N-acetylcysteine selectively protects cerebellar granule cells from 4-hydroxynonenal-induced cell death
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 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 μM) compared with control (7.80 μM) (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 4-hydroxynonenoate (HNEAcid) by the NAD+-dependent aldehyde dehydrogenases (ALDHs) and by conjugation with GSH (Murphy et al., 2003a, Murphy et al., 2003b, Meyer et al., 2004).
N-acetylcysteine (NAC) has been shown to exert survival-promoting 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 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, Sen, 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). α-Tocopherol (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 lipid-soluble seleno-organic compound that exhibits both glutathione peroxidase-like and antioxidant activity (Müller 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 (Müller 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 cholesterol-lowering 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 non-neuronal 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.
Section snippets
Materials
The chemicals used in this study were: Hoechst 33258, MitoRed, LDH-Cytotoxic Test kit, trichloroacetic acid (TCA), 5,5′-dithiobis(nitrobenzoic acid) (DTNB) and glutathione, reduced form (Wako Pure Chemical, Osaka, Japan), HNE (Cayman Chemical, Ann Arbor, MI), NAC, [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) and nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) (Sigma, St. Louis, MO), glutathione reductase (Roche Applied Science, Indianapolis, IN), propidium
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 μM) for 24 h resulted in a concentration-dependent decrease in MTT reduction. A significant decrease in MTT reduction was observed at concentrations of 20 μM HNE
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-DEVD-CHO, a caspase-3 inhibitor, in cerebellar granule neurons (Ito et al., 1999) and hippocampal neurons (Kosuge et al., 2003), suggesting that
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 (54)
- et al.
Role of 4-hydroxynonenal in stress-mediated apoptosis signaling
Mol. Aspects Med.
(2003) - et al.
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.
(1998) - et al.
N-acetylcysteine and glutathione-dependent protective effect of PZ51 (Ebselen) against diquat-induced cytotoxicity in isolated hepatocytes
Biochem. Pharmacol.
(1987) - et al.
β-Amyloid-induced calcium influx induces apoptosis in culture by oxidative stress rather than tau phosphorylation
Brain Res. Mol. Brain Res.
(2000) - et al.
Comparative study of survival signal withdrawal- and 4-hydroxynonenal-induced cell death in cerebellar granule cells
Neurosci. Res.
(1999) - et al.
Protective effect of S-allyl-l-cysteine, a garlic compound, on amyloid β-protein-induced cell death in nerve growth factor-differentiated PC12 cells
Neurosci. Res.
(2003) - et al.
4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes
Neuroscience
(1997) - et al.
S-allyl-l-cysteine selectively protects cultured rat hippocampal neurons from amyloid β-protein- and tunicamycin-induced neuronal death
Neuroscience
(2003) - et al.
Metabolism of 4-hydroxynonenal, a cytotoxic product of lipid peroxidation, in rat precision-cut liver slices
Toxicol. Lett.
(2000) - et al.
Kinetic mechanism and substrate specificity of glutathione peroxidase activity of ebselen (PZ51)
Biochem. Pharmacol.
(1988)