Guiliana Soraya

Chiara Zurzolo
email: zurzolo@pasteur.fr

The spread of prion-like proteins by lysosomes and tunneling nanotubes: Implications for neurodegenerative diseases

Keywords: Aβ, amyloid β, AD, Alzheimer's disease, APP, amyloid precursor protein, ESC RT, endosomal sorting complexes required for transport, HD, Huntington's disease, MVB, multivesicular body, ND, neurodegenerative disease, PD, Parkinson's disease, PMD, protein-misfolding disease, polyQ, polyglutamine, ROS, reactive oxygen species, TFEB, transcription factor EB, TNT, tunneling nanotube, UPR, unfolded protein response

Introduction

The pathological hallmark of a number of neurodegenerative diseases (NDs) is the deposition of protein aggregates in the brain into intracellular or extracellular inclusions. In the case of prion diseases, as well as in Alzheimer's disease (AD), Huntington's disease (HD), and Parkinson's disease (PD), distinct unrelated proteins (prion protein, amyloid precursor protein [APP] and tau, huntingtin [Htt], and α-synuclein, respectively) undergo this process yet have very different conformations, subcellular localizations, and functions [START_REF] Brundin | Prion-like transmission of protein aggregates in neurodegenerative diseases[END_REF]. Upon adoption of the misfolded conformation, these proteins nonetheless share a β-sheet-rich tertiary structure, which renders them prone to the formation of intermediate oligomeric species and higher-order fibrillar aggregates. Protein aggregate accumulation is linked with fatal neuronal dysfunction and toxicity through various mechanisms, though propagation and toxicity may be mediated in distinct phases [START_REF] Sandberg | Prion propagation and toxicity in vivo occur in two distinct mechanistic phases[END_REF]. The nature of the multimeric aggregate species responsible for toxicity is debated; several studies suggest that oligomeric species are more toxic than fibrils for many amyloid proteins [START_REF] Guo | Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases[END_REF] although at least for α-synuclein, this is contested [START_REF] Pieri | Fibrillar α-synuclein and huntingtin exon 1 assemblies are toxic to the cells[END_REF]. Conversely, larger aggregate depositions, such as those in plaques or Lewy bodies, are suggested to be protective to the cell, acting as a sink in which toxic species are held in stable, less toxic formations [START_REF] Verma | Toxic species in amyloid disorders: Oligomers or mature fibrils[END_REF].

These diseases are collectively referred to as proteinmisfolding diseases (PMDs), of which prion diseases are the best characterized [START_REF] Prusiner | Biology and genetics of prions causing neurodegeneration[END_REF]. The term "prion" was coined by [START_REF] Prusiner | Novel proteinaceous infectious particles cause scrapie[END_REF] in the seminal paper identifying the cause of scrapie to denote a proteinaceous infectious particle that was the scrapie-causative protein agent (i.e., misfolded PrP Sc or prion protein). Moreover, although the chemical identity and the topology of the different proteins involved in PMDs are distinct, they are termed "prion-like proteins," "prionoids," or simply "prions" in recognition of the fact that they share several features with prion protein [START_REF] Prusiner | Biology and genetics of prions causing neurodegeneration[END_REF][START_REF] Aguzzi | Cell biology of prions and prionoids: A status report[END_REF]. These features include the ability to self-propagate by inducing the misfolding of native self-molecules and to spread intercellularly in a non-cell-autonomous fashion along the central nervous system. There is, however, much debate about whether these other proteins at the origin of PMDs are true prions [START_REF] Prusiner | Biology and genetics of prions causing neurodegeneration[END_REF][START_REF] Aguzzi | Cell biology of prions and prionoids: A status report[END_REF]Sanders et al., 2016). On one hand, they are prions in the sense that they have the capability to propagate misfolding (see next section), yet on the other hand, they are not, because whereas the prion protein (PrP Sc ) causes infectious (i.e., transmissible) diseases, most PMDs are not, sensu stricto, infectious. We adopt here the term "prion-like" to refer both to the proteins and to the respective diseases. Although this term is not limited to the proteins involved in AD, PD, and HD (Table 1), in this review, we summarize current knowledge of the prion "likeness" of misfolded Tau, amyloid β (Aβ), Htt, and α-synuclein and the mechanisms by which their intercellular transfer is mediated. We specifically focus on the relevance of two common pathways shared by these misfolded proteins that may have important roles in prion-like diseases: tunneling nanotubes (TNTs) and lysosomes (Abounit et al., 2016a,b;[START_REF] Rustom | The missing link: Does tunnelling nanotube-based supercellularity provide a new understanding of chronic and lifestyle diseases?[END_REF]. We furthermore propose a universal role for TNTs, a novel mechanism of intercellular communication, in the spreading of prion-like proteins Progression of pathology in neurodegenerative diseases is hypothesized to be a non-cell-autonomous process that may be mediated by the productive spreading of prion-like protein aggregates from a "donor cell" that is the source of misfolded aggregates to an "acceptor cell" in which misfolding is propagated by conversion of the normal protein. Although the proteins involved in the various diseases are unrelated, common pathways appear to be used for their intercellular propagation and spreading. Here, we summarize recent evidence of the molecular mechanisms relevant for the intercellular trafficking of protein aggregates involved in prion, Alzheimer's, Huntington's, and Parkinson's diseases. We focus in particular on the common roles that lysosomes and tunneling nanotubes play in the formation and spreading of prion-like assemblies.

in PMDs and highlight how the endolysosomal pathway is involved in the generation of and, potentially, the propagation of these aggregates via TNTs.

The prion-like nature of NDs

Seeding. The phenomenon of infectious template-mediated protein conversion is well described for prion diseases, a group of neurodegenerative diseases that were first considered unique because of their transmissibility across individuals and species. The causative agent, cellular prion protein PrP C , misfolds to the disease-related form PrP Sc , which is capable of inducing the further conversion of naive molecules of PrP C , resulting in a pool of potentially toxic PrP Sc molecules that form ordered oligomeric or fibrillar aggregates. These PrP Sc oligomers and aggregates may act as "seeds" for further conversion of naive PrP C [START_REF] Prusiner | Biology and genetics of prions causing neurodegeneration[END_REF]. The intercellular transmission of these misfolded aggregates is believed to be responsible for seeding the further conversion of naive PrP C molecules in neighboring cells, thus contributing to the overall propagation of disease.

This ability to seed conversion is at the heart of the intrinsic nature of prions [START_REF] Prusiner | Biology and genetics of prions causing neurodegeneration[END_REF], but is not restricted to the prion protein [START_REF] Hasegawa | Prion-like mechanisms and potential therapeutic targets in neurodegenerative disorders[END_REF]. When recombinant α-synuclein fibrils were exogenously supplied to cells overexpressing soluble α-synuclein, the authors observed the recruitment and conversion of soluble α-synuclein into Lewy body-like inclusions, the hallmark of PD [START_REF] Luk | Exogenous α-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells[END_REF]. This was later confirmed to occur in vivo in both wild type and transgenic human α-synuclein-expressing mice (Luk et al., 2012a,b;[START_REF] Tyson | Sorting out release, uptake and processing of α-synuclein during prion-like spread of pathology[END_REF]. Evidence also exists showing that tau and Aβ, whose misfolding and aggregation leads to protein tangles and plaques in AD and other tauopathies, can seed conversion [START_REF] Hasegawa | Prion-like mechanisms and potential therapeutic targets in neurodegenerative disorders[END_REF].

In the case of HD, the genetic expansion of a polyglutamine (polyQ) stretch in the N terminus of the Htt protein, to >35 residues, confers on it the tendency to aggregate and is causative of the disease. Aggregative tendency, and thereby disease progression, are related to the length of the repeat. Mutant Htt (mHtt) fragments as well as polyQ peptides were also shown, upon uptake, to seed conversion of a soluble Htt reporter in cells [START_REF] Jansen | Visualization of prion-like transfer in Huntington's disease models[END_REF].

