Peroxynitrite-Induced Neuronal Apoptosis Is Mediated by Intracellular Zinc Release and 12-Lipoxygenase Activation

Materials

3-Morpholino-sydnonimine (SIN-1), peroxynitrite, and diethylenetriamine NONOate (DETA/NO) were obtained from Cayman Chemical (Ann Arbor, MI). Caspase inhibitors [Ac-Asp-Glu-Val-Asp-chloromethylketone (Ac-DEVD-CMK) and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK)], caspase-3 substrate Ac-Asp-Glu-Val-Asp-7-amido-4-(trifluoromethyl)coumarin (Ac-DEVD-AFC), and Mn (III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were obtained from Calbiochem (La Jolla, CA). Staurosporine was purchased from RBI (Natick, MA). 2,7-Dichlorofluorescein diacetate (Newport Green), FluoZin-3, dihydrorhodamine-123 (DHR-123), and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were purchased from Molecular Probes (Eugene, OR). 2,3,5-Trimethyl-6-(12-hydroxy-5-10-dodecadiynyl)-1,4-benzoquinone (AA861), 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol]-2,2-dimethylpropanoic acid (MK-886), 7,7-dimethyleicosadienoic acid (DEDA), and indomethacin were purchased from Biomol (Plymouth Meeting, PA). BMD-122 (N-benzyl-N-hydroxy-5-phenylpentanamide) was a gift from Dr. Lawrence J. Marnett (School of Medicine, Vanderbilt University, Nashville, TN). All other reagents were obtained from Sigma (St. Louis, MO).

Tissue culture

Neuronal cultures were prepared from embryonic day 16 Sprague Dawley rat fetuses using methods similar to those described previously (Dichter, 1978) but modified to produce cultures that contained <1% astrocytes (Rosenberg and Aizenman, 1989; Rosenberg, 1991; Rosenberg et al., 1992; Wang et al., 1998). Cultures were initially plated on poly-l-lysine-coated 24-well plastic plates (Costar, Cambridge, MA) using an 80:10:10 (v/v) mixture of DMEM (11960-010; Invitrogen, San Diego, CA), Ham's F-12 (N-4888; Sigma), and heat-inactivated iron-supplemented calf serum (A2151; HyClone, Logan, UT) containing 2 mm glutamine, 25 mm HEPES, 24 U/ml penicillin, and 24 μg/ml streptomycin and maintained in a 5% CO₂ (balance air) incubator at 36°C. Cell proliferation was inhibited by exposure to 5 μm cytosine arabinoside at 24 hr in vitro for 72 hr. On the fourth day of culture, the medium was removed completely and replaced with 90% MEM, 10% Nuserum IV (Collaborative Research, Bedford, MA), 2 mm glutamine, and 5 mm HEPES, containing 50 U/ml superoxide dismutase (Boehringer Mannheim, Indianapolis, IN), 20 U/ml catalase (CV-40; Sigma), 11 mm total glucose, and 9.3 mm total sodium bicarbonate, plus 2% B27 supplement (17504-036; Invitrogen). Medium was not subsequently changed. To prevent evaporation of water, culture dishes were kept on “wet dishes” containing wet filter paper until they were used.

Application of reagents

In all experiments, 24-well culture plates were first washed two times in HBSS containing 0.1% bovine serum albumin (BSA) and then placed in Earle's balanced salt solution (EBSS), which is composed of the following (in mm): 116 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.8 MgSO₄, 26 NaHCO₃, 1 NaH₂PO₄, and 5.5 d-glucose. Experiments were performed exclusively in EBSS. All drugs were applied 15 min before the cells were exposed to SIN-1, peroxynitrite, NMDA, DETA/NO, or ZnCl₂.

Survival assay

Neurons were exposed to SIN-1 (1 mm) for 24 hr, and the morphological changes were examined by phase-contrast microscopy. Quantitation of survival of cells was determined 24 hr after exposure to the test compounds by assessing the ability of cultures to reduce Alamar Blue (Trek Diagnostic Systems, Westlake, OH) as an index of viability (McGahon et al., 1995). Details of this procedure have been provided previously (Back et al., 1998, 1999) and have been validated in examining the toxicity of SIN-1 in neurons (Zhang and Rosenberg, 2002). In brief, the test medium was removed after 24 hr and then replaced with EBSS plus a 1:100 dilution of Alamar Blue. After 2 hr of exposure, 100 μl of medium from each well (24-well plate) was transferred to a 96-well plate, and the fluorescence of the Alarmar Blue solution in each well was read in a fluorescence plate reader (FluoroCount; Packard, Meriden, CT), with excitation set at λ = 530 nm and emission set at λ = 590 nm at room temperature (RT).

Fluorescence imaging of intracellular liberation of zinc

Assay of direct interaction of peroxynitrite with fluorescent Zn²⁺ indicators. The reaction of peroxynitrite with the Zn²⁺ fluorescent indicators Newport Green and FluoZin-3 was examined in a cell-free system. The hydrolysis of Newport Green and FluoZin-3 AM was performed following the method described by LeBel et al. (1992). Newport Green (1 mm) or FluoZin-3 (1 mm) was dissolved in 50 μl of DMSO, and then an equal volume of methanol was added. After deesterification with 0.4 ml of NaOH (10 mm), 2 ml of NaH₂PO₄ (25 mm; pH 7.4) was added for neutralization. The hydrolyzed Zn²⁺ indicator was diluted 1:100 into EBSS. After addition of peroxynitrite (100 μm), the intensity of fluorescence was measured by a Hitachi (Tokyo, Japan) fluorescence spectrophotometer with an excitation wavelength at 485 nm and an emission wavelength at 530 nm.

Assay of intracellular liberation of zinc using FluoZin-3. Changes in intracellular free Zn²⁺ concentration in neurons were monitored with a high-affinity, zinc-selective indicator, FluoZin-3 (Gee et al., 2002). Neurons were loaded with FluoZin-3 (1 μm) for 30 min and then washed twice with HBSS containing 0.1% BSA. At 30 min after neurons were treated with peroxynitrite (100 μm), fluorescence imaging of intracellular zinc was monitored immediately using digital fluorescence microscopy with a 20× objective (excitation at 485 nm; emission at 530 nm). For all images, the microscope settings, such as brightness, contrast, and exposure time, were held constant to compare the relative intensity of intracellular zinc fluorescence across all treatment conditions.

