Neurochem Res DOI 10.1007/s11064-015-1534-0

ORIGINAL PAPER

NAD+ Treatment Can Prevent Rotenone-Induced Increases in DNA Damage, Bax Levels and Nuclear Translocation of Apoptosis-Inducing Factor in Differentiated PC12 Cells Yunyi Hong • Hui Nie • Xunbin Wei Shen Fu • Weihai Ying



Received: 1 January 2015 / Revised: 27 January 2015 / Accepted: 30 January 2015 Ó Springer Science+Business Media New York 2015

Abstract Nicotinamide adenine dinucleotide (NAD?) plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immunological functions. Our previous studies have found that NAD? administration can profoundly decrease ischemic brain injury and traumatic brain injury. Our recent study has also provided first direct evidence indicating that NAD? treatment can decrease cellular apoptosis, while the mechanisms underlying this protective effect remain unclear. In our current study, we determined the effects of NAD? treatment on several major factors in apoptosis and necrosis, including levels of Bax and nuclear translocation of apoptosis-inducing factor (AIF), as well as levels of DNA double-strand breaks (DSBs) and intracellular ATP in rotenone-treated differentiated PC12 cells. We found that NAD? treatment can markedly attenuate the rotenone-induced increases in the levels of Bax and nuclear translocation of AIF in the cells. We further found that NAD? treatment can significantly attenuate the rotenone-induced Electronic supplementary material The online version of this article (doi:10.1007/s11064-015-1534-0) contains supplementary material, which is available to authorized users. Y. Hong  H. Nie  X. Wei  W. Ying (&) Med-X Research Institute and School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, People’s Republic of China e-mail: [email protected] S. Fu Radiation Oncology Department, Shanghai Proton and Heavy Ion Center, Shanghai 201321, People’s Republic of China W. Ying Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

increase in the levels of DSBs and decrease in the intracellular ATP levels. Collectively, our study has suggested mechanisms underlying the preventive effects of NAD? on apoptosis, which has highlighted the therapeutic potential of NAD? for decreasing apoptotic changes in multiple major diseases. Keywords Apoptosis  Apoptosis-inducing factor  DNA double-strand breaks  Mitochondrial permeability transition  NAD?  Necrosis

Introduction A large number of studies have indicated that NAD? plays important roles in a number of biological functions, including energy metabolism and mitochondrial functions, calcium homeostasis and immune functions [1]. Our study has found that NAD? treatment can decrease DNA alkylating agent- and oxidative stress-induced necrosis of both neurons and astrocytes [2–4]. We have also found that NAD? administration can profoundly decrease both ischemic brain injury and traumatic brain injury [5–7], while the mechanisms underlying the protective effects remain unclear. Our latest study has provided the first direct evidence suggesting that NAD? treatment can also decrease cellular apoptosis, using rotenone-treated PC12 cells as a cellular model [8]. Because apoptosis is a key pathological change in multiple neurological diseases [9], it is of significance to investigate the mechanisms underlying the preventive effects of NAD? treatment on apoptosis. Mitochondrial alterations, including mitochondrial permeability transition (MPT), mitochondrial depolarization, and release of pro-apoptotic proteins, play critical roles in apoptosis [10]. Numerous studies have suggested that

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mitochondrial alterations are important pathological factors in the brain injury of cerebral ischemia and Parkinson’s disease (PD) [11, 12]. The herbicide rotenone is a mitochondrial Complex I inhibitor, which has been widely used as a mitochondrial toxin [13]. Our previous study has shown that NAD? treatment can significantly attenuate rotenone-induced apoptosis of differentiated PC12 cells [8]. In the current study, we investigated the mechanisms underlying the protective effects of NAD? on the rotenoneinduced apoptosis. Our study has shown that NAD? treatment can attenuate rotenone-induced increases in the levels of Bax and nuclear translocation of AIF in the cells, which may underlie the effects of NAD? on the cellular apoptosis.

Materials and Methods Cell Cultures PC12 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The cells were plated in 6-well or 24-well cell culture plates at the initial density of 1 9 105 cells/ml in Dulbecco’s Modified Eagle Medium containing 4,500 mg/L D-glucose, 584 mg/L L-glutamine, 110 mg/L sodium pyruvate (Thermo Scientific, Waltham, MA, USA), which contains 10 % fetal bovine serum (PAA, Linz, Austria) and 1 % penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA). 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 lM rotenone (Sigma, St. Louis, MO, USA) with or without co-treatment with NAD? (Sigma, St. Louis, MO, USA) or N-acetyl cysteine (NAC) (Sigma, St. Louis, MO, USA). The cells were left for 6 or 24 h in humidified atmosphere of 95 % air/5 % CO2 at 37 °C. Immunostaining of c-H2AX, BAX and AIF The cell cultures were fixed in 1 % paraformaldehyde for 30 min, followed by three washes in phosphate buffered saline (PBS). The cells were incubated in 10 % goat serum for 1 h at room temperature, and then incubated with rabbit anti-Bax antibody (1:250 dilution, EPITOMICS, Hangzhou, Zhejiang Province, China), mouse monoclonal anti-AIF antibody (1:300 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or a mouse monoclonal anti-phospho-histone H2AX (anti-c-H2AX) antibody (1:300 dilution,

