Journal of Molecular Neuroscience

, Volume 45, Issue 1, pp 60–67

Inhibition of Endogenous Hydrogen Sulfide Generation is Associated with Homocysteine-Induced Neurotoxicity: Role of ERK1/2 Activation

Authors

    • Department of Physiology, Medical CollegeUniversity of South China
  • Xin-Tian Shen
    • Department of Physiology, Medical CollegeUniversity of South China
    • Department of PhysiologyHuaihua Medical College
  • Yi-E Huang
    • Department of PhysiologyHuaihua Medical College
  • Rong-Qian Chen
    • Department of Physiology, Medical CollegeUniversity of South China
  • Yan-Kai Ren
    • Department of Physiology, Medical CollegeUniversity of South China
  • Heng-Rong Fang
    • Department of Physiology, Medical CollegeUniversity of South China
  • Yuan-Yuan Zhuang
    • Department of Physiology, Medical CollegeUniversity of South China
    • Department of Physiology, Medical CollegeUniversity of South China
Article

DOI: 10.1007/s12031-010-9477-z

Cite this article as:
Tang, X., Shen, X., Huang, Y. et al. J Mol Neurosci (2011) 45: 60. doi:10.1007/s12031-010-9477-z

Abstract

Both elevated homocysteine and decreased hydrogen sulfide (H2S) are observed in the brains of Alzheimer’s disease (AD) patients. Reactive oxygen species (ROS) overproduction contributes to the neurotoxicity of homocysteine; however, H2S is an endogenous antioxidant gas. Therefore, the aim of this study was to investigate whether the imbalance of proportion to this endogenous protective antioxidant gas is involved in homocysteine-caused neurotoxicity. We show that homocysteine inhibits the generation of endogenous H2S and the expression and activity of cystathionine-β-synthetase (CBS), the main enzyme responsible for the generation of H2S in PC12 cells. S-Adenosylmethionine, an activator of CBS, not only prevents homocysteine-induced inhibition of endogenous H2S production but also attenuates homocysteine-triggered cytotoxicity and accumulation of ROS. We find that activation of ERK1/2 occurs in homocysteine-treated PC12 cells and blockade of ERK1/2 with U0126 abolished the homocysteine-induced cytotoxicity and inhibitory effect on endogenous H2S generation. These results indicate that homocysteine neurotoxicity involves reduction of H2S production, which is caused by inhibition of CBS and mediated by activation of ERK1/2. Our study suggests a promising future of H2S-based therapies for neurodegenerative diseases such as AD.

Keywords

HomocysteineHydrogen sulfideCystathionine-β-synthetaseERK1/2Neurotoxicity

Introduction

Homocysteine (Hcy) is a key metabolic intermediate in sulfur amino acid metabolism (Prudova et al. 2006; Selhub 1999). Both in vitro and in vivo studies have shown that Hcy is toxic to neuronal cells (Baydas et al. 2005; Ho et al. 2002; Kim et al. 1987; Kruman et al. 2000; Linnebank et al. 2006; Lipton et al. 1997; Parsons et al. 1998). Evidence has accumulated that elevated plasma Hcy is a strong, independent risk factor of Alzheimer’s disease (AD) (Clarke et al. 1998; Dwyer et al. 2004; Miller 1999; Seshadri et al. 2002; Van Dam and Van Gool 2009). The deleterious influences of hyperhomocysteinemia have been known in neurological abnormalities, such as mental retardation, cerebral atrophy, and seizures (van den Berg et al. 1995). However, the cellular and molecular mechanisms by which Hcy causes neurotoxicity are poorly understood.

Hydrogen sulfide (H2S), a third gaseous mediator, has recently been recognized as an important endogenous neuromodulator (Moore et al. 2003; Wang 2002). In the central nervous system, endogenous H2S is synthesized from l-cysteine, and this process is predominantly catalyzed by cystathionine-β-synthetase (CBS) (Moore et al. 2003; Wang 2002). Interestingly, Hcy is remethylated to methionine by vitamin B12-dependent methionine synthase during methionine cycle and can be converted to cysteine through the transsulfuration pathway. The first and committing step in the transsulfuration pathway is catalyzed by CBS (Prudova et al. 2006). Furthermore, both elevated Hcy and decreased H2S are observed in the brains of AD patients (Eto et al. 2002). Since Hcy and H2S coexist in the same metabolic pathway with CSB, what would be the alterative connection between H2S and Hcy in the pathogenesis of AD? Therefore, we wondered whether Hcy directly impairs endogenous H2S generation and whether inhibition of endogenous H2S generation by Hcy contributes to the toxicity of Hcy to neuronal cells.

