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ROS act as an upstream signal to mediate cadmium-induced mitophagy in mouse brain
NeuroToxicology 46 (2015) 19–24
Contents lists available at ScienceDirect
NeuroToxicology
ROS act as an upstream signal to mediate cadmium-induced mitophagy in mouse brain Xue Wei a, Yongmei Qi a, Xiaoning Zhang a, Xueyan Gu a, Hui Cai b, Jing Yang a, Yingmei Zhang a,* a b
Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China Gansu Provincial Hospital, Lanzhou 730030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 September 2014 Accepted 17 November 2014 Available online 22 November 2014
As a well known generator of reactive oxygen species (ROS), cadmium (Cd) is found to be an effective inducer of mitophagy in mouse kidney and liver cells. Here, we aim to elucidate whether Cd can also initiate mitophagy in mouse brain and what role ROS play in this process. Our results showed that Cd caused overproduction of ROS. Meanwhile, Cd induced mitophagy, as indicated by the collapse of mitochondrial membrane potential (MMP), formation of mitophagosomes, increases of PINK1 level and LC3-II/LC3-I ratio and decrease of mitochondrial mass. Scavenging of ROS by N-acetyl-L-cysteine (NAC) or acetyl-L-carnitine (ALC) rescued MMP and mitochondrial mass, and squelched PINK1 level, mitochondrial accumulation of Parkin and LC3-II/LC3-I ratio, suggesting that ROS were associated with Cd-induced mitophagy. Cyclosporine A (CsA), an inhibitor of mitophagy, blocked Cd-induced mitophagy and PINK1/Parkin pathway but failed to suppress ROS increase, revealing that ROS are the causes rather than the results of Cd-induced mitophagy. In conclusion, this study suggested that ROS functioned on the upstream of PINK1/Parkin pathway to mediate Cd-induced mitophagy. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Cadmium Mitophagy ROS Brain Mouse
1. Introduction Autophagy is an adapting mechanism to respond to the changing environmental stimuli, such as starvation (Komatsu et al., 2005) and oxidative stress (Lemasters, 2005). It can degrade peroxisomes, endoplasmic reticulum, mitochondria and other organelles (Yu et al., 2008). Mitophagy, a specific process that selectively and targetedly removes mitochondria (Lemasters, 2005), can be marked by the collapse of mitochondrial membrane potential (MMP), accumulation of full-length PINK1, formation of mitophagosomes and decrease of mitochondrial mass. MMP collapse is the prerequisite of mitophagy (Elmore et al., 2001; Rodriguez-Enriquez et al., 2006) and also an early event of mitophagy to stabilize PINK1 (Matsuda et al., 2010; Tolkovsky, 2009). PINK1 is usually degraded and maintained at very low level in normal mitochondria, however, it can rapidly accumulate on mitochondrial membrane once mitochondria are damaged and MMP is declined (Youle and Narendra, 2010). The accumulation of
* Corresponding author at: School of Life Sciences, Lanzhou University, 222 South Tianshui R.D., Lanzhou 730000, China. Tel.: +86 13919123067; fax: +86 931 8913631. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.neuro.2014.11.007 0161-813X/ß 2014 Elsevier Inc. All rights reserved.
PINK1 can induce translocation of Parkin from cytosol to damaged mitochondria and reflect the progression of mitophagy (Narendra et al., 2010). Eventually, mitophagy removes damaged mitochondria by regulating mitochondrial mass (Park et al., 2012) and number (Youle and Narendra, 2010). Recent studies have showed that cadmium (Cd) can induce mitophagy in mouse kidney and liver cells (Pi et al., 2013; Wei et al., 2014). However, the impact of Cd on mitophagy in mouse brain and its molecular mechanism have not been explored. Several researches have showed that Cd can enter the brain, causing apoptosis and neurological alterations in clinical and animal studies (Łukawski et al., 2005; Nishimura et al., 2006; Yuan et al., 2013). Cd is also involved in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease (Jiang et al., 2007; Okuda et al., 1997), which are tightly related to mitophagy (Amadoro et al., 2014; Kamat et al., 2014). Notably, accumulating data have pointed that reactive oxygen species (ROS) are implicated in Cd-induced autophagy: Cd induces autophagy through ROS-activated GSK-3b pathway and ROS-dependent LKB1–AMPK signaling pathway (Son et al., 2011; Wang et al., 2009). However, the role of ROS in Cd-induced mitophagy is still unclear. Although some researches focused attention on the function of ROS in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitophagy using HeLa cells, their findings showed