The propensity of the misfolded proteins involved in neurodegenerative disorders to induce a chain reaction of conversion and aggregation of naive molecules seems therefore to be a shared property, regardless of primary and secondary structures, protein topology, and their subcellular localization [START_REF] Aguzzi | Cell biology of prions and prionoids: A status report[END_REF]. However, how specifically this conversion occurs is not clear. Furthermore, because of low solubility and heterogeneous aggregate sizes, the detailed atomic structure of the isoform that can seed the misfolding of the normal conformer counterpart is still unknown for many NDs [START_REF] Riek | The activities of amyloids from a structural perspective[END_REF]. Although a predominant Aβ40 fibrillar form was identified from brains of prolonged duration AD clinical subtypes [START_REF] Qiang | Structural variation in amyloid-β fibrils from Alzheimer's disease clinical subtypes[END_REF], more heterogeneity was observed for rapid forms of the disease. More in vitro studies are thus necessary to understand how, at the molecular level, one protein conformer induces molecules of another conformer of the same protein to refold into the structure of the first. Key topics for exploration include where this occurs inside the cell and what the molecular determinants are in terms of protein sequence and the need for protein and/ or lipid chaperones [START_REF] Gorbenko | The role of lipid-protein interactions in amyloid-type protein fibril formation[END_REF][START_REF] Surewicz | Prion protein and its conformational conversion: A structural perspective[END_REF].

Intercellular spreading. The pathological progression of neurodegenerative diseases is associated with the presence of misfolded aggregated proteins (and associated damage) in an expanding area of the brain, raising the question of cell-tocell transfer. Evidence for intercellular transfer has accumulated for many prion-like proteins (Costanzo and Zurzolo, 2013;[START_REF] Pecho-Vrieseling | Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons[END_REF][START_REF] Calafate | Synaptic contacts enhance cell-to-cell tau pathology propagation[END_REF]Abounit et al., 2016b). Although the underlying mechanisms are not well understood, it is nonetheless clear that there are commonalities in the mechanisms of trafficking of these proteins along neuronal pathways. Furthermore, attempts have been made, using different cell types in vitro, to understand the role of different cells in the progression of the pathology in the brain.

Prions. Cell-to-cell transmission of infectious PrP Sc aggregates has been demonstrated to occur between neuronal cells [START_REF] Kanu | Transfer of scrapie prion infectivity by cell contact in culture[END_REF][START_REF] Gousset | Prions hijack tunnelling nanotubes for intercellular spread[END_REF][START_REF] Zhu | Prion aggregates transfer through tunneling nanotubes in endocytic vesicles[END_REF] as well from neurons to astrocytes, suggesting a role for glia in clearance [START_REF] Victoria | Astrocyteto-neuron intercellular prion transfer is mediated by cell-cell contact[END_REF]. However, astrocytes are very susceptible to prion infection upon uptake of exogenously supplied PrP Sc seed [START_REF] Cronier | Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death[END_REF] and, further, propagate infectivity to neurons [START_REF] Cronier | Endogenous prion protein conversion is required for prion-induced neuritic alterations and neuronal death[END_REF] as well as other naive astrocytes. Astrocytes may therefore be a double-edged sword in disease propagation [START_REF] Victoria | Astrocyteto-neuron intercellular prion transfer is mediated by cell-cell contact[END_REF]. Microglia are activated by neuronal and astrocytic infection [START_REF] Marella | Neurons and astrocytes respond to prion infection by inducing microglia recruitment[END_REF], and their depletion results in disease exacerbation, presumably as their phagocytic role is lost [START_REF] Zhu | A neuroprotective role for microglia in prion diseases[END_REF]. On the other hand, oligodendrocytic cells neither show susceptibility to prions nor seem capable of transferring prion infectivity [START_REF] Prinz | Intrinsic resistance of oligodendrocytes to prion infection[END_REF], though the implications of this resistance remain unclear. [START_REF] Polymenidou | Prion-like spread of protein aggregates in neurodegeneration[END_REF] Yes [START_REF] Feiler | TDP-43 is intercellularly transmitted across axon terminals[END_REF] FUS Yes [START_REF] Polymenidou | Prion-like spread of protein aggregates in neurodegeneration[END_REF] SOD Yes [START_REF] Polymenidou | Prion-like spread of protein aggregates in neurodegeneration[END_REF] Yes [START_REF] Pokrishevsky | TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion[END_REF] Although amyotrophic lateral sclerosis is not discussed in this review, evidence suggests that proteins involved in amyotrophic lateral sclerosis are prion-like. Evidence for seeding and transmissibility of other proteins are described in detail in the text. CJD, Creutzfeldt-Jakob disease; DLB, dementia with Lewy body; FFI, fatal familial insomnia; MSA, multiple system atrophy; PAF, pure autonomic failure; SOD, superoxide dismutase; vCJD, variant Creutzfeldt-Jakob disease.

lysosomes and TNTs in prion-like protein transfer • Victoria and Zurzolo

Different mechanisms have been implicated in intercellular PrP Sc transfer (Fig. 1). Infectivity from primary astrocytes and neuronal cell lines can be released into the tissue culture medium through exosomes or other secretory mechanisms [START_REF] Fevrier | Cells release prions in association with exosomes[END_REF][START_REF] Cronier | Endogenous prion protein conversion is required for prion-induced neuritic alterations and neuronal death[END_REF][START_REF] Arellano-Anaya | Prion strains are differentially released through the exosomal pathway[END_REF]. Exosomes are formed as intraluminal vesicles within multivesicular bodies (MVBs) and are released to the cell exterior by fusion of MVBs with the plasma membrane [START_REF] Colombo | Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles[END_REF]. They have been implicated in the spreading of many prion-like proteins [START_REF] Quek | The role of extracellular vesicles in neurodegenerative diseases[END_REF]. Both PrP C and PrP Sc have been found in exosomes, and exosomes are capable of inducing infection when applied on cells or injected into mouse brains [START_REF] Fevrier | Cells release prions in association with exosomes[END_REF]. However, it is as yet unclear how efficient this is as a mode of prion propagation, as exosome-mediated infection requires exosomes to be purified and highly concentrated [START_REF] Fevrier | Cells release prions in association with exosomes[END_REF][START_REF] Coleman | Prion-infected cells regulate the release of exosomes with distinct ultrastructural features[END_REF], and not all cell types can be infected with conditioned medium from infected cells [START_REF] Kanu | Transfer of scrapie prion infectivity by cell contact in culture[END_REF]. The efficiency of packaging into exosomes can also be prion-strain dependent [START_REF] Arellano-Anaya | Prion strains are differentially released through the exosomal pathway[END_REF]. The mechanisms of exosome uptake and effect on acceptor cells [START_REF] Mckelvey | Exosomes: Mechanisms of uptake[END_REF] are furthermore unclear in this context. Furthermore, work on primary infected astrocytes suggest that although prion infectivity can be released through secretion, the efficiency of neuronal infection via this mode is much lower than when cell-cell contact is allowed [START_REF] Cronier | Endogenous prion protein conversion is required for prion-induced neuritic alterations and neuronal death[END_REF][START_REF] Victoria | Astrocyteto-neuron intercellular prion transfer is mediated by cell-cell contact[END_REF]. In this context, axonal transport and synaptic transfer have also been hypothesized in neurons [START_REF] Shearin | Axonal and transynaptic spread of prions[END_REF]. Furthermore, as described in the next section, TNT-mediated transfer, which depends on direct cell-cell contact, has been shown to have an important role in the intercellular spreading of PrP Sc [START_REF] Gousset | Prions hijack tunnelling nanotubes for intercellular spread[END_REF].

