Mitochondria autophagy is induced after hypoxic/ischemic stress in a Drp1 dependent manner: The role of inhibition of Drp1 in ischemic brain damage
Graphical abstract
Ischemia injury induced bulk autophagy as well as selective mitophagy. Drp-1 mediated mitochondrial fission is the premise of ischemia induced mitophagy that could timely remove of the injured mitochondria and decrease the mitochondria-mediated apoptosis. Mdivi-1 selectively inhibited the translocation of Drp-1 to mitochondria and thus blocked the process of mitophagy. The accumulation of mitochondria exaggerated the ischemia injury by increasing the release of cyt-c, AIF, ROS and so on.
Introduction
Mitochondria are now recognized as a unique and irreplaceable organelle in eukaryotic cells, which not only act as a plant for energy production, but also play important roles in cell death and survival, especially in the condition of hypoxic/ischemic (H/I) injury (Hagberg, 2004, Sas et al., 2007). Mitochondria are the main sources for ROS, Ca2+ and pro-apoptosis factors in cells, and the immediate targets of intracellular and extracellular insults as well. H/I stress is known to induce mitochondrial membrane potential (MMP) changes, mitochondrion fragmentation and dysfunction, which further exaggerate the injury. Many researchers reported that the damaged mitochondria accumulate under conditions of oxidative stress, suggesting that maintaining a pool of healthy mitochondria is crucial for protecting against injury (Lee et al., 2005, Lee et al., 2006).
The researches in the past years have put the emphasis on dysfunctional mitochondria-mediated signaling pathway. In stroke, the therapeutics that have been developed primarily target pro-death signaling pathways up/down-stream of mitochondrial damage, such as death-execution effectors and reactive oxygen species (ROS). However people ignored mitochondria as a whole are dynamic organelles which constitute a dynamic interconnecting network. Mitochondrial functions vary with their changes in morphology (fusion and fission) and abundance (biogenesis and mitophagy) in a variety of diseases (Zorzano et al., 2009a), including cardiovascular disease (Lavandero et al., 2011), metabolic disorders (Yoon et al., 2011, Zorzano et al., 2009b) and neurological diseases (Chen and Chan, 2009). These facts bring up the possibility that novel therapeutics may be designed by better understanding the roles of equilibrium of a healthy mitochondria pool and the mechanism thereof in the pathogenesis of stroke. In this regard, efficient and timely clearance of damaged mitochondria is expected to prevent activation of mitochondria-mediated death pathways, protect against ROS burst and preserve efficient supply of ATP (Thomas and Gustafsson, 2013).
Mitochondria can be degraded by non-selective autophagy, as well as by mitophagy, a selective pattern of autophagy. Non-selective autophagy and mitophagy have been shown to be triggered in response to nutrition deprivation, starvation or rapamycin (Lee et al., 2012, Scherz-Shouval et al., 2007, Sören et al., 2012). Mitochondria engulfed by autophagosome is observed under starvation (Lyamzaev et al., 2004, Priault et al., 2005) and numerous other conditions, particularly when mitochondrial normal function is compromised (Kim et al., 2007, Rodriguez-Hernandez et al., 2009). Several studies have shown that mitophagy plays a major role in the specific recognition and removal of damaged mitochondria in order to ensure mitochondrial quality control (Okamoto and Kondo-Okamoto, 2012). Autophagy is also reported to be involved in OGD cell models (Grishchuk et al., 2011;) or in cerebral ischemia animal models (Sheng et al., 2010, Wang et al., 2012a, Wang et al., 2012b). However, whether mitochondria are targeted by autophagy after brain ischemia is unclear yet.
