Circadian clocks and sleep: impact of rhythmic metabolism and waste clearance on the brain

Circadian

Clocks

and

Sleep:

Impact

of

Rhythmic

Metabolism

and

Waste

Clearance

on

the

Brain

Urs

Albrecht

* and

Jürgen

A.

Ripperger

TherotationoftheEartharounditsaxiscausesperiodicexposureofhalfofits surface to sunlight.This daily recurring event has been internalized in most organismsintheformofcellularcircadianclockmechanisms. Thesecellular clocksaresynchronizedwitheachotherinvariouswaystoestablishcircadian networksthatbuildthecircadianprogramintissuesandorgans,coordinating physiologyandbehaviorin theentireorganism. Inthemammalianbrain, the suprachiasmatic nucleus (SCN) receives lightinformation via the retina and synchronizesitsownneuronalclockstothelightsignal.Subsequently,theSCN transmits this information to the network of clocks in tissues and organs, thereby synchronizing body physiology and behavior. Disruption of cellular clocksand/ordestructionofthesynchronizationbetweentheclocks,as expe-riencedforinstance injetlagandshift-workconditions, affectsnormalbrain functionandcanleadtometabolicproblems,sleepdisturbance,and acceler-atedneurologicaldecline.Inthisreview,wehighlightwaysthroughwhichthe circadiansystemcancoordinatenormalbrainfunction,withafocuson metab-olism and metabolic astrocyte–neuron communications. Recent develop-ments, for example, on how waste clearance in the brain could be modulatedbythecircadianclock,willalsobediscussed.Thissynthesis pro-videsinsightsintotheimpactofmetabolismnotonlyonthecircadianclock,but alsoonsleepandhowthisconnectionmayexacerbateneurologicaldiseases.

TheCircadianClockandMetabolic–NeurophysiologicalAxis

Epidemiologicalstudiesindicatethattherearestronginteractionsbetweenmodernlifestyle, sleepdeprivation, and circadian misalignment.These reciprocal interactionscorrelate with varioushealthproblems,suchasobesity,cancer,addictivebehaviors,cardiovasculardisease, and neurological disorders (reviewed in [1]). Therefore, a detailed understanding of how environmentalconditionssuchaschangesinlightexposure,fooduptake,andstressaffect sleep(seeGlossary)andthecircadiansystemisofhighpriority.

WhilethemechanismsofhowlightaffectstheSCNarewellunderstood[2],theonesbehindthe impacttheSCNhasonsleep,mood,weightregulation,andbrainmetabolismareonlypartly clarified.Amongthekeyquestionsarehowmetabolism(anabolicandcatabolic)isregulatedin thebrain,andwhatrolesdotheneuronalandastrocyticcircadianclocksplayinthe manage-mentofenergyandwasteproductioninordertoorchestratebrainmetabolismandfunction. Metabolismappearstobeattheheartofmostphysiologicalprocessesintheperipheralorgans andmostlikelyalsointhebrain.InthisReview,wesurveytheimpactofmetabolismonthe circadianclock,andconversely,waysthroughwhichthecircadiansystemcancoordinatebrain

Highlights

The physiology and metabolism of

organismsareorganizedinatemporal

fashionusingcellularcircadianclocks

fortiming. These cellularclocks are

synchronized atthe tissueleveland

are in stablephase relationships to

other tissues and organs, thereby

establishinganorganism-wide

circa-diansystem.

Temporal organization ensures ef

fi-cientandhighlycoordinated

metabo-lism,whichisessentialforadaptability

androbustnessofphysiological

func-tions under changing environmental

conditions.Chronicdisturbanceofthis

coordination(e.g.,rotatingshiftwork,

andchronicjetlag)leadsto

interfer-encebetweenanabolicandcatabolic

processesandanincreaseof

meta-bolic and protein waste products

coupledwithinefficientclearance.As

aconsequence,metabolicand

neuro-logicaldiseasesmaydevelop.

Theglymphaticsystemhasemerged

asawasteclearancesystemforthe

central nervous system. Its detailed

function and temporal coordination

foroptimalwasteclearanceissubject

ofintenseresearch.

1

DepartmentofBiology,Universityof

Fribourg,Fribourg,Switzerland

*Correspondence:

urs.albrecht@unifr.ch(U.Albrecht).

http://doc.rero.ch

Published in "Trends in Neurosciences 41 (10): 677–688, 2018"

which should be cited to refer to this work.

metabolismandmetabolicastrocyte–neuroncommunications.Wealsodiscussrecent devel-opmentsinunderstandinghowwasteclearanceinthebrainisrelatedtosleepandthecircadian clock.Findingsonhowthecircadianclockmodulatesthemetabolic–neurophysiologicalaxis willhopefullyprovidenovelinsightsonhowsleep,mood,weightregulation,andneurological disorders can be targeted therapeutically, in order to prevent health problems rooted in dysregulationofbrainfunctions.

