Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease.

1.6 MB·English

by  Costa, A C

Citation:CellDeathandDisease(2013)4,e467;doi:10.1038/cddis.2012.205
&2013MacmillanPublishersLimited Allrightsreserved 2041-4889/13
www.nature.com/cddis
Drosophila Trap1 protects against mitochondrial
dysfunction in a PINK1/parkin model of Parkinson’s
disease
ACCosta1,SHYLoh*,1andLMiguelMartins*,1
Mitochondrialdysfunctioncausedbyproteinaggregationhasbeenshowntohaveanimportantroleinneurologicaldiseases,
such as Parkinson’s disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their
integrityandtheviabilityoftheirhostcell.First,molecularqualitycontrol,throughtheupregulationofmitochondrialchaperones
and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of
defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction
linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial
chaperoneTNFreceptor-associatedprotein1(TRAP1)wasrecentlyreportedtobeacellularsubstrateforthePINK1kinase.Here,
wecharacteriseDrosophilaTrap1nullmutantsanddescribethegeneticanalysisofTrap1functionwithPink1andparkin.We
show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its
upregulationinneuronsofPink1mutantrescuesmitochondrialimpairment.Additionally,theexpressionofTrap1wasableto
partiallyrescuemitochondrialimpairmentinparkinmutantflies;andconversely,expressionofparkinrescuedmitochondrial
impairmentinTrap1mutants.WeconcludethatTrap1worksdownstreamofPink1andinparallelwithparkininDrosophila,and
thatenhancingitsfunctionmayamelioratemitochondrialdysfunctionandrescueneurodegenerationinPD.
CellDeathandDisease(2013)4,e467;doi:10.1038/cddis.2012.205;publishedonline17January2013
SubjectCategory:Neuroscience
Accumulatingevidencesuggeststhatthedisruptionofmito- toactasacellularsubstrateofthePINK1kinase.Incultured
chondrial function and dynamics contributes to age-related cells, PINK1 expression shows a protective effect against
neurodegenerative diseases, such as Parkinson’s disease oxidative stress-induced apoptosis. Upon the induction of
(PD).1 Accordingly, cells have developed complex quality oxidative stress, TRAP1 is phosphorylated in a PINK1-
control mechanisms to cope with the different challenges dependentmanner,andthisphosphorylationisnecessaryto
constantlyimposedontheintegrityofmitochondria.2,3 conferresistancetoapoptosis.10
Molecularqualitycontrol,thefirstlevelofthemitochondrial TRAP1 is a member of the HSP90 family, which has been
defence mechanism, involves the upregulation of nuclear shown to protect cells from oxidative stress and apoptosis
genesthatencodemitochondrialchaperonesandproteases, causedbydifferentagents,11–14anditsupregulationintumours
ensuring the removal of misfolded and non-assembled hasbeenassociatedwithchemotherapyresistance.15,16
polypeptides.Thisstressresponseisknownasthemitochon- The fruit fly, Drosophila melanogaster, has recently
drialunfolded-proteinresponse(UPRmt).4 emerged as a powerful model system for studying the links
Furthermore, the highly dynamic nature of mitochondria between mitochondrial dysfunction and parkinsonian neuro-
facilitatesqualitycontrolthroughfusion andfission.5,6Thus, degeneration.17 In Drosophila, the loss of Pink1 leads to
damaged mitochondria can fuse with healthy organelles to mitochondrialdysfunctionassociatedwithmuscledegenera-
replenishstoresofdeficientcomponents.Whenthedamage tionandthelossofdopaminergicneurons.18,19Inaddition,a
isexcessive,suchprocessesareoverwhelmed;mitochondria decreasedexpressionofTrap1wasfoundtoenhancetheloss
become depolarised, and their components are targeted ofdopaminergicneuronsinfliesexpressingamutantformof
for recycling through a specific form of autophagy, termed humana-synucleinthatcausesPD.20
mitophagy.7,8 Here, we describe the consequences of Trap1 loss-
Recent findings have demonstrated that PTEN-induced of-function in Drosophila and its gain-of-function on mito-
putativekinase1(PINK1)iscapableofdirectlymodulatingthe chondrial integrity. We also genetically address the role of
activity of chaperones.9,10 The TNF receptor-associated Trap1inmitochondrialqualitycontrolthroughthePink1/parkin
protein 1 (TRAP1) is a mitochondrial chaperone suggested pathway.
1CellDeathRegulationLaboratory,MRCToxicologyUnit,Leicester,UK
*Correspondingauthor:LMMartinsorSHYLoh,CellDeathRegulationLaboratory,MRCToxicologyUnit,HodgkinBuilding,LancasterRoad,LeicesterLE19HN,UK.
Tel: þ441162525533or þ441162231501;Fax: þ441162525616or þ441162525616;E-mail:[email protected]@le.ac.uk
Keywords:Drosophila;mitochondria;Parkinson’sdisease;stress
Abbreviations: ATP,adenosinetriphosphate;PD,Parkinson’sdisease;ROS,reactiveoxygenspecies
Received7.11.12;revised5.12.12;accepted6.12.12;EditedbyGMelino.
