Elsevier

Neuroscience Research

Volume 55, Issue 3, July 2006, Pages 255-263
Neuroscience Research

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

https://doi.org/10.1016/j.neures.2006.03.008Get rights and content

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), α-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 μM NAC or 10 and 100 μM 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.

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.

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