Extracellular vesicles in neurodegenerative diseases

Extracellular vesicles in neurodegenerative diseases

Molecular Aspects of Medicine 60 (2018) 52e61 Contents lists available at ScienceDirect Molecular Aspects of Medicine journal homepage: www.elsevier...

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Molecular Aspects of Medicine 60 (2018) 52e61

Contents lists available at ScienceDirect

Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam

Extracellular vesicles in neurodegenerative diseases Tommaso Croese, Roberto Furlan* Clinical Neuroimmunology Unit, Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 August 2017 Received in revised form 31 October 2017 Accepted 10 November 2017 Available online 22 November 2017

Extracellular vesicles (EVs) are released by all neural cells, including neurons, oligodendrocytes, astrocytes, and microglia. The lack of adequate technology has not halted neuroscientists from investigating EVs as a mean to decipher neurodegenerative disorders, still in search of comprehensible pathogenic mechanisms and efficient treatment. EVs are thought to be one of ways neurodegenerative pathologies spread in the brain, but also one of the ways the brain tries to displace toxic proteins, making their meaning in pathogenesis uncertain. EVs, however do reach biological fluids where they can be analyzed, and might therefore constitute clinically decisive biomarkers for neurodegenerative diseases in the future. Finally, if they constitute a physiological inter-cell communication system, they may represent also a very specific drug delivery tool for a difficult target such as the brain. We try to resume here available information on the role of EVs in neurodegeneration, with a special focus on Alzheimer's disease, progressive multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's disease. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Extracellular vesicles Alzheimer's disease Parkinson's disease Multiple sclerosis Amyotrophic lateral sclerosis Huntington's disease

Introduction Neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), the progressive phase of multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), constitute a major challenge for biomedical research since they affect an increasing number of individuals in the aging population, and recognize no treatment (Chiti and Dobson, 2017; Kawachi and Lassmann, 2017; Sanabria-Castro et al., 2017; Saudou and Humbert, 2016; Taylor et al., 2016). Recent years have brought many advances in the clinical definition and in the knowledge on pathogenic mechanisms of neurodegenerative diseases, but translation to effective cure is hampered by several factors. The lack of efficient biomarkers, for example, does not allow diagnosing patients in the early stages of the disease when there is still the possibility to maintain acceptable cognitive performance, impedes to assign patients to their disease subtype, and does not allow monitoring disease progression and thus allow a more efficient clinical trial design. Currently, the gold standard biomarker remains brain imaging, either with magnetic resonance imaging (MRI) or positron emission tomography (PET). At least two years of

* Corresponding author. Clinical Neuroimmunology Unit, Institute of Experimental Neurology - INSpe, Division of Neuroscience, San Raffaele Scientific Institute, Via Olgettina 60, 20132, Milan, Italy. E-mail address: [email protected] (R. Furlan). https://doi.org/10.1016/j.mam.2017.11.006 0098-2997/© 2017 Elsevier Ltd. All rights reserved.

follow-up are needed to appreciate the potential effect of experimental treatments on neurodegeneration with brain imaging, increasing the cost of drug development and dramatically decreasing the number of tested compounds. In general terms, therapeutic strategies for neurodegeneration have been designed to prevent the formation of toxic protein aggregates, present in most neurodegenerative diseases (Shrivastava et al., 2017), to protect neurons from cell death (Hwang et al., 2017), and to modulate concomitant brain inflammation (Heneka et al., 2015; N. P. Rocha et al., 2016). However, despite increasing knowledge on the pathogenic mechanisms leading to formation of toxic protein aggregates, we lack exhaustive information on the final pathways leading to neuronal death and on the role, protective or damaging, of inflammation, to allow the rational design of novel therapeutic strategies. In this scenario, the first reports describing the detection of extracellular vesicles (EVs) released from neural cells raised enormous interest (Rajendran et al., 2006; Scolding et al., 1989; Verderio et al., 2012). EVs, in fact, were immediately investigated as a potential source of information on neural cells involved in the pathological processes causing neurodegenerative diseases. The possibility to shed light on the mechanisms leading to neuronal death, or to develop biomarkers able to measure neurodegenerative processes in real time have attracted attention and resources from a number of neuroscientists. After initial encouraging reports, however, the field has not developed as quickly as expected, although, as described also here, a number of papers have been

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published. There are, clearly, difficulties that the research on EVs in neurodegeneration shares with the whole field of clinical application of EVs: clear definition of the objects (i.e. exosomes, microvesicles, apoptotic bodies, other), nomenclature (Gould and Raposo, 2013), technological limits in the detection and definition of EVs subtypes (Coumans et al., 2017; Witwer et al., 2013), their biological significance (Colombo et al., 2014). In Fig. 1 we have tried to depict what we hypothesize is, in very general terms, the source and path of EVs to biological fluids before analysis. Current knowledge on most steps is, however, extremely limited, hampering our ability to interpret the significance of EVs levels and content in physiological and pathological conditions. We show in Fig. 1 that neurons, astrocytes, oligodendrocytes and microglia are supposed to be the major source of EVs of neural origin during disease, along with ependymal and leptomeningeal cells when searching the CSF, and brain endothelial cells when searching the blood. Blood-derived infiltrating cells contributing to neuroinflammation during neurodegeneration are also a potential cellular source of EVs. We have limited information, however, on the stimuli inducing the release of EVs from neural cells and on their biological significance. We also ignore what kind of EVs have the ability to travel, and how, in the brain parenchyma and reach biological fluids (blood, CSF) where we can detect them. We have no conclusive data on how EVs are able to cross barriers such as the ependymal cell layer or the basal membrane and the brain endothelium, to gain access to CSF or blood. If EVs are messengers, we need to understand when and why cells release them, what message is delivered, what cells are the target of this communication. If EVs are released by cells to discard unwanted molecules, as a defence mechanism or to change phenotype, we need to understand the cellular pathways involved. Without this information, we cannot properly interpret the significance of their presence in biological fluids an their potential as biomarkers. In this review we try to summarize the knowledge gained in neurodegenerative diseases such as AD, PD, MS, ALS, and HD, focusing on human

