Huntingtin inclusion bodies are iron-dependent centers of oxidative events

Huntingtin inclusion bodies are iron-dependent centersof oxidative eventsWance J. J. Firdaus1, Andreas Wyttenbach2, Paola Giuliano3, Carole Kretz-Remy1,R. William Currie1,4 and Andre-Patrick Arrigo1

1 Laboratoire Stress Oxydant, Chaperons et Apoptose, Universite Claude Bernard Lyon-1, Villeurbanne, France

2 Southampton Neuroscience Group, School of Biological Sciences, University of Southampton, UK

3 Department of Clinical Immunology, National Cancer Institute, G. Pascale Foundation, Naples, Italy

4 Department of Anatomy and Neurobiology, Dalhousie University, Halifax, NS, Canada

Huntington’s disease (HD) is a progressive neuro-

degenerative disorder that induces neuronal selective

loss and formation of intraneuronal protein aggregates

in the striatum and cortex. It is caused by abnormally

expanded polyglutamine polyQ tracts (larger than 36

glutamine repeats) in exon1 (Ex1) of huntingtin (htt),

an iron-regulated neuronal protein implicated in vesicle

trafficking [1–6]. In human and transgenic mice, HD

correlates with the appearance of intraneuronal,

intranuclear and perinuclear aggregates ⁄ inclusions

containing the protease-resistant mutated N-terminal

htt fragment [2,7–10]. In spite of the fact that inclusion

bodies formed by mutated N-terminal htt fragment

often correlate with toxicity [11], polyQ proteins can

also be toxic even in the absence of detectable forma-

tion of aggregates ⁄ inclusion bodies [3,12–15]. Inclusion

bodies have also been reported to correlate with cell

survival [16]. Moreover, it has been proposed that

inclusion body formation is probably a cell-mediated

process to concentrate and promote the clearance of

Keywords

Hsps; Huntingtin; inclusion bodies; iron;

ROS

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 0 472432685

Tel: +33 0 472448595

E-mail: [email protected]

(Received 31 August 2006, accepted

11 October 2006)

doi:10.1111/j.1742-4658.2006.05537.x

Recently, we reported that the transient expression of huntingtin exon1

polypeptide containing polyglutamine tracts of various sizes (httEx1-polyQ)

in cell models of Huntington disease generated an oxidative stress whose

intensity was CAG repeat expansion-dependent. Here, we have analyzed

the intracellular localization of the oxidative events generated by the

httEx1-polyQ polypeptides. Analysis of live COS-7 cells as well as neuronal

SK-N-SH and PC12 cells incubated with hydroethidine or dichlorofluoresc-

ein diacetate revealed oxidation of these probes at the level of the inclusion

bodies formed by httEx1-polyQ polypeptides. The intensity and frequency

of the oxidative events among the inclusions were CAG repeat expansion-

dependent. Electron microscopic analysis of cell sections revealed the pres-

ence of oxidation-dependent morphologic alterations in the vicinity of

httEx1-polyQ inclusion bodies. Moreover, a high level of oxidized proteins

was recovered in partially purified inclusions. We also report that the iron

chelator deferroxamine altered the structure, localization and oxidative

potential of httEx1-polyQ inclusion bodies. Hence, despite the fact that the

formation of inclusion bodies may represent a defense reaction of the

cell to eliminate httEx1 mutant polypeptide, this phenomenon appears

inherent to the generation of iron-dependent oxidative events that can be

deleterious to the cell.

Abbreviations

DCFH-DA, dichlorofluorescein diacetate; 2,4-DNBH, 2,4-dinitrophenylhydrazine; EB, ethidium bromide; Ex1, exon1; FACS, fluorescence-

activated cell sorting; FITC, fluorescein isothiocyanate; HA, hemagglutinin; HD, Huntington’s disease; HE, hydroethidine; Hsp, heat shock or

stress protein; htt, huntingtin; NAC, N-acetyl-L-cysteine; ROS, reactive oxygen species.

5428 FEBS Journal 273 (2006) 5428–5441 ª 2006 The Authors Journal compilation ª 2006 FEBS

an undesirable and protease-resistant mutant protein

by activating autophagy [17,18]. Therefore, toxicity

could originate from defects in the cell-mediated pro-

cess leading to inclusion body formation.

In HD transgenic mice and in brains of HD

patients, heat shock or stress proteins (Hsps) are

expressed in affected neurons, where they associate

with polyQ aggregates [19–21]. Analysis performed in

cell models have revealed that Hsps, such as Hsp70

and Hsp40 (Hdj-1), interfere with polyQ toxicity by

decreasing the efficiency of aggregate and inclusion

body formation [11,22–24]. In spite of being less

effective than Hsp70 ⁄Hdj-1 in suppressing polyQ

aggregation, Hsp27 protects neuronal cells against

polyQ-expanded httEx1-mediated oxidative stress [11]

and neuronal apoptotic cell death [25,26].

Neuronal degeneration, including that in Alzheimer’s,

Parkinson’s and Huntington’s diseases, are charac-

terized by oxidative events such as mitochondrial

complex I and IV deficiency, and elevated level of

nitric oxide and reactive oxygen species (ROS) [11,27–

32]. In the case of HD, oxidative modifications are

proportional in intensity with the number of CAG

repeats in the htt-polyQ polypeptide [11,33–36]. In

transgenic HD mice as well as in cellular models of

HD, the expression of mutant httEx1-polyQ polypep-

tide has been reported to generate mitochondrial defi-

ciency as well as elevated levels of nitric oxide, ROS

and protein oxidation [11,33–36] that directly contrib-

ute to cell death [11]. Proteomic analysis aimed at

detecting protein carbonyl residues has revealed that

specific proteins are indeed oxidized in the HD mouse

brain due to httEx1 expression [37]. This recent obser-

vation confirms that oxidative modifications are

observed not only in cellular HD models but also

in vivo in the brains of HD mice. Mitochondrial dys-

function has been proposed to be responsible for the

oxidative events associated with HD, as expression of

proteins containing glutamine repeats usually corre-

lates with mitochondrial depolarization [38–40] and

impaired clearance of oxidized proteins [27]. However,

a comparative analysis of the protection generated by

Hsps with that provided by the antioxidant agent

N-acetyl-l-cysteine (NAC) reveals that, in the cascade

of events responsible for the oxidative stress mediated

by httEx1-polyQ expression, the formation of inclusion

bodies is a phenomenon that is likely to be upstream

of the defects in mitochondria [36].

