Mitochondrial autophagy as a compensatory response to PINK1 deficiency

Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission, and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.     

Mitochondrial fission facilitates mitophagy in Saccharomyces cerevisiae

As a highly dynamic organelle, mitochondria undergo constitutive fusion and fission as well as biogenesis and degradation. Mitophagy, selective mitochondrial degradation through autophagy, is a conserved cellular process used for the elimination of excessive and damaged mitochondria in eukaryotes. Despite the significance of mitophagy in cellular physiology and pathophysiologies, the underlying mechanism of this process is far from clear. In this report, we studied the role of mitochondrial fission during mitophagy, and uncover a direct link between the fission complex and mitophagy machinery in Saccharomyces cerevisiae.     

Inhibition of peroxisome fission,but not mitochondrial fission,increases yeast chronological lifespan

Mitochondria are key players in aging and cell death. It has been suggested that mitochondrial fragmentation, mediated by the Dnm1/Fis1 organelle fission machinery, stimulates aging and cell death. This was based on the observation that Saccharomyces cerevisiae Δdnm1 and Δfis1 mutants show an enhanced lifespan and increased resistance to cell death inducers. However, the Dnm1/Fis1 fission machinery is also required for peroxisome division. Here we analyzed the significance of peroxisome fission in yeast chronological lifespan, using yeast strains in which fission of mitochondria was selectively blocked. Our data indicate that the lifespan extension caused by deletion of FIS1 is mainly due to a defect in peroxisome fission and not caused by a block in mitochondrial fragmentation. These observations are underlined by our observation that deletion of FIS1 does not lead to lifespan extension in yeast peroxisome deficient mutant cells.     

The SUMO protease SENP3 regulates mitochondrial autophagy mediated by Fis1

Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress‐induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP‐induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP‐induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP‐induced mitophagy in SENP3‐depleted cells. Thus, we propose a model in which SENP3‐mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress‐induced mitophagy.     

A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

Mitochondrial fission and remodelling contributes to muscle atrophy

Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro‐fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy‐lysosome and the ubiquitin–proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial‐dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.     

The mitochondrial fission protein hFis1 requires the endoplasmic reticulum gateway to induce apoptosis

Mitochondrial fission ensures organelle inheritance during cell division and participates in apoptosis. The fission protein hFis1 triggers caspase-dependent cell death, by causing the release of cytochrome c from mitochondria. Here we show that mitochondrial fission induced by hFis1 is genetically distinct from apoptosis. In cells lacking the multidomain proapoptotic Bcl-2 family members Bax and Bak (DKO), hFis1 caused mitochondrial fragmentation but not organelle dysfunction and apoptosis. Similarly, a mutant in the intermembrane region of hFis1-induced fission but not cell death, further dissociating mitochondrial fragmentation from apoptosis induction. Selective correction of the endoplasmic reticulum (ER) defect of DKO cells restored killing by hFis1, indicating that death by hFis1 relies on the ER gateway of apoptosis. Consistently, hFis1 did not directly activate BAX and BAK, but induced Ca(2+)-dependent mitochondrial dysfunction. Thus, hFis1 is a bifunctional protein that independently regulates mitochondrial fragmentation and ER-mediated apoptosis.     

Mitochondrial fission,integrity and completion of mitophagy require separable functions of Vps13D in Drosophila neurons

A healthy population of mitochondria, maintained by proper fission, fusion, and degradation, is critical for the long-term survival and function of neurons. Here, our discovery of mitophagy intermediates in fission-impaired Drosophila neurons brings new perspective into the relationship between mitochondrial fission and mitophagy. Neurons lacking either the ataxia disease gene Vps13D or the dynamin related protein Drp1 contain enlarged mitochondria that are engaged with autophagy machinery and also lack matrix components. Reporter assays combined with genetic studies imply that mitophagy both initiates and is completed in Drp1 impaired neurons, but fails to complete in Vps13D impaired neurons, which accumulate compromised mitochondria within stalled mito-phagophores. Our findings imply that in fission-defective neurons, mitophagy becomes induced, and that the lipid channel containing protein Vps13D has separable functions in mitochondrial fission and phagophore elongation.     

hFis1, a novel component of the mammalian mitochondrial fission machinery

The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.     

Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin

Damage to mitochondria can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. However, fusion of uncoupled mitochondria with polarized mitochondria can compensate for damage, reverse membrane depolarization, and obviate mitophagy. Parkin, an E3 ubiquitin ligase that is mutated in monogenic forms of Parkinson's disease, was recently found to induce selective autophagy of damaged mitochondria. Here we show that ubiquitination of mitofusins Mfn1 and Mfn2, large GTPases that mediate mitochondrial fusion, is induced by Parkin upon membrane depolarization and leads to their degradation in a proteasome- and p97-dependent manner. p97, a AAA+ ATPase, accumulates on mitochondria upon uncoupling of Parkin-expressing cells, and both p97 and proteasome activity are required for Parkin-mediated mitophagy. After mitochondrial fission upon depolarization, Parkin prevents or delays refusion of mitochondria, likely by the elimination of mitofusins. Inhibition of Drp1-mediated mitochondrial fission, the proteasome, or p97 prevents Parkin-induced mitophagy.     

综述: 细胞自噬和线粒体质量控制与健康衰老

拥有健康的晚年是每一个人的祈盼,这也是目前应对即将到来的社会老龄化危机而需要解决的重要课题.实现健康衰老需要对人类衰老发生的机制有深入的了解,比如在此过程中扮演着重要角色的线粒体的研究.线粒体是细胞能量和自由基代谢中心,也是细胞凋亡调控中心,并在信号转导和基因表达调控中发挥重要作用.线粒体一旦受损,一方面能量代谢发生紊乱,另一方面产生大量自由基,影响细胞的正常生长,并导致细胞甚至机体的衰老.正常情况下,细胞通过自噬溶酶体机制选择性清除受损伤和不需要的线粒体,这是线粒体质量控制的重要机制.研究发现,线粒体质量控制异常可能在衰老发生过程中起关键作用.限食及增强运动能有效促进线粒体质量控制,改善线粒体功能并延缓衰老.     

Mitochondrial dismissal in mammals,from protein degradation to mitophagy

Mitochondria are double-membraned highly dynamic organelles; the shape, location and function of which are determined by a constant balance between opposing fusion and fission events. A fine modulation of mitochondrial structure is crucial for their correct functionality and for many physiological cell processes, the status of these organelles, being thus a key aspect in a cell's fate. Indeed, the homeostasis of mitochondria needs to be highly regulated for the above mentioned reasons, and since a) they are the major source of energy; b) they participate in various signaling pathways; albeit at the same time c) they are also the major source of reactive oxygen species (ROS, the main damaging detrimental players for all cell components). Elaborate mechanisms of mitochondrial quality control have evolved for maintaining a functional mitochondrial network and avoiding cell damage. The first mechanism is the removal of damaged mitochondrial proteins within the organelle via chaperones and protease; the second is the cytosolic ubiquitin–proteasome system (UPS), able to eliminate proteins embedded in the outer mitochondrial membrane; the third is the removal of the entire mitochondria through mitophagy, in the case of extensive organelle damage and dysfunction. In this review, we provide an overview of these mitochondria stability and quality control mechanisms, highlighting mitophagy, and emphasizing the central role of mitochondrial dynamics in this context. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.     

Regulation by mitophagy

Eukaryotes employ elaborate mitochondrial quality control to maintain the function of the power-generating organelle. Mitochondrial quality control is particularly important for the maintenance of neural and muscular tissues. Mitophagy is specialized version of the autophagy pathway. Mitophagy delivers damaged mitochondria to lysosomes for degradation. Recently, a series of elegant studies have demonstrated that two Parkinson's disease-associated genes PINK1 and parkin are involved in the maintenance of healthy mitochondria as mitophagy. Parkin in co-operation with PINK1 specifically recognizes damaged mitochondria with reduced mitochondrial membrane potential (Δψm), rapidly isolates them from the mitochondrial network and eliminates them through the ubiquitin–proteasome and autophagy pathways. Here we introduce and review recent studies that contribute to understanding the molecular mechanisms of mitophagy such as PINK1 and Parkin-mediated mitochondrial regulation. We also discuss how defects in the PINK1–Parkin pathway may cause neurodegeneration in Parkinson's disease.     