TheCircadianClockattheCellularLevel

Themolecularmechanismofthecircadianclockdrivescirca24-hrhythmsina cell-autono-mousfashion.Itismadeupofatranscription-basedautoregulatoryfeedbackloop.In mam-mals,thecoreclockgenesareBmal1andClock(anditshomologNpas2),whichactivatethe expressionofPer1,Per2,Cry1,andCry2.Thesefactorsinturnrepresstheirownactivation

(Figure 1). The nuclear receptors REV-ERBa or RORa regulate the expression of the

activating clock components Bmal1, Clock, and Npas2 by repressing or activating their transcription, respectively, thus completing the core clock mechanism (tilted grey oval in

Figure1)[3].Additionalnuclearreceptors,suchasPPARacanactivateBmal1transcription

andthisprocesscanbemodulatedbythePER2protein(Figure1).Thecoreclockmechanism canbemodulatedbymolecules(freefattyacids;FFAs)bindingtonuclearreceptors(e.g.,to PPARaand REV-ERBa).Additionally, heme and gases (NOand CO)can affectthe clock components PER, REV-ERBa, and NPAS2, which serve as sensors [4–6]. These small molecule-mediated effects can lead to phase shifts of the circadian oscillator similar to thelightmediatedphaseshiftsoftheclockthataremediatedbyglutamate[2].Additionally, metabolitessuchaslactateandFFAsaffecttheredoxstateofthecellandtheclockmechanism viaNAD+dependentSIRT1[7,8],modulatingtranscriptionalactivityviachromatinremodeling andPER2deacetylation[9,10].Thus,metabolicchangesthataffecttheredoxstatehavean influenceonclockfunction.

Thedescribedclockmechanismcanalsotargetso-calledclock-controlledgenes,whichcode forproteinsimportantforbiologicalfunctionsincludingenzymesinvolvedinmetabolism(blueoval

inFigure1).Hence,thereisamutualinteractionbetweenthecircadianclockandmetabolism.

Researchto identifythe genomic targetsofthecoreclock mechanismhasrevealedthat a substantialfractionofgenes(5–20%)inanycellortissueundergocircadianoscillationsatthe mRNAlevel[11],andunderdiurnalconditions;82%ofgenesarerhythmicinatleastonetissue

[12].Furthermore,circadianregulationofgeneexpressionisnotlimitedtotranscriptional pro-cesses,butincludeseveryregulatorystepingeneexpression,includingsplicing,transcriptional termination,polyadenylation,nuclear/cytoplasmictransport,miRNAregulation,translation, pro-teinphosphorylation,andRNA degradation[3,13].Hence, regulation of circadian gene expression includesbothtranscriptionalandpost-transcriptionalmechanisms,whichinturncanbe modu-latedbyvariousfactors(includingmetabolicones)interactingwiththeseprocesses.

SCNandLocalBrainClocks

Cellularclocksformnetworksandtherebybuildupcircadianprogramsintissues,organs,and subsequently,theentireorganism.Inthebrain,variousfunctionalnucleioperateeitheras self-sustaining clocks (SCN), as semiautonomous clocks (OB, DMH, ARC, HB), or as slave oscillators(BNST,AMY,POA,PVN,NAc)[14](Figure2A).

Theclocksofthenucleicontainingsemiautonomousorslaveclocksarecoordinatedbythe SCN. This coordination ensures optimal function, both physiologically and cognitively, in domainssuchas appetite,mood,memory,andsleep. In theabsence ofan efficientSCN

Glossary

b-Hydroxybutyrate(_bOHB):ketone

bodygeneratedfromFAsduring

fastingandextendedexercise.It

functionsasanenergysourceand

assignalingmoleculethatinduces

theexpressionofBDNFleadingto

mitochondrialbiogenesisand

increasedsynapticplasticity.

Circadianrhythm:rhythmwitha

periodofabout24hthatpersists

underconstantconditions(e.g.,

constantdarkness).

Clockcontrolledgenes:genes

thataretranscriptionallyregulatedby

molecularclockcomponentsbutare

notthemselvespartoftheclock

mechanism.

Diurnalordailyrhythm:rhythm

withaperiodof24hthatdoesnot

persistunderconstantconditions,

butratherrequiresadailysignal

initiatingthenextcycleoftherhythm.

Free-runningperiod:period(cycle

length)determinedunderconstant

conditions.

Intermittentmetabolicswitching

(IMS):repetitivecyclesofa

metabolicchallenge(fastingand/or

exercise)thatleadstodepletionof

liverglycogenstoresandelevates

circulatingketonebodies,followed

byarecoveryperiod(eating,resting

andsleeping).

Nuclearreceptors:classof

proteinsthatbindvarioustypesof

moleculesincludinghormonesand

metabolites.Theyactas

transcriptionalactivatorsor

repressors.

