Olga Corti
email: olga.corti@upmc.fr

Neuronal Mitophagy: Lessons from a Pathway Linked to Parkinson's Disease

Neurons are specialized cells with complex and extended architecture and high energy requirements. Energy in the form of adenosine triphosphate, produced essentially by mitochondrial respiration, is necessary to preserve neuronal morphology, maintain resting potential, fire action potentials and ensure neurotransmission. Pools of functional mitochondria are required in all neuronal compartments, including cell body and dendrites, nodes of Ranvier, growth cones, axons and synapses. The mechanisms by which old or damaged mitochondria are removed and replaced in neurons remain to be fully understood. Mitophagy has gained considerable interest since the discovery of familial forms of Parkinson's disease caused by dysfunction of PINK1 and Parkin, two multifunctional proteins cooperating in the regulation of this process. Over the past ten years, the molecular mechanisms by which PINK1 and Parkin jointly promote the degradation of defective mitochondria by autophagy have been dissected. However, our understanding of the relevance of mitophagy to mitochondrial homeostasis in neurons remains poor. Insight has been recently gained thanks to the development of fluorescent reporter systems for tracking mitochondria in the acidic compartment of the lysosome.

Using these tools, mitophagy events have been visualized in primary neurons in culture and in vivo, under basal conditions and in response to toxic insults. Despite these advances, whether PINK1 and Parkin play a major role in promoting neuronal mitophagy under physiological conditions in adult animals and during aging remains a matter of debate. Future studies will have to clarify in how far dysfunction of neuronal mitophagy is central to the pathophysiology of Parkinson's disease.

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Introduction

Mitophagy is a form of cargo-selective autophagy removing unnecessary mitochondria, or clearing aged or dysfunctional mitochondria to protect the cells from their deleterious effects. More than fifty years ago, electron microscopy images revealed the presence of altered mitochondrial profiles in atypically enlarged lysosomal vesicles in the liver and kidney, under pathological conditions [START_REF] Ashford | Cytoplasmic components in hepatic cell lysosomes[END_REF][START_REF] De Duve | Functions of lysosomes[END_REF][START_REF] Novikoff | The Proximal Tubule Cell in Experimental Hydronephrosis Biophysic and[END_REF][START_REF] Novikoff | Cytolysosomes and mitochondrial degeneration[END_REF]. This phenomenon was interpreted as progressive mitochondrial degeneration [START_REF] Novikoff | Cytolysosomes and mitochondrial degeneration[END_REF]. However, the term mitophagy was coined more recently with the emergence of evidence, initially from yeast, indicating that the autophagy of mitochondria does not occur at random, but rather reflects a selective program [START_REF] Kissova | Uth1p is involved in the autophagic degradation of mitochondria[END_REF][START_REF] Lemasters | Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging[END_REF]. The term was then used to describe the autophagy-dependent degradation of mitochondria in cultured hepatocytes, following nutrient deprivation or laser-induced photodamage [START_REF] Kim | Selective degradation of mitochondria by mitophagy[END_REF][START_REF] Rodriguez-Enriquez | Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes[END_REF]). An earlier precursor study in cultured primary sympathetic neurons reported complete removal of mitochondria under conditions of apoptosis induction in the presence of caspase inhibitors, presumably associated with loss of mitochondrial membrane potential and cyrochrome c release [START_REF] Tolkovsky | Mitochondrial disappearance from cells: a clue to the role of autophagy in programmed cell death and disease?[END_REF].

These studies highlighted a role for mitophagy in the regulation of mitochondrial degradation under conditions of metabolic remodelling or severe mitochondrial damage.

Other types of programmed mitophagy were recognized to be central to specific developmental programs: during the maturation of erythrocytes, whereby the cells get rid of their mitochondria to acquire the ability to transport oxygen [START_REF] Sandoval | Essential role for Nix in autophagic maturation of erythroid cells[END_REF][START_REF] Schweers | NIX is required for programmed mitochondrial clearance during reticulocyte maturation[END_REF]; following fertilization of the oocyte, a stage marked by the removal of paternal mitochondria, underlying the universal principle of maternal mitochondrial inheritance in animals (Al [START_REF] Rawi | Postfertilization autophagy of sperm organelles prevents paternal mitochondrial DNA transmission[END_REF][START_REF] Sato | Degradation of paternal mitochondria by fertilization-triggered autophagy in C. elegans embryos[END_REF]; during cardiomyocyte maturation in the neonatal period, whereby fetal mitochondria are replaced by adult mitochondria to sustain the transition from carbohydrates to fatty acid metabolism [START_REF] Gong | Parkin-mediated mitophagy directs perinatal cardiac metabolic maturation in mice[END_REF]; or during the differentiation of myoblasts into mature myotubes, during which a distinct network of mitochondria replaces the old population to support the metabolic switch from glyocolysis to oxidative phosphorylation [START_REF] Sin | Mitophagy is required for mitochondrial biogenesis and myogenic d i f f e r e n t i a t i o n o f C 2 C 1 2 m y o b l a s t s A u t o p h a g y 1[END_REF].

It was not until 2008 that mitophagy emerged as a mitochondrial quality control mechanism of potential relevance to neuronal cells, with the groundbreaking discovery of the involvement of the RING-IBR-RING E3 ubiquitin ligase Parkin in the clearance of severely damaged mitochondria [START_REF] Narendra | Parkin is recruited selectively to impaired mitochondria and promotes their autophagy[END_REF]. Parkin is encoded by the PARK2 gene, which carries loss-of-function mutations in autosomal recessive forms of Parkinson's disease (PD), a common most often sporadic movement disorder caused by the progressive degeneration of the dopaminergic (DA) neurons in a region of the midbrain termed substantia nigra pars compacta (SNc). Why these neurons die in PD remains a mystery.

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However, mitochondrial dysfunction had been suspected to play a role since the 1980s, when the mitochondrial neurotoxin MPTP was identified as the cause of Parkinsonism in young people consuming drugs [START_REF] Corti | What genetics tells us about the causes and mechanisms of Parkinson's disease[END_REF][START_REF] Exner | Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences[END_REF][START_REF] Langston | Parkinson's disease in a chemist working with 1methyl-4-phenyl-1,2,5,6-tetrahydropyridine[END_REF][START_REF] Schapira | Mitochondrial contribution to Parkinson's disease pathogenesis[END_REF]. The discovery of Parkin-dependent mitophagy provided a cellular mechanism leading to mitochondrial dysfunction in PD. The role of this mechanism was strengthened a few years later, when work from several laboratories demonstrated that the product of another gene responsible of autosomal recessive PD, the mitochondrial serine/threonine kinase PINK1, was central to the activation of Parkin and its mitochondrial recruitment during mitophagy [START_REF] Geisler | PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[END_REF][START_REF] Matsuda | PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy[END_REF][START_REF] Narendra | PINK1 is selectively stabilized on impaired mitochondria to activate Parkin[END_REF]. Thanks to an unprecedented collective effort of the scientific community in the field during the past ten years, enlightened by recent descriptions of the crystal structure of Parkin [START_REF] Byrd | Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase[END_REF][START_REF] Caulfield | Activation of the E3 ubiquitin ligase Parkin[END_REF][START_REF] Gladkova | Mechanism of parkin activation by PINK1[END_REF]Kumar et al. 2017a;[START_REF] Riley | Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases[END_REF]Sauvé et al. 2018;[START_REF] Seirafi | Parkin structure and function[END_REF][START_REF] Spratt | RBR E3 ubiquitin ligases: new structures, new insights[END_REF][START_REF] Trempe | Structure of parkin reveals mechanisms for ubiquitin ligase activation[END_REF][START_REF] Wauer | Structure of the human Pakin ligase domain in an autoinhibited state[END_REF] and PINK1 (Kumar et al. 2017b;[START_REF] Okatsu | Structural insights into ubiquitin phosphorylation by[END_REF][START_REF] Schubert | Structure of PINK1 in complex with its substrate ubiquitin[END_REF], we have gained a comprehensive view of the molecular mechanisms underlying this specific mitophagy program, which have been summarized in excellent recent reviews [START_REF] Mcwilliams | PINK1 and Parkin: emerging themes in mitochondrial homeostasis[END_REF][START_REF] Truban | PINK1, Parkin, and Mitochondrial Quality Control: What can we Learn about Parkinson's Disease Pathobiology?[END_REF].

