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Sirt3 confers protection against neuronal ischemia by inducing autophagy: Involvement of the AMPK-mTOR pathway
Free Radical Biology and Medicine 108 (2017) 345–353
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Original article
Sirt3 confers protection against neuronal ischemia by inducing autophagy: Involvement of the AMPK-mTOR pathway
MARK
Shu-Hui Daia,1, Tao Chena,b,c,1, Xia Lia,1, Kang-Yi Yuea, Peng Luoa, Li-Kun Yangb, Jie Zhub, ⁎ ⁎ Yu-Hai Wangb, Zhou Feia, , Xiao-Fan Jianga, a b c
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China Department of Neurosurgery, The 101th Hospital of PLA, Rescue Center of Craniocerebral Injuries of PLA, Wuxi, Jiangsu 214044, China Department of Neurosurgery, The 123th Hospital of PLA, Bengbu, Anhui 233000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Ischemic stroke Sirt3 Autophagy AMPK mTOR
Sirtuin3 (Sirt3) is a member of the silent information regulator 2 (Sir2) family of proteins located in mitochondria that influences almost every major aspect of mitochondrial biology, including ATP generation and reactive oxygen species (ROS) production. Our previous study showed that Sirt3 exerts protective effects against oxidative stress in neuronal cells. In this study, we investigated the role of Sirt3 in neuronal ischemia using an oxygen and glucose deprivation (OGD) model. Sirt3 was up-regulated by OGD and overexpression of Sirt3 through lentivirus transfection significantly reduced OGD-induced lactate dehydrogenase (LDH) release and neuronal apoptosis. These effects were accompanied by reduced hydrogen dioxide (H2O2) production, enhanced ATP generation and preserved mitochondrial membrane potential (MMP). The results of immunocytochemistry and electron microscopy showed that Sirt3 increased autophagy in OGD-injured neurons, which was also confirmed by the increased expression of Beclin-1 as well as LC3-I to LC3-II conversion. In addition, the autophagy inhibitor 3-MA and bafilomycin A1 partially prevented the effects of Sirt3 on LDH release and apoptosis after OGD. The results of western blotting showed that overexpression of Sirt3 in cortical neurons markedly increased the phosphorylation of AMPK, whereas the phosphor-mTOR (p-mTOR) levels decreased both in the presence and absence of OGD insult. Furthermore, pre-treatment with the AMPK inhibitor compound C partially reversed the protective effects of Sirt3. Taken together, these findings demonstrate that Sirt3 protects against OGD insult by inducing autophagy through regulation of the AMPK-mTOR pathway and that Sirt3 may have therapeutic value for protecting neurons from cerebral ischemia.
1. Introduction Ischemic stroke induced by hypoxic ischemic encephalopathy or cerebrovascular accident is still one of the leading causes of death and permanent disability in the world [1]. Although ischemic stroke has been demonstrated to be associated with many devastating cascades, such as intracellular calcium overload, reactive oxygen species (ROS) generation, excitotoxicity mediated apoptosis and inflammation related necrosis, the exact molecular mechanism underlying ischemic neuronal injury has not been fully elucidated [2–4]. The sirtuins (or Sir2-like proteins) comprise a family of NAD+dependent protein deacetylases and ADP-ribosyltransferases that belong to class III histone deacetylases (HDACs) [5]. Mammals express seven homologues of sirtuins, Sirt1-7. Of those homologues, Sirt3 resides primarily in the mitochondria and has been identified as a
⁎
1
responsive deacetylase that regulates metabolism and oxidative stress [6]. Several studies have shown that Sirt3 plays an important role in regulating cell defence and mediating cell survival [7–9]. Our previous study showed that Sirt3 attenuates hydrogen peroxide-induced oxidative stress through preservation of mitochondrial function in HT22 cells [10]. We also found that overexpression of Sirt3 by lentivirus transfection protects cortical neurons against oxidative stress through regulating mitochondrial Ca2+ and mitochondrial biogenesis [11]. In addition, Sirt3 was shown to act as a pro-survival factor that plays an essential role in protecting neurons experiencing excitotoxicity [12]. However, the exact role of Sirt3 in ischemic neuronal cell injury and the associated molecular mechanisms have not yet been investigated. Autophagy is an evolutionarily conserved process for the bulk degradation and recycling of cytosolic proteins and organelles [13]. Mammalian autophagy exists in three different forms: macroautophagy,
Corresponding authors. E-mail addresses: [email protected] (Z. Fei), [email protected] (X.-F. Jiang). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.005 Received 30 August 2016; Received in revised form 1 April 2017; Accepted 4 April 2017 Available online 07 April 2017 0891-5849/ © 2017 Elsevier Inc. All rights reserved.
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the recombinant Lentivirus LV-Sirt3, 293 T cells were co-transfected with the pGC-FU plasmid (20 μg) with a cDNA encoding Sirt3, a pHelper 1.0 plasmid (15 μg) and a pHelper 2.0 plasmid (10 μg) using Lipofectamine 2000 (100 μl). The supernatant was harvested and the viral titre was calculated by transducing 293 T cells. As a control, we generated a lentiviral vector that expressed GFP alone (LV-control). Cortical neurons were transfected with lentivirus vectors for 72 h and subjected to various treatments.
microautophagy, and chaperone-mediated autophagy. These forms differ in their mechanisms and functions, and macroautophagy has been widely referred to as autophagy [14]. As a degradation/recirculation system, autophagy is believed to play an important role in pathological conditions in many organs, including brain ischemia [15,16]. Although the existence and activation of autophagy after ischemic stroke is undisputable, whether autophagy is a protective or detrimental mechanism for ischemic neuronal injury is still a topic of debate. Previous studies have shown that autophagy is one of several morphological features that occur during cell death after brain ischemia, and inhibition of autophagy exerts protective effects under hypoxic-exicitotoxic conditions [17,18]. In contrast, increasing amounts of evidence have indicated that autophagy may promote neuronal survival through removing damaged organelles to delay apoptosis or preserving energy to prevent necrosis, and the protective role of autophagy has also been demonstrated using chemical inhibitors or inducers under both in vitro and in vivo conditions [19,20]. In the present study, we measured the dynamic changes of Sirt3 expression after neuronal ischemia, and investigated the potential protective effects of Sirt3 against oxygen and glucose deprivation (OGD) in cortical neurons. We also determined the effect of Sirt3 overexpression on neuronal autophagy regulation, and confirmed the involvement of the AMPK-mTOR pathway in autophagy and Sirt3induced protection.
2.4. Oxygen and glucose deprivation (OGD) To initiate OGD, the culture medium was removed and rinsed with phosphate buffered saline (PBS) three times. The cultured neurons were placed into a specialized, humidified chamber containing 5% CO2, 95% N2 at 37 °C with glucose-free DMEM, which was pre-gassed with N2/ CO2 (95%/5%) to remove residual oxygen. After 2 h, the neurons were removed from the anaerobic chamber, and the culture medium was replaced with neurobasal medium containing 2% B27 supplement and 0.5 mM L-Glutamine. The neurons were maintained for 24 h at 37 °C in a humidified 5% CO2 incubator to generate the reperfusion insult. 2.5. Cytotoxicity assay Cytotoxicity was determined by measuring the release of LDH with a diagnostic kit according to the manufacturer's instructions. Briefly, 50 μl of supernatant from each well was collected to assay LDH release. The samples were incubated with a reduced form of nicotinamideadenine dinucleotide (NADH) and pyruvate for 15 min at 37 °C and the reaction was stopped by adding 0.4 M NaOH. The activity of LDH was calculated from the absorbance at 440 nm, and the background absorbance from culture medium that was not used for any cell cultures was subtracted from all of the absorbance measurements. The results are presented as fold increase of the control.
2. Materials and methods 2.1. Antibodies and reagents Antibodies against Sirt3, LC3, and Beclin-1 were obtained from Cell Signaling (Danvers, MA, USA). Antibodies against p-AMPK (Thr172), AMPK, p-mTOR (Ser2448), mTOR and β-actin were obtained from Santa Cruz (Dallas, TX, USA). The secondary antibodies, Rh123, and MitoSox Red were purchased from Sigma (St. Louis, MO, USA). Rapamycin, compound C, 3-MA and bafilomycin A1 were purchased from Tocris Bioscience (Bristol, UK). The TUNEL staining kit was obtained from Promega (Madison, WI, USA). The lactate dehydrogenase (LDH) assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China).
2.6. TUNEL staining Apoptosis in primary cortical neurons subjected to various treatments was detected using TUNEL staining, which is a method to observe DNA strand breaks in nuclei. For TUNEL staining, cortical neurons were seeded on 1.5-cm glass slides at a density of 3×105 cells/cm2. Twentyfour hours after OGD, the cells were fixed by immersing the slides in freshly prepared 4% methanol-free formaldehyde solution in PBS for 20 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min. Neurons were labelled with fluorescein TUNEL reagent mixture for 60 min at 37 °C according to the manufacturer's suggested protocol. Then, the slides were examined by fluorescence microscopy, and the TUNEL-positive (apoptotic) cells were counted. DAPI (10 μg/ ml) was used to stain nuclei.
2.2. Primary culture of cortical neurons All experimental protocols and animal handling procedures were performed according to the National Institutes of Health (NIH) guidelines for the use of experimental animals, and the study was approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University (No. 2014–81371447). Cortical neurons were cultured from Sprague-Dawley rats using a modified method. Briefly, cerebral cortices were removed from embryos at 16–18 days, stripped of meninges and blood vessels and minced. The tissues were dissociated through 0.25% trypsin digestion for 15 min at 37 °C and gentle trituration. Neurons were resuspended in neurobasal medium containing 2% B27 supplement and 0.5 mM L-Glutamine, and then the neurons were plated at a density of 3×105 cells/cm2. Before seeding, the culture vessels, consisting of 96-well plates, 1.5-cm glass slides or 6cm dishes were coated with PLL (50 μg/ml) at room temperature overnight. Neurons were maintained at 37 °C in a humidified 5% CO2 incubator and half of the culture medium was changed every other day.
2.7. Caspase-3 activity Briefly, after being harvested and lysed, 106 cells were mixed with 32 μl of assay buffer and 2 μl of 10 mM Ac-DEVD-pNA substrate. Absorbance at 405 nm was measured after incubation at 37 °C for 4 h. The absorbance of each sample was determined by subtraction of the mean absorbance of the blank and corrected by the protein concentration of the cell lysate.
2.3. Lentivirus construction and transfection
2.8. Measuring intracellular H2O2 by aminotriazole-mediated inactivation of catalase
The coding sequence of Sirt3 was amplified by RT-PCR. The primer sequences were forward, 5′-TACTTCCTTCGGCTGCTTCA-3′; reverse, 5′AAGGCGAAATCAGCCACA −3′. The PCR fragments and the pGC-FU plasmid (GeneChem, Shanghai, China) were digested with Age I and then ligated with T4 DNA ligase to produce pGC-FU-Sirt3. To generate
The intracellular steady-state levels of H2O2 were estimated using a sensitive assay based on 3-aminotriazole inhibition of catalase [21]. After various treatments, neurons were scrape-harvested and the protein concentrations were quantified with the BCA method. The assay was initiated through addition of 100 μl of a 30 mM H2O2 stock 346
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50 nm thickness, stained with lead citrate and then viewed on an electron microscope.
solution in phosphate buffer at pH 7.0, and the loss of absorbance at 240 nm at room temperature over a 2-min time period was monitored. The initial catalase activities were calculated by fitting experimental data to the first-order kinetics as described [22] and expressed as catalase k mU/mg cell protein. The H2O2 concentration was calculated by kinetic analysis of the rate of decrease of catalase activity using the equation: [H2O2]=kinactivation/k1, where kinactivation is the experimental pseudo first-order rate constant of catalase inactivation, and the value k1 is 1.7×107 M−1 S−1, which is the rate constant for the formation of catalase compound I in the presence of H2O2.