Assay of intracellular liberation of zinc by fluorescence resonance energy transfer. Neurons were transfected using Lipofectamine 2000 (Invitrogen) (Pal et al., 2003), with the cDNA of the modified cameleon-2 probe that contains the human metallothionein (MT) IIa cDNA flanked by the cDNA of two mutant green fluorescent proteins, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (Pearce et al., 2000). In brief, 1.5 μg of cDNAs was diluted in 50 μl of Opti-Mem I medium and combined with 50 μl of Opti-Mem I medium containing 4 μl of Lipofectamine 2000. Complexes were allowed to form for 30 min at RT before addition to the cultures. Cells were maintained for 24-48 hr at 37°C, 5% CO₂ before imaging. Dual emission imaging was accomplished by using a 440 DF20 excitation filter, a 455 DRLP dichroic mirror, and alternating emission filters (480DF30 for ECFP; 535DF25 for EYFP). With the cameleon-2 construct, it has been shown that an increase in emission signal at 480 nm that is accompanied by a decrease in emission at 535 nm is reflective of a loss of bound Zn²⁺ ions by the metallothionein construct [i.e., less fluorescence resonance energy transfer (FRET)] (Pearce et al., 2000). Positively transfected cells were monitored for changes in the 535/480 emission intensity ratio after exposure to Zn²⁺ plus pyrithione or the thiol oxidant 2,2′-dithiodipyridine (DTDP), in addition to peroxynitrite. DTDP is an agent that we have shown previously is able to liberate intracellular Zn²⁺ from neurons (Aizenman et al., 2000) and can liberate all seven Zn²⁺ atoms from metallothionein in cell-free assays (Maret and Vallee, 1998).

Assay of intracellular ATP

The Bioluminescent Somatic Cell Assay kit from Sigma (catalog #FL-ASC) was used for assay of ATP, as described previously (Rosenberg et al., 2000; Zhang and Rosenberg, 2002). At selected time points after addition of drug or vehicle, medium was removed and then replaced with 200 μl of cell lysis agent. After brief agitation, the medium was transferred to microcentrifuge tubes that contained 200 μl of water. The tubes were placed on wet ice until assay. ATP assay mix was used at 25-fold dilution, and 100 μl of the extract was assayed. The proteins in cell lysates were quantified using a protein assay kit (Bio-Rad, Hercules, CA).

Gluthathione measurement

Intracellular levels of glutathione were assayed using monochlorobimane (MCB), as described previously (Wang and Joseph, 2000). MCB reacts with gluthathione (GSH) in a reaction catalyzed by glutathione S-transferase (GST) to form a highly fluorescent adduct. Briefly, cultures were exposed to SIN-1 (1 mm) for 30 min and then washed twice with HBSS containing 0.1% BSA. Neurons were then lysed with 0.2% Triton X-100 in EBSS. In 100 μl cell lysates, MCB (50 μm) and GST (0.4 U/ml) were added and incubated at 37°C for 20 min. Fluorescence was measured with the excitation wavelength at 360 nm and emission wavelength at 480 nm. The concentration of GSH was calculated based on the standard curve of GSH and on the protein concentration of the sample.

Assay of 12-lipoxygenase activity

12-Lipoxygenase (12-LOX) activity was measured by quantifying the major metabolic product of 12-LOX, 12-hydroxyeicosatetraenoic acid (12-HETE), by using an ELISA kit (Assay Designs, Ann Arbor, MI). Neurons were treated with SIN-1 or ZnCl₂ for 60 min. After the cultures were washed twice with HBSS containing 0.1 mg/ml BSA, arachidonic acid (AA; 20 μm) in EBSS was added to the culture, which was then incubated for 30 min. The medium was then collected, extracted, and assayed for 12-HETE according to the manufacturer's protocol. The concentration of 12-HETE in each sample was normalized to the protein concentration of the sample.

Measurement of intracellular free-radical generation

Intracellular free-radical generation was evaluated with dihydrorhodamine 123 (Rho123) (Royall and Ischiropoulos, 1993). At 2 hr after zinc exposure (90 min), cells were loaded with Rho123 (10 μm) for 20 min at 37°C and then washed once with EBSS. Fluorescent imaging of oxidized Rho123 was performed immediately using digital fluorescence microscopy with a 20× objective (excitation at 490 nm and emission at 515 nm). All of the images were taken with the same microscope settings.

Assay of the mitochondrial membrane potential

JC-1 (Molecular Probes) was used as an indicator for mitochondrial membrane potential (ψm), as described previously (Salvioli et al., 1997). This carbocyanine exists in a monomeric form in the cytosol, emitting at 527 nm (green) when ψm is low. When JC-1 is concentrated by actively respiring mitochondria, it forms aggregates that are associated with a large shift in emission (590 nm) (red). A final concentration of 1 μg/ml JC-1 was added directly into the culture at 5 hr after 90 min of SIN-1 treatment. For the drug-treated groups, drugs were added during and after 90 min of SIN-1 exposure. At 20 min after the loading with JC-1, cells were visualized using digital fluorescence microscopy with a 20× objective. The green fluorescence and red fluorescence were detected using two optical filters designed for fluorescein and tetramethylrhodamine, respectively. Quantitation of ψm changes in neurons treated with SIN-1 was also performed, using a fluorescent plate reader. The green fluorescence of monomeric JC-1 was read with excitation wavelength at 485 nm and emission wavelength at 530 nm. The red fluorescence of the aggregated JC-1 was read with excitation wavelength at 485 nm and emission wavelength at 590 nm. The percentage decrease in the 590/530 emission intensity ratio in SIN-1-treated neurons compared with control neurons reflects the loss of ψm.

