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reperfusion injury via the inhibition of excessive mitophagy
Accepted Manuscript Title: Inhibition of Mitochondrial Calcium Uniporter Protects Neurocytes From Ischemia/Reperfusion Injury Via The Inhibition of Excessive Mitophagy Author: Shoushui Yu Shengfa Zheng Jing Leng Shilei Wang Tao Zhao Jia Liu PII: DOI: Reference:
S0304-3940(16)30411-6 http://dx.doi.org/doi:10.1016/j.neulet.2016.06.012 NSL 32100
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Neuroscience Letters
Received date: Revised date: Accepted date:
20-4-2016 5-6-2016 6-6-2016
Please cite this article as: Shoushui Yu, Shengfa Zheng, Jing Leng, Shilei Wang, Tao Zhao, Jia Liu, Inhibition of Mitochondrial Calcium Uniporter Protects Neurocytes From Ischemia/Reperfusion Injury Via The Inhibition of Excessive Mitophagy, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of Mitochondrial Calcium Uniporter Protects Neurocytes From Ischemia/Reperfusion Injury Via The Inhibition of Excessive Mitophagy Shoushui Yu a, b, Shengfa Zheng b, Jing Leng b, Shilei Wang c, *, Tao Zhao b, Jia Liu c a
Qingdao University, Qingdao 266003, Shandong, China
b
Department of Anesthesiology, Rizhao People's Hospital, Rizhao 276800, Shandong,
China c
Department of Anesthesiology, Affiliated Hospital of Qingdao University,
Huangdao, Qingdao 266003, Shandong, China * Corresponding author： Shilei Wang, Department of Anesthesiology, Affiliated Hospital of Qingdao University, Wutaishanlu 1677, Huangdao, Qingdao, Shandong, 266003, China. E-mail address: [email protected] Tel: +86 532 82919393
Highlights
OGD/RP induced autophagy and mitophagy in SH-SY5Y cells. Inhibition of MCU can inhibit OGD/RP induced mitophagy. Inhibition of MCU protects neurocytes by inhibiting excessive mitophagy.
Abstract Mitophagy plays an important role in mitochondrial quality control and cell survival during the process of ischemia/reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) is the most important channel responsible for Ca2+ influx into mitochondria and Ca2+ signal plays a potential role in modulating mitophagy. However, the effect of MCU on mitophagy during the process of I/R injury remains unknown. This study constructed an in vitro I/R model by subjecting oxygen and glucose deprivation/reperfusion (OGD/RP) model to SH-SY5Y cells to mimic the cerebral I/R injury and aimed to explore the exact effect of MCU on I/R induced mitophagy. The results showed that OGD/RP induced autophagy and mitophagy in SH-SY5Y cells. Ru360, the inhibitor of MCU, improved mitochondrial morphology and fuctional stability as well as cell viability, significantly reduced OGD/RP induced mitophagy as evidenced by the decrease in Beclin-1 and the increase in Tom20 and P62 expression. Whereas spermine, the agonist of MCU, had no significant impact on the expression of those mitophagy related proteins compared with OGD/RP group. This study indicates that inhibition of MCU can inhibit excessive mitophagy and protect the neurocytes from I/R injury. Keywords: Ischemia/reperfusion injury; MCU; mitophagy; neuroprotection. Abbreviations: I/R, ischemia/reperfusion; MCU, mitochondrial calcium uniporter; OGD/RP, oxygen-glucose deprivation/reperfusion; MMP, mitochondrial membrane potential; MPTP, mitochondrial permeability transition pore; TEM, transmission electron microscopy; Ru360, ruthenium 360; Sper, spermine.
1. Introduction Autophagy is a process for the degradation of cytosolic components and organelles through their delivery to the lysosomes. It is a fundamental cellular process promoting cells survival under various environmental stress conditions. As the crucial organelles for energy production and the major source of reactive oxygen species (ROS) in living cells, mitochondria are also the principal targets of I/R injury [1,2]. I/R injury causes ROS overproduction, cellular components damage and triggers mitochondrial dysfunction. The damaged mitochondria are selectively eliminated by the process of mitophagy [3-5]. Thus, mitophagy plays an important role in mitochondrial quality control and cell survival, and it is also essential for the cells to against I/R injury by the timely elimination of dysfunctional mitochondria. Many studies indicated that both defective and excessive mitophagy are linked to cell death [6,7]. The clearance of damaged mitochondria via mitophagy is beneficial for neuron survival [8], while excessive autophagy can result in removal of too many essential organelles, such as mitochondria, and contribute to the autophagic cell death [9]. MCU is the most important channel responsible for Ca2+ influx into mitochondria [10]. Ca2+ signal plays a potential role in modulating and/or triggering mitophagy [11,12]. It has been proved that MCU can regulate the mitochondrial permeability transition pore (MPTP) in isolated cortical mitochondria [13]. MPTP also plays an important role in the induction of mitophagy [14,15]. Furthermore, mitochondria are dynamic organelles and form a dynamic reticulum that is continuously remodeled by fusion and fission events [16]. Fission can produce
metabolically different daughter units. The daughter mitochondria with healthier membrane potential will continue to participate in fusion and fission cycles, whereas the depolarized daughter mitochondria are unlikely to undergo fusion and degraded through mitophagy. Hence, mitochondrial fission may be the upstream event and essential for mitophagy. Interestingly, MCU can regulate the process of mitochondrial fission and fusion [17]. Inhibition of mitochondrial fission or induction of fusion can abolish mitophagy [18,19]. Even though the exact role of MCU in modulating mitophagy remains unclear, we assume that MCU may have a tight relationship with mitophagy and contribute to the final fate of neurons. In this study, we carried out an in vitro I/R model by subjecting OGD/RP model to SH-SY5Y cells to mimic the process of cerebral I/R injury. We observed the over activation of autophagy and mitophagy and clarified its possible role and mechanism of MCU in modulating mitophagy during the process of OGD/RP.