Although other mitophagy programs have been studied at the molecular level [START_REF] Chu | Multiple pathways for mitophagy: A neurodegenerative conundrum for Parkinson's disease[END_REF] McWilliams and Muqit 2017), PINK1/Parkin-dependent mitochondrial clearance is undoubtedly the best characterized. Comparatively, however, our understanding of the relevance of PINK1/Parkin-dependent mitophagy to mitochondrial homeostasis in neurons, particularly in those that degenerate in PD, remains poor [START_REF] Jang | The role of mitochondria in aging[END_REF]Palikaras et al. 2018).

PINK1/Parkin-dependent mitophagy in brief

Our current understanding of PINK1/Parkin-dependent mitophagy is that it is a program activated by specific stress-related stimuli. Its activation depends on mitochondrial protein import arrest, caused either by toxins leading to mitochondrial depolarization, such as the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) [START_REF] Geisler | PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[END_REF][START_REF] Matsuda | PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy[END_REF][START_REF] Narendra | Parkin is recruited selectively to impaired mitochondria and promotes their autophagy[END_REF][START_REF] Narendra | PINK1 is selectively stabilized on impaired mitochondria to activate Parkin[END_REF], by dysfunction of protein import components or proteases involved in PINK1 processing [START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/ PARKIN-dependent mitochondrial clearance[END_REF][START_REF] Greene | Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment[END_REF][START_REF] Jin | Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by[END_REF], or by unfolded protein stress in the mitochondrial matrix [START_REF] Fiesel | Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy[END_REF][START_REF] Jin | The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria[END_REF]. These conditions interfere with the translocation of PINK1 into the organelle, leading to its accumulation in proximity of the translocase of outer mitochondrial membrane [START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/ PARKIN-dependent mitochondrial clearance[END_REF][START_REF] Hasson | High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy[END_REF][START_REF] Lazarou | Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase[END_REF][START_REF] Okatsu | Mitochondrial hexokinase HKI is a novel substrate of the Parkin ubiquitin ligase[END_REF][START_REF] Okatsu | A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment[END_REF], its dimerization and autophosphorylation, the phosphorylation of ubiquitin moieties associated with the outer mitochondrial membrane (Koyano et al. 2014;[START_REF] Kane | PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity[END_REF][START_REF] Kazlauskaite | Parkin is activated by PINK1-dependent phophorylation of ubiquitin at[END_REF][START_REF] Matsuda | Phospho-ubiquitin: upending the PINK-Parkin-ubiquitin cascade[END_REF], ! 4 the recruitment and activation of the otherwise inactive Parkin ligase [START_REF] Riley | Structure and function of Parkin E3 ubiquitin ligase reveals aspects of RING and HECT ligases[END_REF][START_REF] Trempe | Structure of parkin reveals mechanisms for ubiquitin ligase activation[END_REF][START_REF] Wauer | Structure of the human Pakin ligase domain in an autoinhibited state[END_REF], by binding to phospho-ubiquitin, and its stabilization in an active state through the PINK1-dependent phosphorylation of its Nterminal ubiquitin-like domain [START_REF] Byrd | Compact Parkin only: insights into the structure of an autoinhibited ubiquitin ligase[END_REF][START_REF] Caulfield | Activation of the E3 ubiquitin ligase Parkin[END_REF][START_REF] Chaugule | Autoregulation of Parkin activity through its ubiquitin-like domain[END_REF][START_REF] Gladkova | Mechanism of parkin activation by PINK1[END_REF][START_REF] Kazlauskaite | PINK1 and Parkin -mitochondrial interplay between phosphorylation and ubiquitylation in Parkinson's disease[END_REF][START_REF] Kondapalli | PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65[END_REF][START_REF] Heo | The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy[END_REF]Ordureau et al. 2014;[START_REF] Sauve | Mechanism of parkin activation by phosphorylation[END_REF][START_REF] Shiba-Fukushima | PINK1mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy[END_REF][START_REF] Spratt | RBR E3 ubiquitin ligases: new structures, new insights[END_REF][START_REF] Yamano | Sitespecific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation[END_REF]. As a consequence, Parkin ubiquitylates a number of proteins of the outer mitochondrial membrane by forming different types of ubiquitin chains, which are further phosphorylated by PINK1, recruiting additional Parkin molecules, and thereby feeding an amplification loop [START_REF] Okatsu | Phosphorylated ubiquitin chain is the genuine Parkin receptor[END_REF][START_REF] Heo | The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy[END_REF]Ordureau et al. 2014;[START_REF] Sarraf | Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization[END_REF]). This process is negatively regulated by specific deubiquitylases [START_REF] Bingol | The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy[END_REF][START_REF] Cornelissen | The deubiquitinase USP15 antagonizes Parkin-mediated mitochondrial ubiquitination and mitophagy[END_REF][START_REF] Durcan | USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin[END_REF].

The ubiquitylation of mitochondrial proteins leads to the recruitment of specific ubiquitinbinding autophagy receptors, including NDP52 and optineurin, which in turn promotes association of upstream autophagy-related proteins, thereby priming mitochondria for autophagy [START_REF] Heo | The PINK1-PARKIN Mitochondrial Ubiquitylation Pathway Drives a Program of OPTN/NDP52 Recruitment and TBK1 Activation to Promote Mitophagy[END_REF][START_REF] Itakura | Structures containing Atg9A and the ULK1 complex independently target depolarized mitochondria at initial stages of Parkin-mediated mitophagy[END_REF][START_REF] Lazarou | The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy[END_REF][START_REF] Wei | Prohibitin 2 Is an Inner Mitochondrial Membrane Mitophagy Receptor[END_REF]Yamano et al. 2018). Evidence has been provided that mitophagy is initiated in proximity of contact sites between mitochondria and the endoplasmic reticulum (ER) [START_REF] Gautier | The endoplasmic reticulum-mitochondria interface is perturbed in PARK2 knockout mice and patients with PARK2 mutations[END_REF][START_REF] Gelmetti | PINK1 and BECN1 relocalize at mitochondria-associated membranes during mitophagy and promote ER-mitochondria tethering and a u t o p h a g o s o m e f o r m a t i o n A u t o p h a g y 1[END_REF]Yang and Yang 2013), and that it requires dissociation between the two organelles to proceed efficiently [START_REF] Mclelland | Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy[END_REF]. This is achieved by a rapid burst of ubiquitylation of the Parkin substrate Mitofusin 2 (Mfn2), localized on both outer mitochondrial and ER membranes, which in turn leads to disassembly of Mfn2 complexes and destruction of the mitochondria-ER contact [START_REF] Mclelland | Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy[END_REF].

Neurons: cells on a tight energy budget

The human brain does not exceed 2% of the body's weight but its energy budget represents 20% of that of the body [START_REF] Harris | Synaptic energy use and supply[END_REF][START_REF] Mink | Ratio of central nervous system to body metabolism in vertebrates: its constancy and functional basis[END_REF]. Whilst neural activity drives ATP synthesis by both glycolysis and oxidative phosphorylation, most of the brain ATP is generated by oxidative phosphorylation [START_REF] Hall | Oxidative phosphorylation, not glycolysis, powers presynaptic and postsynaptic mechanisms underlying brain information processing[END_REF]Rangaraju et al. 2014). This makes brain cells highly reliant on mitochondria. Neurons have a uniquely complex cellular architecture intimately linked to their role in information processing and transmission.

They are composed of a cell body containing the nucleus and enriched in other essential organelles, including the Golgi apparatus, the endoplasmic reticulum, lysosomes and mitochondria. A system of highly branched dendrites adapted to receive information from other neurons emerge from the cell body, as well as a single extension of variable length, the axon, responsible for transmitting the electrical signal. The axon ramifies into several terminals that pass the signal to the dendrites of other neurons through the specialized compartment of the synapse. In recent years, thanks to the development of techniques for ! 5 single neurons tracing and 3-dimensional reconstruction of neuronal morphology, it has been calculated that individual neurons can generate axons of tens of centimeters in length in the rodent brain [START_REF] Matsuda | Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum[END_REF][START_REF] Wu | Complete morphologies of basal forebrain cholinergic neurons in the mouse[END_REF]. Extrapolations based on these studies and on available morphometric data predict that in the human brain such axons reach lengths of up to tens of meters [START_REF] Bolam | Living on the edge with too many mouths to feed: why dopamine neurons die[END_REF][START_REF] Matsuda | Single nigrostriatal dopaminergic neurons form widely spread and highly dense axonal arborizations in the neostriatum[END_REF]. DA neurons of the SNc, in particular, have an unmyelinated, extremely large and highly branched axonal arbor. In humans, the average total length of the axonal arbor of a single DA neuron of the SNc has been estimated to exceed 4.5 m [START_REF] Bolam | Living on the edge with too many mouths to feed: why dopamine neurons die[END_REF]. Such an axon can generate up to 2.4 million synapses in the projection region of the striatum.

