Tài liệu Báo cáo khoa học: Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant ppt

Analysis of oxidative events induced by expanded
polyglutamine huntingtin exon 1 that are differentially
restored by expression of heat shock proteins or treatment
with an antioxidant
Wance J. J. Firdaus
1
, Andreas Wyttenbach
2
, Chantal Diaz-Latoud
1
, R. W. Currie
1,3
and Andre
´
-Patrick Arrigo
1
1 Laboratoire Stress Oxydant, Chaperons et Apoptose, Centre de Ge
´
ne
´
tique Mole
´
culaire et Cellulaire, Universite
´
Claude Bernard Lyon-1,
Villeurbanne, France
2 Southampton Neuroscience Group, School of Biological Sciences, University of Southampton, UK
3 Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Canada
Neuronal selective loss and formation of intraneuron-
al protein aggregates are characteristics of Hunting-
ton’s disease (HD), which is one of more than 10

known neurodegenerative disorders caused by abnor-
mally expanded polyglutamine polyQ tracts in the
diseased protein [1]. HD is a progressive, autosomal
dominant and hereditary neurodegenerative disorder
that induces a relatively selective loss of neurons in
striatum and cortex. The mutated gene involved in
HD encodes the 350 kDa huntingtin protein, an iron-
regulated neuronal protein implicated in vesicle traf-
ficking [2,3] that, if inactivated, results in impairment
of basic cellular processes [4]. The mutation is charac-
terized by the expansion of CAG triplets 17 codons
Keywords
heat shock proteins; huntingtin polyQ
inclusion bodies; oxidized proteins;
proteasome; reactive oxygen species
Correspondence
A P. Arrigo, Laboratoire Stress Oxydant,
Chaperons et Apoptose, CNRS UMR 5534,
Centre de Ge
´
ne
´
tique Mole
´
culaire et
Cellulaire, Universite
´
Claude Bernard Lyon-1,
43 Blvd du 11 Novembre, 69622
Villeurbanne Ce

´
dex, France
Fax: +33 472 440555
Tel: +33 472 432685
E-mail:
(Received 16 February 2006, revised
20 April 2006, accepted 12 May 2006)
doi:10.1111/j.1742-4658.2006.05318.x
We recently reported that the transient expression of polyglutamine tracts
of various size in exon 1 of the huntingtin polypeptide (httEx1) generated
abnormally high levels of intracellular reactive oxygen species that directly
contributed to cell death. Here, we compared the protection generated by
heat shock proteins to that provided by the antioxidant agent N-acetyl-l-
cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q),
the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of
the cells with N-acetyl-l-cysteine inhibited not only mitochondrial mem-
brane potential disruption but also the increase in reactive oxygen species,
nitric oxide and protein oxidation. However, only heat shock proteins and
not N-acetyl-l-cysteine reduced the size of the inclusion bodies formed by
httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine
repeats (httEx1-103Q), heat shock proteins neither decreased oxidative
damage nor reduced the size of the inclusions. In contrast, N-acetyl-l-cys-
teine still efficiently decreased the oxidative damage induced by httEx1-
103Q polypeptide without altering the inclusions. N-Acetyl-l-cysteine was
inactive with regard to proteasome inhibition, whereas heat shock proteins
partially restored the caspase-like activity of this protease. These observa-
tions suggest some relationships between the presence of inclusion bodies
and the oxidative damage induced by httEx1-polyQ.
Abbreviations
DCFH-DA, 2¢,7¢-dichlorofluorescein diacetate; 2,4-DNPH, 2,4-dinitrophenyl hydrazine; EGFP, enhanced green fluorescent protein; FCCP,

p-trifluoromethoxy carbonyl cyanide phenylhydrazone; HA, hemagglutinin; HD, Huntington’s disease; HE, dihydroethidine; Hsp, heat shock
protein; NAC, N-acetyl-
L-cysteine; polyQ, polyglutamine tract; ROS, reactive oxygen species.
3076 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS
downstream of the initiator ATG in exon 1 (Ex1) of
the 67 exon-containing htt gene [5]. Pathogenesis in
HD correlates with the cleavage of mutated htt and
the release of an N-terminal fragment bearing the
mutation that is capable of nuclear localization [6,7].
HttEx1-polyQ N-terminal fragments with repeats of
fewer than 38 glutamine residues are soluble and
harmless, but those with more repeats are toxic and
precipitate as insoluble fibers in affected neurons [8].
In human and HD transgenic mice, the disease corre-
lates with the appearance of intraneuronal, intranu-
clear and perinuclear aggregates ⁄ inclusions containing
the abnormal N-terminal htt fragment [1,9,10]. How-
ever, the role of the inclusion bodies is controversial
[8,11,12], since experiments performed in Drosophila
and mouse models have revealed that polyQ proteins
can be toxic even in the absence of detectable forma-
tion of aggregates [13,14]. Experiments performed in
tissue culture cell models have revealed that the pres-
ence of inclusion bodies containing polyQ expanded
httEx1 correlates with the toxicity [15,16] but not
with the cell death induced by this polypeptide [17].
This suggests that inclusion bodies may decrease the
risk of cell death and could have a protective role.
More recent observations support the hypothesis that
inclusion formation is part of a mechanism that pro-

motes the clearance of mutant protein by activating
autophagy [18,19].
Intracellular aggregates containing ubiquitylated
proteins are a prominent cytopathologic feature of
most neurodegenerative disorders. For example, aggre-
gated htt-polyQ in neuronal inclusions of HD mice
and HD patients appears to be ubiquitylated [20]. The
accumulation of ubiquitylated abnormal proteins
results in the formation of pathologic aggregates that
perturb the normal physiology of neurons and lead to
proteotoxicity. The ubiquitin-26S proteasome system
(UPS), which normally degrades short-lived and
abnormal proteins, is probably recruited to eliminate
the pathologic aggregates formed by ubiquitylated htt-
polyQ [21,22]. However, this degradation is likely to
be far from complete [23], because the proteasome can-
not digest polyglutamine sequences and release them
during degradation of polyglutamine-containing pro-
teins [24]. This may interfere with proteasome function
and help explain why long polyQ expansions promote
early disease onset.
Elevated levels of oxidative damage at the level of
DNA, lipids and proteins are evident in numerous neu-
rodegenerative disorders, including Alzheimer’s disease
and HD, suggesting that oxidative stress is inherent to
these neuronal degenerations [25–29]. Recently, we and
others reported that the expression of the expanded
httEx1-polyQ gene product generated mitochondrial
complex IV deficiency, elevated reactive oxygen species
(ROS) levels and elevated nitric oxide [15,30] levels

that directly contributed to cell death. It is of interest
that the increase in ROS levels was found to correlate
with the number of CAG repeats in the httEx1-polyQ
polypeptide [15]. The mechanism responsible for the
appearance of an oxidative stress in response to the
presence of aggregated proteins including expanded
polyQ peptides is unclear [31,32]. Mitochondrial dys-
function may participate in this phenomenon, since
expression of proteins containing glutamine repeats
usually correlates with mitochondrial depolarization
[33,34] and impaired clearance of oxidized proteins
[35].
Heat shock or stress proteins (Hsps) are expressed in
neurons of polyQ diseased brains and have recently
been identified as potent inhibitors of polyQ toxicity
[16,36–38]. In cell models, Hsp70 and Hdj-1(Hsp40)
can inhibit self-assembly of polyglutamine proteins into
amyloid-like fibrils [39] and are associated with aggre-
gates in the brains of HD transgenic mice [40]. Hsp70
and Hdj-1 can inhibit polyQ aggregation and reduce
the size of htt-polyQ inclusion bodies [15,36,37] and
therefore protect against their cytotoxicity. Hsp27 is
less effective than Hsp70 ⁄ Hdj-1 in suppressing polyQ
aggregation [15]. Nevertheless, Hsp27 protects neuronal
cells against apoptosis [41,42], oxidative stress [43,44]
and polyQ-expanded httEx1-mediated oxidative stress
[15].
Several links exist between the proteasome and
oxidative stress. First, the intracellular redox status is
an important parameter that either upregulates (oxi-

