Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation

35 Pages·2012·0.75 MB·English

Human Molecular Genetics, 2012, Vol. 21, No. 13 2936–2945
doi:10.1093/hmg/dds125
Advance Access published on April 5, 2012
Loss of HDAC6, a novel CHIP substrate, alleviates
abnormal tau accumulation
Casey Cook, Tania F. Gendron, Kristyn Scheffel, Yari Carlomagno, Judy Dunmore,
∗
Michael DeTure and Leonard Petrucelli
Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA
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ReceivedJanuary19,2012;RevisedMarch12,2012;AcceptedApril2,2012 lo
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The abnormal accumulation of the microtubule-binding protein tau is associated with a number of neurode- fro
m
generativeconditions,andcorrelateswithcognitivedeclineinAlzheimer’sdisease.Theubiquitinligasecar- h
boxy terminus of Hsp70-interacting protein (CHIP) and the molecular chaperone Hsp90 are implicated in ttps
protein triage decisions involving tau, and have consequently been targeted for therapeutic approaches ://a
c
a
aimed at decreasing tau burden. Here, we present evidence that CHIP binds, ubiquitinates and regulates ex- d
e
pressionofhistonedeacetylase6(HDAC6).AsthedeacetylaseforHsp90,HDAC6modulatesHsp90function m
ic
and determines the favorability of refolding versus degradation of Hsp90 client proteins. Moreover, wedem- .o
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onstratethatHDAC6levelspositivelycorrelatewithtauburden,whileadecreaseinHDAC6activityorexpres- p.c
o
sion promotes tau clearance. Consistent with previous research on Hsp90 clients in cancer, we provide m
evidence thatalossof HDAC6 activityaugmentstheefficacy ofanHsp90inhibitor anddrivesclientdegrad- /hm
g
ation,inthiscasetau.Therefore,ourcurrentfindingsnotonlyidentifyHDAC6asacriticalfactorfortheregu- /a
lationoftaulevels,butalsoindicatethatamulti-facetedtreatmentapproachcouldmoreeffectivelyarresttau rtic
le
accumulation in disease. -a
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INTRODUCTION /13
Previously,theubiquitinligasecarboxyterminusofHsp70- /2
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The microtubule-binding protein tau is central to the regula- interactingprotein(CHIP)andthemolecularchaperoneHsp90 3
6
tion of axonal outgrowth and cellular morphology, as well have been shown to play pivotal roles in protein triage deci- /2
9
asneuronaltransport(1–3).Inanumberofneurodegenerative sions involving tau (6–9). CHIP has a unique binding affinity 00
7
diseases classified as tauopathies, which include frontotem- forabnormallyphosphorylatedtauandisrequiredfortauubiqui- 0
7
poral dementia with parkinsonism associated with chromo- tinationandtargetingtoproteasomesfordegradation(6–8).For b
y
some 17, progressive supranuclear palsy, corticobasal its part, Hsp90, a ubiquitous, constitutively expressed protein g
u
degeneration, and Alzheimer’s disease (AD), tau becomes that constitutes 1–2% of total cellular protein in eukaryotic es
hyperphosphorylated and aggregates into filaments, thus cells (10,11), functions to maintain its client proteins in a t o
n
losing the ability to bind and stabilize microtubules (4,5). properlyfoldedstateandtherebysuppresses their aggregation 1
7
Thesetaufilamentscontinuetoaggregateandformincreasing- (10). During conditions of stress, this dual function of Hsp90 N
o
ly insoluble deposits referred to as neurofibrillary tangles. Al- helps to repair the pool of damaged client proteins, thus ve
though AD is the most common tauopathy and most frequent serving to reestablish a state of cellular equilibrium (12). mb
cause of dementia, the available treatment options only treat Over 100 proteins have been reported to be clients of Hsp90 er 2
the symptoms of AD, and do nothing to alleviate the under- (12,13), including protein kinases, transcriptional regulators, 0
1
lying pathology. Therefore, understanding the mechanism by and steroid receptors (12). Of particular relevance to the 8
which hyperphosphorylated tau is cleared by neurons, and current report, tau is also an Hsp90 client (9,14).
developing therapeutics to eliminate these toxic species are Following binding of Hsp90, client proteins either enter a
of considerable interest. refolding pathway, leading to a functional, properly folded
∗Towhomcorrespondenceshouldbeaddressedat:DepartmentofNeuroscience,MayoClinic,4500SanPabloRoad,Jacksonville,FL32224,USA.
Tel: [phone];Fax: [phone];Email:[email protected]
# The Author 2012. Published by Oxford University Press.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/
licenses/by-nc/2.5),whichpermitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisprop-
erlycited.
Human Molecular Genetics, 2012, Vol. 21, No. 13 2937
client protein or they are targeted for degradation by the species were observed in CHIP-overexpressing cells, likely
ubiquitin-proteasome system (15). The specific components representing ubiquitinated HDAC6.