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that ROS were not required (Narendra et al., 2008) or were indispensible (Wang et al., 2012) for mitophagy. Therefore, the present study sought to elucidate whether ROS are important players in the process of mitophagy, specifically induced by Cd. The role of ROS in Cd-induced mitophagy in mouse brain was examined using ROS scavengers N-acetyl-L-cysteine (NAC) (Downs et al., 2012; Marianna et al., 2013; Qu et al., 2013), acetyl-L-carnitine (ALC) (Akbas¸ et al., 2003; Ferraresi et al., 2006; Karalija et al., 2012) and mitophagy inhibitor cyclosporine A (CsA) (Kim et al., 2007; Rodriguez-Enriquez et al., 2006). Our data showed that ROS functioned as an upstream signal to mediate Cd-induced mitophagy. 2. Materials and methods 2.1. Animals Male Kunming mice (body weight 25.0 2.0 g) from Experimental Animal Center of Lanzhou University were adapted for 1 week before experiment. Mice were housed in controlled conditions of lighting (12 h light/dark cycles), temperature (24 2 8C) and were provided with standard food and water. Mice were randomly divided into eight groups and each contains 8 mice. Mice were intraperitoneally injected with physiological saline solution and Cd (0.05, 0.20, 0.80 mg/kg bw/day) to explore the mitophagic effect. Then 0.20 mg/ kg Cd was selected to co-treat with NAC (100 mg/kg bw/day), ALC (30 mg/kg bw/day) or CsA (10 mg/kg bw/day) to investigate the role of ROS in Cd-induced mitophagy. Mice were sacrificed by cervical dislocation 3 days following the final injection. Brains were harvested for analysis. All of the procedures were approved by the guidelines of Accreditation of Laboratory Animal Care. 2.2. Transmission electron microscopy For the transmission electron microscopy studies, the harvested brains were washed twice with PBS (pH 7.4), cut into pieces, and fixed with 2.5% glutaraldehyde. Then brain pieces were postfixed in 2% osmium tetroxide, embedded and stained with uranyl acetate/lead citrate. Sections were viewed under a JEM 1230 transmission electron microscope (JEOL, Tokyo, Japan) to detect mitophagosomes. 2.3. Single cell isolation Dissected brains were washed with cold PBS, minced with scissors at 4 8C and then filtered with cell strainer. The mixture was centrifuged at 500 rpm for 8 min, washed twice and used for flow cytometry (Hawley and Hawley, 2004). 2.4. Measurement of MMP, mitochondrial mass and ROS The isolated cells were washed twice and counted to 1 106 cells/ml. To measure MMP, brain cells were incubated with 1 mg/ml Rhodamine 123 (excitation, 507 nm; emission, 530 nm) at 37 8C for 30 min. To measure mitochondrial mass, the cells were incubated with 0.5 mM MitoTracker Green (MTG) (excitation, 490 nm; emission, 516 nm) at 37 8C for 30 min. To measure ROS, the cells were incubated with 10 mM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (excitation, 480 nm; emission, 525 nm) at 37 8C for 30 min. Then the fluorescence intensity was monitored by flow cytometer (BD Biosciences, Franklin Lakes, NJ).
mitochondrial proteins were respectively stored at further western blotting detection.
80 8C for
2.6. Western blotting Protein samples that were isolated from brain tissues were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked and incubated with primary antibodies at 4 8C overnight. Anti-LC3 (1:1000), anti-PINK1 (1:1000) and anti-b-actin (1:1000) were purchased from Sigma–Aldrich (St. Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA) and ZhongShan Golden Bridge Biotechnology (Beijing, China), respectively. The secondary antibodies, anti-mouse (1:20,000) and anti-rabbit (1:20,000) were from ZhongShan Golden Bridge Biotechnology (Beijing, China) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. The blots were exposed to Immobilon Western Chemiluminescent HRP Substrate (Millipore, Boston, MA). 2.7. Statistical analysis All data were expressed as mean standard deviation (SD). Oneway analysis of variance (ANOVA) was used for multiple comparisons by SPSS 19.0 software (Chicago, IL). P < 0.01 was considered highly significant. 3. Results 3.1. Cd treatment increased the level of ROS Cd is known to induce ROS production. We first measured ROS level after the mice were treated by Cd for 3 days. Our data showed that ROS levels in 0.20 and 0.80 mg/kg Cd-treated groups were significantly higher than that in control group with 1.4 and 1.5-fold increase, respectively (P < 0.01, Fig. 1). 3.2. Cd induced mitophagy To determine the effects of Cd on mitophagy in mouse brain, we first evaluated the conversion of cellular protein LC3-I to LC3-II, a critical hallmark in autophagy process. As shown in Fig. 2A, compared with control, 0.05, 0.20 and 0.80 mg/kg Cd significantly elevated LC3-II/LC3-I ratio to 1.3, 2.9 and 2.8-fold, respectively (P < 0.01). To address whether mitophagy occurred, we determined the early event of mitophagy, the collapse of MMP. Three days of treatment with 0.20 and 0.80 mg/kg Cd dramatically decreased MMP of brain cells to 81.02% and 55.45% in mice compared with control (P < 0.01, Fig. 2B). Mitochondrial mass was assessed to reflect the amount of remaining mitochondria after mitophagy
2.5. Cytoplasmic and mitochondrial protein extraction To detect Parkin levels in cytoplasm and mitochondria, we used Cytoplasmic and Mitochondrial Protein Extraction Kit (Sangon, Shanghai, China). After extraction, the cytoplasmic and
Fig. 1. Cd treatment increased the level of ROS in mouse brain. Mice were exposed to increasing concentrations of Cd (0–0.80 mg/kg) for 3 days. ROS level was monitored by flow cytometry. 100 mM H2O2 was used as a positive control for ROS. Data were presented as mean SD, n = 8. **P < 0.01 compared with control.
