Musculoskeletal senescence: a moving target ready to be eliminated

Musculoskeletal

senescence:

a

moving

target

ready

to

be

eliminated

Marjolein

P

Baar

,

Eusebio

Perdiguero

,

Pura

Mun˜oz-Ca´noves

and

Peter

LJ

de

Keizer

Agingistheprimeriskfactorforthebroad-baseddevelopmentof diseases.Frailtyisaphenotypicalhallmarkofagingandisoften usedtoassesswhetherthepredictedbenefitsofatherapy outweightherisksforolderpatients.Senescentcellsformasa consequenceofunresolvedmoleculardamageandpersistently secretemoleculesthatcanimpairtissuefunction.Recent evidenceshowssenescentcellscanchronicallyinterferewith stemcellfunctionanddriveagingofthemusculoskeletalsystem. Inaddition,targetedapoptosisofsenescentcellscanrestore tissuehomeostasisinagedanimals.Thus,targetingcellular senescenceprovidesnewtherapeuticopportunitiesforthe interventionoffrailty-associatedpathologiesandcouldhave pleiotropichealthbenefits.

Addresses

1_{DepartmentofMolecularCancerResearch,CenterforMolecular} Medicine,DivisionofBiomedicalGenetics,UniversityMedicalCenter Utrecht,UtrechtUniversity,Universiteitsweg100,3584CGUtrecht,The Netherlands

2_{DepartmentofMolecularGenetics,ErasmusUniversityMedicalCenter,} Rotterdam,TheNetherlands

3

DepartmentofExperimentalandHealthSciences,PompeuFabra University(UPF),CIBERNED,Barcelona,Spain

4_{ICREAandSpanishNationalCenteronCardiovascularResearch} (CNIC),Madrid,Spain

Correspondingauthor:deKeizer,PeterLJ(p.l.j.dekeizer@umcutrecht.nl)

CurrentOpinioninPharmacology2018,40:147–155 ThisreviewcomesfromathemedissueonMusculoskeletal EditedbySJeffreyDixonandPeterChidiac

ForacompleteoverviewseetheIssueandtheEditorial

Availableonline5thJune2018

https://doi.org/10.1016/j.coph.2018.05.007

1471-4892/ã2018TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons. org/licenses/by/4.0/).

Loss

of

cell-intrinsic

and

cell-extrinsic

integrity

perturbs

musculoskeletal

rejuvenation

during

aging

Aged individualscandeteriorateexceptionallyfast after

theonsetofcomplicationsaffectingthemusculoskeletal

system. Tissueerosion dueto life-long mechanical and

biologicalstresscanultimatelyresultinpathologiessuch

as osteoporosis, sarcopenia, and osteoarthritis, and

con-tributetofrailty[1].Whilenotallelderlypeopledevelop

the same age-related diseases, virtually everyone will

experiencemusculoskeletalcomplicationssoonerorlater.

Toextend,andpossiblyevenrestore,healthylife

expec-tancyinoldage,itisessentialtounderstandthecellular

changes underlying musculoskeletal decline. Tissue

regeneration by stem-cell differentiation is critical in

overcoming the relentless day-by-day damage to the

musculoskeletalsystem.Inyoungtissues,differentiation

proceeds without muchhindrance unless one exercises

excessively or suffers undue levels of stress. However,

duringaging,thenumberandfunctionofadultstemcells

declines [2,3]. For example, Pax7-expressing satellite

stem cells, can replace damaged muscle fibers [4].

RemovingPax7-positivecellsfrommiceimpairsmuscle

regenerationafterinjury[5],whereasincreased

availabil-ityofthesecells enhancesmusclerepair[6].

