Laura Le Gall

Stephanie Duguez
email: s.duguez@ulster.ac.uk

William J Duddy

Cecile Martinat

Virginie Mariot

Stephanie Millecamps

Jeanne Lainé

Gisele Ouandaogo

Udaya Geetha Vijayakumar

Susan Knoblach

Cedric Raoul

Olivier Lucas

Jean Philippe Loeffler

Peter Bede

Anthony Behin

Helene Blasco

Gaelle Bruneteau

Maria Del Mar Amador

David Devos

Anne Cecile Durieux

Damien Freyssenet

Alexandre Henriques

Adele Hesters

Lucette Lacomblez

Pascal Laforet

Timothee Langlet

Pascal Leblanc

Nadine Le Forestier

Thierry Maisonobe

Vincent Meininger

Alexandre Mejat

Franscesca Ratti

Laura Robelin

Francois Salachas

Tanya Stojkovic

Giorgia Querin

Julie Dumonceaux

Gillian Butler Browne

Jose-Luis González De Aguilar

Pierre Francois Pradat

Matthew Thorley

Apostolos Malatras

Vincent Mouly

Gillian Butler Browne

Stéphanie Duguez

Sylvain Roquevière

Mariot Virginie

Blandine Madji Hounoum

Romain Joubert

Jose-Luis Gonzales De Aguilar

Stephanie Duguez

Co

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Keywords: ALS, muscle exosomes, RNA, muscle stem cells, intercellular communication, neurotoxic vesicles Litterature Aging, adult stem cells, muscles, skeletal, myoblasts, intercellular signaling peptides and proteins, homeostasis Poster communications

To date, amyotrophic lateral sclerosis (ALS) remain incurable and the causes for motor neuron death are unknown. The primary involvement of skeletal muscle in ALS pathogenesis is still controversial. Several studies suggested that a distal axon degeneration occurs prior to the onset of ALS clinical symptoms.

Moreover, there are growing evidences for a disruption of exosomes biogenesis and secretion pathways in genetic forms of ALS. Knowing that the skeletal muscle can be a source of exosomes, we hypothesised that ALS skeletal muscle could contribute to the toxic environment of motor neurons and thus participate in ALS pathogenesis through their secretion of exosomes. During my PhD, I investigated the secretion of exosomes by muscle cells from sporadic ALS patients and their role in motor neuron death. First, we show that muscle cells present a consistent signature across ALS patients with an accumulation and oversecretion of exosomes. Second, ALS muscle exosomes are toxic toward healthy human iPSCs motor neurons by inducing shorter, reduced branching neurites and cell death. Third, we observed that ALS muscle exosomes contain FUS protein and are enriched in proteins involved in RNA maturation and transport. Fourth, ALS muscle exosomes induced a disruption in RNA transport in healthy human motor neuron. Fifth, the exosome toxicity is dependent on FUS expression level in the recipient cells, as an overexpression of FUS in the recipient cells exacerbated the ALS exosome toxicity while an inhibition of FUS expression decreased their toxic effect. The greater sensitivity of motor neurons to ALS muscle exosomes might thus be explained by their higher expression level of FUS compare to healthy muscle cells.

Altogether, these results suggest that ALS muscle exosomes could contribute to the degeneration of motor neuron in ALS patients.

Laura Le Gall

List of tables and figures

Résumé

A ce jour, les causes de la sclérose latérale amyotrophique (SLA) ne sont pas connues et il n'existe aucun remède. L'implication du muscle dans la pathogénèse de la SLA est toujours controversée, néanmoins il a été démontré qu'il existe une atteinte axonale distale primaire à l'apparition des premiers symptômes de la SLA. De plus, plusieurs données de la littérature suggèrent une perturbation de la voie de biogénèse et de sécrétion des exosomes chez certains patients atteints de forme mono-génique de la SLA. Sachant que le muscle squelettique peut être une source d'exosomes, nous émettons l'hypothèse que le muscle pourrait contribuer à la toxicité de l'environnement des motoneurones, à leur dégénérescence et donc à la pathogénèse de la SLA. J'ai donc étudié au cours de ma thèse la sécrétion d'exosomes par les cellules musculaires de patients sporadiques SLA et leurs rôles dans la SLA. Dans un premier temps, nous avons observé l'existence d'une signature associée à la SLA dans l'ensemble des cellules musculaires analysées, avec une accumulation et sursécrétion d'exosomes. Ces exosomes musculaires de patients SLA étaient toxiques pour les motoneurones dérivés d'iPSCs, induisant une réduction du nombre de branchements, une diminution de la longueur des neurites et une mort des motoneurones. Nous avons ensuite observé que les exosomes ALS contenaient la protéine FUS et d'autres protéines impliquées dans le transport et la maturation des RNAs. Or, le traitement des motoneurones avec des exosomes musculaire SLA induisait une accumulation de RNA dans leurs noyaux. Enfin, nous avons observé que la toxicité des exosomes SLA était dépendante du niveau d'expression de FUS de la cellule réceptrice. En effet, la toxicité des exosomes SLA était exacerbée lorsque FUS était surexprimé dans la cellule réceptrice et était diminuée lorsque l'expression de FUS était éteinte. Or les motoneurones présentent un niveau d'expression de FUS supérieur aux cellules musculaires, suggérant leur sensibilité accrue aux exosomes musculaires SLA. Ensemble ces données suggèrent une implication du muscle dans la dégénérescence des neurones moteurs de patients SLA.

Mots clés : SLA, exosomes musculaires, RNA, cellules souches musculaires, communication intercellulaire, vésicules neurotoxiques.

Chapter 1: ALS I. ALS description and definition

Motor neuron diseases or MND is a group of disorders characterised by a progressive and fatal degeneration of upper and/or lower motor neurons (UMN and LMN respectively) resulting in muscle weakness and wasting. Motor neurons are responsible for movement initiation. LMN directly innervates skeletal muscle fibres and their cell bodies are located either in the spinal cord (their axons stimulate upper and/or lower limb muscles) or in the brainstem (axons stimulating bulbar muscles including throat and tongue muscles).

Descending pathways from the motor cortex comprise the UMN whose cell bodies are located in the cortex and their descending axons synapse with LMN cell bodies in the brainstem or the spinal cord (Figure 1). UMN from the motor cortex are responsible for initiation and transmission of voluntary movements.

ALS or amyotrophic lateral sclerosis is the most common form of MND 1 and specifically associated with the deterioration of both UMN and LMN 2 . Despite the long-established concept of ALS described as a neurodegenerative disease they are growing evidence for a cognitive impairment in some ALS patients 3 . So far, no diagnostic tools have been developed for ALS, however diagnostic criteria (El Escorial 4 and revisions 5 , and Awaji-shima criteria 6 ) help stratifying ALS patients according the severity, fast or slow progressors, and sites of onset.

Figure 1 : Upper and lower motor neuron involvement in ALS

ALS is diagnosed based on combinatorial degeneration of upper and lower motor neurons The upper motor neuron (UMN) cell bodies are located in the motor cortex and the brainstem, and project down to establish synaptic communication directly with the lower motor neuron (LMN) or through interneurons. UMN thus modulate LMN activities. LMN cell bodies reside in the spinal cord and in the brainstem. Their axons project out towards skeletal muscles fibres to make contact at the neuromuscular junction to transmit movement information. UMN and LMN originating from the brainstem are responsible for activating bulbar-innervated muscle (pharyngeal muscles and tongue involved in speech and swallowing). UMN and LMN originating from the spinal cord are responsible for upper and lower limb movement.

Epidemiology

ALS global incidence varies between 1 to 2.6 cases per 100,000 people every year 7 . In Europe, the incidence is ~ 2.6/100,000 person per year 8 , ranging from 0.5/100,000 persons in Serbia to 3.6/100,000 persons in the Faroe Islands annually 9 . Thus the number of newly diagnosed ALS cases reaches about 15,400 every year 10 .

In Asia, the incidence varies dramatically country to country with an extremely low number of new cases in China, ~0.3/100,000 to a higher rate in Japan, with 2.5/100,000 new cases/year 9 . Following the Gulf War, the number of veterans diagnosed with ALS was reported to be 20 out of 690,000 7 , thus a ratio of 2.8 cases per 100,000 while the ALS incidence was ~ 1.7-1.8/100,000 in the US population between 1993-1994 9 .

Because of the short life expectancy -3 to 5 years after the onset -, the number of ALS patients living at a given time can be quite low compare to other rare diseases. Worldwide ALS prevalence is about 6 cases per 100,000 persons with some variation region to region. In Europe, the average prevalence is ~ 8 patients per 100,000 persons, with ~ 40,000 persons affected by ALS in 1995/2011 9 . It varies from 1.1/100,000 in Yugoslavia to 8-8.2/100,000 in the Netherlands, in some Italian regions and in the Faroe Islands respectively 7 . In the US, the prevalence was estimated at ~ 5 /100,000 population in 1994 11 . in Asia, the prevalence ranged from 1.0/100,000 in China to 11.3/100,000 in Japan 9 . ALS affects particularly elderly people and its prevalence increases with ageing, reaching a ratio of 20/100.000 in a population aged between 70-79 years old 11 . Consequently, as the population is ageing, it is expected that the total number of cases observed worldwide in 2005 -number ~ 222,801 -will increase by 69% in 35 years and reached 376,674 in 2040 12 .

Disease onset age for ALS ranges from 54 to 67 years old, with a mean age of 61.8 ± 3.8 years 9 and mean ALS diagnostic age is 64.4 ± 2.9 years 9 . There is an average diagnostic delay of 11-12 months 13 before reaching a definite diagnosis. The age of onset between sporadic and familial cases of ALS slightly differs with peak of age onset at 58-63 years for sporadic cases and 47-52 for familial cases 14 (Table 1). Three categories of onset can be observed in the total ALS population, with extremely rare juvenile cases where the onset occurs before 25 years old, cases with a young-onset from 25 to 45 years old 15,16 , and cases with old-onset where ALS patients are diagnosed in their seventh decade 17 . Worldwide men are diagnosed with ALS at 1.3 to 1.56 times the rate of women 7 , with an exception in China where male:female ratio ranges from 1.45 to 1.98 18 .

Pathological definition of ALS: clinical features and phenotype variability

ALS is a fatal neurodegenerative disorder with an adult onset, characterized by the degeneration of upper and lower motor neurons responsible for muscle denervation, leading to muscle weakness and wasting in ALS patients. Muscles controlling the speech, swallowing and other motor functions are dramatically affecetd 2 . ALS symptoms unceasingly spread and progress quickly causing paralysis and disability as patients lose their ability to control their muscles. With inexorable progression of symptoms appears intercostal and diaphragm muscles weakness following thoracic and cervical spinal cord involvement, responsible for common death due to respiratory failure of ALS patients within 3 years of first symptomatic features onset 2,19 . Despite these generic features observed across ALS patients, ALS phenotypes vary patient to patient as patients can present different site of onset and a variability in clinical features. For example, some patients can develop pure motor dysfunction ALS phenotype while others will present a mixed phenotype associated with extra-motor involvement such as behavioural changes and language difficulties typifying frontotemporal dementia (FTD) disease 3 (see I. 2. 4. Non-motor involvement in ALS: overlap with FTD).

 Clinical symptoms of ALS patients

El Escorial criteria characterises ALS by a progressive and relentless degeneration of both UMN and LMN 4 .

UMN and LMN dysfunction are associated with several clinical features in order to assess the certainty of the disease. UMN dysfunction includes pathological spread of reflexes and hyperreflexia. Various reflexes can be tested for UMN impairment, such as 1) the jaw jerk reflex, 2) Hoffmann's sign, and 3) Babinski reflex 20 . A positive jaw jerk reflex is identified when a sudden and upward jaw contraction is observed due to brisk contraction of the masseter muscles. The Hoffmann's reflex test is based on flexion and adduction of thumb after flicking the finger nail of the middle finger. Finally a positive Babinski reflex is seen when upward movement (or movement towards the top the foot) of the big toe after stroking the sole of the foot occurs. Additionally, patients can present spasticity causing stiffness and tightness in the muscle and involuntary muscle contraction, or clonus. Concerning bulbar-innervated muscles impairment, UMN dysfunction are defined as spastic dysarthria or difficult and laboured speech. LMN signs are characterized by muscle weakness and certain wasting accompanied by fasciculations of wasted limbs. Similarly, tongue wasting often with dysarthria and dysphagia, or difficulty to swallow, are typical features of bulbar LMN dysfunction 20 .

 Site of onset variability

Site of onset varies among ALS patients. In a majority of ALS cases (70%), symptoms initially begins in one limb 20 . Usually muscle weakness is focal and patients will present a foot drop or a clumsy hand 13 . The affected body region progressively become more and more weaken, and finally fasciculate and waste. UMN dysfunction might precede muscle atrophy such as spasms and cramps, and ultimately symptoms spread into the whole body affecting all the limbs. In 25% of ALS cases however, symptoms develop initially in the bulbar-innervated muscles 20 , also referred as bulbar palsy 21 . Usually bulbar-onset ALS is affecting less men than women 13 , especially after 70 years (M:F ratio 1:1,6 17 ). Dysarthria and dysphagia are frequently Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 8 associated with bulbar-onset ALS and cognitive impairment are often present 21 . For approximatively 3% and 5% 1 of ALS cases, respiratory or cognitive onset presentation respectively occurs 14 . Initial trunk or respiratory onset ALS is associated with poor prognosis, the mean survival time is 1.4 years with fast progressing disease only 22,23 . Cognitive onset ALS patients usually present characteristic FTD symptoms, such as a changes in behaviour, personality and cognition suggesting frontal impairment 24 .

 Age of onset variability

Although ALS occurs in patients in their sixth decade, ALS symptoms onset happens at almost all age (Table 1). Extremely rarely, <1/1,000,000 cases, juvenile ALS occurs in patients younger than 25 years old 15 . Juvenile ALS is usually associated with a slower symptoms progression, hence a longer survival time and better prognosis 23 . Some mutations are now described to be specifically related to juvenile ALS such as mutations in FUS, ALS2 or SETX genes 15 . UMN features dysfunction are predominantly represented among juvenile ALS cases. Young-onset ALS includes patients ranging from 25 to 45 years old 15 and upper motor neuron predominant is mainly observed in those patients (60%) 15 . Contrary to "classic ALS phenotype", bulbar-onset is more rare in young-onset ALS and represent ~15% of the cases 23 . In addition, a greater proportion of male are affected with a male : female ratio at 3:1 15 . These young-onset cases are also associated with a better prognosis than older ALS patients. For most patients, ALS symptoms onset peaks approximatively at 61 years old. A last ALS patient subgroup has been identified with an onset after 80 years old 17 . A likelihood of developing UMN predominant phenotype increases ~20% in old-onset ALS patients, as well as initial development of symptoms in bulbar muscles. Moreover, elderly bulbar-onset ALS cases present a greater proportion of females patients (M:F ratio 1-1.6) 15,17 . Symptoms onset after 80 years old is associated with a more aggressive disease and poor prognosis with mean survival time of less than 20 months 17 . Summary of classic clinical features for "Classic ALS" and age variants from Chio et al 25 , Forbes et al 17 , Swinnen et al 23 , Turner at al 15 , Sabetelli et al 16 , and Kiernan et al 14 . In addition to the classical ALS phenotype with age onset ranging from 45 to 70 years old (mean age ~ 61 years old), 3 additional ALSvariants (columns) are characterised depending on age of onset. Male to female ratios, genetic characteristics, site of onset, estimated survival time, and clinical features are further displayed in the following rows if applicable. sALS: sporadic ALS. fALS: familial ALS. UMN: upper motor neurons. LMN: lower motor neurons.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

 Motor neuron involvement in "ALS variants"

ALS patients can also present with either a LMN or UMN predominance (Figure 5). Signs of pure LMN dysfunction are considered as progressive muscular atrophy (PMA) cases whereas predominant UMN signs are associated with primary lateral sclerosis (PLS) 20 . PMA and PLS patients both are rare diseases and represent 5% of MND patients 23 .

UMN-dominant ALS variants:

At first assessment patients can present a "UMN-predominant" dysfunction revealed by UMN signs (see I.

2. 1. Clinical symptoms of ALS) with LMN signs usually initiated in distal upper limb before affecting the proximal upper limbs and in later stage of the disease lower limbs and respiratory muscles 20 . Clinical features showing UMN predominant phenotype can progress to ALS, and represent nearly 40% of PLS cases 26 . However if UMN dysfunction are still observed 4 years after disease onset a pure PLS is diagnosed 20 . Patients diagnosed with PLS for not meeting the diagnostic criteria for ALS can still slowly develop signs of LMN dysfunction and therefore present both UMN and LMN signs 23 . Prognosis of PLS patients is better than classic ALS cases as symptoms progression is slow. Yet if UMN signs are predominant in bulbar-innervated muscles patients are diagnosed with pseudobulbar palsy 27 .

Secretion of neurotoxic vesicles by muscle cells of ALS patients

²LMN-dominant ALS variants:

Conversely patients can also develop a LMN-dominant phenotype that includes PMA, flail-arm and flailleg syndromes "ALS variants". PMA patients are similar to classic ALS patients without obvious signs of UMN. However 50 to 60% PMA patients develop degeneration of upper motor neurons during the progression of the disease 28 . In patients with flail-arm or flail-leg syndromes, LMN dysfunction can remain limited to either the upper or lower limbs for at least 12 months 23 . When pseudobulbar palsy is described as a predominant UMN dysregulation in bulbar muscles, patients with bulbar LMN signs predominantly are diagnosed with progressive bulbar palsy 27 .

 Non motor involvement in ALS and overlap with FTD

For many years ALS was described as an exclusive neurodegenerative disorder with no extra-motor involvement, including cognitive defects. Today it is now accepted that other neurological components are impaired in ALS patients 3 . If early studies suggested low proportions of ALS patients experiencing non motor impairment (3% among sporadic cased and 15% among the familial cases 29 ), studies nowadays propose that approximatively 35% of ALS patients present behavioural and/or cognitive changes (ALSci or ALSbi) and 15% of them meet the Neary criteria 30 for FTD diagnosis (ALS-FTD) 29 . ALS and FTD sometimes are described as part of one continuum with pure ALS patients (without any extra motor involvement) and pure FTD cases (for whom no motor dysfunction have been described) represent opposite sides of the spectrum.

FTD disease comprise 3 variants defined by the Neary criteria 30 : frontal variant FTD (also called behavioural variant FTD 31 ), non-fluent progressive aphasia or semantic dementia. Usually ALS patients meet criteria for behavioural variant FTD characterized by defects in cognitive functions, personality traits and behaviour collapse. Among ALS cases experiencing extra motor dysfunction, language (particularly deficit in verbal fluency 31 ) and cognition are mostly affected, and apathy is the most frequent personality feature impaired encountered 29 in patients.

Dementia in ALS patients -ALS-FTD variants:

ALS-FTD diagnosis is made upon the presence of ALS phenotype associated with behavioural or cognitive defects meeting FTD diagnosis:

-Progressive impairment of behavioural/cognitive functions and observation of at least 3 of behavioural symptoms defined by Rascvosky et al 32 (see Table 2)

-Or loss of insight and/or psychotic features associated with at least 2 of Rascvosky et al 32 symptoms.

-Or language impairment combined with semantic dementia (defined in Neary et al 30 Cognitive changes in non-demented ALS patients -ALSci and ALSbi variants:

Non-demented ALS patients presenting with behavioural impairment are classified as ALSbi-variant, while ALS patients experiencing cognitive deterioration represented by language defects are considered to be ALSci-variant 29 . Based on the revised diagnostic criteria from Strong et al 33 , ALS patients can be diagnosed as ALSci variant if either 1) executive impairment (social cognition) or 2) language dysfunction or 3) a combination of the two features are evident during diagnosis (see Table 3). Diagnostic criteria for ALSbi variant require 1) apathy with or without other behavioural symptoms, or 2) two or more behavioural changes, such as disinhibition, loss of sympathy/empathy, perseverative/stereotype/compulsive behaviour, hyper orality/dietary change, loss of insight and psychotic symptoms (see Table 3).

Secretion of neurotoxic vesicles by muscle cells of ALS patients

ALS-ci Evidence of either executive or language dysfunction, or both

Executive impairment criteria

Impaired verbal fluency (letter). Verbal fluency deficits must control for motor and/or speech impairments to be valid Or impairment on 2 other non-overlapping measures of executive functions:

• Screening (ALS-CBS test ALS CBS is a brief measure of cognition and behaviour in patients with ALS)

 Diagnostic tools

In the absence of a diagnostic tool for ALS, evidence of progressive combination of UMN and LMN signs in patients is associated with positive diagnostic in the event of prior exclusion of ALS-mimic disease.

Since diagnostic criteria have been developed in 1990 by the World Federation of Neurology 4 to establish different level of disease burden (Table 4). ALS diagnosis based on those criteria demand evidence of disease progression within regions of the body that includes: 1) bulbar regions (speech and swallowing function dysregulation), 2) cervical regions (upper limb weakness and deterioration), 3) thoracic regions (abdominal muscles), and 4) lumbar regions (lower limb defect), associated with UMN and LMN Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 14 involvement signs. The original diagnostic criteria ranged diagnostic certainty into 4 categories: suspected ALS, possible ALS, probable ALS, and definite ALS. In the Airlie House criteria (revised El Escorial criteria 5 ) disease certainty is classified as clinically possible ALS to definite ALS, deleting the suspected ALS category from the diagnostic criteria and adding a new clinically probable ALSlaboratory supported.

More recently the Awaji-shima criteria further increased the specificity of ALS diagnosis, allow an earlier detection of LMN loss and simplified ALS diagnosis criteria into 3 categories including clinically possible, probable and definite ALS 6,34 .

The El Escorial diagnosis criteria 4 and its revisions

 ALSFRS

Without specific biomarkers for ALS, it is still challenging to study the rate of progression of ALS patients.

Today, the Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) is the most commonly tool used by clinicians and in clinical trials. The ALSFRS is a questionnaire-based tool establishing the level of impairment in physical function carried out during activities of daily living in ALS patients 36 . However the ALSFRS was lacking establishment of respiratory dysfunction progression. Hence development of revised ALSFRS to equally weight respiratory function disability to limb and bulbar function 37 . Additional questions regarding evaluation of respiratory defects progression have been developed and added to the questionnaire. The new ALSFRS-R questionnaire is composed of 10 questions scale ( In summary, ALS is a clinically heterogeneous disease with variable phenotypes that makes difficult to predict the progression speed of the symptoms as well as survival time. However multiples features of ALS have been associated with a poor prognosis (Figure 3). Classically disease onset in ALS patients is around 61 years old. Young age-associated ALS include juvenile-ALS and young-ALS with disease onset before 25 and 45 years old respectively and are usually associated with slower progression and better survival than "Classic-ALS" 15 . On the contrary, old-onset ALS is characterized by development of ALS symptoms later (after 70 years old) and is associated with poor prognosis, especially among elderly female with bulbaronset phenotype 17 . Initial site of symptoms onset varies among ALS patients from classic limb-onset to rare cognitive-onset phenotypes. Poor prognosis is often associated with bulbar and respiratory onset 14 . Finally, shorter predicted survival of time is also related with disease progression speed. The fastest the symptoms progress the more the survival time shortens. Disease progression can either be assessed depending on diagnostic delay or using ALS rating score (ALSFRS). Poor prognosis is associated with patients whose ALS diagnostic has been given under 8 months after symptoms onset or losing more than 1.4 points on the ALSFRS scale 27 .

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Genetic and sporadic forms of ALS

ALS is mainly sporadic with 90% of ALS cases (sALS) arising at random and approximatively 10% of ALS cases are familial (fALS) 7 . Today more than 30 genes mutations have been related to some sALS (10%) 39 patients as well as fALS cases (70%) 39,40 . The hexanucleotide expansion repeat in C9orf72 gene is the most common genetic cause in fALS (~25%) and sALS (~5%) patients, followed by SOD1 (~20% in fALS and ~2% in sALS cases), TARDBP/TDP43 (~5% in sfALS and ~1% in sALS patients), and FUS (~3% in fALS and 0.5% in sALS cases) 41 .

 Most common ALS associated gene mutations

Superoxide dismutase1, SOD1:

Secretion of neurotoxic vesicles by muscle cells of ALS patients

The first identified gene to be linked with ALS was SOD1 in 1993 42 . SOD1 is ubiquitously expressed in human cells, and is localized in the nucleus, mitochondria, and cytoplasm cell compartments. SOD1 protects cells from ROS (Reactive Oxygen Species) by catalysing the reduction of superoxide anion to oxygen and hydrogen peroxidase. Mutated form of SOD1 present a toxic gain of function responsible for neurotoxicity through ER stress, misfolded protein aggregates, and oxidative stress 40 .

