Hydrogen sulfide antagonizes homocysteine-induced neurotoxicity in PC12 cells

Neuroscience Research 68 (2010) 241–249

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Hydrogen sulfide antagonizes homocysteine-induced neurotoxicity in PC12 cells Xiao-Qing Tang a,∗,1 , Xin-Tian Shen a,c,1 , Yi-E Huang c , Yan-Kai Ren a , Rong-Qian Chen a , Bi Hu a , Jian-Qin He a , Wei-Lan Yin a , Jin-Hua Xu d , Zhi-Sheng Jiang b,∗∗ a

Department of Physiology, Medical College, University of South China, 28 West Changsheng Road, Hengyang 421001, Hunan, PR China Department of Pathophysiology, Medical College, University of South China, 28 West Changsheng Road, Hengyang 421001, Hunan, PR China Department of Physiology, Huaihua Medical College, Huaihua 418000, Hunan, PR China d Laboratory Center of Biochemistry and Molecular Biology, University of South China, 28 West Changsheng Road, Hengyang 421001, Hunan, PR China b c

a r t i c l e

i n f o

Article history: Received 2 December 2009 Received in revised form 2 June 2010 Accepted 21 July 2010 Available online 30 July 2010 Keywords: Hydrogen sulfide Homocysteine Neuroprotection Mitochondrial membrane potential Reactive oxygen species Bcl-2

a b s t r a c t Hydrogen sulfide (H2 S) has been shown to protect neurons against oxidative stress. Lower levels of H2 S as well as accumulation of homocysteine (Hcy), a strong risk of Alzheimer’s disease (AD), are reported in the brains of AD patients. The aim of present study is to explore the protection of H2 S against Hcy-induced cytotoxicity and apoptosis and the molecular mechanisms underlying in PC12 cells. We show that sodium hydrosulfide (NaHS), a H2 S donor, protects PC12 cells against Hcy-mediated cytotoxicity and apoptosis by preventing both the loss of mitochondrial membrane potential (MMP) and the increase in intracellular reactive oxygen species (ROS) induced by Hcy. NaHS not only promotes the expression of bcl-2, but also blocks the down-regulation of bcl-2 by Hcy. These results indicate that H2 S protects neuronal cells against neurotoxicity of Hcy by preserving MMP and attenuating ROS accumulation through up-regulation of bcl2 level. Our study suggests a promising future of H2 S-based therapies for neurodegenerative diseases such as AD. © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction Homocysteine (Hcy) is a key metabolic intermediate in sulfuramino 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 elevate 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), suggesting the potential role of Hcy as a novel therapeutic target for AD (Dwyer et al., 2004). Hcy is remethylated to methionine by vitamin B12dependent methionine synthase during methionine cycle, and can be converted to cysteine through the transsulfuration pathway.

∗ Corresponding author at: Department of Physiology, Medical College, University of South China, 28 West Changsheng Road, Hengyang 421001, Hunan, PR China. Tel.: +86 734 8281389/8282847; fax: +86 734 8281308/8282847. ∗∗ Corresponding author at: Department of Pathophysiology, Medical College, University of South China, 28 West Changsheng Road, Hengyang 421001, Hunan, PR China. E-mail addresses: [email protected] (X.-Q. Tang), [email protected] (Z.-S. Jiang). 1 Both authors contributed equally to this work.

The first and committing step in the transsulfuration pathway is catalyzed by cystathionine-␤-synthetase (CBS). Hydrogen sulfide (H2 S), 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 H2 S is synthesized from l-cysteine and this process is predominantly catalyzed by CBS (Moore et al., 2003; Wang, 2002), the key enzyme in the transsulfuration pathway that processes Hcy. Interestingly, both elevated Hcy and decreased H2 S are observed in the brains of AD patients (Eto et al., 2002). In addition, recent studies have showed opposite effects of homocysteine and H2 S on the viability of neuronal cells: homocysteine induces reactive oxygen species (ROS) formation and stimulates neurotoxicity (Ho et al., 2001; White et al., 2001), while H2 S scavenges ROS formation and prevents oxidative stress-induced neuron death (Kimura and Kimura, 2004; Tang et al., 2008; Whiteman et al., 2004, 2005). Therefore, we wondered whether H2 S directly antagonizes the toxicity of homocysteine to neuronal cells. In the present study, we investigated the effects of H2 S on homocysteine-induced oxidative damage and apoptosis in neuronal cells 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 sodium hydrosulfide

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(NaHS), a H2 S donor, significantly protected PC12 cells against Hcy-induced cytotoxicity and apoptosis by inhibiting dissipation of mitochondrial membrane potential (MMP) and overproduction of ROS through up-regulation of bcl-2 expression. 2. Materials and methods 2.1. Materials Hoechst 33258, propidium iodide (PI), RNase, rhodamine 123 (Rh123), 2 ,7 -dichlorfluorescein-diacetate (DCFH-DA), sodium hydrosulfide (NaHS) and homocysteine were purchased from Sigma Chemical CO (St. Louis, MO, USA). The lactic dehydrogenase (LDH) release assay kit supplied by Uscn Life Science Inc. (Wuhan, China). Specific monoclonal anti-bcl-2 antibody was obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). RPMI-1640 medium, horse serum and fetal bovine serum were supplied by Gibico BRL (Ground Island, NY, USA).