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studies, since the lack of appropriate animal models is another major problem of the field. In Table 1 we report mentioned studies listed according to pathology, if they are human or experimental studies, and we specify techniques and markers used to detect EVs, to provide the reader the possibility to interpret the nature of investigated objects (Table 1). Despite the hurdles and the technical difficulties, all the promises of EVs research in neurodegeneration are still intact. Current knowledge, that we hope to summarize here, sets the stage for exciting developments in the near future. As mentioned, nomenclature is an issue, and as recommended by the reference Society (Gould and Raposo, 2013), we use here the term extracellular vesicles (EVs) whenever we have not clear indication from cited papers on a more specific nature of investigated objects, namely exosomes or microvesicles.

Alzheimer disease and other cognitive impairment While the pathogenesis of Alzheimer disease (AD) remains unclear, all forms of AD appear to share overproduction and/or decreased clearance of amyloid beta peptides. The pathogenesis of AD also involves a second protein, tau. EVs are studied both in the pathogenesis of AD as well as possible biomarker able to predict conversion from Mild Cognitive Impairment (MCI) to overt AD. According to the current view, the accumulation of altered proteins (amyloid beta and tau) is toxic to neurons and EVs-mediated transmission of their pathologic forms between neurons has been proposed to account for the spread of AD in the brain (Guo and Lee, 2011; Iba et al., 2013; Medina and Avila, 2014). The potential role of EVs in AD is object of debate and evidences for both a beneficial and a detrimental role have been reported. More than ten years ago, it was first demonstrated that proteins and peptides (i.e. APP, APPCterminal fragments, APP intra-cellular domain, Ab) associated with AD are released in association with exosomes (Perez-Gonzalez et al., 2012; Rajendran et al., 2006; Sharples et al., 2008; Vingtdeux

Fig. 1. EVs are released by all neural cells, those depicted and labeled here (neurons, oligodendrocytes, astrocytes, microglia, ependymal cells, brain endothelial cells), but in pathological conditions also blood-derived infiltrating inflammatory cells, or activated circulating cells such as monocytes and platelets may modulate EVs release. In the upper left panel we show a scanning electron microscopy of a human microglia cell-line (CHME-5), stimulated with ATP to release EVs. We ignore the precise nature of the stimuli and the biology of EVs release from neural cells in vivo. In the same way, we do not have a clear view on the path followed by EVs to reach biological fluids, namely the cerebrospinal fluid (CSF) or the circulation. Blood or CSF samples can be pre-processed (centrifuged, column-purified, etc.), to enrich for EVs and eliminate objects (proteic aggregates, other cell debris) that might interfere with the analysis. Freezing the sample for preservation will eliminate larger microvesicles and also some exosomes, with an unknown bias. Samples can be then analyzed by flow cytometry, nano tracking, light scattering, resistive pulse sensing, electron microscopy, western blot, next generation sequencing, etc.

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Table 1 FC: Flow Cytometry; WB: Western Blot; AFM: Atomic Force Microscopy; EM: Electron Mycroscopy; IF: Immunofluorescence microscopy; DLSS: Dynamic Light Scattering Spectroscopy; NTA: Nanoparticle Tracking Analysis. Type of Evs

Species

Site and detection

ALZHEIMER DISEASE AND OTHER COGNITIVE IMPAIRMENT Microvesicles Human CSF by FACS Microvesicles Exosomes Exosomes Exosomes Exosomes Exosomes Exosomes Exosomes Exosomes Exosomes Exosomes Exosomes EVs EVs EVs PARKINSON DISEASE Exosomes Exosomes

Cell of origin

Markers

Myeloid cells

IB4 and/or Annexin V Human Cell culture/CSF by FC Microglia IB4 and/or Annexin V Human Cell culture/CSF by WB, AFM, Neurons Alix, Flotillin-1, and Mouse EM, DLSS CD81 Mouse Cell culture/sucrose step Neurons/Microglia e model gradient ultracentrifugation Human Plasma/Serum Exosomal RNA e e Isolation kit Human CSF/Plasma by NTA Neurons PKH26 and Mouse Human Plasma/Serum by ELISA Neurons NCAM, L1CAM, CD82 Mouse Cell culture by WB Neurons Cystatin C Human Plasma by Total exosome e e isolation kit Human Plasma by NTA, WB Neurons L1CAM, CD81 Human Plasma by NTA, WB Astrocytes VD81 Human Plasma by NTA, WB Neurons L1CAM, CD81 Mouse and Cell culture/CSF by WB, EM Neurons Flotillin-1, Monkey Ganglioside GM1 Human Plasma Neurons LCAM, L1CAM Human Cell culture by WB Astrocytes VAMP-2 and Mouse Human Blood/Plasma by FC Platlets CD42