C9orf72:

In 2011, the GGGGCC (G4C2) hexanucleotide repeat expansion mutation (HREM) within C9orf72 gene has been observed in patients with ALS and FTD 43,44 . The HREM is located in intron 1 of C9orf72 and induces pathological repeat in ALS patients. In healthy subjects, G4C2 repeat length ranges from 2 to 23 units 43 . Even if a pathological number of hexanucleotide units clear cut-off has not been established yet, large G4C2 expansion ranges from 30 and above have been observed in ALS patients 43,45 . Considering the localization of the repeat expansion on C9orf72 gene, several hypothesis of loss of function-or gain of function-mediated toxicity from the HREM in C9orf72 have been proposed (Figure 4). The first one is based on reduction of C9orf72 mRNA levels due to G4C2 repeats-mediated inhibition of transcription or happloinsufficiency hypothesis 40 . Sense and antisense RNA generated from the bidirectional transcription of G4C2 repeats have been proposed to induce a toxic gain of function in ALS patients' cells. Thus the G4C2 repeats may become toxic by sequestering RNA binding proteins, and forming RNA foci that will disrupt the RNA metabolism processing in cells 45 . A third hypothesis involves repeat-associated non-AUG translation (RAN) of G4C2 (or G2C4) repeats in dipeptide repeat proteins (DPR). RAN occurs in both sense and antisense reading frame 46 resulting in the production of 5 different DPRs: glycine-alanine (GA), glycine-arginine (GR), proline-arginine (PR), proline-alanine (PA), and glycine-proline (GP) 47 . And finally HREM in C9orf72 is associated with impaired nucleoplasmic transport mediated through repeat-RNA or DPRs 40 . Epidemiologic analysis allowed to establish an age-related penetrance of the pathogenic expansion in C9orf72 gene ranging from non-penetrant in carriers younger than 35 years, to 50% penetrant by mean age of ALS disease onset, and fully penetrant by 80 years. 48

TARDBP/TDP43:

A common pathological features in ALS histology is the cytoplasmic localization and accumulation of TDP43 proteins in ALS and FTD patients 21 . TARDBP gene encodes for TDP43 protein, a ribonucleoprotein that is ubiquitously expressed and described with a range of functions from gene transcription to mRNA biogenesis regulation 40 . In ALS patients, mutations of TARDP gene 49 leads to a mislocalisation of TDP43 from the nucleus to the cytoplasm where it accumulates in pathological inclusions. Both overexpression of mutated TDP43 forming toxic aggregates in the cytosol 50 and knock down inducing a nuclear depletion of TDP43 [50][51][52] lead to an ALS-like phenotype. These studies imply that both toxic gain and loss of function of mutant TDP43 hypothesis play a role in neurodegeneration occuring in ALS 46 .

FUS/TLS:

FUS mutations in ALS cases have been described in 2009 53,54 and induce variable phenotype in patients with a high frequency in juvenile-onset ALS 15 in the ER through activation of the ubiquitin-proteasome system (UPS) 56 .

VCP: Valosin-containing protein (VCP) is an ubiquitous protein belonging to the AAA+ ATPases protein family 57 . VCP is involved in various functions regarding protein homeostasis such as protein degradation, among others including a role in the UPS pathway 58 .

ANG: ANG encodes for angiogenin involved in the maintenance of a normal vascularisation by activating angiogenesis mechanisms in many tissues including the central nervous system 59 .

SETX:

Similarly to some FUS mutations, SETX gene is linked to juvenile-onset forms of ALS with slow progression of the symptoms and a longer life expectancy compared to classic ALS. Patients exhibiting SETX mutations, usually develop a classic ALS phenotypes, however an absence of bulbar and respiratory symptoms have been reported 60 . SETX gene encodes for a ubiquitously expressed protein involved in a various cellular function among them the RNA metabolism regulation.

SQSTM1: SQSTM1 gene encodes for sequestrosome 1 proteins also referred as to ubiquitin-binding protein p62. P62 protein is involved in a range of different function including proteasome-mediated protein degradation, and autophagy through its binding with LC3 autophagosome protein 61 . Mutations in SQSTM1 observed in rare ALS cases and in FTD patients 62 lead to p62 protein inclusions in motor neurons of both patients 63 . SQSTM1 ALS patients present a longer survival compare to most ALS patients.

ALS2 (Alsin): Like FUS and SETX, ALS2 gene mutations is associated with juvenile-onset ALS variant patients. So far, no known ALS2 mutations have been recorded in adult-onset ALS cases 64 . Similarly to VABP, alsin protein is involved in vesicle trafficking and axonal growth maintenance 64 .

OPTN: OPTN mutations mediated ALS cases reveal a high variability in clinical features phenotypes with an age of onset ranging from young-to old-onset ALS patients, and a disease progression varying from aggressive to up to 10 years disease duration 62 . Optineurin protein is an ubiquitous protein required in a Secretion of neurotoxic vesicles by muscle cells of ALS patients

variety of cellular functions such as autophagy maintenance where optineurin acts as a autophagy receptor, vesicle trafficking and Golgi homeostasis 65 .

TBK1: TBK1 mutations have been recently identified in ALS and FTD patients 40 . TBK1 is a multifunctional kinase associated with multiple signalling pathway such as autophagy through the phosphorylation and activation of autophagy adaptors such as optineurin and p62 proteins 66 .

UBQLN2: UBQLN2 mutations have been identified to participate in various ALS phenotypes variants (including familial juvenile-and adult-onset ALS and FTD patients) 67 . It was shown that both fALS and sALS can develop ubiquilin-positive inclusions 68 . Its function in UPS regulation, ER stress associated-ERAD pathway and autophagy suggest a role of ubiquilin in protein homeostasis 67 .

II. Molecular and cellular pathogenesis mechanisms in ALS

Numerous pathological mechanisms have been proposed to explain the neurodegeneration occurring in ALS. However the process is still not fully understood and appears to be multifactorial (Figure 5). The most studied pathological hypotheses in ALS includes mitochondrial dysfunction, glutamate-mediated excitotoxicity, axonal and vesicular trafficking dysregulation, RNA metabolism alteration and ROSassociated oxidative stress [69][70][71][72][73][74][75][76][77][78][79][80] . Numerous pathways dysregulation involving RNA processing have been studied in ALS patients such as RNA metabolism 78 , splicing defects 80 or impaired nucleoplasmic transport 46,81 . Impairment in protein homeostasis is also characteristic of ALS patients, including proteins aggregates toxicity 49 , DPR toxicity 82 , autophagy and lysosomal activity defects 83 and impaired ubiquitin proteasome system 84 . Moreover, neighbouring cells also participate in the toxic environment of motor neurons in ALS. Astrocytes and microglia cells are both involved in the release of inflammatory mediators contributing to neuroinflammation. Multiple pathological mechanisms have been proposed to be involve in ALS pathogenesis. Among the most studied and well known dysregulated pathways are: RNA metabolism, autophagy and protein homeostasis, neuroinflammation and glutamate excitotoxity.

Impaired protein homeostasis

Several gene mutations involve aggregations of misfolded proteins in abnormal cellular compartments accompanied by a failure of the proteasome-based and autophagic-degradation pathways 85,86 (Figure 6), both ubiquitin-proteasome system and autophagy protein degradation pathways being key actors in ALS pathogenesis 82 .

Figure 6 : Protein homeostasis dysregulation.

Protein homeostasis dysregulation is mediated by multiple pathways including defects in autophagy, dysregulation of ubiquitin-proteasome system (UPS) or endoplasmic reticulum stress. In the event of misfolded protein, activation of ERAD lead to a proteasome-mediated degradation to avoid accumulation of misfolded protein in the ER lumen and thus resulting in ER stress. Several ALS-associated gene mutations are reported to inhibit the proteasome-mediated toxicity via sequestration of the UPS pathway components such as ubiquilin and chaperone proteins. Proteolytic activity of the proteasome has also been demonstrated to be targeted by gene mutations in ALS. Once initiated, the autophagy pathway involves the formation and maturation of phagophores that will engulf selected transported-cargo and form autophagosome. Fusion with lysosome enables the degradation of the autophagosome content. Autophagy initiation and expansion via dysregulation of phagophore formation or impaired cargo transport are observed in ALS patients. ER: endoplasmic reticulum. ERAD: endoplasmic reticulum-associated protein degradation.

 Ubiquitinproteasome system (UPS) dysregulation

Evidence for UPS dysregulation in ALS patients started with the identification of mutations of genes encoding proteins such as ubiquilin 2 or VCP 87 , two proteins involved in protein clearance via the ubiquitinproteasome pathway. In addition, SOD1 85 , VABP 56 , HREM in C9orf72 70,88 , and p62 89 mutants are reported to reduce UPS activation. Further studies illustrate the dysregulation of the proteasomal pathway, as ubiquitin-positive inclusions were observed in motor neurons of fALS and sALS 84 patients as well in postmortem neuronal and muscular tissue of C9orf72-associated ALS patients 90 . Concordantly, the sequestration of proteasome component in mutated SOD1 aggregates 91 , and chaperone proteins in ubiquilin

 Autophagy defects

A neuroprotective effect was observed on primary and human iPSC-derived neurons with TDP43 mutations when the autophagy was stimulated and led to a greater clearance of TDP43 proteins [START_REF] Lundberg | Molecular effects of exercise in patients with inflammatory rheumatic disease[END_REF] . C9orf72-ALS patients bear signs of C9orf72 loss of function such as hypersensitivity to autophagy defects and decrease in autophagy function supporting the implication of autophagy dysregulation in ALS patients.

Concordantly, C9orf72 knockdown efficiently inhibits the autophagy and generates ubiquitin-, p62-, and TDP43-positive inclusions in cytoplasm 45,83 , suggesting a significant role of autophagy in protein aggregation feature observed in ALS and the neurodegeneration process. Multiple pathological processes might be involved in autophagy dysregulation. Degradation of ubiquitinated protein through autophagy pathway includes 1) initiation and extension of bilayer vacuole into phagophore, 2) transport of selective cargoes (including ubiquitinated protein, dysregulated mitochondria or protein aggregates), 3) maturation into autophagosome, and 4) fusion with lysosome, forming autolysosome inducing degradation of content.

Several ALS associated genes encode for protein involved in early steps of autophagy regulation such as optineurin 94 , p62 89 , and ubiquilin 67 . Impairment of TBK1 95 and p62 96 functions inhibit the autophagic cargo delivery towards the autophagosome. Finally, the autophagosomes maturation is impaired in ALS cells carrying FUS 97 , VCP 21 , and CHMP2B 98 mutations. Altogether, these studies illustrate numerous autophagic steps impaired in ALS patients (illustrated in Figure 6).

 Protein aggregation

In most cases, TDP43 inclusions 99,[START_REF] Abou-Khalil | Regulation of myogenic stem cell behavior by vessel cells: The "ménage à trois" of satellite cells, periendothelial cells and endothelial cells[END_REF] and neurofilament-positive aggregation 46 are a hallmark of ALS pathology and observed in the cytoplasm of both sALS and fALS. In accordance with UPS defects, ubiquitin-positive protein aggregation are observed in neurons 88 and sometimes in skeletal muscles 43 of ALS patients. These ubiquitin-positive inclusions can also contain/"trap" non-mutated forms of SOD1 101 , TDP43 [START_REF] Liu | TGF-␤ inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3[END_REF] , optineurin 77 , and ubiquilin 2 68 , proteins associated with ALS. In addition, SOD1 [START_REF] Sousa-Victor | Geriatric muscle stem cells switch reversible quiescence into senescence[END_REF] , FUS 53 , and C9orf72-derived dipeptides repeat protein [START_REF] Fukada | Adult stem cell and mesenchymal progenitor theories of aging[END_REF] can generate toxic aggregates. Overall, protein aggregation is a common feature in ALS and underline a defect in protein clearance.

Aberrant RNA metabolism

FUS and TDP43 are RNA-binding proteins involved in multiples steps of RNA metabolism. In ALS patients, both mutant proteins are mislocalised in the cytoplasm resulting in the loss of function and affecting the RNA processing. RNA metabolism is a key feature of ALS pathogenesis and includes transcription defects, alternate splicing changes, miRNA biogenesis, and stress granules formation (Figure 7). In addition, FUS and TDP43 proteins interact with numerous targets expanding the risk of RNA processing dysfunction even further. TDP43 binds to over 6,000 targets in the brain 80 , similarly FUS interacts with numerous binding targets in the brain [START_REF] Kumar | Senescence-associated secretory phenotype and its possible role in chronic obstructive pulmonary disease[END_REF] . Many processes in RNA and miRNA pathways are disrupted in ALS patients, including transcription defects, alternate splicing events, miRNA biogenesis and nucleus-cytosol transport impairment. RNA metabolism defects are particularly relevant in ALS pathogenesis since TDP43 and FUS are both wellknown ALS-associated genes involved in RNA processing. Both FUS and TDP43 mutated proteins misslocalize to the cytoplasm of ALS motor neurons, leading to a probable loss and toxic gain of function of these proteins.

 Transcription defects:

Given the large number of possible protein-protein interaction between FUS or TDP43 and their partners, it is easy to expect important RNA processing defects in ALS patients 21 . Among the differentially expressed binding targets of FUS and TDP43 in ALS, multiple proteins are involved in neuronal physiology such as components of the synaptic plasticity pathways 78,79,[START_REF] Demaria | Cell Autonomous and Non-Autonomous Effects of Senescent Cells in the Skin[END_REF] and dendritic branching process 79,[START_REF] Behringer | Spreading the signal for vasodilatation: Implications for skeletal muscle blood flow control and the effects of ageing[END_REF][START_REF] Leiter | Nitric oxide and voluntary exercise together promote quadriceps hypertrophy and increase vascular density in female 18-mo-old mice[END_REF] [START_REF] Lai | Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy[END_REF] or sequester TDP43 [START_REF] Lai | Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy[END_REF][START_REF] Song | Exercise training modulates the nitric oxide synthase profile in skeletal muscle from old rats[END_REF] and FUS [START_REF] Lai | Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy[END_REF] proteins, indirectly inhibiting transcription of RNA-metabolism associated gene. Similarly to C9orf72-mediated RNA processing defects, FUS mutant has been associated with major transcriptional defects due to its function in transcription with RNA polymerase II [START_REF] Anderson | A role for nitric oxide in muscle repair: Nitric oxide-mediated activation of muscle satellite cells[END_REF] .

 Alternate splicing impairment:

TDP43 knock down in murine tissues results in alternate splicing changes in 965 mRNA transcripts 52,80 and loss of function of FUS induce splicing defects in more than 300 genes 78 , suggesting important alternate splicing event in ALS patients. It is not surprising regarding the ability of FUS to sequester numerous component of the splicing process such as key splicing factors [START_REF] Goldspink | Agerelated changes in collagen gene expression in the muscles of mdx dystrophic and normal mice[END_REF] , U1 snRNP and U11/U12 snRNPs [START_REF] Leiter | Nitric oxide and voluntary exercise together promote quadriceps hypertrophy and increase vascular density in female 18-mo-old mice[END_REF][START_REF] Ferreira | Microfluidic analysis of extracellular matrix-bFGF crosstalk on primary human myoblast chemoproliferation, chemokinesis, and chemotaxis[END_REF] involved in minor introns splicing. Alternative splicing changes have been identified in neuronal genes involved in cytoskeleton organization, axonal growth and guidance in FUS-mutated ALS patients [START_REF] Fannon | Facilitated diffusion of VEGF165 through descemet's membrane with sucrose octasulfate[END_REF][START_REF] Bernet | p38 MAPK signaling underlies a cellautonomous loss of stem cell self-renewal in skeletal muscle of aged mice[END_REF] , and interestingly axonopathy or axon retraction are early stages of ALS [START_REF] Saini | From death, Lead me to immortality"-mantra of ageing skeletal muscle[END_REF] .

 miRNA biogenesis dysfunction:

Evidence for miRNA processing defects have been obtained from studies reporting differential expression of several miRNA in TDP43 (miRNA9, miRNA132, miRNA143 and miRNA558) [START_REF] Liu | Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging[END_REF] and FUS (miRNA125 and miRNA192) [START_REF] Li | Age-Specific Functional Epigenetic Changes in p21 and p16 in Injury-Activated Satellite Cells[END_REF] patients. It has been demonstrated that both TDP43 and FUS, as well as C9orf72mediated DPRs interacts with Drosha [START_REF] Liu | Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging[END_REF][START_REF] Horvath | DNA methylation age of human tissues and cell types[END_REF][START_REF] Bocker | Genome-wide promoter DNA methylation dynamics of human hematopoietic progenitor cells during differentiation and aging[END_REF] . Additionally TDP43 protein associates with the DICER enzyme [START_REF] Liu | Chromatin modifications as determinants of muscle stem cell quiescence and chronological aging[END_REF] , results that are all consistent with a defective miRNA processing in ALS patients.

 Stress granules formation:

In response to stressful conditions, RNA granules also known as stress granules are generated. Under those conditions, FUS and TDP43 can be recruited to stress granules 46,[START_REF] Blaze | Evidence from clinical and animal model studies of the long-term and transgenerational impact of stress on DNA methylation[END_REF] . However in ALS patients, mutations in those RNA binding proteins disrupt stress granules dynamics. Mutants FUS and TDP43 have been shown

to increase the persistence of stress granules in the cytoplasm resulting in a possible toxic gain of function 86 by inhibiting translation and thus contributing to ALS pathological progression. In mutants C9orf72 ALS patients, stress granules also involves sequestration of other proteins such as RAN GAP [START_REF] Cencioni | Oxidative stress and epigenetic regulation in ageing and age-related diseases[END_REF] , a protein required for functional nucleoplasmic transport.

Mitochondrial dysfunction

Mitochondria is the power house of the cell and source of intracellular energy or ATP and are required in cellular respiration, calcium buffering and regulation of apoptosis. Functional mitochondrial defects 69 such as altered morphology and swelling have been identified in neuronal and muscular tissues of ALS patients carrier of SOD1 21 and C9orf72 mutations 21,70 . Several mechanisms can trigger mitochondrial dysfunction in the cell. SOD1 mutant deposition in mitochondria [START_REF] Claflin | Age affects the contraction-induced mitochondrial redox response in skeletal muscle[END_REF] induce the formation of vacuoles in the mitochondrial inter-membrane space as well on the cytoplasmic face of the outer membrane [START_REF] Liu | Microarray analysis reveals novel features of the muscle aging process in men and women[END_REF] .

Consequently, mitochondrial respiration and energy production are defective and lead to oxidative stress generation. Oxidated proteins and lipids in the mitochondria then exacerbate the respiratory chain dysregulation and the oxidative stress [START_REF] Brioche | Growth hormone replacement therapy prevents sarcopenia by a dual mechanism: Improvement of protein balance and of antioxidant defenses[END_REF] . To maintain axonal integrity and survival, neurons transport all components to their distal sites, including mitochondria. Defective mitochondrial transport is mediated through SOD1 mutants in ALS 74 and is responsible for a decrease in ATP production, calcium mishandling 69 at the neuromuscular junction contributing to distal axonopathy. Defective axonal transport induce an accumulation of impaired mitochondria and a decrease in recycling of dysfunctional mitochondria in distal sites [START_REF] Liu | Microarray analysis reveals novel features of the muscle aging process in men and women[END_REF] . Pro-apoptotic signalling activation through impaired mitochondria dysfunction [START_REF] Liu | Microarray analysis reveals novel features of the muscle aging process in men and women[END_REF] such as the caspase-dependent [START_REF] Signer | Mechanisms that regulate stem cell aging and life span[END_REF] or bcl-2-dependant pathways [START_REF] Liu | Microarray analysis reveals novel features of the muscle aging process in men and women[END_REF] might be responsible for motor neuron degeneration. Not only the mitochondria are impaired in SOD1 mutated cells, but also the calcium homeostasis [START_REF] Zampieri | Reconfiguration of DNA methylation in aging[END_REF] . Calcium level dysregulation will then affect many metabolic pathway in motor neurons, as well as the synaptic communication. For instance, a decrease in calcium homeostasis is associated with higher risk of motor neuron damage, and a greater sensitivity to glutamate excitotoxicity [START_REF] Zampieri | Reconfiguration of DNA methylation in aging[END_REF] .

Nucleoplasmic transport defects

Cells actively transport proteins and RNA from the cytoplasm to the nucleus and vice-versa via numerous protein transporters. An impaired nucleoplasmic transport of molecules have been identified in C9orf72 ALS patients 21 . Like FUS and TDP43, C9orf72 mutations can generate stress granules and sequester proteins required for effective nucleoplasmic transport such as RAN GAP [START_REF] Cencioni | Oxidative stress and epigenetic regulation in ageing and age-related diseases[END_REF] or importins and exportins proteins 81 . Generation of repeat-RNA C9orf72 is one of the mechanisms associated with C9orf72 toxicity in ALS patients. By sequestering the nuclear export adaptator serine/arginine-rich splicing factor (SRSF1), the repeat-RNA C9orf72 are efficiently transported in the cytoplasm were translation of DPR can occur [START_REF] Renault | Regenerative potential of human skeletal muscle during aging[END_REF] .

Newly formed DPR like poly PR, in turn binds to nuclear pores transporters impairing the import and export of molecules and thus taking part in impairing nucleoplasmic transport 46,81 .

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Endosomal and vesicular transport impairment

Trafficking of endosome and vesicles in cells is mediated through proteins of the large Rab family. C9orf72 has been proposed to activate Rab proteins because of its structural similarities with the DENN guanine nucleotide exchange factor involved in vesicular trafficking 45 . A reduced endocytosis and defective endosomal/lysosomal trafficking have been described in C9orf72 mice models, and ALS patients 45 . Genetic evidence also suggest impairment of endosomal and vesicular trafficking pathways through several mutations associated with ALS. Thus mutations occurring in the following genes are highly associated with endosomal defects: 1) alsin protein 64 a guanine nucleotide-exchange factor involved in endosomal pathway such as endosome motility and fusion with lysosome, 2) VCP [START_REF] Malmgren | Numerical densities of myonuclei and satellite cells in muscle fiber types in the aging human thyroarytenoid muscle: An immunohistochemical and stereological study using confocal laser scanning microscopy[END_REF] a ubiquitous AAA+ ATPase that interacts with clathrin protein during endocytosis, 3) VABP component of vesicular trafficking pathway, and 4)

CHMP2B or charged multi vesicular protein 2B 98 and optineurin 77 two components of the endosomal sorting complex required for transport (ESCRT) involved in the cargo sorting into intraluminal vesicles.

Axonal transport dysregulation

Motor neurons have particularly long axons up to 1 meter, and their physiological function relies on the transport of organelles such as mitochondria and divers molecules (RNA, proteins and lipids). Protein translation at the neuromuscular junction requires the transport of translation components, including RNA and ribosomal proteins towards synaptic structures [START_REF] Cousin | Regenerative capacity of old muscle stem cells declines without significant accumulation of DNA damage[END_REF] . The anterograde transport in axons, or movement from cytoplasm towards distal sites, depend on microtubule-dependent kinesin and dynein motors 74 . In both sALS and fALS patients, neurofilament network disorganization have been reported 47 . In SOD1 mutant models, both anterograde and retrograde transport are impaired 74 . The high level of tumor necrosis factors in mutated SOD1 motor neurons leads to a disruption of the motor-kinesin dependent axonal transport [START_REF] Alsharidah | Primary human muscle precursor cells obtained from young and old donors produce similar proliferative, differentiation and senescent profiles in culture[END_REF] , results in an axonal accumulation of defective mitochondria and autophagosomes, a decrease in mitochondria-dependent energy production 74 , and ultimately leads to motor neurons death.

Glutamate excitotoxicity

In healthy motor neurons, glutamate, which is the most common neurotransmitter, is released from the presynaptic neurons into the synaptic cleft activating NMDA and AMPA receptors that will mediate an influx of calcium and sodium in the postsynaptic neuronsinflux necessary to generate an action potential.

Glutamate is then removed from the synaptic cleft by glutamate transporters or excitatory amino acid transporter 2 (EEAT2) located at the membrane of the surrounding astrocytes and postsynaptic neuron.

Glutamate excitotoxicity refers to an excessive release of glutamate resulting in abnormal activation of glutamate receptors and thus excessive influx of calcium in the postsynaptic neuron causing an extreme firing of neurons [START_REF] Spalding | Retrospective birth dating of cells in humans[END_REF] . Glutamate excitotoxicity is thought to be mediated through disruption of intracellular Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 30 calcium levels and generation of ROS that will impair normal mitochondrial function. In ALS patients the function of EEAT2 glutamate transporter is disrupted in surrounding astrocytes which contribute to the accumulation of glutamate in synaptic clefts 21 , and higher levels of glutamate in the CSF of patients have been measured [START_REF] Spalding | Retrospective birth dating of cells in humans[END_REF] . Today Riluzole is the only drug validated to slow disease progression, and is believed to target glutamate excitotoxicity damage through its anti-glutamatergic properties 2 .

