Targeting the Intracellular Environment in Cystic Fibrosis: Restoring Autophagy as a Novel Strategy to Circumvent the CFTR Defect.

0.92 MB·English

by  Villella, Valeria Rachela

REVIEWARTICLE
published:21January2013
doi:10.3389/fphar.2013.00001
Targeting the intracellular environment in cystic fibrosis:

restoring autophagy as a novel strategy to circumvent the

CFTR defect

ValeriaRachelaVillella1,SperanzaEsposito1,EmanuelaM.Bruscia1,2,MariaChiaraMaiuri3,4,ValeriaRaia5,
GuidoKroemer4,6,7,8,9*andLuigiMaiuri1,10*

1EuropeanInstituteforResearchinCysticFibrosis,SanRaffaeleScientificInstitute,Milan,Italy

2DepartmentofPediatrics,YaleUniversitySchoolofMedicine,NewHaven,CT,USA

3DepartmentofExperimentalPharmacology,FedericoIIUniversity,Naples,Italy
4INSERMU848,Villejuif,France
5CysticFibrosisUnit,DepartmentofPediatrics,FedericoIIUniversity,Naples,Italy
6UniversitéParisDescartes,Paris,France
7MetabolomicsPlatform,InstitutGustaveRoussy,Villejuif,France

8CentredeRecherchedesCordeliers,Paris,France

9PôledeBiologie,HôpitalEuropéenGeorgesPompidou,AssistancePublique–HôpitauxdeParis,Paris,France
10InstituteofPediatrics,UniversityofFoggia,Foggia,Italy

Editedby: Cysticfibrosis(CF)patientsharboringthemostcommondeletionmutationoftheCFtrans-

MarcChanson,UniversityofGeneva, membraneconductanceregulator(CFTR),F508del,arepoorresponderstopotentiatorsof
Switzerland
CFTRchannelactivitywhichcanbeusedtotreatasmallsubsetofCFpatientswhogenet-

Reviewedby:
icallycarryplasmamembrane(PM)-residentCFTRmutants.ThemisfoldedF508del-CFTR
ValerieChappe,DalhousieUniversity,
Canada protein is unstable in the PM even if rescued by pharmacological agents that prevent its
Jean-MichelSallenave,Université intracellularretentionanddegradation.CFisaconformationaldiseaseinwhichdefective
Paris7DenisDiderot,France CFTRinducesanimpressivederangementofgeneralproteostasisresultingfromdisabled
*Correspondence: autophagy. In this review, we discuss how rescuing Beclin 1 (BECN1), a major player of
GuidoKroemer,INSERMU848,
autophagosomeformation,eitherbymeansofdirectgenetransferorindirectlybyadminis-
InstitutGustaveRoussy,Pavillonde
Recherche1,F-94805Villejuif,France. trationofproteostasisregulators,couldstabilizeF508del-CFTRatthePM.Wefocusonthe
e-mail:[email protected]; relationship between the improvement of peripheral proteostasis and CFTR PM stability
LuigiMaiuri,EuropeanInstitutefor in F508del-CFTR homozygous bronchial epithelia or mouse lungs. Moreover, this article
ResearchinCysticFibrosis,San
reviewsrecentpre-clinicalevidenceindicatingthattargetingtheintracellularenvironment
RaffaeleScientificInstitute,via
Olgettina58,Milan20132,Italy. surroundingthemisfoldedmutantCFTRinsteadofproteinitselfcouldconstituteanattrac-
e-mail:[email protected] tive therapeutic option to sensitize patients carrying the F508del-CFTR mutation to the
beneficialactionofCFTRpotentiatorsonlunginflammation.

