Sirt3 confers protection against acrolein-induced oxidative stress in cochlear nucleus neurons

Neurochemistry International 114 (2018) 1e9

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Sirt3 confers protection against acrolein-induced oxidative stress in cochlear nucleus neurons Juan Qu a, *, Yong-xiang Wu b, Ting Zhang c, Yang Qiu a, Zhong-jia Ding a, Ding-jun Zha a, ** a

Department of Otolaryngology, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi 710032, China Department of Otolaryngology, NO.474 Hospital of China PLA, Urumchi 830011, Xinjiang Province, China c Department of Anatomy, Histology and Embryology, K. K. Leung Brain Research Centre, The Fourth Military Medical University, Xi'an 710000, Shaanxi Province, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 September 2017 Received in revised form 11 December 2017 Accepted 13 December 2017 Available online 14 December 2017

Acrolein is a ubiquitous dietary and environmental pollutant, which can also be generated endogenously during cellular stress. However, the molecular mechanisms underlying acrolein-induced neurotoxicity, especially in ototoxicity conditions, have not been fully determined. In this study, we investigated the mechanisms on acrolein-induced toxicity in primary cultured cochlear nucleus neurons with focus on Sirt3, a mitochondrial deacetylase. We found that acrolein treatment induced neuronal injury and programmed cell death (PCD) in a dose dependent manner in cochlear nucleus neurons, which was accompanied by increased intracellular reactive oxygen species (ROS) generation and lipid peroxidation. Acrolein exposure also significantly reduced the mitochondrial membrane potential (MMP) levels, promoted cytochrome c release and decreased mitochondrial ATP production. In addition, increased ER tracker fluorescence and activation of ER stress factors were observed after acrolein treatment, and the ER stress inhibitors were shown to attenuate acrolein-induced toxicity in cochlear nucleus neurons. The results of western blot and RT-PCR showed that acrolein markedly decreased the expression of Sirt3 at both mRNA and protein levels, and reduced the activity of downstream mitochondrial enzymes. Furthermore, overexpression of Sirt3 by lentivirus transfection partially prevented acrolein-induced neuronal injury in cochlear nucleus neurons. These results demonstrated that acrolein induces mitochondrial dysfunction and ER stress in cochlear nucleus neurons, and Sirt3 acts as an endogenous protective factor in acrolein-induced ototoxicity. © 2017 Published by Elsevier Ltd.

Keywords: Acrolein Mitochondrial dysfunction ER stress Sirt3

1. Introduction Acrolein, an a,b-unsaturated aldehyde, is a ubiquitous dietary and environmental pollutant. It is commonly released during the combustion of petroleum fuels, plastic and tobaccos, and can be absorbed by humans through oral, respiratory and dermal routes (Cahill, 2014; Woodruff et al., 2007). Acrolein can also be generated endogenously during cellular metabolism through lipid peroxidation, degradation of threonine and metabolism of some cancer drugs (Stevens and Maier, 2008). The concentration of acrolein could reach toxic levels in certain cellular microenvironments, and its half-life is on the order of hours to days (Ghilarducci and

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (D.-j. Zha). https://doi.org/10.1016/j.neuint.2017.12.004 0197-0186/© 2017 Published by Elsevier Ltd.

(J.

Qu),

[email protected]

Tjeerdema, 1995). Increased concentrations or prolonged duration of acrolein exposure may overwhelm the anti-oxidative systems, and acrolein-induced oxidative stress has been demonstrated to be involved in several neurological disorder states, such as stroke, Alzheimer's disease and multiple sclerosis (Moghe et al., 2015; Shi et al., 2011). The sirtuins, mammalian homologues of the yeast silent information regulator 2 (Sir2), are a conserved family of NAD-dependent class III histone deacetylases. It comprises seven protein members (Sirt1-Sirt7), among which Sirt3 localizes mostly to the mitochondria where it regulates metabolism and oxidative stress (Jin et al., 2009). Mitochondrial Sirt3 has been shown to regulate almost every major aspect of mitochondrial biology in highly metabolic tissues, including the brain (Bause and Haigis, 2013). Overexpression of Sirt3 in neuronal cells resulted in an increase in cellular respiration and elevated activities of endogenous antioxidative enzymes (Sundaresan et al., 2009; Wang et al., 2014; Yang

