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NAD+ treatment prevents rotenone-induced apoptosis and necrosis of differentiated PC12 cells
Neuroscience Letters 560 (2014) 46–50
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
NAD+ treatment prevents rotenone-induced apoptosis and necrosis of differentiated PC12 cells Yunyi Hong a , Hui Nie a , Danhong Wu c , Xunbin Wei a , Xianting Ding a , Weihai Ying a,b,∗ a b c
Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, PR China Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, PR China Department of Neurology, Third People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 201999, PR China
h i g h l i g h t s • • • •
NAD+ NAD+ NAD+ NAD+
treatment can decrease rotenone-induced early-stage and late-stage apoptosis of PC12 cells. treatment can decrease rotenone-induced necrosis of PC12 cells. treatment can restore the intracellular NAD+ levels of rotenone-treated PC12 cells. treatment can prevent rotenone-induced mitochondrial depolarization of PC12 cells.
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 18 November 2013 Accepted 20 November 2013 Keywords: NAD+ Apoptosis Necrosis PC12 cells Rotenone Mitochondrial membrane potential
a b s t r a c t Nicotinamide adenine dinucleotide (NAD+ ) plays critical roles in not only energy metabolism and mitochondrial functions, but also calcium homeostasis and immunological functions. It has been reported that NAD+ administration can reduce ischemic brain damage. However, the mechanisms underlying the protective effects remain unclear. Because mitochondrial impairments play a key role in the cell death in cerebral ischemia, in this study we tested our hypothesis that NAD+ can decrease mitochondrial damageinduced cell death using differentiated PC12 cells as a cellular model. We found that NAD+ can decrease both early-stage and late-stage apoptosis, as well as necrosis of rotenone-treated PC12 cells, as assessed by FACS-based Annexin V/AAD assay. We also found that NAD+ treatment can restore the intracellular NAD+ levels of the rotenone-treated cells. Moreover, NAD+ treatment can prevent rotenone-induced mitochondria depolarization. In summary, our study has provided first direct evidence that NAD+ treatment can prevent rotenone-induced apoptosis and necrosis. Our study has also indicated that NAD+ treatment can prevent mitochondrial damage-induced cell death, which may at least partially result from its protective effects on rotenone-induced mitochondrial depolarization. Because both mitochondrial damage and apoptosis play key roles in multiple neurological disorders, our study has highlighted the therapeutic potential of NAD+ for brain ischemia and other neurological diseases. © 2013 Elsevier Ireland Ltd. All rights reserved.
Introduction Oxidative stress, mitochondrial impairments, calcium dyshomeostasis, and excessive inflammation are critical pathological factors in multiple major neurological diseases, including stroke, Parkinson’s disease (PD), and Alzheimer’s disease [19,22]. While our understanding regarding the mechanisms of the diseases have significantly improved in the last 30 years, many future studies are required to further investigate the mechanisms
∗ Corresponding author at: Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai, 200030, PR China. Tel.: +86 21 6293 3075; fax: +86 21 6293 2302. E-mail address: [email protected] (W. Ying). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.039
underlying the illnesses, based on which effective therapeutic strategies may be designed. A large number of studies have indicated that NAD+ plays important roles in not only energy metabolism and mitochondrial functions, but also aging, gene expression, calcium homeostasis and immune functions [20,21]. It has also been found that NAD+ treatment can decrease death of astrocytes and neurons induced by oxidative stress [1,2] and DNA alkylating agents [23]. Our previous studies have also found that NAD+ administration can profoundly reduce infarct formation in both rat model [24] and mouse model [25] of transient focal ischemia. However, it is warranted to further elucidate the mechanisms underlying the protective effects of NAD+ , which may not only expose new mechanisms of brain protection, but also establish basis for applying NAD+ for decreasing brain injury. While a number of studies have indicated that NAD+
Y. Hong et al. / Neuroscience Letters 560 (2014) 46–50
47
Fig. 1. Flow cytometry-based annexin V staining showed that NAD+ treatment can significantly attenuate early-stage and late-stage apoptosis, as well as necrosis in rotenone-treated differentiated PC12 cells. (A) The FACS diagrams showed that rotenone induced increases in both early-stage and late-stage apoptosis of PC12 cells, which was decreased by NAD+ treatment. The cells were treated with 0.75 M rotenone, with or without co-treatment 1 mM NAD+ for 24 h. Subsequently, the apoptosis and necrosis of the cells were assessed by FACS assay. In the four fields of the original images from the flow cytometry-based study, the number of the dots indicates the number of annexin V− /7-AAD− (the bottom-left field), annexin V+ /7-AAD− (the bottom-right field), annexin V− /7-AAD+ (the top-left field), and annexin V+ /7-AAD+ cells (the top-right field), respectively. (B) Quantifications of the FACS results indicated that NAD+ treatment can significantly decrease early-stage and late-stage apoptosis, as well as necrosis of the rotenone-treated cells (N = 6). The data were pooled from six independent experiments, **p < 0.01; ***p < 0.001.
