N-acetylcysteine selectively protects cerebellar granule cells from 4-hydroxynonenal-induced cell death

Neuroscience Research 55 (2006) 255–263 www.elsevier.com/locate/neures

N-acetylcysteine selectively protects cerebellar granule cells from 4-hydroxynonenal-induced cell death Motoki Arakawa, Nobuyuki Ushimaru, Nobuhiro Osada, Tetsuro Oda, Kumiko Ishige, Yoshihisa Ito * Department of Pharmacology, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-8555, Japan Received 13 January 2006; accepted 15 March 2006 Available online 3 May 2006

Abstract 4-Hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, has been shown to induce neurotoxicity accompanied by multiple events. To clarify mechanisms of neuroprotective compounds on HNE-induced toxicity, the protective effects of N-acetylcysteine (NAC), a-tocopherol (TOC), ebselen and S-allyl-L-cysteine (SAC) were compared in cerebellar granule neurons. The decrease in MTT reduction induced by HNE was significantly suppressed by pretreatment of the neurons with 1000 mM NAC or 10 and 100 mM TOC; however, lactate dehydrogenase (LDH) release and propidium iodide (PI) fluorescence studies revealed that neuronal death was suppressed by NAC but not by TOC. Treatment of these neurons with HNE resulted in a drastic reduction of mitochondrial membrane potential, and this reduction was also prevented by NAC but not by TOC. Ebselen and SAC, a garlic compound, were unable to protect these neurons against HNE-induced toxicity. Pretreatment with NAC also prevented HNE-induced depletion of intracellular glutathione (GSH) levels in these neurons. These results suggest that NAC, but not other antioxidants such as TOC, SAC and ebselen, exerts significant protective effects against HNE-induced neuronal death in cerebellar granule neurons, and that this neuroprotective effect is due, at least in part, to preservation of mitochondrial membrane potential and intracellular GSH levels. # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: 4-Hydroxynonenal; N-acetylcysteine; a-Tocopherol; Ebselen; S-allyl-L-cysteine; Neuronal death; Mitochondria; Glutathione; Cerebellar granule neurons

1. Introduction Oxidative stress has been shown to play a pivotal role in neuronal dysfunction and death in various neurodegenerative disorders, including spinocerebellar degeneration (SCD), Alzheimer’s disease (AD) and Parkinson’s disease (PD) (Jesberger and Richardson, 1991; Simonian and Coyle, 1996; Yamashita et al., 2000). Reactive oxygen species (ROS) are generated in several metabolic pathways, and a major source of ROS is the superoxide radical anion in mitochondria, which gives rise to hydrogen peroxide. Systems that detoxify ROS include the enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase, and the thiol tripeptide glutathione (GSH). 4-Hydroxynonenal (HNE) is an aldehydic product of membrane lipid peroxidation (Kruman

* Corresponding author. Tel.: +81 47 465 5832; fax: +81 47 465 5832. E-mail address: [email protected] (Y. Ito).

et al., 1997), which is reportedly associated with inhibition of the activity of several cellular functions, such as membrane transport, microtubule formation, and mitochondrial respiration (Keller et al., 1997b; Picklo et al., 1999; Neely et al., 2000). In addition, elevated levels of HNE have been reported in the cerebellum in AD patients (0.67 nmol/mg protein) compared with control (0.44 nmol/mg protein) (Markesbery and Lovell, 1998) and in plasma in AD patients (20.65 mM) compared with control (7.80 mM) (McGrath et al., 2001). An increase in HNE level in the cerebellum has been reported in SCD (Yamashita et al., 2000). HNE is normally detoxified by oxidization to 4hydroxynonenoate (HNEAcid) by the NAD+-dependent aldehyde dehydrogenases (ALDHs) and by conjugation with GSH (Murphy et al., 2003a,b; Meyer et al., 2004). N-acetylcysteine (NAC) has been shown to exert survivalpromoting actions in several cell systems (Shen et al., 1992; Ratan et al., 1994; Mayer and Noble, 1994). Cysteine is transported mainly by alanine-serine-cysteine (ASC) system, a ubiquitous system of Na+-dependent neutral amino acid

0168-0102/$ – see front matter # 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2006.03.008

