Dr Angelo Poletti

D R Philip

M Beart

Dr Olga Corti
email: olga.corti@upmc.fr

Klas Blomgren

Anglo Poletti

Philip M Beart

Autophagy in neurodegeneration: New insights underpinning therapy for neurological diseases

Keywords: autosis, chaperone, hypoxia-ischaemia, mitophagy, motor neuron, Parkinsonism ACD, autophagic cell death AR, androgen receptor ATFS1, Activated Transcription Factor Associated with Stress 1 Atg5, autophagy related protein 5 HIE, hypoxic-ischemic encephalopathy HSD17B10, mitochondrial matrix hydroxysteroid dehydrogenase; HSP, heat

Atg7, autophagy related 7 BAG3, Bcl-2-associated athanogene 3 Bcl-2, B-cell lymphoma 2 BECN1, Beclin1 CASA, chaperone-assisted selective autophagy CCCP, carbonyl cyanide m-chlorophenyl hydrazone

termed autophagosomes, engulf long-lived proteins or organelles such as mitochondria, and transfer them to lysosomes for degradation [START_REF] Ravikumar | Regulation of mammalian autophagy in physiology and pathophysiology[END_REF]. The autophagy machinery is conserved across species, but because the brain possesses precise mechanisms regulating its nutrient and energetic supply, basal autophagic flux was a relatively late discovery in healthy neurons [START_REF] Boland | Neuronal macroautophagy: from development to degeneration[END_REF]. Indeed perhaps the first demonstration of the relevance of autophagy in brain were findings in the autophagy related protein 5 and 7 (Atg5, Atg7) knockout mice [START_REF] Komatsu | Loss of autophagy in the central nervous system causes neurodegeneration in mice[END_REF][START_REF] Hara | Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice[END_REF]. Here Atg7 deficiency caused massive neuronal loss in the cerebral and cerebellar cortices with notable behavioral deficits and accumulation of polyubiquitinated proteins in neurons [START_REF] Komatsu | Loss of autophagy in the central nervous system causes neurodegeneration in mice[END_REF]. There is now a rapidly proliferating literature about the importance of autophagy in brain, with autophagic and associated lysosomal function considered determinant for the maintenance of neuronal health. Autophagy dysfunction caused by gene mutations and/or potentially toxic aggregation-prone proteins is a hallmark of several human neurodegenerative diseases. Autophagic organelle clearance, particulary that of damaged mitochondria (mitophagy), currently attracts appreciable attention because of the brain's unique energy dependence and because the process is suspected to be dysfunctional in various pathologies (e.g. Van Laar & Berman 2013). It is therefore not surprising that drugs that aid the clearance of potentially toxic debris or damaged organelles are now the focus of escalating attention as potential therapeutics [START_REF] Menzies | Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities[END_REF][START_REF] Boland | Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing[END_REF].

On the other hand, more than 2,000 articles in PubMed link autophagy and cell death in neuron or brain, with a number of studies pointing at a causative role of autophagy in cell death. This topic is somewhat controversial, as evidence for such a causative link has often been partial. There has been debate over the ambiguous usage of the terminology "autophagic cell death" (ACD) to define cell death modalities associated with autophagy or triggered by autophagy and occurring in the presence or absence of other standard cell death mechanisms of death, such as apoptosis or necrosis. The use of more precise expressions, such as autophagy-associated cell death, autophagy-mediated cell death and autophagydependent cell death, accompanied by more rigorous analyses to determine the level of involvement of the process, has been recommended [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[END_REF]. Despite these reservations, several studies have demonstrated that autophagy can indeed cause neuronal death, indicating that under specific circumstances autophagy inhibition should considered as a therapeutic option [START_REF] Liu | Autosis and autophagic cell death: the dark side of autophagy[END_REF]. This review follows from a symposium entitled "Autophagy in Neurodegeneration: New Insights Underpinning Therapy for Neurological Diseases" at the 19 th International Neuroscience Winter Conference held at Sölden, Austria, in early 2017. Based on the topics covered by the presentations from the symposium, we here propose to use specific examples to illustrate the critical role of autophagy in pathological conditions affecting the brain, and discuss its potential as a target for therapy. The first and second sections of the review illustrate

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This article is protected by copyright. All rights reserved the contribution of autophagy to hypoxic-ischaemic injury in the adult brain and in the vulnerable immature neonatal brain, and highlight the emergence of autophagy inhibition as a promising approach for treating perinatal asphyxia. The third section describes a type of macroautophagy called chaperone-assisted selective autophagy (CASA), which uses the HSPB8-BAG3 chaperone complex to target misfolded proteins for autophagic degradation and is considered a candidate target for the treatment of neuromuscular and motoneuron diseases (MND). The fourth section is dedicated to the multifunctional proteins PINK1 and Parkin and their role in the regulation of a stress-induced mitophagy program suspected to be central to the physiopathology of certain forms of Parkinson's disease (PD). Finally, the fifth section illustrates the complexity associated with therapeutic approaches targeting autophagy in brain diseases with the concrete example of the autophagy activator rilmenidine and its discordant effects in neuronal and mouse models of MND. The key points addressed in the review and associated take-home messages are summarized schematically in Figure 1.

I. Insult severity, dysfunctional autophagy and hypoxic-ischaemic injury.

There is an existent literature that patterns of neuronal injury are highly dependent upon the type, intensity and duration of insult, especially oxidative stress and excitotoxicity, and presumably load of toxic protein aggregates [START_REF] Galluzzi | Autophagy in acute brain injury[END_REF]. Indeed, we found in cultured cortical neurons that caspase-independent PCD induced by the oxidative stressor hydrogen peroxide was greatly attenuated by 3-methyladenine and knockdown with short interfering RNA (siRNA) directed at Atg7 and Beclin1 (BECN1) as shown by use of specific autophagic and cell death morphological markers. This diverse evidence supports the involvement of autophagy-mediated neuronal death, and also caspase-independent PCD, in a model with different interactive cell death pathways [START_REF] Higgins | Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death[END_REF]. Consistent with the concept for a determinant role in neuronal of insult intensity, in severe oxidative stress there was even more extensive crosstalk between different forms of cell death with siRNA directed at Atg7 being less effective versus injury progression, even though autophagy-associated cell death was found [START_REF] Higgins | Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death[END_REF]. Additionally, we found in a comparative analysis of our extensive microarray data that there are many commonalities in autophagiclysosomal genes regulated downstream of oxidative stress and excitotoxicity, and that these events lead to downstream inhibition of autophagy and the autophagic-lysosomal pathway [START_REF] Yap | Comparative Microarray Analysis Identifies Commonalities in Neuronal Injury: Evidence for Oxidative Stress, Dysfunction of Calcium Signalling, and Inhibition of Autophagy-Lysosomal Pathway[END_REF].

Given that both oxidative stress and excitotoxicity are documented to contribute to hypoxic-ischaemic injury (HI) [START_REF] Khoshnam | Pathogenic mechanisms following ischemic stroke[END_REF], studies of the possible recruitment of autophagy to stroke were a logical extension of our work. Indeed, in post-mortem human brain tissue from patients with a history of stroke we found abundant microtubule-associated protein light chain 3 (LC3)-immunolabelled autophagic vacuoles [START_REF] Frugier | Evidence for the recruitment of autophagic vesicles in human brain after stroke[END_REF]. Dense Sequestosome 1 (SQSTM1) labelling was also found, although immunopositive neurons were further away from the penumbral area unlike those found after LC3 labelling -both SQSTM1 mRNA and protein levels were also increased. This increased level of SQTM1 may be

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This article is protected by copyright. All rights reserved indicative of inhibition of autophagy, however our current understanding of the recruitment of the autophagic-lysosomal system in HI is very much in its infancy with existent evidence suggestive that the "load" of cellular debris and damaged proteins may determine the mode of recruitment of autophagy [START_REF] Wen | Neuronal injury in rat model of permanent focal cerebral ischemia is associated with activation of autophagic and lysosomal pathways[END_REF][START_REF] Li | Lithium reduces apoptosis and autophagy after neonatal hypoxia-ischemia[END_REF][START_REF] Shi | Excessive autophagy contributes to neuron death in cerebral ischemia[END_REF]. Clearly, given the dearth of drugs for the clinical management of HI, autophagy and its management represents an attractive target (vide infra).

II.

Selective neuronal deletion of Atg7 is protective in neonatal brain injury.

The neonatal brain differs from the adult brain.

