Attenuation of Staurosporine-Induced Apoptosis, Oxidative Stress, and Mitochondrial Dysfunction by Synthetic Superoxide Dismutase and Catalase Mimetics, in Cultured Cortical Neurons

Experimental Neurology 171, 84–97 (2001)doi:10.1006/exnr.2001.7747, available online at http://www.idealibrary.com on

Attenuation of Staurosporine-Induced Apoptosis, Oxidative Stress,and Mitochondrial Dysfunction by Synthetic Superoxide Dismutase

and Catalase Mimetics, in Cultured Cortical Neurons

Kevin Pong,1 Susan R. Doctrow,* Karl Huffman,* Christy A. Adinolfi,* and Michel Baudry

Neuroscience Program, University of Southern California, Los Angeles, California; and *Eukarion, Inc., Bedford, Massachusetts

Received August 10, 2000; accepted June 18, 2001

Neuronal apoptosis induced by staurosporine (STS)involves multiple cellular and molecular events, suchas the production of reactive oxygen species (ROS). Inthis study, we tested the efficacy of two synthetic su-peroxide dismutase/catalase mimetics (EUK-134 andEUK-189) on neuronal apoptosis, oxidative stress, andmitochondrial dysfunction produced by STS in pri-mary cortical neuronal cultures. Exposure of culturesto STS for 24 h increased lactate dehydrogenase (LDH)release, the number of apoptotic cells, and decreasedtrypan blue exclusion. Pretreatment with 20 mM EUK-134 or 0.5 mM EUK-189 significantly attenuated STS-induced neurotoxicity, as did pretreatment with thecaspase-1 inhibitor, Ac-YVAD-CHO, but not thecaspase-3 inhibitor, Ac-DEVD-CHO. Posttreatment(1–3 h following STS exposure) with 20 mM EUK-134 or0.5 mM EUK-189 significantly reduced STS-inducedLDH release, in a time-dependent manner. Exposureof cultures to STS for 1 h produced an elevation ofROS, as determined by increased levels of 2,7-dichlo-rofluorescein (DCF). This rapid elevation of ROS wasfollowed by an increase in lipid peroxidation, and boththe increase in DCF fluorescence and in lipid peroxi-dation were significantly blocked by pretreatmentwith EUK-134. STS treatment for 3–6 h increased cy-tochrome c release from mitochondria into the cy-tosol, an effect also blocked by pretreatment withEUK-134. These results indicate that intracellular ox-idative stress and mitochondrial dysfunction are crit-ically involved in STS-induced neurotoxicity. How-ever, there are additional cellular responses to STS,which are insensitive to treatment with radical scav-engers that also contribute to its neurotoxicity.© 2001 Academic Press

1 Present address: Dept. Neuroscience Wyeth-Ayerst ResearchCN-8000, Princeton, NJ 08543-8000. E-mail: [email protected].

840014-4886/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

Key Words: apoptosis; catalase; neurodegenerativediseases; oxidative stress; staurosporine; reactive ox-ygen species; superoxide dismutase.

INTRODUCTION

Apoptosis, or programmed cell death (PCD), is ahighly regulated cellular process that occurs duringanimal development (32, 45) and the development ofthe nervous system, in both vertebrates (24, 48) andinvertebrates (60). Recent evidence has demonstratedthat cell death programs are highly evolutionarily con-served and are also involved in neuronal death occur-ring in both acute and chronic neurodegenerative dis-eases, such as stroke, head trauma, Alzheimer’s dis-ease (AD), amyotrophic lateral sclerosis (ALS) andParkinson’s disease (PD) (5, 64). Although the cellularand molecular mechanisms involved in apoptosis arenot clearly understood, recent studies have begun toelucidate these mechanisms. Mitochondrial dysfunc-tion has been implicated in apoptotic cell death, andpro-apoptotic proteins, such as cytochrome c and apop-tosis-inducing factor (AIF), which are normally con-tained within the mitochondrial intermembrane space,are released into the cytosol (41, 61). Once in the cy-tosol, pro-apoptotic proteins activate several membersof the caspase family of cysteine proteases (7), whichhave been implicated in apoptotic cell death duringimmune (16) and nervous system (39) development andin glutamate-induced apoptosis in cerebellar granulecells (14, 15).

It has also been proposed that oxidative stress, dueto the generation of reactive oxygen species (ROS),such as hydroxyl radicals (•OH) and superoxide anions(O2•2), plays a critical role in apoptosis. Studies fromseveral different laboratories have suggested that ROSinduce apoptosis (4, 21, 22), while other studies havesuggested that ROS accompany apoptotic events (5).

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85SYNTHETIC RADICAL SCAVENGERS ATTENUATE STAUROSPORINE-INDUCED TOXICITY

Furthermore, exposure of cells to oxidative stress hasbeen shown to increase excitatory amino acid (EAA)release (23) and the levels of cytosolic Ca21 (18, 54),resulting in the activation of Ca21-dependent endo-nucleases and apoptotic mechanisms (20).

