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Therapeutic effects of melatonin on cerebral ischemia reperfusion injury: Role of Yap-OPA1 signaling pathway and mitochondrial fusion
Biomedicine & Pharmacotherapy 110 (2019) 203–212
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Therapeutic effects of melatonin on cerebral ischemia reperfusion injury: Role of Yap-OPA1 signaling pathway and mitochondrial fusion Na Weia,b,c,d, Yuehua Pua,b,c,d, Zhonghua Yanga,b,c,d, Yuesong Panb, Liping Liua,b,c,d,
T
⁎
a
Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China China National Clinical Research Center for Neurological Diseases, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China c Center of Stroke, Beijing Institute for Brain Disorders, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China d Beijing Key Laboratory of Translational Medicine for Cerebrovascular Disease, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Melatonin OPA1 Mitochondrial fusion Yap-Hippo pathway Reperfusion stress
The role of OPA1-related mitochondrial fusion in brain reperfusion stress has remained elusive. The aim of our study is to explore whether melatonin alleviates cerebral IR injury by modulating OPA1-related mitochondrial fusion. We found that melatonin reduced infarct area and suppressed neuron death during reperfusion stress. Biological studies have revealed that IR-inhibited mitochondrial fusion was largely reversed by melatonin via upregulated OPA1 expression. Knocking down OPA1 abrogated the protective effects of melatonin on mitochondrial energy metabolism and mitochondrial apoptosis. In addition, we also found that melatonin modified OPA1 expression via the Yap-Hippo pathway; blockade of the Yap-Hippo pathway induced neuron death and mitochondrial damage despite treatment with melatonin. Altogether, our data demonstrated that cerebral IR injury is closely associated with defective OPA1-related mitochondrial fusion. Melatonin supplementation enhances OPA1-related mitochondrial fusion by activating the Yap-Hippo pathway, ultimately reducing brain reperfusion stress.
1. Introduction
phosphorylation and increased ROS production [17]. Moreover, damaged mitochondria liberate pro-apoptotic factors such as cyt-c in the nucleus, where cyt-c launches the mitochondria-dependent apoptosis pathway [18]. Based on previous findings, mitochondrial fission is recognized as a potential target to alleviate cerebral IR injury. In contrast to mitochondrial fission, mitochondrial fusion is the repair system that corrects excessive mitochondrial fission by promoting mitochondrial communication [19]. With the help of optic atrophy 1 (OPA1), a mitochondrial fusion factor, fragmented mitochondria interact with each other, which allows mitochondrial DNA exchange and recovery [20]. Restoration of OPA1 inhibits reperfusion injury in the brain [21] and liver [22] by interfering with mitochondrial fission. This notion is further supported by a cardiac reperfusion model, which demonstrated that OPA1 overexpression sustains mitochondrial homeostasis and cardiomyocyte viability [23]. However, the upstream regulators of OPA1-related mitochondrial fusion remain unclear. Although the hormone melatonin is originally used to regulate body rhythms [24–26], ample evidence supports its therapeutic effects on reperfused brains [27]. Melatonin reduces neuron oxidative stress, attenuates calcium overload, inhibits ER stress and blocks mitochondrial
Revascularization is a procedure used to treat stroke via the timely opening of the occluded vessels [1,2]. However, revascularization itself also induces additional damage to the ischemic brain, which is termed ischemia-reperfusion (IR) injury [3]. Preventing IR injury could further limit infarct size and improve cerebral function [4]. Many studies have focused on the molecular mechanisms underlying neuron death during IR injury and on protective approaches aimed at attenuating IR-induced cerebral damage [5–7]. The role of mitochondria in cerebral IR injury has been explored by several in vitro and in vivo studies [8–11]. Many brain biological processes are handled by mitochondria, including ATP production [12], cellular oxidative stress [13], intracellular calcium balance [14], and apoptosis initiation [15]. In response to cerebral IR injury, mitochondrial morphology initially converts into mostly small fragments, which occurs by a process identified as mitochondrial fission [16]. Subsequently, excessive mitochondrial fission causes mitochondrial DNA damage. Fragmented mitochondria fail to produce sufficient mitochondrial respiratory complexes, leading to decreased oxidative
⁎ Corresponding author at: Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China. E-mail address: [email protected] (L. Liu).
https://doi.org/10.1016/j.biopha.2018.11.060 Received 16 October 2018; Received in revised form 12 November 2018; Accepted 14 November 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase9 (1:1000, Cell Signaling Technology, #9504), pro-caspase3 (1:1000, Abcam, #ab13847), cleaved caspase3 (1:1000, Abcam, #ab49822), c-IAP (1:1000, Cell Signaling Technology, #4952), survivin (1:1000, Cell Signaling Technology, #2808), Bad (:1000; Abcam; #ab90435), cyt-c (1:1000; Abcam; #ab90529), Yap(1:1000; Cell Signaling Technology, #14074), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), GAPDH (1:1000, Cell Signaling Technology, #5174) and β-actin (1:1000, Cell Signaling Technology, #4970).
apoptosis [28,29]. In addition, the inhibitory action of melatonin on mitochondrial fission has been reported in several careful studies [30,31]. Interestingly, no studies have investigated the role of melatonin in mitochondrial fusion, especially OPA1-related mitochondrial fusion. At the molecular level, Yap, a major downstream effector of the Hippo pathway, has been found to be associated with cerebral protection during reperfusion burden [32]. Increased Yap reduces reperfusion-mediated cardiomyocyte apoptosis by repressing Mst1 expression [33]. The Yap-Hippo pathway also alleviates cerebral IR injury by inactivating Drp1-related mitochondrial fission [34]. In rectal cancer and gastric tumors, Yap overexpression promotes cancer survival by diminishing mitochondrial fission and enhancing mitochondrial autophagy [35]. Recent evidence has illustrated cross-talk between mitochondrial fission and Yap-Hippo signaling [36]. However, whether Yap is also involved in reperfusion-related mitochondrial fusion is poorly understood. The aim of our study was to investigate the beneficial effects of melatonin on OPA1-related mitochondrial fusion with a focus on the Yap-Hippo pathway in the setting of cerebral IR injury.
2.4. TUNEL staining and MTT assay Cell death was measured via a TUNEL assay using an in situ cell death detection kit (Roche, Indianapolis, IN, USA). The TUNEL kit stains nuclei containing fragmented DNA. After HR injury, N2a cells were fixed with 3.7% paraformaldehyde for 30 min at room temperature. Subsequently, an equilibration buffer, nucleotide mix and rTdT enzyme were incubated with the samples at 37 °C for 60 min. Then, a saline-sodium citrate buffer was used to stop the reaction. After being loaded with DAPI, the samples were visualized via fluorescence microscopy (Olympus BX-61). In addition, an MTT assay was performed to analyze cell viability according to the methods described in a previous study [40]. Absorbance at 570 nm was determined. The relative cell viability was recorded as the ratio with the control group.
2. Materials and methods 2.1. Animal treatment and cerebral IR injury The surgical protocol used to induce cerebral IR injury was performed according to the methods of a previous study [37]. In brief, 100 mg/kg of pentobarbital was used for anesthesia. Next, the middle cerebral artery was occluded using a 0.22-mm-diameter silicon-covered 6-0 nylon monofilament (Doccol). After 45 min of ischemia, the monofilament was removed to restore the blood flow for approximately 2 h. After IR injury, the brain tissues were isolated and stained with 2,3,5-triphenyltetrazolium chloride (TTC) staining to demonstrate the cerebral infarction zone based on a previous study [38].
