Introduction

Mitochondria are organelles essential for maintaining the energy homeostasis and the survival of eukaryotic cells. The function of mitochondria is especially pivotal for neurons, which have a high energy requirement [1]. Impairment of mitochondrial function is one of the major causes of neuronal toxicity and has been linked to the pathogenesis of several neurodegenerative diseases including Alzheimer’s disease (AD) and Parkinson’s disease (PD) [2, 3]. Mitophagy is a specific form of autophagy which exerts critical role in mitochondrial quality and quantity control by eliminating damaged mitochondria and thus is essential for maintaining mitochondrial homeostasis and cell survival [4, 5]. A deficiency in mitophagy can lead to accumulation of damaged or less efficient mitochondria and has been associated with aging and neurodegenerative diseases; however, increasing evidence has indicated that the excessive autophagy/mitophagy flux reduces mitochondrial content and impairs mitochondrial function, causing cytotoxicity [6, 7]. It has been demonstrated that excessive autophagy/mitophagy contributes to the neuronal apoptosis induced by chronic cerebral hypoperfusion [8]. Moreover, studies have shown that inhibition of autophagy/mitophagy protects neuronal cells from neurotoxic stimuli, alleviating mitochondrial damages and promoting cell survival [9, 10].

Heavy drinking impairs cognitive function and has been linked with the earlier onset of neurodegenerative diseases, such as AD and PD [11]. Acetaldehyde is the most toxic metabolite of ethanol. It has been well documented that acetaldehyde mediates the neurotoxicity induced by overconsumption of alcohol [12,13,14,15]. Aldehyde dehydrogenase 2 (ALDH2) is the main enzyme that catalyzes the conversion of acetaldehyde to acetate, a key step for the detoxification of acetaldehyde in vivo. ALDH2-deficient individuals with chronic alcohol overuse have much higher concentration of acetaldehyde in the blood in comparison with the normal individuals [16]. After a moderate oral dose of ethanol (0.5 g/kg), the peak blood concentration of acetaldehyde in ALDH2-deficent alcoholics can reach 125 μmol/L [17]. It has been shown that acetaldehyde induces cytotoxicity via elevating oxidative stress, decreasing mitochondrial polarization, causing excessive mitochondrial fragmentation, and inducing mitochondria-dependent apoptosis in multiple tissues and organs [18,19,20]. In mice with defective ALDH2, chronic ethanol exposure results in more severe mitochondrial dysfunction and neurotoxicity relative to wild type mice, which can be blunted by pharmacological activation of ALDH2 [21]. Mitochondria are the primary source of reactive oxygen species (ROS), while excessive production of ROS causes damages to mitochondria and has been associated with overactive mitophagy and decreased mitochondrial mass [22]. It has been demonstrated that the accumulation of acetaldehyde in cardiomyocyte leads to autophagy induction and contractile dysfunction [23]. Acetaldehyde-induced autophagic responses also contribute to the loss of skeletal muscle mass following ethanol exposure [24]. In contrast, autophagy activation appears to have protective effects against acetaldehyde or ethanol-induced cell death in mouse primary microvascular endothelial cells [25]. Nonetheless, the precise role of autophagy/mitophagy in acetaldehyde-induced cytotoxicity remains poorly understood.

The aim of the present study was to investigate the role of mitophagy in acetaldehyde-induced cytotoxicity. The occurrence of mitophagic response and the protein levels of important autophagy/mitophagy effectors such as light chain 3 (LC3), Beclin1, and PTEN-induced putative kinase 1 (PINK1)/Parkin were examined in acetaldehyde-treated human neuroblastoma SH-SY5Y cells. In addition, the involvement of ROS in acetaldehyde-induced autophagy/mitophagy was studied.

Materials and Methods

Materials

Fetal bovine serum (FBS), penicillin, streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin, beta tubulin antibody, and nonyl acridine orange (NAO) were purchased from Thermo Fisher Scientific (Rockford, USA). N-acetyl-L-cysteine (NAC) was purchased from Sangon Biotech (Shanghai, China). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) and chloroquine were purchased from Sigma Chemical (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kit was purchased from Wanleibio (Shenyang, China). BCA protein assay kit, mitochondria isolation kit, Beyo ECL moon Western blotting detection system, HRP-labeled donkey anti-goat, goat anti-mouse, and goat anti-rabbit IgG (H + L) were purchased from Beyotime Institute of Biotechnology (Haimen, China). Mitophagy detection kit was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Antibodies for LC3 (12741S), Beclin-1 (D40C5), autophagy-related protein (Atg) 5 (D5F5U), Atg12 (D88H11), Atg16L1 (D6D5), Atg7 (D12B11), phosphorylated Drp1 (Ser616), and Drp1 were purchased from Cell Signaling Technology (Danvers, USA). Antibodies for PINK1 (C-3), Parkin (H-8), and actin were purchased from Santa Cruz Biotechnology (Dallas, USA). COXIV and p62 polyclonal antibodies were purchased from Proteintech (Wuhan, China). 3-methyladenine (3-MA) and mitochondrial division inhibitor 1 (Mdivi-1) were purchased from MedChemExpress (Monmouth Junction, USA).

