)

Notch1f/fxT29-1CamK::cre Synapticplasticityandlearningdeficit Alberietal.(2011)

RBPJKf/fxTgCamK::cre Odordiscriminationdeficit(onlyfemales) Satoetal.(2012)

Notch1f/fxCamKII::cre(CaMcre) Nophenotypedetected Zhengetal.(2012)

Notch2f/fxCamKII::cre(CaMcre) Nophenotypedetected Zhengetal.(2012)

RBPJKf/fxNes::creERT2 _{NeurogenesisdefectinSVZ Basaketal.(2012);Imayoshietal.(2010)}

RBPJKf/fxGLAST::creERT2 _{NeurogenesisdefectinSGZ Lugertetal.(2010)}

Notch1f/fxNes::creERT2 _{DepletionofactiveNSCinSVZ Lugertetal.(2012)}

Notch1GOFmodels

Thy::LSLNICDxG35-3::cre Synapticandvisualplasticitydeficit Dahlhausetal.(2008)

Notchreportermice

RBPJKRE::EGFP Notch/RBPJKsignalingreporter Mizutanietal.(2007)

RBPJKRE::nLacZ Notch/RBPJKsignalingreporter Souilholetal.(2006)

pHes1:;dEGFPpHes5::dEGFP Notch/RBPJKsignalingreporters Ohtsukaetal.(2006)

Hes5::EGFP Notch/RBPJKsignalingreporter BasakandTaylor(2007)

NIP-cre Notchproteolysisreporter Vooijsetal.(2007)

Hes5-nlsLacZ Notch/RBPJKsignalingreporter Imayoshietal.(2010)

Hes1::EmGFPSAT _{Notch/RBPJKsignalingreporter Freetal.(2011)}

Notch-GAL4VP16 Notchactivationdosagereporter Smithetal.(2012)

Abbreviations:LOF,lossoffunction;GOF,gainoffunction;fl,floxallele;cre,crerecombinase;CamKII,calciumcalmodulinkinaseII;ERT2_{,estrogenreceptortamoxifeninducible}

2;Nes,Nestin;GLAST,glutamateaspartatetransporter;Thy,neuronspecificpromoter;LSL,Lox-Stop-Lox;EGFP,enhancedgreenfluorescentprotein;dEGFP,destabilized

enhancedgreenfluorescentprotein;NIP,Notchintramembraneproteolysis;LacZ,␤-galactosidase;EmGFP,emeraldgreenfluorescentprotein;GAL4,galactosidaseactivator

protein;VP16,herpessimplexvirus(HSV)-encodedtranscriptionalactivator.



Fig.1.BrainfunctionanddysfunctionthroughNotchsignaling.SimplisticrepresentationoftheNotch/RBPJKsignalingcascade,whichisinvolved(arrows)inphysiological

brainfunctions(“goodtimes”)orpathologicalconditions(“badtimes”)inthematurebrain.Asketchofamousebrainisrepresented.BoxindicatingNotchcrosstalks,which

areinvolvedinthetwoscenarios.

is thenoperated bydisintegrinand metalloproteinases (ADAM-17andADAM-10),whilethetransmembranedomainisavailable for ␥-secretaseprocessing. Presenilins (PS1 and PS2) of the ␥-secretasecomplexgeneratetheNotchintracellulardomain(NICD) (S3-cleavage)(Mummetal.,2000),andatransmembranepetide (N␤)(S4cleavage)(Okochietal.,2002)(Fig.2).

Inthecanonicalsignaling,onceNICDisgenerated,itis translo-cated tothe nucleusthrough thebinding of importins-␣to its nuclear localization sequence (NLS) domain (Huenniger et al., 2010).NuclearNICDassociateswiththetranscriptionalrepressor RBPJK (recombinationsignalbindingprotein for immunoglobu-lin kappaJ, alsocalled CBF1, suppressorof hairless, lag-1),the co-activatorproteinMAML-1(mastermind-like-1),andp300/CBP (CREB-binding protein),which possesshistoneacetyltransferase (HAT)activityandallows dissociationof therepressor complex formedbySkip(Ski-interactingprotein),SMRT(nuclearreceptor corepressor2)andhistonedeacetylase(HDAC).Thisdisplacement convertsRBPJKfromarepressortoanactivator(KopanandIlagan, 2009)(Fig.2).TheNotch–RBPJKcomplexinitiatetranscriptionof targetgenes,Hes(HairyandEnhancerofSplitinflies),Herp (Hes-relatedproteins)(Isoetal.,2003),Sox2(Ehmetal.,2010)andPax6

(Kumarand Moses,2001), thatareinvolved incellproliferation

and stemcell maintenance.Notch/RBPJKsignalingalsocontrols expressionofcellcycleregulatorssuchascyclinD1(Ronchiniand

Capobianco,2001)andcellgrowthfactorsasc-Myc(Palomeroetal.,

2007).Inaddition,arecentgenome-wideanalysisofNICD/RBPJK targetshasidentified,inembryonicforebrainprogenitors, signal-ingcomponentsoftheWnt,Shh,Hippoandionotropicglutamate receptorpathwaysasdirectcanonicaltargets(Lietal.,2012).Using highthroughputtechnologies,thisstudyhasindicated,forthefirst time,awidearrayofgenesthatareunderNotchregulation.Itis expectedthatNotch/RBPJKsignaling,indevelopmentaswellasin matureneurons,regulatesmanyofthesegenes.

On theother hand,itappears thatNotch alsodisplays non-transcriptional activity that is essential for the crosstalk with othersignalingcomponents,includingRho-GTPase(Giniger,1998), whichmediatesmorphogenesisindifferentiatingneurons.

In the following sections we will discuss critical mech-anisms that regulate Notch expression, transcriptional and non-transcriptionalactivity.

2.2. TheNotchreceptorsinthematurebrain

Notch1 is expressed in pyramidal neurons of the cortex

(Redmondetal.,2000;Sestanetal.,1999;Stumpetal.,2002)and

hippocampus(Stumpetal.,2002).Specificallyinthehippocampus, Notchexpressionisinducedbysensoryexperienceandcanbehave asanovel“plasticitymolecule”:itisexpressedpostsynapticallyand regulatesspinemorphology,synapticplasticityandmemory pro-cessing(Alberietal.,2011;Costaetal.,2003;Wangetal.,2004).On theotherhand,Notch1isstronglyinducedfollowingbraininjury

(Alberietal.,2010;Arumugametal.,2006)andmaycontributeto

celldeath.Notch2expressionpatternissimilartoNotch1, how-ever,itisexpressedatlowerlevelsincorticalandhippocampal neuronsas comparedtoNotch1(Stumpetal., 2002).Notch2is alsocriticallyupregulateduponinjuryandappearstocontribute tobraindamage(Alberietal.,2010;FerrariToninellietal.,2003). Notch3ismostlyexpressedinastroglialprogenitors(Tanigakietal., 2001),choroidplexus(Dangetal.,2006)andvasculature(Joutel etal.,2000), whereasNotch4has,sofar,notbeendetected.On thewhole,itappearsthatNotchexpressionisdynamically regu-latedandthatalterationsinpathwayactivitycaninterferewith physiologicalfunctionsandaffectcellularhomeostasis.

2.3. TheDelta-serrate-lag-2ligandsandothers

TheNotchligandsoftheJaggedandDeltaserratemember fam-iliesareexpressedonthemembrane ofadjacentsignalingcells and contain a DSL-motif that is essential for binding to Notch

(Lindselletal.,1996;NyeandKopan,1995).Inthematurebrain,

theligandsJagged1and Jagged2are prevalentintheforebrain, whereas Delta-like1 and Delta-like3 are expressed at low lev-elsin thecortexand thecerebellum,respectively (Stumpetal., 2002).Ourpreviousstudyconfirmedthat,inmaturehippocampal



Fig.2. WorkingmodelforNotch1signalinginphysiologicalconditions.Notch1isexpressedpostsynapticallyinthereceivingcellwhereastheligandisexpressed

presynap-ticallyinthesignalingcell.ContactofNotch1andJagged1ectodomainsleadstodissociationoftheextracellularportionofNotchandinternalizationinthesignalingcell.

Notch1andJag1maybefoundatthesamemembraneside,in“cis”configuration,inwhichNotchactivationisinhibited.Inaddition,theligandmaygetprocessed/shedded

atthepresynapticsiteandinternalizedwithNotchinthereceivingcell.Notch1expressionisregulatedbyfurincleavage(S1).Furinactivityisundernegativeregulation

ofanovelprotein“Botch”Notch1.ExpressionofNotchfrompre-existingmRNAisactivitydependent.FollowingactivityNotchisrapidlyrecycled(Rab11)possiblyfrom

extrasynaptictosynapticsites.NotchandAPParebothexpressedpostsynapticallyandfollowingsynapticstimulationthroughNMDAreceptorsarecleavedatthemembrane

byproteases,ADAM(S2),Presenilin1and2(PS1/2)(S3)arerepresented.NICDandAICDphysicallyinteractandreciprocallyinhibiteachotherthroughNumb,whichsends

themtodegradation.NotchinteractsalsowithReelinreceptors(ApoER2isrepresented)andtheycrosstalkthroughDab/Ablsignalingcascade.ThetraffickingofArc/Arg3.1

facilitatesNICDgeneration,throughS3cleavage,probablyattheleveloftheendosome.NICDisinternalizedinthenucleus,displacesSMRTandbindstoRBPJKandMAML

toformatranscriptionalcomplexthatinducesexpressionofspecifictargetgenesoftheHesandHerpgenefamily,Sox2andotherknownandyetunknowngenes.NICD

activityisnegativelyregulatedbyCDK8,whichphosphorylatesNICDandsendstodegradationthroughFbw7.Thegrayseparationindicatesthelong-rangedistancebetween

adendriticsynapseandthecellsoma/nucleus.

neurons,Jagged1isprevalentandisexpressedatpresynapticsites, whereaslevelsofDelta-like1 arenegligible(Alberietal.,2011). Thisalsoappearstobethecase inthepostnatalsubventricular zone whereJagged1 regulatesproliferationofNotch-expressing neural stem cells (NSC) (Nyfeler et al., 2005).Interestingly,we haveobservedthat,inmatureneurons,theexpressionofJagged1 increasesinresponsetosynapticstimulationwhereasDelta-like1 remains unchanged (Alberi etal., 2011).On theother hand,in Drosophila, Delta1 seemsto bethe relevant Notch ligand from developmenttothematurebrain(Lieberetal.,2011;Muskavitch,

1994).It isnotclearwhyligandfunctionsandtopologyarenot conserved,buttheseparationofDeltaandJaggedexpressionin dif-ferentmetazoanspecieshasrecentlybeenreported(Gazaveetal.,

2009).