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Results presented a significantly shorter lifespan compared with the
controls (Figure 1c). Given the proposed role of TRAP1 as
LossofTrap1increasessensitivitytostressandcauses a mitochondrial chaperone, we decided to investigate the
a decline in mitochondrial function. To investigate the consequences of exposing Trap14 mutants to increased
in vivo role of Trap1 in Drosophila, we first determined the levelsofstress.WeobservedadecreasedviabilityofTrap14
consequences of its loss. We obtained Trap14 mutant mutants subjected to heat stress (Figure 1d). The Trap14
flies, generated by an imprecise excision of the P-element mutants were also more sensitive to paraquat, a pesticide
insertion line P(EPgy2)Trap1EY10238. The excision deleted linkedtoPDbyepidemiologicalstudies21(Figure1e),andto
the majority of the Trap1 gene (Figure 1a). We next per- mitochondrial poisons such as rotenone (Figure 1f) and
formed quantitative real-time RT-PCR and confirmed that antimycin (Figure 1g). The Trap14 mutants showed an age-
Trap14mutantsshowatotallossofTrap1mRNA,andthatits dependentimpairedclimbingability,suggestingalocomotor
neighbouring genes were not affected by the imprecise deficit (Figure 1h). In Pink1 mutant flies, locomotor defects
excisionoftheP-element(Figure1b).TheTrap1mutantflies result from mitochondrial impairment in the Drosophila
were viable and developed to adulthood; however, they skeletal muscles, including the indirect flight muscle.18,19
Figure1 ThelossofTrap1inDrosophilacausesmotorimpairmentandanincreasedsensitivitytostress.(a)GenomicmapofTrap1(cytologicallocation42B2).Black,
untranslatedregions;lightblue,exons.TheP-elementinsertion(EY10238)isindicatedbytheredtriangle.Theneighbouringgenes(Vha16-1andBap170)areindicatedin
darkblue.Trap14deletion,delimitedbythedashedlines,removesmostoftheTrap1gene.(b)AnalysisoftheexpressionlevelsofTrap1anditsneighbouringgenes.
Expressionlevelsweremeasuredbyreal-timePCRin3-day-oldflieswiththeindicatedgenotypes(meanCt±S.D.,n¼4pergenotype).Expressionofactinwasusedasa
control.NoCtfortheTrap1transcriptwasdetectedinTrap14mutants.(c)Trap14mutants(red)haveareducedlifespancomparedwiththecontrols(black).Flyviabilitywas
scoredoveraperiodof75days,usingaminimumof100fliespergenotype.Thestatisticalsignificanceisindicated(log-rank,Mantel–Coxtest).(d–g)Trap1mutantfliesshow
enhancedsensitivitytostress.(d)Fliesweresubjectedtoheatstress,andviabilitywasassessedafter24h.Atotalof100males(4daysold)wereassayedforeachgenotype.
Theasterisksindicatesignificantvalues(Fisher’sexacttest,two-sided,alphao0.05).(e–g)Fliesweremaintainedonfoodsupplementedwiththeindicateddrugs,andthe
viabilitywasscoredoveraperiodof60days,usingaminimumof100fliespergenotype.Thestatisticalsignificanceisindicatedbytheasterisks(log-rank,Mantel–Coxtest).
(h)Trap14mutantsshowadecreaseinmotorperformance.Flieswiththeindicatedgenotypesandagesweretestedusingastandardclimbingassay(mean±S.D.,nZ80
fliesforeachgenotype).Theasterisksindicatesignificantvalues(two-wayANOVAwiththeBonferronimultiplecomparisontest)
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a b c
e
d
Figure2 ThelossofTrap1inDrosophilaresultsinareductionofmitochondrialfunctionandbraindopaminelevels.(a)DecreasedrespirationinTrap14mutantflies.
Activitywasmeasuredbyhigh-resolutionrespirometryin20-day-oldflies.Dataareshownasthemeans±S.D.(nZ4ineachgenotype).Significantvaluesrelativetothe
controlareindicatedbyasterisks(two-tailedunpairedttest).(b)DecreasedcomplexIproteinlevelsinTrap14mutantflies.Whole-fly(20daysold)proteinlysateswere
subjectedtowesternblotanalysiswiththeindicatedantibodies.(c)Trap14mutantshavelowerlevelsofATPcomparedwiththecontrols.TheATPlevelsweremeasuredusing
abioluminescentassay.Dataareshownasthemeans±S.D.(n¼4foreachgenotype).Thestatisticalsignificanceisindicatedbyasterisks(two-tailedunpairedt-test).