of the Hsp90 complex ultimately determine whether client To further characterize the interaction between CHIP and
refolding or degradation occurs (16). Nucleotide binding to HDAC6, we sought to determine whether CHIP binds
Hsp90 is proposed to alter its conformation and define the HDAC6directlyorwhetherchaperonesHsp70/Hsp90serveto
subsetofchaperoneswithwhichitinteracts(16).IntheADP- bridge the two proteins. Thus, HeLa cells were co-transfected
bound conformation, Hsp90 associates with client-bound tooverexpressHDAC6andeitherwild-type(WT)myc-CHIP,
Hsp70/Hsp40 complexes. At this point, the complex may myc-CHIP(K30A)ormyc-CHIP(H260Q).TheK30Amutation
recruit ubiquitin ligases, such as CHIP, to direct the client to prevents CHIP from docking with chaperones Hsp70/Hsp90,
proteasomes for degradation. The replacement of ADP with whiletheH260Qmutation,presentwithintheU-boxofCHIP,
ATP alters Hsp90 conformation, releasing Hsp70/Hsp40 and obliteratesubiquitinligaseactivity(25).Uponimmunoprecipi-
D
allowing the recruitment of other cochaperones, including tationforHDAC6,bothWTmyc-CHIPandmyc-CHIP(H260Q) ow
p23. This complex folds and stabilizes the client now bound were found to bind HDAC6 (Fig. 1B). In contrast, little myc- nlo
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by Hsp90. Notably, the acetylation state of Hsp90 modulates CHIP(K30A)co-immunoprecipitatedwithHDAC6,suggesting d
e
Hsp90function(17–20);specifically, Hsp90hyperacetylation that either Hsp70 or Hsp90 is required for the interaction d
decreases the affinity of Hsp90 for ATP and oncogenic client betweenCHIPandHDAC6. fro
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proteins, and causes the dissociation of p23 from the Hsp90 SinceCHIPplaysaroleinpromotingtheubiquitinationand h
complex, leading to an impairment in chaperone function proteasomal degradation of several proteins, it was next eval- ttp
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and promoting client degradation (18,21). uatedwhetherlossofCHIPinfluencesHDAC6levels.Tothis ://a
Of importance, inhibition or depletion of histone deacety- end,HeLacellsweretransfectedwitheithersiCONorsiCHIP ca
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lase 6 (HDAC6) promotes the hyperacetylation of Hsp90, for48h,andsubsequentlytransfectedwithmycHDAC6foran e
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thus augmenting the polyubiquitination and subsequent deg- additional 24h. Cells were then treated with cycloheximide ic
.o
radation of Hsp90 client proteins (17–20). Hyperacetylation (CHX) to inhibit new protein translation, and cell lysates u
p
of Hsp90 due to HDAC6 depletion also leads to an increased were collected at various time points thereafter. Interestingly, .c
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binding affinity of Hsp90 for Hsp90inhibitors (21,22). Hsp90 loss of CHIP increased steady-state levels of mycHDAC6 by m
/h
inhibitors disrupt Hsp90 chaperone function such that client 2.2-fold in cultured cells, and also prolonged its half-life m
g
proteins are instead degraded (reviewed in 23). That cotreat- (Fig. 2A–C). /a
ment of leukemia cells with HDAC6 and Hsp90 inhibitors Toinvestigatetheimpactofdecreased CHIPlevelsinvivo, rtic
synergistically promotes the degradation of Hsp90 client pro- brain homogenates from CHIP2/2 and CHIP+/+ mice were le-a
teins suggests that the hyperacetylation of Hsp90 by HDAC6 prepared. In agreement with previous reports, loss of CHIP b
s
augments the pro-degradation effects of Hsp90 inhibitors led to an increase in PHF1-immunopositive tau levels tra
c
(21,24). Therefore, it is possible that alterations in Hsp90 (Fig. 2D) (8). In addition, a significant increase in HDAC6 t/2
acetylation,eitherthroughdifferencesinexpressionoractivity levels was observed in CHIP2/2 mice (Fig. 2D and E). Col- 1/1
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of the deacetylase HDAC6, determine the sensitivity of the lectively, these results indicate that HDAC6 is a substrate of /2
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Hsp90chaperonecomplextochemicalmodulation,ultimately CHIP, and that the loss of CHIP impacts HDAC6 turnover 3
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deciding the fate of Hsp90 client proteins, such as tau. Here, and leads to increased levels of HDAC6 in vitro and in vivo. /2
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we provide evidence that CHIP interacts with and regulates 0
0
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the half-life of HDAC6 in cells and in mice. In addition, we 0
Overexpression of HDAC6 promotes tau accumulation 7
demonstrate that increased levels of HDAC6 lead to an accu- b
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mulation of tau, while decreased HDAC6 levels or activity Given that the loss of CHIP is associated with an increase in g
u
promotes tau clearance and increases the efficacy of Hsp90 both HDAC6 and tau levels (Fig. 2), and that HDAC6 levels e
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inhibitors.Therefore,wehypothesizethatCHIP,byregulating areincreasedinAD(26),theeffectofHDAC6overexpression t o
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HDAC6levels,influencesproteintriagedecisionsbymodulat- ontauaccumulationwasevaluated bytransfectingHeLacells 1
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ing the refolding and degradation activities of Hsp90. to co-express tau with the pathogenic P301L mutation with N
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either GFP or HDAC6. Surprisingly, the co-expression of v
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P301L-tau with HDAC6 resulted in a dramatic upregulation m
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of tau levels, as detected using both PHF1 (pS396/S404) and er 2
RESULTS E1(humantauspecific)antibodies(Fig.3A–D).As theover- 0
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expression of HDAC6 led to a more significant increase in 8
CHIP interacts with and ubiquitinates HDAC6
PHF1-tauthantotalhumanE1tau(Fig.3A–D),HDAC6over-
To investigate whether HDAC6 is a substrate for CHIP, expression may preferentially increase the accumulation of
co-immunoprecipitation experiments were conducted in the pathologically phosphorylated tau species. To verify whether
presence and absence of proteasome inhibition (MG132) to HDAC6 overexpression specifically affects tau levels, cells
slow the degradation of ubiquitinated proteins. HEK293T were transfected to coexpress HDAC6 and either GFP or
cells were transfected to express HA-tagged ubiquitin a-synuclein. No changes in GFP or a-synuclein expression
(HA-ubiquitin) and myc-HDAC6, along with either CHIP or were observed in the presence of HDAC6 (Supplementary
GFP, and HDAC6 was then immunoprecipitated from cell Material, Fig. S1).