Inaddition to cell-intrinsicregulation,musclestemcell

regenerative capacity also depends intimately on the

microenvironment.Duringaging,thelevelsof

inflamma-tion chronically increase, an affect knownas

inflamma-ging[7].Evidenceforthisisprovidedbystudiesshowing

that muscle stem cells (satellite cells) from aged mice

becomemorefibrogenic,aconversionmediatedbyfactors

fromtheagedsystemicenvironment[8].Incontrast,frailty

isreducedbytheJAK/STATinhibitorRuxolitinib,which

reducesinflammationinnaturallyagedmice[9].Stem-cell

impairingcuesdonotnecessarilyhavetocomefromlocal

sourcesbutcantraveloveradistance.Heterochronic

para-biosis experiments showedthattransfusionof oldblood

impairs stem cellfunctionin youngrecipientmice[10],

whilethetransferofyoungbloodfactorsrestoringmuscle

regenerationandmusclestem-cellactivationinaged

ani-mals[11].Therefore,thereisagreatinterestindeveloping

methods tointerfere withtheage-associated

pro-inflam-matorysignalingprofile.Thequestionishow?Toaddress

this question, cellular senescence has recently gained

attentionasapotentialcandidateforintervention.

Signaling

noise

by

senescent

cells

impedes

tissue

homeostasis

during

aging

As we age, each cellin our body accumulates damage.

Earlier in life,thisdamageis usuallyfaithfullyrepaired

[12], but over time more and more damage gets left

behind. This can trigger a molecular chain of events,

be invoked in healthy cells that experience a chronic

damageresponse,eitherinvolvingdirectDNAdamageor

eventsthatmimic themolecular response,suchas

telo-mereshorteningoroncogenicmutations[13].Asa

con-sequence, these cells undergo an irreversiblecell cycle

arrest,effectively limiting thedamage. Sofar, so good,

except that senescent cells secrete a broad range of

growth factors, pro-inflammatory proteins, and matrix

proteinasesthatalterthemicroenvironment:The

Senes-cence-AssociatedSecretoryPhenotype(SASP)[14].

Senescentcellspersistforprolongedperiodsoftimeand

eventuallyaccumulateduringaging[15].Thisalsomeans

thereisagradualand,importantly,ever-presentbuild-up

of deleterious molecules. Thus, senescence can have

continuous detrimental effects on tissue homeostasis

during aging.Thatsenescent cells areadirectcause of

aging was proven beyond a doubt in studies in which

senescent cells were genetically or pharmacologically

removed. In these studies, both rapidly and naturally

aged mice maintained healthspan for much longer, or

evenshowedsignsofagingreversal[16,17,18,19].

Fac-torssecretedbysenescentcellscaninducepluripotency

invivo[20].Assuch,thesecanimpairnormalstemcell

function by forcing a constant state of reprogramming,

somethingwedubbeda‘senescence—stemlock’[13].

Thisissupportedbyobservationsthatfactorssecretedby

senescent cells induce pluripotencyin vivo [20].

Age-associated inflammation may thus deregulate normal

stem cell function at different levels, for instance by

preventing stem cells from producing differentiated

daughter cells. Due to the constant secretion of SASP

factors,senescentcellscouldthusimpairlocalanddistant

stemcellfunction anddifferentiation intimes of need.

Here, we will highlight the interplay between

senes-cence,theSASPandstemnessintheindividual

muscu-loskeletalcompartments:muscle,boneandcartilage.

Skeletal

muscle:

an

intrinsic

interplay

between

senescence

and

stemness

Several reports link senescence to muscle aging and

muscle stemcell dysfunction. For example, expression

ofthemajorsenescencemarkerp16INK4Apreventstissue

regenerationbysatellitecellsafterdamage [21].

Fast-aging BubR1H/H mice develop sarcopenia, and after

geneticremovalofsenescentcells,theyshoweda

reduc-tioninkyphosisandanincreaseinmusclefiberdiameter,

findingssuggestiveofreducedsarcopenia[16].Likewise,

senescenceofmusclestemcellsoccursinmusclesofmice

withdistinctdystrophinopathies,suchasDuchenne

mus-culardystrophyorSteinert’sdiseases[22–25].

Theskeletalmusclestemcellnicheisacandidatethrough

whichsenescentcellsmayexerttheirdeleteriouseffects.

Interleukin6(IL-6)isapleiotropiccytokinethatcanbe

[14], and has been shown to regulate the transition of

satellitecellsfromaquiescenttoanactivatedstate [26].