Additionally,Notchisinducedbyatypicalligands,thatlackthe DSLdomain,suchasDelta/Notch-likeEGFrelatedreceptor(DNER)

(Eirakuetal.,2002)andF3/contactinfamilymembers:MB3and

Contactin1(D‘Souzaetal.,2008).Interestingly,theseligandsare expressedindendrites,_{andaxons(Kurisuetal.,2010;Pierreetal.,}

2001).



Onthewhole,thepresenceofspecificligandsonneuronal pro-cessessuggeststhatNotchsignalingisrequiredatdistalsynapses. 3. RegulationofNotchavailability

NumerousreportshaveindicatedcriticalcheckpointsforNotch expressionandsignaling.Inthissectionwewilldiscusssomeof themostrecentfindingsinNotchexpressionandprocessing regu-lation.

3.1. Translationalandpost-translationalregulation

It hasrecently been shown that in Drosophila embryogene-sis, Notchsignaling canberegulatedat thelevel ofligandand receptor translation(Shepherdetal.,2009,2010).The differen-tialmRNAprocessingensuresthespatio-temporaldosageofthe receptorandligand,whichisnecessaryforproperdevelopment. Atpresent,wedonotknowwhetherRNAprocessingis differen-tiallyregulatedinmammals.Nevertheless,wehaveshownthat Notchisrapidlytranslatedfrompre-existingmRNAuponsynaptic stimulation(Alberietal.,2011)(Fig.2).Interestingly,unpublished evidencefromourgrouphasindicatedthatNotchmRNAis local-izedatdendriticsynapses, suggestinga requirementforreadily availableNotchtranscriptsatplasticitysites.

Moreover,ithasrecentlybeenreportedthatatrans-Golgi pro-tein, Botch,regulatesS1cleavageofNotch, whichis criticalfor maturationoftheNotch1receptor(Chietal.,2012).During neu-ronal development,Botchpromotes neurogenesisbynegatively regulating Notch expression. It remains to be understoodhow BotchfunctionisregulatedandwhetherBotchactivitycan influ-enceNotchexpressioninneurons(Fig.2).

Furthermore, glycosylation of the Notch receptor, by O-fucosylation and O-glycosylation of the EGF repeats, has been showntobeessentialforpropersignalinginDrosophilaand

mam-mals(Stanley,2007).TheO-fucosyltransferase,Pofut,mutantmice

areviablebutdisplayTcelldeficiencyasaresultofreduced Notch-ligandinteractionandsignaling(GeandStanley,2008;Zhouetal., 2008).Twootherreportsusingazebrafishandafucosylationlossof functionmousemodel,showthatneuronaldevelopmentand neu-ronalplasticityareaffected,likelythroughimpairmentinNotch signaling(Songetal.,2010;Yagietal.,2012).

Taken together, several mechanisms regulate Notch protein maturationthusgrantingpropersignaling.Itwillbeofinterestto determinewhetheranyofthesemechanismsareaffectedduring agingorfollowinginjury.

3.2. Ligandmediatedregulation

There havebeen extensiveinvestigations demonstrating the requirementforamembrane-tetheredligandtoinduceNotch acti-vation in the signaling cell (Hicks et al., 2002; Lindsell et al.,

1995; Nicholset al., 2007). It has beendemonstrated that the

E3 ubiquitinligases, neuralizedandmindbomb, promoteNotch signaling byfavoring the internalizationofthe ligandwiththe Notchectodomainintheligand-presentingcell(LeBorgneetal., 2005).Specifically,lossofMindbomb-1impairsNotchsignaling andaffectsneuronaldevelopmentandradialgliaformation(Koo

etal.,2007;Yoonetal.,2008).Thus,transactivationoftheNotch

receptorbytheligand,restrictsthepolarityoftheNotchsignal(del

AlamoandSchweisguth,2009).

SeveralreportsusingDrosophilahaveshownthatwhenthe lig-ands,DeltaorSerrate,andtheNotchreceptorareproximalatthe membrane of thesamecell,in “cis”configuration,theyexerta ligand-mediatedblockadeonNotchactivationthroughtheir intra-cellulardomain(deCelisandBray,1997;Glittenbergetal.,2006;

Milleretal.,2009)(Fig.2).Morerecentlyithasbeenshownthat

alsoNotch,throughitsextracellularportion,canexertablockon SerratemediateNotchactivation(Becametal.,2010).These find-ingsindicateamutualinhibitoryinteractionbetweenligandand receptorwhenpresentatthemembraneofthesignalingcell.The “cis”inhibitionof ligandandreceptor, in mammaliancells,has beenconfirmedusinganeuroblastomacelllinebyoverexpressing Delta,whichleadstoneuriteextension.Conversely,whenDeltais expressedonfibroblasts,itinducesneuritesshorteningby transac-tivatingNotch(Franklinetal.,1999).Inmammalianneurons,the ligandJagged1isexpressedinpresynapticterminalsjuxtaposed toNotch,whichismainlyexpressedandinducedpostsynaptically

(Alberi et al.,2011).Interestingly,it appearsthat, in Drosophila

sensoryneurons,thisconfigurationisinverted:theDeltaligandis expressedpostsynaptically,whereasNotchispresent presynapti-cally(Lieberetal.,2011).Therefore,alsointhematurebrain,spatial separationoftheligandandreceptorappearstograntsignaling directionality.

Furthermore,ithasbeenshownthatsolubleligandscan com-petewiththemembrane-boundcounterpartsandrepresssignaling

(Hicksetal.,2002;Varnum-Finneyetal.,2000).Thereare,however,

severalreportsshowingthatbathapplicationofsolubleligands, containingtheDSLbindingsiteforNotch,canleadtoNotch activa-tioninneurons,neuronalstemcellsandothermammaliancelllines

(Androutsellis-Theotokisetal.,2006;Kyriazisetal.,2010;Wang

etal.,2004).Thepresenceofasolubleligandhasbeenconfirmedin arecentpaperindicatingconstitutiveligandsheddingbyADAM-17

(Parr-Sturgessetal.,2010).

On one side, it appears that a membrane anchored recep-tor–ligandinteractionisessentialforsignaling,while,ontheother hand,experimentalevidencesuggeststhepresenceofa function-allyactivesolubleligand.Thisapparentdiscrepancywillneedto befurtherinvestigated.Nevertheless,itraisesthepossibilitythat internalizationofthe Notch ectodomainwiththeligandin the signalingcellmightnotbeanabsoluterequirementforNotch acti-vation,and thatalternativemechanismofNotchactivationmay takeplace.Inthenextchapter,wewilldiscusssomeofthese puta-tivemechanisms.

3.3. EndocyticinternalizationofNotch

Itremains atopicof debateastowhetherNotch processing occurs at themembrane or in the endosomalcompartment. A recentstudysuggeststhat␥-secretasecleavageattheplasma mem-branegeneratesamoreefficientformofNICD(NICD-V),whereas S3-cleavageintheendosomeproducestwoshort-livedNICDforms: NICD-LandNICD-S(Tagamietal.,2008).Thisworkalsoshowed that someFAD mutationsof Presenilin1 affect theprecision of S3-cleavage,whichmaycontributetodiminishedNotchsignaling

(Tagamietal.,2008).Itwouldbeofinteresttoknowif,inneurons,

theselabileformsaregeneratedwhenNICDsignalisnotrequired. Interestingly,mostoftheNICDweobserveinlong-termneuronal cultureandcorticalneurons,islocalizedinthecytoplasm,andvery littleispresentinthenucleusincontrasttomaturing(Redmond etal.,2000)orinjuredneurons(Arumugametal.,2011).

It has previously been reported that endocytic processing of Notch is associated with weaker signaling, due to ubiq-uitination and degradation via Numb, which is a conserved intracellularmodulatorofNotch(Jafar-Nejadetal.,2002)(Fig.2). Interestingly,Numb associates with Cdc24,EphB2, NMDAR (N-methyl-d-aspartatereceptor)postsynapticallyandcontributesto spinedevelopment(Nishimuraetal.,2006).Itwouldbe interest-ingtoknowwhetherNumbregulatesthelevelsofNotchatthe synapse(Fig.2).

Endosomaltrafficking,however,appearsinstrumentalforNotch activationsincesystematicdeletionofendocyticcomponents,in Drosophila, leads to accumulation and overactivation of Notch



(VaccariandBilder,2005;Vaccarietal.,2008).Thisstudyreveals thatendosomaltraffickingexertsanecessarydampeningeffecton Notchsignaling.Inaddition,recentworkhasindicatedthatNotch activationat theendosome,occurs independentlyof theligand

(Childressetal.,2006;Horietal.,2011).Thismightexplainwhy,in

neurons,Notchprocessingispositivelyregulatedbytheendosomal traffickingmolecule,Arc,regardlessofligandavailability(Alberi etal.,2011)(Fig.2).Additionally,wehavepreliminaryevidence indicatingthat,uponsynapticstimulation,Notchisrapidly inter-nalizedintoearlyendosomesandrecycledtothesurface(Fig.2). WearecurrentlyinvestigatingwhethertherecyclingofNotchis directedfromextrasynaptictosynapticsitesuponphysiological stimulation(Fig.2).

Based on the evidence that Notch is present at distal postsynaptic sites and Presenilins activity is maximal at the endosome (Wu et al., 2011), we argue that under physiolog-ical conditions, endocytic trafficking and processing might be prevalent.

4. Notchsignalingregulation

4.1. ExtrinsicmodulatoryinfluencesonNotchsignaling

ThereisgrowingevidencesuggestingthatNotch signalingis susceptibletoextrinsicfactors.Ithasrecentlybeenreportedthat ADAM activity can be induced by synaptic stimulation (Hoey et al., 2009) and PKC activation (Cisse et al., 2011). Synaptic stimulation can also induce ␥-secretase activity at the endo-some throughthe clustering of thecomplex Arc/Presenilin1 to itssubstrates(Wuetal.,2011).Interestingly,Notchactivation,in responsetoincreasedactivity,occursinmatureneurons(Alberi

et al.,2011;Lieber etal.,2011)and inneuralstem cellsof the

mature brain (Lugert et al., 2010). This suggests that activity dependentNotchsignalingmaybeasignatureof matureNotch function.