Oxidative stress

Oxidative stress results from an imbalance between production and elimination of ROS, and the ability to repair ROS-mediated toxicity. Oxidative stress has been of particular interest in ALS pathogenesis as SOD1 is a major antioxidant proteins. An increase in oxidative damage in ALS patients was among the first hypothesis of how SOD1 mutations could drive toxicity. In fact several body fluids from sporadic ALS patients, including CSF, serum and urine show an elevation of oxidative stress biomarkers 71 . Likewise, TDP43 mutants also induces an increase in free radical damage [START_REF] White | Dynamics of muscle fibre growth during postnatal mouse development[END_REF] . At the cellular level, accumulation of ROS has been linked with oxidative damage to protein, RNA, DNA, and lipids in post-mortem tissue from both sporadic ALS and SOD1 mutation-related familial ALS cases 72,[START_REF] Lee | Aged muscle demonstrates fiber-type adaptations in response to mechanical overload, in the absence of myofiber hypertrophy, Independent of satellite cell abundance[END_REF][START_REF] Fry | Inducible depletion of satellite cells in adult, sedentary mice impairs muscle regenerative capacity without affecting sarcopenia[END_REF] . In healthy cells under oxidative stress, the transcription factor NRF2 (Nuclear erythroid 2-Related Factor) will activate the expression of antioxidant response element (ARE) genes that encode for anti-oxidant responsible for ROS removal [START_REF] Takeuchi | Prednisolone improves walking in Japanese Duchenne muscular dystrophy patients[END_REF] . In ALS patients the NRF2-ARE signalling pathway is impaired [START_REF] Hussein | Steroid therapy is associated with decreased numbers of dendritic cells and fibroblasts, and increased numbers of satellite cells, in the dystrophic skeletal muscle[END_REF] , the oxidative stress is out of control and will induce damages that interact with other pathological mechanisms such as mitochondrial dysfunction, ER stress and impaired protein homeostasis, potentially exacerbating their damage to cells and ultimately contributing to the neuronal loss.

ER stress

During the formation of misfolded protein, unfolded-protein response (UPR) initiate the recognition of these proteins and transport to the ER where ER-resident protein chaperones will properly fold the protein [START_REF] Anderson | Article a micropeptide encoded by a putative long noncoding RNA regulates muscle performance article a micropeptide encoded by a putative long noncoding RNA regulates muscle performance[END_REF] . Accumulation of misfolded proteins in the ER activates the ER stress response pathway or endoplasmic reticulum-associated protein degradation (ERAD) and consists in the translocation of misfolded protein from the ER lumen to the cytosol [START_REF] Anderson | Article a micropeptide encoded by a putative long noncoding RNA regulates muscle performance article a micropeptide encoded by a putative long noncoding RNA regulates muscle performance[END_REF] . Misfolded protein will undergo ubiquitination and degradation through the proteasome machinery [START_REF] Anderson | Article a micropeptide encoded by a putative long noncoding RNA regulates muscle performance article a micropeptide encoded by a putative long noncoding RNA regulates muscle performance[END_REF] . In ALS patient cells, signs of ER stress have been demonstrated through activation of UPR markers and their co-localisation with mutated SOD1 inclusions [START_REF] Barrett | NCBI GEO: Archive for functional genomics data sets -Update[END_REF] . Increase in toxic inclusions number through inhibition of UPR marker suggest protective role of UPR component. Additionally, CSF of ALS patients also displayed an accumulation of ER stress markers [START_REF] Barrett | NCBI GEO: Archive for functional genomics data sets -Update[END_REF] and exposing healthy neurons to ALS patients' CSF induce ER stress with ER fragmentation and caspase-dependent apoptosis activation [START_REF] Clark | The characteristic direction: A geometrical approach to identify differentially expressed genes[END_REF] . ER dysregulation have been associated with C9orf72-

Secretion of neurotoxic vesicles by muscle cells of ALS patients

derived DPRs (such as poly-GA) and mutated SOD1 85 through binding to cytoplasmic face of ER surface and leading to an inhibition of ERAD response.

Motor neuron vulnerability

The primary feature of ALS is the selective loss of motor neurons, however why this neuronal group is more vulnerable than other is not fully understood. A major characteristic of motor neuron includes their large size and long axons with a cell body of 50-60µm and axons length up to 1 meter [START_REF] Chen | Enrichr: Interactive and collaborative HTML5 gene list enrichment analysis tool[END_REF] . Functional motor neurons have high demands in metabolic support and efficient axonal transport to support their demands in energy. Optimal mitochondrial function to supply sufficient energy is thus essential for motor neurons.

However, mitochondria are also responsible for ROS generation, and an increase in mitochondrial demand results in a higher risks of ROS production [START_REF] Chen | Enrichr: Interactive and collaborative HTML5 gene list enrichment analysis tool[END_REF] . Mitochondria in ALS spinal motor neurons are more sensitive to toxicity and cell damage 142 therefore causing a motor-neuron-specific mitochondrial dysfunction risk.

SOD1 mutant-mediated toxicity include numerous mechanisms (mitochondrial function 74,[START_REF] Claflin | Age affects the contraction-induced mitochondrial redox response in skeletal muscle[END_REF][START_REF] Liu | Microarray analysis reveals novel features of the muscle aging process in men and women[END_REF] , oxidative stress 72,143 , ER stress 85 , reduced proteasomal activity 91 ) and a higher physiological expression of SOD1 in motor neurons 144 involves a greater susceptibility to mutant SOD-mediated toxicity. Finally, motor neurons are highly sensitive to glutamate excitotoxicity especially through an increase in intracellular calcium concentration as motor neuron possess a reduced calcium buffering activity and more calcium permeable AMPA receptors 145 .

Introduction

Intercellular communication occurs in many physiological and pathological situations, and has first been characterized by a direct cell-to-cell interaction or secretion of soluble factors. Extracellular vesicles (EVs) are also now considered as mediators of cell-cell communication [146][147][148][149][150] . Based on their physical properties and biogenesis, EVs can be categorized into 3 groups: 1) exosomes, 2) ectosomes or shedding microvesicles and 3) apoptotic bodies (Reviewed in 151 ). They share common features such as a lipid bilayer delimiting a round structure containing a cargo of proteins, genetic materials (RNA, miRNA, DNA), lipids and metabolites. However, their origin (endosomal or from the plasma membrane), biogenesis and buoyant densities differ significantly 151 .

Exosomes are 50-100nm vesicles formed by inward budding of endosomal compartment membranes 152 .

Intraluminal vesicles (ILVs) or exosomes progressively accumulate inside multivesicular structures (called multivesicular bodies, MVBs) that can either fuse with lysosomes for degradation or with the plasma membrane (PM) to release the exosomes into the extracellular space. Once extracted, exosomes present a characteristic cup shape morphology by electron microscopy. Ectosomes (also described as shedding microvesicles or microparticles) are larger vesicles 100-1000nm, and are formed and secreted at the plasma membrane of cell through outward budding. Apoptotic bodies just like ectosomes are a heterogeneous population of 50-500nm vesicles secreted from cells subjected to apoptotic cell clearance. Contrary to the cup-shape appearance of exosomes observed by electron microscopy, microparticles and apoptotic bodies appear as light vesicles presenting heterogeneous sizes 153,154 .

In the 1990's, exosomes secreted by B cells were recurrently observed enriched in MHC class I molecule and consequently suggested to act as antigen-presenter vesicles 148 . Since then, their content has been increasingly investigated (more than 11,000 publications since 1996). It is know that they vehicle functional proteins including diverse signalling proteins and metabolic enzymes, modifying the phenotype of recipient cells [155][156][157] . Nowadays, exosomes are suggested to be important mediators of intercellular communication and organ crosstalk.

I. Exosomes discovery

The secretion of exosomes by cells was first characterized by two studies published in the 1980s 158,159 describing the release in the extracellular space of the transferrin receptor (TfR) by reticulocyte during their maturation process into erythrocytes. The iron transport receptor was secreted in ~50nm vesicles originating from multivesicular organelles that fused with the plasma membrane (PM) of the cell. Using electron microscopy, Harding et al managed to follow the TfR stained with gold particles in the reticulocytes. They Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 34 speculated that the decrease in TfR staining was due to TfR degradation via lysosomes during the maturation of reticulocytes into erythrocytes. However, only rare lysosomes were positive for TfR. Instead, they observed that small 50nm vesicles enclosed inside multivesicular bodies were positive for TfR. These multivesicular bodies fused with the PM, and the vesicles positive for TfR were released in the extracellular space. These vesicles could be then isolated from cell culture supernatant by ultracentrifugation at 100,000g for 90mn. The term exosomes was used for the first time to define the secreted vesicles originating from the invagination of the multivesicular bodies membrane and released by fusion with the plasma membrane of the reticulocytes 160 .

Since 1980's, research on exosome biogenesis and understanding their functions grown exponentially.

Today, many different cell types are known to release exosomes into the extracellular space and found them in biological fluids (Table 1).

II. Physical characteristics of exosomes 1. Exosomes isolation methods

Exosomes have been isolated from conditioned cell culture supernatant and body fluids as plasma, serum or saliva (see Table 1). Yet there is no gold standard protocol for exosomes extraction and the best method must be determined depending on the biological question raised by the study. Exosomes are found in serum used for cell culture. It is therefore important to avoid contamination by serum-exosomes by either growing cells in the absence of foetal bovine serum to produce an exosomes-serum-free medium or deplete the serum from any exosomes by ultracentrifugation if cells do not survive in serum-free conditions 213 .

The most commonly used method for exosomes isolation is ultracentrifugation 214 , allowing small vesicles as exosomes to cluster at the bottom of the tube at 100,000g. Usually prior to exosomes sedimentation, several centrifugation steps are performed to remove larger vesicles and contaminants potentially sedimenting at 100,000g (300g to remove cells, 2000g to pellet dead cells and 10,000g for cell debris and 50,000g for ectosomes and apoptotic bodies) 213 . Various modification have been proposed over the last decade to improve the quality of extracted exosomes such as 1) higher speed ultracentrifugation (110,000g 198,215,216 or 140,000g 217,218 ) and 2) additional purification steps by filtration of large debris and vesicles using 0.1 to 0.22µm filters 213,219 . Ultracentrifugation doesn't allow the purification of exosomes as vesicles and proteins aggregates contaminants can be isolated from the 100,000g pellet as well 219 .

Sucrose (or iodixanol) gradients allow the separation of exosomes from protein aggregates and non-specific associated exosome proteins based on their different capacity to float on sucrose gradient 213 . Due to their small size, exosomes have buoyant densities ranging from 1.13 to 1.19g/ml (See Table 2), while protein aggregates will sediment and pellet at the bottom of the gradient. Similarly to ultracentrifugation modified protocols, additional steps can be added to increase the purity of exosomes by combining floatation on sucrose gradients with filters, concentration columns and/or ultracentrifugation to remove diverse contaminants. Sucrose gradients have been shown to give the purest population of exosomes compare with other extraction protocols such as ultracentrifugation 220 , but it requires more sample and time.

Immunoaffinity based protocols allow the isolation of exosomes depending on their membrane molecular composition. So far, multiple exosomal markers have been used in immunoaffinity protocols and relatively pure vesicle subpopulations have been obtained as no protein contaminants are isolated with the exosomes.

This strategy requires the identification and detection of a membrane protein of interest that may not be expressed across all exosomes, and thus ignoring a portion of the exosome population. Moreover, once bound to affinity-beads, exosomes are not easily eluted, rendering difficult to test their effects on other cells 213 .

Finally commercial kits (eg Total exosomes isolation from Life technologies or ExoQuick) based on polymers base preparations 221,222 allow a rapid and easy extraction of exosomes using short low-speed centrifugation. This rapid strategy decrease the risk of exosomal content degradation and allow to extract exosomes from large fluid volumes -as needed in cell culture condition. However ready-to-use kits are usually more costly than other isolation methods and are still considered as questionable methods despite their efficient extraction of exosomes from various cell culture supernatants 187,194,207 .

Secretion of exosomes subpopulations

Cells secrete a heterogeneous population of exosomes, in terms of molecular composition, physical properties such as the buoyant densities and morphology. During the isolation of secreted vesicles, several subpopulations of exosomes are commonly mixed leading to a bulk analysis of EVs. Depending on the isolation strategy used, the exosome characterization may thus vary. Ultracentrifugation extraction method takes into consideration the size and density of the vesicles while the immunoprecipitation technique is based on the molecular composition of the exosomal membranes and the presence of the protein of interest.

Below will be developed the typical vesicle features explaining the exosome heterogeneity.

Heterogeneity can easily be observed in term of size as exosomes usually range between 30 to 100nm as measured by Théry et al 223 (Table 7 summarizes studies reporting heterogeneous population of exosomes).

Surprisingly, same cells, eg prostate epithelial cells, can produce two populations of exosomes, one fraction larger than the other 224 . Exosomes can be categorized in distinct subpopulation according to their morphological aspect observed by cryo-electron microscopy (cryo-EM) 210,225 . Contrary to EM, the cryo-EM technique does not require the sample to be stained or fixed, preserving the near-atomic resolution of the exosomes and allowing the visualization of both internal and surface features 226 . Different subgroups based on exosomal morphology have been identified: 1) multiple vesicles (corresponding to single/double/triple or more vesicles, i.e. vesicles containing secondary or tertiary vesicles), 2) elongated or oval-like structured vesicles (most of the vesicles have a round shape, but elongated, egg-shaped or even tubular vesicles have also been identified), 3) vesicles coated with external features such as protrusions and 4) electron dense vesicles 225 .

Different exosome subpopulations have been identified using buoyant density properties 224,227,228 .

Depending on their composition, exosomes float at different densities 227 . Moreover, exosome composition will affect the speed to reach the equilibrium buoyant densitywith exosomes enriched in tetraspanins reaching slowly this equilibrium 224 . Not only the exosome composition will affect their migration speed across the sucrose gradient, but also their size. For example, small vesicles (approximatively 60nm) will reach their equilibrium density faster than large vesicles (100nm average vesicles) 224,228 . Consequently, a longer ultracentrifugation (62h) allow the isolation of exosomes having the same buoyant density.

The RNA and protein content varies from exosome to exosome even originating from the same cells, suggesting the existence of different exosomes subpopulation 178,224,225,[227][228][229][230][231] . This is particularly true for polarized cells where two distinct groups of exosomes can be secreted at different cell membrane location (eg apical vs basolateral 178 ). RNA occupancy varies from exosome to exosome, introducing the hypothesis that a subgroup of exosomes will have a high miRNA content while other will have a low concentration.

To illustrate this, one copy of the miRNA (eg miR-126, miR-223 and miR-720) was observed in only one exosome out of hundred 229 . In addition, the pathway involved in the release of the MVB content in the extracellular space will influence the protein composition of the exosome membrane (see III. This table summarizes studies conducted on multiple exosomes including vesicles originating from cell culture supernatant from cancerous and non-cancerous cells or biological fluids. Exosomes are ranked depending on their buoyant flotations (in sucrose g/ml) and associated with the mean vesicle size in nm as measured by electron microscopy (EM) or nanoparticle tracking analysis (NTA) methods. Size ranges and density properties are reported in the table, including classic exosome markers used to identify exosome populations on density gradient separation (listed in third column).

III. Exosomes biogenesis and secretion 1. Molecular content of exosomes

Exosomal protein and lipid composition come from different cellular compartments (cytosol, PM or endocytic compartments) and will vary among different cell types. Lipid bilayer membrane delimitedexosomes are enriched in ceramide, cholesterol, phosphatidylserine andsphingolipids [232][233][234][235][236] , as well as in tetraspanin membrane organisers (CD9, CD81, CD63 and CD82 for example) 223,235 . Membrane transport and fusion proteins (cytoskeletal, annexins or flotillin proteins), adhesion molecules (integrins and lactadherins) are also found at the exosome membrane. Transmembrane proteins will be incorporated during the invagination process of the endosomal membranes while cytosolic cargoes are incorporated within the ILVs. Most abundant proteins identified in the exosome lumen include proteins required for the biogenesis and function of exosomes such as ESCRT proteins machinery and the associated proteins ALIX and TSG101 223,235,237 proteins shared across different exosome subpopulations. Depending on the donor cells, ILVs are loaded with numerous ubiquitous proteins and cell-type-specific proteins, suggesting variations in exosomes molecular composition between cell type and conditions. Additionally, the exosomal lumen contains nucleic acids such as mRNA, miRNA, long non-coding RNAs and t-RNA 149,238- 240 . In summary, exosomes contain various intraluminal cargo including proteins, nucleic acid and lipids.

However, most studies in the literature have performed exosomes molecular composition of bulk populations of secreted vesicles, masking the ILV-specific composition involved in the fate and function of exosomes (reference to paragraph 2.2).

Exosomes cargo sorting

Molecules are not randomly loaded in exosomes, protein cargo sorting inside EV is controlled by specific machineries. Proteins can undergo post-translational modifications (PTM) (including ubiquitination, sumoylation or phosphorylation) and can act as protein cargo sorting signal in exosomes 241 . Ubiquitination of protein is the most important modification involved in sorting of protein in exosomes and involves the endosomal sorting complex required for transport (ESCRT) protein machinery (see paragraph III. 3 Intraluminal vesicles biogenesis). During the ESCRT-dependent exosomes biogenesis, mono-ubiquitinated proteins are recruited and clustered at the limiting membrane of MVB prior to inward budding and ILVs formation inside the large MVBs 242 . Likewise ESCRT-independent mechanisms involving other PTMs are involved in the sorting of protein cargo such as sumoylation 243 and phosphorylation, citrullination or oxidation 241 . It is noteworthy that not all exosomal protein cargo are modified and that not all modified proteins are sorted into exosomes, thus further studies are required to better understand the sorting of specific PTM proteins into the exosomes. There are other mechanisms that will influence the protein composition of exosomes such as the lipid-rafts or the tetraspanins which are proteins present at the exosomal membrane and that interact with cytosolic proteins 233,241,244,245 . To date it is not clear whether all these protein sorting machineries overlap or are completely independent and/or specific to a sub-type of ILVs. The composition of exosome membranes will also vary depending on the biogenesis pathway used to generate exosomes such as ESCRT-, tetraspanins-microdomain-or lipid raft-dependent 241 , contributing to the generation of sub-populations of exosomes (See paragraph II. 2).

The exosomal RNA composition differs from the donor cell's cytoplasm content, and requires specific mechanism(s) for RNA sorting 240 . So far, four main mechanisms are described for exosomes RNA sorting:

1) RNA binding proteins (RBP) 246,247 , such as hnRNA2B1 243 , carry RNA to ILVs; 2) ceramide via its production by neutral sphingomyelinase 2 (nSMase 2) 241 ; 3) RNA binds to the limiting membrane of MVB through raft-binding sequences named EXOmotifs (such as GCCG, UGAC, UCCG, and GGCC), and will be then included in exosome lumen after lipid rafts formation 239 ; and 4) RNA modification such as exosomes-specific D-loop may trigger the RNA sorting toward exosomes 248 .

Intraluminal vesicles biogenesis

During exosomes biogenesis, early endosomes will mature into late endosomes where ILVs are formed and accumulated in their lumen. The process of exosomes formation includes 1) the clustering of sorted cargo at the membrane of the multivesicular body (MVB) forming microdomains and 2) subsequent membrane curvature and fission of vesicles. Generally the fate of MVB is to fuse with lysosomes for degradation of their content, however MVBs can be targeted to the plasma membrane of the cell where ILVs (exosomes) are released in the extracellular space upon membrane fusion 249 . In breast cancer for example, cancerassociated fibroblasts-derived exosomes promote metastasis by stimulating protrusive activity of breast cancer cells. Indeed, once up-taken by breast cancer cells, these fibroblast-derived exosomes are loaded with Wnt11 and are secreted again into the extracellular space to stimulate cell protusions and motility and thus stimulate the invasion capacity of the breast cancer cells 249 .

The paragraphs developed below will attempt to describe the heterogeneity of exosomes, harbouring diverse content and membrane composition, as various actors such as ESCRT family proteins, lipids and /or tetraspanins are involved in their biogenesis and secretion (Figure 1).

 ESCRT-dependent mechanism

The ESCRT proteins are a group of proteins involved in the sorting of ubiquitinated cargo into ILVs.

ESCRT proteins are clustered into 4 complexes: ESCRT-0, I, II and III with associated proteins 242 .

Investigations on the role of ESCRT proteins in the biogenesis of exosomes started when studies identified several members of the ESCRT family in secreted exosomes such as Alix and TSG101 250 . A targeted RNAi screen for 23 ESCRT proteins and associated proteins has further demonstrated the function of ESCRT proteins in exosomes formation 251 (Figure 1). As a result, a set of cargo is sequestered in the inward budding vesicles within the lumen of the MVBs.

ESCRT-0:

The ESCRT-0 complex is composed of HRS (Hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (Signal transducing adaptator molecule) proteins and is recruited to the endosomal membrane via ubiquitinated cargo and phosphatidylinositol 3-phosphate (PI3P). HRS will recognize ubiquitinated protein -ubiquitin acting as a targeting signal for specific incorporation of molecules in ILVs -and binds to PI3P 235,252 . The HRS/STAM complex will then recruit ESCRT-I via TSG101/VPS28 (two components of the ESCRT-I complex) to the endosomal membrane and form an ESCRT-0/ESCRT-I complex.

ESCRT-I: ESCRT-I complex contain one copy of each TSG101, VPS28, VS37 (one of four isotypes VPS37 A-D 252 ) and MVB12. Its recruitment at the endosomal membrane is enhanced by ubiquitinated transmembrane cargo. Both ESCRT-0 and -I are involved in the clustering of selected ubiquitinated cargo into microdomain, and in mobilizing the ESCRT-II complex.

ESCRT-II: ESCRT-II complex is a heterodimer comprising one copy of VPS36 and VPS22 and two VPS25 subunits 253 . Together with ESCRT-I complex, ESCRT-II complex initiate the negative curvature of the emerging ILV at the MVB membrane and uptake of cytosolic cargo 235 . Finally, the association of ESCRT-I and -II will recruit the ESCRT-III complex at the ILV biogenesis site via either ALIX or through a direct interaction with VPS25 -VSP25 belonging to ESCRT-II.

ESCRT-III and ATPase VPS4:

The components of the ESCRT-III complex polymerize into filaments after recruitment at the MVB membrane 253 . The interaction between the VPS25 dimer from ESCRT-II with VPS20 from ESCRT-III initiate the polymerization of SNF7, the most abundant protein in the ESCRT-III Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 43 complex. In turn, SNF7 recruits VPS2 and VPS24 that will form one subunit while SNF7 and VPS20 form another 252 resulting in formation of the last ESCRT component, ESCRT-III. These two substructures forming ESCRT-III inside the nascent neck of the ILV will lead to the closure and detachment of the vesicles containing a specific cargo inside the MVB lumen 235 . Another ESCRT-mediated exosome biogenesis involving the interaction of the ESCRT-III/ALIX with the transmembrane proteoglycan receptor syndecan and its binding partner syntenin has been recently identified 217 . The heparan sulfate proteoglycan syndecans first assemble together at the MVB membrane followed by the cleavage of syndecan autorepulsive domain. Consequently, the syndecans remain clustered and syntenin can bind to the syndecans.

Syntenin will then interact with ALIX, recruit the ECRT-III unit with VPS4 and subsequently lead to the endosomal membrane inward budding and abscission.

The dissociation and recycling of the ESCRT subunits requires the recruitment of the AAA-ATPase VPS4 functioning as a ring shaped oligomers to unfold and separate the ESCRT proteins complexes from the MVB membrane 223,254 . VPS4 protein is composed of 2 hexameric rings stacked on top of each other forming a central core where ESCRT complexes will be disassembled and recycled 223 . ALIX initiate the recruitment of de-ubiquitinating enzymes such as UBPY to remove the ubiquitin from cargoes 242 , contributing to the maintenance of the ubiquitin level in the cells 252 . This de-ubiquitination step occurs before the incorporation of the proteins into the ILV and before the closure of the later 252 .

 ESCRT-independent mechanisms

In addition to the ESCRT-mediated exosomes biogenesis, there is now evidence that ESCRT-independent pathways also promote the sorting of cargo and biogenesis of ILVs. Indeed, the depletion of components of the four ESCRT complexes does not totally abrogate the formation of exosomes 232,251,255 indicating other mechanisms supporting the exosome biogenesis.

Lipid-mediated biogenesis

Similarly to the cell plasma membrane lipid rafts, exosomes are enriched in cholesterol, sphingolipids and ceramide. The neutral sphingomyelinase 232 (nSMase) converting sphingomyelin into ceramide plays a key role in ESCRT machinery-independent ceramide-mediated exosome genesis (Figure 1), as nSMase inhibitor leads to a decrease in the amount of secreted exosome 232 . Phospholipase D2 218 (PLD2) converts phosphatidylcholine into phosphatidic acid (PA) 218 (Figure 1). Both ceramide and PA generated at the limiting membrane of the MVBs possess a cone-shaped structure that may contribute to the negative curvature of the endosomal membrane, leading to inward budding and ultimately the formation of ILVs 256 .

