Research Article 1043

IntroductionGlutathione (GSH) is the most abundant low molecular weightthiol in mammalian cells and acts as the major cellular non-enzymatic antioxidant. This tripeptide is synthesized from its aminoacid precursors in two steps: the enzyme g-glutamylcysteinesynthetase (g-GCS) catalyzes the synthesis of g-glutamyl-cysteinefrom glutamate and cysteine; glutathione synthetase (GS) thencatalyzes the formation of GSH from glycine and g-glutamyl-cysteine. It is present as a reduced form and two oxidized species:glutathione disulfide and glutathione mixed disulfide with proteinthiols. Beside its function as an intracellular redox buffer, GSHparticipates as a cofactor of glutathione peroxidase in thedetoxification of lipid and organic peroxides (Liddell et al., 2006).It also possesses several other roles related to its ability to modulatethe thiol status of cysteine on several proteins, including receptors,molecules involved in signaling and nuclear transcription factors(Filomeni et al., 2002).

In the past few years, many studies have shown that GSHcontent decreases with age in many tissues from different animalspecies, but the underlying mechanism is not still clear (Liu et al.,2004). It has been proposed that GSH decrease could be aconsequence of the formation of protein mixed disulfides. Thisevent can broadly interfere with the catalytic efficiency ofenzymes, which might be reflected in their reduced ability tomount adaptive responses under stressful conditions (Cotgreaveand Gerdes, 1998; Droge, 2002; Rebrin et al., 2003; Rebrin et al.,2005). Moreover, GSH reduction is reported in patients and animal

models of various neurodegenerative disorders. In particular, GSHlevels were found to be lower in the substantia nigra of Parkinson’sdisease patients (Sian et al., 1994; Solano et al., 2008) andpyramidal neurons of Alzheimer’s disease patients (Ballatori etal., 2009; Gu et al., 1998). The most convincing theory is that, inneurodegenerative diseases, the activity of both g-GCS and GSprogressively decreases through downregulation of geneexpression (Ballatori et al., 2009; Sastre et al., 2005; Sethna et al.,1982). Additional roles for the antioxidant function of GSH are inthe context of nitric oxide (NO) physiology and pathology (Hogg,2002; Zhang and Hogg, 2005). NO is a gaseous free radicalendogenously produced from L-arginine by the catalytic activityof NO synthases (NOSs). It has essential roles in diverse biologicalprocesses because of its facile but selective chemical reactivitytowards a number of cellular targets. NO also undergoes reactionswith oxygen, superoxide anion (O2•–) and reducing agents to giveproducts that themselves show selective chemical reactivitytowards a variety of cellular targets, sometimes with amanifestation of toxic effects, such as nitrosative stress (Hughes,2008). Such products of NO reactions include nitroxyl (HNO),oxides (NO2, N2O4 and N2O3), peroxynitrite (ONOO–) and S-nitrosothiols (RSNO). Direct reaction between NO and biologicalthiols can occur in anaerobic conditions, but is a very slowoxidation reaction that yields thiol disulfide and nitroxylanion. Toform S-nitrosoglutathione (GSNO) and protein RSNO, thepresence of oxygen is mandatory. In fact, NO is oxidized to N2O3,which is a better nitrosating agent (Hogg, 2002; Zhang and Hogg,

SummaryGlutathione (GSH) levels progressively decline during aging and in neurodegenerative disorders. However, the contribution of suchevent in mediating neuronal cell death is still uncertain. In this report, we show that, in neuroblastoma cells as well as in primarymouse cortical neurons, GSH decrease, induced by buthionine sulfoximine (BSO), causes protein nitration, S-nitrosylation and DNAstrand breaks. Such alterations are also associated with inhibition of cytochrome c oxidase activity and microtubule networkdisassembly, which are considered hallmarks of nitric oxide (NO) toxicity. In neuroblastoma cells, BSO treatment also induces cellproliferation arrest through the ERK1/2-p53 pathway that finally results in caspase-independent apoptosis, as evident from thetranslocation of apoptosis- inducing factor from mitochondria towards nuclei. A deeper analysis of the signaling processes indicatesthat the NO-cGMP pathway is involved in cell proliferation arrest and death. In fact, these events are completely reversed by L-NAME,a specific NO synthase inhibitor, indicating that NO, rather than the depletion of GSH per se, is the primary mediator of cell damage.In addition, the guanylate cyclase (GC) inhibitor LY83583 is able to completely block activation of ERK1/2 and counteract BSOtoxicity. In cortical neurons, NMDA (N-methyl-D-aspartic acid) treatment results in GSH decrease and BSO-mediated NO cytotoxicityis enhanced by either epidermal growth factor (EGF) or NMDA. These findings support the idea that GSH might represent the mostimportant buffer of NO toxicity in neuronal cells, and indicate that the disruption of cellular redox buffering controlled by GSH makesneuronal cells susceptible to endogenous physiological flux of NO.

Key words: BSO, L-NAME, ERK1/2, AIF, EGF, NO-cGMP

Accepted 10 November 2010Journal of Cell Science 124, 1043-1054 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.077149

Nitric oxide is the primary mediator of cytotoxicityinduced by GSH depletion in neuronal cellsKatia Aquilano1,*, Sara Baldelli2,*, Simone Cardaci1, Giuseppe Rotilio1,2 and Maria Rosa Ciriolo1,2,‡1Department of Biology, University of Rome Tor Vergata, 00133 Rome, Italy2IRCCS San Raffaele ‘La Pisana’, Via dei Bonacolsi, 00165 Rome, Italy*These authors contributed equally to this work‡Author for correspondence (ciriolo@bio.uniroma2.it)

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2005). As GSH is present intracellularly at millimolarconcentrations, the formation of GSNO is plausible. GSNOgenerally has a longer lifetime than NO and protein RSNO, andis considered a more persistent or buffered pool of NO that canbe released when or where it is required for signaling (Stamler etal., 1992). Therefore, GSH might contribute to overwhelmnitrosative stress. In fact, we previously demonstrated that, uponendogenous NO overproduction, a prompt increase in intracellularGSH levels was induced and was pivotal to prevent cell death(Baldelli et al., 2008). Moreover, upon treatment with physiologicalamounts of NO donors, an increase in GSH levels has been alsoreported (Moellering et al., 1999; Moellering et al., 1998).