Autophagy is crucial for maintaining homeostasis in response to stress in most eukaryotic cells, including neurons, mediating neuroprotection following several forms of brain damage. Involvement of autophagy in neuronal cell death has therefore been controversial, and efforts have been made to distinguish cell deathpromoting autophagy from autophagic responses that occur parallel to cell death, or even counteracting degenerative mechanisms. Emerging evidence indicates that inappropriate activation of autophagy can directly mediate neuronal cell death [START_REF] Piras | Inhibition of autophagy delays motoneuron degeneration and extends lifespan in a mouse model of spinal muscular atrophy[END_REF], Ginet et al. 2014b, Ginet et al. 2014a[START_REF] Clarke | Autophagic cell death exists[END_REF]). In the immature, neonatal brain, multiple mechanisms of cell death are often activated simultaneously, even in the same neuron [START_REF] Puka-Sundvall | Subcellular distribution of calcium and ultrastructural changes after cerebral hypoxia-ischemia in immature rats[END_REF][START_REF] Blomgren | Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis[END_REF], and the relative contributions of the various mechanisms are strikingly different from the adult brain [START_REF] Zhu | The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia[END_REF][START_REF] Hu | Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation[END_REF]. In mice and rats, mixed morphological phenotypes indicative of necrotic, apoptotic, and autophagic features are present following HI-induced neuronal cell death in the immature brain, each confirmed by specific biochemical and morphological criteria [START_REF] Li | Lithium reduces apoptosis and autophagy after neonatal hypoxia-ischemia[END_REF][START_REF] Northington | Neuronal cell death in neonatal hypoxiaischemia[END_REF][START_REF] Blomgren | Pathological apoptosis in the developing brain[END_REF], Puyal & Clarke 2009). Levels of caspase-dependent, caspase-independent cell death and lipidation of LC3 provided evidence for greater activity of the apoptotic and autophagic machinery in the immature brain than in the adult brain [START_REF] Zhu | The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia[END_REF]. This is reasonable, given the massive growth and continuous remodelling, including removal of redundant cells and synapses, in the immature brain, where autophagy plays an integral role in maintaining cellular and tissue homeostasis [START_REF] Oppenheim | Cell death during development of the nervous system[END_REF][START_REF] Semple | Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species[END_REF]. Neuronal autophagy has been reported to be enhanced in rodent models of perinatal cerebral ischemia or HI [START_REF] Zhu | The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia[END_REF][START_REF] Liu | Changes in autophagy after traumatic brain injury[END_REF][START_REF] Koike | Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury[END_REF][START_REF] Carloni | Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia[END_REF].

Neuroprotective measures to treat injury in the immature brain should most likely be quite different from those applied in the adult brain [START_REF] Wang | Developmental shift of cyclophilin D contribution to hypoxic-ischemic brain injury[END_REF][START_REF] Zhu | The influence of age on apoptotic and other mechanisms of cell death after cerebral hypoxia-ischemia[END_REF], and autophagy has evolved as a particularly promising strategy in the immature brain

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Methodological aspects.

The role of autophagy in neonatal brain injury is controversial, with independent studies showing protective effects in brain injury in rodents following inhibition or induction of autophagy by pharmacological agents [START_REF] Vaslin | Excitotoxicity-induced endocytosis confers drug targeting in cerebral ischemia[END_REF][START_REF] Carloni | Activation of autophagy and Akt/CREB signaling play an equivalent role in the neuroprotective effect of rapamycin in neonatal hypoxia-ischemia[END_REF]. Confounding aspects are linked to the fact that pharmacological autophagy-modulating agents have poor specificity and that they have been used intraperitoneally or intravenously, although they may not readily cross the blood-brain barrier [START_REF] Galluzzi | Autophagy in acute brain injury[END_REF]). In addition, the methodologies used to monitor autophagic responses in vitro and in vivo, have not always been discriminative. Cellular markers used include expression of LC3, ATG5, BECN1; phosphorylation of AMP-activated protein kinase, Unc-51 Like Autophagy Activating Kinase 1 or substrates of mammalian target of rapamycin complex 1; interaction of BECN1 with B-cell lymphoma 2 (BCL-2) family members;

LC3 lipidation; lysosomal acidification; and the degradation of p62 or autophagic substrates. Cytoplasmic accumulation of autophagosomes and autolysosomes, LC3 lipidation, and other morphological or biochemical features of autophagy have been exploited to imply increased autophagy in neurons and other cells in brain tissue [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[END_REF]). However, cytoplasmic vacuolization and LC3 lipidation can also increase when lysosomal function is impaired, or when autophagosomes cannot fuse with lysosomes. To distinguish between active autophagic responses and autophagy blockade, it is necessary to take into account autophagic flux [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[END_REF], an aspect that has not been considered in many of the reports in the literature. Genetic approaches are more specific, but also have disadvantages. Genetically targeting a component of the autophagic machinery, for example Atg5 or Atg7, is more specific than pharmacological agents, but the targeted protein may also play additional roles unrelated to autophagy.

Furthermore, the genetic deletion is often constitutive and life-long, leading to adaptive changes of the tissue and its metabolism. Making the deletion inducible, not only in terms of which cells that will be targeted, but also when the deletion will occur, will make the interpretations of the results more accurate.

Autophagy is also involved in the release of danger signals and inflammation, both systemically and locally in the central nervous system, and these contribute to the overall short and long term outcomes of any brain injury. Future studies taking these aspects into consideration are needed to more accurately dissect the effects of autophagy in brain injuries.

Transient and specific inhibition of autosis reduces injury to the immature brain.

In 2013, a study was published demonstrating that selective over-activation of autophagy could cause a pattern of cell death with morphological characteristics different from apoptosis or necrosis, termed "autosis" [START_REF] Liu | Autosis is a Na+,K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia[END_REF]) (Table 1). Autosis occurs in vitro and in vivo in cerebral HI of the immature brain, and is inhibited by Na + /K + -ATPase antagonists approved for clinical use. Lithium has been demonstrated to induce autophagy [START_REF] Sarkar | Lithium induces autophagy by inhibiting inositol monophosphatase[END_REF]. However, we found it to be neuroprotective in a rat

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This article is protected by copyright. All rights reserved model of neonatal HI, where it reduced the levels of LC3 lipidation, consistent with autophagy inhibition [START_REF] Li | Lithium-mediated long-term neuroprotection in neonatal rat hypoxia-ischemia is associated with antiinflammatory effects and enhanced proliferation and survival of neural stem/progenitor cells[END_REF][START_REF] Li | Lithium reduces apoptosis and autophagy after neonatal hypoxia-ischemia[END_REF][START_REF] Xie | Therapeutic benefits of delayed lithium administration in the neonatal rat after cerebral hypoxia-ischemia[END_REF]. To circumvent the limited specificity of pharmacological agents, we investigated the effect of HI in mice with neuron-specific deletion of the essential autophagy gene, Atg7. Tissue loss was decreased by more than 40%, and caspase-dependent and -independent cell death was attenuated in several brain regions compared to wild-type mice [START_REF] Xie | Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury[END_REF]. These findings suggest that autophagy inhibition may be a promising therapeutic avenue for the treatment of human neonates developing severe hypoxic-ischemic encephalopathy (HIE). These results extend similar observations in the hippocampus of a milder HI model [START_REF] Koike | Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury[END_REF], supporting a lethal role of autophagy in brain regions highly vulnerable to HI in neonates, such as cortex, thalamus and striatum.

Moreover, investigation of the abundance of LC3 II-and cathepsin D-positive cells in susceptible areas of the brain of human term newborns who died from severe asphyxia with HIE revealed increased neuronal autophagy compared to brains from neonates who died from other causes, without brain injury [START_REF] Xie | Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury[END_REF].