Assay of caspase-3 activation

The activation of caspase-3 was examined using a caspase-3 inhibitor (DEVD-FMK) conjugated to FITC as the fluorescent in situ marker in living cells (catalog #QIA91; Oncogene, San Diego, CA). FITC-DEVD-FMK is cell permeable, nontoxic, and irreversibly binds to activated caspase-3 in apoptotic cells. At 7 hr after exposure of neurons to SIN-1 (1 mm for 90 min) or zinc (75 μm for 90 min) or at 5 hr after staurosporine (STS) (1 μm) exposure, the cultures were washed twice with HBSS containing 0.1% BSA and then incubated with FITC-DEVD-FMK (1 μl/ml in EBSS) for 1 hr in a 37°C incubator with 5% CO₂. The FITC label in apoptotic cells was examined immediately under fluorescence microcopy with excitation and emission at 485 and 535 nm, respectively. All of the images were taken with the same microscope settings.

The activity of caspase-3 was measured following the methods described by Stefanis et al. (1999). At 7 hr after exposure of neurons to SIN-1 (1 mm for 90 min) or at 5 hr after STS (1 μm) exposure, the cultures were washed twice with HBSS containing 0.1% BSA and then lysed with a buffer containing the following (in mm): 25 HEPES, pH 7.5, 5 EDTA, 1 EGTA, 5 MgCl₂, 2 DTT, and a mixture of protease inhibitors (catalog #1836153; Roche, Indianapolis, IN). The cell lysates were put on ice for 20 min and then sonicated on ice. The lysates were centrifuged for 60 min at 16,000 × g. The protein concentrations in the supernatant were determined using a protein assay kit (Bio-Rad). Ten micrograms of protein were incubated in a buffer of 25 mm HEPES, pH 7.5, 10% sucrose, 0.1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid, and 10 mm DTT with the caspase-3 substrate Ac-DEVD-AFC (15 μm) at 37°C for 1 hr. The intensity of fluorescence of each solution was measured by a Hitachi fluorescence spectrophotometer using an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

Western blot analysis of p38 activation

At 1 hr after SIN-1 treatment, neurons were placed on ice. After media aspiration, cells were washed once with ice-cold PBS and lysed with lysis buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm glycerolphosphate, 1 mm Na₃VO₄, and 1 mm PMSF. An aliquot of cell lysate was removed for later protein determination. Cell lysate was mixed with Laemmli buffer, boiled for 5 min, and stored at -20°C. Equal amounts of proteins were separated by SDS-PAGE and electrotransfered to polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in TBS containing 0.1% Tween 20 (TBST) for 1 hr and then incubated overnight at 4°C with the primary antibody for phosphorylated p38 (Biosource, Camarillo, CA) or total p38 (Cell Signaling, Beverly, MA) diluted at 1:1000 in TBST containing 5% BSA. After washing four times with TBST, the membrane was incubated for 1 hr at RT with an HRP-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:4000. Membranes were washed again as above and visualized by ECL according to the manufacturer's protocol.

Statistics

Statistical significance was assessed using ANOVA with the Tukey-Kramer post hoc multiple comparison test. Statistical analysis was performed using the Instat program from GraphPad Software (San Diego, CA). Representative experiments are shown unless noted otherwise. Experiments were performed with triplicate samples, and the data are expressed as mean ± SD or SEM, as appropriate. All experiments were repeated at least three times.

N,N,N′,N′-tetrakis (2-pyridyl-methyl)ethylenediamine, a zinc chelator, protected against neurotoxicity induced by peroxynitrite

SIN-1 is a commonly used peroxynitrite generator, because it can simultaneously generate nitric oxide and superoxide in aerobic conditions (Hogg et al., 1992). Administration of SIN-1 (1 mm) to rat forebrain neurons in culture caused severe neurotoxicity (Fig. 1A). In five experiments, the neuronal survival after 24 hr was 10 ± 5.5% of control levels. N,N,N′,N′-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN), a zinc chelator that has been shown to protect against peroxynitrite-induced toxicity in thymocytes (Virag and Szabo, 1999), was able to attenuate SIN-1-induced toxicity to neurons (Fig. 1A). Neuronal survival in the presence of TPEN (1 μm) was 68 ± 5% (n = 5), which is significantly higher (p < 0.001) than in the SIN-1-treated group. TPEN also blocked the neurotoxicity induced by authentic peroxynitrite (100 μm) (Fig. 1B). The EC₅₀ value of TPEN protection was 0.95 ± 0.03 μm (n = 3). To test the pathophysiological relevance of the protective effect of TPEN against peroxynitrite, we used a model system in which the formation of peroxynitrite might occur in neurons as a result of reaction of exogenous nitric oxide with superoxide generated intracellularly in response to an excitotoxic stimulus (Hewett et al., 1994). It has been shown previously that exposure of neurons to NMDA can generate superoxide (Hewett et al., 1994; Patel et al., 1996; Sengpiel et al., 1998). Therefore, when a nitric oxide donor is applied to the cultures treated with NMDA, nitric oxide may react with superoxide to form peroxynitrite intracellularly. We used conditions of exposure to NMDA (24 hr; 20 μm) or the nitric oxide donor DETA/NO (24 hr; 300 μm) that did not, by themselves, result in toxicity. However, when NMDA and DETA/NO were coapplied, a nearly complete loss of neurons was observed (Fig. 1C). We found that the free-radical spin traps [N-t-butyl-α-phenylnitrone (PBN) and 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO)] and MnTBAP, a scavenger of superoxide as well as peroxynitrite, completely blocked neurotoxicity induced by a 4 hr exposure to NMDA plus DETA/NO (Fig. 1D). The toxicity induced by NMDA and DETA/NO was also significantly attenuated by TPEN at 10 μm (Fig. 1E).

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Figure 1.