On the basis of this knowledge, we investigated the possiblerole of GSH in modulating the level and reactivity of endogenousNO, with the aim of identifying the molecular mechanismsunderlying aging and neurodegenerative disorders. Here, we reportthat, upon GSH depletion, endogenous NO is the primary factoraffecting cell proliferation and viability through the NO-cGMPpathway. We also found that, upon inhibition of GSH synthesis,NO-mediated DNA damage and protein oxidation by NO, in termsof tyrosine nitration and S-nitrosylation, are operative in bothneuroblastoma cells and primary cortical neurons (PCNs). Thissuggests that GSH could be an essential buffer of NO underphysiological conditions and that its imbalance could bedetrimentally linked to harmful effects from NO.

ResultsEffects of GSH depletion on proliferation of SH-SY5Yneuroblastoma cellsGSH decrease is considered to be among the causative factors ofaging and neurodegenerative disorders. Therefore, we investigatedthe effects of GSH depletion by treatment with 1 mM BSO, aspecific inhibitor of GSH synthesis. Intracellular GSH content wasanalyzed in human neuroblastoma SH-SY5Y cells by high-performance liquid chromatography (HPLC). Fig. 1A showssignificant GSH depletion just 3 hours after BSO treatment andalmost undetectable levels after 24 hours. The effect of BSO oncell growth and viability was analyzed by direct count by TrypanBlue staining. We found that, after 24 hours, BSO profoundlyaffected cell number (Fig. 1B) without any increase in dead cells(Fig. 1C), indicating that proliferation arrest was occurring. Thiswas confirmed by anti-BrdU immunohistochemistry, whichdemonstrated a dramatic decrease in BrdU incorporation in BSO-treated cells after 24 hours, indicative of cell-cycle arrest in G1phase (Fig. 1D).

Cell growth arrest upon GSH depletion is induced by theNO-mediated ERK1/2-p53 pathwayIn a previous work, we demonstrated that an increase in GSH levelcould be induced by neuronal NOS (nNOS) overexpression andthat such an increase was efficient in buffering NO toxicity (Baldelliet al., 2008). We therefore attempted to evaluate whether NOimbalance can induce cell-cycle arrest. Indeed, we analyzed NOSactivity by measuring nitrites and nitrates (NOx) in the culturemedium. Fig. 2A shows that BSO treatment leads to significantaccumulation of NOx after 24 hours without any significant increasein nNOS protein (Fig. 2B). To evaluate whether other NOS isoformswere responsible for the NOx increase, we measured endothelialand inducible NOS by western blot, but were not able to finddetectable levels of these enzymes according to SH-SY5Y neuralorigin (data not shown).

These data led us to speculate that NOx accumulates followingNO imbalance because of lack of its efficient removal by GSH andthat an increase in NO could be responsible for cell proliferationarrest. We therefore treated the cells with BSO in the presence ofthe nNOS inhibitor L-NAME (100 M). Fig. 2C,D shows that,after 24 hours, inhibition of nNOS activity was sufficient tocounteract growth arrest elicited by GSH depletion, as assessedthrough cell count by Trypan Blue staining and cell proliferationanalysis by BrdU incorporation. The same results were obtainedusing other inhibitors of the NO pathway, such as the NO scavengercarboxy-PTIO and the nNOS inhibitor 7-nitroindazole (7-Ni) (datanot shown).

Several transcription factors and upstream signaling pathwaysregulating cell proliferation are directly modulated by NO and/orits downstream effector cGMP (Hofseth et al., 2003; Komatsu etal., 2009; Yun et al., 1999). Among these, we assayed for possiblemodulation of p53 using western blot analysis. We found that p53accumulates from 3 hours after BSO administration to 9 hoursafter, finally returning to the basal level after 24 hours (Fig. 2E).Next, we evaluated mitogen-activated protein kinases (MAPKs);significant activation of ERK1/2 by phosphorylation was found,with a time course mirroring that of p53 (Fig. 2E). Phospho-activeforms of p38 and JNK were not detectable in either control cellsor BSO-treated cells up to 48 hours (data not shown). To verifywhether ERK1/2 and p53 modulation depends on NO imbalance,we carried out BSO treatment in the presence of L-NAME. Asreported in Fig. 2E, L-NAME efficiently impedes p53 andphosphorylated ERK1/2 (ERK1/2-P) accumulation, suggesting thatNO is the primary mediator of such effects. cGMP is consideredthe main downstream effector of the signaling pathway governedby NO and has a role in control of the cell cycle and proliferation.This messenger might be involved in phosphorylation processesmediated by MAPKs, including ERK1/2 (Thomas et al., 2008). Wetherefore treated SH-SY5Y cells with BSO in the presence ofLY83583 (LY), an inhibitor of guanylate cyclase (GC), the enzyme

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Fig. 1. GSH depletion causes cell proliferation arrest in SH-SY5Y cells.Cells were treated with BSO (1 mM) for 24 hours. (A)Intracellular GSHcontent was measured by HPLC. Data are expressed as means ± s.d. (n4;*P

that catalyzes the formation of cGMP. Fig. 2F,G shows that LYsignificantly abrogates the effect of BSO on cell proliferation, asassessed by cell count and BrdU assay. LY was able to inhibit p53and ERK1/2-P accumulation (Fig. 2H) similarly to L-NAME,indicating that the NO-cGMP-ERK1/2-p53 signaling axis isinvolved in BSO-mediated effects. Next, we asked whether cGMP-dependent protein kinase (PKG) contributes to cell proliferation

arrest. We treated SH-SY5Y cells with BSO in the presence ofPKG inhibitor (KT5823). KT5823 (KT) was not able to hampercell proliferation arrest at 24 hours, as assessed by cell count(supplementary material Fig. S1A) and BrdU assay (data notshown). In line with this result, the accumulation of p53 andERK1/2-P was still observed in the presence of KT (supplementarymaterial Fig. S1B), demonstrating that NO and cGMP directlyconverge on the ERK1/2-p53 pathway.