2. Materials and methods 2.1. Cell culture All experiments were conducted and approved by the institution of Ethics Committee of Qingdao University Medical College. SH-SY5Y cells were obtained from the Central Laboratory of the Affiliated Hospital of Qingdao University. Cells were incubated in DMEM/F12 (Gibco, USA)，with 10% fetal bovine serum (Gibco, USA), 1% penicillin–streptomycin (100 U/ml of penicillin G and 100 ug/ml of streptomycin) and 2 mM L-glutamine in a humidified incubator (Sanyo, Osaka, Japan)
with 5% (vol/vol) CO2 / 95% (vol/vol) air and maintained at 37 ℃. The medium was replaced for 2 days with the same fresh solution. 2.2. Experimental groups Cells were randomly divided into 6 groups: Control group: cells were cultured in normal condition without any treatment; OGD/RP group: cells were treated with OGD for 6 h and then returned to normoxic conditions for 24 h; OGD/RP + Ru360 group: cells were pretreated with Ru360 (10 uM) for 30 min before OGD and then returned to normoxic conditions for 24 h; OGD/RP + Sper group: cells were pretreated with spermine (10 uM) for 30 min before OGD and then returned to normoxic conditions for 24 h; Ru360 group: cells were pretreated with Ru360 (10 uM) for 30 min and then cultured without drug for 24 h; Sper group: cells were pretreated with spermine (10 uM) for 30 min and then cultured without drug for 24 h. 2.3. OGD/RP model Cells were washed twice with PBS before OGD. Then the culture medium was replaced by glucose-free EBSS (Gibco, USA) at pH 7.4 and the cultivation bottles were shifted to an incubator with 5% CO2 and 95% N2 at a temperature of 37 ℃ for 6 h. OGD was terminated by replacing the glucose-free EBSS with complete medium and incubating the cells in normoxic conditions for 24 h. 2.4. Cell viability assay Cells were plated at a density of 5 × 104 cells per well in 96-well plates and cultured in a humidified incubator with 5% CO2 at 37 ℃. Cell viability was assayed by using cell counting kit-8 (CCK-8, DoJinDo Molecular Technology Inc, Japan)
according to the instructions. 10 uL of CCK-8 solution was added into each well and incubated for 4 h in the dark at 37 ℃. The absorbance was measured using a microplate reader (Model 550, BIO-RAD, USA) at 450 nm. Culture media without cells was used as blank controls. Following the deduction of the blank controls absorbance, cell viability was expressed as a percentage of the absorbance to that of control group. 2.5. Transmission Electron Microscopy (TEM) observation TEM examination was used to observe OGD/RP induced formation of autophagosomes and Ultrastructural changes of cell organelles. Cells were collected and compacted to solid pallets by centrifugation at 1000 r/min for 5 min. Then the cells were fixed in glutaraldehyde with a 1% (w/v) solution of osmium tetroxide and were embedded in Epon812-Araldite. Finally, ultrathin sections (50 nm thick) were cut and observed under a JEM-1200EX transmission electron microscope (JEOL, Tokyo, Japan). 2.6. Mitochondrial membrane potential (MMP) assay MMP assay kit with JC-1 (C2006, Beyotime Company, China) was used to detect MMP. JC-1 accumulates to form J-aggregates and emits red fluorescence (absorption/emission maxima of 585/590 nm) in the mitochondria with higher membrane
potentials,
yet
JC-1
monomers
emit
green
fluorescence
(absorption/emission maxima of 514/529 nm) in those mitochondria with lower membrane potentials. An increase of the ratio (red/green) was thus interpreted as an increase in MMP [20]. For this assay, different groups of cells were incubated with 5
ug/mL of jc-1 working solution in the dark for 20 min at 37 ℃, washed twice with PBS and observed under a fluorescence microscope (Olympus, Japan). Images were analyzed by using Image J software. 2.7. Western blot Cells were collected and protein extraction was carried out as described previously [21]. Protein concentrations were determined by BCA Protein Assay Kit (Beyotime, China). Each sample equivalent to 30 ug of protein was separated by SDS-PAGE (10% for Beclin-1 and P62, 15% for Tom20) at 80 V for 0.5 h and 120 V for 1 h using the Mini-PROTEAN 3 electrophoresis cell system (Bio-Rad). Proteins were then transferred to polyvinylidene fluoride membranes (Bio-Rad). After being blocked in 5% nonfat milk for 2 h, membranes were incubated overnight at 4 ℃ with the primary antibodies: rabbit anti-Beclin-1 (1:1000, Millipore, AB15417), mouse anti-Tom20 (1:1000, Millipore, MABT166), mouse anti-P62 (1:1000, Millipore, MABC32) and mouse anti-β-actin (1:5000, Zhongshan Golden Bridge Biotechnology, China). Then the membranes were washed with PBST for three times and incubated with rabbit/mouse HRP-conjugated secondary antibody for 1 h. The visualization of bands was performed using ECL chemiluminescence system (VILBER Fusion FX5 Spectra, Limoges, France). Images were quantified and analyzed using Quantity One software (Biosoft, CA) and β-actin served as an internal standard [22]. 2.8. Statistical analysis Statistical analysis was performed of SPSS 18.0 statistics package. Data were expressed as means ± SD and obtained from three independent experiments. One-way
ANOVA with Dunnett’s T3 post-hoc test was applied for multiple comparisons. P < 0.05 was considered statistically significant.