Based on the growing body of evidence implicatingROS in apoptosis and neurodegenerative diseases, sev-eral low-molecular-weight molecules that mimic anti-oxidant properties of endogenous enzymes have beendeveloped and characterized, with an emphasis on su-peroxide dismutase (SOD) mimetics. Three majorclasses of these compounds are macrocyclic Mn com-plexes (55), salen Mn complexes (2, 13, 19, 40), and Mnporphyrins (10–12, 17). The metalloporphyrin manga-nese tetrakis (4-benzoic acid) porphyrin (MnTBAP)possesses both SOD and catalase activity (10–12, 17)and has been shown to protect against peroxynitrite-and nitric oxide-induced suppression of mitochondrialrespiration in J774 cells (62), kainate- and paraquat-produced cell death in cortical neuron cultures (49),and hydrogen peroxide-mediated injury in endothelialcells (12). The macrocyclic Mn complexes have SODactivity, but not other ROS scavenging properties, andhave shown efficacy in several biological models fordisease (42, 55, 58). The salen–manganese complexes,employed in the present study, are synthetic com-pounds that also exhibit both SOD and catalase activ-ities (2, 13) and are protective in a wide range ofbiological systems, including models for neurologicaldiseases. For example, the prototype molecule, EUK-8,has been shown to protect hippocampal slice culturesfrom hypoxia-, acidosis-, and Ab-induced cell death (6,44). Furthermore, EUK-134, an analog of EUK-8, sig-nificantly reduced brain infarction volume in a rat focalischemia model (1), kainic acid-induced neuropathol-ogy (56), and oxidative stress and neurotoxicity pro-duced by 1-methyl-4-phenylpyridinium (MPP1) and-hydroxydopamine (6-OHDA) in primary dopaminer-ic neuron cultures (53). Taken together, these resultsuggest a potential role for synthetic SOD/catalase mi-etics as therapeutic agents against various neurolog-

cal disorders.Although initially isolated and characterized as an

nhibitor of protein kinase C (PKC) (63), the bacteriallkaloid compound staurosporine (STS) has since beenhown to inhibit many other protein kinases (25, 57).TS induces apoptosis in nearly all cell types, includ-

ng cell lines from various origins (3, 29), anucleateytoplasts (28), developing epithelial cells (31), andeurons (32) and may therefore activate a common celleath program. The anti-apoptotic gene product bcl-2as been shown to be neuroprotective against STS-

nduced apoptosis (28). Studies have provided evidencehat bcl-2 acts as both an integral intermembrane mi-ochondrial protein (8, 26, 61) and a potent antioxidantolecule (27, 30, 54), suggesting the involvement of

xidative stress and mitochondrial dysfunction. Al-

though several studies have investigated the mecha-nisms underlying STS apoptosis, there is not yet ageneral consensus regarding the relative contributionof oxidative stress and other processes. We thus eval-uated the effects of synthetic SOD/catalase mimeticson several cellular events thought to participate inSTS-induced apoptosis in mixed neuron/glia primarycortical cultures. Most of these studies were conductedwith EUK-134, which, as noted above, has been previ-ously shown to be neuroprotective in several experi-mental models. However, some experiments also em-ployed EUK-189, a newer and more lipophilic analog.

MATERIALS AND METHODS

Materials

EUK-134 and EUK-189 were synthesized as previ-ously described (1) and stock solutions were preparedin water. Unless specified, all other chemicals were ofcell culture grade and purchased from Sigma Chemical(St. Louis, MO).

Cortical Cell Cultures

Experimental protocols involving laboratory animalswere approved by the University Animal Use Commit-tee. Mixed primary cortical cell cultures containingboth neurons and glia were prepared from embryonicday 18 (E18) rat fetuses (Sprague–Dawley, CharlesRiver Laboratories, Wilmington, MA). The fetuseswere collected, their brains were removed, and theneocortices were dissected out in ice-cold phosphate-buffered saline (PBS) without Ca21 and Mg21. Dis-sected pieces of tissue were pooled together and trans-ferred to an enzymatic dissociation medium containing0.2% trypsin (Life Technologies, Rockville, MD) inEarle’s balanced salt solution and incubated for 15 minat 37°C. After enzymatic dissociation, the trypsin so-lution was aspirated and the tissue was mechanicallytriturated with a fire-polished glass Pasteur pipette incomplete medium [equal volumes of minimum essen-tial medium and F-12 nutrient mixture (Life Technol-ogies) supplemented with 0.1 mg/ml apotransferrinand 25 mg/ml insulin] containing 2000 IU/ml DNase.For lactate dehydrogenase (LDH) experiments, single-cell suspensions were seeded on poly-DL-ornithine (0.1mg/ml)- and laminin (1 mg/ml; Life Technologies)-coated 24-well plates at a density of 5.0 3 106 cells/well. For 2,7-dichlorofluorescein diacetate (DCF) ex-periments, single-cell suspensions were seeded on sim-ilarly coated 18-mm glass coverslips at a density of5.0 3 106 cells/slip. For trypan blue uptake, immuno-cytochemistry, and terminal deoxynucleotidyl trans-ferase-mediated dUTP nick-end labeling (TUNEL) ex-periments, single-cell suspensions were seeded on sim-ilarly coated 96-well plates at a density of 0.3 3 105