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transport, in a variety of cells (Bannai and Tateishi, 1986); however NAC is a membrane-permeable cysteine precursor that does not require active transport and delivers cysteine to the cell in this own unique ways (Sen, 1997, 1998; Aoyama et al., 2006). NAC is an antioxidant and a free radical-scavenging agent that increases intracellular GSH, a major component of the pathways by which cells are protected from oxidative stress (Meister, 1988). The efficacy of NAC in protecting cells from apoptosis has generally been interpreted within the context of a mechanism involving oxidative stress (Ferrari et al., 1995). aTocopherol (TOC) is a lipid-soluble free radical scavenger in the vitamin E group. In studies using primary cultures and cell systems, TOC has been demonstrated to protect neurons from oxidative stress (Shea et al., 2002; Osakada et al., 2003). Ebselen, 2-phenyl-1,2-benzisoselenazol-3[2H]-one, is a lipidsoluble seleno-organic compound that exhibits both glutathione peroxidase-like and antioxidant activity (Mu¨ller et al., 1984; Wendel et al., 1984; Maiorino et al., 1988). The mechanism underlying the neuroprotection afforded by ebselen is still not completely understood; however, it is certainly related to its antioxidant and anti-inflammatory properties (Mu¨ller et al., 1984; Takasago et al., 1997). In cultured PC12 cells, ebselen has been shown to inhibit hydrogen peroxide (H2O2)-induced activation of c-Jun N-terminal kinase (JNK) (mitogen-activated protein (MAP) kinase group), which plays a pivotal role in neuronal death (Yoshizumi et al., 2002). S-allyl-L-cysteine (SAC) is one of the organosulfur compounds in aged garlic extract (AGE) obtained by extraction of garlic cloves for more than 10 months. SAC has been shown to have multiple biological activities, such as neurotrophic activity in cultured neurons (Moriguchi et al., 1997), antioxidant and radical scavenging effects (Yamasaki et al., 1994), a protective effect against ischemia and neurotoxicity in rat brain (Numagami and Ohnishi, 2001; Kosuge et al., 2003), anti-cancer activity (Thomson and Ali, 2003), and cholesterollowering activity (Yeh and Liu, 2001). In this study, in order to clarify the protective effects of antioxidants against HNE-induced neurotoxicity, we compared the effects of NAC, TOC, ebselen and SAC on HNE-induced toxicity in a culture containing predominantly a single class of neurons, the cerebellar granule cells, and very few nonneuronal cells (Thangnipon et al., 1983). We found that NAC protected cerebellar granule cells from HNE-induced neurotoxicity, whereas other agents could not protect these neurons but merely delayed the process leading to neuronal death. 2. Materials and methods 2.1. Materials The chemicals used in this study were: Hoechst 33258, MitoRed, LDHCytotoxic Test kit, trichloroacetic acid (TCA), 5,50 -dithiobis(nitrobenzoic acid) (DTNB) and glutathione, reduced form (Wako Pure Chemical, Osaka, Japan), HNE (Cayman Chemical, Ann Arbor, MI), NAC, [3-(4,5)-dimethylthiazol-2yl]-2,5-diphenyltetrazolium (MTT) and nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) (Sigma, St. Louis, MO), glutathione reductase (Roche Applied Science, Indianapolis, IN), propidium iodide (PI), monobromobimane (mBBr), basal medium essential (BME) and minimum

essential medium (MEM) (Invitrogen, Carlsbad, CA), fetal bovine serum (BioSciences Pty. Ltd., Thebarton SA, Australia), and SAC (kindly donated by Wakunaga Pharmaceutical Co. Ltd., Osaka, Japan)

2.2. Cell cultures All efforts were made to minimize the number of animals used and their suffering. All experiments with animals complied with the Ethical Guidelines for Animal Experiments of Nihon University. Cultures rich in cerebellar granule neurons were prepared from 7- to 8-day-old Wistar rats as reported previously (Ito et al., 1995, 1999; Kosuge et al., 2003). The cells were grown in BME supplemented with 25 mM potassium chloride, 10% fetal calf serum and 20 mg/ ml gentamicin on plastic cell wells (Corning, Corning, NY).

2.3. Drug treatment All of the experiments were carried out in vitro using primary cerebellar granule neurons that had been cultured for 7–8 days, and the assessment was performed after addition of HNE. For the evaluation of effects on NAC or TOC, these neurons were pretreated with NAC or TOC for 3 h. Then, the neurons were exposed to vehicle (0.2% EtOH) alone or HNE after transfer to fresh (serumfree) MEM without phenol red. As for the evaluation of effects of ebselen and SAC, these neurons were treated simultaneously with 30 mM HNE and ebselen or SAC for indicated time periods.

2.4. Assessment of cell viability Cell viability was determined by a double-staining procedure using Hoechst 33258 (H258, 25 mg/ml)/propidium iodide (PI, 25 mg/ml) as reported previously (Ito et al., 1999; Kosuge et al., 2003). The stained cells were excited at 330–385 nm, and the fluorescence was imaged with a standard epi-illumination fluorescence microscope with a cold-CCD digital camera system. Cells that were positively stained with PI were considered to be dead. The numbers of dead and surviving neurons were counted from photomicrographs.

2.5. LDH release assay Cytotoxicity was quantified by measurement of lactate dehydrogenase (LDH) released in the medium during exposure to drugs. LDH release was determined by using a LDH-Cytotoxic Test kit according to the manufacturers’ instructions. In this assay, nicotinamide adenine dinucleotide (NAD) is reduced to nicotinamide adenine dinucleotide, reduced form (NADH) through the conversion of lactate to pyruvate by LDH, and then NADH reduces tetrazolium dyes to formazan dyes in the presence of diaphorase. Briefly, an aliquot of 50 ml of culture supernatant was mixed with 50 ml of the LDH substrate mixture in a 96-well plate. After incubation for 0.5 h at room temperature, the reaction was stopped by adding 100 ml of 0.5 M HCl, and the absorbance was measured with a microplate reader (Bio-Rad model 550, Bio-Rad Laboratories, Hercules, CA) at a test wavelength of 570 nm. The background absorbance obtained from the culture medium was subtracted.