To more thoroughly investigate the role played by ERK1/2 andp53 in proliferation arrest, we assessed the effect of U0126, asynthetic ERK1/2 inhibitor. U0126 (260 nM) per se caused asignificant reduction in ERK1/2 phosphorylation, indicating thatthe inhibitor worked effectively (Fig. 3A). Suppression of ERK1/2activation led to significant reduction of BSO-induced p53accumulation, suggesting that ERK1/2 is an upstream modulatorof p53. Cell count showed that U0126 also completely abolishedthe anti-proliferative effect mediated by BSO after 24 hours,suggesting a crucial requirement for the ERK1/2 pathway inpromoting NO-cGMP-mediated growth arrest (Fig. 3B).

1045GSH depletion induces NO cytotoxicity

Fig. 2. Growth arrest is mediated by NO and cGMP signaling, and isassociated with accumulation of ERK1/2-P and p53 in SH-SY5Y cells.(A)Nitrites plus nitrate (NOx) in the culture medium were determined byGriess reaction 24 hours after BSO treatment. Data are reported as micromolesper milligram of protein and expressed as means ± s.d. (n4; *P

BSO-mediated cell growth arrest results in cell death andis associated with widespread nitration stressWe subsequently investigated whether NO-cGMP-mediatedgrowth arrest at 24 hours could finally result in cell death. Wefound that BSO induced a significant cytotoxic effect after 48hours, as evident from the increase in the percentage of deadcells (Fig. 4A). Therefore, we investigated the possible occurrenceof nitration stress upon BSO treatment by analyzing 3-nitrotyrosine (NO2-Tyr), a marker of protein nitration stress,using immunofluorescence microscopy (Abello et al., 2009;Ferrer-Sueta and Radi, 2009; Goldstein and Merenyi, 2008). As

shown in Fig. 4B, BSO induces a time-dependent increase inNO2-Tyr after 9 hours, reaching the maximum level at 48 hours,that was efficiently counteracted by L-NAME. The same resultswere obtained by dot-blot analysis of NO2-Tyr (Fig. 4C). Inaddition, we looked at the level of Ser139-phosphorylated histoneH2A.X (H2A.X-P), a hallmark of stress-induced DNA double-strand breaks (d’Adda di Fagagna et al., 2003). This analysisrevealed that nuclei of BSO-treated cells displayed a parallelincrease in H2A.X-P fluorescence, which was efficiently reversedby L-NAME, indicating that DNA damage was operative andcaused by NO imbalance (Fig. 4B).

Cytochrome c oxidase (CcOX) is considered a preferential targetof NO because of the presence of heme groups for which NO hasgreat binding affinity (Antunes and Cadenas, 2007). Therefore, wemeasured the activity of CcOX after BSO treatment and found thatit was maximally reduced after 48 hours (–60%). Inhibition of NOsynthesis by L-NAME completely reversed the impairment ofCcOX activity (Fig. 4D). As shown in Fig. 4E, the protein level ofCcOX was not altered by BSO, neither was that of othermitochondrial proteins such as cytochrome c and Hsp60, indicatingthat the decrease in its activity is a consequence of NO imbalance.

Other important targets of NO toxicity are microtubules, becausetubulin is highly susceptible to nitration on tyrosine residues(Dremina et al., 2005; Eiserich et al., 1999; Fiore et al., 2006). Weinvestigated by fluorescent microscopy the possible effect of BSOtreatment on microtubule lattices. Fig. 4E shows that, 48 hoursafter BSO treatment, cells display marked changes in themorphology of the microtubule network, and acquire a roundedshape with a reduced increase in the number of neurites. Treatmentwith L-NAME completely prevented microtubule impairment,suggesting that this phenomenon is NO dependent.

Prolonged BSO treatment induces apoptosis through NO-dependent nuclear translocation of AIFWe have demonstrated that ERK1/2 inhibition by U0126 or LY isable to prevent cell growth arrest after 24 hours. We then evaluatedthe effect of such inhibition on SH-SY5Y cells treated with BSOat later time points. This analysis revealed that U0126 was stillable to counteract cell proliferation arrest and death after 48 hours(Fig. 5A); however, NO cytotoxicity was not abolished. In fact,protein nitration and DNA damage were even more pronounced,as assessed by the analysis of NO2-Tyr and H2A.X-P levels (Fig.5B). This condition gives rise to cell death later, at 72 hours, whenthe percentage of dead cells upon BSO and U0126 treatment wascomparable to that observed at 48 hours upon treatment with BSOalone (Fig. 5A). The same results were obtained with LY (data notshown).

To determine the mechanisms by which SH-SY5Y cells dieupon BSO treatment, we examined for apoptosis induction. Wecarried out cytofluorimetric analysis of cell nuclei stained withpropidium iodide after 48 hours. We found that a comparablepercentage of cells in sub-G1 phase (apoptotic cells) were observedin control and BSO-treated cells (Fig. 5C). Also, the level of theprecursors pro-caspase-8 and -9 was not altered at either 24 or 48hours after BSO treatment, thus excluding their activation (Fig.5D). To further validate the absence of caspase-dependent apoptosis,we carried out western blot analysis of poly (ADP-ribose)polymerase (PARP), a well-accepted target of caspase-3, theexecutor caspase downstream of caspase-8 and -9. As shown inFig. 5D, the PARP-cleaved band does not accumulate aftertreatment with BSO.