Here, we have analyzed the intracellular localization

of the oxidative events generated by httEx1-polyQ

expression. Using fluorescent probes and different live

cell types, we observed a high level of oxidative events

at the level of the inclusion bodies formed by HttEx1-

polyQ. These NAC- and Hsp-sensitive oxidative events

were responsible for cellular damage in the vicinity of

the inclusion bodies. Analysis performed in the pres-

ence of the iron chelator deferroxamine revealed that

the oxidation, localization and structural organization

of the inclusion bodies formed by mutant htt were

iron-dependent. Hence, in spite of the fact that inclu-

sion body formation could be a defense reaction of the

cell to eliminate an aberrant polypeptide, our results

suggest that this process is not foolproof, as iron-

dependent oxidative events are often generated that

alter neighboring cellular morphology, including that

of organelles such as mitochondria.

Results

In COS-7 cells as well as in neuronal SK-N-SH

and PC12 cells, HttEx1-polyQ inclusion bodies

are centers of oxidative events

We recently showed [11,36] that the expression of

httEx1-polyQ polypeptide results in the oxidation of

probes [dichlorofluorescein diacetate (DCFH-DA)] and

hydroethidine (HE)] aimed at detecting intracellular

oxidative events. These probes freely penetrate inside

living cells and become fluorescent upon their oxida-

tion by ROS. This study, performed in different types

of cell (i.e COS-7 cells), showed that an abnormal oxi-

dation process, proportional to the number of CAG

repeats, is concomitant with the presence of mutant

httEx1-polyQ polypeptide when it forms inclusion bod-

ies [36]. Illustrations of the inclusion bodies formed by

mutant httEx1 polypeptides fused to enhanced green

fluorescent protein (EGFP) are presented in Fig. 1.

To further increase our knowledge of the oxidation

process generated by httEx1-polyQ–EGFP expression,

we analyzed where this phenomenon was taking place

inside the cell. This was assessed by confocal micro-

scopic analysis of live COS-7 cells incubated for 1 h

with HE (see Experimental procedures). This probe

was used because, once it is oxidized to ethidium bro-

mide (EB), its fluorescent wavelength (590 nm) differs

from that of EGFP (maximum 510 nm). The red EB

fluorescence was recorded in false blue color. As seen

in Fig. 2A, COS-7 cells exposed for 1 h to 10 lm

myxothiazol, an inhibitor of ubiquinol oxidation, dis-

played cytoplasmic blue spots after HE staining. This

staining, which corresponded to high levels of ROS

in mitochondria [41], was not observed in cells not

treated with myxothiazol. COS-7 cells expressing

httEx1-25Q–EGFP were characterized by diffuse green

cytoplasmic EGFP staining and did not display blue

fluorescence (Fig. 2Ba). To confirm that the EB

W. J. J. Firdaus et al. Oxidation in Huntingtin inclusion bodies

FEBS Journal 273 (2006) 5428–5441 ª 2006 The Authors Journal compilation ª 2006 FEBS 5429

fluorescence was independent of the EGFP fluore-

scence, COS-7 cells were transiently transfected with

httEx1-72Q–hemagglutinin (HA) vector devoid of the

EGFP coding sequence (see Experimental procedures).

When these cells were examined without HE incuba-

tion, green and blue fluorescence were present at back-

ground levels (Fig. 2Bb). When COS-7 cells transiently

transfected with httEx1-72Q–HA were incubated with

HE, green fluorescence was not detectable, but the

false blue fluorescence of HE was clearly detectable

and often localized in large spots (Fig. 2Bc). These

spots were similar in size and shape to the httEx1-

72Q–EGFP inclusion bodies shown in Fig. 1. To test

whether the HE-positive spots originated from httEx1-

polyQ inclusion bodies, httEx1-72Q–EGFP-expressing

cells were incubated with HE. As seen in Fig. 2Bd,

some of the green fluorescent inclusion bodies, but not

all, were also stained blue as a consequence of HE oxi-

dation. In the case of httEx1-103Q–EGFP-expressing

cells, all the green spots were also blue when EB fluore-

scence was analyzed (Fig. 2Be). The merged image

clearly showed that the blue color, which appeared

more intense than in httEx1-72Q–EGFP-expressing

cells, was coincident with the green color. A similar

analysis showing cell contours can be seen in Fig. 6A

(see below). Hence, the EB fluorescence is emitted

by inclusions located inside of the cells and is not a

consequence of granules localized outside of the cells

following cell necrosis. A similar analysis was per-

formed using neuronal SK-N-SH cells. We previously

showed that SK-N-SH cells contained elevated levels

of ROS in response to httEx1-polyQ expression,

similar to COS-7 cells [11]. As seen in Fig. 2Bf,

the httEx1-103Q–EGFP inclusions present in these

A

B

a b c

Fig. 1. Confocal analysis of httEx1-polyQ–EGFP-expressing COS-7 cells. (A) COS-7 cells were transiently transfected with vectors encoding

either httEx1-25Q–EGFP (a, denoted 25Q), httEx1-72Q–EGFP (b, denoted 72Q), or httEx1-103Q–EGFP (c, denoted 103Q). Forty-eight hours

after transfection, cells were fixed and analyzed by confocal microscopy, as described in Experimental procedures. Note the presence of

EGFP-positive inclusion bodies in the cytoplasm of httEx1-72Q–EGFP- and httEx1-103Q–EGFP-expressing cells, and rather uniform staining

in cells expressing httEx1-25Q–EGFP polypeptide. In the case of httEx1-72Q–EGFP expression, some cells can still display uniform staining,

due to the lack of granule formation. This phenomenon is less detectable in cells expressing httEx1-103Q–EGFP polypeptides [36]. (B)

COS-7 cells transiently transfected with httEx1-72Q–EGFP vector. Forty-eight hours after transfection, cells were fixed and analyzed by

confocal microscopy. Phase contrast and EGFP fluorescence are shown. Note that the inclusions are localized inside the cells. Some cells

are devoid of EGFP staining as a consequence of a transfection efficiency of only 80%. Scale bar ¼ 20 lm.

