Accumulation of ubiquitin conjugatesin a polyglutamine disease model occurs withoutglobal ubiquitin/proteasome system impairmentChrista J. Maynarda, Claudia Bottchera, Zaira Ortegab, Ruben Smithc, Bogdan I. Floread, Miguel Díaz-Hernandezb,1,Patrik Brundinc, Hermen S. Overkleeftd, Jia-Yi Lic, Jose J. Lucasb, and Nico P. Dantumaa,2

aDepartment of Cell and Molecular Biology, Karolinska Institutet, S-17177 Stockholm, Sweden; bCentro de Biologia Molecular ‘‘Severo Ochoa,’’ ConsejoSuperior de Investigaciones Cientıficas, Universidad Autonoma de Madrid (UAM), and CiberNed, 28049 Madrid, Spain; cNeuronal Survival Unit, WallenbergNeuroscience Center, S-22814, Lund University, Lund, Sweden; and dBio-organic synthesis, Leiden Institute of Chemistry, Leiden University, 2300 RA, Leiden,The Netherlands

Communicated by Huda Y. Zoghbi, Baylor College of Medicine, Houston, TX, June 12, 2009 (received for review February 26, 2009)

Aggregation-prone proteins have been suggested to overwhelmand impair the ubiquitin/proteasome system (UPS) in polyglu-tamine (polyQ) disorders, such as Huntington’s disease (HD). Over-expression of an N-terminal fragment of mutant huntingtin (N-mutHtt), an aggregation-prone polyQ protein responsible for HD,obstructs the UPS in cellular models. Furthermore, based on theaccumulation of polyubiquitin conjugates in brains of R6/2 mice,which express human N-mutHtt and are one of the most severepolyQ disorder models, it has been proposed that UPS dysfunctionis a consistent feature of this pathology, occurring in both in vitroand in vivo models. Here, we have exploited transgenic mice thatubiquitously express a ubiquitin fusion degradation proteasomesubstrate to directly assess the functionality of the UPS in R6/2 miceor the slower onset R6/1 mice. Although expression of N-mutHttcaused a general inhibition of the UPS in PC12 cells, we did notobserve an increase in the levels of proteasome reporter substratein the brains of R6/2 and R6/1 mice. We show that the increase inubiquitin conjugates in R6/2 mice can be primarily attributed to anaccumulation of large ubiquitin conjugates that are different fromthe conjugates observed upon UPS inhibition. Together our datashow that polyubiquitylated proteins accumulate in R6/2 braindespite a largely operative UPS, and suggest that neurons are ableto avoid or compensate for the inhibitory effects of N-mutHtt.

Huntington � neurodegeneration � protein degradation

The primary proteolytic machinery responsible for the turn-over of proteins in the cytosol and nuclei of cells, including

the destruction of misfolded or otherwise abnormal proteins, isthe ubiquitin/proteasome system (UPS) (1). The UPS is inessence a two-step process: the targeting of proteins through thecovalent linkage of polyubiquitin chains (2), and the destructionof ubiquitylated proteins by the proteasome (3). A number ofstudies have implicated UPS dysfunction in a range of polyglu-tamine (polyQ) neurodegenerative diseases (4). The aggrega-tion-prone polyQ proteins are postulated to impair the UPS inthese diseases, either by overloading the capacity of the cell’sUPS machinery (5), by sequestration of essential components ofthe UPS into inclusions (6), or by obstruction of the proteasome(7). If polyQ proteins themselves inhibit the system crucial totheir own degradation, this could elicit a self-perpetuatingpathogenic cascade of events, both accelerating the accumula-tion of the toxic protein, and impairing essential regulatoryfunctions of the UPS (4). With the help of specifically designedreporter substrates, it has been shown that polyQ proteins cancause UPS impairment in cell lines (5, 8, 9). However, since theseexperiments rely on acute overexpression of the polyQ proteinin cells with limited physiological relevance, it is difficult toextrapolate these findings to the status of the UPS during theprogression of the disease in patients.

In contrast to the observation with in vitro models, no signs ofUPS impairment were found in mouse models for spinocere-bellar ataxia 7 (SCA7) (10), or spinal and bulbar muscularatrophy (11), two diseases that are caused by polyQ proteins (12).Huntington’s disease (HD), which is caused by a polyQ repeatexpansion in the protein huntingtin (13), has appeared to be anexceptional case, as several lines of data have implicated UPSimpairment in HD pathology (14). A number of studies haveprovided evidence in favor of UPS impairment in cellularmodels, as a consequence of the expression of the N-terminalfragment of mutant huntingtin (N-mutHtt) (5, 8, 9, 15, 16).Moreover, a recent study suggested that global UPS impairmentis a consistent feature of HD pathology based on a strikingincrease in the levels of various types of polyubiquitin conjugatesin HD postmortem brain and in R6/2 mice, a commonly useddisease model expressing human N-mutHtt (17). The N-mutHttfragment of the human huntingtin protein contains the polyQrepeat, and is more toxic than the full-length protein, resultingin a rapid and aggressive pathogenesis in animal models (18). Inthe present study, we evaluated ubiquitin-dependent proteaso-mal degradation in R6/2 and the milder R6/1 mouse models (18)using transgenic UPS reporter mice (19). These UPS reportermice express a green fluorescent protein (GFP) fusion that isconstitutively targeted for ubiquitin-dependent proteasomaldegradation through the presence of a ubiquitin fusion degra-dation (UFD) signal. Global impairment of the UPS is expectedto give a general accumulation of proteasome substrates, includ-ing UFD substrates, which can be readily assessed in these miceby the levels of the GFP reporter. Our data demonstrate thatglobal impairment of the UPS does not occur in R6 mice andsuggest that the accumulation of polyubiquitin conjugates in HDis not a direct reflection of UPS functionality.