treatment can decrease cell death induced by oxidative stress and DNA alkylating agents [1,2,24], there has been only one study suggesting that NAD+ treatment can attenuate the staurosporineinduced decrease in caspase activity [16]. Because apoptosis is a key pathological change in multiple neurological diseases, it is of significance to investigate the effects of NAD+ treatment on the apoptotic changes of cells under various pathological conditions. Mitochondrial alterations, such as mitochondrial permeability transition (MPT), mitochondrial depolarization, and release of cytochrome c and apoptosis-inducing factor (AIF), play critical roles in apoptosis [4,18]. Mitochondrial impairments are also important pathological factors in the brain injury of cerebral ischemia and PD [5,8,14]. The herbicide rotenone is a mitochondrial complex I inhibitor, which has been used in many studies as a mitochondrial toxin [5]. Previous studies have suggested that rotenone induces cell injury by both decreasing intracellular ATP levels and inducing oxidative stress [6,9]. In this study we determined the effects of NAD+ treatment on both apoptotic changes and necrosis of rotenone-treated differentiated PC12 cells, and to investigate the mechanisms underlying the effects. Our study has provided the first evidence that NAD+ treatment can significantly attenuate rotenone-induced apoptosis and necrosis of PC12 cells.
1.2. Experimental procedures Experiments were initiated by replacing the cell culture medium with medium containing various concentrations of drugs. The cells were treated with 0.75 M rotenone (Sigma, St. Louis, MO, USA) with or without co-treatment with 0.5 mM or 1 mM NAD+ (Sigma, St. Louis, MO, USA). The cells were left for 24 h in an incubator with 5% CO2 at 37 ◦ C. 1.3. Flow cytometry-based annexin V/AAD staining The flow cytometry assay was performed to measure the degrees of both apoptosis and necrosis using ApoScreen Annexin V kit (SouthernBiotech) according to the manufacturer’s protocol. In brief, PC12 cells were digested with 0.1% trypsin and resuspend in cold binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 , 0.1% BSA) to a concentration of 1 × 106 to 1 × 107 cells/mL. 10 L of labeled annexin V was added into 100 L cell buffer, which was then incubated for 15 min on ice, followed by addition of 380 L binding buffer and 10 L AAD solution. Subsequently, the number of stained cells was assessed by a flow cytometer (BD FACSAriaII).
1. Materials and methods
1.4. Nuclear condensation determination
1.1. Cell cultures
The nuclear size of cells was assessed by Hoechst staining [12]. In brief, cells were treated with 20 g/mL Hoechst 33258 in PBS for 20 min. The stained nuclei were photographed under a fluorescence microscope. To quantify the size of the nuclei, three randomly picked fields in each well were photographed.
PC12 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The cells were plated onto 6-well or 24-well cell culture plates at the initial density of 1 × 105 cells/mL in Dulbecco’s Modified Eagle Medium containing 4500 mg/L D-glucose, 584 mg/L L-glutamine, 110 mg/L sodium pyruvate (Thermo Scientific, Waltham, MA, USA) that contains 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (PAA, Linz, Austria). The cells were used when the density of the cell cultures reached 60–80%.
1.5. Determination of intracellular NAD+ concentrations NAD+ concentrations were measured by recycling assay as previously described [23]. Briefly, samples were extracted in 0.5 N perchloric acid. After centrifugation at 12,000 RPM for 5 min, the supernatant was neutralized to pH 7.2 using 3 N potassium
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Fig. 2. Hoechst staining showed that NAD+ treatment can dose-dependently attenuate rotenone-induced nuclear condensation of differentiated PC12 cells. (A) PC12 cells were treated with 0.75 M rotenone, with or without co-treatment with 0.5 or 1 mM NAD+ for 24 h. Subsequently the nuclear size of the cells was assessed by Hoechst staining. (B) Quantifications of the nuclear areas showed that NAD+ can attenuate the rotenone-induced nuclear condensation of PC12 cells (N = 12). The data were pooled from three independent experiments, ***p < 0.001.
hydroxide and 1 M potassium phosphate buffer. Then centrifugation at 12,000 RPM for 5 min, the supernatants were mixed with a reaction medium containing 1.7 mg 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT), 10.6 mg phenazine methosulfate, 1.3 mg alcohol dehydrogenase, 488.4 mg nicotinamide, and 2.4 mL ethanol in 37.6 mL Gly-Gly buffer (65 mM, pH 7.4). The A560 nm was determined immediately and after 10 min, and the readings were calibrated with NAD+ standards. The protein concentration was measured with a BCA Protein Assay Reagent (Thermo Scientific, Waltham, MA, USA). 1.6. FACS-based determinations of mitochondrial membrane potential Mitochondrial dye, JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethylbenzimidazolylcarbocyanine iodideb) (Enzo Life Sciences, Plymouth Meeting, PA, USA) was used to measure mitochondrial membrane potential [3]. In healthy cells, JC-1 accumulates in the mitochondria, forming aggregates that emit red fluorescence (at the wavelength of 590 nm). Upon cell injury, as mitochondrial membrane potential decreases, JC-1 monomers are generated, which emit green fluorescence (at the wavelength of 529 nm). FACS was used to determine both the red and green fluorescence of a cell, and the ratio between the red fluorescence to the green fluorescence was calculated as a measure of mitochondrial membrane potential. 1.7. Statistical analyses All data are presented as mean + SEM. N = number of wells of 24-well plates. Data were assessed by one-way ANOVA, followed by Student–Newman–Keuls post hoc test. P values < 0.05 were considered statistically significant. 2. Results 2.1. NAD+ treatment can significantly attenuate rotenone-induced apoptosis and necrosis of PC12 cells We applied FACS-based annexin V/AAD staining assay to assess the effects of rotenone and NAD+ on the apoptosis and necrosis
of PC12 cells. The assay showed that rotenone induced increases in early-stage apoptosis (annexin V+ /7-AAD− cells), late-stage apoptosis (annexin V+ /7-AAD+ cells) and necrosis (annexin V− /7-AAD+ cells), which were attenuated by NAD+ treatment (Fig. 1A). Quantifications of the FACS results indicated that NAD+ treatment can significantly decrease both early-stage and late-stage apoptosis, as well as necrosis of the rotenone-treated cells (Fig. 1B). Because nuclear condensation is one of the hallmarks of apoptosis, we applied Hoechst staining assay to determine the nuclear size of the cells, so as to further assess the effects of NAD+ treatment on rotenone-induced apoptotic changes. Our study showed that treatment of PC12 cells with rotenone for 24 h induced nuclear condensation, which can be prevented by NAD+ treatment (Fig. 2A). Quantifications of the nuclear size showed that NAD+ treatment can significantly attenuate rotenone-induced nuclear condensation of PC12 cells (Fig. 2B). In contrast, treatment of the rotenone-treated cells with nicotinamide at concentrations up to 1 mM did not affect the nuclear size (data not shown).