Function

References

Prognostic factor

(Agosta et al., 2014)

Spread of pathogenic proteins

(Falker et al., 2016)

Neutralize activity of Ab assemblies Spread of Tau proteins

(An et al., 2013) (Asai et al., 2015)

Prognostic factor

(Cheng et al., 2015)

Spread of pathogenic Ab species

(Eitan et al., 2016; Rajendran et al., 2006) (Fiandaca et al., 2015; Goetzl et al., 2015) (Ghidoni et al., 2011) (Cheng et al., 2013)

Diagnostic tool/Biomarker Neuroprotective role Prognostic factor (miRNA 193b) Biomarker Spread of pathogenic proteins Biomarker Decrease Ab and amyloid depositions Prognostic factor Contribute to neuroinflammation

(Goetzl et al., 2016) (Goetzl et al., 2016) (Mullins et al., 2017) (Yuyama et al., 2015)

Biomarker

(Y. J. Lee et al., 1993) (Chang et al., 2013) (Harischandra et al., 2017)

(Mustapic et al., 2017) (Stenovec et al., 2016)

Mouse Human

Cell culture by EM and FC Cell culture by EM and NTA

Microglia Neurons

CD63 e

Exosomes

Human

Cell culture by NTA

Neurons (iPS)

e

Exosomes

Human

CSF by NTA, EM, WB

e

Flotillin-2

Exosomes

Mouse

Cell culture by NTA and WB

Neurons

Flotillin-1

Exosomes

Human Cell culture by WB and Mouse

Neurons

CD63, Flotillin-1

Increase apoptosis Progression of neurodegeneration/Biomarker Progression of neurodegeneration/Biomarker Accelerate a-synuclein aggregation Accelerate a-synuclein aggregation Spread of a-synuclein

Exosomes EVs

Human Human

e e

CD63, L1CAM e

Biomarker Release of LRRK2

e

Alix

Myeloid cells

IB-4, Annexin V

Sulfatides in Extracellular Vesicles (Moyano et al., 2016) as biomarker Pathogenic, biomarker (Verderio et al., 2012)

Microglia

Annexin V

CSF/Plasma by Urine by step gradient ultracentrifugation

MULTIPLE SCLEROSIS Apoptotic bodies, MVs Human Plasma by EM, WB, NTA and Exososomes CSF MVs Human and mouse MVs Rat Cell culture

Platelets and Monocytes Platelets and Endothelium Monocytes and Platelets Microglia Monocytes

Promoted ceramide and sphingosine production in neurons CD61, CD14 Increase during inflammatory periods Annexin V, CD31, Increase endothelial permeability CD42, CD62E and leukocyte infiltration. Annexin V, Biomarker CD105 Alix, Flotillin Transport of endocannabinoids Alix Biomarker

MVs

Human

Plasma by FC

MVs

Human

Plasma by FC

MVs

Human

Plasma by FC

MVs and Exosomes Exosomes

Rat Human

Exosomes Exosomes

Rat Human

Cell culture by NTA and WB Serum/Cell culture by NTA, WB Cell culture by EM and WB Serum by FC

Exosomes

Human

Serum by NTA, ELISA, WB

PBMC

EVs

Rat

CSF by EM

Oligodendrocytes

Alix, CD63 CD31þ, CD51þ, CD61þ, CD54þ CD9, CD63, CD81, Alix e

EVs

Human

Plasma by FC

Endothelium

CD31, CD51

EVs EVs EVs

Human Human Human

Plasma by FC Cell culture by FC Plasma by FC

Platelets Endothelium e

CD62P CD31, CD62E CD31, CD146, CD54

Dendritic Cells Platelets

(Fernandes et al., 2016) (Stuendl et al., 2016) (Grey et al., 2015) (Danzer et al., 2012; Emmanouilidou et al., 2010) (Shi et al., 2014) (Fraser et al., 2016)

(Antonucci et al., 2012)

enz-Cuesta et al., 2014a) (Sa (Marcos-Ramiro et al., 2014) (Zinger et al., 2016) (Gabrielli et al., 2015) (Selmaj et al., 2017)

Promote remyelination (Pusic et al., 2014) Biomarker of microvascular stress (Alexander et al., 2015) Perpetuate anti-myelin immune reactions Recovery from demyelinating injury Biomarker of endothelial dysfunction Biomarker of platelets activation Enhance monocyte migration Markers of response to IFN-b1a therapy

(Galazka et al., 2017) (Scolding et al., 1989) (Minagar et al., 2001) (Sheremata et al., 2008) (Jimenez et al., 2005) (Lowery-Nordberg et al., 2011)

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Table 1 (continued ) Type of Evs

Species

Site and detection

AMYOTROPHIC LATERAL SCLEROSIS Exosomes Human Cell culture by WB

Cell of origin

Markers

Function

References

Adipose-derived stem cell Adipose-derived stromal cells e

CD9, CD63, HSP70 HSP70, Alix

Therapeutic candidate for ALS

(M. Lee et al., 2016)