A different injury model, genotoxic stress induced through ionizing radiation, was used to model the effects of cranial radiotherapy in children treated for brain malignancies. Irradiation typically targets actively proliferating cells, making it effective in the treatment of high grade brain tumours. Very few cells in the brain proliferate under normal conditions, making the brain relatively radioresistant, but the neurogenic niches in the hippocampus and the subventricular zone, but also in the cerebellum, harbouring neural stem and progenitor cells, display overt cell death after irradiation [START_REF] Naylor | Voluntary running rescues adult hippocampal neurogenesis after irradiation of the young mouse brain[END_REF][START_REF] Roughton | Sex-dependent differences in behavior and hippocampal neurogenesis after irradiation to the young mouse brain[END_REF][START_REF] Fukuda | Irradiation-induced progenitor cell death in the developing brain is resistant to erythropoietin treatment and caspase inhibition[END_REF], Zhou et al. 2017). The loss of neurogenic capacity observed after a single moderate dose of irradiation increased over time, such that the difference compared to controls was bigger the longer we waited, up to more than one year [START_REF] Kalm | Loss of hippocampal neurogenesis, increased novelty-induced activity, decreased home cage activity, and impaired reversal learning one year after irradiation of the young mouse brain[END_REF][START_REF] Bostrom | Irradiation to the young mouse brain caused long-term, progressive depletion of neurogenesis but did not disrupt the neurovascular niche[END_REF]. In paediatric patients, damage to normal brain tissue adjacent to the tumour is a major concern associated with adverse side effects [START_REF] Kahalley | Comparing Intelligence Quotient Change After Treatment With Proton Versus Photon Radiation Therapy for Pediatric Brain Tumors[END_REF]. The young developing brain is more sensitive to irradiation than the adult brain [START_REF] Fukuda | Age-dependent sensitivity of the developing brain to irradiation is correlated with the number and vulnerability of progenitor cells[END_REF][START_REF] Duffner | Risk factors for cognitive decline in children treated for brain tumors[END_REF]). Radiotherapy can cause long-term cognitive impairment, secondary malignancies, and perturb growth and puberty. Irradiation-induced depletion of neural stem and progenitor cells may underlie some of the cognitive deficits. Using the same Atg7 deletion mouse strain as above, we subjected 10-day-old mice to a single dose of 6 Gy whole brain irradiation and found that the number of dying stem and progenitor cells in the dentate gyrus of the hippocampus was reduced by 60% [START_REF] Wang | Inhibition of autophagy prevents irradiation-induced neural stem and progenitor cell death in the juvenile mouse brain[END_REF]. The ensuing inflammatory reaction, as judged by microglial activation and cytokine levels, was also reduced to a similar extent, but since Atg7 was deleted only in neurons it is likely that the reduced inflammation observed was due to the lower number of dying cells rather than autophagy inhibition. The large reduction of neural stem and progenitor cell death was somewhat surprising, given that Atg7 was only deleted in neurons, and the progenitors commit to a neuronal fate approximately 3 days after the stem cells

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This article is protected by copyright. All rights reserved start proliferating [START_REF] Sliter | Type-2 cells as link between glial and neuronal lineage in adult hippocampal neurogenesis[END_REF][START_REF] Brandt | Transient calretinin expression defines early postmitotic step of neuronal differentiation in adult hippocampal neurogenesis of mice[END_REF], and require 4 weeks to be mature neurons [START_REF] Esposito | Neuronal differentiation in the adult hippocampus recapitulates embryonic development[END_REF], van Praag et al. 2002). The deletion of Atg7 was driven by a neuron-specific nestin promoter [START_REF] Komatsu | Loss of autophagy in the central nervous system causes neurodegeneration in mice[END_REF], and it is unclear when this promoter was activated and hence when Atg7

was deleted in the neural stem and progenitor cells. Dying stem and progenitor cells display primarily signs of apoptosis [START_REF] Fukuda | Irradiation-induced progenitor cell death in the developing brain is resistant to erythropoietin treatment and caspase inhibition[END_REF]) and it remains to determine to what extent autophagy is involved in this type of genotoxic cell death, which is very different from the type of neuronal cell death observed after HI.

Furthermore, a recent study suggests that Atg7 deletion merely delayed neural progenitor/neuronal death, since tissue "loss" (lack of growth) and levels of neurogenesis-related markers were similar to WT brains 5 days after irradiation, on postnatal day 15 [START_REF] Wang | Selective Neural Deletion of the Atg7 Gene Reduces Irradiation-Induced Cerebellar White Matter Injury in the Juvenile Mouse Brain by Ameliorating Oligodendrocyte Progenitor Cell Loss[END_REF]. However, prior to irradiation, Atg7-deficient brains displayed a regenerative state, as judged by the higher levels of transcripts related to neurogenesis and oligodendrocyte precursors, indicating that loss of Atg7 in neurons is stressful to the brain tissue, a foreboding of the neurological symptoms that inevitably will develop several weeks later. Importantly, 5 days after irradiation, myelin development was less impaired in the Atg7-deficient animals than in WT mice [START_REF] Wang | Selective Neural Deletion of the Atg7 Gene Reduces Irradiation-Induced Cerebellar White Matter Injury in the Juvenile Mouse Brain by Ameliorating Oligodendrocyte Progenitor Cell Loss[END_REF]. In summary, this relatively recent publication suggests that autophagy inhibition in neurons has indirect protective effects also in other cell types. Autophagy may thus be considered a target for preventing brain injury triggered by irradiation.

Together, these findings point to the importance of elucidating in further detail the mechanisms through which Atg7 deletion renders neurons resistant to ischaemic injury. It also remains to investigate if autosis is an important mechanism of cell death in all types of neurons in the brain, and if it is less important in the adult brain. The role of autophagy in the demise of neural stem and progenitor cells in the neurogenic niches and oligodendrocyte progenitor cells after genotoxic stress through ionizing radiation also requires further investigation, since these cells typically display signs of classic apoptosis rather than the mixed morphologies observed in neurons dying after HI. Prolonged overall inhibition of autophagy, though, would not be a fruitful strategy, since autophagy is of fundamental importance in the homeostasis of the developing brain. This is evidenced by the reduced lifespan of neuronally Atg7-deficient mice; their brains degenerate and they usually die at 6-10 weeks of age [START_REF] Komatsu | Loss of autophagy in the central nervous system causes neurodegeneration in mice[END_REF]). In addition to direct inhibition of cell death-related mechanisms, the lack of autophagic capacity, even if restricted to one cell type, may cause tissue stress, which in turn elicits protective, regenerative responses. In summary, transient and specific autophagy inhibition strategies should be considered to improve clinical outcome after perinatal asphyxia and HIE, and possibly also after radiotherapy-induced brain damage.

III.

Modulation of the selective, chaperone assisted type of autophagy to protect against neuronal death.

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As mentioned above, the term autophagy covers several types of degradative processes, but usually refers to macroautophagy, a pathway generally characterized by high capacity, but lower specificity in the removal of misfolded proteins compared to proteasomal degradation, and also capable of clearing aggresomes or aggregates (aggrephagy) [START_REF] Cuervo | Chaperone-mediated autophagy: Dice's 'wild' idea about lysosomal selectivity[END_REF].

The CASA complex.

Macroautophagy involves the formation of autophagosomes engulfing organelles/aggregate, and their subsequent fusion to lysosomes for content degradation [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[END_REF]). However, autophagosomes can also receive cargoes of aberrant damaged, oxidize or misfolded proteins utilizing more selective pathways. Aberrant proteins that must be cleared from cells can be selectively delivered by specific chaperones [START_REF] Arndt | Chaperone-assisted selective autophagy is essential for muscle maintenance[END_REF]). These aberrant proteins, particularly in their misfolded forms are potentially neurotoxic and chaperones facilitate their degradation in their monomeric/oligomeric form, generally (and possibly) prior to their aggregation [START_REF] Arndt | Chaperone-assisted selective autophagy is essential for muscle maintenance[END_REF]. This type of macroautophagy assisted by specific chaperones has been named chaperone-assisted selective autophagy (CASA) [START_REF] Arndt | Chaperone-assisted selective autophagy is essential for muscle maintenance[END_REF][START_REF] Cuervo | Chaperone-mediated autophagy: Dice's 'wild' idea about lysosomal selectivity[END_REF], Rusmini et al. 2017). CASA differs from other forms of autophagy, like chaperone-mediated autophagy (CMA). In CASA misfolded proteins are delivered to autophagosomes, which then fuse with lysosomes, while in CMA a specific set of chaperones deliver misfolded proteins directly to lysosomes, thus escaping the autophagosome pathway. CASA takes place in muscle and brain, as well as in cancer cells (Crippa et al. 2010b, Crippa et al. 2010a[START_REF] Piccolella | The small heat shock protein B8 (HSPB8) modulates proliferation and migration of breast cancer cells[END_REF][START_REF] Arndt | Chaperone-assisted selective autophagy is essential for muscle maintenance[END_REF][START_REF] Cuervo | Chaperone-mediated autophagy: Dice's 'wild' idea about lysosomal selectivity[END_REF], Rusmini et al. 2017) and CASA involves a peculiar chaperone of the family of the small HSPs, known as HSPB8 [START_REF] Rusmini | The Role of the Heat Shock Protein B8 (HSPB8) in Motoneuron Diseases[END_REF]. In this pathway, HSPB8 dimerizes and form a stable complex with its cochaperone BAG3, a nucleotide exchange factor (NEF) for HSP70s. Of note, mutation found in HSPB8 or in BAG3 have been associated with motor neuronal or neuromuscular diseases [START_REF] Fontaine | Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants[END_REF][START_REF] Irobi | Mutant HSPB8 causes motor neuron-specific neurite degeneration[END_REF][START_REF] Ghaoui | Mutations in HSPB8 causing a new phenotype of distal myopathy and motor neuropathy[END_REF][START_REF] Adriaenssens | Novel insights in the disease biology of mutant small heat shock proteins in neuromuscular diseases[END_REF][START_REF] Bouhy | A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8[END_REF][START_REF] Guilbert | HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency[END_REF][START_REF] Fang | Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy[END_REF][START_REF] Konersman | BAG3 myofibrillar myopathy presenting with cardiomyopathy[END_REF][START_REF] Selcen | Mutation in BAG3 causes severe dominant childhood muscular dystrophy[END_REF], suggesting they play an essential role in the protein quality control system (PQC) in these two tissues. Together, HSPB8 and BAG3 specifically interact with the ATP-dependent chaperone HSP70 conjugated to the E3 ubiquitin ligase carboxyl terminus of Hsc70interacting protein (CHIP). HSPB8 is the limiting factor of the complex and recognizes misfolded protein, while BAG3, via its PxxP, allows the interaction with dynein (and the protein 14-3-3) for delivery of the entire complex to the microtubule organizing center (MTOC) where autophagosomes are assembled.