Notch and the amyloid precursor protein (APP) share com-monprocessingmechanismsthroughADAMand␥-secretase,and both substratesareprocessed inan activitydependentmanner

(Alberietal.,2011;Hoeyetal.,2009).Earlyreportssuggestthat

Notch1andAPPcoprecipitateinbrainlysatesandthatthey inter-actthroughtheirtransmembranedomain(Fassaetal.,2005;Kim etal.,2007).ItwasalsoshownthattheAPPintracellulardomain (AICD),throughinteractionwithNumb,candown-regulateNotch signaling(Roncaratietal.,2002)(Fig.2).Itappears,therefore,that APPandNotchhavecomplementaryfunctions.Nevertheless,ithas beenrecentlyproposedthattheinteractionbetweenNumband Notchcanbereverteduponstressconditions.Thisstudyshowed thattrophicfactorwithdrawalinducesaswitchinNumbisoform toashorter phosphotyrosine-bindingdomain(Numb-SPTB)that favorsendocyticsorting/processingofNotch(Kyriazisetal.,2010) (Fig.3).Therefore,inlightofthisstudy,wemayhypothesizethat APPandNotchinteractionthroughNumbiseithernegativeor pos-itivedependingonthecellularhomeostasis.Itwillbeinteresting toknowwhetheraberrantsynapticactivityasinADandepilepsy

(Palopand Mucke,2009)canfacilitate Notchsignalingthrough

APP-Numb-SPTBinduction.

Oneotherstudyshowedthat,inDrosophilabloodcells,Sima,the proteinorthologoustothemammalianhypoxia-inducible factor-alpha(Hif-1␣),isastrongactivatorofNotchindependentlyofthe ligand(Mukherjeeet al.,2011)(Fig.3).It iscurrentlyunknown whetherhighlevelsof Hif-1␣ canactivateNotch, in the mam-malianbrain,followingcerebralischemia(ChavezandLaManna,

2002).

Additionalstudiesmighthelpunderstandtheunderlying mech-anismofNotchresponsestohomeostaticchanges.

4.2. Regulationwithinthenucleus

Notchtranscriptionalactivityistightlyregulatedalsoatthelevel ofnucleus.Thehalf-lifeofthetranscriptionallyactiveNICD-RBJK complexisdeterminedbyMAML-1.MAML-1recruitscyclin depen-dentkinase-8(CDK8)tonuclearfociandallowsphosphorylationof NICD(NICD-P)andtargetingofNICD-Pforproteosomal degrada-tionbyE3ubiquitinligaseFbw7(Fryeretal.,2004)(Figs.2and3). RecentworkindicatesthatdegradationofNICDbyFbw7is essen-tialforneuralstemrenewalanddifferentiation:geneticdeletionof Fbw7leadstoNICDaccumulation,progenitors’apoptosis(Hoeck etal.,2010)andneuronal–glialfatecommitment(Matsumotoetal., 2011).However,inthesestudiesitwasnotshownwhether accu-mulation ofNICDaffects expressionofNotch1proteinlevelsas partofapositivefeedbackloop.Notch1self-regulationhasbeen reportedinDrosophila(DelMonteetal.,2007)andmurine embry-onicneuralstemcells(Lietal.,2012).

RBPJKformsaconstitutiverepressorcomplexwhenassociated toSkip/SMRT/HDAC.Arecentreportusingmammaliancelllines hasindicatedthatWnt5a,throughCamKII,canfacilitatetheexport ofSMRT,enablingtheinteractionofNotchtotheRBPJK transcrip-tionalcomplex(Annetal.,2012).Interestingly,Wnt5aregulates spinemorphogenesisandsynapticplasticity(Varela-Nallaretal., 2010).It isintriguingtohypothesizethatthiseffectisachieved throughNotchfunction.

In addition,theDrosophila orthologofHif-1␣,hasa stabiliz-ingeffectontheNotch-RBPJKtranscriptionalcomplex(Gustafsson etal.,2005)(Fig.3).ItispossiblethatHif-1␣,which isstrongly inducedbyischemia(ChavezandLaManna,2002),canpotentiate Notchsignalingininjury.

Theseandotheryetundiscoveredmechanismsmightexplain thesignalingpowerofNotchinphysiologicalandpathological con-ditions.

5. Notchcrosstalkingtoothersignalingpathways

Severalstudieshaveshownthatthemembrane-tetheredform of theNotch receptor and theactivatedNICD display signaling capacity through theinteractionwithothersignalingpathways

(Andersenetal.,2012;LeGalletal.,2008;Sanalkumaretal.,2010).

Inthenextsectionswewillhighlightsignificantcrosstalkswith putativeneuronalfunction.Webelievethatstudiesinthisfieldare thefoundationtounderstandthepleiotropicroleofNotchinthe mammalianbrain.

5.1. Cytoplasmiccrosstalk

The workof GinigeronNotch/Abl (Abelson tyrosine-protein kinase)signalinginaxonpatterning,inDrosophila,haspioneered thisresearchavenue.Invariouspapers,theworkfromthisgroup hasdemonstratedthatcytoplasmicNotch,lackingthe transcrip-tionalactivesite,caneffectivelyrepressAblsignaling,bybinding toDisabledandTrio,therebycontrollingaxonalgrowth/guidance

(Crowneretal.,2003;Giniger,1998)(Fig.2).Thissuggeststhatat

distalsitesfromthenucleusNotchcooperateswithlocalsignaling mechanismtodisplaymorphogenicfunctions.

Interestingly in mammals,Disabled is founddownstream of ReelinreceptorsanditisinstrumentalforReelinsignaling(Herz

and Chen,2006).It has,also,beenshown thatReelinstabilizes

NotchsignalingthroughtheinteractionwithDisabled1in devel-opingneurons(Hashimoto-Toriietal.,2008)(Fig.2).Inaddition, dendriticmaturationinReeler(Reelindeficient)micecanbe res-cuedbyNICDexpression(Hashimoto-Toriietal.,2008).Another study revealed that Reelin deficiency leads to a strong reduc-tioninNotchactivationandcanonicalsignalinginthedeveloping



Fig.3.WorkingmodelofNotch1signalinginpathologicalconditions.Followinginjury,asinhypoxia–ischemia,Notch1israpidlyprocessedthroughS2andS3clevage

andinducespathwayactivation.Notch1levelsarealsoincreasedprobablyinresponsetoapositiveNotch-CSLfeedback.ItispossiblethatBotch,whichblocksS1cleavage

influencingNotch1expression,isdirectlyinvolvedinthisprocess.NICDisstabilizedbytheinteractionwithHif-1␣.Astress-dependentNumbisoform(Numb-SPTB)favors

NICD-CSLactivity.Notch1,throughstillunresolvedmechanisms,positivelyinteractswithNF-kappaB/JNKpathwaytomediatemitochondrialtranslocationofcytosolic

enzymesinducingapoptosis.NICDmightbepreferentiallytraffickedtothenucleibyimportin␣/␤,whicharerapidlyinducedfollowinginjury.NICD,onceinthenucleus,

inducesactivationofdownstreamtargetsoftheHes,HerpgenefamiliesandothercontextdependentgenesascyclinD1andp21.Hes1maydownregulateubiquitinligases

MDM2andIDE.Down-regulationofMDM2stabilizesandtherebyincreasesp53levelswhichtriggerstheapoptoticcascade.p53,sensesDNAdamage,cooperateswithNotch

alsoinneuritesretractionthroughRho-GTPases(Rock1/2).Hes1activationleadstodown-regulationofIDE.Thisde-repressesactivationofBACE,throughincreaseinAICD,

andleadstotransitionfromnon-amylogenictoamylogenicA␤production.Thegrayseparationindicatesthelong-rangedistancebetweenadendriticsynapseandthecell

soma/nucleus.

hippocampus(Sibbeetal.,2009).Thisraisesthepossibilitythat someofthetraitsobservedinthevariousReelinmutantmouse models,suchasdecreasedsynapticplasticityandspatiallearning

(Beffertetal.,2005;Weeberetal.,2002)mayunderlieimpaired

Notchsignaling.

AdditionalevidencethatNotchmightbeinstrumentalin neu-ronalfunctionthroughnon-canonicalneuronalmodulatorcomes from the work indicating that Notch interacts with the mam-maliantargetofrapamycin(mTOR)-Aktpathway.Thispathwayis essentialinmemoryformation(Bekinschteinetal.,2007)and con-solidation(Hoefferetal.,2008).Aninterestingparallelbetween Notch and mTOR function can be found in the common LTD

deficiencyobservedinhippocampalslicestreatedwith PI3K-Akt-mTORinhibitors(HouandKlann,2004)andtheNotch1cKOs(Alberi etal.,2011).AmechanisticinteractionhasbeenshowninHeLaand embryonicstemcells.Inthefirststudy,trophicfactorsremovalcan induceAktphosphorylation,throughtheinteractionofNICDwith mTORandtheindependentcompanionofmTOR(Rictor), indepen-dentlyofNotch/RBPJKsignaling(Perumalsamyetal.,2009)(Fig.2). Ontheotherhand,ithasbeenreportedthatapplicationof solu-bleNotchligandscaninducephosphorylationofAktandmTOR instemcells,whichcanbeblockedbyinhibitingNotchprocessing

(Androutsellis-Theotokisetal.,2006).Itisintriguingtohypothesize

thatsuchapositiveinteractionmighttakeplacealsoinneurons.



Atthispoint,itismandatorytounderstandwhetherdysfunction inthemTOR-AktpathwayoccursinNotch1cKOneurons.

Otherrelevantwork,thatputsNotchinconnectionwithanother pathwayinvolvedinsynapticplasticity,indicatesthatNotch nega-tivelyregulatesWntsignalingbyfavoringdegradationof␤-catenin. ThisnegativeinteractionseemstobefacilitatedbyNumb(Kwon et al., 2011). Interestingly, both Notch and Wnt cascade com-ponents areexpressedpostsynaptically,are triggeredby NMDA transmissionandinfluencesynapticplasticity(Alberietal.,2011;

Atamanetal.,2008;Nishimuraetal.,2006).Abehavioral study

hasshownthatduringmemoryconsolidation,levelsofNotchwere reduced, whereas ␤-catenin expression was increased (Conboy et al.,2007).It willbeof interesttounderstandwhetherthese componentscharacterizespecificmemorytraces.