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86 PONG ET AL.

cells/well. For lipid peroxidation experiments, single-cell suspensions were seeded on similarly coated 6-wellplates at a density of 2.0 3 107 cells/well. For Westernblots, single-suspensions were seeded on similarlycoated 100-mm dishes at a density of 2.0 3 107 cells/dish. Cultures were maintained for 7–10 days in vitro(DIV) before experimentation.

Measurement of LDH Release

Mature cultures were treated with control mediumin the absence or presence of different antioxidants for1 h prior to STS exposure. In the delayed administra-tion experiments, cultures were treated with differentantioxidants 1–4 h after starting STS exposure. Cul-tures were then exposed to STS for 24 h. The amount ofLDH released into the culture medium 24 h after STSexposure was measured at room temperature using themethod described by Koh and Choi (34). In brief, ali-quots of media were added to 2.3 mM sodium pyruvatein 0.1 M KH2PO4 buffer (pH 7.4 at 25°C) with 0.2 mg ofNADH. The absorbance of the reaction mixture at 340nm, an index of NADH concentration, was measuredwith a spectrophotometer.

Trypan Blue Exclusion

Mature cultures were treated with control mediumin the absence or presence of 20 mM EUK-134 1 h priorto STS exposure. Cultures were then exposed to vari-ous concentrations of STS for 24 h. The number ofviable cells excluding trypan blue were determined24 h after STS exposure. Sister cultures were washedtwice with prewarmed PBS. Cultures were thenstained with 0.2% trypan blue for 30 min at roomtemperature. Following three washes with PBS, thenumber of trypan blue positive cells/field were counted.Three to five fields were counted for each condition.Values are expressed as percent of values found inuntreated control cultures.

MAP-2 and GFAP Immunocytochemistry

Mature cultures were treated with control mediumin the absence or presence of 20 mM EUK-134 1 h priorto STS exposure. Cultures were then exposed to 100nM STS for 24 h. Cultures were then immunostainedwith MAP-2 or GFAP antibodies 24 h after STS expo-sure. Cultures were fixed with 4% paraformaldehyde(PFA) for 45 min at 37°C. Nonspecific binding wasblocked by incubating with PBS containing 1% NonidetP-40, 1% bovine serum albumin (BSA), and 5% goatserum for 90 min at 37°C. Cultures were then incu-bated overnight at 4°C with an anti-MAP-2 antibody(Sigma) diluted 1:500 in 1% BSA in PBS or an anti-GFAP antibody (Roche Molecular Biochemicals, India-napolis, IN) diluted 1:1000 in 1% BSA in PBS. Follow-ing three washes, cultures were processed and devel-

oped to visualize MAP-2 or GFAP immunoreactivitywith a Vectastain anti-mouse Ig peroxidase ABC kit(Vector Laboratories, Burlingame, CA) and diamino-benzidine substrate.

TUNEL Labeling

Mature cultures were treated with control mediumin the absence or presence of 20 mM EUK-134 1 h priorto STS exposure. Cultures were then exposed to 100nM STS for 24 h. Apoptotic cells were labeled 24 h afterSTS exposure using a TUNEL labeling kit (Roche Mo-lecular Biochemicals). Cultures were fixed with 4%PFA for 30 min at room temperature. Cultures werethen rinsed with PBS and incubated in a permeabili-zation solution containing 0.1% Triton X-100 and 0.1%sodium citrate for 2 min at 4°C. Cultures were thenincubated with the TUNEL reaction mixture for 1 h at37°C. Cultures were finally incubated with the alkalinephosphatase converter for 30 min at 37°C and visual-ized under a light microscope. The number of TUNELpositive cells was counted in 3–5 random fields.

Visualization of ROS Production in Cells with DCF

Levels of ROS production in cells were determinedusing the fluorescent probe DCF (Molecular Probes,Eugene, OR). Aliquots of 10 mM DCF were prepared inDMSO and stored at 220°C. Mature cultures weretreated with control medium in the absence or pres-ence of 20 mM EUK-134 1 h prior to STS exposure.Cultures were then exposed to 100 nM STS for 1–4 h.Following 1–4 h exposure to STS, cells were incubatedfor 1 h in the presence of 10 mM DCF and washed withprewarmed PBS. Cultures were examined under a flu-orescence microscope and images were collected.