Because of its function, the complex HSPB8-BAG3-HSP70-CHIP has been named CASA complex [START_REF] Arndt | Chaperone-assisted selective autophagy is essential for muscle maintenance[END_REF]. The CASA complex component CHIP ubiquittes the misfolded protein cargo for the autophagosomal receptor (SQSTM1/p62) recognition and insertion into autophagosomes [START_REF] Rusmini | The Role of the Heat Shock Protein B8 (HSPB8) in Motoneuron Diseases[END_REF], Cristofani et al. 2017).

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The role of HSPB8 in the CASA complex and in protective activity in NDs.

Interestingly, HSPB8 has been found dramatically increased in spinal cord motor neurons that survive at end stage of disease in amyotrophic lateral sclerosis (ALS) mouse models (Crippa et al. 2010b, Crippa et al. 2010a), as well as in the spinal cord of ALS patients [START_REF] Anagnostou | Vesicle associated membrane protein B (VAPB) is decreased in ALS spinal cord[END_REF]). In the skeletal muscle, another tissue typically affected in motor neuron diseases, like ALS or the polyglutamine disease spinal bulbar and muscular atrophy (SBMA), HSPB8 is robustly upregulated during the course of disease (Crippa et al. 2013a, Crippa et al. 2013b[START_REF] Marino | Differences in protein quality control correlate with phenotype variability in 2 mouse models of familial amyotrophic lateral sclerosis[END_REF], Rusmini et al. 2015). Studies in cellular models of neurodegenerative diseases involving different neuropathogenic proteins, like proteins with elongated glutamine repeat (polyQ) tracts (polyQ-huntingtin, polyQ-ataxin-3 or the SBMA-linked androgen receptor, polyQ-AR), beta-amyloid, alpha-synuclein, superoxide dismutase 1 (SOD1), TAR DNA binding protein of 43 kDa (TDP-43), repeat-associated non-AUG translated dipeptides from the chromosome 9 open reading frame 72 (C9ORF72) gene [START_REF] Chavez Zobel | Distinct chaperone mechanisms can delay the formation of aggresomes by the myopathy-causing R120G alphaB-crystallin mutant[END_REF][START_REF] Wilhelmus | Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity[END_REF], Carra et al. 2008a, Carra et al. 2008b, Crippa et al. 2010b[START_REF] Bruinsma | Inhibition of alpha-synuclein aggregation by small heat shock proteins[END_REF][START_REF] Seidel | The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases[END_REF], Rusmini et al. 2013, Crippa et al. 2016a, Cristofani et al. 2017), demonstrated that HSPB8 possesses a potent anti-aggregant activity via CASA (Crippa et al. 2016a[START_REF] Rusmini | The Role of the Protein Quality Control System in SBMA[END_REF][START_REF] Giorgetti | Synergic prodegradative activity of Bicalutamide and trehalose on the mutant androgen receptor responsible for spinal and bulbar muscular atrophy[END_REF], Rusmini et al. 2013).

Indeed, HSPB8 restores a normal autophagy flux which is found to be blocked in several NDs. Conversely, in most cases, HSPB8 down-regulation resulted in increased accumulation of these mutant proteins (SOD1, TDP-43, dipeptide repeats coded by C9ORF72) supporting its role in the clearance of misfolded proteins (Crippa et al. 2010b, Crippa et al. 2016a, Rusmini et al. 2013, Cristofani et al. 2017). HspB8 also efficiently removes misfolded proteins containing PolyQ tracts, such as polyQ-AR [START_REF] Carra | HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells[END_REF], Carra et al. 2008a[START_REF] Carra | HspB8 participates in protein quality control by a non-chaperone-like mechanism that requires eIF2{alpha} phosphorylation[END_REF], or those involved in Alzheimer's and PD [START_REF] Wilhelmus | Small heat shock protein HspB8: its distribution in Alzheimer's disease brains and its inhibition of amyloid-beta protein aggregation and cerebrovascular amyloid-beta toxicity[END_REF][START_REF] Bruinsma | Inhibition of alpha-synuclein aggregation by small heat shock proteins[END_REF][START_REF] Seidel | The HSPB8-BAG3 chaperone complex is upregulated in astrocytes in the human brain affected by protein aggregation diseases[END_REF]. In most neurodegenerative diseases with alterations of the autophagic pathway the pro-autophagic role of HSPB8 mediated by the enhanced activity of the CASA complex appears to be crucial to prevent misfolded protein accumulation in affected neurons (or surrounding cells).

HSPB8, with BAG3 in the CASA complex mediate the interplay between autophagy and proteasome systems.

It is of note that the CASA complex also serves as routing system of misfolded proteins to autophagy to prevent possible proteasome overwhelming and /or impairment (Figure 2). When dynein mediated retrograde transport is blocked, a condition present in a number of NDs [START_REF] Sau | Dysregulation of axonal transport and motorneuron diseases[END_REF]) (and that we reproduced genetically using siRNAs against dynein or pharmacologically using the selective dynein inhibitor erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA)), the complex HSP70-CHIP cannot bind the HSPB8-BAG3 complex, and the CASA activity is perturbed and cannot dispose of misfolded proteins via autophagy [START_REF] Cristofani | Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases[END_REF]. Under these conditions, specific transcription factors (still unknown)

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This article is protected by copyright. All rights reserved activate the de novo transcription of another nucleotide exchange factor (NEF/BAG) called BAG1. Also BAG1 is capable of selectively binding the complex HSP70/CHIP; but in this case, misfolded proteins are routed to the proteasome instead of autophagy [START_REF] Behl | Breaking BAG: The Co-Chaperone BAG3 in Health and Disease[END_REF][START_REF] Gamerdinger | Emerging roles of molecular chaperones and cochaperones in selective autophagy: focus on BAG proteins[END_REF], Cristofani et al. 2017).

Indeed, BAG1 overexpression facilitates proteasomal removal of ARpolyQ, mutant SOD1 and mutant TDP-43 [START_REF] Cristofani | Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases[END_REF], and this BAG1 activity is fully counteracted by the inhibition of the proteasome activity, but not by inhibition of autophagy [START_REF] Cristofani | Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases[END_REF]. Since, the inhibition of the proteasome also results in the upreguation of both HSPB8 and of BAG3 (Crippa et al. 2010b), while blockage of the delivery of misfolded protein to autophagy results in upregulation of BAG1 (Cristofani et al. 2017), this gives rise to a very nice equilibrium between the proteasome and autophagy selective degradation. The equilibrium is maintained by the ratio BAG3:BAG1, which determines the relative amount of the proteins BAG3 (associated with HSPB8) and BAG1 capable of associating with the dimer HSP70/CHIP and of selecting the proper degradative pathway for a misfolded protein in a given neuron.