Cell Culture

Human neuroblastoma SH-SY5Y cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin and streptomycin. Acetaldehyde was diluted in PBS and added to the cell culture medium to achieve specific concentrations. The cells were maintained at 37 °C in a humidified atmosphere with 5% CO2 and 95% air until being collected for different assays.

MTT Assay

For cell viability measurements, cells (4 × 103/well) were seeded in a 96-well culture plate. After the treatment, cells were incubated with MTT at 37℃ for 4 h. The formazan formed was then dissolved in DMSO and the absorbance was measured at 570 nm using a microplate reader.

Measurement of ROS

The levels of ROS were determined using DCFH-DA as described previously [20]. Cells (1 × 105/well) were seeded in 6-well culture plates. After treatments, cells were harvested by trypsinization and incubated with 10 μmol/L of DCFH-DA for 30 min at 37 °C in the dark. The fluorescence intensity was measured using a Multi-mode Microplate Reader with excitation wavelength at 488 nm and emission wavelength at 525 nm.

Mitochondria Isolation

Mitochondria were prepared using a mitochondria isolation kit following the manufacturer’s instructions as described previously [20]. After the treatment, cells were homogenized in isolation buffer on ice for 60 strokes using a pestle. After centrifugation at 3500 × g for 25 min at 4 °C, the pellets were collected and used as mitochondrial fraction. The supernatant was centrifuged again at 12,000 × g for 10 min at 4 °C, and the resulted supernatant was the cytosolic fraction.

Western Blot Analyses

The whole cell lysates were prepared in cell lysis buffer (Tris 20 mmol/L, NaCl 150 mmol/L, EDTA 1 mmol/L, sodium pyrophosphate 2.5 mmol/L, NaF 20 mmol/L, β-glycerophosphoric acid 1 mmol/L, and Na3VO4 1 mmol/L) after the treatment as described previously [12]. Protein concentrations were measured using BCA protein assay kit. Ten micrograms of protein extracts was resolved by SDS–polyacrylamide gel electrophoresis and then transferred to polyvinylidenedifluoride (PVDF) membrane. After blocking, the PVDF membrane was incubated with a primary antibody, followed by a HRP-coupled secondary antibody. An enhanced chemiluminescence substrate reaction (Beyo ECL moon Western blotting detection system) was used for the detection of the protein bands. The intensities of the bands were then quantified by densitometric analyses.

Mitophagy Detection

For the evaluation of mitophagy, a mitophagy detection kit (Dojindo Molecular Technologies) was used following the manufacturer’s instruction. In the assay, cells are incubated with a small-molecule fluorescent probe (Mtphagy dye), which binds to mitochondria and emits strong fluorescence when damaged mitochondria fuse with acidic lysosomes, and a Lyso dye that stains lysosomes to confirm the fusion of Mtphagy dye–labeled mitochondria with lysosomes [26]. First, the cells were washed twice with FBS-free DMEM and incubated with Mtphagy Dye (200 nM) for 30 min at 37 ℃. The cells were then treated with acetaldehyde for 8 h and incubated with Lyso Dye (2 μmol/L) for 30 min at 37 ℃. The images were captured using an Olympus BX53 fluorescence microscope and analyzed by ImageProPlus6.0 software. The excitation/emission wavelengths for Mtphagy dye and Lyso dye were 577/590 nm and 490/516 nm, respectively. To characterize the co-localization of Mtphagy Dye and Lyso Dye, a total of 30–45 cells were randomly chosen from each treatment group and the Manders overlap coefficient was calculated using the following formula \(\frac{{\sum\limits_{i} {{\text{S}}1_{i} \times {\text{S}}2_{i} } }}{{\sqrt {\sum\limits_{i} {(S1_{i} )^{2} \times \sum\limits_{i} {(S2_{i} )^{2} } } } }}\), where S1i and S2i represent the signal intensity of individual pixels in channels 1 (red) and 2 (green), respectively (i represents single pixel) [27].

