1. Introduction

Parkinson’s disease (PD) is a progressive, mainly sporadic neurodegenerative disorder that is characterized by severe motor symptoms including uncontrollable tremor, slowness of movements, and rigidity. This pathology is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of cytoplasmic inclusions known as Lewy bodies (LB) [1] . Alpha-synu- clein (αS) is a small protein of 140 amino acids with strong links to several neurodegenerative diseases such as PD and is well known as a major component of LB [2] [3] . Moreover, oxidative stress, including the reactive oxygen or nitrogen species generated in the enzymatic oxidation [4] or auto-oxidation of an excess amount of dopamine (DA) [5] , is thought to play an important role in dopaminergic (DAergic) neurotoxicity [6] . DAergic neuron-specific oxidative stress has been a recent focus [7] because DA and its metabolites containing 2 hydroxyl residues exert toxicity in DAergic neuronal cells, primarily due to the generation of highly reactive DA quinone (DAQ) or DOPA quinone [7] . To control the harmful effects of oxidative stress, efficient detoxification pathways in various cells have been identified. Of the various antioxidant systems in the brain, the glutathione (GSH) system is particularly important in controlling cellular redox states and is the primary defense mechanism for the removal of peroxide from the brain [8] [9] . In addition, GSH also has quinone-quenching activities against DA- or L-DOPA-induced neurotoxicity [10] . In the present study, we examined the effects of L-buthionine-SR-sulfoximine (BSO), a typical GSH synthase inhibitor, with or without N-acetylcysteine (NAC), a source of GSH, on αS-induced cell injury in human neuroblastoma SH-SY5Y cells, to clarify the role of GSH on the molecular mechanism of αS-induced cell injury.

2. Materials and Methods

2.1. Construction of Expression Vectors

Human αS cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) amplification and then was ligated into a plasmid vector. Templates for use in the PCR were generated by reverse transcription of total RNA isolated from human neuroblastoma SH-SY5Y cells by the acid guanidinium-phenol- chloroform method using αS gene-specific primers [11] . To construct the αS cDNA expression vector, a fragment of αS cDNA was ligated into pIRES-EGFP (Clontech, Palo Alto, CA) using the calcium phosphate method (Lipofectin® Reagent: Invitrogen, Carlsbad, CA). For selection, 300 μl/ml geneticin disulfate (Sigma, St. Louis, MO) was added to the culture.

2.2. Western Blot Analysis for Confirmation of the αS Expression in SH-SY5Y Cells

Total cell lysates from transformant SH-SY5Y cells were prepared with 10 µg/ml phenylmethylsulfonyl fluoride in ice-cold RIPA buffer [phosphate-buffered saline (pH 7.4), 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)], and analysis using the total cell lysate was performed as described previously [12] . The blots were incubated with rabbit polyclonal antibody for αS (Santa Cruz Biotechnology, Santa Cruz, CA), then reacted with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA). After a washing with 20 mM Tris-buffered saline containing 0.1% Tween 20, the blots were developed using an ECL Western blotting detection system (Amersham, Buckinghamshire, UK) according to the protocol provided by the manufacturer.

2.3. Cell Culture

In the investigations performed in the present study, with the exception of the determination of cell viability, human neuroblastoma SH-SY5Y cells were treated with BSO (final conc. 50 - 500 µM for 144 h) with or without NAC (final conc. 100 - 1000 µM for 144 h), which were all purchased from Sigma. For the preparation of the GSH samples, SH-SY5Y cells were grown for 144 h in the presence or absence of BSO with or without NAC. BSO and NAC were dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in the culture medium was maintained at ≤0.01%. Human neuroblastoma SH-SY5Y (ATCC cell bank; catalogue number CRL-2266: Rockville, MD) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. For the experiments, the cells were plated onto 96-well plates at a density of 4.0 × 10⁴ cells/ml (SH-SY5Y) in order to assess cell viability. In addition, the cells were plated separately onto 6-well plates at 2.0 × 10⁵ cells/ml in order to determine the GSH content. All cultures were maintained at 37˚C in a gas mixture of 5% - 95% CO₂-air. The SH-SY5Y cells were grown for 2 - 3 days before being used in experiments.

2.4. Cell Viability

The cell viability in each well of the 96-well plate was determined by quantitative colorimetric assay using WST-1 [12] , a modification of the standard MTT assay. Viability was expressed as a percentage of the cell viability from each vehicle- treated control culture in the normal-medium-exposed group.