ResultsN-mutHtt Impairs Degradation of Two Unrelated Reporter Substratesin Cells. We generated a PC12 cell line that inducibly expressesa cyan fluorescent protein (CFP)-tagged N-mutHtt, harboring apathogenic polyQ repeat of 94 residues (N-mutHtt-CFP). To thiscell line, we subsequently introduced the UFD reporter substrateubiquitinG76V-yellow fluorescent protein (UbG76V-YFP) or theYFP-CL1 reporter substrate, which carries the CL1 degradation

Author contributions: C.J.M., Z.O., J.-Y.L., J.J.L., and N.P.D. designed research; C.J.M., C.B.,Z.O., R.S., B.I.F., M.D.-H., and N.P.D. performed research; C.J.M., C.B., Z.O., R.S., B.I.F.,M.D.-H., P.B., H.S.O., J.-Y.L., J.J.L., and N.P.D. analyzed data; and C.J.M. and N.P.D. wrote thepaper.

The authors declare no conflict of interest.

1Present address: Deptartment Bioquimica, Fac. Veterinaria, UCM, 28040 Madrid, Spain.

2To whom correspondence should be addressed. E-mail: nico.dantuma@ki.se.

This article contains supporting information online at www.pnas.org/cgi/content/full/0906463106/DCSupplemental.

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signal (20), and for which it has been shown that expression ofN-mutHtt impairs its degradation (5, 8). The functionality of thereporters in the PC12 cell lines was confirmed by treatment witha specific proteasome inhibitor (Fig. S1). Induction of N-mutHtt-CFP expression caused accumulation of both the UbG76V-YFP(Fig. 1 A and C) and YFP-CL1 substrates. (Fig. 1 B and C). Thus,N-mutHtt causes a general inhibition of the UPS in cell lines.

Functional Analysis of UFD Reporter Substrate in UbGFP Mouse Brain.We recently developed two reporter mouse lines, UbGFP/1 andUbGFP/2, that ubiquitously express the UFD substrateubiquitinG76V-green fluorescent protein (UbG76V-GFP) (19). Insitu hybridization showed that the reporter mRNA transcript ispresent throughout the brain including the cortex and striatum,regions that are particularly affected in HD and in R6 mice (Fig.2A). Quantification revealed that UbGFP/1 mice have higher

expression levels than UbGFP/2 mice in all regions examined(Fig. S2). The GFP protein levels in the brains of both lines aretoo low to be detected by native GFP fluorescence (19). Immu-nostaining with a GFP specific antibody revealed a weak butspecific staining in neurons of UbGFP/1 mice (Fig. 2B and Fig.S3), whereas the reporter remained undetectable in UbGFP/2mice (Fig. 2B).

Whilst no native GFP fluorescence was detected in untreatedmice, direct administration of proteasome inhibitor into thebrains of UbGFP/1 and UbGFP/2 mice, which caused an ap-proximate 80% and 50% reduction in the chymotrypsin-like andcaspase-like activities, respectively (Fig. S4A and B), resulted inthe appearance of cells with detectable fluorescence (Fig. 2 Cand D). Subsequent analysis with markers for different cell typesdemonstrated that neurons, microglia, astrocytes, and oligoden-drocytes accumulated the reporter substrate in inhibitor-treated

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Fig. 1. N-mutHtt impairs degradation of two unrelated reporter substrates in cells. PC12 cells expressing UbG76V-YFP (A) and YFP-CL1 (B) in which expressionof N-mutHtt-CFP had not been induced (Left) or had been induced for 4 days by omitting doxycyclin from the medium (Right). (Scale bar, 20 �m.) (C) Quantitationof YFP fluorescence in UbG76V-YFP and YFP-CL1 PC12 cells in the absence or presence of expression of N-mutHtt-CFP. The fluorescence intensity in the absenceof N-mutHtt-CFP expression has been standardized to 1.0. (SEM, n � 75 cells counted per group, t test ***, P � 0.001).