TPEN is protective against neurotoxicity induced by peroxynitrite. A, TPEN protected against SIN-1-induced neurotoxicity. Neurons were exposed to SIN-1 (1 mm) with and without TPEN (1 μm) for 24 hr, and then the neurotoxicity was examined. ^**p < 0.01 was obtained when the TPEN-treated group was compared with the group treated with SIN-1 alone. a, Control; b, SIN-1; c, TPEN; d, TPEN/SIN-1. One representative experiment of five performed is shown. B, TPEN protected against neurotoxicity induced by authentic peroxynitrite. Neurons were exposed to authentic peroxynitrite (100 μm) in the absence or presence of various concentrations of TPEN (1, 3, 10 μm) for 24 hr, and then the neurotoxicity was examined. ^**p < 0.01 and ^***p < 0.001 were obtained when the TPEN-treated groups were compared with the group treated with peroxynitrite alone. One representative experiment of three performed is shown. C, Nitric oxide has a synergistic effect with NMDA in producing neurotoxicity. Neurons were exposed to NMDA (N) (20 μm), DETA/NO (D) (300 μm), or NMDA plus DETA/NO (N+D) for 24 hr, and then the neurotoxicity was examined. ^***p < 0.001 was obtained when the NMDA plus DETA/NO-treated group was compared with other groups. One representative experiment of six performed is shown. D, Free-radical spin traps blocked neurotoxicity induced by NMDA plus DETA/NO. Neurons were exposed to NMDA (20 μm) plus DETA/NO (300 μm) in the absence or presence of free-radical spin traps (10 mm PBN or 1 mm TEMPO) or MnTBAP (100 μm) for 4 hr, and then the neurotoxicity was examined. ^***p < 0.001 was obtained when the drug-treated groups were compared with the group treated with NMDA plus the DETA/NO-treated group alone. One representative experiment of four performed is shown. E, TPEN protected against neurotoxicity induced by NMDA plus DETA/NO. Neurons were exposed to NMDA (20 μm) plus DETA/NO (300 μm) for 1 hr; the toxicity was examined at 24 hr. ^*p < 0.05 was obtained when the TPEN-treated group was compared with the group treated with NMDA plus DETA/NO alone. One representative experiment of three performed is shown. CON, Control. Error bars represent SD.

TPEN is not only a chelator of zinc, it may also chelate other divalent cations, with high affinity for copper and iron and low affinity for calcium and magnesium (Jornot et al., 1998; Virag and Szabo, 1999; Armstrong et al., 2001). An equimolar concentration of ZnCl₂ (1 μm) completely eliminated the neuroprotection of TPEN (1 μm) against SIN-1 (1 mm) toxicity (Fig. 2A). However, an equimolar concentration of FeCl₂ had no effect on the protective effect of TPEN (Fig. 2A). Because TPEN has a higher affinity for copper than zinc (K_D, 10^-20 m for Cu²⁺ and 10^-15 m for Zn²⁺) (McCabe et al., 1993), we examined the effect of a specific copper chelator on the toxicity induced by peroxynitrite. Bathocuprione at 100 μm (Armstrong et al., 2001) was not protective against SIN-1 (Fig. 2B), authentic peroxynitrite, or NMDA plus DETA/NO (data not shown). It is possible that the failure of bathocuprone to protect against peroxynitrite-induced toxicity is attributable to its poor ability to cross membranes. To exclude this possibility, we compared the effect of bathocuprone with a known extracellular metal chelator, EDTA, on neurotoxicity induced by SIN-1. EDTA at 500 μm completely blocked SIN-1 toxicity, but bathocuprone at 500 μm had no effect (data not shown). If the protective effect of EDTA was attributable to its chelation of copper, then bathocuprone, a specific copper chelator, should also be protective, regardless of whether it gets into neurons. These results suggested that copper does not mediate peroxynitrite-induced neurotoxicity. EBSS contains a much higher concentration of calcium (1.8 mm) and magnesium (0.8 mm) than the concentration of TPEN (1 μm) used in these experiments. Therefore, the protective effect of TPEN is probably not attributable to the chelation of calcium or magnesium. These results suggested that TPEN exerts its protective effect by chelating zinc released from intracellular stores.

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Figure 2.

Peroxynitrite-induced neurotoxicity is mediated by zinc but not by iron or copper. A, TPEN (1 μm) protection against SIN-1-induced neurotoxicity was eliminated by coadministration of an equimolar concentration of ZnCl₂ (T/Zn) (1 μm) but not of FeCl₂ (T/Fe) (1 μm). ^**p < 0.01 was obtained when the TPEN-treated group was compared with the group treated with SIN-1 alone. One representative experiment of three performed is shown. B, Copper chelator had no effect on SIN-1-induced neurotoxicity. BCP (100 μm) represents bathocuproine. One representative experiment of three performed is shown. CON, Control. Error bars represent SD.

Peroxynitrite induced intracellular free zinc accumulation

Newport Green and FluoZin-3 have been used as fluorescent Zn²⁺ indicators to monitor the intracellular free Zn²⁺ concentrations under oxidative stress (Aizenman et al., 2000; Gee et al., 2002). To make sure that these indicators do not directly react with peroxynitrite, a strong oxidative agent, we first tested them in the presence of peroxynitrite (100 μm) in EBSS without cells present. Peroxynitrite caused an increase in the fluorescence of Newport Green independent of the Zn²⁺ levels (Fig. 3A). No reaction occurred between peroxynitrite and FluoZin-3 (Fig. 3B). Therefore, FluoZin-3 was used to examine whether peroxynitrite could induce zinc release from neurons.

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Figure 3.