The extracellular signal-regulated kinase (ERK) cascade is a central pathway that transmits signals from many extracellular agents to regulate cellular processes, such as proliferation, differentiation, and cell cycle progression (Yoon and Seger 2006). It has been shown that ERK1/2 mediates Hcy-induced neuronal cell death (Poddar and Paul 2009). Thus, we also determined the role of ERK1/2 activation in Hcy-mediated change in endogenous H2S generation.

In the present study, we investigated the effects of Hcy on endogenous H2S production in neuronal cells and the underlying mechanism by studying PC12 cells, a clonal rat pheochromocytoma cell line that is widely used for studying the cellular biology of neurons (Duan et al. 2009; Li et al. 1998; Wang et al. 2009). We demonstrated for the first time that Hcy significantly inhibits endogenous H2S synthesis in PC12 cells through suppressing the expression and activity of CBS, the major enzyme responsible for endogenous H2S generation in PC12 cells (Tang et al. 2010a), and stimulating the activation of ERK1/2 and that the inhibitory effect of Hcy on endogenous H2S synthesis contributes to its neurotoxicity.

Materials and Methods

Materials

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), S-adenosylmethionine (SAM), dihydrorhodamine 123 (DHR), homocysteine, and U0126 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Monoclonal anti-CBS antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against ERK1/2 and p-ERK1/2 were purchased from Cell Signaling Technology Inc (Beverly, MA, USA). RPMI-1640 medium, horse serum, and fetal bovine serum were supplied by GIBCO BRL (Ground Island, NY, USA).

Cell Culture

PC12 cells, a rat cell line derived from pheochromocytoma cells, were supplied from Sun Yat-sen University Experimental Animal Center (Guangzhou, China) and were maintained on tissue culture plastic in RPMI-1640 medium supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum at 37°C under an atmosphere of 5% CO2 and 95% air. The culture media was changed three times per week.

Determination of Cell Viability

PC12 cells were plated at a density of 1 × 104 cells/well in 96-well plates, and cell viability was determined by the conventional MTT reduction assay. The MTT assay, based on the cleavage of the yellow tetrazolium salt MTT to purple formazan crystal by metabolic active cells, relies primarily on the mitochondrial metabolic capacity of viable cells and reflects the intracellular redox state (Shearman et al. 1995). At the end of treatment, cells were incubated with MTT solution (final concentration, 0.5 mg/mL) for 4 h. The medium was removed, and 100 mL dimethylsulphoxide (DMSO) was added into each well to dissolve the formazan by pipetting up and down several times. The absorbance of formazan was measured at 570 nm with a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability (%) = (the absorbance of cells exposed to drugs/the absorbance of control cells) × 100%.

Measurement of Intracellular ROS Generation

Intracellular ROS were determined by oxidative conversion of cell-permeable DHR to fluorescent Rh123 (Walrand et al. 2003). Cells were collected and washed with PBS. After the addition of 1 mmol/L DHR to cell cultures for 1 h at 37°C, cells were washed twice with PBS. Fluorescence of Rh123 was measured by a flow cytometer (FCM, Beckman-Coulter, Miami, FL, USA). The mean fluorescence intensity (MFI) of the positive cells in 10,000 cells/sample was measured, and the MFI represented the amount of ROS.

Western Blot Analysis for CBS Expression and ERK1/2 Phosphorylation

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was carried out on 5% stacking and 12% resolving gel with low range molecular weight standards (Solarbio, China). Equal amounts of protein were loaded in each lane with loading buffer (Beyotime, China) containing 0.1 M Tris (pH 6.8), 20% glycerol, 10% mercaptoethanol, 4% SDS, and 0.2% bromophenol blue. Samples were heated at 100°C for 5 min before gel loading. Following electrophoresis, the proteins were transferred to a polyvinylidene fluoride transfer membrane (Solarbio, China). After this, the membranes were blocked with TBST (50 mM Tris–HCl, pH 7.4, 0.15 M NaCl, and 0.1% Tween-20) containing 5% bovine serum albumin (Sigma, USA) for 2 h. Following this, the membranes were incubated with primary antibodies diluted 1:1,000 at 4°C over night. After washing with TBST, the membranes were incubated with anti-rabbit IgG labeled with horseradish peroxidase (Zsbio, China) diluted at 1:1,000 at room temperature for 2 h. The membranes were washed again and developed with an enhanced chemiluminescence system (ECL, Zsbio, China) followed by apposition of the membranes with autoradiographic films (Kodak, China). The integrated optical density for the protein band was calculated by Image-J software.