Paravascular(glymphatic)

system:wasteclearancepathwayin

thevertebratecentralnervous

systemthatdependsonglialcells

(astrocytes).CSFentersthebrain

parenchymaviapara-arterialinflux,

whichiscoupledtoamechanism

clearingwasteviaremovalof

interstitialfluidandextracellular

solutesfromthebrainandspinal

cord.Exchangeofsolutesisdriven

byarterialpulsationandtemporal

(circadian?)expansionand

contractionofbrainextracellular

space.Clearanceofwasteand

solubleproteinsisaccomplished

throughconvectivebulkflowof

fluids,facilitatedbyaquaporin-4

waterchannelsonastrocytes.

Phaseshift:displacementofa

rhythm(e.g.,circadianrhythm)to

earliertimes(advance)orlatertimes (delay).

Sleep:recurringstatecharacterized

byreducedorabsent

consciousness,relativelysuspended

sensoryactivity,andinactivityof

nearlyallvoluntarymuscles.Ithasa

circadiancomponentanda

homeostaticcomponent.

Suprachiasmaticnuclei(SCN):

pairednucleiintheventralpartofthe

hypothalamuslyingjustabovethe

opticchiasmandoneithersideof

thethirdventricle.Eachofthem

comprises10000neuronsinmice

andrats.TheSCNisthemaster

circadianpacemakerandcoordinator

ofcentralandperipheralcircadian

rhythms. NO CO Glu FFA AMP ATP PER-CRY PPAR-PER2 NRE RORE BMAL1-CLOCK/NPAS2 Per Cry Rev-erb_ Ror_ SIRT1 SIRT1 Bmal1 BMAL1-CLOCK/NPAS2 REV ROR Heme E-box E-box Lactate Ccg Kinases Kinases NAD+ NADH Output

Figure1.TheMolecularCircadianOscillatorinaCell.Thediagramdepictsasimplifiedmodeloftheautoregulatory

feedbackloopofthecellularcircadianoscillator(greyshadedtiltedoval).Positivefactorsaredepictedinblue,andnegative

onesinred–brown.Thebluehorizontalovaldepictstheregulationofclock-controlledgenes(Ccg;green),whichcodefora

varietyofproteins,includingtranscriptionfactorsandenzymesimportantfortheregulationofphysiologyandmetabolism

(outputoftheclock,greenstar).Variousmoleculesregulateclockproteinsdirectlyorindirectly(salmoncolored).These

moleculesmayentertheorganismfromtheoutsideand/orcanbeproductsofinternalmetabolismfeedingbacktothe

molecularclockmechanism.Abbreviations:BMAL1,brainandmuscleArnt-likeprotein-1;CLOCK,circadianlocomotor

outputcyclesproteinkaput;E-box:bindingsiteforBMAL1andCLOCK/NPAS2;FFA:freefattyacid;NPAS2,neuronal

PASdomainprotein2;NRE:nuclearreceptorelement;PER,periodcircadianregulator;PPAR,peroxisome

proliferator-activatedreceptor;REV,reversethyroidreceptora;ROR,RAR-relatedorphanreceptor;RORE:retinoicorphanreceptor

bindingelement;SIRT1,sirtuin1.

Glucose Glucose Glucose Glucose Gln Gln Glu Glu Glu Glu Glu Pyruvate Lactate Lactate Pyruvate ATP OxPhos FFA FFA FA-CoA `OHB `OHB Tr iacylgly cer ol Lipolysis Adipocyt e Hepa to cyt e Gut Signalling Blood vessel FA-CoA HMG-CoA Aceto-acetate `OHB `OHB Pyruvate Ac-CoA Ac-CoA Ac-CoA Glucose ATP TCA Ox Ph os Glycogen Glycogen depleƟon Exercise fasƟng Feeding (A) (B) Astrocyte Glutamatergic neuron BDNF + Glu Gln GABA GABA GABA GABAergic neuron Signalling TCA TCA ATP Glu PVN ARC DMH POA BNST HB AMY sPVZ OB IGL TMN NAc SCN Figure2.

(Figurelegendcontinuedonthebottomofthenextpage.)

OscillatorsandMetabolismintheMammalianBrain.(A)Theself-sustainingmasterclockintheSCNis

showninred,semiautonomousclocksinyellow,andslaveoscillatorsinblue.Thedifferentnucleiareimportantinthe