Peroxynitrite induced intracellular free zinc accumulation. A, B, Interaction of peroxynitrite with the fluorescent zinc indicators Newport Green and FluoZin-3. A, Peroxynitrite (ONOO) induced an increase in Newport Green fluorescence in EBSS. ^***p < 0.001 when compared with the control (CON). B, Peroxynitrite did not interact with FluoZin-3. The fluorescence intensity of FluoZin-3 was similar in EBSS with or without peroxynitrite. C, TPEN blocked the increase in FluoZin-3 fluorescence in neurons induced by peroxynitrite. Neurons were preloaded with FluoZin-3 for 30 min, washed, and then treated with peroxynitrite in the absence or presence of TPEN. a, Control; b, peroxynitrite (100 μm); c, peroxynitrite with TPEN (10 μm). D-F, FRET demonstrated the binding or liberation of Zn²⁺ from MT in neurons. Cortical neurons were transfected with an MT-FRET construct flanked by ECFP and EYFP. Twenty-four hours after transfection, cells (n = 52) were monitored for changes in the 535/480 emission intensity ratio before, during, and after exposure to 10 μm Zn²⁺ plus 20 μm pyrithione (a zinc ionophore) (D), 3 μm Zn²⁺ plus 20 μm pyrithione followed by 300 μm DTDP (E), or 140 μm peroxynitrite (F). An increase in the 535/480 ratio reflects the binding of Zn²⁺ to protein not saturated by the metal, whereas a decrease in the ratio indicates the liberation of Zn²⁺ from MT-FRET. Inset in D shows the raw, individual emission measurements used to obtain the ratio for the cell shown in that figure. Inset in F denotes the 535/480 ratio (mean ± SEM; n = 7) immediately before (control) and 5 min after the peroxynitrite treatment (^*p < 0.001; two-tailed paired t test). Error bars represent SD.

Neurons were preloaded with FluoZin-3 (1 μm) for 30 min, washed twice with HBSS containing 0.1% BSA, and then treated with peroxynitrite (100 μm) for 30 min in the absence or presence of TPEN (10 μm). Peroxynitrite induced an increase in zinc fluorescence in neurons, and the effect was completely diminished by coapplication of TPEN (Fig. 3C).

We also assayed for intracellular zinc release induced by peroxynitrite using intramolecular FRET. Thiol oxidants and nitric oxide have been shown to induce the release of Zn²⁺ from MTs in a variety of experimental paradigms (Kroncke et al., 1994; Berendji et al., 1997; Aravindakumar et al., 1999; Aizenman et al., 2000; Pearce et al., 2000; Bossy-Wetzel et al., 2004; Pal et al., 2004). Pearce et al. (2000) described a modification of a chimeric “cameleon” probe (Miyawaki et al., 1997), with which conformational changes in MT, resulting from metal binding and unbinding, could be detected by FRET. We used this probe in transfected neurons to determine whether peroxynitrite could induce the release of Zn²⁺ from metallothionein. Because this probe had not been used previously in neurons, we first characterized its behavior in these cells. We reasoned that as a result of the overexpression of MT-FRET in transfected neurons, a substantial portion of the expressed protein would be in the apo or thionein form and that addition of Zn²⁺ would result in an increase in FRET (i.e., an increase in the 535/480 emission intensity ratio resulting from the binding of Zn²⁺; see Materials and Methods). Indeed, this was exactly what was observed after Zn²⁺ exposure (Fig. 3D). Conversely, FRET was readily decreased (i.e., the 535/480 emission ratio decreased after Zn²⁺ unbinding) after exposure to the thiol-oxidizing agent DTDP, as expected from previous studies (Maret and Vallee, 1998; Aizenman et al., 2000) (Fig. 3E). From these data, it appeared that the MT-FRET protein was behaving as predicted (i.e., on binding zinc, FRET increased, whereas releasing zinc resulted in a decrease in FRET). We thus were in a position to be able to test whether an application of peroxynitrite would result in a decrease in FRET, reflective of Zn²⁺ liberation from the MT-containing chimeric indicator. We observed that addition of freshly prepared peroxynitrite (140 μm) produced a relatively slow, albeit reproducible and pronounced decline in the 535/480 emission ratio (Fig. 3F). The time course of the change in FRET is likely attributable to the fact that peroxynitrite is known to interact relatively slowly with most biological molecules (Beckman and Koppenol, 1996). After removal of peroxynitrite, the FRET signal continued to decline for many minutes, suggesting that longer-lived peroxynitrite oxidation products (Beckman and Koppenol, 1996) may contribute to MT oxidation and Zn²⁺ liberation.

TPEN had no effect on peroxynitrite-induced ATP depletion

An increasing body of evidence suggests that zinc can cause inhibition of cellular energy production (Dineley et al., 2003). Sheline et al. (2000) showed that elevation of extracellular zinc could inhibit glycolysis in cultured neurons and that pyruvate, a downstream substrate of glycolysis, had a protective effect. Here, we tested whether pyruvate could block ATP depletion and neurotoxicity induced by peroxynitrite. Pyruvate at 10 mm did not block neurotoxicity induced by 24 hr of SIN-1 exposure (Fig. 4A). However, when neurotoxicity was assessed at 24 hr after a short period (1-3 hr) of SIN-1 exposure, pyruvate had a significant protective effect (Fig. 4B). Pyruvate also blocked ATP depletion after 1-2 hr of SIN-1 exposure but failed to block ATP depletion when neurons were exposed to SIN-1 for 3 hr (Fig. 4C), although significant neuroprotection was observed at 24 hr after 3 hr of SIN-1 exposure (Fig. 4B). To further test whether ATP depletion induced by SIN-1 was mediated by the release of zinc from intracellular stores and responsible for the neurotoxicity, we examined the effect of TPEN on ATP depletion and neurotoxicity induced by a short period of SIN-1 exposure. TPEN at 1 μm had no effect on ATP depletion induced by SIN-1 (1 mm for 1 hr) (Fig. 4D). TPEN at 3 or 10 μm also failed to block ATP depletion (data not shown). However, TPEN (10 μm) completely blocked neurotoxicity at 24 hr after 1 hr of SIN-1 exposure (data not shown). These results suggested that ATP depletion induced by peroxynitrite might be irrelevant to the neurotoxicity of peroxynitrite and intracellular zinc release.

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Figure 4.