Measurement of H2S in Cell Culture Supernatant

The basis of the assay is that H2S produced in the incubate reacts with zinc acetate to form zinc sulphide, which then dissolves in a hydrochloride acid solution of N,N-dimethyl-p-phenylenediamine sulfate (NNDPD) yielding, in the presence of ferric chloride, methylene blue, which is quantitated spectrophotometrically.

Cell culture supernatant (310 μl) were mixed with trichloroacetic acid (20% w/v, 60 μl), zinc acetate (2% w/v, 30 μl), NNDPD (20 mM; 40 μl) in 7.2 M HCl, and FeCl3 (30 mM; 30 μl) in 1.2 M HCl. The absorbance of the resulting solution (670 nm) was measured 15 min thereafter by spectrophotometry. H2S was calculated against a calibration curve of NaHS.

Assay of H2S Synthesizing Activity

PC12 cells were homogenized in 50 mM ice-cold potassium phosphate buffer (pH 6.8). The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.4), l-cysteine (20 μl, 10 mM), pyridoxyal 5′-phosphate (20 μl, 2 mM), saline (30 μl), and 11% w/v tissue homogenate (430 μl). The reaction was performed in tightly stoppered cryovial test tubes and initiated by transferring the tubes from ice to a shaking water bath at 37°C. After incubation for 30 min, 1% w/v zinc acetate (250 μl) was added to trap evolved H2S followed by 10% v/v trichloroacetic acid (250 μl) to denature the protein and stop the reaction. Subsequently, NNDPD (20 μM; 133 μl) in 7.2 M HCl was added, immediately followed by FeCl3 (30 μM; 133 μl) in 1.2 M HCl. The absorbance of the resulting solution at 670 nm was measured by spectrophotometry. The H2S concentration was calculated against a calibration curve of NaHS, and H2S synthesizing activity is expressed as nanomole H2S formed from gram protein (determined using the Bradford assay) per minute (nmol · min−1 · g−1 protein).

Statistical Analysis

Data are expressed as mean ± SEM. The significance of inter-group differences was evaluated by one-way analyses of variance (ANOVA; least-significant difference’s test for post hoc comparisons). Differences were considered significant at P < 0.05.

Results

Homocysteine Inhibits Generation of Endogenous H2S in PC12 Cells

To evaluate the effect of homocysteine on the generation of endogenous H2S in PC12 cells, the content of H2S in culture supernatant was measured. As shown in Fig. 1, after 24 h exposure of PC12 cells to homocysteine (5, 10, and 20 mmol/L), the content of H2S in culture supernatant was significantly decreased (Fig 1b), indicating that homocysteine has an inhibitory effect on the production of endogenous H2S in PC12 cells.
https://static-content.springer.com/image/art%3A10.1007%2Fs12031-010-9477-z/MediaObjects/12031_2010_9477_Fig1_HTML.gif
Fig. 1

Effect of homocysteine on generation of endogenous H2S in PC12 cells. PC12 cells were treated with 5, 10, and 20 mmol/L of homocysteine (Hcy) for 24 h, respectively. The content of H2S in culture supernatant was measured by the N,N-dimethyl-p-phenylenediamine sulfate (NNDPD) method as described in “Materials and methods.” Values are the mean ± SEM (n = 3). *P < 0.05, **P < 0.01 versus control group