2.5. Caspase activity detection and ELISA Caspase-3 and caspase-9 activity were determined using commercial kits (Beyotime Institute of Biotechnology). The levels of antioxidant factors including GPX, SOD, and GSH were measured with ELISA kits, which were purchased from Beyotime Institute of Biotechnology. ELISA Kits (#PM4000B for TNFɑ, #PM6000B for MCP-1; #PM4030B for IL-8, Cusabio Technology, Wuhan, China) were used to measure the concentrations of TNFα, IL-8 and MCP1 after IR injury [41].
2.2. Cell culture and hypoxia-reoxygenation (HR) injury A hypoxia-reoxygenation (HR) model was used in mouse N2a neuroblastoma cells (ATCC® CCL-131™) to mimic cerebral IR injury in vitro. N2a cells were cultured in L-DMEM supplemented with 10% FBS. To induce HR injury, the medium was replaced with L-DMEM without FBS in a hypoxia chamber containing 5% CO2 and 95% N2 for 45 min. Subsequently, the medium was replaced with fresh L-DMEM with 10% FBS, and cells were maintained at 37 °C in a 5% CO2 incubator for another 2 h, according to the methods of a previous study [10]. To prevent Yap activation, verteporfin (Sigma, Cat. No. #SML0534) was added to the medium for 2 h before HR injury. Low-dose (10 μM) and high-dose (20 μM) melatonin were added to the medium 24 h before HR injury.
2.6. Transfection Transfection with siRNA was used to inhibit OPA1 expression in melatonin-treated N2a cell. Two independent siRNAs (siRNA1-OPA1 and siRNA2-OPA2, Yangzhou Ruibo Biotech Co., Ltd. (Yangzhou, China)) were used to infect N2a cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The negative control group was transfected with negative control siRNA. Transfection was performed for approximately 48 h. Then, Western blotting was used to observe the knockdown efficiency after harvesting the transfected cells [42].
2.3. Western blotting
2.7. Flow cytometry for mROS
After treatment, brain tissues and cells were collected, and total protein was isolated. Samples (40–80 μg of protein) were separated via 10% SDS-PAGE and transferred to PVDF membranes. After being washed with TBST, the membranes were blocked with 5% nonfat milk at room temperature for 45 min. Then, the membranes were incubated overnight with the primary antibodies at 4 °C. Subsequently, the membranes were washed again with TBST 3 times at room temperature [39], followed by incubation with secondary antibodies for 45 min at room temperature. After being washed with TBST another three times, the membranes were visualized using an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, China). The following primary antibodies were used for immunoblotting: Drp1 (1:1000, Abcam, #ab56788), OPA1 (1:1000, Abcam, #ab42364), Mfn1 (1:1000, Abcam, #ab57602), Mfn2 (1:1000, Abcam, #ab56889), Mff (1:1000, Cell Signaling Technology, #86668), Bcl2 (1:1000, Cell Signaling
Flow cytometry was used to analyze mitochondrial ROS (mROS) production. After HR injury, N2a cells were washed three times with PBS and then resuspended in PBS using 0.25% trypsin. Subsequently, cells were incubated with the MitoSOX red mitochondrial superoxide indicator (Molecular Probes, USA) for 15 min at 37 °C in the dark. After three washes with PBS, mROS production was analyzed via flow cytometry (Sysmex Partec GmbH, Görlitz, Germany), and the data were analyzed using Flowmax software (Sysmex Partec, Version 2.3, Germany) [43]. 2.8. Immunofluorescent staining and mitochondrial potential detection After HR injury, N2a cells were first fixed with 4% paraformaldehyde for 30 min at room temperature. After incubation with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, 204
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In addition to cell apoptosis, we further observed inflammatory responses during brain IR injury. Using ELISA, we found that serum TNFα, IL-6 and MCP1 levels were rapidly upregulated in response to reperfusion stress (Fig. 1G–I), which was reduced by melatonin supplementation. Altogether, our results demonstrated that melatonin was able to reduce cerebral IR injury in a dose-dependent manner by attenuating neuron apoptosis and repressing the inflammatory response.
the samples were treated overnight with primary antibodies at 4 °C. Afterwards, the slides were washed with PBS and then incubated with secondary antibody (1:500, Invitrogen, Carlsbad, CA, USA) at room temperature for 45 min [44]. Nuclei were stained using DAPI. Images were acquired via fluorescence microscopy (Olympus BX-61). The following primary antibodies were used in the present study: OPA1 (1:1000, Abcam, #ab42364), Yap (1:1000; Cell Signaling Technology, #14074), cyt-c (1:1000; Abcam; #ab90529). Mitochondrial potential was measured using a JC-1 kit (Beyotime Institute of Biotechnology, China). After HR injury, N2a cells were washed three times with PBS and then incubated with fresh medium supplemented with 10 mg/ml JC-1. Thirty minutes later, the samples were washed three times with PBS to remove the free probe, and then fresh medium was added. The samples were observed via fluorescence microscopy (Olympus BX-61). The red/green fluorescence of JC-1 was analyzed using Image Pro Plus version 4.5 (Media Cybernetics, Inc., Rockville, MD, USA).
3.2. Melatonin improves OPA1-related mitochondrial fusion To investigate the beneficial role of melatonin in the reperfused brain, we observed the changes in mitochondrial fusion in vivo, Western blotting demonstrated that IR injury reduced the expression of mitochondrial fusion-related factors, such as Mfn2, Mfn1 and OPA1 (Fig. 2A–F). In contrast, mitochondrial fission factors including Mff and Drp1 were significantly upregulated in response to reperfusion stress (Fig. 2A–F). Interestingly, melatonin supplementation restored the levels of mitochondrial fusion-related proteins and repressed the expression of fission-related factors. This finding was further supported in vitro using qPCR. The transcription of mitochondrial fusion-related factors was rapidly downregulated in HR-treated N2a cells, and this effect was reversed by melatonin treatment (Fig. 2G–I). Interestingly, among the tested proteins, OPA1 expression was increased to the greatest extent by melatonin both in vivo and in vitro. This result indicated that melatonin may activate mitochondrial fusion via OPA1. To further observe mitochondrial fusion, mitochondrial morphology was assessed using immunofluorescence assays. As shown in Fig. 2J, HR injury induced mitochondria division into several fragments, indicative of increased mitochondrial fission and decreased fusion. Interestingly, melatonin treatment reversed the mitochondrial interconnective morphology. To verify whether OPA1 was involved in melatonin-mediated mitochondrial integrity, two independent siRNAs against OPA1 were transfected into melatonin-treated cells. Meanwhile, OPA1 expression was determined using coimmunofluorescence. As shown in Fig. 2J–K, compared with the melatonin group (20 μM), OPA1 siRNAs transfection caused the formation of mitochondrial debris. Subsequently, mitochondrial length was measured and used to quantify mitochondrial fusion. The baseline length of mitochondria was ∼5.3 μm in N2a cells containing abundant OPA1 expression (Fig. 2J–L). However, the average mitochondrial length was rapidly reduced to ∼2.6 μm upon HR stress, which coincided with a drop in OPA1 expression (Fig. 2J–L). Melatonin treatment restored mitochondrial length to ∼4.6 μm, and this effect was abrogated by OPA1 siRNAs transfection. Altogether, our results indicated that melatonin activated OPA1-related mitochondrial fusion in the context of cerebral IR injury.