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Fig. 4. Prolonged GSH depletion induces NO2-Tyr accumulation, H2A.Xphosphorylation, decreased CcOX activity and cell death in SH-SY5Ycells. (A)The percentage of dead cells was determined by Trypan Blueexclusion 48 hours after BSO addition. Data are expressed as means ± s.d.(n6; *P

It has been demonstrated that NO can induce cell death by acaspase-independent pathway (Langer et al., 2008; Uchiyama etal., 2002). We examined whether BSO could activate such apathway, focusing on the mitochondrial protein apoptosis-inducing factor (AIF). AIF is known to translocate into thenucleus, after an apoptotic stimulus, where it participates inconsiderable DNA cleavage (Cande et al., 2002; Lorenzo andSusin, 2007). We performed immunofluorescence analysis and,as expected, found that AIF localized in the mitochondria ofcontrol cells, as evident from cytochrome c counterstaining (Fig.6A). By contrast, in BSO-treated cells, AIF moved into the nuclei,particularly at 48 hours, as demonstrated by the superimpositionof the AIF signal with that of nuclei and the loss of AIFmitochondrial localization. Treatment with BSO in the presenceof L-NAME completely prevented mitochondrial export of AIFtowards the nucleus, strongly indicating that NO toxicity is thesole inducer of caspase-independent apoptosis. In fact, cellcounting by Trypan Blue staining showed that L-NAME and 7-Ni significantly reduced the cytotoxic effect of BSO at 48 hours(Fig. 6B). The involvement of cGMP in BSO toxicity wasinvestigated by analyzing the effect of treatment with BSO incombination with the cGMP inhibitor LY. As reported in Fig. 6B,LY was able to completely counteract BSO-induced cell death at48 hours, further implicating cGMP and NO in BSO toxicity. Bycontrast, the PKG inhibitor KT was not able to prevent BSO-induced cell death (Fig. 6B).

SOD1 overexpression counteracts cell growth arrest andapoptosis induced by BSOTo thoroughly dissect the molecular mechanisms involved in BSOtoxicity, we carried out experiments with SH-SY5Y cells transfectedwith a vector containing a cDNA encoding the human antioxidantenzyme copper-zinc superoxide dismutase (hSOD1 cells). Thesecells are resistant to NO-induced apoptosis (Ciriolo et al., 2000).Cells transfected with the empty vector (mock cells) were used ascontrol. SOD1 can intercept O2•– and inhibit its reaction with NOto form ONOO–, thus limiting cellular stress. In hSOD1 and mockcells, GSH decrease upon treatment with 1 mM BSO followed thesame trend as that observed for untransfected SH-SY5Y cells (datanot shown). We then measured NO2-Tyr levels in the presence ofBSO and/or L-NAME by immunofluorescence analysis. As shownin Fig. 7A, mock cells treated with BSO display an increase inNO2-Tyr with respect to untreated cells that is efficiently

1047GSH depletion induces NO cytotoxicity

Fig. 5. ERK1/2-mediated cell death is not associated with caspase-dependent apoptosis in SH-SY5Y cells. (A)U0126 (260 nM) was added inculture medium 1 hour before BSO treatment and maintained throughout theexperiment. Cells were counted by Trypan Blue exclusion at 48 and 72 hours.Data are expressed as means ± s.d. (n6; #P

counteracted by treatment with L-NAME. By contrast, hSOD1cells, which have increased levels of SOD1 (Fig. 7A), were notaffected by BSO treatment; indeed, no formation of NO2-Tyradducts was evident (Fig. 7A). Furthermore, we analyzed cellproliferation and death of mock and hSOD1 cells after BSOaddition. Fig. 7B,C shows that, in mock cells, BSO inducessignificant growth arrest at 24 hours and death at 48 hours; botheffects are counteracted by L-NAME. On the other hand, BSO did

not induce any detrimental effects on cell proliferation and viabilityof hSOD1 cells (Fig. 7B,C), implying a functional role for ONOO–

in the induction of both cell growth arrest and death.

BSO-mediated cell growth arrest and death depend on thepresence of nNOSTo further evaluate whether GSH depletion is detrimental only inthe presence of NO, we tested other two cell lines: mouse neuronalNSC34 cells expressing high levels of nNOS (Baldelli et al., 2008)and human cervical cancer HeLa cells, which do not express anyNOS isoforms (Bulotta et al., 2001; Nisoli et al., 2003). These cellswere treated with BSO in the presence or absence of L-NAME, andviable and dead cells were counted after 48 hours. Fig. 8A showsthat NSC34 cells are susceptible to BSO as well as SH-SY5Y, andthat L-NAME is able to inhibit both cell death and NO2-Tyraccumulation (Fig. 8B). On the contrary, no toxicity was evident inHeLa up to 48 hours (Fig. 8C), time when significant inhibition ofGSH synthesis (–80%) was achieved (data not shown), indicatingthat GSH depletion alone is not sufficient to cause cytotoxicity.

BSO induces intracellular GSH depletion and NO-mediated cell death in primary mouse cortical neuronsTo assess the validity of our results, we examined the effects ofGSH depletion on an ex vivo experimental model represented bymouse PCNs. In these cells, a different concentration of BSO wasused (25 M), which has previously been reported to be efficientin decreasing GSH levels in mouse PCNs (White and Cappai,2003). Fig. 9A shows that, under our experimental conditions,BSO induces a significant decrease in GSH, starting after 24 hours.

To examine the effect of GSH depletion on viability, PCNs weretreated with BSO in the presence or absence of 100 M L-NAME,and morphological changes were evaluated by optical microscopy.The analysis of PCNs exposed to BSO for 48 hours identifiedwidespread death, and neuritic membrane beading and blebbingconsistent with apoptosis (Fig. 9B). Pre-treatment with L-NAMEprevents these changes, suggesting that NO-mediated toxicity isalso found in PCNs. Moreover, staining with Hoechst 33342(Hoechst) was used to visualize the morphological changes ofnuclei and to estimate cell viability and/or apoptosis commitment.The quantitative data obtained from nuclei counts are reported inFig. 9C. In particular, whereas control cells exhibit uniformlydispersed chromatin and intact nuclei, BSO-treated cells display a

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Fig. 8. Inhibition of NO production completely reverses cell death inducedby BSO in cells of neuronal origin. L-NAME (100M) was added in culturemedium 1 hour before BSO treatment (48 hours) and maintained throughoutthe experiment. (A)NSC34 cells were counted by Trypan Blue exclusion. Dataare expressed as means ± s.d. (n6; *P

significant increase in nuclei with condensed chromatin and theappearance of apoptotic bodies (70%). The presence of L-NAMEmarkedly decreased the number of PCNs with nuclearmorphological changes, as the percentage of condensed-fragmentednuclei returned to control values. The same protective effects wereobtained by treating PCN with BSO in combination with LY (Fig.9C). On the contrary, the use of KT did not prevent death,demonstrating that, in PCNs, BSO toxicity is mediated by the NOand cGMP pathway without the involvement of PKG. Fig. 10Ashows representative images of nuclei stained with Hoechst after48 hours of BSO treatment. Data reported in supplementary materialFig. S2 show that BSO is able to significantly induce PCN deathafter 24 hours, which was efficiently prevented only by L-NAMEand LY co-treatment.