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experiments. Our results showed that NAC or ALC pretreatment markedly decreased the ROS level compared with 0.20 mg/kg Cd alone (P < 0.01, Fig. 3A). Meanwhile, combination of Cd and NAC or ALC dramatically increased MMP and mitochondrial mass (P < 0.01, Fig. 3B), and prominently decreased PINK1 level (P < 0.01, Fig. 3C and D) and mitochondrial accumulation of Parkin (P < 0.01, Fig. 3E and F) compared with Cd alone. NAC or ALC also decreased LC3-II/LC3-I ratio compared with Cd alone (P < 0.01, Fig. 3G and H). Thus, these data indicated that ROS played a pivotal role in Cd-induced mitophagy and PINK1/Parkin pathway. To further explore how ROS mediated Cd-induced mitophagy, we used CsA to block mitophagy. The results showed that MMP and mitochondrial mass were significantly elevated after co-treatment of Cd and CsA compared with Cd treatment alone (P < 0.01, Fig. 4A). PINK1 level, mitochondrial accumulation of Parkin and LC3-II/LC3-I ratio were markedly decreased compared with Cd alone (P < 0.01, Fig. 4B, C and D). However, the ROS level of Cd and CsA co-treated group was not affected compared with Cd alone (Fig. 4E). Collectively, our data clearly showed that ROS functioned as an upstream signal to mediate Cd-induced mitophagy and PINK1/Parkin pathway. 4. Discussion
Fig. 2. Cd induced mitophagy in mouse brain. Mice were exposed to the gradually increased concentrations of Cd (0–0.80 mg/kg) for 3 days. (A) LC3-I and LC3-II levels were analyzed by western blotting. (B) MMP and mitochondrial mass were measured by flow cytometry. (C) Mitophagosome formation was analyzed under the transmission electron microscopy. (D) The level of PINK1 (full length) was detected by western blotting. Data were presented as mean SD, n = 8. ** P < 0.01 compared with control.
process. Compared with control, 0.20 and 0.80 mg/kg Cd exposure extremely decreased mitochondrial mass to 71.11% and 65.77%, respectively (P < 0.01, Fig. 2B). To directly visualize the process of mitophagy, transmission electron microscopy was used to examine mitophagosomes. We found that 0.20 mg/kg Cd exposure induced typical double-membrane and lamellar mitophagosomes in perinuclear area compared with control (Fig. 2C). Protein level of PINK1 was detected to quantify the mitophagy progress. Compared with control, 0.05, 0.20 and 0.80 mg/kg Cd significantly elevated PINK1 level to 2.5, 4.1 and 5.6-fold respectively (P < 0.01, Fig. 2D). 3.3. ROS functioned as an upstream signal to mediate Cd-induced mitophagy To determine whether Cd-induced ROS were essential players in Cd-induced mitophagy, we chose Cd (0.20 mg/kg) and ROS scavenger NAC (100 mg/kg) or ALC (30 mg/kg) for further
Previous studies have showed that various factors such as hypoxia, nutrient deprivation, photoirradiation and ROS were involved in mitophagy (Kim and Lemasters, 2011a,b; Wang et al., 2012; Zhang et al., 2008). As known by many studies, ROS can be induced by Cd. However, the effect of Cd on mitophagy in mouse brain and the role of ROS in this process remain elusive. Recent studies have suggested that Cd can induce mitophagy in mouse kidney and liver cells (Pi et al., 2013; Wei et al., 2014). Our data showed that Cd also could induce mitophagy in mouse brain. The MMP reduction signified that the early event of mitophagy happened. A direct evidence for mitophagy is the formation of mitophagosomes. The progress of mitophagy can be reflected by changes of mitochondrial proteins (Narendra et al., 2010). We found that mitochondrial protein PINK1 and autophagy marker LC3-II/LC3-I ratio were markedly increased by Cd. These data were consistent with previous study that mitophagy was triggered by the loss of MMP, and upon this loss, PINK1 was accumulated on the mitochondrial outer membrane to activate the ubiquitin ligase Parkin to mediate mitophagy (Green and Van Houten, 2011). The reduction of mitochondrial mass can be used to evaluate the result of mitophagy (Klionsky et al., 2012; Park et al., 2012; Zhang and Ney, 2010). In agreement with the report that mitochondrial mass was markedly reduced in CCCP-induced mitophagy (Narendra et al., 2008), Cd treatment grossly lessened mitochondrial mass. ROS can damage mitochondria and are typically associated with mitophagy in yeast and mouse kidney (Kurihara et al., 2012; Wei et al., 2014). The present study also suggested that Cd induced excess ROS production and addressed the role of ROS in Cdinduced mitophagy in mouse brain. The combination of Cd and NAC or ALC significantly suppressed Cd-induced ROS overproduction while reversed Cd-induced mitophagy, indicating that Cdinduced mitophagy was related to the ROS burst triggered by exogenous Cd. Similarly, an acute burst of ROS can cause loss of MMP and subsequently activate mitophagy in HeLa cells after CCCP treatment (Wang et al., 2012). Mitochondria are known to be the main source of ROS production, and high level of ROS could easily cause MMP decrease and mitochondrial depolarization to initiate mitophagy. In addition, the combination of Cd and NAC or ALC also blocked Cd-induced PINK1/Parkin pathway, suggesting that ROS might mediate PINK1/Parkin pathway, which supported the view that the function of PINK1/Parkin pathway appeared to be
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Fig. 3. ROS were important players in Cd-induced mitophagy in mouse brain. Mice were exposed to 0.20 mg/kg Cd alone or in combination with 100 mg/kg NAC or 30 mg/kg ALC. ROS level (A), MMP and mitochondrial mass (B) were measured by flow cytometry. The levels of PINK1 (full length) (C and D), Parkin in cytoplasm and mitochondria (E and F), LC3-I and LC3-II (G and H) were analyzed by western blotting. b-Tubulin was the internal control of cytoplasmic protein loading. HSP60 was the internal control of mitochondrial protein loading. C and M respectively denoted cytoplasm and mitochondria. Data were presented as mean SD, n = 8. **P < 0.01 compared with control. ## P < 0.01 compared with the corresponding Cd treatment alone.