Keywords:cysticfibrosis,CFTR,proteostasisregulators,autophagy,BECN1

INTRODUCTION Althoughmorethan1800differentmutationshavebeenidentified,
Theproteostasisnetworkensuresintracellularhomeostasisinspite one single deletion of phenylalanine at position 508 (F508del-
ofgeneticorepigeneticchangesinproteinconformation,extracel- CFTR), occurs in about 70–90% of CF patients in Northern
lularstress,oraging-associatedperturbations(Balchetal.,2008; EuropeandNorthAmerica(Bobadillaetal.,2002).F508del-CFTR
Huttetal.,2009;Powersetal.,2009;Gidalevitzetal.,2010;Hutt proteincanstillretainapartialchloridechannelactivityifrescued
and Balch, 2010; Roth and Balch, 2011). The accumulation of at the epithelial surface. However, due to its misfold, F508del-
misfolded/modifiedproteinsduetomutationsorduetotheaging- CFTR does not reach the plasma membrane (PM) and is pre-
relateddeclineofproteostasiscontributestoseveralhumancon- maturelydegraded,thusprovokinglocalinflammation,increased
formational diseases including neurodegenerative disorders and susceptibility to respiratory bacterial infections,and progressive
typeIIdiabetes(Balchetal.,2008;Powersetal.,2009;Gidalevitz pulmonaryanddigestiveinsufficiency(O’SullivanandFreedman,
etal.,2010;RothandBalch,2011). 2009;Ratjen,2009).
Cysticfibrosis(CF),themostcommonlife-threateninggenetic ThebirthprevalenceofCFisestimatedtobeonein3500–4500,
diseaseamongCaucasians,constitutesthequintessentialexample with 200-300 new cases each year in Europe. The typical form
ofa“conformationaldisease”(Balchetal.,2011;Okiyonedaetal., of CF is diagnosed during early childhood and is characterized
2011).CFiscausedbymutationsoftheCFtransmembranecon- byrecurrentpulmonaryinfections,pancreaticinsufficiency,and
ductanceregulator(CFTR)genethatencodesacAMP-regulated elevatedchlorideconcentrationsinsweat.AlthoughCFisasys-
chloride channel primarily located at the apical membrane of temicdisease,themaincauseofdeathispersistentanduntreatable
epithelialcells(Quinton,1999;Welshetal.,2001;Parketal.,2010). pulmonaryPseudomonasaeruginosainfection.Lossoffunctional