Neurons require a lot of energy to preserve their architecture, maintain the resting potential, fire action potentials and ensure neurotransmission in the pre-and post-synaptic compartments [START_REF] Attwell | An energy budget for signaling in the grey matter of the brain[END_REF]. Based on a computational model for DA neurons integrating their morphological complexity and electrophysiological properties, it has been calculated that the energy cost associated with axon potential propagation and recovery of resting membrane potential increases exponentially with the number of levels of branches of the axon, and according to a power law of the axonal surface area and number of branch points (Pissadaki and Bolam 2013). The disproportionately higher energy demand associated with the large and highly branched axonal arbor of the SNc DA neurons compared to that of smaller axons, such as those of the DA neurons in the less susceptible ventral tegmental area (VTA), may well be one of the determinants of the special vulnerability of these neurons in PD. In a recent study, it was indeed shown that cultured mouse SNc DA neuron display larger axonal arbors than cultured DA neurons from the VTA of the olfactory bulb (Pacelli et al. 2015). This property correlated with higher density of mitochondria specifically in the axonal compartment, higher oxygen consumption rates, higher levels of reactive oxygen species and greater sensitivity to mitochondrial neurotoxins. Strikingly, treatment of the SNc DA neurons with Semaphorin 7A, a guidance molecule known to modulate axonal arborization in these neurons, reduced the size of the axonal arbour and at the same time diminished oxygen consumption rates and neuronal vulnerability to toxins, highlighting the intimate relationship between the complex morphological architecture of these neurons and their energy requirement and special sensitivity.

Mitochondrial homeostasis in neurons: how are mitochondria rejuvenated?

In contrast to other cell types in the body, neurons do not divide and are therefore destined to last a lifetime. Mitochondria are required in different neuronal compartments and have been shown to accumulate at nodes of Ranvier, in growth cones, axonal branches ! 6 and synapses [START_REF] Hollenbeck | The axonal transport of mitochondria[END_REF]. How do neurons manage to maintain appropriate pools of healthy mitochondria throughout their complex architecture and the lifespan of the organism? A combination of functionally intertwined mechanisms that are active in other cell types are also at play here [START_REF] Misgeld | Mitostasis in Neurons: Maintaining Mitochondria in an Extended[END_REF][START_REF] Rugarli | Mitochondrial quality control: a matter of life and death for neurons[END_REF][START_REF] Shutt | Staying cool in difficult times: mitochondrial dynamics, quality control and the stress response[END_REF]. These include (1) transport from the cell soma to remote peripheral sites in the dendritic arbor, axon and axonal terminals; (2) fusion and fission cycles that shape the morphology and size of the organelle to adapt them to metabolic demand, transport and clearance; (3) pathways for the degradation of individual mitochondrial proteins, portions of mitochondria or whole organelle; and (4) biogenesis of individual components and whole organelles. Studies in cultured neurons and in various in vivo models, including C elegans, Drosophila, zebrafish and mouse, have demonstrated that although most mitochondria are stationary, a fraction of about 10%-40% move along axons at any given time [START_REF] Misgeld | Mitostasis in Neurons: Maintaining Mitochondria in an Extended[END_REF]. To enter the axon, a mitochondrion has to be separated from the network [START_REF] Berthet | Loss of mitochondrial fission depletes axonal mitochondria in midbrain dopamine neurons[END_REF][START_REF] Verstreken | Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions[END_REF], and in the axon, it can move in both anterograde and retrograde directions. However, the proportion of mitochondria moving from the soma to the peripheral arbor is higher than that transported back from the periphery to the cell body [START_REF] R203 Misgeld | Imaging axonal transport of mitochondria in vivo[END_REF]Pilling et al. 2006;Plucinska et al. 2012;[START_REF] Wang | PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility[END_REF], which lends support to the idea that that "old" stationary mitochondria are, at least in part, turned over at axonal terminals.

Calculations of protein turnover rates based on isotopic labeling at the organism level suggest that mitochondria have an average lifetime of several days, a duration that appears to be conserved during evolution and longer in the brain than in other organs (Price et al. 2010;[START_REF] Vincow | The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo[END_REF]. The "rejuvenation" of mitochondria through the replacement of individual components or whole organelles, particularly at distal sites, is probably operated by several parallel pathways. It is admitted that mitochondrial biogenesis is not restricted to the neuronal cell body [START_REF] Harbauer | Mitochondrial health maintenance in axons[END_REF]. Transcripts for nucleus-encoded mitochondrial proteins are present in axons [START_REF] Aschrafi | A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons[END_REF][START_REF] Shigeoka | Dynamic Axonal Translation in Developing and Mature Visual Circuits[END_REF], which are endowed with the protein synthesis machinery [START_REF] Koenig | Protein-synthesizing machinery in the axon compartment[END_REF], and there is evidence for replication of mitochondrial DNA (mtDNA) in axons [START_REF] Amiri | Mitochondrial biogenesis in the axons of vertebrate peripheral neurons[END_REF]. Moreover, the smooth endoplasmic reticulum populates even the most remote neuronal compartments, including the thin branches of the axon terminals, often interacting closely with mitochondria, providing the capacity for local membrane lipid biosynthesis [START_REF] Berridge | Neuronal calcium signaling[END_REF][START_REF] Wu | Contacts between the endoplasmic reticulum and other membranes in neurons[END_REF]. In principle, mitochondrial protein synthesis and mitochondrial biogenesis may thus occur in the neuronal periphery, although the extent to which this is indeed the case has yet to be fully appreciated [START_REF] Saxton | The axonal transport of mitochondria[END_REF]. Fusion with younger mitochondria is another key mechanism by which mitochondrial competence is preserved in neurons, through the redistribution of essential mitochondrial constituents and the dilution of damaged components. The relevance of this process to neurons is illustrated by the consequence of mutations in ! 7

genes encoding components of the mitochondrial fusion machinery, which cause neurodegenerative diseases in humans [START_REF] Chen | Physiological functions of mitochondrial fusion[END_REF]. Finally, new mitochondria may be supplied in neurons by non-canonical pathways, such as through the transfer of mitochondria from astrocytes [START_REF] Hayakawa | Transfer of mitochondria from astrocytes to neurons after stroke[END_REF]), although the extent to which this occurs awaits further experimental corroboration [START_REF] Berridge | Mitochondrial Transfer from Astrocytes to Neurons following Ischemic Insult: Guilt by Association?[END_REF]).

How are "old" or damaged mitochondrial components or whole organelles degraded in neurons? Several complementary pathways for the turnover of mitochondrial components have been identified, and again their importance to neuronal homeostasis is underscored by the neurodegenerative diseases engendered by their dysfunction linked to genetic mutations [START_REF] Misgeld | Mitostasis in Neurons: Maintaining Mitochondria in an Extended[END_REF][START_REF] Rugarli | Mitochondrial quality control: a matter of life and death for neurons[END_REF]. These involve the removal of individual proteins by mitochondrial proteases or by the cytosolic proteasome, the vesicular delivery of mitochondrial cargo to the lysosome through mitochondria-derived vesicles (MDVs), intraneuronal mitophagy, and transcellular mitophagy (Table 1). The relevance of each of these pathways, the physiological circumstances in which they are solicited and the specific subcellular compartments in which they take place remain to be fully understood. Notably, in addition to its role in mitophagy, the PINK1/Parkin pathway has also been involved in the regulation of the degradation of outer mitochondrial membrane proteins by the ubiquitin-proteasome pathway (Tanaka and[START_REF] Tanaka | Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin[END_REF]Youle 2011), and in the delivery of selected damaged mitochondrial components to the lysosome by MDVs (McLelland et al. 2014;[START_REF] Mclelland | Syntaxin-17 delivers PINK1/parkindependent mitochondrial vesicles to the endolysosomal system[END_REF][START_REF] Sugiura | A new pathway for mitochondrial quality control: mitochondrial-derived vesicles[END_REF]) (Table 1).