3. Results 3.1. Inhibition of MCU improved cell viability after OGD/RP treatment To investigating the effect of MCU on OGD/RP induced cell injury, CCK-8 assay was performed to evaluate the cell viability of different groups. It was found that cell viability was significantly lower in OGD/RP group, OGD/RP + Ru360 group and OGD/RP + Sper group compared with control group, no significant difference was observed in Ru360 and Sper group. Cell viability in OGD/RP + Ru360 group was higher, compared with OGD/RP group (Fig. 1 A). These results indicated that OGD/RP treatment caused serious cell injury and inhibition of MCU by Ru360 could attenuate OGD/RP induced cell death effectively, while Ru360 and spermine themselves had no effect on cell viability under normal conditions. 3.2. OGD/RP induced autophagy and mitophagy TEM is a valid and important method for the observation of autophagy [23]. To determine the involvement of autophagy and mitophagy after OGD/RP treatment, TEM examination was used. After OGD/RP treatment, numerous of autophagic vacuoles (AVs) with characteristic morphological features of autophagosomes were detected (Fig. 2 B and D). In addition, some partially degraded mitochondria surrounding by double membranes of typical autophagosomes were observed (Fig. 2 B and D, white arrows). The number of mitochondria was also reduced compared
with control group. These ultrastructural changes indicated the involvement of autophagy and mitophagy after OGD/RP treatment. Furthermore, we examined the expression of Beclin-1, P62 and Tom20 in different groups. Data showed that the expression of Beclin-1 was increased and the expressions of P62 and Tom20 were decreased in OGD/RP group (Fig. 3 A-D), which suggested that autophagic flux was increased and mitochondria degradation through mitophagy was promoted. Taken together, these results indicated that autophagy and mitophagy were activated after OGD/RP treatment. 3.3. Inhibition of MCU reduced OGD/RP induced autophagy and mitophagy To investigate the effect of MCU in modulating OGD/RP induced autophagy and mitophagy, we measured the expressions of Beclin-1, P62 and Tom20 after pretreating with MCU inhibitor Ru360 and agonist spermine before OGD/RP treatment, respectively. As shown in Fig. 3 (A-D), treatment with Ru360 significantly reduced Beclin-1 expression, increased Tom20 and P62 expressions in OGD/RP + Ru360 group compared with OGD/RP group, which suggested that Ru360 pretreatment inhibited OGD/RP induced autophagy and mitophagy. The expressions of these proteins in OGD/RP + Sper group were similar to those in OGD/RP group, which suggested that spermine pretreatment could not further enhance autophagy and mitophagy activation in SH-SY5Y cells after being treated with OGD for 6 h and reperfusion for 24 h. TEM micrographs also showed the reduced amount of AVs and double-membrane autophagosomes in OGD/RP + Ru360 group (Fig. 2 C), which further suggested the reduction of autophagy and mitophagy after Ru360 pretreatment.
Taken together, these results indicated that inhibition of MCU reduced OGD/RP induced autophagy and mitophagy. 3.4. Inhibition of MCU improved mitochondrial morphology and function Mitochondrial function is critical in attenuating I/R induced cerebral injury. To further assess the effect of MCU on mitochondrial function, we measured the MMP in different groups. After OGD/RP treatment, the MMP decreased obviously, Ru360 significantly recovered the MMP in OGD/RP + Ru360 group (Fig. 1 B). Through TEM observation, we found that in OGD/RP group and OGD/RP + Sper group, the mitochondria were swelled and dilated, mitochondria cristae were ambiguous, and an extensive vacuolization was formed, which lost typical mitochondrial structures (Fig. 2 B and D). In contrast, the presences of Ru360 protected the integrality of mitochondrial ultrastructures. Clear cristae could still be observed and the amount of AVs was decreased obviously in OGD/RP + Ru360 group, which showed similar mitochondrial morphology compared with control group (Fig. 2 A and C). These results indicated that the blockage of MCU protected mitochondrial morphology and function after OGD/RP treatment.