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et al., 2016). Sirt3 was demonstrated to act as a pro-survival factor playing an essential role to protect neurons under excitotoxicity (Chen et al., 2018; Dai et al., 2017; Kim et al., 2011). In addition, Sirt3-mediated preservation of mitochondrial function and redox homeostasis are proved to contribute to the protective mechanism of multiple neuroprotective agents and strategies (Cheng et al., 2016; Liu et al., 2017). To date, the effect of acrolein on cell survival in cochlear nucleus neurons, the mechanism of acrolein-induced ototoxicity and the potential role of Sirt3 in acrolein-induced toxicity are not fully determined. In this study, the role of Sirt3 in acrolein-induced neurotoxicity in cochlear nucleus neurons as well as related mechanisms were investigated.

(Jianchen Biological Engineering, Nanjing, China). Briefly, 50 ml 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 value of control group was the absorbance from untreated cells. The results are presented as fold increase of the control. There were six samples in each group, and the experiment was repeated at least 3 times. 2.4. TUNEL staining

2. Materials and methods 2.1. Primary culture of cochlear nucleus neurons and treatment All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Cochlear nucleus neurons were cultured from Sprague-Dawley rats using a modified method (Li et al., 2012). Briefly, Sprague Dawley rats were anesthetized with sodium pentobarbital and decapitated. The brains were quickly removed and stripped of meninges and blood vessels. Brain slices containing the cochlear nucleus (300 mm) were cut and the cochlear nucleus region was identified visually using a dissecting microscope. Mechanical dissociation of cochlear nucleus neurons was performed using a custom-built vibration device with a firepolished glass pipette oscillating at 50 Hz over approximate 0.1e0.2 mm on the surface of the cochlear nucleus region for about 4 min. Neurons were resuspended in neurobasal medium containing 2% B27 supplement and 0.5 mM L-Glutamine and plated in the density of 3
105 cells/cm2 at 37  C in a humidified 5% CO2 incubator. Before seeding, culture vessels, consisting of 96-well plates, 1.5 cm glass slides or 6 cm dishes were coated with poly-Llysine (PLL, 50 mg/ml) at room temperature overnight. Half of the culture medium was changed every other day. Acrolein was obtained from Sigma Chemical (#89116, St. Louis, MO) and diluted in culture medium. Neurons were treated with acrolein at different concentrations for different time periods based on the specific assays. 2.2. Cell viability assay The cell viability assay was performed with Cell Proliferation Reagent WST-1 (Roche), according to the manufacturer's instructions. After treatments, 10 ml of WST-1 was added to each well and incubated for 4 h at 37  C and 5% CO2. One hundred microliters of culture medium and 10 ml of WST-1 were added to one well, and this background control (absorbance of culture medium plus WST1 in the absence of cells) was used as a blank position for the ELISA reader. After thorough shaking for 1 min on a shaker, and the absorbance of the samples was measured against a background control as a blank with a microplate reader. The value of control group was the absorbance from untreated cells. There were six samples in each group, and the experiment was repeated at least 3 times. 2.3. Lactate dehydrogenase (LDH) release assay Cytotoxicity was determined by measuring the release of LDH with a diagnostic kit according to the manufacturer's instructions

