, ,

*Instituto de Biologia Experimental e Tecnologica, Oeiras, Portugal

�Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa, Oeiras, Portugal

Hypoxia-ischemia and reperfusion (HIR), because of strokein adults and because of perinatal complications in newborns,are the main cause of brain damage. Cerebral damage is aresult of oxygen and tissue energy depletion, leading toacidosis, inflammation, glutamate excitotoxicity, and gener-ation of reactive oxygen species (ROS) (Mattson andKroemer 2003; Vannucci and Hagberg 2004).

Carbon monoxide (CO) is an endogenous product ofheme degradation by heme-oxygenase (HO), which alsogenerates free iron and biliverdin (a potent antioxidant)(Ryter et al. 2006). HO plays an important role in cellredox state, acting as an antioxidant enzyme, which can beespecially important for tissues with weak endogenousantioxidant defenses, such as the myocardium and thenervous system (Dore 2002). The neuroprotective activityof HO in the CNS has been extensively described (Doreet al. 1999b, 2000; Schipper 2004; Ahmad et al. 2006).HO-2 isoform is constitutively expressed in neurons and isresponsible for the most of HO activity in the brain. HO-2activity is controlled by post-translational modificationsand/or interactions with other proteins (Dore 2002;

Boehning et al. 2004). HO-1 is a hypoxia and ischemiainducible isoform, confined to glia and small populations ofscattered neurons (Geddes et al. 1996; Koistinaho et al.1996; Schipper 2004) and its over-expression in neuronswas shown to decrease the infarct size in an ischemic-reperfusion model (Panahian et al. 1999).

Low concentrations of CO confer an increased resistanceto apoptosis triggered by several stimuli in different models;

Received April 15, 2008; revised manuscript received June 25, 2008;accepted July 24, 2008.Address correspondence and reprint requests to Dr Paula M. Alves,

Instituto de Tecnologia Quımica e Biologica (ITQB), Instituto de Bio-logia Experimental e Tecnologica (IBET), Apartado 12, Oeiras 2781-901, Portugal. E-mail: marques@itqb.unl.ptAbbreviations used: 5HD, 5-hydroxydecanoate; BHT, butyl-hy-

droxytoluene; CHX, cycloheximide; CO, carbon monoxide; HIR,hypoxia-ischemia and reperfusion; HO, heme-oxygenase; L-NAME,N-nitro-L-arginine methyl ester hydrochloride; mitoKATP, mitochondrialKATP channel; NO, nitric oxide; NOS, nitric oxide synthase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PC, pre-conditioning; ROS,reactive oxygen species; sGC, soluble guanylyl cyclase; t-BHP, tert-butylhydroperoxide.

Abstract

Carbon monoxide (CO) is an endogenous product of mam-

malian cells generated by heme-oxygenase, presenting anti-

apoptotic properties in several tissues. The present work

demonstrates the ability of small amounts of exogenous CO to

prevent neuronal apoptosis induced by excitotoxicity and

oxidative stress in mice primary culture of cerebellar granule

cells. Additionally, our data show that endogenous CO is a

heme-oxygenase product critical for its anti-apoptotic activity.

Despite being neuroprotective, CO also induces reactive

oxygen species generation in neurons. These two phenomena

suggest that CO induces pre-conditioning (PC) to prevent cell

death. The role of several PC mediators, namely soluble

guanylyl cyclase, nitric oxide (NO) synthase, and ATP-

dependent mitochondrial K channel (mitoKATP) was ad-

dressed. Inhibition of soluble guanylyl cyclase or NO synthase

activity, or closing of mitoKATP abolishes the protective effect

conferred by CO. In addition, CO treatment triggers cGMP

and NO production in neurons. Opening of mitoKATP, which

appears to be critical for CO prevention of apoptosis, might be

a later event. We also demonstrated that reactive oxygen

species generation and de novo protein synthesis are nec-

essary for CO PC effect and neuroprotection. In conclusion,

CO induces PC and prevents neuronal apoptosis, therefore

constituting a novel and promising candidate for neuro-

protective therapies.

Keywords: apoptosis, brain, carbon monoxide, neurons,

oxidative stress, pre-conditioning.

