Developmental differences in H2O2-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3

Developmental differences in H2O2-induced oligodendrocyte cell

death: role of glutathione, mitogen-activated protein kinases and

caspase 3

Gabriela Fragoso,* Ana Katherine Martınez-Bermudez,*,� Hsueh-Ning Liu,* Amani Khorchid,*

Sylvain Chemtob,� Walter E. Mushynski� and Guillermina Almazan*

*Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

�Departments of Pediatrics, Ophthalmology and Pharmacology, Research Center of Ste-Justine Hospital, Montreal, Quebec, Canada

�Department of Biochemistry, McGill University, Montreal, Quebec, Canada

Abstract

The molecular mechanisms underlying H2O2-induced toxicity

were characterized in rat oligodendrocyte cultures. While

progenitor cells were more sensitive than mature oligo-

dendrocytes to H2O2, the antioxidant, N-acetyl-L-cysteine,

blocked toxicity at both stages of development. Differentiated

oligodendrocytes contained more glutathione than did pro-

genitors and were less susceptible to decreases in glutathi-

one concentration induced by H2O2 stress. As free radicals

have been considered to serve as second messengers, we

examined the effect of H2O2 on activation of the mitogen-

activated protein kinases (MAPK), extracellular signal-regu-

lated kinases (ERK) 1/2 and p38. H2O2 caused a time- and

concentration-dependent increase in MAPK phosphorylation,

an effect that was totally blocked by N-acetyl-L-cysteine.

Further exploration of potential mechanisms involved in

oligodendrocyte cell death showed that H2O2 treatment

caused DNA condensation and fragmentation at both stages

of development, whereas caspase 3 activation and poly

(ADP-ribose) polymerase cleavage were significantly

increased only in oligodendrocyte progenitors. The pan-ca-

spase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl

ketone, blocked DNA fragmentation in progenitors and pro-

duced a small but significant level of protection from H2O2

toxicity in progenitors and mature oligodendrocytes. In con-

trast, inhibitors of both p38 and MEK reduced H2O2-induced

death most significantly in oligodendrocytes. The poly (ADP-

ribose) polymerase inhibitor, PJ34, reduced H2O2-induced

toxicity on its own but was most effective when combined

with benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone or

PD169316. The finding that molecular mechanisms confer-

ring resistance to reactive oxygen species toxicity are regu-

lated during oligodendrocyte differentiation may be of

importance in designing therapies for certain neurological

diseases affecting white matter.

Keywords: apoptosis, caspases, mitogen-activated pro-

tein kinase, N-acetyl-L-cysteine, oligodendrocytes, oxidative

stress.

J. Neurochem. (2004) 90, 392–404.

During the past three decades there has been a notable

increase in the study of free radicals in biology and medicine.

Under specific conditions and at defined concentrations, free

radicals can damage critical cellular components, such as

DNA, proteins and membrane phospholipids, eventually

leading to cell death (for review see Gutteridge and Halliwell

2000; McCord 2000; Thomas 2000). Oligodendrocytes, the

myelinating cells of the CNS, are very sensitive to oxidative

stress in vitro, apparently due to a low capacity for

antioxidant defence and intrinsic risk factors, such as high

iron content (Juurlink et al. 1998). Accumulating evidence

suggests that free radicals contribute to various diseases that

Resubmitted manuscript received February 20, 2004; accepted February

23, 2004.

Address correspondence and reprint requests to Guillermina Almazan,

Department of Pharmacology and Therapeutics, Room 1321, McGill

University, 3655 Promenade Sir-William-Osler, Montreal, Quebec H3G

1Y6, Canada. E-mail: [email protected]

Abbreviations used: AMC, 7-amido-4-methylcoumarin; CG4, central

glia-4; DEVD, Asp-Glu-Val-Asp; ERK, extracellular signal-regulated

kinase; GSH, glutathione; LDH, lactate dehydrogenase; MAPK, mito-

gen-activated protein kinase; MBP, myelin basic protein; MTT, 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetyl-

L-cysteine; PARP, poly (ADP-ribose) polymerase; PBS, phosphate-buf-

fered saline; TUNEL, terminal deoxynucleotidyl transferase-mediated

dUTP nick end-labeling; zVAD, benzyloxycarbonyl-Val-Ala-Asp fluor-

omethyl ketone.

Journal of Neurochemistry, 2004, 90, 392–404 doi:10.1111/j.1471-4159.2004.02488.x

392 � 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 392–404

affect oligodendrocytes, including multiple sclerosis, a

chronic inflammatory demyelinating disease of the CNS

(Vladimirova et al. 1998; Smith 1999) and cerebral palsy

caused by periventricular leukomalacia (Gilles and Murphy

1969; Leviton and Paneth 1990). Periventricular leukomal-

acia is the principal neuropathology for the constellation of

spastic motor and cognitive deficits that occur in premature

infants (Volpe 1987, 1989).

Recent reports have shown that late oligodendrocyte

progenitors are preferentially damaged by a hypoxic-ischem-

ic insult in neonatal rats (Jelinski et al. 1999; Levison et al.

2001; Ness et al. 2001; Back et al. 2002) and caspase 3

activation under these conditions (Han et al. 2000) suggests

that it is involved in periventricular leukomalacia. Other

reports indicate that oligodendrocyte progenitors in vitro are

significantly more sensitive than mature oligodendrocytes to

toxic insults that include free radical generation induced by

glutathione (GSH) depletion (Volpe 1998), catecholamine

treatment (Khorchid et al. 2002), ischemic injury (Fern and

Moller 2000) or cadmium exposure (Almazan et al. 2000).

Although it has been postulated that apoptosis may differ

in undifferentiated compared with differentiated oligodend-

rocytes, little is known about the molecular mechanisms that

mediate the death of these cells. Reactive oxygen species

can induce delayed cell death or apoptosis and protein

phosphorylation appears to be an important molecular

mechanism for transducing biochemical signals initiated

by free radicals (Kyriakis and Avruch 1996). Thus, reactive

oxygen species have been implicated as second messengers

that activate protein kinase cascades in central glia-4 (CG4)

cells (Bhat and Zhang 1999) and transcription factors in

oligodendrocytes (Vollgraf et al. 1999). Different pro- and

anti-apoptotic factors are up- or down-regulated during the

initiation of apoptosis resulting in caspase activation,

chromatin condensation and DNA fragmentation (Tang

and Porter 1996). However, the role of caspases in

H2O2-induced oligodendrocyte cell death remains to be

determined.