Oxidation in Huntingtin inclusion bodies W. J. J. Firdaus et al.


neuronal cells were also centers of HE oxidation. A

similar observation was made in neuronal PC12 cells

containing nuclear inclusion bodies formed by httEx1-

43Q–EGFP (Fig. 2Bg). These cells were reported by

Giuliano et al. [34] to contain elevated levels of ROS.

It is interesting to note in Fig. 2Bg (see also Fig. 2Bd)

that not all the EGFP-expressing granules displayed

HE-dependent blue staining. These observations sug-

gest that no spillover of EGFP fluorescence is recov-

ered at the level of the blue HE signal, and that in

cells expressing httEx1-72Q and httEx1-103Q, HE oxi-

dation does not take place throughout the cell and ⁄ormitochondria, but is preferentially localized to the

inclusion bodies. Control experiments were therefore

performed to exclude the possibility that HE could

somehow stick to the inclusion bodies unspecifically.

To address this problem, we tested whether DCFH-

DA oxidation also gave a similar signal in COS-7 cells

expressing HA-tagged httEx1-72Q. Figure 2C shows

that DCFH-DA oxidation was concentrated in spots

of similar size to the inclusion bodies (about 10 lm in

diameter). In addition, smaller stained spots were

clearly visible; these may represent oxidative events

that take place in mitochondria, a phenomenon that

was detectable with HE. No fluorescence was detected

in cells not exposed to DCFH-DA. Taken together,

these observations indicate that HE and DCFH-DA

oxidation are concentrated at the level of htt inclusion

bodies, hence suggesting that these structures are

centers of oxidative events.

HE oxidation at the level of httEx1-polyQ

inclusion bodies is decreased by antioxidants

and Hsp overexpression but not by proteasome

inhibitors

As shown in Fig. 2, confocal analysis of httEx1-72Q–

EGFP-expressing live COS-7 cells incubated with HE

revealed a predominant oxidation of this probe (blue

false color) at the level of the inclusion bodies formed

by this protein (fluorescent green). The presence of an

ongoing oxidation process at the level of httEx1-72Q–

EGFP inclusion bodies was further suggested by the

strong decrease in blue staining (HE oxidation) of the

inclusions in cells treated for 12 h with 2 mm NAC

(Fig. 3A) or 5 mm glutathione ethyl ester (not shown)

before being analyzed. In contrast, these antioxi-

dants did not alter EGFP staining and the size of

A

B

C

COS (590 nm)

COS 25Q EGFP

COS 72Q (-EGFP,-HE)

COS 72Q (-EGFP, +HE)

COS 72Q EGFP (+HE)

COS 103Q EGFP (+HE)

SK-N-SH103Q EGFP (+HE)

PC12 43Q EGFP (+HE)

COS 72Q (-EGFP, -HE+ DCFH-DA)

Fig. 2. Huntingtin inclusion bodies are centers of oxidative events.

(A) COS-7 cells were either exposed or not exposed to 10 lM

myxothiazol for 1 h before being incubated for 1 h with 10 lM HE

and analyzed as living cells by confocal microscopy. HE fluores-

cence (blue false color, 590 nm wavength) was recorded. (B) COS-7

cells were transiently transfected with vectors encoding either

httEx1-25Q–EGFP (COS 25Q EGFP, a), httEx1-72Q–HA without

EGFP [COS 72Q (– EGFP), b, c], httEx1-72Q–EGFP (COS 72Q

EGFP, d) or httEx1-103Q–EGFP (COS 103Q EGFP, e). Forty-eight

hours after transfection, cells were incubated for 1 h with 10 lM

HE [except for COS 72Q (– EGFP, – HE), c] before being analyzed

as live cells by confocal microscopy. EGFP (green, 505–530 nm

wavelength) and HE (blue false color, 552–638 nm wavength)

fluorescence as well as the merged result were recorded. SK-N-SH

neuronal cells transfected with httEx1-103Q–EGFP (SK-N-SH 103Q

EGFP, f) and httEx1-43Q-transfected Tet-Off PC12 neuronal cells

(PC12 43Q EGFP, g) were also analyzed. Note the presence of sev-

eral inclusion bodies adjacent to and in the nuclei of PC12 cells.

(C) COS-7 cells, transiently transfected with the EGFP devoid

httEx1-72Q–HA vector [COS 72Q (– EGFP, – HE, + DCFH-DA)],

were incubated for 20 min in NaCl ⁄ Pi containing 5 lgÆlL)1 DCFH-

DA, and fluorescence was recorded at 510 nm. Scale bar ¼ 20 lm.



httEx1-72Q–EGFP inclusions [36]. Proteasome inhibi-

tors have been reported to induce intracellular protein

aggregation and to stimulate oxidative protein modifi-

cations such as increased carbonyl formation [42]. We

therefore analyzed whether proteasome inhibitors

could modulate inclusion body oxidation and ⁄or fur-

ther increase ROS production in httEx1-polyQ-expres-

sing cells. HttEx1-72Q–EGFP-expressing COS-7 cells

were therefore exposed or not exposed for 1 h to

10 lm of the inhibitors lactacystin or MG132 prior to

analysis of HE intracellular oxidation by confocal

microscopy (as shown in Fig. 2). Treatment for only

1 h was used to avoid an increase in the size and num-

ber of the inclusion bodies as described in Wyttenbach

et al. (2002) [11]. It can be seen in Fig. 3B that in the

presence of proteasome inhibitors, HE oxidation still

occurred preferentially at the level of the inclusion

bodies. Analysis of the blue color in the merged images

suggested that HE oxidation was even more intense in

treated than in untreated cells. We then analyzed the

level of HE oxidation in httEx1-Q72–EGFP inclusions

of cells overexpressing either Hsp70 ⁄Hdj-1 or Hsp27.