J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x

JOURNAL OF NEUROCHEMISTRY | 2008 doi: 10.1111/j.1471-4159.2008.05610.x

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x 1

such as, endothelial cells (Brouard et al. 2000), vascularsmooth muscle cells (Liu et al. 2003) or liver and lung(Sarady et al. 2004); CO also mediates other biologicalfunctions, such as anti-inflammatory, anti-proliferative orvasodilatation (Sammut et al. 1998; Otterbein et al. 2000;Taille et al. 2005; Ryter et al. 2006).

Carbon monoxide is also able to inhibit cytochrome coxidase (the terminal enzyme of the electron transport chain)and NADPH oxidase, leading to ROS generation (Sandoukaet al. 2005; Taille et al. 2005; D’Amico et al. 2006; Chinet al. 2007; Wang et al. 2007). CO activates soluble guanylylcyclase (sGC), increasing cGMP levels (Brune and Ullrich1987); however, its efficiency is much lower than that ofnitric oxide (NO) (Stone and Marletta 1994). Recently, it hasbeen demonstrated that CO modulates neuro-inflammatoryresponse in BV-2 microglial cells in vitro, by decreasinginflammation induced by thrombin, interferon-c and/orhypoxia (Bani-Hani et al. 2006). Furthermore, exogenousCO administration suppresses neuroinflammation in animalswith an autoimmune encephalomyelitis disease, a model formultiple sclerosis (Chora et al. 2007). However, the anti-apoptotic role of CO remains weakly explored and theprotective effect of HO is mainly attributed to the antioxidantproperties of biliverdin (Dore et al. 1999a; Boehning andSnyder 2003).

The brain protection triggered by a sublethal ischemia,known as ischemic pre-conditioning (PC), has gainedimportance for potential therapeutic applications (Kirino2002; Dirnagl et al. 2003; Gidday 2006). PC can also beinduced by other stimuli than ischemia, defined aschemical PC. Recently, many factors have been proposedto participate in the brain PC event: (i) production of smallamounts of ROS (Dirnagl et al. 2003); (ii) NO assignaling molecule [produced by activation of neuronalnitric oxide synthase (NOS), and/or expression of endo-thelial NOS] (Gidday et al. 1999; Atochin et al. 2003;Cho et al. 2005); (iii) generation of cGMP by NO-activated GC (Andoh et al. 2000, 2002); and (iv) openingof mitochondrial KATP channel (mitoKATP) (Mayanagiet al. 2007; Gaspar et al. 2008).

The work presented here aims to investigate the ability ofCO to prevent neuronal apoptosis, and provides evidence thatCO mode of action activates a PC-like mechanism.

Material and methods

MaterialsPlastic tissue culture dishes were from Nunc (Roskilde, Denmark);

fetal bovine serum, glutamine, penicillin–streptomycin solution and

Basal Medium Eagle’s (BME) basal medium were obtained from

Gibco, Invitrogen (Paisley, UK); all other chemicals were purchased

from Sigma (Munich, Germany). BALB-C mice were purchased

from Instituto Gulbenkian da Ciencia (Oeiras, Portugal).

Cell culturesPrimary cultures of cerebellar granule cells were prepared as described

by Schousboe et al. (1989). Briefly, cells were isolated from the

cerebella of 7-day-old mice; after mild trypsinization (200 mg/L) of

the tissue, followed by trituration in a Dnase solution (100 lg/mL)

containing soybean trypsin inhibitor (520 mg/L), cells were sus-

pended (1.25 · 106 cells/mL) and cultured in Basal Medium Eagle’s

(BME) basal medium containing 12 mM of glucose, 7.3 lMp-aminobenzoic acid, 4 lg/L insulin, 2 mM glutamine, 1% (v/v)

penicillin–streptomycin solution, and 10% (v/v) fetal bovine serum.

Cells were cultured in 6-, 24-, and 96-well plates coated with poly-D-

lysine (50 mg/L) and maintained in a humidified atmosphere of 7%

CO2 at 37�C. Cytosine arabinoside (20 lM) was added 48 h after

inoculation to prevent glia cell proliferation. The experiments reported

herein were performed on cerebellar granule neurons cultured for

1 week. All experiments were performed at least in triplicate.