signal,regulationofthesubordinatebrainclockscanbecompromisedandaccordinglyaffect brainfunctions.Forexample,studiesinhamstershaveshownthatinductionofarrhythmiabya lightpulsecompromiseshippocampus-dependentmemory,butablationoftheSCNinthese animalsnormalizesthiseffect[15].ThisfindingindicatesthatanaberrantSCNsignalcanhave worseoutcomes–atleastinsomecases–thantheabsenceofsignalingfromtheSCN. Compromisinglocal clockfunctions, inparticular,nuclei candisruptbrainfunctionas well. DeletionofBmal1 inhistaminergicneuronsinthetuberomammillarynucleus(TMN)ofmice leadstoincreasedhistidinedecarboxylaseexpressionandhigherbrainhistaminelevelsduring the day [16]. Becausehistamine enhances wakefulness [17],TMN-Bmal1 knockout mice display more fragmented sleep, prolonged wake periods at night, shallower sleep depth, altered recoveryfrom sleep-deprivation,and impairedmemory [16].Hence, the localTMN clockregulatessleepviahistaminelevelsandmodulatestheSCN-definedsleep–wakecycle. Similarly,a recentstudy indicatesthat the ARCis moreimportant forthe circadian timing systemthanpreviouslythought.InterruptionofSCN–ARCcommunicationdoesnotaffectSCN rhythmicity,butcausesdesynchronizationoftheARC,whichresultsinarrhythmicityofactivity andphysiology[18].AstheARCisanimportantmetabolicintegrator,thisfindinghighlightsan intimateinteractionbetweentheneuralclocksystemandsystemicmetabolism.Itseemslikely thatdisruptionofthisSCN–ARCinteraction,forinstance,byill-timedeatinghabitsorshift-work conditions,mayplayacausalroleinhypothalamicdesynchronizationandpotentially subse-quentdisease.

Inthefuture,itwillbeimportanttountangletheinterdependentnetworkofSCNtiming,local clocktiming,andsleep/wakefulness.Theoverarching,generalunderstandingofthese inter-actions at the moment is that the brain is a temporally resonant system, with circadian oscillationsatitsbasis,anduponthisbasiseachbrainregionislockedtoaspecificphase, tosupportandcomplementotherregions.Accordingtothisintegrativeframework,threatsto any ofthe componentsof the system areexpected to disturbnormalbrain function.This predictionseemstoreceivesupportfromepidemiologicalstudies[1].

CooperationofNeuronsandAstrocytesintheSCN

Asmentionedabove,theSCNarethemajorsynchronizersofcircadianrhythmsinmammals. ThisconclusionwasreachedprincipallybyablatingtheSCNregionandsubsequent reimplan-tationstudies.Forexample,suchanablationstudyinhamstersdemonstratedthatthegrafted tissue–butnottheremaininghosttissue–determinedthefree-runningperiod[19].Hence,

regulationofvariousphysiologicalprocessesincludingfeeding,moodregulation,andsleep.Redarrowsindicatedirect

connectionsfromtheSCN.ThecircadianclocksoftheIGLandsPVZarecontactedbytheSCNbuttheirstatesarenotyet

characterized(grey).(B)Metabolicinteractionbetweenperipheralorgans(bottom,greyarea)andthebrain(top,yellow

area)viabloodvessels(middle,redarea).Feedingtriggersthepathwaysdepictedinblack,whereasexerciseandfasting

triggerthepathwaysdepictedinlightblue.BothpathwaysconvergeatthelevelofAc-CoAtokeepupATPproductionvia

theTCAcycle.Inastrocytes(green)thismaintainstheGln–GlucycleandGln–Glu-GABAcycleinordertokeepupneuronal

signaling(orangecyclicpathwaysinglutamatergicandGABAergicneurons,respectively).Inthisway,thefiringpotentialof

neurons(purpleglutamatergicandblueGABAergic)ismaintained.TheketonebodybOHBisusedinneuronsforATP

productionunderstarvationconditionstomaintainthefunctioningofionicpumpstokeepupiongradients.Also,bOHB

positivelyaffectsthelevelsofBDNF,whichinducesmitochondrialbiogenesisandsynapticplasticity.Abbreviations:

Ac-CoA,acetyl-coenzymeA;AMY,amygdala;ARC,arcuatenucleus;BDNF,brain-derivedneurotrophicfactor;BNST,bed

nucleusofthestriaterminalis;DMH,dorsomedialhypothalamus;FFA,freefattyacid;GABA,g-aminobutyricacid;HB,

habenula;HMG-CoA,3-hydroxy-3-methyl-glutaryl-coenzymeA;IGL,intergeniculateleaflet;NAc,nucleusaccumbens;

OB,olfactorybulb;bOHB,b-hydroxybutyrate;OxPhos,oxidativephosphorylation;POA,preopticarea;PVN,

paraven-tricularnucleus;SCN,suprachiasmaticnucleus;sPVZ,periventricularzone;TCA,tricaboxylicacid;TMN,

tuberomam-millarynucleus.Adapted,withpermission,from[31,53,54].

thesignalsfromtheSCNseemdominantovertherestofthebody.EvenafetalSCN-derived neuronalcelllinerescuedtheablatedSCNregioninrats[20].Nevertheless,thisrescuewasless efficientcomparedtointactordispersedfetalSCNtissue[21].Could,therefore,othercelltypes apartfromneuronscontributetocircadianrhythmgeneration?Immunohistochemicalstudies revealedthattheSCNregionisdenseringlialfibrillaryacidicprotein(GFAP)-like-positivecells compared to thesurrounding tissues[22].Thisobservation suggested(but didnot prove) involvementofGFAP-positiveastrocytesincircadianrhythmgeneration.Plausiblefunctionsof astrocytes would involve the metabolic coupling to neurons and the clearance of neuro-transmitters(seebelow).