Analyses of Mitochondrial Mass

The mitochondrial mass was estimated by staining cells with fluorescent dye NAO, which binds to cardiolipin, a phospholipid specifically present on the inner membrane of mitochondria [28]. After treatment, cells were incubated with 50 nmol/L NAO at 37 °C for 30 min as described previously [29]. The fluorescence intensity was analyzed using a Multi-mode Microplate Reader (excitation at 488 nm, emission at 533 nm).

Statistical Analysis

Quantitative data are analyzed by GraphPad Prism 7.00. Statistical analyses of the data were performed by one-way or two-way ANOVA. p < 0.05 was considered statistically significant.

Results

Acetaldehyde-Induced Cytotoxicity Is Associated with Mitophagy

As shown in Fig. 1a, acetaldehyde treatment induced cytotoxicity in SH-SY5Y cells in a dose-dependent manner. This result was consistent with our previous report that acetaldehyde treatment significantly reduced MTT activity in both primary cultures of rat cortical neurons and SH-SY5Y cells [20]. When the effects of acetaldehyde on cell survival was studied using crystal violet assay, it was found that acetaldehyde treatment up to 24 h did not cause significant cell loss [20]. Thus, the results from MTT assay perhaps reflected the reduced mitochondrial metabolic activity and impaired mitochondrial function [30], which was confirmed by the decrease of mitochondrial membrane potential and the loss of ATP production in acetaldehyde treated cells (Figs. S1 and S2). These results indicated that the impairment of mitochondrial function was a major event in acetaldehyde-induced cytotoxicity.

Fig. 1
figure 1

Acetaldehyde-induced cytotoxicity is associated with autophagy. a SH-SY5Y cells were treated with different concentrations of acetaldehyde for 8 h. MTT assay was then performed. Data represent the mean ± SD of 6 independent experiments. b, c SH-SY5Y cells were exposed to 500 μmol/L acetaldehyde for the indicated times. d SH-SY5Y cells pre-incubated with 10 μmol/L chloroquine for 24 h were treated with 500 μmol/L acetaldehyde for 8 h. The protein levels of LC3 and p62 were determined by Western blot analyses. Data represent the mean ± SD of 3 independent experiments. *p < 0.05 versus control, #p < 0.05 versus acetaldehyde-treated cells

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Our previous study also demonstrated that acetaldehyde treatment induced mitochondrial dysfunction and cytotoxicity by causing increased mitochondrial fission and excessive mitochondrial fragmentation [20]. It has been shown that the fragmentation of mitochondria is required to trigger mitophagy, a selective mitochondrial autophagy that removes damaged mitochondria via lysosomal degradation [4, 5, 31, 32]. In this study, we investigated the role of mitophagy in acetaldehyde-induced cytotoxicity. First, the level of autophagy in acetaldehyde treated cells was determined by examining the levels of LC3-II. LC3-II is formed by conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) upon autophagy induction, which binds to the nascent autophagosome membrane and participates in the formation of autophagosomes [33,34,35]. As shown in Fig. 1b, the levels of LC3-II were significantly increased at 6 h and 8 h after treatment with acetaldehyde, suggesting that autophagic responses were induced by acetaldehyde treatment. To examine the formation of autophagosomes in greater detail, we analyzed the degradation of p62, an autophagy-specific substrate that is selectively incorporated into autophagosomes and efficiently degraded by autophagy [36, 37]. The protein levels of p62 were markedly decreased at 6 h and 8 h after treatment with acetaldehyde by approximately 49% and 51%, respectively (Fig. 1c). Pre-incubation of the cells with autophagy flux inhibitor chloroquine [38] blocked both the degradation of LC3-II and p62, leading to their accumulation (Fig. 1d), confirming the activation of autophagic responses in acetaldehyde-treated cells. The results were similar when SH-SY5Y cells were treated with chloroquine and acetaldehyde simultaneously (Fig. S3). Taken together, these findings suggested that acetaldehyde stimulated autophagy in SH-SY5Y cells.

Next, we examined the effects of acetaldehyde on mitophagy using a mitophagy detection kit. In the assay, the cells were incubated with a fluorescent mitochondrial probe (Mtphagy dye) that emitted strong fluorescence when damaged mitochondria fused with acidic lysosomes, and a Lyso dye that stained lysosomes to confirm the fusion of Mtphagy dye–labeled mitochondria with lysosomes [26]. As shown in Fig. 2a, acetaldehyde treatment increased the fluorescence intensity of the Mtphagy dye by approximately 98%. The co-localization of the Mtphagy dye with the Lyso dye as demonstrated by the Manders overlap coefficient was increased approximately 54% by acetaldehyde treatment. In contrast, the fluorescence intensity of Mtphagy dye increased by acetaldehyde was diminished when cells were co-treated with inhibitors of autophagy, choloroquine, or 3-MA (Fig. S4). The ubiquitin-binding adaptor p62 and LC3-II mediate the sequestration of mitochondria into autophagosomes, facilitating their further fusion with lysosomes and degradation; thus, the elevation of LC3-II and p62 in mitochondria indicates the mitophagosome formation and occurrence of mitophagy [39,40,41,42]. As demonstrated in Fig. 2b, the protein levels of LC3-II and p62 were significantly increased in the mitochondrial fractions of acetaldehyde-treated cells, suggesting the formation of mitophagosomes. Together, these results demonstrated that mitophagic process was induced in acetaldehyde-treated cells.