Neurons, Astrocytes and Microglia Motor Neurons

Flotillin-1, Alix, CD63

Exosomes

Mouse

Cell culture by EM, WB

Exosomes

Human

CSF by WB, IF

Exosomes

Mouse

Cell culture by WB, IF, EM

Exosomes

Mouse

Cell culture by NTA, WB, EM

Exosomes

Human

Plasma and Cell culture by IF, Monocytes WB

HUNTINGTON'S DISEASE Exosomes Mouse Exosomes Human

Cell culture by IF Cell culture by NTA, WB

HEK 293 HEK 293

et al., 2007). The identification of Ab in association with exosomes is an important finding, especially since other exosomal proteins, such as flotillin, have been found to accumulate in the plaques of AD brains. These findings may provide potential explanation for extracellular amyloid deposition in the brain. Concerning myeloid EVs, our group and others have found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and MCI patients converting within 3 years to definite AD (Agosta et al., 2014). Furthermore, Cerebrospinal fluid (CSF) MVs levels correlated with white matter tract damage in MCI, and with hippocampal atrophy in AD (Agosta et al., 2014). Neurotoxicity of MVs may results from the capability of MV lipids to promote formation of soluble Ab species from extracellular insoluble aggregates and from the presence of neurotoxic Ab forms trafficked to MVs after Ab internalization into microglia (Joshi et al., 2013). These finding were further evaluated using an adeno-associated virusebased model exhibiting rapid tau propagation. Using this model, it was found that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo (Asai et al., 2015). EVs may contribute also to impair neuronal Ca2þ handling and mitochondrial function, and may thereby spread neuronal damage (Eitan et al., 2016). Yuyama and co-workers, on the other hand, have suggested a neuroprotective role of EVs in AD describing a novel function for exosomes as scavengers of neurotoxic Ab. Neuronal exosomes, but not glial exosomes, have abundant glycosphingolipids and may capture Ab (Yuyama et al., 2014, 2015). Addition of an exosome/Ab mixture to primary cortical cells significantly suppressed the formation of toxic oligomers and neuronal toxicity revealing the capability of exosomes to trap Ab and to promote its clearance by microglia (Yuyama et al., 2015, 2014). Accordingly, there is in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived Ab (An et al., 2013; Yuyama and Igarashi, 2017). Mechanistically, this effect involves sequestration of synapto-toxic Ab assemblies by exosomal surface proteins such as PrPC rather than Ab proteolysis (An et al., 2013; Yuyama and Igarashi, 2017). Finally, Cystatin C, a protein considered neuroprotective for AD, is also secreted by mouse primary neurons in association with exosomes (Ghidoni et al., 2011). Immunoproteomic analysis revealed the presence in exosomes of at least 9 different cystatin C glycoforms. Moreover, the over-expression of familiar AD-associated presenilin mutations resulted in reduced levels of all cystatin C forms (native and glycosylated) and of APP

Flotillin-1

Protect neurons from oxidative (Bonafede et al., 2016) damage Mediated the propagation of TDP- (Ding et al., 2015) 43 aggregates Neuronal clearance of (Iguchi et al., 2016) pathological TDP-43

Flotillin-1, Alix, CD63 CD63, Flotillin-1

Modulate microglia activation

(Pinto et al., 2017)

Alter monocyte activation

(Zondler et al., 2017)

PKH67 Alix

Delivery of miRNA Biomarker

(S.-T. Lee et al., 2017) (Zhang et al., 2016)

metabolites within exosomes (Ghidoni et al., 2011). As EVs are released into the extracellular space and can be isolated from several body fluids, detecting proteins and other molecules associated with these vesicles may have diagnostic, prognostic and disease monitoring potential. Vesicles have been shown to contain messenger RNA (mRNA) and micro RNA (miRNA) species (Cheng et al., 2014; Hunter et al., 2008; Pant et al., 2012). Analysing enriched exosomal miRNA has advantages over analysing non-exosomal miRNA, considerably improving signal-to-noise ratio as compared to free miRNAs that are essentially diluted in circulating blood. miR-193b, for instance, was predicted to potentially target the 30 -untranslated region of APP. AD patients have lower exosomal miR-193b levels in blood as compared with the MCI and control groups and decreased exosomal miR-193b expression levels were additionally observed in the cerebral spinal fluid (CSF) of AD patients (Liu et al., 2014). miRNAs are also deregulated in brain tissues during the neurodegenerative processes, as is the case for the highly conserved brain miRNA-219 that is down-regulated in brain tissues taken at autopsy from patients with AD and from those with severe primary age-related tauopathy (Santa-Maria et al., 2015). There is, thus, a set of specific miRNAs, including miR-132 and miR-212, that are robustly down-regulated in neurodegenerative disorders, including AD (Cogswell et al., bert et al., 2013; Lau et al., 2014; Wong et al., 2013) and 2008; He bert et al., frontotemporal dementia (Chen-Plotkin et al., 2012; He 2013). Using next-generation deep sequencing, Cheng and others profiled exosomal miRNA from serum of AD patients and find an AD miRNA signature that, added to established risk factors such as age, sex and apolipoprotein ε4 (APOE ε4) allele status, was able to predict AD with a sensitivity and specificity of 87% and 77%, respectively (Cheng et al., 2015; Gui et al., 2015). Despite all these evidences, the pathogenic or protective role of EVs has still not been proven conclusively in vivo for AD. Although there appear to be many pathways that increase the transmission of pathological proteins through exosomes, none provides a direct mechanism for the loading of aggregation-prone proteins into exosomes. EVs, thus, seem to act as double-edged sword. EVs derived from Aß stimulated astrocytes and aggregated tau-treated microglia are involved in Aß aggregation and tau interneuronal propagation, respectively (Asai et al., 2015; Dinkins et al., 2016; Fiandaca et al., 2015; Xiao et al., 2017). Depending on the cell origin and the pathological stage of the disease, EVs may have detrimental roles contributing to worsening or spread of the pathogenesis. Further investigations on the pathophysiological