Finally, we measured the possible occurrence of BSO-mediatednitration stress by examining the NO2-Tyr levels throughimmunofluorescence analysis. As shown in Fig. 10A,B, BSOinduces accumulation of NO2-Tyr levels at 48 hours that isefficiently counteracted by L-NAME. The same results wereobtained by dot-blot analysis of NO2-Tyr levels (Fig. 10B).

Nitration of proteins is considered a less reversible process withrespect to S-nitrosylation (Abello et al., 2009). The former isusually associated with cell death, the latter with signal transductionpathways leading to either cell death or survival (Brune, 2003).Therefore, we investigated the possible occurrence of S-nitrosylation upon BSO treatment by fluorescence microscopyusing a specific S-nitrosocysteine (S-NO) antibody. Fig. 10A showsthat BSO significantly upregulates the levels of S-NO proteins andthat such a phenomenon was abolished by L-NAME. To verifywhether the S-nitrosylation process was selective to some proteinsor nonspecific, we carried out the biotin-switch method on PCNstreated with BSO. Then, we identified S-NO proteins by westernblot analysis of the biotin adducts by incubating cell membranewith HRP-conjugated streptavidin. Fig. 10C shows that a widearray of proteins are S-nitrosylated, indicating that S-nitrosylationis not a triggering event for BSO toxicity.

Growth factors and stimulation of N-methyl-D-aspartic acidreceptor (NMDA-R) are known to activate the synthesis of NO bynNOS in neuronal cells (Clementi et al., 1995). Whereas growthfactors are protective, the excessive stimulation of NMDA-R resultsin cytotoxicity (Gu et al., 2010). We therefore investigated theeffect of NO imbalance mediated by GSH depletion in the presenceof such nNOS activators. In particular, PCNs were treated for 24hours with BSO in the presence of 20 ng/ml epidermal growthfactor (EGF) or 20 M NMDA. The protective effect of EGF andthe harmful effect of NMDA in BSO-untreated cells wereconfirmed. In fact, PCNs treated with EGF or NMDA displayedlower or higher levels of condensed-fragmented nuclei thancontrols, respectively (Fig. 11A). The co-treatment EGF-BSOsignificantly increased cell death with respect to BSO alone (50%versus 30%) (Fig. 11A), indicating that NO increase mediated byEGF (supplementary material Fig. S3A) acts in synergy with thatderiving from BSO. This phenomenon was more evident uponNMDA-BSO co-treatment (BSO 30%; NMDA-BSO 60%),according to the higher rate of NO production upon NMDA-Rstimulation. The same enhanced effect was obtained upon EGF-NMDA co-treatment (NMDA 33%; EGF-NMDA 60%) (Fig. 11A).

The buffering role of GSH against NO was confirmed in PCNsby analyzing the intracellular content of GSH after NMDAtreatment. Fig. 11B shows that GSH is significantly decreased assoon as 15 minutes after NMDA addition. This time correspondsto the maximum increase in NO production, as measured by thevital NO fluorescent probe DAF-2DA (Fig. 11C).

Finally, we determined the levels of NO2-Tyr and S-NO proteinsupon EGF or NMDA treatment. Fig. 11D shows that EGF alonedoes not result in any nitration or nitrosative stress, according toits protective role. However, it exerted an enhancing effect oneliciting NO-mediated protein damage upon co-treatment witheither BSO or NMDA (Fig. 11D). In agreement with its cytotoxicaction, NMDA was able to mediate the accumulation of both S-NO and NO2-Tyr proteins, which was further enhanced by co-treatment with BSO or EGF (Fig. 11D). Addition of L-NAMEcompletely abolished the nitration and S-nitrosylation events in allthe conditions tested (data not shown).

1049GSH depletion induces NO cytotoxicity

Fig. 9. GSH depletion in PCNs induces NO- and cGMP-mediated celldeath. (A)Cells were treated with 25M BSO for the indicated times.Intracellular GSH content was measured by HPLC. Data are expressed asmeans ± s.d. (n4; *P

DiscussionMany neurodegenerative diseases and the physiological agingprocess share oxidative and nitrosative stress as common terminalprocesses. In association with these phenomena, progressivealteration of the GSH pool is also observed (Ballatori et al., 2009),but to date the synergistic effect of such events in favoring agingand its related disorders has not been clarified. Previously, wedemonstrated that chemical inhibition of GSH synthesis results inan increase in free radicals and that either SOD1 or nNOSmodulation are able to affect GSH homeostasis. We suggested thatpossible cross-talk between the two enzymes and GSH wasoperative, especially in assuring neural cell integrity (Aquilano etal., 2006; Baldelli et al., 2008). The data here obtained add anotherpiece to the puzzle of the role of GSH as a direct antioxidant. Inparticular, it has emerged that GSH, besides providing protectionfrom O2•–, counteracts the potential toxicity of physiological levelsof NO. The importance of GSH in counteracting NO-mediatedcytotoxicity has been largely proved in a variety of cell types. Inparticular, upon treatment with NO donors, an increase in GSHand augmented cellular susceptibility to NO challenge, afterchemical depletion of GSH, have been reported (Moellering et al.,1999; Moellering et al., 1998). In a previous work, we found thatGSH was fundamental to preventing the harmful effects of anincrease in NO production after nNOS overexpression (Baldelli etal., 2008). The most intriguing finding of the present work is that,when the GSH pool is altered, NO derived from basal nNOS

