Mitochondrial degradation: Mitophagy and beyond.

Mitochondria are central hubs of cellular metabolism that also play key roles in signaling and disease. It is therefore fundamentally important that mitochondrial quality and activity are tightly regulated. Mitochondrial degradation pathways contribute to quality control of mitochondrial networks and can also regulate the metabolic profile of mitochondria to ensure cellular homeostasis. Here, we cover the many and varied ways in which cells degrade or remove their unwanted mitochondria, ranging from mitophagy to mitochondrial extrusion. The molecular signals driving these varied pathways are discussed, including the cellular and physiological contexts under which the different degradation pathways are engaged.

Unconventional initiation of PINK1/Parkin mitophagy by Optineurin.

Cargo sequestration is a fundamental step of selective autophagy in which cells generate a double-membrane structure termed an "autophagosome" on the surface of cargoes. NDP52, TAX1BP1, and p62 bind FIP200, which recruits the ULK1/2 complex to initiate autophagosome formation on cargoes. How OPTN initiates autophagosome formation during selective autophagy remains unknown despite its importance in neurodegeneration. Here, we uncover an unconventional path of PINK1/Parkin mitophagy initiation by OPTN that does not begin with FIP200 binding or require the ULK1/2 kinases. Using gene-edited cell lines and in vitro reconstitutions, we show that OPTN utilizes the kinase TBK1, which binds directly to the class III phosphatidylinositol 3-kinase complex I to initiate mitophagy. During NDP52 mitophagy initiation, TBK1 is functionally redundant with ULK1/2, classifying TBK1's role as a selective autophagy-initiating kinase. Overall, this work reveals that OPTN mitophagy initiation is mechanistically distinct and highlights the mechanistic plasticity of selective autophagy pathways.

Quality Control: Maintaining molecular order and preventing cellular chaos.

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease.

Programmed autophagy prevents excess organelle baggage during neurogenesis.

By putting a proteomic spin on the analysis of neurogenesis, Ordureau et al. (2021) produce a vast resource of information and reveal a program of mitophagy and organelle remodeling, which may play a key role in adapting organelles to a neuronal lineage.

ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system.

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.

NDP52 acts as a redox sensor in PINK1/Parkin-mediated mitophagy.

Mitophagy, the elimination of mitochondria via the autophagy-lysosome pathway, is essential for the maintenance of cellular homeostasis. The best characterised mitophagy pathway is mediated by stabilisation of the protein kinase PINK1 and recruitment of the ubiquitin ligase Parkin to damaged mitochondria. Ubiquitinated mitochondrial surface proteins are recognised by autophagy receptors including NDP52 which initiate the formation of an autophagic vesicle around the mitochondria. Damaged mitochondria also generate reactive oxygen species (ROS) which have been proposed to act as a signal for mitophagy, however the mechanism of ROS sensing is unknown. Here we found that oxidation of NDP52 is essential for the efficient PINK1/Parkin-dependent mitophagy. We identified redox-sensitive cysteine residues involved in disulphide bond formation and oligomerisation of NDP52 on damaged mitochondria. Oligomerisation of NDP52 facilitates the recruitment of autophagy machinery for rapid mitochondrial degradation. We propose that redox sensing by NDP52 allows mitophagy to function as a mechanism of oxidative stress response.

FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

Rebellious autophagy proteins bypass ATG8 lipidation, taking their own path to autophagic degradation.

LC3/GABARAP (hereafter ATG8) conjugation machineries have long been thought to play an essential role in autophagy by driving ATG8 lipidation on autophagosomal membranes. In this issue, Ohnstad et al (2020) describe an ATG8 lipidation bypass pathway which governs autophagy-dependent turnover of NBR1, highlighting that there is more than one road to autophagic degradation.

A unifying model for the role of the ATG8 system in autophagy.

The formation of autophagosomes and their fusion with lysosomes are key events that underpin autophagic degradation of cargoes. The core ATG8 system, which consists of the ATG8 family of ubiquitin-like proteins and the machineries that conjugate them onto autophagosomal membranes, are among the most-studied autophagy components. Despite the research focus on the core ATG8 system, there are conflicting reports regarding its essential roles in autophagy. Here, we reconcile prior observations of the core ATG8 system into a unifying model of their function that aims to consider apparently conflicting discoveries. Bypass pathways of autophagy that function independently of the core ATG8 system are also discussed.

Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs.

Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill-defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3-interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8-SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.

Parkin inhibits BAK and BAX apoptotic function by distinct mechanisms during mitophagy.

The E3 ubiquitin ligase Parkin is a key effector of the removal of damaged mitochondria by mitophagy. Parkin determines cell fate in response to mitochondrial damage, with its loss promoting early onset Parkinson's disease and potentially also cancer progression. Controlling a cell's apoptotic response is essential to co-ordinate the removal of damaged mitochondria. We report that following mitochondrial damage-induced mitophagy, Parkin directly ubiquitinates the apoptotic effector protein BAK at a conserved lysine in its hydrophobic groove, a region that is crucial for BAK activation by BH3-only proteins and its homo-dimerisation during apoptosis. Ubiquitination inhibited BAK activity by impairing its activation and the formation of lethal BAK oligomers. Parkin also suppresses BAX-mediated apoptosis, but in the absence of BAX ubiquitination suggesting an indirect mechanism. In addition, we find that BAK-dependent mitochondrial outer membrane permeabilisation during apoptosis promotes PINK1-dependent Parkin activation. Hence, we propose that Parkin directly inhibits BAK to suppress errant apoptosis, thereby allowing the effective clearance of damaged mitochondria, but also promotes clearance of apoptotic mitochondria to limit their potential pro-inflammatory effect.

Exercise and Training Regulation of Autophagy Markers in Human and Rat Skeletal Muscle.

Autophagy is a key intracellular mechanism by which cells degrade old or dysfunctional proteins and organelles. In skeletal muscle, evidence suggests that exercise increases autophagosome content and autophagy flux. However, the exercise-induced response seems to differ between rodents and humans, and little is known about how different exercise prescription parameters may affect these results. The present study utilised skeletal muscle samples obtained from four different experimental studies using rats and humans. Here, we show that, following exercise, in the soleus muscle of Wistar rats, there is an increase in LC3B-I protein levels immediately after exercise (+109%), and a subsequent increase in LC3B-II protein levels 3 h into the recovery (+97%), despite no change in Map1lc3b mRNA levels. Conversely, in human skeletal muscle, there is an immediate exercise-induced decrease in LC3B-II protein levels (-24%), independent of whether exercise is performed below or above the maximal lactate steady state, which returns to baseline 3.5 h following recovery, while no change in LC3B-I protein levels or MAP1LC3B mRNA levels is observed. SQSTM1/p62 protein and mRNA levels did not change in either rats or humans following exercise. By employing an ex vivo autophagy flux assay previously used in rodents we demonstrate that the exercise-induced decrease in LC3B-II protein levels in humans does not reflect a decreased autophagy flux. Instead, effect size analyses suggest a modest-to-large increase in autophagy flux following exercise that lasts up to 24 h. Our findings suggest that exercise-induced changes in autophagosome content markers differ between rodents and humans, and that exercise-induced decreases in LC3B-II protein levels do not reflect autophagy flux level.

The influence of aerobic exercise on mitochondrial quality control in skeletal muscle.

Mitochondria are dynamic organelles, intricately designed to meet cellular energy requirements. To accommodate alterations in energy demand, mitochondria have a high degree of plasticity, changing in response to transient activation of numerous stress-related pathways. This adaptive response is particularly relevant in highly metabolic tissues such as skeletal muscle, where mitochondria support numerous biological processes related to metabolism, growth and regeneration. Aerobic exercise is a potent stimulus for skeletal muscle remodelling, leading to alterations in substrate utilisation, fibre-type composition and performance. Underlying these physiological responses is a change in mitochondrial quality control (MQC), a term encompassing the co-ordination of mitochondrial synthesis (biogenesis), remodelling (dynamics) and degradation (mitophagy) pathways. Understanding of MQC in skeletal muscle and the regulatory role of aerobic exercise of this process are rapidly advancing, as are the molecular techniques allowing the study of MQC in vivo. Given the emerging link between MQC and the onset of numerous non-communicable diseases, understanding the molecular regulation of MQC, and the role of aerobic exercise in this process, will have substantial future impact on therapeutic approaches to manipulate MQC and maintain mitochondrial function across health span.

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.

Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.

YoungMito 2018: Report on the 1st International Mitochondria Meeting for Young Scientists.