Tetraspanins-mediated biogenesis

Proteins of the tetraspanins family have also been described as regulators of non-ESCRT dependent exosome biogenesis (Figure 1). Members of the tetraspanin family are very similar, strongly suggesting Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 44 that all tetraspanins proteins present the same configuration. They are transmembrane proteins composed of 4 transmembrane domains resulting in two extracellular domain and three intracellular regions 257 . The crystal structure of the tetraspanin CD81 analysis has recently uncovered an intramembrane pocket in between separated transmembrane regions through and where interaction with cholesterol is possible 258 . Tetraspanin proteins can be glycosylated at various degrees 257 and form homo-and hetero-oligomers with tetraspanins and/or other proteins leading to the formation of protein-enriched microdomain at the PM 259 .

The functional role of tetraspanins glycosylation modifications is still unknown but is possibly contributing to tetraspanin complex formation 260 . Due to their cone-shaped conformation and their ability to cluster into microdomain, tetraspanins could induce the inward budding of the late endosomal membrane and exosomes formation 258 .

 Exosome secretion: MVB and PM fusion

MVBs will be either directed to lysosomes for degradation or to the PM to release the exosomes in the extracellular space (Figure 1). The secretion requires several factors such as the cytoskeleton, associated molecular motors, small GTPases and the fusion machinery including SNARES and tethering proteins 261,262 . Small GTPase Rab and SNAREs protein families are particularly required in the transport of the MVB towards the periphery of the cell, their docking and fusion with the PM 236 . The exosome secretion will be mediated by different Rabs. For example, RAB27 B with its effector exophilin 5 will transport the MVB at the PM along the actin cytoskeleton 263 . The MVBs will then anchore and fuse with the limiting membrane of the cell via RAB27A and its effector synaptotagmin-like protein 4 264 . Different Rabs proteins can be preferably associated with early or late endosomes. For instance, Rab11 264 and Rab35 265 mediate preferably the fusion of early endosome with the PM while Rab27 is involved in the late endosome fusion.

The subsequent fusion of the MVB limiting membrane with the PM requires soluble factors (NSF and SNAP), SNAP-attachment protein receptor (SNARE) protein complexes and protein from the synaptotagmin family 223,236 . The v-SNARE complex present at the vesicles membrane will interact with the t-SNARE complex located at the cell membrane. The SNAREs protein will then form bridges between the opposing membranes and bring them to a sufficient proximity to induce fusion of both lipid bilayers.

Consequently, ILVs present in the MVBs will be released into the extracellular space. (2) Several mechanisms are involved in the biogenesis of exosomes such as ESCRT protein-, lipid-rafts-and tetraspanins microdomain-dependent pathways. Whether one or multiple pathways are required simultaneously on one population of MVB or if each pathway is specific to one population of MVB is still not clear. ESCRT-dependent pathways: A) the most described mechanism involve in the biogenesis of exosomes is the ESCRT-dependent pathway requiring protein of the ESCRT family. Specific transmembrane ubiquitinated cargo are recruited and clustered at the MVB membrane by ESCRT-0 complex, subsequently binding to ESCRT-I structure. ESCRT-II complex is activated and together with ESCRT-I will create and/or stabilize the vesicle neck. Finally, ESCRT-III and its associated proteins will drive the neck constriction. B) The second ESCRT-dependent biogenesis pathway is the syntenin/Alix pathway. The formation of syndecans enriched microdomains leads to the syndecans cleavage, and the formation of syntenin/syndecan complexes that interact with ALIX. The syntenin-syndecan-ALIX complex will then favour the recruitment of ESCRT-III complex to support the MVB membrane curvature and abscission. ESCRT-independent pathways: C) Ceramide-and phosphatidic acid-dependent pathways are based on the formation of lipid-rafts where sphingomyelin is converted to ceramide or phosphatidylcholine to phosphatidic acid. The ceramide-and phosphatidic acid-enriched rafts induce the inward curvature of the MVBs membrane. D) Similarly, tetraspanins enriched microdomains can induce a negative curvature in the MVB membrane. (3) MVBs will either fuse with lysosomes for degradation or with the PM which will consequently release exosomes in the extracellular space (4). Several proteins have been identified in the transport and fusion of the MVB to the PM such as proteins from the Rab protein family and SNAREs complexes. ESCRT: endosomal sorting complex required for transport, SNARE: SNAP (Soluble NSF Attachment Protein) receptor.

IV. Exosomes interactions with recipient cells

The most popular function in physiological or pathological condition of exosomes is to mediate cell-to-cell communication. The hypothesis of exosomes as intercellular messengers suggest that exosomes secreted by one cell communicate with another targeted cell and induce a cellular response. Exosomes are able to deliver biologically active molecules such as lipids, proteins or genetic materials 266 to recipient cells, and they can act as therapeutic vehicles as they potentially convey therapeutic molecules or drugs 267 .

Consequently, it is important to unravel the mechanisms underlying the exosome interactions with its recipient cell. Intercellular communication mediated by secreted exosomes occur in different manners, via 1) indirect signalisation mediated by a soluble fragment cleaved from the vesicles acting as a ligand to cell surface receptor; 2) docking of the exosomes at the PM of the targeted cell; 3) activation of surface proteins by direct ligand-receptor interaction; or 4) internalization or fusion of the vesicle by/with the targeted cell that will release the exosomal content (Figure 2). It is noteworthy that the recipient cell can also uptake its self-produced exosomes in an autocrine mechanism.

Figure 9: Exosome and recipient cell communication

Schematic diagram summarizing exosomes-cell interactions. Once secreted into the extracellular space exosomes mediate cellular responses via distinct pathways. Exosomes are described as messenger of functional cargo altering the physiology of the targeted cell once internalized. However exosomes uptake and direct contact with the targeted cell to mediate cell-exosomes communication are not constantly required. Indirect interaction between secreted vesicles and cells is possible through soluble ligand signalling. Exosomes carry transmembrane protein on their surface, accessible for cleavage by proteases to produce soluble forms of proteins that interact with specific receptor on the PM. Once reaching the targeted recipient cell, exosomes will dock at the PM. Following the anchorage of the vesicles, activation of multiple distinct intercellular signalling pathways might occur via ligand/receptor interaction, also known as juxtacrine signalling. Most of the studies however describe an internalization of exosomes by the recipient cells. Exosomes uptake involved several mechanisms such as endocytosis of exosomes including: 1) clathrin dependent mechanism, 2) caveolin mediated endocytosis, 3) macropinocytosis, 4) phagocytosis and 5) lipid-raft dependent endocytosis. To release their content into the cytoplasm, secreted exosomes can directly fuse with the PM. The vesicle-cell interaction generate distinct cellular responses. Communication mediated by ligand signalling, Juxtacrine signalling or direct fusion with the PM possibly results in intercellular signalling pathway activation since the endocytosis-lysosomal pathways are not involved in the processing of exosomes molecular composition. On the other hand once endocytosed by the cells, exosomes content are systematically release to the endocytic compartments and are more likely to undergo degradation via fusion of endosomes with lysosomes. Some vesicles however have been described to escape degradation by back fusion of the exosomes-containing MVB with the PM of by transport of exosomes towards the golgi apparatus. MVB: Multivesicular bodies.

Evidence for exosomes uptake

Direct (tracking of exosomes inside the recipient cell) and indirect (functional transfer of cargo) methods exists to assess the uptake of exosomes.

 Functional exosomal content transfer

Multiple studies have reported the transfer of exosomal cargo to recipient cells 149,230,267,268 . Interspecies transfer of exosomal cargo from mouse exosomes to human cells 149 demonstrated that both mRNA and miRNA are functionally transported from one cell to another, and was able to mediate a cellular response.

Like genetic material, proteins such as enzymes, can also transmit an enzymatic activity to a recipient cell 154 . Finally, by using exosomes as a vehicle for short interfering RNA, it has been demonstrated that exosomes can carry exogenous molecules to specific cells. For instance, by using a tagged brain-targeted exosomes that is loaded with specific siRNA, BACE1 knockdown (protease involved in formation βamyloid peptide aggregations in Alzheimer's disease pathogenesis) was efficiently achieved suggesting the potential use of exosomes in delivering tissue-specific drugs 267 .

 Exosomes tracking in recipient cells

Direct visualisation of exosomes inside cytoplasm of recipient cells efficiently allows tracking of their uptake in-vitro. Organic dyes such as PKH lipophilic dyes (including PKH67 or PKH26 157,195,[268][269][270][271][272][273][274][275] ) or rhodamine molecules 172,188,276 are fluorescent labelling dyes easy to use to visualise lipid bilayeredstructures such as exosomes. Because of the resolution limitation, researchers can't distinguish single exosomes however cluster of exosomes that are large enough allow the visualisation of fluorescence under a microscope. The use of fluorescent lipophilic dyes present some limitations as it allows the labelling of extracted vesicles and the cell-to-cell transfer of exosomes can't be assessed using this technique.

Genetically encoded fluorescent CD63 are now being developed to illustrate the uptake of vesicles. Because CD63 is specifically expressed in exosomes, labelling of other extracellular vesicles is very unlikely, allowing the tracking of GFP-tagged exosomes uptake in cell-to-cell in live experiments 277 .

Indirect communication: soluble ligand mediated signalling

Exosomes can mediate intercellular communication without direct contact with the recipient cell, by producing soluble ligand resulting from the cleavage of transmembrane proteinligand that will then interact with its receptor at the surface of the targeted cell and activate multiple signalling pathways (Figure 9 ligand signalling). For example, as part of the complement activation pathway, CD46 has been identified as one of the mediators of complement resistance of malignant cells by inactivating C3b and C4b

Exosomes uptake by target cells

Targeting the recipient cells, whether it is a specific and control process or stochastic, is the first step involved in the exosome uptake. This process requires the combination of specific molecules present at the surface of the cell and of the exosome (see Table 3), including proteins (glycoproteins, integrins or tetraspanins), lipids and sugar. Following interaction, exosomes either directly fuse with PM or are internalized by the recipient cell and deliver their content in the cytoplasm.

 Ligandreceptor interaction

Whether exosomes-cell interaction is a controlled and specific mechanism is still unknown, several studies investigated how exosomes could specifically target different cells in different physiological and pathological conditions 196,272,279 . For example, peripheral blood mononuclear cell-derived exosomes from lymphoma patients can specifically target B cells while exosomes derived from natural killer cells and Tlymphocytes cannot 196 . On the other hand, a single cell type can adsorb different exosomal populations 193 .

Among the list of known ligand-receptor interaction, protein-protein interactions are the most abundant.

For instance, when exosomes from human ovarian cells are pre-treated with proteinase K or trypsin to degrade the exosomal transmembrane protein, their uptake by the same cancerous cells is abolished [279][280][281] .

Inhibiting specific interactions using antibodies or soluble ligand prior to treatment of cells with exosomes allowed to discover many specific ligand-receptor involved in the exosomes uptake. On the other hand, negative selection of exosomes can also occurs, as observed with exosomes bearing the CD47 transmembrane proteins inhibiting their phagocytosis by the recipient cells and contributing to a longer retention of exosomes in blood circulation 279 .Targeting specific mediators in exosome-cell interactions would prevent the uptake of specific toxic exosomes allowing the development of therapeutic strategies (see IV. 6 Exosomes functions in intercellular communication).

Exosome ligand Target cell ligand References

Glycoproteins

 Fusion of exosomes with PM

The exosome-recipient cell membrane fusion process is similar to the fusion taking place during a cell-cell fusion, with targetingdockingfusion being the main required steps. One method developed to follow the fusion of exosomes with the PM is the use of self-quenching fluorescent lipid probe R18 230,294 . R18 lipid probe classically used to study the virus-cell fusion mechanims 295 can be applied to exosomes in order to track their direct fusion with the cell membranes 230,294 . Briefly, once R18 is incorporated in the vesicles, exosomes can be applied to the cell culture of interest. When the fusion occurs, R18 probe is diluted into the unlabelled membranes resulting in an increase of its fluorescence. The pH of the extracellular space is an important parameter vesicle-cell fusion process, as a tumorous environment with an acidic pH can enhance the fusion, while a pre-treatment with proton pump inhibitors reducing the extracellular space acidity reversed this phenomenon 294 .

Secretion of neurotoxic vesicles by muscle cells of ALS patients

 Endocytosis

Exosomes uptake by the recipient cells can occur through the endocytic pathways including clathrin-or caveolin dependent endocytosis, and clathrin-independent mechanisms such as lipid-raft mediated endocytosis.

Clathrin-mediated and caveolin-dependent endocytosis

Various studies have highlighted the possibility of exosomes to be internalized by energy-dependent mechanisms involving the cytosqueleton of the recipient cells 275,292 . A well-known endocytosis pathway is the clathrin-mediated endocytosis (CME) involving the internalization of cytoplasm (including exosomes and cytosolic proteins) by means of clathrin coated vesicles 296 . The deformation of the limiting membrane of the cell induced by the clathrin protein, leads to the formation of inward buds growing into a larger vesicle that will mature and pinch off 297 . When chlopromazine was applied to ovarian cancer cells to inhibit CME, the uptake of exosomes by these cells was reduced 298 . Similarly, when dynamin inhibitor is applied to neurons or microglial cellsdynamin being a protein involved in the formation of a ring at the neck of the invaginating membrane to separate the vesicles from the PMthe exosomes uptake was dramatically decreased 157,292 .

Exosomes can be internalized via caveolae, another endocytosis-mediated pathway. Caveolae are invaginations in the plasma membrane enriched in glycoproteins and cholesterol 299 . Caveolin rafts are internalized by the cell through dynamin activity 297 . Specific inhibition of caveolin-dependent endocytosis via the specific knock down of CAV gene reduce the uptake of exosomes in epithelial cells 300 .

Micropinocytosis and phagocytosis

Exosomes can be secreted as a cluster 166 (PS) 297,303 . When several phagocytosis mediators are inhibited using specific drugs (cytochalasin D / latrunculin B, wortmannin / LY294002) and siRNA-targetting dynamin expression, a decrease in exosome uptake is observed. Furthermore, when cells were pre-treated with a soluble analogue of PS, the exosomes uptake decreased suggesting a role for PS found in the bilayer of exosomes membranes to mediate the exosome uptake through phagocytosis pathway 297 . Accordingly, the treatment of recipient cells with TIM4

antibodies -TIM4 being a receptor involved in phagocytosis pathway through its association with PS 234led to the reduction of exosomes uptake via phagocytosis 302 . The pre-treatment of exosomes with annexin V, annexin V binding to PS of the vesicles' membranes, also reduced the exosome uptake 297 . Altogether, these studies the importance of the phagocytic capacity of the recipient cells in the exosomes uptake.

 Lipid raft-mediated endocytosis

Recycling or degradation: fate of exosomes inside the targeted cells

Following their internalization by the recipient cells, exosomes will integrate the endocytic pathway and most probably be targeted towards the lysosome for degradation 236,261,304,305 . For instance, the phagosome containing the exosomes up-taken through phagocytosis-dependent pathway will fuse with the lysosome and their content will be degraded 302 (Figure 9). The co-localization of exosomes with phagolysosome maturation markers (eg LBPA, Rab7 and LAMP proteins) strongly suggest the ability of phagocytic cells such as macrophages to internalize exosomes via phagocytosis forming large exosomes-containing vacuoles that are targeted towards lysosomes 302 . The exosome degradation by the lysosomes can then become a source of metabolites (such as amino acids, lipids, lactate or acetate) for the recipient cells 236,274 .

Exosomal contents could be recycled by back fusion of the MVB with the PM releasing ILVs in the extracellular space 249 .

Secretion of neurotoxic vesicles by muscle cells of ALS patients

How exosomes deliver functional content to the targeted cells

Late endosome accumulate vesicles that contain molecules not destined to be degraded by lysosomes 306 .

To release functional content into the recipient cells' cytoplasm, exosomes escape the lysosomal degradation by back fusion, process where ILVs fuse with the limiting membrane of MVBs 307 . This back fusion process allows the vesicles to release their functional intraluminal content into the cytoplasm of the recipient cell 307 . The vesicular stomatitis virus (VSV) use a similar pathway to deliver its nucleocapsid into the cytoplasm of infected cells. The VSV virus is first internalized by the targeted cells, then directed toward the endocytic pathway where the virus is sorted into intraluminal vesicles. The virus capsid is subsequently released into cytoplasm by back fusion of the limiting membrane of the MVB with newly formed exosomes 308,309 . This virus can stay enclosed in the exosomes for several days, providing the viral content from any degradation before being released as exosomes by integration into new target cells 310 . Once internalized by targeted cells, the secreted VSV-containing exosomes can release their viral content by back fusion. The fusion of the limiting membrane of the MVB with the PM is a another opportunity for the exosomes to escape lysosomal degradation and deliver their functional content 236 . This phenomenon was described to occur with breast cancer cells-derived exosomes 249 and thus contribute to the in the propagation of the disease.

Soluble ligand-and juxtacrine signalling as well as direct fusion of the exosomes with the PM doesn't require the endocytosis of secreted vesicles to modify the phenotype of the recipient cells. In these contexts, exosomes will directly activate intercellular pathway responses.

Exosomes function in intercellular communication

Exosomes mediate multiple intercellular signalling within the recipient cell such as proliferation, angiogenesis, apoptosis and cytokine production among other. Soluble ligand-and juxtacrine signalling and direct fusion with the PM are more likely to induce a cellular signalling activation as a direct interaction between cell and exosomes or release of exosomal content occurs. Internalization of exosomes by endocytosis pathways can sometimes be followed by the release of exosome intraluminal content through back fusion (see Figure 2

 Exosomes-induced signalling and role in the central nervous system

In neuronal tissue, exosomes secreted by various cells including glia and neurons contribute to the physiology of the central nervous system 146,147,156,157,166,315 . Concerning stress protection, oligodendrocytesderived exosomes have been demonstrated to support the metabolism activity of neurons under stress conditions suggesting a role of exosomes in neuroprotection 157 . In addition, through activation of several kinases such as CREB, GSK-3α/β and JNK, oligodendrocytes-derived exosomes mediate the transduction of pro-survival signalling as MEK/Erk and PI3K/Akt in neurons under physiological and stress conditions 156 further supporting the role of neuronal exosomes in the brain. Myelin is the insulating layer of neurons that helps conducting action potential along the axons of neurons. During the maturation of oligodendrocytes, neuronal axons are enwrapped by myelin, and the myelination process seems to be controlled by the exosomes secreted by the oligodendrocytes, activating in an autocrine manner the Rho-ROCK-myosin signalling pathway 146 . In addition to the axonal guidance role of oligodendrocytes-derived exosomes described above 146 , these exosomes can enhance the action potential firing in cortical neurons 156 ,

Secretion of neurotoxic vesicles by muscle cells of ALS patients

but can also ensure a neuroprotective role. For instance, Hsp70 heat shock protein secreted through the oligodendrocytes-derived exosomes 147 that can be internalized by neurons axons mediating stressprotective signal. Finally, cortical neurons can also secrete exosomes 166,315 depending on the glutamatergic activity associated with Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and Nmethyl-D-aspartate (NMDA) glutamate receptors at the synapse. This cortical neurons-derived exosomes contain components of the synapse such as subunits of AMPA receptor and neuron cell adhesion L1, suggesting a role for these exosomes in the synapse plasticity.

In pathological conditions, exosomes have been identified as probable vehicle in disease progression within the nervous system. Alzheimer's disease (AD) is a neurodegenerative disorder characterized by loss of neurons and accumulation of amyloid plaques due to the loss of degradation of β-amyloid peptides in the brain. Aβ-peptides are accumulated in exosomes from AD patients, inducing its propagation in the extracellular space. Spreading of the disease to healthy cells has also been suggested by the colocalization of AD patients-derived exosomes and amyloid plaques 316 . Similarly, in Parkinson's disease (PD) -disease characterized by accumulation of α-synucleinα-synuclein has been identified in exosomal content of neuroblastoma cells 317 , exosomes that might also have a role in the propagation of the disease to healthy neurons.

 Role in protein recycling and intercellular communication

Original studies described the secretion of exosomes as a cellular mechanism to release excess and/or unnecessary proteins in the extracellular space. In early studies, analysis of reticulocyte maturation into erythrocyte unravelled the recycling of TfR through the secretion of exosomes 158,160 . Moreover, exosomes have been extensively studied for their role in immunology signal transduction. An early study by Raposo

Intercellular communication in ALS

Many pathological mechanisms are involved in ALS, as different cell types other than motor neurons also are altered and display dysregulations. In the SOD1 mutant murine model, neuroinflammation signs such as activated microglia and astrocytes were recorded before the symptom-onset 318 . Moreover, muscle cell defects are reported before the motor neuron loss 319,320 . Altogether, these results suggest that a non-cell autonomous toxicity could participate in disease onset and progression and thus be part of the ALS physiopathological mechanism.

Evidence for non-neuronal cell toxicity

There has been several line of evidence implying non-neuronal cells-mediated toxicity. When G37R, G93A

and G85R mutated forms of SOD1proteins were highly expressed in cortical and spinal motor neurons of transgenic murine model, no muscle denervation was observed, motor neuron death was minimal and not sufficient to induce motor deficits 321,322 . It is only in elderly mice (>1 year old) expressing a sufficiently high level of mutant SOD1 that neurodegenerative symptoms appears 323 . In a chimeric murine model, the expression of mutant SOD1 in neuronal cells was not toxic by itself unless the surrounding non-neuronal cells -astrocytes and microglial cells -expressed mutant SOD1 324 . Concordantly, ablating mutant SOD1 expression in motor neurons specifically only lightly the delay onset of symptoms without affecting the the mice survival 69,325 . In addition, when mutant SOD1 motor neurons were surrounded by healthy wild type cells, their death was delayed, while when they were surrounded by mutated SOD1 cells their death was exacerbated 324 . Similarly, when mutant SOD1 expression was decreased in microglial cells at late stage of the disease, symptoms progression declined 69 confirming the microglia-mediated toxicity contribution to the symptom evolution. In vivo and in vitro studies also confirmed that astrocytes expressing mutant SOD1 are toxicity towards motor neurons 326,327 Altogether these studies illustrate the role of glial cells such as astrocytes and microglia in non-autonomous toxicity in ALS patients. Therefore ALS-mediated toxicity is not strictly restricted to motor neurons and affected surrounding non-neuronal cells might contribute to pathogenesis.

Non-cell autonomous toxic mechanisms

In the central nervous system, microglia represent the immune cells pool 328 . Upon damage, microglial cells become active and secrete ROS and toxic molecules that may participate to the toxic environment of motor neurons in ALS 329,330 . In physiological conditions, astrocytes are supportive cells that will provide nutrients for motor neurons as well as ion buffering and neurotransmitter recycling 331 . In ALS patients, astrocytes undergo modifications switching from a neuroprotective to neurotoxic property against motor neurons 332 .

Astrogliosis and microgliosis are the main pathological indications of neuroinflammation in ALS patients 328 Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 59 and signs of active microglia and astrocytes have been reported in numerous studies [333][334][335] .

Oligodendrocytes also protect upper motor neurons by myelinating their axons and providing metabolic support such as lactate through the action of their monocarboxylate transporter 1 (MCT1). Expression of mutated form of SOD1 is accompanied by a reduction of the MCT1 expression in oligodendrocytes resulting in impaired axons and neuron loss 336 . Concordantly, deleting mutant SOD1 expression in oligodendrocytes of SOD1 mice re-establish their function and slow down symptoms progression 337 .

II. Axonopathy mechanisms and muscle involvement

More recently there has been increasing interest in determining whether motor neuron degeneration is driven by toxic mechanisms arising from the cell body of neurons or by destruction of axonal projections.

The role of muscle in keeping the neuromuscular junction hence suggest a possible role for musclemediated toxicity in motor neuron degeneration.

The neuromuscular junction

Motor neurons and skeletal muscle fibers interacts at the neuromuscular junction (NMJ), to transmit action potentials and induce muscular contraction. The NMJ is composed of the presynaptic cell (motor neuron), the postsynaptic cell (skeletal muscle cell) and the terminal glial cell providing support. The axons arising from the motor neuron innervate multiple skeletal muscle fibers ranging from 10 to over 1,000 fibers 338 .

Each presynaptic cell contains many synaptic vesicles (50nm diameter vesicles), normally highly dense in presynaptic membrane structures called "active zone" 339 . Those specialized membrane section are particularly enriched in voltage-gated calcium channels involve in the release of the synaptic vesicles in the synaptic cleft. The muscle fibers at their membrane accumulates neurotransmitter receptors and voltagegated sodium channels hollow projections that crosses the fiber called tubules T. The process of neurotransmission includes several mechanisms to transmit action potentials from motor neurons to muscle fibers and induce muscle contraction. 1) Acetylcholine, which is the neurotransmitter in vertebrates, is stored in the synaptic vesicles present in the motor neuron. 2) Following depolarization voltage-gated calcium channels opens resulting in an increase in intracellular calcium concentration. 3) Acetylcholine is released in the synaptic cleft and binds to its receptors at the muscle fiber membrane. 4) The induced depolarization results in a new action potential that propagates along the muscle fibers, allow the muscle to contract. 5) Acetylcholine is rapidly recycled from the synaptic cleft through the acetylcholinesterase activity, hence stopping the neuromuscular transmission (Figure 10). Following an action potential that reaches the per-synaptic neuron, the neurotransmitter acetylcholine (ACh) is released in the synaptic cleft. Ach binds to its receptor on the post-synaptic nerve and triggers influx of Na + and 1) produce a synaptic potential that 2) activates voltage-gated sodium channels and produce an action potential that propagates along the membrane of the myofiber. 3) When it reaches the Tubule T invagination, calcium is released from the sarcoplasmic reticulum. 4) Finally calcium triggers muscle contraction through binding to troponin. 5) Myosin binds to the exposed binding site on the troponin protein and form a cross-bridge responsible for muscle contraction.