Fig. 2
figure 2

Acetaldehyde induced mitophagy. a SH-SY5Y cells were exposed to 500 μmol/L acetaldehyde for 8 h. Fluorescence intensity of Mtphagy dye and the co-localization of Mtphagy dye and Lyso dye were then analyzed. Scale bar, 10 μm. The histograms on the right illustrated the fluorescence intensity of Mtphagy dye and the Manders overlap coefficient in control and acetaldehyde-treated groups, respectively. Data represent the mean ± SD of 4 independent experiments. b SH-SY5Y cells were exposed to 500 μmol/L acetaldehyde for 6 h. The mitochondrial and cytoplastic fractions were prepared and the protein levels of LC3 and p62 were determined by Western blot analyses. Tubulin and COXIV were used as internal controls of cytoplasm and mitochondria, respectively. Data represent the mean ± SD of 3 independent experiments. *p < 0.05 versus control

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Effects of Acetaldehyde on Key Proteins Involved in Autophagy/Mitophagy Initiation

A wide variety of proteins are involved in the regulation of autophagy. Among them, Beclin1 has been considered the “gatekeeper” of autophagy signaling pathway, which initiates the process of autophagy [43]. As shown in Fig. 3a, acetaldehyde treatment significantly increased the protein levels of Beclin1 as early as 4 h after the treatment, leading to an approximate 40% increase in cells treated with 500 μmol/L acetaldehyde when compared with that in control cells (Fig. 3b). There are about 20 core Atgs involved in the membrane dynamics during autophagy [44]. Atg5, Atg7, Atg12, and Atg16L1 are well-known for participating in the formation and expansion of autophagosome membrane [45]. As demonstrated in Fig. 3c and d, the protein levels of Atg5 and Atg16L1 were elevated at 4 h after acetaldehyde treatment in a dose–response manner. The protein levels of Atg5 and Atg16L1 were increased approximately 70% and 75%, respectively, by treatment with 500 μmol/L acetaldehyde. Meanwhile, the protein levels of Atg7 and Atg12 were not affected by acetaldehyde treatment (Fig. 3e and f). These results indicated that acetaldehyde treatment resulted in the elevation of proteins involved in autophagy initiation and autophagosome membrane formation and expansion.

Fig. 3
figure 3

Effects of acetaldehyde on key proteins involved in autophagy initiation and autophagosome membrane formation and expansion. a SH-SY5Y cells were treated with 500 μmol/L acetaldehyde for the indicated times. b, c, d, e, f SH-SY5Y cells were treated with the indicated concentrations of acetaldehyde for 4 h. The protein levels of Beclin1, Atg5, Atg16L1, Atg7, and Atg12 were determined by Western blot analyses. Data represent the mean ± SD of 3 independent experiments. *p < 0.05 versus control

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It is well established that PINK1 can be selectively accumulated on the depolarized mitochondria, recruiting and activating Parkin and the downstream signals that drive mitophagy [46]. It has been shown that the elevated expression of PINK1 and Parkin leads to the overactivation of mitophagy and declined cell viability [47, 48]. As shown in Fig. 4a, acetaldehyde dramatically increased the protein levels of PINK1 and Parkin as early as 4 h after the treatment and in a dose-dependent manner. The protein levels of PINK1 and Parkin in cells treated with 500 μmol/L of acetaldehyde were increased by approximately 0.7 and 1.9 folds, respectively, when compared with those in control cells (Fig. 4b and c). PINK1 is constitutively expressed in neurons and can be stabilized on the depolarized mitochondria that have lost membrane potential [49]. Parkin is then recruited by PINK1 to the dysfunctional mitochondria to initiate mitophagy [50, 51]. Further analyses of the levels of PINK1 and Parkin in mitochondrial and cytoplastic fractions of the cells showed that acetaldehyde treatment significantly promoted the accumulation of PINK1 and Parkin on mitochondria (Fig. 4d). Thus, the elevation of PINK1 and Parkin protein levels and their accumulation on mitochondria might be a major mechanism that mediated mitophagy in acetaldehyde treated SH-SY5Y cells.