(d)AnalysisofTHlevelsinTrap1mutantflies.Fly-headproteinlysatesfrom20-day-oldflieswereusedforwesternblottinganalysiswiththeindicatedantibodies.(e)Trap1
mutantflieshavedecreaseddopamineandincreasedserotoninlevels.NeurotransmitterlevelswereassessedbyHPLCwithelectrochemicaldetection.Dataareshownasthe
means±S.D.(nZ5foreachgenotype).Thestatisticalsignificanceisindicatedbyasterisks(two-tailedunpairedt-test)
To determine if the loss of Trap1 lead to mitochondrial mutants revealed a significant decrease in the dopamine
impairment, we compared the respiration rates in the controls content,comparedtothecontrols(Figure2e).
and mutants. This analysis revealed a significant decrease in
the respiratory function of the Trap1 mutants (Figure 2a). Trap1 expression enhances motor performance and
Trap14 mutants also showed a decrease in the levels of protects against oxidative stress. Recently, TRAP1 was
the mitochondrial complex I (Figure 2b). Mitochondria are suggestedtoworkdownstreamofPINK1inthepreventionof
responsiblefortheproductionofthemajorityofcellularenergy mitochondrial dysfunction associated with PD pathogenesis.10
intheformofATP.ThemeasurementofATPlevelsinTrap14 Wethereforedecidedtotesttheepistaticrelationshipbetween
mutants revealed a significant decrease when compared with Trap1 and Pink1 in flies. We generated transgenic flies
controls(Figure2c).Takentogether,theseresultssuggestthat expressing full-length Trap1, and using UAS-GAL4 system23
the loss of the mitochondrial chaperone Trap1 results in a weconfirmedtheexpressionofthetransgenebywesternblot
decrease inmitochondrial function, whichis associatedwitha analysis(Figure3a).Toinvestigateitsintracellularlocalisation,
lossofATPinadultflies.Thelossofdopaminergicneuronscan we crossed UAS-Trap1 flies with a transgenic line expressing
be indirectly assessed through the analysis of the expression mitochondria-targeted green-fluorescent protein (mitoGFP).
levels of tyrosine hydroxylase (TH), an enzyme expressed ThisanalysisrevealedthatTrap1localisestothemitochondria
indopaminergicneurons.22Wefailedtodetectanydifferences (Figure 3b). To further investigate the phenotypic conse-
in the TH levels of Trap14 mutants (Figure 2d); however quences of Trap1 expression, we compared the locomotor
measuring neurotransmitter levels in the heads of Trap14 performanceofTrap1flieswiththatofcontrolsusingaclimbing
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Figure3 ExpressionofDrosophilaTrap1confersresistancetostress.(a)AnalysisoftheTrap1expressionlevelsintransgenicflies.Whole-flylysateswereanalysedby
westernblottingwiththeindicatedantibodies.(b)Trap1localisestothemitochondria.ConfocalanalysisoftheposteriorcompartmentoflarvalwingdiscscoexpressingTrap1
andamitoGFPunderthecontroloftheenGAL4driver.Inthemergedcolourimage,redcorrespondstoTrap1,bluetonucleiandgreentomitoGFP.(c)Theexpressionof
Trap1resultsinincreasedmotorperformance.Flieswiththeindicatedgenotypesandagesweretestedusingastandardclimbingassay(mean±S.D.,nZ120fliesforeach
genotype).Theasterisksindicatesignificantvaluesrelativetothecontrol(two-wayANOVAwiththeBonferronimultiplecomparisontest).(d)TheexpressionofTrap1reversed
thedecreaseinmotorperformanceinTrap14mutants.Flieswiththeindicatedgenotypesandagesweretestedusingastandardclimbingassay(mean±S.D.,nZ80fliesfor
eachgenotype).Theasterisksindicatesignificantvaluesrelativetothecontrol(two-wayANOVAwiththeBonferronimultiplecomparisontest).(e)Trap1expressiondoesnot
affectthetotallifespan.Atotalof100fliespergenotypeweremaintainedonnormalfoodforaperiodof80days.(f)Trap1-expressingflies(greenandblue)showenhanced
resistancetoparaquattoxicitycomparedwiththecontrols(black).Flyviabilitywasscoredoveraperiodof60daysusingaminimumof100fliespergenotype.Thestatistical
significanceisindicatedbytheasterisks(log-rank,Mantel–Coxtest)
assay.ThisanalysisrevealedthatTrap1expressionleadstoa TRAP1 phosphorylation was reported to be enhanced in
significantly enhanced climbing performance in aged flies responsetooxidativestress.10FliesexpressingTrap1showa
(Figure 3c) and suppressed the climbing defects of Trap14 normal lifespan compared with the controls (Figure 3e).
mutants (Figure 3d). We previously showed that treating flies However,Trap1expressionsignificantlyincreasedthelifespan
with paraquat leads to the upregulation of mitochondrial of paraquat-treated flies (Figure 3f), indicating that the
chaperones as a means to protect mitochondria from the expression of this mitochondrial chaperone protects against
oxidative stress triggered by this pesticide.24 Additionally, oxidativestressinvivo.