lysates using an anti-myc antibody. As shown in Figure 1A, HDAC6-mediated tau accumulation may depend on the
CHIP co-immunoprecipitated with HDAC6, and multiple deacetylase activity of HDAC6. Alternatively, given that
HDAC6- and HA-ubiquitin-positive high molecular weight HDAC6 contains a ubiquitin-binding domain and has been
2938 Human Molecular Genetics, 2012, Vol. 21, No. 13
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Figure1.CHIPinteractswithandubiquitinatesHDAC6invitro.(A)HEK293TcellsweretransfectedwitheitherGFPorCHIPinthepresenceofmycHDAC6 ad
andHA-ubiquitin,andtreatedwiththeproteasomeinhibitor,MG132,for24h.Ananti-mycantibodywasusedtoimmunoprecipitatemycHDAC6fromcell ed
lysates, and samples were analyzed by western blot. (B) HEK293T cells were transfected with untagged HDAC6 or vector, and either GFP, myc-CHIP, fro
mycCHIP (K30A mutant) or mycCHIP (H260Qmutant). An HDAC6 antibody was used for immunoprecipitation, and cell lysates and immunoprecipitates m
wereprobedforanti-myc. h
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Figure2.LossofCHIPprolongsthehalf-lifeofHDAC6invitroandincreasesHDAC6levelsinvivo.(A)HeLacellstransfectedwithmycHDAC6weretreated b
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withnon-targetingsiRNA(siCON)orsiCHIPtoknock-downendogenousCHIPfor48h.Cellswerethenexposedtocycloheximide(CHX;0.1mg/ml)for4,8or g
12htoinhibitthecontinuedtranslationofmycHDAC6,andmycHDAC6levelswereexaminedbywesternblotusingananti-mycantibody.(B)Steady-state ue
levels(0hCHX)ofmycHDAC6weresignificantlyhigherinculturestreatedwithsiCHIPcomparedwithsiCON-treatedcultures(P,0.005,t-test,n¼3).(C) st o
TherateofdegradationofmycHDAC6post-CHXtreatmentinsiCON-orsiCHIP-treatedcultureswascomparedbynormalizingvaluesateachtimepointto n
their respective levels at 0h CHX treatment. There was a significant difference in the rate of degradation between siCON- and siCHIP-treated samples as 1
7
assessed by two-way ANOVA (effect of siCHIP F¼388.3; P,0.0001; effect of CHX treatment F¼72.39; P,0.0001; interaction F¼10.71; P¼ N
0.0004).Estimatedhalf-lifewasthefollowing:siCON,4.7h;siCHIP,7.9h.(D)HDAC6andphosphorylatedtaulevelswereexaminedinbrainhomogenates ov
fromCHIP+/+andCHIP2/2miceusinganti-HDAC6andPHF1antibody,respectively.LossofCHIPleadstoincreasedHDAC6levelsandanti-PHF1immu- em
noreactivephospho-tauinmice.(E)HDAC6levelsweresignificantlyhigherinCHIP2/2comparedwithCHIP+/+brainhomogenates(t¼3;P,0.05,n¼4). b
Alldataarepresentedasmean+SEM.∗∗∗P,0.001∗P,0.05. er 2
0
1
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showntoplayaroleinsequesteringubiquitinatedproteinsand combination with previous reports demonstrating that
targeting them to the aggresome, it is possible that HDAC6 HDAC6 regulates the acetylation state of Hsp90 (18,20–22),
directly binds and sequesters tau, thereby preventing tau deg- suggest that HDAC6 overexpression increases the affinity of
radation. Thus, to determine whether the deacetylase activity the Hsp90 chaperone complex for pathological tau species
ofHDAC6isrequiredtopromotetauaccumulation,acatalyt- bydecreasingHsp90acetylation,therebyblockingtaudegrad-
icallyinactivemutant(H216A/H611A)ofHDAC6wasgener- ation.
atedandcellsweretransfectedtoexpresseitherthismutantor
WT HDAC6. As shown in Figure 3E–G, loss of deacetylase
HDAC6 deficiency promotes tau degradation
activity prevented HDAC6 from promoting tau
accumulation, indicating that the catalytic activity of We and others have shown that inhibitors of Hsp90 enhance
HDAC6 is required for this effect. These results, in the degradation of mutated or hyperphosphorylated tau in
Human Molecular Genetics, 2012, Vol. 21, No. 13 2939
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Figure3.OverexpressionofHDAC6promotestheaccumulationoftauinvitro.(A)HeLacellsweretransfectedwithGFP,P301L-V5orHDAC6expression ://ac
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constructsfor48h.LevelsofphosphorylatedandtotaltaupresentincelllysateswerethenexaminedbyimmunoblottingwithPHF1andE1antibodies,respect- d
e
ively.HDAC6overexpressionwasconfirmedbyprobingforHDAC6.(B)AnalysisofPHF1-positivetaulevelsbystudent’st-testverifiedthattherewasasig- m
nificant increaseincells overexpressingHDAC6(t¼14.5;P,0.0001).(C)QuantificationofE1immunoreactivitynormalizedtoGAPDHalsorevealed a ic
significantincreaseincellsoverexpressingHDAC6(t¼11.04;P¼0.0004).(D)TheratioofPHF1-positivetautoE1-positivetauwassignificantlyincreased .ou
uponoverexpressionofHDAC6(t¼5.9;P¼0.004),indicatingthatPHF1-tauisspecificallyincreasedincellsoverexpressingHDAC6.(E)HeLacellswere p.c
cotransfectedwithP301L-V5andeitherGFP,mycHDAC6oracatalyticallyinactivemycHDAC6construct(H216/611Amutant).Celllysateswereimmuno- o
m
blotted for PHF1-tau, myc or actin to control for protein loading. (F) PHF1-tau levels were quantified and normalized to actin, and analyzed by one-way /h
ANOVA. OverexpressionofmycHDAC6causeda significantincrease inPHF1-taulevels (P,0.001),butthere wasno effectof the catalytically inactive m
g
mycHDAC6mutantonPHF1-tau(P.0.05).(G)Anti-myclevelswereassessedandnormalizedtoactin,verifyingthatbothmycHDAC6andthecatalytically /a
inactivemutantwereexpressedatsimilarlevels.Alldataarepresentedasmean+SEM(n¼3).∗∗∗P,0.0001,∗∗P,0.001,∗P,0.01. rtic
le
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vitroandinvivo,aneffectthatisdependentuponthepresence (Fig. 4E and F). Taken together, these results suggest that stra
of CHIP (9,14,27). More specifically, our work reveals that HDAC6 deficiency enhances tau degradation induced by c
Hsp90 inhibition does not simply facilitate the proteasomal Hsp90 inhibitors, which is consistent with previous reports t/21
degradation of tau, but selectively targets aberrantly phos- showing that HDAC6 deficiency leads to an increased acetyl- /13
phorylated tau, indicating that the surveillance mechanism of ationofHsp90,therebyincreasingthesensitivityofHsp90for /29
3
the chaperone network is a highly sensitive and controlled Hsp90 inhibitors (21,22). 6