Fig. 2. Functional analysis of UFD reporter substrate in the UbGFP mouse brain. (A) In situ hybridization of UbG76V-GFP reporter mRNA expression in 30-to40-week-old non-transgenic (NTg), UbGFP/1, and UbGFP/2 brain. Sagittal sections are shown. [Labels, cortex (Ctx), striatum (Str), hippocampus (Hc), andcerebellum (Cb)]. (B) Anti-GFP immunostaining of UbG76V-GFP reporter protein expression in 12-week NTg, UbGFP/1, and UbGFP/2 cortex. Nuclei are stained withHoechst. (C) Native GFP fluorescence in striatum of UbGFP/1 (Upper) and UbGFP/2 mice (Lower) 24 h after stereotactic injection of vehicle only (Left) or 50 nmolof the proteasome inhibitor lactacystin (Right). (Scale bar, 50 �m.) (D) Quantitation of number of GFP-positive cells/mm2 in striata from lactacystin treatedUbGFP/1 and UbGFP/2 mice. Sections from lactacystin treated UbGFP/1 (E), and UbGFP/2 (F) mice were probed with antibodies specific for neurons (NeuN),microglia (Iba1), astrocytes (GFAP), and oligodendrocytes (CNPase). The percentage of GFP-positive cells double stained for the respective markers werequantitated. Average counts of 2 sections from each of 2 mice � SD. (G) Western blot analysis of GFP products in homogenates of forebrain, cerebellum andpancreas of UbGFP/1 and UbGFP/2 mice. Blots were probed with a monoclonal GFP antibody. Astrices mark non-specific bands. (H) GFP products wereimmunoprecipitated (IP) from forebrain, cerebellum, and pancreas homogenates with a polyclonal GFP antibody before detection of the products by Westernblotting with a monocolonal GFP antibody.

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UbGFP/1 (Fig. 2E and Fig. S4C) and UbGFP/2 mice (Fig. 2Fand Fig. S4D).

We next analyzed the levels of the reporter substrate in theforebrain and cerebellum of UbGFP/1 and UbGFP/2 mice byWestern blotting. As a reference tissue, we included the pancreassince it exhibits particularly high reporter levels in UbGFP/1mice. In tissue homogenates, the reporter protein could only bedetected in UbGFP/1 mice (Fig. 2G), whereas concentrating thereporter by immunoprecipitation before Western blotting wasrequired to detect the reporter in tissues from UbGFP/2 mice(Fig. 2H). Notably, two specific GFP bands were detected in allthree analyzed tissues of both mouse lines. The upper bandcorresponded in molecular weight with the full-length UbG76V-GFP fusion and the lower band corresponded in size withunmodified GFP (Fig. S5A). We reported earlier that expressionin cell lines gave rise to small amounts of a similar truncatedproduct (21). In primary fibroblast cultures from UbGFP/1 mice,the full length UbG76V-GFP had a very short half-life whereas thetruncated product was long-lived and remained stable during an8-h chase (Fig. S5B). Western blot analysis of UbG76V-GFP/1striatal tissue after injection with proteasome inhibitor showedthat the full length UbGFP but not the smaller GFP fragmentaccumulated (Fig. S4E).

Together our data show that the mouse lines express the UPSreporter throughout the brain and that both neuronal andnon-neuronal cell types accumulate the reporter substrate inresponse to UPS dysfunction. Moreover, in addition to theshort-lived UbG76V-GFP fusion, which accumulates in responseto UPS inhibition, the tissues also contain a long-lived fragmentcorresponding in size with GFP.

Absence of Global UPS Impairment in R6/2 and R6/1 Mice. Next, wecrossed the UPS reporter mice with the R6/2 and R6/1 mousemodels, which express human N-mutHtt (22). Due to differences inexpression levels and repeat length, R6/2 and R6/1 mice exhibitdifferent rates of disease onset and pathogenesis, developing late-stage symptoms at 12 and 40 weeks of age, respectively (22).

We first analyzed the effect of N-mutHtt on expression of thereporter in UbGFP mice. Although we observed no grosschanges in the expression pattern of the reporter transcript inbrains of late stage R6/2 and R6/1 mice (Fig. S6A), levels of thetranscripts were slightly elevated in the striatum and cortex (Fig.S6B). To investigate the effect of N-mutHtt on the levels of thereporter protein substrate, we first used the UbGFP/2 reportermouse line, because the absence of a constitutively detectableGFP signal enables clear detection of impairment of the UPS.Detailed immunocytochemical analysis of the brains of UbGFP/2:R6/2 (6–14 weeks) and UbGFP/2:R6/1 (6–40 weeks) mice,revealed however, no accumulation of the GFP reporter early orlate in the disease process (Fig. 3A and Fig. S7A).

Consolidating these findings, we also investigated proteasomeactivity using the activity probe MV151 that covalently labels theproteolytically active subunits of the proteasome (23). Earlierstudies based on cleavage of fluorogenic substrates reported anincrease in proteasome activity in N-mutHtt mice including R6/2(24, 25). Using the activity probe, we observed however no differ-ences in the activities of the constitutive subunits of the proteasomein brain lysates from late-stage R6/2 mice compared with age-matched controls (Fig. 3 B and C). We cannot exclude the possi-bility that the levels of inducible proteasome subunits are elevated,as reported previously for a similar N-mutHtt mouse model (25),since they remained below the detection level in our assay.