4. Discussion SH-SY5Y cells are widely used as an in vitro model to study neuronal injury due to their remarkable features of normal neuronal cells in neurochemical, morphological and electrophysiological properties [24]. OGD/RP model is also well-established for ischemic studies. Therefore, an OGD/RP model of SH-SY5Y cells was carried to
imitate I/R injury in brain in the present study. It was reported that inhibition of MCU can reduce Ca2+ transport into mitochondria and protect the brain from I/R injury[17]. In the present study, we found that pretreated with Ru360 improved mitochondrial morphology and function stability as well as cell viability obviously, which further confirmed the idea that inhibition of MCU can protect neurocytes from I/R injury. Mitophagy is a selective form of autophagy. We explored the involvement of mitophagy and detected the effect of MCU on mitophagy during the process of OGD/RP. Through TEM observation, mitophagy was characterized by the presence of abnormal mitochondria within autophagosomes or fused with AVs. These morphological features suggested the involvement of mitophagy after OGD/RP treatment. Beclin-1, an autophagy related protein, is formerly found to promote autophagy [25]. The decline of Beclin-1 can decrease autophagic activity in cells [26]. P62, an ubiquitin-binding adaptor protein, can recruit to depolarized mitochondria and facilitate recruitment of damaged mitochondria to autophagosomes by binding to LC3. The declining abundance of P62 is always used as a marker of increased autophagic flux [27]. Tom20, a mitochondrial outer membrane protein, is an important receptor subunit of the Tom complex and the decreased Tom20 level always indicates the drop of mitochondria number [28]. The relative number of mitochondria can be assessed to reflect the dynamics of mitophagy [29]. Our data showed that Tom20 expression was decreased after OGD/RP treatment, which further suggested that autophagy-related mitochondrial loss occured after OGD/RP treatment. We also found that the expression of Beclin-1 decreased and P62 and Tom20 increased in OGD/RP + Ru360
group compared with OGD/RP group, which suggested that Inhibition of MCU inhibited OGD/RP induced mitophagy. TEM micrographs also showed less damaged mitochondria ultrastructures and reduced number of mitochondria fusing with autophagosomes in OGD/RP + Ru360 group compared with OGD/RP group, which further suggested that inhibition of MCU protected mitochondrial morphology and inhibited mitophagy after OGD/RP treatment. In contrast, 10 uM spermine had no obvious effect on mitophagy and mitochondrial morphology in our model. We suspected that OGD for 6 h with the subsequent reperfusion for 24 h had completely stimulated MCU activation and induced excessive mitochondrial damage, further stimulation of MCU could not enhance mitochondrial damage and mitophagy anymore. Inhibition of MCU by Ru360 can inhibit mitochondrial fission[17]. A recent study reported that inhibition of mitochondrial fission can promote the expression of Bcl-2 [30]. Thus, we assume that Ru360 can inhibit mitochondrial fission and promote the expression of Bcl-2. It has been demonstrated that Bcl-2 and Bcl-XL can bind to Beclin-1 at the BH3 region and prevent formation of the omegasome, an ER-associated platform for the initial formation of pre-autophagosomal vesicles [31]. Therefore, the increased Bcl-2 can bind to Beclin-1 to form Beclin-1-Bcl-2/Bcl-XL complexes and inhibit autophagy activity [32]. This may be one aspect of the mechanisms of MCU in modulating mitophagy. The PINK1-Parkin pathway is important in regulating clearance of dysfunctional mitochondria via mitophagy[3]. In our study, OGD/RP caused an obvious loss of
MMP in SH-SY5Y cells (Fig. 1 B). The loss of MMP stabilized intact PINK1 on the mitochondrial outer membrane. The accumulation of PINK1 on the mitochondrial surface induced translocation of Parkin from the cytosol to damaged mitochondria, then the recruited Parkin promoted the degradation of mitochondria through mitophagy [33]. Thus, the loss of MMP might be the event that activated this pathway acutely. Conversely, Ru360 pretreatment reduced the loss of MMP induced by OGD/RP (Fig. 1 B). Ru360 inhibited the activation of MCU and reduced calcium overload and inhibited the opening of MPTP thus to preserve the MMP at a high level. As a result, Ru360 reduced the recruitment of Parkin to mitochondria and inhibited the elemilation of mitochondria via mitopgagy. Therefore, it is possilbe that MCU can modulate mitophagy via PINK1-Parkin pathway with the regulation of MMP.
5. Conclusion Collectively, the present study indicates that MCU can modulate OGD/RP induced
mitophagy. Inhibition of MCU can inhibit excessive mitophagy by reducing mitochondria fission and maintaining mitochondrial morphology and function, thus protecting the neurocytes from I/R injury.
Conflict of Interest The authors declare no financial conflict of interest.
Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC) Grant Number 81371448. The authors gratefully acknowledge Central Laboratory of the Affiliated Hospital of Qingdao University Medical College for equipment support and technical assistance.
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Figure legends Fig. 1. Measurements of cell viability and MMP (A) Cell viability was measured by using cell counting kit-8 (CCK-8, DoJinDo Molecular Technology Inc, Japan). (B) MMP was measured by using mitochondrial membrane potential assay kit with JC-1. Images were analyzed using Image J software. Data are expressed as mean ± SD, n = 6. ***P< 0.001 vs. Control group; ###
P < 0.001 vs. OGD/RP group.
Fig. 2. Comparison of Ultrastructural changes in SH-SY5Y cells Images were captured using a JEM-1200EX transmission electron microscope (Jeol, Tokyo, Japan). Black arrows represent mitochondria structures (A, B, C, E, F); White arrows represent autophagy and mitophagy structures (B, D). N = nucleus, scale bar = 0.5 um. A: Control group; B: OGD/RP group; C: OGD/RP + Ru360 group; D: OGD/RP + Sper group; E: Ru360 group; F: Sper group. Fig. 3. Comparison of the expressions of Tom20, P62 and Beclin-1 by Western blot (A) Expressions of Tom20, P62 and Beclin-1 in different groups by western blot . Bands were analyzed using Image J software and β-actin was used as a loading control. Quantification of (B) Tom20, (C) P62 and (D) Beclin-1 by Western blot. Protein levels are relative to the Control group. Results are typical of three independent experiments. Data are represented as mean ± SD, (n = 6). ***P<0.001 vs. Control group; ###P < 0.001 vs. OGD/RP group.