Measurement of Lipid Peroxidation

Mature cultures were treated with control mediumin the absence or presence of 20 mM EUK-134 1 h priorto STS exposure. Cultures were then exposed to 100nM STS for 24 h. Lipid peroxidation was determined24 h after STS exposure by using the thiobarbituricacid-reactive substances (TBARS) assay described byOhkawa et al. (47), with modifications for use in cellulture (51). Following removal of the culture medium,ells were washed with PBS and subsequently lysedith lysis buffer (2.5% sodium dodecyl sulfate contain-

ng 2.5 mM deferoxamine and 5 mM probucol) to pre-vent the occurrence of lipid peroxidation after cell lysis.Lysate pH was adjusted to 3.5 by the addition of aceticacid with sodium hydroxide. An aqueous solution ofthiobarbituric acid (final concentration of 0.3%) wasadded to the lysates, which were then heated at 95°Cfor 1 h. Samples were then transferred to a room tem-perature water bath for 10 min and allowed to cool.TBARS were extracted into an organic layer from the

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reaction mixture through the addition of 1-butanol/pyridine (15:1) and centrifugation (4000g, 10 min).

evels of TBARS were quantified by spectrofluorom-try (excitation 5 515 nm, emission 5 553 nm).

DS–PAGE and Western Blotting for CytosolicCytochrome c

SDS–PAGE and Western Blots were performed asescribed previously (52). Cultures were grown in00-mm dishes as described above. Forty-eight hoursfter plating, medium was exchanged and culturesere returned to the incubator. Cultures were pre-

reated with either 20 mM EUK-134 or control mediumfor 1 h and then exposed to 100 nM STS for 3–6 h.Cultures were washed with ice-cold PBS and subse-quently lysed with lysis buffer (137 mM NaCl, 20 mMTris, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM vanadate, 10%glycerol, 1% NP-40, and 1 mM phenylmethylsulfoxylfluoride). The lysates were collected and centrifuged at12,000 rpm for 20 min. The supernatants were col-lected as the cytosol (46). Protein concentrations weredetermined with a Bradford protein assay. Superna-tants (50 mg of protein) were fractionated by SDS–PAGE on a 15.0% acrylamide gel and transferred tonitrocellulose membranes. Membranes were blockedwith 3% gelatin and probed with an anti-cytochrome cantibody (1:400, Pharmingen, San Diego, CA) over-night, at room temperature. Secondary antibodies con-jugated to alkaline-phosphatase (Bio-Rad, Hercules,CA) were used to visualize cytochrome c. Bands repre-senting cytochrome c were quantified using the ImageQuant System (Molecular Devices, Sunnyvale, CA).

Lipophilicity Measurements

The relative lipophilicities of EUK-134 and EUK-189were assessed using octanol partitioning. An aliquot of100 ml of an aqueous solution (200 mM) of compoundwas mixed with 500 ml n-octanol. The mixture wasvortexed for 30 s and then centrifuged at 8000g for 5min to facilitate phase separation. An aliquot of eachlayer was diluted into mobile phase and analyzed byreversed phase HPLC, using an octadecyl-silica col-umn eluted isocratically with a mobile phase consistingof 60% methanol:40% 100 mM NaCl in water, monitor-ing elution with an absorbance wavelength of 240 nm.For each compound, the percentage partitioned intothe octanol layer was calculated.

RESULTS

1. STS-Induced Neurotoxicity Is Attenuatedby EUK-134

Cortical neuron cultures were prepared and main-tained for 7–10 days prior to performing experiments.An initial dose–response analysis was done with STS

to determine its neurotoxicity under our culture condi-tions. Cultures were exposed to various concentrationsof STS for 24 h. At the end of the exposure period,neuronal viability was determined. Treatment withSTS resulted in a dose-dependent decrease in neuronalviability, as measured by LDH release (Fig. 1A). Sim-ilarly, an initial dose–response analysis was done withEUK-134 to determine relative levels of toxicity ortrophic activity in our cultures. In these cortical neu-ron cultures, EUK-134 showed no neurotoxicity or neu-rotrophic activity at concentrations up to 50 mM (Fig.1B).

To determine whether EUK-134 could protectagainst STS-induced neurotoxicity, we examined LDHrelease, trypan blue exclusion, MAP-2 immunostain-ing, and TUNEL labeling. Cultures were pretreated for1 h with various concentrations of EUK-134, followedby a 24-h exposure to 100 nM STS. At the end of the24-h exposure, LDH released into the medium wasmeasured. The optimal concentration of EUK-134 wasdetermined to be 20 mM (Fig. 1B). Pretreatment with20 mM EUK-134 significantly reduced the amount ofLDH released into the medium from 250 to 155% ofuntreated control values (i.e., a 66% reduction in LDHrelease). Higher concentrations of EUK-134 were lesseffective, although these concentrations were not neu-rotoxic by themselves. In another set of experiments,cultures were pretreated for 1 h with 20 mM EUK-134and then exposed to various concentrations of STS for24 h. At the end of the 24-h exposure, LDH release andtrypan blue exclusion were determined. Pretreatmentwith 20 mM EUK-134 significantly reduced STS-in-duced neurotoxicity at all STS concentrations (Figs. 1Cand 1D), although the degree of protection slightlydecreased with increasing STS concentration. Since 20mM EUK-134 provided the optimal protection againstSTS-induced neurotoxicity, this concentration wasused for all subsequent experiments.