Fig. 4
figure 4

Effects of acetaldehyde on proteins involved in initiation of mitophagy. a SH-SY5Y cells were treated with 500 μmol/L acetaldehyde for the indicated times. b, c SH-SY5Y cells were treated with the indicated concentrations of acetaldehyde for 4 h. The protein levels of PINK1 and Parkin were determined by Western blot analyses. d SH-SY5Y cells were exposed to 500 μmol/L acetaldehyde for indicated times. The mitochondrial and cytoplastic fractions were prepared and the protein levels of PINK1 and Parkin were determined by Western blot analyses. Tubulin and COXIV were used as internal controls of cytoplasm and mitochondria, respectively. Data represent the mean ± SD of 3 independent experiments. *p < 0.05 versus control

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Overactive Mitophagy Induced by Acetaldehyde Caused the Reduction of Mitochondrial Mass and Cell Viability

Overactivation of mitophagy has been shown to cause significant decrease of cell survival [52]. To determine whether autophagy/mitophagy is associated with the cytotoxicity induced by acetaldehyde, SH-SY5Y cells were pre-incubated with autophagy inhibitor chloroquine or 3-MA before the acetaldehyde treatment. As shown in Fig. 5a and b, pre-incubation with chloroquine or 3-MA significantly alleviated the reduction of MTT activity in acetaldehyde-treated cells, suggesting that inhibition of mitophagy reduced acetaldehyde-induced cytotoxicity. Overactivation of mitophagy has been shown to cause significant reduction of mitochondrial mass [52]. The mitochondrial mass was then examined by NAO staining, which is widely used to assess the changes of mitochondrial content [53,54,55]. The result demonstrated that acetaldehyde significantly reduced the mitochondrial mass by about 30% as early as 6 h after the treatment (Fig. 5c). As the mitochondrial protein level is correlated with the mitochondrial mass [56, 57], we next isolated mitochondria from control and acetaldehyde-treated cells and determined the protein concentration of mitochondrial fraction. It was found that the mitochondrial protein levels were significantly reduced by approximately 30% and 50% after acetaldehyde treatment for 6 h and 8 h, respectively (Fig. 5d). Consistently, the protein level of COXIV, which is considered an indicator for mitochondrial mass [58,59,60], was significantly reduced by acetaldehyde treatment (Fig. 5e). Furthermore, pre-incubation of cells with autophagy inhibitor 3-MA before acetaldehyde treatment significantly attenuated the loss of COXIV induced by acetaldehyde treatment (Fig. 5f). Overall, these results suggested that acetaldehyde treatment triggered overactivation of mitophagy, instead of restoring the quality of mitochondria, causing disruption of mitochondrial homeostasis and reduction of mitochondrial mass, leading to cytotoxicity and decrease of the cell viability.

Fig. 5
figure 5

Acetaldehyde-induced overactive mitophagy leads to the reduction of mitochondrial mass and cell viability. a SH-SY5Y cells were incubated with 10 μmol/L chloroquine for 24 h before treatment with 500 μmol/L acetaldehyde for 8 h. b SH-SY5Y cells were incubated with 2.5 mmol/L 3-MA for 24 h before treatment with 500 μmol/L acetaldehyde for 8 h. MTT assay was then performed. Data represent the mean ± SD of 6 independent experiments. c SH-SY5Y cells were exposed to 500 μmol/L acetaldehyde for the indicated times. The mitochondrial mass was determined by NAO staining assay. d SH-SY5Y cells were exposed to 500 μmol/L acetaldehyde for the indicated times and the protein concentrations of mitochondrial fraction were determined. e SH-SY5Y cells were exposed to 500 μmol/L acetaldehyde for the indicated times. f SH-SY5Y cells were pre-incubated with 2.5 mmol/L 3-MA and then exposed to 500 μmol/L acetaldehyde for 8 h. The protein levels of COXIV were determined by Western blot analyses. Data represent the mean ± SD of 3 independent experiments. *p < 0.05 versus control, #p < 0.05 versus acetaldehyde-treated cells

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It has to be noted that the mitochondrial mass in cells treated with acetaldehyde for 24 h was not significantly altered as shown by both MitoTracker Green and NAO staining assays (data not shown). It was possible that compensatory mechanisms promoting mitochondrial homeostasis were activated by acetaldehyde treatment, eventually resulting in the recovery of mitochondrial mass. However, this assumption has yet to be proved.