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dent inhibition of oxidative stress-induced cytochrome c
release and cell death.10 The mitochondrial dysfunction in
Pink1mutantsisassociatedwithmusclepathology,affecting
the indirect flight muscles, leading to the appearance of
thoracic indentations and impaired motor performance.18,19
The expression of Trap1 partially rescued the degree of
thoracic indentations (Figure 4a) and the impaired motor
performance in the Pink1B9 mutants (Figure 4b). Mito-
chondrial defects present in the Pink1 mutants are also
associated with a decreased lifespan.18 We noted that the
expression of Trap1 increased the lifespan of the Pink1
mutants (Figure 4c) and decreased their sensitivity to
paraquat (Figure 4d). Taken together, these observations
suggest that the ubiquitous expression of Trap1 protects
against the phenotypic consequences of Pink1 mutations,
and that Trap1 acts genetically downstream of Pink1. The
mitochondrial dysfunction in the Pink1 mutant flies is reflec-
ted by a loss of mitochondrial proteins and decreased
respiration. By expressing Trap1 in Pink1B9 mutants, we
observed a recovery of the mitochondrial protein content,
indirectly assessed through the analysis of the levels of a
complexIsubunit(Figure5a);apartialrecoveryoftherespi-
ration rates (Figure 5b) and ATP levels (Figure 5c). Taken
together, these observations indicate that Trap1 expression
results in a recovery of the mitochondrial function in Pink1
mutants.
Neuronal expression of Trap1 is sufficient to revert the
phenotype of Pink1 mutants. The genetic removal of the
function of Drosophila Pink1 results in apoptotic muscle
degeneration and a decrease in the brain dopamine levels
as a consequence of the degeneration of dopaminergic
neurons.19
The loss of mitochondria at neuronal synapses causes
defects in neurotransmission of the Drosophila neuro-
muscular junction (NMJ).25 Given that one of the most
important features of Pink1 mutant flies is the degeneration
ofboththeindirectflightmuscleanddopaminergicneurons,
wenexttestedwhetherthedegenerationoftheindirectflight
muscle involves a presynaptic component, and is therefore
precededbyneuronalloss.
WedetectedadecreaseintheTHlevelsinPink1B9mutants
thatwasreversedupontheexpressionofTrap1usingapan-
neuralelav-GAL4driver(Figure6a).Thisresultindicatesthat
theneuronalexpressionofTrap1cansuppressneurodegen-
Figure4 Trap1gain-of-functionrescuesPink1mutantflies.(a)Expressionof eration inPink1mutantflies.Next,we evaluatedtheconse-
Trap1rescuesthethoracicdefectsofPink1B9mutants.Thoracicindentationswere quence of the neuronal expression of Trap1 on phenotypes
countedupto24haftereclosion(n4900foreachgenotype).Theasterisksindicate associated withmuscle degeneration.The neuronalexpres-
significance(w2,two-sided,alphao0.05).(b)Trap1expressionsuppressesmotor sionofTrap1wassufficienttosuppressmuscledegeneration
impairmentinthePink1B9mutants.10-day-oldflieswiththeindicatedgenotypes inPink1B9mutants,whichwasreflectedinadecreaseinthe
were tested using a standard climbing assay (mean±S.D., n¼120 flies per
genotype).ThestatisticalsignificancerelativetoPink1B9isindicatedbyasterisks degreeofthoracicindentations(Figure6b)andanimproved
(one-way ANOVA with Dunnett’s multiple comparison test). (c and d) Trap1 climbing performance (Figure 6c). Finally, the neuronal
expressionenhancesthelifespanofPink1B9mutants.Aminimumof80fliesper expression of Trap1 significantly reversed the respiration
genotypeweremaintainedoneithernormal(c)orparaquat-containingfood(d).Fly deficit present in Pink1 mutant flies (Figure 6d). Taken
viabilitywasscoredoveraperiodof70days.Thestatisticalsignificanceisindicated together, these results indicate that the neuronal rescue of
bytheasterisks(log-rank,Mantel–Coxtest)
mitochondrialfunctioninPink1mutantsbyTrap1issufficient
tosuppressmuscledegenerationandmotorimpairment.
Expression of Trap1 suppresses mitochondrial dys-
function in Pink1 mutants. The expression of TRAP1 in Genetic epistasis analysis of parkin and Trap1. Upon
cultured cells was shown to be required for PINK1-depen- mitochondrial damage, PINK1 has been shown to recruit
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Figure5 DrosophilaTrap1reversesmitochondrialdysfunctioninPink1mutants.(a)Trap1expressionrestoresthemitochondrialcomplexIproteinlevelsinthePink1B9
mutantflies.Whole-flyproteinlysateswereusedforwesternblottinganalysiswiththeindicatedantibodies.(b)Trap1expressionpartiallyrescuesthemitochondrialrespiratory
defectsinthePink1B9mutants.MeasurementsofstatethreerespirationofcomplexIandcomplexII,andcomplexIIuncoupledrespirationbyhigh-resolutionrespirometry.The
dataareshownasthemeans±S.D.(n¼6foreachgenotype).Thestatisticalsignificanceisindicatedbytheasterisks(one-wayANOVAwithDunnett’smultiplecomparison
test).(c)ExpressionofTrap1increasedtheATPlevelsinthePink1B9mutants.ATPlevelsweremeasuredusingabioluminescentassay.Dataareshownasthemeans±S.D.