Effect of pyruvate on SIN-1-induced ATP depletion and neurotoxicity. A, Pyruvate did not prevent neurotoxicity induced by 24 hr of SIN-1 exposure. Neurons were exposed to SIN-1 (1 mm) in the absence or presence of pyruvate (10 mm) for 24 hr, and then the toxicity was examined. PYR/S, Pyruvate plus SIN-1. One representative experiment of three performed is shown. B, Pyruvate (PYR; 10 mm) protected against neurotoxicity at 24 hr after 1-3 hr of SIN-1 exposure. ^**p < 0.01 and ^***p < 0.001 were obtained when pyruvate-treated groups were compared with the corresponding groups treated with SIN-1 alone. One representative experiment of three performed is shown. C, Pyruvate (10 mm) blocked ATP depletion induced by 1-2 hr of SIN-1 (1 mm) exposure but not by 3 hr of SIN-1 exposure. The cellular levels of ATP were examined in neurons treated with SIN-1 or SIN-1 together with pyruvate at 1, 2, and 3 hr. ^***p < 0.001 was obtained when pyruvate-treated groups were compared with the corresponding groups treated with SIN-1 alone. One representative experiment of three performed is shown. D, TPEN did not block ATP depletion induced by SIN-1. Neurons were exposed to SIN-1 with or without TPEN for 1 hr, and then the cellular levels of ATP were examined. One representative experiment of three performed is shown. CON, Control; TPEN/S, TPEN plus SIN-1. Error bars represent SD.

TPEN did not block SIN-1-induced GSH depletion

It has been reported that zinc causes GSH depletion in mixed cortical cultures containing neurons and astrocytes, which later leads to neuronal death (Chen and Liao, 2003). We next tested whether peroxynitrite causes GSH depletion and, if so, whether this depletion could be blocked by TPEN. SIN-1 (1 mm) exposure to neurons for 30 min caused a significant drop in GSH (p < 0.001) that was not blocked by TPEN at 10 μm (Fig. 5). In four experiments, the cellular levels of GSH in SIN-1 and TPEN plus SIN-1 groups were 49 ± 7 and 47 ± 9% of control (p < 0.001), respectively. This result suggested that GSH depletion was not induced by zinc. Zinc release and GSH depletion may both contribute to the neurotoxicity induced by peroxynitrite.

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Figure 5.

TPEN had no effect on GSH depletion induced by SIN-1. Neurons were exposed to SIN-1 (1 mm) in the absence or presence of TPEN (10 μm) for 30 min, and then the neurons were lysed and assayed for GSH activity. One representative experiment of four performed is shown. ^***p < 0.001 was obtained when the SIN-1 group was compared with the control group. CON, Control. Error bars represent SD.

12-LOX activation is involved in peroxynitrite-induced neurotoxicity

Glutathione depletion has been shown to cause 12-LOX activation (Li et al., 1997). It has been suggested that zinc can also participate in the activation of 12-LOX leading to free-radical generation and neuronal injury (Aizenman et al., 2000; Du et al., 2002). We found that two 12-LOX inhibitors, AA861 at 10 μm (Li et al., 1997) and BMD-122 at 1 μm (Honn et al., 1994; Nie et al., 2000), did not block neurotoxicity induced by 24 hr of SIN-1 exposure (data not shown). However, they significantly blocked the neurotoxicity at 24 hr after a short period (1 hr) of SIN-1 exposure (Fig. 6A). In the presence of AA861 and BMD-122, neuronal survival was 73 ± 7 and 94 ± 5%, respectively, compared with neuronal survival in the presence of SIN-1 alone (37 ± 4%; p < 0.01; n = 5). To rule out the possibility that the protective effects of AA861 and BMD-122 might be attributable to their chelation of zinc, equimolar concentrations of zinc together with AA861 or BMD-122 were also tested and found to still protect against SIN-1-induced toxicity (data not shown). This result suggested that the metabolism of arachidonic acid via 12-LOX is involved in peroxynitrite-induced neurotoxicity. To examine the specificity of 12-LOX activation, we also examined the effect of two 5-LOX inhibitors, MK-886 (1 μm) and caffeic acid (30 μm), on the neurotoxicity induced by SIN-1 and found that both drugs had no protective effect (data not shown). The inhibitor of cyclooxygenase (COX), indomethacin, and the inhibitors of cytochrome P450, proadifen and clotrimazole, also had no protective effect (data not shown), suggesting that the metabolic pathways of arachidonic acid via COX and cytochrome P450 (Shimizu and Wolfe, 1990) were not involved in the neurotoxicity induced by peroxynitrite. To further examine the involvement of 12-LOX in peroxynitrite-induced neurotoxicity, we assayed the cellular levels of 12-LOX activity after 60 min of SIN-1 (1 mm) exposure to neurons. SIN-1 caused significant activation of 12-LOX (Fig. 6B). TPEN at 10 μm blocked 12-LOX activation (Fig. 6 B). In four experiments, the levels of 12-LOX activity were 170 ± 16% of the control levels (p < 0.01).

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Figure 6.

SIN-1-induced neurotoxicity was mediated by 12-LOX activation. A, 12-LOX inhibitors blocked neurotoxicity induced by SIN-1. Neurons were exposed to SIN-1 (1 mm) for 1 hr, and the toxicity was examined at 24 hr. For AA861 (10 μm)- and BMD-122 (1 μm)-treated groups, the drugs were applied before, during, and after SIN-1 treatment. ^**p < 0.01 was obtained when drug-treated groups were compared with the group treated with SIN-1 alone. One representative experiment of five performed is shown. B, TPEN blocked 12-LOX activation induced by SIN-1. Neurons were exposed to SIN-1 (1 mm) in the absence or presence of TPEN (10 μm) for 60 min, and then the 12-LOX activity was examined. ^***p < 0.001 was obtained when the SIN-1-treated group was compared with the control group. ^##p < 0.01 was obtained when the TPEN-treated group was compared with the SIN-1 alone group. One representative experiment of four performed is shown. CON, Control. Error bars represent SD.