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Millipore, Billerica, MA, USA) in PBS containing 1 % goat serum overnight at 4 °C. After three washes with PBS, the cells were incubated with Alexa Fluor 488 goat anti-rabbit or goat anti-mouse secondary antibody (Molecular Probes, Eugene, Oregon, USA) diluted at 1:300 containing in 1 % goat serum for 1 h at room temperature. After counter-staining of the cells with DAPI, the fluorescence images of the cell cultures were photographed under a Leica confocal fluorescence microscope (Leica TCS SP5 II). Western Blots Cells were lysed in RIPA buffer (Millipore, Temecula, CA, USA) containing Complete Protease Inhibitor Cocktail (Roche Diagnostics, Mannheim, Germany) plus 1 mM PMSF. Thirty lg of total protein (for Western blot of Bax) or 60 lg of total protein (for Western blot of c-H2AX) was electrophoresed through a 10 % SDS–polyacrylamide gel, and then transferred to 0.45 lm nitrocellulose membranes (Millipore, Billerica, MA, USA) on a semi-dry electro transferring unit (Bio-Rad Laboratories, Hercules, CA, USA). The blots were incubated overnight at 4 °C with a rabbit polyclonal anti-Bax antibody (1:2000 dilution, Epitomics, Hangzhou, Zhejiang Province, China) or a mouse anti-c-H2AX antibody (1:2000 dilution, Millipore, Millipore, Billerica, MA, USA), then incubated with appropriate HRP-conjugated secondary antibody (Epitomics, Hangzhou, Zhejiang Province, China). Protein signals were detected using an ECL detection system (Pierce Biotechonology, Rockford, IL, USA). A rabbit anti-actin antibody (Sigma, St. Louis, MO, USA) or mouse anti-btubulin (AbMart, Shanghai, China) was used to normalize sample loading and transfer. The intensities of the bands were quantified by densitometry using Gel-Pro Analyzer. ATP Assay ATP levels were determined by Roche ATP Bioluminescence Assay Kit (HS II) following the manufactory’s protocol. Briefly, the cells were washed twice with PBS and lysed with the Cell Lysis Reagent. Then 50 lL of the lysates was mixed with 50 lL of the Luciferase Reagent, and the luminescence was detected using a plate reader (Biotek Synergy 2). The protein concentrations of the samples were assessed using the BCA assay. The ATP concentrations of the sample were calibrated using ATP standard, and normalized to the protein concentrations of the samples. Statistical Analyses All data are presented as mean ± SEM. Statistical analyses were conducted by one-way ANOVA, followed by

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Student–Newman–Keuls post hoc test. P values \ 0.05 were considered statistically significant.

Results NAD? can Significantly Attenuate Rotenone-Induced Increases in the Bax Level of Differentiated PC12 Cells We determined the effects of rotenone and NAD? on the Bax level of differentiated PC12 cells by both Western blot and immunostaining assays. Our immunostaining assays showed that rotenone treatment induced an obvious increase in the Bax level of the cells, which was attenuated by treatment of the cells with 1 mM NAD? (Fig. 1a). Our Western blot assays also showed that rotenone treatment induced a significant increase in the Bax level of the cells, which was significantly attenuated by the NAD? treatment (Fig. 1b, c). NAD? Treatment can Significantly Attenuate Rotenone-Induced Nuclear Translocation of AIF of Differentiated PC12 Cells PARP-1 cleavage is one of the hallmarks of caspase-3 activation [14]. Our Western blot on the PARP-1 level did not show that rotenone can induce a significant decrease in the PARP-1 level (data not shown), thus suggesting that