/2
system (9,14,27,28). Since loss of HDAC6 activity has been 9
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shown to increase the affinity of Hsp90 for Hsp90 inhibitors, 07
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suchas17AAG,aswellasenhance17AAG-mediateddegrad- 7
b
ation of Hsp90 client proteins (21,22), we sought to evaluate y
HDAC6 deficiency modulates the expression g
whether HDAC6 depletion would likewise enhance the deg- u
of the cochaperone p23 e
radation of tau induced by Hsp90 inhibition. HeLa cells st o
were treated with siCON or siHDAC6, and then exposed to The cochaperone p23 locks Hsp90 in a conformational state n
1
17AAG. To confirm efficient Hsp90 inhibition, Hsp70 induc- that has a high affinity for client proteins (31). Hsp90 com- 7
N
tion, a consequence of Hsp90 inhibition (15,29), was also plexes that contain p23 promote the folding and stabilization o
v
monitored(Fig.4AandC).IncontrasttoHDAC6overexpres- of clients, rather than their degradation. Rao et al. demon- e
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sion, HDAC6 knockdown resulted in a decrease in P301L tau stratedthatHDAC6deficiencyleadstoadecreasedinteraction b
e
levels compared with tau levels in siCON-treated cells, as between Hsp90 and p23 (21). In addition, we have reported r 2
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assessed using the PHF1 antibody (Fig. 4A and B, compare that the loss of p23 expression significantly decreases tau 1
8
vehicle-treated samples). In addition, treatment with 17AAG levels (9). Intriguingly, a significant decrease in p23 was
led to a dose-dependent decrease in tau levels, which was detected upon both HDAC6 knockdown (Fig. 5A and B) and
enhanced in cells treated with siHDAC6 (Fig. 4A and B). In HDAC6inhibitionwithM344(Fig.5CandD),demonstrating
a similar fashion, enhanced tau clearance was observed upon that the loss of HDAC6 function is associated with decreased
HDAC6 inhibition. Specifically, treatment with M344, an levels of p23. Given that decreased HDAC6 activity has been
HDAC6 inhibitor (30), led to a significant decrease in tau shown to decrease the interaction between Hsp90 and p23
levels, and also augmented the ability of 17AAG to promote (21), the current results suggest that HDAC6 deficiency pro-
tau degradation (Fig. 4C and D). As expected, the half-life motes client degradation through the increased formation of
of tau is decreased in cells treated with M344, an effect that Hsp90 chaperone complexes that lack p23, which are charac-
is reversed in the presence of a proteasome inhibitor terized by a very low affinity for client proteins, such as tau.
2940 Human Molecular Genetics, 2012, Vol. 21, No. 13
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Figure4.HDAC6deficiencyorinhibitiondecreasestaulevels,andfunctionssynergisticallywithHsp90inhibitiontopromotethedegradationoftau.(A)HeLa 6
cellsweretransfectedwithsiCONorsiHDAC6for24h,andsubsequentlytransfectedwithP301L-V5foranother24h.Cellswerethentreatedwith0.1or1mM /29
of17AAGfor24h,andcelllysatesimmunoblottedforPHF1-tau,HDAC6,Hsp70andGAPDHtocontrolforproteinloading.(B)AnalysisofPHF1-taulevelsby 00
two-wayANOVAverifiedasignificanttreatmenteffectof17AAG(F¼21.1,P¼0.0001)andsiHDAC6(F¼24.5,P¼0.0003),aswellasasignificantinter- 70
actionbetween17AAGandsiHDAC6(F¼5.75,P¼0.02).(C)HeLacellsweretransfectedtooverexpressP301L-V5for24h,pretreatedwiththeHDAC6 7 b
ienxhaimbiitnoerdMb3y44imfmorun1ohblaonttdinsgu.b(sDeq)uAenntalylystriesaotefdPwHiFth1-0ta.1u–l1evmeMls1b7y-AtwAoG-wfaoyraAnNaOdVdiAtiocnoanlfi2r4mhe.dLaevseiglsniofifcPanHtFt1re-taatumeanntdeHffsepc7t0opfrbeosethnt1i7nAcAelGlly(Fsa¼tes33w.7er3e, y gu
P,0.0001)andM344(F¼153.3,P,0.0001).Alldataarepresentedasmean+SEM(n¼3).(E)HeLacellsweretransfectedtooverexpressP301L-V5 es
for 24h, pretreated with 1mM MG132 for 1h and subsequently treated with 1mM M344 and CHX for 4, 8 or 12h to inhibit the continued translation of t o
n
tau.LevelsofPHF1-tauandGAPDHpresentincelllysateswereexaminedbyimmunoblotting.(F)TherateofdegradationofPHF1-taupost-CHXtreatment 1
winavseahiscilgen,iMfic3a4n4tdoirffMerGen1c3e2i+ntMhe34ra4t-etroefatdeedgcraedllastiwoansbceotmwepeanrevdebhyicnleo,rMma3l4iz4inagndvaMluGes13a2te+acMht3im44e-tpreoaintetdtosathmepirlersesapseacstisveessleedveblysatwt0o-hwCayHAXNtrOeaVtmAe(netf.fTechteoref 7 No
M344orMG132+M344treatmentF¼6.4;P¼0.01;effectofCHXtreatmentF¼95.3;P,0.0001;interactionF¼3.4;P¼0.04).Estimatedhalf-lifewas ve
thefollowing:vehicle,5h;M344,3.8h;MG132+M344,6.4h).Datafromthisexperimentarepresentedasmean+SEM(n¼2).∗∗P,0.001,∗P,0.05. m
b
e
r 2
0
Inhibition of HDAC6 decreases tau levels in primary 1
M344 (Fig. 6D), suggesting that M344 drives the formation 8
neuronal cultures
of Hsp90 chaperone complexes favoring degradation, which
To evaluate the significance of these findings to neurodegen- is sufficient to promote tau clearance in neurons. In addition,
erative diseases characterized by the accumulation of tau in consistent with reports demonstrating that the loss of
neurons of the brain, the impact of HDAC6 inhibition on tau HDAC6 activity increases the affinity of Hsp90 for Hsp90
levels was examined in primary neuronal cultures. Consistent inhibitors (21,22), we observed an enhanced induction of the
withtheresultsabove,asignificantdecreaseinPHF1-positive inducible Hsp70 isoform in response to 17AAG in the pres-
taulevelswasobservedinneuronstreatedwithM344(Fig.6A enceofM344(Fig.6EandF).Giventhattheincreasedinduc-
and B). Treatment with M344 also led to an increase in the tion of inducible Hsp70 is observed at both the RNA and
acetylation of tubulin, confirming that M344 did in fact protein levels, this indicates the upregulation is due to
inhibitthedeacetylaseactivityofHDAC6(Fig.6C).Asignifi- increased HSF1-mediated gene transcription, and not due to
cant decrease in p23 levels was also seen upon exposure to an altered half-life of Hsp70.