2.9. RNA isolation and qPCR Total RNA was extracted from reperfused brains using RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Subsequently, cDNA was reverse-transcribed according to a previous study. mRNA expression was measured via qRT-PCR (SYBR Green method). The SYBR Green reagent was obtained from Solarbio (Beijing, China). Relative mRNA levels were normalized to GAPDH and calculated using the 2−ΔΔCt methods based on the methods of a previous study [45]. The following primers were used in the present study: TNFα (Forward, 5'’AGATGGAGCAACCTAAGGTC-3'; Reverse, 5’GCAGACCTCGCTGTTCT AGC-3'), IL6 (Forward, 5’-CAGACTCGCGCCTCTAAGGAGT3'; Reverse, 5'-GATAGCCGATCCGTCGAA-3'), MCP1 (Forward, 5’-GGATGGATTGC ACAGCCATT-3'; Reverse, 5’-GCGCCGACTCAGAGGTGT-3'), GAPDH, (Forward: 5’-AAGTTGTGFATTAGTCA-3’, Reverse: 5’-AGAATAGTCCTA TAATCA-3’), Mfn1 (Forward 5’- CATGGACGAGCTGGCCTTC-3’, Reverse 5’-ATCCTGTAGTGATGTATCAGG-3’), Mfn2 (Forward 5’-CCTCTT GATCCTGATCTTAACGT-3’, Reverse 5’-GGACTACCTGATTGTCATTC3’), and OPA1 (Forward 5’-GCTACTTGTGAGGTCGATTC-3’, Reverse 5′-GCCGTATACCGTGGTATGTCTG-3’). 2.10. Statistical analysis Statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). All results in the present study were analyzed by oneway analysis of variance followed by Tukey's test. P < 0.05 was considered statistically significant. 3. Results 3.1. Melatonin attenuates reperfusion stress-mediated cerebral damage
3.3. OPA1 knockdown abrogates the protective effects of melatonin on mitochondrial energy metabolism
In our study, 45 min of ischemia and 4 h of reperfusion were used to establish cerebral IR injury, and low- and high-dose melatonin were administered 24 h before cerebral IR injury. Subsequently, the infarction area was measured to evaluate the role of melatonin in brain protection. As shown in Fig. 1A–B. the cerebral infarct area was obviously increased in reperfused brains, and this effect was repressed by melatonin in a concentration-dependent manner (Fig. 1A–B). The formation of the infarct zone entails neuron death in response to reperfusion stress. As shown in Fig. 1C–D, TUNEL staining revealed that the number of the TUNEL-positive neurons was markedly elevated after IR injury and was reduced by melatonin treatment in a dose-dependent manner. This finding was supported in vitro using N2a cell in a hypoxiareoxygenation (HR) model. Caspase-3 activity was significantly increased in HR-treated Na2 cells (Fig. 1E), an effect that was accompanied by a drop in cell viability, as assessed by MTT assay (Fig. 1F). However, melatonin was able to reverse HR-mediated neuron damage in a dose-dependent manner.
To explain the protective mechanism of OPA1-related mitochondrial fusion on reperfused brains, mitochondrial function was determined. First, cellular ATP content was reduced in response to HR treatment, and this effect was reversed by melatonin (Fig. 3A). However, OPA1 knockdown abolished the ability of melatonin to promote ATP production. At the molecular level, mitochondria are the energy center of N2a cells, and ATP is primarily generated in mitochondria via conversion of the mitochondrial potential energy into chemical energy. Interestingly, mitochondrial potential, as assessed by JC-1 staining, was significantly dissipated in response to HR stress (Fig. 3B–C). However, melatonin stabilized the mitochondrial potential depending on OPA1related mitochondrial fusion. We also found that expression of the mitochondrial respiratory complex was downregulated in HR-treated N2a cell (Fig. 3D–G); this effect was reversed by melatonin supplementation via enhanced OPA1-related mitochondrial fusion. Furthermore, the remaining glucose in the medium was detected to analyze mitochondrial metabolism. Compared with the control group, 205
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Fig. 1. Melatonin treatment alleviates cerebral IR injury by reducing neuron death and repressing the inflammatory response. Forty-five min of ischemia and 2 h of reperfusion were used to establish the cerebral IR injury model. Low-dose (10 mg/kg) and high-dose (20 mg/kg) melatonin were administered intraperitoneally 24 h before cerebral IR injury. A–B. The infarct area was observed using TTC staining. C–D. TUNEL assays were performed to quantify neuron death in response to cerebral IR injury. E. in vitro, N2a cell were used and 45 min of hypoxia and 2 h of reoxygenation were used to establish HR injury. Then, cell apoptosis was determined by analyzing caspase-3 activity. F. Cell viability was measured using MTT assays. G–I. Blood was collected after cerebral IR injury, and the concentrations of inflammatory factors were analyzed using ELISA. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
the decline of antioxidant factors. OPA1 knockdown blocked the antioxidative properties of melatonin during cerebral reperfusion stress. Excessive mROS production was accompanied by cyt-c translocation into the cytoplasm/nucleus, and this process was verified via immunofluorescence (Fig. 4F). Interestingly, melatonin treatment repressed cyt-c liberation, and this effect was achieved via OPA1-related mitochondrial fusion. This finding was also supported by Western blotting. HR increased the level of cytoplasmic cyt-c (cyto cyt-c) and reduced the expression of mitochondrial cyt-c (mito cyt-c) (Fig. 4G–K); these phenotypic alterations were nullified by melatonin in a manner dependent on OPA1-related mitochondrial fusion. As a consequence of cyt-c liberation, the levels of pro-apoptotic proteins, including Bax and caspase-9, were significantly increased by HR treatment (Fig. 4G–K). In contrast, the expression of anti-apoptotic proteins, such as Bcl-2 and survivin, was drastically downregulated (Fig. 4G–K). However, melatonin treatment reversed the changes in anti-apoptotic protein expression and prevented the activation of pro-apoptotic factors, and this effect was blocked by OPA1 siRNAs transfection. Overall, our data illustrated that melatonin inhibited HR-mediated N2a cell mitochondrial apoptosis via OPA1-related mitochondrial fusion.
HR treatment increased the concentration of glucose in the medium (Fig. 3H), indicating decreased glucose uptake by N2a cell. This effect was closely associated with a decline in lactic acid production (Fig. 3I). However, melatonin supplementation improved glucose uptake and promoted lactic acid generation, and these effects were nullified by OPA1 knockdown. Taken together, our results indicated that melatonin sustained mitochondrial function by modulating OPA1-related mitochondrial fusion. 3.4. Loss of OPA1 induces mitochondrial damage Irreversible mitochondrial damage initiates the mitochondria-dependent endogenous apoptosis pathway, which is defined as mitochondrial oxidative stress, cyt-c liberation, pro-apoptotic protein upregulation and caspase-9 activation. As shown in Fig. 4A–B, using flow cytometry, we observed a significant increase in mitochondrial ROS (mROS) production after HR treatment, indicating mitochondrial oxidative stress. This effect was followed by a steep drop in cellular antioxidant factors, such as GSH, GOD and GPX (Fig. 4C–E). Interestingly, melatonin treatment neutralized excessive mROS and reversed 206
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Fig. 2. Melatonin enhances OPA1-related mitochondrial fusion. A–F. in vivo, western blotting of mitochondrial fusion- and fission-related proteins. G–I. in vitro, qPCR was used to analyze the transcription of mitochondrial fusion-related genes. J–K. Mitochondrial fusion and OPA1 expression were observed using immunofluorescence. Two independent siRNAs against OPA1 were transfected into melatonin-treated cells. L. The average mitochondrial length was determined to quantify mitochondrial fusion. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
and was restored to near-normal levels by melatonin supplementation (Fig. 5A–C). To verify whether melatonin modulated OPA1 expression via Yap-Hippo signaling, the Yap antagonist verteporfin (ver) was used in melatonin-treated N2a cells. Verteporfin treatment prevented melatonin-mediated Yap upregulation, and this effect was accompanied by a drop in OPA1 expression (Fig. 5A–C). These results indicated that the Yap-Hippo pathway is required for melatonin-mediated OPA1