2.2. NAD+ can attenuate rotenone-induced decrease in the intracellular NAD+ level of PC12 cells In order to elucidate the mechanisms underlying the protective effects of NAD+ on the rotenone-induced apoptosis of PC12 cells, we first determined if the NAD+ treatment was capable of normalizing the intracellular NAD+ of the rotenone-treated cells. We found that rotenone induced a significant decrease in the intracellular NAD+ levels of the cells, which was significantly attenuated by the NAD+ treatment (Fig. 3).
2.3. NAD+ treatment can attenuate rotenone-induced decrease in the mitochondrial membrane potential of differentiated PC12 cells We determined the effects of rotenone and NAD+ treatment on the mitochondrial membrane potential of the PC12 cells by applying FACS-based assay using JC-1 as a probe. The FACS diagrams showed that rotenone induced a decrease in the mitochondrial membrane potential, which was attenuated by NAD+ treatment (Fig. 4A). Quantifications of the JC-1 staining showed that rotenone induced a significant decrease in the mitochondrial membrane
Y. Hong et al. / Neuroscience Letters 560 (2014) 46–50
Fig. 3. NAD+ can attenuate rotenone-induced decrease in the intracellular NAD+ level of PC12 cells. PC12 cells were treated with 0.75 M rotenone, with or without co-treatment with 1 mM NAD+ for 24 h. Subsequently the intracellular NAD+ level of the cells was assessed (N = 24). The data were pooled from six independent experiments, **p < 0.01.
potential, which was significantly attenuated by NAD+ treatment (Fig. 4B). 3. Discussion The major findings of our current study include: first, NAD+ can decrease rotenone-induced early-stage and late-stage apoptosis of differentiated PC12 cells; second, NAD+ can also decrease rotenoneinduced necrosis of differentiated PC12 cells; third, rotenone can induce a significant decrease in the intracellular NAD+ levels of the cells, which can be restored by the NAD+ treatment; and fourth, NAD+ treatment can prevent rotenone-induced mitochondrial depolarization of the cells. In summary, our study has provided the first evidence that NAD+ treatment can prevent mitochondria damage-induced apoptosis and necrosis, which may at least partially result from its protective effects on rotenone-induced mitochondrial depolarization. Our study showed that rotenone can induce both early-stage and late-stage apoptosis of differentiated PC12 cells, which can be significantly attenuated by NAD+ treatment. The preventive effect
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of NAD+ on rotenone-induced apoptosis has been further supported by our observations that NAD+ can decrease rotenone-induced nuclear condensation – a hallmark of apoptosis. Because apoptotic changes belong to major pathological changes in such neurological disorders as cerebral ischemia, our study has suggested that NAD+ produced prevention of apoptosis may be one of the mechanisms underlying the protective effects of NAD+ on ischemic brain injury [24,25]. We reported our finding regarding the protective effects of NAD+ on rotenone-induced apoptotic changes in abstract form in 2010, which was the first report that NAD+ is capable of decreasing apoptosis [7]. In 2011, Pittelli et al. also reported that NAD+ treatment can decrease staurosporine-induced caspase activation [16]. However, because caspase activation was the only apoptotic index assessed in their study, which could also produce physiological effects in addition to executing apoptosis [10], their study had not produced sufficient evidence indicating that NAD+ can decrease apoptosis. Because our current study has conducted FACS-based annexin V/AAD assay–a widely accepted approach for determining early-stage and late-stage apoptosis, as well as Hoechst stainingbased nuclear condensation assay, our study could be the first study that has solidly indicated that NAD+ treatment can decrease apoptosis of cells under certain conditions. Our study using FACS-based AAD assay has also shown that NAD+ treatment can decrease rotenone-induced cell necrosis. Therefore, our study has suggested that NAD+ treatment can decrease both apoptosis and necrosis of rotenone-treated cells. Because necrosis and apoptosis are two major forms of death in multiple neurological diseases including cerebral ischemia, our current study has highlighted the therapeutic potential of NAD+ for neurological diseases. Mitochondrial alterations play critical roles in apoptosis [4,18], which are also important pathological factors in the brain injury of cerebral ischemia and PD [5,8,14]. Our current study, using rotenone as a model mitochondrial toxin, has indicated that NAD+ is effective in preventing rotenone-induced cell death. This observation has suggested the therapeutic potential of NAD+ for multiple diseases in which mitochondrial impairments play critical roles. It has been indicated that rotenone induces the apoptosis of differentiated PC12 cells by inducing mitochondrial AIF release and subsequent AIF translocation into the nucleus [13], which has
Fig. 4. NAD+ treatment can attenuate rotenone-induced decrease in the mitochondrial membrane potential of differentiated PC12 cells. (A) The FACS diagrams showed that rotenone induced a decrease in the mitochondrial membrane potential, which was attenuated by NAD+ treatment. PC12 cells were treated with 0.75 M rotenone, with or without co-treatment with 1 mM NAD+ for 24 h. Subsequently the mitochondrial membrane potential of the PC12 cells was assessed by FACS-based assay using JC-1 as a probe. (B) Quantifications of the JC-1 staining showed that rotenone induced a significant decrease in the mitochondrial membrane potential, which was significantly attenuated by NAD+ treatment (N = 6). The data were pooled from six independent experiments, ***p < 0.001.