S0304-3940(16)30411-6 http://dx.doi.org/doi:10.1016/j.neulet.2016.06.012 NSL 32100
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
20-4-2016 5-6-2016 6-6-2016
Please cite this article as: Shoushui Yu, Shengfa Zheng, Jing Leng, Shilei Wang, Tao Zhao, Jia Liu, Inhibition of Mitochondrial Calcium Uniporter Protects Neurocytes From Ischemia/Reperfusion Injury Via The Inhibition of Excessive Mitophagy, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2016.06.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Inhibition of Mitochondrial Calcium Uniporter Protects Neurocytes From Ischemia/Reperfusion Injury Via The Inhibition of Excessive Mitophagy Shoushui Yu a, b, Shengfa Zheng b, Jing Leng b, Shilei Wang c, *, Tao Zhao b, Jia Liu c a
Qingdao University, Qingdao 266003, Shandong, China
b
Department of Anesthesiology, Rizhao People's Hospital, Rizhao 276800, Shandong,
China c
Department of Anesthesiology, Affiliated Hospital of Qingdao University,
Huangdao, Qingdao 266003, Shandong, China * Corresponding author： Shilei Wang, Department of Anesthesiology, Affiliated Hospital of Qingdao University, Wutaishanlu 1677, Huangdao, Qingdao, Shandong, 266003, China. E-mail address: [email protected] Tel: +86 532 82919393
Highlights
OGD/RP induced autophagy and mitophagy in SH-SY5Y cells. Inhibition of MCU can inhibit OGD/RP induced mitophagy. Inhibition of MCU protects neurocytes by inhibiting excessive mitophagy.
Abstract Mitophagy plays an important role in mitochondrial quality control and cell survival during the process of ischemia/reperfusion (I/R) injury. Mitochondrial calcium uniporter (MCU) is the most important channel responsible for Ca2+ influx into mitochondria and Ca2+ signal plays a potential role in modulating mitophagy. However, the effect of MCU on mitophagy during the process of I/R injury remains unknown. This study constructed an in vitro I/R model by subjecting oxygen and glucose deprivation/reperfusion (OGD/RP) model to SH-SY5Y cells to mimic the cerebral I/R injury and aimed to explore the exact effect of MCU on I/R induced mitophagy. The results showed that OGD/RP induced autophagy and mitophagy in SH-SY5Y cells. Ru360, the inhibitor of MCU, improved mitochondrial morphology and fuctional stability as well as cell viability, significantly reduced OGD/RP induced mitophagy as evidenced by the decrease in Beclin-1 and the increase in Tom20 and P62 expression. Whereas spermine, the agonist of MCU, had no significant impact on the expression of those mitophagy related proteins compared with OGD/RP group. This study indicates that inhibition of MCU can inhibit excessive mitophagy and protect the neurocytes from I/R injury. Keywords: Ischemia/reperfusion injury; MCU; mitophagy; neuroprotection. Abbreviations: I/R, ischemia/reperfusion; MCU, mitochondrial calcium uniporter; OGD/RP, oxygen-glucose deprivation/reperfusion; MMP, mitochondrial membrane potential; MPTP, mitochondrial permeability transition pore; TEM, transmission electron microscopy; Ru360, ruthenium 360; Sper, spermine.
1. Introduction Autophagy is a process for the degradation of cytosolic components and organelles through their delivery to the lysosomes. It is a fundamental cellular process promoting cells survival under various environmental stress conditions. As the crucial organelles for energy production and the major source of reactive oxygen species (ROS) in living cells, mitochondria are also the principal targets of I/R injury [1,2]. I/R injury causes ROS overproduction, cellular components damage and triggers mitochondrial dysfunction. The damaged mitochondria are selectively eliminated by the process of mitophagy [3-5]. Thus, mitophagy plays an important role in mitochondrial quality control and cell survival, and it is also essential for the cells to against I/R injury by the timely elimination of dysfunctional mitochondria. Many studies indicated that both defective and excessive mitophagy are linked to cell death [6,7]. The clearance of damaged mitochondria via mitophagy is beneficial for neuron survival [8], while excessive autophagy can result in removal of too many essential organelles, such as mitochondria, and contribute to the autophagic cell death [9]. MCU is the most important channel responsible for Ca2+ influx into mitochondria [10]. Ca2+ signal plays a potential role in modulating and/or triggering mitophagy [11,12]. It has been proved that MCU can regulate the mitochondrial permeability transition pore (MPTP) in isolated cortical mitochondria [13]. MPTP also plays an important role in the induction of mitophagy [14,15]. Furthermore, mitochondria are dynamic organelles and form a dynamic reticulum that is continuously remodeled by fusion and fission events [16]. Fission can produce
metabolically different daughter units. The daughter mitochondria with healthier membrane potential will continue to participate in fusion and fission cycles, whereas the depolarized daughter mitochondria are unlikely to undergo fusion and degraded through mitophagy. Hence, mitochondrial fission may be the upstream event and essential for mitophagy. Interestingly, MCU can regulate the process of mitochondrial fission and fusion [17]. Inhibition of mitochondrial fission or induction of fusion can abolish mitophagy [18,19]. Even though the exact role of MCU in modulating mitophagy remains unclear, we assume that MCU may have a tight relationship with mitophagy and contribute to the final fate of neurons. In this study, we carried out an in vitro I/R model by subjecting OGD/RP model to SH-SY5Y cells to mimic the process of cerebral I/R injury. We observed the over activation of autophagy and mitophagy and clarified its possible role and mechanism of MCU in modulating mitophagy during the process of OGD/RP.
