Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 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 ´ ´ ´ ´ Laboratoire Stress Oxydant, Chaperons et Apoptose, Centre de Genetique Moleculaire et Cellulaire, Universite Claude Bernard Lyon-1, Villeurbanne, France Southampton Neuroscience Group, School of Biological Sciences, University of Southampton, UK Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Canada 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 Genetique Moleculaire et ´ Cellulaire, Universite Claude Bernard Lyon-1, 43 Blvd du 11 Novembre, 69622 ´ Villeurbanne Cedex, 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 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-lcysteine 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 membrane 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-cysteine still efficiently decreased the oxidative damage induced by httEx1103Q 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 observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ Neuronal selective loss and formation of intraneuronal protein aggregates are characteristics of Huntington’s disease (HD), which is one of more than 10 known neurodegenerative disorders caused by abnormally 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 ironregulated neuronal protein implicated in vesicle trafficking [2,3] that, if inactivated, results in impairment of basic cellular processes [4] The mutation is characterized by the expansion of CAG triplets 17 codons 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 W J J Firdaus et al downstream of the initiator ATG in exon (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 correlates with the appearance of intraneuronal, intranuclear and perinuclear aggregates ⁄ inclusions containing the abnormal N-terminal htt fragment [1,9,10] However, 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 formation of aggregates [13,14] Experiments performed in tissue culture cell models have revealed that the presence 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 promotes 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, aggregated 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 httpolyQ [21,22] However, this degradation is likely to be far from complete [23], because the proteasome cannot digest polyglutamine sequences and release them during degradation of polyglutamine-containing proteins [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 neurodegenerative 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 Huntingtin inclusions and oxidation 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 dysfunction 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 aggregates 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 (oxidative stress conditions) [45] or downregulates (antioxidant 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) inactivates 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 membrane potential and increase cell vulnerability to oxidative injury [32] Moreover, Hsps can confer resistance to oxidative stress by preserving proteasome function and attenuating the toxicity of proteasome inhibition [31] It is, however, not yet known if the oxidative stress generated by polyglutamine-containing httEx1 polypeptides is due to alterations in proteasome activities FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3077 Huntingtin inclusions and oxidation W J J Firdaus et al 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) Formation 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-polyQmediated inclusion body formation whereas treatment with the antioxidant agent NAC does not To compare the oxidative effects induced by httEx1polyQ expression, we used monkey kidney COS-7 cells, which are characterized by a very high yield of transfection efficiency (more than 80%; Fig 1B) Transfections 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 displayed inclusion bodies was up to 80% following 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 following analyses were performed 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 3078 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, Hdj1, Hdj-2 or Hsp27 Hdj-2 is an isoform of Hdj-1 that has been previously shown not to decrease htt inclusion 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 Hdj2, whereas the upregulation of Hsp27 and Hsp70 levels was more modest (Fig 1E) The effects mediated by Hsp overexpression on the formation of inclusion bodies 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 transfection, 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 lm) formed by httEx1-72Q-EGFP This finding 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 httEx1103Q-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 polypeptide 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 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS W J J Firdaus et al Huntingtin inclusions and oxidation A E B C D F Fig (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 cytoplasm 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-acetylL-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 described 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 mM) 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 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 performed in COS-7 cells transiently transfected as described above Forty-eight hours after transfection, FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3079 Huntingtin inclusions and oxidation W J J Firdaus et al C 25Q 25Q +NAC Counts 40 80 120 160 200 A 101 102 FL2-H 103 104 72Q 72Q+NAC Counts 40 80 120 160 200 100 101 102 FL2-H 103 104 B 103Q 103Q+NAC Counts 40 80 120 160 200 100 100 101 102 FL2-H 103 104 Fig 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 transfection, cells were treated or not