We also compared the effects of EUK-134 with thoseof other ROS scavengers, i.e., trolox and a-tocopherol(Fig. 2). All three compounds were equally effective inreducing neurotoxicity elicited by 10 nM STS, buttrolox and a-tocopherol appeared considerably morepotent. We hypothesized that the protection providedby these agents might require intracellular scavengingof oxygen free radicals and that, consequently, a morelipophilic, cell-permeable compound would have an ad-vantage. To test this hypothesis, we employed a morelipophilic salen manganese complex, EUK-189. EUK-189 is structurally similar to EUK-134 (Fig. 3A) andhas identical SOD and catalase activities (Fig. 3B).However, it is more lipophilic, as indicated by its struc-ture and confirmed by its octanol partitioning behavior(Fig. 3B). These two salen manganese complexes werecompared for their ability to protect neurons againststaurosporine. As shown in Fig. 4A, both EUK-134 andEUK-189 provided a similar degree of maximal protec-

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88 PONG ET AL.

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tion of about 65–70% against staurosporine, but EUK-189 was about 100 times more potent than EUK-134.In contrast (Fig. 4B), EUK-134 and EUK-189 showedidentical activity in a cytoprotective model (2), involv-ing toxicity by an extracellular hydrogen peroxide in-sult.

In a similar treatment paradigm, the caspase-1 in-hibitor Ac-YVAD-CHO provided a dose-dependent re-duction in STS-induced neurotoxicity whereas thecaspase-3 inhibitor Ac-DEVD-CHO did not provide anyprotection (Table 1). The maximal protection affordedby the caspase-1 inhibitor was about 90%. To deter-mine whether treatment with ROS scavengers could beneuroprotective when started after STS addition, wetreated cultures with various scavengers at varioustime points after the initiation of STS exposure. Cul-tures were treated with 20 mM EUK-134 or 0.5 mMEUK-189, 1 mM trolox, or 1mM a-tocopherol 1–4 hfollowing exposure to 10 nM STS. Twenty-four hourslater, neuronal viability was determined by measuringLDH release (Fig. 5). All four compounds were effec-tive, albeit to various degrees, in reducing neurotoxic-ity elicited by 10 nM STS. Treatments with 20 mMEUK-134, 1 mM trolox, or 1 mM a-tocopherol wereeffective in protecting against STS-induced neurotox-icity up to 3 h following STS exposure. However, troloxwas less effective than EUK-134 and a-tocopherol.Posttreatment with 20 mM EUK-134, 1 mM trolox, or1mM a-tocopherol at 4 h and beyond was not protectiveagainst STS-induced toxicity. EUK-189 afforded moreprotection than the other three compounds and wasstill significantly protective even at the 4-h time point.

FIG. 2. Effects of the antioxidants, EUK-134, trolox, and a-tocoere pretreated with 20 mM EUK-134, 1 mM trolox, or 1 mM a-tocop4-h period, LDH release was measured. Values are expressed as a peD of three independent experiments.

pherol, on staurosporine-induced cell death. Cortical neuronal culturesherol for 1 h and then exposed to 10 nM STS for 24 h. At the end of thercentage of values found in untreated control cultures and are means 6

FIG. 3. Comparative structures and properties of EUK-134 andEUK-189. (A) Structures of EUK-134 and EUK-189. The axial ligand(X) is chloride for EUK-134 and acetate for EUK-189. (B) Catalyticactivities and lipophilicity of EUK-134 and EUK-189. SOD and cata-lase activities were assayed as described previously (1). All valueshave been normalized, and are expressed as percentage activity ofEUK-134 (means 6 SD for three to five determinations). The abso-lute catalytic activities of EUK-134 have been reported previously(1). Lipophilicity was determined by octanol partitioning as de-scribed under Materials and Methods. The results were calculated asthe percentage compound partitioning into octanol and were ex-pressed as percentage of the value for EUK-134 (means 6 SD for fivedeterminations). The absolute partitioning values were 7.6 6 0.7 and14.2 6 0.4% for EUK-134 and EUK-189, respectively.

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90 PONG ET AL.