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Villellaetal. Targetingautophagyincysticfibrosis
CFTRexpressionisthoughttodisturbthebalancebetweenfluid (Sharmaetal.,2004;Okiyonedaetal.,2010).Thisseminalobser-
secretion and absorption into the epithelial layer,leading to net vation of Lukacs’ group can explain why CF patients carrying
volume depletion of mucus, increased viscosity, and ineffective the misfolded F508del-CFTR respond poorly to molecules that
bacterialclearance.Bacterialinfectioninturninducesanincreased increasetheactivityofCFTRchannel(CFTRpotentiator)(Davis,
inflammatoryresponseandsignaling,thusfuelingaviciouscycle 2011;Ramseyetal.,2011).Indeed,therescuedF508del-CFTRis
ofmucusretention,infection,andinflammation. nolongeravailableatthePMfortheactionofCFTRpotentiators.
Mounting evidences indicate that a constitutive inflamma- Therefore,combiningCFTRcorrectorsandpotentiatorsmaybe
tory condition characterizes CF airways regardless of bacter- asuitableapproachforF508del-CFTRpatients,providedthatthe
ial exposure. CFTR dysfunction results in constitutive, elevated correctormoleculesareeffectiveinincreasingF508del-CFTRPM
NF-κB activation resulting in increased production of the pro- stabilityafterrescue.Currently,phaseIIclinicalstudiesevaluat-
inflammatorychemokine,interleukin-8(Vijetal.,2009;Belcher ingthecombinationofVX-809andthepotentiatorVX-770inCF
andVij,2010;BodasandVij,2010;Hunteretal.,2010).Moreover, patientsthatexpressF508del-CFTRareunderway(Elborn,2012).
thelackoffunctionalCFTRinmacrophageshasbeenreportedto Restorationofafunctionalproteostasisnetworkbytheadmin-
increasetheirresponsivenesstoinflammatorystimuliviauncon- istrationof proteostasisregulators(PRs)hasemergedasanovel
trolledTLR4signaling(Brusciaetal.,2009,2011)andtoaffecttheir approachtocorrectproteinmisfoldinginconformationaldiseases
capacitytokillPseudomonasaeruginosa(Dietal.,2006;Deriyetal., (Muetal.,2008;Powersetal.,2009;Gidalevitzetal.,2010;Balch
2009;Zhangetal.,2010;DelPortoetal.,2011).Thesefindingssup- etal.,2011).Therefore,strategiesaimingatmanipulatingperiph-
porttheroleofCFTRdysfunctioninfavoringbronchopulmonary eralproteostasiscouldrepresentapromisingareaof researchin
inflammation. CFdrugdiscovery.Understandingthemechanismsunderlyingthe
AdvancesinCFtreatmenthaveincreasedthemedianpredicted derangementofproteostasisconsequenttodefectiveCFTRfunc-
survival age from less than 5years in the 1940s to over 37years tioncouldhelpimprovingthesearchofnewdrugcandidatesfor
presently(Davis,2006).Inadditiontotherapeuticapproachesthat CFpatientscarryingF508del-CFTRmutants.
target cellular events downstream of the CFTR defect (Mozzillo
et al., 2009; Anderson, 2010; Belcher and Vij, 2010; Ratjen and THREETOTANGOINCYSTICFIBROSIS:CFTR,
Grasemann, 2012), other strategies focused on the basic CFTR TRANSGLUTAMINASE2,ANDAUTOPHAGY
defecthaveemerged(Riordan,2008;SloaneandRowe,2010;Ama- DEFECTIVECFTRFUNCTIONPERTURBSTHEPOST-TRANSLATIONAL
ral,2011;LukacsandVerkman,2012).Todate,genetherapyhas NETWORKOFCFEPITHELIALCELLS
failed to demonstrate a clinical benefit for CF (Riordan, 2008; An impressive derangement of cellular homeostasis takes place
Amaral,2011).Thus,pharmacologicalstrategiesaimedatcorrect- in CF airways. Tissue transglutaminase (TG2) is upregulated in
ing mutation-specific CFTR defects (CFTR-repairing therapies) CF epithelial cells at the transcriptional and even more at the
havegainedaprominentroleinCFdrugdiscovery. post-transcriptional levels (Maiuri et al., 2008). TG2 is a versa-
ThestillpartiallyfunctionalF508del-CFTRproteincanberes- tilemultifunctionalproteinthatchangesitsfunctiondepending
cuedatthePMbymeansofexperimentallowthermalconditions onexternalandinternalsignals(NurminskayaandBelkin,2012).
(Denning et al.,1992),as well as by so-called correctors,which In the presence of high Ca2+ levels, TG2 works as a crosslink-
aremoleculesthatavoidtheintracellularretentionanddegrada- ingenzyme,catalyzingseveralpost-translationalmodificationsof
tionofF508del-CFTRprotein(Pedemonteetal.,2005;Verkman target proteins. At low Ca2+ concentrations,TG2 may function
etal.,2006;VerkmanandGalietta,2009),asextensivelyreviewedby as a G-protein or as a protein disulfide isomerase, thus con-
Molinskietal.andPedemonteetal.inotherchaptersofthisSpecial tributingtothefunctionalityof mitochondrialrespiratorychain
Topic.AnumberofCFTRcorrectormoleculeshavebeenidentified complexes (Nurminskaya and Belkin, 2012). Increased levels of
byhigh-throughputscreening(Galiettaetal.,2001;Pedemonte TG2 are observed in several human pathologies including neu-
etal.,2005;VanGooretal.,2006,2011).SeveralCFTRcorrectors rodegenerative diseases such as Alzheimer’s, Huntington’s, and
haveprovedtheirefficacyinrescuingF508del-CFTRinvitro.How- Parkinson’s diseases, as well as in chronic inflammatory condi-
ever,theirefficacyinamelioratingtheCFlungphenotype,either tions(Tayloretal.,2003;Malornietal.,2008;Iismaaetal.,2009;
inpre-clinicalmodelsorinCFpatients,hasnotyetestablished. MastrobernardinoandPiacentini,2010).Mostproteinsinvolved
A recent clinical trial with the most promising CFTR correc- inthepathogenesisofneurodegenerativediseases,ashuntingtin,
tor,VX-809 (Van Goor et al.,2011),in F508del-CFTR homozy- ataxin1,tau,andalpha-synuclein,werereportedtobeTG2sub-
gous patients demonstrated modest dose-dependent reductions strates (Mastrobernardino and Piacentini,2010). Increased TG2
in sweat chloride (Clancy et al., 2012). However, beyond this expression has also been reported for glioblastomas, malignant
laboratory parameter, no improvement in lung function or CF melanomas, and pancreatic ductal adenocarcinomas. Moreover,
complicationswasreported(Clancyetal.,2012;Elborn,2012). TG2 expression is often associated with an increased metastatic
Thepoolof F508del-CFTRmoleculesthatcanreachthePM activity or acquisition of drug-resistance (Antonyak et al.,2004;
after treatment with currently available corrector molecules is KarinandGreten,2005;Kimetal.,2006;Satpathyetal.,2007).
unstable.Thisinstabilitycanbeexplainedbycarboxyl-terminus In CF airway epithelial cells, TG2 undergoes small ubiqui-
heat shock cognate 70 (HSP70)–interacting protein (CHIP)- tin like-modifier (SUMO)ylation (Luciani et al., 2009), a post-
mediated Ubiquitination of F508del-CFTR (Okiyoneda et al., translationalmodificationthataffectsthestabilityandfunctions
2010), followed by redirection of the protein from endosomal ofproteins.SUMOylationisakeyplayerofthepost-translational
recyclingtowardlysosomaldeliveryandsubsequentdegradation networkasitregulatestranscription,nucleartranslocation,stress