An advantage of the UbGFP/1 reporter mouse line is that itcan be used for detection of more subtle differences in the levelsof the reporter, since the higher transgene expression levelsresult in detectable basal levels of the reporter. However,consistent with our data obtained using UbGFP/2 mice, weobserved no increase in fluorescence intensity in late-stage

Fig. 3. Absence of global UPS impairment in R6/2 mice. (A) Representativecryosections from brains of non-transgenic (NTg), UbGFP/2 and UbGFP/2:R6/2mice co-immunostained for Htt and GFP. Nuclei were counterstained withHoechst. Micrographs show 12-week non-transgenic (NTg), UbGFP/2 andUbGFP/2:R6/2 Cortex (Ctx) and striatum (Str). (Scale bar, 20 �m.) (B) Activitylabeling of proteolytic active proteasome subunits in brain homogenates offour 14-week-old NTg controls and R6/2 mice using the fluorescent activityprobe MV151. Image shows in-gel fluorescence readout. Labeled �-1, �-2, and�-5 proteasome subunits are indicated. (C) Densitometric analysis of bandintensities from B (n � 4, SEM).

Fig. 4. No changes in UbG76V-GFP and decrease in long-lived GFP product inR6/2 mice. (A) Cortex (Ctx) and striatum (Str) of 12-week-old NTg, UbGFP/1,and UbGFP/1:R6/2 mice co-immunostained for N-mutHtt and GFP. Nucleicounterstained with Hoechst (Lower). (Scale bar, 20 �m.) (B) Western blotanaysis of UbGFP and GFP protein products in forebrain homogenates of14-week UbGFP/1 and UbGFP/1:R6/2 mice using �-GFP antibody. (C) Quanti-fication of band intensities in B (n � 6, SEM; t test, ***, P � 0.001).

13988 � www.pnas.org�cgi�doi�10.1073�pnas.0906463106 Maynard et al.

UbGFP/1:R6/2 (Fig. 4A) or UbGFP/1:R6/1 mice (Fig. S7B).Instead, we noticed that the neuronal GFP staining was reducedcompared to age-matched UbGFP/1 control mice (Fig. 4A andFig. S7B). Western blot analysis confirmed this finding andrevealed that despite there being no significant increase in thelevels of full-length UbG76V-GFP, consistent with the absence ofUPS impairment, the levels of the truncated product weresignificantly reduced in UbGFP/1:R6/2 mice (Fig. 4 B and C).Our data therefore suggest that N-mutHtt does not affect theclearance of the short-lived reporter but reduces the levels of thelong-lived GFP fragment. We conclude that global UPS impair-ment does not occur in the R6 mouse models.

Polyubiquitylated Proteins Accumulating in R6/2 Mice Differ fromThose Observed upon Proteasome Inhibition. It has been recentlyshown that the levels of ubiquitin conjugates are significantlyincreased in R6/2 mice, which has been interpreted as evidencefor the occurrence of UPS dysfunction in R6/2 mice (17). Weindeed found that the levels of ubiquitin conjugates were dra-matically increased in late stage R6/2 mice (Fig. 5A). Ubiquitinconjugates and the GFP reporter accumulated in primary Ub-GFP/1 fibroblasts at a similar level of proteasome inhibitionexcluding the possibility that ubiquitin conjugates are a moresensitive readout for UPS impairment (Fig. 5B and Fig. S8A andB). A more detailed analysis of the ubiquitin conjugates in thesoluble fraction of R6/2 brain lysates showed that the increasewas primarily due to an elevation in the levels of large ubiquitincomplexes (Fig. 5 C and D). This was even more striking whenthe brain lysates were separated using a stacking gel consisting

of a low-percentage of agarose to enable separation of largerprotein complexes (Fig. 5E). Notably, the N-mutHtt also mi-grated as large complexes (Fig. 5F). In contrast, administrationof proteasome inhibitor to the brain (Fig. 5G) or cells (Fig. S8C)caused an increase in high molecular weight polyubiquitinconjugates whereas no increase was detected in large complexesremaining in the stacking gel. We conclude that the polyubiq-uitin conjugates that accumulate in brains of R6/2 mice do notcorrespond with those observed upon proteasome inhibition butare primarily attributed to a pool of large polyubiquitylatedcomplexes. These findings suggest that the elevation in ubiquitinconjugates in R6/2 mice is not a consequence of global UPSimpairment, consistent with the absence of accumulation of UPSreporter substrates.