In order to test the cellular specificity of staurospor-ine neurotoxicity, we determined the effects of stauro-sporine treatment on a neuronal marker (MAP-2) anda glial marker (GFAP). Representative micrographswere taken of sister cultures fixed and stained for themicrotubule marker, MAP-2 (Fig. 6). Cultures exposedto 100 nM STS for 24 h showed significantly reducedMAP-2 staining of cell bodies and neurites (Fig. 6B)when compared to untreated control cultures (Fig. 6A).Pretreatment for 1 h with 20 mM EUK-134 completelyblocked the morphological changes induced by STS(Fig. 6C). Cultures treated with 20 mM EUK-134 aloneshowed no morphological changes when compared to

FIG. 4. Effects of EUK-134 and EUK-189 on staurosporine- andhydrogen peroxide-induced cell death. (A) Cortical neuronal cultureswere pretreated with various concentrations of EUK-134 or EUK-189 for 1 h and then exposed to 10 nM STS for 24 h. At the end of the24-h period, LDH release was measured. Values are expressed as apercent of maximal LDH release found in 10 nM STS treated cul-tures and are means 6 SD. (B) Human skin fibroblasts were sub-jected to glucose and glucose oxidase, a hydrogen peroxide generat-ing system, and cytotoxicity was assessed using the XTT reagent asdescribed previously (1). In this system, cytotoxicity is extracellular,with complete protection afforded by the high-molecular-weight pro-tein bovine liver catalase (1). The figure shows the mean 6 SD (n 5

per dose). Results were calculated as percentage of maximumoxicity, determined with glucose plus glucose oxidase in the absencef EUK-134 and EUK-189, with zero toxicity determined in thebsence of glucose oxidase.

untreated control cultures (Fig. 6D). Cultures exposedto 100 nM STS showed no significant changes in GFAPstaining (data not shown).

Representative micrographs were also taken of sis-ter cultures fixed and stained with a TUNEL labelingsolution. Cultures exposed to 100 nM STS for 24 hshowed increased TUNEL labeling (Fig. 7B) when com-pared to untreated control cultures (Fig. 7A). Pretreat-ment for 1 h with 20 mM EUK-134 significantly atten-uated STS-induced DNA fragmentation (Fig. 7C). Cul-tures treated with 20 mM EUK-134 alone showed nochanges in TUNEL labeling (Fig. 7D). Random fields(3-5 per group) were chosen to quantify the number ofTUNEL-positive cells. Exposure of cultures to 100 nMSTS increased the number of TUNEL-positive cells to355% of untreated control values. Pretreatment with20 mM EUK-134 significantly reduced the number ofTUNEL positive cells to 237% of untreated controlvalues (i.e., about a 50% reduction in the number ofTUNEL-positive cells) (Fig. 8).

2. STS-Induced Oxidative Stress Is Blockedby EUK-134

To determine whether EUK-134 could prevent STS-induced ROS generation and the resulting oxidativestress, levels of ROS production in cells were deter-mined using the fluorescent probe DCF and the result-ant oxidative stress was determined by measuring thelevels of lipid peroxidation. Cultures were treated withcontrol media or 20 mM EUK-134 1 h prior to STSexposure. Following 1–4 h exposure to STS, cells wereincubated for 1 h in the presence of 10 mM DCF andwashed with prewarmed PBS. Cultures were exam-ined under a fluorescence microscope and representa-

TABLE 1

The Caspase-1 Inhibitor Ac-YVAD-CHO, But Not theaspase-3 Inhibitor Ac-DEVD-CHO Attenuate Staurospor-

ne-Induced Neurotoxicity

Treatment LDH release (% of control)

Control 100 6 810 mM YVAD 101 6 1100 mM YVAD 104 6 910 mM DEVD 102 6 2100 mM DEVD 101 6 310 nM STS 168 6 12STS 1 10 mM YVAD 131 6 4STS 1 100 mM YVAD 108 6 4STS 1 10 mM DEVD 171 6 2STS 1 100 mM DEVD 166 6 5

Note. Cortical neuron cultures were prepared from E18 rat em-bryos and maintained for 7–10 days prior to experimentation. Cul-tures were pretreated with 10 and 100 mM YVAD or DEVD andexposed to 10 nM STS for 24 h. At the end of the 24-h period, LDHrelease was measured. Values are expressed as percentage of valuesfound in untreated control cultures and are means 6 SD of three tofive independent experiments.

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tive images were collected. Cultures exposed to STS for1 h showed increased intensity and number of DCF-labeled cells (Figs. 9B1 and 9B2) when compared tountreated control cultures (Figs. 9A1 and 9A2). Thisncrease in number and intensity was a prolonged ef-ect, which was still visible after 4 h of exposure to STSdata not shown). Cultures pretreated with 20 mMUK-134 showed reduced intensity and number ofCF labeled cells (Figs. 9C1 and 9C2) when compared

to STS-treated cultures. Cultures treated with 20 mMEUK-134 alone showed mild DCF labeling (Figs. 9D1

and 9D2). In addition, there was no difference in DCFlabeling between cultures treated with EUK-134 withor without STS treatment. As reported previously byBruce et al. (6) EUK-8, also caused a mild increase inDCF fluorescence, even in the absence of cells. Thisphenomenon is most likely caused by the peroxidaseactivity of catalase mimetics such as EUK-134 (13),since DCF is a peroxidase substrate.