Distal axon degeneration

Any dysregulation of the NMJ impairs the synaptic transmission and is likely to induce muscle weakness or wasting 339 . Today it's still not clear whether the degeneration of motor neurons is a dying forward process, where the motor neuron lying in the cortex are the first impaired and then damages are transmitted to their axons, or if ALS is an axonopathy in which the primary injury starts at the neuron projection endings and extends toward the cell bodies (see Moloney et al [START_REF] Saini | From death, Lead me to immortality"-mantra of ageing skeletal muscle[END_REF] for review). It is likely that both anterograde and retrograde pattern of degeneration are taking place. In the SOD1 mouse model, pathological changes at the

Secretion of neurotoxic vesicles by muscle cells of ALS patients

NMJ are reported before the degeneration of motor neuron axons and cell bodies 341,342 . These results illustrate the progressive retrograde degeneration affecting distal regions earlier than neuronal degeneration, suggesting that muscle tissues could potentially be affected before the loss of motor neurons. Consequently, the muscle weakness and wasting observed in SOD1 mice could result from a combination of both independent and dependent-denervation features.

The role of skeletal muscle in ALS patients

Additionally to the trophic support of glial cell towards the motor neurons, skeletal muscle also appear to regulate the homeostasis of the NMJ. Muscle cells plays crucial role in maintaining neuronal survival and axonal growth, especially for developing neurons. A dysfunctional muscle metabolism may thus contribute to NMJ impairment 343,344 . Interestingly signs of muscles dysregulation have been reported in ALS patients [345][346][347] , including early mitochondrial dysfunction 345 , decrease in NMJ trophic support molecules such as the fibroblast growth factor binding protein 1 (FGFBP1) levels 346 or oxidative stress 347 . When human mutant SOD1 expression is restricted to the skeletal muscle, it induced an ALS-like phenotype in mice with progressive muscle weakness and wasting and mitochondrial dysfunction 347,348 . On the other hand, several studies tempted to knock down mutant SOD1 expression in skeletal murine muscle and show no significant decreases in the progression of symptoms 349,350 . However the silencing techniques used in both studies -lentivirus well known to be inefficient to target the skeletal muscle 351 and low dose of adenoassociated virus serotype 6 (10

Secreted vesicles and role in pathogenesis

Intercellular communications happens among neurons and their surrounding cells in the central nervous system through secretion of soluble molecules, the release of exosomes containing neurotrophic support molecules in the extracellular space 156,353 C9orf72 DPRs spread from cell to cell and strengthen the propagation of misfolded proteins and toxic aggregates in recipient cells 358,359,361 . Similarly, skeletal muscle cells also acts as a secretory organ and release myokines 362 and extracellular vesicles under physiological 268 and physiopathological conditions 363 , such as ectosomes (or microparticles) and exosomes.

Conclusion

Regarding 1) the emergence of ALS as a multisystemic disease, as pathological alteration are observed in different cell types including in muscles), 2) the distal axonal degeneration preceding loss of motor neurons implying a toxic intercellular communication between muscle and nerve and 3) the pathological potential of exosomes in ALS disease, we hypothesize that vesicle trafficking is disrupted in ALS muscle cells and influence the communication at the NMJ. This phenomenon could be a key element in disease progression.

Objectives

In addition to its classical role in locomotion, the skeletal muscles can act as a secretory organ. The muscle secretome is composed of exosomes, but also of microvesicles and secreted proteins called myokines 268,363 .

Exosomes originate from multivesicular bodies or MVB (also called late endosome) that bud off internal vesicles. The MVB then fuse with the plasma membrane of the cell, releasing the exosomes in the extracellular space 364 . If they were first thought to be garbage vesicles, they are now seen as powerful intercellular messengers that can spread information from cell to cell [146][147][148]160,223,317,365,366 . Hence, exosome influence the homeostasis of recipient cells and interestingly muscle exosomes have been demonstrated to be able to deliver functional proteins, miRNA and microRNA to target cells 268,367 .

ALS is an adult-onset fatal neurodegenerative disorder characterised by a progressive, rapid and fatal deterioration of both upper-and lower motor neurons 2 . ALS is mainly sporadic with 90% of ALS cases with no apparent family history, and only 5-10% of ALS cases are familial 7 . Today ALS remains incurable and the causes of muscle denervation are still unknown. Numerous mechanisms involving the neighbouring cells, including astrocytes and microglial cells are suggested to contribute to the motor neuron death in ALS patients and therefore is considered as multisystemic disease 69,324,326,327 . It has now been demonstrated that pathological molecular and cellular changes occur at the neuromuscular junction prior to motor neurons degenerations and symptom onset 342,368 . In addition, since the identification of SOD1 mutations in ALS patients 42 several gene associated with the vesicle trafficking have been described in ALS patients, such as the vesicle-associated membrane protein B gene 76 (VABP), alsin protein 64 or the valosin containing protein [START_REF] Malmgren | Numerical densities of myonuclei and satellite cells in muscle fiber types in the aging human thyroarytenoid muscle: An immunohistochemical and stereological study using confocal laser scanning microscopy[END_REF] (VCP). These results highlight the potential disruption of the exosomes biogenesis and secretion pathways in ALS patients. More recently hnRNA2B1 involved in the sorting of RNA into exosomes 243 has also been identified in ALS cases 369 suggesting a RNA processing dysregulation in ALS patients.

In light of these data, we hypothesise that vesicle trafficking is disturbed in ALS muscle cells and might influence the intercellular communication between the muscle and the nerve. This phenomenon could be a key element in the disease progression.

The purpose of this PhD project was to 1) confirm an altered exosomes secretion by ALS muscle cells, 2) determine the effects of the ALS exosomes on motor neuron survival, and 3) unravel the toxicity mechanism mediated by muscle ALS exosomes.

Experimental protocol n°1

Muscle stem cells extraction and culture (1) -Muscle biopsy dissection: micro-explant culture

Since 1961 the muscle fiber, the multinucleated contractile unit of muscle, is known to result from the fusion of numerous mononucleated myoblasts 370 . In adult healthy muscles, the muscle stem cells called satellite cells are the mononucleated muscle precursor responsible for muscle grow and repair following trauma. Satellite cells are located peripherally between the basal lamina and the muscle fibers. Following tissue damage, activation of these satellite cells induce their proliferation and are referred to as myoblasts. The myoblasts consequently differentiate and fuse into myotubes that will further elongate by fusing with other myotubes.

The approach we used to extract the muscle stem cells from biopsy is the microexplant culture technique to allow cells to proliferate out the biopsy during culture 371 . To mimic a muscle trauma, the muscle biopsies are cut to trigger satellite cells activation, migration, and proliferation. This technique is an easy and reliable procedure that minimise primary cell extraction trauma and produce a cell population composed of approximatively 85% of proliferative myoblasts 371 .

Dr G Oudaogo, Dr S D Duguez and L Le Gall extracted primary muscle stem cells from human biopsy as described below.

Procedure:

1. One 10cm petri dish is prepared for culture of explants:

 1-2ml of serum is added to the petri dish  The excess is removed by aspiration using a glass pipette.

2. To get rid of as much blood as possible the muscle biopsy is rinsed in DMEM 3 times in small 6cm petri dishes, and transfer in a petri dish containing serum

Procedure

I. Myoblasts proliferation:

Following the migration of cells out of the muscle biopsy explant, cells proliferate until the required number of cells is achieved.

II. CD56+ MACS cell sorting:

Before performing a CD56 MACS cell sorting, cells count should reach at least 1.10 6 cells per culture.

Magnetic labelling

1. The cell number and division is determined as described above.

2. MACS buffer is prepared as follows : PBS pH7.

Experimental protocol n°2

Muscle stem cells extraction and culture (2) -Myoblasts proliferation, sorting and differentiation

Magnetic separation

1. An appropriate number of MACS columns are placed on the magnetic field of a MACS separator with collection tube and rinsed with 500µl MACS buffer.

2. The cell suspension is added to the MACS column and washed 3 times with 500µl MACS buffer.

3. The collection tube is removed and unlabelled CD56 -cells (nonmyogenic cells) which passed through the columns can be collected and counted for storage in -80°C freezer for further use.

III. Myoblasts differentiation:

Once the cell count has reached the appropriate number of proliferative myoblasts per flask, differentiation into myoblasts is induced.

Differentiation induction:

1. The proliferative medium is carefully withdrawn from flasks.

Experimental protocol n°3

Human iPSCs motor neurons culture 3. Carefully, 100 of fresh 100L N2B27 ++ 2 is added to each well and cells are incubated at 37°C for 3 days.

Procedure

Mycoplasma screening were performed using 600µl culture media from approximatively 80% confluent cells, cultivated without antibiotics for at least a week.

I. DNA extraction and purification

DNA extraction from culture media was completed with PureLink Genomic DNA Kits from Life Techologies.

Sample preparation

1. The culture media (600µl) is mixed with 20µl Proteinase K, and an equal volume (600µl) of PureLink genomic Lysis/Binding Buffer is added/ 2. The mix is briefly vortexed and incubated at 55°C for 10min.

3. Lysed samples are centrifuged for 5sec and 200µl of 96-100% ethanol is added.

DNA purification

Wash Buffer I and Wash Buffer II are prepared as mentioned by supplier.

1. The lysate is added to the column and centrifuge at 10,000g for 1min at room temperature.

2. The column is placed on a new collection tube, and step 1 is repeated until the entire sample has been through the column.

3. DNA is washed with 500µl of Wash Buffer I and columns are centrifuged at 10,000g for 1min at room temperature.

III. PCR and results evaluation

Thermal cycle profile

The thermal cycle profile includes:



Experimental protocol n°5

Exosomes extraction from culture medium

Exosomes are 50-100nm secreted vesicles release by numerous cell types including skeletal muscle cells 268 .

In order to have access to the secreted exosomes in culture medium of 3 days differentiated myotubes, the Total exosomes isolation kit from Life Technologies is used. This kit is based on polymers-containing preparations 221,222 , allowing a rapid extraction of exosomes with low-speed centrifugations. However myotubes also secrete microparticles (or ectosomes) that are larger than exosomes (100-1000nm), so clearing the culture medium of larger vesicles and other cell debris is essential before performing extraction of exosomes.

Procedure

I. Culture medium preparation

Exosomes are extracted from culture medium from 3 days differentiated myotubes. To obtain pure exosomes pellets, larger vesicles and cell debris must be cleared from the culture medium.

Experimental protocol n°8

Protein extraction and quantification from exosomal samples

Total proteins quantification from exosomes sample was performed using the bicinchoninic acid (BCA) based assay, allowing for a colorimetric quantification of proteins.

Procedure

I. Exosomes extraction and protein extraction

1. Exosomes are extracted as described previously (Experimental procedure n°5) and resuspended in 100µ PBS.

2. 25µl are used to perform quantification and 10µl of Urea 8M buffer (supplemented with 10µL/mL proteases and phosphatases inhibitors cocktail).

3. Exosome samples are incubated 15 min at 4°C for complete lysis.

4. Lysed exosomal samples are centrifuged at 14,000g for 10 min at 4°C.

Soluble proteins contained in the supernatant are transferred to a new

Eppendorf tube and stored at -80°C for further applications.

II. Protein quantification

1. BSA standards are prepared as previously described using a 2mg/ml BSA stock solution 5. The plate is incubated at 37°C for 30 min before absorbance can be read at 562nm using a plate reader.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Experimental protocol n°9

Western blotting -Cell lysate samples

The western blotting, or immunoblotting (in reference to antibodies used to specifically detect proteins of interest) is a technique based on the separation of proteins on a gel and their subsequent transfer to a membrane for antibody-based detection. The first step includes preparation of proteins samples in reducing and denaturing condition to allow proteins to unfold and acquire a negative charge. Usually, ßmercaptoethanol and DTT are common reducing agent responsible for the breaking of disulphide bonds.

Moreover, sodium dodecyl sulfate SDS (SDS) is responsible for negatively charging the proteins by attachment of SDS anions to proteins samples. Following a boiling step at 70°C, proteins samples are fully unfolded and ready for electrophoresis, allowing their separation based on their molecular weight. The second step consists in transferring the proteins to a PVDF membrane using the iBlot 2 Dry Blotting System (Life Technologies). Finally, membrane are stained with primary antibodies specific for antigen of interest and Horseradish Peroxidase (HRP)-conjugated secondary antibodies targeting the primary antibody. In the presence of its substrate hydrogen peroxide and luminol, HRP produces a chemiluminescent signal that is acquired to detect proteins.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

II. Electrophoresis

The electrophoresis is performed using the XCell SureLock MiniCell and 4-12% polyacrylamide Bis Tris gels from LifeTechnologies.

1. The 1X SDS Running buffer is prepared as follows:

 50mL 20X

Experimental protocol n°9

Western blotting -Cell lysate samples III.

Transfer using iBlot TM 2 Dry Blotting system

The transfer stack provided with the iBlot 2 system includes a PVDF membrane.

1. Once electrophoresis is completed, the gel is removed from its cassette and is incubated in 20% ethanol for equilibration and optimal transfer.

2. One filter paper is soaked in deionized water and the transfer is assembled following the subsequent order:

 The bottom stack is placed first in the iBlot 2 system, the PVDF membrane should be on top of the bottom stack.

 The wet gel is carefully added to the stack followed by the pre-soaked filter paper. Air bubbles are gently removed.

 The top stack is placed on top and air bubbles are removed.

 The absorbent pad is finally added on top of the stack, making electrical contact with the iBlot 2 system.

3. The transfer of proteins to the PVDF membrane is run at 23V for 6min.

Following transfer, the PVDF membrane is removed from the transfer stack and Ponceau staining is performed to check transfer efficiency.

4. The PVDF membrane is quickly re-activated for 2 min in methanol.

5. The membrane is rinsed with distilled water for 10min.

6. The membrane is incubated in Red Ponceau staining until clear bands are visible. The excess of staining is washed by rinsing the membrane quickly with deionized water.

7. The membrane is ready for staining.

IV. Blotting membrane using iBind TM Flex Western system

The iBind Flex system allows an automated immunodetection of proteins. 2. 2ml of Amersham ECL mix is added per midi membrane, 1ml per mini membrane, and incubated for 5 mins at room temperature.

3. Chemiluminescent signal is acquired using the UVP ChemiDoc-It² Imager and UVP software.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Experimental protocol n°10

Exosomes samples preparation for immunoblotting

The western blotting performed on exosomal samples requires supplemental steps to allow detection of antigens. The total exosomes isolation kit is a polymer based reagent and additional washings of exosomes are necessary to allow an efficient staining of samples.

Procedure

Exosomes protein extraction

Experimental protocol n°11

Immunocytochemistry (ICC)

Immunocytochemistry technique allows fluorescent labelling of a specific antigen in cultured cells. The antigen of interest is bound by a non-fluorescent primary antibody which is than targeted by a fluorescentlylabelled secondary antibody that will amplify the signal. The signal produced by the fluorophore conjugated to the secondary antibody is detected using a fluorescent microscope.

Procedure

I. Cells fixation

Fixation of the cells using formaldehyde allows the preservation of cellular structures.

1. 4% formaldehyde solution is prepared in PBS, and cell culture are fixed for 10min.

2. Excess of formaldehyde is washed from cells with 3 times with approximatively 500µl PBS before staining.

3. If necessary, cultured cell can be stored in PBS (to avoid the wells to dry) at 4°C for several weeks until staining is performed.

II. Non-specific sites blocking.

To avoid the acquisition of background signal produced by non-specific binding of antibodies, a blocking step is required. The blocking buffer is composed of serum and BSA allowing the masking of all sites, and are replaced by the primary antibodies applied to cultured cells carrying a higher affinity for the antigen. The blocking buffer also includes detergents to permeabilize cell membrane and allow access to intercellular targets.

1. The blocking buffer is prepared as follows in PBS:

 20% FBS  0.5% Triton  0.5% Tween 20  5% BSA 2.
Cells are incubated in blocking buffer for 30 min at room temperature.

Products:

Formaldehyde (Sigma #252549-100ml)
Triton (Sigma T8787-50ml) Tween 20 (Sigma P2287-500ml)

Experimental protocol n°11

Immunocytochemistry (ICC)

III.

Antibodies staining

The table below summarizes the primary antibodies used.

1. The blocking buffer is gently removed and primary antibodies are diluted as required in blocking buffer. Primary antibodies solution are slowly added to the cells and incubated for 2 hours.

2. After 3 washes in PBS, labelled-secondary antibodies diluted in blocking buffer are added to the cells and incubated for 1h30 at room temperature and protected from the light.

3. Cells are washed with PBS, counter-stained with 1µg/ml DAPI for 1 min, and rinsed twice in PBS.

4. One drop of ibidi® mounting medium is added in each well to preserve fluorophores conjugated with secondary antibodies.

5. Pictures were acquired with Zeiss Axio Imager A2 microscope.

IV. Immunohistochemistry (IHC)

Similarly to cultured cells, tissue sections can be fluorescently labelled, technique known as immunohistochemistry. Muscles biopsies were embed in OCT prior to cryo-sectioning. 8µm transverse muscle sections were cut using a mircrotome-cryostat and were similarly permeabilised, blocked and stained as described above.

V. RNA staining

Acridine orange allows the differential labelling of DNA and RNA. In fact, when bound to double stranded nuclei acid such as DNA, acridine orange will emit a green fluorescence, while bound to single stranded nucleic acid such as RNA, acridine orange (AO) emits an orange fluorescence.

1. Following cell fixation, cell cultures were permeabilized for 1hr at room temperature.

2. Cells are stained with 20µg/ml AO diluted in PBS for 10 min.

3. Pictures were acquired using Zeiss Axio Imager A2 microscope.

Products: Experimental protocol n°12

Ibidi®

RT-qPCR and relative gene expression analysis

The real time reverse transcriptase chain reaction (RT-qPCR) allows the quantification of mRNA in cell samples. RT-qPCR combines 3 steps: 1) following RNA extraction, reverse transcription is performed to convert RNA into cDNA, 2) the amplification of cDNA using the PCR technique, and 3) signal acquisition and quantification of PCR products in real time.

Procedure

I. RNA extraction and purification

RNA from muscle samples were prepared using TRIzol combines with PureLink RNA Mini Kit.

RNA extraction

1. Cell pellets were dissolved in 100µl TRIzol and complete cell lysis was achieved by adding 900µl TRIzol and pipetting up and down until mixture is homogeneous, and incubated at room temperature for 5min.

2. 200µl chloroform are added and tubes are vigorously shook before a 5min incubation.

3. Lysed cells are centrifuged at 12,000g for 15min at 4°C and the top aqueous phase containing RNA was transferred into a new Eppendorf tube for RNA purification.

4. One volume of 75% ethanol was added and mixed for a few seconds.

RNA purification

1. Up to 700µl of samples is transferred to the spin cartridge and centrifuged at 12,000g for 15sec. This step was repeated until the entire RNA sample was added.

2. The filtrate is discarded and 700µl of Wash Buffer is added to the spin cartridge and centrifuged at 12,000g for 15sec, filtrate is discarded.

3. RNA is washed a second time by adding 500µl of Wash Buffer II and spin cartridge centrifuged at 12,000g for 15 sec, twice.

Products:

TRIzol (Life Technologies #15596018)

Chloroform (Sigma #25666-100ml)

PureLink RNA Mini Kit (ThermoFisher #12183020)

Experimental protocol n°12

RT-qPCR and relative gene expression analysis

4. Spin cartridge were dried out by centrifuging it at 12,000g for 2min at room temperature and RNase free water was warmed up to 45°C.

5. RNA was eluted by adding 30µl warmed-up RNase free water.

Spin columns were incubated for 2/3 min at room temperature before RNA was eluted at 12,000g for 2min at room temperature.

6. RNA purity, quantity, and quality was measured using the Nanodrop spectrophotometer (Thermo FisherScientific), and RNA was used for cDNA synthesis

II. cDNA synthesis

cDNA synthesis was achieved using the Moloney Murine Leukeia Virus

Reverse Transcriptase (M-MLV-RT, Life Technologies).

1. 1µg of RNA was mixed with 0.5µl oligod(dT) (500µg/ml), 0.5µl random primers, 1µl 10nM dNTPs Mix (10nM each dATP, dGTP, dTTP, and dCTP at neutral pH) and 12µl of sterile and distilled water, and warmed at 65°C for 5 min before being quickly chilled an ice and centrifuged 2. 4µl First Strand Buffer, 2µl 0.1M DTT, and 1µl RNase OUT Recombinant Ribonuclease Inhibitor (40 units/µl) are gently added to the sample and incubated at 37°C for 2min.

Finally 1µl M-MLV-RT is added to the mix and incubated at 25°C

for 10min and at 37°C for 50min.

4. The reaction was stopped by heating the samples at 70°C for 15min.

5. cDNA purity and quantity was checked using the Nanodrop, and the newly synthetize cDNA was used for RT-qPCR

III. RT-qPCR

Quantitative RT-PCR of exosomal markers analysis and housekeeping gene selection completed on muscles cells were performed on LightCycler 480 Instrument Roche using LightCycler 480 DNA SYBR Green I Master. 1. RT-qPCR was performed in 96 wells plate, and each well contained 15µl 2. A SYBR Green mix was set-up for each gene, SYBR Green mix for 1 well is prepared as follows:  3.9µl distiller water  7.5µl SYBR Green  0.6µl reverse and forward primers (20pmol/µl) for the gene of interest (see Table 1 for primer sequence).

3. 12µl of mix and 3µl of cDNA were added per well, in triplicate, and the plate was quickly centrifuged.

The cycling protocol includes:

 Initial step: 50°C for 2 min, and 95°C for 2min  50 cycles of : 94°C for 15sec, 60°C for 30sec, and 72°C for 5sec

Products:

Platinium SYBR Green qPCR Super-Mix (Life Technologies #11733-013)

Experimental protocol n°12 RT-qPCR and relative gene expression analysis

IV.

Comparative method analysis

The gene expression quantification was performed following the comparative Ct method, also referred to as ΔΔCt. The PCR amplification efficiency of the reaction was calculated using the slope of the standard curve using PCR products from healthy muscle cells as reference samples (1:20, 1:100, 1:500, and 1:2500 dilutions were used) as follows:

𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 (𝐸) = 10 -1/𝑠𝑙𝑜𝑝𝑒
The expression level in arbitrary unit was then calculated as follows:

𝐸

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Experimental protocol n°13

siRNA-based FUS silencing

Protein synthesis follows 2 steps, transcription of DNA into mRNA and translation of mRNA into proteins.

To silence a gene, small interfering RNA (siRNA) induce the degradation of mRNA before it can be translated into proteins. siRNA molecules are introduced to the cytoplasm of the recipient cells via cationiclipid-dependent transfection. The transfectant reagent (Lipofectamine) forms liposomes containing the siRNA that will be uptaken by the cells via endocytosis and delivers the siRNA to the cytoplasm of the cell.

Once in the cytoplasm, the double stranded siRNA that is complementary to the target mRNA forms a complex with the multiprotein complex RISC (RNA induced silencing complex). The RISC complex cuts the double stranded RNA into 2 single stranded RNA, the sense and antisense strands. RISC stays bound to the antisense strand to find complementary sequence with the mRNA. Once the target mRNA exits the nucleus for translation into proteins, the RISC complex identifies the complementary sequence and associate with the mRNA via complementary base pairing. The mRNA strand is cleaved by RISC and is rapidly degraded before translation into proteins can happen.

We performed Lipofectamine-mediated transfection on 1 day differentiated muscle cells to silence FUS gene. Three days after transfection induction and PKH26 labelled-exosomes treatment, fresh media is added to the 4 days differentiated muscle cells, and cells were cultured for 3 more days before fixation of cells.

Procedure

I. siRNA preparation

1. 100µM siRNA solutions, including siRNA of target genes and negative control siRNA (siRNA with sequence that don't have any sequence target) are prepared in nuclease-free water from dried siRNA.

2. siRNA solution at 10µM are diluted for utilization during transfection.

Products:

siRNA FUS sS5401 (Ambion #4392420)

Negative Control Silencer -Scramble (Ambion AM4635)

Nucleases-free water (Ambion #AM9937) Alternative if exosomes treatment is performed as well:

1. The Lipofectamine/siRNA is prepared as described in step 2.

2. Exosomes samples are labelled with PKH26 and resuspended in 450µl of DMEM per well treated.

Experimental protocol n°14

Exosomes treatment

Analysis:

1. Cell death: Following fixation, cells are stained with DAPI and cell death after treatment is defined by nuclei loss, total nuclei were counted in each treatment conditions and compared to non-treated cells.