Programmed cell death (PCD) in neurons subjected to various treatments was detected using TUNEL staining (Roche). Briefly, neurons were seeded on 1.5-cm glass slides and treated with acrolein. Neurons were fixed by immersing the slides in 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 using 20 times objective lens, and the TUNEL-positive (apoptotic) cells and DAPI-positive cells were counted. DAPI (10 mg/ml) was used to stain the nuclei. A blind researcher counted the number of TUNELpositive cells in six visions field per slides, and the mean number of TUNEL-positive cells in the six visions was regarded as the data of each section. A total of five slides from each group were used for quantification. The apoptotic rate was calculated as a percentage of total number of DAPI-positive cells. 2.5. Measurement of ROS generation Briefly, neurons were incubated with 10 mM 2,7dichlorofluorescein diacetate (DCF-DA) for 1 h at 37  C in the dark, and then resuspended in PBS. Intracellular ROS production was detected using the fluorescence intensity of the oxidantsensitive probe 2,7-dichlorodihydro-fluorescein diacetate (H2DCF-DA) in a microscope using 20 times objective lens and the fluorescence was read using an excitation wavelength of 480 nm and an emission wavelength of 530 nm. The level of ROS was illustrated by the value of mean average fluorescent intensity, which was quantified using the Image J densitometry software. Five visions field per slides and four slides from each group were used for measurement. The data of the treatment groups were expressed as a fold of values in control group. 2.6. Measurement of lipid peroxidation Malonyl dialdehyde (MDA) and 4-hydroxynonenal (4-HNE), two index of lipid peroxidation, were determined using assay kits from Cell Bio labs and strictly following the manufacturer's instruction. The absorbance of the samples was measured by a microplate reader. The results were presented as the fold of control. There were six samples in each group, and the experiment was repeated at least 3 times. 2.7. Measurement of mitochondrial membrane potential (MMP) MMP was measured using the fluorescent dye Rh123 as reported previously (Chen et al., 2012). Rh123 was added to the culture medium to achieve a final concentration of 10 ìM for 30 min at 37  C after the neurons were treated and washed with

J. Qu et al. / Neurochemistry International 114 (2018) 1e9

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). The loss of MMP was illustrated by the value of mean average fluorescent intensity, which was quantified using the Image J densitometry software. Five visions field per slides and four slides from each group were used for measurement. The data of the treatment groups were expressed as a percentage of MMP in control group.

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2.12. Mitochondrial enzyme activity The enzyme activities of MnSOD and CAT in the mitochondria were determined by commercial assay kits purchased from Nanjing Jiancheng Bioengineering (Nanjing, China), according to the manufacturer's instructions, respectively. There were six samples in each group, and the experiment was repeated at least 3 times. 2.13. Lentivirus construction and transfection

2.8. Mitochondrial isolation and purification Neurons were lysed with a lysis buffer containing protease inhibitors. The cell lysate was centrifuged for 10 min at 750 g at 4  C, and the pellets containing the nuclei and unbroken cells were discarded. The supernatant was then centrifuged at 15 000 g for 15 min. The resulting supernatant was removed and used as the cytosolic fraction. The pellet fraction containing mitochondria was further incubated with PBS containing 0.5% Trition X-100 for 10 min at 4  C. After centrifugation at 16 000 g for 10 min, the supernatant was collected as mitochondrial fraction.

2.9. Measurement of ATP synthesis Isolated mitochondria were utilized to measure ATP synthesis with a luciferase/luciferin-based system as described elsewhere (Parone et al., 2013). Thirty mg of mitochondria-enriched pellets were resuspended in 100 ml 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 at 530 nm. There were six samples in each group, and the experiment was repeated at least 3 times.

2.10. Real-time RT-PCR Total RNA was prepared from neurons with the Trizol Reagent method (Chen et al., 2013). The mRNA levels were determined by real-time RT-PCR, and the primer set is: XBP1u: forward, 50 -GAA TGC CCT GGT TAC TGA AGA G-3'; reverse, 50 -CCA AAA GGA TAT CAG ACT CAG AAT C-3'; GAPDH: forward, 50 -ATG TAT CCG TTG TGG ATC TGA C-3’; reverse, 50 -CCT GCT TCA CCA CCT TCT TG-3’. The relative expression value was normalized to the expression value of GAPDH. There were six samples in each group, and the experiment was repeated at least 3 times.