Cellular treatmentsApoptosis was induced with tert-butylhydroperoxide (t-BHP) at

concentrations ranging from 16 to 28 lM or with glutamate at

concentrations from 20 to 40 lM for 20 h. For the cell death

inhibition experiments, cerebellar granule cells were continuously

exposed to 250 ppm of CO for 90 min in a chamber, as described in

(Otterbein et al. 2000), before treatment with t-BHP or glutamate.

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 5 lM), 5-hy-

droxydecanoate (5HD, 500 lM), N-nitro-L-arginine methyl ester

hydrochloride (L-NAME, 200 lM), butyl-hydroxytoluene (BHT,

10 lM), zinc protoporphyrin IX (5 lM), or cycloheximide (CHX,

1 lg/mL) used to modulate CO effect were applied to cells 1 h prior

to CO treatment (Appendix S1).

Assessment of apoptosis-associated parametersCells cultured on a poly-D-lysine-coated coverslip were stained with

Hoechst 33342 (2 lM; Sigma) and propidium iodide (1 lM;

Molecular Probes, Eugene, OR, USA) followed by fluorescence

microscopic qualitative and quantitative assessment of chromatin

condensation and cell viability, respectively. The cells were observed

on a Leica DMRB microscope (Leica, Wetzlar, Germany) using a

filter cube giving an UV excitation range with a bandpass of 340–

380 nm of wavelength. For each coverslip, 8–10 fields containing

around 200 cells were counted (a total of at least 1500 counted cells).

Measurements of intracellular ROS generation, NO, and cGMP

production, as well as data analysis procedures are described in

Supporting information section (Appendex S1).

Results

Anti-apoptotic effect of carbon monoxide treatment inneuronal apoptosisPrimary culture of cerebellar granule cells was treated witheither glutamate or t-BHP to mimic two consequences ofHIR in the brain: excitotoxicity and oxidative stress,respectively. The hallmarks of apoptosis, chromatin conden-sation, and loss of viability (based on the loss of plasmaticmembrane integrity), are qualitatively represented in Fig. 1aand b, respectively. Quantification of chromatin condensation

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x� 2008 The Authors

2 | H. L. A. Vieira et al.

and loss of viability induced by glutamate or t-BHP with orwithout CO pre-treatment are shown in Fig. 1c–f. Glutamateappeared to be a weaker cell death inducer than t-BHP.

Pre-treatment with CO protected neurons from apoptosis inall ranges of glutamate concentrations, especially at 30 and40 lM (Fig. 1c and d). In the case of t-BHP, CO was able to

Control0 µM glutamate

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Fig. 1 Effect of carbon monoxide treatment in neuronal apoptosis

induced by excitotoxicity and oxidative stress. Primary cultures of

neuronal cells were pre-treated during 90 min with CO in a closed

chamber under a continuous flux of 250 ppm of CO gas, followed by

20 h of glutamate (20–40 lM) or t-BHP (16–28 lM) treatment.

Apoptotic hallmarks were analyzed by fluorescent microscopy. Rep-

resentative micrographs of neurons treated or not with glutamate and

CO are shown for (a) chromatin condensation, measured by Hoechst

33342 (blue fluorescence) and (b) loss of viability, measured by pro-

pidium iodide (red fluorescence). Quantification of apoptotic para-

meters by fluorescent microscopy was based on counting the normal

nuclei (nuclei presenting no chromatin condensation) and unviable

cells (presenting red stained nuclei): for each coverslip, at least 1500

cells were counted. All values are mean ± SD, n = 5. (c and d) For

glutamate apoptosis induction, *p < 0.05 compared with control with

glutamate at 20 lM, **p < 0.05 compared with control with glutamate

at 30 lM and #p < 0.05 compared with control with glutamate at

40 lM). (e and f) For t-BHP apoptosis induction, *p < 0.05 compared

with control with t-BHP at 16 lM and #p < 0.05 compared with control

with t-BHP at 20 lM).

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x

Carbon monoxide as neuroprotector | 3

decrease neuronal apoptosis at 16 and 20 lM of the oxidantagent, but not at 28 lM; in this case, cell death induction wasalready too extensive (Fig. 1e and f). It is important to noticethat the percentage of normal nuclei (presenting no chroma-tin condensation) was always lower than the viable cellpopulation percentage, as the chromatin condensation eventtakes place upstream of the plasmatic membrane loss ofpermeability.