2.14. Western blot analysis Equivalent amounts of protein (40 μg per lane) were loaded and separated by 10% sodium dodecyl sulphate (SDS)-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk solution in tris-buffered saline with 0.1% Triton X-100 for 1 h, and then incubated overnight at 4 °C with primary antibodies diluted in TBST. After the incubation, the membranes were washed and incubated with secondary antibody for 1 h at room temperature. Immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Image J software was used to quantify the optical density of each band.
2.9. ROS-GLO H2O2 assay The ROS-GLO H2O2 assay uses a modified luciferin substrate, based on boronate oxidation, which reacts directly with H2O2 to generate a luciferin precursor. Upon addition of the detection reagent, the precursor is converted to luciferin, and the Ultra-Glo™ Recombinant Luciferase included in the detection reagent produces a light signal proportional to the level of H2O2 in the sample. Before OGD treatment, 20 μl of a mixture of H2O2 substrate and H2O2 dilution buffer was immediately added to the culture medium. After OGD exposure, 100 μl ROS-GLO™ Detection Solution was added to the neurons, and all samples were incubated at room temperature for 20 min. Luminescence was read during an integration time of 1000 ms.
2.15. Statistical analysis All of the experiments were performed a minimum of three times. The statistical evaluation was carried out with GraphPad Prism software, version 6.0. Significant differences between experiments were assessed by univariate ANOVA (more than two groups), followed by Bonferroni's multiple comparisons or an unpaired t-test (two groups).
2.10. Measurement of mitochondrial membrane potential (MMP)
3. Results
MMP was measured using the fluorescent dye Rh123 as reported previously [23]. Rh123 was added to the culture medium to achieve a final concentration of 10 μM for 30 min at 37 °C after the neurons were treated and washed with PBS. The fluorescence was observed using an Olympus BX60 microscope with the appropriate fluorescence filters (excitation wavelength of 480 nm and emission wavelength of 530 nm).
3.1. Sirt3 protects against neuronal ischemia in cortical neurons
Cultured neurons were homogenized in a mitochondria isolation buffer, and mitochondria were isolated through multiple centrifugation steps as detailed earlier [11]. Isolated mitochondria were used to measure ATP synthesis with a luciferase/ luciferin-based system, as described previously [24]. Thirty micrograms of mitochondria-enriched pellets was resuspended in 100 μl of buffer A (150 mM KCl, 25 mM TrisHCl, 2 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4) with 0.1% BSA, 1 mM malate, 1 mM glutamate and buffer B (containing 0.8 mM luciferin and 20 mg/ml luciferase in 0.5 M Tris-acetate, pH 7.75). The reaction was initiated by addition of 0.1 mM ADP and monitored for 120 min using a microplate reader.
To investigate the effects of ischemia on Sirt3, cortical neurons were treated with OGD for 2 h, and the levels of Sirt3 protein at different time points were determined by western blot. The results showed that OGD significantly increased the levels of Sirt3 protein at 6, 12, 24 and 48 h after OGD (Fig. 1A). Lentiviral transduction was used to determine the effects of Sirt3 on neuronal survival and death after OGD, and the results of western blotting showed that transfection with a Sirt3targeted lentivirus (LV-Sirt3) significantly increased the levels of Sirt3 protein (Fig. 1B). Representative phase photomicrographs showed that LV-Sirt3-transduced neurons exhibited lower cell death relative to LVControl-transduced cultures (Fig. 1C). As shown in Fig. 1D, we observed a similar effect using the LDH release assay, as demonstrated by the reduced LDH release after Sirt3 overexpression. In addition, TUNEL staining was used to detect apoptotic cell death in our in vitro model (Fig. 1E). The results showed that Sirt3 overexpression obviously decreased the number of TUNEL-positive cells after OGD insult (Fig. 1F). As shown in Fig. 1G, a similar result for caspase-3 activity was also observed.
2.12. Immunocytochemistry (ICC)
3.2. Sirt3 preserves mitochondrial function and cellular bioenergetics
The neurons were fixed for 30 min with 4% paraformaldehyde, rinsed twice with PBS and subsequently incubated with 1% hydrogen peroxide for 10 min. Following two PBS rinses, the cells were incubated with blocking solution for 20 min and incubated with a primary antiLC3 antibody (1:50) at 4 °C overnight. The cells were then rinsed twice with PBS and incubated with fluorescein isothiocyanate (FITC) labelled secondary antibody (1:500) for 1 h at room temperature. Coverslips were mounted in mounting medium and visualized using a fluorescence microscope.
We also determined whether Sirt3-induced protection against OGD was associated with decreased oxidative stress in vitro. The intracellular steady-state levels of H2O2 were estimated using a sensitive assay based on 3-aminotriazole inhibition of catalase. There was a significant increase in the steady-state level of intracellular H2O2 after OGD injury. As expected, Sirt3 overexpression profoundly attenuated the intracellular H2O2 steady state levels produced by OGD (Fig. 2A). As shown in Fig. 2B, a similar result for total H2O2 production, which was measured using a ROS-GLO probe, was also observed. Furthermore, to confirm the effects of Sirt3 on mitochondrial function, we measured mitochondrial O.-2 levels using MitoSOX Red fluorescence (Fig. 2C). Consistent with the data we collected above, increased mitochondrial O.-2 levels were clearly observed following OGD treatment, which was partially prevented by Sirt3 overexpression. In addition, ATP levels were significantly greater in the homogenates of cortical neurons transfected with LV-Sirt3 compared to control neurons after OGD (Fig. 2E). To further
2.11. Measurement of ATP synthesis
2.13. Electron microscopy The neurons were harvested and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at room temperature. The cell pellets were postfixed with 1% osmium tetroxide, dehydrated through acetone and then immersed in resin. After hardening, the blocks were thinly sectioned at 347
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Fig. 1. Sirt3 protects against neuronal ischemia in cortical neurons. Cortical neurons were treated with OGD for 2 h, and the expression of Sirt3 at different time points (3, 6, 12, 24 or 48 h) was determined by western blot (A). Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. The expression of Sirt3 was detected by western blot at 24 h after OGD (B). Representative phase photomicrographs are shown (C), and neuronal cell death was quantified by an LDH release assay (D). Apoptotic cell death was detected by TUNEL staining (E), and the number of TUNEL positive cells was counted (F). The activity of caspase-3 was measured with a colorimetric assay kit (G). The data are represented as means ± SEM. #p < 0.05, *p < 0.05 vs. Control, & p < 0.05 vs. OGD+LV-Control.
and absence of OGD (Fig. 3E). As shown in Fig. 3E, there was a similar result for Beclin-1 expression.
determine the roles of Sirt3 in mitochondrial function and stress resistance after OGD, the fluorescence of Rh123 was used to identify changes in MMP levels. The results demonstrated that there was an obvious decrease in MMP after OGD exposure, which was partially prevented by Sirt3 overexpression (Fig. 2F).
3.4. Autophagy contributes to the neuroprotective effects of Sirt3 Cortical neurons were treated with the autophagy inducer rapamycin to determine the role of autophagy in our in vitro ischemia model. The results of the western blotting showed that the increased conversion of LC3-I to LC3-II induced by OGD was further enhanced by rapamycin treatment (Fig. 4A). Treatment with rapamycin significantly decreased LDH release (Fig. 4B) and reduced the number of TUNEL positive cells (Fig. 4C) after OGD. To determine whether autophagy was involved in Sirt3-induced protection, cortical neurons were transfected with LV-Sirt3 and/or treated with the autophagy inhibitor 3-MA. As shown in Fig. 4D, the increased conversion of LC3-I to LC3-II induced by Sirt3 overexpression was significantly decreased by 3-MA. Moreover, treatment with 3-MA or bafilomycin A1, another autophagy inhibitor, partially prevented the decrease in LDH release (Fig. 4E) and led to a decrease in the number of TUNEL positive cells (Fig. 4F) after OGD.
3.3. Sirt3 regulates autophagy following neuronal ischemia Next, we tested whether Sirt3 regulates autophagy in our in vitro model. The number of LC3-positive puncta per neuron was determined by using immunohistochemistry to measure autophagy 6 h after OGD (Fig. 3A). There were no LC3-positive puncta in control neurons, and overexpression of Sirt3 significantly increased the number of LC3positive puncta after OGD compared to the LV-Control group (Fig. 3B). We also performed electron microscopy to examine the autophagosomes in neurons (Fig. 3C). Compared to the LV-Control group, there were more autophagosomes in the LV-Sirt3 group (Fig. 3D). In addition, as a marker of autophagy, the LC3-I to LC3-II conversion was significantly increased by LV-Sirt3 transfection, both in the presence 348
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Fig. 2. Sirt3 preserves mitochondrial function and cellular bioenergetics. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. The intracellular steady-state levels of H2O2 (A) and total H2O2 production (B) were measured. The mitochondrial O.-2 levels were evaluated by imaging of MitoSOX Red fluorescence (C and D). Scale bar =50 µm. The total ATP levels (E) were measured after OGD, and MMP levels was analyzed by Rh123 staining (F). The data are represented as means ± SEM. *p < 0.05 vs. OGD+LV-Control.
Fig. 3. Sirt3 regulates autophagy following neuronal ischemia. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. LC3-positive puncta in neurons were detected by fluorescent staining (A), and the number of LC3 puncta (arrows) in neurons were calculated (B). Scale bar =10 µm. Neuronal autophagy was observed by electron microscopy (C), and the number of autophagosomes (red arrows) in neurons was calculated (D). Scale bar =100 nm. N: nucleus. The expression of LC3 and Beclin-1 was detected by western blot (E). The data are represented as means ± SEM. #p < 0.05 vs. Control, *p < 0.05 vs. OGD+LV-Control.
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Fig. 4. Autophagy contributes to the neuroprotective effect of Sirt3. Cortical neurons were treated with 1 μM rapamycin and exposed to OGD for 2 h. The expression of LC3 was detected by western blot (A). Neuronal cell death was quantified with an LDH release assay (B), and apoptotic cell death was detected by TUNEL staining (C). Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h with or without 10 mM 3-MA or 5 μM bafilomycin A1 and exposed to OGD for 2 h. The expression of LC3 was detected by western blot (D). Neuronal cell death was quantified by an LDH release assay (E), and apoptotic cell death was detected by TUNEL staining (F). The data are represented as means ± SEM. #p < 0.05 vs. Control, * p < 0.05 vs. OGD, & p < 0.05 vs. OGD + LV-Control, #p < 0.05 vs. OGD+LV-Sirt3.