The coding sequence of Sirt3 was amplified by RT-PCR. The primer sequences were forward, 50 -TACTTCCTTCGGCTGCTTCA-3'; reverse, 50 -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-FUSirt3. To generate the recombinant Lentivirus LV-Sirt3, 293T cells were co-transfected with the pGC-FU plasmid (20 ìg) with a cDNA encoding Sirt3, a pHelper 1.0 plasmid (15 ìg) and a pHelper 2.0 plasmid (10 ìg) using Lipofectamine 2000 (100 ìl). The supernatant was harvested and the viral titre was calculated by transducing 293T cells. As a control, we generated a lentiviral vector that expressed GFP alone (LV-control). Cortical neurons were transfected with lentivirus vectors for 48 h and subjected to various treatments. 2.14. Western blot analysis Total protein concentrations were measured using the Pierce BCA method (Sigma, USA). Equivalent amounts of protein (40 mg per lane) were loaded and separated by 10% SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skimmed milk solution in trisbuffered saline with 0.1% Triton X-100 (TBST) for 1 h, and then incubated overnight at 4  C with the primary cytochrome c (1:800, sc-13561, Santa Cruz, USA), tubulin (1:1500, #5335, Cell Signaling, USA), COX V (1:500, sc-376907, Santa Cruz, USA), CHOP (1:1000, #5554, Cell Signaling, USA), cleaved-caspase-12 (1:200, #2202, Cell Signaling, USA), Sirt3 (1:1000, sc-36517, Santa Cruz, USA) or b-actin (1:500, #4970, Cell Signaling, USA) antibody dilutions in TBST. After that the membranes were washed and incubated with secondary antibodies (Santa Cruz, USA), for 1 h at room temperature. Immunoreactivity was detected with Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA). Image J (Scion Corporation) was used to quantify the optical density of each band. The expression of each protein was calculated from the optical density of each band normalized against the optical density of b-actin, tubulin or COX V, and expressed as the fold of control levels. 2.15. Statistical analysis

2.11. Immunocytochemistry (ICC) 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 neurons were incubated with blocking solution (5% Normal Goat serum in PBS, pH 7.4) for 20 min and incubated with a primary anti-Sirt3 antibody (1:50, sc-36517, Santa Cruz, USA) at 4  C overnight. The cells were then rinsed twice with PBS and incubated with fluorescein isothiocyanate (FITC) labelled secondary antibody (1:500, sc-365175 AF488, Santa Cruz, USA) for 1 h at room temperature. Coverslips were mounted in mounting medium (VectaMount #H5000, Vector Laboratories, USA) and visualized using a fluorescence microscope using 20 times objective lens. Five visions field per slides and four slides from each group were analyzed.

Statistical analysis was performed using SPSS 16.0. Statistical evaluation of the data was performed by one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons. A value of p < .05 was considered statistically significant. 3. Results 3.1. Acrolein induces neurotoxicity in cochlear nucleus neurons To investigate the neurotoxic effects of acrolein in vitro, cochlear nucleus neurons were treated with acrolein at different concentrations. The WST assay was performed at 24 h, and the results showed that 1, 5 and 10 mM acrolein significantly decreased the cell viability as compared to control (Fig. 1A). Acrolein at 0.1 or 0.5 mM

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Fig. 1. Acrolein induces neurotoxicity in cochlear nucleus neurons. The neurons were treated with acrolein at different concentrations (0.1, 0.5, 1, 5 or 10 mM) for 24 h. The cell viability (A) was determined by WST-1 assay and LDH release (B) were measured. TUNEL staining was performed to detect PCD (C) and the number of TUNEL positive cells was counted (D). Scale bar: 50 mm. Data are shown as mean ± SEM from at least three separate experiments. #p < .05 vs. Control.