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properties of these vesicles may open the door to develop new diagnostic and therapeutic strategies for AD. Parkinson disease Parkinson disease (PD) is a chronic, progressive neurodegenerative disorder characterized by any combination of four cardinal signs: bradykinesia, rest tremor, rigidity, and postural instability (E. M. Rocha et al., 2017). The main pathological characteristics of PD are cell death in the brain's basal ganglia and accumulations of the protein alpha-synuclein in many of the remaining neurons. These insoluble protein aggregates do not themselves appear to have a prominent neurotoxic effect, whereas various a-synuclein oligomers appear harmful (E. M. Rocha et al., 2017). Although it is incompletely known how the prefibrillar species may be pathogenic, they have been detected on exosomes and other EVs, suggesting that such structures may mediate toxic a-synuclein propagation between neurons (Emmanouilidou et al., 2010). In vitro studies support the idea of transcellular spread of extracellular, secreted alpha-synuclein across membranes. Exosomeassociated alpha-synuclein oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free alpha-synuclein oligomers. Specifically, alpha-synuclein oligomers are present on both the outside as well as inside of exosomes and their secretion serves to amplify and propagate Parkinson disease related pathology (Chang et al., 2013; Danzer et al., 2012; Emmanouilidou et al., 2010). Exosomes, in fact, reduce the lag time of aggregation kinetics indicating that they provide catalytic environments for nucleation. Vesicles prepared from extracted exosome lipids accelerate aggregation as well, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise (Fernandes et al., 2016; Grey et al., 2015). The balance between the cell's internal protein degradative systems and the secreted or EVmediated release of a-synuclein has been explored and inhibition of lysosomal protein degradation resulted in an increase in the levels of exosomal alpha-synuclein released from cultured cells (AlvarezErviti et al., 2011; Guerreiro et al., 2013; Kong et al., 2014). Additional PD-related proteins may contribute to pathology by affecting the secretion/release and uptake of a-synuclein. Patients with mutations in LRRK2 (Leucine- Rich Repeat Kinase 2) manifest dominantly inherited forms of PD, although the apparent molecular link between LRRK2 and a-synuclein has not yet been elucidated (Burgos et al., 2014). LRRK2 interactors, including 14-3-3, are some of the most abundant proteins found in exosomes (Fraser et al., 2013) and urinary exosome auto-phosphorylated Ser(P)-1292 LRRK2 levels are elevated in idiopathic PD and correlate with the severity of cognitive impairment and difficultly in accomplishing activities of daily living (Fraser et al., 2016). The quantification of cerebrospinal fluid exosomal alpha-synuclein seems to be a useful diagnostic marker for PD and, importantly, cerebrospinal fluid exosomes derived from Parkinson disease induce oligomerization of a-synuclein in a reporter cell line in a dose-dependent manner, suggesting that cerebrospinal fluid exosomes from patients with Parkinson disease contain a pathogenic species of a-synuclein, which could initiate oligomerization of soluble a-synuclein in target cells and confer disease pathology (Stuendl et al., 2016). On the other hand, similar serum-derived exosome numbers were detected between PD and other patients, while 23 exosomeassociated proteins were differentially abundant in PD suggesting that they may reflect exosome subpopulations with distinct functions (Tomlinson et al., 2015). Finally, from a therapeutic point of view, exosomes secreted by monocytes and macrophages have been exploited to enhance delivery of incorporated PD drugs to target cells, avoiding entrapment in mononuclear phagocytes, and increasing potential drug