Aβ. Aβ are amyloid fragments resulting from the proteolytic cleavage of the amyloid precursor protein (APP) by the β and γ secretase. They accumulate, mainly extracellularly, in amyloid plaques in AD [START_REF] De Strooper | The cellular phase of Alzheimer's disease[END_REF]. Importantly, a recent seminal study reported that although a less toxic version (Aβ40) can be released extracellularly, the production of a large intracellular pool of the toxic Aβ42 is restricted to late endosomes and lysosomes because of the localization of the secretase enzyme [START_REF] Sannerud | Restricted location of PSEN2/γ-secretase determines substrate specificity and generates an intracellular Aβ pool[END_REF], highlighting the importance of intracellular trafficking on the fate and toxicity of the protein.

Several studies have reported the occurrence of intercellular spreading of Aβ. Fluorescent oligomeric Aβ microinjected into hippocampal neurons spread to neighboring neurons as well as astroglia in a manner that did not appear to correspond to simple diffusive dissemination and might be cell-cell contact mediated [START_REF] Nath | Spreading of neurodegenerative pathology via neuron-to-neuron transmission of β-amyloid[END_REF]. The authors reported that neuritic connections appeared to mediate interneuronal transfer and that oligomeric Aβ appeared to travel within these neurites in granules that moved at speeds similar to lysosomes and late (1) Synaptic transfer. Misfolded proteins (red) such as mHtt, Tau, and α-synuclein, which have been shown to bind to synaptic vesicles (green), may be released from cells through synaptic vesicle fusion with the plasma membrane and thereafter be taken up by the postsynaptic neuron. (2) TNTs. TNTs have been implicated in the transfer of prion, mHtt, Aβ, tau, and α-synuclein. (3) Phagocytosis. The engulfment of aggregate-containing damaged/dying cells or synaptic terminals by glial cells may represent a mechanism that propagates transfer between cells of different types and has been demonstrated for mHtt and Aβ. This process may also occur for extracellular naked or exosomally localized aggregates in close apposition to the engulfing cell. ( 4) Exocytosis of exosomes. The fusion of MVBs with the plasma membrane may release exosomes containing intraluminal aggregate-containing vesicles (exosomes) that can be internalized by neighboring cells.

(5) Lysosomal exocytosis. Fusion of lysosomes (yellow) with the plasma membrane can release naked aggregates (such as Aβ) into the extracellular space that may form plaques or be taken up by neighboring cells.

endosomes [START_REF] Nath | Spreading of neurodegenerative pathology via neuron-to-neuron transmission of β-amyloid[END_REF]. Aβ peptides were also released in exosomes [START_REF] Rajendran | Alzheimer's disease β-amyloid peptides are released in association with exosomes[END_REF], and perturbations in lysosomal function increased its presence in MVBs [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF], suggesting that this organelle could be a sorting point for the eventual fate of aggregated proteins, promoting either their degradation through fusion with lysosomes or their secretion through fusion with the plasma membrane releasing exosomes. Interfering with the endosomal sorting complexes required for transport (ESC RT) machinery, which is involved in MVB biogenesis [START_REF] Mckelvey | Exosomes: Mechanisms of uptake[END_REF] can tip the balance between intracellular accumulation of Aβ and its exosomal release through MVBs. However, the role of ESC RT-mediated sorting of APP in MVBs remains unclear. Perturbing MVB sorting of APP via disruption of interactions with early ESC RT components, (e.g., Hrs and Tsg101) are reported both to increase secretion of Aβ [START_REF] Morel | Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system[END_REF] as well as decrease it [START_REF] Choy | Amyloid precursor protein (APP) traffics from the cell surface via endosomes for amyloid β (Aβ) production in the trans-Golgi network[END_REF][START_REF] Edgar | ESC RTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-β accumulation[END_REF], whereas depletion of the late ESC RT protein CHMP-6 increases Aβ secretion [START_REF] Choy | Amyloid precursor protein (APP) traffics from the cell surface via endosomes for amyloid β (Aβ) production in the trans-Golgi network[END_REF].

Studies have also suggested that astrocytes can take up external Aβ plaques, possibly through phagocytic mechanisms [START_REF] Wyss-Coray | Adult mouse astrocytes degrade amyloid-β in vitro and in situ[END_REF], suggesting a role for these cells, as well as for microglia, in Aβ clearance. Oligodendrocytes are sensitive to Aβ-induced toxicity [START_REF] Lee | Amyloid-β peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway[END_REF] and, interestingly, NG2 cells (a subset of oligodendrocytic precursor cells) also cluster around amyloid plaques in mice brains and are capable of taking up external Aβ42 peptides [START_REF] Li | Autophagy is involved in oligodendroglial precursor-mediated clearance of amyloid peptide[END_REF]. It is possible that impaired repair of oligodendrocyte precursor cells by Aβ pathology might enhance disease progression. However, the roles of these cells in Aβ pathology need to be further explored [START_REF] De Strooper | The cellular phase of Alzheimer's disease[END_REF].

Tau. Tau, the major component of intraneuronal neurofibrillary tangles, is a microtubule-stabilizing protein found predominantly in axons. Hyperphosphorylation or truncation of the protein correlates with its misfolding and neuropathology [START_REF] Frost | Propagation of tau misfolding from the outside to the inside of a cell[END_REF]. Work in vivo and in vitro has demonstrated that pathological tau transfers between cells and in between anatomically connected brain regions, causing seeding of the protein in neurons and oligodendrocytes [START_REF] Clavaguera | Transmission and spreading of tauopathy in transgenic mouse brain[END_REF][START_REF] Clavaguera | Intercellular transfer of tau aggregates and spreading of tau pathology: Implications for therapeutic strategies[END_REF][START_REF] Frost | Propagation of tau misfolding from the outside to the inside of a cell[END_REF]. Evidence also exists that trans-synaptic spread of tau occurs in vivo [START_REF] Liu | Trans-synaptic spread of tau pathology in vivo[END_REF][START_REF] Wu | Neuronal activity enhances tau propagation and tau pathology in vivo[END_REF]. A recent study using a three-chambered microfluidic system demonstrated that synaptic contact facilitated trans-neuronal tau propagation, which is relevant in the context that both normal and pathogenic tau have been found localized in synaptic terminals [START_REF] Calafate | Synaptic contacts enhance cell-to-cell tau pathology propagation[END_REF]. Interestingly, misfolded tau propagates in vivo between synaptically connected neurons even in the absence of endogenous tau expression, although its neurotoxicity was reduced, suggesting that conversion is necessary for toxicity, but not for transfer [START_REF] Wegmann | Removing endogenous tau does not prevent tau propagation yet reduces its neurotoxicity[END_REF]. Strikingly, several studies suggest that tau is secreted as naked aggregates from cells through synaptic release and nonconventional secretion [START_REF] Chai | Constitutive secretion of tau protein by an unconventional mechanism[END_REF][START_REF] Kfoury | Trans-cellular propagation of Tau aggregation by fibrillar species[END_REF][START_REF] Pooler | Physiological release of endogenous tau is stimulated by neuronal activity[END_REF], but exosomal release also occurs [START_REF] Saman | Exosomeassociated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid in early Alzheimer disease[END_REF]. Taken together, these data support the contribution of both synaptic and nonsynaptic mechanisms to the propagation of tau pathology, although increase in synaptic connections or activation enhances the process.