Acetaldehyde Induced Cytotoxicity via promoting Drp1 Phosphorylation

The fusion-fission dynamic balance of mitochondria regulates both the quantity and the function of mitochondria. In our previous study, it was found that acetaldehyde treatment caused excessive mitochondrial fragmentation and impaired mitochondrial function by increasing the activation of mitochondrial fission–related protein Drp1 [20]. Excessive mitochondrial fragmentation can induce mitophagic cascade, impair mitochondrial function, and cause cytotoxicity [61]. To examine the role of Drp1 activation in acetaldehyde-induced cytotoxicity, cells were pre-incubated with Mdivi-1, a chemical inhibitor of Drp1 that inhibits mitochondrial division [62], before acetaldehyde treatment. As shown in Fig. 6a and b, Mdivi-1 pretreatment almost completely blocked the elevation of Drp1 phosphorylation at Ser616 induced by acetaldehyde, while significantly ameliorating the reduction of MTT activity in acetaldehyde-treated cells, suggesting that the phosphorylation of Drp1 at Ser616 was closely associated with acetaldehyde-induced cytotoxicity.

Fig. 6
figure 6

The phosphorylation of Drp1 at Ser616 is closely associated with acetaldehyde-induced cytotoxicity. a SH-SY5Y cells pre-incubated with 25 μmol/L Mdivi-1 for 24 h were treated with 500 μmol/L acetaldehyde for 4 h. The protein levels of p-Drp1 (Ser616) and Drp1 were determined by Western blot analyses. Data represent the mean ± SD of 3 independent experiments. b SH-SY5Y cells were exposed to 25 μmol/L Mdivi-1 for 24 h and then 500 μmol/L acetaldehyde for 8 h. MTT assay was then performed. Data represent the mean ± SD of 6 independent experiments. *p < 0.05 versus control, #p < 0.05 versus acetaldehyde-treated cells

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Interestingly, the phosphorylation of Drp1 induced by acetaldehyde was also attenuated by 3-MA pretreatment (Fig. S5). Further investigation is required to understand the significance of the interaction between autophagy responses and cell signals leading to Drp1 phosphorylation. Nonetheless, Drp1 phosphorylation is a critical event in the cytotoxicity induced by acetaldehyde.

Inhibition of Oxidative Stress Attenuates the Mitophagy and Cytotoxicity Induced by Acetaldehyde

Mitochondria are the major source of intracellular ROS. Dysfunction of mitochondria has been shown to cause intracellular redox imbalance and oxidative stress [63]. Acetaldehyde has been shown to induce oxidative stress in neuronal cells [12, 14, 20, 64]. As shown in Fig. 7a, the exposure of 500 μmol/L acetaldehyde caused a quick and pronounced increase of intracellular ROS in SH-SY5Y cells, while pretreatment of antioxidant NAC significantly inhibited acetaldehyde-induced ROS production. Furthermore, the decrease of COXIV in acetaldehyde-treated cells was mitigated by NAC pretreatment (Fig. 7b), suggesting that the reduction of mitochondrial mass induced by acetaldehyde was attenuated by inhibition of oxidative stress. The effect of NAC on cell viability was further examined. As shown in Fig. 7c, NAC pretreatment markedly alleviated the suppression of cell activity induced by acetaldehyde. These results indicated that ROS-mediated cell signaling is a key mediator in acetaldehyde-induced loss of mitochondrial mass and cytotoxicity.

Fig. 7
figure 7

Inhibition of oxidative stress attenuates the effects of acetaldehyde on mitochondrial mass and cytotoxicity. a SH-SY5Y cells pre-incubated with 1 mmol/L NAC for 1 h were treated with 500 μmol/L acetaldehyde for 0.5 h. The cells were then stained with DCFH-DA and the fluorescence intensity was measured. b SH-SY5Y cells pre-incubated with 1 mmol/L NAC for 1 h were treated with 500 μmol/L acetaldehyde for 8 h. The protein level of COXIV was determined by Western blot analyses. Data represent the mean ± SD of 3 independent experiments. c SH-SY5Y cells pre-incubated with 1 mmol/L NAC for 1 h were treated with 500 μmol/L acetaldehyde for 24 h. MTT assay was then performed. Data represent the mean ± SD of 6 independent experiments. *p < 0.05 versus control, #p < 0.05 versus acetaldehyde-treated cells

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Meanwhile, NAC pretreatment decreased acetaldehyde-induced elevation of Beclin1 protein level by approximately 30% (Fig. 8a). NAC pretreatment also significantly decreased the protein levels of Atg5 and Atg16L1 in acetaldehyde-treated cells, respectively (Fig. 8b and c). Similarly, NAC pretreatment decreased acetaldehyde-induced elevation of PINK1 and Parkin protein levels by 20% and 32%, respectively (Fig. 8d and e). Besides, the accumulation of PINK1 and Parkin on mitochondria induced by acetaldehyde was inhibited by NAC (Fig. 8f). In conclusion, the results demonstrated that ROS-mediated cell signals play important roles in acetaldehyde-induced mitophagic responses.