2.6. MTT reduction assay The method is based on the ability of living cells to reduce MTT tetrazolium salt into MTT formazan by the mitochondrial enzyme succinate deshydrogenase, as reported previously (Ishige et al., 2001). Briefly, the cells were incubated with MTT (0.15 mg/ml) for 4 h at 37 8C, and the reaction was stopped by adding a solubilization solution (50% dimethylformamide, 20% sodium dodecyl sulfate, pH 4.8). The next day, the amount of MTT formazan product was determined by measuring absorbance with a microplate reader (Bio-Rad model 550) at a test wavelength of 570 nm and reference wavelength of 655 nm.

2.7. Mitochondrial membrane potential Mitochondrial membrane potential was determined by a staining procedure using MitoRed (Romancino et al., 2004)/H258. For studies with MitoRed, an excitation wavelength of 510–550 nm and emission wavelength of 580 nm were

M. Arakawa et al. / Neuroscience Research 55 (2006) 255–263 used. Fluorescence images were obtained using a standard epi-illumination fluorescence microscope with a cold-CCD digital camera system. Cells that were positively stained with MitoRed were considered to have active mitochondrial membrane potential, and the numbers of such neurons were counted. The total numbers of neurons were defined by counting cells that were stained by H258.

2.8. Analysis of intracellular sulfhydryl level Intracellular sulfhydryl levels (GSH, NAC, etc.) were assessed using the fluorescent sulfhydryl probe, (mBBr) (40 mM), according to the method described previously (Kosuge et al., 2003). mBBr was added to the cultures for 45 min in serum-free medium without phenol red. Cells were excited at 330– 385 nm, and the fluorescence was imaged with a standard epi-illumination fluorescence microscope with a cold-CCD digital camera system (emission, 488 nm).

2.9. Measurement of total GSH Intracellular GSH levels were measured by the enzymatic recycling procedure (Tietze, 1969). Cells (1
106) were washed with phosphate-buffered saline (PBS), and lysed with ice-cold 10% TCA. The supernatant was obtained for assay after centrifugation (12,000
g for 15 min at 4 8C). Determination of GSH levels was carried out following removal of the protein precipitated from the supernatant solutions by extraction with diethylether. Residual traces of diethylether were removed by shaking under nitrogen gas. GSH levels were

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measured by the rate of colorimetric change of 625 mM DTNB at 415 nm in the presence of 0.6 units of glutathione reductase and 0.25 mM NADPH. GSH levels were adjusted to sample protein content.

2.10. Statistical analysis The data are given as the mean  S.E.M. Significance testing was performed using one-way analysis of variance (ANOVA) followed by Tukey’s test.

3. Results 3.1. Characterization of HNE-induced death in cerebellar granule neurons In order to investigate HNE-induced neuronal death, cultured cerebellar granule neurons were exposed to vehicle (0.2% EtOH) alone or various concentrations of HNE for 24 h after transfer to fresh serum-free MEM, and MTT assay and double-staining with H258/PI was performed. As shown in Fig. 1A, exposing these cultured neurons to HNE (1–50 mM) for 24 h resulted in a concentration-dependent decrease in MTT reduction. A significant decrease in MTT reduction was

Fig. 1. HNE-induced cell death in cerebellar granule neurons. (A) Cerebellar granule neurons were exposed to vehicle (0.2% EtOH) alone or various concentrations of HNE after transfer to fresh (serum-free) medium, then 24 h later, the levels of MTT reduction were examined. Each value represents the mean  S.E.M. for 12 separate experiments. *P < 0.001 as compared with HNE 0 mM (vehicle). (B) Phase contrast photomicrographs (upper panels) and double-staining with Hoechst 33258 (H258, 25 mg/ml)/propidium iodide (PI, 25 mg/ml) of cerebellar granule neurons (lower panels) 24 h after treatment with or without HNE. a and b, HNE 0 mM; c and d, HNE 10 mM; e and f, HNE 30 mM; g and h, HNE 50 mM.

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observed at concentrations of 20 mM HNE and higher. Photomicrographs of phase contrast and double-staining with H258/PI of these neurons 24 h after treatment with HNE (Fig. 1B) showed complete loss of viability at concentrations of 30 mM HNE and higher.

3.2. Effect of pretreatment with NAC and TOC on HNEinduced cell death To investigate whether NAC or TOC could protect against HNE-induced cell death, cerebellar granule neurons were

Fig. 2. Effect of pretreatment with NAC and TOC on HNE-induced cell death in cultured cerebellar granule neurons. (A and B) Cerebellar granule neurons were exposed to various concentrations of NAC or TOC. Three hours later, the cells were treated with or without 30 mM HNE after transfer to fresh (serum-free) medium. The levels of MTT reduction (A) and LDH release (B) were assessed 24 h after the treatment. All the results for NAC and TOC are expressed as the mean  S.E.M. for three separate experiments. *P < 0.05, **P < 0.01 as compared with NAC 0 mM or TOC 0 mM + HNE 0 mM. #P < 0.05, ##P < 0.01 as compared with NAC 0 mM or TOC 0 mM + HNE 30 mM. (C) Phase contrast photomicrographs and double-staining with Hoechst 33258 (H258, 25 mg/ml)/propidium iodide (PI, 25 mg/ml) of cerebellar granule neurons 24 h after treatment with or without HNE. a and b, HNE 0 mM; c and d, HNE 30 mM; e and f, NAC 1000 mM 3 h pretreatment + HNE 30 mM; g and h, TOC 100 mM 3 h pretreatment + HNE 30 mM.