The objective of this studywas to examine the susceptibility

of cultured oligodendrocyte progenitors and mature cells to

H2O2 toxicity and to establish whether activation of the

mitogen-activated protein kinases (MAPKs), extracellular

signal-regulated kinase (ERK) 1/2 and p38, plays a critical

role in this process. We also addressed the question of whether

there is a correlation between GSH concentration and suscep-

tibility to H2O2-induced toxicity in oligodendrocyte progen-

itors compared with mature cells. In addition, the activation of

caspase 3 and involvement of poly (ADP-ribose) polymerase

(PARP) were explored in an effort to unravel the potential

mechanisms mediating H2O2-induced oligodendrocyte cell

death. Finally, a general caspase inhibitor, benzyloxycarbonyl-

Val-Ala-Asp fluoromethyl ketone (zVAD), the antioxidant N-

acetyl-L-cysteine (NAC) and inhibitors of p38 (PD169316),

MEK (U0126 and PD098059) and PARP (PJ34) were

evaluated for their ability to reduce H2O2-mediated toxicity

and nuclear fragmentation.

Materials and methods

Dulbecco’s modified Eagle’s medium and Ham’s F12 medium as

well as phosphate-buffered saline (PBS), Hank’s balanced salt

solution, 7.5% bovine serum albumin fraction V, fetal calf serum,

calf serum, penicillin and streptomycin were purchased from

Invitrogen Canada (Toronto, ON, Canada). Other reagents were

purchased from the following suppliers: 4,6-diamidino-2-pheny-

lindole dihydrochloride from Polysciences Inc. (Warrington, PA,

USA); Immobilon-P membranes from Millipore (Mississauga,

ON, Canada); ECL Western Blotting Detection Kit from Amer-

sham Canada Ltd (Oakville, ON, Canada); Kodak XRP-5 film

from Mandel (Guelph, ON, Canada); platelet-derived growth

factor AA and basic fibroblast growth factor from PeproTech, Inc.

(Rocky Hill, NJ, USA); GSH reductase, lactate dehydrogenase

(LDH) and terminal deoxynucleotidyl transferase-mediated dUTP

nick end-labeling (TUNEL) kits from Roche Diagnostics (Laval,

QC, Canada); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT), monoclonal anti-glial fibrillary acidic protein,

poly D-lysine, poly L-ornithine, Triton-X-100, human transferrin,

insulin, oxidized GSH, 5,5¢-dithiobis-2-nitrobenzoic acid, NADPH

and NAC from Sigma-Aldrich (Oakville, ON, Canada); mono-

clonal anti-complement receptor C3b (OX-41) from Serotec

(Raleigh, NC, USA); monoclonal anti-myelin basic protein

(MBP) from Sternberger Monoclonals (Lutherville, MD, USA);

zVAD and PJ34 from Calbiochem (San Diego, CA, USA);

phospho-specific ERK1/2 (Thr183 and Tyr185) and p38 (Thr 180

and Tyr 182) antibodies and anti-caspase 3 fragment from New

England Biolabs (Mississauga, ON, Canada); monoclonal anti-

PARP from Biomol Res. Laboratory, Inc. (Plymouth Meeting, PA,

USA) and secondary antibodies used for immunoblotting were

from BIO-RAD (Mississauga, ON, Canada).

Cell culture

Primary cultures were generated from newborn rat brains as

described by Almazan et al. (1993) according to a technique of

McCarthy and de Vellis (1980). Oligodendrocyte progenitors were

plated on poly D-lysine-coated culture dishes and grown in serum-

free medium consisting of a Dulbecco’s modified Eagle’s medium-

F12 mixture (1 : 1), 10 mM HEPES, 0.1% bovine serum albumin,

25 lg/mL human transferrin, 30 nM tri-iodothyronine, 20 nM

hydrocortisone, 20 nM progesterone, 10 nM biotin, 5 lg/mL insulin,

16 lg/mL putrescine, 30 nM selenium and the growth factors

platelet-derived growth factor AA or basic fibroblast growth factor

at a concentration of 2.5 ng/mL. Cultures were characterized

immunocytochemically using specific markers for different cell

types (Cohen and Almazan 1994; Radhakrishna and Almazan

1994). More than 95% of the cells reacted positively with mouse

monoclonal antibody against A2B5, a marker for oligodendrocyte

progenitors in culture, and less than 5% consisted of galactocere-

broside-positive oligodendrocytes, glial fibrillary acidic protein-

positive astrocytes or complement type 3-positive microglia (Cohen

et al. 1996). At this stage progenitor cells are bipolar or poorly

branched, as shown in Fig. 1(a) (cells labeled with A2B5), and

proliferate actively in the presence of the mitogens platelet-derived

H2O2-induced toxicity in oligodendrocytes 393

� 2004 International Society for Neurochemistry, J. Neurochem. (2004) 90, 392–404

growth factor AA and basic fibroblast growth factor. Progenitor

cultures were differentiated to oligodendrocytes in serum-free

medium without platelet-derived growth factor and basic fibroblast

growth factor, which was supplemented with 3% calf serum after

day 3. Morphologically mature cells displayed a profuse network of

processes and membranes (Fig. 1b) and were immunolabeled with

anti-MBP (�90%) while a few progenitors kept dividing and were

A2B5 positive (�5%). The number of astrocytes or microglia did

not increase with differentiation of the cultures as previously

reported (Cohen and Almazan 1994; Khorchid et al. 2002).

All experiments were conducted in serum-free medium with

progenitor cells or 12 d differentiated oligodendrocytes in the

absence or presence of the indicated pharmacological agents.

Immunofluorescence staining

For detection of surface antigens, unfixed cells were incubated with

monoclonal antibodies A2B5, O1 (anti-GalC) or OX-42 in culture

medium. After rinsing with culture medium, the cells were

incubated for 20 min with secondary goat anti-mouse IgM or

IgG2a-fluorescein isothiocyanate conjugates. To visualize MBP or

glial fibrillary acidic protein, cells were fixed with 4% paraformal-

dehyde in PBS for 20 min at room temperature and then with

methanol for 5 min at )20�C. Afterwards the cells were washed

three times with PBS and blocked for 15 min in PBS containing

0.2% bovine serum albumin, 5% goat serum, 5% rabbit serum and

0.2% Triton X-100. Monoclonal anti-MBP or anti-glial fibrillary

acidic protein were diluted in the same solution and applied for

45 min at room temperature. The secondary goat anti-mouse IgG2b-

TxR or IgG1-TxR was applied for 20 min at room temperature.

Coverslips were mounted with Immu-Mount from Shandon

(Pittsburgh, PA, USA) and examined under a Leitz Diaplan

epifluorescent microscope from Leica Microsystems (Richmond

Hill, ON, Canada) and photographed with TX 400 ASA film

(Eastman Kodak Company; Rochester, NY, USA).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

assay of cell viability

Cell viability was estimated by reduction of MTT by mitochondrial

dehydrogenases in living cells. The amount of formazan produced is

proportional to the number of cells present (Denizot and Lang 1986).

Progenitors and mature cells growing in 24-well dishes were

incubated with different concentrations of H2O2 for 1–3 h, washed

and allowed to recover for 18 h. Once the experiments were

concluded, MTT (0.5 mg/mL in PBS, pH 7.2) was added and the

cultures were incubated for 3 h at 37�C. Afterwards, the medium was

removed, the formazan product dissolved with acidified isopropanol

and the optical density determined at 600 nm.Results are expressed as

a percentage of the values obtained with untreated cultures.