Monitoringof[Ca2+]inastrocytesanding-aminobutyricacid(GABA)secretingneuronsofthe dorsal SCN revealedthat thesecelltypes had roughly opposite phasesof activity [23].In addition,glutamatewasreleasedbytheastrocytesandtakenuprhythmicallybytheneurons viatime-of-daydependentactivityoftheexcitatoryaminoacidtransporter(EAAT)3. Interfer-encewitheitherthecircadianactivityofastrocytesinthedorsalSCN,orthefluxofglutamate betweenthedifferentcelltypesinterferedwiththeproperfunctioningofthecircadiantiming system.Asapossibleinterpretation,itwasproposedthatduringthecircadiannight,glutamate isnottakenupbytheneuronsbutCa2+is,provokingahyperpolarizationoftheGABAergicSCN neurons [23]. In contrast, during the circadian day, the conditions reverse, leading to a depolarizationofthesameneuronsandconsequentlyincreasedneuronalfiringrates. What is the potential advantage of using astrocytes to synchronize neurons? In theory, inhibitionof neuronalactivity couldresult from the secretionof GABAby GABAergicSCN neurons (Figure 2B, right, orange pathway). However, this would be dependent on the individualneuronalactivityandconsequentlywouldprobablybetoounreliable.Hence, astro-cytesmaybenecessarytosynchronizemultipleneuronswithoutinterferingwiththeiroverall activityandconsequentlytofinetunethesystem.AstrocytesintheSCNhavebeendescribed asaconnectednetworkcommunicatingviaconnexin43gapjunctions[24].Thistightcoupling couldcoordinatetheactivity ofthe entireastrocyte network. Similarly,spikesofglutamate releasedbyastrocytesinthecortexhavebeensuggestedtosynchronizetheactivityofneurons and generate slow wave sleep (1Hz) oscillations [25]. Taken together, astrocytes and neuronsarerhythmicallyinterconnected,andtheircouplingaffectsthepolarizationstate of SCN neurons in order to optimize circadian rhythms in the dorsal part of the SCN [23]

(Figure2B,orangepathways).

Two additional studies analyzed the impact of arrhythmic astrocytes in the SCN on the circadiantimingsystemofmice[26,27].Bothstudiesfoundalengtheningofthefree-running perioduponspecificdeletionofBmal1inastrocytesoftheSCN.Hence,therhythmicityofthe astrocytesisnotaprerequisiteforthefunctioningofthecircadiantimingsystem,butrather necessary to adjust the period length. An additional function of astrocytes is to absorb neurotransmitterssuchasGABAreleasedfromtheneuronsintothesynapticcleft.Thisuptake wasimpairedinBmal1-deficientastrocytes,andhigherlevelsofGABAweremeasuredinthe SCNofastrocyte-specificknockoutmice[26].Finally,theapplicationofseveralGABAreceptor antagonistscouldreversetheperiodlengtheningofthefree-runninglocomotoractivityofthese mice[26].Hence,astrocytescanfine-tunetheperiodofSCN neuronsbyneurotransmitter reuptake.

Takentogether,theseresults,fromthreeindependentstudies,confirmthatastrocyteshave circadianrhythms thatare an integralpart ofthe circadiantiming system inthe SCN and thereforeaffectthesynchronizationofrhythmsacrosstheentireanimal.Assuch,therhythmic

activityofastrocytesintheSCNcouldhavealsoanimpactonthecircadiancomponentofthe sleep–wakecycle[23,28].

MetabolismintheBrain:ModulationbyDailyActivityandFeeding,and

DependenceonPeripheralOrgans

Itisgenerallyacceptedthatthecircadianclockenablesorganismstopredictdailyrecurring events. This view is supported, for example, by a recent observation, that clock-driven vasopressin(VP)neuronsmediateanticipatorythirstinmicejustbeforesleep.Notably,thirst wasnotmotivatedbyphysiologicalneed,butratherwasdependentontheSCNprojectionsto theorganumvasculosumlamina terminalis(OVLT),where VPactivates nonselectivecation channelsviaV1areceptorstoinducewaterintake.ThisSCNclockregulatingthirstanticipation isprobablyimportantinordertoprotectagainstovernightdehydration[29].