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pretreated with NAC or TOC for 3 h, and then the cells were treated with 30 mM HNE after transfer to fresh MEM. As shown in Fig. 2, a drastic decrease in MTT reduction and a significant increase in LDH release was observed 24 h after addition of 30 mM HNE alone (Fig. 2A and B). The decrease in MTT reduction was significantly suppressed by 1000 mM NAC or TOC at 10 and 100 mM ((a) in Fig. 2A). Although the increase in HNE-induced LDH release was partially but significantly suppressed by 1000 mM NAC, TOC had no effect on the HNE-induced increase in LDH release (Fig. 2B). Photomicrographs of these neurons using phase contrast and double-staining with H258/PI 24 h after treatment with HNE (Fig. 2C) showed that pretreatment with 1000 mM NAC suppressed HNE-induced neuronal death completely, and that

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neuronal integrity was comparable to that of control cells (e and f in Fig. 2C). However, in the case of 100 mM TOC, photomicrographs showed many dying cells (purple) and dead cells (red) that were double-stained with H258/PI, neuronal integrity being completely destroyed after the HNE treatment (g and h in Fig. 2C). 3.3. Mitochondrial membrane potential Experiments were performed to investigate whether pretreatment with NAC or TOC could protect against the HNE-induced decrease in mitochondrial membrane potential in cultured cerebellar granule neurons. The neurons were pretreated with 1000 mM NAC or 100 mM TOC, and then

Fig. 3. HNE-induced decrease in mitochondrial membrane potential. (A) Cerebellar granule neurons were exposed to 1000 mM NAC or 100 mM TOC, then 3 h later, the cells were treated with 30 mM HNE after transfer to fresh (serum-free) medium. Eight hours after this treatment, neuronal survival (a) and mitochondrial membrane potential activity (b) were examined. The cells were stained with Hoechst 33258 (H258, 25 mg/ml)/propidium iodide (PI, 25 mg/ml) (a) or MitoRed (25 nM)/H258, MitoRed (b) at the time points indicated, and examined immediately with a standard epi-illumination fluorescence microscope. The numbers of dead and surviving cells (a) or cells with active mitochondrial membrane potential (b) were counted from photomicrographs. The results of H258/PI and MitoRed are each expressed as the mean  S.E.M. for 3–5 separate experiments. *P < 0.001 as compared with 0 h. #P < 0.001 as compared with HNE 30 mM alone. (B) Doublestaining with H258/PI and staining with MitoRed of cerebellar granule neurons. Photomicrographs taken 0 or 4 h after treatment with 30 mM HNE.

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Fig. 4. Effects of ebselen and SAC on HNE-induced cell death and mitochondrial membrane potential in cultured cerebellar granule neurons. These neurons were treated simultaneously with 30 mM HNE and ebselen (EB) or SAC after transfer to fresh (serum-free) medium, the 24 h after the treatment, neuronal survival (A) and mitochondrial membrane potential activity (B) were examined. The cells were stained with Hoechst 33258 (H258, 25 mg/ml)/propidium iodide (PI, 25 mg/ml) (A) or MitoRed (25 nM)/H258 (B) and examined immediately with a standard epi-illumination fluorescence microscope. The numbers of dead and surviving cells (A) or cells with active mitochondrial membrane potential (B) were counted from photomicrographs. Each value represents the mean  S.E.M. for 3–9 (A) and 3–8 (B) experiments. *P < 0.001 as compared with HNE 0 mM. #P < 0.05, ##P < 0.001 as compared with HNE 30 mM alone.

stained with H258/PI or MitoRed/H258 after exposure to 30 mM HNE. A time-course study revealed significant decreases in viability using H258/PI, and MitoRed-positive cells were observed 4 h and later after HNE treatment ((a) and (b) in Fig. 3A). This decrease in cell viability due to HNE

treatment was significantly suppressed by pretreatment with 1000 mM NAC and 100 mM TOC up to 8 h after exposure ((a) in Fig. 3A). Although NAC protected neurons from the HNEinduced decrease in mitochondrial membrane potential, TOC had no effect on the potential ((b) in Fig. 3A). Photomicrographs

Fig. 5. Effect of NAC against HNE-induced depletion of intracellular sulfhydryl levels in cerebellar granule neurons. Cerebellar granule neurons were exposed to 1000 mM NAC, then 3 h later, the cells were treated with or without 30 mM HNE for 4 h after transfer to fresh (serum-free) medium. (A) After these treatments, the cells were stained with mBBr (40 mM), and fluorescence was examined immediately with a standard epi-illumination fluorescence microscope. (B) After these treatments, intracellular GSH levels were measured. The results of intracellular GSH levels are each expressed as the mean  S.E.M. for five separate experiments. * P < 0.01, **P < 0.001 as compared with NAC 0 mM (0 h after removal of NAC), #P < 0.001 as compared with NAC 0 mM + HNE 30 mM (4 h after removal of NAC).