Blebbingcellular stress:

The number of cell blebs are counted on live pictures at 4hrs, 24hrs, 48hrs, 72hrs, 96hrs, and 168hrs and normalized per cell area. 𝑀𝑦𝑜𝑛𝑢𝑐𝑙𝑒𝑎𝑟 𝑑𝑜𝑚𝑎𝑖𝑛 = 𝛴 𝑀𝐹20 𝑎𝑟𝑒𝑎 𝛴 𝑛𝑢𝑐𝑙𝑒𝑖 𝑖𝑛 𝑚𝑦𝑜𝑡𝑢𝑏𝑒𝑠 6. Exosomes integration: Exosomes integration was assessed on live pictures following integration of PKH26 staining in cytoplasm of myotubes.

Fusion index:

Healthy muscle cells treatment with ALS and healthy exosomes (2):

1. 100,000 healthy muscle cells are cultured in ibidi 4 well plate. 2. 50,000 cells were plated in ibidi 8 well plate and treated with 0.5µg of ALS or healthy exosomes at the time of differentiation.

3. After 3 days of differentiation, fresh DMEM is added to each well and cells were fixed at 6 days of differentiation.

Experimental protocol n°14

Exosomes treatment

Analysis:

Cell death and cell blebbing levels are determined as described above.

KO-FUS muscle cells treatment:

1. 100,000 healthy muscle cells are plated in 4 well Ibidi and transfected with either a 200nM s5401 FUS siRNA (experimental protocol n°13) or a negative control siRNA and treated with 4µg of ALS or healthy exosomes at day 1 of differentiation (see experimental protocol n° 8 for protein quantification).

2. Exosomes are integrated for 3 days before fresh DMEM is added.

3. Cells were fixed at 7 days of differentiation.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Experimental protocol n°14

Exosomes treatment

Analysis:

1. Cell death and RNA localisation: Analysis are following instructions as previously described.

2. RPL5 aggregation: Cells are stained for RPL5 and percentage of RPL5 aggregates-positive nuclei is determined.

II.

iPSCs MN treatment

Experimental protocol n°14

Exosomes treatment

Analysis:

-Cell death: Nuclei loss defining cell death is calculated following staining of cell cultures with DAPI as previously described.

-Neurites length: Cells are stained with anti-tubulin, beta III isoform antibody, or TUJ1 to label neurites. The length of all the neurites per picture is manually measured for each motor neuron and compared to non-treated cells.

-Neurites branching: Similarly, the number of branching for each motor neuron is manually counted and compared to non-treated cells.

-Isl1/2 proportion: To characterize the proportion neuronal cells altered by exosomes treatment, cell cultures are stained with a motor neuron marker (Isl1/2) and is used to calculate the number of motor neurons per picture. The percentage of motor neuron death is normalized by the total number of nuclei per picture.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Experimental protocol n°14

Exosomes treatment

Results

To date, ALS pathogenesis is still unknown and several studies suggest involvement of neighbouring cells such as astrocytes and microglial cells responsible for the degeneration of motor neurons in ALS patient 69,324,326,327 . Hence it is believed that ALS is a multisystemic disease. The primary involvement of muscle is still controversial, however multiple signs for an axonopathy in ALS patients prior to MN degeneration and symptom onset [START_REF] Saini | From death, Lead me to immortality"-mantra of ageing skeletal muscle[END_REF]342,368 suggest that in addition to its role in NMJ stabilization 346 , the muscle could participate to a toxic MN environment 343,344 . It is known that the muscle is acting as an endocrine organ via its secretory properties 154 , so we hypothesised that the muscle secretome contribute to the degeneration of motor neurons in ALS.

Thanks to the in silico secretome performed using transcriptomic data from two independent previously published studies 375,376 , the team observed that both endosome and lysosomes pathways were significantly enriched in ALS muscle -pathways known to be part of the exosomes genesis and secretion. The team then confirmed by electron microscopy imaging and immunostaining that there is a significant accumulation of MVB (TSG101) and exosomes (CD63) under the sarcolemma on muscle sections from sporadic ALS.

Exosomes originate from the inward budding of late endosomes and accumulate in endosomes called multivesicular bodies or MVB 152,377 . The MVBs full of exosomes then fuses either with the lysosome for degradation or with the plasma membrane to release their intraluminal vesicles in the extracellular space.

First thought to be the "cell's garbage" 158,159 , the role of exosomes in intercellular signalling was rapidly unravelled [147][148][149][150] , suggesting their important function in physiology and homeostasis. Thus we hypothesized that ALS muscle might influence intercellular communication between muscle and nerve through a disrupted exosomes pathway.

I. Exosomal pathway disruption in ALS patients.

ALS patients are characterized by progressive degeneration of upper and lower motor neurons leading to a gradual denervation responsible for muscle weakness and wasting 2 . In order to determine if the exosomal pathway up-regulation in ALS patients is dependent, or not, from muscle denervation, we extracted muscle stem cells (also known as satellite cells) -cells located at the surface of the myofiber 378 and naïve for any innervation/denervation process. To conduct this project, the team had access to muscle biopsies from sporadic ALS patients (including three C9orf72 mutation-and one ATXN2 mutation-associated patients)

and healthy subjects (including control diseases such as polyneurithis, SBMA and SMA patients, see Table 14). Because of the limited lifespan of human primary cells, cells from different patients were used for different experiment along the study: transcriptomic analysis, or for imaging (including electron microscopy and histology), or for exosomes extraction and cell treatments (muscle cells and iPSCs-derived The ALS and healthy muscle stem cells have the same mitotic age 379 as no differences in their proliferative capacities were observed. ALS muscle stem cells had a lower fusion index (~10% less) and formed thinner myotubes, characteristics already established by two studies 379,380 . The transcriptomic analysis of differentiated myoblasts performed by the team show that all ALS patient myotubes cluster together and are separated not only from the healthy subjects but also from the disease controls. ALS myotubes present thus a specific gene expression profile. Cell Where is an online tool developed by the team that allows a graphical display of protein subcellular localization (https://sys-myo.com/cellwhere/). Following the groundbreaking transcriptomic analysis results and using the Cell Where tool, the list of top 30 genes mostly contributing to this ALS-specific signature, revealed among others an enrichment of genes encoding for proteins that are secreted through exosomes proteins. The present study shows for the first time that: 1) the ALS signature is shared across muscle stem cells from ALS patients (including monogenic form of ALS and ALS with non-known causes) and 2) this strong signature has never been observed in motor neurons, astrocytes or inflammatory cells 381,382 . Accordingly, a specific gene expression profile was observed in muscles cells of an ALS murine model and was different from axotomy-induced dennervated muscles transcriptome profile 383 .

II. Muscle exosomes characterization and toxicity study

This project is based on extraction of exosomes from muscle cells cultures. There is no gold standard protocol for optimal separation of exosomes, classically exosomes extraction is performed by differential centrifugation 148 . Because the exosomes extracted after ultracentrifugation form a loose invisible pellet and consequently lead to the loss of most of the exosomal material, it thus requires a large volume of starting material, approximatively 100-250 million muscle stem cells 268 , to assure an efficient exosomal extraction for one experiment without any replicates. Our muscle stem cells are primary cells possessing a limited maximum number of division (38-46 divisions) before reaching senescence (Table 15). In order to avoid working with pre-senescent cells, we are working with a limited number of cells forcing us to use a polymer based extraction kit from Life technologies that allows a rapid exosomes isolation from smaller starting material samples. Single vesicle analysis, including electron microscopy immunostaining, and NanoSight analysis and protein content composition through western blotting allowed us to validate that the extraction kit isolate cupshaped vesicles, positive for exosome markers as recommended by the field 390 . The quantity of exosomes secreted by ALS muscle cells was greater than the healthy cells (see Figure 2H and supplemental Figure 3F in paper). To follow the integration of exosomes by recipient cells, we labelled exosomes with an organic fluorescent dye PKH26. We first studied the effect of ALS muscles exosomes on muscle cells in a muscleto-muscle ratio of 1:1. Labelled ALS and healthy exosomes were rapidly uptaken by the muscle cells, four hours after adding the exosomes to the culture medium. The differentiation of myoblasts into myotubes was not affected by the exosome treatment, as the fusion index and the number of myonuclei per myotube were similar in all conditions (Figure 11). ALS exosomes were toxic for muscle cells by inducing muscle atrophy, cell stress and cell death (see supplemental Figure 4A-E in paper). When we loaded either 8µg or 4µg of ALS and healthy exosomes, we consistently observed a lower death when myotubes were treated with 4µg ALS exosomes compare to 8 µg. In addition, the lowest concentration of ALS exosomes -4µg -induced a greater cell death than the highest concentration of healthy exosomes -8µg-, suggesting that both quantity and quality of exosomes are important for the toxicity of ALS exosomes.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Secretion of neurotoxic vesicles by muscle cells of ALS patients

As ALS is a motor neurone disease affecting motor neuron survival, we next characterized the effect of muscle exosomes on healthy human iPSC-MN. For this purpose we had to determine how much exosomes amount would be added to the MN cultures. A single motor neuron innervates several muscle fibers, forming together one motor unit. However, the number of muscle fibers per motor unit is highly variable 338,391 , ranging from 180 muscle fibers in the soleus muscle 392 to several hundred in the gastrocnemius muscle 338 . Moreover, a single myofiber contains in average 500 myonuclei 393,394 . If we take the example of the soleus muscle, one motor unit would contain approximatively 90,000 myonuclei.

Translated to cell cultures where 3,000 MN are seeded in a single well from a 384 well plate, exosomes isolated from the culture medium of 270,000,000 myoblasts would be necessary for a single well treatment.

Because it is technically impossible to meet these criteria we decided to apply a muscle-to-neuron ratio of

Secretion of neurotoxic vesicles by muscle cells of ALS patients

1. Similarly MN were treated with ALS and healthy muscle exosomes and 72h after treatment 60% of neuronal death was observed. In collaboration with Dr Hélène Blasco and Dr Blandine Madjihounoum, we treated primary murine motor neurons with ALS and healthy human exosomes and observed 96hrs after treatment a higher cell death (see Figure 13).

Figure 13 : Primary motor neuron survival after ALS or healthy exosomes treatment

To further study the role the toxic effect of exosomes, we treated the human iPSCs motor neurons with equal amount of ALS and healthy exosomes following BCA-based protein quantification. The same low dose corresponding to 0.5µg of exosomes was added to the culture medium of 3,000 iPSCs MN per well.

Likewise, both ALS and healthy exosomes were integrated by the recipient cells, and 72h after the treatment of the motor neurons with exosomes we observed signs of cell stress such as shorter neurites and reduced branching in iPSCs MN treated with ALS muscle exosomes. Interestingly, even low concentrations of ALS muscle exosomes induced a higher cell death compare with healthy exosomes treatments. As previously described 373 , during the motor neuron progenitors' differentiation other neuronal cell type such as interneurons can be find among the motor neurons population. Approximatively 80% of the progenitors will differentiate into motor neurons. To characterize the proportion of motor neurons affected by the ALS exosomes treatment, we used a motor neuron specific marker Isl1/2. Our results show that treatment of MN with ALS exosomes didn't affect the proportion of non-neuronal cells or Isl1/2 negative cells suggesting that ALS exosomes are specifically targeting MN. The cell death we measured (approximatively 65%) is representing the loss of MN. In addition, these exosomes also induced shortened, less branched neurites.

Numerous studies have described protein aggregation in ALS patients either positive for SOD1, FUS or TDP43 and others 43,53,63,99,[START_REF] Liu | TGF-␤ inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3[END_REF][START_REF] Sousa-Victor | Geriatric muscle stem cells switch reversible quiescence into senescence[END_REF] . Based on these data we wanted to explore whether 1) we could detect these proteins in muscle exosomes, and 2) determine if these ALS candidate proteins are involved in ALS muscle exosomes toxicity. Interestingly only FUS was found in ALS muscle exosomes. Based on these data we investigated the role of exosomes on the FUS pathway. We then generated four stable cell lines expressing

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Laura Le Gall 117 either a Flagged form of wild-type FUS (FUSFLAG), or wild-type SOD1FLAG or wild-type TDP43FLAG. At equal low concentration (0.5µg of exosomes per well), we observed a greater cell stress and death after treatment with ALS exosomes in all four cell lines, but the effect was markedly greater in the FUSoverexpression cells (see Figure 4G in the paper).

The toxic effect of ALS exosomes is thus exacerbated in the presence of an over-expression of FUS (see Figure 4G in the paper). On the other hand, when we knock down FUS expression in the recipient cells, the cells were less sensitive to ALS exosomes (see Figure 4H in the paper). These data suggest that cells that express a high level of FUS will be more sensitive to ALS muscle exosomes. Interestingly iPSCs MN express higher level of FUS further highlighting the role of FUS in exosomes toxicity (See figure 4I in the Human proteome database). FUS is a multifunctional DNA/RNA-binding protein involved in multiples steps of gene expression, including mRNA transcription and RNA transport 53 and many RNA metabolism pathways are believed to be disrupted in ALS patients. Interestingly, when we treated the cells with ALS exosomes, we observed a significant increased number of nuclei positive for RNA (see Figure 4D and supplemental Figure 5B in the paper), suggesting a disruption of RNA transport following ALS muscle exosomes treatment.

In parallel, we also observed that ALS myotubes present an altered distribution of FUS (FUS granulation study performed using FindFoci algorithm on ImageJ/Fiji software 395 ) and TDP43 (see Figure 12), they accumulate RNA in their myonuclei, and they present a mislocalization of the two FUS partners, RPL5 and Caprin-1 (see Figure 4C and supplemental Figure 5A in the paper). Altogether, these data suggest that RNA transport is affected in ALS muscle cellsphenomenon described to be affected in different ALS models 396 . In summary, the present study we show that (1) ALS myotubes present a specific signature with accumulation and over-secretion of exosomes and (2) these muscle exosomes induce toxic effects on both human healthy muscle cells and iPSCs MN. We also unraveled that ALS muscle exosomes toxicity involves the FUS pathway.

Abstract:

The abstract should be about 100-150 words, and organized in this structure: An opening sentence that sets the question that you address and is comprehensible to the general reader, background content specific to this study, results, and a concluding sentence. It should be one paragraph only.

The cause of the motor neuron death that drives terminal pathology in Amyotrophic Lateral Sclerosis (ALS) remains unknown, and is increasingly thought to result from the interplay of multiple mechanisms and cell types. We report that the skeletal muscle of ALS patients secretes exosomal vesicles that are specifically toxic to motor neurons. Among muscle biopsies and biopsyderived denervation-naïve differentiated muscle stem cells (myotubes) from 66 human subjects, including healthy and disease controls, ALS myotubes had a consistent signature of disrupted exosome biogenesis and RNA-processing. Exosomes from ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins, in motor neurons. Some 37% of known protein binding partners of the RNA-processing protein FUS were present in the proteome of ALS muscle exosomes, and exosomal toxicity was dependent on the presence of FUS, which is highly expressed in recipient motor neurons.

One Sentence Summary: Muscle cells of ALS patients secrete exosomal vesicles that are toxic to motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disorder affecting 3-5/100,000 individuals per year (1). The cause of pathology is likely complex with onset resulting from some combination of genetic mutations, DNA damage, environmental risk factors, viral infections, or other factors (2-4), leading to diverse cellular dysfunction such as glutamatemediated excitotoxicity (5), abnormal protein aggregation (6), and mitochondrial disorganization and dysfunction (7, 8) contributing to oxidative stress (9).

Not only motor neurons (MN) are affected in ALS, but also glial cells, muscle fibers (10) and immune cells (11), each of which may participate actively to ALS onset and progression. In a FUS murine model of motor neurone disease, where the FUS mutation is expressed in all tissues except the MN, the motor deficits still appear at a late stage (12). In addition, when the human mutated SOD1(hSOD1) is selectively knocked-down in MN and astrocytes of newborn transgenic hSOD1 mice, their life expectancy is prolonged by only 65-70 days, and there is still significant astrogliosis and microglial activation (13). These studies suggest that the local environment has a key role in "non-cell autonomous" MN death (14).

Numerous cell and tissue types, including skeletal muscle, can secrete exosomes and other types of vesicle (15, 16). Such extracellular vesicles may be involved in cell-cell communication in the central nervous system (17, 18), where they can carry out intercellular transport of functional proteins, mRNA, miRNA, and lipids, and may have key roles in spreading of proteinopathies (17, 18) or neurotoxic elements (19). For instance, astrocytes extracted from the SOD1 murine model of ALS secrete exosomes that contain hSOD1, and propagate this toxic protein to neighboring MNs (20). As intensive efforts remain underway to determine the role of cell to cell communication in ALS, we hypothesized that the muscle secretome may be an important component of the disease, based on long-standing observations that disassembly of the neuromuscular junction is an early critical event in the onset of the clinical pathology of ALS (10), and that motor neuron degeneration may start at the neuromuscular junction (21-26).

Accumulation of exosomes in ALS muscle biopsies

To explore previous data for evidence of muscle vesicle involvement in ALS, we tested for dysregulation of secretory compartments in a reanalysis of two published independent studies of gene expression in muscle biopsies from sporadic ALS patients (27, 28). These showed a significant enrichment in endosomal and lysosomal compartments (Fig. S1A, Table S1) -two structures that are implicated in exosomal biogenesis and secretion. Prompted by this finding, we analysed patient muscle biopsies, and observed an increased frequency of multi-vesicular bodies (MVBs) in ALS muscles (1 MVB for every 62.09±13.3 sarcomeres, vs 1 MVB for every 330.8±127 sarcomeres in healthy controls; Fig. S1B) and an accumulation of exosomal markers at the periphery of the myofibers (Fig. 1, Fig. S1C-D).

ALS patient muscle cells accumulate and secrete exosomes

To investigate the pathological consequences of increased exosomal accumulation in ALS muscle, we extracted myoblast cells from biopsies of confirmed ALS patients at an early stage of pathology. These were compared to healthy subjects, and to SMA-III/IV and SBMA which are two lower motor neuron disorders that mimic ALS clinically (see Table S2 for patient description, and a breakdown of which samples were used in which experiment). Transcriptomic analysis showed that ALS myotubes had gene expression patterns significantly different not only from healthy control subjects but also from SMA-III/IV and SBMA (Fig. 2A), and many of the dysregulated mRNAs encode proteins known to be secreted or localized in exosomes (Fig. 2B).

Differentiated ALS myoblasts formed smaller myotubes with less nuclei per myotube (Fig. S2C), a smaller nuclear domain (Fig. S2E) and lower fusion index (Fig. S2F), compared to controls, whereas mitotic age and proliferative capacity were the same as controls (Fig. S2A andB). These observations suggest that ALS muscle cells are not subject to increased rates of regeneration throughout patient lifespan, and that observed differences with healthy controls are not due to ageor disease-induced cell senescence (29).

Immunostaining (Fig. 2C and Fig. S2G), RT-qPCR (Fig. 2D) and electron microscopy (Fig. 2E) all showed a consistent accumulation of exosomal markers in ALS myotubes. This feature is not a characteristic of muscle denervation or repeated re-innervation as no accumulation of exosomal markers was observed in SBMA, SMA-III/IV and polyneuritis cells by immunostaining (Fig. 2C).

Similarly, no upregulation of exosomal markers was observed in either murine cachectic muscle or muscle denervated for 7 days (Fig. S3A-C). Exosome secretion by ALS myotubes was confirmed by electron microscopy (Fig. 2F). Both ALS and healthy vesicles were positive for exosomal markers CD63, CD82, Flotillin and both were negative for calnexin, an endoplasmic reticulum marker normally absent in exosome fraction (Fig. 2G andS3D). The size of the exosomes was also typical (90-200 nm) (Fig. S3E). The exosome-enriched fraction secreted by ALS myotubes contained twice as much protein as that secreted by an equal number of healthy control myotubes (Fig. 2H, Fig. S3F).

Secreted ALS exosomes are toxic and their toxicity is driven by FUS

To test exosomal toxicity, 0.5 µg of ALS or healthy exosomes were added to the culture medium of healthy human iPSC-derived motor neurons (hiPSC-MN). Following exosomal uptake (Fig. 3A), only ALS exosomes and not healthy exosomes resulted in shorter neurites (Fig. 3B), with less branching (Fig. 3C) and a greater cell death (Fig. 3D) 72h post-treatment. Similarly, when added to the culture medium of healthy human myotubes, ALS exosomes induced myotube atrophy (Fig. S4A), and cell stress (Fig. S4B) leading to cell death (Fig. S4C-D). The quantity of cell death was decreased when less exosomal protein was loaded, though ALS exosome toxicity remained greater than healthy exosome toxicity at either dose (Fig. S4E-G), suggesting that toxicity is dependent on both the quantity and content of exosomes. Since a leading theory is that disruption of RNA metabolism, including of its translation, transport, storage, and degradation, contributes to ALS physiopathology by affecting neuronal function and viability (30), we hypothesized an involvement of RNA processing in exosome toxicity. Human ALS myotubes presented a greater granulation of the RNA-processing protein FUS within the nucleus (Fig. 4A), as well as a nuclear accumulation of RNA (Fig. 4B), and mislocalization of two FUS protein binding partners, RPL5 and caprin 1, that are involved in RNA processing and stress granule formation (Fig. 4C and S5A) (31). Furthermore, proteomic analysis revealed that ALS muscle exosomes contain many known protein binding partners of the RNA-processing proteins FUS (31) and TDP43 (32) (P< 0.00001; Fig. S6A), with some 37% (40 of 109) of FUS binding partners being detected. Genes encoding FUS and TDP43 binding proteins were upregulated in the myotubes of ALS patients and these genes were shared with many RNA-processing pathways that were similarly upregulated (Fig. S6B andS6C). Interestingly, when human iPSC-MNs were treated with ALS exosomes, RNA accumulated in their nuclei (Fig. 4D), an event that could lead to cell death (33). Similar results were obtained when ALS exosomes were added to cultures of healthy human myotubes (Fig. S5B). Furthermore, exosomes contained FUS and FUS binding partners (Fig. 4E-F, Fig. S5C, Fig. S6). Therefore, ALS exosomal toxicity in recipient cells may be mediated through the FUS pathway. To test this, we added 0.5 µg of ALS or healthy exosomes to the culture medium of a human muscle cell line that over-expressed a previously published tagged form of wild-type FUS (FUS-FLAG, (34)). This induced a dramatic increase in cell death (Fig. 4G), accompanied with greater cellular stress (Fig. S5D) -the same was not observed in cell lines that over-expressed tagged forms of wild-type TDP43 and SOD1 (Fig. 4G). Conversely, when ALS exosomes were added to the culture medium of cells in which FUS was knocked down, a lower proportion of RPL5 granules was observed (Fig. S5E) and the quantity of cell death was reduced (Fig. 4H), suggesting that the toxicity of ALS exosomes was partially reduced in the absence of FUS expression in the recipient cells. These data implicate the FUS pathway in exosome toxicity.

Consequently, cells that express a high level of FUS, as observed in hiPSC-MN (Fig. 4I), may be particularly sensitive to ALS muscle exosome toxicity.

Discussion

Exosomes are suspected to carry toxic elements from astrocytes towards motor neurons (13), and pathological proteins have recently been identified in the extracellular vesicles of sALS patients (35). Here muscle exosomes are shown to be toxic to motor neurons, thereby establishing the skeletal muscle as a participant to a vesicle-mediated toxicity model of ALS pathology. The observation that ALS muscle exosomes act on RNA transport, and that RNA transport protein mislocalization is partially corrected and cell death reduced when FUS expression was abolished in recipient cells, is consistent with a body of literature suggesting an RNA processing blockade mechanism in ALS MN (36, 37). High expression levels of FUS in motor neurons may render them especially susceptible to the toxic contents of exosomes, corroborating with the previously observed selective vulnerability of motor neurons to endocytosis of pathogenic proteins (38, 39).

The proportionate abundance of muscle tissue in the body would suggest that, as a source of exosomal toxicity, muscle could make an important contribution to motor neuron pathology.

The accumulation and secretion of neurotoxic muscle exosomes was observed across all sporadic ALS patients of our cohort, including patients carrying mutations in C9orf72 or ATXN2, suggesting that this is a common feature across most or all genetic backgrounds of ALS. The consistent observation of a pronounced RNA processing and protein mislocalization phenotype in the ALS myotubes suggests that these cells recapitulate aspects of the disease mechanism that have been observed in motor neurons. Since this phenotype was preserved in primary myoblasts after several divisions and differentiation ex vivo, it must be due to some property of the cells that is heritable between generations of cell division, suggesting DNA mutation or epigenetic imprinting, but it is also possible that some transmissible agent is responsible. Moreover, muscle vesicle-mediated toxicity may represent a common target for novel ALS therapeutics, and muscle exosomes, which have the advantage of being released outside of the blood-brain barrier, and their contents may act as biomarkers with mechanistic links to this toxicity.