Zinc-induced neurotoxicity was mediated by activation of 12-LOX

The finding that TPEN blocked SIN-1-induced 12-LOX activation suggests that the liberation of intracellular zinc by peroxynitrite is upstream of 12-LOX. Therefore, inhibition of 12-LOX activity might be able to block the toxicity of exogenously applied zinc. Indeed, zinc caused concentration-dependent neurotoxicity at 24 hr after neurons were exposed to ZnCl₂ for 90 min (Fig. 7A). The EC₅₀ value of zinc-induced neurotoxicity was 58 ± 5 μm (n = 5). The toxicity induced by ZnCl₂ (75 μm) was significantly attenuated by AA861 (10 μm) (Fig. 7B). When ZnCl₂ was applied to neurons, a significant increase in 12-LOX activity was observed (Fig. 7C). In three experiments, the 12-LOX activity was 197 ± 14% of control levels (p < 0.01).

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Figure 7.

12-LOX activation is involved in neurotoxicity induced by exogenous zinc. A, Concentration dependence of zinc toxicity to neurons. Neurons were exposed to a various concentration of ZnCl₂ (50, 75, and 100 μm) for 90 min, and the neurotoxicity was examined at 24 hr. One representative experiment of five performed is shown. B, 12-LOX inhibitor blocked neurotoxicity induced by zinc. Neurons were exposed to ZnCl₂ (75 μm) for 90 min, and the toxicity was examined at 24 hr. For the drug-treated group, AA861 (10 μm) was applied before, during, and after zinc treatment. ^*p < 0.05 was obtained when the drug-treated group was compared with the group treated with zinc alone. One representative experiment of three performed is shown. C, Zinc induced 12-LOX activation. Neurons were exposed to ZnCl₂ (75 μm) for 1 hr, and then the 12-LOX activity was examined. ^**p < 0.01 was obtained when the zinc-treated group was compared with the control group. One representative experiment of three performed is shown. D, Inhibition of 12-LOX blocked ROS generation induced by zinc. At 2 hr after zinc exposure (75 μm for 90 min), neurons were loaded with DHR-123 for 20 min, and the image was examined under a fluorescent microscope. a, Control; b, ZnCl₂; c, AA861/ZnCl₂; d, vitamin E/ZnCl_2. One representative experiment of three performed is shown. CON, Control. Error bars represent SD.

Oxidation of arachidonic acid by 12-LOX can generate lipid peroxides (Chamulitrat et al., 1991; Dikalov and Mason, 2001) and cause neurotoxicity (Li et al., 1997). At 2 hr after 90 min of ZnCl₂ exposure, increased ROS generation was observed in neurons (Fig. 7D), indicated by enhanced DHR-123 fluorescence. AA861 significantly attenuated ROS generation after zinc exposure (Fig. 7D). BMD-122 also had a similar effect (data not shown). As a positive control, we found that the commonly used antioxidant vitamin E (10 μm) also blocked ROS generation induced by zinc (Fig. 7D).

Peroxynitrite-induced mitochondrial membrane depolarization was partially blocked by zinc chelation and 12-LOX inhibition

It has been reported that both peroxynitrite and zinc can cause mitochondrial membrane depolarization, permeability transition pore opening, and the release of factors capable of initiating apoptosis (Jiang et al., 2001; Sensi et al., 2003; Szabo, 2003). Whether the mitochondrial membrane depolarization induced by peroxynitrite is mediated by intracellular zinc release is unknown. We found that SIN-1 induced a significant loss of ψm, indicated by enhanced green fluorescence of JC-1 and diminished red fluorescence formed by JC-1 aggregates in mitochondria of neurons compared with control neurons (Fig. 8). TPEN and BMD-122 partially attenuated the loss of ψm (Fig. 8). In three experiments, the loss of ψm in the presence of SIN-1 was 67 ± 4%; when TPEN and BMD-122 were coapplied, the loss of ψm was 27 ± 3% (p < 0.001) and 48 ± 3% (p < 0.05), respectively.

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Figure 8.

The zinc chelator and a 12-LOX inhibitor attenuated the mitochondrial membrane depolarization induced by peroxynitrite. TPEN (10 μm) and BMD-122 (1 μm) attenuated mitochondrial membrane depolarization induced by SIN-1. A, At 5 hr after SIN-1 exposure (1 mm for 90 min), neurons were loaded with JC-1 for 20 min, and the image was examined under a fluorescent microscope. a, Control; b, SIN-1; c, TPEN/SIN-1; d, BMD-122/SIN-1. The percentage decrease of JC-1 ratio reflecting the loss of mitochondrial membrane potential is shown in B. ^*p < 0.05 and ^***p < 0.001 were obtained when BMD-122- or TPEN-treated neurons were compared with neurons treated with SIN-1 alone. One representative experiment of three performed is shown. Error bars represent SD. TPEN/S, TPEN/SIN-1; BMD/S, BMD-122/SIN-1.

Peroxynitrite- or zinc-induced neuronal apoptosis

The duration of SIN-1 exposure is critical for the mode of cell death (Bonfoco et al., 1995; Zhang and Rosenberg, 2004). We examined whether caspase activation is involved in the neurotoxicity induced by a long period (24 hr) as well as a short period (1 hr) of exposure to SIN-1. A caspase-3 inhibitor, Ac-DEVD-CMK at 100 μm, had no effect on neurotoxicity induced by 24 hr of SIN-1 exposure (Fig. 9A) but significantly blocked neurotoxicity at 24 hr after 1 hr of SIN-1 exposure (Fig. 9B). Neuronal survival in the presence of Ac-DEVD-CMK was 78 ± 6% (n = 4) of control, which was significantly higher (p < 0.001) than neuronal survival with SIN-1 treatment alone (29 ± 6%). A pancaspase inhibitor, zVAD-FMK (100 μm), also failed to block the neurotoxicity induced by SIN-1 after 24 hr of exposure (Fig. 9A). However, when neurons were exposed to SIN-1 for 1 hr, the toxicity at 24 hr was blocked by zVAD-FMK (Fig. 9B). Neuronal survival in the presence of zVAD-FMK was 52 ± 7%, significantly higher than in the SIN-1-treated group (p < 0.05; n = 4). Ac-DEVD-CMK and zVAD-FMK also protected against neurotoxicity at 24 hr after 90 min of zinc (75 μm) exposure (Fig. 9C). Consistent with these pharmacological data, caspase activation was observed at 7 hr after 90 min of exposure of neurons to SIN-1 or zinc (Fig. 9D). As a positive control, the activation of caspase induced by staurosporine (1 μm for 5 hr), an apoptosis-inducing agent, was also examined (Fig. 9D). These results suggested that peroxynitrite causes caspase-dependent apoptosis, which is likely mediated by zinc release from intracellular stores.