Fig. 1 NAD? can attenuate rotenone-induced increases in the Bax level of differentiated PC12 cells. a Immunostaining assay showed that rotenone treatment led to an increase in the Bax level of PC12 cells at 6 h after rotenone treatment, while co-treatment with 1 mM NAD? attenuated the increase in the Bax level. b Western blot assay showed that NAD? attenuated the rotenone-induced increase in the Bax level. The cells were treated with 0.75 lM rotenone, with or

rotenone may induce caspase-3-independent apoptosis. AIF-dependent apoptosis is a major form of caspase-3-independent apoptosis [15]. Because nuclear translocation of AIF is a hallmark of AIF-dependent apoptosis [15], we conducted immunostaining assays to determine if rotenone may induce nuclear translocation of AIF. We found that rotenone treatment induced an obvious increase in the nuclear translocation of AIF (Fig. 2), which is consistent with previous findings [16]. NAD? treatment was shown to significantly attenuate the rotenone-induced nuclear translocation of AIF of the cells (Fig. 2). NAD? Treatment can Significantly Attenuate Rotenone-Induced DSBs of Differentiated PC12 Cells The level of phospho-histone H2AX (c-H2AX) is a widely used marker of DSBs [3, 17]. We determined the effects of rotenone and NAD? on the c-H2AX level of differentiated PC12 cells by both immunostaining assays and Western blot. Our immunostaining assays showed that rotenone treatment induced an obvious increase in the c-H2AX level of the cells, which was decreased by treatment of the cells with 1 mM NAD? (Fig. 3a). Our Western blot assays also showed that rotenone treatment induced a significant increase in the c-H2AX level of the cells, which were significantly attenuated by the NAD? treatment (Fig. 3b, c). We also investigated the roles of oxidative stress in the rotenone-induced increase in c-H2AX, showing that the

without co-treatment with 1 mM NAD? for 6 h. Subsequently, Western blot assays on the Bax levels of the cells were conducted. c Quantifications of the Western blots showed that NAD? significantly attenuated the rotenone-induced increase in the Bax level. N = 12. The data were pooled from four independent experiments. *p \ 0.05; **p \ 0.01

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Neurochem Res Fig. 2 NAD? treatment can attenuate rotenone-induced nuclear translocation of AIF of PC12 cells. AIF immunostaining assay showed that NAD? treatment attenuated rotenone-induced nuclear translocation of AIF, assessed at 24 h after rotenone treatment. The data were representative of the results from four independent experiments

rotenone-induced increase in the c-H2AX level was significantly attenuated by N-acetyl cysteine (NAC)—a widely used antioxidant (Supplemental Fig. 1). NAD? Treatment can Significantly Attenuate Rotenone-Induced Decreases in the Intracellular ATP Level of PC12 Cells We assessed the effects of rotenone and NAD? on the intracellular ATP level of the cells. Our study showed that rotenone treatment induced a significant decrease in the intracellular ATP level of the cells, which was significantly attenuated by the NAD? treatment (Fig. 4).

Discussion The major findings of our current study include: First, NAD? treatment can significantly attenuate the rotenoneinduced increases in the level of Bax in differentiated PC12 cells; second, NAD? treatment can markedly decrease nuclear translocation of AIF in the cells; third, NAD? treatment can significantly attenuate the rotenone-induced increase in the DSBs levels of the cells; and fourth, NAD? treatment can significantly attenuate the rotenone-induced decrease in the intracellular ATP level. Collectively, our study has suggested that NAD? could prevent rotenoneinduced alterations of the levels of mitochondrial pro-

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apoptotic proteins, which may at least partially underlie the preventive effects of NAD? treatment on rotenone-induced apoptosis. Moreover, our study showing the protective effects of NAD? treatment on rotenone-induced DNA damage and decreases in the intracellular ATP level have further suggested the mechanisms underlying the effects of NAD? treatment on cell death. Our previous 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 [8]. However, the mechanisms underlying the protective effects of NAD? on rotenone-induced apoptosis remain unclear. Our current study has shown that NAD? treatment can significantly decrease the rotenone-induced increase in the Bax level of the cells. Because Bax is one of the major pro-apoptotic proteins [18], our study has suggested that NAD? attenuates the rotenone-induced apoptosis at least partially by preventing rotenone-induced increase in the Bax level. Previous studies have suggested that NAD? prevents mitochondrial damage induced by oxidative stress or DNA alkylating agents mainly by preventing glycolytic inhibition [2–4]. Our current study has suggested that NAD? could also decrease mitochondrial alterations by preventing an increase in Bax level. Nuclear translocation of AIF is a hallmark of caspase 3-independent apoptosis [15]. Our study has suggested that rotenone induces nuclear translocation of AIF, instead of caspase-3 activation. This result is consistent with previous