had no effect on LDH release, whereas 1, 5 and 10 mM acrolein markedly increased LDH release in neurons (Fig. 1B). As shown in Fig. 1C, TUNEL staining was used to detect apoptotic cell death, and DAPI was used to stain the nucleus. The results showed that there was few TUNEL positive cells in control, 0.1 and 0.5 mM acrolein group, and the number of TUNEL positive cells in 1, 5 and 10 mM acrolein group were higher than that in control group (P < .05). 3.2. Acrolein induces oxidative stress in cochlear nucleus neurons DCF staining was performed to detect intracellular ROS generation in cochlear nucleus neurons (Fig. 2A). The results showed that 1 and 5 mM acrolein significantly increased intracellular ROS levels, but 0.5 mM acrolein had no such effect (Fig. 2B). We also measured the levels of MDA and 4-HNE to detect lipid peroxidation, and the results showed that 1 and 5 mM acrolein, not 0.5 mM acrolein, markedly increased MDA and 4-HNE levels in cochlear nucleus neurons (Fig. 2C and D). To confirm the involvement of ROS in acrolein-induced neurotoxicity, neurons were pretreated with the ROS scavenger N-acetylcysteine (NAC, 10 mM). As shown in Fig. 2E, we found that the decrease in cell viability after exposure to 1 and 5 mM acrolein was significantly attenuated by NAC pretreatment. 3.3. Acrolein induces mitochondrial dysfunction in cochlear nucleus neurons Rh123 staining was performed to detect the MMP levels (Fig. 3A). The results showed that 1 and 5 mM acrolein significantly decreased MMP levels in cochlear nucleus neurons, whereas the

MMP levels between control and 0.5 mM acrolein group were not statistically different (Fig. 3B). We also detected the distribution of cytochrome c in mitochondrial and cytosolic fractions, and the results showed that 1 and 5 mM acrolein, not 0.5 mM acrolein, significantly increased the release of cytochrome from mitochondria to cytoplasm (Fig. 3C). To determine changes of energy levels, ATP synthesis in isolated and purified mitochondria was monitored up to 120 min after acrolein exposure (Fig. 3D). We found that the mitochondrial ATP generation was markedly inhibited by 1 and 5 mM acrolein.

3.4. Acrolein induces ER stress in cochlear nucleus neurons ER tracker staining was performed to detect morphological changes of the ER in cochlear nucleus neurons (Fig. 4A). An increase in ER tracker fluorescence and some vacuoles formed from the destruction of ER structural integrity were observed after exposure to 1 or 5 mM acrolein. RT-PCR assay showed that the mRNA levels of XBP1u was decreased by acrolein at 0.5, 1 and 5 mM (Fig. 4B). In addition, the expression of ER stress associated pro-apoptotic factors was detected by western blot (Fig. 4E). The results showed that acrolein markedly increased the expression of CHOP and the cleavage of caspase-12 in cochlear nucleus neurons (Fig. 4D and E). To further confirm the involvement of ER stress, salubrinal (Sal) and AEBSF (AEB) were used to inhibit ER stress in vitro. The results showed that acrolein-induced decrease in cell viability (Fig. 5A), increase in LDH release (Fig. 5B) and apoptotic cell death (Fig. 5C) were all partially prevented by Sal and AEB.

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Fig. 2. Acrolein induces oxidative stress in cochlear nucleus neurons. The neurons were treated with acrolein at different concentrations (0.5, 1 or 5 mM) for 24 h. The ROS generation was detected by DCF staining (A and B), and the MDA (C) and 4-HNE (D) levels were measured. The neurons were treated with 1 or 5 mM acrolein in the presence or absence of N-acetylcysteine (NAC, 10 mM), and the cell viability was assayed by WST-1 assay (E). Scale bar: 20 mm. Data are shown as mean ± SEM from at least three separate experiments. # p < .05 vs. Control. *p < .05.

Fig. 3. Acrolein induces mitochondrial dysfunction in cochlear nucleus neurons. The neurons were treated with acrolein at different concentrations (0.5, 1 or 5 mM) for 24 h. Rh123 staining was performed to detect MMP (A), and the relative MMP levels were calculated (B). Mitochondria in each group were isolated and purified. The expression of cytochrome c was detected by western blot analysis in mitochondrial fraction and cytosolic fraction (C). Mitochondrial ATP synthesis was measured up to 120 min (D). Scale bar: 10 mm. Data are shown as mean ± SEM from at least three separate experiments. #p < .05 vs. Control.