The 1st International Mitochondria Meeting for Young Scientists (International YoungMito 2018) was held at Hotel Co-op Inn Kyoto in Kyoto, Japan, from 20 to 22 April 2018. The meeting was attended by 130 mitochondrial researchers from 15 countries. International YoungMito 2018 was the first international mitochondria meeting held in Japan organized by and for young mitochondrial researchers. Over the 3-day period, there were 28 oral presentations including two keynote lectures, 20 presentations from invited speakers, and six short talks selected from abstract submissions. Many different topics were covered including quality control pathways acting against mitochondrial stresses, mitochondrial dynamics, protein/lipid transport, cristae organization, respiration/ATP synthesis, mtDNA maintenance, mitochondrial disease models, and pharmacological approaches. In addition, we had 64 posters, a number which represented almost half of all attendees. Thanks to the cutting-edge information and high-quality unpublished data that were presented, there were many lively discussions during oral and poster sessions that continued into the coffee breaks, lunchtime, and nighttime discussions. The 1st international YoungMito meeting was successful in promoting intellectual exchange among all participants, facilitating collaborations beyond national boundaries, and closed with great success. It was a great pleasure that many participants were looking forward to the next YoungMito meeting.

Keeping the immune system in check: a role for mitophagy.

Mitochondria play a central role in many facets of cellular function including energy production, control of cell death and immune signaling. Breakdown of any of these pathways because of mitochondrial deficits or excessive reactive oxygen species production has detrimental consequences for immune system function and cell viability. Maintaining the functional integrity of mitochondria is therefore a critical challenge for the cell. Surveillance systems that monitor mitochondrial status enable the cell to identify and either repair or eliminate dysfunctional mitochondria. Mitophagy is a selective form of autophagy that eliminates dysfunctional mitochondria from the population to maintain overall mitochondrial health. This review covers the major players involved in mitophagy and explores the role mitophagy plays to support the immune system.

Tissue-specific splicing of an Ndufs6 gene-trap insertion generates a mitochondrial complex I deficiency-specific cardiomyopathy.

Mitochondrial complex I (CI) deficiency is the most common mitochondrial enzyme defect in humans. Treatment of mitochondrial disorders is currently inadequate, emphasizing the need for experimental models. In humans, mutations in the NDUFS6 gene, encoding a CI subunit, cause severe CI deficiency and neonatal death. In this study, we generated a CI-deficient mouse model by knockdown of the Ndufs6 gene using a gene-trap embryonic stem cell line. Ndufs6(gt/gt) mice have essentially complete knockout of the Ndufs6 subunit in heart, resulting in marked CI deficiency. Small amounts of wild-type Ndufs6 mRNA are present in other tissues, apparently due to tissue-specific mRNA splicing, resulting in milder CI defects. Ndufs6(gt/gt) mice are born healthy, attain normal weight and maturity, and are fertile. However, after 4 mo in males and 8 mo in females, Ndufs6(gt/gt) mice are at increased risk of cardiac failure and death. Before overt heart failure, Ndufs6(gt/gt) hearts show decreased ATP synthesis, accumulation of hydroxyacylcarnitine, but not reactive oxygen species (ROS). Ndufs6(gt/gt) mice develop biventricular enlargement by 1 mo, most pronounced in males, with scattered fibrosis and abnormal mitochondrial but normal myofibrillar ultrastructure. Ndufs6(gt/gt) isolated working heart preparations show markedly reduced left ventricular systolic function, cardiac output, and functional work capacity. This reduced energetic and functional capacity is consistent with a known susceptibility of individuals with mitochondrial cardiomyopathy to metabolic crises precipitated by stresses. This model of CI deficiency will facilitate studies of pathogenesis, modifier genes, and testing of therapeutic approaches.

Assembly of the Bak apoptotic pore: a critical role for the Bak protein α6 helix in the multimerization of homodimers during apoptosis.

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore.

The covalent attachment of ubiquitin-like LC3 proteins (microtubule-associated proteins 1A/1B light chain 3) prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligaseautophagy-related 12 (ATG12)-ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (i) the phosphatidylinositol 3-phosphate effector protein WD repeat domain phosphoinositide-interacting protein 2 (WIPI2), (ii) helix α2 of ATG16L1, and (iii) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12-ATG5-ATG16L1 machinery establishes a high level of regulatory control.

A RAB7A phosphoswitch coordinates Rubicon Homology protein regulation of Parkin-dependent mitophagy.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A-positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.