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Fig. 4. ROS functioned as an upstream signal to mediate Cd-induced mitophagy in mouse brain. Mice were exposed to 0.20 mg/kg Cd alone or in combination with 10 mg/kg CsA for 3 days. MMP and mitochondrial mass (A) were then measured by flow cytometry. The levels of PINK1 (full length) (B), Parkin in cytoplasm and mitochondria (C), LC3-I and LC3-II (D) were analyzed by western blotting. b-Tubulin was the internal control of cytoplasmic protein loading. HSP60 was the internal control of mitochondrial protein loading. C and M respectively denoted cytoplasm and mitochondria. ROS level (E) was monitored by flow cytometry. Data were presented as mean SD, n = 8. ** P < 0.01 compared with control. ##P < 0.01 compared with the corresponding Cd treatment alone.
dependent on ROS (Dagda et al., 2009; Joselin et al., 2012). Based on these findings and our results, we conclude that ROS are tightly implicated in Cd-induced mitophagy and PINK1/Parkin pathway. Previous studies suggested that an increase in cellular ROS, either mitochondrial ROS or exogenous ROS, could upregulate general autophagy (Chen et al., 2009; Scherz-Shouval et al., 2007). How ROS functioned in Cd-induced mitophagy? Several studies show that ROS function downstream of mitophagy pathway in human neuronal cells where knockdown of the essential mitophagy proteins Atg7 and Atg8/LC3B inhibits mitophagy without affecting the increased mitochondrial superoxide (Dagda et al.,
2009) and mitophagy contributes a lot to preventing excess mitochondrial ROS production in yeast and mouse embryo fibroblasts (Kurihara et al., 2012; Zhang et al., 2008). However, Our study showed that CsA and Cd co-treatment markedly inhibited mitophagy without affecting Cd-induced ROS level. This finding suggested that ROS were the upstream signal of Cdinduced mitophagy in mouse brain, which was in accordance with our previous report in which ROS mediated Cd-induced mitophagy through PINK1/Parkin pathway in mouse kidney (Wei et al., 2014). This study was also consistent with the report that ROS work as an important upstream activator to induce mitochondrial damage
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and further promote mitophagy under CCCP exposure, in which, ROS-induced PARK2 recruitment triggered mitophagy (Wang et al., 2012). Besides, ROS can also induce the opening of mitochondrial permeability transition pore, simultaneously cause MMP collapse and serve as an upstream signal to initiate mitophagy (Kim et al., 2007; Lemasters, 2005). In summary, our data demonstrated that Cd induced mitophagy in mouse brain, and Cd-induced ROS functioned as an upstream signal on PINK1/Parkin pathway to mediate mitophagy progress. Our results may contribute to further understanding of the Cdcaused neurotoxicity. Transparency document The transparency document associated with this article can be found in the online version. Conflict of interests The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (31300941), the Fundamental Research Funds for the Central Universities (lzujbky-2014-91), the Traditional Chinese Medicine Research Fund of Gansu (GZK-2012-61) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1137). References ¨ zcan C, et al. The effect of L-carnitine on the Akbas¸ Y, Pata YS, Go¨ru¨r K, Polat G, Polat A, O prevention of experimentally induced myringosclerosis in rats Hear Res 2003;184:107–12. Amadoro G, Corsetti V, Florenzano F, Atlante A, Bobba A, Nicolin V, et al. Morphological and bioenergetic demands underlying the mitophagy in post-mitotic neurons: the pink-parkin pathway. Front Aging Neurosci 2014;6:18. Chen Y, Azad M, Gibson S. Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ 2009;16:1040–52. Dagda RK, Cherra SJ, Kulich SM, Tandon A, Park D, Chu CT. Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J Biol Chem 2009;284:13843–55. Downs I, Liu J, Aw TY, Adegboyega PA, Ajuebor MN. The ROS scavenger, NAC, regulates hepatic Va14iNKT cells signaling during Fas mAb-dependent fulminant liver failure. PLoS ONE 2012;7:e38051. Elmore SP, Qian T, Grissom SF, Lemasters JJ. The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J 2001;15:2286–7. Ferraresi R, Troiano L, Roat E, Nemes E, Lugli E, Nasi M, et al. Protective effect of acetylL-carnitine against oxidative stress induced by antiretroviral drugs. FEBS Lett 2006;580:6612–6. Green DR, Van Houten B. Mitochondrial quality control. Cell 2011;147:950. Hawley TS, Hawley RG. Flow cytometry protocols. 2nd ed. New Jersey: Humana Press; 2004. Jiang LF, Yao TM, Zhu ZL, Wang C, Ji LN. Impacts of Cd (II) on the conformation and selfaggregation of Alzheimer’s tau fragment corresponding to the third repeat of microtubule-binding domain Biochim Biophys Acta – Proteins Proteom 2007;1774:1414–21. Joselin AP, Hewitt SJ, Callaghan SM, Kim RH, Chung YH, Mak TW, et al. ROS-dependent regulation of Parkin and DJ-1 localization during oxidative stress in neurons. Hum Mol Genet 2012;21:4888–903. Kamat PK, Kalani A, Kyles P, Tyagi SC, Tyagi N. Autophagy of mitochondria: a promising therapeutic target for neurodegenerative disease. Cell Biochem Biophys 2014;70: 707–19. Karalija A, Novikova LN, Kingham PJ, Wiberg M, Novikov LN. Neuroprotective effects of N-acetyl-cysteine and acetyl-L-carnitine after spinal cord injury in adult rats. PLoS ONE 2012;7:e41086.