Lactate dehydrogenase assay of cell death

An increase in membrane damage by toxic insults causes the release

of LDH into the culture supernatant fluid. The LDH release was

assessed with a cytotoxicity detection kit (Roche Molecular

Biomedicals, Laval, QC, Canada). The LDH values were calculated

relative to the total LDH content measured after the cells were lyzed

completely by 1% Triton-X-100.

Western blot analysis

Cells grown in six-well culture plates were harvested, after treatment,

in 60 lL of ice-cold lysis buffer which contained 20 mM Tris-HCl

(pH 8), 1% Nonidet P-40, 10% glycerol, 137 mM NaCl, 1 mM

phenylmethylsulphonyl fluoride, 1 mM aprotinin, 0.1 mM sodium

vanadate and 20 mM NaF. Protein content of the cell lysates was

determined with the Protein Assay Kit (BIO-RAD) and samples were

adjusted to contain 2% sodium dodecyl sulfate, 5% glycerol, 5%

b-mercaptoethanol and 0.01% bromophenol blue and boiled for

5 min. Aliquots containing 50 lg of protein were resolved by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis and transferred

to Immobilon-P membranes which were blocked and probed with the

appropriate phospho-epitope-specific antibodies. Bands were visu-

alized with horseradish peroxidase-conjugated secondary antibody

used in conjunction with an ECL western blotting detection kit. The

resultant bands were quantified by densitometry. To normalize for

sample loading and protein transfer, the membranes were stripped

and reprobed with an antibody for total p38.

Glutathione measurement

Intracellular GSH was determined as described previously (Almazan

et al. 2000) using a well-established kinetic assay (Tietze 1969).

Under all experimental conditions presented here the fraction of

Fig. 1 Immunocytochemical properties of oligodendrocyte cultures.

Oligodendrocyte cultures were characterized by immunofluorescence

microscopy. (a) Progenitor cells were labeled with A2B5 antibody and

displayed a typical bipolar or poorly branched morphology. (b) 12-day

differentiated oligodendrocytes expressed myelin based protein (MBP)

and displayed complex cellular processes.

394 G. Fragoso et al.


GSH disulfide was always < 1% of the total GSH, hence the results

are presented as GSH only.

Caspase activation and poly (ADP-ribose) polymerase cleavage

Caspase 3 activity was assessed with the synthetic substrate,

Ac-Asp-Glu-Val-Asp(DEVD)-7-amido-4-methylcoumarin (AMC)

(Yin et al. 1999). Aliquots of oligodendrocyte progenitor lysates

containing 30 lg protein were incubated for 60 min at 37�C in

buffer (50 mM HEPES, pH 7.4, 5 mM EDTA, 1% Triton X-100

and 2 mM dithiothreitol) containing 50 lM peptide substrate. The

AMC fluorescence was measured with an excitation wavelength

of 380 nm and an emission wavelength of 460 nm using a Tecan

Spectra Fluor Plus multiwell scanner from Tecan US (Research

Triangle Park, NC, USA). The DEVD-aldehyde (CHO) caspase 3

inhibitor was used at 1 lM to test the specificity of the reaction.

The activated caspase 3 fragment (17 kDa) and PARP cleavage

were detected by western blotting of cell lysates prepared as

described for MAPKs.

Visualization of apoptotic nuclei

Progenitor and mature oligodendrocyte cultures growing in 24-well

dishes on poly D-lysine-coated coverslips were incubated with 0.1 or

0.5 mM H2O2 for 1 h, washed and maintained for 16 h in serum-free

medium. Cells were fixed with 4% paraformaldehyde in phosphate

buffer, incubated in PBS containing 4,6-diamidino-2-phenylindole

dihydrochloride at a concentration of 5 lg/mL for 10 min at room

temperature and mounted with Immu-Mount (Shandon) to identify

cells undergoing apoptosis. Fluorescence was observed with a

Diaplan microscope (Leica Microsystems). The TUNEL assay was

performed using a commercial kit from Roche Diagnostics and

following the manufacturer’s instructions. A 3,3¢-diaminobenzidine

substrate kit (Vector Laboratories, Burlingame, CA, USA) was used

to detect peroxidase activity. Stained cells were visualized by light

microscopy.

Statistical analysis

Unless otherwise indicated, results are represented as the mean

± SEM of at least three separate experiments performed in triplicate.

Differences between group means were established by one-way

ANOVA followed by the Tukey test to determine statistical signifi-

cance; p-values less than 0.05 were considered significant.

Results

Cell toxicity induced by H2O2

Figure 2(a) shows the decreases in cell viability (MTT

reduction) following exposure of oligodendroglial cells to

increasing concentrations of H2O2 for 1 h and an 18- h

recovery period. Oligodendrocyte progenitors were more

sensitive than mature cells, with half-maximal decline in

viability occurring at �0.05 and �0.5 mM H2O2, respect-

ively. H2O2 caused both concentration- and time-dependent

membrane damage in oligodendrocyte cultures as shown by

LDH release into the medium. The H2O2-induced LDH

release was not evident at 2 h, increased significantly by

4 h and reached maximal levels by 18 h in both progenitors

and mature cells (Figs 2b and c). In progenitor cells, a

significant increase inLDH releasewas obtainedwith 0.05 mM

H2O2 with the maximal effect occurring at 0.1 mM. Similar to

the MTT viability curve, mature oligodendrocytes required

about 10 times more H2O2 (�1 mM) for maximal release of

LDH.

Fig. 2 H2O2 toxicity in progenitors and differentiated oligodendro-

cytes. Cultures (progenitors and 12-d differentiated cells) were ex-

posed to increasing concentrations of H2O2 for 1 h, washed and

allowed to recover for 2, 4 or 18 h. (a) Cell viability at 18 h was

measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) reduction as described in Materials and methods. (b and

c) Released lactate dehydrogenase (LDH) was measured in cultured

supernatant fluids at 2, 4 and 18 h after H2O2 treatment. Data points

are the mean ± SEM of four independent experiments performed in

triplicate. H2O2 caused a concentration-dependent decrease in MTT

reduction and a time- and concentration-dependent increase in LDH

release (p < 0.001 by one-way ANOVA).



Effect of N-acetyl-L-cysteine on H2O2-induced toxicity

and intracellular glutathione levels

Glutathione is the major cellular antioxidant and functions to

protect cells from oxidative damage caused bymany toxins. To

determine whether intracellular GSH levels play a role in the

differential susceptibility of oligodendrocyte progenitors

compared with mature cells, cultures were treated with H2O2

in the presence and absence of NAC, a GSH precursor. As

shown in Fig. 3(a), 0.1 or 0.5 mM H2O2 caused significant

inhibition of mitochondrial dehydrogenase activity in progen-

itor cells (71.4%) and oligodendrocytes (68.6%), respectively.