2.2. Cell culture

2.7. Measurement of the mitochondrial membrane potential (MMP) MMP was monitored using the fluorescent dye Rh123, a cell permeable cationic dye, which preferentially enters into mitochondria based on the highly negative MMP. Depolarization of MMP results in the loss of Rh123 from the mitochondria and a decrease in intracellular fluorescence (Mattson et al., 1993). Rh123 (100 ␮g/L) was added to cell cultures for 45 min at 37 ◦ C. Rh123 fluorescence was measured over the whole field of vision using a fluorescent microscope connected to an imaging system (BX50-FLA, Olympus, Tokyo, Japan). In addition, ten thousand cells per sample were analyzed by flow cytometry (FCM, Beckman-Coulter Co., USA), the mean fluorescent intensity (MFI) in the positive cells represents the level of MMP. 2.8. Measurement of intracellular ROS generation Intracellular ROS were determined by oxidative conversion of cell permeable 2 ,7 -dichlorfluorescein-diacetate (DCFH-DA) to fluorescent 2 ,7 -dichlorfluorescein (DCF) (Cathcart et al., 1983; Grieve et al., 1992). The cells were collected by pipetting and were washed one time with PBS. After DCFH-DA (2.5 ␮M) was added to cell cultures for 20 min at 37 ◦ C, the cells were washed twice with PBS. DCF fluorescence was measured over the whole field of vision using a fluorescent

PC12 cells, a rat cell line derived from a 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 (FBS) at 37 ◦ C under an atmosphere of 5% CO2 and 95% air. The culture media was changed three times per week.

2.3. Determination of cell viability The viability of PC12 cell line was determined by trypan blue exclusion analysis. At the end of treatment, 0.2 mL of the cells suspension were transferred to test tubes with 0.5 mL of 0.4% (w/v) trypan blue solution and 0.3 mL of PBS and mixed thoroughly at room temperature. Allow to stand for 5–15 min. Only dead cells with a damaged cell membrane are permeable to trypan blue. The numbers of trypan blue-permeable blue cells and viable white cells were counted in six randomly chosen fields per well under a phase contrast microscope (BX50-FLA; Olympus, Tokyo, Japan). The percentage of viable cells was evaluated as follows: the percentage of viable cells = the numbers of viable white cells/(the numbers of trypan blue-permeable blue cells + the numbers of viable white cells) × 100%. 2.4. Lactate dehydrogenase (LDH) release assay At the end of treatment, cell culture medium was collected and briefly centrifuged. The supernatants (50 ␮L) were transferred into wells in 96-well plates pre-coated with an antibody specific to LDH. Avidin conjugated to horseradish peroxidase (HRP) (100 ␮L) is added to each well and incubated for 60 min at 37 ◦ C. The liquid of each well is removed. Then the substrate solution (100 ␮L) is added to each well and incubated for 15 min at 37 ◦ C. The enzyme–substrate reaction is terminated by the addition of a sulfuric acid solution. Spectrophotometrical absorbance was measured at a wavelength of 450 nm. The concentration of LDH in the samples is then determined by comparing the O.D. of the samples to the standard curve.

2.5. Nuclear staining for assessment of apoptosis Chromosomal condensation and morphological changes in the nucleus of PC12 cells were observed using the chromatin dye Hoechst 33258. The PC12 cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 10 min. After three rinses with PBS, the cells were stained with 5 mg/L Hoechst 33258 for 10 min. Slides were rinsed briefly with PBS, air dried, then mounted in an antifluorescein fading medium (Perma Fluor, Immunon, PA, USA). Slides were visualized under a fluorescent microscope (BX50-FLA, Olympus, Tokyo, Japan). Viable cells displayed normal nuclear size and uniform fluorescence, whereas apoptotic cells showed condensed nuclei or nuclear condensations. The percentage of apoptotic cells was evaluated as follows: the percentage of apoptotic cells = the number of apoptotic cells/(the number of apoptotic cells + the numbers of viable cells) × 100%. 2.6. Flow cytometry analysis of apoptosis Treated PC12 cells were digested with trypsin (2.5 g/L) and centrifuged at 250 × g for 10 min and the supernatant removed. Cells were washed twice with PBS and fixed with 70% ethanol. Cells were then centrifuged at 250 × g for 10 min, washed in PBS twice and adjusted to a concentration of 1 × 106 cells/mL. To a 0.5 mL cell sample, 0.5 mL RNase (1 mg/mL in PBS) was added. After gentle mixing with PI (at a final concentration of 50 mg/L), mixed cells were filtered and incubated in the dark at 4 ◦ C for 30 min before flow cytometric (FCM, Beckman-Coulter, Miami, FL, USA) analysis. In the DNA histogram, the amplitude of the sub-G1 DNA peak represents the number of apoptotic cells.