Fig. 8
figure 8

Inhibition of oxidative stress attenuates the effect of acetaldehyde on the protein levels of mitophagy related proteins. a, b, c, d, e SH-SY5Y cells pre-incubated with 1 mmol/L NAC for 1 h were treated with 500 μmol/L acetaldehyde for 4 h. The protein levels of Beclin1, Atg5, Atg16L1, PINK1, and Parkin were determined by Western blot analyses. f SH-SY5Y cells were exposed to 1 mmol/L NAC for 1 h and then 500 μmol/L acetaldehyde for 6 h. The mitochondrial and cytoplastic fractions were prepared and the protein levels of PINK1 and Parkin were determined by Western blot analyses. Tubulin and COXIV were used as internal controls of cytoplasm and mitochondria, respectively. Data represent the mean ± SD of 3 independent experiments. *p < 0.05 versus control, #p < 0.05 versus acetaldehyde-treated cells

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Discussion

Efficient mitochondrial function is essential for the function of the brain. Mitochondrial damages and deficit of mitophagy have been consistently observed in the neurodegenerative diseases [2, 3]. Although mitophagy is an important mechanism for mitochondrial quality control, overactive mitophagy can lead to excessive mitochondrial degradation and decreased mitochondrial membrane potential, causing cytotoxicity and neuronal death [52]. Previously, it was shown that acetaldehyde treatment significantly elevated the phosphorylation of Drp1, causing mitochondrial fragmentation and dysfunction [20]. The findings of the present study demonstrated that mitophagic responses were activated in acetaldehyde-treated SH-SY5Y cells. Moreover, acetaldehyde treatment led to a reduction of mitochondrial mass, indicating that an excessive mitophagy might occur in acetaldehyde-treated cells, which could potentiate the cytotoxicity. Consistently, the inhibition of autophagy prevented the decline of mitochondrial mass and alleviated the cytotoxicity in acetaldehyde treated cells, suggesting that overactive mitophagy played a pivotal role in acetaldehyde-induced cytotoxicity.

Beclin1 participates in the formation of autophagosomes and is a key molecule in autophagy initiation [43]. Subsequent expansion of autophagosome membrane is mediated by ubiquitin-like conjugating systems and the Atgs [44]. Atg12, a ubiquitin-like modifier required for macroautophagy, covalently attaches to Atg5 and Atg16L1 with the help of ubiquitin-like E1 activating enzyme Atg7 [65, 66]. The ubiquitin-like conjugating system formed by Atg12-Atg5-Atg16L1 plays an indispensable role in the early steps of autophagosome membrane formation [45]. These proteins, which are essential for the initiation and progression of autophagy, have also been shown to mediate the pro-death role of autophagy [67, 68]. Here, it was found that acetaldehyde dramatically increased the protein levels of Beclin1, Atg5, and Atg16L1. Similarly, cardiomyocyte contractile dysfunction caused by acetaldehyde was associated with the elevation of Beclin1 protein levels and the induction of autophagy, which could be ablated by 3-MA treatment [23]. Thus, the elevation of these key players in autophagy appears to promote the cytotoxic effect of acetaldehyde. However, it has to be noted that there are a wide variety of proteins involved in the regulation of autophagic/mitophagic process, only several of which were investigated in the current study. It would be interesting to find out in the future studies whether acetaldehyde affects the expression of other autophagy-related proteins, for example, serine/threonine protein kinase ULK1 (unc-51-like kinase 1), which is a conserved mediator for autophagosome formation.

PINK1 and Parkin function together to sense mitochondrial depolarization and label-damaged mitochondria for autophagic degradation [46]. Parkin is recruited to mitochondria in a PINK1-dependent manner to ubiquitinate several mitochondrial proteins such as mitochondrial fusion proteins (Mfn)1/2 and translocase of outer mitochondrial membrane 20, subsequently recruiting the ubiquitin- and LC3-binding adaptor protein p62 to mitochondria and inducing mitophagy [69, 70]. Thus, PINK1 and Parkin play crucial roles in mitochondrial homeostasis. Recently, it has been reported that Aβ1-42-induced cytotoxicity in SH-SY5Y cells is associated with a marked increase of PINK1 and Parkin expression [71]. Similarly, the activation of PINK1/Parkin signaling causes abnormal mitophagy and cytotoxicity in liver cells treated with silica nanoparticles [72]. In cells treated with toxic metabolic derivative of di-2-ethylhexyl phthalate, PINK1 and Parkin-mediated mitophagic signals lead to overactive mitophagy, causing excessive mitochondrial elimination, loss of mitochondrial function, and cell death [73]. Here, it was demonstrated that acetaldehyde increased the levels of PINK1 and Parkin and promoted the accumulation of PINK1 and Parkin on mitochondria in SH-SY5Y cells. It was possible that PINK1 and Parkin–mediated mitophagic signals also contributed to the overactivation of mitophagic responses and cytotoxicity in acetaldehyde-treated cells. As 3-MA and chloroquine are not specific inhibitors of mitophagy, studies using cells in which mitophagy is inhibited by targeting mitophagy-specific regulators such as pink1 or parkin may further confirm the role of mitophagy in acetaldehyde-induced mitochondrial dysfunction and cytotoxicity.