dative stress conditions) [45] or downregulates (anti-
oxidant conditions) [46] the chymotrypsin-like activity
of the 20S proteasome. Second, the 20S proteasome
appears to be responsible for the degradation of
oxidized proteins [47–51], probably without the need
for a ubiquitylation step [52,53]. Indeed, relatively
mild oxidative stress rapidly (but reversibly) inacti-
vates both the ubiquitin-activating ⁄ conjugating system
and 26S proteasome activity but does not affect 20S
proteasome activity [52,54,55]. Third, it has been
observed that proteasome inhibitors can mimic the
effects of oxidative stressors on mitochondrial mem-
brane potential and increase cell vulnerability to oxi-
dative injury [32]. Moreover, Hsps can confer
resistance to oxidative stress by preserving protea-
some function and attenuating the toxicity of protea-
some inhibition [31]. It is, however, not yet known if
the oxidative stress generated by polyglutamine-con-
taining httEx1 polypeptides is due to alterations in
proteasome activities.
W. J. J. Firdaus et al. Huntingtin inclusions and oxidation
FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3077
The analysis presented here was performed in
COS-7 cells because of their very high transfection
efficiency. Using transiently transfected COS cells
expressing mutated Ex1 of htt (httEx1-polyQ), we have
compared the protective activity provided by Hsps and
the antioxidant agent N-acetyl-l-cysteine (NAC). For-
mation of inclusion bodies, mitochondrial membrane
potential ( DYm), ROS, protein oxidation, iron and

nitric oxide levels as well as proteasome activities were
examined.
Results
Hsp overexpression impedes HttEx1-polyQ-
mediated inclusion body formation whereas treat-
ment with the antioxidant agent NAC does not
To compare the oxidative effects induced by httEx1-
polyQ expression, we used monkey kidney COS-7 cells,
which are characterized by a very high yield of trans-
fection efficiency (more than 80%; Fig. 1B). Transfec-
tions were performed with either a control vector
(pCIneo-EGFP) expressing enhanced green fluorescent
protein (EGFP) alone, or vectors expressing polyQ
mutants of httEx1 (25 repeats, 25Q; 72 repeats, 72Q;
and 103 repeats, 103Q) fused to EGFP. Two days after
transfection, the corresponding polypeptides (denoted
EGFP, 25Q-EGFP, 72Q-EGFP and 103Q-EGFP) were
analyzed in immunoblots probed with anti-EGFP.
Figure 1A shows comparable levels of accumulation of
these polypeptides.
Two days after transfection, COS-7 cells were also
analyzed by confocal microscopy as described in
Experimental procedures. As we previously reported
[15], httEx1-25Q-EGFP polypeptide had a diffuse
cytoplasmic distribution and did not form inclusion
bodies (Fig. 1B,Ca). In contrast, httEx1-72Q-EGFP
polypeptide expression resulted in the formation of
perinuclear inclusion bodies in about 55% of the cells
(Fig. 1B,Cb,D). The percentage of cells that dis-
played inclusion bodies was up to 80% follow-

ing transfection with httEx1-103Q-EGFP polypeptide
(Fig. 1B,Cc,D). In both cases (72Q and 103Q), a
broad distribution of the size of the inclusions was
noticed. Moreover, the percentage of cells presenting
inclusions as well as the distribution of the size of the
inclusions were dependent on when the analysis was
performed after transfection. Therefore, all the follow-
ing analyses were performed 2 days after transfection.
At that time point, the size of the inclusions formed
by either httEx1-72Q-EGFP or httEx1-103Q-EGFP
polypeptide was heterogeneous but averaged around
10 lm.
We and others have already reported that the
expression of either Hsp70 ⁄ Hdj-1 or Hsp27 induces
protection against httEx1-polyQ-induced cell death
[15,56]. The Hsp70 ⁄ Hdj-1 chaperone machine acts by
decreasing htt aggregation [39,40], whereas Hsp27,
which is less effective than Hsp70 ⁄ Hdj-1 at reducing
aggregation, appears to interfere with cell death
through its antioxidant-related properties [15]. Hsp
overexpression in COS-7 cells was assessed by transient
transfection using vectors encoding either Hsp70, Hdj-
1, Hdj-2 or Hsp27. Hdj-2 is an isoform of Hdj-1 that
has been previously shown not to decrease htt inclu-
sion body formation in COS-7 cells [39]. Immunoblot
analysis of the intracellular level of Hsps revealed an
apparent large increase in the level of Hdj-1 and Hdj-
2, whereas the upregulation of Hsp27 and Hsp70 levels
was more modest (Fig. 1E). The effects mediated by
Hsp overexpression on the formation of inclusion bod-

ies were assessed by transient transfection of COS-7
cells with httEx1-72Q-EGFP or httEx1-103Q-EGFP
vectors in combination with vectors encoding for either
Hsp70 ⁄ Hdj-1 or Hsp27. Forty-eight hours after trans-
fection, confocal analysis was performed to analyze the
EGFP-containing inclusions. Figure 1F shows that the
expression of Hsp70 together with Hdj-1 (Hsp40) did
decrease the average size of the EGFP-containing
inclusion bodies (average size of 10 lm reduced to
about 2 lm) formed by httEx1-72Q-EGFP. This find-
ing is consistent with previous studies that showed a
decrease of aggregate ⁄ inclusion body formation by
these chaperones [39,40,57]. Hsp27 overexpression also
decreased the size of the inclusions but the effect was
less intense (average size of 10 lm reduced to about
4–5 lm). In contrast, the size of the inclusions was not
significantly altered by the presence of the antioxidant
NAC. Concerning the inclusions formed by httEx1-
103Q-EGFP, it can be seen in Fig. 1F that the
overexpression of either Hsp70 + Hsp40 or Hsp27, or
treatment with NAC, did not significantly alter their
size. Similar observations were made when cells were
treated with another antioxidant drug, glutathione
ethyl ester, instead of NAC (not shown). These results
indicate that in COS-7 cells, Hsps are not effective in
reducing the size of the inclusions if httEx1 polypep-
tide contains 103 CAG repeats.
NAC treatment reverses mitochondrial
membrane potential (DYm) disruption induced
by httEx1-polyQ but Hsps are only active towards

httEx1-72Q
The expression of httEx1-polyQ is known to alter
mitochondrial activity, leading to mitochondrial
Huntingtin inclusions and oxidation W. J. J. Firdaus et al.
3078 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS
membrane potential (DYm) disruption and ROS
production [15,58]. The phenomenon was measured in
our cell system to compare the protective effects medi-
ated by Hsps and NAC. Analysis of DYm was per-
formed in COS-7 cells transiently transfected as
described above. Forty-eight hours after transfection,
AB
EF
CD
Fig. 1. (A–D) Characterization of httEx1-polyQ-EGFP expression in COS-7 cells. (A) Immunoblot analysis performed 48 h after transfection of
total protein extracts of COS-7 cells transfected with either the pCIneo-EGFP control vector (denoted EGFP) or the same vector bearing
either the httEx1-25Q-EGFP (denoted 25Q-EGFP), httEx1-72Q-EGFP (denoted 72Q-EGFP) or httEx1-103Q-EGFP (denoted 103Q-EGFP) coding
sequence. The immunoblots were probed with anti-EGFP and visualized with ECL as described in Experimental procedures. (B) Confocal
immunofluorescence analysis of transfected cell population. COS-7 cells were transfected with vectors encoding either (a) httEx1-25Q-EGFP,
(b) httEx1-72Q-EGFP) or (c) httEx1-103Q-EGFP. Forty-eight hours after transfection, cells were fixed and analyzed by confocal microscopy as
described in Experimental procedures. Bar, 100 lm. (C) As (B) but enlarged fields are shown. Note the presence of the granules in the cyto-
plasm of the cells. Bar, 20 lm. (D) The percentage of EGFP-containing cells displaying granules is shown. The average percentages,
including standard deviations calculated from three independent experiments, are shown. (E,F) Heat shock proteins (Hsps), but not N-acetyl-
L-cysteine (NAC), decrease the size of httEx1-72Q-EGFP inclusion bodies but are not efficient in decreasing the size of those containing
httEx1-103Q-EGFP. (E) Immunoblot analysis performed 48 h after transfection of total protein extracts of COS-7 cells transfected with
either (a) control vector (pCIneo) or (b) vectors bearing the Hsp70 ⁄ Hdj-1, Hdj-2 or Hsp27 coding sequence. The immunoblots were probed
with the corresponding antibodies. Control of gel loading was performed with anti-actin. Immunoblots were visualized by ECL as des-
cribed in Experimental procedures. (F) Confocal immunofluorescence analysis of COS-7 cells transfected with vectors encoding either
httEx1-72Q-EGFP (72Q-EGFP) or httEx1-103Q-EGFP (103Q-EGFP) together with pCIneo vector or vectors bearing the Hsp70 ⁄ Hdj-1
(+ Hsp40 + Hsp70) or Hsp27 (+ Hsp27) coding sequence. NAC (2 m