Mitophagy in cultured neurons: evidence for or against

Most studies investigated mitophagy in immortalized cell lines, following treatment with chemical uncouplers or inhibitors of the mitochondrial respiratory chain. The question of whether this process is relevant to primary cells, particularly to neurons, under physiological conditions, has been highly debated [START_REF] Grenier | Parkin-and PINK1-Dependent Mitophagy in Neurons: Will the Real Pathway Please Stand Up?[END_REF]. Relatively quickly after the discovery of Parkin-depedent mitophagy, studies in different primary neuronal models, including DA neurons differentiated from induced pluripotent stem cells (iPSCs), provided evidence for mitochondrial translocation of exogenously expressed Parkin [START_REF] Cai | Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons[END_REF][START_REF] Joselin | ROS-dependent regulation of Parkin and DJ-1 localization during oxidative stress in neurons[END_REF][START_REF] Seibler | Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells[END_REF][START_REF] Wang | PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility[END_REF]) in cells treated with various mitochondrial toxins. As in other cell types, recruitment of Parkin to mitochondria was dependent on the presence of PINK1 [START_REF] Seibler | Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells[END_REF]. Very recently, it was also shown that exposure to mitochondrial uncouplers stabilizes endogenous PINK1 [START_REF] Oh | S-Nitrosylation of PINK1 Attenuates PINK1/Parkin-Dependent Mitophagy in hiPSC-Based Parkinson[END_REF][START_REF] Soutar | AKT signalling selectively regulates PINK1 mitophagy in SHSY5Y cells and human iPSC-derived neurons[END_REF]) and activates endogenous Parkin in primary rodent and human neurons, according to the same molecular mechanisms identified in cell lines overexpressing Parkin [START_REF] Barini | The Anthelmintic Drug Niclosamide and Its Analogues Activate the Parkinson's Disease Associated Protein Kinase PINK1[END_REF][START_REF] Oh | S-Nitrosylation of PINK1 Attenuates PINK1/Parkin-Dependent Mitophagy in hiPSC-Based Parkinson[END_REF]Ordureau et al. 2018;McWilliams 2018a).

! 8 Some studies provided direct evidence for mitophagic events in neurons. By co-expressing fluorescent proteins targeted to autophagic vesicles (GFP-LC3) or lysosomes (GFP-LAMP1), is was shown that autophagosomes are co-recruited to Parkin-positive mitochondria, and that mitochondria are engulfed by lysosomal vesicles in primary mouse cortical neurons treated with the protonophore carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP) [START_REF] Cai | Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons[END_REF]). These authors also showed accumulation of depolarized mitochondria in cells silenced for endogenous Parkin, strongly supporting the involvement of mitophagy in mitochondrial maintenance in neurons subjected to acute mitochondrial stress. At the same time, however, other researchers reported absence of Parkin tanslocation to mitochondria or recruitment of autophagic vesicles in primary rat cortical neurons exposed to a mitochondrial uncoupler, using similar approaches [START_REF] Van Laar | Bioenergetics of neurons inhibit the translocation response of Parkin following rapid mitochondrial depolarization[END_REF]). These authors proposed that the bioenergetic properties of neurons, their strong reliance on oxidative phosphorylation and reluctance to switch to glyocolytic metabolism under mitochondrial stress, preclude the occurrence of mitophagy. Consistent with this possibility, mitochondrial depolarization triggered by the potassium ionophore valinomycin did not lead to appreciable loss of mitochondrial markers in human iPSc-derived neurons, even following overexpression of Parkin (Rakovic & Klein 2012). However, reductions in mtDNA copy number were reported under these conditions, a phenomenon that was not observed in neurons from patients with PINK1 mutations [START_REF] Seibler | Mitochondrial Parkin recruitment is impaired in neurons derived from mutant PINK1 induced pluripotent stem cells[END_REF]. Others have confirmed that mitochondrial proteins are also cleared to a certain extent in human iPScderived neurons treated with CCCP [START_REF] Soutar | AKT signalling selectively regulates PINK1 mitophagy in SHSY5Y cells and human iPSC-derived neurons[END_REF]. Careful inspection of the culture conditions across the different studies [START_REF] Grenier | Parkin-and PINK1-Dependent Mitophagy in Neurons: Will the Real Pathway Please Stand Up?[END_REF]) revealed that authors reporting massive mitochondrial Parkin translocation or signs of mitophagy in neurons had supplemented the medium with apoptosis inhibitors [START_REF] Cai | Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons[END_REF] or antioxidants [START_REF] Joselin | ROS-dependent regulation of Parkin and DJ-1 localization during oxidative stress in neurons[END_REF]). Thus, massive neuronal death may mask the occurrence of mitophagy, which is in line with the recent discovery that Parkin not only promotes mitophagy but also sensitized towards apoptosis under conditions of acute mitochondrial stress [START_REF] Carroll | Parkin sensitizes toward apoptosis induced by mitochondrial depolarization through promoting degradation of Mcl-1[END_REF]Zhang et al. 2014).

Global depolarization of the mitochondrial network is unlikely to occur under physiological conditions. In such conditions, mitophagy would rather occur as a local process removing depolarized mitochondrial fragments. To mimic such a situation, some researchers used the mitochondrion-targeted red fluorescent protein mt-KillerRed to photosensitize small subsets of mitochondria in the axonal compartment of rat hippocampal neurons cultured in microfluidic devices [START_REF] Ashrafi | Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin[END_REF]. Using time-lapse video microscopy, they observed local recruitment of GFP-LC3-positive autophagic vesicles and LAMP1-YFPpositive lysosomes to damaged mitochondria as early as 20 minutes following irradiation. In some cases they also reported disappearance of single mitochondria from these structures. These events were observed in the absence of antioxidants in the culture medium, and occurred to a much lesser extent in neurons from Parkin-or PINK1-deficient mice, ! 9 demonstrating reliance on the PINK1/Parkin pathway. This study demonstrated that mitophagy occurs locally in the distal axonal compartment. Similar observations were made more recently by Hsieh and colleagues in iPSC-derived neurons exposed to the mitochondrial complex III inhibitor antimycin A [START_REF] Hsieh | Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson[END_REF]). These authors also showed a delay in axonal mitophagy in neurons from PD patients with mutations in LRRK2 or from sporadic PD patients. This contrasts with the conclusion drawn by Cai and colleagues, who observed accumulation of Parkin-positive mitochondria and associated autophagosomes essentially in the somatodendritic compartment of neurons treated with CCCP for 24 hours [START_REF] Cai | Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons[END_REF]. Based on these observations, it was suggested that autophagic vesicles containing damaged mitochondria are transported back to the soma to be degraded by cytoplasmic lysosomes. This later response may however reflect a compensatory mechanism due the overwhelming of the axonal lysosomes.

All studies mentioned above investigated mitophagy under conditions of acute and severe mitochondrial stress, but what about neuronal mitophagy under conditions of mild mitochondrial stress, which is more relevant to the chronic progressive mitochondrial dysfunction that characterizes neurodegenerative diseases? Lin and colleagues [START_REF] Lin | Releasing Syntaphilin Removes Stressed Mitochondria from Axons Independent of Mitophagy under Pathophysiological Conditions[END_REF]) recently proposed that mitophagy may not be the main mechanism by which defective mitochondria are removed from axons under conditions of reversible mitochondrial depolarization triggered by low doses of antimycin A. In this case, damaged mitochondria were removed from axons by retrograde transport to the soma, a process that was enhanced by the release from mitochondria of the axonal mitochondrial anchoring protein syntaphilin. Finally, basal mitophagy was explored in cultured hippocampal neurons, using the ratiometric mitochondrion-targeted pH-sensitive biosensor mt-Keima, enabling differentiation between mitochondria in the cytoplasm and mitochondria in the acidic microenvironment of the lysosome [START_REF] Bingol | The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy[END_REF]). This study showed progressive accumulation of the biosensor in lysosomes, mostly in the neuronal soma, consistent with ongoing mitophagy in the absence of Parkin overexpression or toxins. The delivery of mitochondria to lysosomes was reduced upon silencing of PINK1 or Parkin using small hairpin RNAs, suggesting that these proteins are required for basal mitophagy, in addition to stress-induced mitophagy. However, this view has been challenged by recent in vivo studies, as will be discussed in the next section.

In vivo mitophagy in the nervous system: a controversial issue

Early attempts to determine the turnover rates of mitochondria in different tissues in the 1960ies were based on the in vivo radiolabeling of protein and lipid components in rats.