Pre-treatment for 30 min with increasing concentrations of

NAC reduced H2O2 toxicity in a concentration-dependent

manner. Maximal protection of mature oligodendrocytes was

afforded with 10 mMNAC although even 20 mMNAC did not

fully protect progenitor cells from H2O2 toxicity.

Figure 3(b) shows the GSH content in oligodendroglial

progenitors and 12-d differentiated cells 4 h after addition to

the culture medium of H2O2 at concentrations ranging from 0

to 1 mM. Statistically significant decreases in intracellular

GSH were observed in progenitors and mature cells treated

with 0.1 and 0.5 mM H2O2, respectively. These H2O2 concen-

trations caused significant increases in LDH release at this

time-point (Fig. 2). As previously reported, the basal level of

GSH was 47% higher in differentiated cells compared with

progenitors (Almazan et al. 2000). In the presence of 1 mM

H2O2, the GSH concentration decreased by �64% in progen-

itors and by �35% in differentiated cells. Figure 3(c) shows

that NAC not only prevented the decrease in intracellular GSH

provoked by H2O2 but actually increased GSH significantly

above basal levels. This is more noticeable for mature cells

where 20 mM NAC increased the GSH level by 100%.

Concentration and time dependency of MAPK activation

by H2O2 in oligodendrocyte cultures

In our attempts to identify downstream events that might

mediate H2O2-induced oligodendrocyte cell death, we used

phospho-specific MAPK antibodies to assess the possibility

that MAPKs were being activated. Figure 4 shows the

phosphorylation status of MAPKs when progenitors (Fig 4a)

and 12-d mature cells (Fig. 4b) were exposed for 15 min to

increasing concentrations of H2O2. This time-point was

Fig. 3 Effect of H2O2 on intracellular levels of glutathione (GSH) and

protective effects of N-acetyl-L-cysteine (NAC). (a) Progenitor and

differentiated oligodendrocyte cultures were incubated with different

concentrations of NAC (from 0 to 20 mM) for 30 min, prior to a 3-h

exposure to H2O2 (0.1 and 0.5 mM, respectively). Cell viability was

measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT) reduction 18 h after removing H2O2 as described in

Materials and methods. Results shown in the graphs are the mean

± SEM of three independent experiments performed in triplicate. Sta-

tistical differences are indicated for progenitors: H2O2 treatment ver-

sus H2O2 + 5 mM NAC (p < 0.05); H2O2 treatment versus H2O2 + 10–

20 mM NAC (p < 0.001); and for oligodendrocytes: H2O2 treatment

versus H2O2 + 5–20 mM NAC (p < 0.001). In (b) progenitor and

oligodendrocyte cultures were exposed to different concentrations of

H2O2 alone for 4 h. In (c) cultures were incubated with different con-

centrations of NAC for 30 min, prior to a 4-h exposure to 0.1 mM H2O2

for progenitors and 0.5 mM for oligodendrocytes. Intracellular GSH

levels were determined as described in Materials and methods. Re-

sults are expressed as nmol GSH/mg protein and represent the mean

± SEM of three independent experiments performed in triplicate. Sta-

tistical differences from control levels in (b) were as follows: in pro-

genitors: 0.1 mM H2O2 (p < 0.01), 0.5 and 1 mM H2O2 (p < 0.001) and

in oligodendrocytes: 0.1 mM H2O2 (p < 0.05), 0.5 mM H2O2 (p < 0.01),

1 mM H2O2 (p < 0.001). Statistical differences in (c) from H2O2 alone

were as follows: in progenitors: H2O2 + 5 mM NAC (p < 0.01), + 10 mM

and + 20 mM NAC (p < 0.001) and in oligodendrocytes: H2O2 + 5 mM,

+ 10 mM and + 20 mM NAC (p < 0.001). H2O2 versus control

(p < 0.01) in progenitors and oligodendrocytes.



selected because it lies between the maximal activation times

for p38 and the ERKs (Fig. 5). Significant increases in

phosphorylation were observed for p38, ERK1 and ERK2 at

0.1 mM H2O2, reaching maximal levels at 1 mM H2O2 in

both progenitor and oligodendrocyte cultures. However, the

relative activation levels were significantly higher for

progenitors than for mature cells. The time-course of MAPK

phosphorylation was studied in cultures exposed to 0.1 mM

H2O2 (Fig. 5). A rapid and transient phosphorylation of p38

was observed in progenitors, reaching a maximum at 5 min

(290%) but still remaining activated at 30 min. In contrast,

ERK1 and ERK2 were maximally activated (180 and 495%,

respectively) at 30 min and returned to basal levels at 2 h. In

addition, the time-course of MAPK activation in mature cells

was similar to that of progenitors (results not shown).

Effect of N-acetyl-L-cysteine on H2O2-stimulated MAPK

activation

In an effort to establish a link between MAPK activation and

H2O2 toxicity, cultures were treated with NAC, a precursor of

GSH. The NAC was added 30 min prior to the 15-min

treatment with 0.1 or 0.5 mM H2O2 for progenitors and

mature cells, respectively. Figure 6(a) shows that activation

in progenitors of p38 and ERK1/2 by H2O2 was significantly

reduced with 10 mM NAC while complete blockage was

attained with 20 mM NAC. In contrast, 10 mM NAC

completely blocked H2O2-induced p38 and ERK1/2 activa-

tion in differentiated cells (Fig. 6b).

Fig. 4 Concentration dependency of p38 and extracellular signal-

regulated kinase (ERK)1/2 activation by H2O2 in oligodendrocyte

progenitors and oligodendrocyte cultures. Cell cultures were exposed

to various concentrations (0.01–1 mM) of H2O2 for 15 min and MAPK

activation was determined by immunoblot analysis as described in

Materials and methods. The top panels in (a and b) show western blots

of duplicates or triplicates from a typical experiment. The blots were

analyzed by densitometry and the values are expressed as the mean

± SEM of three independent experiments performed in triplicate. (a) In

progenitors: p38 and ERK1 (0.01 mM, p > 0.05; 0.1 mM, p < 0.001;

1 mM, p < 0.001). (b) In mature cells: p38 and ERK1 (0.1 mM,

p > 0.05; 0.5 mM, p < 0.001; 1 mM, p < 0.001; 5 mM, p < 0.001).

Fig. 5 Time dependency of p38 and extracellular signal-regulated

kinase (ERK)1/2 activation in H2O2-treated oligodendrocyte progenitor

cultures. Cell cultures were exposed to 0.1 mM H2O2 for the indicated

times. MAPK activation was determined by immunoblot analysis as

described in Materials and methods. The top panel shows western

blots of triplicate samples from a typical experiment. The blots of two

independent experiments performed in triplicate were analyzed by

densitometry and the values are expressed as the mean ± SEM of

percent difference from non-stimulated control cultures. p38 (5 min,

p < 0.001; 30 min, p < 0.01; 120 min, p > 0.05). ERK1 (5 min,

p > 0.05; 30 min, p < 0.01; 120 min, p < 0.001).