Thisisbeneficialuponacutetissuestress,whereIL-6is

transientlyreleasedbygrowingmyofiberstoactivate

satel-litecellsandtherebystimulatemyogenesis[26].However,

the chronicIL-6 signalingcaused bysenescenceduring

aging would have very detrimental effects on muscle

function.Indeed, muscle atrophyislinked to highIL-6

levelsinpatientswithinflammatorydiseasessuchascancer

[27].Inaddition,persistentIL-6expressionwasshownto

increase muscle degradation in combination with other

circulating factors in mice [28,29]. Interestingly, when

IL-6receptors were blocked in mice with ectopic IL-6

expression,atrophycouldbeattenuated,indicatingadirect

regulationof musclewastingbyIL-6[30].ChronicIL-6

signalingcausesproteindegradationinmuscle,explaining

age-relatedmusclewasting[31].Additionally,IL-6

depen-dentmuscledegradationmaybelinkedtostemcell

func-tion. For example, senescence induction after muscle

injurycan promotePax7positiveunipotentcells toundergo

reprogrammingandregainpluripotency[32].This

pro-cessisdependentonIL-6secretedbythesenescentcells.

Furtherunderscoringtherolebetweenthesenescentniche

andstemnessinthemuscleisprovidedbyelegantwork

employingasysteminwhichthefourYamanakastemcell

factors,Oct4,Sox2, Klf4and c-Myc(OSKM)were

tran-sientlyexpressedinvivo.Thisresultedinamarked

reduc-tion in senescence, SASP factors as IL6 and improved

recoveryin muscle injury experiments [33]. Together,

this supports a model we postulated previously that

becausesenescenceincreaseslocallyduringaginghotspots

are formed of high IL6 concentrations. This cancause

neighboringcells to become pluripotent. However, due

tothechronicnatureoftheSASP,senescentcellsprovidea

continuoussourceofIL6causingthesecellsremain

per-manentlylockedinapluripotentstateandrenderingthem

unabletorejuvenatethetissueafterinjury[13](Figure1).

Althoughsatellitedysfunctionhasbeenlinkedto

sarco-penia, this relationship is controversial. Recent studies

suggestthatthedeclineinsatellitecellfunction during

agingisnotthecauseofsarcopenia[34,35].Whensatellite

cellsweregeneticallyremovedoveraprolongedperiod,

no difference in muscle mass was observed compared

withmicethatmaintainedtheirsatellitecells.However,

there was a clear increase in fibrosis, indicating that

satellitecellsareindeedcrucialformusclehomeostasis.

Furthermore,severalstudiesshowthatsarcopenicmuscle

has a reduced ability to recover after injury, which is

dependentonsatellitecellfunction[5,21,35,36].

Over-all,while the role of satellite cells in sarcopenia is still

debated,there is consensusthat Pax7positive cells are

required for regeneration after muscle injury and that

reducedfunctionofthesestemcellsleadstoage-related

Themyokinesreleasedbymusclecellsnotonlysignalto

stemcells,butalsoattractimmunecellsthatcanfacilitate

tissuerepairandregulateimmunecellfunction.IL-15is

released by muscle cells in response to exercise and

promotes survival of NK cells [37,38]; in contrast, NK

cellsareinhibitedbyIL-6andTNFa[39].Anage-related

decrease in muscle mass could therefore lead to a

decreaseinIL-15andtherebyadecreaseinthenumber

of NK cells, an effect aggravated by an increase in

systemic IL-6 levels (Reviewed in [40]). Importantly,

NK cells arenatural eliminatorsof senescentcells [41].

Muscleatrophyduringagingthusaddstothebuild-upof

senescencebyreducingtheabilityoftheimmunesystem

toclearsenescentcells.This,inturn,furtheraccelerates

musclelossandage-relatedfrailty.Studiesareunderway

to determine whether anti-senescence treatment can

overcome muscle loss. Aging is the greatest risk factor

formostchronicdiseases,andmechanisticlinksbetween

aginganddiseasearestartingtoemerge.Severalstudies

show an involvement of cellular senescence, and in

particular,musclestemcellsenescence,indistincttypes

of muscular dystrophies.In Myotonic dystrophytype 1

(DM1 or Steinert’s disease), entry into senescence of

human satellitecell-derivedmyoblastscorrelateswith a

lowerproliferativeratethanage-matchedcontrolsandhas

been causallyimplicatedin theprogressiveatrophyand

degenerationofDM1muscles[22,23].Similarly,cellular

senescence traits have been described in mdx mice, a

widely used model of Duchenne muscular dystrophy

(DMD), correlating with poor regenerative capacity

[24,25,42].Prematurecellularsenescencealso underlies

myopathy in a mouse model of limb-girdle muscular

dystrophy [43]. Whether interference in cellular

senes-cencecanprovideatherapeuticapproachforthese

mus-cle diseasesisunknown.