therapeutic efficacy (Hall et al., 2016). Exosomal-based delivery system for molecules such as catalase or GDNF to treat PD were developed and provided significant neuroprotective effects in in vitro and in vivo models of PD (Garbayo et al., 2016; Haney et al., 2015). In conclusion, exosomes have been shown to possess the ability to propagate PD pathology as well as to be used to hinder it. Several steps, however, are still to be done before EVs can be used for PD in a clinical setting, as biomarker or drug delivery tool. Multiple sclerosis Diseases that affect central nervous system myelin can be categorized as demyelinating and dysmyelinating. The most common immune-mediated inflammatory demyelinating disease of the central nervous system is multiple sclerosis (MS). Inflammation, demyelination, and axonal degeneration are the major pathologic mechanisms that cause the clinical manifestations (Compston and Coles, 2008). However, the cause of MS remains unknown. The most widely accepted theory is that MS begins as an inflammatory immune-mediated disorder characterized by autoreactive lymphocytes (Compston and Coles, 2008; Dendrou et al., 2015). Later, the disease is dominated by microglial activation and chronic neurodegeneration (Kawachi and Lassmann, 2017). Given the demonstrated important role of EVs in immune regulation, it is not surprising that many studies on this topic have appeared in attempts to specifically define their involvement in autoimmune diseases like MS. Scolding firstly demonstrated, in 1989, that recovery of oligodendrocytes from injury involves the release of membrane-attack complex-enriched vesicles from the surface of viable cells (Scolding et al., 1989). Other studies in vitro have revealed that brain endothelium-derived microvesicles were involved in the activation of CD4þ and CD8þ lymphocytes through expression of b2-microglobulin, MHC II, CD40 and ICOSL (Wheway et al., 2014). Minagar et al. hypothesized that plasma from MS patients contains factors that can induce endothelial activation, as suggested by the release into circulation of CD31 EVs from a BBB model cell culture treated with plasma from patients both in exacerbation and remission (Jimenez et al., 2005; Minagar et al., 2001). Together with endothelial-derived MVs, platelet-derived MVs from MS patients have been shown to increase the permeability of endothelial layers in vitro, suggesting their involvement in the disruption of the BBB (Alexander et al., 2015; Marcos-Ramiro et al., 2014; Sheremata et al., 2008). Furthermore, EVs found in plasma seem to be able to interact and form complexes with monocytes and induce their activation, playing an important role in the trans-endothelial migration of inflammatory cells (Jy et al., 2004). The injection of microvesicles from microglial cells into the brains of mice with EAE resulted in enhanced inflammation and exaggerated disease (Jy et al., 2004). Accordingly, mice with impaired secretion of microvesicles (aSMase deficient) were resistant to EAE although these genetic mutant mice may have defects also in other compartments relevant to the disease (Verderio et al., 2012). Microglia-derived MVs are able also to influence synaptic activity through promotion of ceramide and sphingosine production in neurons (Antonucci et al., 2012) and carrying on their surface N-arachidonoylethanolamine (AEA), which can in turn stimulate type-1 cannabinoid receptors (CB1) inhibiting presynaptic transmission in target GABAergic neurons (Gabrielli et al., 2015). Also dendritic cells (DCs) release EVs and after stimulation with low-level IFNg, released exosomes are taken up by oligodendrocytes increasing baseline myelination following acute lysolecithin-induced demyelination (Pusic et al., 2014). As the evidences above show, the association between EVs concentration and the pathological condition of MS patients is easily established.

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There are significant differences in CD61þ, CD45þ and CD14þ EVs counts in blood samples from MS patients compared to those from healthy controls. Relapsing-remitting patients had the highest counts for the three subtypes of EVs while secondary progressive patients were found to have similar numbers to those in healthy enz-Cuesta et al., 2014a, 2014b). In human CSF, the controls (Sa numbers of EVs have been found to be higher in patients than controls and MVs counts correlated linearly with gadoliniumenhancing MRI lesions, a sign of active disease (Verderio et al., 2012). Moyano found that EVs of different sizes display C16:0 sulfatide and suggests them as candidate biomarker (Moyano et al., 2016). Recently, Galatzka found that exosomes isolated not in the CNS, but in the serum, express myelin proteins, and the presence of MOG fragments strongly correlates with disease activity (Galazka et al., 2017). Finally, Selmaj using Next Generation Sequencing (NGS) to define the global RNA profile of serum exosomes in MS patients, found that 4 circulating exosomal sequences within the miRNA category were differentially expressed in RRMS patients versus HC: hsa-miR-122-5p, hsa-miR-196b-5p, hsa-miR-301a-3p, and hsa-miR-532-5p. Serum exosomal expression of these miRNAs was significantly decreased during relapse in RRMS. These miRNAs were also decreased in patients with a gadolinium enhancement on brain magnetic resonance imaging (Selmaj et al., 2017). Considering EVs as biomarkers of therapeutic efficacy, Jimenez report that IFN-b 1b reduces the release of endothelial EVs induced by plasma from MS patients (Jimenez et al., 2005). LoweryNordberg et al., during a prospective 1-year study, used flow cytometry to measure changes in plasma microparticles (PMP) bearing CD31, CD146, and CD54 in patients with relapsingremitting MS before and after 3, 6, and 12 months of subcutaneous therapy with interferon-beta1a. They found that plasma levels of CD31þ EVs, and CD54þ EVs were significantly reduced by treatment with IFN-b1a. In addition, the decrease in plasma levels of CD31þ and CD54þ EVs levels at 12 months were associated with a significant decrease in the number and volume of contrast enhancing T1-weigthed lesions (Lowery-Nordberg et al., 2011). On the contrary, another study, focusing probably on MVs, reported higher counts of MVs in IFN-b and natalizumab-treated patients. [64] Dawson et al. demonstrated that fingolimod inhibits aSMase (Dawson and Qin, 2011), the enzyme that controls MVs production. Zinger found that in non-treated MS patients compared to healthy and fingolimod-treated patients, endothelial microparticles were higher, while B-cell-microparticle numbers were lower. Furthermore, Fingolimod dramatically reduced tumour necrosis factor (TNF)-induced endothelial microparticles release in vitro (Zinger et al., 2016). Regardless of all the evidence cited above, also for MS application of EV research findings to daily clinical practice seems still difficult. There is evidence that their levels, in plasma or CSF, reflect disease progression, but we need to better understand the molecular mechanisms that are involved in the secretion of membrane vesicles and to have more robust detection methods to determine precisely how and with what types of cell secreted vesicles we are dealing, what their significance, what their interactors are in vivo ry et al., 2009). (The Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS), first described by Charcot in the nineteenth century (Rowland, 2001), is a relentlessly progressive neurodegenerative disorder that causes muscle weakness, disability, and eventually death, with a median survival of three to five years. Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron degeneration and death with gliosis replacing lost