Mechanism of cell death

In order to determine whether cell death elicited by H2O2

involved apoptosis, DNA condensation/fragmentation,

caspase 3 activation and PARP cleavage were examined.

Evidence for apoptosis was provided by measurements of

chromatin fragmentation or condensation, as determined by

examining 4,6-diamidino-2-phenylindole dihydrochloride-

labeled nuclei, as well as by the TUNEL assay (Fig. 7,

Table 1). Comparison of the results obtained clearly showed

that both methods provided reliable estimates of chromatin

damage when cells were exposed to H2O2 for 1 h and

allowed to recover for 18 h. A concentration of 0.1 mM H2O2

increased the proportion of TUNEL-positive progenitors

from 8 to 52% and the cells lost their processes, formed

clusters and many nuclei were fragmented (Table 1 and

Fig. 7). Mature oligodendrocytes, treated with 0.5 mM H2O2,

showed chromatin condensation and the number of TUNEL-

positive cells increased from 8% in controls to 55%.

H2O2 treatment of oligodendrocyte progenitors resulted in

a concentration-dependent activation of caspase 3 as shown

by cleavage of the 32-kDa pro-caspase to produce the active

17-kDa fragment (Fig. 8). A significant increase in caspase 3

activation was observed with 0.01 mM H2O2, reaching

maximal levels at 0.025 mM and declining when concentra-

tions of H2O2 ranging from 0.1 to 0.5 mM were used. These

results suggest that the lower concentrations of H2O2 (0.01–

0.025 mM) cause caspase 3-dependent apoptosis of progen-

itors while higher concentrations (0.1–0.5 mM) also cause

necrosis. This conclusion is supported by the results of

Fig. 6 N-acetyl-L-cysteine (NAC) prevents MAPK activation by H2O2

in oligodendrocyte cultures. (a) Progenitor and (b) oligodendrocyte

cultures were incubated with different concentrations of NAC for

30 min, prior to a 15-min exposure to H2O2 (0.1 mM for progenitors

and 0.5 mM for oligodendrocytes). MAPK activation was determined

by immunoblot analysis as described in Materials and methods. The

top panels in (a and b) shows western blots of duplicate samples from

a typical experiment. The blots were analyzed by densitometry and the

values are expressed as the mean ± SEM of three independent

experiments performed in triplicate. The values for the cultures treated

with H2O2 were set at 100%. ERK, extracellular signal-regulated kin-

ase; cont, control.

Fig. 7 Effect of H2O2 on DNA fragmentation. DNA fragmentation and

condensation were assessed in oligodendrocyte progenitor cultures

treated with 0.1 mM H2O2 or mature cells treated with 0.5 mM H2O2 for

1 h, washed and allowed to recover for 18 h. (a, c, e and g) In situ

labeling of DNA by the terminal deoxynucleotidyl transferase-mediated

dUTP nick end-labeling method. (b, d, f and h) 4,6-diamidino-2-

phenylindole dihydrochloride-labeled nuclei. H2O2 caused DNA frag-

mentation and condensation in more than 50% of the H2O2-treated

cells (c and d for progenitors; g and h for mature oligodendrocytes)

compared with negligible values for controls (a and b for progenitors; e

and f for mature oligodendrocytes). Arrowheads point to condensed or

fragmented nuclei.



experiments measuring LDH release, which showed statis-

tically significant increases at 4 h after exposure in progen-

itors treated with 0.05 mM H2O2 (Fig. 2).

Caspase 3 activation was also time dependent, a small

increase being detected at 4 h and maximal activation at 12 h

(results not shown). To correlate the appearance of the 17-

kDa caspase fragment with the onset of activity, we assayed

AMC release from the peptide DEVD-AMC, which mimics

the target sequence of the substrate. Caspase activity

increased almost 10-fold (control, 38 F.U./lg protein;

H2O2, 380 F.U./lg protein) 12 h after treatment with

0.1 mM H2O2 and was inhibited in the presence of 1 lM

Ac-DEVD-aldehyde (CHO). In contrast to the results

obtained with progenitor cultures, mature cells exposed to

H2O2 (0.01–1 mM) did not show a significant increase in

procaspase 3 cleavage by western blot analysis and only a

small increase in caspase 3 activity was observed through

enzymatic cleavage of DEVD-AMC (control, 42 F.U./lgprotein; H2O2, 100 F.U./lg protein). These results suggest

that caspase 3 activation is not an important event in H2O2-

induced death of mature oligodendrocytes. The small

increase in DEVD-AMC cleavage could be due to the

presence of a small number of progenitor cells (� 5%),

which continue to proliferate in the mature cultures.

Furthermore, to determine whether caspases are involved in

DNA fragmentation, progenitors and mature cells were pre-

treated with zVAD (100 lM), a pan-caspase inhibitor, prior to

H2O2 exposure. The TUNEL assays (Table 1) showed that

zVAD reduced the percentage of chromatin-damaged pro-

genitors from 52 to 14% while the number of chromatin-

damaged oligodendrocytes was reduced from 55 to 48%.

Caspases thus play an important role in effecting chromatin

damage in progenitors with only a negligible effect in mature

cells. In contrast to zVAD, the free radical scavenger, NAC

(10 mM), afforded full protection as shown by reduction of

the number of TUNEL-positive cells to control levels in both

progenitors and mature cells.

Another measure of caspase 3 activation is the cleavage of

specific substrates, including PARP. This nuclear enzyme is

activated by binding to DNA breaks and appears to have an

important function in DNA repair and cell death (Ueda and

Hayaishi 1985). Poly (ADP-ribose) polymerase is cleaved by

caspase 3 in response to many apoptotic stimuli (Kaufmann

et al. 1993). In progenitor cultures, further support for

caspase activation was provided by the selective cleavage of

PARP (116-kDa protein) to generate a 85-kDa fragment that

was clearly observed 4 h after treatment with 0.1 mM H2O2

(Fig. 9). By 12 h after H2O2 treatment, almost all of the

PARP was degraded as both the 116- and 85-kDa bands were

absent. In contrast, mature oligodendrocytes exposed to

Table 1 Effects of benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ke-

tone (zVAD) and N-acetyl-L-cysteine (NAC) on H2O2-induced cell

death

Treatment TUNEL-positive cells (%)

Progenitors Oligodendrocytes

Control 8.0 ± 0.7 7.6 ± 1.2

H2O2 52.0 ± 1.8a 54.8 ± 1.5a

+ zVAD (100 lM) 13.6 ± 0.6b 48.25 ± 2.3c

+ NAC (10 mM) 15.3 ± 1.4b 10.5 ± 1.7b

The H2O2 concentration used was 0.1 mM for progenitors and 0.5 mM

for mature oligodendrocytes. Drugs were added 30 min before 1 h

H2O2 treatment. Data represent the mean ± SEM of three separate

experiments performed in triplicate. ap < 0.001 compared with corre-

sponding control, bp < 0.001; cp < 0.05 compared with corresponding

values of cells treated with H2O2 only.

TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick

end-labeling.

Fig. 8 Exposure to H2O2 activates caspase 3 in oligodendrocyte pro-

genitors. Here we demonstrate by western blotting that treatment of

progenitors with H2O2 is accompanied by a concentration-dependent

cleavage of the 32-kDa pro-caspase into the active 17-kDa fragment of

caspase 3, reaching maximal levels at 25 lM H2O2 after 16 h. In con-

trast, no significant increases were detected in mature oligodendrocyte

cultures. OL, oligodendrocytes; OP, oligodendrocyte progenitors.

Fig. 9 Exposure to H2O2 causes poly (ADP-ribose) polymerase

(PARP) cleavage in oligodendrocyte progenitors. Cleavage of the

caspase 3 substrate PARP (116-kDa protein) generated an 85-kDa

fragment that was detectable 4 h after H2O2 treatment of progenitor

cultures but not oligodendrocyte cultures.



H2O2 (0.5 mM) did not show a significant increase in PARP

cleavage, in agreement with the results for caspase 3 (Fig. 8).

Effect of p38, MEK, caspase and poly (ADP-ribose)

polymerase inhibitors on H2O2-induced oligodendrocyte

cell death

A conflicting body of literature exists on the potential role of

MAPKs in oxidative stress. Thus, inhibitors of these kinases

can reduce, enhance or have no effect on cell death.

Oligodendroglial cultures were pre-treated for 30 min with

the different inhibitors, followed by 1 h exposure to H2O2

(0.1 mM for progenitors and 0.5 mM for oligodendrocytes)

and recovery for 18 h before LDH release or MTT reduction

was assayed. U0126 (10 lM) or PD098059 (30 lM), specific

inhibitors of the MAPK/ERK kinase, MEK, afforded signi-

ficant protection from H2O2 toxicity in mature oligodendro-

cytes. Thus, LDH release was decreased from 47 to �23%

while MTT levels increased from 30 to 50–60%. Similarly,

the p38 inhibitor, PD169316 (5 lM), significantly reduced

the toxic effect of H2O2 on mature oligodendrocytes with a

50% decrease in LDH release and a 50% increase in MTT

values. Neither the MEK nor p38 inhibitors provided

significant protection from H2O2 toxicity in progenitors. In

contrast to the MAPK inhibitors, zVAD (100 lM) caused a

small but significant decrease in cell death both in oligo-

dendrocytes as well as in progenitors.

As PARP inhibitors have been shown to protect PC12

cells, which are a model of sympathetic neurons, from H2O2-

induced injury (Cole and Perez-Polo 2002), we pre-treated

oligodendroglial cultures with PJ34 prior to H2O2 exposure.

This compound has been shown to inhibit PARP activity and

peroxynitrite-induced cell necrosis in mouse thymocytes

(Garcia Soriano et al. 2001). PJ34 (5 lM) caused a small but

significant decrease in LDH release in progenitors but the

MTT survival assay was not significantly different from

H2O2 alone. In mature oligodendrocytes, PJ34 was protective

as shown by both assays.

The effect of all inhibitors was further explored by testing

their ability to protect oligodendroglial cultures from higher

concentrations of H2O2 (0.25 mM for progenitors and 2 mM

for mature cells). These concentrations caused the release of

50% of LDH at the 4 h time-point (Fig. 2 and Table 3).

Interestingly, MEK, p38, PARP and caspase inhibitors all

afforded a significant reduction in LDH release caused by

H2O2 treatment (p < 0.01). Furthermore, combinations of

two drugs (PD169316 and PJ34 or z-VAD and PJ34) were

more effective than the individual drugs in protecting both

progenitors and mature oligodendrocytes from H2O2 toxicity

(p < 0.001).

Table 2 Effect of MEK, p38, caspase and poly (ADP-ribose) polymerase inhibitors on H2O2-induced cell death

Treatment

Released LDH (% of total) MTT assay (% of total)

Progenitors Oligodendrocytes Progenitors Oligodendrocytes

H2O2 82.2 ± 1.7 47.2 ± 1.0 21.5 ± 0.7 30.5 ± 2.3

+ UO126 (10 lM) 86.1 ± 1.4ns 23.6 ± 0.7b 24.0 ± 1.7ns 60.3 ± 0.8b

+ PD098059 (30 lM) 83.4 ± 1.3ns 22.5 ± 0.8b 23.6 ± 1.6ns 48.4 ± 4.1a

+ PD169316 (5 lM) 87.3 ± 1.7ns 21.7 ± 0.8b 18.6 ± 1.2ns 61.3 ± 3.2b

+ zVAD (100 lM) 70.0 ± 0.9a 27.9 ± 0.9b 28.8 ± 1.7a 43.2 ± 2.1a

+ PJ34 (3 lM) 72.5 ± 1.2a 19.6 ± 0.8b 22.4 ± 1.4ns 44.5 ± 3.5a

The H2O2 concentrations used were 0.1 and 0.5 mM for progenitors and mature oligodendrocytes, respectively. Inhibitors were added 30 min prior

to the 1-h H2O2 treatment. Cells were washed and allowed to recover for 18 h in serum-free medium. Data represent the mean ± SEM of three

separate experiments performed in triplicate. ap < 0.01, bp < 0.001 compared with corresponding values for H2O2-treated cells; ns, not significant.

LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; zVAD, benzyloxycarbonyl-Val-Ala-Asp fluoro-

methyl ketone.

Table 3 Effect of MEK, caspase and poly (ADP-ribose) polymerase

inhibitors on lactate dehydrogenase (LDH) release induced by H2O2

Condition

Released LDH (% of total)

Progenitors Oligodendrocytes

H2O2 50.0 ± 4.5 50.0 ± 4.0

+ U0126 (10 lM) 34.2 ± 3.0a 25.7 ± 3.4a

+ PD098059 (30 lM) 35.1 ± 2.0a 23.4 ± 3.0a

+ PD169316 (5 lM) 30.2 ± 3.6a 22.3 ± 3.8a

+ zVAD (100 lM) 25.1 ± 2.0a 25.5 ± 3.0a

+ PJ34 (3 lM) 30.3 ± 2.1a 22.3 ± 2.5a

+ PD169316 and PJ34 17.4 ± 2.4b 15.7 ± 2.0b

+ zVAD and PJ34 15.0 ± 1.1b 7.7 ± 0.9b

The H2O2 concentration used was 0.25 mM for progenitors and 2 mM

for mature oligodendrocytes. Inhibitors were added 30 min prior to

addition of H2O2; cell cultures were then treated with H2O2 for 1 h after

which culture medium was changed and cells were allowed to recover

for 4 h in serum-free medium. Data represent the mean ± SEM of

three separate experiments performed in triplicate. ap < 0.01;bp < 0.001 compared with corresponding values for H2O2-treated

cells.

zVAD, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone.