Homocysteine Inhibits Expression and Activity of CBS in PC12 Cells

CBS is the major enzyme responsible for endogenous H2S generation in PC12 cells (Tang et al. 2010a). Western blot analysis was performed to evaluate whether homocysteine decreases endogenous H2S production by inhibiting expression of CBS. As shown in Fig. 2a, treatment with homocysteine (5, 10, and 20 mmol/L) for 24 h caused a significant down-regulation of CBS expression in PC12 cell. In addition, we also examined the effect of homocysteine on CBS activity. Exposure of PC12 cells to homocysteine (5, 10, and 20 mmol/L) for 24 h reduced the activity of CBS (Fig. 2b). These data suggested that homocysteine-induced inhibitions of CBS expression and activity in PC12 contribute to homocysteine-elicited decrease in endogenous H2S production.
https://static-content.springer.com/image/art%3A10.1007%2Fs12031-010-9477-z/MediaObjects/12031_2010_9477_Fig2_HTML.gif
Fig. 2

Effects of homocysteine on the expression and activity of CBS in PC12 cells. PC12 cells were treated with 5, 10, and 20 mmol/L of homocysteine (Hcy) for 24 h, respectively. a The expression levels of CBS in PC12 cells were examined by Western blot and β-actin was used as internal control. b The activities of CBS in PC12 cells were measured by the N,N-dimethyl-p-phenylenediamine sulfate (NNDPD) method as described in “Materials and methods.” Values are the mean ± SEM (n = 3). *P < 0.05, **P < 0.01 versus control group

SAM, an Activator of CBS, Prevents Homocysteine-Induced Inhibition of Endogenous H2S Generation and Attenuates Homocysteine-Triggered Cytotoxicity and ROS Accumulation in PC12 Cells

To confirm whether the inhibitory effect of homocysteine on endogenous H2S production contributes to its neurotoxicity, an activator of CBS, SAM (0.8 mmol/L), was applied to the cell culture 30 min before addition of homocysteine (10 mmol/L), and the effects of SAM on homocysteine-mediated inhibition of endogenous H2S generation and cytotoxicity were examine. As shown in Fig. 3a, co-treated with 0.8 mmol/L SAM significantly reversed the homocysteine-exerted suppression in generation of H2S in PC12 cells, suggesting that SAM can overcome homocysteine-induced inhibition of endogenous H2S production. Furthermore, we found that the cytotoxic effects of homocysteine on cell viability (Fig. 3b) were extenuated by stimulation of endogenous H2S generation with SAM (0.8 mmol/L). These data indicated that the cytotoxicity caused by homocysteine is associated with its inhibitory effect on endogenous H2S generation.
https://static-content.springer.com/image/art%3A10.1007%2Fs12031-010-9477-z/MediaObjects/12031_2010_9477_Fig3_HTML.gif
Fig. 3

Effects of SAM, an agonist of CBS, on homocysteine-induced inhibition of endogenous H2S production, cytotoxicity, and ROS accumulation. PC12 cells were treated with 10 mmol/L homocysteine (Hcy) in the absence or presence of SAM (0.8 mmol/L) for 24 h. The content of H2S in culture supernatant was measured by the N,N-dimethyl-p-phenylenediamine sulfate (NNDPD) method (a), cell viability was determined by MTT assay (b), and the levels of ROS were evaluated by DHR staining (c). Values are the mean ± SEM (n = 3). **P < 0.01 versus control group; #P < 0.05, ##P < 0.01 versus 10 mmol/L Hcy-treated alone group

Cytotoxicity of homocysteine is mainly mediated by oxidative stress (Heinecke et al. 1987), so we further tested whether SAM affects homocysteine-induced ROS formation. DHR staining results revealed an increase in fluorescence intensity in PC12 cells after 24 h exposure to 10 mmol/L of homocysteine, indicating elevated level of intracellular ROS (Fig. 3c). However, when PC12 cells were pretreated with SAM (0.8 mmol/L) for 30 min before the treatment of homocysteine, the MFI of DHR fluorescence was significantly decreased (Fig. 3c), suggesting that homocysteine-induced intracellular ROS accumulation is attenuated by SAM-stimulated H2S generation.

ERK1/2 Mediates the Inhibitory Effect of Homocysteine on Endogenous H2S Generation in PC12 Cells

To determine the involvement of ERK1/2 in the inhibitory effect of homocysteine on endogenous H2S production, ERK1/2 phosphorylation was measured at the end of 24 h exposure of homocysteine. Western blot analysis showed that treatment with homocysteine (5, 10, and 20 mmol/L) caused a concentration-dependent phosphorylation of ERK1/2 (Fig. 4a), indicating that homocysteine can induce the activation of ERK1/2 in PC12 cells.
https://static-content.springer.com/image/art%3A10.1007%2Fs12031-010-9477-z/MediaObjects/12031_2010_9477_Fig4_HTML.gif
Fig. 4

