Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells

Mitophagy, the selective removal of mitochondria through the autophagic pathway, is involved in cellular mitochondria quality control. Dysfunctional mitochondria can be selectively eliminated through Parkin-mediated mitophagy. Parkin is a ubiquitin E3 ligase that selectively translocates onto impaired mitochondria to initiate mitophagy, and mutations in Parkin have been identified in autosomal recessive forms of Parkinson disease. Here with the use of a genetically encoded, mitochondria-matrix targeting photosensitizer, we established a robust strategy that allows for spatiotemporally controlled initiation of Parkin-mediated mitophagy in single cells with light. The method can specifically target varying numbers of mitochondria into the Parkin-mediated mitophagy pathway for clearance. Combined with live cell imaging, we demonstrated that mitochondria can be cleared by Parkin-mediated mitophagy without juxtanuclear mito-aggresome formation. Autophagy proceeded with the asynchronous appearance of small LC3B-coated structures on Parkin-labeled mitochondria subsections in a nucleation-expansion manner. Our method allows for quantitative measurement on the Parkin-mediated mitophagy process, and can be multiplexed in imaging for higher throughput studies.     

Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells

Mitophagy, the selective removal of mitochondria through the autophagic pathway, is involved in cellular mitochondria quality control. Dysfunctional mitochondria can be selectively eliminated through Parkin-mediated mitophagy. Parkin is a ubiquitin E3 ligase that selectively translocates onto impaired mitochondria to initiate mitophagy, and mutations in Parkin have been identified in autosomal recessive forms of Parkinson disease. Here with the use of a genetically encoded, mitochondria-matrix targeting photosensitizer, we established a robust strategy that allows for spatiotemporally controlled initiation of Parkin-mediated mitophagy in single cells with light. The method can specifically target varying numbers of mitochondria into the Parkin-mediated mitophagy pathway for clearance. Combined with live cell imaging, we demonstrated that mitochondria can be cleared by Parkin-mediated mitophagy without juxtanuclear mito-aggresome formation. Autophagy proceeded with the asynchronous appearance of small LC3B-coated structures on Parkin-labeled mitochondria subsections in a nucleation-expansion manner. Our method allows for quantitative measurement on the Parkin-mediated mitophagy process, and can be multiplexed in imaging for higher throughput studies.     

Deubiquitinating enzymes regulate PARK2-mediated mitophagy

The selective degradation of mitochondria by the process of autophagy, termed mitophagy, is one of the major mechanisms of mitochondrial quality control. The best-studied mitophagy pathway is the one mediated by PINK1 and PARK2/Parkin. From recent studies it has become clear that ubiquitin-ligation plays a pivotal role and most of the focus has been on the role of ubiquitination of mitochondrial proteins in mitophagy. Even though ubiquitination is a reversible process, very little is known about the role of deubiquitinating enzymes (DUBs) in mitophagy. Here, we report that 2 mitochondrial DUBs, USP30 and USP35, regulate PARK2-mediated mitophagy. We show that USP30 and USP35 can delay PARK2-mediated mitophagy using a quantitative mitophagy assay. Furthermore, we show that USP30 delays mitophagy by delaying PARK2 recruitment to the mitochondria during mitophagy. USP35 does not delay PARK2 recruitment, suggesting that it regulates mitophagy through an alternative mechanism. Interestingly, USP35 only associates with polarized mitochondria, and rapidly translocates to the cytosol during CCCP-induced mitophagy. It is clear that PARK2-mediated mitophagy is regulated at many steps in this important quality control pathway. Taken together, these findings demonstrate an important role of mitochondrial-associated DUBs in mitophagy. Because defects in mitochondria quality control are implicated in many neurodegenerative disorders, our study provides clear rationales for the design and development of drugs for the therapeutic treatment of neurodegenerative diseases such as Parkinson and Alzheimer diseases.     

ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy

Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1, phosphorylating it at serine 17, which enhances FUNDC1 binding to LC3. A ULK1‐binding‐deficient mutant of FUNDC1 prevents ULK1 translocation to mitochondria and inhibits mitophagy. Finally, kinase‐active ULK1 and a phospho‐mimicking mutant of FUNDC1 rescue mitophagy in ULK1‐null cells. Thus, we conclude that FUNDC1 regulates ULK1 recruitment to damaged mitochondria, where FUNDC1 phosphorylation by ULK1 is crucial for mitophagy.     

Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons

Mitochondria are essential for neuronal survival and function. Proper degradation of aged and damaged mitochondria through mitophagy is a key cellular pathway for mitochondrial quality control. Recent studies have indicated that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Translocation of Parkin to damaged mitochondria induces mitophagy in many nonneuronal cell types. However, evidence showing Parkin translocation in primary neurons is controversial, leaving unanswered questions as to how and where Parkin-mediated mitophagy occurs in neurons. Here, we report the unique process of dissipating mitochondrial Δψ(m)-induced and Parkin-mediated mitophagy in mature cortical neurons. Compared with nonneuronal cells, neuronal mitophagy is a much slower and compartmentally restricted process, coupled with reduced anterograde mitochondrial transport. Parkin-targeted mitochondria are accumulated in the somatodendritic regions where mature lysosomes are predominantly located. Time-lapse imaging shows dynamic formation and elimination of Parkin- and LC3-ring-like structures surrounding depolarized mitochondria through the autophagy-lysosomal pathway in the soma. Knocking down Parkin in neurons impairs the elimination of dysfunctional mitochondria. Thus, our study provides neuronal evidence for dynamic and spatial Parkin-mediated mitophagy, which will help us understand whether altered mitophagy contributes to pathogenesis of several major neurodegenerative diseases characterized by mitochondrial dysfunction and impaired transport.     

ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy

Reactive oxygen species (ROS) have been implicated as a signal for general autophagy. Both mitochondrial-produced and exogenous ROS induce autophagosome formation. However, it is unclear whether ROS are required for the selective autophagic degradation of mitochondria, a process called mitophagy. Recent work using carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial-uncoupling reagent, has been shown to induce mitophagy. However, CCCP treatment may not be biologically relevant since it causes the depolarization of the entire mitochondrial network. Since mitochondria are the main ROS production sites in mammalian cells, we propose that short bursts of ROS produced within mitochondria may be involved in the signaling for mitophagy. To test this hypothesis, we induced an acute burst of ROS within mitochondria using a mitochondrial-targeted photosensitizer, mitochondrial KillerRed (mtKR). Using mtKR, we increased ROS levels in the mitochondrial matrix, which resulted in the loss of membrane potential and the subsequent activation of PARK2-dependent mitophagy. Importantly, we showed that overexpression of the mitochondrial antioxidant protein, superoxide dismutase-2, can squelch mtKR-induced mitophagy, demonstrating that mitochondrial ROS are responsible for mitophagy activation. Using this assay, we examined the impact of mitochondrial morphology on mitophagy. It was shown recently that elongated mitochondria are more resistant to mitophagy through unknown mechanisms. Here, we show that elongated mitochondria are more resistant to ROS-induced damage and mitophagy compared with fragmented mitochondria, suggesting that mitochondrial morphology has an important role in regulating ROS and mitophagy. Together, our results suggest that ROS-induced mitochondrial damage may be an important upstream activator of mitophagy.     

Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells

Accumulating evidence has shown that dysfunctional mitochondria can be selectively removed by mitophagy. Dysregulation of mitophagy is implicated in the development of neurodegenerative disease and metabolic disorders. How individual mitochondria are recognized for removal and how this process is regulated remain poorly understood. Here we report that FUNDC1, an integral mitochondrial outer-membrane protein, is a receptor for hypoxia-induced mitophagy. FUNDC1 interacted with LC3 through its typical LC3-binding motif Y(18)xxL(21), and mutation of the LC3-interaction region impaired its interaction with LC3 and the subsequent induction of mitophagy. Knockdown of endogenous FUNDC1 significantly prevented hypoxia-induced mitophagy, which could be reversed by the expression of wild-type FUNDC1, but not LC3-interaction-deficient FUNDC1 mutants. Mechanistic studies further revealed that hypoxia induced dephosphorylation of FUNDC1 and enhanced its interaction with LC3 for selective mitophagy. Our findings thus offer insights into mitochondrial quality control in mammalian cells.     