Lipid peroxidation induced by STS was assessed byusing the TBARS assay. Cultures were pretreated with20 mM EUK-134 or control medium for 1 h then ex-posed to 100 nM STS for 24 h. STS exposure produceda significant increase in lipid peroxidation, represent-ing a 72% increase above untreated control levels. Pre-treatment with 20 mM EUK-134 almost completely

revented STS-induced lipid peroxidation (Fig. 10).

. STS-Induced Cytochrome c Release fromMitochondria Is Blocked by EUK-134

It has been demonstrated that exposure to STS in-uces mitochondrial dysfunction, including the releasef cytochrome c, a pro-apoptotic protein. To determine

FIG. 5. Effects of delayed EUK-134, EUK-189, trolox, and a-tocopcultures were first treated with 10 nM STS for 1–4 h. Cultures were1 mM a-tocopherol or control medium at the indicated times followin

TS in the medium for 24 h. At the end of the 24-h period, LDH relen untreated control cultures and are means 6 SD of three to five in

whether EUK-134 was effective in preventing mito-chondrial dysfunction, we measured cytochrome c re-lease from the mitochondria inner membrane into thecytosol. Exposure to STS for 3–6 h dramatically in-creased cytochrome c release up to 17.9-fold above un-treated control values. Pretreatment with 20 mM EUK-134 almost completely blocked the increase in cyto-chrome c release (Fig. 11).

DISCUSSION

Several studies have investigated the mechanismsunderlying STS-induced apoptosis in neurons. How-ever, the use of different culture conditions, STS con-centrations, and experimental protocols has notyielded a definite answer regarding the respective con-tribution and time-course of different cellular pro-cesses proposed to be involved in neuronal death. Ourstudy provides evidence that oxidative stress and mi-tochondrial dysfunction are associated with STS expo-sure, and that treatment with SOD/catalase mimeticsalmost completely blocks these cellular events. Morespecifically, we demonstrated that STS exposurecaused a rapid and prolonged increase in ROS produc-tion, as determined by the dramatic increase in thenumber and intensity of DCF-labeled cells. The rapidand prolonged production of ROS is likely to be respon-sible for the increase in lipid peroxidation observed24 h after staurosporine treatment. Our studies arethus in good agreement with those of Patel (50) andKrohn et al. (37), which showed that various types ofadical scavengers could protect neurons from STS-nduced oxidative stress and apoptosis. Interestingly,

rol treatment on staurosporine-induced cell death. Cortical neuronalen treated with 20 mM EUK-134, 0.5 mM EUK-189, 1 mM trolox, orTS treatment. Cultures were maintained with both compounds andwas measured. Values are expressed as percentage of values found

pendent experiments.

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92 PONG ET AL.

the comparison of the potency and efficacy of twosalen–manganese complexes with identical catalyticactivities but differing lipophilicities, clearly indicatedthat oxidative stress is produced intracellularly, as thecompound with greater lipophilicity and, presumably,greater membrane permeability, exhibited a large in-crease in potency. To further support this interpreta-tion, the two compounds exhibited identical potenciesagainst an extracellular hydrogen peroxide insult.

In addition to producing oxidative stress, STS expo-sure caused mitochondrial dysfunction. Under normalphysiological conditions, pro-apoptotic proteins, suchas cytochrome c, are contained within the intermem-brane space of the mitochondria. STS exposure causeda significant release of cytochrome c from the mito-chondria into the cytosol. Pretreatment with EUK-134completely blocked STS-induced ROS production, lipidperoxidation, and mitochondrial dysfunction. This sug-gests that mitochondrial dysfunction is a consequenceof oxidative stress. However, EUK-134 and EUK-189,were unable to completely prevent STS-induced neuro-

FIG. 6. Effects of EUK-134 on MAP-2-positive neurons in staurUK-134 or control medium for 1 h and then exposed to 100 nM STSAP-2 immunoreactivity with a Vectastain anti-mouse Ig peroxidase

re shown. (A) Control, (B) 100 nM STS, (C) 100 nM STS 1 20 mM

toxicity, as determined by LDH release, trypan blueexclusion, and TUNEL labeling. The maximal degreeof protection provided by a concentration of EUK-134that completely prevented oxidative stress was about60–70%. This result contrasts with the report byKrohn et al. (37) that indicates that trolox or a-tocoph-erol almost completely prevents STS-induced apopto-sis. In our study, trolox and a-tocopherol provided thesame degree of protection as EUK-134. We also notethat in the Krohn et al. study (37), STS was morepotent than in our study. It is interesting to note thatthese authors used a pure hippocampal neuron culture,suggesting that, under different culture conditions,STS-induced apoptosis might engage various cellularevents. It is therefore likely that there are additionalcellular responses to STS, which are insensitive toEUK-134 treatment and contribute to STS neurotoxic-ity. In addition, our data indicate that cortical neuronscan be subdivided into (at least) two groups, a groupthat STS kills predominantly by oxidative stress and agroup that STS kills through a different mechanism.

orine-induced toxicity. Cultures were pretreated with either 20 mM24 h. Cultures were then fixed, processed, and developed to visualizeC kit and diaminobenzidine substrate. Representative micrographsK-134, (D) 20 mM EUK-134 alone.