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Fig. 4. Acrolein induces ER stress in cochlear nucleus neurons. The neurons were treated with acrolein at different concentrations (0.5, 1 or 5 mM) for 24 h. The morphological changes of ER were detected by ER tracker (A), and the expression of XBP1u mRNA was assayed by RT-PCR (B). The expression of CHOP and cleavage of caspase-12 were detected by western blot (CeE). Scale bar: 10 mm. Data are shown as mean ± SEM from at least three separate experiments. #p < .05 vs. Control.

Fig. 5. Effects of ER stress inhibitors on acrolein-induced neurotoxicity. The neurons were treated with 5 mM acrolein in the presence or absence of the ER stress inhibitors (Salubrinal, Sal, 25 mM; AEBSF, AEB, 300 mM) for 24 h. The cell viability (A) was determined by WST-1 assay and LDH release (B) were measured. PCD was detected by TUNEL staining (C). Data are shown as mean ± SEM from at least three separate experiments. #p < .05 vs. Control. *p < .05. vs. Acrolein.

3.5. Acrolein inhibits Sirt3 activity in cochlear nucleus neurons Immunostaining was performed to detect the changes of Sirt3 protein after acrolein exposure (Fig. 6A). The results showed that acrolein treatment reduced the fluorescence intensity of Sirt3, but the subcellular distribution of Sirt3 protein was unaffected. As shown in Fig. 6B, the mRNA levels of Sirt3 significantly decreased from 3 to 24 h after acrolein exposure. The results of western blot showed that the expression of Sirt3 protein decreased at 6, 12 and 24 h, but not at 3 h after acrolein treatment (Fig. 6C). In addition, the enzymatic activities of MnSOD and CAT, two downstream targets of Sirt3, were assayed, and the results showed that acrolein exposure markedly reduced the activities of MnSOD and CAT (Fig. 6D and E). 3.6. Overexpression of Sirt3 attenuates acrolein-induced neurotoxicity To investigate the biological functions of Sirt3 in acroleininduced neurotoxicity, cochlear nucleus neurons were transfected with lentivirus expressed Sirt3 (LV-Sirt3) or control lentivirus (LV-

control). As shown in Fig. 7A, LV-Sirt3 significantly increased the expression of Sirt3 protein in the presence of acrolein. The results showed that acrolein-induced decrease in cell viability (Fig. 7B), increase in LDH release (Fig. 7C) and apoptotic cell death (Fig. 7D) were all partially prevented by Sirt3 overexpression. LV-Sirt3 also preserved MMP levels after acrolein exposure (Fig. 7E). Furthermore, the acrolein-induced CHOP expression and caspase-12 cleavage were both partially reversed by LV-Sirt3 transfection as compared to LV-control group (Fig. 7FeH). 4. Discussion Elucidation of the underlying mechanisms associated with the acrolein-induced toxicity has been of great interest in the field of research. The results of this study provide evidence that ER stress and mitochondrial dysfunction-mediated oxidative stress contribute to acrolein-induced neurotoxicity in cochlear nucleus neurons. We found that (a) acrolein treatment results in PCD in a dose-dependent manner in cochlear nucleus neurons; (b) acrolein induces intracellular ROS generation and lipid peroxidation; (c)

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Fig. 6. Acrolein inhibits Sirt3 activity in cochlear nucleus neurons. The neurons were treated with 5 mM acrolein for 24 h, and the distribution of Sirt3 was detected by immunofluorescence staining (A). The mRNA levels of Sirt3 at different time points (3, 6, 12 or 24 h) were measured by PCR (B), and the protein levels of Sirt3 at each time point were detected by western blot (C). The enzymatic activities of MnSOD (D) and CAT (E) were assayed. Scale bar: 10 mm. Data are shown as mean ± SEM from at least three separate experiments. #p < .05 vs. Control.