DiscussionSince the UPS is important for protein quality control (26), butunable to degrade aggregated proteins (27), it is feasible thatfutile attempts to degrade protein aggregates hinders the pro-teasome from fulfilling other tasks, which in turn, may causecellular dysfunction or death. We indeed found, in line withearlier studies (5, 8, 9), that N-mutHtt caused functional im-pairment of ubiquitin-dependent proteasomal degradation incell lines but did not observe global UPS impairment in brainsof mice expressing N-mutHtt. Although we cannot exclude theoccurrence of spatially or temporally confined impairment of theUPS (28) or more subtle changes affecting a selective subpopu-lation of substrates, our data unequivocally shows that the UPSis largely operative and not globally impaired. This conclusion is

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Fig. 5. Polyubiquitylated proteins accumulating in R6/2 mice differ from those observed upon proteasome inhibition. (A) A slot blot of 14-week-old UbGFP/1and UbGFP/1:R6/2 brain homogenates using a monoclonal antibody specific for conjugated polyubiquitin (FK1). (B) Accumulation of UbG76V-GFP reporter andpolyubiquitin conjugates in UbGFP/1 primary fibroblast cultures treated with increasing concentrations of epoxomycin. Standardised signals from densitometricanalysis of the Western blot in Fig. S8A. Representative of 3 independent experiments. (C) Separation of high and low MW polyubiquitinylated material fromthe same samples as in A by SDS/PAGE Western blotting. Ubiquitin conjugates in the stacking gel (a); upper resolving gel �250kDa (b); resolving gel 20–150 kDa(c) are indicated. (i) shorter exposure, (ii) longer exposure. (D) Densitomentric analysis of the ubiquitin conjugates in the stacking gel, upper resolving gel andresolving gel shown in C (n � 6, SEM; t test ***, P � 0.001). (E) Polyacrylamide stacking gel was replaced by a 1% agarose stacking gel to allow entry of largerprotein aggregates. Blot was probed with the FK1 antibody. (F) Immunostaining of the same samples as shown in E with �-Htt antibody. (G) Western blot analysisof polyubiquitin conjugates in striatal tissue of UbGFP/1 mice 24 h after lactacystin injection or vehicle control. Far right lane, 14-week R6/2 brain homogenate.

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strengthened by a recent study demonstrating that the degrada-tion of a CL1-based UPS reporter is also unconpromised in R6/2mice (29). An important difference between the in vitro and invivo models is that in the cellular model, the cells are acutelychallenged with high levels of the N-mutHtt, whereas the R6mice are chronically exposed to more moderate expression levelsof this protein (22). As global impairment of the UPS is lethalfor cells (30), it is not unlikely that the chronic presence ofN-mutHtt may trigger adaptive responses that allow the cells tocope with this toxic protein. It has indeed been shown thatchronic exposure of cell cultures to a high dose of proteasomeinhibitor results in the appearance of adapted cells that havecompensated for the curtailed proteasome activity by up-regulating other proteases (31), such as the tripeptidyl peptidaseII (32). In respect to polyQ proteins, the puromycin-sensitiveaminopeptidase is also of interest since it has been shown thatthis protease is able to degrade expanded polyQ repeats (33).However, we did not detect any striking differences in the levelsof these proteases in R6/2 mice (Fig. S9 A and B).

Our data suggest that polyubiquitylated proteins accumulate notas a consequence of UPS impairment but despite the presence ofa largely functional UPS. If ubiquitylated substrates are sequesteredinto larger protein aggregates before reaching the proteasome fordegradation, this could account for the observed elevation in highmolecular weight polyubiquitin conjugates. Furthermore, sincepolyubiquitin modifications are involved in a large number ofcellular processes other than proteasomal degradation (34), it isfeasible that the increase in polyubiquitylated material reflectschanges in ubiquitin-dependent processes unrelated to UPS func-tion, either as a consequence of impairment of these processes, oralternatively, as part of an adaptive response. An interesting pos-sibility can be found in the fact that ubiquitylation plays pivotal rolesin the formation of aggresomes (35) and the clearance of aggre-gated proteins by macroautophagy (36, 37). Both events are stim-ulated by polyQ proteins (38) and are believed to be largelyprotective responses that defend the cell against the toxic insults ofaggregation-prone proteins (38, 39) and can restore UPS function(16, 40). Since macroautophagy is primarily responsible for theturnover of long-lived proteins (41), and induction of this pathwaydoes not affect the levels of UFD substrates (16), this may alsoprovide an explanation for the selective decrease in the long-livedGFP product in the brains of R6/1 and R6/2 mice.

The same reporter mice that were applied in our study haveconfirmed UPS dysfunction in prion-infected mice (42) and intransgenic mice overexpressing a mutant SOD1 responsible forfamilial amyotrophic lateral sclerosis (43) showing that these miceare suited for in vivo assessment of the UPS. In contrast, in threepolyQ disease models, namely SCA7 (10), spinal and bulbar mus-cular atrophy (11) and mice expressing N-mutHtt, the UPS remainsoperative. Although it may be premature to generalize theseobservations as pertaining to all polyQ pathologies, these observa-tions together suggest that the UPS manages to escape globalimpairment even in the face of a pathological polyQ challenge. Weconclude that global UPS impairment is not a unanimous casualtyamongst protein misfolding diseases. The fact that the UPS appearsto be functional, even at late stages of the pathology in R6 micewhen many cellular pathways are disturbed, underscores the ro-bustness of this proteolytic machinery and suggests that the UPSmay be a reliable partner to exploit for reducing the load of the toxicproteins in these pathologies.