Although some of these reports investigated the turnover of different categories of mitochondrial proteins, e.g. water-soluble, water-insoluble, structural, contractile and/or included the analysis of cytochrome c [START_REF] Fletcher | Turnover of rat-liver mitochondria[END_REF][START_REF] Beattie | The turnover of the protein components of mitochondria from rat liver, kidney, and brain[END_REF], in ! 10 general these studies did not take into account the half-lives of individual mitochondrial proteins. Based on the observation that insoluble and soluble protein, lipid and cytochrome c from rat liver mitochondria turned over at near identical rates, Fletcher and Sanadi proposed for the first time that mitochondria are synthesized and broken down as discrete entities [START_REF] Fletcher | Turnover of rat-liver mitochondria[END_REF]. Later reports of some heterogeneity in the turnover of mitochondrial components in different tissues, including the brain, lend support to the possibility that individual mitochondrial components may be degraded independently, but did not fundamentally challenge the idea that a mitochondrial structural unit is turned over as an entity [START_REF] Beattie | The turnover of the protein components of mitochondria from rat liver, kidney, and brain[END_REF][START_REF] Cuzner | Turnover of brain mitochondrial membrane lipids[END_REF][START_REF] Gross | Thymidine content and turnover in the rat[END_REF]).

Together with first observations of mitochondria inside lysosomes by electron microscopy [START_REF] Ashford | Cytoplasmic components in hepatic cell lysosomes[END_REF][START_REF] De Duve | Functions of lysosomes[END_REF][START_REF] Novikoff | The Proximal Tubule Cell in Experimental Hydronephrosis Biophysic and[END_REF][START_REF] Novikoff | Cytolysosomes and mitochondrial degeneration[END_REF][START_REF] Swift | Focal Degradation as a Biological Process[END_REF], these pioneering studies anticipated the discovery of mitophagy, and strongly supported the existence of a mechanism for the degradation of mitochondrial entities in vivo. This issue was reinvestigated more recently in Drosophila melanogaster, in a proteomic assay based on the feeding of adult flies with deuterated leucine and the use of mass spectrometry analyses to monitor simultaneously the half-lives of numerous mitochondrial and non-mitochondrial proteins [START_REF] Vincow | The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo[END_REF]. Parallel studies in wild type, parkin null and autophagy-deficient Atg7 null flies showed prolonged half-lives for nearly 150 mitochondrial proteins in both mutant strains, and significant correlation between the effects of Atg7 and parkin mutations, specifically for this set of proteins, and not for proteins targeted to other organelles known to be degraded by autophagy. This study provided the first evidence for a role of parkin in the regulation of mitophagy in vivo. Intriguingly, the turnover of a subset of 40 mitochondrial proteins, including 19 subunits of respiratory chain components representative of the five respiratory complexes, appeared to depend more on Parkin than on Atg7, and there was strong correlation between the effect of parkin mutation and the effect of pink1 mutation on the turnover of these components [START_REF] Vincow | The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo[END_REF]. This suggested the existence of a degradation process dependent on PINK1 and Parkin but independent of autophagy, possibly involving the MDV pathway [START_REF] Vincow | The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo[END_REF]). On the other hand, no change in mitochondrial density was observed in Drosophila larval motor neurons axons or cell bodies, as would have been predicted by the reported role of the PINK1/Parkin pathway in mitophagy [START_REF] Devireddy | The Organization of Mitochondrial Quality Control and Life Cycle in the Nervous System In Vivo in the Absence of PINK1[END_REF]. Abnormally large, discrete mitochondria did, however, accumulate in the neuronal cell bodies, suggesting that mitochondrial degradation may be limited to this neuronal compartment in the nervous system in vivo.

Several groups made efforts towards clarifying the relevance of PINK1/Parkin-dependent mitophagy to dopaminergic neurons in mouse models, specifically in the context of accelerated accumulation of mtDNA defects (Pickrell et al. 2015;Pinto et al. 2018;[START_REF] Song | Parkin deficiency accelerates consequences of mitochondrial DNA deletions and Parkinsonism[END_REF][START_REF] Sterky | Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo[END_REF]). The rationale for these studies came from the observation that nigral DA neurons in humans are more prone to accumulate mtDNA deletions than ! 11 neurons in other brain regions [START_REF] Bender | High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease[END_REF][START_REF] Kraytsberg | Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons[END_REF]. Such defects are compensated for by an increase in the number of normal mtDNA copies in neurologically healthy individuals but not in PD patients [START_REF] Dolle | Defective mitochondrial DNA homeostasis in the substantia nigra in Parkinson disease[END_REF], possibly due to alterations in mitochondrial biogenesis [START_REF] Grunewald | Mitochondrial DNA Depletion in Respiratory Chain-Deficient Parkinson Disease Neurons[END_REF] [START_REF] Ekstrand | Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons[END_REF]Pickrell et al. 2011;[START_REF] Song | Mutant Twinkle increases dopaminergic neurodegeneration, mtDNA deletions and modulates Parkin expression[END_REF], the accumulation of somatic mtDNA mutations in Mutator mice led to premature aging in the absence of overt neurodegeneration [START_REF] Kujoth | Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging[END_REF][START_REF] Trifunovic | Premature ageing in mice expressing defective mitochondrial DNA polymerase[END_REF]). Suprisingly, Parkin-deficiency affected differently the phenotypes in these mice. There was no modification of the mitochondrial morphological alterations or neurodegenerative process characteristic of the MitoPark model [START_REF] Sterky | Impaired mitochondrial transport and Parkin-independent degeneration of respiratory chain-deficient dopamine neurons in vivo[END_REF]. In contrast, mtDNA mutation load or predicted pathogenicity, mitochondrial dysfunction, DA neurodegeneration and behavioral defects were anticipated or exacerbated in the three other models. Despite different interpretations about whether the observed effects reflected or not a physiological role of Parkin in the clearance of mitochondria with high mtDNA mutation loads, none of these studies were based on the direct exploration of the mitophagy process.

Direct investigation of mitophagy in vivo has only recently been rendered possible by the development of specific fluorescent reporters with acid-labile components, including the already mentioned mt-Keima and mito-QC, a mitochondrion-targeted tandem mCherry-GFP protein [START_REF] Rodger | Mammalian mitophagy -from in vitro molecules to in vivo models[END_REF]). These reporters have been used to generate transgenic Drosophila and mouse models and explore the presence of mitochondria within lysosomes under basal conditions across organs and tissues [START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila Elife 7[END_REF][START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF][START_REF] Mcwilliams | mito-QC illuminates mitophagy and mitochondrial architecture in vivo[END_REF]Sun et al. 2015). It should be noted, however, that these reporters cannot formally discriminate between autophagy-dependent and autophagy-independent events, such as those mediated by the MDV pathway, unless systematic parallel analyses ! 12 are performed in corresponding autophagy-deficient models (Table 1). Nevertheless, in all these studies, quenching of the acid-labile fluorescent component in the lysosome has been exclusively interpreted in terms of mitophagy. In Drosophila, such structures were abundant in various tissues during development, including in the nervous tissue, and were not observed following deletion of Atg5 or overexpression of a kinase-dead version of Atg1 [START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila Elife 7[END_REF][START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF]. Importantly, in the adult brain, mitophagy events were readily detected in DA neuron clusters reported to degenerate in pink1 and parkin mutant flies, albeit with some differences depending on the reporter used. For example, using mito-QC, the steady-state levels of the supposed mitophagy were found to be stable during aging in the DA neurons from the PPL1 cluster of the posterior inferiorlateral protocerebrum [START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF], whereas in mt-Keima flies, the mitophagy index increased by 30 % between one and four weeks of age [START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila Elife 7[END_REF]). In addition, in mito-QC flies mitophagy events were not detectable in muscle tissue, which is also affected in pink1 and parkin mutants [START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF]. In contrast, mitolysosomes were observed in the indirect flight muscles of mt-Keima flies, where they were also found to increase fourfold in abundance between week 1 and week 4 [START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila Elife 7[END_REF]).

These discrepancies highlight potential differences in the sensitivities of the biosensors used to track mitochondria in lysosomes. Consistent with this possibility, the use of mt-Keima revealed a 20-fold higher mitophagy index in the DA neurons of the PPL1 cluster than in the indirect flight muscles [START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila Elife 7[END_REF], indicating that mitophagy occurs only rarely in muscles, where it may remain below the detection limit when monitored with mito-QC [START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF]. Notably, mt-Keima is a mitochondrial matrix protein, whereas mito-QC is targeted to the outer mitochondrial membrane. As reported for proteins of this submitochondrial compartment (Karbowski and Youle 2011), mito-QC may thus by subject to degradation by the ubiquitin-proteasome system in addition to mitophagy, which could act as another source of bias. For example, mitophagy may be artificially underestimated, as a subset of mitochondrial units potentially undergoing mitophagy will be missed because of prior degradation of the fusion protein. On the other hand, mt-Keima cannot be analyzed in fixed samples, because fixation compromises the lysosomal pH gradient, and this may engender signal variability. Moreover, there is partial overlap in the Keima excitation spectrums for red and green fluorescence, which can complicate the interpretation of the data obtained with this probe.