2. Materials and methods 2.1. Cell culture All experiments were conducted and approved by the institution of Ethics Committee of Qingdao University Medical College. SH-SY5Y cells were obtained from the Central Laboratory of the Affiliated Hospital of Qingdao University. Cells were incubated in DMEM/F12 (Gibco, USA)，with 10% fetal bovine serum (Gibco, USA), 1% penicillin–streptomycin (100 U/ml of penicillin G and 100 ug/ml of streptomycin) and 2 mM L-glutamine in a humidified incubator (Sanyo, Osaka, Japan)
with 5% (vol/vol) CO2 / 95% (vol/vol) air and maintained at 37 ℃. The medium was replaced for 2 days with the same fresh solution. 2.2. Experimental groups Cells were randomly divided into 6 groups: Control group: cells were cultured in normal condition without any treatment; OGD/RP group: cells were treated with OGD for 6 h and then returned to normoxic conditions for 24 h; OGD/RP + Ru360 group: cells were pretreated with Ru360 (10 uM) for 30 min before OGD and then returned to normoxic conditions for 24 h; OGD/RP + Sper group: cells were pretreated with spermine (10 uM) for 30 min before OGD and then returned to normoxic conditions for 24 h; Ru360 group: cells were pretreated with Ru360 (10 uM) for 30 min and then cultured without drug for 24 h; Sper group: cells were pretreated with spermine (10 uM) for 30 min and then cultured without drug for 24 h. 2.3. OGD/RP model Cells were washed twice with PBS before OGD. Then the culture medium was replaced by glucose-free EBSS (Gibco, USA) at pH 7.4 and the cultivation bottles were shifted to an incubator with 5% CO2 and 95% N2 at a temperature of 37 ℃ for 6 h. OGD was terminated by replacing the glucose-free EBSS with complete medium and incubating the cells in normoxic conditions for 24 h. 2.4. Cell viability assay Cells were plated at a density of 5 × 104 cells per well in 96-well plates and cultured in a humidified incubator with 5% CO2 at 37 ℃. Cell viability was assayed by using cell counting kit-8 (CCK-8, DoJinDo Molecular Technology Inc, Japan)
according to the instructions. 10 uL of CCK-8 solution was added into each well and incubated for 4 h in the dark at 37 ℃. The absorbance was measured using a microplate reader (Model 550, BIO-RAD, USA) at 450 nm. Culture media without cells was used as blank controls. Following the deduction of the blank controls absorbance, cell viability was expressed as a percentage of the absorbance to that of control group. 2.5. Transmission Electron Microscopy (TEM) observation TEM examination was used to observe OGD/RP induced formation of autophagosomes and Ultrastructural changes of cell organelles. Cells were collected and compacted to solid pallets by centrifugation at 1000 r/min for 5 min. Then the cells were fixed in glutaraldehyde with a 1% (w/v) solution of osmium tetroxide and were embedded in Epon812-Araldite. Finally, ultrathin sections (50 nm thick) were cut and observed under a JEM-1200EX transmission electron microscope (JEOL, Tokyo, Japan). 2.6. Mitochondrial membrane potential (MMP) assay MMP assay kit with JC-1 (C2006, Beyotime Company, China) was used to detect MMP. JC-1 accumulates to form J-aggregates and emits red fluorescence (absorption/emission maxima of 585/590 nm) in the mitochondria with higher membrane
potentials,
yet
JC-1
monomers
emit
green
fluorescence
(absorption/emission maxima of 514/529 nm) in those mitochondria with lower membrane potentials. An increase of the ratio (red/green) was thus interpreted as an increase in MMP [20]. For this assay, different groups of cells were incubated with 5
ug/mL of jc-1 working solution in the dark for 20 min at 37 ℃, washed twice with PBS and observed under a fluorescence microscope (Olympus, Japan). Images were analyzed by using Image J software. 2.7. Western blot Cells were collected and protein extraction was carried out as described previously [21]. Protein concentrations were determined by BCA Protein Assay Kit (Beyotime, China). Each sample equivalent to 30 ug of protein was separated by SDS-PAGE (10% for Beclin-1 and P62, 15% for Tom20) at 80 V for 0.5 h and 120 V for 1 h using the Mini-PROTEAN 3 electrophoresis cell system (Bio-Rad). Proteins were then transferred to polyvinylidene fluoride membranes (Bio-Rad). After being blocked in 5% nonfat milk for 2 h, membranes were incubated overnight at 4 ℃ with the primary antibodies: rabbit anti-Beclin-1 (1:1000, Millipore, AB15417), mouse anti-Tom20 (1:1000, Millipore, MABT166), mouse anti-P62 (1:1000, Millipore, MABC32) and mouse anti-β-actin (1:5000, Zhongshan Golden Bridge Biotechnology, China). Then the membranes were washed with PBST for three times and incubated with rabbit/mouse HRP-conjugated secondary antibody for 1 h. The visualization of bands was performed using ECL chemiluminescence system (VILBER Fusion FX5 Spectra, Limoges, France). Images were quantified and analyzed using Quantity One software (Biosoft, CA) and β-actin served as an internal standard [22]. 2.8. Statistical analysis Statistical analysis was performed of SPSS 18.0 statistics package. Data were expressed as means ± SD and obtained from three independent experiments. One-way
ANOVA with Dunnett’s T3 post-hoc test was applied for multiple comparisons. P < 0.05 was considered statistically significant.