Materials and MethodsCell Lines. Exon 1 of human Htt containing 94 polyQ repeats was ligated to theN terminus of CFP (N-mutHtt-CFP), cloned into the pTRE-tight vector (Clon-tech) and introduced into PC12 cells carrying the tet-transactivator (Clontech).Subsequently, the UbG76V-YFP or YFP-CL1 reporter (44) was integrated togenerate double stable cell lines. Cells were analyzed with a confocal laser

scanning microscope (Zeiss LSM510 META). Images were quantified usingVolocity software (Improvision).

Transgenic Mice. All animal experiments were approved by local ethical commit-tees and conformed to international animal welfare guidelines. The UbGFPmouse lines were maintained in a heterozygous state on a C57/BL6 background,and R6/1 and R6/2 mice on a BL6/CBA background. Double transgenic UbGFP:R6mice were obtained by crossing R6/1 or R6/2 males with UbGFP females. Micewere decapitated or perfused transcardially with PBS. For immunohistochemicalanalyses, mice were perfused transcardially with PBS followed by 4% parafor-maldehyde. Tissues were postfixed in 4% paraformaldehyde, cryopreserved in30% sucrose/PBS, and mounted in Tissue-Tec O.C.T. compound (Sakura).

In Situ Hybridization. In situ hybridization was performed as described in SI Textusingthespecificantisenseoligonucleotidetogreenfluorescentprotein (GFP) (5�CAC CTA CGG CAA GCT GAC CCT GAA GTT CAT CTG CAC CAC CGG C 3�) and thecorresponding sense sequence used as a negative control. Quantitation wasperformed using Image J software (National Institutes of Health). For each brainregion analyzed, the signal intensity was taken as the average of at least threesections for each mouse analyzed, and corrected for the background signal fromNTg controls.

Immunostaining. Immunostaining was performed on cryotome-cut brain sec-tions using rabbit polyclonal anti-GFP (Invitrogen); mouse monoclonal anti-human Htt antibody, MAB5374 (Millipore); mouse monoclonal anti-NeuN,MAB377 (Chemicon), in PBS and 0.2% Triton X-100, pH 7.4. Alexafluor con-jugated goat anti-mouse and goat anti-rabbit secondary antibodies wereapplied in PBS and 0.2% Triton X-100. Immunofluorescence staining com-bined with native GFP fluorescence detection was performed by incubatingfree-floating slices at room temperature in PBS, 1% BSA, and 0.1% TritonX-100 with: anti-NeuN antibody; anti-GFAP polyclonal antibody (Promega);anti-CNPase monoclonal antibody (11–5B, AbCam); or anti-Iba1 polyclonal anti-body (Wako). The reaction was visualized with TexasRed-conjugated anti-mouseor anti-rabbit IgG (Molecular Probes). Sections were examined with a confocallaser scanning microscope (Zeiss LSM510 META).

Western Blotting. Frozen tissues were homogenized in 5–10� wt/vol ice coldhomogenization buffer [20 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM NaF, 1%Triton X-100, 1 mM orthovanadate, 10 mM EDTA, 20 mM N-ethylmaleimide(Sigma-Aldrich), and complete miniprotease inhibitor mixture (Roche)]. Homog-enization was carried out by sonication on a Bandelin Sonopuls ultrasonic ho-mogenizer. Lysates were cleared by centrifugation at 14,000 � g. Equal amountsof protein were separated on Tris-glycine polyacrylamide gels or NuPAGE (Bio-Rad) gels, and electroblotted onto 0.45-�M nitrocellulose membranes. Agarosestacking gel (1% agarose and 0.05% SDS in 200 mM Tris, pH 8.8) preparation wasadapted from Warren et al. (45). Briefly, stacking gels were poured at 65 °C, gelswere run at 15 mA and transferred at 40 mV for 4 h on ice. GFP protein, ubiquitinconjugates, and GAPDH were detected using a mixed-mouse monoclonal anti-GFP antibody (Roche), the monoclonal antibody FK1 (Chemicon), and GAPDHantibody (Fitzgerald Industries International), respectively, followed by horseradish peroxidase-conjugated goat-anti-mouse secondary antibody (GE Health-care). Enhanced chemiluminescence (ECL) and Fuji Super RX film were used fordetection. Band intensities were measured using Image J software (NIH). Slot-blots were performed on an SDS minifold I (Bio-Rad) using 0.2-�m nitrocelluloseand 0.1% SDS in PBS for equilibration and washing.

Immunoprecipitation. Brain homogenates were prepared as for Western blot-ting, and immunoprecipitation was performed on 2 mg total protein in a 1 mLfinal volume. Samples were precleared with protein A Sepharose (GE Health-care), followed by incubation with 1 �L rabbit �-GFP antibody (A6455, Mo-lecular Probes) overnight 4 °C, and immunoprecipitation with protein ASepharose. GFP immunoreactivity was detected after immunoblotting using amonoclonal �-GFP antibody as for western blotting.