AnotherremarkableinteractionisthecrosstalkbetweenNotch andNF-␬Bsignaling.NF-␬Bdenotesadimerictranscriptional com-plexcomposedbyp55:p65.Interestingly,this dimerisfoundat synapses and can respond to increase in local Ca2+ _transients

(Meffertetal.,2003).Ithasbeenshownthatthesepathwayshave

synergisticfunctionsinphysiologicalconditionsandinbraininjury. TheNotchantisensemice(Notch1as),inwhichNotchispartially deleted(50–70%)usinganantisenseinterferencestrategy(Cheng etal.,2001),displayspecificsynapticplasticitydefectscoupledwith reductioninNF-␬Bbindingactivity(Wangetal.,2004), suggest-ingthatNotchregulatesNF-␬Bsignaling.Interestingly,p65mutant micedisplaydefectsinlearning/memoryprocessing(Meffertetal., 2003)similartotheNotch1+/−andNotch1cKOmice(Alberietal.,

2011;Costaetal.,2003).Thispositiveinteractionhasbeen

inves-tigatedmoreextensivelyinahypoxia–ischemiainjuryparadigm. ArumugamandcolleagueshaveshownthatNotchactivationinan ischemia-likeinjurymodeliscoupledwithactivationoftheNF-␬B pathwayandcanbeblockedbyapplicationofseveral␥-secretase inhibitorsbothinvitroandinvivo(Arumugametal.,2006,2011) (Fig.3).The lateststudyfromthis groupindicatedthat nuclear NICDisessentialforNF-␬B/JNKactivity,althoughthemolecular mechanismremainsyetunraveled.

Itappears,however,thatNF-␬BactivitycanalsoregulateNotch signaling.ReportsrevealedthatJagged1isadirecttargetof NF-␬B(Bashetal.,1999)andthatNF-␬BinfluencesNotchsignaling byfavoringthetranslocationofthenuclearcorepressorofRBPJK

(Espinosaetal.,2002).ThereforeNotchandNF-␬Bpathwayappear

tosynergizeinapositivefeedforwardandfeedbackmechanism. Nevertheless,a recent investigationfromBonini haschallenged thishypothesisandhasshownthat,inhippocampalculturesfrom p50mutantmice,Jagged1isoverexpressedandtriggersincreased Notchsignaling.Inaddition,theauthorsclaimthat,asaresultof Notchactivation,lessdendriticbranchingisobserved(Boninietal., 2011).Thisis somewhatsurprisingconsideringthatNotch pro-motesdendriticbranching(Breunigetal.,2007;Redmondetal., 2000).It remainspossiblethat theeffectof Notch ondendritic morphologyispartiallymediatedbyNF-␬Bsignaling.Another pos-sibilityisthatoveractivationofNotchresultsinneuritesretraction andcelldeath.Thislatterpointisoffurtherinterestbecause,ifthe authorsdidnotobserveanysignofcelldeath,itwouldemphasize thatthesepathwaysfunctiontogetherinmediatingneuronalcell demise.

StudieslookingatcytoplasmicinteractionsbetweenNotchand other signalingcomponents will advanceour understandingof Notchsignalinginthematurebraindramatically.

5.2. Nuclearcrosstalk

We,andothers,haveobservedthatunderphysiological condi-tion,Hes1andHes5expression(asreadoutofNotch-CSLsignaling) appear unresponsivetochanges in Notchactivity(Alberi etal.,

2011;Yuetal.,2001),whereasunderischemicconditionthereis

astrongup-regulationofNotch/RBPJKtargets(Alberietal.,2010;

Arumugametal.,2006).Wemayspeculatethat,followinginjury,

NotchundergoesrapidImportin-mediatedtraffickingandnuclear signalingisfavored(Huennigeretal.,2010)(Fig.3).Afteraxonal injury,Importins-␣/␤arerapidlytranslatedinsituandfunctionas proteins’carrierfromtheperipherytothenucleus,mediatingeither prosurvivalordeathsignals(Hanzetal.,2003).Thiswouldbeinline withanotherworkindicatingthatanotherNotchtarget,cyclinD1, isinducedfollowingstroke(Timsitetal.,1999)(Fig.3). Interest-ingly,cyclinD1heterozygousmicedisplaypartialneuroprotection following kainic acid injury (Koeller et al., 2008), suggesting that Notch signaling might be instrumental in the life/death choice.

Inaddition,NotchandAPPproteinsareaberrantlyregulatedin Alzheimer’sdisease(Wooetal.,2009).NotchandAPPappearto haveamutualpositivefeedbackondownstreamtargets(Fischer etal.,2005).ThiswouldsuggestthatAPPandNotchcooperateat theleveloftranscription.Indeed,ithasbeenshownthatAICDand NICDarefoundinnuclearfactoriesinassociationwiththeeffector proteinFe65(Konietzkoetal.,2010).Anothermorerecentstudy indicatesthatNotchtranscriptionalactivity,throughHes1, regu-latesAPPmetabolismleadingtode-repressionofBACE-1andan increaseintheproductionofA␤42insolublepeptidesthat

concen-tratesinamyloidplaques(Lealetal.,2012)(Fig.3).Thisreportis veryintriguingandindicatesforthefirsttimeadirectregulationof APPprocessingbyNotchandapotentialcriticalinteractioninthe pathobiologyofAlzheimer’sdisease.

Thereisadditionalevidenceemergingfromworkin neurode-velopmentanddifferentiationthatNotchcooperateswithanother celldeathsignal,Trp53(p53).MiceconditionallyexpressingNICD undertheNestinpromoter,displayedincreasedapoptosisof neu-ronalprogenitorsduringdevelopment.Apoptosiswascoupledwith aberrant increase in p53pathway activity and wasrescued by geneticablationofp53(Yangetal.,2004).Thepositivefeedback ofNotchonp53hasalsobeenobservedinmousefibroblasts.One studyindicatedthatNotchactivation,throughNotch/RBPJK activ-ity,de-repressesp53expressionbyinhibitingtheubiquitinligase, MDM2,whichdegradesp53(Huangetal.,2004)(Fig.3).Another investigationusingmousefibroblasts,lackingtheubiquitinligase Fbw7,showedthatNotchactivationislinkedtop53upregulationin mediatingcellcyclearrest(Ishikawaetal.,2008).Amutualpositive feedbackofp53onNotchactivityhasalsobeenrecentlyreported: p53,bysensing DNAdamage,activatesanapoptosiscascadein neuronalstemcellsandcontributes,togetherwithNotch,to reduc-ingneurites’lengththroughtheactionofthesmallGTPaseRhoA kinases,Rock1andRock2(Ferronetal.,2009)(Fig.3).Similarresults havebeenobtainedinkeratinocytes(Lefortetal.,2007).Fromthese studiesitappearsthatp53andNotchfunctionsareclosely corre-latedinlife/deathdecisionandneurites’morphogenesis.

Onthewhole,itappearsthatNotchsignalingisintertwinedwith multipleconservedpathwaysalsoatthenuclearlevel.Future stud-ieswillhelpaddresshowthesedifferentcrosstalksarefine-tuned tomediateadaptivechangesinneurons.

6. Notchingoodtimes 6.1. Notchinsynapticplasticity

WhenlookingatthetemporalexpressionpatternofNotchin thecortex,itisinterestingtoobservethat,aroundbirth,Notch1 expressionisattenuatedascomparedtodevelopment.However, afterthefirstpostnatalweek,expressionbecomesmoreabundant

(Tokunagaetal.,2004).Thistimecoincideswiththecriticalperiod

when synaptic connections are being refined and spontaneous synchronized activitypatterns synapticconnections (Garaschuk



et al.,2000).We mighttherefore hypothesizethat during post-natal development,Notch is induced and requiredfor network maturation. Severalpapers have confirmedthis hypothesis and haveshown thatNotchhasa directeffectondendritic

pattern-ing(Berezovskaetal.,1999;Breunigetal.,2007;Redmondetal.,

2000;Sestanetal.,1999)andaxonaloutgrowth(Crowneretal.,

2003).Wenowknow,fromseveralstudies,thatreductioninNotch signalingaffects sensoryand memoryprocessingacrossseveral species(Alberietal.,2011;Chaoetal.,2005;Costaetal.,2003;

Geetal.,2004;Wangetal.,2004).Thiseffectdoesnotappeartobe

theresultofaberrantconnectivity,butratherdependsonchanges insynapticplasticity(Alberietal.,2011).Interestingly,the plastic-itydeficitintheNotch1cKOdoesnotcompletelyphenocopythe lossofPresenilin1/2(PS1/PS2;PsdcKO)(Sauraetal.,2004).Inthe PsdcKOmice,theLTPdeficitisaccompaniedbyreduced presynap-ticrelease,whichcanneitherbeobservedintheNotch1cKOsnorin Notch1asmice.ThissuggeststhatPresenilin1/2mayalsoregulate essentialfunctionsofpresynapticsubstratesthatinfluence neu-rotransmitterrelease.ThepostsynapticfunctionofNotchisalso supportedbythefactthatbathapplicationofJagged1potentiates LTP(Wangetal.,2004).

Interestingly,a morerecentreportindicatesthatconditional deletionofMindbomb-1,whichisinstrumentalforligand endo-cytosisandNotchsignaling(Kooetal.,2005),leadstoasynaptic plasticityand memorydeficit(Yoon etal.,2012).Thisprovides additionalevidencethat,in thematurebrain,Jagged1mightbe instrumental forNotch-dependentplasticity.Interestingly, Alag-illepatients, carrying geneticmutationfor Jagged1,besidesthe liverandheartpathology,alsodisplaysignsofmentalretardation

(Alagilleetal.,1975).Itwouldbeofinteresttoknowwhetherthis

istheresultofimpairedconnectivityorplasticityinthebrain. Canonical Notch signalinghasbeen shown tobecritical for spatiallearning andmemory,basedontheevidencethat RBPJK heterozygousmicedisplayasimilardeficitastheNotch

heterozy-gousmice(Costaetal., 2003).Nevertheless,a recentpaperhas

shownthatpostnataldeletionofRBPJKdoesnotaffectmemoryand learning(Satoetal.,2012).ThisstudyconcludesthatNotch/RBPJK signalingisdispensableforsynapticplasticity.Inaddition,NICD overexpressioninpyramidalneuronsofthevisualcortexreduces spinedensityandrepresseslong-termpotentiation(Dahlhausetal., 2008).ThissuggeststhatectopicNICDinterfereswith physiologi-calNotchfunctionsand/orthatnon-transcriptionalNotchactivity isinstrumentalinneuronalphysiology.Inthisdirection,astudyin Drosophilahasindicatedthattheadhesionmolecule,Klingon,isa directnon-canonical/non-transcriptionaltargetofNotchina mem-oryparadigm,andthatitisinstrumentalforlongtermmemory formation(Matsunoetal.,2009).Thisstudyisveryintriguingand suggeststhatnon-canonicalmediatorsofNotchmaybeinvolved insynapticplasticityandmemoryprocessing.

On thewhole, it appearsthattransientincrease of Notchin responsetoactivityisessentialinsynapticplasticitymodulation

(Alberietal.,2011;deBivortetal.,2009;Lieberetal.,2011).