studies [11,25,26]. In the current study, we revealed that Sirt3 confers protection against ischemic neuronal injury by regulating autophagy. First, the upregulation of Sirt3 attenuated the neuronal injury induced by OGD through reducing apoptosis. Second, the beneficial effects on neuronal survival induced by Sirt3 were associated with activation of autophagy in cortical neurons. Third, the discovery that altered activation of the AMPK-mTOR pathway was responsible for much of the regulation of autophagy in our in vitro model demonstrated that AMPK-mTOR was involved in Sirt3-induced neuroprotection. As a member of the sirtuins family of proteins, Sirt3 is expressed at high levels in the brain and other nervous system tissues [27]. It resides primarily in the mitochondria and influences almost every major aspect of mitochondrial function, including ATP generation, nutrient oxidation, ROS production and mitochondrial dynamics [28]. Previous studies have shown that Sirt3 was dynamically regulated by cellular stress factors at both the transcriptional and translational levels under pathological conditions. Increased expression of Sirt3 was observed in hearts under stress conditions, whereas Sirt3 levels were reduced in hypertrophied or failing hearts [29,30]. Our previous studies showed that Sirt3 was upregulated by hydrogen peroxide treatment and that overexpression of Sirt3 protected HT22 cells and cortical neurons against oxidative stress [10,11]. More recently, Cheng et al. found that exercise and synaptic activity induced hippocampal Sirt3 expression to modulate mitochondrial protein acetylation and bolster neuronal resistance to oxidative stress and apoptosis [25]. In this study, our results showed that the levels of Sirt3 protein increased after OGD treatment in a time-dependent manner in primary culture cortical
3.5. Involvement of the AMPK-mTOR pathway in Sirt3-induced protection To further elucidate the molecular mechanisms through which Sirt3 mediates autophagy and protection, we detected the phosphorylation of AMPK and mTOR after Sirt3 overexpression using western blotting. The results showed that in the OGD group, the phosphorylation of AMPK increased, while the expression of p-mTOR was reduced compared to the control group (Fig. 5A and B). Overexpression of Sirt3 significantly increased the phosphorylation of AMPK but decreased the phosphorylation of mTOR both in the presence and absence of OGD injury. In addition, the increased expression of p-AMPK induced by Sirt3 was partially reversed by the AMPK inhibitor compound C, but not by the mTOR inhibitor rapamycin (Fig. 5C). As shown in Fig. 5D, both compound C and rapamycin significantly increased the phosphorylation of mTOR compared to LV-Sirt3-transfected neurons. The results of the western blot showed that the increased expression of Beclin-1 and conversion of LC3-I to LC3-II induced by Sirt3 overexpression were abolished by compound C treatment (Fig. 5E). Consistent with those results, Sirt3-induced deceases in LDH release (Fig. 5F) and neuronal apoptosis (Fig. 5G) were both partially nullified by compound C, which indicated that the AMPK-mTOR pathway was involved in Sirt3-induced protection. 4. Discussion The involvement of Sirt3 in adaptive responses of neuronal cells to stress conditions has been well established in several previous in vitro 350
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Fig. 5. Involvement of the AMPK-mTOR pathway in Sirt3-induced protection. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. The expression and phosphorylation of AMPK (A) and mTOR (B) were detected by western blot. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h with or without 1 μM rapamycin or 5 μM compound C and exposed to OGD for 2 h. The expression and phosphorylation of AMPK (C) and mTOR (D) were detected by western blot. Cortical neurons were transfected with LV-Sirt3 with or without 5 μM compound C for 72 h, and the expression of LC3 and Beclin-1 was detected by western blot (E). Cortical neurons were transfected with LVSirt3 with or without 5 μM of compound C for 72 h and exposed to OGD for 2 h. Neuronal cell death was quantified by an LDH release assay (F), and apoptotic cell death was detected by TUNEL staining (G). *p < 0.05.
involved in mitochondrial energy production. Thus, how autophagy is regulated by Sirt3 knockdown or overexpression is of great interest to researchers. Sirt3 activation attenuated oxidized low-density lipoprotein-induced apoptosis through sustaining autophagy in human umbilical vein endothelial cells [38]. A more recent study found that Sirt3SOD2-mROS-dependent autophagy was involved in melatonin-induced protection against cadmium-induced hepatotoxicity [39]. In this study, we found that Sirt3 overexpression significantly increased the number of LC3-positive puncta and increased the expression of LC3, the level of which was shown to be proportional to the abundance of autophagosomes [40]. In addition, Sirt3-overexpressing neurons exhibited a lower level of mitochondrial O.-2 and preserved cellular bioenergetics. Previous studies have demonstrated that Sirt3 could directly bind and deacetylate SOD2, which in turn led to significant effects on mitochondrial ROS homeostasis and autophagic flux [41,42]. Thus, our results strongly indicated that Sirt3 protect against OGD-induced neuronal injury and mitochondrial dysfunction by inducing autophagy which was also confirmed by the reversed effects of 3-MA and bafilomycin A1 in Srit3-induced protection. Sirt3 is involved in suppressing the onset of many neurological diseases, but the mechanism through which it regulates neuronal autophagy has yet to be determined. As for one of the clues, our results from western blotting showed that Sirt3 overexpression increased the phosphorylation of AMPK, which is a sensor of cellular energy status. AMPK serves as a regulator of cell survival or death in response to multiple pathological stresses, including hypoxia, ischemia and oxidative stress [43]. Previous studies showed that Sirt3 maintains bone homeostasis by regulating AMPK-PGC-1β axis in mice, and activation of acetyl-CoA synthetase 2 (AceCS2) by Sirt3 may elevate the AMP/ATP ratio and consequently activate AMPK [44–46]. As expected, the AMPK inhibitor compound C partially abolished the Sirt3-induced protective effects in our in vitro model. It is well known that activated AMPK
neurons. Overexpression of Sirt3 significantly increased neuronal survival and decreased neuronal apoptosis after OGD, which strongly suggested that Sirt3 might be an endogenous protective mechanism under neuronal ischemia conditions. Autophagy is an evolutionarily conserved process that degrades and recycles proteins and organelles through an intracellular bulk degradation pathway. It is well accepted that autophagy is rapidly activated in neurons exposed to hypoxic stimuli and focal cerebral ischemia [31,32]. However, whether enhanced autophagy is part of a death or survival mechanism is still a matter of debate [13]. It may aid cell death through excessive self-digestion and degradation of essential cellular constituents, but increasing amounts of evidence suggests that autophagy may promote cell survival by removing toxic metabolites and intracellular pathogens. In our in vitro model, moderate protection, as demonstrated by the decreased LDH release and reduced number of TUNEL positive cells, was found in rapamycin-treated neurons after OGD exposure, which was consistent with previous data showed that rapamycin reduced apoptotic cell death by decreasing the activation of the intrinsic apoptotic mitochondrial pathway [33]. All of these results provide further evidence that autophagy is beneficial for neurons injured by OGD. In contrast, recent studies showed that treatment with 3MA protected against brain and spinal cord ischemia in in vivo models [34,35]. The differences between our results and previous studies might be explained by the different models and time points as well as the in vitro conditions. The sirtuins (Sirt1-7) are a conserved class of protein deacylases, and lysine acetylation has recently emerged as an important posttranslational modification that is employed to regulate autophagy [36]. A previous study showed that overexpression of Sirt1 stimulated autophagy while Sirt1 deficiency attenuated autophagy under starvation conditions [37]. Among the seven sirtuins members, Sirt3 resides primarily in the mitochondria and it directs biological functions 351
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81371447, No. 81671303, No. 81430043, No. 81301037) and Shaanxi Province Natural Science Foundation Research Program (No. 2014JM4131). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2017.04. 005. References [1] D. Mozaffarian, E.J. Benjamin, A.S. Go, D.K. Arnett, M.J. Blaha, M. Cushman, S.R. Das, S. de Ferranti, J.P. Despres, H.J. Fullerton, V.J. Howard, M.D. Huffman, C.R. Isasi, M.C. Jimenez, S.E. Judd, B.M. Kissela, J.H. Lichtman, L.D. Lisabeth, S. Liu, R.H. Mackey, D.J. Magid, D.K. McGuire, E.R. Mohler 3rd, C.S. Moy, P. Muntner, M.E. Mussolino, K. Nasir, R.W. Neumar, G. Nichol, L. Palaniappan, D.K. Pandey, M.J. Reeves, C.J. Rodriguez, W. Rosamond, P.D. Sorlie, J. Stein, A. Towfighi, T.N. Turan, S.S. Virani, D. Woo, R.W. Yeh, M.B. Turner, Heart Disease and Stroke Statistics-2016 Update: a Report From the American Heart Association, Circulation 133 (4) (2016) e38–e360. [2] R. Jin, G. Yang, G. Li, Inflammatory mechanisms in ischemic stroke: role of inflammatory cells, J. Leukoc. Biol. 87 (5) (2010) 779–789. [3] T. Chen, W. Liu, X. Chao, Y. Qu, L. Zhang, P. Luo, K. Xie, J. Huo, Z. Fei, Neuroprotective effect of osthole against oxygen and glucose deprivation in rat cortical neurons: involvement of mitogen-activated protein kinase pathway, Neuroscience 183 (2011) 203–211. [4] R.S. Pandya, L. Mao, H. Zhou, S. Zhou, J. Zeng, A.J. Popp, X. Wang, Central nervous system agents for ischemic stroke: neuroprotection mechanisms, Cent. Nerv. Syst. Agents Med. Chem. 11 (2) (2011) 81–97. [5] R.N. Dutnall, L. Pillus, Deciphering NAD-dependent deacetylases, Cell 105 (2) (2001) 161–164. [6] L. Jin, H. Galonek, K. Israelian, W. Choy, M. Morrison, Y. Xia, X. Wang, Y. Xu, Y. Yang, J.J. Smith, E. Hoffmann, D.P. Carney, R.B. Perni, M.R. Jirousek, J.E. Bemis, J.C. Milne, D.A. Sinclair, C.H. Westphal, Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3, Protein Sci.: a Publ. Protein Soc. 18 (3) (2009) 514–525. [7] C.J. Chen, Y.C. Fu, W. Yu, W. Wang, SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-kappaB, Biochem. Biophys. Res. Commun. 430 (2) (2013) 798–803. [8] Y. Kawamura, Y. Uchijima, N. Horike, K. Tonami, K. Nishiyama, T. Amano, T. Asano, Y. Kurihara, H. Kurihara, Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest, J. Clin. Investig. 120 (8) (2010) 2817–2828. [9] Y. Chen, J. Zhang, Y. Lin, Q. Lei, K.L. Guan, S. Zhao, Y. Xiong, Tumour suppressor SIRT3 deacetylates and activates manganese superoxide dismutase to scavenge ROS, EMBO Rep. 12 (6) (2011) 534–541. [10] S.H. Dai, T. Chen, Y.H. Wang, J. Zhu, P. Luo, W. Rao, Y.F. Yang, Z. Fei, X.F. Jiang, Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells, Int. J. Mol. Med. 34 (4) (2014) 1159–1168. [11] S.H. Dai, T. Chen, Y.H. Wang, J. Zhu, P. Luo, W. Rao, Y.F. Yang, Z. Fei, X.F. Jiang, Sirt3 protects cortical neurons against oxidative stress via regulating mitochondrial Ca2+ and mitochondrial biogenesis, Int. J. Mol. Sci. 15 (8) (2014) 14591–14609. [12] S.H. Kim, H.F. Lu, C.C. Alano, Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture, PloS One 6 (3) (2011) e14731. [13] W. Balduini, S. Carloni, G. Buonocore, Autophagy in hypoxia-ischemia induced brain injury: evidence and speculations, Autophagy 5 (2) (2009) 221–223. [14] W. Balduini, S. Carloni, G. Buonocore, Autophagy in hypoxia-ischemia induced brain injury, J. Matern.-Fetal Neonatal Med.: Off. J. Eur. Assoc. Perinat. Med., Fed. Asia Ocean. Perinat. Soc., Int. Soc. Perinat. Obstet. 25 (Suppl 1) (2012) 30–34. [15] C. Huang, S. Yitzhaki, C.N. Perry, W. Liu, Z. Giricz, R.M. Mentzer Jr., R.A. Gottlieb, Autophagy induced by ischemic preconditioning is essential for cardioprotection, J. Cardiovasc. Transl. Res. 3 (4) (2010) 365–373. [16] D.D. Esposti, M.C. Domart, M. Sebagh, F. Harper, G. Pierron, C. Brenner, A. Lemoine, Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis, Autophagy 6 (1) (2010) 172–174. [17] V. Ginet, A. Spiehlmann, C. Rummel, N. Rudinskiy, Y. Grishchuk, R. Luthi-Carter, P.G. Clarke, A.C. Truttmann, J. Puyal, Involvement of autophagy in hypoxicexcitotoxic neuronal death, Autophagy 10 (5) (2014) 846–860. [18] F. Adhami, G. Liao, Y.M. Morozov, A. Schloemer, V.J. Schmithorst, J.N. Lorenz, R.S. Dunn, C.V. Vorhees, M. Wills-Karp, J.L. Degen, R.J. Davis, N. Mizushima, P. Rakic, B.J. Dardzinski, S.K. Holland, F.R. Sharp, C.Y. Kuan, Cerebral ischemiahypoxia induces intravascular coagulation and autophagy, Am. J. Pathol. 169 (2) (2006) 566–583. [19] K. Wei, P. Wang, C.Y. Miao, A double-edged sword with therapeutic potential: an updated role of autophagy in ischemic cerebral injury, CNS Neurosci. Ther. 18 (11)
Fig. 6. A proposed diagram tying together the observations involved in Sirt3-induced neuroprotection against OGD. OGD increased Sirt3 protein levels, which might be associated with PGC-1-mediated transcription. Sirt3 increased phosphorylation of AMPK, which in turn inhibited mitochondrial dysfunction through regulating MMP, ROS generation and ATP production. In addition, Sirt3 inhibited phosphorylation of mTOR, which led to increased expression of LC3, Beclin-1, and formation of autophagosomes.