M) was added (+ NAC) to the culture medium 24 h after transfection
of the cells. Two days after transfection, cells were fixed and analyzed by confocal microscopy as described in Experimental procedures.
Bar, 20 lm.
W. J. J. Firdaus et al. Huntingtin inclusions and oxidation
FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3079
cells were incubated with the fluorescent probe
MitoTracker
tm
Red (CM-H
2
XRos), and the resulting
red fluorescence was analyzed in a FACS calibur
Cytometer (see Experimental procedures). As seen in
Fig. 2A, CM-H
2
XRos fluorescence was not altered
in cells transfected with httEx1-25Q-EGFP vector,
25Q
AC
B
10
0
04080
Counts
120 160 200
10
1
10
2
FL2-H

10
3
10
4
25Q
+NAC
72Q
72Q+NAC
103Q
103Q+NAC
04080
Counts
120 160 200
10
0
10
1
10
2
FL2-H
10
3
10
4
10
0
10
1
10
2

FL2-H
10
3
10
4
04080
Counts
120 160 200
Fig. 2. Analysis of mitochondrial membrane potential (DYm) and morphology. (A) DYm analysis. COS-7 cells were transiently transfected
with vectors encoding either httEx1-25Q-EGFP (25Q), httEx1-72Q-EGFP (72Q) or httEx1-103Q-EGFP (103Q). Twenty-four hours after trans-
fection, cells were treated or not treated with 2 m
M N-acetyl-L-cysteine (NAC). Forty-eight hours after transfection, cells were incubated with
MitoTracker
TM
Red CM-H
2
XRos and analyzed by cytometry as described in Experimental procedures. The intensity of MitoTracker
TM
Red
fluorescence is shown on the FL2-H axis. Black curve, untreated cells; light curve, NAC-treated cells. (B) Quantitative analysis of the protect-
ive effect of heat shock proteins (Hsps) and NAC against httEx1-polyQ-mediated DYm disruption. Transfections were performed with a com-
bination of either httEx1-72Q-EGFP or httEx1-103Q-EGFP vectors with those encoding Hsp70 ⁄ Hdj-1 and Hsp27. As in (A), httEx1-103Q-
EGFP-expressing cells were treated or not treated with 2 m
M NAC. COS-7 cells transiently transfected with pCIneo-EGFP vector were also
treated for 15 min with 10 l
M of the mitochondria uncoupler p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) before being ana-
lyzed. Analysis was performed with MitoTracker
TM
Red CM-H
2

XRos and cytometry was performed as described in (A). The percentage of
the cell population with an FL2-H fluorescence greater than 2 · 10
1
was recorded during the FACS analysis. The percentage of decrease of
DYm was calculated as the ratio of the percentage of cells with FL2-H fluorescence greater than 2 · 10
1
in the samples to that observed in
control cells (transfected with pCIneo-EGFP). A representative experiment is presented. The data from three independent experiments were
used to perform statistical analysis (see Experimental procedures). (C) Electron microscopy analysis of mitochondrial morphology of COS-7
cells transfected with either pCIneo-EGFP vector (pCIneo), httEx1-72Q-EGFP vector (72Q) or httEx1-103Q-EGFP vector (103Q). Transfections
were performed with a combination of those encoding Hsp70 ⁄ Hdj-1 (Hsp70 + Hdj-1) and Hsp27 (Hsp27). Cells transfected with httEx1-
103Q-EGFP vector were also exposed to 2 m
M NAC before being analyzed (as described in the previous figures). Bar, 1 lm.
Huntingtin inclusions and oxidation W. J. J. Firdaus et al.
3080 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS
whereas it was slightly decreased by the transfection of
httEx1-72Q-EGFP vector. This suggests an alteration
of DYm as a consequence of the expression of httEx1-
72Q-EGFP polypeptide. A similar effect was noticed
when the experiment was carried out with httEx1-
polyQ-HA vectors [15] encoding httEx1 polypeptides
with 23 or 74 glutamine repeats not fused to EGFP,
but to a hemagglutinin (HA) tag (data not shown).
This control experiment suggests that no significant
green fluorescent spillover or cytotoxicity was induced
by EGFP expression. The mitochondrial depolariza-
tion mediated by httEx1-103Q-EGFP expression was
more drastic and was roughly similar to that induced
by a 15 min incubation of control cells with a 10 lm
solution of the mitochondrial depolarizer p-trifluoro-

methoxy carbonyl cyanide phenylhydrazone (FCCP)
(Fig. 2B). These observations confirm that, in our cell
system, httEx1-polyQ expression decreases and can
even abolish DYm in a polyQ repeat-dependent man-
ner. If, after transfection, cells were treated with NAC
before being analyzed, the fluorescence of MitoTrack-
er
tm
Red was almost normal, suggesting that DYm
was not altered. Similar observations were made when
cells were treated with glutathione ethyl ester (not
shown). To analyze the effects mediated by Hsp over-
expression on DYm disruption induced by httEx1-
polyQ, COS-7 cells were transiently transfected with
vectors encoding httEx1-72Q-EGFP or httEx1-103Q-
EGFP and either Hsp70 ⁄ Hdj-1 or Hsp27. As shown in
Fig. 2B, in cells expressing httEx1-72Q-EGFP, an
almost complete reversal of the 15% decrease in DYm
was induced by Hsp27 or Hsp70 ⁄ Hdj-1 expression. In
contrast, in cells expressing httEx1-103Q-EGFP, no
significant protective effect of Hsps was detected
against the 65% loss in MitoTracker
tm
Red fluores-
cence. Electron microscopy analysis (see Experimental
procedures) was performed as a control. This experi-
ment confirms that COS-7 cells transiently transfected
with vectors encoding either httEx1-72Q-EGFP or
httEx1-103Q-EGFP have mitochondria with damaged
morphology (Fig. 2C), a phenomenon not observed in

the presence of NAC. In this respect, Hsps were active
only in the case of cells transfected with httEx1-72Q-
EGFP vector. In cells expressing httEx1-103Q-EGFP,
the presence of Hsps did not restore normal morphol-
ogy of the mitochondria (Fig. 2C).
This suggests that ROS are probably responsible for
the DYm disruption and damage to mitochondrial
morphology in httEx1-72Q-EGFP-expressing or
httEx1-103Q-EGFP-expressing cells. In contrast,
httEx1-25Q-EGFP expression did not alter DYm
(Fig. 2A) or the morphology of mitochondria (not
shown).
Comparative analysis of the protective effect of
NAC and Hsps against ROS, protein oxidation,
iron and nitric oxide level upregulation caused
by expanded httEx1 expression
DYm disruption usually causes an intracellular burst
of ROS [58] that induce oxidative damage, such as that
observed in cells expressing expanded httEx1 [15].
Recently, we showed, using different cell lines, inclu-
ding COS-7 cells, incubated with the fluorescent probe
DCFH-DA, that peroxide production was induced by
the expression of httEx1-polyQ-HA polypeptides [15].
An increase in the number of CAG repeats from 23 to
74 correlated with an increase in the oxidation process.
Here, we have performed similar experiments using the
httEx1-polyQ-EGFP vectors described above that con-
tain a broader range of polyQ repeats: 25, 72 and 103.
As seen in Fig. 3A, 48 h after transfection, the fluores-
cence of DCFH-DA (see Experimental procedures)

increased by 30% in cells expressing httEx1-25Q-
EGFP compared to the value observed in cells expres-
sing EGFP only. An almost two-fold increase
(P<0.001) was then observed in httEx1-72Q-EGFP-
expressing cells compared to httEx1-25Q-EGFP-
expressing cells. The fluorescence index was further
increased by about 17% in cells expressing the httEx1-
103Q-EGFP polypeptides. As shown in Fig. 2, cells
were treated with NAC before being analyzed to deter-
mine if the increase in fluorescence described above
was indeed due to ROS accumulation. In the presence
of the antioxidant, the fluorescence of httEx1-103Q-
EGFP-expressing cells decreased and was roughly
similar to that observed in cells expressing httEx1-
25Q-EGFP. Immunoblot analysis revealed a constant
level of expression of httEx1-polyQ-EGFP polypep-
tides in the presence of NAC (not shown) and, as
shown in Fig. 1, NAC did not change the size and
EGFP fluorescence of inclusion bodies. Similar obser-
vations were made in cells treated with glutathione
ethyl ester (not shown). We also tested the effects
mediated by the pan-caspase inhibitor z-VAD-fmk to
verify that the increase in ROS did not arise from the
low percentage (about 20%) of cells that underwent
apoptosis in response to 48 h of expression of httEx1-
polyQ-EGFP polypeptides. We have previously
reported that z-VAD-fmk completely suppressed h ttEx1-
polyQ-induced death in COS-7 cells [39]. As seen in
Fig. 3A, z-VAD-fmk did not significantly modify the
increased fluorescent signal in cells transiently expres-