Fig. 1. Effect of hydrogen sulfide on homocysteine-induced cytotoxicity in PC12 cells. (A) PC12 cells were treated with indicated concentrations of homocysteine in the absence or presence of 200 ␮mol/L NaHS for 24 h. (B) PC12 cells were treated with 10 mmol/L homocysteine in the absence or presence of 50, 100, or 200 ␮mol/L NaHS for 24 h. Cell viability was determined by trypan blue exclusion analysis. (C) PC12 cells were treated with 10 mmol/L homocysteine in the absence or presence of 200 ␮mol/L NaHS for 24 h and the lactic dehydrogenase (LDH) release was detected by LDH release assay. Values are the mean ± SEM (n = 5). **p < 0.01 vs. control group; ## p < 0.01 vs. homocysteine-treated alone group.

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Fig. 2. Hydrogen sulfide ameliorates homocysteine-induced morphological defect in PC12 cells. PC12 cells were treated with control, NaHS (200 ␮mol/L), homocysteine (Hcy, 10 mmol/L) and NaHS (200 ␮mol/L) + homocysteine (Hcy, 10 mmol/L) and then examined under a bright field microscope (10× objective, BX50-FLA, Olympus) after 24 h of treatment. microscope connected to an imaging system (BX50-FLA, Olympus, Tokyo, Japan). In addition, the mean fluorescent intensity (MFI) of the positive cells in ten thousand cells per sample was measured by FCM, and the MFI represents the amount of ROS. 2.9. Western blot analysis for bcl-2 expression SDS-polyacrylamide gel electrophoresis (PAGE) 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 PVDF transfer membrane (Solarbio, China). After this, the membranes were blocked with TBST (50 mM Tris–HCl, pH 7.4, 0.15 M NaCl, 0.1% Tween-20) containing 5% BSA (Sigma, USA) for 2 h. Following this, the membranes were incubated with rabbit monoclonal anti-bcl2 primary antibodies (Cell Signaling Technology, USA) diluted 1:1000 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 1:1000 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. 2.10. 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.

3. Results 3.1. Hydrogen sulfide protects PC12 cells against homocysteine-induced cytotoxicity To determine the protection of H2 S against homocysteineinduced cytotoxicity, cell viability was evaluated by trypan blue exclusion analysis. As shown in Fig. 1(A), the cytotoxic effects of 5, 10 and 20 mmol/L homocysteine were significantly blocked by 200 ␮mol/L NaHS. Furthermore, PC12 cells were treated with 10 mmol/L homocysteine together with various amount of NaHS (50, 100 and 200 ␮mol/L), and the counteractive effect of NaHS was concentration dependent (Fig. 1(B)).

NaHS alone (50, 100 and 200 ␮mol/L) did not significantly affect the viability of PC12 cells (data not shown). These results indicate that H2 S protects PC12 cells homocysteine-caused cytotoxicity. The beneficial effects of H2 S against homocysteine-exerted cytotoxicity were further examined by the lactic dehydrogenase (LDH) release assay. Exposure of PC12 cells to 10 mmol/L homocysteine for 24 h induced an increase in LDH release compared with that in the untreated group (Fig. 1(C)). Co-treated with NaHS (200 ␮mol/L) significantly attenuated homocysteine-induced LDH release (Fig. 1(C)). NaHS (200 ␮mol/L) alone had no effect on LDH release (Fig. 1(C)). In addition, we observed the effect of NaHS on morphology of PC12 cells treated with homocysteine. As shown in Fig. 2, at 24 h after treatment of 10 mmol/L homocysteine, PC12 cells lost their normal spindle-like morphology, shrank or became round, detached and floated in the media. Co-treatment with NaHS (200 ␮mol/L) markedly ameliorated homocysteine-induced morphological alteration. Untreated and NaHS-treated cells displayed normal morphology. 3.2. Hydrogen sulfide protects PC12 cells against homocysteine-induced apoptosis The nuclear staining assay was used to assess the morphological changes of apoptosis in PC12 cells. As illustrated in Fig. 3, the untreated cells and the cells treated with 200 ␮mol/L NaHS exhibited uniformly dispersed chromatin and intact cell membrane. On the other hand, the homocysteine-treated cells (10 mmol/L, for 24 h) appeared typical characteristics of apoptosis, including apoptotic nuclear condensation. When PC12 cells were co-treated with 10 mmol/L homocysteine and 200 ␮mol/L NaHS for 24 h, however, the number of cells with nuclear condensation was significantly reduced, suggesting that NaHS protects PC12 cells against apoptosis induced by homocysteine. Similarly, the statistical finding from FCM analysis after PI staining for apoptosis also indicated that H2 S protects PC12 cells against homocysteine-induced apoptosis. As shown in Fig. 4, exposure of