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of these neurons double-stained with H258/PI and MitoRed/ H258 4 h after exposure to HNE also revealed that mitochondrial membrane potential was almost completely retained in NACpretreated cells as compared with the cells treated with HNE alone (e and f in Fig. 3B). In contrast, pretreatment with TOC did not affect the HNE-induced reduction of mitochondrial membrane potential (g and h in Fig. 3B). 3.4. Effects of ebselen and SAC on HNE-induced cell death and mitochondrial membrane potential We also investigated whether ebselen, a lipid-soluble selenoorganic compound, and SAC could protect these neurons from HNE-induced neurotoxicity. These neurons were treated with 30 mM HNE and these agents simultaneously after transfer to fresh MEM (serum-free), and viability was measured using H258/PI 4 h after the treatment. As shown in Fig. 4(A), the HNE-induced decrease in viability was significantly suppressed by 1 and 10 mM ebselen 4 h, but not 24 h, after the treatment. SAC did not show any effects on the HNE-induced decrease in viability. Furthermore, neither ebselen nor SAC treatment exhibited a protective effect on mitochondrial membrane potential (Fig. 4(B)). 3.5. Effect of NAC on HNE-induced depletion of intracellular sulfhydryl and GSH levels Cerebellar granule neurons were exposed to 1000 mM NAC for 3 h, and then treated with 30 mM HNE after transfer to fresh BME. Four hours after this treatment, the cells were stained with mBBr (40 mM), which forms a fluorescent adduct with sulfhydryl groups (GSH, NAC, etc.). The compounds with a sulfhydryl base were completely depleted by 30 mM HNE. Pretreatment with 1000 mM NAC protected neurons against HNE-induced depletion of intracellular sulfhydryl levels (e and f in Fig. 5A). In addition, intracellular GSH levels were measured by the enzymatic recycling procedure. As shown in Fig. 5B, NAC also significantly protected neurons from HNEinduced depletion of intracellular GSH. 4. Discussion HNE has been shown to alter cellular signaling and to exhibit cytotoxicity through alkylation (Blanc et al., 1997; Keller et al., 1997a; Keller and Mattson, 1998; Awasthi et al., 2003). Site of action of HNE is multiple, and it is summarized in a report (Keller and Mattson, 1998). We have already shown that HNE-induced neurotoxicity is suppressed by Ac-DEVDCHO, a caspase-3 inhibitor, in cerebellar granule neurons (Ito et al., 1999) and hippocampal neurons (Kosuge et al., 2003), suggesting that HNE-induced neuronal death is attributable to activation of the caspase-3-dependent pathway. In order to compare the protective effects of NAC and TOC, these cells were pretreated with these agents, and HNE-induced cell death was examined by various methods. Present results showed crearly that NAC provided almost complete protection against HNE-induced injury while TOC could not protect these

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neurons but merely delayed the process leading to neuronal death (Fig. 2). Pretreatment of these neurons with TOC apparently reversed the HNE-induced decrease in MTT reduction activity, whereas the LDH release and morphological and H258/PI staining analyses revealed that TOC could not protect these neurons against HNE-induced injury. It has been shown that mitochondria play an important role in the disposition of aldehydes derived from ethanol, neurotransmitters, and lipid peroxidation. The mitochondrial detoxification of HNE can take the form of phases I and II processes. In the phase I process, brain mitochondria oxidize HNE to HNEAcid by the ALDHs, ALDH2 and ALDH5A (Murphy et al., 2003a,b; Meyer et al., 2004). The formation of HNEAcid is a major pathway of HNE detoxification, comprising approximately 30–40% of HNE metabolism in multiple systems including the heart, liver, and brain (Srivastava et al., 1998; Laurent et al., 2000; Murphy et al., 2003b). ALDH2 and ALDH5A have been shown to be localized in the mitochondrial matrix. Mitochondrial membrane potential is maintained by the respiratory chain, which contains four electron transporting complexes I–IV and one H+translocating adenosine triphosphate (ATP) synthetic complex (complex V), and electron transporting complex I has NADHubiquinone oxidoreductase activity (Raha and Robinson, 2000). Therefore, experiments were performed to compare the protective effects of NAC and TOC against HNE-induced mitochondrial injury in cerebellar granule neurons. The results demonstrated clearly that HNE-induced mitochondrial injury as measured by MitoRed was significantly suppressed by the pretreatment with NAC, but not by TOC ((b) in Fig. 3A), suggesting that the protective effect of NAC on mitochondrial function plays a significant role in its neuroprotective effects. These results also suggest that mechanistic differences exist between the effects of NAC and those of TOC, although both agents have been shown to have antioxidant activity in various cells (Ferrari et al., 1995; Yan et al., 1995; Rappeneau et al., 2000; Ekinci et al., 2000; Shea et al., 2002; Osakada et al., 2003). The present results also demonstrated that pretreatment of the cells with 1000 mM NAC tended to increase intracellular GSH levels, and that it partially but significantly restored the levels of sulfhydryl base and GSH depleted by 30 mM HNE, suggesting that this effect also plays a pivotal role in the neuroprotective action of NAC. The mitochondrial detoxification phase II process has been shown to involve glutathione conjugation to the electrophilic C3 carbon directly or through the action of glutathione-Stransferases (GSTs), forming the GSH-HNE adduct S-(4hydroxy-l-oxononan-3-yl)glutathione (GSHNE) (Xie et al., 1998). Mitochondria in rat brain possess GST A4-4, an inducible GST isoform with high activity toward HNE (Bhagwat et al., 1998). We also examined the protective effects of ebselen, a lipid-soluble seleno-organic compound that has been shown to exhibit both glutathione peroxidase-like activity and antioxidant activity (Mu¨ller et al., 1984; Wendel et al., 1984; Maiorino et al., 1988) against HNE-induced cell death. These results suggest that although ebselen delayed the