3. Results 3.1. Inhibition of MCU improved cell viability after OGD/RP treatment To investigating the effect of MCU on OGD/RP induced cell injury, CCK-8 assay was performed to evaluate the cell viability of different groups. It was found that cell viability was significantly lower in OGD/RP group, OGD/RP + Ru360 group and OGD/RP + Sper group compared with control group, no significant difference was observed in Ru360 and Sper group. Cell viability in OGD/RP + Ru360 group was higher, compared with OGD/RP group (Fig. 1 A). These results indicated that OGD/RP treatment caused serious cell injury and inhibition of MCU by Ru360 could attenuate OGD/RP induced cell death effectively, while Ru360 and spermine themselves had no effect on cell viability under normal conditions. 3.2. OGD/RP induced autophagy and mitophagy TEM is a valid and important method for the observation of autophagy [23]. To determine the involvement of autophagy and mitophagy after OGD/RP treatment, TEM examination was used. After OGD/RP treatment, numerous of autophagic vacuoles (AVs) with characteristic morphological features of autophagosomes were detected (Fig. 2 B and D). In addition, some partially degraded mitochondria surrounding by double membranes of typical autophagosomes were observed (Fig. 2 B and D, white arrows). The number of mitochondria was also reduced compared
with control group. These ultrastructural changes indicated the involvement of autophagy and mitophagy after OGD/RP treatment. Furthermore, we examined the expression of Beclin-1, P62 and Tom20 in different groups. Data showed that the expression of Beclin-1 was increased and the expressions of P62 and Tom20 were decreased in OGD/RP group (Fig. 3 A-D), which suggested that autophagic flux was increased and mitochondria degradation through mitophagy was promoted. Taken together, these results indicated that autophagy and mitophagy were activated after OGD/RP treatment. 3.3. Inhibition of MCU reduced OGD/RP induced autophagy and mitophagy To investigate the effect of MCU in modulating OGD/RP induced autophagy and mitophagy, we measured the expressions of Beclin-1, P62 and Tom20 after pretreating with MCU inhibitor Ru360 and agonist spermine before OGD/RP treatment, respectively. As shown in Fig. 3 (A-D), treatment with Ru360 significantly reduced Beclin-1 expression, increased Tom20 and P62 expressions in OGD/RP + Ru360 group compared with OGD/RP group, which suggested that Ru360 pretreatment inhibited OGD/RP induced autophagy and mitophagy. The expressions of these proteins in OGD/RP + Sper group were similar to those in OGD/RP group, which suggested that spermine pretreatment could not further enhance autophagy and mitophagy activation in SH-SY5Y cells after being treated with OGD for 6 h and reperfusion for 24 h. TEM micrographs also showed the reduced amount of AVs and double-membrane autophagosomes in OGD/RP + Ru360 group (Fig. 2 C), which further suggested the reduction of autophagy and mitophagy after Ru360 pretreatment.
Taken together, these results indicated that inhibition of MCU reduced OGD/RP induced autophagy and mitophagy. 3.4. Inhibition of MCU improved mitochondrial morphology and function Mitochondrial function is critical in attenuating I/R induced cerebral injury. To further assess the effect of MCU on mitochondrial function, we measured the MMP in different groups. After OGD/RP treatment, the MMP decreased obviously, Ru360 significantly recovered the MMP in OGD/RP + Ru360 group (Fig. 1 B). Through TEM observation, we found that in OGD/RP group and OGD/RP + Sper group, the mitochondria were swelled and dilated, mitochondria cristae were ambiguous, and an extensive vacuolization was formed, which lost typical mitochondrial structures (Fig. 2 B and D). In contrast, the presences of Ru360 protected the integrality of mitochondrial ultrastructures. Clear cristae could still be observed and the amount of AVs was decreased obviously in OGD/RP + Ru360 group, which showed similar mitochondrial morphology compared with control group (Fig. 2 A and C). These results indicated that the blockage of MCU protected mitochondrial morphology and function after OGD/RP treatment.