Discussion

The main objectives of this study were to elucidate factors

responsible for the differential sensitivity of progenitors and

mature oligodendrocytes to H2O2 exposure and to evaluate

potential protective agents for reducing toxic damage. We

demonstrate that oligodendrocyte progenitors are more

vulnerable than mature cells to H2O2 exposure as determined

by monitoring LDH release, mitochondrial dehydrogenase

activity and decreases in intracellular GSH levels. In

progenitors, a low concentration of H2O2 (10–25 lM) caused

activation of caspase 3 without LDH release, suggesting cell

death by apoptosis, while higher concentrations of H2O2

decreased caspase 3 activation and increased LDH release,

implicating both necrosis and apoptosis. Neither caspase 3

nor PARP cleavages were observed in mature cells. At both

stages of development, H2O2 caused activation of the

MAPKs, p38 and ERK1/2, with maximal increases occurring

at high H2O2 concentrations. Pre-treatment of cells with the

antioxidant, NAC, suppressed H2O2-induced MAPK activa-

tion, the decrease in intracellular GSH concentration and cell

death at both stages of development. Inhibitors of MEK1

(PD098059 and U0126), p38 (PD169316), PARP and zVAD

provided progenitors and mature oligodendrocytes with

varying levels of protection against H2O2 toxicity, while

combinations of drugsweremore effective than either drug alone.

Glutathione levels regulate H2O2 toxicity in

oligodendrocytes

Glutathione, the major free thiol in most living cells,

participates in diverse biological processes such as removal

of hydroperoxides (Arias and Jakoby 1976). Intracellular

GSH is effectively maintained in the reduced state by GSH

disulfide reductase via NADPH and reduced GSH, either

alone or in conjunction with oxygen radical-scavenging

enzymes, is important in protecting cells against reactive

oxygen species (Richter and Kass 1991; Koppenol 1993;

Winterbourn 1993).

Several reports have provided evidence that oligodendro-

cyte progenitors are significantly more sensitive to toxic

insults than mature cells (Back et al. 1998; Volpe 1998;

Almazan et al. 2000; Fern and Moller 2000; Molina-

Holgado et al. 2001; Khorchid et al. 2002). In line with

these reports, we show that progenitor cultures were 10 times

more sensitive to H2O2-induced toxicity than mature oligo-

dendrocytes, as determined by assaying mitochondrial

dehydrogenase activity to monitor cell viability, LDH release

to detect damage to plasma membrane or cell death and

changes in intracellular GSH levels as an index of response

to oxidative stress. In our cultures the basal level of GSH was

significantly lower in progenitor cells than in mature

oligodendrocytes, in agreement with previous reports (Juur-

link et al. 1998; Almazan et al. 2000). Furthermore, higher

concentrations of H2O2 were required to significantly reduce

intracellular GSH levels, suggesting that mature cells can

dispose of exogenous H2O2 more effectively than progenitor

cells (Hirrlinger et al. 2002). The susceptibility of

oligodendrocyte progenitors to free radical damage has been

proposed to be due to the low levels of GSH and high levels

of free iron in these cells (Husain and Juurlink 1995; Connor

and Menzies 1996; Thorburne and Juurlink 1996; Juurlink

et al. 1998). In addition, oligodendrocytes, like neurons, are

more sensitive to oxidative stress because they utilize high

levels of oxygen for normal function while their antioxidant

mechanisms are poorly developed (Halliwell 1992; Wood

and Youle 1994; Husain and Juurlink 1995; Juurlink 1997).

That intracellular GSH constitutes an important defence

against H2O2-mediated toxicity is further supported by the

ability of the antioxidant, NAC, to block toxicity in a

concentration-dependent manner in both oligodendrocyte

progenitors and mature cells. In our cultures NAC not only

prevented GSH decreases provoked by H2O2 but also

increased intracellular GSH to higher than control levels. In

addition to its action as an artificial precursor of GSH (Bernard

1991), NAC acts as a powerful scavenger of oxygen free

radicals, yielding NAC-disulfide end products (Zhang et al.

1995). The enzyme, catalase, can also scavenge H2O2 but

occurs at low levels in oligodendrocytes. Whether catalase

plays a role in the differential susceptibility of progenitors to

H2O2 is not clear at present because different groups have

reported higher, lower or equal levels than in mature cells

(Adamo et al. 1986; Bernardo et al. 2003; Baud et al. 2004).

However, catalase has been proposed to cooperate with GSH

peroxidase in conferring resistance to H2O2 toxicity by mature

oligodendrocytes (Baud et al. 2004). The H2O2-induced

killing of maturing oligodendrocytes (differentiated for

6 days) was also reduced by the antioxidants, pyrrolidine

dithiocarbamate and vitamin E, as well as by iron chelators

(Vollgraf et al. 1999). In addition to preventing oligoden-

droglial cell death, NAC was capable of blocking other H2O2

effects, including MAPK activation and DNA fragmentation.

H2O2-induced MAPK activation

Numerous studies have shown that H2O2 activates signaling

pathways associated with protein tyrosine kinases and their

downstream signaling components, such as MAPKs and

transcription factors regulating cell survival (for review see

Kamata and Hirata 1999). Bhat and Zhang (1999) demon-

strated that H2O2 increased tyrosine phosphorylation of the

platelet-derived growth factor receptor and activation of the

three MAPK subgroups, ERK 1/2, p38 and c-Jun N-terminal

kinase, in an oligodendrocyte progenitor cell line, CG4.

Interestingly, these authors found that cell death induced by

high concentrations of H2O2 (0.25–1 mM) involved cell

necrosis and ERK activation as it could be blocked by

inhibition of the upstream activator, MEK, with PD098059.

Hence, one of our objectives was to determine whether

MAPKs were also activated in primary oligodendrocyte



cultures and whether they played a role in the differential

susceptibility of progenitors and mature oligodendrocytes to

H2O2 exposure.

In our study, both progenitors and mature oligodendro-

cytes responded to H2O2 treatment with increased activation

(phosphorylation) of ERK1/2 and p38, the maximal increase

occurring at 1 mM H2O2 for both developmental stages. At

this concentration, H2O2 was also shown to cause a greater

than 50% increase in LDH release in progenitors and 40% in

mature cells, as early as 4 h after treatment. Our results,

therefore, suggest that activation of ERK1/2 and p38

correlates with cell necrosis rather than apoptosis.

Inhibitors of MEK1 (PD098059 and U0126) and p38

(PD169316) partially protected mature oligodendrocytes

from H2O2 toxicity at concentrations of 0.5 and 2 mM,

respectively, both at 4 and 18 h. In contrast, progenitor cells

were only partially protected by these inhibitors at the 4 h

time-point with 0.25 mM H2O2, a concentration causing 50%

release of LDH. Our results are in partial agreement with

those of Bhat and Zhang (1999) in showing that the MEK

inhibitor, PD098059, could reduce H2O2 toxicity but, in

contrast to these authors, we also found that some protection

was afforded by the p38 inhibitor. Although the differences

between results obtained with our primary cultures compared

with those for CG4 cells are not easily explained, the latter

displayed sensitivity to H2O2 similar to that of mature

oligodendrocytes suggesting that CG4 cells are more resist-

ant than progenitors to oxidative stress.

