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 i
Trang 1polyglutamine huntingtin exon 1 that are differentially
restored by expression of heat shock proteins or treatment with an antioxidant
Wance J J Firdaus1, Andreas Wyttenbach2, Chantal Diaz-Latoud1, R W Currie1,3
and Andre´-Patrick Arrigo1
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: arrigo@univ-lyon1.fr
(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 relaobserva-tionships 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.
Trang 2downstream 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 perinuintranu-clear 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 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
Trang 3The 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 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
Trang 4membrane 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,
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.
Trang 5cells were incubated with the fluorescent probe
MitoTrackertm
Red (CM-H2XRos), and the resulting
red fluorescence was analyzed in a FACS calibur
Cytometer (see Experimental procedures) As seen in Fig 2A, CM-H2XRos fluorescence was not altered
in cells transfected with httEx1-25Q-EGFP vector,
25Q
B
FL2-H
25Q +NAC
72Q
72Q+NAC
103Q
103Q+NAC
FL2-H
FL2-H
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.
Trang 6whereas 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-ertm
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 MitoTrackertm
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-httEx1-72Q-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 httEx1-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
Trang 7cells 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 O2•–(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 (httEx1-72Q 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.
Trang 8The 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 O2•–
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
Trang 9expression 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
Control Hsp70/Hdj-1 Hsp27 Control Hsp70/Hdj-1 Hsp27 Control Hsp70/Hdj-1 Hsp27 Control Hsp70/Hdj-1 Hsp27 NA
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.
Trang 10in 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
2 m 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.