Supplemental Table S1 No. x 61 M Table summarizing the subject samples used for each type of experiment. Subject age at time of biopsy is indicated in the column "Age". All ALS patients were confirmed to be ALS according to El Escorial. The ALSFRS-R and Muscle Testing results measured at the time of the biopsies are given. Manual muscle testing was scored from 0, representing total paralysis, to 5, representing normal strength, according to the Medical Research Council Score. Enrichment map showing that genes encoding FUS and TDP43 binding proteins are upregulated in ALS myotubes compared to healthy controls, and that these genes are shared with many RNA-processing pathways that are similarly upregulated (red = upregulated).

N/ A N/ A N/ A N/ A N/ A x 75 M N/ A N/ A N/ A N/ A N/ A x 56 F N/ A N/ A N/ A N/ A N/ A x 64 F N/ A N/ A N/ A N/ A N/ A x 51 M N/ A N/ A N/ A N/ A N/ A x 52 F N/ A N/ A N/ A N/ A N/ A x x 47 F N/ A N/ A N/ A N/ A N/ A x x 59 M N/ A N/ A N/ A N/ A N/ A x x 19 M N/ A N/ A N/ A N/ A N/ A x x 59 M N/ A N/ A N/ A N/ A N/ A x x x x 54 M N/ A N/ A N/ A N/ A N/ A x x x 59 F N/ A N/ A N/ A N/ A N/ A x x x 49 F N/ A N/ A N/ A N/ A N/ A x x x 53 M N/ A N/ A N/ A N/ A N/ A x x x x 70 M N/ A N/ A N/ A N/ A N/ A x x x x 21 M N/ A N/ A N/ A N/ A N/ A x x 21 M N/ A N/ A N/ A N/ A N/ A x x 53 M N/ A N/ A N/ A N/ A N/ A x 69 F N/ A N/ A N/ A N/ A N/ A x 66 M N/ A N/ A N/ A N/ A N/ A x 21 M N/ A N/ A N/ A N/ A N/ A x x x x 33 M N/ A N/ A N/ A N/ A N/ A x x x 22 M N/ A N/ A N/ A N/ A N/ A x x x x 25 M N/ A N/ A N/ A N/ A N/ A x x 70 M N/ A N/ A N/ A N/ A 13 x x 45 F N/ A N/ A N/ A N/ A

Material and Methods

Participants and ethical approvals

An open biopsy was performed on deltoid muscles of 18 ALS patients with probable or definite ALS according to the revised El Escorial criteria (Brooks BR et S1.

The protocols (NCT01984957) and (NCT02360891) were approved by the local Ethical Committee and all subjects signed an informed consent in accordance with institutional guidelines.

Immunolabelling

-Immunocytochemistry: 200,000 cells or 100,000 cells were respectively plated on u-dish 35mm high ibidiTreat or 4 wells plate ibidiTreat (ibidi®) in proliferative medium. The following day, muscle stem cells were washed with PBS and myogenic differentiation was induced by cultivating the cells in DMEM only.

Muscle cells were fixed at 3 days of differentiation using 4% formaldehyde. The cells were permeabilised, blocked and stained as described (42).

-Immunohistology: 8 μm muscle transverse sections were cut from human biopsies on a cryostat microtome at -20ºC, permeabilised, blocked and stained as previously described (29) Primary antibodies used are listed in the Table below and

Electron microscopy

-Electron Microscopy for extracted exosomes -Purified vesicles were fixed in 2% paraformaldehyde and were counterstained with uranyl and lead citrate and analysed as previously described ( 16).

-Electron microscopy for human myotubes -Human myotubes were plated on plastic (Thermanox, Nalge Nunc, Rochester, NY, USA) coverslips and fixed in

Muscle stem cell extraction and culture.

Briefly, muscles biopsies were dissociated mechanically as previously described in (29), and plated in proliferation medium [1 volume of M199, 4 volumes of Dulbecco's modified Eagle's medium (DMEM), 20% foetal bovine serum (v:v), 25 ug.ml -1 Fetuin, 0.5 ng.ml -1 bFGF, 5 ng.ml -1 EGF, 5 ug.ml-1 Insulin]. The myogenic cell population was enriched using CD56 magnetic beads, and for their myogenicity using anti-desmin antibodies as described before (29). A minimum of 80% of the cell population were positive for desmin. After rinsing 3 times the proliferative myoblasts with PBS, and 3 times with DMEM to remove any FBS residual, the human muscle stem cells were differentiated into myotubes by culturing them in DMEM for 3 days.

Number of divisions per day and life span.

Primary myoblast proliferation was followed until proliferative exhaustion. The number of divisions was calculated as follows:

RNA extraction.

Purified muscle stem cells were differentiated for 3 days into myotubes. RNA from muscle cells was extracted as described in (29). The quality of RNA samples was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA).

Gene expression profiling.

-mRNA gene expression profiling -Aliquotes of high-quality total RNA from each sample (ALS n=6, SBMA n=6, SMA n=5, and healthy n=6, muscle stem cells)

were used for mRNA expression profiling using GeneChip Human Exon 1.0 ST arrays (Affymetrix) as previously described (27).

- The amplification efficiency of the reaction was calculated using data from a standard curve using RT products from control cells as reference samples

(1:20, 1:100, 1:500 and 1:2500 dilutions used). The gene expression level was estimated using the comparative Ct method -Ct representing the cycle at which the fluorescence signal crosses the threshold as previously described (29).

Primers

Sequence (5' → 3')

Lamp1

Forward

Exosome extraction

Cells were differentiated into myotubes for 3 days in DMEM. The conditioned media were then harvested, centrifuged at 260 g for 10 min at room temperature, and then at 4000 g for 20 min at 4°C to remove any dead cells and cell debris. The subsequent supernatant was filtered through 0.22 um filter to remove microparticles. The filtered medium was then mixed with total exosome isolation reagent (Life technologies™;

2:1, v:v), incubated overnight at 4°C, and then centrifuged at 10,000 g at 4°C for 1 h.

The supernatant was discarded and the pellet containing the exosomes was resuspended in 100 µl PBS. The exosome suspensions were kept at -80°C until needed. Exosomal protein was extracted using 8M Urea and quantified using BCA kit.

NanoSight

Size and distribution of ALS and control exosomes (n=3 per group) were evaluated by a NanoSight LM10 instrument (NanoSight) equipped with NTA 2.0 analytic software.

Exosome labelling

The exosomes were labelled using PKH26 kit (Sigma-Aldrich®). Briefly, after adding 100 µl of Diluent C to the exosome suspension, 100 µl of 4 µM PKH26 solution were added to the sample. After 5 min of incubation, 1 ml PBS was added and the exosomes were washed using a 100K concentrators, 15,000 g at 4°C for 10 min. The exosomes were washed 3 times in PBS using the 100K concentrators before being mixed with the cell media for treatment.

Exosomes added to iPSC motor neurons

hiPSC-derived motor neurons were obtained as previously described (44). 6,000 MN progenitors differentiated for 9 days were then plated in poly-l-ornithine (SIGMA)

Exosomes added to healthy human muscle cells.

Labelled exosomes were added to the differentiation medium of 200,000 control cells cultured in Ibidi 35mm µ-Dishes. Exosome absorption occurred during the first 3 days of differentiation. The myotubes were then rinsed 3 times with PBS, and fresh DMEM was added to the petri-dishes. The cells were fixed with 3,6% formaldehyde for 15min

at room temperature at day 3 or day 7 of differentiation, then washed 3 times in PBS and stored at 4C until subsequent analysis:

-Exosome integration -The exosome integration was checked by live imaging at 4h, 24h, 48h, 72h, 96h and 168h.

-Myonuclear domain -The myotubes were fixed and stained for DAPI and MF20

as described above.

o The myonuclear domain was calculated using the following formula:

Myonuclear domain = ( Σ MF20 area) (Σ nuclei in myotubes) -Stress blebbing -Blebs were counted on live images.

-Cell death inducing an increase in H2Ax expression level -The myotubes were fixed and stained for DAPI and H2AX as described above. To measure H2AX signal per myonucleus, the total area of H2AX signal was divided by the total number of myonuclei.

-Cell loss -The total number of nuclei were counted in each field using ImageJ 1.37v, and summed for each well.

RNA staining

ALS and healthy muscle cells were differentiated for 3 days before being fixed using 4% paraformaldehyde and permeabilized for 1 hour at RT (5% BSA; 20% FBS; 0,5% Triton X100; 0,5% Tween20). Cells were then stained with a 20 µg/mL acridine orange solution for 10 min at RT.

Muscle cell lines overexpressing wild type forms of FUS, SOD1 and TDP43.

Healthy human muscle cell lines were transduced with previously published plasmid coding either for a tagged form of wild type FUS (FUS-3xFLAG, (34)), or wild type SOD1 (SOD1-3xFLAG, modified WT-SOD1 plasmid from ( 45)) or TDP43 (TDP43-3xFLAG, modified WT-TDP43-YFP plasmid from ( 46)) and a selection was done with hygromycin. 50,000 Cont, FUSFLAG, TDP43FLAG and SOD1FLAG muscle cells were plated in 8 well Ibidi® plates in proliferative medium. The next day, myogenic differentiation was induced by replacing the medium with DMEM, and cells were treated with 0.5µg of healthy and ALS exosomes. Exosomes were applied for 3 days, before fresh DMEM medium was applied to the cultures. At 6 days of differentiation, cells were fixed and analyzed for cell death and cell blebbing as described above.

Proteomic

-Protein extraction from exosomes -A volume of cell culture medium was mixed to 0.5 volumes of Total Exosome Isolation Reagent from cell culture media (lifeTechnologies®) and incubated overnight at 4°C. The samples were then centrifugated at 10,000 × g for 1 hr at 4°C. The exosome pellets were resuspended in 25 μl 8M Urea, 50 mM ammonium bicarbonate, pH 8.5, and reduced with DTT for 1 h at 4˚C. Protein concentrations were then quantified using Pierce BCA Protein Assay kit (ThermoFisher®). Exosomal proteins were kept at -80°C.

-Proteome profile determined by Mass spectrometry -Approximately 20 µg of exosome protein were trypsin digested using a SmartDigest column (Thermo) for 2 hours at 70oC and 1400 rpm. Peptides were then fractionated into 8 fractions using a high pH reverse phase spin column (Thermo). Fractioned Exosomes were integrated by the cells for 3 days before fresh differentiation medium was added to the 5 days differentiated cells. The cells were harvested at day 8 of differentiation to check for RNA levels by RT-qPCR and to perform cell death analysis, immunostaining for RPL5, and distribution analysis of RNA/DNA using acridine orange staining.

Western Blotting

Extracted ALS and healthy exosomes were washed three times with PBS using 100K MWCO concentrator columns (LifeTechnologies

Discussion

Knowing that neuromuscular junction disassembly is a key step in the pathology 341,342 , we investigated the role of muscle and reported that the skeletal muscle of ALS patients secretes exosomal vesicles that are specifically toxic to motor neurons. The increased secretion of exosomes by ALS muscle cells might be related to an increased calcium concentration in ALS muscle 397 , calcium known to stimulate the exosome secretion in cells 398 .

Do muscle exosomes contribute to the proteinopathy observed in ALS?

Numerous proteins pathways dysregulation have been reported such as proteins aggregation in the cytoplasm of the cells, misfolded proteins, proteasome activity defects and autophagy failure 85,86 and correspond to key pathogenesis pathways in ALS patients.

Exosomes have already been suggested in other neurological diseases to propagate neurotoxic elements such as -amyeloide peptides or synuclein in Alzheimers's and Parkinson's diseases 317,366 , respectively.

Similarly, ALS associated proteins such as SOD1, FUS, TDP43 are observed in circulating extracellular vesicles from ALS serum 399 and CSF samples 400 . These vesicles could thus participate to the propagation of protein aggregatesaggregates that are a common feature among ALS patients 43,53,63,99,[START_REF] Liu | TGF-␤ inhibits muscle differentiation through functional repression of myogenic transcription factors by Smad3[END_REF][START_REF] Sousa-Victor | Geriatric muscle stem cells switch reversible quiescence into senescence[END_REF] and contribute to motor neuron death.

Muscle atrophy has been generally linked with dysregulated proteasome activity 401 and several studies have demonstrated that exosomes contain components of the proteolytic machinery 363,402 . In ALS patients, a protein homeostasis dysregulation has been studied in many ALS associated mutations and sporadic cases 70,84,85,88,89,403 , and muscles exosomes could deliver proteasome components to nearby muscle cells and contribute to this abnormal proteolytic activity. Concordantly, in our study ALS exosomes induced myotube atrophy once added to the culture medium of healthy myotubes (See Figure supplemental 4A).

How exosomes contribute to RNA processing disruption?

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Following the identification of mutations in two RNA binding protein in ALS patients, FUS and TDP43, RNA metabolism has been vastly studied. Among the RNA processing pathways that are altered in ALS is transcription defects, splicing dysregulation and nucleoplasmic transport impairment 22,46,78,81,404 . RNA accumulation in the nuclei has already been demonstrated in SOD1 mutant and C9orf72 mutation associated ALS patients 405,406 . Interestingly, when ALS muscle exosomes are added to the culture medium of motor neurons, they contribute to an increase level of RNA accumulation in MN nuclei (Figure 16).

ALS exosomes contain FUS and binding partners (see Figure 4E and Figure supplemental 5C and 6). An increase in wild type FUS expression level is sufficient to induce ALS pathogenesis in motor neuron 407 .

Moreover FUS contribute to DNA damage response 408,409 , as a knock down of FUS is responsible for an increase in DNA damage levels. FUS is also suggested to be involved in the

How can exosomes participate to the propagation of disease?

Knowing that the pathology starts in one member before spreading to the other non-affected limb, and then ultimately into the entire body, one can hypothesize that exosomes toxicity could be spread towards healthy part of the body through the bloodstream (Figure 16). Indeed, muscle exosomes have been isolated from blood samples 411 (and results in the team) (Figure 16, 7-). Moreover several studies have already demonstrated the ability of exosomes to cross the blood-brain-barrier 267,412 suggesting that once in the bloodstream, ALS muscle exosomes can easily target non-affected UMN and by extend LMN or muscle fibers contributing to the relentless and fast progression of symptoms in ALS patients. Furthermore, muscle exosomes could possibly exploit viral routes to target the CNS (Figure 16, 5-). The NMJ is a highly specialized synapse between nerve and muscles, and most motor neurons synapsing with muscle have their soma in the spinal cord where a synaptic contact with a motor neuron that have their cell body in the cortex 413 . It is not surprising that viruses and bacteria take advantage of the NMJ as a gateway to spread in the CNS. The Rabies virus (RABV) is a neurotrophic virus that induce a fatal and acute myeloencephalitis in human following infection 413 . The RABV transport depends on long distance axonal spread between synaptically connected neurons using retrograde transport machinery as well as endosomal trafficking pathway 414,415 . This infection mechanism allows the spreading of the infection from post-synaptic to presynaptic motor neuron until reaching the CNS. Similarly it has been demonstrated that the canine adenovirus type 2 once injected in the muscles is transduced in motor neurons in the same manner 416 This project demonstrated that 1) ALS muscle stem cells share a specific ALS signature that is independent from any innervation/denervation process. 2) Multiple RNA processing dysregulation have been observed in ALS muscle cells such as nuclear accumulation of RNA, high FUS nuclear granulation, and aggregation of FUS partner, RPL5 in myonuclei. Here illustrated as brown nuclei from altered muscle fiber in dark red colour. 3) We showed a toxic secretion of exosomes by muscle cells of ALS patients that are toxic and induce cell death in 4) muscle cells and 5) motor neurons. 5) ALS muscle exosomes are toxic towards MN and could potentially be retrogradely transported towards to cell body of the affected MN (here in red dashed line versus green full line for healthy non-altered MN) and 6) induce an accumulation of RNA in MN nuclei. On the other hand, exosomes have been isolated from blood samples meaning they can 7) have access to the bloodstream and 8) pass the brain-blood-barrier. 9) Exosomes from motor neuron neighbouring cells such as astrocytes, microglia and oligodendrocytes also secrete exosomes that participate in the toxic motor neuron environment. 10) Once the upper motor neurons are altered by ALS muscle cells, toxicity could easily be spread to non-affected motor neurons and/or muscle cells and contribute to the progression of ALS pathology.

Conclusion

For many years now the muscle involvement in ALS has been controversial and several studies have suggested contradictory effects of muscles. This project based on human primary muscle cells from sporadic ALS, healthy subjects and control diseases has demonstrated for the first time the significant role of muscle stem cells in exosomes in ALS pathology. During this study, we analysed bulk isolations of muscle exosomes from ALS patients, however multiples exosomes subpopulations are secreted at the same time by the same cell 178,224,225,[227][228][229][230] . Using immunoaffinity-based isolation technique we could determine if the entire population of ALS muscle exosomes or a specific sub-population is toxic and would allow future studies to specifically target the muscle-induced toxicity.

INTRODUCTION

Over the last 60 years, work performed on animal models, chiefly mouse, rat, and avian, and on human samples, has revealed and explored the capacity of adult stem cells -also called somatic stem cells -to self-renew and to differentiate into unipotent progeny within their residing tissue [1], generally for the purpose of repair. Resident stem cell populations have now been described in most tissues, including bone marrow Fax: +33 1 42 16 57 00; E-mail: stephanie.duguez@upmc.fr.

[2], blood vessels [3], peripheral blood [4], skin [5], teeth [6], gut [7], liver [8], heart [9], brain [10] and skeletal muscle [11]. Once body growth has stopped and adulthood is reached, most of these stem cells become quiescent, and will only be activated for tissue turnover. Although this turnover can be very active as in circulating blood or gut epithelium in other tissues such as liver the stem cells usually remain unsolicited as hepatic damage rarely occurs in healthy adults [8]. Despite this heterogeneity, a decline in number and properties is universally observed in aged stem cells, a phenomenon which alters the maintenance of tissue homeostasis with aging. In aged skeletal muscle, a tissue with low turnover, this decline in the adult stem cell (also called satellite cell), which is responsible for muscle repair [12], is associated with muscle atrophy and muscle weakness [13-15], although their depletion in the mouse has differential effects depending on the muscle [12].

Muscle stem cells or satellite cells are localized beneath the basal lamina, peripheral to the muscle fibers [11], and express Pax7 [16] and Notch3 [17]. After muscle injury, satellite cells are driven out of their quiescent state, and start to proliferate. Most of the activated satellite cells rapidly co-express MyoD or Myf5 [16, 18]. The proliferating satellite cells -also called myogenic precursor cells or myoblasts -expand under the control of Notch3 [17] and Notch1/Hey1 pathways [19, 20]. They divide asymmetrically, with self-renewal of the stem cell pool being maintained by a minor population of myogenic precursor cells that down-regulate their expression of MyoD and Myf5 and return to a quiescent state [18, 21-23]. This asymmetrical division involves Numb, an antagonist of the Notch signalling pathway [19, 24]. Numb is asymmetrically localized during myoblast mitosis and it is the cell that has a high level of Numb that goes back to quiescence for self-renewal [19, 24-26]. After several rounds of proliferation, activated myoblasts decrease their expression levels of Pax7, Myf5 [16, 18] and Notch3 [17]. The Notch1 pathway is then repressed by Stra13 [20] through the CBF1 pathway [20, 27]. Simultaneously, the Wnt pathway is activated and promotes myoblast differentiation through ␤-catenin [28]. Myoblasts exit the cell cycle by expressing p57 [29], and then cyclin inhibitors -p21 and hypophosphorylated pRb [30-32] -together with higher levels MyoD followed by myogenin, a driver which triggers the expression of the differentiation genes [33, 34]. The myoblasts consequently undergo differentiation into myocytes, and fuse either with each other or with existing multi-nucleated myofibers in order to repair injured muscle [35, 36]. The differentiation and maturation process is regulated by MEF2, MEF3, and Mrf4 pathways [37-39], while other factors, such as Myomaker, are involved in fusion [40]. Muscle precursor cell proliferation, fusion and differentiation are tightly orchestrated by circulating hormones (e.g. growth hormone [41], testosterone [42, 43] and thyroid hormones [44, 45]), growth factors (e.g. IGF system [41], FGF system [46-48], TGF-b [49, 50]), G-CSF [51], chemokines (e.g. interleukines [52-55], MPC [55, 56]) and other secreted components (e.g. vesicles [57, 58]) present in the muscle stem cell environment.

Aged human [59] or murine [60-62] muscle can regenerate and repair, although the rate of regeneration declines [60-62]. This slower regeneration can be explained by: (1) changes in the muscle stem cell environment (growth factors, growth hormones, inflammation, and extracellular matrix content); (2) a lowered responsiveness of progenitor cells to repair stimuli; and (3) decrease in the number of muscle stem cells with aging. Each of these factors may impact on muscle homeostasis and each may both participate to, and be affected by, age-associated changes in intercellular communication. The subsequent sections will describe the different roles that intercellular communication may play in muscle aging, from hormonal and other circulating endocrine factors to local paracrine and autocrine secretory environment of the stem cell niche that may also modify the intrinsic properties of the stem cells themselves.

HORMONAL AND OTHER CIRCULATING FACTORS: CHANGE IN ENDOCRINE COMMUNICATION WITH AGING

The decline in muscle regenerative capacity with age [63] has been partly attributed to a decline in extrinsic environmental cues (see Fig. 1). Levels of circulating hormones, such as testosterone or IL-6 or growth hormone (GH) or IGF-1, are low in serum samples of aged subjects [64-66].

The endocrine hypothalamic-pituitary axis is altered with aging, leading to changes in hormone secretion that can contribute to cognitive decline or depression. Epidemiological studies have also shown a correlation between the decrease in growth hormone (GH) secretion and sarcopenia as well as other signatures of aging (e.g. intra-abdominal adiposity, osteopenia, etc.) [67]. GH is a stress hormone produced by the hypothalamus. It plays a key role in muscle mass maintenance through life [66]. It acts on myoblasts through its receptor GHR and activates NFATc2 that in turn stimulates the expression and secretion of IL-4 [41, 68, 69] -IL-4 being critical for myoblast fusion [68, 69]. GH also stimulates IGF-1 secretion by both liver and muscle [66]. IGF-1 and its splice variants -IGF-1Ea and IGF-1Ebmodulate myoblast proliferation [70] and differentiation [71] through MAPK and ERK1/2 signalling [70]. The latter regulates myogenesis, for example by interacting with p38␣␤ MAPK and the asymmetric division and self-renewal of satellite cells [72].

These age-associated changes in the endocrine hypothalamic-pituitary axis can have further effects on the gonadotropic axis. Sex-steroid privation associated with age participates to, among other phenomena, Fig. 1. Age alters serum composition and thereby affects intercellular communication at distance. The endocrine hypothalamic-pituitary axis is altered with aging, affecting the composition of circulating hormones in the serum. For instance, the secretion of growth hormone is decreased, leading to loss of muscle mass. In addition, the lower level of growth hormone will also stimulate less the secretion of IGF-1 -IGF-1 being involved in muscle mass maintenance and in the satellite cell myogenic program. The endocrine hypothalamic-gonadotropic axis is also affected, leading to a decrease of sex steroids such as Testosterone, another hormone involved in muscle mass maintenance. Similarly, a decrease in oestrogen can act on the myogenic program through IGF-1 signaling. The decrease in circulating hormones affects the capacity of the satellite cells to respond to muscle damage. Aging is also associated with an increase in inflammation. The cytokines secretion by aged inflammatory cells as well as their ROS production is modified and can also affect the capacity of the satellite cells to respond to muscle damage. The modification of the entire serum composition with aging has negative effects on muscle mass and on muscle regeneration capacity. loss of muscle mass [67]. The sex-steroid testosterone, secreted by the testis, has been extensively studied in muscle, and can be considered as a double-sided blade, acting both on myoblast proliferation and differentiation. It acts on myoblasts through the androgen receptor localized in the nucleus [73] or through G protein-coupled receptors [74]. It promotes myoblast proliferation through protein kinase C (PKC) signaling [74] -for instance through nPKCδ and extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation [75]. Once ERK1/2 is phosphorylated, it is accompanied by an increase in cyclin E and Cdk2 -which are involved in myoblast proliferation [76]. Testosterone acts also on myoblast differentiation via protein kinase A (PKA) signaling [74] -PKA being required for myoblast fusion [77, 78]. Interestingly, oestrogens act similarly on the myogenic program through IGF-1 signaling [79].