(n¼4foreachgenotype).Thestatisticalsignificanceisindicatedbytheasterisks(one-wayANOVAwithDunnett’smultiplecomparisontestrelativetothecontrol)
Figure6 TargetedneuronalexpressionofTrap1rescuesmitochondrialdysfunctioninPink1mutants.(a)DecreasedTHlevelsobservedinthePink1B9mutantscanbe
partiallyrestoredbytheexpressionofTrap1inneurons.Fly-headproteinlysatesfrom20-day-oldflieswereusedforwesternblottinganalysiswiththeindicatedantibodies.(b)
NeuronalexpressionofTrap1iscapableofdiminishingthedegreeofthoracicindentationsinthePink1B9mutants.Thoracicindentationswerecountedupto24haftereclosion
(n4300foreachgenotype).TheasterisksindicatethesignificancerelativetoPink1B9(w2,two-sided,alphao0.05).(c)TargetedneuronalexpressionofTrap1improvesthe
climbingperformanceofPink1B9mutants.10-day-oldflieswiththeindicatedgenotypesweretestedusingastandardclimbingassay(mean±S.D.,n¼120fliesper
genotype).ThestatisticalsignificancerelativetoPink1B9isindicatedbytheasterisks(one-wayANOVAwithDunnett’smultiplecomparisontest).(d)ExpressionofTrap1
specifically in neurons is sufficient to improve mitochondrial respiration in the Pink1B9 mutants. Complex II-uncoupled respiration was measured by high-resolution
respirometry.Dataareshownasthemeans±S.D.(n¼6foreachgenotype).Thestatisticalsignificanceisindicatedbytheasterisks(one-wayANOVAwithDunnett’smultiple
comparisontestrelativetothePink1B9mutant)
Parkin,acytosolicE3ubiquitinligase,todamagedmitochon- factor Mfn to label terminally damaged mitochondria for
dria.26,27 Parkin recruitment to damaged mitochondria has autophagic degradation,28 a process that has been defined
been proposed to cause the ubiquitination of the profusion asorganellarqualitycontrol.3,29TheimportanceofParkinas
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a downstream effector of PINK1 was confirmed in a series respect, the serine protease HtrA2, a downstream target of
of genetic studies in Drosophila, which showed that the PINK19,33 involved in mitochondrial molecular quality con-
expression of parkin could rescue the phenotype of Pink1 trol,34 could potentially partially compensate for any distur-
mutant flies.18,19 To address the epistatic relationship bet- bances in protein folding in the intermembrane space of
ween parkin and Trap1, we first tested whether Trap1 mitochondria.
expression could also rescue the parkin mutant phenotype. The expression of Trap1 was sufficient to enhance the
The expression of Trap1 partially rescued the degree of climbingperformanceofagedfliesandpromoteresistanceto
thoracic indentations (Figure 7a) but not the impaired motor oxidativestress(Figure3).Mitochondriainskeletalmuscles
performance in the parkin mutants (Figure 7b). We also arehighlyspecialisedtowardsenergyproductionforcontrac-
observed that the expression of Trap1 had a modest effect tileactivity,35andtherefore,theexpressionofthischaperone
onthesuppressionoftheATPlosspresentinparkinmutants mightcontributetothemaintenanceofmitochondrialperfor-
(Figure 7c) and caused a recovery in the mitochondrial manceintissueswithhigh-energydemand,suchasskeletal
protein content, indirectly assessed through the analysis of muscles,therebyenhancingthemotorperformance.
the levels of a complex I subunit (Figure 7d). Moreover, OurresultsindicatethattheneuronalexpressionofTrap1in
Trap1expressioninparkinmutantsledtoanincreasedlevel Pink1mutantsissufficienttosuppressmuscledegeneration.
ofsurvivalinfliesmaintainedonnormalfood(Figure7e)and Mitochondriahavebeenshowntobeconcentratedinboththe
on paraquat-containing food (Figure 7f). We next tested presynapticnerveterminalandthepostsynapticendplatesin
whether parkin expression could also rescue the Trap1 muscle,36 indicating that a decline in mitochondrial bioener-
mutant phenotype. When we expressed parkin in Trap14 geticfunctionmayaffectsynaptictransmissionandresultina
mutant flies, we observed a significant suppression of their lossofNMJinnervation.Ourdatasuggestthatmitochondrial
climbingdefects(Figure7g),aswellasamarkedrecoveryof dysfunctionatthepresynapticterminaloftheNMJisamajor
the levels of mitochondrial complex I (Figure 7h). Taken determinantofthepathologiesoftheskeletalmusclepresent
together,theseresultsshowthattheenhancedexpressionof in the Pink1 mutants, and that the compromise of the
Trap1 can partially compensate for the loss of parkin, and motor neuron function in the Pink1 mutants causes muscle
reciprocally, the enhanced expression of parkin can also dysfunction; therefore, is likely to precede muscle
ameliorate some of the Trap1 mutant-associated pheno- degeneration.