treated with mM N-acetyl-L-cysteine (NAC) Forty-eight hours after transfection, cells were incubated with MitoTrackerTM Red CM-H2XRos and analyzed by cytometry as described in Experimental procedures The intensity of MitoTrackerTM Red fluorescence is shown on the FL2-H axis Black curve, untreated cells; light curve, NAC-treated cells (B) Quantitative analysis of the protective effect of heat shock proteins (Hsps) and NAC against httEx1-polyQ-mediated DYm disruption Transfections were performed with a combination of either httEx1-72Q-EGFP or httEx1-103Q-EGFP vectors with those encoding Hsp70 ⁄ Hdj-1 and Hsp27 As in (A), httEx1-103QEGFP-expressing cells were treated or not treated with mM NAC COS-7 cells transiently transfected with pCIneo-EGFP vector were also treated for 15 with 10 lM of the mitochondria uncoupler p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) before being analyzed Analysis was performed with MitoTrackerTM Red CM-H2XRos and cytometry was performed as described in (A) The percentage of the cell population with an FL2-H fluorescence greater than · 101 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 · 101 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 httEx1103Q-EGFP vector were also exposed to mM NAC before being analyzed (as described in the previous figures) Bar, lm cells were incubated with the fluorescent probe MitoTrackertm Red (CM-H2XRos), and the resulting red fluorescence was analyzed in a FACS calibur 3080 Cytometer (see Experimental procedures) As seen in Fig 2A, CM-H2XRos fluorescence was not altered in cells transfected with httEx1-25Q-EGFP vector, FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS W J J Firdaus et al 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 httEx172Q-EGFP polypeptide A similar effect was noticed when the experiment was carried out with httEx1polyQ-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 depolarization mediated by httEx1-103Q-EGFP expression was more drastic and was roughly similar to that induced by a 15 incubation of control cells with a 10 lm solution of the mitochondrial depolarizer p-trifluoromethoxy 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 manner If, after transfection, cells were treated with NAC before being analyzed, the fluorescence of MitoTrackertm 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 overexpression on DYm disruption induced by httEx1polyQ, COS-7 cells were transiently transfected with vectors encoding httEx1-72Q-EGFP or httEx1-103QEGFP 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 fluorescence Electron microscopy analysis (see Experimental procedures) was performed as a control This experiment 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 httEx1-72QEGFP vector In cells expressing httEx1-103Q-EGFP, the presence of Hsps did not restore normal morphology 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) Huntingtin inclusions and oxidation 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, including 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 contain a broader range of polyQ repeats: 25, 72 and 103 As seen in Fig 3A, 48 h after transfection, the fluorescence of DCFH-DA (see Experimental procedures) increased by 30% in cells expressing httEx1-25QEGFP compared to the value observed in cells expressing EGFP only An almost two-fold increase (P < 0.001) was then observed in httEx1-72Q-EGFPexpressing cells compared to httEx1-25Q-EGFPexpressing cells The fluorescence index was further increased by about 17% in cells expressing the httEx1103Q-EGFP polypeptides As shown in Fig 2, cells were treated with NAC before being analyzed to determine if the increase in fluorescence described above was indeed due to ROS accumulation In the presence of the antioxidant, the fluorescence of httEx1-103QEGFP-expressing cells decreased and was roughly similar to that observed in cells expressing httEx125Q-EGFP Immunoblot analysis revealed a constant level of expression of httEx1-polyQ-EGFP polypeptides 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 observations 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 httEx1polyQ-EGFP polypeptides We have previously reported that z-VAD-fmk completely suppressed httEx1polyQ-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 expressing 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 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3081 Huntingtin inclusions and oxidation W J J Firdaus et al Fig httEx1-polyQ expression enhances reactive oxygen species (ROS) levels (A) Analysis of ROS induced by httEx1-polyQ expression 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 ⁄ Pi, 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 vectortransfected cells A representative experiment is presented As control, 36 h after transfection, cells transfected with httEx1-103QEGFP were exposed or not exposed to 20 lM z-VAD-fmk before being analyzed Treatment with mM 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 httEx172Q EGFP (72Q EGFP) In addition, cotransfections were performed 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 analysis (see Experimental procedures) In (A) and (B) the asterisks denote statistical significance when compared with respective controls: *P < 0.05; **P < 0.001 cells expressing httEx1-72Q or httEx1-103Q polypeptides Similar to the use of DCFH-DA, upregulated fluorescence was detected using dihydroethidine (HE), 3082 a probe that is preferentially oxidized to ethidium bromide 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 similar 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 overexpression, 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) overexpression 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 httEx1-72QEGFP-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 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS W J J Firdaus et al The Hsp27(C137A) mutant is characterized by the substitution 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 presence 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 macromolecules inside the cell is the hydroxyl radical (OH•), which originates from the Harber–Weiss ⁄ Fenton reactions [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 ability 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, httEx1polyQ-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 migration of httEx1-polyQ-EGFP polypeptides were detected, 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 control cells Expression of Hsp70 ⁄ Hdj-1 or Hsp27 did not significantly change the pattern and level of oxidized proteins in control cells, whereas the overexpression of these chaperones correlated with a decreased level of oxidized proteins in response to httEx1-72QEGFP expression (Fig 4A) Analysis of httEx1-103QEGFP-expressing cells revealed that in this case Hsp70 ⁄ Hdj-1 or Hsp27 were not efficient in counteracting the increased level of protein oxidation In contrast, 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-polyQEGFP polypeptides Huntingtin inclusions and oxidation Iron regulates huntingtin polypeptide [2] and catalyzes OH• formation through Fenton reactions [61– 63] Since elevated levels of OH• appear to be produced in cells expressing httEx1-polyQ-EGFP polypeptides, we have analyzed whether the phenomenon correlated with increased levels of Fe(II) The intracellular level of Fe(II) was determined (see Experimental procedures) in COS-7 cells transfected as described above As seen in Fig 5, the expression of httEx1-25Q-EGFP and httEx1-72Q-EGFP polypeptides induced only a weak increase in the absorbance of the ferrozine–Fe(II) complex In contrast, expression 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 ⁄ and R6 ⁄ model) that may contribute to pathogenesis 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 analyze NO• levels in COS-7 cells expressing httEx1polyQ-EGFP polypeptides and to test whether Hsps or NAC could modulate NO• levels A comparison of NO• levels in COS-7 cells transfected with either control or httEx1-polyQ-EGFP vectors was performed Figure shows that the transient expression of httEx1-25Q-EGFP did not much change the intracellular level of NO• In contrast, httEx1-72QEGFP 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 significant 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 httEx1-25QEGFP vectors When the Hsp-encoding vectors were transfected together with that encoding httEx1-72QEGFP, 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 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3083 Huntingtin inclusions and oxidation W J J Firdaus et al NAC Hsp27 Hsp70/Hdj-1 103Q EGFP vector Control Hsp27 Hsp70/Hdj-1 72Q EGFP vector Control Hsp27 25Q EGFP vector Hsp70/Hdj-1 Hsp27 Control Hsp70/Hdj-1 EGFP vector Control A kDa 54 37 29 20 B C Fig (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 Quantitatively 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 mM 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 representative experiment are shown 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 completely abolished the increase in NO• level generated by httEx1-103Q-EGFP expression 3084 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 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS W J J Firdaus et al Fig 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 ⁄ Pi Following 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 mM N-acetyl-L-cysteine (NAC) is presented The data from three independent experiments were used to performed statistical analysis (see Experimental procedures) *P < 0.05 in proteins [31,69] Hence, we first investigated the possibility that proteasome inhibition could be responsible for the oxidative stress mediated by the expression of httEx1-polyQ-EGFP polypeptides Control pCIneoEGFP-transfected COS-7 cells were exposed for h to 10 lm of the proteasome inhibitor lactacystin In these cells, 80% inhibition of proteasome activities correlated with a 50% increase in ROS levels and with a 1.7fold 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 proteasome 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 and 4) We next analyzed the effects mediated by the expression 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 proteasome 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 Huntingtin inclusions and oxidation Fig 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 mM N-acetyl-L-cysteine (NAC) before being analyzed (as described in the previous figures) The data from three independent experiments 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 Hsps as described above Forty-eight hours after transfection, the chymotrypsin-like activity of the 20S proteasome was determined in cell extracts with fluoropeptide suc-LLVY-MCA, and the trypsin-like and caspase-like activities were determined using N-bocLSTR-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 httEx172Q-EGFP and httEx1-103Q-EGFP expression (Fig 7A) No significant effects were induced by either Hsp70 ⁄ Hdj-1 or Hsp27 overexpression or NAC treatment The trypsin-like activity of 20S proteasome was more altered than the chymotrypsin-like activity, since a 30% decrease was noticed in httEx1-103Q-EGFPexpressing 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 FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3085 Huntingtin inclusions and oxidation W J J Firdaus et al 40 polyQ-EGFP expression, and the level of this inhibition 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 (particularly Hsp70 + Hdj-1), and a partial but significant restoration could be observed in httEx1-103Q-EGFPexpressing cells, which then displayed an inhibition