Proteasome Activity Measurements. For proteasome subunit activity labeling,brain samples were homogenized in buffer containing 50 mM Tris (pH 7.5), 5mM MgCl2, 250 mM sucrose, 1 mM DTT, and 2 mM ATP, centrifuged at14,000 � g and the supernatant used for analysis. For labeling reactions, 50 �gprotein was incubated for 1 h at 37 °C with MV151 (500 nM). The reaction wasceased by boiling in Laemmli’s buffer and 5 �g total protein was resolved by12.5% SDS/PAGE. In-gel visualization of labeled proteasome subunits wasperformed as described previously (23). Proteasome activities in brain tissueand cell lysates were measured with fluorogenic substrates as describedpreviously (46). Non-proteasomal activity was determined by performing the

13990 � www.pnas.org�cgi�doi�10.1073�pnas.0906463106 Maynard et al.

assay after addition of high concentrations of proteasome inhibitor (20 �Mepoxomycin for cell lysates; 50 �M lactacystin or 10 �M MG-132 for striatalhomogenates) to the in vitro reaction.

ACKNOWLEDGMENTS. We thank Dr. Paul Taylor and members of the Dan-tuma laboratory for helpful suggestions. This work was supported by theSwedish Research Council and the Nordic Center of Excellence Neurodegen-

eration (J.Y.L., P.B., N.P.D.); the HighQ Foundation (N.P.D., J.J.L.); the SwedishCancer Society, the Hereditary Disease Foundation, the Marie Curie ResearchTraining Network (MRTN-CT-2004-512585), and the Karolinska Institute(N.P.D.); Loo and Hans Ostermans foundation and the Foundation for Geri-atric Diseases (C.J.M.); the Wenner-Gren Foundation (C.B.); the Spanish Min-istry of Science/MEC, CiberNed, Comunidad de Madrid, Fundacion ‘‘La Caixa,’’and Fundacion Ramon Areces (J.J.L).

1. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479.2. Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–

533.3. Baumeister W, Walz J, Zuhl F, Seemuller E (1998) The proteasome: Paradigm of a

self-compartmentalizing protease. Cell 92:367–380.4. Ciechanover A, Brundin P (2003) The ubiquitin proteasome system in neuordegenera-

tive diseases: Sometimes the chicken, sometimes the egg. Neuron 40:427–446.5. Bence NF, Sampat RM, Kopito RR (2001) Impairment of the ubiquitin-proteasome

system by protein aggregation. Science 292:1552–1555.6. Cummings CJ, et al. (1998) Chaperone suppression of aggregation and altered subcel-

lular proteasome localization imply protein misfolding in SCA1. Nat Genet 19:148–154.7. Venkatraman P, et al. (2004) Eukaryotic proteasomes cannot digest polyglutamine

sequences and release them during degradation of polyglutamine-containing pro-teins. Mol Cell 14:95–104.

8. Bennett EJ, Bence NF, Jayakumar R, Kopito RR (2005) Global impairment of theubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedesinclusion body formation. Mol Cell 17:351–365.

9. Duennwald ML, Lindquist S (2008) Impaired ERAD and ER stress are early and specificevents in polyglutamine toxicity. Genes Dev 22:3308–3319.

10. Bowman AB, Yoo SY, Dantuma NP, Zoghbi HY (2005) Neuronal dysfunction in apolyglutamine disease model occurs in the absence of ubiquitin-proteasome systemimpairment and inversely correlates with the degree of nuclear inclusion formation.Hum Mol Genet 20:20.

11. Tokui K, et al. (2009) 17-DMAG ameliorates polyglutamine-mediated motor neurondegeneration through well-preserved proteasome function in an SBMA model mouse.Hum Mol Genet 18:898–910.

12. Orr HT, Zoghbi HY (2007) Trinucleotide repeat disorders. Annu Rev Neurosci 30:575–621.

13. Davies SW, et al. (1997) Formation of neuronal intranuclear inclusions underlies theneurological dysfunction in mice transgenic for the HD mutation. Cell 90:537–548.

14. Landles C, Bates GP (2004) Huntingtin and the molecular pathogenesis of Huntington’sdisease. Fourth in molecular medicine review series. EMBO Rep 5:958–963.

15. Jana NR, Zemskov EA, Wang G, Nukina N (2001) Altered proteasomal function due tothe expression of polyglutamine-expanded truncated N-terminal huntingtin inducesapoptosis by caspase activation through mitochondrial cytochrome c release. Hum MolGenet 10:1049–1059.

16. Mitra S, Tsvetkov AS, Finkbeiner S (2009) Single neuron ubiquitin-proteasome dynam-ics accompanying inclusion body formation in huntington disease. J Biol Chem284:4398–4403.

17. Bennett EJ, et al. (2007) Global changes to the ubiquitin system in Huntington’s disease.Nature 448:704–708.

18. Beal MF, Ferrante RJ (2004) Experimental therapeutics in transgenic mouse models ofHuntington’s disease. Nat Rev Neurosci 5:373–384.

19. Lindsten K, Menendez-Benito V, Masucci MG, Dantuma NP (2003) A transgenic mousemodel of the ubiquitin/proteasome system. Nat Biotechnol 21:897–902.