α-Synuclein. Like tau, α-synuclein is an intrinsically unstructured monomeric protein, which has been shown to misfold and aggregate in PD and other synuclopathies [START_REF] Davidson | Stabilization of α-synuclein secondary structure upon binding to synthetic membranes[END_REF]. α-Synuclein is the major component of Lewy bodies, along with ubiquitin and neurofilaments [START_REF] Spillantini | α-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with lewy bodies[END_REF]. Evidence that prion-like spreading propagates the disease arose when Lewy body pathology was detected in graft tissue from PD patients who had received therapeutic grafts [START_REF] Kordower | Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease[END_REF][START_REF] Li | Characterization of Lewy body pathology in 12-and 16-year-old intrastriatal mesencephalic grafts surviving in a patient with Parkinson's disease[END_REF]. Using grafts of embryonic mesencephalic tissue into mouse and rat brains expressing human α-synuclein, the protein was shown to transfer between neurons in vivo [START_REF] Hansen | α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells[END_REF][START_REF] Angot | α-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo[END_REF]. α-Synuclein also transfers to oligodendrocytes in ventral mesencephalon tissue grafted into rat brains [START_REF] Reyes | α-Synuclein transfers from neurons to oligodendrocytes[END_REF]. Additionally, transfer of α-synuclein from neurons to astrocytes was demonstrated in vitro and in vivo, and astroglia that endocytosed the infectious seed initiated an inflammatory response in microglia, providing insight into the mechanisms of pathogenicity in PD [START_REF] Lee | Direct transfer of α-synuclein from neuron to astroglia causes inflammatory responses in synucleinopathies[END_REF]. Interestingly in vitro co-cultures of primary neurons and astrocytes and organotypic brain slices confirm that α-synuclein fibrils are transferred efficiently from neurons to astrocytes but not the reverse, and support a major role of astrocytes in degradation of the protein and not in spreading of the fibrils [START_REF] Loria | α-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading[END_REF]. Microglial cells are capable of endocytosing α-synuclein and have the highest rate of degrading α-synuclein compared with neurons and astrocytes, consistent with a role in clearance [START_REF] Lee | Clearance and deposition of extracellular α-synuclein aggregates in microglia[END_REF]. α-synuclein has been demonstrated to be released from neuronal cells in exosomes as well as in a naked form [START_REF] Desplats | Inclusion formation and neuronal cell death through neuron-to-neuron transmission of α-synuclein[END_REF][START_REF] Jang | Nonclassical exocytosis of α-synuclein is sensitive to folding states and promoted under stress conditions[END_REF][START_REF] Danzer | Exosomal cell-to-cell transmission of α synuclein oligomers[END_REF], suggesting that a secretory mechanism of transfer operates for this protein. The internalization of exosomally packaged α-synuclein has been reported to be more efficient than that of naked aggregates [START_REF] Danzer | Exosomal cell-to-cell transmission of α synuclein oligomers[END_REF]; however, the mechanism of this uptake, whether through plasma membrane fusion or phagocytosis, is unclear. Interestingly, using microfluidic chambers, [START_REF] Freundt | Neuron-toneuron transmission of α-synuclein fibrils through axonal transport[END_REF] observed axonal transport of exogenously uptaken α-synuclein fibrils within neurons and the subsequent transfer of these aggregates to recipient neurons in a secondary chamber. Because transfer occurred between immature neurons, this is considered to be mediated through secretion rather than synaptic contacts [START_REF] Brahic | Axonal transport and secretion of fibrillar forms of α-synuclein, Aβ42 peptide and HTTExon 1[END_REF].

mHtt. In HD, expansion of the CAG repeat in the HTT gene results in the intracellular accumulation of polyQ Htt aggregates. These aggregates were shown to be transmissible to daughter nonneuronal cells after mitosis through an undefined mechanism [START_REF] Ren | Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates[END_REF]. Subsequently, transfer of mHtt fragments between neuronal cells and between primary neurons was first demonstrated in vitro to occur predominantly through cell-cell contact (Costanzo et al., 2013). Later work in vivo demonstrated that the non-cell-autonomous propagation of mHtt in the central nervous system also occurs between neurons and glial cells [START_REF] Pearce | Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain[END_REF] as well as between neurons [START_REF] Pecho-Vrieseling | Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons[END_REF][START_REF] Babcock | Transcellular spreading of huntingtin aggregates in the Drosophila brain[END_REF]. An elegant set of experiments, using both organotypic mouse brain slices and in vivo models, demonstrated that corticostriatal neuron-neuron mHtt propagation was predominantly mediated through synapses and did not appear to be secretion mediated [START_REF] Pecho-Vrieseling | Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons[END_REF]. However, other modes of transmission may apply, as Drosophila melanogaster glial cells were shown to phagocytose mHtt from neurons [START_REF] Pearce | Prion-like transmission of neuronal huntingtin aggregates to phagocytic glia in the Drosophila brain[END_REF]. This event was followed by recruitment of soluble Htt in the glial cytoplasm into aggregates, thus suggesting that normal protective clearance mechanisms can be subverted to spread pathogenicity.

Common pathways for different diseases. Although synaptic transfer and exosomal secretion have long been investigated as potential mechanisms of aggregate transfer, the discovery of TNTs in 2004 as a mechanism for direct cell-cell contact [START_REF] Rustom | Nanotubular highways for intercellular organelle transport[END_REF] opened new avenues in the study of intercellular aggregate transfer. Here, we discuss two aspects of the mechanism of transfer that we propose to have a major impact on the propagation of amyloidogenic prion-like seeds: TNTs and lysosomes.

TNTs: A direct, fast route for prion-like aggregates?

TNTs and aggregate transfer. TNTs are emerging as a mechanism for long-range communication between cells [START_REF] Rustom | Nanotubular highways for intercellular organelle transport[END_REF]. These dynamic structures create direct transient cytoplasmic continuity between otherwise unconnected cells. TNTs are membranous tubes that can extend up to 100 µm in length, with diameters ranging between 50 and 800 nm, and are supported by actin and, in certain cases, microtubules [START_REF] Abounit | Wiring through tunneling nanotubes: From electrical signals to organelle transfer[END_REF]. It was recently demonstrated that despite their similarity with filopodial protrusions, TNTs and filopodia are distinct structures. Indeed, although the formation of both these membrane extensions appear to rely on actin polymerization inside the protrusion, the same actin regulators (e.g., CDC42-IRSp53-VASP network and EPS8) exert contrasting effects on the two structures. This indicates that a switch in the molecular composition in common actin regulatory complexes is critical for driving the formation of either type of membrane protrusion [START_REF] Delage | Differential identity of filopodia and tunneling nanotubes revealed by the opposite functions of actin regulatory complexes[END_REF].

TNTs mediate the transfer of a variety of cellular organelles, including mitochondria and lysosomes, from one cell to another [START_REF] Rustom | Nanotubular highways for intercellular organelle transport[END_REF][START_REF] Wang | Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells[END_REF][START_REF] Zhu | Prion aggregates transfer through tunneling nanotubes in endocytic vesicles[END_REF]. They may perform protective roles, such as correcting a genetic lysosomal defect that causes cystinosis by supplying functional lysosomes from macrophages to cystinosin-deficient cells while concomitantly mediating transfer of dysfunctional cystine-loaded lysosomes away from the diseased cell [START_REF] Naphade | Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes[END_REF]. Of specific interest, they also mediate the transfer of pathogens such as viruses [START_REF] Eugenin | Tunneling nanotubes (TNT) are induced by HIV-infection of macrophages: A potential mechanism for intercellular HIV trafficking[END_REF] and have been shown to effectively propagate PrP Sc and several prion-like proteins intercellularly [START_REF] Gousset | Prions hijack tunnelling nanotubes for intercellular spread[END_REF]Wang et al., 2011;Costanzo et al., 2013;[START_REF] Ding | Exposure to ALS-FTD-CSF generates TDP-43 aggregates in glioblastoma cells through exosomes and TNTslike structure[END_REF]Abounit et al., 2016a,b). Specifically, PrP Sc transfers from dendritic cells to neurons and between neurons in co-cultures through TNTs [START_REF] Gousset | Prions hijack tunnelling nanotubes for intercellular spread[END_REF], wherein it colocalized with late endosomes/lysosomes and recycling endosomes [START_REF] Zhu | Prion aggregates transfer through tunneling nanotubes in endocytic vesicles[END_REF], both of which contain the propitious environment for prion conversion as well as infectious seeds [START_REF] Marijanovic | Identification of an intracellular site of prion conversion[END_REF].