3.5. Melatonin modulates OPA1 via the Yap-Hippo pathway The Yap-Hippo pathway has been associated with cerebral and cardiac IR injury by sustaining mitochondrial homeostasis. In the present study, we investigated whether melatonin improves OPA1-related mitochondrial fusion via the Yap-Hippo pathway. Western blotting revealed that Yap expression was downregulated in response to HR injury 207
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Fig. 3. OPA1 knockdown abolishes the protective effects of melatonin on mitochondrial energy metabolism. A. ATP production was evaluated using ELISA. Two independent siRNAs against OPA1 were transfected into melatonin-treated cells. B–C. The JC-1 probe was used to measure the mitochondrial membrane potential. Quantification of the mitochondrial membrane potential was performed by detecting the red-to-green fluorescence intensity ratio. D–G. After HR injury, N2a cells were isolated, and western blotting was performed to quantify the expression of the mitochondrial respiratory complex. H–I. Glucose uptake and lactic acid production were determined using ELISA. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
to increased mitochondrial fission and decreased fusion. Interestingly, melatonin improved mitochondrial fusion, and this effect was achieved via upregulation of OPA1 expression. Increased OPA1-related mitochondrial fusion suppressed mitochondrial oxidative stress and disrupted mitochondrial apoptosis, favoring N2a cell survival in the context of cerebral IR injury. However, loss of OPA1 abolished the protective effects of melatonin on mitochondrial homeostasis and N2a cell viability. Although imbalanced mitochondrial dynamics (mitochondrial fission and fusion) have been acknowledged as a pathogenic factor contributing to the progression of cerebral IR injury, little attention has been paid to the role of mitochondrial fusion in reperfusion-related brain damage. Accordingly, this study is the first to explore the molecular features of melatonin-induced mitochondrial fusion in cerebral reperfusion stress. In addition, our study provided evidence to support the regulatory role of melatonin in OPA1-related mitochondrial fusion via the Yap-Hippo pathway. Excessive mitochondrial fission has been noted in reperfused brains in several animal and cell studies [46,47]. The primary consequence of mitochondrial fission is mitochondrial malfunction and N2a cell apoptosis. In contrast to mitochondrial fission, mitochondrial fusion corrects the aberrant fission by promoting mitochondrial communication [48,49]. The beneficial effects of mitochondrial fusion on mitochondrial genome integrity, mitochondrial oxidative stress, mitochondrial autophagy, mitochondrial calcium homeostasis and mitochondrial apoptosis has been explored in several disease models, such as amyotrophic lateral sclerosis [25], pulmonary arterial hypertension [50], heart failure [51] and Parkinson's disease [52]. In addition, several cell biological processes are highly modulated by mitochondrial fusion, such as the cell cycle [53], cell apoptosis [54], stem cell differentiation [55], and mitochondrial biogenesis [56]. Most
expression. This finding was further supported by immunofluorescence, as both Yap and OPA1 expression were downregulated in response to HR injury and rescued to near-normal levels by melatonin treatment (Fig. 5D–F). However, verteporfin application inhibited melatoninmediated OPA1 upregulation, recapitulating the essential role played by the Yap-Hippo pathway in melatonin-induced OPA1 activation. 3.6. Yap-Hippo signaling is also implicated in reperfusion-mediated N2a cell mitochondrial damage Finally, we explored whether the Yap-Hippo pathway was involved in reperfusion-mediated mitochondrial stress and N2a cell death. HRmediated cyt-c liberation (Fig. 6A–B) and ATP depletion (Fig. 6C) were reversed by melatonin in a Yap-Hippo pathway-dependent manner. In addition, N2a cell death, as evaluated via TUNEL staining (Fig. 6D–E) and caspase-9 activation (Fig. 6F), was significantly triggered by HR injury and repressed by melatonin treatment. However, blockade of Yao-Hippo signaling via verteporfin abolished the anti-apoptotic effects of melatonin in HR-treated N2a cell. Altogether, these data indicated that activation of the Yap-Hippo pathway by melatonin promoted N2a cell survival and mitochondrial integrity. 4. Discussion In the present study, we demonstrated that melatonin alleviated cerebral IR injury by activating OPA1-related mitochondrial fusion. Biological analyses illustrated that melatonin reduced N2a cell death, maintained brain function and corrected cell energy metabolism disorders. At the molecular level, cerebral IR injury was characterized by mitochondrial stress, highlighted by mitochondrial fragmentation due 208
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Fig. 4. OPA1 deficiency activates mitochondrial apoptosis. A–B. Mitochondrial ROS (mROS) were analyzed using flow cytometry. C–E. The concentrations of cellular antioxidant factors were determined using ELISA. F. Immunofluorescence assay for cyt-c translocation from the mitochondria to the nucleus. DAPI was used to label the nucleus. G–K. After HR injury, N2a cells were isolated, and western blotting was used to quantify the proteins related to mitochondrial apoptosis. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
fusion was largely inhibited by IR injury, as evidenced by the reduced transcription and expression of mitochondrial fusion factors. This finding was similar to those of previous studies in which enhanced mitochondrial fusion alleviated heart [57] and liver IR injury [58].
studies of cerebral IR injury have focused on the influence of mitochondrial fission in reperfusion-induced N2a cell damage, but few studies have explored the contribution of mitochondrial fusion in cerebral IR injury. In the present study, we reported that mitochondrial
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Fig. 5. Melatonin improves OPA1 expression by activating the Yap-Hippo pathway. A–C. Western blotting was used to analyze the changes in OPA1 and Yap. Verteporfin, a Yap-Hippo pathway antagonist, was added to the medium. D–F. Immunofluorescence assay for OPA1 and Yap. The fluorescence intensity of OPA1 and Yap was determined in response to melatonin and/or verteporfin treatment. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
Funding
These results implicate mitochondrial fusion as an important target to modify mitochondrial homeostasis and N2a cell viability in response to cerebral reperfusion stress. Our data demonstrated that melatonin activated OPA1-related mitochondrial fusion via the Yap-Hippo pathway. In cerebral IR injury, Yap overexpression attenuates reperfusion-mediated neuronal apoptosis by inhibiting mitochondrial fission [59]. Similarly, in human rectal cancer, Yap interrupts Drp1-dependent mitochondrial fission, contributing to cancer survival [35]. This evidence highlights a strong correlation between Yap expression and mitochondrial homeostasis. Notably, no studies have investigated the role of Yap in mitochondrial fusion. Our data may provide some answers, showing that Yap activation is required for OPA1 stabilization and that this process is closely regulated by melatonin. However, the molecular mechanisms by which Yap controls OPA1 expression remain to be elucidated. In view of the central role played by Yap in promoting oncogene expression [60], whether Yap transcriptionally modulates OPA1 activity is an open question. Overall, our results show that OPA1-related mitochondrial fusion is repressed by cerebral IR injury due to inactive Yap-Hippo signaling. Restoring OPA1-related mitochondrial fusion via melatonin treatment attenuates mitochondrial damage and contributes to neuron survival in the context of cerebral reperfusion stress. This work identifies the YapHippo pathway and OPA1-related mitochondrial fusion as novel targets for the potential development of therapeutic interventions against acute cerebral reperfusion stress.
Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Author contributions SXM and ZMD were involved in the conception and design, performance of experiments, data analysis and interpretation, and manuscript writing. ZMD was involved in data analysis and interpretation. Ethics approval and consent to participate Animal studies were performed in accordance with the Declaration of Helsinki. All protocols were approved by the Ethics Committee of the Second Hospital of Hebei Medical University (ethics number: SHHMU2018-24S). Availability of data and materials All data generated or analyzed during this study are included in this published article. 210
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Fig. 6. Inhibition of the Yap-Hippo pathway induces neuron death and mitochondrial stress. A–B. Cyt-c immunofluorescence. DAPI was used to label the nucleus. The nuclear expression of cyt-c was analyzed. C. ATP production was analyzed using ELISA. D–E. Neuron death was analyzed using TUNEL assays. The number of TUNELpositive cells was observed. F. Caspase-9 activity was measured via ELISA. Verteporfin was used to prevent Yap-Hippo pathway activation. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
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Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Therapeutic effects of melatonin on cerebral ischemia reperfusion injury: Role of Yap-OPA1 signaling pathway and mitochondrial fusion Na Weia,b,c,d, Yuehua Pua,b,c,d, Zhonghua Yanga,b,c,d, Yuesong Panb, Liping Liua,b,c,d,
T
⁎
a
Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China China National Clinical Research Center for Neurological Diseases, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China c Center of Stroke, Beijing Institute for Brain Disorders, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China d Beijing Key Laboratory of Translational Medicine for Cerebrovascular Disease, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Melatonin OPA1 Mitochondrial fusion Yap-Hippo pathway Reperfusion stress
The role of OPA1-related mitochondrial fusion in brain reperfusion stress has remained elusive. The aim of our study is to explore whether melatonin alleviates cerebral IR injury by modulating OPA1-related mitochondrial fusion. We found that melatonin reduced infarct area and suppressed neuron death during reperfusion stress. Biological studies have revealed that IR-inhibited mitochondrial fusion was largely reversed by melatonin via upregulated OPA1 expression. Knocking down OPA1 abrogated the protective effects of melatonin on mitochondrial energy metabolism and mitochondrial apoptosis. In addition, we also found that melatonin modified OPA1 expression via the Yap-Hippo pathway; blockade of the Yap-Hippo pathway induced neuron death and mitochondrial damage despite treatment with melatonin. Altogether, our data demonstrated that cerebral IR injury is closely associated with defective OPA1-related mitochondrial fusion. Melatonin supplementation enhances OPA1-related mitochondrial fusion by activating the Yap-Hippo pathway, ultimately reducing brain reperfusion stress.
1. Introduction
phosphorylation and increased ROS production [17]. Moreover, damaged mitochondria liberate pro-apoptotic factors such as cyt-c in the nucleus, where cyt-c launches the mitochondria-dependent apoptosis pathway [18]. Based on previous findings, mitochondrial fission is recognized as a potential target to alleviate cerebral IR injury. In contrast to mitochondrial fission, mitochondrial fusion is the repair system that corrects excessive mitochondrial fission by promoting mitochondrial communication [19]. With the help of optic atrophy 1 (OPA1), a mitochondrial fusion factor, fragmented mitochondria interact with each other, which allows mitochondrial DNA exchange and recovery [20]. Restoration of OPA1 inhibits reperfusion injury in the brain [21] and liver [22] by interfering with mitochondrial fission. This notion is further supported by a cardiac reperfusion model, which demonstrated that OPA1 overexpression sustains mitochondrial homeostasis and cardiomyocyte viability [23]. However, the upstream regulators of OPA1-related mitochondrial fusion remain unclear. Although the hormone melatonin is originally used to regulate body rhythms [24–26], ample evidence supports its therapeutic effects on reperfused brains [27]. Melatonin reduces neuron oxidative stress, attenuates calcium overload, inhibits ER stress and blocks mitochondrial
Revascularization is a procedure used to treat stroke via the timely opening of the occluded vessels [1,2]. However, revascularization itself also induces additional damage to the ischemic brain, which is termed ischemia-reperfusion (IR) injury [3]. Preventing IR injury could further limit infarct size and improve cerebral function [4]. Many studies have focused on the molecular mechanisms underlying neuron death during IR injury and on protective approaches aimed at attenuating IR-induced cerebral damage [5–7]. The role of mitochondria in cerebral IR injury has been explored by several in vitro and in vivo studies [8–11]. Many brain biological processes are handled by mitochondria, including ATP production [12], cellular oxidative stress [13], intracellular calcium balance [14], and apoptosis initiation [15]. In response to cerebral IR injury, mitochondrial morphology initially converts into mostly small fragments, which occurs by a process identified as mitochondrial fission [16]. Subsequently, excessive mitochondrial fission causes mitochondrial DNA damage. Fragmented mitochondria fail to produce sufficient mitochondrial respiratory complexes, leading to decreased oxidative
⁎ Corresponding author at: Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, No. 119, Nansihuan West Road, Fengtai District, Beijing, 100070, China. E-mail address: [email protected] (L. Liu).
https://doi.org/10.1016/j.biopha.2018.11.060 Received 16 October 2018; Received in revised form 12 November 2018; Accepted 14 November 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase9 (1:1000, Cell Signaling Technology, #9504), pro-caspase3 (1:1000, Abcam, #ab13847), cleaved caspase3 (1:1000, Abcam, #ab49822), c-IAP (1:1000, Cell Signaling Technology, #4952), survivin (1:1000, Cell Signaling Technology, #2808), Bad (:1000; Abcam; #ab90435), cyt-c (1:1000; Abcam; #ab90529), Yap(1:1000; Cell Signaling Technology, #14074), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), GAPDH (1:1000, Cell Signaling Technology, #5174) and β-actin (1:1000, Cell Signaling Technology, #4970).
apoptosis [28,29]. In addition, the inhibitory action of melatonin on mitochondrial fission has been reported in several careful studies [30,31]. Interestingly, no studies have investigated the role of melatonin in mitochondrial fusion, especially OPA1-related mitochondrial fusion. At the molecular level, Yap, a major downstream effector of the Hippo pathway, has been found to be associated with cerebral protection during reperfusion burden [32]. Increased Yap reduces reperfusion-mediated cardiomyocyte apoptosis by repressing Mst1 expression [33]. The Yap-Hippo pathway also alleviates cerebral IR injury by inactivating Drp1-related mitochondrial fission [34]. In rectal cancer and gastric tumors, Yap overexpression promotes cancer survival by diminishing mitochondrial fission and enhancing mitochondrial autophagy [35]. Recent evidence has illustrated cross-talk between mitochondrial fission and Yap-Hippo signaling [36]. However, whether Yap is also involved in reperfusion-related mitochondrial fusion is poorly understood. The aim of our study was to investigate the beneficial effects of melatonin on OPA1-related mitochondrial fusion with a focus on the Yap-Hippo pathway in the setting of cerebral IR injury.
2.4. TUNEL staining and MTT assay Cell death was measured via a TUNEL assay using an in situ cell death detection kit (Roche, Indianapolis, IN, USA). The TUNEL kit stains nuclei containing fragmented DNA. After HR injury, N2a cells were fixed with 3.7% paraformaldehyde for 30 min at room temperature. Subsequently, an equilibration buffer, nucleotide mix and rTdT enzyme were incubated with the samples at 37 °C for 60 min. Then, a saline-sodium citrate buffer was used to stop the reaction. After being loaded with DAPI, the samples were visualized via fluorescence microscopy (Olympus BX-61). In addition, an MTT assay was performed to analyze cell viability according to the methods described in a previous study [40]. Absorbance at 570 nm was determined. The relative cell viability was recorded as the ratio with the control group.
2. Materials and methods 2.1. Animal treatment and cerebral IR injury The surgical protocol used to induce cerebral IR injury was performed according to the methods of a previous study [37]. In brief, 100 mg/kg of pentobarbital was used for anesthesia. Next, the middle cerebral artery was occluded using a 0.22-mm-diameter silicon-covered 6-0 nylon monofilament (Doccol). After 45 min of ischemia, the monofilament was removed to restore the blood flow for approximately 2 h. After IR injury, the brain tissues were isolated and stained with 2,3,5-triphenyltetrazolium chloride (TTC) staining to demonstrate the cerebral infarction zone based on a previous study [38].
2.5. Caspase activity detection and ELISA Caspase-3 and caspase-9 activity were determined using commercial kits (Beyotime Institute of Biotechnology). The levels of antioxidant factors including GPX, SOD, and GSH were measured with ELISA kits, which were purchased from Beyotime Institute of Biotechnology. ELISA Kits (#PM4000B for TNFɑ, #PM6000B for MCP-1; #PM4030B for IL-8, Cusabio Technology, Wuhan, China) were used to measure the concentrations of TNFα, IL-8 and MCP1 after IR injury [41].