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neurons. Cortical motor cells (pyramidal and Betz cells) disappear leading to retrograde axonal loss and gliosis in the corticospinal tract. Intracellular inclusions in degenerating neurons and glia are frequent neuropathological findings of ALS: phosphorylated and nonphosphorylated neurofilament inclusions are prominent in spinal motor neurons; Bunina bodies are unique to ALS and consist of eosinophilic aggregates that are positive for cystatin C; Ubiquinated inclusions are seen in ALS and several other neurodegenerative disorders, including frontotemporal dementia (FTD) with ubiquitin positive/tau negative inclusions; TDP-43 accumulation and inclusion formation is observed in most sporadic cases of ALS, FTD and overlapping ALS with frontotemporal dementia (Taylor et al., 2016). The etiology of ALS is unknown. A number of potential mechanisms have been proposed including abnormal RNA processing, SOD1-mediated toxicity, excitotoxicity, cytoskeletal derangements, mitochondrial dysfunction, viral infections, apoptosis, growth factor abnormalities, inflammatory responses and others (Peters et al., 2015). Given the complexity of ALS pathogenesis and since EVs can act at the same time via multiple mechanisms, in the last years an increasing interest has been addressed to these molecules. In 2015, Ding et al. demonstrated that intracellular TDP-43 mislocalization and aggregates were induced in the human glioma U251 cells following exposure to ALSFTD-CSF but not ALS-CSF and normal control-CSF for 21 days. The exosomes derived from ALS-FTD CSF were enriched in TDP-43 Cterminal fragments. Incubation of ALS-FTD CSF induced the increase of mislocated TDP-43 positive exosomes and exposure to ALS-FTD CSF induced the generations of tunneling nanotubes-like structure and exosomes at different stages, which mediated the propagation of TDP-43 aggregates in the cultured U251 cells (Ding et al., 2015). One year later, Iguchi and collaborators detected TDP43 in secreted exosomes from Neuro2a cells and primary neurons but not from astrocytes or microglia. Exposure of Neuro2a cells to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, caused cytoplasmic redistribution of TDP-43, suggesting that secreted exosomes might contribute to propagation of TDP-43 proteinopathy. Yet, inhibition of exosome secretion by inactivation of neutral sphingomyelinase 2 with GW4869 or by silencing RAB27A provoked formation of TDP-43 aggregates in Neuro2a cells. Moreover, administration of GW4869 exacerbated the disease phenotypes of transgenic mice expressing human TDP43A315T mutant. Thus, even though results suggest that exosomes containing pathological TDP-43 may play a key role in the propagation of TDP-43 proteinopathy, in vivo data suggest that exosome secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43 (Iguchi et al., 2016). While glia-derived exosomes and their effects in neurons have been addressed by several studies, only a few investigated the influence of motor neuron (MN)-derived exosomes in other cell function. Pinto et al. assessed a set of “inflamma-miRNAs” in NSC-34 MN-like cells transfected with mutant SOD1 (G93A) and extended the study into their derived exosomes (mSOD1 exosomes). The effects produced by mSOD1 exosomes in the activation and polarization of the recipient N9 microglial cells were investigated. Increased miR-124 expression was found in mSOD1 NSC-34 cells and in their derived exosomes. Incubation of mSOD1 exosomes with N9 cells determined a sustained 50% reduction in the cell phagocytic ability. It also caused a persistent NF-kB activation and an acute generation of NO, MMP2, and MMP-9 activation, as well as upregulation of IL-1b, TNFa, MHC-II, and iNOS gene expression, suggestive of induced M1 polarization. Marked elevation of IL-10, Arginase 1, TREM2, RAGE, and TLR4 mRNA levels, together with increased miR-124, miR-146a, and miR-155, at 24 h incubation, suggest the switch to mixed M1 and M2 subpopulations in the exosome-treated N9 microglial cells. Exosomes from mSOD1 NSC-34 MNs also enhanced the number of