directly phosphorylates PGC-1α, which is required for Sirt3-mediated mitochondrial biogenesis [47,48]. Thus, the activation of AMPK might cause an increase in the cellular NAD+/NADH ratio in OGD-injured neurons, which provides the positive feedback loop needed for prolonged activation of Sirt3 (as shown in Fig. 1A). In addition, decreased activation of mTOR was also observed after Sirt3 overexpression. mTOR is a serine/threonine kinase that promotes anabolic metabolism and inhibits autophagy induction [49]. There are mTOR inhibitors already in clinical trials or that have been approved for the treatment of diseases associated with autophagy defects [50,51]. Accumulating evidence has shown that AMPK activates autophagy via inhibition of mTOR [52,53], which was an effect we also demonstrated in our present study. Intriguingly, our results showed that the Sirt3-induced increase in p-AMPK was partially prevented by compound C but not rapamycin, which could form a complex with FKB12 to bind to mTOR and allosterically inhibit its kinase activity [54]. These data suggested that mTOR was one of the downstream signaling cascades involved in Sirt3-induced regulation of AMPK and autophagy. One limitation of this study was that the protective effect of Sirt3 against ischemia was only investigated in cultured cortical neurons. In this in vitro condition, the cellular microenvironment is missing and neuronal and glial cells associated signaling cascades cannot be completely mimicked. More experiments in cerebral tissues or in in vivo conditions, such as middle cerebral artery occlusion (MCAO) models, need to be done to confirm these observations in the future. In conclusion, we propose an intriguing mechanism whereby Sirt3 protects against OGD-induced neuronal injury via activation of autophagy. Importantly, Sirt3 showed a compensatory and protective role in eliminating intracellular H2O2, attenuating mitochondrial O.-2 and promoting autophagy through the AMPK-mTOR pathway in vitro (Fig. 6). Taken together, these findings provide new insights about the link between Sirt3 and autophagy signaling in neurons under ischemic conditions.
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autophagy, Autophagy 9 (6) (2013) 819–829. [37] I.H. Lee, L. Cao, R. Mostoslavsky, D.B. Lombard, J. Liu, N.E. Bruns, M. Tsokos, F.W. Alt, T. Finkel, A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy, Proc. Natl. Acad. Sci. USA 105 (9) (2008) 3374–3379. [38] X. Luo, Z. Yang, S. Zheng, Y. Cao, Y. Wu, Sirt3 activation attenuated oxidized lowdensity lipoprotein-induced human umbilical vein endothelial cells' apoptosis by sustaining autophagy, Cell Biol. Int. (2014). [39] H. Pi, S. Xu, R.J. Reiter, P. Guo, L. Zhang, Y. Li, M. Li, Z. Cao, L. Tian, J. Xie, R. Zhang, M. He, Y. Lu, C. Liu, W. Duan, Z. Yu, Z. Zhou, SIRT3-SOD2-mROSdependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin, Autophagy 11 (7) (2015) 1037–1051. [40] Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (21) (2000) 5720–5728. [41] X. Qiu, K. Brown, M.D. Hirschey, E. Verdin, D. Chen, Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation, Cell Metab. 12 (6) (2010) 662–667. [42] Q. Liang, G.A. Benavides, A. Vassilopoulos, D. Gius, V. Darley-Usmar, J. Zhang, Bioenergetic and autophagic control by Sirt3 in response to nutrient deprivation in mouse embryonic fibroblasts, Biochem. J. 454 (2) (2013) 249–257. [43] G.V. Ronnett, S. Ramamurthy, A.M. Kleman, L.E. Landree, S. Aja, AMPK in the brain: its roles in energy balance and neuroprotection, J. Neurochem. 109 (Suppl 1) (2009) 17–23. [44] J.E. Huh, J.H. Shin, E.S. Jang, S.J. Park, D.R. Park, R. Ko, D.H. Seo, H.S. Kim, S.H. Lee, Y. Choi, S.Y. Lee, Sirtuin 3 (SIRT3) maintains bone homeostasis by regulating AMPK-PGC-1beta axis in mice, Sci. Rep. 6 (2016) 22511. [45] W.C. Hallows, S. Lee, J.M. Denu, Sirtuins deacetylate and activate mammalian acetyl-coa synthetases, Proc. Natl. Acad. Sci. USA 103 (27) (2006) 10230–10235. [46] B.J. North, D.A. Sinclair, Sirtuins: a conserved key unlocking AceCS activity, Trends Biochem. Sci. 32 (1) (2007) 1–4. [47] B. Schwer, J. Bunkenborg, R.O. Verdin, J.S. Andersen, E. Verdin, Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2, Proc. Natl. Acad. Sci. USA 103 (27) (2006) 10224–10229. [48] 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 (4) (2003) 809–813. [49] Y.C. Kim, K.L. Guan, mTOR: a pharmacologic target for autophagy regulation, J. Clin. Investig. 125 (1) (2015) 25–32. [50] S.A. Wander, B.T. Hennessy, J.M. Slingerland, , Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy, J. Clin. Investig. 121 (4) (2011) 1231–1241. [51] D.W. Lamming, L. Ye, D.M. Sabatini, J.A. Baur, Rapalogs and mTOR inhibitors as anti-aging therapeutics, J. Clin. Investig. 123 (3) (2013) 980–989. [52] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2) (2011) 132–141. [53] C.S. Yang, J.J. Kim, H.M. Lee, H.S. Jin, S.H. Lee, J.H. Park, S.J. Kim, J.M. Kim, Y.M. Han, M.S. Lee, G.R. Kweon, M. Shong, E.K. Jo, The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy, Autophagy 10 (5) (2014) 785–802. [54] M. Wong, Mammalian target of rapamycin (mTOR) pathways in neurological diseases, Biomed. J. 36 (2) (2013) 40–50.
(2012) 879–886. [20] S. Carloni, G. Buonocore, W. Balduini, Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury, Neurobiol. Dis. 32 (3) (2008) 329–339. [21] Q. Tong, Y. Zhu, J.W. Galaske, E.A. Kosmacek, A. Chatterjee, B.C. Dickinson, R.E. Oberley-Deegan, MnTE-2-PyP modulates thiol oxidation in a hydrogen peroxide-mediated manner in a human prostate cancer cell, Free Radic. Biol. Med. 101 (2016) 32–43. [22] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. [23] T. Chen, F. Fei, X.F. Jiang, L. Zhang, Y. Qu, K. Huo, Z. Fei, Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons, Free Radic. Biol. Med. 52 (1) (2012) 208–217. [24] P.A. Parone, S. Da Cruz, J.S. Han, M. McAlonis-Downes, A.P. Vetto, S.K. Lee, E. Tseng, D.W. Cleveland, Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis, J. Neurosci.: Off. J. Soc. Neurosci. 33 (11) (2013) 4657–4671. [25] A. Cheng, Y. Yang, Y. Zhou, C. Maharana, D. Lu, W. Peng, Y. Liu, R. Wan, K. Marosi, M. Misiak, V.A. Bohr, M.P. Mattson, Mitochondrial SIRT3 Mediates Adaptive Responses of Neurons to Exercise and Metabolic and Excitatory Challenges, Cell Metab. 23 (1) (2016) 128–142. [26] W. Song, Y. Song, B. Kincaid, B. Bossy, E. Bossy-Wetzel, Mutant SOD1G93A triggers mitochondrial fragmentation in spinal cord motor neurons: neuroprotection by SIRT3 and PGC-1alpha, Neurobiol. Dis. 51 (2013) 72–81. [27] E. McDonnell, B.S. Peterson, H.M. Bomze, M.D. Hirschey, SIRT3 regulates progression and development of diseases of aging, Trends Endocrinol. Metab.: TEM 26 (9) (2015) 486–492. [28] A.S. Bause, M.C. Haigis, SIRT3 regulation of mitochondrial oxidative stress, Exp. Gerontol. 48 (7) (2013) 634–639. [29] V.B. Pillai, N.R. Sundaresan, G. Kim, M. Gupta, S.B. Rajamohan, J.B. Pillai, S. Samant, P.V. Ravindra, A. Isbatan, M.P. Gupta, Exogenous, NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-amp-activated kinase pathway, J. Biol. Chem. 285 (5) (2010) 3133–3144. [30] N.R. Sundaresan, M. Gupta, G. Kim, S.B. Rajamohan, A. Isbatan, M.P. Gupta, Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice, J. Clin. Investig. 119 (9) (2009) 2758–2771. [31] C. Zhu, X. Wang, F. Xu, B.A. Bahr, M. Shibata, Y. Uchiyama, H. Hagberg, K. Blomgren, The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia, Cell death Differ. 12 (2) (2005) 162–176. [32] A. Rami, A. Langhagen, S. Steiger, Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death, Neurobiol. Dis. 29 (1) (2008) 132–141. [33] S. Carloni, G. Buonocore, M. Longini, F. Proietti, W. Balduini, Inhibition of rapamycin-induced autophagy causes necrotic cell death associated with Bax/Bad mitochondrial translocation, Neuroscience 203 (2012) 160–169. [34] X. Wei, Z. Zhou, L. Li, J. Gu, C. Wang, F. Xu, Q. Dong, X. Zhou, Intrathecal injection of 3-Methyladenine reduces neuronal damage and promotes functional recovery via autophagy attenuation after spinal cord ischemia/reperfusion injury in rats, Biol. Pharm. Bull. 39 (5) (2016) 665–673. [35] J.Y. Wang, Q. Xia, K.T. Chu, J. Pan, L.N. Sun, B. Zeng, Y.J. Zhu, Q. Wang, K. Wang, B.Y. Luo, Severe global cerebral ischemia-induced programmed necrosis of hippocampal CA1 neurons in rat is prevented by 3-methyladenine: a widely used inhibitor of autophagy, J. Neuropathol. Exp. Neurol. 70 (4) (2011) 314–322. [36] A. Banreti, M. Sass, Y. Graba, The emerging role of acetylation in the regulation of
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Original article
Sirt3 confers protection against neuronal ischemia by inducing autophagy: Involvement of the AMPK-mTOR pathway
MARK
Shu-Hui Daia,1, Tao Chena,b,c,1, Xia Lia,1, Kang-Yi Yuea, Peng Luoa, Li-Kun Yangb, Jie Zhub, ⁎ ⁎ Yu-Hai Wangb, Zhou Feia, , Xiao-Fan Jianga, a b c
Department of Neurosurgery, Xijing Institute of Clinical Neuroscience, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi 710032, China Department of Neurosurgery, The 101th Hospital of PLA, Rescue Center of Craniocerebral Injuries of PLA, Wuxi, Jiangsu 214044, China Department of Neurosurgery, The 123th Hospital of PLA, Bengbu, Anhui 233000, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Ischemic stroke Sirt3 Autophagy AMPK mTOR
Sirtuin3 (Sirt3) is a member of the silent information regulator 2 (Sir2) family of proteins located in mitochondria that influences almost every major aspect of mitochondrial biology, including ATP generation and reactive oxygen species (ROS) production. Our previous study showed that Sirt3 exerts protective effects against oxidative stress in neuronal cells. In this study, we investigated the role of Sirt3 in neuronal ischemia using an oxygen and glucose deprivation (OGD) model. Sirt3 was up-regulated by OGD and overexpression of Sirt3 through lentivirus transfection significantly reduced OGD-induced lactate dehydrogenase (LDH) release and neuronal apoptosis. These effects were accompanied by reduced hydrogen dioxide (H2O2) production, enhanced ATP generation and preserved mitochondrial membrane potential (MMP). The results of immunocytochemistry and electron microscopy showed that Sirt3 increased autophagy in OGD-injured neurons, which was also confirmed by the increased expression of Beclin-1 as well as LC3-I to LC3-II conversion. In addition, the autophagy inhibitor 3-MA and bafilomycin A1 partially prevented the effects of Sirt3 on LDH release and apoptosis after OGD. The results of western blotting showed that overexpression of Sirt3 in cortical neurons markedly increased the phosphorylation of AMPK, whereas the phosphor-mTOR (p-mTOR) levels decreased both in the presence and absence of OGD insult. Furthermore, pre-treatment with the AMPK inhibitor compound C partially reversed the protective effects of Sirt3. Taken together, these findings demonstrate that Sirt3 protects against OGD insult by inducing autophagy through regulation of the AMPK-mTOR pathway and that Sirt3 may have therapeutic value for protecting neurons from cerebral ischemia.