These results confirmed that CO does prevent neuronalapoptosis triggered by either excitotoxicity or oxidativestress.

Role of endogenous carbon monoxideThe actual hypothesis for the anti-apoptotic activity of HOarises mainly from the bilirubin antioxidant property. Toclarify the role of endogenous CO, the chemical inhibitor ofHO activity, zinc protoporphyrin IX, was added to cellcultures before CO and/or glutamate treatment. Figure 2

shows that inhibition of HO activity presented a synergisticeffect with glutamate, increasing cell death levels (Fig. 2a forapoptotic nuclei and Fig. 2b for cell viability). However, COpre-treatment reversed the toxic effect caused by inhibition ofHO (especially at glutamate concentration of 20 and 30 lM),indicating that the enzyme activity was substituted byexogenous CO. These data suggest that endogenous COhas a relevant role in the anti-apoptotic activity conferred byHO.

Oxidative stress generation by carbon monoxideThe ability of CO to generate oxidative stress was tested inthe present system of primary culture of neuronal cells.Indeed, an increase in neuronal intracellular ROS levels ofabout 50% was observed after CO exposure (Fig. S1).Moreover, treatment with hydrogen peroxide (positive con-trol) increased intracellular ROS levels by 300% (data notshown).

Assessment of a pre-conditioning pathway to protectneurons from apoptosis by COAs CO seemed to confer two distinct, and apparentlycontradictory, effects in neurons i.e. protection against celldeath and oxidative stress generation (by inducing ROSformation), the possible existence of a PC mechanism had tobe evaluated. Therefore, cerebral PC-related factors, such assGC, NOS, and ATP-dependent mitoKATP were tested in thepresent model. For that purpose, the chemicals ODQ,L-NAME, and 5HD, which are inhibitors of sGC, NOS,and mitoKATP, respectively, were used to verify their role inthe neuroprotection conferred by CO.

As shown in Fig. 3, inhibition of sGC activity (Fig. 3a andb), inhibition of NOS activity (Fig. 3c and d) or closingmitoKATP channels (Fig. 3e and f) removed the protectiveeffect of CO against neuronal cell death, in particular in thecase of glutamate at 20 and 30 lM. Under a challenge with40 lM of glutamate in the presence of either ODQ,L-NAME, or 5HD, the percentage of neurons with normalnuclei chromatin morphology (Fig. 3a, c, and e) and viablecells (Fig. 3b, d, and f) were much lower than for controlcells (without CO treatment). This indicated that inhibition ofsGC, NOS, or closing mitoKATP potentiated the toxic effectof glutamate at 40 lM. However, control cells (without pre-incubation with CO) treated with ODQ, L-NAME, or 5HD inthe presence of 20 and 30 lM of glutamate did not presentany significant change at the apoptotic hallmark parameters(data not shown), confirming that these three chemicals didnot present any additional toxic effect to excitotoxicity, atthese concentrations of glutamate. Thus, sGC, NOS, andmitoKATP are involved in the pathways of apoptosis inhibi-tion induced by CO.

To confirm the role of NO and cGMP (products of sGCand NOS) in CO neuroprotection, as well as to betterdescribe the involved pathways, quantification of ROS, NO,

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Fig. 2 Role of endogenous CO on the neuroprotection conferred by

HO activity. Cells grown in 24-well plates were pre-incubated with

5 lM of zinc protoporphyrin IX (ZnPP) for 60 min, followed by CO

treatment (90 min) or no treatment in the case of control neurons. Cell

death was induced with glutamate for 20 h and apoptotic hallmarks,

such as chromatin condensation (a) and cell viability (b), were ana-

lyzed by fluorescent microscopy. Quantification was performed as in

Fig. 1. #p < 0.05 compared with control with glutamate at 20 lM,

*p < 0.05 compared with control with glutamate at 30 lM and

**p < 0.05 compared with control with glutamate at 40 lM.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x� 2008 The Authors

4 | H. L. A. Vieira et al.

and cGMP were performed after treatment with CO, and inthe presence or absence of the chemical inhibitors ODQ,L-NAME, and 5HD.