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Villellaetal. Targetingautophagyincysticfibrosis
responses,andchromatinstructure.Moreover,itinfluencesintra- ubiquitination of IκBα inhibits IκBα SUMOylation and favors
cellular localization and stability of modified proteins (Geiss- NF-κBactivationandnucleartranslocation(Lucianietal.,2009).
FriedlanderandMelchior,2007;MeulmeesterandMelchior,2008; Both events were reported to favor inflammation in CF air-
Tempè et al., 2008). SUMOylation is accomplished by an enzy- ways. Therefore, TG2 can function as a rheostat of the post-
maticcascadethatinvolvesE3ligaseswhichorchestrateSUMO- translationalnetworkinresponsetoCF-associatedoxidativestress.
modificationsinresponsetostress.Wediscoveredthattheprotein TG2 SUMOylation with sustained TG2 activation switches off
inhibitor of activated STAT (PIAS)y, which is induced by reac- the post-translational regulatory mechanisms and perturbs the
tive oxygen species (ROS) and participates in the SUMOylation intracellularenvironment(Figure1).
of NF-κB essential modulator (NEMO) upon genotoxic stress TG2-mediated protein ubiquitination and crosslinking may
(MabbandWuerzberger-Davis,2006),isupregulatedinCFepithe- leadtoproteinaggregationandproteasomeoverload,thusfavor-
lia in response to oxidative stress and then mediates SUMOyla- ing aggresome formation (Muma, 2007; Dohm et al., 2008).
tion of TG2 (Luciani et al., 2009). Indeed, TG2 contains three Misfolded or post-translationally modified proteins that can-
SUMOacceptorsites(consensussequence:ψ_kxE)initssequence. not be degraded by the proteasome machinery can be stocked
SUMOylationoflysinesisincompatiblewiththeUbiquitination in the cytoplasm in the form of aggresomes (Kawaguchi et al.,
oftheseresidues(MullerandHoege,2001).Thus,TG2SUMOy- 2003; Kirkin et al., 2009). Accordingly, ubiquitylated PPARγ
lationultimatelyresultsintheinhibitionof TG2ubiquitination, and IκBα aggregates are sequestered within histone-deacetylase
therebypreventingitsproteasomaldegradation.Thissustainshigh (HDAC)6+/vimentin+ intracellular aggresomes in CF epithelial
intracellularTG2proteinlevels,coupledtoprolongedTG2enzyme cells(Figure2).
activation as the result of the elevated Ca2i+ content. Indeed, Therefore,proteostasis of F508del-CFTR epithelia is affected
emergingevidencesupporttheroleof elevatedintracellularcal- byacombinationofgeneticdefect(resultingfromthemisfolded
ciumconcentrationinmediatingthesignalingeventsthatimpair CFTR protein) and post-translational alterations (through the
homeostasisinCFepithelia,asreviewedbyAntignyetal.(2011a) ROS/TG2axis).
in another chapter of this Special Topic. Although the mecha- TG2islocalizedinmultiplecellularcompartmentsincluding
nismsunderlyingthedisturbedcalciumhomeostasisobservedin cellsurfaceandextracellularmatrix.Besidesitscrosslinkingactiv-
CF remain incompletely understood,recent studies suggest that ity on ECM substrates, extracellular TG2 is also endowed with
impairedcalciumsignalingmaybetheresultof eitherincreased PDI,orGTPasefunctions(NurminskayaandBelkin,2012).How-
agonist-mediated activation of G-protein-coupled receptors or ever,thepotentialrelevanceoftheextracellularTG2inCFisstill
abnormalregulationofcalciumstoragecompartments(Eganetal., unknown.
2002,2004;Ribeiroetal.,2005a,b;Norezetal.,2006a,b;Martino
etal.,2009).Moreover,theabnormalCa2+ responseobservedin DEFECTIVECFTRDISABLESAUTOPHAGY
CFcellsdependsonthepresenceofCFTRatthecellsurfaceand GiventheoverproductionofROStogetherwiththeendoplasmic
this reciprocal regulation of CFTR and Ca2+ channels has been reticulum (ER) stress induced by the mutant CFTR,one would
describedintheliterature(Antignyetal.,2011b). expect an activation of autophagy in F508del-CFTR homozy-
Therefore,inCF,increasedintracellularlevelsofROS,induced gous epithelial cells. Autophagy is pivotal in promoting cellular
bydefectiveCFTRfunction,leadtotheupregulationoftheSUMO clearance of protein aggregates and removal of ROS sources,
E3-ligase PIASy, which facilitates TG2 SUMOylation, persistent such as damaged mitochondria (Mizushima et al., 2008; Kirkin
high TG2 protein levels, and sustained TG2 activation as the etal.,2009;Korolchuketal.,2009;Kroemeretal.,2010;Moreau
resultof“permissive”elevatedCa2+ levels.Thepresenceof high et al., 2010). Surprisingly, however, human and mouse CF air-
TG2 levels might in turn sustain ROS, as it is known that TG2 ways exhibit a pronounced defect in autophagy, as indicated
maystimulatetheactivityofthemitochondrialrespiratorychains byreducedautophagosomeformation,andtheaccumulationof
(Malornietal.,2008).Remarkably,inhibitingCFTR,eitherbygene sequestosome 1 (SQSTM1), a major autophagic substrate also
silencingorbymeansofpharmacologicalinhibitors,recapitulates known as p62. This occurs in spite of the normal expression of
these post-translational modifications of TG2 through upregu- major autophagy genes (Luciani et al., 2010, 2011). A defective
lating ROS levels in cell lines expressing wild-type (wt)-CFTR autophagicresponsetobacterialinfectionhasalsobeenreported
(Lucianietal.,2009). inmurineCFmacrophages.Reducedautophagosomeformation
These post-translational changes of TG2 protein,induced by in CF macrophages promotes Burkholderia cenocepacia survival
defective CFTR, may have functional implications in epithe- andhypersecretionofIL-1β(Abdulrahmanetal.,2011).
lial homeostasis. Sustained TG2 activation leads to crosslinking, Autophagy results in the lysosomal degradation of cytoplas-
increasedubiquitination,andfunctionalsequestrationoftheTG2 micorganellesorcytosoliccomponentsaftertheirsequestration
substrates peroxisome proliferator-activated receptor (PPAR)γ intwo-membranedvesicles(Kroemeretal.,2010;YangandKlion-
andIκBα(DaynesandJones,2002;Kimetal.,2006;Maiurietal., sky,2010;Codognoetal.,2011;Mizushimaetal.,2011).Inthelast
2008). Indeed, the anti-inflammatory molecule PPARγ under- few years,autophagy has emerged not just as a simply degrada-
goes SUMOylation in response to its agonists, thus interacting tive process, but also as a cellular mechanism essential for the
withtheN-CoR-histonedeacetylase(HDAC)3co-repressorcom- maintenanceofcellularhomeostasisandoftheenergeticbalance
plex to maintain a repressor condition (Pascual et al., 2005). (Kroemeretal.,2010).Thus,disabledautophagyisassociatedwith
TG2-mediated ubiquitination of PPARγ inhibits its SUMOyla- andisrelevanttoseveralhumandiseasesincludingcancer,viral
tion and interaction with N-CoR. Similarly, crosslinking and infection,neurodegenerativediseases,respiratorypathologies,and
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Villellaetal. Targetingautophagyincysticfibrosis