Human Molecular Genetics, 2012, Vol. 21, No. 13 2941
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Figure5.HDAC6knockdownandinhibitiondecreasecochaperonep23levels.(A)HeLacellsweretransfectedwithsiCONorsiHDAC6for48h,andthen d
treatedwith vehicle(DMSO)or 1mM of 17-AAGfor an additional24h. Cell lysateswere immunoblottedfor p23 andGAPDH,thelatter asa control for fro
m
proteinloading.(B)Levelsofp23werequantifiedandnormalizedtoGAPDH,andanalysisbytwo-wayANOVArevealedasignificanteffectofsiHDAC6
on p23 (F¼82.02; P,0.0001), as well as a significant treatment effect of 17AAG (F¼47.07; P¼0.0001), but no interaction between siHDAC6 and http
17AAG (F¼0.25; P¼0.63). (C) HeLa cells were pretreated with the HDAC6 inhibitor M344 for 1h, and subsequently treated with vehicle (DMSO) or s
1mM17AAGforanadditional24h.Levelsofp23andGAPDHincelllysateswereexaminedbyimmunoblotting.(D)Levelsofp23werequantifiedandnormal- ://a
izedtoGAPDH,andanalysisbytwo-wayANOVArevealedasignificanteffectofM344leadingtoadecreaseinp23(F¼52.6;P,0.0001),aswellasa ca
significanttreatmenteffectof17AAG(F¼9.6;P¼0.01).Alldataarepresentedasmean+SEM(n¼3).∗∗P,0.001,∗P,0.01. de
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Figure6.HDAC6inhibitiondecreasestaulevelsinprimaryneuronalcultures.(A)Primaryneuronsfromnon-transgenicmice(129SVE×FVB)werecultured gu
for9days,treatedwithvehicle(50%ethanol)orM344(1mM)for24handharvestedonthe10thdayinvitro(DIV).Celllysateswereimmunoblottedfor es
PHF1-tau, acetylated tubulin, p23, and total tubulin. (B) Treatment with 1mM M344 for 24h led to a significant decrease in PHF1-tau levels in primary t o
neurons (t¼4.7; P¼0.01). (C) Assessment of acetylated-tubulin levels verified that M344 does inhibit the deacetylase activity of HDAC6 and promotes n 1
increasedacetylationoftubulin(t¼29.7;P,0.0001).(D)Levelsofp23werealsoquantifiedandnormalizedtotubulin,andstatisticalanalysisrevealeda 7
significanteffectofM344leadingtoadecreaseinp23(t¼13.74;P¼0.0002).(E)OnDIV9,primaryneuronsweretreatedwithvehicle(50%ethanol)or No
M344(1mM)for1h,andsubsequentlytreatedwithvehicle(DMSO)ortheindicatedconcentrationof17AAGforanadditional23h.Celllysateswereimmuno- ve
blottedforHsp70,acetylatedtubulinandtotaltubulin.(F)HSPA1AmRNAexpressionwasevaluatedinprimaryneuronalculturespretreatedwithvehicle(50% m
b
elothaadninogl)coornMtro3l4f4or(1eamcMh)safomrp1leh(nan¼d3supbesreqcuoenndtiltyiotnr)e,aatenddweaitchhvseahmicpllee(wDaMsaSnOa)lyozred17iAnAquGad(r1umplMic)aftoe.rRanesaudltdsitwioenraela2n3alhy.zGedAbPyDtHwow-wasayusAedNaOsVaAn,ernedvoegaelinnoguas er 2
0
significanteffectof17AAG(F¼62.31;P,0.0001),aswellasasignificanteffectofM344(F¼29.82;P,0.0001)onHsp70mRNAlevels.Alldataare 1
presentedasmean+SEM(n¼3).∗∗P≤0.0002,∗P¼0.01. 8
DISCUSSION clearance byubiquitination,CHIPmayalsofacilitatetaudeg-
radation by abrogating the protein folding function of Hsp90.