Bone:

senescence

distorts

the

balance

between

resorption

and

formation

During aging,there isan increase in senescence in the

bone.This,inturn,canleadtochangesinbonedensity.

Boneconsistsofmultiplecelltypes,includingosteoblasts

thatformbone,osteoclaststhatbreakdownbonetissue,

and osteocytesthat makeup themajorityof bonecells

(reviewedin[44]).Outof thevariouscelltypesthatare

affected, the main SASPproducing cells are senescent

osteocytes [45]. Osteocytes are known to influence

Figure1 Vascular compartment Muscle Fiber Sarcopenic/Atrophic Fiber Systemic factors Resident inflammatory cell Senescent cells Interstitial cell NK cell Satellite cell Satellite cell (p16INK4a₎

AGED/DYSTROPHIC

YOUNG

Current Opinion in Pharmacology

Agedmusclefibersshowatrophythatislinkedtoanage-relatedincreaseincellularsenescence.Satellitecellsloseproliferationcapacitythrough senescenceinductionorthechronicpresenceofSASPfactorssuchasIL-6.Thus,regenerationofdamagedtissueisprevented.Additionally, IL-15secretedbymuscletissuefacilitatesNKcellsurvivalinyoungorganisms,whileIL-6repressestheseimmunecellsduringagingandthereby reducesthenaturalablationofsenescentcells,aggravatinglossofmusclemassobservedduringaging.

osteoblastandosteoclastfunction[46],andSASPfactors

secreted by osteocytes, such as IL-1 and MMP13,

increase osteoclast differentiation and thereby increase

boneresorptiontocausetheage-relatedboneloss

associ-atedwithosteoporosis[47–49].Theconditionedmedium

ofsenescentcellscandecreaseosteoblastfunctioninvitro

andpromote osteoclastactivity[50].Furthermore,

inhi-bition of senescence induction stimulates osteogenesis

andpreventsosteoporosis[51].Theseobservations

indi-cateacausalroleofsenescenceindisruptingthebalance

betweenboneformationandresorption,leadingto

oste-oporosis(Figure 2).

Bonestemcellfunctionduringagingislikelyinfluenced

bysecreted SASP factors. Osteoblasts have a relatively

short lifespan and are derived frommesenchymal stem

cells in the bone marrow (BMSCs), periosteum and

elsewhere[52].BMSCscangiveriseto bothosteoblasts

andadipocytes[53].Thisbalanceisheavilyinfluencedby

themicroenvironment[54],andduringosteoporosis

oxi-dative stress and inflammatory cytokines influence

BMSCsto favoradipogenesisoverosteogenesis[55,56].

Thereforeadipose tissue accumulation isa hallmark of

osteoporosisandislinkedtosenescenceinthe

microen-vironment.Furthermore,BMSCsshowareduced

differ-entiationcapacityduringaging.Forexample,serumfrom

aged individualsinhibits differentiation of BMSCsinto

osteoblasts [57]. Additionally, BMSCs can become

observations indicate that targeting senescent cells in

bonewouldlikelyimprovebone stemcellfunction.

Thereare several mouse modelsthat show accelerated

aging and are known to have an increased number of

senescentcells,suchasmicewithDNArepairor

telome-rasedeficiency; such miceoften show osteoporosis and

othermusculoskeletalafflictions[60,61].Theyare

there-fore ideal model organisms for studying the effect of

senescenceinthesedisorders.Forexample,

Klotho-defi-cient mice show accelerated senescence and a wide

variety of age-related diseases, including osteoporosis.