α-Synuclein fibrils can also transfer between neuronal cells inside TNTs within lysosomes (Abounit et al., 2016b) and seed soluble α-synuclein in acceptor cells in co-culture. Notably, we found that in neuronal co-culture systems, transfer of α-synuclein was not significantly affected by transfecting acceptor cells with a dominant-negative dynamin construct shown before to inhibit α-synuclein endocytosis (Abounit et al., 2016b), thus suggesting that endocytic uptake of α-synuclein was not necessary for transfer. Although evidence also supports the localization and transfer in TNTs of Aβ, tau, and polyQ Htt [START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF]Costanzo et al., 2013;Abounit et al., 2016a), it is unclear whether these proteins are also associated with lysosomes, as shown for PrP Sc and α-synuclein, during TNT mediated transfer.

TNT induction: A cellular stress response. Evidence shows that increasing TNT formation through the use of established TNT inducers enhances the intercellular transfer of α-synuclein and prion [START_REF] Gousset | Myo10 is a key regulator of TNT formation in neuronal cells[END_REF]Abounit et al., 2016b). Congruently, inhibiting the formation of TNTs reduces intercellular transfer of these aggregates [START_REF] Gousset | Prions hijack tunnelling nanotubes for intercellular spread[END_REF]Abounit et al., 2016b). Strikingly, intracellular accumulation of the aggregates themselves induces an increase in TNT formation and thereby intercellular transfer of the respective protein to neighboring cells (Costanzo et al., 2013;[START_REF] Zhu | Prion aggregates transfer through tunneling nanotubes in endocytic vesicles[END_REF]Abounit et al., 2016a,b). This was demonstrated directly for PrP and Htt to be caused by the pathogenic conformer, as overexpression of the native conformer did not result in increased intercellular transfer (Costanzo et al., 2013;[START_REF] Zhu | Prion aggregates transfer through tunneling nanotubes in endocytic vesicles[END_REF]. We also have direct evidence that this is also the case for αsynuclein and tau (unpublished data). Collectively, these data suggest that TNTs may be induced by the stress that accompanies intracellular aggregate buildup (Abounit et al., 2016a). Indeed, although oxidative stress increases TNT formation in neuronal cells and primary neurons [START_REF] Gousset | Prions hijack tunnelling nanotubes for intercellular spread[END_REF][START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF], it is also associated with the presence of aggregated proteins in neurodegeneration. Thus, we propose that reactive oxygen species (ROS), generated as a consequence of prion-like protein aggregation, may stimulate TNT formation through an as yet unknown mechanism (Fig. 2).

It is also well known that actin changes its polymerization status as a consequence of different types of stress [START_REF] Schiffhauer | Mechanochemical signaling directs cell-shape change[END_REF]. For example, a cofilin-actin rod stress response whereby cofilin saturates actin filaments causing them to bundle into rod structures occurs under a variety of stress conditions and specifically in PMDs (including AD, PD, and HD; [START_REF] Munsie | The role of the cofilin-actin rod stress response in neurodegenerative diseases uncovers potential new drug targets[END_REF]. Another possibility for stimulating TNT formation could be the up-regulation of membrane biosynthetic genes through the unfolded protein response (UPR), which is activated in protein misfolding diseases [START_REF] Moreno | Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice[END_REF]. Alternatively, stress mediated through lysosomes may be involved. As discussed in more detail in the Intercellular transfer of aggregates via lysosomal dysregulation section, prion-like aggregates are dispatched to lysosomes for degradation, but their accumulation causes lysosomal damage that may induce a stress-response pathway [START_REF] Butler | Oxidative stress and lysosomes: CNS-related consequences and implications for lysosomal enhancement strategies and induction of autophagy[END_REF]. This in turn could result in increased numbers of TNTs, thus favoring the transfer of damaged lysosomes and prion-like aggregates from one cell to another and contribute to the spreading of the pathogenic aggregates (Fig. 2). We propose that TNTs may represent a common "rescue" mechanism adopted by the cell to rid itself of toxic material such as protein aggregates or damaged organelles. Meanwhile, because it has been shown that TNTs can also rescue functions by transferring healthy organelles such as lysosomes and mitochondria to damaged cells [START_REF] Naphade | Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes[END_REF][START_REF] Rustom | The missing link: Does tunnelling nanotube-based supercellularity provide a new understanding of chronic and lifestyle diseases?[END_REF], we can envisage that a reciprocal transfer of such organelles from healthy to damaged cells can also occur. An alternative hypothesis could therefore be that stressed cells send out TNTs to be rescued and that the aggregates would simply hijack these highways to transfer and spread between cells. Essential experiments to understand the direction of transfer (uni-or bidirectional) and the mechanism of TNT formation, TNT structure, and TNT composition are needed to understand which of these non-mutually exclusive hypotheses is correct.

Lysosomal impairment in NDs: A link with intercellular propagation?

Risk factors in ND. Although more than 90% of NDs are sporadic, genome-wide association studies and whole-genome and whole-exome sequencing have identified different loci correlated with an increased risk for late-onset PMDs. These unbiased analyses surprisingly converge on abnormalities in the endolysosomal pathway in early, preclinical stages of the diseases [START_REF] Peric | Early etiology of Alzheimer's disease: Tipping the balance toward autophagy or endosomal dysfunction?[END_REF][START_REF] Abeliovich | Defects in trafficking bridge Parkinson's disease pathology and genetics[END_REF]. Variants of endocytic regulators with increased risk for late-onset AD include sortilin-related receptor 1 (SORL1), phosphatidylinositol-binding clathrin assembly protein (PIC ALM), bridging integrator 1 (BIN1), and CD2-associated protein (CD2AP).

There is also accumulating evidence that failure of the retromer complex could be a risk factor for NDs [START_REF] Morel | Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system[END_REF][START_REF] Small | Retromer in Alzheimer disease, Parkinson disease and other neurological disorders[END_REF]. The retromer complex plays a primary role in sorting endosomal cargo back to the cell surface for reuse, to the TGN, or, alternatively, to the lysosomes (Seaman, 2012). Defects such as haploinsufficiency or mutations in one or several units of the retromer complex have been associated with AD and PD in addition to other NDs [START_REF] Wen | VPS35 haploinsufficiency increases Alzheimer's disease neuropathology[END_REF][START_REF] Zimprich | A mutation in VPS35, encoding a subunit of the retromer complex, causes late-onset Parkinson disease[END_REF][START_REF] Zavodszky | Mutation in VPS35 associated with Parkinson's disease impairs WASH complex association and inhibits autophagy[END_REF]. Furthermore, reduced retromer levels were found in several parts of the brains of patients with AD or PD, suggesting that reduced retromer abundance may have been associated with these conditions, and retromer has also been suggested to play a role in Creutzfeldt-Jakob disease [START_REF] Kipkorir | Highly infectious CJD particles lack prion protein but contain many viral-linked peptides by LC-MS/MS[END_REF]. These retromer deficiencies could potentially lead to lysosomal dysfunction, which in turn can be both a cause and a consequence of NDs.