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Contents lists available at ScienceDirect
NeuroToxicology
ROS act as an upstream signal to mediate cadmium-induced mitophagy in mouse brain Xue Wei a, Yongmei Qi a, Xiaoning Zhang a, Xueyan Gu a, Hui Cai b, Jing Yang a, Yingmei Zhang a,* a b
Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China Gansu Provincial Hospital, Lanzhou 730030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 September 2014 Accepted 17 November 2014 Available online 22 November 2014
As a well known generator of reactive oxygen species (ROS), cadmium (Cd) is found to be an effective inducer of mitophagy in mouse kidney and liver cells. Here, we aim to elucidate whether Cd can also initiate mitophagy in mouse brain and what role ROS play in this process. Our results showed that Cd caused overproduction of ROS. Meanwhile, Cd induced mitophagy, as indicated by the collapse of mitochondrial membrane potential (MMP), formation of mitophagosomes, increases of PINK1 level and LC3-II/LC3-I ratio and decrease of mitochondrial mass. Scavenging of ROS by N-acetyl-L-cysteine (NAC) or acetyl-L-carnitine (ALC) rescued MMP and mitochondrial mass, and squelched PINK1 level, mitochondrial accumulation of Parkin and LC3-II/LC3-I ratio, suggesting that ROS were associated with Cd-induced mitophagy. Cyclosporine A (CsA), an inhibitor of mitophagy, blocked Cd-induced mitophagy and PINK1/Parkin pathway but failed to suppress ROS increase, revealing that ROS are the causes rather than the results of Cd-induced mitophagy. In conclusion, this study suggested that ROS functioned on the upstream of PINK1/Parkin pathway to mediate Cd-induced mitophagy. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Cadmium Mitophagy ROS Brain Mouse
1. Introduction Autophagy is an adapting mechanism to respond to the changing environmental stimuli, such as starvation (Komatsu et al., 2005) and oxidative stress (Lemasters, 2005). It can degrade peroxisomes, endoplasmic reticulum, mitochondria and other organelles (Yu et al., 2008). Mitophagy, a specific process that selectively and targetedly removes mitochondria (Lemasters, 2005), can be marked by the collapse of mitochondrial membrane potential (MMP), accumulation of full-length PINK1, formation of mitophagosomes and decrease of mitochondrial mass. MMP collapse is the prerequisite of mitophagy (Elmore et al., 2001; Rodriguez-Enriquez et al., 2006) and also an early event of mitophagy to stabilize PINK1 (Matsuda et al., 2010; Tolkovsky, 2009). PINK1 is usually degraded and maintained at very low level in normal mitochondria, however, it can rapidly accumulate on mitochondrial membrane once mitochondria are damaged and MMP is declined (Youle and Narendra, 2010). The accumulation of
* Corresponding author at: School of Life Sciences, Lanzhou University, 222 South Tianshui R.D., Lanzhou 730000, China. Tel.: +86 13919123067; fax: +86 931 8913631. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.neuro.2014.11.007 0161-813X/ß 2014 Elsevier Inc. All rights reserved.
PINK1 can induce translocation of Parkin from cytosol to damaged mitochondria and reflect the progression of mitophagy (Narendra et al., 2010). Eventually, mitophagy removes damaged mitochondria by regulating mitochondrial mass (Park et al., 2012) and number (Youle and Narendra, 2010). Recent studies have showed that cadmium (Cd) can induce mitophagy in mouse kidney and liver cells (Pi et al., 2013; Wei et al., 2014). However, the impact of Cd on mitophagy in mouse brain and its molecular mechanism have not been explored. Several researches have showed that Cd can enter the brain, causing apoptosis and neurological alterations in clinical and animal studies (Łukawski et al., 2005; Nishimura et al., 2006; Yuan et al., 2013). Cd is also involved in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease (Jiang et al., 2007; Okuda et al., 1997), which are tightly related to mitophagy (Amadoro et al., 2014; Kamat et al., 2014). Notably, accumulating data have pointed that reactive oxygen species (ROS) are implicated in Cd-induced autophagy: Cd induces autophagy through ROS-activated GSK-3b pathway and ROS-dependent LKB1–AMPK signaling pathway (Son et al., 2011; Wang et al., 2009). However, the role of ROS in Cd-induced mitophagy is still unclear. Although some researches focused attention on the function of ROS in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitophagy using HeLa cells, their findings showed