Never-thelessithasbeenshownthatNotchsignalingisattenuatedduring the periodof memoryconsolidation (Conboy et al.,2007).This raisestheinteresting possibilitythatNotch signalingis respon-sibleforearlyratherthanlatemolecularchangesatthesynapse followingpotentiation.

Ultimately,thesestudieshighlightthatNotch signalingis an essentialrequirementforneuronalfunctionandmemory forma-tion.

6.2. Notchinpostnatalneurogenesis

Inthematurebrain,Notchsignalingisadditionallyimplicated inadultneurogenesisofthesubventricularzone(SVZ)(Basaketal.,

2012;Imayoshietal.,2010;Nyfeleretal.,2005)andsubgranular

zoneofthehippocampus(SGZ)(Lugertetal.,2010).Neurogenesis, andintegrationofnewbornneuronsisthoughttocontributeto olfaction(SVZ)(Sakamotoetal.,2011)andspatialmemory(SGZ)

(KempermannandGage,2002).

CanonicalNotch/RBPJKsignalingisessentialinmaintaininga neurogenicpoolinthematurebrain(Basaketal.,2012;Imayoshi

et al., 2010; Lugertet al., 2010).It hasbeen shown that

abla-tionofRBPJKinadultneuralstemcells(NSCs)oftheSVZinduces atransientincrease in neurogenesisbutleadsin thelongterm toa permanent depletionof theprogenitorpool(Imayoshi and

Kageyama,2011).Anotherrecentstudy,using conditional

dele-tionmousemodelsforNotch1andRBPJKinadultNSCoftheSVZ, hasconfirmedthatlossoffunctionalNotchsignalingexhauststhe progenitorpool(Basaketal.,2012).Thissamestudyindicatesa functionalseparationbetweenNotch1andRBPJKfunctionand indi-catesthatNotch1doesnotregulatethecellfateofquiescentNSCs, which,instead,appears tobeunderRBPJK regulation,probably throughtheotherheterologousreceptors,Notch2andNotch3.The groupofTaylorhasalsodemonstrated,atleastintwopapers,that Notch1isimportantinmaintaininganactivelyproliferatingNSCs pool,whichselectivelylostduringaging(Basaketal.,2012;Lugert

etal.,2010).

Workfromthesamegrouphasindicatedthat,similartothe SVZ,ablation of RBPJK in NSCsof the postnatal DGleads toa strongdepletioninprogenitorcells(Lugertetal.,2010).Usinga Notchreporterline,inwhichcellswithongoingNotch/RBPJKare GFPlabeled(Hes5::GFP),theauthorsfoundthatNSCsofSGZwith Notch/RBPJKsignalingarethemostmultipotentofthetotal progen-itors.InbasalconditionstheseNSCsare,forthelargepartquiescent, butcanrapidlyswitchintoaneurogenicmodeinresponseto max-imalactivityorexercise(Lugertetal.,2010,2012).Thisimportant studyindicatesthattheregenerativepotentialofneuronal progen-itorsisresponsivetoincreasedsynchronizedactivity.Asamatter offact,neurogenesisintheSGZcanbeinducedbyexercise(van

Praagetal.,1999),environmentalenrichment(Brownetal.,2003)

andsynapticpotentiation(Kamedaetal.,2012).Otherextrinsic sensoryfactors,asolfaction,canincreaseneurogenesisintheSVZ

(Rochefortetal.,2002).Wemayhypothesizethatthisprocessis

mediatedbyactivity-dependentNotchactivation,althoughother signaling pathways, such as PI3K/AKT (Bruel-Jungerman et al., 2009), BDNF/TrkB(Bergamiet al.,2009)and Wntsignaling(Lie etal.,2005)havealsobeenimplicatedintheseprocesses.

Besidetheroleinneuralstemmaintenance,Notchalsoregulates integrationofnewbornneuronsinthegranulecelllayer(Breunig etal., 2007).Various studiesusing animal models have shown that,duringaging(BenAbdallahetal.,2010;Lugertetal.,2010) and Alzheimer’s disease, neurogenesis is considerably reduced

(Jaskelioffetal.,2011;Rodriguezetal.,2008,2009).Inaddition,

severallossoffunctionofPresenilinsdisplayreduced

neurogene-sis(Chenetal.,2008b;Chevallieretal.,2005;Faureetal.,2011).

Despitethemultipletargetsof␥-secretase,webelievethat,inlight oftherecentfindings,perturbationinNotchmaycontributetothe deficitinneurogenesisobservedinthesestudies.

ThereforeNotchsignalingappearsinstrumentalforthe regen-erativecapacityofthematurebrain.

7. Notchinbadtimes 7.1. NotchinAlzheimer’sdisease

NotchsignalinghasoftenbeenimplicatedinAD,however,at present,themechanismsinvolvingNotchinprogressive neurode-generationremainselusive.SeveralfamilialAD(FAD)mutations of Presenilin1, leading to early onset dementia, are associated withdiminished Notch activity (Chavez-Gutierrez et al., 2012;

Moehlmann et al., 2002; Steineret al., 2001).In addition, FAD mutationsof presenilinproduce longerand morelipophilic N␤ peptides in analogy to theamylogenic A␤42 at theexpense of

thenon-amylogenic A␤40 forms (Okochi etal., 2002).A␤42 are

responsibleforinsolubleamyloidplaqueformation.Theefficiency of␥-secretaseappearstobeaffectedduringaging.Infact,inthe brainsofagedmice,␥-secretaseactivityincreasedAPP process-ingandA␤42production,whereasNotch1processingisreduced

(Placanicaetal.,2009).

In contrasttothe previousreports indicatinga reduction in Notch activity in AD, two other studies showed a significant increaseinNotchexpression/activityinsporadicAlzheimer’s

dis-ease(Berezovskaetal.,1998;Nagarshethetal.,2006).Inaddition,

otherneurodegenerativediseasessuchasDownsyndrome(Fischer etal.,2005),Pick’s(Nagarshethet al.,2006)andPrion’sdisease

(Ishikuraetal.,2005),arecharacterizedbyconcomitantincreases

inNotchexpressionandamyloidplaquesformation.Itremainsto beunderstoodwhyprogressiveneurodegenerativediseaseshave differentmechanismsintermsofNotchprocessingandsignaling, andwhetherNotcheffectivelycontributestotheirpathobiology.

BesidetheunresolvedroleofNotchinAD,thecommon pro-cessingmechanismofNotchandAPPhasposedseriouschallenges in the development of drugs that can counteract the progres-sion of AD: Pre-clinical trials using ␥-secretase inhibitorshave reducedamyloidplaquesdepositionbuthaveoftencaused intesti-nalbleedingandimmunosuppressionduetotheinhibitionofNotch signaling(Wongetal.,2004).Therefore,inrecentyears,more com-poundshavebeendevelopedthattargetAPPmetabolismwithout affectingNotchsignaling(Wolfe,2012).Thesenoveldrugsmight improvenotonlytheperipheralactionsofthecompounds,butalso spareNotchsignalinginthebrain.

Furtherstudiesaimedatunravelingthemolecularmediators ofNotch-dependentprocessesinprogressiveneurodegenerative diseasewillfurthercontributetothedevelopmentofbetter preven-tiveandtherapeuticstrategiestocounteractimbalancesinNotch signalinginagingandAD.

7.2. Notchinischemicinjury

Several studies have shown that, following ischemic injury, Notch signalingis aberrantlyup-regulated (Alberi et al., 2010;

Arumugam etal., 2006;Louet al.,2012;Shimadaet al.,2011).

Notchinductionfollowinginjuryresultsinaplethoraofeffectsand cancontributetodamageaswellasregeneration.Inmature neu-rons,Notch1(Arumugametal.,2006)andNotch2(Arumugametal.,

2011;FerrariToninellietal.,2003)arebothinduced,suggestinga

summationofdownstreameffects.

TheinvolvementofNotchinischemia-inducedcelldeathhas beenfurthershownbytheworkofArumugametal.(2011),where NotchappearstointeractwiththeNF-␬Bcomponentsp65,p50and Bimintheapoptoticprocessinvitroandinvivo(Fig.3).Theonly caveatofthislatterstudyisthechemicalinvivolossoffunction modelutilized(␥-secretaseinhibitors[␥GSI]).Thesedrugsinhibit theprocessingofseveral␥-secretasesubstratesatthesametime

(SelkoeandWolfe,2007)anditisthereforedifficulttostudyspecific

implicationsofNotchsignalinginanyoftheseprocesses. Employ-inggeneticlossoffunctionalongwithGSIisstronglyrecommended tounderstandtheclinicalrelevanceofthispathway.

Another recent study, using both GSI and a Notch loss-of-function model, has shown that Notch signaling is crucial for inducingNF-␬Bdrivenmicrogliaresponses,similartowhatoccurs in mature neurons (Wei et al., 2011). The microglial response uponinjuryisnotoriousforproducinganimmuno-inflammatory responsethatcanworsenandamplifyneuronaldamage(Stolletal., 1998).Similarly, another recent study indicatesthat Notch1is

directlyinvolvedin reactivegliogenesis,contributing tothecell invasionintheperi-infarctarea(Shimadaetal.,2011).

Followingcerebralischemia,angiogenesisandvesselsprouting takesplace.Notch1isanimportantangiogenicfactorboth dur-ingdevelopment(Limbourgetal.,2005)andpostnatally(Takeshita et al.,2007).Itappearsthat,following cerebralischemia,Notch playsanimportantroleinreperfusionandthereorganizationof the microcirculation(Louet al., 2012).Besides Notch1, Notch3 playsafundamentalroleinthevasculature,whereitisspecifically expressedinsmoothmusclecellsoftheendothelium.Mutationsin theNotch3genesinduceCADASIL,whichischaracterizedby pro-gressivebrainischemiaandvasculardementia(Dichgans,2007). Notch3 mutant micehave confirmed the fundamental require-mentofNoch3invasculaturereorganizationfollowingischemic challenge(Arboleda-Velasquezet al.,2008).Morerecently,two transgenicmouselinescarryingdistinctCADASILmutationsshow hypomorphicNotch3activityandwithagedevelopsmoothmuscle cellsabnormalitiessimilartohumanCADASIL(Arboleda-Velasquez etal.,2011).Therefore,itappearsthatinhibitingNotchsignaling early after injury can provide some beneficial effects by limit-ingtheincreasedinflammatoryresponse,providedbyreperfusion

(Arumugametal.,2006).Ontheotherhand,itmightbeexpected

thatlong-termblockadeofNotchcanworsenthescenarioby pre-ventingregeneration(HofmannandIruela-Arispe,2007).