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Fig. 3. Nuclear staining to evaluate the anti-apoptotic effect of hydrogen sulfide. PC12 cells exposed to 10 mmol/L homocysteine (Hcy) in the absence or presence of 200 ␮mol/L NaHS for 24 h and incubated with 5 mg/L Hoechst 33258 for 30 min. (A) Representative morphology visualized under a fluorescence microscope (10× objective, BX50-FLA, Olympus). Cells with brightly fluorescent and fragmented nuclei were apoptotic. (B) Quantitative analysis of the percentage of apoptotic cells. Values are the mean ± SEM (n = 5). **p < 0.01 vs. control group; ## p < 0.01 vs. homocysteine-treated alone group.

PC12 cells to homocysteine (10 mmol/L, 24 h) caused significant apoptosis and the apoptotic effects induced by homocysteine were inhibited by co-treatment with NaHS (200 ␮mol/L) for 24 h. 3.3. Hydrogen sulfide prevents homocysteine-induced dissipation of the mitochondrial membrane potential (MMP) Dissipation of MMP is a critical event in the process of apoptosis (Petronilli et al., 2001). To examine preservation of MMP involve in the anti-apoptotic effect of H2 S, we used Rh123 staining to assess the level of MMP in PC12 cells. After 24 h exposure to 10 mmol/L homocysteine, the MMP was obviously reduced, as shown by the decrease in Rh123 fluorescence (Fig. 5(A)) and the MFI of Rh123 quantified by FCM analysis (Fig. 5(B) and (C)), compared with non-treated control cells. Although NaHS exposure alone (200 ␮mol/L) has no effect on MMP of PC12 cells, the cells co-

treated with NaHS (200 ␮mol/L) and homocysteine (10 mmol/L) for 24 h showed enhanced intensity of Rh123 fluorescence compared to the homocysteine-treated cells (Fig. 5). These results suggested that homocysteine-induced dissipation of MMP is inhibited by H2 S. 3.4. Hydrogen sulfide attenuates homocysteine-induced an increase in intracellular reactive oxygen species (ROS) level As the cytotoxicity of homocysteine is mainly mediated by oxidative stress (Ho et al., 2001; White et al., 2001), we investigated the effect NaHS on homocysteine-induced ROS formation using DCFH-DA staining. Compared with non-treated control cells, the level of intracellular ROS was increased in PC12 cells treated with 10 mmol/L homocysteine for 24 h, as shown by the increase in DCF fluorescence (Fig. 6(A)) and the MFI of DCF quantified by

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FCM analysis (Fig. 6(B) and (C)). However, when PC12 cells were co-treated with NaHS (200 ␮mol/L) and homocysteine (10 mmol/L) for 24 h, both the DCF fluorescence (Fig. 6(A)) and the MFI of DCF (Fig. 6(B) and (C)) were significantly decreased, suggesting that homocysteine-induced intracellular ROS accumulation is attenuated by H2 S. The cells treated with NaHS (200 ␮mol/L) alone showed weak DCF fluorescence similar to that in the vehicle control (Fig. 6).

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bcl-2 were measured by Western blot analysis. As illustrated in Fig. 7, 24 h treatment with 200 ␮mol/L NaHS increased the amount of bcl-2 expression by 69% (p < 0.01). Exposure to homocysteine (10 mmol/L, 24 h), on the other hand, reduced bcl-2 by 39% (p < 0.01). However, the homocysteine-induced decrease of bcl-2 was significantly abolished by co-treatment with 200 ␮mol/L NaHS for 24 h (p < 0.01). These results indicate that H2 S is able to not only promote the expression of bcl-2, but also block the homocysteineinduced down-regulation of bcl-2.

3.5. Hydrogen sulfide promotes bcl-2 expression and blocks homocysteine-induced down-regulation of bcl-2 expression

4. Discussion

Bcl-2 is an anti-apoptotic protein. To explore whether H2 S modulate the expression of bcl-2 in PC12 cells and its response to the effect of homocysteine on bcl-2 expression, the levels of

In the present work, we provided evidence that NaHS, a H2 S donor, obviously protects PC12 cells against homocysteine (Hcy)induced cytotoxicity and apoptosis. It prevents Hcy-mediated loss

Fig. 4. Flow cytometric analysis after PI staining to evaluate the anti-apoptotic effect of hydrogen sulfide. PC12 cells were treated with homocysteine (Hcy, 10 mmol/L) in the absence or presence of NaHS (200 ␮mol/L) for 24 h and apoptosis was assessed by flow cytometry after PI staining. (A) Representative DNA histogram of PC12 cells exposed to different treatments. In the DNA histogram, the amplitude of the sub-G1 DNA peak represents the number of apoptotic cells. (B) Quantitative analysis of the rate of apoptosis. Values are the mean ± SEM (n = 3), **p < 0.01 vs. control group; ## p < 0.01 vs. Hcy alone group.