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also been confirmed in our cellular model (data not shown). It has been indicated that mitochondrial depolarization plays a key role in AIF-mediated cell death [11]. Mitochondrial depolarization promotes MPT, and MPT can in turn trigger mitochondrial AIF release [1,2,15,17]. Our study has shown that NAD+ treatment can significantly attenuate rotenone-induced mitochondrial depolarization. Collectively, these observations have suggested that NAD+ treatment may prevent rotenone-induced, AIF-mediated apoptosis at least partially by preventing mitochondrial depolarization. 4. Conclusion In summary, our study has provided first evidence that NAD+ treatment can prevent rotenone-induced apoptosis and necrosis of differentiated PC12 cells. This study has also indicated that NAD+ treatment can attenuate mitochondrial damage-induced cell death. Because apoptosis, necrosis, and mitochondrial impairments belong to key pathological changes in multiple neurological diseases, our study has suggested that NAD+ may be used to prevent mitochondrial impairment-induced apoptosis and necrosis in neurological disorders. Acknowledgments This study was supported by Chinese National Science Foundation Grants #81171098 and #81271305 (to W. Y.), a National Key Basic Research ‘973 Program’ Grant #2010CB834306 (to W.Y.), and a Shanghai Engineering Center Grant #11DZ2211000 (to W.Y.) References [1] C.C. Alano, P. Garnier, W. Ying, Y. Higashi, T.M. Kauppinen, R.A. Swanson, NAD+ depletion is necessary and sufficient for poly (ADP-ribose) polymerase1-mediated neuronal death, J. Neurosci. 30 (2010) 2967–2978. [2] C.C. Alano, W. Ying, R.A. Swanson, Poly (ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition, J. Biol. Chem. 279 (2004) 18895–18902. [3] C.A. Baker, S. Bousheri, I. Ssewanyana, N.G. Jones, O. K’Aluoch, D. Baliruno, F. Ssali, C. Huyen, HIV subtypes distribution and implication for antiretroviral treatment in a Ugandan population, J. Int. Assoc. Physicians AIDS Care (Chic.) 6 (2007) 260–263. [4] V. Borutaite, Mitochondria as decision-makers in cell death, Environ. Mol. Mutagen 51 406–416. [5] J.T. Greenamyre, R. Betarbet, T.B. Sherer, The rotenone model of Parkinson’s disease: genes, environment and mitochondria, Parkinsonism Relat. Disord. 9 (Suppl. 2) (2003) S59–S64.
[6] J.T. Greenamyre, G. MacKenzie, T.I. Peng, S.E. Stephans, Mitochondrial dysfunction in Parkinson’s disease, Biochem. Soc. Symp. 66 (1999) 85–97. [7] Y. Hong, J. Shao, W. Xia, W. Ying, NAD+ treatment can block rotenone-induced nuclear apoptotic changes of PC12 cells, FASEB J. 24 (2010) (2010’ Experimental Biology Meeting Abstracts) 965.6. [8] B.S. Kristal, I.G. Stavrovskaya, M.V. Narayanan, B.F. Krasnikov, A.M. Brown, M.F. Beal, R.M. Friedlander, The mitochondrial permeability transition as a target for neuroprotection, J. Bioenerg. Biomembr. 36 (2004) 309–312. [9] Y. Kushnareva, A.N. Murphy, A. Andreyev, Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation–reduction state, Biochem. J. 368 (2002) 545–553. [10] C. Lu, Y. Wang, K. Furukawa, W. Fu, X. Ouyang, M.P. Mattson, Evidence that caspase-1 is a negative regulator of AMPA receptor-mediated long-term potentiation at hippocampal synapses, J. Neurochem. 97 (2006) 1104–1110. [11] J.D. Ly, D.R. Grubb, A. Lawen, The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update, Apoptosis 8 (2003) 115–128. [12] Y. Ma, J. Jiang, L. Wang, H. Nie, W. Xia, J. Liu, W. Ying, CD38 is a key enzyme for the survival of mouse microglial BV2 cells, Biochem. Biophys. Res. Commun. 418 (2012) 714–719. [13] M. Marella, B.B. Seo, A. Matsuno-Yagi, T. Yagi, Mechanism of cell death caused by complex I defects in a rat dopaminergic cell line, J. Biol. Chem. 282 (2007) 24146–24156. [14] M.D. Norenberg, K.V. Rao, The mitochondrial permeability transition in neurologic disease, Neurochem. Int. 50 (2007) 983–997. [15] P.X. Petit, M. Goubern, P. Diolez, S.A. Susin, N. Zamzami, G. Kroemer, Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition, FEBS Lett. 426 (1998) 111–116. [16] M. Pittelli, R. Felici, V. Pitozzi, L. Giovannelli, E. Bigagli, F. Cialdai, G. Romano, F. Moroni, A. Chiarugi, Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis, Mol. Pharmacol. 