sing httEx1-103Q-EGFP. A similar observation was
made in the case of cells expressing httEx1-72Q-EGFP
(not shown). Hence, upregulation of ROS levels
appears to be an intrinsic property of living COS-7
W. J. J. Firdaus et al. Huntingtin inclusions and oxidation
FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3081
cells expressing httEx1-72Q or httEx1-103Q polypep-
tides. Similar to the use of DCFH-DA, upregulated
fluorescence was detected using dihydroethidine (HE),
a probe that is preferentially oxidized to ethidium bro-
mide by superoxide anions O
2
•–
(data not shown) and
has a different fluorescent emission wavelength from
EGFP (EGFP, 510 lm; HE, 590 lm). Hence, despite
the fact that EGFP and DCFH-DA have quite sim-
ilar emission wavelengths, it is possible to detect an
NAC-sensitive increase in fluorescence that reflects
accumulation of intracellular ROS levels in COS-7
cells transiently transfected with httEx1-polyQ-EGFP
vectors.
To analyze the effects on ROS mediated by Hsp over-
expression, COS-7 cells were transiently transfected
with vectors encoding httEx1-72Q-EGFP and either
Hsp70 ⁄ Hdj-1 or Hsp27. In control cells transfected with
the EGFP vector, the overexpression of Hsp70 ⁄ Hdj-1
decreased ROS levels slightly but not significantly
(Fig. 3B). In contrast, overexpression of Hsp27 was
more efficient and induced a significant decrease

(P<0.001; Fig. 3B). Similar observations were made
by analyzing cells expressing httEx1-25Q-EGFP (not
shown), confirming our previous observations that, even
in unstressed cells, Hsp27 transient overexpression can
decrease intracellular ROS levels [15,59]. We also show
here that the effect is specific to Hsp27, since it is not
observed in the case of Hsp70 ⁄ Hsp40(Hdj-1) overex-
pression. In cells expressing httEx1-72Q-EGFP, the
coexpression of Hsp70 ⁄ Hdj-1 inhibited the mutant
htt-induced increase in ROS levels by 65% (P<0.001)
(Fig. 3B). Coexpression of Hsp27 also significantly
reduced the httEx1-72Q-EGFP-mediated increase in
ROS levels (about 35%, P < 0.001). Coexpression of
Hsp70 ⁄ Hdj-1 and Hsp27 together in httEx1-72Q-
EGFP-expressing COS-7 cells significantly decreased
ROS level upregulation by about 80% (P<0.001,
compared to the cells expressing httEx1-72Q-EGFP).
We also analyzed the activity of the Hsp70 ⁄ Hdj-1
reconformation machine by cotransfecting COS-7
cells with vectors encoding Hsp70 and the nonactive
isoform of Hsp40, Hdj-2. In the presence of Hsp70 ⁄
Hdj-2, no significant decrease (P > 0.05) in ROS levels
was observed. However, under these conditions,
Hsp27 was still able to decrease ROS levels (30%
decrease; P < 0.001). Similarly, coexpression of the
Hsp27(C137A) mutant with either Hsp70 ⁄ Hdj-1 or
Hsp70 ⁄ Hdj-2 was less effective at reducing the ROS
levels as compared to wild-type Hsp27. This means that
in httEx1-72Q-EGFP-transfected COS-7 cells, both
Hsp70 ⁄ Hdj-1 and Hsp27 are efficient in buffering the

ROS burst generated by httEx1-72Q-EGFP expression,
and when all three Hsps were overexpressed, a more
intense decrease (P<0.001) in ROS levels was
observed, an effect that was reversed if Hsp27(C137A)
mutant was overexpressed instead of wild-type Hsp27.
Fig. 3. httEx1-polyQ expression enhances reactive oxygen species
(ROS) levels. (A) Analysis of ROS induced by httEx1-polyQ expres-
sion. COS-7 cells were transfected with either control pCIneo-EGFP
vector (EGFP vector), or vectors encoding httEx1-25Q-EGFP (25Q
EGFP), httEx1-72Q EGFP (72Q EGFP) or httEx1-103Q EGFP (103Q
EGFP). Forty-eight hours after transfection, cells were washed with
NaCl ⁄ P
i
, and incubated with 2¢,7¢-dichlorofluorescein (DCFH-DA),
and fluorescence was monitored by FACS cytometry as described
in Experimental procedures. The fluorescence index was
determined as the ratio of the fluorescence of cells expressing
httEx1-polyQ polypeptides to that of control pCIneo-EGFP vector-
transfected cells. A representative experiment is presented. As
control, 36 h after transfection, cells transfected with httEx1-103Q-
EGFP were exposed or not exposed to 20 l
M z-VAD-fmk before
being analyzed. Treatment with 2 m
M N-acetyl-L-cysteine (NAC)
was as previously described. (B) ROS induced by httEx1-72Q-EGFP
expression in COS-7 cells expressing different sets of heat shock
proteins (Hsps). COS-7 cells were transfected with either control
pCIneoEGFP vector (EGFP vector) or the vector encoding httEx1-
72Q EGFP (72Q EGFP). In addition, cotransfections were per-
formed using vectors encoding Hsp70 ⁄ Hdj-1, Hsp70 ⁄ Hdj-2, Hsp27

or mutant Hsp27(C137A). (C) Same as (B), except that cells were
transfected with httEx1-103Q-EGFP vector (103Q EGFP) and that
Hsp27 C137A and Hdj-2 mutants were not analyzed. The data from
four independent experiments were used to perform statistical ana-
lysis (see Experimental procedures). In (A) and (B) the asterisks
denote statistical significance when compared with respective con-
trols: *P < 0.05; **P < 0.001.
Huntingtin inclusions and oxidation W. J. J. Firdaus et al.
3082 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS
The Hsp27(C137A) mutant is characterized by the sub-
stitution of the unique cysteine residue of Hsp27 by an
alanine residue, and is unable to protect against cell
death induced by different agents, including oxidative
stress [15,44,60].
In cells expressing httEx1-103Q-EGFP, the efficiency
of Hsps was less marked, since Hsp70 + Hdj-1
decreased ROS production by 37% and Hsp27 by only
18% (Fig. 3C). In contrast, NAC completely abolished
ROS production (Fig. 3A).
Hence, these observations support the hypothesis
that the expression of expanded httEx1 and the pres-
ence of httEx1 aggregates ⁄ inclusion bodies correlate
with elevated ROS levels, and that NAC and Hsps
have different abilities to counteract this phenomenon.
One of the most potent ROS that oxidize macromol-
ecules inside the cell is the hydroxyl radical (OH
•
),
which originates from the Harber–Weiss ⁄ Fenton reac-
tions [61–63]. One of the major and easily detectable

oxidative modifications mainly induced by OH
•
is the
formation of carbonyl residues on amino acid side
chains of proteins [43,64]. In order to explore the abil-
ity of httEx1-polyQ to oxidize cellular proteins, we
performed immunoblot detection of protein carbonyl
residues in 2,4-dinitrophenylhydrazine (2,4-DNPH)-
treated extracts of COS-7 cells expressing the different
httEx1-polyQ-EGFP polypeptides (see Experimental
procedures) (Fig. 4A). Quantitative analysis of the
oxyblots (in the 10–40 kDa molecular mass range) is
presented in Fig. 4B,C. As seen in Fig. 4A, httEx1-
polyQ-EGFP expression increased the detection of
protein carbonyl residues in cellular polypeptides in a
polyQ expansion size-dependent manner. No specific
oxidized protein bands corresponding to the gel migra-
tion of httEx1-polyQ-EGFP polypeptides were detec-
ted, suggesting that httEx1-polyQ-EGFP expression
mainly enhances the oxidation of cellular proteins
(particularly in the 10–40 kDa molecular mass range)
that already display a basal level of oxidation in con-
trol cells. Expression of Hsp70 ⁄ Hdj-1 or Hsp27 did
not significantly change the pattern and level of oxid-
ized proteins in control cells, whereas the overexpres-
sion of these chaperones correlated with a decreased
level of oxidized proteins in response to httEx1-72Q-
EGFP expression (Fig. 4A). Analysis of httEx1-103Q-
EGFP-expressing cells revealed that in this case
Hsp70 ⁄ Hdj-1 or Hsp27 were not efficient in counter-