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process leading to cell death, it did not have a neuroprotective effect against HNE-induced toxicity in cerebellar granule neurons, possibly due to lack of a protective effect on mitochondrial activity. Unlike NAC, ebselen has been shown to lack the ability to synthesize GSH which react HNE (Cotgreave et al., 1987). In our previous study, we found that SAC had a neuroprotective effect against amyloid b-peptide (Ab)- or tunicamycin-induced cell death in cultured rat hippocampal neurons and PC12 cells when these cells were treated simultaneously (Ito et al., 2003; Kosuge et al., 2003). However, SAC was unable to protect cerebellar granule neurons against Ab-induced neurotoxicity (Kosuge et al., 2003). We also speculated that SAC might protect against the neuronal cell death that is triggered by endoplasmic reticulum (ER) dysfunction in the hippocampus, and that it has no effect on neuronal cell death that is dependent on the caspase-3-mediated oxidative pathway. The present study also showed that SAC did not preserve mitochondrial membrane potential or exert a protective effect against HNE-induced toxicity in cerebellar granule neurons. Taken together, it is suggested that the neuroprotective effect of SAC is region-specific, and that it does not affect neuronal death induced by mitochondrial dysfunction in cerebellar granule neurons. In conclusion, the present results have revealed that loss of mitochondrial membrane potential is one of the important mechanisms involved in HNE-induced neurotoxicity in cerebellar granule cells, and that pretreatment of these neurons with NAC detoxifies HNE-induced neuronal toxicity, possibly due to protection of the cells against HNE-induced inhibition of mitochondrial activity. It is also suggested that endogenous GSH plays a key role in the protective effects of NAC against HNE-induced cell death. Acknowledgements We are grateful to Wakunaga Pharmaceutical Co. (Osaka, Japan) for supplying SAC. This work was supported by a Nihon University Multidisciplinary Global Research Grant. References Aoyama, K., Suh, S.W., Hamby, A.M., Liu, J., Chan, W.Y., Chen, Y., Swanson, R.A., 2006. Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse. Nat. Neurosci. 9, 119–126. Awasthi, Y.C., Sharma, R., Cheng, J.Z., Yang, Y., Sharma, A., Singhal, S.S., Awasthi, S., 2003. Role of 4-hydroxynonenal in stress-mediated apoptosis signaling. Mol. Aspects Med. 24, 219–230. Bannai, S., Tateishi, N., 1986. Role of membrane transport in metabolism and function of glutathione in mammals. J. Membr. Biol. 89, 1–8. Bhagwat, S.V., Vijayasarathy, C., Raza, H., Mullick, J., Avadhani, N.G., 1998. Preferential effects of nicotine and 4-(N-methyl-N-nitrosamine)-1-(3-pyridyl)-1-butanone on mitochondrial glutathione S-transferase A4-4 induction and increased oxidative stress in the rat brain. Biochem. Pharmacol. 56, 831–839. Blanc, E.M., Kelly, J.F., Mark, R.J., Waeg, G., Mattson, M.P., 1997. 4-Hydroxynonenal, an aldehydic product of lipid peroxidation, impairs signal transduction associated with muscarinic acetylcholine and metabotropic glutamate receptors: possible action on Gaq/11. J. Neurochem. 69, 570–580.