80 (2011) 1136–1146. [17] B.M. Polster, G. Basanez, A. Etxebarria, J.M. Hardwick, D.G. Nicholls, Calpain I induces cleavage and release of apoptosis-inducing factor from isolated mitochondria, J. Biol. Chem. 280 (2005) 6447–6454. [18] Y. Tsujimoto, S. Shimizu, Role of the mitochondrial membrane permeability transition in cell death, Apoptosis 12 (2007) 835–840. [19] W. Ying, Deleterious network: a testable pathogenetic concept of Alzheimer’s disease, Gerontology 43 (1997) 242–253. [20] W. Ying, NAD+ and NADH in cellular functions and cell death, Front. Biosci. 11 (2006) 3129–3148. [21] W. Ying, NAD+ /NADH and NADP+ /NADPH in cellular functions and cell death: regulation and biological consequences, Antioxid. Redox Signal. 10 (2008) 179–206. [22] W. Ying, A new hypothesis of neurodegenerative diseases: the deleterious network hypothesis, Med. Hypotheses 47 (1996) 307–313. [23] W. Ying, P. Garnier, R.A. Swanson, NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes, Biochem. Biophys. Res. Commun. 308 (2003) 809–813. [24] W. Ying, G. Wei, D. Wang, Q. Wang, X. Tang, J. Shi, P. Zhang, H. Lu, Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia, Front. Biosci. 12 (2007) 2728–2734. [25] C. Zheng, J. Han, W. Xia, S. Shi, J. Liu, W. Ying, NAD+ administration decreases ischemic brain damage partially by blocking autophagy in a mouse model of brain ischemia, Neurosci. Lett. 512 (2012) 67–71.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
NAD+ treatment prevents rotenone-induced apoptosis and necrosis of differentiated PC12 cells Yunyi Hong a , Hui Nie a , Danhong Wu c , Xunbin Wei a , Xianting Ding a , Weihai Ying a,b,∗ a b c
Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai 200030, PR China Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, PR China Department of Neurology, Third People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 201999, PR China
h i g h l i g h t s • • • •
NAD+ NAD+ NAD+ NAD+
treatment can decrease rotenone-induced early-stage and late-stage apoptosis of PC12 cells. treatment can decrease rotenone-induced necrosis of PC12 cells. treatment can restore the intracellular NAD+ levels of rotenone-treated PC12 cells. treatment can prevent rotenone-induced mitochondrial depolarization of PC12 cells.
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 18 November 2013 Accepted 20 November 2013 Keywords: NAD+ Apoptosis Necrosis PC12 cells Rotenone Mitochondrial membrane potential
a b s t r a c t Nicotinamide adenine dinucleotide (NAD+ ) plays critical roles in not only energy metabolism and mitochondrial functions, but also calcium homeostasis and immunological functions. It has been reported that NAD+ administration can reduce ischemic brain damage. However, the mechanisms underlying the protective effects remain unclear. Because mitochondrial impairments play a key role in the cell death in cerebral ischemia, in this study we tested our hypothesis that NAD+ can decrease mitochondrial damageinduced cell death using differentiated PC12 cells as a cellular model. We found that NAD+ can decrease both early-stage and late-stage apoptosis, as well as necrosis of rotenone-treated PC12 cells, as assessed by FACS-based Annexin V/AAD assay. We also found that NAD+ treatment can restore the intracellular NAD+ levels of the rotenone-treated cells. Moreover, NAD+ treatment can prevent rotenone-induced mitochondria depolarization. In summary, our study has provided first direct evidence that NAD+ treatment can prevent rotenone-induced apoptosis and necrosis. Our study has also indicated that NAD+ treatment can prevent mitochondrial damage-induced cell death, which may at least partially result from its protective effects on rotenone-induced mitochondrial depolarization. Because both mitochondrial damage and apoptosis play key roles in multiple neurological disorders, our study has highlighted the therapeutic potential of NAD+ for brain ischemia and other neurological diseases. © 2013 Elsevier Ireland Ltd. All rights reserved.