Reactive oxygen species production induced by CO wasnot altered by the presence of sGC or mitoKATP inhibitors(Fig. 4), indicating that ROS generation takes place upstreamof the activation of these players. Inhibition of NOS activitypartially prevented ROS production (ROS increase induced

by CO, with or without L-NAME treatment, were signifi-cantly different at a confidence level of 95%), indicating thatNO also contributes to ROS formation, through an ampli-fication loop between CO and NO generating more ROS.

Carbon monoxide treatment induced cGMP formation inneurons (Fig. 5), confirming the role of sGC in the mech-anisms involved in CO inhibition against neuronal cell death.Moreover, cGMP generation was prevented by ODQ and by

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Fig. 3 Role of soluble guanylyl cyclase (sGC), nitric oxide synthase

(NOS), and ATP-dependent mitochondrial K channel (mitoKATP) in the

CO neuroprotective mechanisms. Primary cultures of cerebellar

granule cells grown in 24-well plates were pre-incubated with 5 lM of

ODQ, 200 lM of L-NAME, and 500 lM of 5HD (inhibitors of sGC,

NOS, and mitoKATP, respectively) for 60 min before CO treatment

(90 min). Cell death was induced with glutamate for 20 h, then

apoptotic hallmarks, such as chromatin condensation and cell viability,

were analyzed by fluorescent microscopy: (a and b) role of sGC, (c

and d) role of NOS, and (e and f) role of mitoKATP in the neuropro-

tective effect induced by CO. Quantification was performed as in

Fig. 1. #p < 0.05 compared with control with glutamate at 20 lM,

*p < 0.05 compared with control with glutamate at 30 lM, and

**p < 0.05 compared with control with glutamate at 40 lM.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x

Carbon monoxide as neuroprotector | 5

L-NAME, inhibitors of sGC and NOS, respectively. Thus,sGC activation is totally dependent upon NO generation,confirming that, in the neuronal system, sGC activation byCO takes place through NO synthesis. On the other hand,closing mitoKATP did not change cGMP formation (Fig. 5),suggesting that the opening of this channel is a downstreamevent of the process.

Finally, NO generation was also triggered by CO (Fig. 6),confirming the involvement of NOS activity in the neuro-protective effect of CO. No effect on NO formation wasobserved when mitoKATP was closed by 5HD (Fig. 6), whichindicates once again that the opening of mitoKATP is adownstream step within the pathway. In addition, ODQpartially prevented NO formation (Fig. 6), which suggeststhat cGMP also controls, at least in part, NO generation.

Probably, a feedback control between sGC and NOSactivities exists.

Altogether, these data indicate that CO induces neuronalPC protection against apoptosis and that sGC, NOS andmitoKATP are implicated in this process. Moreover, ROSgeneration appears to be the first step, while opening ofmitoKATP seems to be the last event of the proposed pathwaytriggered by CO.

Implication of ROS generation for the pre-conditioningeventSince it has been described in other models that CO maydirectly activate NOS (Ryter et al. 2006), it is imperative toverify if ROS generation is necessary for CO to preventneuronal apoptosis or if the CO anti-apoptotic activity isdirectly dependent upon NOS activation. For this purpose,the antioxidant BHT was used to address the role of ROS in(i) the NO generation and (ii) the anti-apoptotic effect of CO.BHT treatment prevented ROS increase induced byCO (Fig. 7a). BHT also prevented NO production inducedby CO treatment (Fig. 7b). Moreover, the anti-apoptoticeffect of CO is partially reversed by BHT (Fig. 7c and d), inparticular in the presence of glutamate at 30 and 40 lM.Thus, these data indicate that CO induces PC and neuropro-tection against apoptosis via ROS generation.

Role of protein synthesis in neuroprotection induced by COTo clarify the necessity of protein synthesis for CO to induceneuronal protection against apoptosis, neurons were pre-treated with CHX for 1 h before CO exposure. Indeed, CHXtreatment abolished apoptosis inhibition induced by CO(Fig. S2a and b). Control cells (without pre-incubation withCO) treated with CHX in the presence of glutamate did notpresent any significant change at the apoptotic hallmark

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Cells cultured in 96-well plates were pre-treated for 60 min with 5 lM

of ODQ, 200 lM of L-NAME, and 500 lM of 5HD (inhibitors of sGC,

NOS, and mitoKATP, respectively), followed by incubation with CO

during 90 min; and ROS were quantified using 2¢,7¢-dichlorofluores-

cein diacetate. All values are mean ± SD, n = 3. #p < 0.05 compared

with control without CO treatment and *p < 0.05 compared with cells

treated only with CO.