HSPB8 protects against misfolded protein toxicity in animal models of ALS.

Few animal model studies have been performed to study the effect of HSPB8 overexpression in NDs.

However, by enhancing the expression of the fly functional ortholog of HSPB8 (HSP67Bc) in two

Drosophila melanogaster models of ALS, we found that HSP67Bc prevents the mislocalization of a neurotoxic mutant TDP-43 protein [START_REF] Ritson | TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97[END_REF]. As a proof of principle, HSP67Bc downregulation resulted in TDP-43 and polyubiquitinated proteins accumulation and worsened the eye phenotype of mutant TDP-43 flies (Crippa et al. 2016a). The HSPB8 fly ortholog also extended survival of the fly ALS models, since it rescued from pupae lethality the flies expressing an ALS-associated 35 kDa TDP-43 fragment (TDP-35) (Crippa et al. 2016a). Surprisingly, no major effects of HSPB8 silencing were observed in a transgenic mouse model with a functional knock-out of HSPB8 which showed motor behaviour performances similar to those of wild-type animals [START_REF] Bouhy | A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8[END_REF], thus suggesting that the proautophagic activity of HSPB8 may be relevant specifically when aberrant proteins are generated in affected cells. Conversely, mice expressing a Charcot-Marie-Tooth disease related mutation of HSPB8 showed clear sign of motor deficits due to degeneration of peripheral nerves, which were accompanied by severe muscle atrophy and protein inclusions [START_REF] Bouhy | A knock-in/knock-out mouse model of HSPB8-associated distal hereditary motor neuropathy and myopathy reveals toxic gain-of-function of mutant Hspb8[END_REF].

On these bases, a large screen aimed to identify commercially available drugs that are able to enhance HSPB8 expression in motor neuronal cells for therapeutic purposes (Crippa et al. 2016b) led to the identification of the drug colchicine as a potent inducer of HSPB8 together with a series of other genes involved in autophagy activation (transcription factor EB, p62, LC3) suggesting that colchicine could represent a useful candidate to be tested in misfolded protein associated NDs (Crippa et al. 2016b). Of note, estrogens are also potent inducers of HSPB8 expression [START_REF] Piccolella | The small heat shock protein B8 (HSPB8) modulates proliferation and migration of breast cancer cells[END_REF], confirming previous

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This article is protected by copyright. All rights reserved published data (Sun et al. 2007), and this might in part explain some of the gender differences described in misfolded protein associated NDs neurodegenerative diseases, including ALS.

IV. Mitochondrial autophagy and beyond in Parkinson's disease.

Parkinson's disease-linked proteins promote mitophagy at the endoplasmic reticulum-mitochondria interface.

Mitophagy is a relatively recent term describing the selective autophagy of mitochondria under specific stress conditions, including during apoptosis induction in the presence of caspase inhibitors, following nutrient deprivation or toxin-induced mitochondrial damage (Tolkovsky et al. 2002[START_REF] Elmore | The mitochondrial permeability transition initiates autophagy in rat hepatocytes[END_REF][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], or during specific developmental programs, such as metabolic reshuffling in the developing heart [START_REF] Gong | Parkinmediated mitophagy directs perinatal cardiac metabolic maturation in mice[END_REF] or red blood cell maturation [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]. Mitophagy became relevant to neurodegeneration when the mitochondrial serine/threonine kinase PINK1 and the ubiquitin-protein ligase Parkin, the products of two genes (PINK1 and PARK2) mutated in autosomal recessive forms of PD, were found to jointly promote the clearance of depolarized mitochondria in cell lines treated with the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) [START_REF] Narendra | Parkin is recruited selectively to impaired mitochondria and promotes their autophagy[END_REF], Narendra et al. 2010[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].

We now know that a number of stress-related stimuli activate this program, including specific inhibitors of the respiratory chain, unfolded protein stress in the mitochondrial matrix, and impairment of the mitochondrial protein import pathway [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][START_REF] Greene | Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment[END_REF][START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance[END_REF], Wang et al. 2011, Barini et al. 2018, Jin et al. 2010[START_REF] Fiesel | Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy[END_REF]). These conditions impair PINK1 import into mitochondria, promoting its accumulation on the outer mitochondrial membrane (OMM), in proximity of the translocase of the outer mitochondrial membrane (TOM) through which the majority of the mitochondrial proteins enter the organelle [START_REF] Lazarou | Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin[END_REF][START_REF] Hasson | High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy[END_REF][START_REF] Okatsu | A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment[END_REF].

This triggers a cascade of events, including the PINK1-dependent recruitment and activation of Parkin, the ubiquitylation of a number of proteins of the OMM, and the recruitment of autophagy receptors and These events are probably concomitant to the separation of the damaged mitochondrion from the rest of the

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This article is protected by copyright. All rights reserved network, operated by the dynamin-related GTPase Drp1 and regulated by the ER-mitochondria interface (Twig et al. 2008[START_REF] Narendra | PINK1 is selectively stabilized on impaired mitochondria to activate Parkin[END_REF][START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF][START_REF] Friedman | ER tubules mark sites of mitochondrial division[END_REF]. Drp1 is cooperatively recruited by PINK1 and Parkin on depolarized mitochondria, suggesting that mitochondrial fission and mitophagy are orchestrated at the ER-mitochondria interface [START_REF] Buhlman | Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance[END_REF][START_REF] Erpapazoglou | The endoplasmic reticulum/mitochondria interface: a subcellular platform for the orchestration of the functions of the PINK1-Parkin pathway?[END_REF].

PINK1 and Parkin keep mitochondria functional by multiple mechanisms.

PINK1 and Parkin jointly regulate various mitochondrial quality control mechanisms in addition to mitophagy: the delivery of damaged mitochondrial components to the lysosome by mitochondria-derived vesicles (MDV), mitochondrial biogenesis and local translation on the OMM of transcripts for nuclearencoded respiratory chain components [START_REF] Mclelland | Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control[END_REF][START_REF] Sugiura | A new pathway for mitochondrial quality control: mitochondrial-derived vesicles[END_REF][START_REF] Gehrke | PINK1 and Parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane[END_REF][START_REF] Shin | PARIS (ZNF746) repression of PGC-1alpha contributes to neurodegeneration in Parkinson's disease[END_REF][START_REF] Lee | PINK1 Primes Parkin-Mediated Ubiquitination of PARIS in Dopaminergic Neuronal Survival[END_REF]) (Figure 3). This latter mechanism requires functional interaction between PINK1 and the TOM complex to target the transcripts to the OMM during cotranslational import [START_REF] Gehrke | PINK1 and Parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane[END_REF]. Considering the proximity of the activated PINK1/Parkin system to the TOM complex [START_REF] Lazarou | Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin[END_REF][START_REF] Hasson | High-content genome-wide RNAi screens identify regulators of parkin upstream of mitophagy[END_REF][START_REF] Okatsu | A dimeric PINK1-containing complex on depolarized mitochondria stimulates Parkin recruitment[END_REF][START_REF] Bertolin | The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance[END_REF], and the increasing importance gained by local translation on the mitochondrial surface (Golani-Armon & Arava 2016), this mechanism may be of broader relevance. Supporting this hypothesis, we found that Parkin participates in maintaining mitochondrial levels of the multifunctional mitochondrial matrix hydroxysteroid dehydrogenase, HSD17B10 [START_REF] Bertolin | Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-beta hydroxysteroid dehydrogenase type 10[END_REF], shown to be depleted in a PD mouse model and PD patients (Tieu et al. 2004). HSD17B10 was co-recruited with Parkin near the TOM complex and PINK1, suggesting regulation of its mitochondrial import by a PINK1/Parkin-dependent mechanism [START_REF] Bertolin | Parkin maintains mitochondrial levels of the protective Parkinson's disease-related enzyme 17-beta hydroxysteroid dehydrogenase type 10[END_REF]. Using an original biosensor for exploring the presequence-mediated protein import pathway in living cells, we recently showed that this process is more generally facilitated by the PINK1/Parkin system [START_REF] Jacoupy | The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells[END_REF].