2.2. Cell culture and hypoxia-reoxygenation (HR) injury A hypoxia-reoxygenation (HR) model was used in mouse N2a neuroblastoma cells (ATCC® CCL-131™) to mimic cerebral IR injury in vitro. N2a cells were cultured in L-DMEM supplemented with 10% FBS. To induce HR injury, the medium was replaced with L-DMEM without FBS in a hypoxia chamber containing 5% CO2 and 95% N2 for 45 min. Subsequently, the medium was replaced with fresh L-DMEM with 10% FBS, and cells were maintained at 37 °C in a 5% CO2 incubator for another 2 h, according to the methods of a previous study [10]. To prevent Yap activation, verteporfin (Sigma, Cat. No. #SML0534) was added to the medium for 2 h before HR injury. Low-dose (10 μM) and high-dose (20 μM) melatonin were added to the medium 24 h before HR injury.
2.6. Transfection Transfection with siRNA was used to inhibit OPA1 expression in melatonin-treated N2a cell. Two independent siRNAs (siRNA1-OPA1 and siRNA2-OPA2, Yangzhou Ruibo Biotech Co., Ltd. (Yangzhou, China)) were used to infect N2a cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The negative control group was transfected with negative control siRNA. Transfection was performed for approximately 48 h. Then, Western blotting was used to observe the knockdown efficiency after harvesting the transfected cells [42].
2.3. Western blotting
2.7. Flow cytometry for mROS
After treatment, brain tissues and cells were collected, and total protein was isolated. Samples (40–80 μg of protein) were separated via 10% SDS-PAGE and transferred to PVDF membranes. After being washed with TBST, the membranes were blocked with 5% nonfat milk at room temperature for 45 min. Then, the membranes were incubated overnight with the primary antibodies at 4 °C. Subsequently, the membranes were washed again with TBST 3 times at room temperature [39], followed by incubation with secondary antibodies for 45 min at room temperature. After being washed with TBST another three times, the membranes were visualized using an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology, China). The following primary antibodies were used for immunoblotting: Drp1 (1:1000, Abcam, #ab56788), OPA1 (1:1000, Abcam, #ab42364), Mfn1 (1:1000, Abcam, #ab57602), Mfn2 (1:1000, Abcam, #ab56889), Mff (1:1000, Cell Signaling Technology, #86668), Bcl2 (1:1000, Cell Signaling
Flow cytometry was used to analyze mitochondrial ROS (mROS) production. After HR injury, N2a cells were washed three times with PBS and then resuspended in PBS using 0.25% trypsin. Subsequently, cells were incubated with the MitoSOX red mitochondrial superoxide indicator (Molecular Probes, USA) for 15 min at 37 °C in the dark. After three washes with PBS, mROS production was analyzed via flow cytometry (Sysmex Partec GmbH, Görlitz, Germany), and the data were analyzed using Flowmax software (Sysmex Partec, Version 2.3, Germany) [43]. 2.8. Immunofluorescent staining and mitochondrial potential detection After HR injury, N2a cells were first fixed with 4% paraformaldehyde for 30 min at room temperature. After incubation with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, 204
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In addition to cell apoptosis, we further observed inflammatory responses during brain IR injury. Using ELISA, we found that serum TNFα, IL-6 and MCP1 levels were rapidly upregulated in response to reperfusion stress (Fig. 1G–I), which was reduced by melatonin supplementation. Altogether, our results demonstrated that melatonin was able to reduce cerebral IR injury in a dose-dependent manner by attenuating neuron apoptosis and repressing the inflammatory response.
the samples were treated overnight with primary antibodies at 4 °C. Afterwards, the slides were washed with PBS and then incubated with secondary antibody (1:500, Invitrogen, Carlsbad, CA, USA) at room temperature for 45 min [44]. Nuclei were stained using DAPI. Images were acquired via fluorescence microscopy (Olympus BX-61). The following primary antibodies were used in the present study: OPA1 (1:1000, Abcam, #ab42364), Yap (1:1000; Cell Signaling Technology, #14074), cyt-c (1:1000; Abcam; #ab90529). Mitochondrial potential was measured using a JC-1 kit (Beyotime Institute of Biotechnology, China). After HR injury, N2a cells were washed three times with PBS and then incubated with fresh medium supplemented with 10 mg/ml JC-1. Thirty minutes later, the samples were washed three times with PBS to remove the free probe, and then fresh medium was added. The samples were observed via fluorescence microscopy (Olympus BX-61). The red/green fluorescence of JC-1 was analyzed using Image Pro Plus version 4.5 (Media Cybernetics, Inc., Rockville, MD, USA).
3.2. Melatonin improves OPA1-related mitochondrial fusion To investigate the beneficial role of melatonin in the reperfused brain, we observed the changes in mitochondrial fusion in vivo, Western blotting demonstrated that IR injury reduced the expression of mitochondrial fusion-related factors, such as Mfn2, Mfn1 and OPA1 (Fig. 2A–F). In contrast, mitochondrial fission factors including Mff and Drp1 were significantly upregulated in response to reperfusion stress (Fig. 2A–F). Interestingly, melatonin supplementation restored the levels of mitochondrial fusion-related proteins and repressed the expression of fission-related factors. This finding was further supported in vitro using qPCR. The transcription of mitochondrial fusion-related factors was rapidly downregulated in HR-treated N2a cells, and this effect was reversed by melatonin treatment (Fig. 2G–I). Interestingly, among the tested proteins, OPA1 expression was increased to the greatest extent by melatonin both in vivo and in vitro. This result indicated that melatonin may activate mitochondrial fusion via OPA1. To further observe mitochondrial fusion, mitochondrial morphology was assessed using immunofluorescence assays. As shown in Fig. 2J, HR injury induced mitochondria division into several fragments, indicative of increased mitochondrial fission and decreased fusion. Interestingly, melatonin treatment reversed the mitochondrial interconnective morphology. To verify whether OPA1 was involved in melatonin-mediated mitochondrial integrity, two independent siRNAs against OPA1 were transfected into melatonin-treated cells. Meanwhile, OPA1 expression was determined using coimmunofluorescence. As shown in Fig. 2J–K, compared with the melatonin group (20 μM), OPA1 siRNAs transfection caused the formation of mitochondrial debris. Subsequently, mitochondrial length was measured and used to quantify mitochondrial fusion. The baseline length of mitochondria was ∼5.3 μm in N2a cells containing abundant OPA1 expression (Fig. 2J–L). However, the average mitochondrial length was rapidly reduced to ∼2.6 μm upon HR stress, which coincided with a drop in OPA1 expression (Fig. 2J–L). Melatonin treatment restored mitochondrial length to ∼4.6 μm, and this effect was abrogated by OPA1 siRNAs transfection. Altogether, our results indicated that melatonin activated OPA1-related mitochondrial fusion in the context of cerebral IR injury.