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Fig. 3 NAD? can attenuate rotenone-induced DSBs in PC12 cells. a Immunostaining assays showed that NAD? attenuated rotenoneinduced increases in c-H2AX foci in the nuclei of PC12 cells, assessed at 6 h after rotenone treatment. b Western blot study showed that NAD? attenuated rotenone-induced increase in the c-H2AX level, assessed at 6 h after the drug treatment. c Quantifications of the

Fig. 4 NAD? treatment can significantly attenuate rotenone-induced decrease in the intracellular ATP level of PC12 cells. PC12 cells were treated with 0.75 lM rotenone, with or without NAD? co-treatment for 24 h. Subsequently, the intracellular ATP level of the cells was assessed by luciferin/luciferase-based assay. N = 12. The data were pooled from three independent experiments. ***p \ 0.001

reports regarding the pathways of rotenone-induced apoptosis [16]. Multiple studies have indicated that increased Bax levels could lead to MPT, which has been shown to induce mitochondrial depolarization and AIF release from mitochondrial [2, 3]. Therefore, the rotenone-induced increase in Bax may lead to the rotenone-induced mitochondrial alterations observed in our current study and our previous study, including nuclear translocation of AIF and mitochondrial depolarization [8]. Our current observation that NAD? can prevent rotenone-induced increases in DSBs is consistent with our previous observations that NAD? administration can decrease synchrotron radiation X-ray-induced DSBs in rodent testes [19] as well as doxorubicin-induced DSBs in mouse

Western blots results showed that NAD? significantly attenuated the rotenone-induced c-H2AX increase in the PC12 cells. The cells were treated with 0.75 lM rotenone and 1 mM NAD? for 6 h. Subsequently, immunostaining assays and Western blot assays on the cH2AX levels of the cells were conducted. N = 16. The data were pooled from four independent experiments. *p \ 0.05; **p \ 0.01

livers [20]. A previous study has also indicated that NAD? repletion can restore DNA repair activity by inhibiting serine-specific phosphorylation of AP endonuclease and DNA polymerase-b in neuronal cultures exposed to oxygen-glucose deprivation [17]. Because a number of studies have found that DSBs can induce apoptosis by activating such pathways as p53-dependent pathway [21], our current study has suggested that NAD? may prevent the DSBsinitiated cell death pathway, which may partially underlie our observations that NAD? treatment can decrease rotenone-induced apoptosis. However, future studies are needed to further investigate the relationship between the protective effect of NAD? on DSBs and that of NAD? on Bax levels. Our previous study using FACS-based AAD assay has also shown that NAD? treatment can decrease rotenoneinduced cell necrosis [8]. Our current study has found that NAD? treatment can prevent rotenone-induced decrease in the intracellular ATP levels. Because decreased intracellular ATP levels play a key role in determining the switch from apoptosis to necrosis [22], our current study has suggested that NAD? treatment could attenuate the rotenone-induced cell necrosis at least partially by preventing the decrease in the intracellular ATP level. Mitochondrial membrane potential is one of the major factors mediating mitochondrial ATP production [23]. Our previous study showed that NAD? treatment can prevent rotenone-induced mitochondrial depolarization [8]. Therefore, NAD? may prevent the rotenone-induced decrease in the intracellular ATP levels by attenuating rotenone-induced mitochondrial depolarization.

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In the current study, we used rotenone treatment to mimic the pathological insults that produce the brain injury in such neurological diseases as stroke. However, we noticed the limitations of rotenone to mimic the pathological conditions. In order to elucidate the mechanisms underlying the neuroprotective effects of NAD?, future studies are warranted to determine the effects of NAD? on the mitochondrial alterations in animal models of stoke and other neurological diseases.

Conclusions Our study has found that NAD? can prevent rotenoneinduced alterations of mitochondrial pro-apoptotic proteins, thus suggesting new mechanisms underlying the preventive effects of NAD? on rotenone-induced apoptosis. Our study has also shown that NAD? can attenuate rotenone-induced DNA damage and decrease in the intracellular ATP level. Collectively, these pieces of information have provided valuable information for understanding the mechanisms underlying the significant preventive effects of NAD? treatment on rotenone-induced apoptosis and necrosis. Because mitochondrial alterations are important pathological factors in the brain injury of cerebral ischemia and Parkinson’s disease (PD) [12, 24], our study has highlighted the therapeutic potential of NAD? for these major neurological disorders. Acknowledgments This study was supported by Chinese National Science Foundation Grants #81171098 and #81271305 (to W. Y.), and two Shanghai Jiao Tong University Grants for Interdisciplinary Research on Medicine and Engineering (to W. Y.).

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NAD+ treatment can prevent rotenone-induced increases in DNA damage, Bax levels and nuclear translocation of apoptosis-inducing factor in differentiated PC12 cells.

Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immunological f...
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