Characterization of a natural variant of human NDP52 and its functional consequences on mitophagy

The role of mitophagy, a process that allows the removal of damaged mitochondria from cells, remains unknown in multiple sclerosis (MS), a disease that is found associated with dysfunctional mitochondria. Here we have qualitatively and quantitatively studied the main players in PINK1-mediated mitophagy in peripheral blood mononuclear cells (PBMCs) of patients with relapsing–remitting MS. We found the variant c.491G>A (rs550510, p.G140E) of NDP52, one of the major mitophagy receptor genes, associated with a MS cohort. Through the characterization of this variant, we discovered that the residue 140 of human NDP52 is a crucial modulator of NDP52/LC3C binding, promoting the formation of autophagosomes in order to drive efficient mitophagy. In addition, we found that in the PBMC population, NDP52 is mainly expressed in B cells and by ensuring efficient mitophagy, it is able to limit the production of the proinflammatory cytokine TNF-α following cell stimulation. In sum, our results contribute to a better understanding of the role of NDP52 in mitophagy and underline, for the first time, a possible role of NDP52 in MS.Subject terms: Autophagy, Molecular modelling, Immunological disorders     

Atg9A-mediated mitophagy is required for decidual differentiation of endometrial stromal cells

Endometrial decidualization is the foundation of a healthy pregnancy. Mitochondrial dysfunction is an independent cause of disease for energy-intensive organs. Mitochondrial homeostasis plays a key role in the differentiation processes of many cell types. We showed increased activation of mitophagy (mitochondrial autophagy) in the decidua compared with proliferative or secretory endometrium. To better comprehend the mechanisms underlying healthy conception, understanding the mechanism of endometrial stromal cell decidualization is of great importance. Here, we artificially induced decidualization of a human endometrial stromal cell line (T HESCs) and characterized subsequent activation of mitophagy using immunofluorescence assay, electron microscopy and Western blot assay. Knockdown of autophagy-related 9A (ATG9A) led to an obvious reduction of mitophagy and deficiencies in decidualization. Our findings demonstrate a key role for proper mitochondrial dynamics in decidual differentiation and identify ATG9A-mediated mitophagy as a novel therapeutic target for repeated implantation failure or recurrent abortion.     

The molecular mechanism of mitochondria autophagy in yeast

Mitochondria are critical for supplying energy to the cell, but during catabolism this organelle also produces reactive oxygen species that can cause oxidative damage. Accordingly, quality control of mitochondria is important to maintain cellular homeostasis. It has been assumed that autophagy is the pathway for mitochondrial recycling, and that the selective degradation of mitochondria via autophagy (mitophagy) is the primary mechanism for mitochondrial quality control, although there is little experimental evidence to support this idea. Recent studies in yeast identified several mitophagy‐related genes and have uncovered components involved in the molecular mechanism and regulation of mitophagy. Similarly, studies of Parkinson disease and reticulocyte maturation reveal that Parkin and Nix, respectively, are required for mitophagy in mammalian cells, and these analyses have revealed important physiological roles for mitophagy. Here, we review the current knowledge on mitophagy, in particular on the molecular mechanism and regulation of mitophagy in yeast. We also discuss some of the differences between yeast and mammalian mitophagy.     

The BCL2L1 and PGAM5 axis defines hypoxia-induced receptor-mediated mitophagy

Receptor-mediated mitophagy is one of the major mechanisms of mitochondrial quality control essential for cell survival. We previously have identified FUNDC1 as a mitophagy receptor for selectively removing damaged mitochondria in mammalian systems. A critical unanswered question is how receptor-mediated mitophagy is regulated in response to cellular and environmental cues. Here, we report the striking finding that BCL2L1/Bcl-xL, but not BCL2, suppresses mitophagy mediated by FUNDC1 through its BH3 domain. Mechanistically, we demonstrate that BCL2L1, but not BCL2, interacts with and inhibits PGAM5, a mitochondrially localized phosphatase, to prevent the dephosphorylation of FUNDC1 at serine 13 (Ser13), which activates hypoxia-induced mitophagy. Our results showed that the BCL2L1-PGAM5-FUNDC1 axis is critical for receptor-mediated mitophagy in response to hypoxia and that BCL2L1 possesses unique functions distinct from BCL2.     