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in cGMP formation. Cells grown in six-well plates were pre-treated for

60 min with 5 lM of ODQ, 200 lM of L-NAME, and 500 lM of 5HD

(inhibitors of sGC, NOS, and mitoKATP, respectively), followed by

incubation with CO during 90 min; then cGMP content was quantified.

All values are mean ± SD, n = 3. #p < 0.05 compared with control and

*p < 0.05 compared with cells treated only with CO.

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Fig. 6 Effect of CO treatment and sGC, NOS, and mitoKATP activities

in NO generation. Neuronal cells were pre-treated for 60 min with

5 lM of ODQ, 200 lM of L-NAME, and 500 lM of 5HD (inhibitors of

sGC, NOS, and mitoKATP, respectively), followed by incubation with

CO during 90 min, and nitrite (NO2)) content in the culture supernatant

was quantified as an indirect measure of intracellular NO generation.

All values are mean ± SD, n = 3. #p < 0.05 compared with control

cells and *p < 0.05 compared with cells treated only with CO.

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x� 2008 The Authors

6 | H. L. A. Vieira et al.

parameters (data not shown). Therefore, this result points outthat de novo protein synthesis is involved in CO mode ofaction.

Discussion

Heme-oxygenase activity has been suggested to modulateand prevent cerebral cell death in several models; this

neuroprotective property has been mainly attributed to thebilirubin antioxidant activity (Dore et al. 1999a; Boehningand Snyder 2003). The present work has shown that COdirectly triggers protection of cerebellar granule neuronalcells against apoptosis induced by excitotoxicity and oxida-tive stress (Fig. 1). Moreover, our data also demonstratedthat endogenous CO is critical for the anti-apoptotic role ofHO, as the toxicity produced by HO activity inhibition is

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Fig. 7 Effect of the anti-oxidant BHT on ROS increase, NO production

and neuroprotection conferred by CO. Neuronal cells cultured in

96-well plates were pre-treated for 60 min with 10 lM of BHT, followed

by incubation of CO during 90 min; and (a) ROS were quantified using

2¢,7¢-dichlorofluorescein diacetate and (b) nitrite (NO2)) content in the

culture supernatant was quantified as an indirect measure of intra-

cellular NO generation. #p < 0.05 compared with control and *p < 0.05

compared with CO treated cells. Cells grown in 24-well plates were

pre-incubated with 10 lM of BHT for 60 min before CO treatment

(90 min). Cell death was induced with glutamate for 20 h and apoptotic

hallmarks, such as chromatin condensation (c) and cell viability (d),

were analyzed by fluorescent microscopy. Quantification was per-

formed as in Fig. 1. #p < 0.05 compared with control cells at the same

concentration of glutamate and *p < 0.05 compared with CO treated

cells.

cGMPgeneration

(sGC)

mitoKATPopening

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(NOS)

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Fig. 8 Proposed pathways for CO to prevent neuronal apoptosis. Filled lines represent the pathways proposed by experimental data and dashed

lines represent possible pathways for the opening of mitoKATP.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 10.1111/j.1471-4159.2008.05610.x

Carbon monoxide as neuroprotector | 7

reverted by exogenous CO administration (Fig. 2). Thus, HOprotection against apoptosis is also as a result of COproduction.

It has been described that CO is able to bind to cytochromec oxidase and generate ROS in muscle tissue (Alonso et al.2003), renal isolated mitochondria (Sandouka et al. 2005)and airway smooth muscle cells (Taille et al. 2005); our datashowed that this effect was also present in neuronal cells(Fig. S1). Thus, CO induced protection against cell death andalso formation of ROS, suggesting a PC-like mode of action:an apparent insult (like generation of small amounts of ROS)can promote cell protection. Additionally, it was observedthat other PC key players, such as sGC, NO synthase andmitoKATP also participate in the CO neuroprotective induc-tion (Figs 3–6). As these results were obtained usingchemical inhibitors, further data based on genetic strategiesis needed for confirmation.