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Figure 9.

Peroxynitrite or zinc induced caspase-3 activation. A, Caspase inhibitors did not block neurotoxicity induced by 24 hr of SIN-1 exposure. Neurons were exposed to SIN-1 for 24 hr, and then toxicity was examined. Ac-DEVD-CMK and zVAD-FMK were administered before and during SIN-1 exposure. B, Caspase inhibitors blocked neurotoxicity induced by short exposure of SIN-1. Neurons were exposed to SIN-1 for 90 min, and toxicity was examined at 24 hr. Ac-DEVD-CMK and zVAD-FMK were administered before, during, and after SIN-1 exposure. ^*p < 0.05 and ^***p < 0.001 were obtained when drug-treated groups were compared with the group treated with SIN-1 alone. One representative experiment of four performed is shown. C, Caspase inhibitors blocked neurotoxicity induced by zinc. Neurons were exposed to ZnCl₂ for 90 min, and toxicity was examined at 24 hr. Ac-DEVD-CMK and zVAD-FMK were administered before, during, and after ZnCl₂ exposure. ^**p < 0.01 and ^***p < 0.001 were obtained when drug-treated groups were compared with the group treated with ZnCl₂ alone. One representative experiment of four performed is shown. D, SIN-1 or ZnCl₂ induced caspase-3 activation. At 7 hr after SIN-1 exposure (1 mm for 90 min), neurons were loaded with FITC-DEVD-FMK for 1 hr, and then the image was examined under fluorescent microcopy. a, Control; b, SIN-1; c, ZnCl₂; d, staurosporine. One representative experiment of three performed is shown. CON, Control. Error bars represent SD.

Phosphorylation of p38 mitogen-activated protein kinase is downstream of zinc release and 12-LOX activation and upstream of caspase activation

Activation of mitogen-activated protein kinases (MAPKs) has been found to link oxidative stress and neurotoxicity (McLaughlin et al., 2001; Du et al., 2002; Choi et al., 2004). However, specific MAPKs, for example, extracellular signal-regulated kinase (ERK)42/44 or p38, might be involved in neuronal necrosis or apoptosis induced by different oxidizing agents (McLaughlin et al., 2001; Du et al., 2002). It has been shown previously that an oxidizing agent, DTDP, causes neuronal apoptosis via a Zn²⁺-p38-K⁺ efflux pathway (McLaughlin et al., 2001), whereas methylisothiazolinone, another oxidizing agent, causes nonapoptotic neuronal death via a Zn²⁺-12-LOX-ROS pathway (Du et al., 2002). We tested whether inhibition of ERK42/44 or p38 activation could block the neurotoxicity induced by SIN-1. 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580) (10 μm), an inhibitor of p38 MAPK (Saklatvala et al., 1996), significantly blocked the neurotoxicity induced by SIN-1 (Fig. 10A). Neuronal survival in the presence of SB203580 was 79 ± 4% of control (n = 3), significantly higher (p < 0.01) than the survival of neurons treated with SIN-1 alone (32 ± 4%). Neuronal survival in the presence of 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene (U0126), an inhibitor of ERK42/44 phosphorylation (Du et al., 2002), plus SIN-1 was 36 ± 2%, which is not significantly different from SIN-1 alone.

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Figure 10.

Peroxynitrite induced phosphorylation of p38 MAPK. A, Inhibition of p38, but not ERK42/44, blocked neurotoxicity induced by SIN-1. Neurons were exposed to SIN-1 for 1 hr, and toxicity was examined at 24 hr. SB203580 (SB) (10 μm) and U0126 (5 μm) were administered before, during, and after SIN-1 exposure. ^*p < 0.05 was obtained when the SB-treated group was compared with the SIN-1 group. One representative experiment of four performed is shown. B, SIN-1 induced phosphorylation of p38. Neurons were exposed to SIN-1 in the absence or presence of TPEN, AA861, and Ac-DEVD-CMK for 1 hr, and the phosphorylation of p38 was examined by immunoblot. SIN-1 (1 mm) induced significant phosphorylation of p38 (P-p38). TPEN (10 μm) and AA861 (10 μm) blocked the phosphorylation, but Ac-DEVD-CMK (DEVD) (100 μm) had no effect. One representative experiment of three performed is shown. C, Inhibition of zinc release, 12-LOX activation, and p38 phosphorylation blocked SIN-1-induced caspase activation. At 7 hr after SIN-1 exposure (1 mm for 90 min), neurons were lysed. After centrifugation, the caspase-3 activity in the supernatant was examined. ^***p < 0.001 was obtained when the SIN-1-treated group was compared with the control or the drug-treated groups. One representative experiment of three performed is shown. CON, Control. Error bars represent SD.

At 1 hr after SIN-1 exposure to neurons, significant elevation of p38 phosphorylation was observed (Fig. 10B). The activation of p38 was also observed when zinc or authentic peroxynitrite was used (data not shown). TPEN and AA861 completely blocked SIN-1-induced phosphorylation of p38, but the caspase-3 inhibitor Ac-DEVD-CMK had no effect (Fig. 10B). This result suggests that caspase activation is downstream of p38 activation. To further test this idea, we measured cellular caspase-3 activity 7 hr after neurons were exposed to SIN-1 (90 min exposure) in the absence or presence of TPEN, AA861, and SB203580 (Fig. 10C). The activity of caspase-3 was significantly elevated by SIN-1 and attenuated by the coapplication of TPEN, AA861, and SB203580 with SIN-1. As a positive control, Ac-DEVD-CMK completely blocked SIN-1-induced activation of caspase-3 (Fig. 10C). These results suggested that activation of p38 is downstream of zinc release and 12-LOX activation induced by peroxynitrite.