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of MMP as well as Hcy-induced increase in intracellular ROS. NaHS not only promotes the expression of bcl-2, but also blocks Hcyinduced down-regulation of bcl-2 level. Hcy, a metabolite of the essential amino acid methionine, is toxic to neuron (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). Elevation of plasma Hcy is correlated with Alzheimer’s disease (AD) (Miller, 1999; Van Dam and Van Gool, 2009) and elevated brain homocysteine has been reported in AD (Eto et al., 2002). Antagonizing the Hcy neurotoxicity could be a new therapeutic way for AD. Our previous study demonstrated that H2 S protects neurons against beta-amyloid-induced cytotoxicity and apoptosis (Tang et al., 2008). Therefore, we suggested that H2 S may be a potential therapeutic approach for treatment of AD. Hcy is known to induce neurotoxicity and apoptosis (Baydas et al., 2005; Kruman et al., 2000) and cause oxidative damage (Yan et al., 2006). In the present work, we examined the effect of Hcy

on the viability and apoptosis of PC12 cells. Similar to the findings by Linnebank et al. (2006), we found that exposure of PC12 cells to homocysteine resulted in decrease of viability and increase of apoptotic cells. These results indicated that Hcy induces significant neurotoxicity and apoptosis in PC12 cell. Furthermore, in agreement with previous report that H2 S protects myocardium and vascular smooth muscle cells against Hcy-induced damage (Chang et al., 2008; Yan et al., 2006), our results demonstrated that H2 S protects PC12 cells against Hcy-exerted cytotoxicity and apoptosis. To our knowledge, this is the first report that treatment with H2 S ameliorates the neurotoxicity of Hcy. It has been reported that mitochondrial damage is an important factor associated with cell death and some models of apoptosis (Petronilli et al., 2001). Mitochondrial damage is consistent with intracellular ROS production and changes in MMP during apoptosis (Kim et al., 2004). MMP has been shown to be involved in a variety of pathophysiological conditions, in particular for apopto-

Fig. 5. Effects of H2 S on homocysteine-induced loss of mitochondrial membrane potential (MMP) in PC12 cells. PC12 cells were exposed to 10 mmol/L homocysteine (Hcy) in the presence or absence of 200 ␮mol/L NaHS for 24 h and stained with Rh123 for 20 min. The changes of MMP in different treatment groups were visualized under fluorescence microscope (10× objective, BX50-FLA, Olympus) (A) and quantified by fluorescent sorting FCM analysis (B, C). (A) Representative micrographs of Rh123-derived fluorescence in PC12 cells exposed to different treatments. (B) Representative histogram of Rh123-derived fluorescence in PC12 cells exposed to different treatments measured by FCM. (C) Quantitative analysis of the mean fluorescence intensity (MFI) of Rh123 measured by FCM. Values are the mean ± SEM (n = 3). **p < 0.01 vs. control; ## p < 0.01 vs. 10 mmol/L homocysteine-treated alone group.

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Fig. 6. Effects of H2 S on homocysteine-exerted accumulation of intracellular reactive oxygen species (ROS) in PC12 cells. PC12 cells were exposed to 10 mmol/L homocysteine (Hcy) in the presence or absence of 200 ␮mol/L NaHS for 24 h and stained with DCFH-DA for 20 min. The changes of ROS in different treatment groups were visualized under fluorescence microscope (10× objective, BX50-FLA, Olympus) (A) and quantified by fluorescent sorting FCM analysis (B, C). (A) Representative micrographs of DCF-derived fluorescence in PC12 cells exposed to different treatments. (B) Representative histogram of DCF-derived fluorescence in PC12 cells exposed to different treatments measured by FCM. (C) Quantitative analysis of the mean fluorescence intensity (MFI) of DCF measured by FCM. Values are the mean ± SEM (n = 3). **p < 0.01 vs. control; ## p < 0.01 vs. 10 mmol/L homocysteine-treated alone group.