Figure 3C shows that in both cases, HE oxidation was

less intense when compared to cells expressing httEx1-

Q72–EGFP without these Hsps. This phenomenon was

correlated, particularly in the case of Hsp70 ⁄Hdj-1-

overexpressing cells, with reduced size of the inclusion

bodies.

NAC treatment decreases the intracellular

morphologic alterations that occur in the vicinity

of httEx1-polyQ inclusion bodies

The morphology of httEx1-polyQ inclusion bodies was

analyzed by electron microscopy. This was assessed by

analyzing COS-7 cells that were transfected with either

httEx1-Q72–EGFP- or httEx1-Q103–EGFP-encoding

vectors. Two days after transfection, COS-7 cells

were processed for electron microscopy as described

in Experimental procedures. Cells that expressed

HttEx1-72Q–EGFP (Fig. 4A) or HttEx1-103Q–EGFP

(Fig. 4C) polypeptides contained fibrilous inclusion

bodies surrounded by mitochondria and organelles that

showed pathologic morphologies. Moreover, an empty

halo was often detected around the inclusions, partic-

ularly in cells transfected with httEx1-Q103–EGFP-

encoding vector. High magnification of the electron

micrograph presented in Fig. 4C (see Fig. 4E) suggests

that this halo represents a zone where the cell structures

have been destroyed. Indeed, we have recorded several

pictures of dying cells where a large cell domain around

the inclusion bodies is empty and has probably been

destroyed (Fig. 4G). To test whether the halo, or empty

domain, is a consequence of the oxidative stress that

occurs at the surface of inclusion bodies, a similar ana-

lysis was performed, with the difference that COS-7

cells were treated with 2 mm NAC for 12 h before

being processed for electron microscopy. In the pres-

ence of this antioxidant, cells still contained fibrillar

inclusion bodies, but the cellular morphology surround-

ing the inclusions was less altered. Particularly, no

empty halo or empty space could be detected (Fig. 4B,D

and the higher magnification presented in Fig. 4F).

A

B

C

Fig. 3. Analysis of factors that could modulate oxidation of HE at

the level of htt inclusion bodies.(A) COS-7 cells were transiently

transfected with vector encoding httEx1-72Q–EGFP. Thirty-six

hours after transfection, cells were either kept untreated (NT) or

treated for 12 h with 2 mM NAC before being processed to analyze

HE oxidation at the level of htt inclusion bodies (as described in the

legend of Fig. 2 and in Experimental procedures). (B) Same as (A),

but in this case, 47 h after transfection, cells were treated or not

treated for 1 h with 10 lM lactacystin or MG132 before being ana-

lyzed. (C) Cotransfection of the cells with vectors encoding either

Hsp70 ⁄ Hdj-1 or Hsp27 was also performed. Note the reduced size

of the inclusions. Confocal microscopy of live cells is presented.

EGFP (green) and HE (blue false color) fluorescence as well as the

merged result were recorded. Scale bar ¼ 20 lm.



These cellular alterations close to inclusions may induce

a necrotic type of death (Fig. 4G) or, if they are less

intense, apoptotic death characterized by nuclear dis-

ruption and apoptotic body formation (Fig. 4H).

Partially purified inclusion bodies are enriched

in oxidized proteins

To further analyze the oxidative stress that occurs at

the level of httEx1-polyQ inclusions, experiments were

performed to obtain and analyze cell extracts enriched

in these structures. We did not plan to obtain pure

inclusion bodies, as we were interested in analyzing the

oxidation (using an oxyblot approach) of the cellular

material surrounding these structures. As inclusion

body purification necessitated a large number of cells,

we used stably transfected PC12 43Q–EGFP Tet-Off

cells, which, upon stimulation in doxycycline hydro-

chloride-devoid medium, allow the nuclear accumu-

lation of httEx1-43Q–EGFP polypeptide. Like the

cytoplamic inclusions observed in COS-7 cells, these

nuclear granules are also centers of oxidative events

(Fig. 2Bg). HttEx1-43Q–EGFP inclusion bodies were

partially purified as described in Experimental proce-

dures. Fluorescent and phase contrast microscope ana-

lysis findings are presented in Fig. 5A. The partially

purified material shows the presence of EGFP granules

surrounded by cellular material. Analysis of the pro-

tein content revealed that the partially purified mater-

ial is enriched in several polypeptides, particularly in

the 20 kDa range (Fig. 5B). Immunoblot analysis

using anti-EGFP serum also revealed that the purified

A72Q 72Q+NAC

103Q 103+NAC

5 µm

5 µm

5 µm

gr

chap

gr

1 µm

B G

H

DC

E F

Fig. 4. Electron microscopic analysis of htt inclusion bodies in transfected COS-7 cells. COS-7 cells transiently expressing httEx1-72Q–EGFP

(A, B) or httEx1-103Q–EGFP (C, D, E, F) were either kept untreated (A, C, E) or treated (B, D, F) with 2 mM NAC. The NAC treatment started

36 h after transfection and lasted for 12 h. At the end of the NAC treatment, i.e. 48 h after transfection, cells were processed for electron

microscopy analysis as described in Experimental procedures. High-magnification micrographs are shown to illustrate the cellular morphology

in the vicinity of httEx1-polyQ–EGFP inclusion bodies (E, F). Arrowheads in (C) and (D) denote the regions of the inclusion that were enlarged

in the (E) and (F) magnification panels. Note, in (A), (C) and (E), the altered cellular morphology in the vicinity of httEx1-polyQ–EGFP inclusion

bodies. In NAC-treated cells (B, D, F), the halo surrounding the inclusions is no longer detectable and the cellular morphology is almost nor-

mal. In (G) and (H), images of dying httEx1-72Q–EGFP-expressing COS-7 cells are presented. G, necrotic morphology with enhanced cellular

destruction in the vicinity of the inclusion; H, apoptotic death; ap, apoptotic body; ch, chromatin; gr, htt granule or aggregate. Scale bars are

indicated in the figure.



material was enriched (about 2.5-fold, see Experimen-

tal procedures) in httEx1-43Q–EGFP polypeptide

(about 45 kDa). Immunoblot detection of protein car-

bonyl residues using 2,4-dinitrophenylhydrazine (2,4-

DNPH)-treated extracts was performed as described in

Experimental procedures. Quantification analysis of

the blot revealed a three-fold overall increase in the

level of oxidized proteins in the partially purified

material. Moreover, it was also possible to detect oxid-

ized polypeptides in the enriched fraction that were

undetectable in total cell extracts. Note that no major

oxidized polypeptide had an SDS gel migration similar

to that of httEx1-polyQ, suggesting that the oxidation

process occurs mainly at the level of the cellular pro-

teins surrounding or trapped in the inclusions rather

than at the level of httEx1 polypeptide itself.