Lysosomes and aggregate-mediated pathology. Prion and prion-like proteins are targeted to the lysosome for degradation through both endocytic and autophagosomal routes, and these pathways are implicated in pathogenic protein conversion [START_REF] Victoria | Trafficking and degradation pathways in pathogenic conversion of prions and prion-like proteins in neurodegenerative diseases[END_REF]. As discussed in detail in the next section, aggregate accumulation in this organelle can cause its dysfunction through different mechanisms, such as membrane permeabilization and loss of lysosomal integrity (Fig. 3), and lysosomes may be involved in intercellular aggregate transfer, including through lysosomal exocytosis of their contents, or travel through TNTs for direct cell-cell transfer [START_REF] Annunziata | Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-β secretion via deregulated lysosomal exocytosis[END_REF]Abounit et al., 2016b). How the aggregates transfer in lysosomes is also a question of interest. Upon endocytosis, α-synuclein and prion colocalize with lysosomal proteins, presumably within the organelle [START_REF] Freeman | α-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis[END_REF][START_REF] Victoria | Astrocyteto-neuron intercellular prion transfer is mediated by cell-cell contact[END_REF][START_REF] Loria | α-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading[END_REF]. However, cytosolic aggregates of α-synuclein are also translocated through the lysosomal membrane through chaperone-mediated autophagy, and evidence indicates that pathogenic α-synuclein mutants bind to receptors on the cytosolic face of lysosomal membrane but are poorly translocated to the interior for degradation [START_REF] Cuervo | Impaired degradation of mutant α-synuclein by chaperone-mediated autophagy[END_REF]. Thus, aggregates may also associate with the cytosolic face of lysosomes, where they may interfere with lysosomal translocation function and signaling complexes. In neurons, internalized tau is found in lysosomes and may travel through axons in these compartments [START_REF] Wu | Small misfolded Tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons[END_REF]. A growing body of evidence further indicates that tau is degraded by the autophagic-lysosomal system and that disturbances to this system result in the enhanced formation of tau aggregates [START_REF] Hamano | Autophagic-lysosomal perturbation enhances tau aggregation in transfectants with induced wild-type tau expression[END_REF]. Furthermore, truncated tau products are described to remain initially associated to the cytosolic surface of the lysosomal membrane. This would favor oligomerization at or near the surface of the organelle, and eventually resulting in membrane disruption, lysosomal leakage, and release of other fragments into the cytosol, where they can induce aggregation [START_REF] Wang | Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing[END_REF]. From these and many other studies [START_REF] Alvarez-Erviti | Lysosomal dysfunction increases exosomemediated α-synuclein release and transmission[END_REF][START_REF] Lee | Autophagic failure promotes the exocytosis and intercellular transfer of α-synuclein[END_REF][START_REF] Söllvander | Accumulation of amyloid-β by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons[END_REF], it is evident that perturbations in the protein degradation machinery have consequences for the intercellular transfer of infectious aggregates. Lysosomal dysfunction is associated with many NDs in two different ways: (1) impairment of lysosomal function (e.g., because of genetic factors or ageing) leads to increased protein aggregation, and (2) accumulation of aggregated proteins leads to lysosomal impairment, thus originating a vicious circle [START_REF] Fraldi | Brain disorders due to lysosomal dysfunction[END_REF].

Intercellular transfer of aggregates via lysosomal dysregulation. Apart from mediating degradation, lysosomes are significant hubs for stress signaling to the nucleus and mitochondria and regulate a variety of cellular processes, from nutrient homeostasis to plasma membrane repair to oxidative stress signaling and apoptosis [START_REF] Butler | Oxidative stress and lysosomes: CNS-related consequences and implications for lysosomal enhancement strategies and induction of autophagy[END_REF][START_REF] Lim | The lysosome as a command-and-control center for cellular metabolism[END_REF]. The intracellular accumulation of amyloidogenic proteins, diminished degradative capacity, and damage to the lysosome membrane caused by their buildup [START_REF] Wyss-Coray | Adult mouse astrocytes degrade amyloid-β in vitro and in situ[END_REF][START_REF] Freeman | α-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis[END_REF]Abounit et al., 2016b) can therefore potentially misregulate lysosomes and their signaling pathways (Fig. 3), leading to their intercellular propagation by various means in an attempt to relieve the cell of The aggregation and accumulation of prions or prion-like proteins in an "infected" cell (1) induces an increase in TNT number via an unknown mechanism, e.g., via ROS-induced stress pathways (2). The prion/prion-like aggregates may then be propagated via TNTs from infected cells to naive cells (3), wherein they effect the conversion and seeding of naive self-protein molecules. Adapted from Abounit et al. (2016a).

their toxic burden. Diminishing lysosomal capacity can induce a compensatory increase of exosomal release [START_REF] Vingtdeux | Alkalizing drugs induce accumulation of amyloid precursor protein by-products in luminal vesicles of multivesicular bodies[END_REF][START_REF] Alvarez-Erviti | Lysosomal dysfunction increases exosomemediated α-synuclein release and transmission[END_REF]. Deficiency or mutations in the lysosomal hydrolase β-glucocerebrosidase, which cause the lysosomal storage disease known as Gaucher's disease, have been linked to PD and cause lysosomal dysfunction and increased α-synuclein aggregate accumulation [START_REF] Bae | Loss of glucocerebrosidase 1 activity causes lysosomal dysfunction and α-synuclein aggregation[END_REF], which may result in increased intercellular transfer.

There are several indications that perturbations of the autophagy-lysosomal pathway increase extracellular vesicle/exosome release, in contexts such as lysosomal storage diseases, viral infections, and neurodegenerative diseases [START_REF] Eitan | Impact of lysosome status on extracellular vesicle content and release[END_REF]. The study by [START_REF] Annunziata | Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-β secretion via deregulated lysosomal exocytosis[END_REF] demonstrated that lysosomal enzymatic deficiency resulted in not only impaired processing of Aβ but also increased lysosomal exocytosis through the oversialylation of Lamp1, which acts as a signal for exocytosis. Indeed, the lysosomal accumulation of most of these amyloidogenic proteins causes abnormal morphology and physical damage [START_REF] Rockenstein | Lysosomal pathology associated with α-synuclein accumulation in transgenic models using an eGFP fusion protein[END_REF][START_REF] Freeman | α-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis[END_REF][START_REF] Domert | Spreading of amyloid-β peptides via neuritic cell-tocell transfer is dependent on insufficient cellular clearance[END_REF][START_REF] Victoria | Astrocyteto-neuron intercellular prion transfer is mediated by cell-cell contact[END_REF]. Notwithstanding, there are few data regarding whether lysosomal exocytosis is a generalized mechanism of extracellular release of these aggregates. Lysosomal exocytosis is regulated by increased Ca 2+ levels; intriguingly, Ca 2+ homeostasis is perturbed by Aβ, α-synuclein, and mHtt [START_REF] Danzer | Different species of α-synuclein oligomers induce calcium influx and seeding[END_REF][START_REF] Lim | Calcium homeostasis and mitochondrial dysfunction in striatal neurons of Huntington disease[END_REF][START_REF] Small | Dysregulation of calcium homeostasis in Alzheimer's disease[END_REF].