sis (Fiskum et al., 2003; Knott et al., 2008). ROS is responsible for the homocysteine-induced neurotoxicity (Ho et al., 2001; White et al., 2001). To investigate the mechanisms of the cytoprotective effect of H2 S on Hcy-induced toxicity in PC12 cells, we examined the effects of NaHS on the Hcy-mediated changes in MMP and ROS. The dissipation of MMP and overproduction of ROS were significantly induced in Hcy-exposed PC12 cells, while co-treated with NaHS prevented both phenomena. In agreement with previous studies that H2 S prevents the loss of MMP induced by rotenone (Hu et al., 2009) and that H2 S decreases ROS generation induced by homocysteine in vascular smooth muscle cells (Yan et al., 2006), our results suggested that H2 S protects cells against Hcy-induced neurotoxicity by blocking the loss of MMP and the increase in ROS level. Recently, the antioxidant efficacy of bcl-2 has been emphasized. Bcl-2 has been shown to prevent apoptosis by regulating an

antioxidant pathway (Hockenbery et al., 1993). Kane et al. (1993) indicated that bcl-2 inhibits neural death by reducing the generation of ROS. Additionally, over-expression of bcl-2 increases stability of MMP (Wu et al., 2004), and blocks cytochrome C release from mitochondria prior to mitochondrial membrane depolarization by preventing mitochondrial pore opening (Yang et al., 1997). These findings suggested that the cytoprotective effects associated with decrease in ROS generation and stability of MMP may be the results of over-expressed bcl-2. In the present study, we demonstrated that H2 S not only promoted the expression of bcl2, but also blocked the down-regulation of bcl-2 induced by Hcy, which implied that H2 S-induced up-regulation of bcl-2 expression may be involved in the neuroprotective effects of H2 S against Hcy. H2 S exists in the body in two forms. In physiological saline, about one-third of the H2 S exists in undissociated form and the remain-

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References

Fig. 7. Effects of hydrogen sulfide on the expression of bcl-2 in PC12 cells. The levels of bcl-2 expression in PC12 cells, following exposure to 10 mmol/L homocysteine (Hcy) for 24 h in the presence or absence of 200 ␮mol/L NaHS, were determined by Western blot using an anti-bcl-2 antibody. Western blot images show representative results from three independent experiments. In all blots, staining for ␤-actin was used as a loading control. The level of bcl-2 expression obtained in each experimental condition was calculated as a fold of the control. Values are the mean ± SEM (n = 3). **p < 0.01 vs. control; ## p < 0.01 vs. 10 mmol/L homocysteine-treated alone group.

ing two-thirds in HS− form at equilibrium with H2 S (Reiffenstein et al., 1992). Its difficult to calculate concentration of H2 S when applying H2 S by bubbling H2 S gas into the solution. Instead, NaHS dissociates to Na+ and HS− in solution and then HS− associates with H+ to produce H2 S. Using NaHS as the donor of H2 S makes it easy to define the concentration of H2 S in solution more accurately and reproducibly. Therefore, NaHS solution has been widely used to provide exogenous H2 S for studies of H2 S (Abe and Kimura, 1996; Chang et al., 2008; Hu et al., 2009; Kimura and Kimura, 2004). In conclusion, we demonstrate that NaHS, a H2 S donor, protects PC12 cells against homocysteine-induced cytotoxicity and apoptosis. The underlying mechanism may involve inhibition of both loss of MMP and accumulation of ROS by preventing Hcy-induced down-regulation of bcl-2 expression. Previous studies have shown that the lowest antioxidant activity of apolipoprotein E4 involve Alzheimer’s disease (AD) (Tamaoka et al., 2000) and that oxidative stress up-regulates the expression of presenilin 1 in neuronal cells (Oda et al., 2010), clarifying that oxidative stress plays an extremely important role in the pathomechanism of AD. We have previously reported that H2 S protects neurons against beta-amyloid-induced cytotoxicity and apoptosis (Tang et al., 2008). Taken together, these findings suggest a promising role of H2 S supplement as a novel therapeutic strategy for AD.

Acknowledgements We thank Y. Wang for excellent revising. This study was supported by Natural Science Foundation of China (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).