Inaddition,Notchactivationhasbeenshowntobeessentialin neuralstemcellproliferationinseveralhypoxia–ischemiarodent models(Androutsellis-Theotokisetal.,2006;Carlenetal.,2009;

Chenetal.,2008a;Oyaetal.,2009;Wangetal.,2009).Canonical

Notchsignalingisactivatedthroughahypoxia-dependent mech-anism (Johnson, 2011; Seidel et al., 2010), as well as through overexpressionoftheligand(Androutsellis-Theotokisetal.,2006; Liuetal.,2011).NeuralstemcellsoftheSGZandSVZ,whichare quiescentunderphysiologicalconditions,havebeenshownto pro-liferatefollowinginjury(Kawaietal.,2005;Lugertetal.,2010), probablyinanattempttorepairbraindamage.Notchsignaling, which isa knownneurogenicfactor (GaianoandFishell,2002), hasbeenshowntobecentralinthisprocess,asdemonstratedby geneticandchemicallossoffunctionmodels(Kawaietal.,2005;

Oyaetal.,2009;Wangetal.,2009;Xinetal.,2006).Interestingly,

thisregenerativepotential,inresponsetoinjury,isdiminishedwith aging(Lugertetal.,2010).

Sometimeafterstroke,attenuationofNotchsignalingappears to be required for integration of newly born neurons in the hippocampal network (Oya et al., 2009). On the other hand, a recent report has shown that 1 week after kainate injury, Notch-dependent stem cell proliferation is short-lived and cell fatecommitment,mainlyofastroglialtype,occurs (Sibbeet al., 2012). This discrepancy might beexplained by distinct micro-environmentalreactionsinhypoxia–ischemiaandischemiawhere Notch–DSL interactions with either Jagged1 or Deltalike1 lead to neurogenic (Nyfeler et al., 2005) or gliogenic commitment

(Grandbarbeetal.,2003),respectively.Applicationofexogenous

Jagged1mightpromotetheneurogenicpotentialinthesclerotic scarand provideabeneficial influencein termsofregeneration andneuronalreplenishment.

Interestingly,ithasrecentlybeenproposedthatcanonicalNotch signalingisinducedbyisofluoranepreconditioningandcan medi-ateneuroprotectivefunctionsfollowingfocalischemia(Yangetal., 2012).Ithasbeenproposedthatpreconditioning,usingsub-lethal insults,canactivatesignalingcascadessuchasNF-␬BandHIFthat haveneuroprotectivefunctions(Gidday,2006).Itispossiblethat Notchactivation,throughayetunknownmechanism,takespart inthesepro-survivalprocesses.Interestingly,arecentlyidentified neuroprotectivegene,Botch(Daietal.,2010),canregulateNotch expression(Chietal.,2012).Similardualfunctionshavealsobeen describedforanotherpathway:PIK3/AKT(Brown,2007;Saweetal.,



2008),whichhasbeenintimatelyconnectedtoNotchinthe sur-vival/death choiceincancer(Calzavara etal.,2008;Chanet al.,

2007;GutierrezandLook,2007;Palomeroetal.,2007).

On thewhole, it appearsthat severalNotch-dependent pro-cessesaretriggeredbycerebralhypoxia–ischemia.Understanding themoleculardynamicsofthissignalingcascadewillhelpdevise moreeffectivetherapeuticapproachesthatinfluencethispathway andcanlimitbraindamageandfavorregeneration.

8. Conclusions

Inthisreview,wehavefocusedontheroleofNotchsignaling inthematurebrain.Wehaveattemptedtogiveanoverview col-lectingsignificantfindingsfrompastand presentinvestigations. ItemergesthatNotchiscriticalforphysiologicalbrainfunctions from neurogenesistomemoryprocessing. Inaddition, aberrant activationfollowingbraininjuryappearstocontributeononeside toneuronaldemise,triggeringapoptoticprocessesandincreasing inflammatoryresponses,andontheothersidetoregeneration.The molecularmechanismsbeyondsuchdiversefunctionsarecurrently asubjectofintensestudyandweexpectittoincreaseinthefuture. Fortheyetunsolvedmechanisms,wehavelookedatother biologi-calmodelssuchasdevelopmentandcancer,whereNotchhasbeen extensivelystudied.Inthisway,wehavetriedtofillthecurrent bio-logicalgapinordertofindpossibleexplanationsforNotchfunction inthematurebrain.Wehavealsoreportedbraindiseases,suchas ADandstroke,whereNotchsignalingalterationsarereported.We havealsomentionedsomeofthechallengesindrugdevelopment thattarget␥-secretasedysfunctionsduetooff-targeteffectsonthe Notchpathway.Webelievethatthiscascade,atthepaceofother highlyconservedpathways,playsacrucialroleinbrainphysiology andpathology.Weexpectthatinthenextdecade,several break-throughdiscoverieswillbemade,connectingthedotsinthebrain “signalome”.Theseworkswillhelpthedevelopmentofnew thera-peuticstrategiesforthepreventionandtreatmentofbraindisease inwhichNotchisdirectlyimplicated.

Acknowledgments

We would like to thank Nicholas Gaiano, Cedric Wesley, ShuxiLiu andRobertKretzfortheirhelpfulcontributiontothis manuscript. This research is supported entirely by the Swiss NationalFoundation,SynapsisFoundationandSwissHeart Asso-ciation.

References

Alagille,D.,Odievre,M.,Gautier, M.,Dommergues,J.P.,1975. Hepatic

ductu-larhypoplasiaassociatedwithcharacteristicfacies,vertebralmalformations, retardedphysical,mental,andsexualdevelopment,andcardiacmurmur. Jour-nalofPediatrics86,63–71.

Alberi,L.,Chi,Z.,Kadam,S.D.,Mulholland,J.D.,Dawson,V.L.,Gaiano,N.,Comi,A.M.,

2010. Neonatalstrokeinmicecauseslong-termchangesinneuronalNotch-2

expressionthatmaycontributetoprolongedinjury.Stroke41,S64–S71.

Alberi,L.,Liu,S.,Wang,Y.,Badie,R.,Smith-Hicks,C.,Wu,J.,Pierfelice,T.J.,Abazyan,

B.,Mattson,M.P.,Kuhl,D.,Pletnikov,M.,Worley,P.F.,Gaiano,N.,2011.

Activity-inducedNotchsignalinginneuronsrequiresArc/Arg3.1andisessentialfor synapticplasticityinhippocampalnetworks.Neuron69,437–444.

Andersen,P.,Uosaki,H.,Shenje,L.T.,Kwon,C.,2012.Non-canonicalNotchsignaling:

emergingroleandmechanism.TrendsinCellBiology22,257–265.

Androutsellis-Theotokis,A.,Leker,R.R.,Soldner,F.,Hoeppner,D.J.,Ravin,R.,Poser,

S.W.,Rueger,M.A.,Bae,S.K.,Kittappa,R.,McKay,R.D.,2006. Notchsignalling

regulatesstemcellnumbersinvitroandinvivo.Nature442,823–826.

Ann,E.J.,Kim,H.Y.,Seo,M.S.,Mo,J.S.,Kim,M.Y.,Yoon,J.H.,Park,H.S.,2012. Wnt5a

controlsNotch1signalingthroughCaMKIImediateddegradationoftheSMRT corepressorprotein.JournalofBiologicalChemistry.

Arboleda-Velasquez,J.F.,Manent,J.,Lee,J.H.,Tikka,S.,Ospina,C.,Vanderburg,C.R.,

Frosch,M.P.,Rodriguez-Falcon,M.,Villen,J.,Gygi,S.,Lopera,F.,Kalimo,H.,

Moskowitz,M.A.,Ayata,C.,Louvi,A.,Artavanis-Tsakonas,S.,2011.Hypomorphic

Notch3alleleslinkNotchsignalingtoischemiccerebralsmall-vesseldisease.

ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica 108,E128–E135.

Arboleda-Velasquez,J.F.,Zhou,Z.,Shin,H.K.,Louvi,A.,Kim,H.H.,Savitz,S.I.,Liao,

J.K.,Salomone,S.,Ayata,C.,Moskowitz,M.A.,Artavanis-Tsakonas,S.,2008.

Link-ingNotchsignalingtoischemicstroke.ProceedingsoftheNationalAcademyof SciencesoftheUnitedStatesofAmerica105,4856–4861.

Artavanis-Tsakonas,S.,Rand,M.D.,Lake,R.J.,1999.Notchsignaling:cellfatecontrol

andsignalintegrationindevelopment.Science284,770–776.

Arumugam,T.V.,Chan,S.L.,Jo,D.G.,Yilmaz,G.,Tang,S.C.,Cheng,A.,Gleichmann,

M.,Okun,E.,Dixit,V.D.,Chigurupati,S.,Mughal,M.R.,Ouyang,X.,Miele,L.,

Magnus,T.,Poosala,S.,Granger,D.N.,Mattson,M.P.,2006. Gamma

secretase-mediatedNotchsignalingworsensbraindamageandfunctionaloutcomein ischemicstroke.NatureMedicine12,621–623.

Arumugam,T.V.,Cheng,Y.L.,Choi,Y.,Choi,Y.H.,Yang,S.,Yun,Y.K.,Park,J.S.,Yang,

D.K.,Thundyil,J.,Gelderblom,M.,Karamyan,V.T.,Tang,S.C.,Chan,S.L.,

Mag-nus,T.,Sobey,C.G.,Jo,D.G.,2011. Evidencethatgamma-secretase-mediated

Notchsignalinginducesneuronalcelldeathviathenuclear factor-kappaB-Bcl-2-interactingmediatorofcelldeathpathwayinischemicstroke.Molecular Pharmacology80,23–31.

Ataman,B.,Ashley,J.,Gorczyca,M.,Ramachandran,P.,Fouquet,W.,Sigrist,S.J.,

Bud-nik,V.,2008.Rapidactivity-dependentmodificationsinsynapticstructureand

functionrequirebidirectionalWntsignaling.Neuron57,705–718.

Basak,O.,Giachino,C.,Fiorini,E.,Macdonald,H.R.,Taylor,V.,2012. Neurogenic

subventricularzonestem/progenitorcellsareNotch1-dependentintheiractive butnotquiescentstate.JournalofNeuroscience32,5654–5666.

Basak,O.,Taylor,V.,2007.Identificationofself-replicatingmultipotentprogenitors

intheembryonicnervoussystembyhighNotchactivityandHes5expression. EuropeanJournalofNeuroscience25,1006–1022.

Bash,J.,Zong,W.X.,Banga,S.,Rivera,A.,Ballard,D.W.,Ron,Y.,Gelinas,C.,1999.

Rel/NF-kappaBcantriggertheNotchsignalingpathwaybyinducingthe expres-sionofJagged1,aligandforNotchreceptors.EMBOJournal18,2803–2811.