Another factor that could contribute to the greater

susceptibility of progenitors to H2O2 toxicity is the balance

between expression of proapoptotic and anti-apoptotic genes

that determines the sensitivity to apoptosis-inducing insults.

As we have shown previously, expression levels of proca-

spase 3 and the ratio of the proapoptotic protein bax to the

anti-apoptotic protein bcl-xl are several-fold higher in

progenitors than in mature oligodendrocytes (Khorchid et al.

2002). Other differences between oligodendrocyte progeni-

tors and mature cells in the levels of bcl-2 family members

have been recently reported (Itoh et al. 2003).

Role of caspase 3 in the mechanism of oligodendrocyte

cell death

Prominent features of apoptosis include caspase activation

and DNA condensation and fragmentation. Caspases, the

crucial executors of apoptosis (Nicholson 1996), are syn-

thesized as inactive precursors that are proteolytically

cleaved to generate active species. Among the 14 identified

caspases, caspase 3 is a potent effector of apoptosis and

promotes oligodendrocyte death in cultures exposed to

hypoxic injury (Shibata et al. 2000).

Several observations support the conclusion that cell death

in oligodendrocyte progenitors involves apoptosis, including

activation of caspase 3, cleavage of PARP and chromatin

condensation and fragmentation. Thus, we show that clea-

vage of procaspase 3 to generate the active 17-kDa fragment

depended on concentration and time of exposure to H2O2

with maximal activation in oligodendrocyte progenitor

cultures occurring 12 h after treatment. It is interesting to

note that maximal activation of caspase 3 occurred at a

concentration of H2O2 (0.025 mM) which did not induce

LDH release while higher concentrations (0.1–0.25 mM)

caused early membrane damage and a decrease in the level of

caspase 3 activation, suggesting that cells are also undergo-

ing necrosis. A dual mechanism of cell death inflicted by

H2O2 has been reported in other systems as well as a switch

from apoptosis to necrosis with higher concentrations

(Gardner et al. 1997).

Apoptosis is associated with the proteolytic cleavage of

the 116-kDa caspase 3 substrate, PARP, at a characteristic

DEVD sequence in the DNA-binding domain of the enzyme

to yield 89- and 24-kDa fragments (Kaufmann et al. 1993;

Ha and Snyder 2000). We found evidence of PARP cleavage

4 h after H2O2 treatment while the protein was practically

undetectable in oligodendrocyte progenitors at later time-

points (8 and 12 h). Further evidence of apoptosis in

progenitors was provided by the significant increase in the

number of TUNEL-positive cells after H2O2 treatment.

Chromatin condensation and fragmentation were also

assessed by 4,6-diamidino-2-phenylindole dihydrochloride

staining and by the appearance of DNA laddering on an

agarose gel (results not shown). Treatment of oligodendro-

cyte progenitor cultures with H2O2 caused a significant

increase in DNA fragmentation, which was almost abolished

(85%) by a pan-caspase inhibitor, zVAD. In contrast, z-VAD

only partially protected progenitors from H2O2 toxicity as

determined by decreases in MTT or release of LDH. This

apparent discrepancy has been reported in PC12 cells by

other investigators (Jiang et al. 2001) and we cannot exclude

the possibility that some of the progenitor cells are dying by

necrosis as H2O2 can induce cell death by both necrotic and

apoptotic mechanisms, depending on the concentration used

to treat cells (Gardner et al. 1997).

Other workers have observed that H2O2-induced DNA

strand breaks can activate PARP (Hyslop et al. 1988), while

PARP inhibitors reduced the degree of tissue injury after

ischemia and reperfusion in the brain (Eliasson et al. 1997) and

in neural cell lines exposed to H2O2 in vitro (Cole and Perez-

Polo 2002). We show that both oligodendrocyte progenitors

and mature cells are partially protected from H2O2 toxicity by

PJ34, a PARP inhibitor that blocks peroxynitrite-induced cell

necrosis in mouse thymocytes (Garcia Soriano et al. 2001).

Furthermore, the combination of PJ34 with zVAD or the p38

inhibitor was more effective than either drug alone, suggesting

that more than one pathway or mechanism of cell death is

responsible for the toxic effects of H2O2.

Significant differences in cell death mechanisms involving

progenitors and mature cells were also observed. Thus, H2O2

(0.01–0.5 mM) did not cause an increase in caspase 3 or PARP



cleavage in mature cells although TUNEL results clearly

showed the occurrence of DNA damage. Another interesting

observation stemming from our work was the ability of zVAD

to reduce the killing of mature oligodendrocytes. As caspase 3

activation was not detected, these results could suggest that

other caspases may be involved in oligodendrocyte death.

Alternatively, most mature oligodendrocytes in our cultures

may be dying by a different mechanism involving DNA

damage. Other investigators have shown that 1 mM H2O2 can

induce rapid cleavage of chromatin into highmolecular weight

fragments in mature oligodendrocytes in a process that is

independent of caspase activation (Mouzannar et al. 2001)

while the same H2O2 concentration caused necrotic death of

CG4 cells (Bhat and Zhang 1999). In contrast, 0.1 mM H2O2

caused DNA condensation and fragmentation in maturing

oligodendrocyte cultures into characteristic internucleosomal

fragments as assessed by electron microscopy and agarose gel

electrophoresis (Vollgraf et al. 1999).

In conclusion, the results presented here provide evidence

that oligodendrocyte progenitors are more sensitive than

mature cells to H2O2-induced toxicity. Factors contributing

to the vulnerability of progenitors to free radical damage

could include low levels of intracellular GSH and the

selective action of caspase 3. Furthermore, damage induced

in oligodendrocyte progenitors and mature cells by H2O2 can

be prevented by the antioxidant, NAC, as we have previously

shown to be the case for toxicity induced by a-amino-3-

hydroxy-5-methylisoxazole-4-propionate receptor activation,

cadmium or dopamine (Almazan et al. 2000; Khorchid et al.

2002; Liu et al. 2002). In addition, p38, MEK, caspase and

PARP inhibitors appear to protect oligodendrocytes more

efficiently when applied in combination, suggesting a

complex cell death mechanism. Our combined studies

highlight the crucial role of free radicals in oligodendrocyte

pathology and the necessity to develop antioxidant thera-

peutics, such as NAC or combined therapies, for the

treatment of diseases involving oligodendrocyte death.

Acknowledgements

This work was funded by the Canadian Institutes of Health and

Research and the Multiple Sclerosis Society of Canada to GA. H-NL

and AK held studentships from the Multiple Sclerosis Society of

Canada.

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Developmental differences in H2O2-induced oligodendrocyte cell death: role of glutathione, mitogen-activated protein kinases and caspase 3

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