In mice, events interpreted as mitophagy were detected using the same reporters in a range of tissues with high metabolic activity, such as heart and skeletal muscle, kidney, liver, pancreas and brain [START_REF] Mcwilliams | mito-QC illuminates mitophagy and mitochondrial architecture in vivo[END_REF]McWilliams et al. 2018b;Sun et al. 2015). Here, mitolysosomes were particularly abundant in the Purkinje cell layer of the cerebellum and in regions enriched with neural stem cells, such as the lateral ventricle and the dentate gyrus in the hippocampus [START_REF] Mcwilliams | mito-QC illuminates mitophagy and mitochondrial architecture in vivo[END_REF]Sun et al. 2015).

Moreover, mitolysosomes were abundant in different DA neuron populations, including the ! 13 highly vulnerable A9 SNc neurons that degenerate in PD, but also the less susceptible A10 DA neurons of the VTA, and the A16 periglomerular DA neurons of the olfactory bulb, known to increase in number in PD (McWilliams et al. 2018b). In these neurons, mitolysosomes were rare in the axonal arbors, whereas they were enriched in the somata and axon initial segments. In slices of the mesencephalon prepared ex vivo from mice following viral vector-mediated delivery of mt-Keima, the basal rate of mitophagy turned out to be significantly higher in DA neurons of the SNc than in DA neurons of the VTA, or in other basal ganglia neurons [START_REF] Guzman | Systemic isradipine treatment diminishes calcium-dependent mitochondrial oxidant stress[END_REF]. Remarkably, chronic treatment of the mice with the Cav1 channel inhibitor isradipine significantly reduced basal mitophagy in SNc DA neurons. Cav1 channel-mediated calcium entry into the somatodendritic compartment stimulates mitochondrial intermediary metabolism and oxidative phosphorylation in these neurons to sustain the bioenergetic demand associated with their characteristic autonomous pacemaking activity; this occurs at the cost of an increase in the generation of mitochondrial reactive oxygen species [START_REF] Dragicevic | Dopamine midbrain neurons in health and Parkinson's disease: emerging roles of voltage-gated calcium channels and ATPsensitive potassium channels[END_REF]. In addition to normalizing basal mitophagy, israpidine mitigated oxidative stress in SNc neuron, suggesting that this mechanism acts as a key determinant of vulnerability in SNc DA neurons [START_REF] Guzman | Systemic isradipine treatment diminishes calcium-dependent mitochondrial oxidant stress[END_REF].

In mito-QC mice, the levels of basal mitophagy did not appear to be affected by loss-offunction mutations of PINK1 or PARK2, supporting the idea that the PINK1/Parkindependent mitochondrial clearance program is not active under basal conditions (McWilliams et al. 2018a, b). Alternatively, complementary mitophagy pathways may be activated in the absence of PINK1, as previously suggested [START_REF] Dagda | Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission[END_REF][START_REF] Vincow | The PINK1-Parkin pathway promotes both mitophagy and selective respiratory chain turnover in vivo[END_REF]. However, future studies will have to more carefully investigate the possible impact of PINK1/Parkin deficiency on basal mitophagy, particularly during aging and under stress. Sun et al. reported a 70% decrease in mitophagy in dentate gyrus neurons in 21month-old mice compared to 3-month-old animals (Sun et al. 2015), but mitophagy in the absence of PINK1 was only investigated up to 9.5 months of age (McWilliams et al. 2018b).

Moreover, although Lee and colleagues did not observe changes in basal mitophagy in the absence of PINK1 in Drosophila [START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF]), Cornelissen and his team reported impairment of the age-dependent increase in mitolysosome abundance in flight muscles and dopaminergic neurons of pink1 and parkin mutant flies [START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila Elife 7[END_REF].

Remarkably,

Conclusions

Since early studies in the 1960s based on classical in vivo radiolabeling approaches in rodents to investigate the turnover of protein and lipid components and postulating the existence of a mechanism ensuring the degradation of mitochondria as discrete entities, this hypothesis has been verified in the past fifteen years by the identification of specific programs for the autophagy-dependent destruction of mitochondria. One of these ! 14 programs is activated in cell culture by mitochondrial stress and regulated by the protein products of two genes responsible for familial forms of one of the most common neurodegenerative diseases, providing the impetus for the direct investigation of mitophagy in neuronal cells. Despite many debates, fueled by the idea that the metabolic and bioenergetic properties of neurons are not compatible with such a mechanism, several laboratories have provided evidence for its occurrence, not only in cultured neurons exposed to toxins, but also in neurons in the brain of model organisms under basal conditions. Many open questions still persist: what is the relative contribution of mitophagy to mitochondrial quality control in neurons, compared to other mechanisms involving degradation of mitochondrial components by intramitochondrial proteases, or direct delivery to the lysosome through the MDV pathway [START_REF] Sugiura | A new pathway for mitochondrial quality control: mitochondrial-derived vesicles[END_REF])? What are the physiological conditions that activate this process in neurons, and what are the mechanisms that regulate basal versus evoked mitophagy? How is mitophagy interconnected with mitochondrial biogenesis, how is it linked to mitochondrial transport fusion and fission in neurons? In which neuronal compartments does it occur primarily? Does it contribute in any way to the modulation of neuronal activity, for example by limiting energy supply to fundamental processes, such as synaptic vesicle recycling? Finally, with respect to PINK1/Parkin-dependent mitophagy, we still need to understand in how far this pathway is central to neuronal degeneration in Parkinson's disease. This will imply solving present controversies as to the involvement of this specific mechanism in whole organisms, particularly during aging, which is acknowledged to be the greatest risk factor for Parkinson's disease [START_REF] Collier | Aging and Parkinson's disease: Different sides of the same coin?[END_REF], or under stress conditions. These studies will have to be paralleled by the in-depth investigation of the mitophagy-independent functions of the PINK1/Parkin signaling pathway [START_REF] Jang | The role of mitochondria in aging[END_REF]Palikaras et al. 2018).

PINK1 and Parkin have been reported to exert other functions in relation to mitochondrial maintenance, be it cooperatively or independently, including in mitochondrial biogenesis, the derepression of transcripts encoding specific mitochondrial respiratory chain subunits on the outer mitochondrial membrane, the regulation of the activity of mitochondrial respiratory chain complex I, and the MDV pathway (reviewed by Alves Da Costa and Checler, 2012, [START_REF] Charan | Pathologic and therapeutic implications for the cell biology of parkin[END_REF], Scarffe et al. 2015[START_REF] Sugiura | A new pathway for mitochondrial quality control: mitochondrial-derived vesicles[END_REF][START_REF] Voigt | The mitochondrial kinase PINK1: functions beyond mitophagy[END_REF][START_REF] Winklhofer | Parkin and mitochondrial quality control: toward assembling the puzzle[END_REF][START_REF] Mouton-Liger | PINK1/Parkin-Dependent Mitochondrial Surveillance: From Pleiotropy to Parkinson's[END_REF][START_REF] Chu | Multiple pathways for mitophagy: A neurodegenerative conundrum for Parkinson's disease[END_REF]

Compliance with ethical standards

Conflict of interest

There is no conflict of interest.

Table 1. Pathways for the degradation of mitochondrial components or whole mitochondria.

Pathway Description and key references

Mitochondrial Proteolytic systems of the ATP-dependent AAA+ enzyme family, degrading irreversibly misfolded, proteases damaged or oxidatively modified proteins in the mitochondrial matrix and inner mitochondrial membrane. The genetic alteration of these proteases causes neurological disorders in humans (Rugarli e t a l . 2 0 1 2 ; V o o s e t a l . 2 0 1 6 ) .

Ubiquitin-

This pathway is involved in the degradation of proteins of the outer mitochondrial membrane, proteasome including pro-and anti-apoptotic proteins, such as Mcl1, and proteins regulating mitochondrial system dynamics, such as Mfn2 (Karbowski and Youle 2011). The degradation of some of these proteins is regulated by PINK1 and Parkin (Carrol et al. 2014;[START_REF] Mclelland | Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy[END_REF][START_REF] Tanaka | Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin[END_REF].