types. This suggests that both Trap1 and parkin act in a In Drosophila, the expression of parkin can rescue the
parallelpathwaythatmodulatesmitochondrialcompetence. phenotype of Pink1 mutants, presumably by enhancing the
organellar quality control through autophagic disposal of
defective mitochondria.29 Our data suggest that Trap1
Discussion
expression can partially compensate for the mitochondrial
We have characterised the Drosophila orthologue of the defects present in parkin mutant flies. Given that Trap1 is
TRAP1 chaperone and found that, similar to its mammalian likely to act as a molecular chaperone in the clearance of
counterpart,itlocalisestothemitochondria.Usingaloss-of- misfolded proteins, its expression may lead to enhanced
function approach, through the generation of Trap1 mutant molecular quality control in mitochondria, and therefore
flies that lack Trap1 expression, we determined that its loss decrease the demand for organellar quality control as a
compromises mitochondrial function. Mutations in either clearance mechanism for mitochondria overwhelmed by
Pink1 or parkin influence mitochondrial dynamics and func- excessiveproteinmisfolding.EnhancedexpressionofTrap1
tion, causing an impairment of fission, which affects male mayinturndecreasethedemandonorganellarqualitycontrol
fertility by compromising the normal development of mito- in parkin mutants, leading to a decrease in the degree of
chondrial derivatives (also known as nebenkern).18,19,30,31 dysfunctional mitochondria in these insects. We also
During spermatogenesis, Drosophila mitochondria undergo observed that the expression of parkin was capable of
significantmorphologicalchangesthatrequirefusiontoforma suppressing some of the phenotypic consequences asso-
large spherical nebenkern and subsequent fission when the ciated with Trap1 mutations. This suggests that both the
nebenkern unfurls to yield two mitochondrial derivatives.32 molecular and organellar pathways for mitochondrial quality
TheTrap1mutantsdevelopednormallytoadulthoodandwere controlworkinparallel,andtheenhancementofeitheroneof
both viable and fertile, suggesting that their mitochondrial these can relieve pressure on the other and contribute to a
defects do not compromise the mitochondrial fission and bettermitochondrialhealth.
fusionprocessesrequiredfornormalspermatogenesis.
AlthoughTrap1mutantsshownodefectsindevelopment,
MaterialsandMethods
theyhadadecreasedlifespan,progressivelocomotordefects Antibodies. The following primary antibodies were used in this study: anti-
andadecreaseinthebraindopaminelevels.Takentogether, TRAP1mouseIgG(clone30)(612342),BDTransductionLaboratories,Franklin
these results suggest that this mitochondrial chaperone is Lakes, NJ, USA; anti-NDUFS3 mouse monoclonal (ab14711), Mitosciences,
Eugene, OR, USA; anti-TH rabbit polyclonal (AB152), Millipore (Billerica, MA,
important to minimise the age-dependent decline of mito-
USA);andanti-a-tubulinIgG1cloneB-5-1-2,Sigma-Aldrich(StLouis,MO,USA).
chondrialfunction in post-mitotic cells, such as muscle cells
and neurons, which rely heavily on mitochondrial function.
Genetics and Drosophila strains. Transgenic strains with UAS-Trap1
The milder phenotype of Trap1 mutants compared with the
were generated by P-element-mediated transformation. Transgenic flies were
Pink1 mutants suggest that other effectors of molecular
generatedatBestGeneInc.(ChinoHills,CA,USA).Threeindependenttransgenic
quality control acting downstream of Pink1 can compensate lineswereobtained,andallweretestedforproteinexpression.Pink1B9,park25,
forthepotentialdefectscausedbythelossofTrap1.Inthis UASparkin62Bandda-GAL4flieswereagiftfromAWithworth(MRC,Centrefor
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Figure7 EpistaticrelationshipbetweenTrap1andparkin.(a)Trap1expressiondecreasesthedegreeofthoracicindentationsinparkinmutantflies,park25.Thoracic
indentationswerecountedupto24haftereclosion(n4250foreachgenotype).Theasterisksindicatethesignificance(w2,two-sided,alphao0.05).(b)Theexpressionof
Trap1failstorescuetheclimbingdefectofthepark25mutants.3-day-oldflieswiththeindicatedgenotypesweretestedusingastandardclimbingassay(mean±S.D.,n4120
fliespergenotype).(c)Trap1expressionincreasesthelevelsofATPinthepark25mutants.TheATPlevelsweremeasuredusingabioluminescentassay.Dataareshownas