1. Introduction Ischemic stroke induced by hypoxic ischemic encephalopathy or cerebrovascular accident is still one of the leading causes of death and permanent disability in the world [1]. Although ischemic stroke has been demonstrated to be associated with many devastating cascades, such as intracellular calcium overload, reactive oxygen species (ROS) generation, excitotoxicity mediated apoptosis and inflammation related necrosis, the exact molecular mechanism underlying ischemic neuronal injury has not been fully elucidated [2–4]. The sirtuins (or Sir2-like proteins) comprise a family of NAD+dependent protein deacetylases and ADP-ribosyltransferases that belong to class III histone deacetylases (HDACs) [5]. Mammals express seven homologues of sirtuins, Sirt1-7. Of those homologues, Sirt3 resides primarily in the mitochondria and has been identified as a
⁎
1
responsive deacetylase that regulates metabolism and oxidative stress [6]. Several studies have shown that Sirt3 plays an important role in regulating cell defence and mediating cell survival [7–9]. Our previous study showed that Sirt3 attenuates hydrogen peroxide-induced oxidative stress through preservation of mitochondrial function in HT22 cells [10]. We also found that overexpression of Sirt3 by lentivirus transfection protects cortical neurons against oxidative stress through regulating mitochondrial Ca2+ and mitochondrial biogenesis [11]. In addition, Sirt3 was shown to act as a pro-survival factor that plays an essential role in protecting neurons experiencing excitotoxicity [12]. However, the exact role of Sirt3 in ischemic neuronal cell injury and the associated molecular mechanisms have not yet been investigated. Autophagy is an evolutionarily conserved process for the bulk degradation and recycling of cytosolic proteins and organelles [13]. Mammalian autophagy exists in three different forms: macroautophagy,
Corresponding authors. E-mail addresses: [email protected] (Z. Fei), [email protected] (X.-F. Jiang). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.005 Received 30 August 2016; Received in revised form 1 April 2017; Accepted 4 April 2017 Available online 07 April 2017 0891-5849/ © 2017 Elsevier Inc. All rights reserved.
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the recombinant Lentivirus LV-Sirt3, 293 T cells were co-transfected with the pGC-FU plasmid (20 μg) with a cDNA encoding Sirt3, a pHelper 1.0 plasmid (15 μg) and a pHelper 2.0 plasmid (10 μg) using Lipofectamine 2000 (100 μl). The supernatant was harvested and the viral titre was calculated by transducing 293 T cells. As a control, we generated a lentiviral vector that expressed GFP alone (LV-control). Cortical neurons were transfected with lentivirus vectors for 72 h and subjected to various treatments.
microautophagy, and chaperone-mediated autophagy. These forms differ in their mechanisms and functions, and macroautophagy has been widely referred to as autophagy [14]. As a degradation/recirculation system, autophagy is believed to play an important role in pathological conditions in many organs, including brain ischemia [15,16]. Although the existence and activation of autophagy after ischemic stroke is undisputable, whether autophagy is a protective or detrimental mechanism for ischemic neuronal injury is still a topic of debate. Previous studies have shown that autophagy is one of several morphological features that occur during cell death after brain ischemia, and inhibition of autophagy exerts protective effects under hypoxic-exicitotoxic conditions [17,18]. In contrast, increasing amounts of evidence have indicated that autophagy may promote neuronal survival through removing damaged organelles to delay apoptosis or preserving energy to prevent necrosis, and the protective role of autophagy has also been demonstrated using chemical inhibitors or inducers under both in vitro and in vivo conditions [19,20]. In the present study, we measured the dynamic changes of Sirt3 expression after neuronal ischemia, and investigated the potential protective effects of Sirt3 against oxygen and glucose deprivation (OGD) in cortical neurons. We also determined the effect of Sirt3 overexpression on neuronal autophagy regulation, and confirmed the involvement of the AMPK-mTOR pathway in autophagy and Sirt3induced protection.
2.4. Oxygen and glucose deprivation (OGD) To initiate OGD, the culture medium was removed and rinsed with phosphate buffered saline (PBS) three times. The cultured neurons were placed into a specialized, humidified chamber containing 5% CO2, 95% N2 at 37 °C with glucose-free DMEM, which was pre-gassed with N2/ CO2 (95%/5%) to remove residual oxygen. After 2 h, the neurons were removed from the anaerobic chamber, and the culture medium was replaced with neurobasal medium containing 2% B27 supplement and 0.5 mM L-Glutamine. The neurons were maintained for 24 h at 37 °C in a humidified 5% CO2 incubator to generate the reperfusion insult. 2.5. Cytotoxicity assay Cytotoxicity was determined by measuring the release of LDH with a diagnostic kit according to the manufacturer's instructions. Briefly, 50 μl of supernatant from each well was collected to assay LDH release. The samples were incubated with a reduced form of nicotinamideadenine dinucleotide (NADH) and pyruvate for 15 min at 37 °C and the reaction was stopped by adding 0.4 M NaOH. The activity of LDH was calculated from the absorbance at 440 nm, and the background absorbance from culture medium that was not used for any cell cultures was subtracted from all of the absorbance measurements. The results are presented as fold increase of the control.
2. Materials and methods 2.1. Antibodies and reagents Antibodies against Sirt3, LC3, and Beclin-1 were obtained from Cell Signaling (Danvers, MA, USA). Antibodies against p-AMPK (Thr172), AMPK, p-mTOR (Ser2448), mTOR and β-actin were obtained from Santa Cruz (Dallas, TX, USA). The secondary antibodies, Rh123, and MitoSox Red were purchased from Sigma (St. Louis, MO, USA). Rapamycin, compound C, 3-MA and bafilomycin A1 were purchased from Tocris Bioscience (Bristol, UK). The TUNEL staining kit was obtained from Promega (Madison, WI, USA). The lactate dehydrogenase (LDH) assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China).
2.6. TUNEL staining Apoptosis in primary cortical neurons subjected to various treatments was detected using TUNEL staining, which is a method to observe DNA strand breaks in nuclei. For TUNEL staining, cortical neurons were seeded on 1.5-cm glass slides at a density of 3×105 cells/cm2. Twentyfour hours after OGD, the cells were fixed by immersing the slides in freshly prepared 4% methanol-free formaldehyde solution in PBS for 20 min at room temperature and permeabilized with 0.2% Triton X-100 for 5 min. Neurons were labelled with fluorescein TUNEL reagent mixture for 60 min at 37 °C according to the manufacturer's suggested protocol. Then, the slides were examined by fluorescence microscopy, and the TUNEL-positive (apoptotic) cells were counted. DAPI (10 μg/ ml) was used to stain nuclei.
2.2. Primary culture of cortical neurons All experimental protocols and animal handling procedures were performed according to the National Institutes of Health (NIH) guidelines for the use of experimental animals, and the study was approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University (No. 2014–81371447). Cortical neurons were cultured from Sprague-Dawley rats using a modified method. Briefly, cerebral cortices were removed from embryos at 16–18 days, stripped of meninges and blood vessels and minced. The tissues were dissociated through 0.25% trypsin digestion for 15 min at 37 °C and gentle trituration. Neurons were resuspended in neurobasal medium containing 2% B27 supplement and 0.5 mM L-Glutamine, and then the neurons were plated at a density of 3×105 cells/cm2. Before seeding, the culture vessels, consisting of 96-well plates, 1.5-cm glass slides or 6cm dishes were coated with PLL (50 μg/ml) at room temperature overnight. Neurons were maintained at 37 °C in a humidified 5% CO2 incubator and half of the culture medium was changed every other day.
2.7. Caspase-3 activity Briefly, after being harvested and lysed, 106 cells were mixed with 32 μl of assay buffer and 2 μl of 10 mM Ac-DEVD-pNA substrate. Absorbance at 405 nm was measured after incubation at 37 °C for 4 h. The absorbance of each sample was determined by subtraction of the mean absorbance of the blank and corrected by the protein concentration of the cell lysate.
2.3. Lentivirus construction and transfection
2.8. Measuring intracellular H2O2 by aminotriazole-mediated inactivation of catalase
The coding sequence of Sirt3 was amplified by RT-PCR. The primer sequences were forward, 5′-TACTTCCTTCGGCTGCTTCA-3′; reverse, 5′AAGGCGAAATCAGCCACA −3′. The PCR fragments and the pGC-FU plasmid (GeneChem, Shanghai, China) were digested with Age I and then ligated with T4 DNA ligase to produce pGC-FU-Sirt3. To generate
The intracellular steady-state levels of H2O2 were estimated using a sensitive assay based on 3-aminotriazole inhibition of catalase [21]. After various treatments, neurons were scrape-harvested and the protein concentrations were quantified with the BCA method. The assay was initiated through addition of 100 μl of a 30 mM H2O2 stock 346
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50 nm thickness, stained with lead citrate and then viewed on an electron microscope.
solution in phosphate buffer at pH 7.0, and the loss of absorbance at 240 nm at room temperature over a 2-min time period was monitored. The initial catalase activities were calculated by fitting experimental data to the first-order kinetics as described [22] and expressed as catalase k mU/mg cell protein. The H2O2 concentration was calculated by kinetic analysis of the rate of decrease of catalase activity using the equation: [H2O2]=kinactivation/k1, where kinactivation is the experimental pseudo first-order rate constant of catalase inactivation, and the value k1 is 1.7×107 M−1 S−1, which is the rate constant for the formation of catalase compound I in the presence of H2O2.