activity can be equally detrimental both in neuroblastoma cells andin PCNs. Interestingly, in PCNs, we observed that NO produceddownstream of NMDA-R stimulation promptly causes a decreasein the GSH pool, thus reinforcing the idea of a buffering role forGSH against NO. In fact, NO2-Tyr accumulation is significantlyinduced in neuronal cells and prevented by using an NO scavengeror inhibiting NO synthesis. Also, the accumulation of H2A.X-P,which reflects the occurrence of DNA strand breaks, is totallyprevented upon NO inhibition. The effectiveness of SOD1overexpression in avoiding BSO-mediated nitration damage toproteins finally highlights the synergistic activity of O2•– and NOin triggering oxidative stress. This suggests that ONOO– could bethe primary cause of cell damage upon GSH depletion, althoughother pathways of protein nitration could be operative, such asthose governed by its derivatives or by NO2• produced by variousheme-peroxidases or by the interaction of NO with tyrosyl radicals(Abello et al., 2009).

Being a heme-containing enzyme, CcOX is an important sensorof NO, strongly inhibiting its activity. In 1994, four independentlaboratories reported that NO, either exogenously added to severalpreparations containing mitochondria or endogenously producedin intact cells, inhibited CcOX. However, in the three laboratoriesthat used exogenous NO, the effect on CcOX was found to bereversible (Bolanos et al., 1994; Brown and Cooper, 1994; Cleeteret al., 1994; Schweizer and Richter, 1994) and in competition withO2, whereas in the other study, which relied upon endogenous or

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Fig. 10. GSH depletion in PCNs induces proteinnitration and S-nitrosylation. L-NAME (100M)was added in culture medium 1 hour before BSOtreatment and maintained throughout the experiment.(A)After 48 hours BSO treatment, cells wereincubated for fluorescence microscopy analysis withanti-NO2-Tyr (green), anti-S-NO (red) and Hoechstto stain nuclei (blue). (B)Cells were treated withBSO for 48 hours and proteins (20g) were spottedon nitrocellulose membrane and subjected to dot-blotanalysis using a polyclonal NO2-Tyr antibody.Density of immunoreactive dots was normalized forPonceau Red and reported as arbitrary units (a.u.).Data are expressed as means ± s.d. (n3; *P

sustained NO formation, inhibition of CcOX activity was found tobe progressive and persistent. The molecular mechanismresponsible for the irreversible inhibition of CcOX by ONOO– wasfurther investigated and revealed by Sharpe and Cooper, and Pearceet al. (Pearce et al., 1999; Sharpe and Cooper, 1998). Regardlessof the specific mechanism leading to irreversible damage of CcOXby ONOO–, it is now clear that this interaction might have as yetunconsidered patho-physiological implications (Cooper et al.,2003).

This work also revealed that NO imbalance and the consequentactivation of the NO and cGMP pathway, rather than alteration ofthe GSH redox system, are the main cause of cellular homeostasis

impairment. The involvement of NO is also supported by the factthat other neural cell lines expressing nNOS, such as NSC34(Aquilano et al., 2007), but not NOSs-lacking cells, such as HeLa(Bulotta et al., 2001; Nisoli et al., 2003), are susceptible to BSOtoxicity. Surprisingly, PKG, which is known to be the main proteinactivated by NO and cGMP, does not seem to be involved in ourexperimental systems, as the use of a PKG inhibitor does notchange the cytotoxic effect of BSO. In the absence of GSH,prevention of proliferation arrest and neuronal death is completelyachieved by inhibiting NOS and GC, implying that alternativepathway(s) can be activated. In this context, we found thatimpairment of cell proliferation is due to the NO- and cGMP-mediated activation of the ERK1/2-p53 pathway. Actually, the useof the MEK1/2 inhibitor U0126 and the GC inhibitor LY clearlydemonstrated that the transient activation of ERK1/2 is fundamentalto inducing p53 and arresting cell proliferation. In fact, by impedingERK1/2 phospho-activation, both p53 accumulation and growtharrest are inhibited. However, both ERK1/2 and p53 rapidly returnto basal levels, excluding their contribution to the induction ofBSO-mediated apoptosis. This assumption is also confirmed bythe fact that cell death, even if at later time points, is equallyobtained upon BSO treatment in the presence of ERK1/2 or GCinhibitors, probably due to the irreversible and additionalaccumulation of nitration damage.

According to previous studies, NO is able to inhibit at nanomolarconcentration the activity of cysteine protease caspases through S-nitrosylation (Kim et al., 1997). However, apoptosis can proceedin the absence of caspase activation by a caspase-independentpathway (Tait and Green, 2008). A master regulator of such apathway is AIF, which, after alteration of mitochondrialhomeostasis, migrates into the nucleus, where it participates withendonuclease G in nuclear fragmentation (Lorenzo and Susin,2007). It has been established that NO is able to triggermitochondrial alteration and promote AIF release frommitochondria (Vieira and Kroemer, 2003). Our data are consistentwith NO-dependent induction of AIF nuclear translocation, asinhibition of NO production upon BSO treatment is able tocompletely prevent this phenomenon. Inhibitor of GC and L-NAME are effective in inhibiting cell death; therefore, it is likelythat cGMP plays a crucial function in transducing the signal to theapoptotic machinery. To the best of our knowledge, cross-talkbetween cGMP signaling and AIF has scarcely been investigatedand deserves further research. The strength of this present study isthat PCNs show the same detrimental effects upon GSH depletion.Indeed, the increase in NO-mediated damage and cell death wasefficiently prevented by inhibiting NOS. In particular, both nitrationand nitrosylation stress on proteins is strongly induced after GSHdepletion, with S-nitrosylation probably representing a side-effectof NO imbalance and not being directly involved in signaltransduction leading to cell death. This hypothesis is supported bythe widespread increase in S-nitrosylated proteins upon BSOchallenge. From the results obtained, it can be assumed that GSHdeficiency could have a comparable detrimental effect with respectto the excitotoxic activity of NMDA-R stimulation. Likewise,although EGF is implicated in cell protection, it can enhance thetoxic activity of NO upon BSO and NMDA treatment.