Abe, K., Kimura, H., 1996. The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. 16, 1066–1071. Baydas, G., Reiter, R.J., Akbulut, M., Tuzcu, M., Tamer, S., 2005. Melatonin inhibits neural apoptosis induced by homocysteine in hippocampus of rats via inhibition of cytochrome c translocation and caspase-3 activation and by regulating proand anti-apoptotic protein levels. Neuroscience 135, 879–886. Cathcart, R., Schwiers, E., Ames, B.N., 1983. Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay. Anal. Biochem. 134, 111–116. Chang, L., Geng, B., Yu, F., Zhao, J., Jiang, H., Du, J., Tang, C., 2008. Hydrogen sulfide inhibits myocardial injury induced by homocysteine in rats. Amino Acids 34, 573–585. Clarke, R., Smith, A.D., Jobst, K.A., Refsum, H., Sutton, L., Ueland, P.M., 1998. Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch. Neurol. 55, 1449–1455. Duan, W.G., Shang, J., Jiang, Z.Z., Yao, J.C., Yun, Y., Yan, M., Shu, B., Lin, Q., Yu, Z.P., Zhang, L.Y., 2009. Rho kinase inhibitor Y-27632 down-regulates norepinephrine synthesis and release in PC12 cells. Basic Clin. Pharmacol. Toxicol. 104, 434–440. Dwyer, B.E., Raina, A.K., Perry, G., Smith, M.A., 2004. Homocysteine and Alzheimer’s disease: a modifiable risk? Free Radic. Biol. Med. 36, 1471–1475. Eto, K., Asada, T., Arima, K., Makifuchi, T., Kimura, H., 2002. Brain hydrogen sulfide is severely decreased in Alzheimer’s disease. Biochem. Biophys. Res. Commun. 293, 1485–1488. Fiskum, G., Starkov, A., Polster, B.M., Chinopoulos, C., 2003. Mitochondrial mechanisms of neural cell death and neuroprotective interventions in Parkinson’s disease. Ann. N. Y. Acad. Sci. 991, 111–119. Grieve, A., Butcher, S.P., Griffiths, R., 1992. Synaptosomal plasma membrane transport of excitatory sulfur amino acid transmitter candidates: kinetic characterisation and analysis of carrier specificity. J. Neurosci. Res. 32, 60–68. Ho, P.I., Collins, S.C., Dhitavat, S., Ortiz, D., Ashline, D., Rogers, E., Shea, T.B., 2001. Homocysteine potentiates beta-amyloid neurotoxicity: role of oxidative stress. J. Neurochem. 78, 249–253. Ho, P.I., Ortiz, D., Rogers, E., Shea, T.B., 2002. Multiple aspects of homocysteine neurotoxicity: glutamate excitotoxicity, kinase hyperactivation and DNA damage. J. Neurosci. Res. 70, 694–702. Hockenbery, D.M., Oltvai, Z.N., Yin, X.M., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241– 251. Hu, L.F., Lu, M., Wu, Z.Y., Wong, P.T., Bian, J.S., 2009. Hydrogen sulfide inhibits rotenone-induced apoptosis via preservation of mitochondrial function. Mol. Pharmacol. 75, 27–34. Kane, D.J., Sarafian, T.A., Anton, R., Hahn, H., Gralla, E.B., Valentine, J.S., Ord, T., Bredesen, D.E., 1993. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 262, 1274–1277. Kim, J.P., Koh, J.Y., Choi, D.W., 1987. L-homocysteate is a potent neurotoxin on cultured cortical neurons. Brain Res. 437, 103–110. Kim, W.H., Park, W.B., Gao, B., Jung, M.H., 2004. Critical role of reactive oxygen species and mitochondrial membrane potential in Korean mistletoe lectin-induced apoptosis in human hepatocarcinoma cells. Mol. Pharmacol. 66, 1383–1396. Kimura, Y., Kimura, H., 2004. Hydrogen sulfide protects neurons from oxidative stress. FASEB J. 18, 1165–1167. Knott, A.B., Perkins, G., Schwarzenbacher, R., Bossy-Wetzel, E., 2008. Mitochondrial fragmentation in neurodegeneration. Nat. Rev. Neurosci. 9, 505–518. Kruman, I.I., Culmsee, C., Chan, S.L., Kruman, Y., Guo, Z., Penix, L., Mattson, M.P., 2000. Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity to excitotoxicity. J. Neurosci. 20, 6920–6926. Li, R., Kong, Y., Ladisch, S., 1998. Nerve growth factor-induced neurite formation in PC12 cells is independent of endogenous cellular gangliosides. Glycobiology 8, 597–603. Linnebank, M., Lutz, H., Jarre, E., Vielhaber, S., Noelker, C., Struys, E., Jakobs, C., Klockgether, T., Evert, B.O., Kunz, W.S., Wullner, U., 2006. Binding of copper is a mechanism of homocysteine toxicity leading to COX deficiency and apoptosis in primary neurons, PC12 and SHSY-5Y cells. Neurobiol. Dis. 23, 725–730. Lipton, S.A., Kim, W.K., Choi, Y.B., Kumar, S., D’Emilia, D.M., Rayudu, P.V., Arnelle, D.R., Stamler, J.S., 1997. Neurotoxicity associated with dual actions of homocysteine at the N-methyl-d-aspartate receptor. Proc. Natl. Acad. Sci. U.S.A. 94, 5923–5928. Mattson, M.P., Zhang, Y., Bose, S., 1993. Growth factors prevent mitochondrial dysfunction, loss of calcium homeostasis, and cell injury, but not ATP depletion in hippocampal neurons deprived of glucose. Exp. Neurol. 121, 1–13. Miller, J.W., 1999. Homocysteine and Alzheimer’s disease. Nutr. Rev. 57, 126–129. Moore, P.K., Bhatia, M., Moochhala, S., 2003. Hydrogen sulfide: from the smell of the past to the mediator of the future? Trends Pharmacol. Sci. 24, 609–611. Oda, A., Tamaoka, A., Araki, W., 2010. Oxidative stress up-regulates presenilin 1 in lipid rafts in neuronal cells. J. Neurosci. Res. 88, 1137–1145. Parsons, R.B., Waring, R.H., Ramsden, D.B., Williams, A.C., 1998. In vitro effect of the cysteine metabolites homocysteic acid, homocysteine and cysteic acid upon human neuronal cell lines. Neurotoxicology 19, 599–603. Petronilli, V., Penzo, D., Scorrano, L., Bernardi, P., Di Lisa, F., 2001. The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ. J. Biol. Chem. 276, 12030–12034. Prudova, A., Bauman, Z., Braun, A., Vitvitsky, V., Lu, S.C., Banerjee, R., 2006. S-adenosylmethionine stabilizes cystathionine beta-synthase and modulates redox capacity. Proc. Natl. Acad. Sci. U.S.A. 103, 6489–6494.