The structural organization and localization of

httEx1-polyQ inclusion bodies, as well as the

oxidative stress associated with these structures,

are iron-dependent phenomena

A paradoxical role is believed to be played by transition

metals such as copper and iron in the pathology of

neurodegenerative diseases [43,44]. For example, htt is

an iron-regulated protein [4], and the formation of ROS

is detected in in vitro assays in which aggregated a-syn-uclein [45], b-amyloid [46,47] or prion protein fragment

[48] are incubated in the presence of redox-active

transition metals. We therefore tested the effect of

deferroxamine, an iron-chelating agent, on the oxidative

stress generated by inclusion bodies in COS-7 cells tran-

siently transfected with httEx1-103Q–EGFP vector and

in PC12 43Q–EGFP-Tet-Off cells grown in doxycycline

hydrochloride-devoid medium. Twenty-four hours after

transfection, COS-7 cells were treated with 5 mm defer-

roxamine for 24 h before being analyzed. As seen in

Fig. 6A, these cells displayed a drastic decrease in the

size of most inclusion bodies formed by httEx1-103Q,

suggesting that, in COS-7 cells, iron plays a key role in

the formation and ⁄or structural integrity of httEx1-

polyQ inclusion bodies. Moreover, most of the deferrox-

amine-generated small inclusions were unable to oxidize

HE. In some cells, both the original large inclusions

stained with HE and the small inclusions, usually less

prone to oxidation, coexisted (Fig. 6Ab, arrows). The

antioxidant effect generated by deferroxamine was

further quantified by analyzing the fluorescence emitted

by oxidized HE by fluorescence-activated cell sorting

(FACS) analysis (see Experimental procedures). As seen

A Ba

b

Fig. 5. Analysis of the oxidative protein status of partially purified httEx1-43Q–EGFP nuclear inclusions isolated from stably transfected PC12

neuronal cells. httEx1-43Q–EGFP nuclear inclusions were partially purified from httEx1-43Q–EGFP-transfected Tet-Off PC12 cells grown for

6 days in culture medium devoid of doxycycline hydrochloride to induce the accumulation of httEx1-43Q–EGFP nuclear inclusions as des-

cribed in Experimental procedures. (A) Detection of EGFP-positive inclusions: (a) starting material; (b) enriched nuclear fraction. Merged ima-

ges of EGFP fluorescence (arrows) and phase contrast analysis are presented. Arrows point to EGFP-positive inclusions. Bar: 10 lm (a) and

5 lm (b). (B) SDS ⁄ PAGE of proteins. Quantitatively equivalent amounts of proteins present in total cell lysate (Tot) and the enriched nuclear

fraction (NF) were analyzed. Coomassie blue-stained gels, red Ponceau staining of nitrocellulose membrane after blotting, immunoblot detec-

tion of EGFP and oxyblot analysis (see Experimental procedures) are presented. The immunoblots were probed with anti-EGFP serum and

the oxyblot was probed with anti-2,4-DNPH serum. Visualization was with enhanced chemiluminescence (ECL), as described in Experimental

procedures. The samples obtained from the derivation-control solution (negative controls of oxyblots; see Experimental procedures) were

devoid of any signals and are not presented in the figure. A, actin; H, histones.



in Fig. 6B, in COS cells expressing httEx1-103Q–EGFP,

oxidized HE fluorescence was more intense (about 2.5-

fold) than in httEx1-25Q–EGFP-expressing cells. The

effect was not observed if httEx1-103Q–EGFP-expres-

sing cells were exposed to deferroxamine (24 h, 5 mm)

prior to analysis. In httEx1-25Q–EGFP-expressing cells,

deferroxamine had no significant effect (not shown).

Hence, HE oxidation in httEx1-103Q–EGFP-expressing

COS-7 cells appears to be iron-dependent.

Similar analysis performed in PC12 43Q–EGFP Tet-

Off cells revealed that deferroxamine interfered with

the nuclear localization of the inclusion bodies formed

by httEx1-43Q–EGFP and strongly reduced their abil-

ity to oxidize HE (Fig. 7). However, no significant

changes in the size of the 43Q–EGFP inclusions were

detectable, probably because the inclusions formed by

httEx1-43Q–EGFP are rather small compared to those

formed by httEx1-103Q in COS-7 cells. It is also inter-

esting to note that some HE-negative inclusions were

recovered outside of PC-12 cells.

A B C

FEDDEF

NT

EGFP HE Merge

Fig. 7. Iron as a key modulator of the localization and oxidation of

htt inclusion bodies present in the nucleus of PC12 neuronal

cells. httEx1-43Q-transfected Tet-Off PC12 cells were grown for

6 days in culture medium devoid of doxycycline hydrochloride to

induce the accumulation of httEx1-43Q–EGFP nuclear inclusions.

Cells were either kept untreated (NT) or treated for 24 h with

5 mM deferroxamine (DEF) before being processed to analyze

HE oxidation at the level of EGFP-positive htt nuclear inclusion

bodies (as described in Experimental procedures). Note the intra-

cellular redistribution of the inclusions in the cytoplasm or outside

of the cells (arrow) and their decreased ability to oxidize HE.

Scale bar ¼ 20 lm.