When these mice were crossed with p16ink4a knockout

mice, osteoporosis was attenuated [61], indicating that

senescentcellablationcanpotentiallypreventthis

dete-rioration. Indeed, osteoporosis was delayed in naturally

aged INK-ATTAC mice when senescent cells, which

continuouslydevelop,wereablatedtwiceaweek.

More-over,thesemicehadanimprovedmicroarchitectureand

strength [62]. The reductionof senescent cells likely

leadstoalowerlevelofinflammationin thebone.This

then reduces theformation of osteoclasts and prevents

bone degradation.Indeed, in INK-ATTAC mice, bone

resorptionwasloweredandboneformationimproved.In

conclusion,senescent cellremoval preventsage-related

bonelossin mice.

Cartilage:

senescence-associated

chronic

inflammation

perturbs

cartilage

regeneration

Articular cartilage — a flexible connective tissue that

protectstheendsofboneswithinajoint—affordssmooth

surfaceswith low frictionfor movement,and facilitates

transmissionofloadstotheunderlyingbone.Thistissue

mainlyconsistsofextracellularmatrixproducedby

chon-drocytes,thecelltypepresentincartilage.The

regenera-tivepotentialofcartilageafterdamageislimited,possibly

becausethetissuecontains alow numberof

mesenchy-malstemcells[63].Furthermore,likemusclestemcells,

these stem cells are less able to regenerate damaged

tissue with age. This is in part due to intrinsic MSC

agingandsenescenceinduction[64,65],butisalsodueto

thealteredtissuemicroenvironmentandchronic

inflam-mation[66].Additionally,chondrocytescanexpress

stem-ness markersin osteoarthritis [67,68]. Again,

inflamma-toryfactorspromoteachronicdedifferentiatedstateand

thereby prevent tissue repair during aging [69].

Alto-gether, this leads to thinning of cartilage during aging,

resulting in stiffness and pain in the joints that are

characteristicof osteoarthritis[70](Figure3).

Acausalroleofsenescenceinosteoarthritiswasshownby

transplantingsenescentcellsintomousejoints,resulting

inpainandmorphologicalchangesindicativeof

osteoar-thritis[71].

SASP

Current Opinion in Pharmacology

Inagedbone,thebalancebetweenboneformationbyosteoblasts andboneresorptionbyosteoclastsisdistorted.Anaccumulationof senescentcellsisobservedthatpromoteanincreasedosteoclast activationthroughtheSASP.Bonelossisalsoworsenedbythe inhibitionofosteoblastformationbypro-inflammatoryfactors.For example,knownSASPfactorscausemesenchymalstemcellstofavor adipogenesisoverosteoblastproduction.

Furthermore,chondrocytesshowanage-relatedincrease

insenescence,andduringosteoarthritispro-inflammatory

cytokinessuchastheprominentSASPfactorIL-1induce

excessexpressionofmatrixmetalloproteinases(MMPs),

leadingtocartilageloss[72].Increasedlevelsof

circulat-ingSASPfactorssuchasIL-6arelinkedtofrailtyandrisk

of osteoarthritis [73].Additionally,in amouse modelof

osteoarthritis, overexpression of SIRT6 prevents

senes-cence induction and concurrent inflammation, thereby

reducing cartilage degeneration [74]. Thisfinding

indi-cates that eliminating senescent cells from cartilage

wouldattenuateosteoarthritisandimprovejointfunction,

especiallysincechondrocytedeathdoesnotseemtodrive

cartilagedamageinresponsetoinjury[75].Several

stud-ieshaveexaminedtheeffectofsenescentcellremovalon

osteoarthritis development. For example, osteoarthritis

wassurgicallyinducedin micethroughanteriorcruciate

ligament transection (ACLT) in the kneejoint. In this

model, genetic removal of senescent cells delayed the

development of osteoarthritis, evidenced by reduced

inflammationinthekneejointandanincreaseincartilage

development,indicatingbetterjointfunction[76].The

micehadlesspainafterthesenescentcellswereremoved.

Furthermore,osteoarthritisoccursnaturallyinaged

INK-ATTACmice,andcartilagedegenerationwasattenuated

after removalof senescentcellsin thismodel.