FIGURE1|DefectiveCFTR-inducedperturbationofthe proteosomaldegradation,thussustainingincreasedTG2proteinlevels.
post-translationalnetworkinCFepithelialcells.DefectiveCFTR SustainedTG2activationmediatescrosslinkingofPPARγandIκBα,
leadstoincreasedlevelsofreactiveoxygenspecies(ROS)that whichundergoubiquitinationandproteasomedegradation.Thisinhibits
increasethelevelsoftheSUMOE3-ligasePIASy,causingTG2 nucleartranslocationofPPARγandfavorsnucleartranslocationof

SUMOylation,that,inturn,inhibitsTG2ubiquitination,andavoidsits NF-κB,stimulatinginflammation.

chronic inflammatory disease (Levine et al., 2011; Rubinsztein autophagyinduction.Moreover,BECN1interactswiththeessen-
etal.,2011;Sridharetal.,2012;Pateletal.,2013). tialcomponentsofthePI3KcomplexIIIhVps34,hVps15,Ambra1,
Through which mechanisms is autophagy inhibited in CF? aswellaswithAtg14L,aBECN1interactorthatdivertshVps/Class
We have demonstrated that the inhibition of autophagy in CF III PI3K into an autophagic role (Liang et al.,2008; Matsunaga
epithelial cells is part of the complex perturbation of the post- etal.,2009).However,inCFepithelialcells,theBECN1interac-
translational network consequent to defective CFTR function. tomeisdislodgedawayfromtheERasaconsequenceofBECN1
DisabledautophagyinCFepithelialcellsisaconsequenceofTG2- crosslinkingandissequesteredwithinHDAC6+aggresomes.This
mediatedcrosslinkingandfunctionalsequestrationofBECN1,a impairs autophagosome formation in CF cells (Luciani et al.,
majorplayerofautophagosomeformation,whichexhibitstarget 2010).
sites(QP,QxxP)forcrosslinkingbyTG2(Lucianietal.,2010). Autophagy deficient CF cells accumulate SQSTM1 (p62), an
BECN1 is a haploinsufficient tumor suppressor protein that ubiquitin-binding (and LC3-binding) protein (Bjørkøy et al.,
is essential for autophagy (Sinha and Levine, 2008; He and 2005; Kirkin et al., 2009; Mathew et al., 2009; Duran et al.,
Levine, 2010; Maiuri et al., 2010). Accumulating evidence indi- 2011) that is selectively degraded by autophagy. Autophagy
catethatBECN1dissociatesfromBcl-2duringstressconditions, upregulation has been reported as a compensatory response to
such as starvation, thus promoting autophagy (Pattingre et al., proteasome inhibition, thus revealing a crosstalk between the
2005;Maiurietal.,2007,2010;Axeetal.,2008;Hayashi-Nishino proteasome-based and the autophagy-based degradation path-
et al., 2009). Subsequently, BECN1 interacts with the class III ways (Komatsu et al., 2007; Kirkin et al., 2009; Korolchuk
phosphatidyl-inositol 3 kinase (PI3K), human vacuolar protein et al., 2009; Lamark and Johansen, 2010). SQSTM1 accumu-
sorting (hVps)34 (Matsunaga et al., 2009; Zhong et al., 2009), lation resulting from autophagy inhibition contributes to pro-
facilitatingitsactivation.TheER-associatedclassIIIPI3Kactiv- teasome overload and favors aggresome formation, while dis-
ityiscrucialfortheinitiationofautophagosomeformation(Axe abledautophagyinhibitstheclearanceofsuchproteinaggregates.
etal.,2008;Hayashi-Nishinoetal.,2009). Altogether, the combined inhibition of protein and aggresome
ReducedBECN1/Bcl-2interactionuponstarvationisobserved turnovermayalsoinfluencethefateofmisfoldedCFTR.Indeed,
inCFcells,suggestinganintracellularenvironmentfavorableto the enforced expression of F508del-CFTR in CF epithelial cell
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FIGURE2|TG2-mediatedinhibitionofautophagyinCFepithelial sequestrationofcross-linkedTG2substrates(PPARγ,IκBα,BECN1)within
cells.DefectiveCFTR-mediatedTG2activationleadstoBECN1 HDAC6+aggresomes.Thecombinedinhibitionofproteinandaggresome
crosslinkinganddisplacesBECN1interactomeawayfromthe turnovermayalsofavortheaccumulationofF508del-CFTR(togetherwith