2.9. RNA isolation and qPCR Total RNA was extracted from reperfused brains using RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Subsequently, cDNA was reverse-transcribed according to a previous study. mRNA expression was measured via qRT-PCR (SYBR Green method). The SYBR Green reagent was obtained from Solarbio (Beijing, China). Relative mRNA levels were normalized to GAPDH and calculated using the 2−ΔΔCt methods based on the methods of a previous study [45]. The following primers were used in the present study: TNFα (Forward, 5'’AGATGGAGCAACCTAAGGTC-3'; Reverse, 5’GCAGACCTCGCTGTTCT AGC-3'), IL6 (Forward, 5’-CAGACTCGCGCCTCTAAGGAGT3'; Reverse, 5'-GATAGCCGATCCGTCGAA-3'), MCP1 (Forward, 5’-GGATGGATTGC ACAGCCATT-3'; Reverse, 5’-GCGCCGACTCAGAGGTGT-3'), GAPDH, (Forward: 5’-AAGTTGTGFATTAGTCA-3’, Reverse: 5’-AGAATAGTCCTA TAATCA-3’), Mfn1 (Forward 5’- CATGGACGAGCTGGCCTTC-3’, Reverse 5’-ATCCTGTAGTGATGTATCAGG-3’), Mfn2 (Forward 5’-CCTCTT GATCCTGATCTTAACGT-3’, Reverse 5’-GGACTACCTGATTGTCATTC3’), and OPA1 (Forward 5’-GCTACTTGTGAGGTCGATTC-3’, Reverse 5′-GCCGTATACCGTGGTATGTCTG-3’). 2.10. Statistical analysis Statistical analyses were performed using SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). All results in the present study were analyzed by oneway analysis of variance followed by Tukey's test. P < 0.05 was considered statistically significant. 3. Results 3.1. Melatonin attenuates reperfusion stress-mediated cerebral damage
3.3. OPA1 knockdown abrogates the protective effects of melatonin on mitochondrial energy metabolism
In our study, 45 min of ischemia and 4 h of reperfusion were used to establish cerebral IR injury, and low- and high-dose melatonin were administered 24 h before cerebral IR injury. Subsequently, the infarction area was measured to evaluate the role of melatonin in brain protection. As shown in Fig. 1A–B. the cerebral infarct area was obviously increased in reperfused brains, and this effect was repressed by melatonin in a concentration-dependent manner (Fig. 1A–B). The formation of the infarct zone entails neuron death in response to reperfusion stress. As shown in Fig. 1C–D, TUNEL staining revealed that the number of the TUNEL-positive neurons was markedly elevated after IR injury and was reduced by melatonin treatment in a dose-dependent manner. This finding was supported in vitro using N2a cell in a hypoxiareoxygenation (HR) model. Caspase-3 activity was significantly increased in HR-treated Na2 cells (Fig. 1E), an effect that was accompanied by a drop in cell viability, as assessed by MTT assay (Fig. 1F). However, melatonin was able to reverse HR-mediated neuron damage in a dose-dependent manner.
To explain the protective mechanism of OPA1-related mitochondrial fusion on reperfused brains, mitochondrial function was determined. First, cellular ATP content was reduced in response to HR treatment, and this effect was reversed by melatonin (Fig. 3A). However, OPA1 knockdown abolished the ability of melatonin to promote ATP production. At the molecular level, mitochondria are the energy center of N2a cells, and ATP is primarily generated in mitochondria via conversion of the mitochondrial potential energy into chemical energy. Interestingly, mitochondrial potential, as assessed by JC-1 staining, was significantly dissipated in response to HR stress (Fig. 3B–C). However, melatonin stabilized the mitochondrial potential depending on OPA1related mitochondrial fusion. We also found that expression of the mitochondrial respiratory complex was downregulated in HR-treated N2a cell (Fig. 3D–G); this effect was reversed by melatonin supplementation via enhanced OPA1-related mitochondrial fusion. Furthermore, the remaining glucose in the medium was detected to analyze mitochondrial metabolism. Compared with the control group, 205
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Fig. 1. Melatonin treatment alleviates cerebral IR injury by reducing neuron death and repressing the inflammatory response. Forty-five min of ischemia and 2 h of reperfusion were used to establish the cerebral IR injury model. Low-dose (10 mg/kg) and high-dose (20 mg/kg) melatonin were administered intraperitoneally 24 h before cerebral IR injury. A–B. The infarct area was observed using TTC staining. C–D. TUNEL assays were performed to quantify neuron death in response to cerebral IR injury. E. in vitro, N2a cell were used and 45 min of hypoxia and 2 h of reoxygenation were used to establish HR injury. Then, cell apoptosis was determined by analyzing caspase-3 activity. F. Cell viability was measured using MTT assays. G–I. Blood was collected after cerebral IR injury, and the concentrations of inflammatory factors were analyzed using ELISA. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
the decline of antioxidant factors. OPA1 knockdown blocked the antioxidative properties of melatonin during cerebral reperfusion stress. Excessive mROS production was accompanied by cyt-c translocation into the cytoplasm/nucleus, and this process was verified via immunofluorescence (Fig. 4F). Interestingly, melatonin treatment repressed cyt-c liberation, and this effect was achieved via OPA1-related mitochondrial fusion. This finding was also supported by Western blotting. HR increased the level of cytoplasmic cyt-c (cyto cyt-c) and reduced the expression of mitochondrial cyt-c (mito cyt-c) (Fig. 4G–K); these phenotypic alterations were nullified by melatonin in a manner dependent on OPA1-related mitochondrial fusion. As a consequence of cyt-c liberation, the levels of pro-apoptotic proteins, including Bax and caspase-9, were significantly increased by HR treatment (Fig. 4G–K). In contrast, the expression of anti-apoptotic proteins, such as Bcl-2 and survivin, was drastically downregulated (Fig. 4G–K). However, melatonin treatment reversed the changes in anti-apoptotic protein expression and prevented the activation of pro-apoptotic factors, and this effect was blocked by OPA1 siRNAs transfection. Overall, our data illustrated that melatonin inhibited HR-mediated N2a cell mitochondrial apoptosis via OPA1-related mitochondrial fusion.
HR treatment increased the concentration of glucose in the medium (Fig. 3H), indicating decreased glucose uptake by N2a cell. This effect was closely associated with a decline in lactic acid production (Fig. 3I). However, melatonin supplementation improved glucose uptake and promoted lactic acid generation, and these effects were nullified by OPA1 knockdown. Taken together, our results indicated that melatonin sustained mitochondrial function by modulating OPA1-related mitochondrial fusion. 3.4. Loss of OPA1 induces mitochondrial damage Irreversible mitochondrial damage initiates the mitochondria-dependent endogenous apoptosis pathway, which is defined as mitochondrial oxidative stress, cyt-c liberation, pro-apoptotic protein upregulation and caspase-9 activation. As shown in Fig. 4A–B, using flow cytometry, we observed a significant increase in mitochondrial ROS (mROS) production after HR treatment, indicating mitochondrial oxidative stress. This effect was followed by a steep drop in cellular antioxidant factors, such as GSH, GOD and GPX (Fig. 4C–E). Interestingly, melatonin treatment neutralized excessive mROS and reversed 206
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Fig. 2. Melatonin enhances OPA1-related mitochondrial fusion. A–F. in vivo, western blotting of mitochondrial fusion- and fission-related proteins. G–I. in vitro, qPCR was used to analyze the transcription of mitochondrial fusion-related genes. J–K. Mitochondrial fusion and OPA1 expression were observed using immunofluorescence. Two independent siRNAs against OPA1 were transfected into melatonin-treated cells. L. The average mitochondrial length was determined to quantify mitochondrial fusion. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
and was restored to near-normal levels by melatonin supplementation (Fig. 5A–C). To verify whether melatonin modulated OPA1 expression via Yap-Hippo signaling, the Yap antagonist verteporfin (ver) was used in melatonin-treated N2a cells. Verteporfin treatment prevented melatonin-mediated Yap upregulation, and this effect was accompanied by a drop in OPA1 expression (Fig. 5A–C). These results indicated that the Yap-Hippo pathway is required for melatonin-mediated OPA1
3.5. Melatonin modulates OPA1 via the Yap-Hippo pathway The Yap-Hippo pathway has been associated with cerebral and cardiac IR injury by sustaining mitochondrial homeostasis. In the present study, we investigated whether melatonin improves OPA1-related mitochondrial fusion via the Yap-Hippo pathway. Western blotting revealed that Yap expression was downregulated in response to HR injury 207