that was similar to that observed in httEx1-72Qexpressing 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 differentially 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 20 20 Discussion 0 60 60 suc-LLVY-MCA (A.U.) × 10 –3 A * 40 40 20 20 0 60 60 N-boc-LSTR-MCA (A.U.) × 10 –3 B 40 * * * 40 20 20 60 N-Cbz-LLEβ-NA (A.U.) × 10 –3 C * * * * 60 * 40 EGFP vector 25Q EGFP 72Q EGFP 103Q EGFP Hsp70/Hdj-1 Hsp27 NAC * + - + + - + + - + - + + - + + - + - + + - + + - + - + + - + + - + - + + Fig Analysis of 20S proteasome activities in httEx1-polyQexpressing 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, sucLLVY-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 mM 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 httEx1-polyQ expression (Fig 7B) A similar observation was made in cells treated with NAC In contrast, the caspase-like activity was strongly altered by httEx1 3086 We have already shown that the expression of httEx1polyQ polypeptides modulates the redox status of several 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 compounds on inclusion formation [15] An almost complete 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 mitochondria increased in a CAG repeat expansion-dependent manner to reach a point where it could not be restored FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS W J J Firdaus et al by Hsp overexpression The protective effect provided by Hsp expression was then compared to that mediated by NAC It is of interest that even in cells expressing 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 disruption and the presence of inclusion bodies We have observed that polyQ-mediated DYm disruption correlated, in a CAG repeat expansiondependent manner, with an increased level of intracellular ROS and with increased formation of carbonyl residues in proteins The enhanced oxidation of proteins 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] Overexpression 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 expressing httEx1-103Q-EGFP In contrast, NAC was still effective This further confirms that the httEx1-polyQmediated 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 huntingtin-expressing COS-7 cells It has been proposed that intramitochondrial peroxynitrite formation from NO• is the causative agent that stimulates ROS production by mitochondria [72] Indeed, despite the fact that NO• can be protective, particularly against H2O2 cytotoxicity [73], the elevated level of NO• that is produced in cells expressing mutated htt is toxic and precedes 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 httEx172Q-EGFP-expressing cells, we have observed that Hsp70 ⁄ Hsp40 or Hsp27 overexpression abolished the increase in NO• levels In contrast, Hsps were not efficient in httEx1-103Q-EGFP-expressing cells, but NAC did abolish the NO• increase induced by both httEx1- Huntingtin inclusions and oxidation 72Q-EGFP and httEx1-103Q-EGFP polypeptides Hence, correlations again exist between NO• and ROS upregulation and the presence of aggregated httEx1polyQ It has been reported that proteasome inhibitors induce intracellular protein aggregation and stimulate oxidative protein modifications such as increased carbonyl 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 confirmed 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% inhibition) 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 or not during Huntington pathology We also observed that, in httEx1-polyQ-EGFPexpressing 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 phenomenon 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 proteasome activities generated by httEx1-polyQ expression 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 not depend on inclusion body formation Moreover, 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 bodies generate ROS by themselves? Intriguing observations may favor this hypothesis For example, in vitro, aggregated a-synuclein (Parkinson) and b-amyloid FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3087 Huntingtin inclusions and oxidation W J J Firdaus et al (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 whether it shares the ability to produce ROS in the presence 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 mitochondria concentrated in the area surrounding the inclusions [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 therefore represent a problem that the cell has to challenge to promote the formation and clearance of these bodies 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 amounts of each vector (1–2 lg of DNA to a total of 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) Experimental procedures Membrane potential (DYm) and electron microscopy analysis of mitochondria 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 (104 L)1) (Gibco, Invitrogen), and 250 lgỈmL)1 Fungizone (Gibco, Invitrogen) The cells were maintained at 37 °C in a 5% CO2 atmosphere with 95% humidity The EGFP-encoding DNA vector (pCIneo-EGFP) as well as the same vector containing 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 glutamine 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 encoding wild-type Hsp27 and mutant Hsp27(C137A) [44,78] For transfection experiments, exponentially growing COS-7 cells were plated in 60 mm plates (5 · 105 cells per plate) (TPP, Zurich, Switzerland) day before transfection Each ă transfection experiment was performed with lg of DNA encoding httEx1-polyQ or the various chaperones (Hsp70 ⁄ Hdj-1 and Hsp27) using lipofectamine (Gibco, Invitrogen) according to the manufacturer’s instructions In the case of transfection with multiple vectors, we used identical 3088 Immunoblot analysis COS-7 cells were harvested and the cell pellet was solubilized in boiling · SDS sample buffer Protein concentration 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 ⁄ Interchim, Montlucon, France), anti-Hdj-1(Hsp40) (1 : 1000) ¸ (Stressgen, Victoria, Canada), anti-Hsp27 (1 : 200) (Stressgen), 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 ECLtm system (Amersham Biosciences, GE Healthcare, Chalfont St Giles, UK) Autoradiographs were recorded on X-Omat LS films (Eastman Kodak Co., Rochester, NY) Twenty-four hours after transfection, · 105 COS-7 cells were plated in dishes (45 mm diameter ⁄ mL of medium) and further grown for 24 h After this time period, cells were either kept untreated or incubated for 15 with 10 lm of the mitochondrial uncoupler FCCP (SigmaAldrich) Cells were washed in NaCl ⁄ Pi, resuspended in growth medium and exposed for 15 to 50 nm of MitoTrackertm Red CM-H2XRos (Molecular Probes) Cells were washed in NaCl ⁄ Pi before being analyzed in a FACS calibur Cytometer (Becton Dickinson, Mountain View, CA) equipped with an argon ion laser emitting at 488 nm CM-H2XRos fluorescence was detected in the FL2-H channel The percentage of cells that displayed a FL2-H fluorescence value higher than · 101 was automatically determined and used to calculate the percentage of mitochondrial membrane potential disruption induced by httEx1-polyQ or FCCP For electron microscope analysis, transfected COS-7 cells were grown for 48 h in 60 mm diameter plates before being fixed for 30 at °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 °C in 0.1 m Na-cacodylate ⁄ HCl buffer, pH 7.4, containing 0.2 m sucrose, before being postfixed for 30 at °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 graded ethanol, scraped and pelleted in 70% ethanol and embedded in Epon as a cell pellet After polymerization FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS W J J Firdaus et al at 60 °C for days, ultrathin sections (60–80 nm) were cut using an RMC MTX ultramicrotome (Ventana Medical 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 software (Soft Imaging System GmbH, Munster, Germany) ă was used to analyze the images Determination of intracellular ROS levels After transfection, · 104 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 ⁄ Pi before being incubated for 20 in NaCl Pi containing lgặmL)1 2Â,7Â-dichlorouorescein 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 estimated using HE In this case, cells were incubated for 10 in NaCl ⁄ Pi containing 40 lgỈmL)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 ⁄ Pi Following centrifugation, pelleted cells were resuspended in mL of water An equivalent of mg of total cell protein was used per assay After addition of 500 lL of solution A (0.6 m HCl, 0.142 m KMnO4), the samples were incubated for 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-(2pyridyl)-5,6-bis(4-phenylsulfonate)-1,2,4-triazine (Sigma P5338), m ascorbic acid, m ammonium acetate] and a further 60 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 ⁄ Pi devoid of calcium and magnesium and fixed for 10 with 3.7% formaldehyde, pH 7.4, in NaCl ⁄ Pi Permeabilization was for 3.5 in 0.2% Triton X-100 Examination of samples was performed in a LSM510 laser scanning confocal Zeiss Huntingtin inclusions and oxidation 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 Oxyblottm 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 mm dithiothreitol Ten microliters of each sample lysate was transferred into each of two eppendorf tubes and treated for 15 with either 10 lL of the 1· DNPH solution or 10 lL of the derivation-control solution (negative control) After incubation, 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 ECLtmsystem (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 (Dynatech, 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 ⁄ Pi and lyzed by a 30 min-incubation in 0.5 mm dithiothreitol 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 carried out with 100 lg of protein cell extracts in a total volume of 200 lL of proteasome buffer (50 mm Tris ⁄ HCl, pH 7.8, 20 mm KCl, mm MgOAc, 0.5 mm dithiothreitol) The fluorogenic substrates suc-LLVY-MCA, N-boc-LSTRMCA and N-Cbz-LLEb-NA (Sigma-Aldrich) were added to the cell extracts to measure chymotrypsin-like, trypsin-like FEBS Journal 273 (2006) 3076–3093 ª 2006 The Authors Journal compilation ª 2006 FEBS 3089 Huntingtin inclusions and oxidation W J J Firdaus et al and caspase-like activities, respectively Following incubation at 37 °C for h, the reaction was stopped by addition of sodium borate ⁄ ethanol (9 : 1) The fluorescence of the samples was measured with a Victor Wallach cytofluorimeter (EG & G Instruments, Evry, France) The excitation 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 ± 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 microscopy This work was supported by the Association pour la recherche sur le cancer (grant number 4602) ´ ´ and the Region Rhone-Alpes (Thematique Cancer) (to ˆ 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 support William Currie was a Visiting Professor, from Dalhousie University, Halifax, Canada and held a CIHR ⁄ CNRS International Scientific Exchange Scholarship from the Canadian Institutes of Health Research and the Centre National de la Recherche Scientifique, France 10 11 12 13 References Ross CA (1997) Intranuclear neuronal inclusions: a common pathogenic mechanism for glutamine-repeat neurodegenerative diseases? 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10 0 10 1 10 2 FL2-H 10 3 10 4 Fig Analysis of mitochondrial... vector (72Q) or httEx1 -10 3Q-EGFP vector (10 3Q) Transfections were performed with a combination of those encoding Hsp70 ⁄ Hdj -1 (Hsp70 + Hdj -1) and Hsp27 (Hsp27) Cells transfected with httEx 110 3Q-EGFP... mediated by Hsp overexpression on the formation of inclusion bodies were assessed by transient transfection of COS-7 cells with httEx1-72Q-EGFP or httEx1 -10 3Q-EGFP vectors in combination with vectors