2.14. Western blot analysis Equivalent amounts of protein (40 μg per lane) were loaded and separated by 10% sodium dodecyl sulphate (SDS)-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk solution in tris-buffered saline with 0.1% Triton X-100 for 1 h, and then incubated overnight at 4 °C with primary antibodies diluted in TBST. After the incubation, the membranes were washed and incubated with secondary antibody for 1 h at room temperature. Immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Image J software was used to quantify the optical density of each band.
2.9. ROS-GLO H2O2 assay The ROS-GLO H2O2 assay uses a modified luciferin substrate, based on boronate oxidation, which reacts directly with H2O2 to generate a luciferin precursor. Upon addition of the detection reagent, the precursor is converted to luciferin, and the Ultra-Glo™ Recombinant Luciferase included in the detection reagent produces a light signal proportional to the level of H2O2 in the sample. Before OGD treatment, 20 μl of a mixture of H2O2 substrate and H2O2 dilution buffer was immediately added to the culture medium. After OGD exposure, 100 μl ROS-GLO™ Detection Solution was added to the neurons, and all samples were incubated at room temperature for 20 min. Luminescence was read during an integration time of 1000 ms.
2.15. Statistical analysis All of the experiments were performed a minimum of three times. The statistical evaluation was carried out with GraphPad Prism software, version 6.0. Significant differences between experiments were assessed by univariate ANOVA (more than two groups), followed by Bonferroni's multiple comparisons or an unpaired t-test (two groups).
2.10. Measurement of mitochondrial membrane potential (MMP)
3. Results
MMP was measured using the fluorescent dye Rh123 as reported previously [23]. Rh123 was added to the culture medium to achieve a final concentration of 10 μM for 30 min at 37 °C after the neurons were treated and washed with PBS. The fluorescence was observed using an Olympus BX60 microscope with the appropriate fluorescence filters (excitation wavelength of 480 nm and emission wavelength of 530 nm).
3.1. Sirt3 protects against neuronal ischemia in cortical neurons
Cultured neurons were homogenized in a mitochondria isolation buffer, and mitochondria were isolated through multiple centrifugation steps as detailed earlier [11]. Isolated mitochondria were used to measure ATP synthesis with a luciferase/ luciferin-based system, as described previously [24]. Thirty micrograms of mitochondria-enriched pellets was resuspended in 100 μl of buffer A (150 mM KCl, 25 mM TrisHCl, 2 mM potassium phosphate, 0.1 mM MgCl2, pH 7.4) with 0.1% BSA, 1 mM malate, 1 mM glutamate and buffer B (containing 0.8 mM luciferin and 20 mg/ml luciferase in 0.5 M Tris-acetate, pH 7.75). The reaction was initiated by addition of 0.1 mM ADP and monitored for 120 min using a microplate reader.
To investigate the effects of ischemia on Sirt3, cortical neurons were treated with OGD for 2 h, and the levels of Sirt3 protein at different time points were determined by western blot. The results showed that OGD significantly increased the levels of Sirt3 protein at 6, 12, 24 and 48 h after OGD (Fig. 1A). Lentiviral transduction was used to determine the effects of Sirt3 on neuronal survival and death after OGD, and the results of western blotting showed that transfection with a Sirt3targeted lentivirus (LV-Sirt3) significantly increased the levels of Sirt3 protein (Fig. 1B). Representative phase photomicrographs showed that LV-Sirt3-transduced neurons exhibited lower cell death relative to LVControl-transduced cultures (Fig. 1C). As shown in Fig. 1D, we observed a similar effect using the LDH release assay, as demonstrated by the reduced LDH release after Sirt3 overexpression. In addition, TUNEL staining was used to detect apoptotic cell death in our in vitro model (Fig. 1E). The results showed that Sirt3 overexpression obviously decreased the number of TUNEL-positive cells after OGD insult (Fig. 1F). As shown in Fig. 1G, a similar result for caspase-3 activity was also observed.
2.12. Immunocytochemistry (ICC)
3.2. Sirt3 preserves mitochondrial function and cellular bioenergetics
The neurons were fixed for 30 min with 4% paraformaldehyde, rinsed twice with PBS and subsequently incubated with 1% hydrogen peroxide for 10 min. Following two PBS rinses, the cells were incubated with blocking solution for 20 min and incubated with a primary antiLC3 antibody (1:50) at 4 °C overnight. The cells were then rinsed twice with PBS and incubated with fluorescein isothiocyanate (FITC) labelled secondary antibody (1:500) for 1 h at room temperature. Coverslips were mounted in mounting medium and visualized using a fluorescence microscope.
We also determined whether Sirt3-induced protection against OGD was associated with decreased oxidative stress in vitro. The intracellular steady-state levels of H2O2 were estimated using a sensitive assay based on 3-aminotriazole inhibition of catalase. There was a significant increase in the steady-state level of intracellular H2O2 after OGD injury. As expected, Sirt3 overexpression profoundly attenuated the intracellular H2O2 steady state levels produced by OGD (Fig. 2A). As shown in Fig. 2B, a similar result for total H2O2 production, which was measured using a ROS-GLO probe, was also observed. Furthermore, to confirm the effects of Sirt3 on mitochondrial function, we measured mitochondrial O.-2 levels using MitoSOX Red fluorescence (Fig. 2C). Consistent with the data we collected above, increased mitochondrial O.-2 levels were clearly observed following OGD treatment, which was partially prevented by Sirt3 overexpression. In addition, ATP levels were significantly greater in the homogenates of cortical neurons transfected with LV-Sirt3 compared to control neurons after OGD (Fig. 2E). To further
2.11. Measurement of ATP synthesis
2.13. Electron microscopy The neurons were harvested and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at room temperature. The cell pellets were postfixed with 1% osmium tetroxide, dehydrated through acetone and then immersed in resin. After hardening, the blocks were thinly sectioned at 347
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Fig. 1. Sirt3 protects against neuronal ischemia in cortical neurons. Cortical neurons were treated with OGD for 2 h, and the expression of Sirt3 at different time points (3, 6, 12, 24 or 48 h) was determined by western blot (A). Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. The expression of Sirt3 was detected by western blot at 24 h after OGD (B). Representative phase photomicrographs are shown (C), and neuronal cell death was quantified by an LDH release assay (D). Apoptotic cell death was detected by TUNEL staining (E), and the number of TUNEL positive cells was counted (F). The activity of caspase-3 was measured with a colorimetric assay kit (G). The data are represented as means ± SEM. #p < 0.05, *p < 0.05 vs. Control, & p < 0.05 vs. OGD+LV-Control.
and absence of OGD (Fig. 3E). As shown in Fig. 3E, there was a similar result for Beclin-1 expression.
determine the roles of Sirt3 in mitochondrial function and stress resistance after OGD, the fluorescence of Rh123 was used to identify changes in MMP levels. The results demonstrated that there was an obvious decrease in MMP after OGD exposure, which was partially prevented by Sirt3 overexpression (Fig. 2F).
3.4. Autophagy contributes to the neuroprotective effects of Sirt3 Cortical neurons were treated with the autophagy inducer rapamycin to determine the role of autophagy in our in vitro ischemia model. The results of the western blotting showed that the increased conversion of LC3-I to LC3-II induced by OGD was further enhanced by rapamycin treatment (Fig. 4A). Treatment with rapamycin significantly decreased LDH release (Fig. 4B) and reduced the number of TUNEL positive cells (Fig. 4C) after OGD. To determine whether autophagy was involved in Sirt3-induced protection, cortical neurons were transfected with LV-Sirt3 and/or treated with the autophagy inhibitor 3-MA. As shown in Fig. 4D, the increased conversion of LC3-I to LC3-II induced by Sirt3 overexpression was significantly decreased by 3-MA. Moreover, treatment with 3-MA or bafilomycin A1, another autophagy inhibitor, partially prevented the decrease in LDH release (Fig. 4E) and led to a decrease in the number of TUNEL positive cells (Fig. 4F) after OGD.
3.3. Sirt3 regulates autophagy following neuronal ischemia Next, we tested whether Sirt3 regulates autophagy in our in vitro model. The number of LC3-positive puncta per neuron was determined by using immunohistochemistry to measure autophagy 6 h after OGD (Fig. 3A). There were no LC3-positive puncta in control neurons, and overexpression of Sirt3 significantly increased the number of LC3positive puncta after OGD compared to the LV-Control group (Fig. 3B). We also performed electron microscopy to examine the autophagosomes in neurons (Fig. 3C). Compared to the LV-Control group, there were more autophagosomes in the LV-Sirt3 group (Fig. 3D). In addition, as a marker of autophagy, the LC3-I to LC3-II conversion was significantly increased by LV-Sirt3 transfection, both in the presence 348
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Fig. 2. Sirt3 preserves mitochondrial function and cellular bioenergetics. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. The intracellular steady-state levels of H2O2 (A) and total H2O2 production (B) were measured. The mitochondrial O.-2 levels were evaluated by imaging of MitoSOX Red fluorescence (C and D). Scale bar =50 µm. The total ATP levels (E) were measured after OGD, and MMP levels was analyzed by Rh123 staining (F). The data are represented as means ± SEM. *p < 0.05 vs. OGD+LV-Control.
Fig. 3. Sirt3 regulates autophagy following neuronal ischemia. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. LC3-positive puncta in neurons were detected by fluorescent staining (A), and the number of LC3 puncta (arrows) in neurons were calculated (B). Scale bar =10 µm. Neuronal autophagy was observed by electron microscopy (C), and the number of autophagosomes (red arrows) in neurons was calculated (D). Scale bar =100 nm. N: nucleus. The expression of LC3 and Beclin-1 was detected by western blot (E). The data are represented as means ± SEM. #p < 0.05 vs. Control, *p < 0.05 vs. OGD+LV-Control.
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Fig. 4. Autophagy contributes to the neuroprotective effect of Sirt3. Cortical neurons were treated with 1 μM rapamycin and exposed to OGD for 2 h. The expression of LC3 was detected by western blot (A). Neuronal cell death was quantified with an LDH release assay (B), and apoptotic cell death was detected by TUNEL staining (C). Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h with or without 10 mM 3-MA or 5 μM bafilomycin A1 and exposed to OGD for 2 h. The expression of LC3 was detected by western blot (D). Neuronal cell death was quantified by an LDH release assay (E), and apoptotic cell death was detected by TUNEL staining (F). The data are represented as means ± SEM. #p < 0.05 vs. Control, * p < 0.05 vs. OGD, & p < 0.05 vs. OGD + LV-Control, #p < 0.05 vs. OGD+LV-Sirt3.