Dissecting the various mechanisms by which the PINK1/Parkin sensor/effector system maintains mitochondrial quality has highlighted the central role played by mitochondrial protein import in monitoring the mitochondrial functional state. There is a remarkable parallel between the characteristics of the PINK1/Parkin system and a key regulatory component of the mitochondrial unfolded protein response (UPR mt ) in the small invertebrate organism C. elegans, Activated Transcription Factor Associated with Stress 1 (ATFS-1). ATFS-1 is constitutively imported into functional mitochondria by an N-terminal mitochondrial targeting signal, but under mitochondrial stress it translocates to the nucleus to activate a protective transcriptional response mediated by hundreds of genes with various functions, including coping with the accumulation of unfolded proteins in mitochondria, fighting against an excess of reactive oxygen species, and remodelling of cellular metabolism [START_REF] Nargund | Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation[END_REF]. By analogy, it is likely that PINK1

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This article is protected by copyright. All rights reserved and Parkin are more broadly involved in the modulation of the mitochondrial stress response in mammalian cells, considering the well-known cell-protective properties of Parkin [START_REF] Imai | Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity[END_REF][START_REF] Darios | Parkin prevents mitochondrial swelling and cytochrome c release in mitochondria-dependent cell death[END_REF][START_REF] Johnson | The ubiquitin E3 ligase parkin regulates the proapoptotic function of Bax[END_REF][START_REF] Bouman | Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress[END_REF]

Fluorescent reporters with pH-sensitive components illuminate PINK1/Parkin-dependent mitophagy in cultured neurons and in vivo.

We still lack sufficient evidence for a role of PINK1/Parkin-dependent mitophagy in mitochondrial maintenance and neuronal survival in vivo and in the context of PD. The question of whether mitophagy is relevant to neurons has been intensively debated [START_REF] Grenier | Parkin-and PINK1-Dependent Mitophagy in Neurons: Will the Real Pathway Please Stand Up?[END_REF], Van Laar et al. 2011). Studies based on the simultaneous expression in primary neurons of fluorescent reporters targeted to the mitochondrial compartment, the autophagosome and the lysosome have, however, provided evidence in neurons for events of engulfment of mitochondria into autophagosomes, or of fusion of autophagosomes containing mitochondria with lysosomes [START_REF] Cai | Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons[END_REF][START_REF] Ashrafi | Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin[END_REF][START_REF] Hsieh | Functional Impairment in Miro Degradation and Mitophagy Is a Shared Feature in Familial and Sporadic Parkinson's Disease[END_REF]). These events were observed to a lesser degree in neurons from Parkin-and PINK1-deficient mice, supporting their relation to PINK1/Parkin-dependent mitophagy [START_REF] Ashrafi | Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin[END_REF]. More convincingly, mitophagy has been investigated in cultured primary cells, including neurons and in vivo, in Drosophila and mice, using ratiometric or dual-fluorescence reporters with pH-sensitive components for direct tracking of mitochondria in lysosomes [START_REF] Bingol | The mitochondrial deubiquitinase USP30 opposes parkin-mediated mitophagy[END_REF][START_REF] Bonello | LRRK2 impairs PINK1/Parkin-dependent mitophagy via its kinase activity: pathologic insights into Parkinson's disease[END_REF][START_REF] Mcwilliams | mito-QC illuminates mitophagy and mitochondrial architecture in vivo[END_REF][START_REF] Mcwilliams | Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand[END_REF][START_REF] Gehrke | PINK1 and Parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane[END_REF][START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF][START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila[END_REF][START_REF] Shin | Inhibition of bioenergetics provides novel insights into recruitment of PINK1-dependent neuronal mitophagy[END_REF]. These studies have demonstrated the occurrence of mitophagy under physiological conditions in neurons, including the highly vulnerable dopaminergic (DA) neuron in the mouse [START_REF] Mcwilliams | Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand[END_REF]) and fly brains [START_REF] Lee | Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin[END_REF][START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila[END_REF]). However, it remains controversial whether PINK1 and Parkin coregulate this type of physiological mitophagy detected in the absence of exogenous insults, particularly during aging [START_REF] Cornelissen | Deficiency of parkin and PINK1 impairs age-dependent mitophagy in Drosophila[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 | Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand[END_REF], which is recognized today as the major risk factor for PD [START_REF] Collier | Aging and Parkinson's disease: Different sides of the same coin?[END_REF]. Notably, a previous study reported strong immunostaining for

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This article is protected by copyright. All rights reserved phosphorylated ubiquitin partially colocalizing with mitochondria and lysosomes in the aged and PDaffected human brain, but it remains to be clarified whether this reflects impaired or increased mitophagy [START_REF] Fiesel | Patho-)physiological relevance of PINK1dependent ubiquitin phosphorylation[END_REF].

Beyond neurons: a role in innate immunity.

Finally, there is mounting evidence that PINK1 and Parkin jointly regulate immune-related mechanisms, which is of particular interest, considering the neuroinflammatory component in PD [START_REF] Greene | Genetic and genomic studies of Drosophila parkin mutants implicate oxidative stress and innate immune responses in pathogenesis[END_REF][START_REF] Manzanillo | The ubiquitin ligase parkin mediates resistance to intracellular pathogens[END_REF][START_REF] Matheoud | Parkinson's Disease-Related Proteins PINK1 and Parkin Repress Mitochondrial Antigen Presentation[END_REF], Torres-Odio et al. 2017, Sun et al. 2018[START_REF] Kim | Hepatitis C virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence[END_REF][START_REF] Kang | A novel PINK1-and PARK2-dependent protective neuroimmune pathway in lethal sepsis[END_REF][START_REF] Zhong | NF-kappaB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria[END_REF][START_REF] Sumpter | Fanconi Anemia Proteins Function in Mitophagy and Immunity[END_REF], Mouton-Liger et al. 2017[START_REF] Sliter | Type-2 cells as link between glial and neuronal lineage in adult hippocampal neurogenesis[END_REF]. In some cases, these regulatory roles of PINK1 and Parkin have been clearly linked to modulation of mitophagy [START_REF] Kim | Hepatitis C virus triggers mitochondrial fission and attenuates apoptosis to promote viral persistence[END_REF][START_REF] Zhong | NF-kappaB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria[END_REF][START_REF] Sumpter | Fanconi Anemia Proteins Function in Mitophagy and Immunity[END_REF]. Specifically, PINK1/Parkindependent mitophagy has been recognized to counterbalance the action of the nucleotide-binding domainlike receptor protein 3 (NLRP3) inflammasome, a proinflammatory multiprotein complex activated in response to pathogens as well as endogenous signals associated with cell and tissue damage [START_REF] Sumpter | Fanconi Anemia Proteins Function in Mitophagy and Immunity[END_REF][START_REF] Zhong | NF-kappaB Restricts Inflammasome Activation via Elimination of Damaged Mitochondria[END_REF]. This complex plays a central role in a number of inflammatory/autoimmune disorders, and is emerging as key player in neurodegenerative diseases, including PD [START_REF] Labzin | Innate Immunity and Neurodegeneration[END_REF].

Specifically, the neurotransmitter DA has been shown to attenuate NLRP3 inflammasome signalling in immune cells, whereas mitochondrial toxins, such as MPTP or rotenone, exacerbate NLRP3-dependent inflammation, leading to DA neurodegeneration in cell and animal models [START_REF] Yan | Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome[END_REF][START_REF] Sarkar | Mitochondrial impairment in microglia amplifies NLRP3 inflammasome proinflammatory signaling in cell culture and animal models of Parkinson's disease[END_REF]. We have recently reported exacerbation of the NLRP3 inflammasome pathway in bone-marrowderived macrophages and microglial cells from Parkin-and PINK1-deficient mouse models, and confirmed this defect in PD patients with PARK2 mutations (Mouton-Liger et al. 2018).

V. Focus on autophagy as therapeutic target in brain pathologies.

With our improved understanding of the regulation of autophagy, its diverse regulatory mechanisms and a greater awareness that it represents a unique target with multiple therapeutic options for the clearance of toxic aggregates and damaged organelles, there has been an extraordinary rise of interest in its clinical applicability and options for translation (e.g. [START_REF] Feng | Reactive nitrogen species as therapeutic targets for autophagy: implication for ischemic stroke[END_REF], Tramutola et al. 2017) [START_REF] Lipinski | Function and Mechanisms of Autophagy in Brain and Spinal Cord Trauma[END_REF], Rubinsztein et al. 2015[START_REF] Maiese | Targeting molecules to medicine with mTOR, autophagy and neurodegenerative disorders[END_REF][START_REF] Boland | Promoting the clearance of neurotoxic proteins in neurodegenerative disorders of ageing[END_REF]. Indeed these reviews are pertinent to topics as different in pathology as Alzheimer's disease and traumatic brain injury, reflecting the wide applicability of such therapeutic options. A seminal publication that initiated cognisance of this area, indeed a veritable tour de force, overviewed multiple classes of drugs and potential molecular targets regulating autophagic mechanisms [START_REF] Rubinsztein | Potential therapeutic applications of autophagy[END_REF].