X.-Q. Tang et al. / Neuroscience Research 68 (2010) 241–249 Reiffenstein, R.J., Hulbert, W.C., Roth, S.H., 1992. Toxicology of hydrogen sulfide. Annu. Rev. Pharmacol. Toxicol. 32, 109–134. Selhub, J., 1999. Homocysteine metabolism. Annu. Rev. Nutr. 19, 217–246. Seshadri, S., Beiser, A., Selhub, J., Jacques, P.F., Rosenberg, I.H., D’Agostino, R.B., Wilson, P.W., Wolf, P.A., 2002. Plasma homocysteine as a risk factor for dementia and Alzheimer’s disease. N. Engl. J. Med. 346, 476–483. Tamaoka, A., Miyatake, F., Matsuno, S., Ishii, K., Nagase, S., Sahara, N., Ono, S., Mori, H., Wakabayashi, K., Tsuji, S., et al., 2000. Apolipoprotein E allele-dependent antioxidant activity in brains with Alzheimer’s disease. Neurology 54, 2319–2321. Tang, X.Q., Yang, C.T., Chen, J., Yin, W.L., Tian, S.W., Hu, B., Feng, J.Q., Li, Y.J., 2008. Effect of hydrogen sulfide on beta-amyloid-induced damage in PC12 cells. Clin. Exp. Pharmacol. Physiol. 35, 180–186. Van Dam, F., Van Gool, W.A., 2009. Hyperhomocysteinemia and Alzheimer’s disease: a systematic review. Arch. Gerontol. Geriatr. 48, 425–430. Wang, R., 2002. Two’s company, three’s a crowd: can H2 S be the third endogenous gaseous transmitter? FASEB J. 16, 1792–1798. Wang, S., Hu, C.P., Jiang, D.J., Peng, J., Zhou, Z., Yuan, Q., Nie, S.D., Jiang, J.L., Li, Y.J., Huang, K.L., 2009. All-trans retinoic acid inhibits cobalt chloride-induced apoptosis in PC12 cells: role of the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine pathway. J. Neurosci. Res. 87, 1938–1946. White, A.R., Huang, X., Jobling, M.F., Barrow, C.J., Beyreuther, K., Masters, C.L., Bush, A.I., Cappai, R., 2001. Homocysteine potentiates copper- and amyloid

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beta peptide-mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer’s-type neurodegenerative pathways. J. Neurochem. 76, 1509–1520. Whiteman, M., Armstrong, J.S., Chu, S.H., Jia-Ling, S., Wong, B.S., Cheung, N.S., Halliwell, B., Moore, P.K., 2004. The novel neuromodulator hydrogen sulfide: an endogenous peroxynitrite ‘scavenger’? J. Neurochem. 90, 765– 768. Whiteman, M., Cheung, N.S., Zhu, Y.Z., Chu, S.H., Siau, J.L., Wong, B.S., Armstrong, J.S., Moore, P.K., 2005. Hydrogen sulfide: a novel inhibitor of hypochlorous acidmediated oxidative damage in the brain? Biochem. Biophys. Res. Commun. 326, 794–798. Wu, L.Y., Ding, A.S., Zhao, T., Ma, Z.M., Wang, F.Z., Fan, M., 2004. Involvement of increased stability of mitochondrial membrane potential and over-expression of Bcl-2 in enhanced anoxic tolerance induced by hypoxic preconditioning in cultured hypothalamic neurons. Brain Res. 999, 149–154. Yan, S.K., Chang, T., Wang, H., Wu, L., Wang, R., Meng, Q.H., 2006. Effects of hydrogen sulfide on homocysteine-induced oxidative stress in vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 351, 485–491. Yang, J., Liu, X., Bhalla, K., Kim, C.N., Ibrado, A.M., Cai, J., Peng, T.I., Jones, D.P., Wang, X., 1997. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275, 1129–1132.