It is still unclear how acetaldehyde induces mitophagy. Previously, we have reported that the acetaldehyde treatment caused mitochondrial fragmentation via elevating the phosphorylation of Drp1 [20]. Drp1 is a dynamin-related GTPase that plays a central role in mitochondrial fission [61]. The fragmentation of mitochondria is a prerequisite for the induction of mitophagy to eliminate the dysfunctional mitochondria [31, 32]. Thus, the mitophagy observed in acetaldehyde-treated cells might be induced initially as a stress-response mechanism in response to the accumulation of fragmented mitochondria. However, overproduction of fragmented mitochondria can lead to excessive mitophagy and eventually cause cytotoxicity. It was recently reported that in hexavalent chromium-induced hepatotoxicity, Mdivi-1 blocked Drp1 activation and prevented the overactive mitophagy, subsequently ameliorating the cytotoxicity [74]. Similarly, as shown in Fig. 6, pretreatment with Mdivi-1 significantly inhibited the phosphorylation of Drp1 at Ser616 while attenuating acetaldehyde-induced reduction of MTT activity. The results indicated that acetaldehyde-induced Drp1 activation and mitochondrial fragmentation were important contributors to acetaldehyde-induced cytotoxicity. However, additional research is needed to determine whether Drp1 or other fission proteins such as mitochondrial fission 1 (Fis1) is required for mitophagy over-stimulation upon acetaldehyde treatment.

Our previous study demonstrated that oxidative stress is a major factor mediating Drp1 activation and the cytotoxicity caused by acetaldehyde [20]. It has been shown that oxidative stress also contributes to Drp1-mediated excessive mitophagy, leading to persistent mitochondrial loss and energy shortage, eventually resulting in neuronal cytotoxicity [75]. Indeed, ROS are important mediators for autophagy induction to remove the damaged mitochondria and restore mitochondrial homeostasis [76]. However, overproduction of ROS can lead to oxidative stress. When the oxidative stress levels exceed certain thresholds, excessive mitophagy occurs in response to the increased accumulation of mitochondrial damages, promoting cytotoxicity and autophagic cell death instead of exerting protective effects [73, 77]. Antioxidants have been documented to reduce the expression of autophagy-related proteins and inhibit PINK1-Parkin-mediated overactive mitophagy, thus promoting cell survival following exposure to oxidative stress [48, 78]. Here, it was shown that acetaldehyde caused a quick and significant increase of intracellular ROS production. Treatment with antioxidant NAC decreased the production of intracellular ROS and inhibited the elevation of mitophagy related proteins Beclin1, Atg5, Atg16L1, PINK1, and Parkin, while alleviating acetaldehyde-induced cytotoxicity. Moreover, NAC was found to prevent acetaldehyde-induced accumulation of PINK1 and Parkin on mitochondria and attenuated the decline of mitochondrial mass. Therefore, oxidative stress promoted the mitophagic responses and disrupted mitochondrial biogenesis in cells treated with acetaldehyde. These results indicated that oxidative stress was an early event and a key mediator for acetaldehyde-induced overactivation of mitophagy and cytotoxicity.

In summary, acetaldehyde treatment induces ROS production and impairs mitochondrial function, causing the elevation of mitophagy-related proteins and initiation of mitophagic responses (Fig. 9). When the oxidative stress persists, the damaged mitochondria generate more ROS, leading to a vicious cycle that exacerbates the oxidative stress, resulting in the accumulation of defective mitochondria and excessive mitophagy, eventually causing autophagic cytotoxicity. Taken together, oxidative stress and overactivated mitophagy are two major factors implicated in acetaldehyde-induced cytotoxicity. The results also indicated that inhibition of oxidative stress or mitigation of the overactive mitophagy may be beneficial for preventing the neurotoxicity associated with alcohol abuse or acetaldehyde.

Fig. 9
figure 9

The mitochondrial damages and elevation of ROS production activate mitophagic responses in acetaldehyde-treated cells

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