A B

Fig. 6. Iron chelation alters the structure and oxidation of htt inclusion bodies present in the cytoplasm of COS-7 cells. (A) Effect of iron on

the shape and oxidation of htt inclusions. COS-7 cells were transiently transfected with httEx1-103Q–EGFP-encoding vector. Twenty-four

hours after transfection, cells were either kept untreated or treated for 24 h with 5 mM deferroxamine before being processed for confocal

microscopy analysis of HE oxidation at the level of EGFP-positive htt cytoplasmic inclusion bodies (as described in the legend of Fig. 2 and

in Experimental procedures). (a) Untreated cells. Two examples of deferroxamine-treated cells are presented in (b) and (c). Note the

decreased size of most inclusions and their weak ability to oxidize HE. In (b), the disruption of the inclusion is not complete: the arrows point

to inclusions that are still large and positive for HE oxidation. In (c), only small granules are detected. In (a), (b) and (c), the EGFP background

was artificially increased to allow better detection of the cell contours. The images shown are representative of three independent analyses.

Bar ¼ 20 lm. (B) ROS produced by htt inclusion bodies are iron-dependent. COS-7 cells were transiently transfected with vectors encoding

either httEx1-25Q–EGFP (25Q) or httEx1-103Q–EGFP (103Q). Twenty-four hours after transfection, cells were either kept untreated or trea-

ted for 24 h with 5 mM deferroxamine (DEF) before being processed for ROS analysis using HE as a probe and fluorescence detection by

FACS cytometry (see Experimental procedures). A representative experiment is presented.



Discussion

In transiently transfected mammalian cells, the forma-

tion of cytoplasmic perinuclear inclusion bodies in

response to httEx1-polyQ expression correlates, in a

CAG repeat expansion-dependent manner, with

increased levels of intracellular ROS [11]. A similar

observation was made in yeast cells expressing mutant

httEx1 with 103 CAG repeats [35]. This oxidative

stress, which appears to be linked to the presence of

perinuclear inclusion bodies [36], alters the cellular

environment, particularly mitochondria [49] concentra-

ted in the vicinity of the inclusion bodies [50,51]. Here

we have analyzed the intracellular localization of the

oxidative events generated in response to httEx1-polyQ

expression. Using confocal microscopic analysis of live

cells incubated with HE, we observed that this probe

was oxidized at the level of httEx1 inclusion bodies.

In httEx1-72Q–EGFP cells, the intensity and size of

oxidized HE fluorescent spots were often less than

those of spots showing EGFP fluorescence, but the

intensity of fluorescence was more intense than, and

the size closely matched, those of EGFP fluorescent

spots in httEx1-103Q–EGFP-expressing cells. Similar

HE oxidation has been observed in nuclear httEx1

bodies of neuronal SK-N-SH and PC12 cells, suggest-

ing that the oxidative events induced by this neuronal

protein can occur in the cytoplasm or nucleus and are

not cell type-specific. It is of interest that in httEx1-

72Q–EGFP-expressing cells, some inclusion bodies

appeared to be devoid of oxidative events. In contrast,

inclusion bodies formed by httEx1-103Q polypeptide

were all positive. Is the oxidation of HE a consequence

of a high level of ROS in the vicinity of the inclusion

bodies? Experiments performed in the presence of anti-

oxidants, such as NAC, suggest that this is indeed the

case, as in cells exposed to NAC, a drastic loss of

oxidized HE fluorescence was noticed, confirming that

the phenomenon recorded is representative of oxidative

events at the level of inclusion bodies. We observed

that Hsp70 ⁄Hdj-1 or Hsp27 overexpression decreased

the overall cellular intensity of HE oxidation. This

phenomenon may be a consequence of the decreased

size of httEx1-polyQ bodies and ⁄or of the antioxidant

chaperone activity of some of the Hsps, particularly

Hsp27, which maintains glutathione in its reduced

form and decreases intracellular levels of oxidized pro-

teins [52–54] and redox-sensitive Fe(II) [55,56]. In cells

exposed to proteasome inhibitors, which are known to

stimulate oxidative protein modifications [42], a higher

level of HE oxidation at the level of htt inclusion bod-

ies was noted (this study), as well as an increased level

of oxidized proteins [36].

To further investigate the oxidative events taking

place at the level of htt inclusion bodies, we performed

electron microscope analysis. We show here that the

cell structure is destroyed in the vicinity of the inclu-

sion bodies, a phenomenon that was CAG repeat

expansion-dependent and did not develop in cells

exposed to antioxidants. Furthermore, analysis of par-

tially purified inclusion bodies, which still contained

cellular material tags, revealed the high level of oxid-

ized proteins associated or copurifying with the inclu-

sions. These observations confirm that oxidative events

are taking place at the level of htt inclusion bodies and

that these events could be deleterious to the cell.

How can inclusion bodies formed by httEx1-polyQ

generate oxidative events? No easy answer can be given

to this question; however, several recent observations

point to a role played by redox-active transition metals,

such as iron and copper, which are important factors in

the pathology of neurodegenerative diseases [43,44].

For example, the brain regions that are most affected in

these diseases also often contain deposits of iron and

copper [43,44,57–59]. Membrane-associated oxidative

stress, which is a metal-catalyzed oxidative disruption

of membrane protein and lipid signaling, occurs in the

pathogenesis of Alzheimer’s, disease, Parkinson’s dis-

ease and Huntington’s diseases [60]. Moreover, purified

aggregated a-synuclein and b-amyloid, which do not

spontaneously form free radicals [61], can do so if they

are in contact with redox-active Fe(II) [45,47]. Finally,

htt is an iron-regulated polypeptide [4]. This prompted

us to analyze the role played by the iron chelator defer-

roxamine in the oxidative events associated with inclu-

sion bodies. We observed that chelatable iron played a

key role in the formation and ⁄or structural integrity

and localization of httEx1-polyQ inclusion bodies.

Chelatable iron was also necessary to promote the oxi-

dative events associated with these aggregated structu-

res. These observations are reminiscent of an earlier

observation [47] suggesting that the formation of aggre-

gated a-synuclein and b-amyloid is a metal-dependent

mechanism that releases ROS as side products of the

process. ROS produced during aggregate formation are

subsequently converted to hydroxyl radicals by an

Fe(II)-catalyzed Fenton reaction. Our results support

the hypothesis that the formation of htt inclusion bodies

is also an iron-dependent process that generates ROS.