studies [11,25,26]. In the current study, we revealed that Sirt3 confers protection against ischemic neuronal injury by regulating autophagy. First, the upregulation of Sirt3 attenuated the neuronal injury induced by OGD through reducing apoptosis. Second, the beneficial effects on neuronal survival induced by Sirt3 were associated with activation of autophagy in cortical neurons. Third, the discovery that altered activation of the AMPK-mTOR pathway was responsible for much of the regulation of autophagy in our in vitro model demonstrated that AMPK-mTOR was involved in Sirt3-induced neuroprotection. As a member of the sirtuins family of proteins, Sirt3 is expressed at high levels in the brain and other nervous system tissues [27]. It resides primarily in the mitochondria and influences almost every major aspect of mitochondrial function, including ATP generation, nutrient oxidation, ROS production and mitochondrial dynamics [28]. Previous studies have shown that Sirt3 was dynamically regulated by cellular stress factors at both the transcriptional and translational levels under pathological conditions. Increased expression of Sirt3 was observed in hearts under stress conditions, whereas Sirt3 levels were reduced in hypertrophied or failing hearts [29,30]. Our previous studies showed that Sirt3 was upregulated by hydrogen peroxide treatment and that overexpression of Sirt3 protected HT22 cells and cortical neurons against oxidative stress [10,11]. More recently, Cheng et al. found that exercise and synaptic activity induced hippocampal Sirt3 expression to modulate mitochondrial protein acetylation and bolster neuronal resistance to oxidative stress and apoptosis [25]. In this study, our results showed that the levels of Sirt3 protein increased after OGD treatment in a time-dependent manner in primary culture cortical
3.5. Involvement of the AMPK-mTOR pathway in Sirt3-induced protection To further elucidate the molecular mechanisms through which Sirt3 mediates autophagy and protection, we detected the phosphorylation of AMPK and mTOR after Sirt3 overexpression using western blotting. The results showed that in the OGD group, the phosphorylation of AMPK increased, while the expression of p-mTOR was reduced compared to the control group (Fig. 5A and B). Overexpression of Sirt3 significantly increased the phosphorylation of AMPK but decreased the phosphorylation of mTOR both in the presence and absence of OGD injury. In addition, the increased expression of p-AMPK induced by Sirt3 was partially reversed by the AMPK inhibitor compound C, but not by the mTOR inhibitor rapamycin (Fig. 5C). As shown in Fig. 5D, both compound C and rapamycin significantly increased the phosphorylation of mTOR compared to LV-Sirt3-transfected neurons. The results of the western blot showed that the increased expression of Beclin-1 and conversion of LC3-I to LC3-II induced by Sirt3 overexpression were abolished by compound C treatment (Fig. 5E). Consistent with those results, Sirt3-induced deceases in LDH release (Fig. 5F) and neuronal apoptosis (Fig. 5G) were both partially nullified by compound C, which indicated that the AMPK-mTOR pathway was involved in Sirt3-induced protection. 4. Discussion The involvement of Sirt3 in adaptive responses of neuronal cells to stress conditions has been well established in several previous in vitro 350
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Fig. 5. Involvement of the AMPK-mTOR pathway in Sirt3-induced protection. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h and exposed to OGD for 2 h. The expression and phosphorylation of AMPK (A) and mTOR (B) were detected by western blot. Cortical neurons were transfected with LV-Control or LV-Sirt3 for 72 h with or without 1 μM rapamycin or 5 μM compound C and exposed to OGD for 2 h. The expression and phosphorylation of AMPK (C) and mTOR (D) were detected by western blot. Cortical neurons were transfected with LV-Sirt3 with or without 5 μM compound C for 72 h, and the expression of LC3 and Beclin-1 was detected by western blot (E). Cortical neurons were transfected with LVSirt3 with or without 5 μM of compound C for 72 h and exposed to OGD for 2 h. Neuronal cell death was quantified by an LDH release assay (F), and apoptotic cell death was detected by TUNEL staining (G). *p < 0.05.
involved in mitochondrial energy production. Thus, how autophagy is regulated by Sirt3 knockdown or overexpression is of great interest to researchers. Sirt3 activation attenuated oxidized low-density lipoprotein-induced apoptosis through sustaining autophagy in human umbilical vein endothelial cells [38]. A more recent study found that Sirt3SOD2-mROS-dependent autophagy was involved in melatonin-induced protection against cadmium-induced hepatotoxicity [39]. In this study, we found that Sirt3 overexpression significantly increased the number of LC3-positive puncta and increased the expression of LC3, the level of which was shown to be proportional to the abundance of autophagosomes [40]. In addition, Sirt3-overexpressing neurons exhibited a lower level of mitochondrial O.-2 and preserved cellular bioenergetics. Previous studies have demonstrated that Sirt3 could directly bind and deacetylate SOD2, which in turn led to significant effects on mitochondrial ROS homeostasis and autophagic flux [41,42]. Thus, our results strongly indicated that Sirt3 protect against OGD-induced neuronal injury and mitochondrial dysfunction by inducing autophagy which was also confirmed by the reversed effects of 3-MA and bafilomycin A1 in Srit3-induced protection. Sirt3 is involved in suppressing the onset of many neurological diseases, but the mechanism through which it regulates neuronal autophagy has yet to be determined. As for one of the clues, our results from western blotting showed that Sirt3 overexpression increased the phosphorylation of AMPK, which is a sensor of cellular energy status. AMPK serves as a regulator of cell survival or death in response to multiple pathological stresses, including hypoxia, ischemia and oxidative stress [43]. Previous studies showed that Sirt3 maintains bone homeostasis by regulating AMPK-PGC-1β axis in mice, and activation of acetyl-CoA synthetase 2 (AceCS2) by Sirt3 may elevate the AMP/ATP ratio and consequently activate AMPK [44–46]. As expected, the AMPK inhibitor compound C partially abolished the Sirt3-induced protective effects in our in vitro model. It is well known that activated AMPK
neurons. Overexpression of Sirt3 significantly increased neuronal survival and decreased neuronal apoptosis after OGD, which strongly suggested that Sirt3 might be an endogenous protective mechanism under neuronal ischemia conditions. Autophagy is an evolutionarily conserved process that degrades and recycles proteins and organelles through an intracellular bulk degradation pathway. It is well accepted that autophagy is rapidly activated in neurons exposed to hypoxic stimuli and focal cerebral ischemia [31,32]. However, whether enhanced autophagy is part of a death or survival mechanism is still a matter of debate [13]. It may aid cell death through excessive self-digestion and degradation of essential cellular constituents, but increasing amounts of evidence suggests that autophagy may promote cell survival by removing toxic metabolites and intracellular pathogens. In our in vitro model, moderate protection, as demonstrated by the decreased LDH release and reduced number of TUNEL positive cells, was found in rapamycin-treated neurons after OGD exposure, which was consistent with previous data showed that rapamycin reduced apoptotic cell death by decreasing the activation of the intrinsic apoptotic mitochondrial pathway [33]. All of these results provide further evidence that autophagy is beneficial for neurons injured by OGD. In contrast, recent studies showed that treatment with 3MA protected against brain and spinal cord ischemia in in vivo models [34,35]. The differences between our results and previous studies might be explained by the different models and time points as well as the in vitro conditions. The sirtuins (Sirt1-7) are a conserved class of protein deacylases, and lysine acetylation has recently emerged as an important posttranslational modification that is employed to regulate autophagy [36]. A previous study showed that overexpression of Sirt1 stimulated autophagy while Sirt1 deficiency attenuated autophagy under starvation conditions [37]. Among the seven sirtuins members, Sirt3 resides primarily in the mitochondria and it directs biological functions 351
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Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81371447, No. 81671303, No. 81430043, No. 81301037) and Shaanxi Province Natural Science Foundation Research Program (No. 2014JM4131). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2017.04. 005. References [1] D. Mozaffarian, E.J. Benjamin, A.S. Go, D.K. Arnett, M.J. Blaha, M. Cushman, S.R. Das, S. de Ferranti, J.P. Despres, H.J. Fullerton, V.J. Howard, M.D. Huffman, C.R. Isasi, M.C. Jimenez, S.E. Judd, B.M. Kissela, J.H. Lichtman, L.D. Lisabeth, S. Liu, R.H. Mackey, D.J. Magid, D.K. McGuire, E.R. Mohler 3rd, C.S. Moy, P. Muntner, M.E. Mussolino, K. Nasir, R.W. Neumar, G. Nichol, L. Palaniappan, D.K. Pandey, M.J. Reeves, C.J. Rodriguez, W. Rosamond, P.D. Sorlie, J. Stein, A. Towfighi, T.N. Turan, S.S. Virani, D. Woo, R.W. Yeh, M.B. Turner, Heart Disease and Stroke Statistics-2016 Update: a Report From the American Heart Association, Circulation 133 (4) (2016) e38–e360. [2] R. Jin, G. Yang, G. Li, Inflammatory mechanisms in ischemic stroke: role of inflammatory cells, J. Leukoc. Biol. 87 (5) (2010) 779–789. [3] T. Chen, W. Liu, X. Chao, Y. Qu, L. Zhang, P. Luo, K. Xie, J. Huo, Z. Fei, Neuroprotective effect of osthole against oxygen and glucose deprivation in rat cortical neurons: involvement of mitogen-activated protein kinase pathway, Neuroscience 183 (2011) 203–211. [4] R.S. Pandya, L. Mao, H. Zhou, S. Zhou, J. Zeng, A.J. Popp, X. Wang, Central nervous system agents for ischemic stroke: neuroprotection mechanisms, Cent. Nerv. Syst. Agents Med. Chem. 11 (2) (2011) 81–97. [5] R.N. Dutnall, L. Pillus, Deciphering NAD-dependent deacetylases, Cell 105 (2) (2001) 161–164. [6] L. Jin, H. Galonek, K. Israelian, W. Choy, M. Morrison, Y. Xia, X. Wang, Y. Xu, Y. Yang, J.J. Smith, E. Hoffmann, D.P. Carney, R.B. Perni, M.R. Jirousek, J.E. Bemis, J.C. Milne, D.A. Sinclair, C.H. Westphal, Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3, Protein Sci.: a Publ. Protein Soc. 18 (3) (2009) 514–525. [7] C.J. Chen, Y.C. Fu, W. Yu, W. Wang, SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-kappaB, Biochem. Biophys. Res. Commun. 430 (2) (2013) 798–803. [8] Y. Kawamura, Y. Uchijima, N. Horike, K. Tonami, K. Nishiyama, T. Amano, T. Asano, Y. Kurihara, H. Kurihara, Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest, J. Clin. Investig. 120 (8) (2010) 2817–2828. [9] Y. Chen, J. Zhang, Y. Lin, Q. Lei, K.L. Guan, S. Zhao, Y. Xiong, Tumour suppressor SIRT3 deacetylates and activates manganese superoxide dismutase to scavenge ROS, EMBO Rep. 12 (6) (2011) 534–541. [10] S.H. Dai, T. Chen, Y.H. Wang, J. Zhu, P. Luo, W. Rao, Y.F. Yang, Z. Fei, X.F. Jiang, Sirt3 attenuates hydrogen peroxide-induced oxidative stress through the preservation of mitochondrial function in HT22 cells, Int. J. Mol. Med. 34 (4) (2014) 1159–1168. [11] S.H. Dai, T. Chen, Y.H. Wang, J. Zhu, P. Luo, W. Rao, Y.F. Yang, Z. Fei, X.F. Jiang, Sirt3 protects cortical neurons against oxidative stress via regulating mitochondrial Ca2+ and mitochondrial biogenesis, Int. J. Mol. Sci. 15 (8) (2014) 14591–14609. [12] S.H. Kim, H.F. Lu, C.C. Alano, Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture, PloS One 6 (3) (2011) e14731. [13] W. Balduini, S. Carloni, G. Buonocore, Autophagy in hypoxia-ischemia induced brain injury: evidence and speculations, Autophagy 5 (2) (2009) 221–223. [14] W. Balduini, S. Carloni, G. Buonocore, Autophagy in hypoxia-ischemia induced brain injury, J. Matern.-Fetal Neonatal Med.: Off. J. Eur. Assoc. Perinat. Med., Fed. Asia Ocean. Perinat. Soc., Int. Soc. Perinat. Obstet. 25 (Suppl 1) (2012) 30–34. [15] C. Huang, S. Yitzhaki, C.N. Perry, W. Liu, Z. Giricz, R.M. Mentzer Jr., R.A. Gottlieb, Autophagy induced by ischemic preconditioning is essential for cardioprotection, J. Cardiovasc. Transl. Res. 3 (4) (2010) 365–373. [16] D.D. Esposti, M.C. Domart, M. Sebagh, F. Harper, G. Pierron, C. Brenner, A. Lemoine, Autophagy is induced by ischemic preconditioning in human livers formerly treated by chemotherapy to limit necrosis, Autophagy 6 (1) (2010) 172–174. [17] V. Ginet, A. Spiehlmann, C. Rummel, N. Rudinskiy, Y. Grishchuk, R. Luthi-Carter, P.G. Clarke, A.C. Truttmann, J. Puyal, Involvement of autophagy in hypoxicexcitotoxic neuronal death, Autophagy 10 (5) (2014) 846–860. [18] F. Adhami, G. Liao, Y.M. Morozov, A. Schloemer, V.J. Schmithorst, J.N. Lorenz, R.S. Dunn, C.V. Vorhees, M. Wills-Karp, J.L. Degen, R.J. Davis, N. Mizushima, P. Rakic, B.J. Dardzinski, S.K. Holland, F.R. Sharp, C.Y. Kuan, Cerebral ischemiahypoxia induces intravascular coagulation and autophagy, Am. J. Pathol. 169 (2) (2006) 566–583. [19] K. Wei, P. Wang, C.Y. Miao, A double-edged sword with therapeutic potential: an updated role of autophagy in ischemic cerebral injury, CNS Neurosci. Ther. 18 (11)
Fig. 6. A proposed diagram tying together the observations involved in Sirt3-induced neuroprotection against OGD. OGD increased Sirt3 protein levels, which might be associated with PGC-1-mediated transcription. Sirt3 increased phosphorylation of AMPK, which in turn inhibited mitochondrial dysfunction through regulating MMP, ROS generation and ATP production. In addition, Sirt3 inhibited phosphorylation of mTOR, which led to increased expression of LC3, Beclin-1, and formation of autophagosomes.