20. Gilon T, Chomsky O, Kulka RG (1998) Degradation signals for ubiquitin system prote-olysis in Saccharomyces cerevisiae. EMBO J 17:2759–2766.

21. Dantuma NP, et al. (2000) Short-lived green fluorescent proteins for quantifyingubiquitin/proteasome-dependent proteolysis in living cells. Nat Biotechnol 18:538–543.

22. Mangiarini L, et al. (1996) Exon 1 of the HD gene with an expanded CAG repeat issufficient to cause a progressive neurological phenotype in transgenic mice. Cell87:493–506.

23. Verdoes M, et al. (2006) A fluorescent broad-spectrum proteasome inhibitor forlabeling proteasomes in vitro and in vivo. Chem Biol 13:1217–1226.

24. Bett JS, et al. (2006) Proteasome impairment does not contribute to pathogenesis inR6/2 Huntington’s disease mice: Exclusion of proteasome activator REGgamma as atherapeutic target. Hum Mol Genet 15:33–44.

25. Diaz-Hernandez M, et al. (2003) Neuronal induction of the immunoproteasome inHuntington’s disease. J Neurosci 23:11653–11661.

26. Sherman MY, Goldberg AL (2001) Cellular defenses against unfolded proteins: A cellbiologist thinks about neurodegenerative diseases. Neuron 29:15–32.

27. Verhoef LG, Lindsten K, Masucci MG, Dantuma NP (2002) Aggregate formation inhibitsproteasomal degradation of polyglutamine proteins. Hum Mol Genet 11:2689–2700.

28. Wang J, et al. (2008) Impaired ubiquitin-proteasome system activity in the synapses ofHuntington’s disease mice. J Cell Biol 180:1177–1189.

29. Bett JS, Cook C, Petrucelli L, Bates GP (2009) The ubiquitin-proteasome reporter GFPudoes not accumulate in neurons of the R6/2 transgenic mouse model of Huntington’sdisease. PLoS ONE 4:e5128.

30. Kisselev AF, Goldberg AL (2001) Proteasome inhibitors: From research tools to drugcandidates. Chem Biol 8:739–758.

31. Glas R, et al. (1998) A proteolytic system that compensates for loss of proteasomefunction. Nature 392:618–622.

32. Geier E, et al. (1999) A giant protease with potential to substitute for some functionsof the proteasome. Science 283:978–981.

33. Bhutani N, Venkatraman P, Goldberg AL (2007) Puromycin-sensitive aminopeptidase isthe major peptidase responsible for digesting polyglutamine sequences released byproteasomes during protein degradation. EMBO J 26:1385–1396.

34. Welchman RL, Gordon C, Mayer RJ (2005) Ubiquitin and ubiquitin-like proteins asmultifunctional signals. Nat Rev Mol Cell Biol 6:599–609.

35. Kawaguchi Y, et al. (2003) The deacetylase HDAC6 regulates aggresome formation andcell viability in response to misfolded protein stress. Cell 115:727–738.

36. Bjorkoy G, et al. (2005) p62/SQSTM1 forms protein aggregates degraded by autophagyand has a protective effect on huntingtin-induced cell death. J Cell Biol 171:603–614.

37. Kim PK, Hailey DW, Mullen RT, Lippincott-Schwartz J (2008) Ubiquitin signals autoph-agic degradation of cytosolic proteins and peroxisomes. Proc Natl Acad Sci USA105:20567–20574.

38. Ravikumar B, et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicityof polyglutamine expansions in fly and mouse models of Huntington disease. NatGenet 36:585–595.

39. Arrasate M, et al. (2004) Inclusion body formation reduces levels of mutant huntingtinand the risk of neuronal death. Nature 431:805–810.

40. Pandey UB, et al. (2007) HDAC6 rescues neurodegeneration and provides an essentiallink between autophagy and the UPS. Nature 447:859–863.

41. Ciechanover A, Finley D, Varshavsky A (1984) Ubiquitin dependence of selectiveprotein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell37:57–66.

42. Kristiansen M, et al. (2007) Disease-associated prion protein oligomers inhibit the 26Sproteasome. Mol Cell 26:175–188.

43. Cheroni C, et al. (2008) Functional alterations of the ubiquitin proteasome system inmotor neurons of a mouse model of familial Amyotrophic Lateral Sclerosis. Hum MolGenet 18:82–96.

44. Menendez-Benito V, Verhoef LG, Masucci MG, Dantuma NP (2005) Endoplasmic retic-ulum stress compromises the ubiquitin-proteasome system. Hum Mol Genet 14:2787–2799.

45. Warren CM, Krzesinski PR, Greaser ML (2003) Vertical agarose gel electrophoresis andelectroblotting of high-molecular-weight proteins. Electrophoresis 24:1695–1702.

46. Kisselev AF, Goldberg AL (2005) Monitoring activity and inhibition of 26S proteasomeswith fluorogenic peptide substrates. Methods Enzymol. 398:364–378.

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