What is the role of the oxidative events initiated by

httEx1-polyQ inclusion bodies? Do they lead to the

oxidation of mutant htt and therefore enhance its de-

gradation? This is probably not the case, as oxyblot

analysis of httEx1-polyQ-expressing cells or partially

purified inclusions revealed no specific oxidation of

httEx1-polyQ polypeptides but only enhanced oxida-



tion of cellular proteins (our results and [36]). Hence,

our results are probably not suggestive of an ‘oxido-

some type’ of mechanism that involves the specific

oxidation of aggregated httEx1-polyQ proteins.

The provocative idea has been proposed that htt

inclusion bodies are in fact beneficial to the cell, by

promoting the clearance of mutant protein by activa-

ting autophagy through the inhibition of mammalian

target for rapamycin (mTOR) [18] and by neutralizing

the toxicity of dispersed filamentous htt aggregates,

such as proteasome inhibition [62–64]. The fact that

htt inclusion bodies can be centers of iron-dependent

oxidative events may therefore represent a problem

that the cell has to deal with to promote the formation

and clearance of these bodies. Inclusion clearance

could therefore be balanced between the rate of htt

aggregation and the oxidative stress that is generated

by this phenomenon. Both phenomena depend on the

intracellular level of iron that can be chelated by defer-

roxamine. However, the level of iron required in each

case is not known, and the levels may be different,

allowing only a few possibilities for the cell to promote

inclusion formation without generating oxidative side-

effects. Further studies will be required to test whether

inclusions (formed by httEx1-43Q or httEx1-72Q) that

are devoid of oxidative events are better able to

undergo an autophagy process than those that are

oxidized, and whether antioxidants or iron chelators

that abolish httEx1-polyQ toxicity could stimulate the

autophagy process.

Experimental procedures

Cell culture, reagents, DNA vectors and

transfection

African green monkey kidney (COS-7) and neuronal SK-

N-SH and Tet-Off PC12 cells were cultured, seeded in

DMEM (Gibco, InVitrogen, Paisley, UK) supplemented

with 10% heat-inactivated neonatal bovine serum. The

medium included penicillin and streptomycin (50 UÆmL)1)

(Gibco, InVitrogen), and 1 lgÆmL)1 Fungizone (Gibco,

InVitrogen). The growing medium of PC12 cells was

further supplemented with 10 lgÆmL)1 of doxycycline

hydrochloride (Gibco, InVitrogen). The cells were main-

tained at 37 �C in a 5% CO2 atmosphere with 95%

humidity. EGFP-encoding DNA control vector (called

pCineo-EGFP) as well as the same vector containing

httEx1 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 [11]. We have

also used vectors encoding Ex1 of htt fused to HA that

contain 25 or 72 glutamine repeats (called httEx1-25Q–HA

and httEx1-72Q–HA) [11]. Control vector (pCineo) and the

same vector bearing Hsp27 (pCIneohsp27) have already

been characterized [65], as well as vectors encoding human

Hdj-1(Hsp40), Hdj-2, and Hsp70 [11]. For transfection

experiments, exponentially growing COS-7 and SK-N-SH

cells were plated in 60 mm dishes (5 · 105 cells per dish) 1

day before transfection. Each transfection experiment was

performed with 3 lg of DNA encoding httEx1-polyQ or

the various chaperones (Hsp70 ⁄Hdj-1 and Hsp27) using

lipofectamine (Gibco, InVitrogen) according to the manu-

facturer’s instructions. In the case of transfection with mul-

tiple vectors, we used identical amounts of each vector

(1–2 lg of DNA to a total of 4 lg). Forty-eight hours aftertransfection, the cell medium was removed and replaced

with DMEM supplemented with 10% fetal bovine serum.

PC12 cells expressing httEx1-Q43–EGFP polypeptide under

the control of a Tet-Off promoter have already been

described [34]. Induction was performed by growing PC12

Tet-Off cells for 6 days in culture medium devoid of doxy-

cycline hydrochloride (Sigma–Aldrich, St-Quentin-Fallavier,

France). Deferroxamine, NAC, lactacystin and MG132

were obtained from Sigma–Aldrich.

Determination of intracellular ROS levels

Forty-eight hours after transfection, COS-7 cells were pla-

ted in triplicate at a concentration of 5 · 104 cells per well

(96-well culture plates) and allowed to grow for 24 h at

37 �C. Cells were then washed three times in NaCl ⁄Pi

(devoid of calcium and magnesium) before being incubated

for 10 min in NaCl ⁄Pi containing 40 lgÆmL)1 of HE. Flow

cytometric analysis was performed using a FACScalibur

cytometer (Becton Dickinson, Mountain View, CA) using a

488 nm excitation wavelength. The emission filter was

610 nm bandpassed for EB fluorescence (FL2-H).

In vivo intracellular EGFP and ROS localization

by confocal microscopy

Transiently transfected COS-7 and neuronal SK-N-SH cells

were grown on coverslips in 60 mm dishes, as were stably

transfected PC-12 cells. Forty-eight hours after transfection,

cells were rinsed once in DMEM supplemented with 10%

fetal bovine serum. Control of oxidative stress in mitochon-

dria was assessed by treating cells for 1 h with 10 lLÆmL)1

of an inhibitor of ubiquinol oxidation, myxothiazol

(Sigma–Aldrich). Stock solutions of 1 mm HE were prepared

in dimethylsulfoxide on the day of the experiments and were

kept in the dark. HE at a concentration of 10 lm was added,

and the cells were incubated for 1 h at 37 �C. We also used

DCFH-DA to detect ROS in live cells transfected with

HA-tagged httEx1-72Q. DCFH-DA at a concentration of

5 lm was added, and the cells were incubated for 20 min at

37 �C. After incubation with HE or DCFH-DA probes, cells



were washed once with NaCl ⁄Pi devoid of calcium and mag-

nesium before being observed alive (in NaCl ⁄Pi devoid of

calcium and magnesium) using a confocal inverted micro-

scope equipped with 40· oil immersion objective lens (Zeiss

LSM 510 Meta, Axiovert 200X, Jena, Germany) in k mode.