A number of factors that are responsible for mitophagy initiation have been identified such as Atg32 (Kanki et al., 2009), Uth1p (Kissova et al., 2004), Atg32 (Kanki et al., 2009) and Aup1p (Tal et al., 2007). Studies showed that Parkin, a kinase, and PINK1, an E3-ubiquitin ligase, play key roles in pathologic process of Parkinson's disease (PD), because recruiting Parkin to the impaired mitochondria leads to its autophagy in eukaryotic cells (Karbowski et al., 2007). In reticulocytes, BNIP3L, a Bcl-2-related protein, is reported to act as a mitochondrial receptor required for mitochondrial clearance (Novak et al., 2010). Here an open question is how dysfunctional mitochondria are distinguished from the healthy ones after H/I injury once the mitophagy has occurred. It has been hypothesized that there exists a mechanism capable of separating the dysfunctional mitochondria from the functional ones for removal of damaged mitochondria after ischemic-hypoxic injury. The dynamic characteristic of mitochondrial morphology has been reported to be involved in this process (Bereiter-Hahn and Vöth, 1994). Current investigations indicate that mitochondrial fission/fusion is kept under tight control of conserved dynamin-related GTPases (Westermann, 2010). Among these ones, dynamin-related protein 1 (Drp1) is a critical regulator of mitochondrial fission which brings into play after translocating to mitochondrial outer-membrane from cytoplasm. On the contrary fusion is controlled by mitofusin-1/2 (Mfn-1/2) (Lackner and Nunnari, 2008). It has been proved in in vitro studies that during starvation (Rambold et al., 2011) or normal (Parone et al., 2008) conditions, mitophagy is blocked when mitochondrial fission is interrupted. Mitochondrial fission enhancement by hFis1 over-expression is reported to promote mitophagy (Gomes and Scorrano, 2008). On the other hand, some studies have produced conflicting evidences regarding the role of mitochondrial pro-fission factors in mitophagy. For example, it was reported that starvation-stimulated mitophagy is uninfluenced in a condition lacking the mitochondrial pro-fission factors such as Mdv1, Fis1 or Caf4 (Kanki et al., 2009). Thus, it is until now obscure whether mitochondrial fission is involved in the clearance of dysfunctional mitochondria under H/I conditions and other conditions.
H/I injury induced by pMCAO in rats or OGD in vitro leads to bulk autophagy that is known to remove mitochondria either. However, it is not known whether in these cases mitochondrial fission allows induction of mitophagy while non-selective autophagy is not triggered or changed simultaneously.
In the present study, mitophagy was investigated in a rat model of pMCAO and PC12 cells exposed to oxygen–glucose deprivation (OGD). Using immunofluorescence staining and electron microcopy, we showed that H/I injury leads to the induction of mitophagy as well as mitochondrial fragmentation. We also provided evidence by inhibition of Drp1 that mitochondrial fission is necessary for the induction of mitophagy while without affecting non-selective autophagy. Drp1 inhibitors were found to exaggerate neurological deficits and increase the infarct volume in pMCAO by promoting mitochondria-mediated cell death pathway.
Section snippets
Rat pMCAO model and drug administration
Adult male Sprague-Dawley rats weighing 260–280 g were purchased from VITAL RIVER Laboratories, Beijing, China. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Peking Union Medical College and Chinese Academy of Medical Sciences, and were in accordance with the principles outlined in the NIH Guide for the Care and Use of Laboratory Animals. Permanent cerebral ischemia was induced by middle cerebral artery occlusion (pMCAO) for 1 (M1), 3 (M3), 6
Statistical analysis
Data were presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using analysis of one way or two-way ANOVA. Tukey's post hoc test was used for multiple comparisons. T-test was used for testing between two groups. Differences were considered significant if P values were less than 0.05.
Mitochondrial autophagy is induced by pMCAO injury
Beclin-1 and LC3B are involved in the formation of autophagolysosomes, while p62 can constantly be degraded by autophagy. The pMCAO model used for H/I stress was well known to induce autophagy (Qi et al., 2012). In our study, the results showed that the levels of LC3B and Beclin-1 were increased in the ischemic cortex, peaking at 3 h and 1 h, respectively (Fig. 1A–C, p < 0.01). A reduction in p62 protein levels was observed with a minimum level at 3 h after pMCAO (Fig. 1A, D, p < 0.01), which
Discussion
Mitochondrial dysfunction is linked with aging (Wang et al., 2013) and various CNS diseases such as PD (Ziviani et al., 2010). The molecular mechanisms for removal of dysfunctional mitochondria are thought of as crucial. However, the specific mechanism responsible for mitochondrial selective mitophagy and dynamics remains elusive. For example, which conditions induce mitophagy specifically? Are the mechanistic details for mitophagy identical to that for the bulk autophagy? Can mitophagy occur
Acknowledgments
National Natural Science Foundation of China Grants (No. 81274122, 81102831, 81073078, 81373997, 81173578). Special Purpose for New Drug Development (2012ZX09301002-004, 2012ZX09103101-006). Studies on Structure and function of Bioactive Substances from Natural Medicines (IRT1007). Beijing Natural Science Foundation (7131013), Research Fund for the Doctoral Program of Higher Education of China (20121106130001). Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study
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