Lysosomal stress/damage caused by the accumulation of amyloidogenic proteins may also contribute to TNT formation (Abounit et al., 2016a,b). A recent model proposed that TNTs could be an intermediate cell survival response to cope with increased oxidative stress, allowing the bidirectional transfer of material between cells in an attempt to rescue damage before cell death [START_REF] Rustom | The missing link: Does tunnelling nanotube-based supercellularity provide a new understanding of chronic and lifestyle diseases?[END_REF]. Dissection of signaling pathways involved in TNT formation identified several molecules that overlap between mitochondrial and lysosomal signaling, notably the mTOR [START_REF] Martini-Stoica | The autophagylysosomal pathway in neurodegeneration: A TFEB perspective[END_REF][START_REF] Rustom | The missing link: Does tunnelling nanotube-based supercellularity provide a new understanding of chronic and lifestyle diseases?[END_REF] and Akt-PI3K signaling pathways (Fig. 4; [START_REF] Wang | Tunneling-nanotube development in astrocytes depends on p53 activation[END_REF]. Oxidative stress, although associated with the mitochondria, is also regulated by lysosomal damage [START_REF] Butler | Oxidative stress and lysosomes: CNS-related consequences and implications for lysosomal enhancement strategies and induction of autophagy[END_REF]. The coupling of mitochondria and lysosomes in regulating oxidative stress (the mitochondria-lysosome axis) could therefore be involved in regulating TNT formation induced by protein aggregates (Fig. 3). The intracellular accumulation of prions, α-synuclein, mHtt, and tau has been shown to increase TNT formation (Costanzo et al., 2013;[START_REF] Zhu | Prion aggregates transfer through tunneling nanotubes in endocytic vesicles[END_REF]Abounit et al., 2016a,b), and these proteins are associated with increased ROS production and oxidative stress [START_REF] Kim | The role of oxidative stress in neurodegenerative diseases[END_REF]. Evidence suggests that lysosomes are damaged in various ways by these proteins, including diminished lysosomal degradation [START_REF] Wyss-Coray | Adult mouse astrocytes degrade amyloid-β in vitro and in situ[END_REF][START_REF] Cuervo | Impaired degradation of mutant α-synuclein by chaperone-mediated autophagy[END_REF][START_REF] Butler | Oxidative stress and lysosomes: CNS-related consequences and implications for lysosomal enhancement strategies and induction of autophagy[END_REF][START_REF] Wang | Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing[END_REF][START_REF] Jackson | Translation of the prion protein mRNA is robust in astrocytes but does not amplify during reactive astrocytosis in the mouse brain[END_REF] and lysosomal membrane permeabilization and rupture in the case of Aβ and synuclein and tau [START_REF] Ditaranto | Lysosomal membrane damage in soluble Aβ-mediated cell death in Alzheimer's disease[END_REF][START_REF] Wang | Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing[END_REF][START_REF] Freeman | α-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis[END_REF]. Diminished lysosomal capacity results in the lysosomal buildup of oxidized proteins and other damaged material, causing lysosomal stress which can cause increased feedback loops of mitochondrial dysfunction and ROS production [START_REF] Cleeter | Glucocerebrosidase inhibition causes mitochondrial dysfunction and free radical damage[END_REF], as well as increased autophagy, a phenotype that is well documented in neurodegenerative disease [START_REF] Butler | Oxidative stress and lysosomes: CNS-related consequences and implications for lysosomal enhancement strategies and induction of autophagy[END_REF]. Lysosomal membrane permeabilization may also (1) Lysosomal enzyme activity may decrease with the natural processes of aging or by the accumulation of resistant protein aggregates within the structure. The resultant diminished degradative capacity can lead to increased intracellular burden, not only of the toxic aggregate but also of partially degraded/oxidized lipids such as cholesterol, resulting in lysosomal stress and buildup of ROS. Insufficiency in β-glucocerebroside, for example, is implicated in α-synuclein accumulation. (2) The cytosolic tails of transport receptor proteins such as Lamp2a are involved in the recognition of protein substrates of chaperone-mediated autophagy. Recognition of the misfolded protein results in translocation of the protein across the lysosomal membrane to be degraded. However, misfolded α-synuclein mutant aggregates can bind to Lamp2a receptors and block their ability to transport, thereby causing decreased degradation and increased cytosolic burden of aggregates that can result in neurotoxicity through a variety of pathways. Other receptors have yet to be investigated. (3) Calcium channels such as MCO LN1 are involved in mediating lysosomal exocytosis. They have been identified as being affected in lysosomal storage diseases, dysregulating normal Ca 2+ homeostasis in the cell. Although Ca 2+ homeostasis is disturbed in these diseases, it remains unclear whether channels like these are directly perturbed by the buildup of protein.

(4) Proteins involved in membrane fusion, such as NSF1, have been shown to be important for clearance of toxic protein aggregates such as Htt. These proteins may regulate fusion with autophagosomes or with the plasma membrane for lysosomal exocytosis. Aggregate buildup on the surface also affects trafficking of these vesicles. (5) Lysosome-nucleus signaling is regulated by nutrient-sensing machinery, which includes the mTORC1 complex. Binding of the master transcription factor TFEB to mTORC1 normally sequesters TFEB in the cytoplasm. However, upon certain signals such as nutrient starvation, TFEB is released, whereupon it enters the nucleus and up-regulates genes involved in lysosome biogenesis, lysosomal exocytosis and autophagy. Synuclein has been shown to bind directly to TFEB, and therefore, cytosolic aggregates can potentially dysregulate this signaling during neurodegenerative disease. ( 6) Direct breaching of the integrity of the lysosomal membrane by aggregates has been proposed to cause lysosomal damage, release of aggregates into the cytosol thereby leading to propagation of seeding as well as causing apoptosis through the release of cathepsins into the cytoplasm. Adapted from [START_REF] Settembre | Signals from the lysosome: a control centre for cellular clearance and energy metabolism[END_REF].

trigger oxidative stress and eventual apoptosis [START_REF] Ghosh | Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages[END_REF]. Aggregate-damaged lysosomes therefore may contribute to the localized ROS production proposed to be involved in TNT formation (Figs. 2 and3).

There are several other indications that incomplete or insufficient lysosomal activity is particularly important in creating neurotoxicity and neurodegeneration. Transcription factor EB (TFEB), a major regulator of the autophagy-lysosomal pathway, is a cytosolic transcription factor that can associate with the lysosome surface under normal conditions [START_REF] Sardiello | A gene network regulating lysosomal biogenesis and function[END_REF][START_REF] Martina | MTO RC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB[END_REF]. It is itself regulated by the mTOR pathway and, upon nutrient starvation or other lysosomal stresses, translocates to the nucleus, up-regulating lysosome biogenesis and exocytosis [START_REF] Settembre | TFEB links autophagy to lysosomal biogenesis[END_REF][START_REF] Settembre | A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB[END_REF][START_REF] Martina | MTO RC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB[END_REF][START_REF] Medina | Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB[END_REF]. Several studies point to it being affected in PD patients and, to a less well-defined extent, in AD pathologies [START_REF] Martini-Stoica | The autophagylysosomal pathway in neurodegeneration: A TFEB perspective[END_REF]. Restoring lysosomal activity through the overexpression of TFEB has been shown to rescue neurotoxicity resulting from tau, Htt, and α-synuclein aggregation [START_REF] Tsunemi | PGC-1α rescues Huntington's disease proteotoxicity by preventing oxidative stress and promoting TFEB function[END_REF][START_REF] Decressac | TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity[END_REF][START_REF] Polito | Selective clearance of aberrant tau proteins and rescue of neurotoxicity by transcription factor EB[END_REF]. Congruently, miRNA repression of TFEB exacerbated synuclein-mediated toxicity in dopaminergic neurons [START_REF] Decressac | TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity[END_REF]. Additionally, TFEB-induced astrocytic clearance reduced external amyloid plaque deposition [START_REF] Xiao | Enhancing astrocytic lysosome biogenesis facilitates Aβ clearance and attenuates amyloid plaque pathogenesis[END_REF], highlighting a link between impaired degradation and disease propagation through extracellular release of toxic material. It is therefore increasingly clear that perturbations of degradation or clearance organelles, especially the lysosome, affect intercellular transfer. This is especially significant in light of the emerging role of this organelle as a signaling hub that regulates cellular homeostasis, and so it is tempting to consider them as new therapeutic targets. However, it should be noted that these pathways are not solely responsible for toxicity and that responses like the UPR are also up-regulated. Therapeutic treatment targeting the UPR, for instance, has been shown to relieve prion infections in mice [START_REF] Moreno | Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice[END_REF]; therefore, several cellular pathways are simultaneously affected. Additionally, therapeutics aimed at increasing or rescuing lysosomal activity should also take into account the role of this organelle in the production of amyloidogenic aggregates [START_REF] Pasternak | Presenilin-1, nicastrin, amyloid precursor protein, and gamma-secretase activity are co-localized in the lysosomal membrane[END_REF][START_REF] Wang | Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing[END_REF][START_REF] Sannerud | Restricted location of PSEN2/γ-secretase determines substrate specificity and generates an intracellular Aβ pool[END_REF].