4. Discussion SH-SY5Y cells are widely used as an in vitro model to study neuronal injury due to their remarkable features of normal neuronal cells in neurochemical, morphological and electrophysiological properties [24]. OGD/RP model is also well-established for ischemic studies. Therefore, an OGD/RP model of SH-SY5Y cells was carried to
imitate I/R injury in brain in the present study. It was reported that inhibition of MCU can reduce Ca2+ transport into mitochondria and protect the brain from I/R injury[17]. In the present study, we found that pretreated with Ru360 improved mitochondrial morphology and function stability as well as cell viability obviously, which further confirmed the idea that inhibition of MCU can protect neurocytes from I/R injury. Mitophagy is a selective form of autophagy. We explored the involvement of mitophagy and detected the effect of MCU on mitophagy during the process of OGD/RP. Through TEM observation, mitophagy was characterized by the presence of abnormal mitochondria within autophagosomes or fused with AVs. These morphological features suggested the involvement of mitophagy after OGD/RP treatment. Beclin-1, an autophagy related protein, is formerly found to promote autophagy [25]. The decline of Beclin-1 can decrease autophagic activity in cells [26]. P62, an ubiquitin-binding adaptor protein, can recruit to depolarized mitochondria and facilitate recruitment of damaged mitochondria to autophagosomes by binding to LC3. The declining abundance of P62 is always used as a marker of increased autophagic flux [27]. Tom20, a mitochondrial outer membrane protein, is an important receptor subunit of the Tom complex and the decreased Tom20 level always indicates the drop of mitochondria number [28]. The relative number of mitochondria can be assessed to reflect the dynamics of mitophagy [29]. Our data showed that Tom20 expression was decreased after OGD/RP treatment, which further suggested that autophagy-related mitochondrial loss occured after OGD/RP treatment. We also found that the expression of Beclin-1 decreased and P62 and Tom20 increased in OGD/RP + Ru360
group compared with OGD/RP group, which suggested that Inhibition of MCU inhibited OGD/RP induced mitophagy. TEM micrographs also showed less damaged mitochondria ultrastructures and reduced number of mitochondria fusing with autophagosomes in OGD/RP + Ru360 group compared with OGD/RP group, which further suggested that inhibition of MCU protected mitochondrial morphology and inhibited mitophagy after OGD/RP treatment. In contrast, 10 uM spermine had no obvious effect on mitophagy and mitochondrial morphology in our model. We suspected that OGD for 6 h with the subsequent reperfusion for 24 h had completely stimulated MCU activation and induced excessive mitochondrial damage, further stimulation of MCU could not enhance mitochondrial damage and mitophagy anymore. Inhibition of MCU by Ru360 can inhibit mitochondrial fission[17]. A recent study reported that inhibition of mitochondrial fission can promote the expression of Bcl-2 [30]. Thus, we assume that Ru360 can inhibit mitochondrial fission and promote the expression of Bcl-2. It has been demonstrated that Bcl-2 and Bcl-XL can bind to Beclin-1 at the BH3 region and prevent formation of the omegasome, an ER-associated platform for the initial formation of pre-autophagosomal vesicles [31]. Therefore, the increased Bcl-2 can bind to Beclin-1 to form Beclin-1-Bcl-2/Bcl-XL complexes and inhibit autophagy activity [32]. This may be one aspect of the mechanisms of MCU in modulating mitophagy. The PINK1-Parkin pathway is important in regulating clearance of dysfunctional mitochondria via mitophagy[3]. In our study, OGD/RP caused an obvious loss of
MMP in SH-SY5Y cells (Fig. 1 B). The loss of MMP stabilized intact PINK1 on the mitochondrial outer membrane. The accumulation of PINK1 on the mitochondrial surface induced translocation of Parkin from the cytosol to damaged mitochondria, then the recruited Parkin promoted the degradation of mitochondria through mitophagy [33]. Thus, the loss of MMP might be the event that activated this pathway acutely. Conversely, Ru360 pretreatment reduced the loss of MMP induced by OGD/RP (Fig. 1 B). Ru360 inhibited the activation of MCU and reduced calcium overload and inhibited the opening of MPTP thus to preserve the MMP at a high level. As a result, Ru360 reduced the recruitment of Parkin to mitochondria and inhibited the elemilation of mitochondria via mitopgagy. Therefore, it is possilbe that MCU can modulate mitophagy via PINK1-Parkin pathway with the regulation of MMP.
5. Conclusion Collectively, the present study indicates that MCU can modulate OGD/RP induced
mitophagy. Inhibition of MCU can inhibit excessive mitophagy by reducing mitochondria fission and maintaining mitochondrial morphology and function, thus protecting the neurocytes from I/R injury.
Conflict of Interest The authors declare no financial conflict of interest.
Acknowledgments This work was supported by the National Natural Science Foundation of China (NSFC) Grant Number 81371448. The authors gratefully acknowledge Central Laboratory of the Affiliated Hospital of Qingdao University Medical College for equipment support and technical assistance.
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Figure legends Fig. 1. Measurements of cell viability and MMP (A) Cell viability was measured by using cell counting kit-8 (CCK-8, DoJinDo Molecular Technology Inc, Japan). (B) MMP was measured by using mitochondrial membrane potential assay kit with JC-1. Images were analyzed using Image J software. Data are expressed as mean ± SD, n = 6. ***P< 0.001 vs. Control group; ###
P < 0.001 vs. OGD/RP group.
Fig. 2. Comparison of Ultrastructural changes in SH-SY5Y cells Images were captured using a JEM-1200EX transmission electron microscope (Jeol, Tokyo, Japan). Black arrows represent mitochondria structures (A, B, C, E, F); White arrows represent autophagy and mitophagy structures (B, D). N = nucleus, scale bar = 0.5 um. A: Control group; B: OGD/RP group; C: OGD/RP + Ru360 group; D: OGD/RP + Sper group; E: Ru360 group; F: Sper group. Fig. 3. Comparison of the expressions of Tom20, P62 and Beclin-1 by Western blot (A) Expressions of Tom20, P62 and Beclin-1 in different groups by western blot . Bands were analyzed using Image J software and β-actin was used as a loading control. Quantification of (B) Tom20, (C) P62 and (D) Beclin-1 by Western blot. Protein levels are relative to the Control group. Results are typical of three independent experiments. Data are represented as mean ± SD, (n = 6). ***P<0.001 vs. Control group; ###P < 0.001 vs. OGD/RP group.