Fig. 7. Overexpression of Sirt3 attenuates acrolein-induced neurotoxicity. The neurons were transfected with LV-Sirt3 or LV-control for 48 h and exposed to 5 mM acrolein for 24 h, and the protein levels of Sirt3 were detected by western blot (A). The cell viability (B) was determined by WST-1 assay and LDH release (C) were measured. Neuronal PCD was detected by TUNEL staining (D). The MMP level was assayed by Rh123 staining (E), and the expression of CHOP and cleavage of caspase-12 were detected by western blot (FeH). Data are shown as mean ± SEM from at least three separate experiments. *p < .05. vs. LV-control.

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acrolein exposure leads to mitochondrial dysfunction and ER stress; (d) the expression of Sirt3 mRNA and protein markedly decreases in acrolein-treated neurons; and (e) mechanistically, acrolein-induced mitochondrial dysfunction, ER stress and PCD are partially prevented by Sirt3 overexpression. Acrolein is a ubiquitous pollutant that is also generated endogenously under pathological conditions. This compound is hundreds of times more reactive and persists in solution for much longer than many other known free radicals (Shi et al., 2011). Exposure to acrolein at concentrations as low as 1 mM for 4 h could increase cell membrane permeability to ethidium bromide (400 Da), and even to LDH (144 Kd) (Luo and Shi, 2004). Here, we found that acrolein at the concentrations higher than 1 mM exerted apparent neurotoxicity in cochlear nucleus neurons. Induction of oxidative stress is proposed to be one of the main underlying mechanisms of acrolein-induced toxicity, and we also observed increased intracellular ROS generation and lipid peroxidation after acrolein exposure in our in vitro cellular model. Our results showed that acrolein induced MMP collapse, cytochrome c release, morphological changes of the ER and activation of ER associated apoptotic factors, some of which were similar to the findings in hepatocytes (Mohammad et al., 2012). These results elucidate some basic mechanisms underlying acrolein-induced toxicity, and point out some potential therapeutic targets for further research. Accumulating evidence supports the concept that ER stress is involved in neuronal injury in various neurological disorders (Valenzuela et al., 2016). Recent experiments indicate that ER stress also contribute to neuronal loss in cochlea and damage of hair cells in the organ of corti under auditory disorders. Increased expression of calreticulin, a calcium binding chaperone of the ER, and enhanced phosphorylation of the ER stress-induced protein GRP58, were observed in a rat model of cisplatin-induced ototoxicity (Coling et al., 2007). Intra-tympanic aminoglycoside treatment was shown to cause high-frequency hearing loss in XBP1(±) mice but not in wild-type littermates (Oishi et al., 2015). In this study, reduced expression of XBP1u mRNA and increased expression of CHOP were observed after acrolein treatment, indicating the involvement of ER stress and unfolded protein response (UPR) pathways. In addition, elevated cleavage of caspase-12, the ER specific apoptosis executer molecular, was also observed in acrolein-treated neurons. Our hypothesis was further confirmed by the results that acrolein-induced neurotoxicity was significantly attenuated by the ER stress inhibitor salubrinal and AEBSF. As a conserved family of histone deacetylases, the sirtuins dynamically change under stress conditions and differently regulate cell death and survival. Previous studies showed that Sirt1 was upregulated in both neuroblastoma SK-N-SH cells and rat primary astrocytes following acrolein exposure (Dang et al., 2010, 2011), but the effect of acrolein on Sirt3 expression and activity has not been determined. Our results showed that acrolein treatment decreased the expression of Sirt3 in both mRNA and protein levels in cochlear nucleus neurons, and these changes could be observed up to 24 h after acrolein exposure. Similarly, decreased Sirt3 expression was also observed in neurons and endothelia in experimental subarachnoid hemorrhage (SAH) (Huang et al., 2016). However, previous experiments showed that hydrogen peroxide (H2O2) treatment significantly upregulated Sirt3 mRNA and protein, which was also found in H2O2-treated neuronal HT22 cells (Dai et al., 2014a, b). These contrary results might be attribute to the differences in cell types and toxic agents. As a mitochondrial specific deacetylase, Sirt3 regulates several mitochondrial proteins, among which MnSOD and CAT are two vital antioxidative enzymes (Kincaid and Bossy-Wetzel, 2013; Zhu et al., 2012). Thus, we also detected the enzymatic activities of MnSOD and CAT, and found that acrolein significantly reduced the activities of these enzymes,