Introduction Oxidative stress, mitochondrial impairments, calcium dyshomeostasis, and excessive inflammation are critical pathological factors in multiple major neurological diseases, including stroke, Parkinson’s disease (PD), and Alzheimer’s disease [19,22]. While our understanding regarding the mechanisms of the diseases have significantly improved in the last 30 years, many future studies are required to further investigate the mechanisms
∗ Corresponding author at: Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai, 200030, PR China. Tel.: +86 21 6293 3075; fax: +86 21 6293 2302. E-mail address: [email protected] (W. Ying). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.11.039
underlying the illnesses, based on which effective therapeutic strategies may be designed. A large number of studies have indicated that NAD+ plays important roles in not only energy metabolism and mitochondrial functions, but also aging, gene expression, calcium homeostasis and immune functions [20,21]. It has also been found that NAD+ treatment can decrease death of astrocytes and neurons induced by oxidative stress [1,2] and DNA alkylating agents [23]. Our previous studies have also found that NAD+ administration can profoundly reduce infarct formation in both rat model [24] and mouse model [25] of transient focal ischemia. However, it is warranted to further elucidate the mechanisms underlying the protective effects of NAD+ , which may not only expose new mechanisms of brain protection, but also establish basis for applying NAD+ for decreasing brain injury. While a number of studies have indicated that NAD+
Y. Hong et al. / Neuroscience Letters 560 (2014) 46–50
47
Fig. 1. Flow cytometry-based annexin V staining showed that NAD+ treatment can significantly attenuate early-stage and late-stage apoptosis, as well as necrosis in rotenone-treated differentiated PC12 cells. (A) The FACS diagrams showed that rotenone induced increases in both early-stage and late-stage apoptosis of PC12 cells, which was decreased by NAD+ treatment. The cells were treated with 0.75 M rotenone, with or without co-treatment 1 mM NAD+ for 24 h. Subsequently, the apoptosis and necrosis of the cells were assessed by FACS assay. In the four fields of the original images from the flow cytometry-based study, the number of the dots indicates the number of annexin V− /7-AAD− (the bottom-left field), annexin V+ /7-AAD− (the bottom-right field), annexin V− /7-AAD+ (the top-left field), and annexin V+ /7-AAD+ cells (the top-right field), respectively. (B) Quantifications of the FACS results indicated that NAD+ treatment can significantly decrease early-stage and late-stage apoptosis, as well as necrosis of the rotenone-treated cells (N = 6). The data were pooled from six independent experiments, **p < 0.01; ***p < 0.001.
treatment can decrease cell death induced by oxidative stress and DNA alkylating agents [1,2,24], there has been only one study suggesting that NAD+ treatment can attenuate the staurosporineinduced decrease in caspase activity [16]. Because apoptosis is a key pathological change in multiple neurological diseases, it is of significance to investigate the effects of NAD+ treatment on the apoptotic changes of cells under various pathological conditions. Mitochondrial alterations, such as mitochondrial permeability transition (MPT), mitochondrial depolarization, and release of cytochrome c and apoptosis-inducing factor (AIF), play critical roles in apoptosis [4,18]. Mitochondrial impairments are also important pathological factors in the brain injury of cerebral ischemia and PD [5,8,14]. The herbicide rotenone is a mitochondrial complex I inhibitor, which has been used in many studies as a mitochondrial toxin [5]. Previous studies have suggested that rotenone induces cell injury by both decreasing intracellular ATP levels and inducing oxidative stress [6,9]. In this study we determined the effects of NAD+ treatment on both apoptotic changes and necrosis of rotenone-treated differentiated PC12 cells, and to investigate the mechanisms underlying the effects. Our study has provided the first evidence that NAD+ treatment can significantly attenuate rotenone-induced apoptosis and necrosis of PC12 cells.
1.2. Experimental procedures Experiments were initiated by replacing the cell culture medium with medium containing various concentrations of drugs. The cells were treated with 0.75 M rotenone (Sigma, St. Louis, MO, USA) with or without co-treatment with 0.5 mM or 1 mM NAD+ (Sigma, St. Louis, MO, USA). The cells were left for 24 h in an incubator with 5% CO2 at 37 ◦ C. 1.3. Flow cytometry-based annexin V/AAD staining The flow cytometry assay was performed to measure the degrees of both apoptosis and necrosis using ApoScreen Annexin V kit (SouthernBiotech) according to the manufacturer’s protocol. In brief, PC12 cells were digested with 0.1% trypsin and resuspend in cold binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 , 0.1% BSA) to a concentration of 1 × 106 to 1 × 107 cells/mL. 10 L of labeled annexin V was added into 100 L cell buffer, which was then incubated for 15 min on ice, followed by addition of 380 L binding buffer and 10 L AAD solution. Subsequently, the number of stained cells was assessed by a flow cytometer (BD FACSAriaII).
1. Materials and methods
1.4. Nuclear condensation determination
1.1. Cell cultures
The nuclear size of cells was assessed by Hoechst staining [12]. In brief, cells were treated with 20 g/mL Hoechst 33258 in PBS for 20 min. The stained nuclei were photographed under a fluorescence microscope. To quantify the size of the nuclei, three randomly picked fields in each well were photographed.
PC12 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The cells were plated onto 6-well or 24-well cell culture plates at the initial density of 1 × 105 cells/mL in Dulbecco’s Modified Eagle Medium containing 4500 mg/L D-glucose, 584 mg/L L-glutamine, 110 mg/L sodium pyruvate (Thermo Scientific, Waltham, MA, USA) that contains 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% fetal bovine serum (PAA, Linz, Austria). The cells were used when the density of the cell cultures reached 60–80%.