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that ROS were not required (Narendra et al., 2008) or were indispensible (Wang et al., 2012) for mitophagy. Therefore, the present study sought to elucidate whether ROS are important players in the process of mitophagy, specifically induced by Cd. The role of ROS in Cd-induced mitophagy in mouse brain was examined using ROS scavengers N-acetyl-L-cysteine (NAC) (Downs et al., 2012; Marianna et al., 2013; Qu et al., 2013), acetyl-L-carnitine (ALC) (Akbas¸ et al., 2003; Ferraresi et al., 2006; Karalija et al., 2012) and mitophagy inhibitor cyclosporine A (CsA) (Kim et al., 2007; Rodriguez-Enriquez et al., 2006). Our data showed that ROS functioned as an upstream signal to mediate Cd-induced mitophagy. 2. Materials and methods 2.1. Animals Male Kunming mice (body weight 25.0 2.0 g) from Experimental Animal Center of Lanzhou University were adapted for 1 week before experiment. Mice were housed in controlled conditions of lighting (12 h light/dark cycles), temperature (24 2 8C) and were provided with standard food and water. Mice were randomly divided into eight groups and each contains 8 mice. Mice were intraperitoneally injected with physiological saline solution and Cd (0.05, 0.20, 0.80 mg/kg bw/day) to explore the mitophagic effect. Then 0.20 mg/ kg Cd was selected to co-treat with NAC (100 mg/kg bw/day), ALC (30 mg/kg bw/day) or CsA (10 mg/kg bw/day) to investigate the role of ROS in Cd-induced mitophagy. Mice were sacrificed by cervical dislocation 3 days following the final injection. Brains were harvested for analysis. All of the procedures were approved by the guidelines of Accreditation of Laboratory Animal Care. 2.2. Transmission electron microscopy For the transmission electron microscopy studies, the harvested brains were washed twice with PBS (pH 7.4), cut into pieces, and fixed with 2.5% glutaraldehyde. Then brain pieces were postfixed in 2% osmium tetroxide, embedded and stained with uranyl acetate/lead citrate. Sections were viewed under a JEM 1230 transmission electron microscope (JEOL, Tokyo, Japan) to detect mitophagosomes. 2.3. Single cell isolation Dissected brains were washed with cold PBS, minced with scissors at 4 8C and then filtered with cell strainer. The mixture was centrifuged at 500 rpm for 8 min, washed twice and used for flow cytometry (Hawley and Hawley, 2004). 2.4. Measurement of MMP, mitochondrial mass and ROS The isolated cells were washed twice and counted to 1 106 cells/ml. To measure MMP, brain cells were incubated with 1 mg/ml Rhodamine 123 (excitation, 507 nm; emission, 530 nm) at 37 8C for 30 min. To measure mitochondrial mass, the cells were incubated with 0.5 mM MitoTracker Green (MTG) (excitation, 490 nm; emission, 516 nm) at 37 8C for 30 min. To measure ROS, the cells were incubated with 10 mM 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (excitation, 480 nm; emission, 525 nm) at 37 8C for 30 min. Then the fluorescence intensity was monitored by flow cytometer (BD Biosciences, Franklin Lakes, NJ).
mitochondrial proteins were respectively stored at further western blotting detection.
80 8C for
2.6. Western blotting Protein samples that were isolated from brain tissues were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked and incubated with primary antibodies at 4 8C overnight. Anti-LC3 (1:1000), anti-PINK1 (1:1000) and anti-b-actin (1:1000) were purchased from Sigma–Aldrich (St. Louis, MO), Santa Cruz Biotechnology (Santa Cruz, CA) and ZhongShan Golden Bridge Biotechnology (Beijing, China), respectively. The secondary antibodies, anti-mouse (1:20,000) and anti-rabbit (1:20,000) were from ZhongShan Golden Bridge Biotechnology (Beijing, China) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. The blots were exposed to Immobilon Western Chemiluminescent HRP Substrate (Millipore, Boston, MA). 2.7. Statistical analysis All data were expressed as mean standard deviation (SD). Oneway analysis of variance (ANOVA) was used for multiple comparisons by SPSS 19.0 software (Chicago, IL). P < 0.01 was considered highly significant. 3. Results 3.1. Cd treatment increased the level of ROS Cd is known to induce ROS production. We first measured ROS level after the mice were treated by Cd for 3 days. Our data showed that ROS levels in 0.20 and 0.80 mg/kg Cd-treated groups were significantly higher than that in control group with 1.4 and 1.5-fold increase, respectively (P < 0.01, Fig. 1). 3.2. Cd induced mitophagy To determine the effects of Cd on mitophagy in mouse brain, we first evaluated the conversion of cellular protein LC3-I to LC3-II, a critical hallmark in autophagy process. As shown in Fig. 2A, compared with control, 0.05, 0.20 and 0.80 mg/kg Cd significantly elevated LC3-II/LC3-I ratio to 1.3, 2.9 and 2.8-fold, respectively (P < 0.01). To address whether mitophagy occurred, we determined the early event of mitophagy, the collapse of MMP. Three days of treatment with 0.20 and 0.80 mg/kg Cd dramatically decreased MMP of brain cells to 81.02% and 55.45% in mice compared with control (P < 0.01, Fig. 2B). Mitochondrial mass was assessed to reflect the amount of remaining mitochondria after mitophagy
2.5. Cytoplasmic and mitochondrial protein extraction To detect Parkin levels in cytoplasm and mitochondria, we used Cytoplasmic and Mitochondrial Protein Extraction Kit (Sangon, Shanghai, China). After extraction, the cytoplasmic and
Fig. 1. Cd treatment increased the level of ROS in mouse brain. Mice were exposed to increasing concentrations of Cd (0–0.80 mg/kg) for 3 days. ROS level was monitored by flow cytometry. 100 mM H2O2 was used as a positive control for ROS. Data were presented as mean SD, n = 8. **P < 0.01 compared with control.