endoplasmicreticulum(ER).Thismislocalizationinhibitsautophagosome SQSTM1)withinHDAC6+/ubiquitin+intracellularaggregates.Defective

formation,disablesautophagy,andinducesaccumulationofSQSTM1 autophagyinhibitstheclearanceofdamagedmitochondriathatcontribute
(p62).SQSTM1accumulationleadstoproteasomeoverloadandfavors tothegenerationofpro-inflammatoryROS.

linesfavorstheaccumulationof misfoldedCFTR(togetherwith inflammationinCftrF508delmice(Lucianietal.,2010,2012).Simi-

SQSTM1) within HDAC6+/ubiquitin+ intracellular aggregates larly,cysteaminehasalreadybeensuccessfullyusedinmousemod-

(Lucianietal.,2010). elsofHuntington’sdiseasetoimprovedisease-relatedphenotype

We suggest that this cascade of events can generate a vicious (Karpujetal.,2002).

feed-forwardloop,asitimpairstheclearanceof damagedmito- Importantly, we demonstrated that amelioration of lung

chondria,thusincreasingROSgenerationthatinturnenhances inflammation in CftrF508del mice secondary to cystamine treat-

TG2 activation and BECN1 sequestration, further sustaining mentpersistsupto10daysbeyondcystaminewithdrawal,unless