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We became aware of the FDA approved drug rilmenidine, considered to act via a mTOR-independent mechanism, after it was reported to have beneficial actions in a mouse model of Huntington's disease [START_REF] Rose | Rilmenidine attenuates toxicity of polyglutamine expansions in a mouse model of Huntington's disease[END_REF]. At the time we were working on 3,4-methylene-dioxymethamphetamine (ecstasy) induced injury of serotonin (5-HT) neurons, and (i) since autophagy was considered to be involved in the brain's response to amphetamines [START_REF] Galluzzi | Autophagy in acute brain injury[END_REF], and (ii) rilmenidine binding sites exist in 5-HT raphe nuclei [START_REF] King | Quantitative autoradiographic localization in rat brain of alpha 2-adrenergic and non-adrenergic I-receptor binding sites labelled by [3H]rilmenidine[END_REF], the inclusion of rilmenidine in our study was an obvious step forward. Indeed ecstasy-induced neurotoxicity of 5-HT neurons involved an autophagic component and rilmenidine completely blocked the toxicity of ecstasy [START_REF] Mercer | MDMA-induced neurotoxicity of serotonin neurons involves autophagy and rilmenidine is protective against its pathobiology[END_REF]. Thus this FDA-approved molecule, subsequently documented to be well-tolerated and suitable for human usage (Underwood et al. 2017), likely also possesses a profile suitable for use in drug abuse. Success here led to our further studies exploring the potential of rilmenidine in cellular and animal models of MND. We found beneficial effects on rilmenidine in a MN cell line and in MNs derived from human stem cells expressing mutant superoxide dismutase 1 (SOD1): there was an increase in autophagic flux following treatment, as assessed by monitoring LC3 lipidation levels and abundance of autolysosomes, using a tandem mCherry-GFP-LC3B reporter construct, accompanied by a 20% reduction in the load of mutant SOD1 [START_REF] Perera | Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression[END_REF].

However, we were disappointed at its lack of effectiveness in the SOD1 G93A mouse, where it actually worsened the pathological and functional outcome [START_REF] Perera | Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression[END_REF]. What our in vivo study does highlight is that rilmenidine was certainly efficacious enhancing autophagic flux, but that there was a concurrent accumulation of misfolded SOD1, and mitochondrial depletion with autophagosome accumulation in MNs. In ongoing work in a transgenic mouse model overexpressing the normal (WT) and ALS-linked Q331K variants of the human TDP-43 protein in neurons (TDP-43 WTxQ331K mice) [START_REF] Mitchell | Wild type human TDP-43 potentiates ALS-linked mutant TDP-43 driven progressive motor and cortical neuron degeneration with pathological features of ALS[END_REF], we have found similar findings with rilmenidine with reduced TDP-43 immunoreactivity in MNs (Supplementary Fig. 1), so moving forward it will be interesting to determine whether the enhanced auto-/mitophagic flux found with rilmenidine (Perera et al. 2017) will prove useful in other TDP-43 models. Given the positive outcomes here rilmenidine may prove effective in less aggressive forms of MND and certainly a key issue is that the timepoint of drug intervention may well be critical relative to toxic aggregate load and stage of disease progression. Indeed, our evidence for activation of autophagy in TARDBP in cell culture models, when taken the finding that rapamycin produces benefits in a mouse TARDBP model [START_REF] Wang | Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNAbinding protein 43[END_REF], strongly suggests that autophagy is likely to be a valid target in MND.

Although toxic aggregates and dysfunctional proteostasis [START_REF] Yerbury | Walking the tightrope: proteostasis and neurodegenerative disease[END_REF] have been linked to diverse proteins in MND, emergent reports increasingly suggest that successful management may be possible by targeting autophagic signalling [START_REF] Deng | Is amyotrophic lateral sclerosis/frontotemporal dementia an autophagy disease?[END_REF]. For example, recent elegant work screening clinically approved drugs revealed a number of small molecules with beneficial actions using FUS-expressing stem cells and a Drosophila model [START_REF] Marrone | Isogenic FUS-eGFP iPSC Reporter Lines Enable Quantification of FUS Stress Granule Pathology that Is Rescued by Drugs Inducing Autophagy[END_REF].

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In addition to the current great interest in potentially clinically effective small molecules, numerous strategies, including gene therapy, RNA interference or antisense oligonucleotides, continue to attract attention focusing on for example mTOR-dependent and-independent pathways, and broadly on signalling linked to Beclin1, Atg5, AMP-dependent protein kinase, PICALM and histone deacetylases [START_REF] Shoji-Kawata | Identification of a candidate therapeutic autophagy-inducing peptide[END_REF][START_REF] Hu | Up-regulation of autophagy-related gene 5 (ATG5) protects dopaminergic neurons in a zebrafish model of Parkinson's disease[END_REF][START_REF] Menzies | Autophagy and Neurodegeneration: Pathogenic Mechanisms and Therapeutic Opportunities[END_REF][START_REF] Rahman | Therapeutic implication of autophagy in neurodegenerative diseases[END_REF]). Although at the present time subsets of such published data are relatively small as they relate to neurodegeneration and human brain pathologies, there continue to be rapid developments in this area with new advances in our understanding of the regulation and dynamics of autophagic flux, and its interface with the ubiquitin proteasome, as highlighted by the preceding sections.

Conclusions and perspectives.

The examples discussed in this review are representative of a growing body of literature showing how autophagy has not only physiological roles in neurodevelopment and the maintenance of neuronal homeostasis, but also different levels of involvement in pathological processes affecting the brain, including neurodegenerative disorders and stroke. As we have seen here, a set of observations involve dysfunction of selective types of autophagy in specific neurodegenerative disease. For example, defective CASA has been linked to motor neuron disorders such as ALS and SBMA and mitophagy is suspected to be impaired in specific forms of Parkinson's disease, as well as in other neurodegenerative diseases, according to studies not discussed in this review [START_REF] Wong | Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation[END_REF][START_REF] Cai | Mitophagy in Alzheimer's Disease and Other Age-Related Neurodegenerative Diseases[END_REF]. In other pathological conditions of the brain, autophagy appears to be uncontrollably activated and to cause neuronal death, as in the case of perinatal asphyxia. From an assessment of the current state of knowledge, it is clear that we are still some distance from a holistic understanding of how autophagy is recruited in different brain diseases and in which cases it is merely associated with cell death rather than mediating or even causing cell death [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[END_REF]. Thus, we still need appropriate in vivo documentation of the various forms of autophagy in different disease contexts and across different disease stages, as well as their relationship to different forms of cell death to fully appreciate whether and when they play beneficial or detrimental roles. Findings presented herein argue that steps to take advantage of the beneficial modulation of autophagy by pharmacological strategies, and the specific modalities of such strategies, are likely to be quite different depending on the age of the affected individual, the type of injury/disease, the specific stage of progression, as well as the overall duration and severity of the disease. For instance, it has already been reported in the SOD1 G93A mouse model of MND that short-term treatment with clemastine is beneficial, but long-term treatment fails to alleviate disease [START_REF] Apolloni | Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression[END_REF]. As an additional take-home message, we need to better understand

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This article is protected by copyright. All rights reserved the complex interplay between autophagy and other intracellular processes that may also at some point be recruited by the pathological process, either as contributing factors or protective responses. As illustrated with PINK1 and Parkin, many proteins involved in autophagy and its regulation have multiple functions in the cell, and these have to be taken into consideration if we want to explore meaningful therapeutic avenues for neurological diseases. The case of CASA, on the other hand, illustrates the interplay between autophagy and the proteasome system, leaving room for envisaging strategies aimed at promoting alternative degradation routes when autophagy is impaired. Finally, while autophagy and mitophagy are emerging as central modulators of inflammatory and immune-related mechanisms, which are recognized as a component of the pathological process in various brain diseases, work discussed here also shows that the selective modulation of autophagy in neurons has broader impact on brain tissue homeostasis. Therefore, a better knowledge of the different cell types affected by the disease process and their complex interactions will provide invaluable insight into the most appropriate ways of using autophagy modulation for therapeutic purposes in the brain.

--Human subjects --

Figure 2

The HSC70/CHIP-mediated routing system between proteasome and autophagy. BAG1 is a nucleotide exchange factor which is capable to complex the chaperone HSC70 and E3 ubiquitine ligase CHIP. This complex recognize monomeric misfolded proteins to be driven to proteasome degradation.