CHIP is intimately involved in the mechanisms of Indeed,CHIPhasbeenshowntoelicitthereleaseofp23from
Hsp-mediated client degradation, and seems to be an integral Hsp90 upon binding (32), thereby causing a shift in the func-
participant in the removal of hyperphosphorylated tau. It has tionofHsp90fromrefoldingclientproteinstopromotingtheir
a unique binding affinity for tau phosphorylated at disease- degradation. We have now identified an additional, novel
specific phospho-epitopes, and is required for the ubiquitina- mechanism by which CHIP regulates protein triage decisions
tionofthesetauspecies,leadingtotheirproteasome-mediated involving Hsp90. Specifically, our data suggest that CHIP
degradation (6–8). In addition to directly promoting tau facilitates tau clearance by decreasing the steady-state levels
2942 Human Molecular Genetics, 2012, Vol. 21, No. 13
Currently, modulation of the Hsp90 chaperone complex
with chemical inhibitors to promote the degradation of
Hsp90 client proteins is being tested in clinical trials as a
therapeutic approach for the treatment of cancer (reviewed
in 35). In cancer, cell death pathways are often blocked by
the overexpression of prosurvival Hsp90 client proteins that
promote uncontrolled cell proliferation (16,36,37). Hsp90 in-
hibition results in the ubiquitination and proteasomal degrad-
ation of these deleterious client proteins. It is noteworthy
that Hsp90 chaperone complexes from tumor cells exhibit a
high affinity for Hsp90 inhibitors when compared with
D
Figure 7. CHIP and HDAC6 levels modulate Hsp90 chaperone complex. Hsp90 chaperone complexes from normal cells (38,39), dem- ow
SticohneomfaatincHdsiapg9r0amchdapepericotninegcohmowpleCxHeIiPthaenrdfaHvDorAinCg6relefovledlisnignflourednecgerafdoarmtioan- onstrating thattreatment withHsp90inhibitors can be usedto nloa
ofclientproteins,andtheresultingimpactontauaccumulation.Specifically, selectivelytargetdiseasedcells.Thesefindingsformthebasis de
ddericvrienagsetdauHcDleAarCan6cea.ctCivointyverfsaevloyr,sufporremgualtaiotinonooffaH‘DdAegCra6doartiolons’scoofmCpHleIxP, fimorptohretaunscee,otfhHersepi9s0ainshigibniitfiocrasnitnatmheoutrnetaotmf eevnitdoefncceanicnedri.caOt-f d fro
m
promotesformationofa‘refolding’complex,whichleadstotauaccumulation. ing that Hsp90 inhibitors may also hold clinical relevance for h
the treatment of neurodegenerative diseases characterized by ttp
s
of HDAC6, thereby increasing the sensitivity of Hsp90 to abnormaltauaccumulation.Forinstance,theincreaseinphos- ://a
17-AAGthroughacetylationandpromotingclientdegradation phorylatedtauassociatedwithdisease,aswellastheabilityof ca
d
(Fig. 7). Hsp90 inhibition to promote the preferential degradation of e
m
Inthisreport,wepresentevidencedemonstratingthatCHIP phosphorylatedtauspeciesinpathogenicmodels,conclusively ic
.o
interacts with and ubiquitinates HDAC6 (Fig. 1). HDAC6 identifiestauasanHsp90clientprotein,andfurtherimplicates u
p
likely undergoes CHIP-mediated proteasomal degradation, Hsp90dysfunctioninthepathogenesisofdisease(9,14,27,28). .c
o
given that knocking-down CHIP in cultured cells and Moreover, the demonstration that the Hsp90 chaperone m
/h
knocking-out CHIP in mice both result in elevated HDAC6 complex exhibits a high binding affinity for Hsp90 inhibitors m
g
levels(Fig.2).Increases inHDAC6,inturn,have detrimental only in affected brain regions of AD patients, and not in un- /a
consequences on tau levels. Indeed, we show that HDAC6 affected regions (9), suggests that similarly to cancer, Hsp90 rtic
overexpression promotes the accumulation of tau (Fig. 3); inhibitors can be used to selectively target Hsp90 chaperone le-a
conversely, HDAC6 deficiency decreases tau (Figs 4 and 6). complexes in diseased areas of the brain. Certainly, with the bs
We propose that changes in HDAC6 indirectly influence potential clinical applications of Hsp90 inhibitors for tauopa- tra
c
whether or not tau is degraded by modulating the acetylation thies, deciphering the mechanisms that cause Hsp90 to adopt t/2
1
state of Hsp90. As mentioned, Hsp90 hyperacetylation pro- a high affinity state for Hsp90 inhibitors is of considerable /1
3
motes client degradation (18,21). Since Hsp90 is a substrate interest.Hsp90hasahigherbindingaffinityforHsp90inhibi- /2
9
for the deacetylase activity of HDAC6, loss of HDAC6 ex- tors when acetylated (21,22), and we hypothesize that CHIP 3
6
pression would promote Hsp90 acetylation, thereby favoring modulates the affinity of Hsp90 for Hsp90 inhibitors by regu- /2
9
taudegradation.Incontrast,anincreaseinHDAC6wouldde- latinglevelsofthedeacetylase,HDAC6.Inadditiontoubiqui- 0
0
7
creaseHsp90acetylation,andthereforecauseadecreaseintau tinating clients for targeting to the proteasome (6–8), CHIP 0
7
degradation. Given that HDAC6 can form an Hsp90/HDAC6/ may regulate protein triage decisions by modulating the b
y
HSF1 complex (33,34), it is possible that alterations in folding and degradation functions of Hsp90 via HDAC6. g
u
HDAC6 expression disrupt Hsp90 chaperone function due to Given that a treatment paradigm combining HDAC6 and e
s
changes in complex formation, and not due to HDAC6- Hsp90inhibitionhasbeenshowntosynergisticallyandselect- t o
n
mediated deacetylation of Hsp90. However, because the cata- ively augment Hsp90 client degradation in leukemia cells but 1
7
lytically inactive HDAC6 mutant (H216A/H611A) fails to not normal cells (21), it is possible that a similar approach N
o
promote tau accumulation (Fig. 3D), it is most likely that could be utilized in neurodegenerative disease to selectively v
e
HDAC6-mediated tau accumulation is dependent on the dea- targetHsp90chaperone complexespresentincells expressing m
b
cetylase function of HDAC6. pathologicalclientproteins,suchasphosphorylatedtau.While er 2
Not only do HDAC6 depletion and inhibition enhance tau exploringthetherapeutic benefit ofmodulatingHDAC6func- 0
1
clearance, they also augment Hsp90 inhibitor-mediated tau tion, the relationship among tau pathology, HDAC6 activity 8
degradation (Figs 4 and 6). This is consistent with findings and Hsp90 acetylation should be more fully characterized. It
that hyperacetylation of Hsp90 due to HDAC6 depletion has been demonstrated that tau overexpression inhibits
leadstoanincreasedbindingaffinityofHsp90forHsp90inhi- HDAC6inacellculturemodel(40).Additionally,theoverex-
bitors(21,22).Indeed,weshowthatHsp70induction,aconse- pressionoftauleadstoanincreaseintubulinacetylation(41),
quence of effective Hsp90 inhibition by 17AAG, is enhanced whichisindicativeofalossofHDAC6function.Byinhibiting
in primary neurons treated with the HDAC6 inhibitor, M344 HDAC6,taumayindirectlyregulatetheacetylationofHsp90.