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experiments. Our results showed that NAC or ALC pretreatment markedly decreased the ROS level compared with 0.20 mg/kg Cd alone (P < 0.01, Fig. 3A). Meanwhile, combination of Cd and NAC or ALC dramatically increased MMP and mitochondrial mass (P < 0.01, Fig. 3B), and prominently decreased PINK1 level (P < 0.01, Fig. 3C and D) and mitochondrial accumulation of Parkin (P < 0.01, Fig. 3E and F) compared with Cd alone. NAC or ALC also decreased LC3-II/LC3-I ratio compared with Cd alone (P < 0.01, Fig. 3G and H). Thus, these data indicated that ROS played a pivotal role in Cd-induced mitophagy and PINK1/Parkin pathway. To further explore how ROS mediated Cd-induced mitophagy, we used CsA to block mitophagy. The results showed that MMP and mitochondrial mass were significantly elevated after co-treatment of Cd and CsA compared with Cd treatment alone (P < 0.01, Fig. 4A). PINK1 level, mitochondrial accumulation of Parkin and LC3-II/LC3-I ratio were markedly decreased compared with Cd alone (P < 0.01, Fig. 4B, C and D). However, the ROS level of Cd and CsA co-treated group was not affected compared with Cd alone (Fig. 4E). Collectively, our data clearly showed that ROS functioned as an upstream signal to mediate Cd-induced mitophagy and PINK1/Parkin pathway. 4. Discussion
Fig. 2. Cd induced mitophagy in mouse brain. Mice were exposed to the gradually increased concentrations of Cd (0–0.80 mg/kg) for 3 days. (A) LC3-I and LC3-II levels were analyzed by western blotting. (B) MMP and mitochondrial mass were measured by flow cytometry. (C) Mitophagosome formation was analyzed under the transmission electron microscopy. (D) The level of PINK1 (full length) was detected by western blotting. Data were presented as mean SD, n = 8. ** P < 0.01 compared with control.
process. Compared with control, 0.20 and 0.80 mg/kg Cd exposure extremely decreased mitochondrial mass to 71.11% and 65.77%, respectively (P < 0.01, Fig. 2B). To directly visualize the process of mitophagy, transmission electron microscopy was used to examine mitophagosomes. We found that 0.20 mg/kg Cd exposure induced typical double-membrane and lamellar mitophagosomes in perinuclear area compared with control (Fig. 2C). Protein level of PINK1 was detected to quantify the mitophagy progress. Compared with control, 0.05, 0.20 and 0.80 mg/kg Cd significantly elevated PINK1 level to 2.5, 4.1 and 5.6-fold respectively (P < 0.01, Fig. 2D). 3.3. ROS functioned as an upstream signal to mediate Cd-induced mitophagy To determine whether Cd-induced ROS were essential players in Cd-induced mitophagy, we chose Cd (0.20 mg/kg) and ROS scavenger NAC (100 mg/kg) or ALC (30 mg/kg) for further
Previous studies have showed that various factors such as hypoxia, nutrient deprivation, photoirradiation and ROS were involved in mitophagy (Kim and Lemasters, 2011a,b; Wang et al., 2012; Zhang et al., 2008). As known by many studies, ROS can be induced by Cd. However, the effect of Cd on mitophagy in mouse brain and the role of ROS in this process remain elusive. Recent studies have suggested that Cd can induce mitophagy in mouse kidney and liver cells (Pi et al., 2013; Wei et al., 2014). Our data showed that Cd also could induce mitophagy in mouse brain. The MMP reduction signified that the early event of mitophagy happened. A direct evidence for mitophagy is the formation of mitophagosomes. The progress of mitophagy can be reflected by changes of mitochondrial proteins (Narendra et al., 2010). We found that mitochondrial protein PINK1 and autophagy marker LC3-II/LC3-I ratio were markedly increased by Cd. These data were consistent with previous study that mitophagy was triggered by the loss of MMP, and upon this loss, PINK1 was accumulated on the mitochondrial outer membrane to activate the ubiquitin ligase Parkin to mediate mitophagy (Green and Van Houten, 2011). The reduction of mitochondrial mass can be used to evaluate the result of mitophagy (Klionsky et al., 2012; Park et al., 2012; Zhang and Ney, 2010). In agreement with the report that mitochondrial mass was markedly reduced in CCCP-induced mitophagy (Narendra et al., 2008), Cd treatment grossly lessened mitochondrial mass. ROS can damage mitochondria and are typically associated with mitophagy in yeast and mouse kidney (Kurihara et al., 2012; Wei et al., 2014). The present study also suggested that Cd induced excess ROS production and addressed the role of ROS in Cdinduced mitophagy in mouse brain. The combination of Cd and NAC or ALC significantly suppressed Cd-induced ROS overproduction while reversed Cd-induced mitophagy, indicating that Cdinduced mitophagy was related to the ROS burst triggered by exogenous Cd. Similarly, an acute burst of ROS can cause loss of MMP and subsequently activate mitophagy in HeLa cells after CCCP treatment (Wang et al., 2012). Mitochondria are known to be the main source of ROS production, and high level of ROS could easily cause MMP decrease and mitochondrial depolarization to initiate mitophagy. In addition, the combination of Cd and NAC or ALC also blocked Cd-induced PINK1/Parkin pathway, suggesting that ROS might mediate PINK1/Parkin pathway, which supported the view that the function of PINK1/Parkin pathway appeared to be
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Fig. 3. ROS were important players in Cd-induced mitophagy in mouse brain. Mice were exposed to 0.20 mg/kg Cd alone or in combination with 100 mg/kg NAC or 30 mg/kg ALC. ROS level (A), MMP and mitochondrial mass (B) were measured by flow cytometry. The levels of PINK1 (full length) (C and D), Parkin in cytoplasm and mitochondria (E and F), LC3-I and LC3-II (G and H) were analyzed by western blotting. b-Tubulin was the internal control of cytoplasmic protein loading. HSP60 was the internal control of mitochondrial protein loading. C and M respectively denoted cytoplasm and mitochondria. Data were presented as mean SD, n = 8. **P < 0.01 compared with control. ## P < 0.01 compared with the corresponding Cd treatment alone.