Role of ERK1/2 in homocysteine-induced inhibition of endogenous H2S generation and cytotoxicity in PC12 cells. a PC12 cells were treated with 5, 10, and 20 mmol/L of homocysteine (Hcy) for 24 h, respectively, and the phosphorylation levels of ERK1/2 in PC12 cells were examined by Western blot. b, c PC12 cells were pretreated with 20 μmol/L U0126, a specific ERK1/2 inhibitor, for 30 min before 24 h exposure to 10 mmol/L Hcy. The content of H2S in culture supernatant was measured by the N,N-dimethyl-p- phenylenediamine sulfate (NNDPD) method (b) and the cell viability was determined by MTT assay (c). Values are the mean ± SEM (n = 3). *P < 0.05, **P < 0.01 versus control group; ##P < 0.01 versus 10 mmol/L Hcy-treated alone group

To further confirm the contribution of ERK1/2 activation to homocysteine-mediated inhibition of H2S generation, cells were treated with U0126 (20 μmol/L), a specific ERK1/2 inhibitor, 30 min before administration of homocysteine (10 mmol/L), and the effect of pretreatment with U0126 on homocysteine-caused inhibition of H2S generation was determined. As shown in Fig. 4b, U0126 significantly attenuated the inhibitory effect of homocysteine on H2S production in PC12 cells, suggesting that blockade of ERK1/2 activation abolishes homocysteine-induced inhibition in H2S production. These data indicate that homocysteine inhibits endogenous H2S production by activation of ERK1/2.

We further examine whether pretreatment with U0126 prevents homocysteine-induced cytotoxicity. As shown in Fig. 4c, pretreatment of PC12 cells with U0126 (20 μmol/L) for 30 min significantly prevented the cytotoxicity induced by 10 mmol/L of homocysteine for 24 h, indicating that abolishment of homocysteine-induced inhibition in endogenous H2S production protects PC12 cells against homocysteine-mediated cytotoxicity.

Discussion

Hcy is a strong, independent risk factor of AD (Clarke et al. 1998; Dwyer et al. 2004; Miller 1999; Seshadri et al. 2002; Van Dam and Van Gool 2009). Elucidating the molecular mechanisms of Hcy-induced neurotoxicity will lead to important insights into the pathogenesis and treatment of AD. H2S is a well-known toxic gas with the smell of rotten eggs. However, evidence has accumulated in recent years, which suggests that H2S is formed naturally in mammalian tissues and exhibits a range of biological and physiological effects (Kimura 2002; Lowicka and Beltowski 2007; Szabo 2007; Wang 2002). Now, H2S is recognized as the third member of the family of “gasotransmitters” (Lowicka and Beltowski 2007; Wang 2002). H2S deficiency was observed in arterial and pulmonary hypertension, gastric mucosal injury, and liver cirrhosis (Lowicka and Beltowski 2007). We have demonstrated the contribution of deficits in endogenous H2S generation to 1-methy-4-phenylpyridinium ion-induced neurotoxicity (Tang et al. 2010a). To the best of our knowledge, there is no information about the potential role of H2S generation in Hcy-induced neuronal cell death. Furthermore, recent studies have showed opposite effects of homocysteine and H2S on the viability of neuronal cells: Homocysteine induces ROS formation and stimulates neurotoxicity (Ho et al. 2001; White et al. 2001), while H2S scavenges ROS formation and prevents oxidative stress-induced neuron death (Kimura and Kimura 2004; Tang et al. 2008; Whiteman et al. 2004; Whiteman et al. 2005). Therefore, the main aim of the present work was to elucidate whether Hcy impairs endogenous H2S generation and whether H2S deficiency contributes to Hcy-mediated neurotoxicity.

PC12 cells, originally derived from a transplantable rat pheochromocytoma, are accepted as a model system for primary neuronal cells because of their ability to respond to nerve growth factor (Greene and Tischler 1976). We have demonstrated that PC12 cells can generate H2S, and CBS is the main enzyme responsible for the generation of H2S (Tang et al. 2010a). In the present work, we used PC12 cells to study the effect of Hcy on endogenous H2S generation and its role in the neurotoxicity of Hcy. We demonstrated that exposure of PC12 cells to Hcy led to the significant decrease in H2S generation. We further found that Hcy inhibited the expression and activity of CBS in PC12 cells. These data indicated that Hcy reduces H2S generation by down-regulating the expression and activity of CBS.