Incontrasttoanticipatorythirst,foodanticipationisindependentofafunctionalSCN[30].Food anticipationisobservedunderrestrictedfoodavailability(oftencoupledwithcaloricrestriction) when foodis available only ataspecific time.This wayof feedingcan also beviewed as intermittentfasting,whichisknowntohaveprofoundeffectsonneuroplasticityandbrainhealth (reviewedin[31]).Time-restrictedfeedingeverydayduringtheearlylightphasewithcaloric restriction (30% less than ad libitum consumption) is inducing intermittent metabolic switching(IMS).Thefastingand/orexerciseperioddepletesliverglycogenstoresandelevates circulatingketonebody levels.Thefollowingrecoveryperiod(eating,resting,andsleeping) restoresglycogenintheliverandreducesketonebodylevelsinthecirculation.Hence,thereisa switchbetweenglucoseandketonebodyusage.Ketonebodylevels,especiallyof b-hydrox-ybutyrate(bOHB),risebeforefeedingtimeinratsandmiceundertheseconditions(reviewed in[31]).

MicethatlacktheclockgenePer2intheliverdidnotdisplayariseinbOHBlevelsbeforefood availabilityandshowednofoodanticipatoryactivity.Thephenotype,however,wasrescuedby perfusingbOHBintothebloodpriortofoodavailability[32].Thisobservationindicatedthat bOHBis important for food anticipationand that IMSand the clockare linkeddirectly or indirectlyviathePer2gene. Thisfinding alsocorrelatedwiththeobservation thatthe rate-limiting enzymes for ketone body synthesis were altered in their expressionin Per2 liver knockoutmice[32].bOHBmayserveasadailysignalinordertoinducefood-seekingbehavior beforeglucoselevelscannolongerbemaintainedataconstantlevel.Hence,peripheraltissues suchastheliverinfluencethebrain,viametabolicsignaling,toinducespecific,time-regulated behaviors.

Thefuelsupplyofthebrainisentirelydependentonperipheraltissues,mostlytheliverand adipose tissues (Figure 2B). To sustain neuronal signaling, the glutamine–glutamate cycle betweenastrocytes andneurons (orangepathway inFigure2B) needs aconstant energy supply.Thishomeostasisisachievedthroughcouplingthisprocesstousageofglucoseand FAsviathetricarboxylicacid(TCA)cycle(blackandbluepathwaysinFigure2B).Underfeeding conditions,glucoseisused(blackpathwaysinFigure2B),ensuringATPproduction.Under fastingand/orexerciseconditions(bluepathwaysinFigure2B),lipolysisleadsto releaseof FFAsfromadiposetissueintothecirculation,fromwheretheyaretakenupbyastrocytesand usedforATPgenerationandbOHBproductiontoruntheglutamine–glutamatecycle,and/or provideenergy to neurons.Hepatocytes (the main cells of the liver, andwhich have high metabolicactivity)takeupFFAsaswellandsupplyneuronswithenergyinformofbOHB(blue pathwaysinFigure2B).Ofnote,bOHBappearstohaveapositiveinfluenceonbrain-derived neurotrophic factor (BDNF) production, correlating with the positive effects of IMS on

neuroplasticity(reviewedin[31]).Hence,dailycyclesinthelevelsofmetabolitesintheblood affectneuronalmetabolismandfunction.

TimedClearanceofMetabolicWaste fromtheBrain

Thefunctionality of the braindepends on the delivery of nutrients, salts, and vitamins by peripheral organs. Nutrients are transported via the vascular system that branches into capillariestopenetratethebrain.Theblooditself,however,isnotindirectcontactwithbrain cells.Itisseparatedfromthebrainparenchymabytwodistinctbarriers:theblood–brainbarrier (BBB)andtheblood–cerebrospinalfluidbarrier(B-CSF).Thesebarriersarecriticalinorderto maintaintheionicandbiochemicalcompositionofthefourfluidcompartmentsofthebrain: intracellularfluid(ICF)(60–68%),interstitialfluid(ISF)(extracellularfluid,12–20%),blood(10%), andCSF(10%)(Figure3A).Toreachthebrainandnourishbraincellssuchasneuronsand astrocytes,nutrientshavetopasstheBBBandtheB-CSF.Thistransportisaccomplishedvia activetransportsystemsinvolvingvariousreceptorsandtransporters.

Likewise, clearance of metabolic waste from the brain is essential in order to keep the biochemicalmilieuofthe braincapableofcontrollingimportantphysiological actions.Daily fluctuationsintherelativelevelsofmetabolites,leptin,andcytokinesbetweenthebloodandthe CSFhavebeenobserved[33,34],suggestingthatthereisadailyrhythminthepermeabilityof theB-CSF barrier.Thisclaim issupportedby the observationthat the choroidplexus,an importantcomponentoftheB-CSFbarrier,containscircadianclockactivity[35],potentially regulatingtimedfluidexchangebetweenthebloodandCSF.Arecentstudyusingthefruitflyas amodelsystemrevealedthatacircadianclockinglialcells ofthehemolymph–brainbarrier regulatedxenobioticefflux[36].Therefore,itistemptingtospeculatethatinmammals,similarto fruit flies, glial cells including astrocytes may regulate BBB function, thereby modulating metabolicwasteclearance(Figure3B).