Becam,I.,Fiuza,U.M.,Arias,A.M.,Milan,M.,2010.AroleofreceptorNotchinligand

cis-inhibitioninDrosophila.CurrentBiology20,554–560.

Beffert,U.,Weeber,E.J.,Durudas,A.,Qiu,S.,Masiulis,I.,Sweatt,J.D.,Li,W.P.,

Adel-mann,G.,Frotscher,M.,Hammer,R.E.,Herz,J.,2005. Modulationofsynaptic

plasticityandmemorybyReelininvolvesdifferentialsplicingofthelipoprotein receptorApoer2.Neuron47,567–579.

Bekinschtein,P.,Katche,C.,Slipczuk,L.N.,Igaz,L.M.,Cammarota,M.,Izquierdo,I.,

Medina,J.H.,2007.mTORsignalinginthehippocampusisnecessaryformemory

formation.NeurobiologyofLearningandMemory87,303–307.

BenAbdallah,N.M.,Slomianka,L.,Vyssotski,A.L.,Lipp,H.P.,2010.Earlyage-related

changesinadulthippocampalneurogenesisinC57mice.NeurobiologyofAging 31,151–161.

Berezovska,O.,McLean,P.,Knowles,R.,Frosh,M.,Lu,F.M.,Lux,S.E.,Hyman,B.T.,

1999.Notch1inhibitsneuriteoutgrowthinpostmitoticprimaryneurons.

Neu-roscience93,433–439.

Berezovska,O.,Xia,M.Q.,Hyman,B.T.,1998. Notchisexpressedinadultbrain,is

coexpressedwithpresenilin-1,andisalteredinAlzheimerdisease.Journalof NeuropathologyandExperimentalNeurology57,738–745.

Bergami,M.,Berninger,B.,Canossa,M.,2009. ConditionaldeletionofTrkBalters

adulthippocampalneurogenesisandanxiety-relatedbehavior.Communicative andIntegrativeBiology2,14–16.

Bonini,S.A.,Ferrari-Toninelli,G.,Uberti,D.,Montinaro,M.,Buizza,L.,Lanni,C.,Grilli,

M.,Memo,M.,2011. NuclearfactorkappaB-dependentneuriteremodelingis

mediatedbyNotchpathway.JournalofNeuroscience31,11697–11705.

Breunig,J.J.,Silbereis,J.,Vaccarino,F.M.,Sestan,N.,Rakic,P.,2007.Notchregulates

cellfateanddendritemorphologyofnewbornneuronsinthepostnataldentate gyrus.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesof America104,20558–20563.

Brown,I.R.,2007.Heatshockproteinsandprotectionofthenervoussystem.Annals

oftheNewYorkAcademyofSciences1113,147–158.

Brown,J.,Cooper-Kuhn,C.M.,Kempermann,G.,VanPraag,H.,Winkler,J.,Gage,

F.H.,Kuhn,H.G.,2003. Enrichedenvironmentandphysicalactivitystimulate

hippocampalbutnotolfactorybulbneurogenesis.EuropeanJournalof Neuro-science17,2042–2046.

Bruel-Jungerman,E.,Veyrac,A.,Dufour,F.,Horwood,J.,Laroche,S.,Davis,S.,2009.

InhibitionofPI3K-Aktsignalingblocksexercise-mediatedenhancementofadult neurogenesisandsynapticplasticityinthedentategyrus.PLoSONE4,e7901.

Calzavara,E.,Chiaramonte,R.,Cesana,D.,Basile,A.,Sherbet,G.V.,Comi,P.,2008.

ReciprocalregulationofNotchandPI3K/AktsignallinginT-ALLcellsinvitro. JournalofCellularBiochemistry103,1405–1412.

Carlen,M.,Meletis,K.,Goritz,C.,Darsalia,V.,Evergren,E.,Tanigaki,K.,Amendola,

M.,Barnabe-Heider,F.,Yeung,M.S.,Naldini,L.,Honjo,T.,Kokaia,Z.,Shupliakov,

O.,Cassidy,R.M.,Lindvall,O.,Frisen,J.,2009. Forebrainependymalcellsare

Notch-dependentandgenerateneuroblastsandastrocytesafterstroke.Nature Neuroscience12,259–267.

Chan,S.M.,Weng,A.P.,Tibshirani,R.,Aster,J.C.,Utz,P.J.,2007. Notchsignals

pos-itivelyregulateactivityofthemTORpathwayinT-cellacutelymphoblastic leukemia.Blood110,278–286.

Chao,M.Y.,Larkins-Ford,J.,Tucey,T.M.,Hart,A.C.,2005. lin-12Notchfunctionsin

theadultnervoussystemofC.elegans.BMCNeuroscience6,45.

Chavez,J.C.,LaManna,J.C.,2002. Activationofhypoxia-induciblefactor-1inthe

ratcerebralcortexaftertransientglobalischemia:potentialroleofinsulin-like growthfactor-1.JournalofNeuroscience22,8922–8931.

Chavez-Gutierrez,L.,Bammens,L.,Benilova,I.,Vandersteen,A.,Benurwar,M.,

Borg-ers,M.,Lismont,S.,Zhou,L.,VanCleynenbreugel,S.,Esselmann,H.,Wiltfang,J.,



Serneels,L.,Karran,E.,Gijsen,H.,Schymkowitz,J.,Rousseau,F.,Broersen,K.,De

Strooper,B.,2012.Themechanismofgamma-Secretasedysfunctioninfamilial

Alzheimerdisease.EMBOJournal.

Chen,J.,Zacharek,A.,Li,A.,Cui,X.,Roberts,C.,Lu,M.,Chopp,M.,2008a.

Atorvas-tatinpromotespresenilin-1expressionandNotch1activityandincreasesneural progenitorcellproliferationafterstroke.Stroke39,220–226.

Chen,Q.,Nakajima,A.,Choi,S.H.,Xiong,X.,Sisodia,S.S.,Tang,Y.P.,2008b. Adult

neurogenesisisfunctionallyassociatedwithAD-likeneurodegeneration. Neu-robiologyofDisease29,316–326.

Cheng,P.,Zlobin,A.,Volgina,V.,Gottipati,S.,Osborne,B.,Simel,E.J.,Miele,L.,

Gabrilovich,D.I.,2001. Notch-1regulatesNF-kappaBactivityinhemopoietic

progenitorcells.JournalofImmunology167,4458–4467.

Chevallier,N.L.,Soriano,S.,Kang,D.E.,Masliah,E.,Hu,G.,Koo,E.H.,2005.Perturbed

neurogenesisintheadulthippocampusassociatedwithpresenilin-1A246E mutation.AmericanJournalofPathology167,151–159.

Chi,Z.,Zhang,J.,Tokunaga,A.,Harraz,M.M.,Byrne,S.T.,Dolinko,A.,Xu,J.,Blackshaw,

S.,Gaiano,N.,Dawson,T.M.,Dawson,V.L.,2012. Botchpromotesneurogenesis

byantagonizingNotch.DevelopmentalCell22,707–720.

Childress,J.L.,Acar,M.,Tao,C.,Halder,G.,2006. Lethalgiantdiscs,anovel

C2-domainprotein,restrictsnotchactivationduringendocytosis.CurrentBiology 16,2228–2233.

Cisse,M.,Duplan,E.,Guillot-Sestier,M.V.,Rumigny,J.,Bauer,C.,Pages,G.,

Orze-chowski, H.D.,Slack, B.E.,Checler,F.,Vincent,B.,2011. Theextracellular

regulatedkinase-1(ERK1)controlsregulatedalpha-secretase-mediated pro-cessing,promotertransactivation,andmRNAlevelsofthecellularprionprotein. JournalofBiologicalChemistry286,29192–29206.

Conboy,L.,Seymour,C.M.,Monopoli,M.P.,O‘Sullivan,N.C.,Murphy,K.J.,Regan,C.M.,

2007.Notchsignallingbecomestransientlyattenuatedduringlong-term

mem-oryconsolidationinadultWistarrats.NeurobiologyofLearningandMemory 88,342–351.

Costa,R.M.,Honjo,T.,Silva,A.J.,2003.LearningandmemorydeficitsinNotchmutant

mice.CurrentBiology13,1348–1354.

Crowner,D.,LeGall,M.,Gates,M.A.,Giniger,E.,2003. NotchsteersDrosophila

ISNbmotoraxonsbyregulatingtheAblsignalingpathway.CurrentBiology13, 967–972.

D‘Souza,B.,Miyamoto,A.,Weinmaster,G.,2008.ThemanyfacetsofNotchligands.

Oncogene27,5148–5167.

Dahlhaus,M.,Hermans,J.M.,VanWoerden,L.H.,Saiepour,M.H.,Nakazawa,K.,

Mansvelder,H.D.,Heimel,J.A.,Levelt,C.N.,2008.Notch1signalinginpyramidal

neuronsregulatessynapticconnectivityandexperience-dependent modifica-tionsofacuityinthevisualcortex.JournalofNeuroscience28,10794–10802.

Dai,C.,Liang,D.,Li,H.,Sasaki,M.,Dawson,T.M.,Dawson,V.L.,2010. Functional

identificationofneuroprotectivemolecules.PLoSONE5,e15008.

Dang,L.,Fan,X.,Chaudhry,A.,Wang,M.,Gaiano,N.,Eberhart,C.G.,2006. Notch3

signalinginitiateschoroidplexustumorformation.Oncogene25,487–491.

de Bivort, B.L., Guo, H.F., Zhong, Y., 2009. Notch Signaling Is Required for

Activity-DependentSynapticPlasticityattheDrosophilaNeuromuscular Junc-tion.JournalofNeurogenetics,1–10.

deCelis,J.F.,Bray,S.,1997.Feed-backmechanismsaffectingNotchactivationatthe

dorsoventralboundaryintheDrosophilawing.Development124,3241–3251.

delaPompa,J.L.,Wakeham,A.,Correia,K.M.,Samper,E.,Brown,S.,Aguilera,R.J.,

Nakano,T.,Honjo,T.,Mak,T.W.,Rossant,J.,Conlon,R.A.,1997. Conservationof

theNotchsignallingpathwayinmammalianneurogenesis.Development124, 1139–1148.

delAlamo,D.,Schweisguth,F.,2009. Notchsignalling:receptorcis-inhibitionto

achievedirectionality.CurrentBiology19,R683–R684.

DelMonte,G.,Grego-Bessa,J.,Gonzalez-Rajal,A.,Bolos,V.,DeLaPompa,J.L.,2007.

MonitoringNotch1activityindevelopment:evidenceforafeedbackregulatory loop.DevelopmentalDynamics236,2594–2614.