Is inhibition of H2S generation a novel mechanism underlying the cytotoxicity of Hcy? To answer this question, we investigated the effects of stimulation of endogenous H2S production on the cytotoxicity of Hcy. PC12 cells were treated with SAM, an agonist of CBS, for 30 min, before they were exposed to Hcy. Our results demonstrated that administration of SAM not only extenuated Hcy-induced inhibition in endogenous H2S production in PC12 cells but also protected PC12 cells against cytotoxicity induced by Hcy. Together with the previous reports that exogenous administration of H2S protected PC12 cells against Hcy-induced neurotoxicity(Tang et al. 2010b), we suggest that reduced H2S generation contributes to PC12 cells injury caused by Hcy and that abolishment of Hcy-induced inhibition in endogenous H2S production protects PC12 cells against Hcy-mediated cytotoxicity. The toxicity of Hcy is believed to be due to the generation of ROS, including superoxide anion (\( {\hbox{O}}_2^{{ \bullet - }} \)) and hydrogen peroxide (H2O2; Heinecke et al. 1987). H2S is a highly reactive molecule and reacts with \( {\hbox{O}}_2^{{ \bullet - }} \) (Mitsuhashi et al. 2005) and H2O2 (Geng et al. 2004). In the present work, we also found that pretreatment with SAM extenuates Hcy-induced ROS accumulation. In addition, we have demonstrated that exogenous administration of H2S extenuates Hcy-induced ROS accumulation (Tang et al. 2010b). Thus, we hypothesize that inhibited CBS expression and activity and reduced H2S generation by Hcy lead to the scavenging of ROS deficiency, resulting in excessive levels of ROS, and ultimately promote cell to death.

Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that regulate the diversity of cellular activities. Three major classes have been described: ERKs, involved in proliferation and differentiation, and the JNKs and p38 MAPK both involved in stress response. Several in vitro models of the Hcy toxicity in hippocampus slices, primary cortical neurons, and cerebellar granule cells in culture have reported an activation of the ERK1/2 by Hcy (Gu et al. 2010; Poddar and Paul 2009; Robert et al. 2005). Therefore, we investigated whether activation of ERK1/2 occurs in Hcy-treated PC12 cells and the role of Hcy-triggered ERK1/2 activation in Hcy-induced inhibition of endogenous H2S generation. Our results demonstrated that Hcy stimulated a concentration-dependent ERK1/2 activation in PC12 cells and pretreatment with U0126, a special ERK1/2 inhibitor, abolished Hcy-induced inhibition in H2S production. These data indicated that inhibition of endogenous H2S generation by Hcy is mediated by activation of ERK1/2. In addition, a decreased neurotoxicity of Hcy was demonstrated when ERK1/2 activity was prevented by U0126. This finding is consistent with the above notation that preventing the inhibition of endogenous H2S production induced by Hcy protects PC12 cells against the neurotoxicity of Hcy.

In conclusion, the present work showed that Hcy inhibited endogenous H2S generation and down-regulated the expression and activity of CBS, the main enzyme responsible for the generation of H2S in PC12 cells. Prevention of Hcy-induced inhibition in endogenous H2S production by pretreatment with SAM, an activator of CBS, protected PC12 cells against Hcy-caused damage and accumulation of ROS. Hcy activated ERK1/2 and blockade of ERK1/2 with U0126 abolished the Hcy-induced cytotoxicity and inhibitory effect on endogenous H2S generation. These findings indicated that the neurotoxicity of Hcy is associated with the inhibited production of H2S, which is mediated by activation of ERK1/2. These data also suggest that H2S is an important endogenous substance to reduce neurotoxicity induced by Hcy and that enhancement of H2S synthesis may be a useful therapeutic strategy against AD associated with Hcy.

Acknowledgments

This study was supported by Natural Science Foundation of China (81071005 and 30770740), Natural Science Foundation of Hunan Province, China (06JJ2074), China Postdoctoral Science Foundation (2005038233), Plan Project for Scientific Research, Department of Science and Technology, Hunan Province (05FJ3039), and the Research Foundation of Education Bureau of Hunan Province (06C700).

Copyright information

© Springer Science+Business Media, LLC 2010