Integrating all the data presented here, it is possible topropose a pathway for CO to prevent neuronal apoptosisdescribed in Fig. 8. Exogenous CO gas administrationinduces intracellular ROS generation, activating NOS, whichcan also amplify oxidative stress as NO promotes more ROSproduction (Fig. 4). sGC appeared to be fully controlled byNO, because inhibition of NOS totally prevented cGMPformation (Fig. 5), while cGMP seemed to also modulate theactivity of NOS, as prevention of sGC partially inhibited NOsynthesis (Fig. 6).

Nitric oxide generation occurred early in the pathway, only2 h after CO pre-treatment (Fig. 6), which is in accordancewith the fact that neurons contain constitutive isoform ofneuronal NOS.

Closure of ATP-dependent mitoKATP reversed the protec-tive effect of CO (Fig. 3e and f), thus opening this channelseems to be important for the CO neuroprotective effect.Closure of mitoKATP did not alter ROS, cGMP, or NOgeneration (Figs 4–6), indicating that the opening of thischannel might be a late event of the process. Further data areessential to clarify if opening of mitoKATP is dependent of thesignaling molecules NO and/or cGMP, or if it is a processthat can be held in parallel.

Although CO has been described as a signaling moleculeacting directly on NOS and sGC in several cellular models(Brune and Ullrich 1987; Stone and Marletta 1994; Ryteret al. 2006), in neuronal cells CO seemed to act via ROSgeneration as prevention of ROS increase totally inhibitedNO generation (Fig. 7b) and partially prevented CO neuro-protection (Fig. 7c and d). Additionally, the recent literaturesupports more and more that CO signals via ROS (Bilbanet al. 2008).

Finally, de novo protein synthesis is imperative for CO toconfer neuroprotection, as pre-treatment with CHX (a proteinsynthesis inhibitor) reversed apoptosis prevention induced byCO (Fig. S2), indicating that a late PC event is involved inthis anti-apoptotic process.

As apoptosis occurs during hours or even days after theHIR insult, originating a region called penumbra, anti-apoptotic therapies can reduce brain damage caused byHIR; CO appears to be a strong candidate for an anti-apoptotic therapy. Compared with other pharmacologicaldrugs, CO presents the main advantage of being anendogenous molecule to which the organism is fullyadapted. Additionally, the direct administration of CO as atherapeutic molecule is preferable to therapies based onactivation of HO, as HO hyperactivity can contribute topathological iron deposition into mitochondria, pro-oxidantstate and bioenergetic failure, which amplifies agingand degenerates human neural tissues (Schipper 2004).However, because of the high affinity of CO to hemoglo-bin, therapies based on CO inhalation are not applicablefor obvious oxygen transport inhibition. Thus, moleculesable to deliver CO – CO-releasing molecules (Motterliniet al. 2005) – exerting their therapeutic action belowthe toxicity levels of CO and specific to the target tissues,are now being explored in the present model of neuronalcell.

In summary, CO induces PC events in primary culture ofneurons, providing protection against apoptosis.

Acknowledgements

This workwas supported by the Portuguese Fundacao para a Ciencia e

Tecnologia (PTDC/SAU-NEU/64327/2006) and the Portuguese

Fundacao para a Ciencia e Tecnologia, for HLAV’s SFRH/BPD/

27125/2006 fellowship. The authors express their gratitude to Dr

Werner Haas and Dr Jan Andersson from Alfama, Portugal, for the

critical reading of the manuscript, as well as for fruitful discussions;

and to Dr Miguel Soares from Instituto Gulbenkian de Ciencia,

Portugal, for providing the CO gas chamber.

Supporting information

Additional supporting information may be found in the online

version of this article.

Fig. S1 Effect of CO in the intracellular ROS generation.

Fig. S2 Role of de novo protein synthesis in the CO neuropro-

tective mechanisms.

Appendix S1 Supplementary Materials and methods.

Please note: Wiley-Blackwell are not responsible for the content

or functionality of any supporting materials supplied by the authors.

Any queries (other than missing material) should be directed to the

corresponding author for the article.

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