themeans±S.D.(n¼4foreachgenotype).Thestatisticalsignificanceisindicatedbytheasterisks(one-wayANOVAwithDunnett’smultiplecomparisontestrelativeto
park25).(d)Trap1expressionrestoresthemitochondrialcomplexIproteinlevelsinthepark25mutantflies.Whole-flyproteinlysateswereusedforwesternblottinganalysis
withtheindicatedantibodies.(e)Trap1expressionenhancesthelifespanofthepark25mutants.Flyviabilitywasscoredoveraperiodof40daysusingaminimumof100flies
pergenotype.Thestatisticalsignificanceisindicatedbytheasterisks(log-rank,Mantel–Coxtest).(f)ExpressionofTrap1increasestheresistanceofpark25mutantsto
paraquat-inducedstress.Flyviabilitywasscoredoveraperiodof15daysusingaminimumof80fliespergenotype.Thestatisticalsignificanceisindicatedbytheasterisks
(log-rank,Mantel–Coxtest).(g)TheexpressionofparkinrescuestheclimbingdefectsofTrap14mutants.Flieswiththeindicatedgenotypesandagesweretestedusinga
standardclimbingassay(mean±S.D.,n460fliespergenotype).Theasterisksindicatesignificantvalues(two-wayANOVAwiththeBonferronimultiplecomparisontest).(h)
parkinexpressionrestoresthemitochondrialcomplexIproteinlevelsinTrap14mutantflies.Whole-flyproteinlysatesfrom20-day-oldflieswereusedforwesternblotting
analysiswiththeindicatedantibodies
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Trap1involvementinthePink1/parkinpathway
ACCostaetal
9
DevelopmentalandBiomedicalGenetics,UniversityofSheffield,Sheffield,UK). respiration buffer (20mM HEPES, 10mM KHPO, 110mM sucrose, 20mM
2 4
Trap14linewaskindlyprovidedbyDr.JETreisman(NewYorkUniversitySchool taurine, 60mM K-lactobionate, 0.5mM EGTA, 3mM MgCl, 1g/l fatty acid-free
2
ofMedicine,NY,USA).w1118,Act5C-GAL4,enGAL4andelav-GAL4lineswere BSA). The basal respiration rates were assayed in the absence of exogenous
obtained from Bloomington Stock Centre (Indiana University, Bloomington, IN, respiration substrates. For coupled (state 3) assays, complex I activity was
USA).Flystocksandcrossesweremaintainedonstandardcornmealagarmedia assayed in MiR05 respiration buffer in the presence of 2mM malate, 10mM
at251C.Thegenotypesofthefliesusedineachindividualfigurearelistedin glutamate and 5mM ADP. Complex II was assayed in respiration buffer
SupplementaryTableS1. supplementedwith1mMrotenone,10mMsuccinateand5mMADP.ADPwas
notaddedforthemeasurementofrespirationinuncoupledmitochondriaobtained
Molecular biology. Expression constructs encoding Drosophila Trap1 byonefreeze(liquidnitrogen)–thawcycleoftheflytissue.
sequence were generated by polymerase chain reaction using appropriate
adaptor primers. The PCR fragments were cloned into the GAL4-responsive Lifespan analysis. Groups of 10 newly eclosed males of each genotype
pUASTexpressionvectorforthegenerationoftransgenicflystrains. wereplacedintoseparatevialswithfoodandmaintainedat251C.Theflieswere
transferredtovialscontainingfreshfoodevery2–3days,andthenumberofdead
HPLC measurement of dopamine and serotonin. For sample flieswasrecorded.ThedataarepresentedasKaplan–Meiersurvivaldistributions,
preparation, 15 male fly heads (20 days old) were dissected under mild CO andthesignificancewasdeterminedbylog-ranktests.
2
anaesthesia,andquicklyfrozenindryice.Theheadswerethenhomogenisedin
Drugtreatments. Allthedrugsusedinthisstudywereincorporatedintothe
50ml chilled 0.1M perchloric acid using a motorised, hand-held tissue
flyfood.Theconcentrationsofeachofthedrugsinthefoodwerethefollowing:
homogeniser.Thechilledhomogenatesofflyheadswerefilteredthroughalow-
5mMforparaquat,600mMforrotenoneand50mMforantimycinA.Thenewly
binding Durapore (0.22mm) PVDF membrane using Ultrafree-MC centrifugal
eclosedmaleswereplacedwithnormalfoodingroupsof10for1daypriortotheir
devices.Thedopamineandserotoninmeasurementswereperformedimmediately
transfertovialscontainingdrug.Thefliesweretransferredtonewvialsevery2–3
after sample preparation. Supernatant fluid (30ml) was eluted at a flow rate of
days,andthenumberofdeadflieswasrecorded.Theassaywascompletedafter
50ml/min through a 150(cid:2)1.0mm C18 column. The mobile phase contained
allthefliesdied.Aminimumof80fliespergenotypewereanalysed.Theresults
50mM phosphoric acid, 8mM NaCl, 0.1mM EDTA, 12.5% (v/v) methanol,
are presented as Kaplan–Meier survival distributions, and the significance was
500mg/l octanesulfonic acid (OSA) (pH 6.0). Analysis was performed usingan
determinedbylog-ranktests.
AlexysLC-ECsystemequippedwithaDECADEIIelectrochemicaldetector.