The BCL2L1 and PGAM5 axis defines hypoxia-induced receptor-mediated mitophagy

Receptor-mediated mitophagy is one of the major mechanisms of mitochondrial quality control essential for cell survival. We previously have identified FUNDC1 as a mitophagy receptor for selectively removing damaged mitochondria in mammalian systems. A critical unanswered question is how receptor-mediated mitophagy is regulated in response to cellular and environmental cues. Here, we report the striking finding that BCL2L1/Bcl-xL, but not BCL2, suppresses mitophagy mediated by FUNDC1 through its BH3 domain. Mechanistically, we demonstrate that BCL2L1, but not BCL2, interacts with and inhibits PGAM5, a mitochondrially localized phosphatase, to prevent the dephosphorylation of FUNDC1 at serine 13 (Ser13), which activates hypoxia-induced mitophagy. Our results showed that the BCL2L1-PGAM5-FUNDC1 axis is critical for receptor-mediated mitophagy in response to hypoxia and that BCL2L1 possesses unique functions distinct from BCL2.     

线粒体自噬的研究进展

线粒体自噬(mitophagy)是指细胞通过自噬机制选择性清除多余或损伤线粒体的过程,对于线粒体质量控制以及细胞生存具有重要作用。在线粒体自噬的过程中,线粒体自噬受体FUNDCl、Nix、BNIP3,接头蛋白OPTN、NDP52以及去泛素化酶UPS30、UPS8等发挥了重要的调控作用。近年来,研究发现线粒体自噬与神经退行性疾病、脑损伤以及胶质瘤相关。因此,研究线粒体自噬的分子机制具有重要意义。本文就与哺乳动物相关的线粒体自噬分子机制及最新研究进展做一综述。     

Two MAPK-signaling pathways are required for mitophagy in Saccharomyces cerevisiae

Macroautophagy (hereafter referred to simply as autophagy) is a catabolic pathway that mediates the degradation of long-lived proteins and organelles in eukaryotic cells. The regulation of mitochondrial degradation through autophagy plays an essential role in the maintenance and quality control of this organelle. Compared with our understanding of the essential function of mitochondria in many aspects of cellular metabolism such as energy production and of the role of dysfunctional mitochondria in cell death, little is known regarding their degradation and especially how upstream signaling pathways control this process. Here, we report that two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1, are required for mitophagy in Saccharomyces cerevisiae. Slt2 is required for the degradation of both mitochondria and peroxisomes (via pexophagy), whereas Hog1 functions specifically in mitophagy. Slt2 also affects the recruitment of mitochondria to the phagophore assembly site (PAS), a critical step in the packaging of cargo for selective degradation.     

Molecular mechanisms and physiological functions of mitophagy

Degradation of mitochondria via a selective form of autophagy, named mitophagy, is a fundamental mechanism conserved from yeast to humans that regulates mitochondrial quality and quantity control. Mitophagy is promoted via specific mitochondrial outer membrane receptors, or ubiquitin molecules conjugated to proteins on the mitochondrial surface leading to the formation of autophagosomes surrounding mitochondria. Mitophagy‐mediated elimination of mitochondria plays an important role in many processes including early embryonic development, cell differentiation, inflammation, and apoptosis. Recent advances in analyzing mitophagy in vivo also reveal high rates of steady‐state mitochondrial turnover in diverse cell types, highlighting the intracellular housekeeping role of mitophagy. Defects in mitophagy are associated with various pathological conditions such as neurodegeneration, heart failure, cancer, and aging, further underscoring the biological relevance. Here, we review our current molecular understanding of mitophagy, and its physiological implications, and discuss how multiple mitophagy pathways coordinately modulate mitochondrial fitness and populations.     

Phospholipid methylation controls Atg32‐mediated mitophagy and Atg8 recycling

Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy‐inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria‐anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8–PMME, a mitophagy‐incompetent lipid conjugate of the autophagy‐related ubiquitin‐like modifier. Amelioration of Atg32 expression and attenuation of Atg8–PMME conjugation markedly rescue mitophagy in opi3‐null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32‐mediated mitophagy.     