The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.     

Fis1, Mff,MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission

Several mitochondrial outer membrane proteins—mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)—have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.     

Mitochondrial quality control in diabetic cardiomyopathy: from molecular mechanisms to therapeutic strategies

In diabetic cardiomyopathy (DCM), a major diabetic complication, the myocardium is structurally and functionally altered without evidence of coronary artery disease, hypertension or valvular disease. Although numerous anti-diabetic drugs have been applied clinically, specific medicines to prevent DCM progression are unavailable, so the prognosis of DCM remains poor. Mitochondrial ATP production maintains the energetic requirements of cardiomyocytes, whereas mitochondrial dysfunction can induce or aggravate DCM by promoting oxidative stress, dysregulated calcium homeostasis, metabolic reprogramming, abnormal intracellular signaling and mitochondrial apoptosis in cardiomyocytes. In response to mitochondrial dysfunction, the mitochondrial quality control (MQC) system (including mitochondrial fission, fusion, and mitophagy) is activated to repair damaged mitochondria. Physiological mitochondrial fission fragments the network to isolate damaged mitochondria. Mitophagy then allows dysfunctional mitochondria to be engulfed by autophagosomes and degraded in lysosomes. However, abnormal MQC results in excessive mitochondrial fission, impaired mitochondrial fusion and delayed mitophagy, causing fragmented mitochondria to accumulate in cardiomyocytes. In this review, we summarize the molecular mechanisms of MQC and discuss how pathological MQC contributes to DCM development. We then present promising therapeutic approaches to improve MQC and prevent DCM progression.     

Mitophagy,a potential therapeutic target for stroke

Mitochondria autophagy, termed as mitophagy, is a mechanism of specific autophagic elimination of mitochondria. Mitophagy controls the quality and the number of mitochondria, eliminating dysfunctional or excessive mitochondria that can generate reactive oxygen species (ROS) and cause cell death. Mitochondria are centrally implicated in neuron and tissue injury after stroke, due to the function of supplying adenosine triphosphate (ATP) to the tissue, regulating oxidative metabolism during the pathologic process, and contribution to apoptotic cell death after stroke. As a catabolic mechanism, mitophagy links numbers of a complex network of mitochondria, and affects mitochondrial dynamic process, fusion and fission, reducing mitochondrial production of ROS, mediated by the mitochondrial permeability transition pore (MPTP). The precise nature of mitophagy’s involvement in stroke, and its underlying molecular mechanisms, have yet to be fully clarified. This review aims to provide a comprehensive overview of the integration of mitochondria with mitophagy, also to introduce and discuss recent advances in the understanding of the potential role, and possible signaling pathway, of mitophagy in the pathological processes of both hemorrhagic and ischemic stroke. The author also provides evidence to explain the dual role of mitophagy in stroke.     

Mff is an essential factor for mitochondrial recruitment of Drp1 during mitochondrial fission in mammalian cells

The cytoplasmic dynamin-related guanosine triphosphatase Drp1 is recruited to mitochondria and mediates mitochondrial fission. Although the mitochondrial outer membrane (MOM) protein Fis1 is thought to be a Drp1 receptor, this has not been confirmed. To analyze the mechanism of Drp1 recruitment, we manipulated the expression of mitochondrial fission and fusion proteins and demonstrated that (a) mitochondrial fission factor (Mff) knockdown released the Drp1 foci from the MOM accompanied by network extension, whereas Mff overexpression stimulated mitochondrial recruitment of Drp1 accompanied by mitochondrial fission; (b) Mff-dependent mitochondrial fission proceeded independent of Fis1; (c) a Mff mutant with the plasma membrane-targeted CAAX motif directed Drp1 to the target membrane; (d) Mff and Drp1 physically interacted in vitro and in vivo; (e) exogenous stimuli-induced mitochondrial fission and apoptosis were compromised by knockdown of Drp1 and Mff but not Fis1; and (f) conditional knockout of Fis1 in colon carcinoma cells revealed that it is dispensable for mitochondrial fission. Thus, Mff functions as an essential factor in mitochondrial recruitment of Drp1.