acting the increased level of protein oxidation. In con-
trast, NAC efficiently interfered with the accumulation
of oxidized proteins in httEx1-103Q-EGFP-expressing
cells. These observations suggest that elevated levels of
OH
•
are produced in cells expressing httEx1-polyQ-
EGFP polypeptides.
Iron regulates huntingtin polypeptide [2] and cata-
lyzes OH
•
formation through Fenton reactions [61–
63]. Since elevated levels of OH
•
appear to be pro-
duced in cells expressing httEx1-polyQ-EGFP poly-
peptides, we have analyzed whether the phenomenon
correlated with increased levels of Fe(II). The intra-
cellular level of Fe(II) was determined (see Experi-
mental procedures) in COS-7 cells transfected as
described above. As seen in Fig. 5, the expression of
httEx1-25Q-EGFP and httEx1-72Q-EGFP polypep-
tides induced only a weak increase in the absorbance
of the ferrozine–Fe(II) complex. In contrast, expres-
sion of httEx1-103Q-EGFP polypeptide resulted in a
1.7-fold increase in absorbance, which was abolished
when cells were cotransfected with vectors encoding
either Hsp70 ⁄ Hdj-1 or Hsp27 or were treated with
NAC. These observations suggest that the increase in
Fe(II) levels observed in cells transiently expressing

httEx1-103Q-EGFP polypeptide is a consequence
rather than a cause of the deleterious effect generated
by the oxidative stress.
Another important parameter of oxidative stress is
nitric oxide (NO
•
). Indeed, elevated levels of NO
•
have
been observed in HD [65] and transgenic HD mice
(R6 ⁄ 2 and R6 ⁄ 1 model) that may contribute to patho-
genesis and precede neuronal cell death [66,67]. The
pathology of NO
•
results from its reaction with O
2
•–
to form peroxynitrite (ONOO
•–
), which can diffuse for
several micrometers before decomposing to form the
powerful and cytotoxic oxidants OH
•
and nitrogen
dioxide [68]. These observations prompted us to ana-
lyze NO
•
levels in COS-7 cells expressing httEx1-
polyQ-EGFP polypeptides and to test whether Hsps or
NAC could modulate NO

•
levels.
A comparison of NO
•
levels in COS-7 cells trans-
fected with either control or httEx1-polyQ-EGFP vec-
tors was performed. Figure 6 shows that the transient
expression of httEx1-25Q-EGFP did not much change
the intracellular level of NO
•
. In contrast, httEx1-72Q-
EGFP expression increased the intracellular level of
NO
•
by about 38% (P<0.001). The increase was up
52% (P<0.001) in the case of httEx1-103Q-EGFP
expression. When the vectors encoding either Hsp70 ⁄
Hdj-1 or Hsp27 were cotransfected, a small but signifi-
cant decrease in the basal level of NO
•
was observed
(P<0.001) compared to the level observed in COS-7
cells transfected with control EGFP and httEx1-25Q-
EGFP vectors. When the Hsp-encoding vectors were
transfected together with that encoding httEx1-72Q-
EGFP, the level of NO
•
was the same as in control
cells (P<0.001). Under these conditions, both
Hsp70 ⁄ Hdj-1 and Hsp27 expression abolished the

increase in NO
•
level generated by httEx1-72Q-EGFP
W. J. J. Firdaus et al. Huntingtin inclusions and oxidation
FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3083
expression. Concerning the elevation of NO
•
induced
by httEx1-103Q-EGFP polypeptide, Hsp70 ⁄ Hdj-1
overexpression had no significant effects, whereas
Hsp27 reduced the increase in NO
•
level by more than
50% (P<0.001). It is of interest that NAC com-
pletely abolished the increase in NO
•
level generated
by httEx1-103Q-EGFP expression.
Analysis of httEx1-polyQ expression with regard
to the three major proteolytic activities of 20S
proteasome, a phenomenon partially restored
by Hsp expression but not by NAC
Proteasome inhibition is known to induce intracellular
protein aggregation and increased carbonyl formation
EGFP
vector
A
BC
Control
Hsp70/Hdj-1

Hsp27
Control
Hsp70/Hdj-1
Hsp27
Control
Hsp70/Hdj-1
Hsp27
Control
Hsp70/Hdj-1
Hsp27
NAC
kDa
54
37
29
20
25Q EGFP
vector
72Q EGFP
vector
103Q EGFP
vector
Fig. 4. (A) Oxyblot analysis. COS-7 cells were transfected with either control pCIneo-EGFP vector (EGFP vector) or the vector encoding
httEx1-25Q-EGFP (25Q EGFP vector), httEx1-72Q-EGFP (72Q EGFP vector) or httEx1-103Q-EGFP (103Q EGFP vector). Cotransfections were
performed using the vectors encoding Hsp70 ⁄ Hdj-1 or Hsp27. Forty-eight hours after transfection, cells were lysed and the carbonyl content
present in proteins was determined using 2¢,4¢-dinitrophenyl hydrazine (2,4-DNPH) as described under Experimental procedures. Quantita-
tively equivalent amounts of each fraction were analyzed. The immunoblots were probed with anti-DNPH, and gel loading was verified by
immunological detection of actin (not shown). Immunoblots were visualized by ECL as described in Experimental procedures. The samples
from the derivation-control solution (negative controls, see Experimental procedures) were devoid of any signals and are not presented in
the figure. As a control, 24 h after transfection, cells transfected with httEx1-103Q-EGFP were exposed to 2 m

M N-acetyl-L-cysteine (NAC)
before being analyzed. The arrow indicates the position of the more intensively oxidized polypeptide in the assay. The bracket underlines the
domain (molecular mass range 10–40 kDa) of the oxyblots that contains the greatest changes in protein oxidation. (B) Quantitative analysis
of the oxyblots presented in (A) (see Experimental procedures). The domains of the blots indicated by a bracket (see Fig. 4A) were scanned
and the signals quantified (see Experimental procedures). This approach was used to avoid the major oxidized protein (about 45 kDa), which
shows a rather unaltered signal throughout the experiment. The level of protein oxidation (arbitrary units) is presented. (C) Protein oxidation
index. The values in (B) were divided by the value determined for the control cells transfected with the EGFP vector. The results from a rep-
resentative experiment are shown.
Huntingtin inclusions and oxidation W. J. J. Firdaus et al.
3084 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS
in proteins [31,69]. Hence, we first investigated the pos-
sibility that proteasome inhibition could be responsible
for the oxidative stress mediated by the expression of
httEx1-polyQ-EGFP polypeptides. Control pCIneo-
EGFP-transfected COS-7 cells were exposed for 1 h to
10 lm of the proteasome inhibitor lactacystin. In these
cells, 80% inhibition of proteasome activities correla-
ted with a 50% increase in ROS levels and with a 1.7-
fold increase in the level of oxidized proteins (ranging
between 10 and 40 kDa, as defined above in Fig. 4)
(not shown). The oxidative stress induced by protea-
some inhibition therefore seems to be less intense than
that induced by the expression of httEx1-72Q-EGFP
and httEx1-103Q-EGFP polypeptides (see above;
Figs 3 and 4).
We next analyzed the effects mediated by the expres-
sion of the different httEx1-polyQ-EGFP polypeptides
and Hsps as well as those induced by NAC treatment
on the three major proteolytic activities of the 20S pro-
teasome. Indeed, Hsps (particularly, Hdj-1 ⁄ Hsp40) can

confer resistance to oxidative stress by preserving
proteasome function and by attenuating the toxicity
induced by proteasome inhibition [31]. To perform this
analysis, COS-7 cells were transiently transfected with
the different vectors encoding httEx1-polyQ-EGFP or
Hsps as described above. Forty-eight hours after trans-
fection, the chymotrypsin-like activity of the 20S
proteasome was determined in cell extracts with fluoro-
peptide suc-LLVY-MCA, and the trypsin-like and
caspase-like activities were determined using N-boc-
LSTR-MCA and N-Cbz-LLEb-NA fluoropeptides,
respectively (Fig. 7; see Experimental procedures). No
alteration of the chymotrypsin-like activity was
induced by httEx1-25Q-EGFP expression, and only a
slight decrease (about 10%) was induced by httEx1-
72Q-EGFP and httEx1-103Q-EGFP expression
(Fig. 7A). No significant effects were induced by either
Hsp70 ⁄ Hdj-1 or Hsp27 overexpression or NAC treat-
ment. The trypsin-like activity of 20S proteasome was
more altered than the chymotrypsin-like activity, since
a 30% decrease was noticed in httEx1-103Q-EGFP-
expressing cells (P<0.01). Despite a small increase in
the trypsin-like activity mediated by Hsp70 ⁄ Hdj-1 and
Hsp27 in control EGFP cells, these chaperones were
not effective in restoring the inhibition mediated by
Fig. 5. Analysis of intracellular level of iron [Fe(II)]. Forty-eight hours
after transfection using the vectors described in Fig. 4A, COS-7
cells were washed and scraped off the culture dish in NaCl ⁄ P
i
. Fol-