airwayinflammation(Figure2). therescueofBECN1andautophagyareinhibitedbytheadmin-

istration of 3-MA during washout (Luciani et al., 2012). These

RESTORINGPROTEOSTASISAMELIORATESLUNGINFLAMMATION data suggest the provocative hypothesis that, once the cellular
THROUGHRESCUINGAUTOPHAGYINCF environment has been re-directed toward a physiological status,
InhibitingTG2activitybycystamine(anditsreducedformcys- a driving force is re-established within the cell, so as to pro-
teamine)ortargetingROScanreduceinflammationinF508del- long these beneficial effects. Could this “newly re-established”
CFTRairways,bothinvivo inF508del-CFTRhomozygousmice player be the functional CFTR itself at the epithelial surface?
(CftrF508del mice) and in ex vivo using explanted human polyp Indeed, manipulating proteostasis might actually improve the
biopsiesfromCFpatients(Raiaetal.,2005;Lucianietal.,2010). functionofmisfoldedproteins(Balchetal.,2008;RothandBalch,
The effects of cystamine on airway inflammation are medi- 2011).
ated by its ability to rescue BECN1 and autophagy, as either
BECN1 depletion used in vitro or administration of the PI3K TARGETINGAUTOPHAGYASANEWSTRATEGYTOENABLE
complex III inhibitor 3-methyl-adenine (3-MA) used in vivo in THEACTIONOFCFTRPOTENTIATORSONF508del-CFTR
CftrF508del mice,abrogated these beneficial effects of cystamine. F508del-CFTRrescuedatthePMbymeansofcorrectorstrategies
Either enforced BECN1 overexpression or SQSTM1 depletion is rapidly dismissed and re-directed to lysosomes for degrada-
invivorecapitulatedtheeffectsofcystamineinamelioratinglung tion (Sharma et al., 2004; Okiyoneda et al., 2010, 2011; Lukacs
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Villellaetal. Targetingautophagyincysticfibrosis
andVerkman,2012).Accordingly,thebiochemicalhalf-lifeofPM TARGETINGAUTOPHAGYENABLESTHEBENEFICIALACTIONOF
F508del-CFTRislowerthan4h(Lukacsetal.,1993;Hedaetal., POTENTIATORSONF508del-CFTR
2001). Therefore,F508del-CFTR is no longer present at the PM ThesefindingsindicatethatPRsmaybeusedtorescueandstabilize
andcannotinteractwithCFTRpotentiatorsafterrescue. F508del-CFTRatthePMof CFepithelialcells.Inprinciple,this
Recently, we have reported that overexpression of BECN1, strategycouldlowertheERqualitycontrol(QC)thresholdofall
administration of cystamine, or depletion of SQSTM1 by RNA misfoldedproteins,thusinterferingwiththeQCfidelity.However,
interference, can favor the trafficking of F508del-CFTR pro- besidesitseffectsonF508del-CFTRrescueatthePM,cystamine
tein to the epithelial cell surface in vitro in CF epithelial can also delay the disposal of PM resident F508del-CFTR pro-
cell lines (CFBE41o- or IB3-1, carrying F508del/F508del or tein.Theevidencediscussedinthisreviewsupportsthatdefective
F508del/W1282XCFTR,respectively),exvivoinnasalpolypbiop- CFTRsuppressesautophagywithintheCFepithelialenvironment
siesfromCFpatients,andinvivoinCftrF508delmice.Interestingly, (though the ROS/TG2 pathway), and, conversely, that rescuing
these treatments can restore a functional CFTR in CF cell lines autophagycanrestoreafunctionalCFTRatthePM.Altogether,
andinprimarybrushednasalepithelialcellsfromF508del-CFTR these insights suggest the existence of a vicious cycle in which
homozygouspatients(Lucianietal.,2012). defectiveCFTRfunctionsdestabilizestheCFTRproteinandthat
Therefore,PR-basedstrategiesinCF(asadministrationofcys- canbeinterruptedbycystamine.
tamineorgeneticrestorationofBECN1)mayhaveadualeffect,as Considering these factors, one would expect that sustaining
theyreducelunginflammationwhilerescuingafunctionalmutant PMresidenceofF508del-CFTRbyPRs,couldallowpotentiators
CFTRtotheepithelialsurface. toimproveCl− transportthoughPMresidentCFTRmolecules.
Indeed,ourrecentdataindicatethat,ifproteostasishasbeenpre-
TARGETINGAUTOPHAGYIMPROVESF508del-CFTRPMSTABILITYIN viouslyrestoredbycystamine,potentiatorscanbecomeeffective
AIRWAYCFEPITHELIAWELLBEYONDDRUGWASHOUT in sustaining the anti-inflammatory effects of cystamine in vivo
In addition to its ability to rescue F508del-CFTR, cystamine is in CftrF508del mice. Importantly, genistein, which has no effects
effective in delaying the disposal of PM resident F508del-CFTR onitsowninCftrF508del mice,synergisticallyinteractswithcys-
proteinandgeneratespermissiveconditionstoprolongF508del- tamine to reduce lung inflammation triggered by the challenge
CFTRPMresidencewellbeyondcystaminewashout.Theseeffects withlipopolysaccharide(LPS)fromPseudomonasaeruginosa.The
are mediated by the ability to restore BECN1 and autophagy,as same effects are observed if proteostasis has been previously re-
bothBECN1depletionand3-MAabrogatethebeneficialeffectsof established in CftrF508del mice by means of genetic manipula-
cystamine.PMresidentmutantF508del-CFTRisstillfunctional tions,suchaslentiviralexpressionofBECN1orshRNA-mediated