This configuration was used to achieve separation of the

different fluorescences. The microscope was able to scan

the whole visible spectrum (detection every 10 nm). Using

this configuration, the spectrum of each fluorochrome was

determined, and these spectra were used to separate, in the

final image, the fluorescence of each signal by spectral de-

convolution. The fluorochromes were excited at 488 nm,

and the emission wavelength was 590 nm for oxidized HE

and 510 nm for EGFP or oxidized DCFH-DA. For con-

venient visualization of the phenomenon, a false color

(blue) was used to monitor the red fluorescence generated

by oxidized HE (EB). Groups of cells as well as single cells

were randomly selected from the microscope field and ana-

lyzed for the fluorescence of either HE (blue), EGFP

(green), DCFH-DA (green) or the superimposed merged

images. In the merged images, a blue color is indicative of

intense oxidation of HE. Image processing was performed

using lsm 510 meta software (Zeiss, Jena, Germany) and

photoshop 7.0 (Adobe Inc., Mountainview, CA, USA).

Partial purification of httEx1-43Q–EGFP nuclear

inclusions from PC12 cells

HttEx1-43Q–EGFP nuclear inclusions were partially puri-

fied according to the method of Mitsui et al. [66], with some

modifications. PC12 43Q–EGFP Tet-Off cells (5 · 107) were

plated in 10 100 mm tissue culture dishes (5 · 106 cells per

dish) and cultured for 6 days at 37 �C under 5% CO2 in

DMEM containing 10% fetal bovine serum, 50 UÆmL)1

penicillin and streptomycin (Gibco, InVitrogen) and

1 lgÆmL)1 Fungizone (Gibco, InVitrogen). During the incu-

bation, the medium was devoid of doxycycline hydrochlo-

ride to allow expression of httEx1-43Q–EGFP polypeptide.

Cells were collected in an ice-cooled homogenization glass

potter with 3 mL of lysis buffer composed of NaCl ⁄Pi con-

taining 10 mm MgCl2, 1500 U of DNase I (Sigma–Aldrich)

and 3 U of RNase A (Sigma–Aldrich). Homogenization

was performed with a Potter–Elvehjem-type homogenizer

with 30 up–down strokes on ice. Lysates were transferred in

eppendorf tubes and centrifuged at 1500 g for 30 min with

an Eppendorf centrifuge (Hamburg, Germany), rotor type

10 · 1.5 mL ⁄ 2 mL aerosol tight motor with lid. The pres-

ence of inclusion bodies in the pellets was detected using a

fluorescence inverted microscope equipped with a 40· oil

immersion objective lens (Zeiss Axiovert 200M; Zeiss, Jena,

Germany) using a fluorescein isothiocyanate filter. The pel-

lets were then washed several times in NaCl ⁄Pi containing

4% sarcosyl. The partially purified inclusion bodies were

then stored at ) 80 �C prior to gel electrophoresis and

immunoblot analysis.

Electron microscopy

Forty-eight hours after transfection, COS-7 cells grown in

60 mm diameter dishes (TPP, Zurich, Switzerland) were

fixed for 30 min at 4 �C in a buffer containing 2% glutaral-

dehyde in 0.1 m sodium cacodylate ⁄HCl buffer (pH 7.4).

Cells were then rinsed three times (overnight) at 4 �C in

0.1 m sodium cacodylate ⁄HCl buffer (pH 7.4) containing

0.2 m sucrose before being postfixed for 30 min at 4 �C in a

buffer composed of 1% osmium tetroxide and 0.15 m

sodium 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 at 60 �C for 3 days, ultrathin sections

(60–80 nm) were cut using an RMC MTX ultramicrotome

(Ventana, France), collected on 200 mesh copper grids,

stained with uranyl acetate and lead citrate, and observed

with a JEOL 1200 CX transmission electron microscope

(Tokyo, Japan). Images were recorded with a Megaview II

numeric camera (New York, NY, USA), and analysis

software (Eloise, Roissy, France) was used to analyze the

images.

Immunoblot analysis and protein carbonyl

residue determination (oxyblot)

Protein concentration was determined in aliquots using the

Bradford protein assay. Total protein samples were separ-

ated in 12% SDS ⁄PAGE before being analyzed in immu-

noblots probed with anti-EGFP (1 : 1000) (Molecular

Probes ⁄ Interchim, Montlucon, France) and peroxidase-

labeled secondary antibody (1 : 1000) (Tebu, Le Perray en

Yvelines, France). Protein bands were visualized with the

ECLTM system (GE Healthcare, Chalfont St Giles, UK)

and autoradiographs were recorded onto X-Omat LS films

(Eastman Kodak Co., Rochester, NY). Immunoblot detec-

tion of carbonyl residues was done as previously described

[67] using the S7150 OxyblotTM Protein Oxidation Detec-

tion Kit from Chemicon International (Temecula, CA).

In brief, 48 h after transfection, cells were lysed in SDS

(6% final concentration) 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 min with either 10 lL of the 1· 2,4-DNPH solution

or 10 lL of the derivation-control solution (negative con-

trol). 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-2,4-DNPH according to the manufacturer’s

instructions. Immune complexes were detected by

chemiluminescence using the ECLTMsystem (Amersham-

Biosciences). Autoradiographs were recorded onto X-Omat

LS films (Eastman Kodak Co.). Quantification of the blots

was performed using nih image version 1.62 software

(NIH, Bethesda, MD).



Acknowledgements

We wish to thank Dominique Guillet for excellent tech-

nical assistance, Dr Simone Peyrol at CeCiL (Centre

Commun de Microscope Electronique et d’Imagerie

Laennec, Faculte de Medicine RTH Laennec, UCB-

Lyon1, France) for assistance with electron microscopy,

and Dr Beatrice Burdin (Centre Technologique des

Microstructures, UCBL-Lyon1, France) for assistance

with confocal microscopy. This work was supported by

the Association pour la recherche sur le cancer (grant

no. 4602) and the Region Rhone-Alpes (Thematique

Cancer) (to APA). Wance Firdaus was a postgraduate

scholarship holder from CNOUS (Centre National des

Oeuvres Universitaires et Scolaires), Paris. Dr Andreas

Wyttenbach thanks the HighQ Foundation and the

Medical Research Council (MRC) for financial support.

William Currie was a Visiting-Professor from Dalhousie

University, NS, 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.

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Huntingtin inclusion bodies are iron-dependent centers of oxidative events

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