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Fig. 3. OPA1 knockdown abolishes the protective effects of melatonin on mitochondrial energy metabolism. A. ATP production was evaluated using ELISA. Two independent siRNAs against OPA1 were transfected into melatonin-treated cells. B–C. The JC-1 probe was used to measure the mitochondrial membrane potential. Quantification of the mitochondrial membrane potential was performed by detecting the red-to-green fluorescence intensity ratio. D–G. After HR injury, N2a cells were isolated, and western blotting was performed to quantify the expression of the mitochondrial respiratory complex. H–I. Glucose uptake and lactic acid production were determined using ELISA. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
to increased mitochondrial fission and decreased fusion. Interestingly, melatonin improved mitochondrial fusion, and this effect was achieved via upregulation of OPA1 expression. Increased OPA1-related mitochondrial fusion suppressed mitochondrial oxidative stress and disrupted mitochondrial apoptosis, favoring N2a cell survival in the context of cerebral IR injury. However, loss of OPA1 abolished the protective effects of melatonin on mitochondrial homeostasis and N2a cell viability. Although imbalanced mitochondrial dynamics (mitochondrial fission and fusion) have been acknowledged as a pathogenic factor contributing to the progression of cerebral IR injury, little attention has been paid to the role of mitochondrial fusion in reperfusion-related brain damage. Accordingly, this study is the first to explore the molecular features of melatonin-induced mitochondrial fusion in cerebral reperfusion stress. In addition, our study provided evidence to support the regulatory role of melatonin in OPA1-related mitochondrial fusion via the Yap-Hippo pathway. Excessive mitochondrial fission has been noted in reperfused brains in several animal and cell studies [46,47]. The primary consequence of mitochondrial fission is mitochondrial malfunction and N2a cell apoptosis. In contrast to mitochondrial fission, mitochondrial fusion corrects the aberrant fission by promoting mitochondrial communication [48,49]. The beneficial effects of mitochondrial fusion on mitochondrial genome integrity, mitochondrial oxidative stress, mitochondrial autophagy, mitochondrial calcium homeostasis and mitochondrial apoptosis has been explored in several disease models, such as amyotrophic lateral sclerosis [25], pulmonary arterial hypertension [50], heart failure [51] and Parkinson's disease [52]. In addition, several cell biological processes are highly modulated by mitochondrial fusion, such as the cell cycle [53], cell apoptosis [54], stem cell differentiation [55], and mitochondrial biogenesis [56]. Most
expression. This finding was further supported by immunofluorescence, as both Yap and OPA1 expression were downregulated in response to HR injury and rescued to near-normal levels by melatonin treatment (Fig. 5D–F). However, verteporfin application inhibited melatoninmediated OPA1 upregulation, recapitulating the essential role played by the Yap-Hippo pathway in melatonin-induced OPA1 activation. 3.6. Yap-Hippo signaling is also implicated in reperfusion-mediated N2a cell mitochondrial damage Finally, we explored whether the Yap-Hippo pathway was involved in reperfusion-mediated mitochondrial stress and N2a cell death. HRmediated cyt-c liberation (Fig. 6A–B) and ATP depletion (Fig. 6C) were reversed by melatonin in a Yap-Hippo pathway-dependent manner. In addition, N2a cell death, as evaluated via TUNEL staining (Fig. 6D–E) and caspase-9 activation (Fig. 6F), was significantly triggered by HR injury and repressed by melatonin treatment. However, blockade of Yao-Hippo signaling via verteporfin abolished the anti-apoptotic effects of melatonin in HR-treated N2a cell. Altogether, these data indicated that activation of the Yap-Hippo pathway by melatonin promoted N2a cell survival and mitochondrial integrity. 4. Discussion In the present study, we demonstrated that melatonin alleviated cerebral IR injury by activating OPA1-related mitochondrial fusion. Biological analyses illustrated that melatonin reduced N2a cell death, maintained brain function and corrected cell energy metabolism disorders. At the molecular level, cerebral IR injury was characterized by mitochondrial stress, highlighted by mitochondrial fragmentation due 208
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Fig. 4. OPA1 deficiency activates mitochondrial apoptosis. A–B. Mitochondrial ROS (mROS) were analyzed using flow cytometry. C–E. The concentrations of cellular antioxidant factors were determined using ELISA. F. Immunofluorescence assay for cyt-c translocation from the mitochondria to the nucleus. DAPI was used to label the nucleus. G–K. After HR injury, N2a cells were isolated, and western blotting was used to quantify the proteins related to mitochondrial apoptosis. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
fusion was largely inhibited by IR injury, as evidenced by the reduced transcription and expression of mitochondrial fusion factors. This finding was similar to those of previous studies in which enhanced mitochondrial fusion alleviated heart [57] and liver IR injury [58].
studies of cerebral IR injury have focused on the influence of mitochondrial fission in reperfusion-induced N2a cell damage, but few studies have explored the contribution of mitochondrial fusion in cerebral IR injury. In the present study, we reported that mitochondrial
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Fig. 5. Melatonin improves OPA1 expression by activating the Yap-Hippo pathway. A–C. Western blotting was used to analyze the changes in OPA1 and Yap. Verteporfin, a Yap-Hippo pathway antagonist, was added to the medium. D–F. Immunofluorescence assay for OPA1 and Yap. The fluorescence intensity of OPA1 and Yap was determined in response to melatonin and/or verteporfin treatment. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
Funding
These results implicate mitochondrial fusion as an important target to modify mitochondrial homeostasis and N2a cell viability in response to cerebral reperfusion stress. Our data demonstrated that melatonin activated OPA1-related mitochondrial fusion via the Yap-Hippo pathway. In cerebral IR injury, Yap overexpression attenuates reperfusion-mediated neuronal apoptosis by inhibiting mitochondrial fission [59]. Similarly, in human rectal cancer, Yap interrupts Drp1-dependent mitochondrial fission, contributing to cancer survival [35]. This evidence highlights a strong correlation between Yap expression and mitochondrial homeostasis. Notably, no studies have investigated the role of Yap in mitochondrial fusion. Our data may provide some answers, showing that Yap activation is required for OPA1 stabilization and that this process is closely regulated by melatonin. However, the molecular mechanisms by which Yap controls OPA1 expression remain to be elucidated. In view of the central role played by Yap in promoting oncogene expression [60], whether Yap transcriptionally modulates OPA1 activity is an open question. Overall, our results show that OPA1-related mitochondrial fusion is repressed by cerebral IR injury due to inactive Yap-Hippo signaling. Restoring OPA1-related mitochondrial fusion via melatonin treatment attenuates mitochondrial damage and contributes to neuron survival in the context of cerebral reperfusion stress. This work identifies the YapHippo pathway and OPA1-related mitochondrial fusion as novel targets for the potential development of therapeutic interventions against acute cerebral reperfusion stress.
Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Author contributions SXM and ZMD were involved in the conception and design, performance of experiments, data analysis and interpretation, and manuscript writing. ZMD was involved in data analysis and interpretation. Ethics approval and consent to participate Animal studies were performed in accordance with the Declaration of Helsinki. All protocols were approved by the Ethics Committee of the Second Hospital of Hebei Medical University (ethics number: SHHMU2018-24S). Availability of data and materials All data generated or analyzed during this study are included in this published article. 210
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Fig. 6. Inhibition of the Yap-Hippo pathway induces neuron death and mitochondrial stress. A–B. Cyt-c immunofluorescence. DAPI was used to label the nucleus. The nuclear expression of cyt-c was analyzed. C. ATP production was analyzed using ELISA. D–E. Neuron death was analyzed using TUNEL assays. The number of TUNELpositive cells was observed. F. Caspase-9 activity was measured via ELISA. Verteporfin was used to prevent Yap-Hippo pathway activation. Data represent the mean ± SEM (n = 6 for each group). *p < 0.05.
Consent for publication
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