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senescent-like positive N9 cells (Pinto et al., 2017). Zondler et al., instead, hypothesize that circulating blood exosomes are putative mediators of monocytic deregulation in ALS. They characterized exosomal uptake and the respective immunological reaction of peripheral monocytes from ALS patients and healthy donors using both serum-derived exosomes and TDP-43-loaded exosomes produced in cell culture and found the pro-inflammatory cytokine secretion by ALS monocytes upon exosomal stimulation to be impaired compared with control monocytes. Moreover, exosomal TDP-43 induces increased monocytic activation compared with non-aggregation-prone cargo (Zondler et al., 2017). In human CSF, an increased number of leukocyte-derived microparticles with normal cell counts was also reported in one patient affected by ALS (Zachau et al., 2012). Given the complexity of ALS pathogenesis, to date there is no effective treatment to cure or significantly ameliorate the quality of life of patients. Since exosomes and microvesicles can transfer biological information over long distance, increasing attention has been paid to these vesicles as suppressors of pathological processes. As exosomes are smaller than microvesicles and possibly capable to cross the BBB, most of the studies present in literature on EVs and ALS focus on the use of exosomes as a potential therapeutic tool (Bonafede and Mariotti, 2017). Bonafede et al. investigated, in vitro, the efficacy of the use of exosomes derived from murine adiposederived stromal cells on motoneuron-like NSC-34 cells expressing ALS mutations, used to model the disease. Data presented indicate that exosomes from adipose-derived stromal cells may protect NSC-34 cells from oxidative damage, which is one of the main mechanism of damage in ALS, increasing cell viability (Bonafede et al., 2016). These data were further validated by Lee who found that exosomes produced by adipose-derived stem cells alleviate aggregation of superoxide dismutase 1 in G93A ALS mice model and help to normalize phospho-CREB/CREB ratio and PGC-1a expression level in neurons (M. Lee et al., 2016). It is certainly interesting that both vesicles and free aggregates of pathogenic proteins may be involved in releasing SOD1 from affected cells consistent with contiguous propagation (Silverman et al., 2016); it remains to be determined, however, which of these mechanisms results in the effective seeding of template-directed misfolding or the acquisition of toxic properties in the recipient cell. Huntington's disease Huntington disease (HD) is an inherited progressive neurodegenerative disorder characterized by choreiform movements, psychiatric problems, and dementia. It is caused by a cytosineadenine-guanine (CAG) trinucleotide repeat expansion in the huntingtin (HTT) gene on chromosome 4p and inherited in an autosomal-dominant pattern (Richards, 2001). The pathophysiology of HD is not fully understood, although it is thought to be related to toxicity of the mutant huntingtin protein. Accumulated research implicates both the polyQ protein and the expanded repeat RNA in causing toxicity leading to neurodegeneration in HD. Different theories have emerged as to how the neurodegeneration spreads throughout the brain, with one possibility being the transport of toxic protein and RNA in EVs. Zhang used a model culture system with an overexpression of HTT-exon 1 polyQ-GFP constructs in human 293T cells and found that the EVs did incorporate both the polyQ-GFP protein and expanded repeat RNA. Striatal mouse neural cells were able to take up these EVs with a consequent increase in the green fluorescent protein (GFP) and polyQ-GFP RNAs, but with no evidence of uptake of polyQ-GFP protein or any apparent toxicity, at least over a relatively short period of exposure. A differentiated striatal cell line expressing endogenous levels of HTT mRNA containing the expanded repeat

incorporated more of this mRNA into EVs as compared to similar cells expressing this mRNA with a normal repeat length. These findings support the potential of EVs to deliver toxic expanded trinucleotide repeat RNAs from one cell to another (Zhang et al., 2016). On the other hand, two recent papers hypothesized a therapeutic role for EVs in HD. Lee, as he already did for ALS, demonstrated that adipose-derived stem cells exosomes significantly decrease HTT aggregates in R6/2 mice-derived neuronal cells. These exosomes seem also to ameliorate abnormal apoptotic protein level and reduce mitochondrial dysfunction and cell apoptosis of in vitro HD model (S.-T. Lee et al., 2017). Another treatment option, recently developed, is an exosome-based delivery method to treat this neurodegenerative disease. miR-124, one of the key miRNAs that is repressed in HD, was stably overexpressed in a stable cell line, and its exosomes (Exo-124) exhibited a high level of miR-124 expression and were taken up by recipient cells. When Exo-124 was injected into the striatum of R6/2 transgenic HD mice, expression of the target gene, RE1-Silencing Transcription Factor, was reduced. However, Exo-124 treatment did not produce significant behavioral improvement (S.-T. Lee et al., 2017). These preliminary studies support further work both on the transfer of CAG-repeat RNA and polyQ proteins via EVs over longer time intervals and on in vivo models in the brain to assess the fate of transferred RNA/proteins and potential toxicity to recipient cells. In addition, these data evoke the potential to evaluate the levels of expanded repeat RNA and polyQ proteins in EVs in biofluids of HD patients as biomarkers of disease progression and response to therapy. Conclusion EVs research in neurodegenerative diseases has ignited enormous interest and hype. Neuroscientists deal with the absence of appropriate animal models of most neurological diseases, and with all the limitations associated with human brain studies, possible either through imaging techniques or post-mortem, since brain biopsies are performed in very limited cases. Thus, the possibility to gain information on inaccessible neural cells through the tiny messengers we call extracellular vesicles has clearly created very high expectations. Extracellular vesicles represent, however, a relatively new field of research also for basic cell biology, and especially neurobiology. This means that we miss crucial information to correctly interpret our findings, to appreciate the kind of information that EVs can provide when we investigate them as biomarkers, but also their contribution, in detrimental or protective terms, to pathogenic mechanisms. Limits are of knowledge, but also technical. Current methodologies to measure and analyze EVs in biological fluids have several limitations, and it is very difficult to separate the different types of EVs and thus to optimize also cargo analysis. But, none of these hurdles is prospectively insuperable. Considering EVs as potential biomarker, the most likely possibility is to develop new, non-specific, indicators of neuroinflammation or neurodegeneration. It is difficult to predict if these potential biomarkers will be clinical more useful, or complementary, or more handy and cost-effective than traditional biomarkers such as MRI. EVs may become disease-specific, i.e. diagnostic, biomarkers for neurological disorders if research will reveal that indeed their content differs in different pathologies and technologies will be developed to allow the routine detection of this content in individual samples. Nevertheless, the information that can be gained by the detailed examination of EVs content is already contributing to understand the pathogenesis of neurological diseases, and will do so even more in the future. Further, EVs are being developed also as highly versatile drug-delivery tools, endowed with new features, and their use in neurology, to reach selective targets in the nervous system, is under active investigation. Thus EVs constitute one of the

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