Peripheral organsclear their waste via the lymphatic system. Thebrain is a highly active metabolictissueanddoesnothavedirectaccesstothelymphaticsystem.Giventhat,one wonders how the brain removes its waste products. First indications for possible waste-clearance routes have come from experiments in which large proteins were injected into the CSF.These proteins were identifiedaftersome time inthe perivascular space, which surroundsthecerebralbloodvessels[37,38].Thisfindingsuggeststhatfluidcirculationthrough the central nervoussystem (CNS)may occurvia paravascularpathways, surrounding the vasculatureinthebrain.Morerecentstudieshavecharacterizedspecificsystemsthatremove wasteproductssuchasglucose,lactate,andb-amyloidfromthebrain[39,40].Itappearedthat specializedastrocyteshaveacentralfunctioninthiscontext,servingasfiltersthatcontrolthe fluxofcompounds smallerthan100kDaviatheiraquaporin(AQP)-4channels(reviewedin

[41,42])(Figure3B).Thevascularendothelialcellsborderingtheparavascularspacedonot

haveAQP4channelsand,hence,fluidfromthebloodstreamcannotmovedirectlyintothe brain. Therefore, liquid hasto flow through the paravascularspace and then throughthe astrocytestogainaccesstothebraintissue(Figure3B).

Instudiesaimedtoexamineproductclearanceviaparavascularpathways,pumpingofblood throughthearteriesappearedtopropellargequantitiesofCSFintotheparavascularspace. TheCSFthenmovedviaastrocytesthroughthebraintissue,pickingupdiscardedproteins suchasb-amyloid[43,44].Thevolumeoftheparavascularsystem(alsotermedglymphatic system),andwithittheflowrateoftheCSFappearedtovaryaccordingtotimeofday.These dailyvariationswerecorrelatedwiththesleepstate.Duringsleep,thevolumeoftheinterstitial space increased substantially (60%, according to the study) with concomitantly faster

(B) (A)

Aquaporin 4 Waste Nutrients

Arterial blood Ňow Venous blood Ňow

Cellular clocks Paravascular Ňow

Perivascular Ňow Para-arterial space Paravenous space Perivascular space ? Blood capillary Fec ISF TJ CPE Blood–CSF barrier Blood–brain barrier CSF

Key:

(Figurelegendcontinuedonthebottomofthenextpage.)

SchematicoftheParavascular(Glymphatic)WasteClearanceSystemwithPotentialCircadian Clocks.(A)Representationofthefluidcompartmentsandbarriersofthebrain.(B)Wasteclearanceroutethroughthe

clearanceofwasteproductssuchasb-amyloid[45].Thesechangesappearedtobecontrolled bynoradrenergicsignaling.Itisnotknown,however,whetherandhowsuchsignalingaffects astrocytesortheirAQP4channelstoallow aninfluxofwaterintotheinterstitialspace,and whetherthisprocessmightbeundercontrolofthecircadianclock.

DiurnalVariationsofLiquidFlowintheBrain

Fluidsenteringthebrainviathepara-arterialspace,flushingthroughthebrainandcollecting waste,leavethe braintissueviatheparavenousspace(Figure3B)[41,42]. Eventually,the combined paravascularwaste liquidsreach the neck region, where they enter the lymph system to flow back into the general blood circulation (reviewed in [41]). Alternatively, a perivascularpathway,locatedwithinthearterialtunicamedia(Figure3B),hasbeendescribed withasolutefluxalongarteriesindirectionoppositetothatoftheblood[46,47].Whetherthis pathwaycouldclearwasteproductsefficientlyisunderdebate(reviewedin[41]).Onepossibility isthat the paravascularand the perivascular systemsare two independent but somehow intermingledtransportsystems.Itisalsopossiblethatthereisarhythmiccomponentinvolved intheselectionofthedirectionofwastedisposal.Theflowratesanddirectionofthecilia-based flow of CSF in the third ventricledisplayed a diurnal change [48], suggesting time-of-day dependenceuponCSFflow.Recently,thecochleahasbeenshowntobeundercircadian controlaffectingitssensitivitytonoisetrauma.Sincetheinnerearfluidsofthecochleaarein continuitywiththeCSF,therecouldbesimilarmechanismscontrollingcochlearfluids[49,50]. However,furtherresearch isnecessaryto understandtheregulationofflowdirections and henceclearanceofCSFandwastefromthebrain.