MDV pathway

A vesicular mitochondrial quality control pathway involving the PINK1/Parkin-regulated formation of small vesicles emerging from mitochondria (MDVs) under conditions of oxidative stress, and transporting selected oxidized proteins directly to the lysosome for degradation. Contrary to mitophagy, this pathway does not require mitochondrial fission mediated by dynamin-related protein 1 (Drp1), and is independent from canonical autophagy [START_REF] Soubannier | A vesicular transport pathway shuttles cargo from mitochondria to lysosomes[END_REF][START_REF] Mclelland | Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control[END_REF][START_REF] Mclelland | Syntaxin-17 delivers PINK1/parkindependent mitochondrial vesicles to the endolysosomal system[END_REF][START_REF] Sugiura | A new pathway for mitochondrial quality control: mitochondrial-derived vesicles[END_REF].

Mitophagy

A term designating various mechanisms for the selective delivery of whole mitochondria to the lysosome within autophagic vesicles. This pathway operates under basal conditions, during specific developmental programs or in response to stress or mitochondrial damage (McWilliams andMuqit 2017, Chu et al. 2018). Among the different mitophagy mechanisms described so far, PINK1/Parkin-dependent mitophagy is the most comprehensively characterized at the molecular level [START_REF] Geisler | PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[END_REF][START_REF] Matsuda | PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy[END_REF][START_REF] Mcwilliams | PINK1 and Parkin: emerging themes in mitochondrial homeostasis[END_REF][START_REF] Narendra | Parkin is recruited selectively to impaired mitochondria and promotes their autophagy[END_REF][START_REF] Narendra | PINK1 is selectively stabilized on impaired mitochondria to activate Parkin[END_REF][START_REF] Truban | PINK1, Parkin, and Mitochondrial Quality Control: What can we Learn about Parkinson's Disease Pathobiology?[END_REF].

Transmitophagy A cell non-autonomous pathway for the autophagy-dependent degradation of neuronal mitochondria by adjacent glial cells. The existence of this pathway has been demonstrated in vivo at the optic nerve head, where protrusions shed from axons originating from retinal ! 16 ganglion cells were found to be phagocytosed by neighboring astrocytes. Using a mitochondrion-targeted fluorescent reporter combining an acid-labile EGFP and mCherry, it was shown that mitochondria contained in these axonal protrusions were degraded in lysosomal vesicles within the astrocytes (Davis et al. 2014).

! 17 ! 29

  . It has thus been suggested that PINK1/Parkin-dependent mitophagy may play a key role in counterselecting mitochondria with high mtDNA mutational loads. To address the relevance of this mechanism in vivo, Parkin knockout mice were crossed with different transgenic mice modeling the accumulation of mtDNA deletions or mutations: the MitoPark mouse, with SN DA neuron-specific knockout of mitochondrial transcription factor A, an integral component of the basal mitochondrial transcription machinery with an additional role in the regulation of mtDNA copy number (Sterky et al. 2011); the Mutator mouse, homozygous for a mutation affecting the proofreading activity of DNA polymerase γ, responsible for mtDNA replication (Pickrell et al. 2015); a mouse model expressing a mutant version of the Twinkle helicase, involved in mtDNA replication, specifically in SN DA neurons (Song et al. 2017); and the PD-mito-Pst I mouse, in which a mitochondrion-targeted version of the restriction enzyme PstI is selectively expressed in SN DA neurons (Pinto et al. 2018). While the MitoPark, mutant Twinkle and PD-mito-Pst I models developed age-dependent PD-like phenotpes associated with progressive degeneration of SN DA neurons and defects in locomotor behavior

  . Importantly, PINK1 and Parkin play also central roles in maintaining neuronal viability in response to stress by various, only partially elucidated mechanisms, independent of their roles in mitochondrial quality control (Alves Da Costa and Checler 2012, Charan and LaVoie 2015, Winklhofer 2014), and they can even exert proapoptotic functions under specific circumstances (Carrol et al. 2014, Zhang et al. 2014). Considering this complexity, there is still a way to go before we can fully appreciate the relative contribution of each of these specific mechanisms to the pathophysiology of Parkinson's disease. ! 15 Funding information Our work is supported by Institut national de la santé et de la recherche médicale (INSERM), Fondation Institutdu Cerveau et de la Moelle épinière and Agence Nationale pour la Recherche ("Investissements d'avenir," grant ANR-10-IAIHU-06), Fondation de France, Association France Parkinson and Michael J. Fox Foundation.

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EDITORIAL

Back to plastic pollution in COVID times

Giuliana Gorrasi 1 • Andrea Sorrentino 2 • Eric Lichtfouse 3,4 The great problem of social chemistry we call politics, is to discover what desires of mankind may be gratified, and what must be suppressed, if the highly complex compound, society, is to avoid decomposition Thomas Huxley (1871) In this article, we show that the chaos and urgency induced by the coronavirus disease 2019 (COVID-19) pandemic has led to massive fossil fuel-derived plastic production, largely ignoring recent environmental policies. Solutions for a sustainable and circular economy require radical innovations, consumer education and most importantly political willingness.

The plastic evil

The world health crisis caused by the spread of COVID-19 is not only damaging people's health and the world economy but is also threatening environmental sustainability [START_REF] Sharma | Environmental chemistry is most relevant to study coronavirus pandemics[END_REF]. Numerous media have warned about the ecological disaster caused by this pandemic [START_REF] Canning-Clode | Will COVID-19 containment and treatment measures drive shifts in marine litter pollution?[END_REF][START_REF] Aragaw | Surgical face masks as a potential source for microplastic pollution in the COVID-19 scenario[END_REF]. For instance, there is a sharp increase in plastic waste from personal protective equipment such as masks and gloves, and from the massive return to using plastic shopping bags to prevent cross-contamination, whereas many nations had started to ban at least partly the use of non-biodegradable bags before the COVID era [START_REF] Klemeš | Minimising the present and future plastic waste, energy and environmental footprints related to COVID-19[END_REF][START_REF] Fadare | Covid-19 face masks: a potential source of microplastic fibers in the environment[END_REF]. Moreover, the lockdown has highly fostered e-commerce, resulting in massive consumption of materials for containment and packaging [START_REF] Silva | Increased plastic pollution due to COVID-19 pandemic: challenges and recommendations[END_REF][START_REF] Han | Can the coronavirus disease be transmitted from food? A review of evidence, risks, policies and knowledge gaps[END_REF]. Medical waste has grown up to 370% and packaging plastic demand up to 40% (Patrício [START_REF] Silva | Increased plastic pollution due to COVID-19 pandemic: challenges and recommendations[END_REF]. The fight against the virus has thus overshadowed environmental protection policies. For example, the recent drop of oil prices, which may appear as a benefit for drivers, has in fact prompted a real setback of policies aimed at reducing plastic materials of synthetic origin [START_REF] Sun | Unflushable or missing toilet paper, the dilemma for developing communities during the COVID-19 episode[END_REF]. We even observe the rise of opposing factions ready to challenge each other to prove their ideas: on one hand, some people think that plastic is the absolute evil to be eliminated in any way; on the other hand, others argue that it is impossible to change the current lifestyle [START_REF] Czigany | The coronavirus and plastics[END_REF]. The truth is that both sides aim to solve a very complex problem with simplistic solutions. Indeed, plastic waste is a problem that simultaneously involves economic, social and technological aspects [START_REF] Vanapalli | Challenges and strategies for effective plastic waste management during and post COVID-19 pandemic[END_REF][START_REF] Prata | COVID-19 Pandemic Repercussions on the Use and Management of Plastics[END_REF]. In other words, all society issues cannot be solved anymore by the painkiller approach in a globalized word, but required a new world system involving fast evaluation and actions taking into account all aspects of a problem [START_REF] Lichtfouse | Climate change, intercropping, pest control and beneficial microorganism[END_REF][START_REF] Lichtfouse | Society issues, painkiller solutions, dependence and sustainable agriculture[END_REF][START_REF] Lichtfouse | Social chemistry[END_REF]).