lowing centrifugation, pelleted cells were used to determine the
Fe(II) level as described in Experimental procedures. All samples
contained similar amounts of protein. Absorbance of the ferrozine–
Fe(II) complex (AU, arbitrary units) was read at 562 nm. The results
for cells treated, as described in the previous figures, with 2 m
M
N-acetyl-L-cysteine (NAC) is presented. The data from three inde-
pendent experiments were used to performed statistical analysis
(see Experimental procedures). *P < 0.05.
Fig. 6. Nitric oxide level determination. COS-7 cells were transfected
with either control pCIneo-EGFP vector (EGFP vector), or the same
vector encoding httEx1-25Q-EGFP (25Q EGFP), httEx1-72Q-EGFP
(72Q EGFP) or httEx1-103Q-EGFP (103Q EGFP). Cotransfections
were performed using the vectors encoding Hsp70 ⁄ Hdj-1 or Hsp27.
Forty-eight hours after transfection, cells were processed for nitric
oxide level determination as described in Experimental procedures.
Cells transfected with httEx1-103Q-EGFP were also exposed to
2m
M N-acetyl-L-cysteine (NAC) before being analyzed (as described
in the previous figures). The data from three independent experi-
ments were used to performed statistical analysis (see Experimental
procedures). The asterisks denote statistical significance when
compared with respective controls: *P < 0.05; **P < 0.001.
W. J. J. Firdaus et al. Huntingtin inclusions and oxidation
FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3085
httEx1-polyQ expression (Fig. 7B). A similar observa-
tion was made in cells treated with NAC. In contrast,
the caspase-like activity was strongly altered by httEx1
polyQ-EGFP expression, and the level of this inhibi-
tion increased with polyQ expansion size, reaching

more than 60% in cells expressing httEx1-103Q-EGFP
polypeptide (P<0.01; Fig. 7C). With regard to this
activity, the Hsps were slightly more active (partic-
ularly Hsp70 + Hdj-1), and a partial but significant
restoration could be observed in httEx1-103Q-EGFP-
expressing cells, which then displayed an inhibition
that was similar to that observed in httEx1-72Q-
expressing cells (about 40% inhibition instead of 60%,
P < 0.01). In contrast, NAC was still inefficient in
restoring this activity. These observations indicate that
the proteolytic activities of 20S proteasome are differ-
entially altered by httEx1-polyQ-EGFP expression.
Hsps can partially restore the caspase-like activity, and
the phenomenon is observed even in cells expressing
httEx1-103Q-EGFP. Once again, NAC has no effect,
suggesting that the inhibition of proteasome activities
by httEx1-polyQ-EGFP expression is oxidative stress
independent. Taken together, these results also suggest
that proteasome partial inhibition by httEx1-polyQ is
not the major cause of the oxidative stress observed in
cells expressing these proteins.
Discussion
We have already shown that the expression of httEx1-
polyQ polypeptides modulates the redox status of sev-
eral cell types, including, SK-N-SH neuronal precursor
cells and non-neuronal HeLa and COS-7 cells [15].
Here, we have used COS-7 cells, which have the
advantages of being transfected with a high level of
efficiency and of displaying a flat morphology that
facilitates polyQ inclusion detection. We have observed

that the expression of either Hsp70 + Hdj-1 or Hsp27
reduced inclusion size in COS-7 cells expressing httEx1
with 72Q repeats. However, Hsps were ineffective
towards the inclusions formed by httEx1-103Q. No
significant modulation of inclusion size by NAC was
detected. These findings confirm the results of several
other studies performed in cellular models of polyQ
disease that showed no effect of antioxidant com-
pounds on inclusion formation [15]. An almost com-
plete reversal of httEx1-72Q-EGFP-mediated DYm
disruption and mitochondrial morphology alteration
was observed in cells that overexpress Hsp70 ⁄ Hdj-1 or
Hsp27. In contrast, the overexpression of these Hsps
did not attenuate the drastic mitochondrial defects
generated by httEx1-103Q-EGFP. Hence, despite the
fact that unaggregated mutant htt may already be
toxic [34,70], httEx1-polyQ toxicity towards mitochon-
dria increased in a CAG repeat expansion-dependent
manner to reach a point where it could not be restored
60
A
B
C
*
*
*
*
*
*
*

*
*
40
suc-LLVY-MCA (A.U.) × 10
–3
N-boc-LSTR-MCA (A.U.) × 10
–3
N-Cbz-LLEβ-NA (A.U.) × 10
–3
20
0
60
40
20
0
60
40
20
0
60
40
20
0
60
40
20
0
EGFP vector
25Q EGFP
72Q EGFP

103Q EGFP
Hsp70/Hdj-1
Hsp27
NAC
+++





+
-+-
60
40
20
0
+++





+
-+-
+++




+

-+-

++ +



+
-+-


++

-+
-
-

*
Fig. 7. Analysis of 20S proteasome activities in httEx1-polyQ-
expressing COS-7 cells. COS-7 cells were transiently transfected
with either control pCIneo-EGFP vector (EGFP vector), or the same
vector encoding httEx1-25Q-EGFP (25Q EGFP), httEx1-72Q-EGFP
(72Q EGFP) or httEx1-103Q-EGFP (103Q EGFP). Cotransfections of
the cells with vectors encoding either Hsp70 ⁄ Hdj-1 or Hsp27 were
also performed. Forty-eight hours after transfection, cells were
lysed and processed for the analysis of the three major activities
of the 20S proteasome using the fluorogenic substrates, suc-
LLVY-MCA, N-boc-LSTR-MCA and N-Cbz-LLEb-NA to measure
chymotrypsin-like (A), trypsin-like (B) and caspase-like (C) activities,
respectively. Cells transfected with httEx1-103Q-EGFP were also
exposed to 2 m

M N-acetyl-L-cysteine (NAC) before being analyzed
(as described in the previous figures). Treatment with lactacystin
abolished suc-LLVY-MCA fluorescence by more than 80% (not
shown). The data from three independent experiments were used
to perform statistical analysis (see Experimental procedures). The
asterisks denote statistical significance when compared with
respective controls: *P < 0.05; **P < 0.001.
Huntingtin inclusions and oxidation W. J. J. Firdaus et al.
3086 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS
by Hsp overexpression. The protective effect provided
by Hsp expression was then compared to that media-
ted by NAC. It is of interest that even in cells expres-
sing httEx1-103Q-EGFP, the presence of NAC
counteracted DYm disruption. Hence, mitochondrial
disfunction in httEx1-polyQ-expressing cells appears to
be ROS dependent. Taking into account that NAC did
not reduce the size of inclusion bodies, the difference
in protection mediated by Hsps and NAC suggests a
correlation between the ROS-dependent DYm disrup-
tion and the presence of inclusion bodies.
We have observed that polyQ-mediated DYm
disruption correlated, in a CAG repeat expansion-
dependent manner, with an increased level of intracel-
lular ROS and with increased formation of carbonyl
residues in proteins. The enhanced oxidation of pro-
teins observed in httEx1-polyQ-expressing cells was
not found to correlate with an increased intracellular
level of iron. Interestingly, a recent proteomic analysis
detecting protein carbonyl residues confirmed that,
in vivo, some proteins are indeed oxidized in the HD

mouse brain, due to httEx1 expression [71]. Overex-
pression of Hsp70 ⁄ Hdj-1 or Hsp27 attenuated the
increase in ROS generated by httEx1-72Q-EGFP and
decreased the intracellular level of oxidized proteins,
whereas these Hsps were less effective in cells expres-
sing httEx1-103Q-EGFP. In contrast, NAC was still
effective. This further confirms that the httEx1-polyQ-
mediated increase in ROS levels and protein oxidation
correlate with the presence of large inclusions. In our
study, we have not probed the effect of htt precursors
such as htt oligomeric species on oxidative stress and
mitochondrial dysfunction, but this will be important
for future studies.
We next analyzed the protective activity of Hsps
and NAC against NO
•
upregulation in mutant hunt-
ingtin-expressing COS-7 cells. It has been proposed
that intramitochondrial peroxynitrite formation from
NO
•
is the causative agent that stimulates ROS pro-
duction by mitochondria [72]. Indeed, despite the fact
that NO
•
can be protective, particularly against H
2
O
2
cytotoxicity [73], the elevated level of NO