Targeting

senescence

to

counteract

age-related

frailty

Theencouragingresultsobtainedupongenetic

elimina-tionofsenescentcellshaveimportantimplicationsforthe

treatmentofmusculoskeletaldeterioration.Since

senes-cenceisthoughttoplayasignificantroleinthe

progres-sionof age-relatedfrailty, anti-senescence drugscanbe

predicted to benefit patients with musculoskeletal

dis-orders(Table 1).

Currently, drugs that targetinflammatory cytokinesare

tested in patients with musculoskeletal diseases. For

example,severalstrategiesforIL1inhibitionin

osteoar-thritishavebeenexplored.ThesetherapiesincludeIL1

receptorantagonistproteins(IRAP), monoclonal

antibo-diestargetingfreeIL1ortheIL1-receptor,andan

inhib-itor of IL1b production called Diacerein (reviewed in

[77]). Most of these therapies show a trend of pain

reduction versus placebo. However, these results were

oftennotstatisticallysignificant,possiblyduetotheshort

half-lifeoftheantagonistproteinsorblockingantibodies.

Only Diacerein treatment has shown significant

anti-inflammatoryeffects andpainreductioninmoststudies

[77].TreatmentofmdxdystrophicmicewiththeNAD+

precursor nicotinamide riboside (NR) prevented

senes-cence of muscle stem cells, and this rejuvenated their

regenerativecapacity[24].TheNotchpathwayis

chroni-cally activated in severely dystrophic muscles of mdx

mice double mutant for dystrophin and utrophin, and

blocking this pathway with the g-Secretase inhibitor

DAPTreducedstemcellsenescenceandthe

histopath-ological featuresofDMD[42].Importantly, abolitionof

p16INK4a,whichaccumulatesabnormallyinsatellitecells

ofDM1muscles,partiallyrestoresearlygrowtharrestand

reducessenescenceinvitro[22],reinforcingtheideathat

Figure3

IL-6 MMPs

IL1

Youn

g

Old

Current Opinion in Pharmacology

Age-relatedcartilagedegenerationleadstoosteoarthritis.Senescentchondrocytespresentinagedcartilagecannotproliferatetoregenerate damagedcartilageandinduceextracellularmatrixdegenerationthroughtheSASP.Furthermore,cartilageregenerationisinhibitedduringaging duetosenescentmesenchymalstemcells.

thismechanismmightparticipateintheimpaired

regen-erationofDM1muscles.Notably,theregenerativedeficit

ofsatellitecellsfromdystrophicmusclesresemblesthat

of geriatric mice, which also show p16INK4a-induced

senescence and can berejuvenated by silencingof the

gene encoding p16INK4a [21]. Overall, these studies

show limitedeffects, and the long-term safety of these

drugsand/orgeneticapproacheshasyet tobeassessed.

However, it is unlikely that essential molecules and

pathways such as Notch or p16INK4a can be targeted

systemically without severe secondary effects. In

addi-tion,thesestrategiesareaimedatreducingsymptomsand

donottreattheunderlyingcausesofdiseaseprogression.

Removal of senescentcells isexpected to reducethese

inflammatoryproteinswhilepreservingstemcellfunction

andisthereforeexpectedtobesaferandhavemore

long-lastingeffects.

Theresults obtainedaftergeneticremovalofsenescent

cells prompted a search for therapeutically applicable

anti-senescence compounds. A small number of these

compoundshavebeendiscovered,withvaryingdegrees

of success. One example is Navitoclax, a BCL2 family

inhibitor.Inthemusculoskeletalsystem,Navitoclaxwas

found to decreasetheexpression of cytokinesthat

pro-moteosteoclastactivityinvitro,suchasIL-1aand

MMP-13 [58]. Furthermore, muscle stem cells isolated from

naturally aged, Navitoclax-treated mice showed

improvedclonogenicity[78].