(Fig. 6E). That a similar increase in 17AAG-mediated induc- Given that increased acetylation of Hsp90 increases the
tion of Hsp70 following HDAC6 inhibition in HeLa cells binding affinity of the Hsp90 chaperone complex for Hsp90
was not observed (Fig. 4) is most likely due to the difference inhibitors, it is possible that the increased binding affinity of
in basal levels of inducible Hsp70 between HeLa cells (in the Hsp90 chaperone complex observed in affected regions
which it is high) and primary neurons (in which it is low). of AD brain (9) could be due to hyperacetylation of Hsp90
Human Molecular Genetics, 2012, Vol. 21, No. 13 2943
asaresultofpathologicaltau-mediatedinhibitionofHDAC6. 2ml. Forty-eight hours after transfection, the complete
However, the level of Hsp90 acetylation in affected areas of medium was removed and replaced with serum-free
the AD brain remains to be determined. Regardless, given Opti-Mem for subsequent plasmid transfection.
theimportanceofproteinqualitycontrolinneurodegenerative For plasmid transfections, 1–2mg plasmid DNA was com-
diseases,thecurrentfindingswillnotonlybenefitthetaufield, bined with Lipofectamine 2000 reagent for 15min in 500ml
but are also expected to have broad implications for all dis- Opti-Mem, and this mixture was added to either untreated or
eases characterized by the accumulation and aggregation of siRNA-transfected cells for 4h. The transfection mixture
abnormal Hsp90 client proteins. was then replaced with fresh complete media.
MATERIALS AND METHODS Immunoprecipitation
D
Antibodies, chemicals and reagents For coimmunoprecipitation studies, 90% of confluent cells in ow
10cmdishesweretransfectedusingLipofectamine2000with nlo
PHF1 (1:500; anti-S396/S404 p-tau) was provided by the combinations of HA-ubiquitin, HDAC6, myc-HDAC6, ad
P. Davies (Albert Einstein College of Medicine, New York, GFP, CHIP and myc-CHIP (WT, K30A and H260Q mutants) ed
NY, USA). Anti-myc (1:1000) and anti-HA (1:1000) were for4handthen maintained incomplete mediafor48h.Cells fro
m
obtained from Roche Applied Science. Anti-HDAC6 were collected in coimmunoprecipitation buffer (50mM Tris, h
(1:1000) was purchased from Millipore. Anti-GFP (1:2000) 274mM NaCl, 5mM KCl, 5mM EDTA, 1% Triton X-100, ttps
and anti-V5 (1:1000) were obtained from Life Technologies. 1mMPMSFandaproteaseandphosphataseinhibitorcocktail), ://a
Anti-actin (1:10000) was obtained from Sigma Aldrich. supernatantswerepreclearedwithproteinGandsubsequently ca
Anti-hsp70 (1:1000) was obtained from Enzo Life Sciences. incubated with anti-myc antibody or anti-HDAC6 antibody de
m
Anti-GAPDH (1:10000) was obtained from Meridian Life and 20ml protein G overnight at 48C. Protein G beads were ic
Science,Inc.Anti-p23(1:1000)wasobtainedfromFisherSci- washed three times in coimmunoprecipitation buffer, resus- .ou
entific. Anti-CHIP (1:1000) and E1 (1:1000; human specific pended in 2× tris-glycine SDS sample buffer with 5% beta- p.c
o
tau antibody) were generated by our group. 17AAG was pur- mercaptoethanol, heated for 5min at 958C and subjected to m
chased from A.G. Scientific, M344 and cycloheximide were westernblotanalysesfollowingSDS–PAGEelectrophoresis. /hm
obtained from Sigma-Aldrich and MG132 was purchased g
/a
froAmllCsaiRymNaAnsCwheermeicoablt.ained from Qiagen, with the propri- Mice rticle
-a
etary sequence of the non-silencing control not disclosed by CHIP–/– mice were generated as previously described (42). bs
the company. The target sequence for siCHIP (STUB1) is CHIP–/– and CHIP+/+ mice were humanely euthanized at tra
TGCCGCCACTATCTGTGTAAT, and the target sequence 30daysofage,andtheirbrainswerequicklyremovedfordis- ct/2
for siHDAC6 is CACCGTCAACGTGGCATGGAA. siRNA sectionandfrozenondryiceforsubsequentbiochemicalana- 1/1
efficiencyforproteinknockdownwasvalidatedbywesternblot. lyses. Brain tissue was homogenized in buffer containing 3/2
9
50mM Tris, 274mM NaCl, 5mM KCl, 5mM EDTA, 1% 36
Expression plasmids SDS,1%TritonX-100,1mMPMSFandaproteaseandphos- /29
phataseinhibitorcocktail(pH7.4).Homogenateswere centri- 0
0
GFP, untagged and myc-tagged CHIP, P301L-V5 tau and fuged at 15400g for 15min, and supernatants were collected 70
7
HA-ubiquitin were generated by our lab. A human HDAC6 and subjected to western blot analyses. b
y
clone in a pCMV SPORT6 vector was purchased from Life g
u
Technologies. The QuikChange Mutagenesis kit (Stratagene) e
was utilized to generate a catalytically inactive HDAC6 Sample preparation and immunoblotting procedure st o
n
mutant (H216A/H611A) following the manufacturer’s proto- Cell samples for immunoblotting were suspended in 100– 1
7
col.TheQuikChangeMutagenesiskitwasalsoutilizedtogen- 200ml of homogenate buffer [50mM Tris–HCl, pH 7.4, N
erateK30AandH260Qmutationsinmyc-CHIP.Theintegrity 300mM NaCl, 5mM EDTA, 1% Triton X-100, 1% SDS, ove
of all constructs was confirmed by automated sequencing. 1mM PMSF, protease inhibitor cocktail, phosphatase inhibi- mb
taonrds hIoamnodgIeIni(zFeidshienr1S0c×ienvtiofilcu)m].eMoficheobmroaignesnawteerbeuwffeeirg.hAeldl er 20
Cell culture and transient transfections 1
samples were sonicated, and following sonication, samples 8
HeLa and HEK-293T were maintained in Opti-Mem (Life were centrifuged at 16000g for 15min, and a BCA protein
Technologies) supplemented with 10% heat-inactivated FBS assay (Fisher Scientific) performed on the supernatant.