•
that is pro-
duced in cells expressing mutated htt is toxic and pre-
cedes neuronal death [65–67]. We show here for the
first time in a cell culture model that an elevated level
of NO
•
is generated in COS-7 cells in response to
httEx1-polyQ-EGFP expression, indicating that this
effect is not restricted to neuronal cells. In httEx1-
72Q-EGFP-expressing cells, we have observed that
Hsp70 ⁄ Hsp40 or Hsp27 overexpression abolished the
increase in NO
•
levels. In contrast, Hsps were not effi-
cient in httEx1-103Q-EGFP-expressing cells, but NAC
did abolish the NO
•
increase induced by both httEx1-
72Q-EGFP and httEx1-103Q-EGFP polypeptides.
Hence, correlations again exist between NO
•
and ROS
upregulation and the presence of aggregated httEx1-
polyQ.
It has been reported that proteasome inhibitors
induce intracellular protein aggregation and stimulate
oxidative protein modifications such as increased car-
bonyl formation, similar to those seen with hydrogen
peroxide treatment [69]. Proteasome inhibition has

been proposed to favor mitochondrial dysfunction
through oxidative stress induction [32] and to increase
inclusion body formation by httEx1-polyQ polypeptide
in various cell types [39]. At the protein level, we con-
firmed that lactacystin increased the level of oxidized
proteins in control cells; however, this increase was
smaller than that induced by httEx1-103Q-EGFP
expression. We also observed a decrease in the three
major 20S proteasome activities that was CAG repeat
expansion dependent. This may reflect the fact that
proteasome has difficulty in degrading CAG repeats
[74]. However, the trypsin-like activity (30% inhibi-
tion) and particularly the caspase-like activity (60%
inhibition) appeared to be more affected than the
chymotrypsin-like activity (10% inhibition only). This
observation may have implications for what the
proteasome can do or not do during Huntington
pathology.
We also observed that, in httEx1-polyQ-EGFP-
expressing cells, overexpression of Hsp70 ⁄ Hdj-1 or
Hsp27 had a beneficial effect on the more altered
activity, i.e. the caspase-like activity. Whether the phe-
nomenon results from a direct effect of Hsps on the
proteasome or these proteins stimulate some of its
activities because of better substrate availability is not
yet known. The partial restoration of proteasome
activities by Hsps contrasts with the lack of effect
induced by NAC. Hence, the partial inhibition of pro-
teasome activities generated by httEx1-polyQ expres-
sion does appear to be a consequence of the oxidative

stress induced by this mutant protein. The effects
induced by Hsps in httEx1-72Q-EGFP-expressing cells
suggest that the interferences in proteasome activities
do not depend on inclusion body formation. More-
over, the comparison with the oxidative stress induced
by lactacystin suggests that the rather weak alterations
in proteasome activities induced by httEx1-polyQ
expression are not responsible for the oxidative stress
generated by these polypeptides.
Hence, the results presented in this study suggest the
following question: could httEx1-polyQ inclusion bod-
ies generate ROS by themselves? Intriguing observa-
tions may favor this hypothesis. For example, in vitro,
aggregated a-synuclein (Parkinson) and b-amyloid
W. J. J. Firdaus et al. Huntingtin inclusions and oxidation
FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3087
(Alzheimer) polypeptides generate spin-trap detectable
ROS when incubated with redox-active transition
metals, such as iron or copper [75]. Since htt is an
iron-regulated protein, it will be of interest to test whe-
ther it shares the ability to produce ROS in the pres-
ence of metals. If the hypothesis is true, it will mean
that oxidative events are generated by httEx1-polyQ
inclusion bodies that alter the function of mitochon-
dria concentrated in the area surrounding the inclu-
sions [76], leading to a subsequent burst of ROS of
mitochondrial origin.
Several reports have proposed the provocative idea
that inclusion bodies are in fact beneficial to the cell
[17,19] by, for example, promoting the clearance of

mutant protein by activating autophagy through the
inhibition of mTOR [19]. Oxidative events may there-
fore represent a problem that the cell has to challenge
to promote the formation and clearance of these bod-
ies. Aggregate clearance could therefore be balanced
between the rate of huntingtin aggregation and the
deleterious oxidative stress that correlates with the
presence of these aggregates.
Experimental procedures
Cell culture, DNA vectors, transfection and
reagents
African green monkey kidney (COS-7) cells were cultured,
and seeded in DMEM (Gibco, Invitrogen, Paisley, UK)
supplemented with 10% heat-inactivated FBS. The medium
included penicillin and streptomycin (10
4
UÆL
)1
) (Gibco,
Invitrogen), and 250 lgÆmL
)1
Fungizone (Gibco, Invitro-
gen). The cells were maintained at 37 °C in a 5% CO
2
atmosphere with 95% humidity. The EGFP-encoding DNA
vector (pCIneo-EGFP) as well as the same vector contain-
ing Ex1htt with 25, 72 or 103 glutamine repeats fused to
EGFP (called httEx1-25Q-EGFP, httEx1-72Q-EGFP and
httEx1-103Q-EGFP) have already been described [15]. A
vector encoding Ex1htt fused to HA containing 72 gluta-

mine repeats (called httEx1-72Q-HA) was also used [15].
The control vector (pCIneo) and Hsp27 wild-type-bearing
vector (pCIneohsp27) have already been characterized [77].
Vectors encoding human Hdj-1(Hsp40), Hdj-2 and Hsp70
have already been described [15], as well as vectors enco-
ding wild-type Hsp27 and mutant Hsp27(C137A) [44,78].
For transfection experiments, exponentially growing COS-7
cells were plated in 60 mm plates (5 · 10
5
cells per plate)
(TPP, Zu
¨
rich, Switzerland) 1 day before transfection. Each
transfection experiment was performed with 3 lgof
DNA encoding httEx1-polyQ or the various chaperones
(Hsp70 ⁄ Hdj-1 and Hsp27) using lipofectamine (Gibco, Invi-
trogen) according to the manufacturer’s instructions. In the
case of transfection with multiple vectors, we used identical
amounts of each vector (1–2 lg of DNA to a total of
4 lg). Forty-eight hours after transfection, the cell medium
was removed and replaced with DMEM supplemented with
10% FBS. z-VAD-fmk and NAC were from Sigma–Aldrich
(St-Quentin-Fallavier, France).
Immunoblot analysis
COS-7 cells were harvested and the cell pellet was solubi-
lized in boiling 2 · SDS sample buffer. Protein concentra-
tion was determined in aliquots using the Bradford protein
assay. Total protein samples were separated by 12%
SDS ⁄ PAGE before being analyzed on immunoblots probed
with either anti-EGFP (1 : 1000) (Molecular Probes ⁄ Inter-

chim, Montluc¸ on, France), anti-Hdj-1(Hsp40) (1 : 1000)
(Stressgen, Victoria, Canada), anti-Hsp27 (1 : 200) (Stress-
gen), anti-Hsp70 (1 : 1000) (Stressgen) or anti-actin (Tebu,
Le Perray en Yvelines, France). Blots were probed with
peroxidase-labeled anti-mouse or anti-rabbit IgG (1 : 1000)
(Tebu). Protein bands were visualized with the ECL
tm
sys-
tem (Amersham Biosciences, GE Healthcare, Chalfont
St Giles, UK). Autoradiographs were recorded on X-Omat
LS films (Eastman Kodak Co., Rochester, NY).
Membrane potential (DYm) and electron
microscopy analysis of mitochondria
Twenty-four hours after transfection, 3 · 10
5
COS-7 cells
were plated in dishes (45 mm diameter ⁄ 2 mL of medium)
and further grown for 24 h. After this time period, cells
were either kept untreated or incubated for 15 min with
10 lm of the mitochondrial uncoupler FCCP (Sigma-
Aldrich). Cells were washed in NaCl ⁄ P
i
, resuspended in
growth medium and exposed for 15 min to 50 nm of
MitoTracker
tm
Red CM-H
2
XRos (Molecular Probes).
Cells were washed in NaCl ⁄ P