Preparation of Drosophila protein lysates and western blot
Immunofluorescenceandconfocalmicroscopy. Thirdinstarlarvae
analysis. Singleadultflies(3,10or20daysold)werehomogenisedusinga
were dissected, fixed in 4% paraformaldehyde, stained with the anti-TRAP1
motorised pestle in lysis buffer (0.1% Triton-X100, 10mM EDTA, 1mM DTT,
antibodyandincubatedwithHoechst33342.Wingimaginaldiscsweremounted
100mMKCl,20mMHEPES(pH7.5),5%glycerol,1mg/mlchymostatin,1mg/ml
onglassslideswithVectashield(VectorLaboratories,Burlingame,CA,USA)and
leupeptin, 1mg/ml pepstatin A and 1mg/ml antipain). The lysates were
imagedonaZeissLSM510confocalmicroscope.
subsequently cleared by centrifugation. The proteins were separated on 12 or
10%SDS-PAGEgelsandtransferredtoImmobilonPVDFmembranes(Millipore).
Statisticalanalysis. Thedataarepresentedasmeanvalues,andtheerror
Themembraneswereblockedin5%milkandthenincubatedwiththeindicated
barsindicate±S.D.InferentialstatisticalanalysiswasperformedusingthePrism
primary antibody prior to incubation with the appropriate HRP-conjugated
andStatMatesoftwarepackages(www.graphpad.com).Thesignificancelevelis
secondary antibody. The antibody complexes were visualised by enhanced
indicatedas****forPo0.0001,***forPo0.001,**forPo0.01,*forPo0.05
chemiluminescence (ECL). The results are representative of at least three
andnsforP40.05.
biologicalreplicatespergenotype.
ATPassays. Fivemaleflies(10or20daysold)werehomogenisedin100ml
RNAextractionandquantitativereal-timeRT-PCR. Theisolationof
of6Mguanidine-HClinextractionbuffer(100mMTrisand4mMEDTA,pH7.8)to
totalRNAfromthreeadultfliespersamplewasperformedusingtheRNeasyMini
inhibitATPases.
Kit (Qiagen, Hilden, Germany). Quantitative real-time RT-PCR was performed
Homogenised samples were subjected to rapid freezing in liquid nitrogen,
on an M(cid:2)4000 (Stratagene, La Jolla, CA, USA) real-time cycler using the
followed by boiling for 5min. Samples were cleared by centrifugation, and
QuantiTect SYBR Green RT-PCR system (Qiagen). Primers used (QuantiTect
supernatantwasdiluted(1/50)withextractionbufferandmixedwithaluminescent
Primer Assay, Qiagen) were Cat. No. QT00967393 (actin 79B, used as a
solution(CellTiter-GloLuminescentCellViabilityAssay,Promega,Fitchburg,WI,
housekeeping gene), Cat. No. QT00940520 (Trap1), Cat. No. QT00501130,
USA).LuminescencewasmeasuredonanInfiniteM200Promultifunctionreader
(Vha16-1)andCat.No.QT00940527,(Bap170).Thequantificationwasperformed
(TECAN, Ma¨nnedorf, Switzerland). The relative ATP levels were calculated by usingthecomparativeCtmethod.38
dividingtheluminescencebythetotalproteinconcentration,whichwasdetermined
bytheBradfordmethod.
Heatstress. Forheatsensitivity,4-day-oldmalefliesweredividedintogroups
of15andplacedinemptyvials(nofoodorwater).Thefliesweremaintainedat
Climbing assays. The climbing assays were performed as previously
251Cfor30min,afterwhichthethermostresswasappliedbyplacingthevialsin
described,37 using a countercurrent apparatus developed initially for phototaxis
awaterbathat371Cfor1h.Thisstepwasfollowedbya30-minrecoveryat251C
experiments.Atotalof20maleflieswereplacedintothefirstchamber,tapedto
andanewheatstressfor1hat371C.Theflieswerethentransferredtovials
the bottom and then given 20s to climb a distance of 10cm. Those flies that
containing fresh food and maintained at 251C. The number of living flies
successfullyclimbed10cmorbeyondin20swerethenshiftedtoanewchamber,
wasrecorded24hlater.Atotalof100fliespergenotypeweretestedforthermo
andbothsetsoffliesweregivenanotheropportunitytoclimbthe10-cmdistance.
stress.
Thisprocedurewasrepeatedatotaloffivetimes.Afterfivetrials,thenumberof
fliesineachchamberwascounted.Atleast120flieswereusedforeachgenotype
tested. ConflictofInterest
Theauthorsdeclarenoconflictofinterest.
Respirometryanalysis. Mitochondrialrespirationwasassayedat371Cby
high-resolution respirometry using an OROBOROS Oxygraph. DatLab software
package (OROBOROS, Innsbruck, Austria) was used for the data acquisition Acknowledgements. WewouldliketothankAlexWhitworth,Dr.JETreisman
(2-s-timeintervals)andanalysis,includingthecalculationofthetimederivativeof andtheBloomingtonDrosophilaStockCentrefortheflystocks,andJalpaParmar
theoxygenconcentration,signaldeconvolutiondependentontheresponsetime fortheflyfoodpreparation.WethankIneˆsPCastro,NicoletaMoisoiandRoberta
of the oxygen sensor and correction for instrumental background oxygen flux. Tufiforreagentsandadvice.WealsothankDr.AaronVoigtforsharingunpublished
Respiration was assayed by homogenising two flies using a pestle in MiR05 data.ThisworkisfundedbytheMedicalResearchCouncil,UK.
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