(Life Technologies) and passaged every 3–4 days based on Thirty microgram of protein from each sample was diluted
90% confluency. in dH O, 2× tris-glycine SDS sample buffer (Life Technolo-
2
siRNAexperimentswerecarriedoutinsix-wellplatesusing gies), 5% beta-mercaptoethanol (Sigma Aldrich) and heat-
human gene-specific validated and genome-wide siRNAs. denaturedfor5minat958C.Sampleswererunon4–20%tris-
Final siRNA concentration per well was 20nM in Opti-Mem, glycine gels (Life Technologies), and transferred to PVDF
with 4ml of siLentFect transfection reagent (Bio-Rad) used membrane (Millipore). Membranes were blocked in 5% milk
per well. This mixture was incubated in a final volume of in TBS/0.1% Triton X-100, and incubated overnight in
500ml for 20min and then added to 40–50% confluent primary antibody diluted in 5% milk in TBS/0.1% Triton
HeLa cells in six-well plates for a final in-well volume of X-100 rocking at 48C. Membranes were incubated in
2944 Human Molecular Genetics, 2012, Vol. 21, No. 13
HRP-conjugated secondary antibodies (1:5000; Jackson 4. Buee,L.,Bussiere,T.,Buee-Scherrer,V.,Delacourte,A.andHof,P.R.
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neurodegenerativedisorders.BrainRes.BrainRes.Rev.,33,95–130.
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Primary neuronal culture DeLucia,M.,McGowan,E.,Lewis,J.,Prihar,G.etal.(2004)CHIPand
Hsp70regulatetauubiquitination,degradationandaggregation.Hum.
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HIBERNATETM A media without calcium (BrainBits), sup- CHIP-Hsc70complexubiquitinatesphosphorylatedtauandenhancescell
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W.C.,Zehr,C.,West,G.,Cao,S.,Clark,A.M.etal.(2006)Deletionof o
Excised hippocampi were digested in papain (1mg/ml; theubiquitinligaseCHIPleadstotheaccumulation,butnotthe wn
Fisher Scientific), triturated with a Pasteur pipet (bore size aggregation,ofbothendogenousphospho-andcaspase-3-cleavedtau loa
0in.8N–e1umroMba),sacleAntr(iLfuifgeedTetochcnoollloecgtiecse)l,lspueplplelet,maenndterdeswusitphenBd2e7d, 9. DDspiuecnckmieeyos.,reCJ,..JAN.,.e,AuKrsoahsm,cPia..l,,,2SA6h.,,o6rL9auk8na5d,–gS6r.e9,n9Z,6lK.at.k,oKvlioc,saJk.,,ENc.k,mBaanil,eyC,.BR..Met.,al. ded fro
GMAX,gentamicinandbFGF(LifeTechnologies).Following m
determination of cell number, neurons were plated on (d2e0g0ra7d)eTshpehhoisgphh-oafrfiylnaitteydHtSauP9c0li-eCnHtIpProctoeminps.leJx.rCelciong.nInizveessta.n,d11se7l,ectively http
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SUPPLEMENTARY MATERIAL 13. Lattouf,J.B.,Srinivasan,R.,Pinto,P.A.,Linehan,W.M.andNeckers,L. .co
m
(2006)Mechanismsofdisease:theroleofheat-shockprotein90in
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ACKNOWLEDGEMENTS proline-directedSer/ThrsitesbutnotKXGS(MARK)sites.FASEBJ., -a
b
20,753–755. s
Dr Cam Patterson kindly provided the CHIP knockout mice 15. Kamal,A.,Boehm,M.F.andBurrows,F.J.(2004)Therapeuticand tra
which were used in our previous studies (4,5). Dr Peter diagnosticimplicationsofHsp90activation.TrendsMol.Med.,10, ct/2
Davies kindly provided us with the PHF-1 antibodies. 283–290. 1
16. Isaacs,J.S.,Xu,W.andNeckers,L.(2003)Heatshockprotein90asa /13
moleculartargetforcancertherapeutics.CancerCell,3,213–217. /2
Conflict of Interest statement. None declared. 9
17. Bali,P.,Pranpat,M.,Bradner,J.,Balasis,M.,Fiskus,W.,Guo,F.,Rocha, 3
6
K.,Kumaraswamy,S.,Boyapalle,S.,Atadja,P.etal.(2005)Inhibitionof /2
9
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0
FUNDING heatshockprotein90:anovelbasisforantileukemiaactivityofhistone 70
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This work was supported by Mayo Clinic Foundation (L.P.), 18. Kovacs,J.J.,Murphy,P.J.,Gaillard,S.,Zhao,X.,Wu,J.T.,Nicchitta, by
National Institutes of Health/National Institute on Aging C.V.,Yoshida,M.,Toft,D.O.,Pratt,W.B.andYao,T.P.(2005)HDAC6 gu
regulatesHsp90acetylationandchaperone-dependentactivationof e
[5R01AG026251-04 (L.P.) and AG17216-10JP3 (L.P.)], Na- s
glucocorticoidreceptor.Mol.Cell,18,601–607. t o
tional Institutes of Health/National Institute of Neurological 19. Murphy,P.J.,Morishima,Y.,Kovacs,J.J.,Yao,T.P.andPratt,W.B. n 1
Disorders and Stroke [R01 NS 063964-01 (L.P.), R01 (2005)Regulationofthedynamicsofhsp90actionontheglucocorticoid 7
NS077402 (L.P.)], PSP Foundation (L.P.) and ADRC receptorbyacetylation/deacetylationofthechaperone.J.Biol.Chem., No
AG016574 (C.C. and T.F.G.). Funding to pay the Open 280,33792–33799. ve
20. Scroggins,B.T.,Robzyk,K.,Wang,D.,Marcu,M.G.,Tsutsumi,S., m
Access publication charges for this article was provided by b
Beebe,K.,Cotter,R.J.,Felts,S.,Toft,D.,Karnitz,L.etal.(2007)An e
Mayo Clinic. acetylationsiteinthemiddledomainofHsp90regulateschaperone r 2
0
function.Mol.Cell,25,151–159. 1
8
21. Rao,R.,Fiskus,W.,Yang,Y.,Lee,P.,Joshi,R.,Fernandez,P.,
Mandawat,A.,Atadja,P.,Bradner,J.E.andBhalla,K.(2008)HDAC6
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