Conclusion and perspectives

Recent data suggest that transfer of PrP Sc , α-synuclein, tau, polyQ aggregates, and Aβ assemblies between co-cultured neuronal cells and primary neurons occurs through TNTs. Despite differences in structure, they are all prion-like, supporting the hypothesis that other prion-like protein aggregates may use TNTs as one mechanism of intercellular transmission. We propose that TNTs have a relevant role in the spreading of the pathology of prions and other NDs. As intracellular accumulation of misfolded proteins causes lysosomal impairments, cells try to dispose of damaged material through TNTs, which enhances their formation. We also postulate that accumulation of aggregates in lysosomes changes their signaling/positioning, making them more prone to entering the newly formed TNTs. As a consequence of lysosomal transfer and damage, proteins escape from lysosomes, leading to further seeding and propagation of the diseases (Fig. 2).

Characterizing the mechanism of TNT formation and how misfolded protein aggregates trigger their formation along with their intercellular transport, as well as the mechanisms leading to lysosomal impairment, may lead to new therapeutic avenues to inhibit the spreading of prion-like proteins and the progression of these devastating, fatal diseases.

Figure 4. Signaling pathways that can cross talk with TNT formation. The Akt-dependent TNT formation pathway cross talks with signaling complexes from the lysosome and the mitochondria. Increased oxidative stress, through increased ROS formation from aggregates within the cytosol or from lysosomal aggregation of protein, can cause mitochondrial dysfunction and lysosomal dysfunction, which act in a feedback loop to create increased ROS. Increased localized ROS is believed to be responsible for TNT formation [START_REF] Rustom | The missing link: Does tunnelling nanotube-based supercellularity provide a new understanding of chronic and lifestyle diseases?[END_REF] through the Akt pathway. Lysosomal stress can also alter TFEB signaling, which is directly responsible for increasing lysosomal exocytosis [START_REF] Martini-Stoica | The autophagylysosomal pathway in neurodegeneration: A TFEB perspective[END_REF]. Direct interaction between α-synuclein and TFEB has been demonstrated [START_REF] Decressac | TFEB-mediated autophagy rescues midbrain dopamine neurons from α-synuclein toxicity[END_REF], and thus, cytosolic aggregates may influence TFEB signaling in a way that affects its intercellular transfer.

Figure 1 .

 1 Figure 1. Proposed routes of intercellular transfer.(1) Synaptic transfer. Misfolded proteins (red) such as mHtt, Tau, and α-synuclein, which have been shown to bind to synaptic vesicles (green), may be released from cells through synaptic vesicle fusion with the plasma membrane and thereafter be taken up by the postsynaptic neuron. (2) TNTs. TNTs have been implicated in the transfer of prion, mHtt, Aβ, tau, and α-synuclein. (3) Phagocytosis. The engulfment of aggregate-containing damaged/dying cells or synaptic terminals by glial cells may represent a mechanism that propagates transfer between cells of different types and has been demonstrated for mHtt and Aβ. This process may also occur for extracellular naked or exosomally localized aggregates in close apposition to the engulfing cell. (4) Exocytosis of exosomes. The fusion of MVBs with the plasma membrane may release exosomes containing intraluminal aggregate-containing vesicles (exosomes) that can be internalized by neighboring cells.(5) Lysosomal exocytosis. Fusion of lysosomes (yellow) with the plasma membrane can release naked aggregates (such as Aβ) into the extracellular space that may form plaques or be taken up by neighboring cells.

Figure 2 .

 2 Figure2. Model of prion-like protein aggregate-induced formation of TNTs and intercellular spreading. The aggregation and accumulation of prions or prion-like proteins in an "infected" cell (1) induces an increase in TNT number via an unknown mechanism, e.g., via ROS-induced stress pathways (2). The prion/prion-like aggregates may then be propagated via TNTs from infected cells to naive cells (3), wherein they effect the conversion and seeding of naive self-protein molecules. Adapted fromAbounit et al. (2016a).

Figure 3 .

 3 Figure3. Multiple factors within lysosome structures can affect proteopathic disease propagation. Lysosomes are complex vesicular structures that regulate cellular homeostasis as well as perform a significant part of cellular degradation. (1) Lysosomal enzyme activity may decrease with the natural processes of aging or by the accumulation of resistant protein aggregates within the structure. The resultant diminished degradative capacity can lead to increased intracellular burden, not only of the toxic aggregate but also of partially degraded/oxidized lipids such as cholesterol, resulting in lysosomal stress and buildup of ROS. Insufficiency in β-glucocerebroside, for example, is implicated in α-synuclein accumulation. (2) The cytosolic tails of transport receptor proteins such as Lamp2a are involved in the recognition of protein substrates of chaperone-mediated autophagy. Recognition of the misfolded protein results in translocation of the protein across the lysosomal membrane to be degraded. However, misfolded α-synuclein mutant aggregates can bind to Lamp2a receptors and block their ability to transport, thereby causing decreased degradation and increased cytosolic burden of aggregates that can result in neurotoxicity through a variety of pathways. Other receptors have yet to be investigated. (3) Calcium channels such as MCO LN1 are involved in mediating lysosomal exocytosis. They have been identified as being affected in lysosomal storage diseases, dysregulating normal Ca 2+ homeostasis in the cell. Although Ca 2+ homeostasis is disturbed in these diseases, it remains unclear whether channels like these are directly perturbed by the buildup of protein.(4) Proteins involved in membrane fusion, such as NSF1, have been shown to be important for clearance of toxic protein aggregates such as Htt. These proteins may regulate fusion with autophagosomes or with the plasma membrane for lysosomal exocytosis. Aggregate buildup on the surface also affects trafficking of these vesicles. (5) Lysosome-nucleus signaling is regulated by nutrient-sensing machinery, which includes the mTORC1 complex. Binding of the master transcription factor TFEB to mTORC1 normally sequesters TFEB in the cytoplasm. However, upon certain signals such as nutrient starvation, TFEB is released, whereupon it enters the nucleus and up-regulates genes involved in lysosome biogenesis, lysosomal exocytosis and autophagy. Synuclein has been shown to bind directly to TFEB, and therefore, cytosolic aggregates can potentially dysregulate this signaling during neurodegenerative disease. (6) Direct breaching of the integrity of the lysosomal membrane by aggregates has been proposed to cause lysosomal damage, release of aggregates into the cytosol thereby leading to propagation of seeding as well as causing apoptosis through the release of cathepsins into the cytoplasm. Adapted from[START_REF] Settembre | Signals from the lysosome: a control centre for cellular clearance and energy metabolism[END_REF].

Table 1 .

 1 Misfolding of specific proteins causes a spectrum of neurodegenerative disorders

	Disorder	Protein aggregate	Seeding	Intercellular transmissibility
	Prion diseases (CJD, vCJD, Kuru, and FFI)	PrP Sc	Yes	Yes
	AD	Aβ, Tau	Yes	Yes
	Tauopathies (tauopathy, progressive supranuclear palsy,	Tau	Yes	Yes
	Pick's disease, and frontotemporal dementia)
	HD	mHtt	Yes	Yes
	Synucleinopathies (PD, DLB, MSA, and PAF)	α-Synuclein	Yes	Yes
	ALS	TDP-43	Yes

lysosomes and TNTs in prion-like protein transfer • Victoria and Zurzolo

Acknowledgments

We thank Drs. Ayşegül Dilsizoğlu-Şenol, Yuan-Ju Wu, and Frida Loria Salinas for their critical reading of the manuscript.

C. Zurzolo was supported by the Agence Nationale de Recherche (grants Joint Programme-Neurodegenerative Disease Neutargets: ANR-14-JPCD-0002-01 and ANR-16 CE160019-01 NEU ROT UNN)