1.5. Determination of intracellular NAD+ concentrations NAD+ concentrations were measured by recycling assay as previously described [23]. Briefly, samples were extracted in 0.5 N perchloric acid. After centrifugation at 12,000 RPM for 5 min, the supernatant was neutralized to pH 7.2 using 3 N potassium
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Fig. 2. Hoechst staining showed that NAD+ treatment can dose-dependently attenuate rotenone-induced nuclear condensation of differentiated PC12 cells. (A) PC12 cells were treated with 0.75 M rotenone, with or without co-treatment with 0.5 or 1 mM NAD+ for 24 h. Subsequently the nuclear size of the cells was assessed by Hoechst staining. (B) Quantifications of the nuclear areas showed that NAD+ can attenuate the rotenone-induced nuclear condensation of PC12 cells (N = 12). The data were pooled from three independent experiments, ***p < 0.001.
hydroxide and 1 M potassium phosphate buffer. Then centrifugation at 12,000 RPM for 5 min, the supernatants were mixed with a reaction medium containing 1.7 mg 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazolium bromide (MTT), 10.6 mg phenazine methosulfate, 1.3 mg alcohol dehydrogenase, 488.4 mg nicotinamide, and 2.4 mL ethanol in 37.6 mL Gly-Gly buffer (65 mM, pH 7.4). The A560 nm was determined immediately and after 10 min, and the readings were calibrated with NAD+ standards. The protein concentration was measured with a BCA Protein Assay Reagent (Thermo Scientific, Waltham, MA, USA). 1.6. FACS-based determinations of mitochondrial membrane potential Mitochondrial dye, JC-1 (5,5 ,6,6 -tetrachloro-1,1 ,3,3 tetraethylbenzimidazolylcarbocyanine iodideb) (Enzo Life Sciences, Plymouth Meeting, PA, USA) was used to measure mitochondrial membrane potential [3]. In healthy cells, JC-1 accumulates in the mitochondria, forming aggregates that emit red fluorescence (at the wavelength of 590 nm). Upon cell injury, as mitochondrial membrane potential decreases, JC-1 monomers are generated, which emit green fluorescence (at the wavelength of 529 nm). FACS was used to determine both the red and green fluorescence of a cell, and the ratio between the red fluorescence to the green fluorescence was calculated as a measure of mitochondrial membrane potential. 1.7. Statistical analyses All data are presented as mean + SEM. N = number of wells of 24-well plates. Data were assessed by one-way ANOVA, followed by Student–Newman–Keuls post hoc test. P values < 0.05 were considered statistically significant. 2. Results 2.1. NAD+ treatment can significantly attenuate rotenone-induced apoptosis and necrosis of PC12 cells We applied FACS-based annexin V/AAD staining assay to assess the effects of rotenone and NAD+ on the apoptosis and necrosis
of PC12 cells. The assay showed that rotenone induced increases in early-stage apoptosis (annexin V+ /7-AAD− cells), late-stage apoptosis (annexin V+ /7-AAD+ cells) and necrosis (annexin V− /7-AAD+ cells), which were attenuated by NAD+ treatment (Fig. 1A). Quantifications of the FACS results indicated that NAD+ treatment can significantly decrease both early-stage and late-stage apoptosis, as well as necrosis of the rotenone-treated cells (Fig. 1B). Because nuclear condensation is one of the hallmarks of apoptosis, we applied Hoechst staining assay to determine the nuclear size of the cells, so as to further assess the effects of NAD+ treatment on rotenone-induced apoptotic changes. Our study showed that treatment of PC12 cells with rotenone for 24 h induced nuclear condensation, which can be prevented by NAD+ treatment (Fig. 2A). Quantifications of the nuclear size showed that NAD+ treatment can significantly attenuate rotenone-induced nuclear condensation of PC12 cells (Fig. 2B). In contrast, treatment of the rotenone-treated cells with nicotinamide at concentrations up to 1 mM did not affect the nuclear size (data not shown).
2.2. NAD+ can attenuate rotenone-induced decrease in the intracellular NAD+ level of PC12 cells In order to elucidate the mechanisms underlying the protective effects of NAD+ on the rotenone-induced apoptosis of PC12 cells, we first determined if the NAD+ treatment was capable of normalizing the intracellular NAD+ of the rotenone-treated cells. We found that rotenone induced a significant decrease in the intracellular NAD+ levels of the cells, which was significantly attenuated by the NAD+ treatment (Fig. 3).
2.3. NAD+ treatment can attenuate rotenone-induced decrease in the mitochondrial membrane potential of differentiated PC12 cells We determined the effects of rotenone and NAD+ treatment on the mitochondrial membrane potential of the PC12 cells by applying FACS-based assay using JC-1 as a probe. The FACS diagrams showed that rotenone induced a decrease in the mitochondrial membrane potential, which was attenuated by NAD+ treatment (Fig. 4A). Quantifications of the JC-1 staining showed that rotenone induced a significant decrease in the mitochondrial membrane
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Fig. 3. NAD+ can attenuate rotenone-induced decrease in the intracellular NAD+ level of PC12 cells. PC12 cells were treated with 0.75 M rotenone, with or without co-treatment with 1 mM NAD+ for 24 h. Subsequently the intracellular NAD+ level of the cells was assessed (N = 24). The data were pooled from six independent experiments, **p < 0.01.