directly phosphorylates PGC-1α, which is required for Sirt3-mediated mitochondrial biogenesis [47,48]. Thus, the activation of AMPK might cause an increase in the cellular NAD+/NADH ratio in OGD-injured neurons, which provides the positive feedback loop needed for prolonged activation of Sirt3 (as shown in Fig. 1A). In addition, decreased activation of mTOR was also observed after Sirt3 overexpression. mTOR is a serine/threonine kinase that promotes anabolic metabolism and inhibits autophagy induction [49]. There are mTOR inhibitors already in clinical trials or that have been approved for the treatment of diseases associated with autophagy defects [50,51]. Accumulating evidence has shown that AMPK activates autophagy via inhibition of mTOR [52,53], which was an effect we also demonstrated in our present study. Intriguingly, our results showed that the Sirt3-induced increase in p-AMPK was partially prevented by compound C but not rapamycin, which could form a complex with FKB12 to bind to mTOR and allosterically inhibit its kinase activity [54]. These data suggested that mTOR was one of the downstream signaling cascades involved in Sirt3-induced regulation of AMPK and autophagy. One limitation of this study was that the protective effect of Sirt3 against ischemia was only investigated in cultured cortical neurons. In this in vitro condition, the cellular microenvironment is missing and neuronal and glial cells associated signaling cascades cannot be completely mimicked. More experiments in cerebral tissues or in in vivo conditions, such as middle cerebral artery occlusion (MCAO) models, need to be done to confirm these observations in the future. In conclusion, we propose an intriguing mechanism whereby Sirt3 protects against OGD-induced neuronal injury via activation of autophagy. Importantly, Sirt3 showed a compensatory and protective role in eliminating intracellular H2O2, attenuating mitochondrial O.-2 and promoting autophagy through the AMPK-mTOR pathway in vitro (Fig. 6). Taken together, these findings provide new insights about the link between Sirt3 and autophagy signaling in neurons under ischemic conditions.
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autophagy, Autophagy 9 (6) (2013) 819–829. [37] I.H. Lee, L. Cao, R. Mostoslavsky, D.B. Lombard, J. Liu, N.E. Bruns, M. Tsokos, F.W. Alt, T. Finkel, A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy, Proc. Natl. Acad. Sci. USA 105 (9) (2008) 3374–3379. [38] X. Luo, Z. Yang, S. Zheng, Y. Cao, Y. Wu, Sirt3 activation attenuated oxidized lowdensity lipoprotein-induced human umbilical vein endothelial cells' apoptosis by sustaining autophagy, Cell Biol. Int. (2014). [39] H. Pi, S. Xu, R.J. Reiter, P. Guo, L. Zhang, Y. Li, M. Li, Z. Cao, L. Tian, J. Xie, R. Zhang, M. He, Y. Lu, C. Liu, W. Duan, Z. Yu, Z. Zhou, SIRT3-SOD2-mROSdependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin, Autophagy 11 (7) (2015) 1037–1051. [40] Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (21) (2000) 5720–5728. [41] X. Qiu, K. Brown, M.D. Hirschey, E. Verdin, D. Chen, Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation, Cell Metab. 12 (6) (2010) 662–667. [42] Q. Liang, G.A. Benavides, A. Vassilopoulos, D. Gius, V. Darley-Usmar, J. Zhang, Bioenergetic and autophagic control by Sirt3 in response to nutrient deprivation in mouse embryonic fibroblasts, Biochem. J. 454 (2) (2013) 249–257. [43] G.V. Ronnett, S. Ramamurthy, A.M. Kleman, L.E. Landree, S. Aja, AMPK in the brain: its roles in energy balance and neuroprotection, J. Neurochem. 109 (Suppl 1) (2009) 17–23. [44] J.E. Huh, J.H. Shin, E.S. Jang, S.J. Park, D.R. Park, R. Ko, D.H. Seo, H.S. Kim, S.H. Lee, Y. Choi, S.Y. Lee, Sirtuin 3 (SIRT3) maintains bone homeostasis by regulating AMPK-PGC-1beta axis in mice, Sci. Rep. 6 (2016) 22511. [45] W.C. Hallows, S. Lee, J.M. Denu, Sirtuins deacetylate and activate mammalian acetyl-coa synthetases, Proc. Natl. Acad. Sci. USA 103 (27) (2006) 10230–10235. [46] B.J. North, D.A. Sinclair, Sirtuins: a conserved key unlocking AceCS activity, Trends Biochem. Sci. 32 (1) (2007) 1–4. [47] B. Schwer, J. Bunkenborg, R.O. Verdin, J.S. Andersen, E. Verdin, Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2, Proc. Natl. Acad. Sci. USA 103 (27) (2006) 10224–10229. [48] 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 (4) (2003) 809–813. [49] Y.C. Kim, K.L. Guan, mTOR: a pharmacologic target for autophagy regulation, J. Clin. Investig. 125 (1) (2015) 25–32. [50] S.A. Wander, B.T. Hennessy, J.M. Slingerland, , Next-generation mTOR inhibitors in clinical oncology: how pathway complexity informs therapeutic strategy, J. Clin. Investig. 121 (4) (2011) 1231–1241. [51] D.W. Lamming, L. Ye, D.M. Sabatini, J.A. Baur, Rapalogs and mTOR inhibitors as anti-aging therapeutics, J. Clin. Investig. 123 (3) (2013) 980–989. [52] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2) (2011) 132–141. [53] C.S. Yang, J.J. Kim, H.M. Lee, H.S. Jin, S.H. Lee, J.H. Park, S.J. Kim, J.M. Kim, Y.M. Han, M.S. Lee, G.R. Kweon, M. Shong, E.K. Jo, The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy, Autophagy 10 (5) (2014) 785–802. [54] M. Wong, Mammalian target of rapamycin (mTOR) pathways in neurological diseases, Biomed. J. 36 (2) (2013) 40–50.
(2012) 879–886. [20] S. Carloni, G. Buonocore, W. Balduini, Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury, Neurobiol. Dis. 32 (3) (2008) 329–339. [21] Q. Tong, Y. Zhu, J.W. Galaske, E.A. Kosmacek, A. Chatterjee, B.C. Dickinson, R.E. Oberley-Deegan, MnTE-2-PyP modulates thiol oxidation in a hydrogen peroxide-mediated manner in a human prostate cancer cell, Free Radic. Biol. Med. 101 (2016) 32–43. [22] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. [23] T. Chen, F. Fei, X.F. Jiang, L. Zhang, Y. Qu, K. Huo, Z. Fei, Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons, Free Radic. Biol. Med. 52 (1) (2012) 208–217. [24] P.A. Parone, S. Da Cruz, J.S. Han, M. McAlonis-Downes, A.P. Vetto, S.K. Lee, E. Tseng, D.W. Cleveland, Enhancing mitochondrial calcium buffering capacity reduces aggregation of misfolded SOD1 and motor neuron cell death without extending survival in mouse models of inherited amyotrophic lateral sclerosis, J. Neurosci.: Off. J. Soc. Neurosci. 33 (11) (2013) 4657–4671. [25] A. Cheng, Y. Yang, Y. Zhou, C. Maharana, D. Lu, W. Peng, Y. Liu, R. Wan, K. Marosi, M. Misiak, V.A. Bohr, M.P. Mattson, Mitochondrial SIRT3 Mediates Adaptive Responses of Neurons to Exercise and Metabolic and Excitatory Challenges, Cell Metab. 23 (1) (2016) 128–142. [26] W. Song, Y. Song, B. Kincaid, B. Bossy, E. Bossy-Wetzel, Mutant SOD1G93A triggers mitochondrial fragmentation in spinal cord motor neurons: neuroprotection by SIRT3 and PGC-1alpha, Neurobiol. Dis. 51 (2013) 72–81. [27] E. McDonnell, B.S. Peterson, H.M. Bomze, M.D. Hirschey, SIRT3 regulates progression and development of diseases of aging, Trends Endocrinol. Metab.: TEM 26 (9) (2015) 486–492. [28] A.S. Bause, M.C. Haigis, SIRT3 regulation of mitochondrial oxidative stress, Exp. Gerontol. 48 (7) (2013) 634–639. [29] V.B. Pillai, N.R. Sundaresan, G. Kim, M. Gupta, S.B. Rajamohan, J.B. Pillai, S. Samant, P.V. Ravindra, A. Isbatan, M.P. Gupta, Exogenous, NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-amp-activated kinase pathway, J. Biol. Chem. 285 (5) (2010) 3133–3144. [30] N.R. Sundaresan, M. Gupta, G. Kim, S.B. Rajamohan, A. Isbatan, M.P. Gupta, Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice, J. Clin. Investig. 119 (9) (2009) 2758–2771. [31] C. Zhu, X. Wang, F. Xu, B.A. Bahr, M. Shibata, Y. Uchiyama, H. Hagberg, K. Blomgren, The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia, Cell death Differ. 12 (2) (2005) 162–176. [32] A. Rami, A. Langhagen, S. Steiger, Focal cerebral ischemia induces upregulation of Beclin 1 and autophagy-like cell death, Neurobiol. Dis. 29 (1) (2008) 132–141. [33] S. Carloni, G. Buonocore, M. Longini, F. Proietti, W. Balduini, Inhibition of rapamycin-induced autophagy causes necrotic cell death associated with Bax/Bad mitochondrial translocation, Neuroscience 203 (2012) 160–169. [34] X. Wei, Z. Zhou, L. Li, J. Gu, C. Wang, F. Xu, Q. Dong, X. Zhou, Intrathecal injection of 3-Methyladenine reduces neuronal damage and promotes functional recovery via autophagy attenuation after spinal cord ischemia/reperfusion injury in rats, Biol. Pharm. Bull. 39 (5) (2016) 665–673. [35] J.Y. Wang, Q. Xia, K.T. Chu, J. Pan, L.N. Sun, B. Zeng, Y.J. Zhu, Q. Wang, K. Wang, B.Y. Luo, Severe global cerebral ischemia-induced programmed necrosis of hippocampal CA1 neurons in rat is prevented by 3-methyladenine: a widely used inhibitor of autophagy, J. Neuropathol. Exp. Neurol. 70 (4) (2011) 314–322. [36] A. Banreti, M. Sass, Y. Graba, The emerging role of acetylation in the regulation of
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