Cotgreave, I.A., Sandy, M.S., Berggren, M., Molde´us, P.W., Smith, M.T., 1987. N-acetylcysteine and glutathione-dependent protective effect of PZ51 (Ebselen) against diquat-induced cytotoxicity in isolated hepatocytes. Biochem. Pharmacol. 36, 2899–2904. Ekinci, F.J., Linsley, M.D., Shea, T.B., 2000. b-Amyloid-induced calcium influx induces apoptosis in culture by oxidative stress rather than tau phosphorylation. Brain Res. Mol. Brain Res. 76, 389–395. Ferrari, G., Yan, C.Y., Greene, L.A., 1995. N-acetylcysteine (D- and L-stereoisomers) prevents apoptotic death of neuronal cells. J. Neurosci. 15, 2857– 2866. Ishige, K., Chen, Q., Sagara, Y., Schubert, D., 2001. The activation of dopamine D4 receptors inhibits oxidative stress-induced nerve cell death. J. Neurosci. 21, 6069–6076. Ito, Y., Arakawa, M., Ishige, K., Fukuda, H., 1999. Comparative study of survival signal withdrawal- and 4-hydroxynonenal-induced cell death in cerebellar granule cells. Neurosci. Res. 35, 321–327. Ito, Y., Ishige, K., Zaitsu, E., Anzai, K., Fukuda, H., 1995. g-Hydroxybutyric acid increases intracellular Ca2+ concentration and nuclear cyclic AMPresponsive element- and activator protein 1 DNA-binding activities through GABAB receptor in cultured cerebellar granule cells. J. Neurochem. 65, 75–83. Ito, Y., Kosuge, Y., Sakikubo, T., Horie, K., Ishikawa, N., Obokata, N., Yokoyama, E., Yamashina, K., Yamamoto, M., Saito, H., Arakawa, M., Ishige, K., 2003. Protective effect of S-allyl-L-cysteine, a garlic compound, on amyloid b-protein-induced cell death in nerve growth factor-differentiated PC12 cells. Neurosci. Res. 46, 119–125. Jesberger, J.A., Richardson, J.S., 1991. Oxygen free radicals and brain dysfunction. Int. J. Neurosci. 57, 1–17. Keller, J.N., Mark, R.J., Bruce, A.J., Blanc, E., Rothstein, J.D., Uchida, K., Waeg, G., Mattson, M.P., 1997a. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience 80, 685–696. Keller, J.N., Mattson, M.P., 1998. Roles of lipid peroxidation in modulation of cellular signaling pathways, cell dysfunction, and death in the nervous system. Rev. Neurosci. 9, 105–116. Keller, J.N., Pang, Z., Geddes, J.W., Begley, J.G., Germeyer, A., Waeg, G., Mattson, M.P., 1997b. Impairment of glucose and glutamate transport and induction of mitochondrial oxidative stress and dysfunction in synaptosomes by amyloid b-peptide: role of the lipid peroxidation product 4hydroxynonenal. J. Neurochem. 69, 273–284. Kosuge, Y., Koen, Y., Ishige, K., Minami, K., Urasawa, H., Saito, H., Ito, Y., 2003. S-allyl-L-cysteine selectively protects cultured rat hippocampal neurons from amyloid b-protein- and tunicamycin-induced neuronal death. Neuroscience 122, 885–895. Kruman, I., Bruce-Keller, A.J., Bredesen, D., Waeg, G., Mattson, M.P., 1997. Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis. J. Neurosci. 17, 5089–5100. Laurent, A., Perdu-Durand, E., Alary, J., Debrauwer, L., Cravedi, J.P., 2000. Metabolism of 4-hydroxynonenal, a cytotoxic product of lipid peroxidation, in rat precision-cut liver slices. Toxicol. Lett. 114, 203–214. Maiorino, M., Roveri, A., Coassin, M., Ursini, F., 1988. Kinetic mechanism and substrate specificity of glutathione peroxidase activity of ebselen (PZ51). Biochem. Pharmacol. 37, 2267–2271. Markesbery, W.R., Lovell, M.A., 1998. Four-hydroxynonenal, a product of lipid peroxidation, is increased in the brain in Alzheimer’s disease. Neurobiol. Aging 19, 33–36. Mayer, M., Noble, M., 1994. N-acetyl-L-cysteine is a pluripotent protector against cell death and enhancer of trophic factor-mediated cell survival in vitro. Proc. Natl. Acad. Sci. U.S.A. 91, 7496–7500. McGrath, L.T., McGleenon, B.M., Brennan, S., McColl, D., McILroy, S., Passmore, A.P., 2001. Increased oxidative stress in Alzheimer’s disease as assessed with 4-hydroxynonenal but not malondialdehyde. QJM 94, 485– 490. Meister, A., 1988. Glutathione metabolism and its selective modification. J. Biol. Chem. 263, 17205–17208. Meyer, M.J., Mosely, D.E., Amarnath, V., Picklo, M.J., 2004. Metabolism of 4hydroxy-trans-2-nonenal by central nervous system mitochondria is dependent on age and NAD+ availability. Chem. Res. Toxicol. 17, 1272–1279.