potential, which was significantly attenuated by NAD+ treatment (Fig. 4B). 3. Discussion The major findings of our current study include: first, NAD+ can decrease rotenone-induced early-stage and late-stage apoptosis of differentiated PC12 cells; second, NAD+ can also decrease rotenoneinduced necrosis of differentiated PC12 cells; third, rotenone can induce a significant decrease in the intracellular NAD+ levels of the cells, which can be restored by the NAD+ treatment; and fourth, NAD+ treatment can prevent rotenone-induced mitochondrial depolarization of the cells. In summary, our study has provided the first evidence that NAD+ treatment can prevent mitochondria damage-induced apoptosis and necrosis, which may at least partially result from its protective effects on rotenone-induced mitochondrial depolarization. Our study showed that rotenone can induce both early-stage and late-stage apoptosis of differentiated PC12 cells, which can be significantly attenuated by NAD+ treatment. The preventive effect
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of NAD+ on rotenone-induced apoptosis has been further supported by our observations that NAD+ can decrease rotenone-induced nuclear condensation – a hallmark of apoptosis. Because apoptotic changes belong to major pathological changes in such neurological disorders as cerebral ischemia, our study has suggested that NAD+ produced prevention of apoptosis may be one of the mechanisms underlying the protective effects of NAD+ on ischemic brain injury [24,25]. We reported our finding regarding the protective effects of NAD+ on rotenone-induced apoptotic changes in abstract form in 2010, which was the first report that NAD+ is capable of decreasing apoptosis [7]. In 2011, Pittelli et al. also reported that NAD+ treatment can decrease staurosporine-induced caspase activation [16]. However, because caspase activation was the only apoptotic index assessed in their study, which could also produce physiological effects in addition to executing apoptosis [10], their study had not produced sufficient evidence indicating that NAD+ can decrease apoptosis. Because our current study has conducted FACS-based annexin V/AAD assay–a widely accepted approach for determining early-stage and late-stage apoptosis, as well as Hoechst stainingbased nuclear condensation assay, our study could be the first study that has solidly indicated that NAD+ treatment can decrease apoptosis of cells under certain conditions. Our study using FACS-based AAD assay has also shown that NAD+ treatment can decrease rotenone-induced cell necrosis. Therefore, our study has suggested that NAD+ treatment can decrease both apoptosis and necrosis of rotenone-treated cells. Because necrosis and apoptosis are two major forms of death in multiple neurological diseases including cerebral ischemia, our current study has highlighted the therapeutic potential of NAD+ for neurological diseases. Mitochondrial alterations play critical roles in apoptosis [4,18], which are also important pathological factors in the brain injury of cerebral ischemia and PD [5,8,14]. Our current study, using rotenone as a model mitochondrial toxin, has indicated that NAD+ is effective in preventing rotenone-induced cell death. This observation has suggested the therapeutic potential of NAD+ for multiple diseases in which mitochondrial impairments play critical roles. It has been indicated that rotenone induces the apoptosis of differentiated PC12 cells by inducing mitochondrial AIF release and subsequent AIF translocation into the nucleus [13], which has
Fig. 4. NAD+ treatment can attenuate rotenone-induced decrease in the mitochondrial membrane potential of differentiated PC12 cells. (A) The FACS diagrams showed that rotenone induced a decrease in the mitochondrial membrane potential, which was attenuated by NAD+ treatment. PC12 cells were treated with 0.75 M rotenone, with or without co-treatment with 1 mM NAD+ for 24 h. Subsequently the mitochondrial membrane potential of the PC12 cells was assessed by FACS-based assay using JC-1 as a probe. (B) Quantifications of the JC-1 staining showed that rotenone induced a significant decrease in the mitochondrial membrane potential, which was significantly attenuated by NAD+ treatment (N = 6). The data were pooled from six independent experiments, ***p < 0.001.
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also been confirmed in our cellular model (data not shown). It has been indicated that mitochondrial depolarization plays a key role in AIF-mediated cell death [11]. Mitochondrial depolarization promotes MPT, and MPT can in turn trigger mitochondrial AIF release [1,2,15,17]. Our study has shown that NAD+ treatment can significantly attenuate rotenone-induced mitochondrial depolarization. Collectively, these observations have suggested that NAD+ treatment may prevent rotenone-induced, AIF-mediated apoptosis at least partially by preventing mitochondrial depolarization. 4. Conclusion In summary, our study has provided first evidence that NAD+ treatment can prevent rotenone-induced apoptosis and necrosis of differentiated PC12 cells. This study has also indicated that NAD+ treatment can attenuate mitochondrial damage-induced cell death. Because apoptosis, necrosis, and mitochondrial impairments belong to key pathological changes in multiple neurological diseases, our study has suggested that NAD+ may be used to prevent mitochondrial impairment-induced apoptosis and necrosis in neurological disorders. Acknowledgments This study was supported by Chinese National Science Foundation Grants #81171098 and #81271305 (to W. Y.), a National Key Basic Research ‘973 Program’ Grant #2010CB834306 (to W.Y.), and a Shanghai Engineering Center Grant #11DZ2211000 (to W.Y.) References [1] C.C. Alano, P. Garnier, W. Ying, Y. Higashi, T.M. Kauppinen, R.A. Swanson, NAD+ depletion is necessary and sufficient for poly (ADP-ribose) polymerase1-mediated neuronal death, J. Neurosci. 30 (2010) 2967–2978. [2] C.C. Alano, W. Ying, R.A. Swanson, Poly (ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition, J. Biol. Chem. 279 (2004) 18895–18902. [3] C.A. Baker, S. Bousheri, I. Ssewanyana, N.G. Jones, O. K’Aluoch, D. Baliruno, F. Ssali, C. Huyen, HIV subtypes distribution and implication for antiretroviral treatment in a Ugandan population, J. Int. Assoc. Physicians AIDS Care (Chic.) 6 (2007) 260–263. [4] V. Borutaite, Mitochondria as decision-makers in cell death, Environ. Mol. Mutagen 51 406–416. [5] J.T. Greenamyre, R. Betarbet, T.B. Sherer, The rotenone model of Parkinson’s disease: genes, environment and mitochondria, Parkinsonism Relat. Disord. 9 (Suppl. 2) (2003) S59–S64.
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