2.5. Glutathione (GSH) Content

GSH levels were determined using the enzymatic recycling method with some modifications [12] . Briefly, SH-SY5Y transformant cells were homogenized in 0.1-M phosphate buffer (pH 7.4); then 10% trichloroacetic acid was added in an equivalent volume to the cellular homogenates. Acid extracts were mixed with 0.01 M phosphate buffer (pH 7.4, 580 μL), NADPH (4 mM, 50 μL) and glutathione reductase (6 U/mL, 100 μL) and incubated for 5 min at 37˚C. 3-Carboxy- 4-nitrophenyl disulfide (10 mM, 50 μL) was added just before the absorbance was read. The formation of 2-nitro-5-thiobenzoic acid was measured at 450 nm for 6 min. Total GSH was determined from a standard curve constructed using known amounts of GSH.

2.6. Statistical Analysis

Data are presented as the means ± SEM. Differences between groups were tested for statistical significance using one-way analysis of variance (ANOVA) followed by the post hoc Tukey-Kramer Multiple Comparisons test. P-values <0.05 were considered to indicate a statistically significant difference.

3. Results

Expression of αS proteins was detected in both αS-transfected and empty vector- transfected SH-SY5Y cells, although that in αS-transfected cells were stronger than that in control cells (Figure 1). BSO induced a reduction of cell viability in αS-transfected SH-SY5Y cells; in particular, treatment with BSO (100 μM) for 144 h resulted in markedly reduced cell viability in αS-transfected cells compared with the control cells (Figure 2; p < 0.01). However, treatment with NAC (100 - 1000 μM) dose-dependently prevented the BSO-induced reduction of cell viability in αS-transfected cells (Figure 3). BSO induced a reduction of GSH content in αS-transfected SH-SY5Y cells, but treatment with NAC prevented the BSO-induced reduction of GSH contents (Figure 4).

4. Discussion

αS protein has been directly linked to PD-associated neurodegeneration, and in

patients exhibiting such neorodegeneration the αS protein is aberrantly folded, forming abnormal oligomers and amyloid fibrils within neurons or oligodendrocytes. Thus, the toxicity of αS is generally attributed to the formation of these toxic oligomers [3] . Oxidative conjugation of DA with αS inhibits the transition of αS from protofibrils to mature fibrils, leading to the potential accumulation of cytotoxic soluble protofibrils in DAergic neurons. The antioxidants have the ability to reverse the formation of these adducts, suggesting that catechol oxidation can contribute to the accumulation of αS protofibrils [6] . Moreover, the levels of GSH are lower in the SN early in PD, and this may contribute to mitochondrial dysfunction and oxidative stress, with the oxidative stress in turn increasing the accumulation of toxic forms of αS [13] . Several reports have suggested that established risk factors such as oxidative stress, DA and αS, may in combination stabilize protofibrillar αS and promote the pathogenesis of PD [14] . However, the reason why the molecular basis of αS-induced cytotoxicity can be highly toxic to cells has remained unclear [3] .

To examine the above hypotheses, we determined the effect of BSO on cell injury in the αS-transfected cells. BSO induced a reduction of both cell viability and total GSH content in αS-transfected SH-SY5Y cells; in particular, treatment with BSO (100 μM) for 144 h markedly reduced cell viability in αS-transfected cells compared with vector-transfected control cells. Thus, it could at least be concluded that GSH-related oxidative stress induced cell injury in αS-transfected cells. A previous report also suggested that αS aggregation disrupts mitochondria together with the enhanced oxidation [15] . In addition, we also hypothesized that supplementation with NAC would protect against αS-induced cytotoxicity. NAC (100 - 1000 μM) dose-dependently prevented the BSO-induced reduction of both cell viability and GSH content in αS-transfected cells, although the protective effects of NAC on BSO-induced reduction of GSH content were only partial. It is known that NAC not only raises GSH levels by acting as a cysteine donor in the synthesis of GSH, but also has several additional effects including direct antioxidant activity [16] . Systemic administration of NAC has been shown to increase brain levels of GSH in mice, reduce markers of oxidative damage, and increase brain synaptic mitochondrial complex I activity [17] . Oral treatment with NAC decreases αS levels in the brain and partially protects against loss of DAergic neurons associated with overexpression of αS in αS-overexpressing mice [13] . Moreover, manganese-induced αS oligomerization was also partially alleviated by pretreatment with GSH and aggravated by H₂O₂ pretreatment [18] .

Considering these reports and our results regarding the partial effect of NAC on GSH content, the protective property of NAC may be mainly dependent on additional effects such as direct antioxidant activity. Thus, GSH would play important roles in αS-induced cytotoxicity, such as the reduction of cell viability, although further studies are required to clarify the molecular basis of αS-induced cytotoxicity.

Acknowledgements

The authors would like to thank Dr. Y. Higashi, Ms. N. Fujita and Ms. A. Tamura for their technical support.

Conflict of Interest

The authors confirm that this article content has no conflict of interest.

Author Contribution

Ken-ichi Tanaka designed experiments, provided technical support, analyzed date and wrote manuscript; Kanako Sonoda co-designed and performed experiments; Masato Asanuma interpreted results and provided other related support.