M. Arakawa et al. / Neuroscience Research 55 (2006) 255–263 Moriguchi, T., Matsuura, H., Itakura, Y., Katsuki, H., Saito, H., Nishiyama, N., 1997. Allixin, a phytoalexin produced by garlic, and its analogues as novel exogenous substances with neurotrophic activity. Life Sci. 61, 1413–1420. Mu¨ller, A., Cadenas, E., Graf, P., Sies, H., 1984. A novel biologically active seleno-organic compound-I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen). Biochem. Pharmacol. 33, 3235–3239. Murphy, T.C., Amarnath, V., Gibson, K.M., Picklo, M.J., 2003a. Oxidation of 4hydroxy-2-nonenal by succinic semialdehyde dehydrogenase (ALDH5A). J. Neurochem. 86, 298–305. Murphy, T.C., Amarnath, V., Picklo, M.J., 2003b. Mitochondrial oxidation of 4hydroxy-2-nonenal in rat cerebral cortex. J. Neurochem. 84, 1313–1321. Neely, M.D., Zimmerman, L., Picklo, M.J., Ou, J.J., Morales, C.R., Montine, K.S., Amaranth, V., Montine, T.J., 2000. Congeners of Na-acetyl-L-cysteine but not aminoguanidine act as neuroprotectants from the lipid peroxidation product 4-hydroxy-2-nonenal. Free Radic. Biol. Med. 29, 1028–1036. Numagami, Y., Ohnishi, S.T., 2001. S-allylcysteine inhibits free radical production, lipid peroxidation and neuronal damage in rat brain ischemia. J. Nutr. 131, 1100S–1105S. Osakada, F., Hashino, A., Kume, T., Katsuki, H., Kaneko, S., Akaike, A., 2003. Neuroprotective effects of a-tocopherol on oxidative stress in rat striatal cultures. Eur. J. Pharmacol. 465, 15–22. Picklo, M.J., Amarnath, V., McIntyre, J.O., Graham, D.G., Montine, T.J., 1999. 4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple sites. J. Neurochem. 72, 1617–1624. Raha, S., Robinson, B.H., 2000. Mitochondria, oxygen free radicals, disease and ageing. Trends Biochem. Sci. 25, 502–508. Rappeneau, S., Baeza-Squiban, A., Jeulin, C., Marano, F., 2000. Protection from cytotoxic effects induced by the nitrogen mustard mechlorethamine on human bronchial epithelial cells in vitro. Toxicol. Sci. 54, 212–221. Ratan, R.R., Murphy, T.H., Baraban, J.M., 1994. Macromolecular synthesis inhibitors prevent oxidative stress-induced apoptosis in embryonic cortical neurons by shunting cysteine from protein synthesis to glutathione. J. Neurosci. 14, 4385–4392. Romancino, D.P., Montana, G., Di Carlo, M., 2004. Maternal Paracentrotus lividus RNAs are differentially localized during the first cell division. Arch. Biochem. Biophys. 429, 164–170. Sen, C.K., 1997. Nutritional biochemistry of cellular glutathione. J. Nutr. Biochem. 8, 660–672. Sen, C.K., 1998. Redox signaling and the emerging therapeutic potential of thiol antioxidants. Biochem. Pharmacol. 55, 1747–1758. Shea, T.B., Ekinci, F.J., Ortiz, D., Dawn-Linsley, M., Wilson, T.O., Nicolosi, R.J., 2002. Efficacy of vitamin E, phosphatidyl choline, and pyruvate on buffering neuronal degeneration and oxidative stress in cultured cortical neurons and in central nervous tissue of apolipoprotein E-deficient mice. Free Radic. Biol. Med. 33, 276–282.

263

Shen, W., Kamendulis, L.M., Ray, S.D., Corcoran, G.B., 1992. Acetaminopheninduced cytotoxicity in cultured mouse hepatocytes: effects of Ca2+-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death. Toxicol. Appl. Pharmacol. 112, 32–40. Simonian, N.A., Coyle, J.T., 1996. Oxidative stress in neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol. 36, 83–106. Srivastava, S., Chandra, A., Wang, L.F., Seifert, W.E., DaGue, B.B., Ansari, N.H., Srivastava, S.K., Bhatnagar, A., 1998. Metabolism of the lipid peroxidation product, 4-hydroxy-trans-2-nonenal, in isolated perfused rat heart. J. Biol. Chem. 273, 10893–10900. Takasago, T., Peters, E.E., Graham, D.I., Masayasu, H., Macrae, I.M., 1997. Neuroprotective efficacy of ebselen, an anti-oxidant with anti-inflammatory actions, in a rodent model of permanent middle cerebral artery occlusion. Br. J. Pharmacol. 122, 1251–1256. Thangnipon, W., Kingsbury, A., Webb, M., Balazs, R., 1983. Observations on rat cerebellar cells in vitro: influence of substratum, potassium concentration and relationship between neurones and astrocytes. Brain Res. 313, 177–189. Thomson, M., Ali, M., 2003. Garlic [Allium sativum]: a review of its potential use as an anti-cancer agent. Curr. Cancer Drug. Targets 3, 67–81. Tietze, F., 1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522. Wendel, A., Fausel, M., Safayhi, H., Tiegs, G., Otter, R., 1984. A novel biologically active seleno-organic compound-II. Activity of PZ 51 in relation to glutathione peroxidase. Biochem. Pharmacol. 33, 3241–3245. Xie, C., Lovell, M.A., Markesbery, W.R., 1998. Glutathione transferase protects neuronal cultures against four hydroxynonenal toxicity. Free Radic. Biol. Med. 25, 979–988. Yamasaki, T., Li, L., Lau, B.H.S., 1994. Garlic compounds protect vascular endothelial cells from hydrogen peroxide-induced oxidant injury. Phytother. Res. 8, 408–412. Yamashita, T., Ando, Y., Obayashi, K., Terazaki, H., Sakashita, N., Uchida, K., Ohama, E., Ando, M., Uchino, M., 2000. Oxidative injury is present in Purkinje cells in patients with olivopontocerebellar atrophy. J. Neurol. Sci. 175, 107–110. Yan, C.Y., Ferrari, G., Greene, L.A., 1995. N-acetylcysteine-promoted survival of PC12 cells is glutathione-independent but transcription-dependent. J. Biol. Chem. 270, 26827–26832. Yeh, Y.Y., Liu, L., 2001. Cholesterol-lowering effect of garlic extracts and organosulfur compounds: human and animal studies. J. Nutr. 131, 989S– 993S. Yoshizumi, M., Kogame, T., Suzaki, Y., Fujita, Y., Kyaw, M., Kirima, K., Ishizawa, K., Tsuchiya, K., Kagami, S., Tamaki, T., 2002. Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun Nterminal kinase and activator protein-1 signalling pathway in PC12 cells. Br. J. Pharmacol. 136, 1023–1032.