In PCNs, as well as the condensation of nuclear chromatin,typical nuclear apoptotic bodies are formed; this deserves furtherstudy to better characterize the apoptotic process (caspase dependentor caspase independent). It is now attractive to verify whether theobserved NO imbalance can also be effective in mice in which

1051GSH depletion induces NO cytotoxicity

Fig. 11. NO toxicity induced by GSH depletion is enhanced upontreatment with EGF or NMDA in PCNs. NMDA (20M) or EGF (20ng/ml) were added together with BSO and maintained throughout theexperiment. (A)After 24 hours, condensed-fragmented nuclei were countedafter staining with Hoechst. Data are expressed as means ± s.d. (n6;*P

BSO and L-NAME have been successfully used to inhibit GSHand NO synthesis in several tissues and organs (Cattan et al., 2008;Orucevic and Lala, 1996; Stevanovic et al., 2009; Tunctan et al.,2006; Watanabe et al., 2003). Work is in progress in our laboratoryto dissect this issue.

It has been reported that the level of GSH in peripheral bloodleukocytes is a useful biomarker to detect redox imbalance ininherited disorders affecting mitochondrial function (Atkuri et al.,2009). Therefore, we can speculate on the great importance ofmeasuring GSH levels to monitor disease status and response toantioxidants therapies in order to avoid its decrease and theconsequent nitrative or oxidative stress.

In conclusion, our work demonstrates that neuronal cells arehighly vulnerable to physiological flux of NO in the absence ofGSH. In particular, our study showed that the intracellular depletionof GSH is able to induce cellular stress in NO-producing cellsthrough a NO-dependent mechanism, such as inhibition of CcOXactivity, DNA damage, and S-NO and NO2-Tyr proteinaccumulation. Moreover, NO seems to be the only mediator of cellproliferation arrest through the ERK1/2-p53 signaling pathwayand apoptosis through AIF nuclear translocation. The resultsobtained give support to the idea that GSH represents an importantbuffer of NO toxicity, both under physiological conditions andupon excitotoxic NMDA-R stimulation. They also help tounderstand the molecular mechanisms underlying neuronal cellloss during aging and its related neurodegenerative disorders.

Materials and MethodsReagentsProtease inhibitor cocktail, rabbit polyclonal anti-S-NO, mouse monoclonal anti-catalase, anti-SOD1, anti--tubulin, anti-actin and anti-p53, Triton X-100, propidiumiodide, poly-L-lysine, dimethylsulfoxide (DMSO), NMDA, EGF andparaformaldehyde were from Sigma. Rabbit polyclonal anti-iNOS, anti-eNOS, anti-Hsp60 and anti-PARP, mouse monoclonal anti-cytochrome c, anti-nNOS and anti-GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Nitrocellulosemembrane and IgG (H + L) HRP-conjugated mouse and rabbit secondary antibodieswere from Bio-Rad Laboratories (Hercules, CA). 7-Ni, L-NAME, carboxy-PTIO,MEK1/2 inhibitor U0126, GC inhibitor LY83583 and the PKG inhibitor KT5823were from Merck-Chemicals (Darmstadt, Germany). Anti-CcOX subunit IV, Hoechst33342, and AlexaFluor568- and AlexaFluor488-conjugated secondary antibodieswere from Invitrogen (Carlsbad, CA). Polyclonal rabbit anti-ERK1/2-P and anti-NO2-Tyr were from Cell Signaling Technology (Danvers, MA). Mouse monoclonalanti-H2A.X-P, anti-procaspase-8 and -9 were from Millipore (Billerica, MA).ChemiGlow chemiluminescence substrate was from Alpha Innotech Corporation(San Leandro, CA). BrdU Cell Proliferation assay kit was from GE Healthcare(Buckinghamshire, UK). Rabbit polyclonal anti-AIF was from Biomol ResearchLaboratories (Plymouth Meeting, USA). All other chemicals were obtained fromMerck Ltd (Darmstadt, Germany).

Cell cultures and treatmentsSH-SY5Y neuroblastoma cells and HeLa cervix carcinoma cells were purchasedfrom the European Collection of Cell Cultures (Salisbury, UK). The NSC34 cellswere a kind gift of Neil Cashman (Montreal Neurological Institute, Montreal,Quebec, Canada). SH-SY5Y, HeLa and NSC34 cells were grown in DMEM-F12,RPMI and D-MEM, respectively, supplemented with 10% fetal bovine serum (FBS),2 mM glutamine, 100 U/ml penicillin/streptomycin (Lonza Sales, Basel, Switzerland)and maintained at 37°C in an atmosphere of 5% CO2 in air. SH-SY5Y cellsoverexpressing human SOD1 (named hSOD1) were obtained as previously described(Aquilano et al., 2003).

BSO, a highly selective and potent inhibitor of the enzyme g-GCS, was added inculture medium at a concentration of 1 mM unless otherwise stated. The NOSinhibitors L-NAME and 7-Ni were added in culture medium at concentrations of 100M and 10 M, respectively. The GC inhibitor LY and the PKG inhibitor KT wereadded at concentrations of 2 M and 4 M, respectively. The NO scavenger carboxy-PTIO was added at a concentration of 2 M. MEK1/2 inhibitor U0126 was addedat a concentration of 260 nM. All these chemicals were added 1 hour before BSOtreatment and maintained throughout the experiment. EGF and NMDA were used atconcentrations of 20 ng/ml and 20 M, respectively, and added together with BSO.All the concentrations of the chemicals used and the duration of the experiment wereselected after performing time-course and dose-response curves.

Primary mouse cortical neuronsMouse PNC cultures were obtained from cerebral cortices of E15 C57BL-6 miceembryos, as previously described (Filomeni et al., 2010). All the experiments wereperformed according to the Animal Research Guidelines of the EuropeanCommunities Council Directive (86/609/EEC).