Alternatively, misfolded monomeric, oligomeric and/or aggregated misfolded proteins can be recognized by another nucleotide exchange factor BAG3 associated to the small HSP named HSPB8. Once the complex HSPB8/BAG3 recognizes target misfolded proteins, the entire complex can associate to the HSC70/CHIP complex, thus sequestering it from the BAG1 binding. The HSPB8/BAG3/HSC70/CHIP complex has been named the CASA complex, since it mediates a peculiar form of autophagy known as "Chaperone-Assisted Selective Autophagy (CASA)". In this case misfolded aggregating proteins, bound to the CASA complex, are routed by a dynein-dependent transport to the microtubule organization center (MTOC) where aggresomes are formed and inserted into nascent autophagosomes. The HSC70/CHIPmediated routing system thus generates a delicate equilibrium which is responsible for the choice of two alternative pathways for damaged/misfolded protein degradation. Of note, the blockage of the dyneinmediated transport of the CASA complex correlates which enhanced BAG1 expression. Conversely, proteasome inhibition results in HSPB8 and BAG3 overexpression. This transcriptionally regulated expression of the routing system factors maintains the fine tuned equilibrium between proteasome and autophagy degradation in cells.

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This figure was drawn using illustrations adapted from the image bank of Servier Medical Art (Cellular Biology; https://smart.servier.com/). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License. https://creativecommons.org/licenses/by/3.0/.

Mechanism of execution

Disposal of cell corpses

• Phagocytic uptake and lyososmal degradation

  upstream autophagy-related protein to prime mitochondria for mitophagy (reviewed by[START_REF] Sekine | PINK1 import regulation; a fine system to convey mitochondrial stress to the cytosol[END_REF], Truban et al. 2017[START_REF] Mcwilliams | PINK1 and Parkin: emerging themes in mitochondrial homeostasis[END_REF]. These events occur in proximity of the interface between mitochondria and the endoplasmic reticulum (ER)[START_REF] Yang | Bit-by-bit autophagic removal of parkin-labelled mitochondria[END_REF][START_REF] Gelmetti | PINK1 and BECN1 relocalize at mitochondriaassociated membranes during mitophagy and promote ER-mitochondria tethering and autophagosome formation[END_REF]), a subcellular compartment increasingly involved in neurodegeneration (reviewed by[START_REF] Erpapazoglou | From dysfunctional endoplasmic reticulummitochondria coupling to neurodegeneration[END_REF]). This interface is perturbed in cells from Parkin-deficient mice and patients with PARK2 mutations, due to accumulation of the Parkin substrate and ER-mitochondria tethering protein Mitofusin 2 (Mfn2)[START_REF] Gautier | The endoplasmic reticulum-mitochondria interface is perturbed in PARK2 knockout mice and patients with PARK2 mutations[END_REF]. Ubiquitylation of Mfn2 by Parkin, disassembly of Mfn2 complexes and dissociation of mitochondria from the ER are instrumental for the initiation of mitophagy[START_REF] Mclelland | Mfn2 ubiquitination by PINK1/parkin gates the p97dependent release of ER from mitochondria to drive mitophagy[END_REF].

  , and the fact the PINK1/Parkin system is induced during the UPR mt[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]. Establishing the consequence of PINK1 and Parkin dysfunction on the mitochondrial stress response, and investigating the possibility that these proteins coactivate transcription factors near dysfunctional mitochondria, are major challenges for future research. Activating Transcription Factor 5 (ATF5) may mediate the UPR mt response in mammals, but whether this transcription factor interacts with the PINK1/Parkin system is unknown(Fiorese et al. 2016). Notably, PINK1 and Parkin contribute to the activation of transcription factors of the microphtalmia/transcription factor E (MiT/TFE) family during mitophagy, including the master regulator of lysosomal biogenesis, transcription factor EB (TFEB)[START_REF] Nezich | MiT/TFE transcription factors are activated during mitophagy downstream of Parkin and Atg5[END_REF]. Morover Parkin acts as transcription factor itself, although the precise mechanisms regulating its nuclear translocation and transcriptional activity remain to be determined[START_REF] Da Costa | Transcriptional repression of p53 by parkin and impairment by mutations associated with autosomal recessive juvenile Parkinson's disease[END_REF][START_REF] Alves Da Costa | Parkin: much more than a simple ubiquitin ligase[END_REF].

Figure 3

 3 Figure 3

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Tab le 1: Characteristic features of autosis comparatively to other forms of cell death Autosis Autophagy-dependent cell death Apoptosis Necrosis

	Morphological features	•	Nuclear shrinkage; focal separation of inner and outer nuclear membranes	• •	Nuclear compaction and fragmentation Marked chromatin condensation`	•	Swelling of organelles
			with focal expansion of perinuclear
		• •	space Extensive cytoplasmic vacuolization Enhanced cell-substrate adhesion	• •	Cell shrinkage, membrane blebbing (apoptotic bodies) Cell rounding up, detachment from	•	Cell swelling
					substrate
	Molecular markers			•	DNA fragmentation (laddering)

Acknowledgements and conflict of interest disclosure

Accepted Article

This article is protected by copyright. All rights reserved Philip M. Beart is the ISN Historian and a former Editor of the Journal of Neurochemistry. Philip M. Beart was supported in part by a NHMRC Research Fellowship (APP1019833) and by NHMRC Grants APP509217 and APP1104295, and thanks collaborators Drs Nirma Perera and Bradley Turner from the provision of the photomicrographs shown in Supplementary Figure 1. The Florey Institute of Neuroscience and Mental Health receives infrastructure support from the Victorian State Government (Australia). Olga Corti received support from Institut national de la santé et de la recherche médicale (INSERM), Fondation Institut du Cerveau et de la Moelle épinière and Agence Nationale pour la Recherche ("Investissements d'avenir," grant ANR-10-IAIHU-06), Innovative Medicines Initiative Joint Undertaking under grant agreement n•115568, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' kind contribution, Fondation de France (Grants ID Engt 2012-00034508 and 2016-00066513) and the Michael J. Fox Foundation (Target Validation Awards Spring 2016 Program, Grant ID12095). Angelo Poletti is an editor for Journal of Neurochemistry. Angelo Poletti acknowledges support from Fondazione Telethon, Italy (GGP14039), Fondazione Cariplo Italy (2014-0686), Fondazione AriSLA Italy (ALS-HSPB8., ALS-Granulopathy, MLOpathy, Target-RAN), Association Française contre les Myopathies France (AFM Telethon 16406), Fondazione Regionale per la Ricerca Biomedica (Regione Lombardia, TRANS-ALS, 2015-0023) Italy, Italian Ministry of University and Research (MIUR) PRIN -Progetti di ricerca di interesse nazionale (2015LFPNMN ), Agenzia Italiana del Farmaco (AIFA) (Co-ALS ), EU Joint Programme -Neurodegenerative Disease Research project funded by European Union's Horizon 2020 Research and Innovation Programme (643417, Grant ID: 01ED1601A, CureALS) and MIUR Progetto Dipartimenti di Eccellenza. Klas Blomgren acknowledges support from the Swedish Childhood Cancer Found, the Swedish Research Council, the Swedish Cancer Foundation, the Stockholm County Council, the Frimurare Barnhus Foundation of Stockholm, the Märta and Gunnar V. Philipson Foundation, the Swedish Brain Foundation and the Swedish Radiation Safety Authority.

Accepted Article

This article is protected by copyright. All rights reserved Sun, L., Shen, R., Agnihotri, S. K., Chen, Y., Huang, Z. and Bueler, H. (2018) Lack of PINK1 alters glia innate immune responses and enhances inflammation-induced, nitric oxide-mediated neuron death. Sci Rep, 8, 383. Sun, N., Yun, J., Liu, J. et al. (2015) Measuring In Vivo Mitophagy. Mol Cell, 60, 685-696. Sun, X., Fontaine, J. M., Bartl, I., Behnam, B., Welsh, M. J. andBenndorf, R. (2007) Induction of Hsp22 (HspB8) by estrogen and the metalloestrogen cadmium in estrogen receptor-positive breast cancer cells.

Cell Stress Chaperones, 12, 307-319. Tanaka, A., Cleland, M. M., Xu, S., Narendra, D. P., Suen, D. F., Karbowski, M. and Youle, R. J. ( 2010

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