after cystamine withdrawal, as it retains the ability to respond depletionofSQSTM1(Lucianietal.,2012).
to forskolin added together with CFTR potentiators (as genis- These observations indicate that pharmacological measures
teinorVX-532orVX-770)wellbeyondthewashoutperiod.This thatamelioratethecellularenvironmentinwhichmutantCFTR
prolonged function of F508del-CFTR was observed in CF cell traffics,insteadofspecificallytargetingthemisfoldedproteinitself,
lines, as well as in primary brushed nasal epithelial cells from canresultinimproveddiseaseoutcome.
F508del-CFTR homozygous patients. Moreover, cystamine sus-
tainsF508del-CFTRre-locationatthelungepithelialsurfaceafter PERSPECTIVES
10daysfollowingwashoutinvivoinCftrF508del mice,unlessthat CFTR-REPAIRINGSTRATEGIESANDANTI-INFLAMMATORYTHERAPIES:
cystaminewascombinedwith3-MA(Lucianietal.,2012). TWOSIDESOFTHESAMECOIN?
These effects of cystamine can explain how the anti- Thedatadiscussedinthisreviewsuggestthatdifferentapproaches
inflammatory effects as a result of the restoration of autophagy couldbeenvisagedtocontrolCFlunginflammation.Somether-
(eitherviapharmacologicalintervention,ascystamine,orbythe apiesfocusonconventionaloremerginganti-inflammatorymol-
enforcedexpressionofBECN1)persistwellbeyonditswithdrawal ecules, downstream of CFTR. Others, as cystamine and other
inCftrF508delmice.TheyprobablyrelyonCFTRitself.Indeed,the yet-to-be-developed PRs,rely on the rescue and stabilization of
pro-autophagiceffectsofcystaminepersistafter10daysfollowing functionalCFTRatthePM.Apparently,cystaminecaninterrupt
cystaminewithdrawalunlessCFTRwasdepletedduringwashout, thedangerouscycleleadingtolunginflammation,thusopeninga
suggesting that these anti-inflammatory effects are mediated by newscenariointhesearchofthemostappropriateCFTR-repairing
theabilitytosustainafunctionalCFTRatthecellsurface(Luciani strategy.
etal.,2012).Therefore,sustainedCFTRfunctionatthePMcan SearchingtheappropriateCFTRcorrectorisachallengingissue
interruptthecascadeofROSgeneration,TG2activation,BECN1 indrugdevelopment.Anidealdrugcandidateforthetreatmentof
sequestration, and autophagy inhibition, and ultimately reduce F508del-CFTRpatientsshouldnotonlyaimatrescuingtrafficking
lunginflammation. ofmutantCFTR,beitthroughtheconventionalGolgi-mediated
Thesefindingscouldalsoexplainhowcystamine,whichisnot exocyticpathway(Wardetal.,1995;Quinton,1999;Amaral,2004,
an autophagy inducer,is highly effective in restoring autophagy 2011) or the unconventional GRASP-dependent secretory path-
withina“CFenvironment.”Thefactthatcystaminecanregulate way(Geeetal.,2011),butalsoatsustainingtherescuedmutant
peripheralproteostasisisalsosupportedbytheobservationthat CFTRatthePM,toallowthecombinedactionofpotentiatorson
cystamine (but not CFTR correctors as VX-325 or Corr-4a) is PMresidentF508del-CFTR.Ideally,onesinglemoleculeshouldbe
effectiveinsustainingPMstabilityofF508del-CFTRevenifithas endowedwithallthesepropertiestominimizeundesirableeffects.
previouslybeenrescuedatthePMbylowtemperature. OurrecentobservationssuggestthatF508del-CFTRpatientscould
FrontiersinPharmacology|PharmacologyofIonChannelsandChannelopathies January2013|Volume4|Article1|6
Villellaetal. Targetingautophagyincysticfibrosis
be sequentially treated with two single pharmacological agents, Maiuri), the Programma di Ricerca Scientifica di Rilevante
firstwithcystamineandthenwithCFTRpotentiators. Interesse Nazionale (2008RMJB3A_004, 2008) of the Minis-
Thefindingsdiscussedinthisreviewalsohighlighttheimpor- terodell’Istruzione,dell’UniversitàedellaRicerca(LuigiMaiuri,
tanceoftestingF508del-CFTRcorrectorsfortheircapacitytoexert Valeria Raia), RO1 HL093004 (Emanuela M. Bruscia), Telethon
a prolonged control of lung inflammation in pre-clinical mod- Grant # GGP12128 (Luigi Maiuri, Emanuela M. Bruscia, Vale-
els, before initiating clinical trials. So far, our strategy has been ria Raia, Maria Chiara Maiuri), Ligue Nationale contre le
successfully tested in nasal polyp biopsies from F508del-CFTR Cancer (Equipe labellisée; Guido Kroemer), AXA Chair for
homozygouspatientsaswellasinvivoinF508del-CFTRhomozy- Longevity Research,Agence Nationale pour la Recherche (ANR;
gousmice(Lucianietal.,2012).Atthisstage,clinicaltrialsonCF Guido Kroemer), European Commission (Active p53, Apo-
patientsarejustified. Sys, ChemoRes, ApopTrain; Guido Kroemer), Foundation pour
la Recherche Médicale (FRM), Institute National du Cancer
ACKNOWLEDGMENTS (INCa),CancéropôleIle-de-France(GuidoKroemer),Fondation
This was supported by the European Institute for Research in Bettencourt-Schueller,andtheLabExOnco-Immunology(Guido
Cystic Fibrosis and Italian Cystic Fibrosis Association (Luigi Kroemer).

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