Dichgans,M.,2007.Geneticsofischaemicstroke.TheLancetNeurology6,149–161.

Ehm,O.,Goritz,C.,Covic,M.,Schaffner,I.,Schwarz,T.J.,Karaca,E.,Kempkes,B.,

Krem-mer,E.,Pfrieger,F.W.,Espinosa,L.,Bigas,A.,Giachino,C.,Taylor,V.,Frisen,J.,Lie,

D.C.,2010. RBPJkappa-dependentsignalingisessentialforlong-term

mainte-nanceofneuralstemcellsintheadulthippocampus.JournalofNeuroscience 30,13794–13807.

Eiraku,M.,Hirata,Y.,Takeshima,H.,Hirano,T.,Kengaku,M.,2002.

Delta/notch-likeepidermalgrowthfactor(EGF)-relatedreceptor,anovelEGF-like repeat-containingproteintargetedtodendritesofdevelopingandadultcentralnervous systemneurons.JournalofBiologicalChemistry277,25400–25407.

Espinosa,L., Santos,S.,Ingles-Esteve, J., Munoz-Canoves, P., Bigas, A.,2002.

p65-NFkappaBsynergizeswithNotchtoactivatetranscriptionbytriggering cytoplasmictranslocationofthenuclearreceptorcorepressorN-CoR.Journal ofCellScience115,1295–1303.

Fassa,A.,Mehta,P.,Efthimiopoulos,S.,2005. Notch1interactswiththeamyloid

precursorproteininaNumb-independentmanner.JournalofNeuroscience Research82,214–224.

Faure,A.,Verret,L.,Bozon,B.,ElTannirElTayara,N.,Ly,M.,Kober,F.,Dhenain,

M.,Rampon,C.,Delatour,B.,2011. Impairedneurogenesis,neuronalloss,and

brainfunctionaldeficitsintheAPPxPS1-KimousemodelofAlzheimer’sdisease. NeurobiologyofAging32,407–418.

FerrariToninelli,G.,Bernardi,C.,Quarto,M.,Lozza,G.,Memo,M.,Grilli,M.,2003.

Long-lastinginductionofNotch2inthehippocampusofkainate-treatedadult mice.Neuroreport14,917–921.

Ferron,S.R.,Marques-Torrejon,M.A.,Mira,H.,Flores,I.,Taylor,K.,Blasco,M.A.,

Farinas,I.,2009. Telomereshorteninginneuralstemcellsdisruptsneuronal

differentiationandneuritogenesis.JournalofNeuroscience29,14394–14407.

Fischer,D.F.,vanDijk,R.,Sluijs,J.A.,Nair,S.M.,Racchi,M.,Levelt,C.N.,vanLeeuwen,

F.W.,Hol,E.M.,2005. ActivationoftheNotchpathwayinDownsyndrome:

cross-talkofNotchandAPP.FASEBJournal19,1451–1458.

Fortini,M.E.,Artavanis-Tsakonas,S.,1994. Thesuppressorofhairlessprotein

par-ticipatesinnotchreceptorsignaling.Cell79,273–282.

Franklin,J.L.,Berechid,B.E.,Cutting,F.B.,Presente,A.,Chambers,C.B.,Foltz,D.R.,

Ferreira,A.,Nye,J.S.,1999. Autonomousandnon-autonomousregulationof

mammalianneuritedevelopmentbyNotch1andDelta1.CurrentBiology9, 1448–1457.

Fre,S.,Hannezo,E.,Sale,S.,Huyghe,M.,Lafkas,D.,Kissel,H.,Louvi,A.,Greve,J.,

Lou-vard,D.,Artavanis-Tsakonas,S.,2011. Notchlineagesandactivityinintestinal

stemcellsdeterminedbyanewsetofknock-inmice.PLoSONE6,e25785.

Fryer,C.J.,White,J.B.,Jones,K.A.,2004. MastermindrecruitsCycC:CDK8to

phos-phorylatetheNotchICDandcoordinateactivationwithturnover.MolecularCell 16,509–520.

Gaiano,N.,Fishell,G.,2002. Theroleofnotchinpromotingglialandneuralstem

cellfates.AnnualReviewofNeuroscience25,471–490.

Garaschuk,O.,Linn,J.,Eilers,J.,Konnerth,A.,2000. Large-scaleoscillatorycalcium

wavesintheimmaturecortex.NatureNeuroscience3,452–459.

Gazave,E.,Lapebie,P.,Richards,G.S.,Brunet,F.,Ereskovsky,A.V.,Degnan,B.M.,

Borchiellini,C.,Vervoort,M.,Renard,E.,2009.OriginandevolutionoftheNotch

signallingpathway:anoverviewfromeukaryoticgenomes.BMCEvolutionary Biology9,249.

Ge,C.,Stanley,P.,2008.TheO-fucoseglycanintheligand-bindingdomainofNotch1

regulatesembryogenesisandTcelldevelopment.ProceedingsoftheNational AcademyofSciencesoftheUnitedStatesofAmerica105,1539–1544.

Ge,X.,Hannan,F.,Xie,Z.,Feng,C.,Tully,T.,Zhou,H.,Zhong,Y.,2004. Notch

sig-nalinginDrosophilalong-termmemoryformation.ProceedingsoftheNational AcademyofSciencesoftheUnitedStatesofAmerica101,10172–10176.

Gidday,J.M., 2006. Cerebralpreconditioning andischaemictolerance.Nature

ReviewsNeuroscience7,437–448.

Giniger,E.,1998.AroleforAblinNotchsignaling.Neuron20,667–681.

Glittenberg,M.,Pitsouli,C.,Garvey,C.,Delidakis,C.,Bray,S.,2006.Roleofconserved

intracellularmotifsinSerratesignalling,cis-inhibitionandendocytosis.EMBO Journal25,4697–4706.

Grandbarbe,L.,Bouissac,J.,Rand,M.,HrabedeAngelis,M.,Artavanis-Tsakonas,

S.,Mohier,E.,2003. Delta-Notchsignalingcontrolsthegenerationof

neu-rons/glia fromneural stemcells inastepwiseprocess.Development130, 1391–1402.

Gustafsson,M.V.,Zheng,X.,Pereira,T.,Gradin,K.,Jin,S.,Lundkvist,J.,Ruas,J.L.,

Poellinger,L.,Lendahl,U.,Bondesson,M.,2005.Hypoxiarequiresnotchsignaling

tomaintaintheundifferentiatedcellstate.DevelopmentalCell9,617–628.

Gutierrez,A.,Look,A.T.,2007.NOTCHandPI3K-AKTpathwaysintertwined.Cancer

Cell12,411–413.

Hanz,S.,Perlson,E.,Willis,D.,Zheng,J.Q.,Massarwa,R.,Huerta,J.J.,Koltzenburg,M.,

Kohler,M.,van-Minnen,J.,Twiss,J.L.,Fainzilber,M.,2003.Axoplasmicimportins

enableretrogradeinjurysignalinginlesionednerve.Neuron40,1095–1104.

Hashimoto-Torii,K.,Torii,M.,Sarkisian,M.R.,Bartley,C.M.,Shen,J.,Radtke,F.,

Grid-ley,T.,Sestan,N.,Rakic,P.,2008.InteractionbetweenReelinandNotchsignaling

regulatesneuronalmigrationinthecerebralcortex.Neuron60,273–284.

Herz,J.,Chen,Y.,2006.Reelin,lipoproteinreceptorsandsynapticplasticity.Nature

ReviewsNeuroscience7,850–859.

Hicks,C.,Ladi,E.,Lindsell,C.,Hsieh,J.J.,Hayward,S.D.,Collazo,A.,Weinmaster,G.,

2002. AsecretedDelta1-Fcfusionproteinfunctionsbothasanactivatorand

inhibitorofNotch1signaling.JournalofNeuroscienceResearch68,655–667.

Hoeck,J.D.,Jandke,A.,Blake,S.M.,Nye,E.,Spencer-Dene,B.,Brandner,S.,Behrens,A.,

2010. Fbw7controlsneuralstemcelldifferentiationandprogenitorapoptosis

viaNotchandc-Jun.NatureNeuroscience13,1365–1372.

Hoeffer,C.A.,Tang,W.,Wong,H.,Santillan,A.,Patterson,R.J.,Martinez,L.A.,

Tejada-Simon,M.V.,Paylor,R.,Hamilton,S.L.,Klann,E.,2008. RemovalofFKBP12

enhancesmTOR-Raptorinteractions,LTP,memory,andperseverative/repetitive behavior.Neuron60,832–845.

Hoey,S.E.,Williams,R.J.,Perkinton,M.S.,2009.SynapticNMDAreceptoractivation

stimulatesalpha-secretaseamyloidprecursorproteinprocessingandinhibits amyloid-betaproduction.JournalofNeuroscience29,4442–4460.

Hofmann,J.J.,Iruela-Arispe,M.L.,2007. Notchsignalinginbloodvessels:whois

talkingtowhomaboutwhat?CirculationResearch100,1556–1568.

Hori,K.,Sen,A.,Kirchhausen,T.,Artavanis-Tsakonas,S.,2011. Synergybetween

theESCRT-IIIcomplexandDeltexdefinesaligand-independentNotchsignal. JournalofCellBiology195,1005–1015.

Hou, L., Klann, E., 2004. Activation of the phosphoinositide

3-kinase-Akt-mammaliantargetofrapamycinsignalingpathwayisrequiredformetabotropic glutamatereceptor-dependentlong-termdepression.JournalofNeuroscience 24,6352–6361.

Huang,Q.,Raya,A.,DeJesus,P.,Chao,S.H.,Quon,K.C.,Caldwell,J.S.,Chanda,S.K.,

Izpisua-Belmonte,J.C.,Schultz,P.G.,2004. Identificationofp53regulatorsby

genome-widefunctionalanalysis.ProceedingsoftheNationalAcademyof Sci-encesoftheUnitedStatesofAmerica101,3456–3461.

Huenniger,K.,Kramer,A.,Soom,M.,Chang,I.,Kohler,M.,Depping,R.,Kehlenbach,

R.H.,Kaether,C.,2010. Notch1signalingismediatedbyimportinsalpha3,4,

and7.CellularandMolecularLifeSciences67,3187–3196.

Imayoshi,I.,Kageyama,R.,2011. TheroleofNotchsignalinginadultneurogenesis.

MolecularNeurobiology44,7–12.

Imayoshi,I.,Sakamoto,M.,Yamaguchi,M.,Mori,K.,Kageyama,R.,2010. Essential

rolesofNotchsignalinginmaintenanceofneuralstemcellsindevelopingand adultbrains.JournalofNeuroscience30,3489–3498.