Receptor-mediated mitophagy in yeast and mammalian systems

Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease.     

After the banquet

Mitochondria are highly dynamic organelles of crucial importance to the proper functioning of neuronal, cardiac and other cell types dependent upon aerobic efficiency. Mitochondrial dysfunction has been implicated in numerous human conditions, to include cancer, metabolic diseases, neurodegeneration, diabetes, and aging. In recent years, mitochondrial turnover by macroautophagy (mitophagy) has captured the limelight, due in part to discoveries that genes linked to Parkinson disease regulate this quality control process. A rapidly growing literature is clarifying effector mechanisms that underlie the process of mitophagy; however, factors that regulate positive or negative cellular outcomes have been less studied. Here, we review the literature on two major pathways that together may determine cellular adaptation vs. cell death in response to mitochondrial dysfunction. Mitochondrial biogenesis and mitophagy represent two opposing, but coordinated processes that determine mitochondrial content, structure, and function. Recent data indicate that the capacity to undergo mitochondrial biogenesis, which is dysregulated in disease states, may play a key role in determining cell survival following mitophagy-inducing injuries. The current literature on major pathways that regulate mitophagy and mitochondrial biogenesis is summarized, and mechanisms by which the interplay of these two processes may determine cell fate are discussed. We conclude that in primary neurons and other mitochondrially dependent cells, disruptions in any phase of the mitochondrial recycling process can contribute to cellular dysfunction and disease. Given the emerging importance of crosstalk among regulators of mitochondrial function, autophagy, and biogenesis, signaling pathways that coordinate these processes may contribute to therapeutic strategies that target or regulate mitochondrial turnover and regeneration.     

Mitophagy: the life-or-death dichotomy includes yeast

The degradation and recycling of mitochondria is an important household chore in eukaryotic cells. It is thought that mitochondrial autophagy, or mitophagy, is the major route by which mitochondria are degraded. In this view, the cell would selectively induce mitophagy to expunge malfunctioning mitochondria, thus ridding the cell of troublesome sources of reactive oxygen species, apoptosis-inducing factors, or unnecessary metabolic burden. This standard view of mitophagy, in addition to some experimental reports, points to a pro-survival role of mitophagy. However, there is also a significant amount of evidence that suggests a pro-death role of this process, some of it coming from studies in yeast. Aup1 is a protein phosphatase homolog that shows a genetic interaction with the Atg1 protein kinase, localizes to mitochondria, and is required for mitophagy under stationary phase conditions in lactate medium. In contrast with previous yeast studies on mitophagy, deletion of AUP1 results in decreased viability under mitophagy-inducing conditions, suggesting a pro-survival role under physiologically relevant conditions. Thus, the Janus-faced nature of mitophagy is conserved between yeast and mammalian systems.     

PINK1/Parkin介导的线粒体自噬

线粒体自噬（mitochondrial autophagy, or mitophagy）指的是细胞通过自吞噬作用,降解与清除受损线粒体或者多余线粒体,其对整个线粒体网络的功能完整性和细胞存活具有重要作用。线粒体自噬过程受多种途径调控,PINK1/Parkin通路是其中的一条,其异常与多种疾病的发生密切相关,如心血管疾病、肿瘤和帕金森病等。在去极化线粒体中,磷酸酶及张力蛋白同源物(PTEN)诱导的激酶1（PTEN-induced kinase 1,PINK1）作为受损线粒体的分子传感器,触发线粒体自噬的起始信号,并将Parkin募集至线粒体;Parkin作为线粒体自噬信号的“增强子”,通过对线粒体蛋白质进一步泛素化介导自噬信号的扩大;去泛素化酶和PTEN-long蛋白参与调控该过程,并对维持线粒体稳态具有重要作用。本文主要对PINK1与Parkin蛋白质的分子结构和其介导线粒体自噬发生的分子机制,以及参与调控该途径的关键蛋白质进行综述,为进一步研究以线粒体自噬缺陷为特征的疾病治疗提供理论基础。