A major challenge when developing anti-senescence

therapies is to avoid toxicity to healthy non-senescent

cells. It is therefore important to identify the unique

characteristics of senescent cells that can be targeted

byatherapeuticcompound.Senescentcellsoftenexpress

persistent nuclear damage foci called DNA-SCARS

(DNASegmentswithChromatinAlterationsReinforcing

Senescence)thatcontainDDRproteins suchas 53BP1,

gH2AXandactivatedp53[79].TheseDNA-SCARSplay

a role in maintaining permanent growth arrest and are

critical for SASP expression. In addition, we recently

showed that the transcription factor FOXO4 resides

withinPMLbodiesfusedtothesepersistentdamagefoci

[19].Here,FOXO4bindsp53andprevents

p53-depen-dentapoptosis.Inordertodisruptthisinteractionandto

induceapoptosis,weprospectivelygenerateda

D-Retro-Inverso peptide mimicking the FOXO4 p53-binding

domain.Thispeptide,FOXO4-DRI, causestherelease

of p53 to the cytoplasm, where p53 indeed induces

apoptosisinatranscriptionindependentmanner.Indeed,

invivouseofFOXO4-DRIshowspromisingresults.For

theseexperimentswemadeuseofXpdTTD/TTDmicethat

showacceleratedagingandage-relatedailmentssuchas

osteoporosisandarethereforeanidealmodelfor

muscu-loskeletal diseases [60]. FOXO4-DRI treatment

improvedoverallfitnessandrenalfunctioninthesemice,

includinganimprovedrunningwheelperformance[19],

anespeciallypromisingresultforthetreatmentof

mus-culoskeletal diseases. FOXO4-DRI showed around

10 fold selectivity for eliminating senescent vs. control

cells.Whileenough forexperiments inrodents,

transla-tiontotheclinicrequiresfurtherimprovementto

elimi-natetoxicity,whichwouldbeintolerableinthissetting.

Sucheffortsarenowunderway inourlaboratory.

Unanswered

questions

Aswehighlightedhere,thetissuesofthemusculoskeletal

systemaredamagedbyinflammationduringaging.

Cel-lular senescence, by driving a persistent inflammatory

response,isamajorcontributortotheseeffects.However,

itremainsunclearwhichsenescentcelltypesarethemain

producersofthesepro-inflammatoryfactors.Agingofthe

musculoskeletalsystemisduetobothlocalandsystemic

factors. For example, senescent cells transplanted into

cartilagecanindependentlycauseosteoarthritis[71].On

the other hand, systemically increased IL-6 levels are

linkedto muscle wasting,and theimmune system also

seemstobecrucialinthisprocess[28,29].Thissystemic

inflammation can be caused by many cell types. For

example,adipose tissue significantlycontributes to

sys-temicinflammation[80].Fatpresentinjointscanproduce

factorsthatpromote osteoarthritis [81].In turn, cells of

Tissue Modelsystem Senescencecleared/delayedby: Improvementsmusculoskeletalsystem Ref. FastagingBubR1H/H_mice

Naturallyagedmice FastagingLAKI(LmnaG609G₎

INK-ATTAC Navitoclax

TransientOSKMexpression

Kyphosisreduction,increaseinmuscle fiberdiameter

Improvedmusclestemcellfunction Improvedregenerationaftermuscleinjury

[16] [78] [33]

Klothodeficientmice Naturallyagedmice

p16INK4AKnockout INK-ATTAC

Delayinosteoporosis

Improvedbonestructureandstrength, improvedboneformation,reductioninbone resorption

[61] [62]

ACLTinthemouseKneejoint Naturallyagedmice

FastagingXpdTTD/TTD_mice

p16::3MR INK-ATTAC

FOXO4-p53interferingpeptide

Reducedinflammation,painreduction, increaseincartilagedevelopment Reducedcartilagedegeneration Improvedrunningwheelperformance

[76] [62] [19]

themusculoskeletalsystemalsosecretesystemicfactors

andinfluenceoveralltissueintegrity.Forexample,

mus-clecellsaffectNKcellsduringagingand,asNKcellsare

responsible for clearance of senescent cells [41], these

would also influence the systemic senescence burden.

Sincevariousanti-senescencecompoundspotentiallykill

distinctsubsetsofsenescentcells,itisvitaltoknowwhich

celltypetotarget;knowledgeaboutwhichsenescentcells

contribute most to musculoskeletal degeneration will

ultimatelyguidethedevelopmentofeffectivetreatment.