An old problem

Long before the world knew about COVID-19, millions of tons of plastic ended up daily in the sea with serious damage to the environment and with an unacceptable waste of precious resources [START_REF] Haward | A critical analysis of the impacts of COVID-19 on the global economy and ecosystems and opportunities for circular economy strategies[END_REF]. Macroscopic residues from the degradation of common objects such as bottles or food packaging are only a small percentage of these wastes [START_REF] Wang | Microplastic contamination in freshwater: first observation in Lake Ulansuhai, Yellow River Basin, China[END_REF][START_REF] Tofa | Visible light photocatalytic degradation of microplastic residues with zinc oxide nanorods[END_REF]. The main part is represented by micro-and nanoparticles, e.g., microplastics, produced by common operations such as the discharge of wastewater from our washing machines, the use of cosmetics and detergents, and the consumption of car tires [START_REF] Padervand | Removal of microplastics from the environment. A review[END_REF][START_REF] Naqash | Interaction of freshwater microplastics with biota and heavy metals: a review[END_REF]. Certainly, the solution cannot be solely to eliminate plastic materials, including those of synthetic origin. This would be a utopian solution, technically unsustainable and destined to succumb to multiple socioeconomic interests. Of course, this does not mean that any kind of behavior can be excused. There are millions of cotton swabs, toy balloons and drinking straws that end up on our beaches every year.

Forgotten bioplastics

The emergency caused by COVID-19 is highlighting, once again, the extreme fragility of our systems of management, disposal and recycling of plastic waste (Nanda and Berruti 2020a). To fight this health emergency, we have quickly needed large quantities of inexpensive, safe and easy-tofind materials. In the rush, the only choice available was to use petroleum-derived polymers such as polypropylene and polyethylene as disposable materials. This does not mean that alternatives based on biodegradable materials, often of natural origin, were not available [START_REF] Brandelli | Nanostructured bioactive compounds for ecological food packaging[END_REF]. Indeed, scientists have recently made available various biodegradable polymers [START_REF] Qasim | Renewable cellulosic nanocomposites for food packaging to avoid fossil fuel plastic pollution: a review[END_REF][START_REF] De Amorim | Plant and bacterial nanocellulose: production, properties and applications in medicine, food, cosmetics, electronics and engineering. A review[END_REF]. Some biopolymers even display high performances and unique new functions compared to traditional materials [START_REF] Morin-Crini | Applications of chitosan in food, pharmaceuticals, medicine, cosmetics, agriculture, textiles, pulp and paper, biotechnology, and environmental chemistry[END_REF]). However, these biopolymers are not yet the first choice when there is a need for a quick and efficient response to a real problem. This is because, from a commercial point of view, these materials are still poorly known, expensive and often difficult to handle. Moreover, there are still no biorefineries capable of supplying high volumes of bioplastics fast, in a sustainable way and at competitive costs. Similarly, there are still too few companies able to recycle these materials. Furthermore, many scientists argue that the massive use of biodegradable materials could create serious issues for the few existing treatment plants [START_REF] Wojnowska-Baryła | Effect of biobased products on waste management[END_REF][START_REF] Meeks | Compostable biopolymer use in the real world: stakeholder interviews to better understand the motivations and realities of use and disposal in the US[END_REF]. Indeed, when working with expiring materials, it is not enough to produce and market them, but it is also necessary to plan their correct management, use and disposal. In the absence of adequate management plans, the massive use of biopolymers, in addition to being dangerous for consumers, may also become a problem for the environment.

Political will

The COVID-19 pandemic has disclosed the limits of the current economic system and challenged us to rethink and redesign this system into a more resilient, circular and lowcarbon economic model [START_REF] Wuyts | Circular economy as a COVID-19 cure?[END_REF]). The COVID-19 crisis has revealed the brittleness of many global supply chains (Ibn-Mohammed et al. 2021;[START_REF] Sarkis | A brave new world: lessons from the COVID-19 pandemic for transitioning to sustainable supply and production[END_REF]). Here, circular principles such as recyclability and reusability offer credible solutions in terms of resilience and competitiveness [START_REF] Fawzy | Strategies for mitigation of climate change: a review[END_REF]. Maintaining the value of products, materials and resources for as long as possible by reinserting materials into the product cycle at the end of their use allows to minimize waste generation. However, the buildup of plastic recyclability is still a challenge because it requires specific regulations. In many countries, the use of waste as a raw material for other products is shrunk by regulations that prevent material transport and marketing. Changing these regulations typically leads to conflicts between stakeholders [START_REF] Gunarathne | Biochar from municipal solid waste for resource recovery and pollution remediation[END_REF]. Similar conflicts arise when diverting biogenic waste streams from use in energy and composting to other uses (Nanda and Berruti 2020b). Today, we have all research and development capabilities to set up rapidly to new solutions and technologies on a large scale. Yet, what is still missing is a real political willingness to do so. As a consequence, the circular economy is much more than minimizing the production of waste or inventing biodegradable materials; the circular economy is a radically different economy, which overcomes the traditional conflicts between socioeconomic and environmental interests [START_REF] Aldaco | Food waste management during the COVID-19 outbreak: a holistic climate, economic and nutritional approach[END_REF]. Only when governments will set a clear direction for circular innovation in the private sector, will it be possible to combine economic regeneration, better social outcomes and climate ambitions.

Additive manufacturing

Much more effort must be dedicated to new materials and alternative production processes. Biodegradable materials must certainly continue to be developed. Low-cost, renewable materials with adequate properties and capable of rapid degradation in environment must be developed. New assembly and processing techniques are also necessary to avoid the inevitable shortcomings of new materials [START_REF] Olivieri | Additive manufacturing for biodegradable polymers[END_REF]. For instance, additive manufacturing is a revolutionary technology that offers new freedom in materials employed and expands the range of possibilities for design, production and performances of novel products and systems [START_REF] Sanchez-Rexach | Sustainable materials and chemical processes for additive manufacturing[END_REF]). In additive manufacturing, complex three-dimension objects are obtained with high precision by depositing materials in a layer-by-layer fashion. This provides several benefits such as the complete control of the object properties and significant savings in terms of time and cost [START_REF] Balla | Additive manufacturing of natural fiber reinforced polymer composites: processing and prospects[END_REF].

Circular economy

From an economic point of view, it must be clear that any solution will fail without taking into account the benefit for all. For instance, incentive policies for the use of bio-sustainable raw materials must be strengthened. Moreover, the calculated cost of a material cannot be only that of the raw material, but should take into account the impact on the environment. To do so, the whole production system must be considered, including waste generation, collection, transport, recycling and treatment, use of recovered resource and disposal of remains. Here, the circular approach allows both to reduce dependency on resource markets and decreases disposal costs [START_REF] You | COVID-19: resource recovery from plastic waste against plastic pollution[END_REF]) (Fig. 1). From a social point of view, the implementation of circular economies on a local basis can solve many problems related to the disasters produced by the uncontrolled globalization in recent decades. Bio-based products need new supply chains [START_REF] Kosheleva | Synthesis of activated carbon from food waste[END_REF] because the reduced life of bioproducts requires a denser location of production plants and strong collaborations between goods companies, retailers, consumers, waste management companies and legislators. This should induce social benefits such as increasing job creation at the local level and better employment conditions.

Radical innovation

We have to fundamentally rethink the value we assign to the objects of our everyday life [START_REF] Prideaux | Lessons from COVID-19 can prepare global tourism for the economic transformation needed to combat climate change[END_REF]) and get out of the 'I shop therefore I am' mantra evidenced by Barbara Kruger in 1987, to go back to the original quote 'I think therefore I am' from René Descartes in 1637. To do this, we need to understand the social and environmental values of products, and how to keep them in circulation for as long as possible [START_REF] Milios | Plastic recycling in the Nordics: a value chain market analysis[END_REF]. Designing products that are easier to disassemble and recycle and creating infrastructures that facilitate the return of products to manufacturers are also needed [START_REF] Nithya | Electronic waste generation, regulation and metal recovery: a review[END_REF]. Radical innovation in an holistic way is thus critical to enable businesses and governments to reach their goals.

Consumers psychology

In the post-pandemic world, some of our COVID-19 habits will probably hang on for a long time. For example, e-commerce sales will continue to grow as many consumers will not give up on the benefits in terms of convenience and choice. This growth will lead to a significant increase in packaging material and associated problems of treatment and recycling. The COVID-19 pandemic is expected to spur a rapid transition to new models of circular economy. In particular, we need urgent and coordinated efforts to develop larger and more efficient systems and infrastructures for plastic waste management. Strict policies must force companies to reduce the use of natural materials in favor of those coming from recycling. In addition, working on the psychological aspects of consumers and encouraging sustainable products and processes through efficient tax policies would help to achieve sustainable development. Plastics are important because they provide many advantages, yet instead of demonizing, we should learn to use plastics responsibly.