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This idea is further supported by the fact that thecaspase-1 inhibitor YVAD almost completely pre-vented STS-induced neurotoxicity, a result in good

FIG. 7. Effects of EUK-134 on staurosporine-induced TUNEL lamedium for 1 h and then exposed to 100 nM STS for 24 h. Culturesvisualized under a light microscope. Representative micrographs are(D) 20 mM EUK-134 alone.

FIG. 8. Effects of EUK-134 on the number of TUNEL-positivpretreated with either 20 mM EUK-134 or control medium for 1 h anncubated with the TUNEL reaction mixture and visualized underandom fields. Values are expressed as percentage of values found

independent experiments. *P # 0.01 vs 100 nM STS; #P # 0.01 vs

agreement with the study by Krohn et al. (37). It haseen previously proposed that caspase-1 activationepresents an initiator or an early event in STS-in-

ng. Cultures were pretreated with either 20 mM EUK-134 or controlre then fixed and incubated with the TUNEL reaction mixture andwn. (A) Control, (B) 100 nM STS, (C) 100 nM STS 1 20 mM EUK-134,

ells following staurosporine-induced neurotoxicity. Cultures werehen exposed to 100 nM STS for 24 h. Cultures were then fixed and

ght microscope. TUNEL-positive cells were counted in three to fiveuntreated control cultures and are means 6 SD of three to five

ntrol.

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94 PONG ET AL.

FIG. 9. Effects of EUK-134 on staurosporine-induced ROS generation. Cultures were treated with 20 mM EUK-134 or control mediumfor 1 h and then exposed to 100 nM STS for 1–4 h. Cells were then incubated for 1 h in the presence of 10 mM DCF and washed withprewarmed PBS. Cultures were examined under a fluorescence microscope and images were collected. Representative micrographs of 1-hexposure to STS are shown. Micrographs A1–D1 are low magnification images (43) while A2–D2 are higher magnification images (103). (A)Control, (B) 100 nM STS, (C) 100 nM STS 1 20 mM EUK-134, (D) 20 mM EUK-134 alone.

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duced apoptosis (37) and thus, in a fraction of corticalneurons it could precede the activation of events af-fected by EUK-134.

FIG. 11. Effects of EUK-134 on staurosporine-induced cytochromedium for 3 h then exposed to 100 nM STS for 3–6 h. Cultures weand Western blot. Values are expressed as fold-increase above valuefive independent experiments. *P # 0.01 vs 100 nM STS, #P # 0.0

FIG. 10. Effects of EUK-134 on staurosporine-induced lipid peroxor 1 h and then exposed to 100 nM STS for 24 h. Cultures were thalues are expressed as percentage of values found in untreatedxperiments. *P # 0.01 vs 100 nM STS; #P # 0.01 vs control.

Apoptosis is critical in regulating the number andtypes of neurons in various regions of the developingnervous system. This biological phenomenon also ap-

-C release. Cultures were treated with 20 mM EUK-134 or controlhen lysed and cytochrome-c release was determined by SDS–PAGEound in untreated control cultures and are means 6 SD of three tos control.

tion. Cultures were treated with 20 mM EUK-134 or control mediumlysed and lipid peroxidation was measured with the TBARS assay.ntrol cultures and are means 6 SD of three to five independent

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pears to be involved in many neurodegenerative dis-eases, such as AD, ALS, and PD, which are character-ized by the gradual loss of specific neuronal popula-tions (5, 64). As STS induces apoptosis in a variety ofcell types (3, 28, 31, 35), it has been proposed that ittriggers a common cell death cascade and that STS-induced apoptosis might provide a powerful system tounderstand the cellular events that take place duringapoptosis. In this study, we report that STS-inducedneurotoxicity is mediated by oxidative stress, mito-chondrial dysfunction, and other apoptotic processes.Oxidative stress and mitochondrial dysfunction occurrelatively rapidly following the exposure to STS andtreatment with SOD/catalase mimetics completely pre-vented these cellular events, and provided a very sub-stantial degree of neuroprotection. The salen manga-nese complexes have been shown to be protective inseveral animal models of degenerative diseases (13)and to increase life span in C. elegans (43). More re-cently, these molecules have been reported to prolongsurvival and to prevent spinal cord oxidative stress ina mouse model of amyotrophic lateral sclerosis (33).The present results further indicate that the therapeu-tic applications of these molecules might encompassconditions associated with apoptotic cell death.

ACKNOWLEDGMENTS

We thank Dr. Wei Liu, Dr. Christian Pike and Walid Soussou fortheir technical advice.

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Documents

Attenuation of Staurosporine-Induced Apoptosis, Oxidative Stress, and Mitochondrial Dysfunction by Synthetic Superoxide Dismutase and Catalase Mimetics, in Cultured Cortical Neurons