Analysis of cell viability and apoptosisCells were directly counted by optical microscopy on a hemocytometer after TrypanBlue staining. Alternatively, cells were stained with 50 g/ml propidium iodideaccording to Nicoletti et al. (Nicoletti et al., 1991) and analyzed by a FACScaliburinstrument (Beckton and Dickinson, San Josè, CA). The percentage of cells in eachphase of the cell cycle was evaluated by WinMdi 2.9 software (Joseph Trotter,Scripps, San Diego, CA). Cell proliferation was assayed by a BrdU Cell Proliferationassay kit, as previously described (Baldelli et al., 2008).

Cell viability in BSO-treated primary mouse neurons cultures was monitored byphase-contrast microscopy or staining the cells with the nuclear dye Hoechst(Lieberthal et al., 1998). Cells were seeded on glass cover slips, fixed with 4%paraformaldehyde and incubated with Hoechst for 10 minutes at room temperature.Fluorescence images were acquired on Olympus IX 70 equipped with Nanomover®and softWoRx DeltaVision (Applied Precision, WA) with a U-PLAN-APO 60�objective. Nuclei of ten different images were counted and the results were reportedas the percentage of condensed-fragmented nuclei with respect to total cell number.

Preparation of cell lysates, western blot and dot-blot analysesCell pellets were resuspended in lysis buffer containing 10 mmol/L Tris-HCl, pH7.4, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.5% IGEPAL CA-630 and proteaseinhibitor cocktail. 20 g total protein was electrophoresed on 8.5% or 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membrane. Membranes werestained with primary antibodies against NO2-Tyr (1:200), H2A.X-P (1:1000), pro-caspase-8 (1:1000), pro-caspase-9 (1:1000), p53 (1:5000), ERK1/2-P (1:200), catalase(1:5000), GAPDH (1:5000), -tubulin (1:5000), Hsp60 (1:5000), actin (1:5000),CcOX subunit IV (1:1000), cytochrome c (1:2000), nNOS (1:500), eNOS (1:200),iNOS (1:200) and PARP (1:500). For dot-blot analysis, 20 g protein was vacuumtransferred to nitrocellulose membrane using a Bio-Rad dot-blot apparatus (Bio-RadLaboratories). Nitrocellulose membrane was stained with anti-NO2-Tyr (1:200).After incubation with the appropriate HRP-conjugated secondary antibodies, proteinswere detected using a Fluorchem Imaging system (Alpha Innotech-Corporation, StLeandro, CA) upon staining with ChemiGlow chemiluminescence substrate.Densitometric analyses of protein bands were performed by the Quantity one software(Bio-Rad Laboratories, Hercules, CA). Immunoblots reported in the figures arerepresentative of at least four experiments that gave similar results. Catalase, -tubulin or GAPDH were used as loading control.

Determination of GSHIntracellular GSH was determined as previously described (Baldelli et al., 2008).Data are expressed as nanomoles of GSH per milligram of protein.

Measurement of NOxAccumulation of NOx in culture medium was measured by the Griess reaction, aspreviously described (Aquilano et al., 2007).

Biotin switch assayBiotin switch assay was performed as previously described (Jaffrey and Snyder,2001). After protein separation by non-reducing SDS-PAGE and western blot,biotinylated proteins were detected by incubation of nitrocellulose membrane withHRP-conjugated streptavidin.

ImmunofluorescenceImmunofluorescence analysis was performed as previously described (Aquilano etal., 2010). Cells were incubated with anti-NO2-Tyr, anti-AIF, anti--tubulin, anti-cytochrome c, anti-pH2A.X or anti-SOD1 (1:50). The levels of S-NO proteins weredetected according to Iwakiri et al. (Iwakiri et al., 2006). Fluorescence images wereacquired on Olympus IX 70 equipped with Nanomover (60� objective) or with aNikon Eclipse TE200 epifluorescence microscope with a 100� objective (Nikon,Firenze, Italy). All images were captured under constant exposure time, gain andoffset. The images reported in the figures are representative of at least fourexperiments that gave similar results.

CcOX activity assayCcOX was assayed spectrophotometrically as previously described (Ciriolo et al.,2000). Activity was expressed as Units (micromol cytochrome c oxidized min–1) permilligram protein.

Proteins were assayed by the method of Lowry et al. (Lowry et al., 1951).

Statistical analysisThe results are presented as means ± s.d. Statistical evaluation was conducted byANOVA, followed by the post Student-Newman-Keuls analysis. Differences wereconsidered to be significant at P

We gratefully acknowledge Palma Mattioli (Department of Biology,University of Rome Tor Vergata, Italy) for her assistance in fluorescentmicroscopy and image analysis. We are also grateful to the animalhouse staff of the University of Rome Tor Vergata, Italy. This workwas partially supported by grants from Ministero della Salute andMIUR.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/7/1043/DC1

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1054 Journal of Cell Science 124 (7)

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SummaryKey words: BSO, L-NAME, ERK1/2, AIF, EGF, NO-cGMPIntroductionResultsEffects of GSH depletion on proliferation of SH-SY5Y neuroblastoma cellsCell growth arrest upon GSH depletion is induced by theBSO-mediated cell growth arrest results in cell death and isProlonged BSO treatment induces apoptosis through NO-dependent nuclear translocation ofSOD1 overexpression counteracts cell growth arrest and apoptosis induced byBSO-mediated cell growth arrest and death depend on the presenceBSO induces intracellular GSH depletion and NO-mediated cell death in

Fig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8.Fig. 9.DiscussionFig. 10.Fig. 11.Materials and MethodsReagentsCell cultures and treatmentsPrimary mouse cortical neuronsAnalysis of cell viability and apoptosisPreparation of cell lysates, western blot and dot-blot analysesDetermination of GSHMeasurement of NOxBiotin switch assayImmunofluorescenceCcOX activity assayStatistical analysis

Supplementary materialReferences