indicating that acrolein attenuate both expression and activity of Sirt3 in cochlear nucleus neurons. The beneficial effects of Sirt3 in delaying aging and in cellular repair process under stress conditions have been extensively studied. For example, Sirt3 was shown to deacetylate Ku70 and protect cardiomyocytes from stress-mediated cell death by hindering the translocation of Bax to mitochondria (Sundaresan et al., 2008). Sirt3 regulates subunits of mitochondrial electron transport chain (ETC) complexes and tricarboxylic acid (TCA) cycle enzymes to increase production of energy equivalents during caloric restriction (CR), fasting and exercise (Kincaid and Bossy-Wetzel, 2013). The protective effects of Sirt3 was also demonstrated in neurological disorders. A previous study showed that Sirt3 protected cortical neurons against oxidative stress via regulating mitochondrial Ca2þ and mitochondrial biogenesis (Dai et al., 2014b). Han and colleagues showed that the age-related decline in Sirt3 function is a major factor underlying mitochondrial dysfunction and loss of SNc dopaminergic neurons in PD (Shi et al., 2017). In this study, decreased expression of Sirt3 was accompanied by mitochondrial dysfunction and ER stress. Using lentivirusmediated overexpression technology, we found that overexpression of Sirt3 partially reversed the neuronal injury induced by acrolein. Previous studies showed that increased expression and activity of Sirt3 contributed to the beneficial effects of many neuroprotective agents, such as ZL006 and fucoidan (Liu et al., 2017; Wang et al., 2016). All these data suggest that acrolein-induced neurotoxicity in cochlear nucleus neurons was associated with reduced Sirt3 function, which might act as an endogenous protective mechanism in our in vitro model. In this study, we found that NAC, salubrinal, AEBSF or overexpression of Sirt3 could partially prevent neuronal injury after acrolein treatment in cochlear nucleus neurons. Whether combined treatments of NAC with ER stress inhibitors and/or Sirt3 overexpression exert additive protective effects against acrolein-induced toxicity needs to be determined in the future. In summary, our results showed that acrolein treatment induced mitochondrial dysfunction and ER stress, which contribute to its toxic effects in cochlear nucleus neurons. In addition, acrolein exposure resulted in reduced expression and function of Sirt3, and overexpression of Sirt3 attenuated acrolein-induced neuronal injury. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81570922). The authors would like to thank Dr. Terry Chen for his technical support for the experiments and the preparation of the manuscript. References Bause, A.S., Haigis, M.C., 2013. SIRT3 regulation of mitochondrial oxidative stress. Exp. Gerontol. 48, 634e639. Cahill, T.M., 2014. Ambient acrolein concentrations in coastal, remote, and urban regions in California. Environ. Sci. Technol. 48, 8507e8513. Chen, T., Dai, S.H., Li, X., Luo, P., Zhu, J., Wang, Y.H., Fei, Z., Jiang, X.F., 2018. Sirt1-Sirt3 axis regulates human blood-brain barrier permeability in response to ischemia. Redox Biol 14, 229e236. Chen, T., Fei, F., Jiang, X.F., Zhang, L., Qu, Y., Huo, K., Fei, Z., 2012. Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons. Free Radic. Biol. Med. 52, 208e217. Chen, T., Zhu, J., Zhang, C., Huo, K., Fei, Z., Jiang, X.F., 2013. Protective effects of SKF96365, a non-specific inhibitor of SOCE, against MPPþ-induced cytotoxicity in PC12 cells: potential role of Homer1. PLoS One 8, e55601. Cheng, A., Yang, Y., Zhou, Y., Maharana, C., Lu, D., Peng, W., Liu, Y., Wan, R., Marosi, K., Misiak, M., Bohr, V.A., Mattson, M.P., 2016. Mitochondrial SIRT3 mediates adaptive responses of neurons to exercise and metabolic and excitatory challenges. Cell Metab 23, 128e142.

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