Anti-senescence therapymayalsobebeneficialfor

sev-eral incurable muscular dystrophies andfor wasting,by

reducinginflammaging and henceboosting thesatellite

cellregenerativefunctions.Interestingly,cellular

senes-cence has been shown to mediate fibrotic pulmonary

disease,andsenescentcellablationimprovespulmonary

functioninthissetting[82].Mostdystrophinopathiesalso

feature increasedmuscle fibrosis[83],which aggravates

disease progression by substituting muscle with scar

tissue, and it is plausible thatanti-senescence cocktails

will also haltfibrosis andimprove patienthealth status.

Thus,eliminationofsenescentcellsmayhavebenefitsfor

tissue repairbyreversing severaldetrimentalprocesses;

however, it remains to be determined whether

senes-cenceshouldbeblockedpartiallyortotallyoreliminated

onlyonceearlypotentialstemness-relatedfunctionshave

beencompleted.Theanswerstothesequestionsmaynot

beeasytoobtain,yetwearerapidlyobtainingtoolsthat

allowmanipulationofthesenescenceprocess(for

remov-ing senescentcells, neutralizingtheSASP, or both

pro-cesses). The final goal is to preservestem cellbenefits

while minimizing the deleterious consequences of

senescence.

Italsoremainsunclearhowtissuesrejuvenateafter

senes-centcellablationandwhethersideeffectsorunexpected

challenges will occur. For example, in addition to its

potentialtoeliminatesenescentcells,tissueengineering

is being explored as a treatment for musculoskeletal

diseases. In this scenario, stem cells are isolated and

healthy tissue is generated ex vivo to replace damaged

tissuessuchascartilage andbone.For example,

mesen-chymal stem cells can be isolated and cultured on a

biodegradable scaffold where they are stimulated with

TGFbto induce differentiationinto chondrocytes[84].

This newly formed cartilage could then be used for

surgical reconstructionof joints.However,amajor

chal-lengeintissueengineeringistopreventstemcell

senes-cence[85].Itremainsunclearwhethersimilarissueswill

arise aftersenescenceclearance.Sofar,tissue

regenera-tion seems efficient after these cells are removed. For

example, although cartilage has a weak regenerative

potential, it is rejuvenated after senescent cells are

removed.Tissue-specificstemcellsarelikelykeytothis

regeneration. It is possible that the reduction of SASP

proteins in the tissue microenvironment releases these

cells from their ‘stemcell lock’, resultingin arestored

regenerative potential. In addition, cells that are

dedif-ferentiated due to senescence, such as chondrocytes,

couldhelprejuvenatemusculoskeletaltissue.Ingeneral,

multiple factors likely contribute to this rejuvenation.

Both local and systemic inflammation are expected to

decline, affecting immune system functioning, natural

senescent cellclearance, stem cell function, and tissue

regeneration.

In conclusion,targeting senescencehasthepotentialto

prevent or reverse multiple age-related diseases and to

reduce frailty. Furthermore, it seems likely that

thera-peuticallyapplicableanti-senescencecompoundswillbe

available in the future. However, the toxicity of these

drugsremainsamajorconcern.Periodic treatmentswill

likelybenecessarytomaintainpossiblebeneficialeffects

anditisstilllargelyunknownwhattheeffectofmultiple

treatment rounds will be. Therefore, the timing and

frequencyofthesetreatmentsshouldbestudied,aswell

as the long-termeffect of senescence clearanceon

bio-logicalprocessessuchasstemcellfunction.

Conflict

of

interest

PDKisco-founder,shareholderandconsultantforCleara

BiotechB.V., theNetherlands.

Acknowledgements

TheauthorsacknowledgesupportforMBfromDutchCancerSocietyGrant UMCU-7141awardedtoPdK,andforEPandPMCfrom ERC-2016-AdG-741966(STEM-AGING),SAF2015-67369-R,MDAandAFM.The DCESX/UPFisrecipientofa‘Marı´adeMaeztu’ProgramforUnitsof ExcellenceinR&DMDM-2014-0370(GovernmentofSpain).

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