As mentioned, the glymphatic system seems to be regulated by sleep state, raising the interestinghypothesisthatamajorfunctionofsleepwouldbetoclearoutmetabolicwaste productsfromthebrainthroughthissystem[38,45].Similar totheprocessesfoundatthe circuitlevelintheSCNandneocortex,itistemptingtospeculatethatastrocytes–ratherthan neurons–arethemainsensorsandcoordinatorsofwaste disposal.Bythesametoken,a changeintheconcentrationofwasteproductsintheISFcouldrepresentasignalindicating sleepneed.Basedonseveralobservationsshowingthatmetabolismandsleepmayberelated, itispostulatedthatrestorationofbrainenergymetabolismmaybeoneofthefunctionsofsleep

[51].Clearanceoflactatebytheglymphaticsystemcorrelateswellwiththesleepphase[40], andthisclearancecouldbegovernedbylocalastrocytesaccordingtotheircircadianstate.It willbeinterestingtochallengethisconceptinthefuture.

ConcludingRemarksandFuturePerspectives

Themammalian circadian system isoften seen as a networkof circadian clocksthat are governedbytheSCN.Recentdevelopments,however,indicatethatperipheralclocksstrongly influencethecircadiansystem, mainlythroughmetabolism,whichisinamutualregulatory feedbackloopwiththecellularcircadianclocks(Figure1).Sincethebrainisentirelydependent onenergysuppliedbyperipheralorgans,properfunctioningofthebraindependsonthese organsandthedailyenergysuppliedbythem.Hence,effectivecommunicationmechanisms betweenthebrainandtheperiphery,forinstance,viahormonalandsmall-moleculesignals

blood–brainbarrierwithaparavascularflow(red–blueshadedarrow)fromarteries(red)toveins(purple).Astrocytes(green)

takeupfluidfromthepara-arterialspaceviaaquaporin4channelsanddistributeittoneurons(orange)andtheintercellular

space.Wasteloadedfluidisthenclearedbyastrocytesviaaquaporin4intotheparavenousspaceandtransportedoutof

thebrain.Aperivascularflow(yellowarrows)isalsohypothesized.Abbreviations:CPE,choroidplexusepithelialcell;CSF,

cerebrospinalfluid;Fec,fenestratedendothelialcell;ISF,interstitialfluid;TJ,tightjunction.Adapted,withpermission,from

[41,55].

(e.g.,metabolites),toensureasteadyflowequilibriumofnutrientsandofwasteclearance,are crucialforoptimalbrainfunctionandultimatelyforsurvival.Thebrainhasaprivilegedpositionof regulatinguptakeofmoleculesfromthebloodstreamthroughspecificmechanismsintheBBB andtheB-CSF.Recentevidencehasemerged[35,36]showingthatcircadianclocksexistat thesebarriersbetweentheperipheryandthebrain.Theseclocksmaygateinatemporally controlled manner bywhich molecules can enter and/or exit the brain, and possibly also synchronize the entry/exit of subsets of these molecules. This regulation appears to be essentialfor normalbrainfunction.Overloading thebrainwithnutrients whenthey are not needed, for instance, could lead to excess waste production and accumulation of toxic substances, suchas reactiveoxygen species.These can modifyproteinsandmay impair brainfunctionandevendestroyneurons.Theimpactofsuchtoxicaccumulationmaybefirst noticedasmemorylossandsleepdisturbance.Inthisframework,sleepcanbeseen(atleastin part)asaconsequenceofmetabolicregulation,aimedtoprotectcellsinthebrain[52]. Overall, brain fluid management is emerging as a key factor in brain function [41]. Our understandingofthesystemsmediatingliquidfluxthroughthebrain,forinstance,the para-vascular(glymphatic)system, isstillevolving, andfuture studieswill help clarifythe driving forcesbehindbrainfluidinfluxandoutflow.Thecircadianclocklikelyplaysanimportantrolein this context, which relates to the more general purpose of the clock of managing and coordinatingdailycyclesinmost,ifnotallphysiologicalandbiochemicalprocessesacross theorganism.Anothergoalforfutureexperimentsistofurtherelucidatetransportmechanisms actingattheBBBandB-CSF,includingwhetherandhowtheseareregulatedbythecircadian clock(seeOutstandingQuestions).

Altogether,thecircadianclockregulatesavarietyofcrucialprocessesinthebrain,including sleep,brainmetabolism,andmaintenanceofflowbalanceofcompoundsintoandoutofthe brain.Disruptionofthecircadianclock,and/oranyofthesignalingpathwaysregulatedbyit, canleadtoinefficientanabolicandcatabolicbiochemicalprocesses.Thesemetabolic impair-mentsandotherclock-relateddysfunctionsareincreasinglyrecognizedaskeyplayersinthe etiology of sleep disturbances, mood alternations, and a variety of neuropsychiatric and neurologicaldiseases.

Acknowledgments

WethankCressidaHarveyforcriticallyreadingthemanuscript.TheworkwassupportedbytheSwissNationalScience

Foundation(31003A_166682),theVeluxFoundation(995),andtheUniversityofFribourg.

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