i
before being analyzed in a
FACS calibur Cytometer (Becton Dickinson, Mountain
View, CA) equipped with an argon ion laser emitting at
488 nm. CM-H
2
XRos fluorescence was detected in the
FL2-H channel. The percentage of cells that displayed a
FL2-H fluorescence value higher than 2 · 10
1
was auto-
matically determined and used to calculate the percentage
of mitochondrial membrane potential disruption induced
by httEx1-polyQ or FCCP. For electron microscope ana-
lysis, transfected COS-7 cells were grown for 48 h in
60 mm diameter plates before being fixed for 30 min at
4 °C in a buffer composed of 2% glutaraldehyde in 0.1 m
Na-cacodylate ⁄ HCl buffer, pH 7.4. Cells were then rinsed
three times (overnight) at 4 °C in 0.1 m Na-cacody-
late ⁄ HCl buffer, pH 7.4, containing 0.2 m sucrose, before
being postfixed for 30 min at 4 °C in a buffer composed
of 1% osmium tetroxide and 0.15 m Na-cacodylate ⁄ HCl,
adjusted to pH 7.4. Cells were then dehydrated with gra-
ded ethanol, scraped and pelleted in 70% ethanol and
embedded in Epon as a cell pellet. After polymerization
Huntingtin inclusions and oxidation W. J. J. Firdaus et al.
3088 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS
at 60 °C for 3 days, ultrathin sections (60–80 nm) were
cut using an RMC MTX ultramicrotome (Ventana Medi-
cal Systems, Tucson, AZ, USA), collected on 200 mesh

copper grids, stained with uranyl acetate and lead citrate,
and observed with a JEOL 1200 CX transmission electron
microscope (Jeol, Tokyo, Japan). Images were recorded
with a MegaviewII numeric camera, and analysis soft-
ware (Soft Imaging System GmbH, Mu
¨
nster, Germany)
was used to analyze the images.
Determination of intracellular ROS levels
After transfection, 5 · 10
4
COS-7 cells per well were plated
in 96-well tissue culture plates and allowed to grow for
48 h at 37 °C. Cells were then washed three times in
NaCl ⁄ P
i
before being incubated for 20 min in NaCl ⁄ P
i
containing 5 lgÆmL
)1
2¢,7¢-dichlorofluorescein diacetate
(DCFH-DA) (Molecular Probes). DCFH-DA oxidation
was monitored in a cytofluorometer (Wallac, Victor,
Finland) with an excitation wavelength of 485 nm and an
emission wavelength of 530 nm. ROS levels were also esti-
mated using HE. In this case, cells were incubated for
10 min in NaCl ⁄ P
i
containing 40 lgÆmL
)1

HE and analyzed
by flow cytometry with an excitation wavelength of
488 nm. The emission filter was 610 nm bandpassed for
ethidium bromide fluorescence (FL2-H).
Determination of intracellular iron levels
The method used to estimate intracellular levels of total
Fe(II) was based on the Fish colorimetric assay [1]. In brief,
cells were washed and scraped off the culture dish in
NaCl ⁄ P
i
. Following centrifugation, pelleted cells were resus-
pended in 1 mL of water. An equivalent of 2 mg of total
cell protein was used per assay. After addition of 500 lL
of solution A (0.6 m HCl, 0.142 m KMnO
4
), the samples
were incubated for 2 h at 60 °C. During this incubation,
iron was released in soluble form. Subsequent to addition
of 100 lL of buffer B [6.5 mm ferrozine (disodium 3-(2-
pyridyl)-5,6-bis(4-phenylsulfonate)-1,2,4-triazine (Sigma
P5338), 2 m ascorbic acid, 5 m ammonium acetate] and a
further 60 min incubation, absorbance of the ferrozine–
Fe(II) complex was read at 562 nm.
Immunofluorescence of EGFP inclusions
Transfected COS-7 cells were grown on coverslips in
60 mm plates. Forty-eight hours after transfection, cells
were rinsed once in DMEM supplemented with 10% FBS
before being washed once with NaCl ⁄ P
i
devoid of calcium

and magnesium and fixed for 10 min with 3.7% formalde-
hyde, pH 7.4, in NaCl ⁄ P
i
. Permeabilization was for 3.5 min
in 0.2% Triton X-100. Examination of samples was
performed in a LSM510 laser scanning confocal Zeiss
microscope using a 63· (NA 1,4) Zeiss Plan Neo Fluor
objective (Carl Zeiss SAS, Le Pecq, France).
Analysis of protein carbonyl residues
Immunoblot detection of carbonyl residues was done as
previously described [64] using the S7150 Oxyblot
tm
Protein
Oxidation Detection Kit from Chemicon International
(Temecula, CA). In brief, 48 h after transfection, cells were
lysed in 12% SDS in the presence of 50 m m dithiothreitol.
Ten microliters of each sample lysate was transferred into
each of two eppendorf tubes and treated for 15 min with
either 10 lL of the 1· DNPH solution or 10 lL of the
derivation-control solution (negative control). After incuba-
tion, 7.5 lL of neutralization solution was added to both
tubes. Proteins were then analyzed by gel electrophoresis,
and immunoblotting was performed using anti-DNPH
according to the manufacturer’s instructions. Immune
complexes were detected by chemiluminescence using the
ECL
tm
system (Amersham Biosciences). Autoradiographs
were recorded on X-Omat LS films (Eastman Kodak).
Quantitative analysis of the oxyblots was performed using

NIH image 1.62 software (NIH, Bethesda, MD, USA).
Determination of NO
•
levels
The Nitric Oxide Quantification Kit from Active Motif
Europe (Rixensart, Belgium) was used. In brief, 48 h after
transfection, cells were trypsinized and lysed in the buffer
provided with the kit. Since large proteins can interfere
with the Griess reaction, cell lysates were filtered through
micropore filters (10 kDa cut) using centrifugal filter tubes
(Amicon-Milipore, St-Quentin en Yvelines, France). One
hundred microliters of the filtered lysates was then analyzed
according to the manufacturer’s instructions. Absorbance
was read on a Dynatech MR5000 microplate reader (Dyna-
tech, Chantilly, VA, USA) at 540 nm with a reference
wavelength of 620 nm.
Proteasome activities
20S proteasome activity was measured in cell extracts 48 h
after transfection. COS-7 cells were washed twice with cold
NaCl ⁄ P
i
and lyzed by a 30 min-incubation in 0.5 mm dithio-
threitol as already described [79]. Unlysed cells, membranes
and nuclei were eliminated by centrifugation at 14 000 g
(Biofuge Fresno centrifuge, rotor #3325, Heraeus SAS,
Courtabeouf, France). A typical proteasome assay was car-
ried out with 100 lg of protein cell extracts in a total vol-
ume of 200 lL of proteasome buffer (50 mm Tris ⁄ HCl,
pH 7.8, 20 mm KCl, 5 mm MgOAc, 0.5 mm dithiothreitol).
The fluorogenic substrates suc-LLVY-MCA, N-boc-LSTR-

MCA and N-Cbz-LLEb-NA (Sigma-Aldrich) were added to
the cell extracts to measure chymotrypsin-like, trypsin-like
W. J. J. Firdaus et al. Huntingtin inclusions and oxidation
FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3089
and caspase-like activities, respectively. Following
incubation at 37 °C for 1 h, the reaction was stopped by
addition of sodium borate ⁄ ethanol (9 : 1). The fluorescence
of the samples was measured with a Victor Wallach cytoflu-
orimeter (EG & G Instruments, Evry, France). The excita-
tion and emission wavelengths for aminomethyl coumarine
(chymotrypsin-like and trypsin-like activities) were 365 nm
and 460 nm. In the case of naphthalamide (caspase-like
activity), the wavelength settings were excitation 355 nm
and emission 420 nm.
Statistical analysis
Data are expressed as means ± 1 SD. The significance of
differences was determined by anova and post hoc multiple
comparison test with spss 11.5 software (SPSS, Chicago,
IL, USA). P < 0.05 was considered to be statistically
significant. Probability is reported at either P < 0.05 or
P < 0.001.
Ackowledgements
We wish to thank Dominique Guillet for excellent
technical assistance and Dr Beatrice Burdin (Centre
Technologique des Microstructures, Claude Bernard
University-Lyon1, France) for assistance with micros-
copy. This work was supported by the Association
pour la recherche sur le cancer (grant number 4602)
and the Re
´

gion Rhoˆ ne-Alpes (The
´
matique Cancer) (to
APA). Wance Firdaus was a French postgraduate
scholarship holder from CNOUS (Centre National des
Oeuvres Universitaires et Scolaires), Paris. Andreas
Wyttenbach thanks the HighQ Foundation and the
Medical Research Council (MRC) for financial sup-
port. William Currie was a Visiting Professor, from
Dalhousie University, Halifax, Canada and held a
CIHR ⁄ CNRS International Scientific Exchange Schol-
arship from the Canadian Institutes of Health
Research and the Centre National de la Recherche Sci-
entifique, France.
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