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Fig. 4. ROS functioned as an upstream signal to mediate Cd-induced mitophagy in mouse brain. Mice were exposed to 0.20 mg/kg Cd alone or in combination with 10 mg/kg CsA for 3 days. MMP and mitochondrial mass (A) were then measured by flow cytometry. The levels of PINK1 (full length) (B), Parkin in cytoplasm and mitochondria (C), LC3-I and LC3-II (D) were analyzed by western blotting. b-Tubulin was the internal control of cytoplasmic protein loading. HSP60 was the internal control of mitochondrial protein loading. C and M respectively denoted cytoplasm and mitochondria. ROS level (E) was monitored by flow cytometry. Data were presented as mean SD, n = 8. ** P < 0.01 compared with control. ##P < 0.01 compared with the corresponding Cd treatment alone.
dependent on ROS (Dagda et al., 2009; Joselin et al., 2012). Based on these findings and our results, we conclude that ROS are tightly implicated in Cd-induced mitophagy and PINK1/Parkin pathway. Previous studies suggested that an increase in cellular ROS, either mitochondrial ROS or exogenous ROS, could upregulate general autophagy (Chen et al., 2009; Scherz-Shouval et al., 2007). How ROS functioned in Cd-induced mitophagy? Several studies show that ROS function downstream of mitophagy pathway in human neuronal cells where knockdown of the essential mitophagy proteins Atg7 and Atg8/LC3B inhibits mitophagy without affecting the increased mitochondrial superoxide (Dagda et al.,
2009) and mitophagy contributes a lot to preventing excess mitochondrial ROS production in yeast and mouse embryo fibroblasts (Kurihara et al., 2012; Zhang et al., 2008). However, Our study showed that CsA and Cd co-treatment markedly inhibited mitophagy without affecting Cd-induced ROS level. This finding suggested that ROS were the upstream signal of Cdinduced mitophagy in mouse brain, which was in accordance with our previous report in which ROS mediated Cd-induced mitophagy through PINK1/Parkin pathway in mouse kidney (Wei et al., 2014). This study was also consistent with the report that ROS work as an important upstream activator to induce mitochondrial damage
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and further promote mitophagy under CCCP exposure, in which, ROS-induced PARK2 recruitment triggered mitophagy (Wang et al., 2012). Besides, ROS can also induce the opening of mitochondrial permeability transition pore, simultaneously cause MMP collapse and serve as an upstream signal to initiate mitophagy (Kim et al., 2007; Lemasters, 2005). In summary, our data demonstrated that Cd induced mitophagy in mouse brain, and Cd-induced ROS functioned as an upstream signal on PINK1/Parkin pathway to mediate mitophagy progress. Our results may contribute to further understanding of the Cdcaused neurotoxicity. Transparency document The transparency document associated with this article can be found in the online version. Conflict of interests The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (31300941), the Fundamental Research Funds for the Central Universities (lzujbky-2014-91), the Traditional Chinese Medicine Research Fund of Gansu (GZK-2012-61) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1137). References ¨ zcan C, et al. The effect of L-carnitine on the Akbas¸ Y, Pata YS, Go¨ru¨r K, Polat G, Polat A, O prevention of experimentally induced myringosclerosis in rats Hear Res 2003;184:107–12. Amadoro G, Corsetti V, Florenzano F, Atlante A, Bobba A, Nicolin V, et al. Morphological and bioenergetic demands underlying the mitophagy in post-mitotic neurons: the pink-parkin pathway. Front Aging Neurosci 2014;6:18. Chen Y, Azad M, Gibson S. Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ 2009;16:1040–52. Dagda RK, Cherra SJ, Kulich SM, Tandon A, Park D, Chu CT. Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission. J Biol Chem 2009;284:13843–55. Downs I, Liu J, Aw TY, Adegboyega PA, Ajuebor MN. The ROS scavenger, NAC, regulates hepatic Va14iNKT cells signaling during Fas mAb-dependent fulminant liver failure. PLoS ONE 2012;7:e38051. Elmore SP, Qian T, Grissom SF, Lemasters JJ. The mitochondrial permeability transition initiates autophagy in rat hepatocytes. FASEB J 2001;15:2286–7. Ferraresi R, Troiano L, Roat E, Nemes E, Lugli E, Nasi M, et al. Protective effect of acetylL-carnitine against oxidative stress induced by antiretroviral drugs. FEBS Lett 2006;580:6612–6. Green DR, Van Houten B. Mitochondrial quality control. Cell 2011;147:950. Hawley TS, Hawley RG. Flow cytometry protocols. 2nd ed. New Jersey: Humana Press; 2004. Jiang LF, Yao TM, Zhu ZL, Wang C, Ji LN. Impacts of Cd (II) on the conformation and selfaggregation of Alzheimer’s tau fragment corresponding to the third repeat of microtubule-binding domain Biochim Biophys Acta – Proteins Proteom 2007;1774:1414–21. Joselin AP, Hewitt SJ, Callaghan SM, Kim RH, Chung YH, Mak TW, et al. ROS-dependent regulation of Parkin and DJ-1 localization during oxidative stress in neurons. Hum Mol Genet 2012;21:4888–903. Kamat PK, Kalani A, Kyles P, Tyagi SC, Tyagi N. Autophagy of mitochondria: a promising therapeutic target for neurodegenerative disease. Cell Biochem Biophys 2014;70: 707–19. Karalija A, Novikova LN, Kingham PJ, Wiberg M, Novikov LN. Neuroprotective effects of N-acetyl-cysteine and acetyl-L-carnitine after spinal cord injury in adult rats. PLoS ONE 2012;7:e41086.
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