Aging is associated with an increase in low grade chronic and systemic inflammation, also called inflammaging [80]. Inflammaging could be due to microbial infection, cell debris, over-activated coagulation system, or an increase in cellular senescence with the associated changes in secretion [80]. This increased inflammation is generally attributed to a modified immune partner. Indeed, while young macrophages have been shown to have a beneficial effect to clear muscle debris after injury and stimulate myogenesis [81-83], aged macrophages can release a higher level of osteopontin that inhibits the muscle regeneration process [84]. Not only macrophages are involved in the muscle regeneration process, but also neutrophils, lymphocytes, dendritic cells, etc. These inflammatory cells secrete numerous chemokines and cytokines, but little is known about the impact of aging on cytokine secretion [85]. In the literature, it is described that IL-6 serum level is decreasing during aging [65]. IL-6 originates from the inflammatory cells, but also from the skeletal muscle itself [86]. It has been shown to be an important regulator of muscle stem cells [53], as it activates janus kinase 2 (Jak2) that will in turn phosphorylate STAT3 [52]. Once STAT3 is phosphorylated, it homodimerizes and translocates to the nucleus to bind to the ␥-interferon activation sequence [87] in the promoter regions of genes involved in myoblast proliferation such as c-myc [52]. IL-6 not only regulates myoblast proliferation, but also promotes myoblast differentiation through the p38 MAPK pathway [88]. A decrease in IL-6 serum level could thus impact muscle regeneration efficacy.

The tissues from which circulating factors originate, such as muscle, hypothalamus, gonads, and liver, become atrophic and less active with age [8, 66, 89]. This change in body composition and activity with aging can thus participate to the decrease in circulating hormones (see Fig. 1). Consequently, when muscle damage occurs in an aged person, satellite cells be less prone to activation and differentiation, leading to a less efficient repair. Ten years ago, Conboy et al. elegantly showed that muscle regeneration could be partly rescued in aged mice exposed to serum from young mice through a parabiosis system [90]. Similarly, hormones released during pregnancy rescued the muscle regenerative capacity of aged female mice [91]. When aged subjects are trained, a rejuvenating effect is observed on muscle. This benefit effect could probably be due to a decrease of the inflammation for instance, as observed in exercised patients affected by myositis [92, 93]. When aged muscle stem cells were engrafted into young mice [94], their capacities to proliferate and differentiate were partly restored. Together these data suggest that circulating agents, which can originate from different tissues, impinge on muscle regeneration efficiency. Aging affects both the size and function of each tissue and consequently tissue secretory capacity. This alters the composition of circulating serum effecting intercellular communication at distance.

SECRETORY ENVIRONMENT OF THE STEM CELL NICHE: CHANGE IN PARACRINE AND AUTOCRINE CELL-CELL COMMUNICATION WITH AGING

In addition to its classical role as a locomotive system, skeletal muscle has recently been shown to have a secretory activity. For instance, IL-6 [86] and musculin [95] have been identified to originate from and be secreted by skeletal muscle in vivo. In vitro, the secretome profile of C2C12 myotubes [55, 56], human myotubes [57] and rat muscle explants [96] suggest that muscle cells secrete numerous growth factors (e.g. follistatin like protein 1, IGF-2, TGF, etc) and cytokines. Secreted proteins -also named myokines [95] -may act in an autocrine/paracrine manner on neighboring muscle cells and contribute to muscle growth and regeneration. This local muscle secretome can be altered with aging. For instance, Chakkalakal et al. have shown that an increased secretion of FGF-2 by aged myofibers in mice inhibits sprouty1 expression in satellite cells, and consequently reduces their capacity to go back to quiescence and replenish the pool of the muscle stem cell [47]. The muscle secretome includes not only hormones, but also extracellular matrix components (ECM, e.g. TIMP2, fibronectin), miRNAs, and vesicles (exosomes and microvesicles) [57, 58, 97, 98]. Interestingly, exosomes originating from differentiated myocytes stimulate the myogenic program of proliferating myoblasts [58]. Myocyte exosomes contain miRNAs that inhibit Sirtuin expression, and thus stimulate the myoblast differentiation into myotubes [98]. A decrease in muscle mass with aging may thus reduce muscle secretory output. In a transcriptomic analysis performed on quadriceps muscle from young (15-24 years old) and elderly (72-80 years old) subjects, we indeed observed down-regulation of secretome markers in aged muscle [99]. However, little is known about the changes in the composition of the muscle secretome of aged muscle and further investigation is needed.

The local niche of muscle stem cells includes growth factors and cytokines secreted not only by the myofibers themselves, but also potentially by other cell types present within muscle, such as fibroblasts, endothelial or peri-endothelial cells [100, 101]. This local secretome can also be altered with aging (Fig. 2). For instance, fibroblasts present in aged skeletal muscle express a high level of TGF-␤ [101] -a growth factor that inhibits differentiation of myoblasts [102], and thus slows down the regeneration process. In addition, aging is described to be associated with an increase in senescent or pre-senescent cells in muscle and other tissue [103, 104]. During the last decade, the secretome of senescent cells from different tissues has been investigated and has been described to have an impact on the inflammatory response (by stimulating it in chronic obstructive pulmonary disease [105]) and to be instrumental in poor tissue regeneration (as observed in aged skin [106]). Altogether, these data suggest that the presence of senescent cells distinct from satellite cells within muscle tissue could alter these microenvironment of the satellite cells, and thus their behavior.

Muscle perfusion is decreased with aging [107], which may render myofibers and satellite cells less accessible to circulating hormones. This loss of perfusion may be maintained by the muscle loss itself. Indeed, sarcopenic muscle presents a disruption of the dystroglycan complex [108], leading to NOS-1 mislocalization, due to the link of NOS-1 to the dystrophin protein [109]. The mis-localization of NOS-1 results in decreased NO production, thereby diminishing muscle perfusion [110]. A second effect of decreased NO production is a reduction in satellite cell activation [111].

Aged skeletal muscle presents a thickening of the ECM and a general increase in fibrosis [112]. Even if muscle fibers can secrete collagens and other components of the ECM [57, 97], little is known about their role in ECM thickening. A recent study shows that fibroblasts present in aged rat muscles express a higher level of collagen IVa2 and laminin 2 -which may participate in the thickening of the ECM [101]. This increase in the ECM thickness can interfere with the muscle regeneration process by modifying myoblast activation, proliferation and migration [48, 113]. Finally, a thickened ECM may act as a partial barrier, reducing the accessibility of the satellite cells to circulating growth factors, as observed for smooth muscle cells [114], and thus impair satellite cell activation and differentiation during muscle repair.

Together, these data suggest that the changes in the secretory composition of the muscle stem cell's local niche with aging can slow down the regeneration process and decrease the replenishment of the pool of reserve cells. Repetitive iterations of this could contribute to the loss of muscle stem cells with aging.

CHANGE IN THE INTRINSIC PROPERTIES OF STEM CELLS WITH AGING

Exposure to a young environment by engraftment into young subjects or by parabiosis experiments only partly rescues the properties of aged satellite cells [90]. For instance their capacity to replenish the pool of reserve cells is not rescued (our unpublished data and [115]). These data suggest that some intrinsic properties of satellite cells are altered with aging and are not easily manipulated by external cues.

Intrinsic properties rely at least partly on DNA methylation, which may regulate gene expression in two ways [116]: (1) the accessibility of methylated enhancer regions to transcription factors is reduced, resulting in gene expression repression;

(2) methyl-CpG-binding proteins bind to methylated DNA and alter the activity of histone deacetylases and methyltransferases. Consequently, local histones are hypermethylated, stabilizing the nucleosomes, so that DNA in methylated regions is tightly packed preventing binding of transcription factors or RNA polymerases. A recent study shows that histone methylation patterns are different between aged and young satellite cells in mice [117], and that the methylation profile can be modified by the presence of local growth factors such as FGF-2 [118]. The authors associated this histone methylation profile to a slower capacity of aged satellite cells to re-enter the cell cycle for aged satellite cells [117]. Interestingly, this study [117], as well as our own observations on culture of aged human muscle stem cells, show that once activated, aged satellite cells have a similar myogenic potential to young satellite cells. This indicates that muscle stem cells do not lose their differentiation potency with age, suggesting that the decrease with aging in the differentiation program during muscle regeneration is strongly related to changes in circulating factors.

DNA methylation has been shown to be increased in several tissues with aging, and the skeletal muscle is no exception [119, 120]. We have observed a higher level of DNA methylation in satellite cells of aged subjects (unpublished data). This hypermethylation could impact on the satellite cell fate and interfere with their capacity for self-renewal as observed in previously published studies (our data and [47, 115]). How DNA methylation is regulated with aging is not well known. Repeated stress over time can be one of the parameters implicated [121], involving for instance reactive oxygen species (ROS) [122]. Increased ROS production with age can be due to an increase in inflammation with aging [80] or to mitochondrial dysfunction in aged muscle [123, 124]. A decrease of circulating GH is also associated with a higher level of ROS and a lower level of anti-oxidants [125]. This overproduction of ROS could participate to increased DNA damage observed with aging [126]. Consequently, DNA methyltransferases (DNMT) are recruited to the DNA damage site, potentially inducing DNA silencing of the region nearby [127]. When we re-analyzed the transcriptome data available online (GSE9103), we indeed confirm a significant enrichment in the cellular response to oxidative stress in aged muscles (Fig. 3), suggesting a higher stress in aged muscle. ROS diffuse easily through membranes of fibers, thus potentially affecting DNA damage in neighboring satellite cells, and modifying their methylation status.

Factors discussed above -changes in the composition of circulating hormones in serum, as well as in the microenvironment -could also modify the epigenetic status of satellite cells and thus their behavior during regeneration, slowing satellite cell activation and decreasing their capacity to go back to quiescence

THE LOSS OF MUSCLE STEM CELLS WITH AGING AND ITS CONSEQUENCES ON MUSCLE HOMEOSTASIS

The number of muscle stem cells declines with age in mouse [13, 47, 90, 94] and humans [128, 129]. Although this loss can be caused by an increase in cell death, cellular senescence, or a deficiency in re-quiescence, apoptosis is rarely observed in aged murine and human muscle stem cells [94, 130, 131], suggesting that this cannot by itself explain the loss of muscle stem cells with aging. However, we cannot exclude the fact that apoptosis is a short punctual event that may be missed experimentally. Cellular senescence -also called replicative senescence -is defined as a phenomenon by which normal diploid cells cease to divide. It can be induced by telomere shortening that occurs during cell proliferation, and has been proposed to contribute to the loss of satellite cell function with aging [103]. However, shortened telomere length has not been reported in aged human satellite cells. Fig. 3. Increase of oxidative stress response in aged muscles. For this analysis we retrieved the gene expression matrix of old and young muscles of sedentary subjects (series GSE9103 from the Gene Expression Omnibus [139]). We discarded three samples as they did not pass the quality control threshold. To identify differentially expressed genes we used the characteristic direction method [140] followed by gene set enrichment with Enrichr [141] Furthermore, there may be insufficient activation and turnover of satellite cells to allow senescence to be a major contributor to stem cell decline. Satellite cells are rarely activated in healthy adult human or mouse, and once muscle growth is complete in young human adults [14], subsequent myonuclear turnover is slow, being estimated at 15 years during adulthood [132]. In mouse models, myofiber growth by the addition of new nuclei through satellite cell fusion is completed by 21 days postnatally [133], and there is little evidence to suggest significant turnover. As discussed earlier, the capacity to re-quiesce through the sprouty1 pathway is decreased in aged stem cells [47, 117]. Consequently, when satellite cells are activated for muscle repair in elderly subjects, they do not replenish the pool of reserve cells. This failure of re-quiescence is a likely contributor to stem cell population decline.

The decrease in the number of satellite cells with aging can affect muscle homeostasis by altering the ECM composition. Indeed, aged muscle depleted in satellite cells in a Pax7 CreER -DTA murine model shows an increase in fibrotic deposition [134], while fiber size was unaffected [135]. Resulting thickening of the ECM may increase myofiber fragility, and reduce the response of satellite cells to muscle damage, as discussed earlier. Therefore a loss of satellite cells could Fig. 4. Links between whole-body composition changes, the decline of muscle size and function, and the loss of muscle stem cells and their functions with aging. Modifications with age of the endocrine systems (hypothalamus-pituitary system, gonad glands, liver, etc.) affect the quantity and the content of circulating serum hormones and impact muscle mass maintenance (atrophy of the muscle fibers and decrease in the regenerative capacity of the muscle). Increased inflammation with age -also called inflammaging -affects whole body stress level and is accompanied by modification in secreted cytokine content. Increased oxidative stress of the muscle leads to DNA damage and epigenetic changes, and consequently affects the regenerative capacity of the muscle. The composition of the microenvironment of the satellite cells is affected with age, through the presence of aged fibroblasts, of senescent cells, and aged myofibers. These local changes contribute to the fragility of the myofibers and to a decrease in the regenerative potency.

impinge directly upon muscle homeostasis, and exacerbate muscle fragility with aging.

CONCLUSION

The interplay between whole-body tissue composition, the quantity and content of circulating serum hormones, and whole-body stress, such as ROS production, changes with age, and contributes to the decline in muscle mass and function (Fig. 4). Increased stress can act through epigenetic marking of satellite cells, changing their intrinsic properties with ageaged satellite cells show a decrease in their activation rate due to epigenetic changes. In addition, satellite cell number is decreased during aging, a loss that can contribute not only to a decreased regenerative capacity, but also to an increase in fibrotic deposition and ECM thickening. Increased muscle stiffness renders myofibers more fragile, requiring the activation of an already reduced and less responsive satellite cell population. Age-associated changes in the local signalling environment can affect the myogenic program causing a lower regeneration efficacy, a decrease in myonuclear turnover, and a failure in replenishing the pool of reserve cells, further contributing to the loss of muscle mass. Changes in the whole system of intercellular communication -both at the whole-body scale and in muscle microenvironment -may thus act as a vicious circle to exacerbate sarcopenia as the body ages. It is noteworthy that most studies on muscle aging in the literature have been done on muscle stem cells, rather than myofibers, and thus emphasize the role of satellite cells in muscle mass maintenance. The effect of hormones and cytokines and more generally the effect of aging on myofibers is difficult to assess directly and should not be neglected. The myofibers themselves comprise the bulk of the muscle mass and are clearly a key part of the maintenance of their own mass with aging, as indicated by disequilibrium of protein synthesis and degradation or the expression of micropeptides such as myoregulin that have a key role in muscle performance [138].

Introduction: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with an adult onset and characterized by a rapid and lethal deterioration of cortical, brainstem and spinal cord motor neurons. This denervation leads to a gradual muscle weakness characterized by muscle atrophy and stiffness, and muscle cramps. Patients experience paralysis and die within 3 to 5 years after the symptoms have reached the intercostal and the diaphragm muscle. To date, numerous mutations have been identified such as SOD1, FUS and the intronic hexanucleotide expansion in C9orf72 gene. Today ALS remains incurable and the causes of muscle denervation are still unknown. Numerous mechanisms involving the neighbouring non-neuronal cells are suggested to affect motor neurons death in ALS. It has been demonstrated that pathological molecular and cellular changes occur at the neuromuscular junction prior to motor neurons degeneration and symptom onset. Based on these studies we hypothesised that muscle can participate to the ALS pathogenesis. Moreover, using transcripomic data previously published we performed an in-silico secretome revealing that the endosome and lysosome secretion compartments are significantly enriched in ALS muscle. These pathways are known to be part of the exosomes genesis and Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 192 secretion. Thus, we hypothesised that the vesicle trafficking is disturbed in ALS muscle and might influence intercellular communication between muscle and nerve. Methods: We extracted human primary cells from deltoid muscle biopsies from ALS (n=18), SBMA (n=12), SMA (n=12) and healthy subjects (n=15). The muscle cells were sorted using CD56 MACS beads. Muscle exosomes were extracted after removing cell debris and microparticles using the Life Technologies kit.

Results: We demonstrated that there is an ALS signature in muscle stem cells as we observed an accumulation of MVBs is independent from the denervation process that is a characteristic of the ALS myotubes. The disruption of the exosomal pathway is associated with a 2 fold higher amount of secreted exosomes by ALS muscle cells. And finally we could demonstrate that ALS exosomes are toxic for muscle cells and toward neurons as they can induce a greater cellular stress, nuclear loss and cell death after treatment with healthy muscle stem cells or healthy human iPSC-motor neurons or healthy primary motor neuron.

Discussion Altogether, the present study suggests that muscle cells from sporadic patient secrete toxic agents through their exosomes. The secretion of toxic exosomes may influence the intercellular communication between the muscle and its environment, including motor neurons, and may have a key role in the ALS pathology The cause of ALS remains unknown, and several studies performed on muscle cells from patients have suggested an involvement of skeletal muscle. We hypothesized that the muscle secretome was disrupted in ALS patients. To test this hypothesis, we extracted primary muscle stem cells from the deltoid muscle biopsies of sporadic ALS patients (n=18) and aged-matched healthy subjects (n=21). After differentiating the cells for 3 days into myotubes, we observed by RT-qPCR a significant upregulation of CD81, CD63, TSG101 and Rab5genes that are involved in exosomal biogenesis. When muscle stem cells were differentiated into myotubes, we observed a significantly greater amount of exosomes that were released into the culture medium of ALS myotubes. To test the effect of the exosomes on surrounding cells, we labelled them with PKH26 and tested their effect on healthy muscle cells over time. ALS and healthy exosomes started to be integrated into muscle cells as soon as 4 hintegration that gradually increased over 48h. Cell blebsa signature of cellular stressappeared as soon as 4h in ALS exosome-treated muscle cells and increased until 168h. Consequently, we observed a greater cell death at day 3 after treatmentas shown by a significantly higher level of H2Ax in nuclei and a greater loss of nuclei. In addition, we observed an atrophy in myotubes as the myonuclear domain was smaller in myotubes treated with ALS exosomes.

Together the present data suggest that muscle stem cells have a disruption of exosomal biogenesis, and that ALS muscle exosomes are toxic to muscle cellsphenomena that are independent to muscle denervation.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

We are now characterizing the content of these exosomes and determining whether the exosome content can act with a prion-like effect. Background :

To date ALS remains incurable and the primary involvement of muscle in ALS pathogenesis is still controversial. However, there is now evidence implying a perturbation of pathways involved in the exosomal pathway in sporadic as well as familial cases. Moreover, it has been demonstrated that pathological molecular and cellular changes occur at the neuromuscular junction prior to motor neuron degeneration and symptoms onset. Altogether these data suggest that the skeletal muscle could participate to ALS.

?

Motor neuron environment is toxic in ALS It is easy for us to move our legs when we want to, right? We can stimulate our muscles by impulses that travel from our brain towards our muscles. Critically important are the nerves that innervate our muscles, called motor neurons as they create a valuable bridge between our brain and muscles. These neurons are essential, they allow us to walk, talk, speak and more importantly breathe.

Secretion of neurotoxic vesicles by muscle cells of ALS patients

Unfortunately for ALS patients, these motor neurons are killed, breaking this vital line of communication. Eventually, the ability to start and control voluntary movement is lost, patients' muscles no longer receive stimulation and will gradually weaken, resulting in paralysis and ultimately death. It is challenging to predict how fast ALS will progress, in fact Dr Stephen Hawking's longevity was quite rare since ALS patients are only given 2 to 5 years to live.

Today there is no cure available for ALS and it is critical to identify the murderer among a long list of suspects. As a PhD student I am now taking part in this investigation and my work aims to determine if the muscle is indeed a prime suspect.

You may be wondering, "why the muscle?" when many researchers are more interested in other cells surrounding the motor neurons in the brain. Well, as surprising as it may sound, your muscles are very chatty and one way for them to communicate is by releasing molecules packaged inside vesicles. A vesicle could be compared to a very small soap bubble that is surrounded by a membrane and filled with molecules, just like a soap bubble is surrounded by a film of soap and filled with air.

My PhD project aims to investigate the effect of vesicles released by ALS muscle and more particularly, identify whether their contents are toxic. If this is the case, then my project may provide an explanation of how the muscle may be involved in the death of motor neurons in ALS.

ALS is not an incurable disease! It is just a misunderstood one and our best chances to identify a cure is to catch the killer.

FIGURE 1 :

 1 FIGURE 1 : UPPER AND LOWER MOTOR NEURON INVOLVEMENT IN ALS............................................................................... FIGURE 2 : UPPER AND LOWER MOTOR NEURONS ROLE IN DIFFERENT "ALS VARIANTS. ......................................................... FIGURE 3 : CLINICAL FEATURES OF ALS AND ROLE IN PROGNOSIS. .................................................................................... FIGURE 4: HYPOTHESIS FOR GGGGCC REPEAT EXPANSION IN C9ORF72 GENE-MEDIATED PATHOLOGY. .................................... FIGURE 5 : MOLECULAR AND CELLULAR MECHANISMS INVOLVED IN ALS PATHOGENESIS. ....................................................... FIGURE 6 : PROTEIN HOMEOSTASIS DYSREGULATION. ................................................................................................... FIGURE 7 : RNA AND MIRNA BIOGENESIS DEFECTS IN ALS. ........................................................................................... FIGURE 8 : BIOGENESIS AND SECRETION OF EXOSOMES. ................................................................................................ FIGURE 9: EXOSOME AND RECIPIENT CELL COMMUNICATION .......................................................................................... FIGURE 10 : THE NEUROMUSCULAR JUNCTION AND MUSCLE CONTRACTION........................................................................ FIGURE 11 : HOUSEKEEPING GENE EXPRESSION LEVEL MEASUREMENT. ............................................................................. FIGURE 12 : SIMILAR MYOTUBES DIFFERENTIATION AFTER ALS AND CONTROL EXOSOMES TREATMENT. ................................... FIGURE 13 : PRIMARY MOTOR NEURON SURVIVAL AFTER ALS OR HEALTHY EXOSOMES TREATMENT ........................................ FIGURE 14 : TDP43 GRANULATION IN ALS AND HEALTHY MUSCLE CELLS. ........................................................................ FIGURE 15 : ALS MUSCLE CELLS SECRETE NEUROTOXIC EXOSOMES. ................................................................................ FIGURE 16 : GRAPHICAL ABSTRACT. SPECULATION ON HOW MUSCLE EXOSOMES CAN HAVE A ROLE IN THE PROPAGATION OF THE DISEASE. .................................................................................................................................................

Figure 2 :

 2 Figure 2 : Upper and lower motor neurons role in different "ALS variants. ALS is a disease with high clinical phenotype variability. "Classic ALS" patients will present with signs of both UMN and LMN degeneration. However patients with progressive muscular atrophy (PMA) and primary lateral sclerosis (PLS) ALS variants present with LMN-predominant or UMN-predominant signs respectively. LMN-predominant patients also includes flail-arm syndrome and flail-leg syndrome ALS variants where LMN signs are present in upper or lower limbs respectively. ALS patients might present symptoms in bulbar-innervated muscles, if LMN are predominantly affected the term progressive bulbar palsy is used. However if UMN signs are predominant, patients are diagnosed with pseudobulbar-palsy. Blue colour indicate non-affected neurons. Red colour indicate the UMN/LMN signs associated with each ALS variants. ALS: Amyotrophic lateral sclerosis. PLS: Primary lateral sclerosis. PMA: Progressive muscular atrophy.

Figure 3 :

 3 Figure 3 : Clinical features of ALS and role in prognosis. Diagram displaying ALS features associates with typical good or bad prognosis. ALSFRS: Amyotrophic lateral sclerosis functional rating scale.

Figure 4 :

 4 Figure 4: Hypothesis for GGGGCC repeat expansion in C9orf72 gene-mediated pathology. The most pathogenic mutation represented among both sALS and fALS is the hexanucleotide repeat expansion in the C9orf72 gene. 1) Inhibition of C9orf72 transcription by repeat expansion induce a toxic reduction of C9orf72 mRNA levels in ALS patients. 2) Generation of toxic repeat-containing RNA assembling in RNA foci. 3) Repeat-associated non-AUG translation mediated DPR toxic effects. 4) Finally both repeat-RNA and DPR products interact with the nucleus-cytoplasm machinery, inducing a toxic nucleoplasmic dysregulation. RBP: RNA-binding protein. RNA foci and DPR accumulation pictures from Balendra et al 45 .

Figure 5 :

 5 Figure 5 : Molecular and cellular mechanisms involved in ALS pathogenesis.Multiple pathological mechanisms have been proposed to be involve in ALS pathogenesis. Among the most studied and well known dysregulated pathways are: RNA metabolism, autophagy and protein homeostasis, neuroinflammation and glutamate excitotoxity.

Figure 7 :

 7 Figure 7 : RNA and miRNA biogenesis defects in ALS.Many processes in RNA and miRNA pathways are disrupted in ALS patients, including transcription defects, alternate splicing events, miRNA biogenesis and nucleus-cytosol transport impairment. RNA metabolism defects are particularly relevant in ALS pathogenesis since TDP43 and FUS are both wellknown ALS-associated genes involved in RNA processing. Both FUS and TDP43 mutated proteins misslocalize to the cytoplasm of ALS motor neurons, leading to a probable loss and toxic gain of function of these proteins.

-

  ESCRT-0 complex binds and clusters ubiquitinated transmembrane proteins at the membrane of the endosomes -ESCRT-I and -II complexes induce the deformation of the endosomal membrane into nascent ILVs containing sorted cargo -ESCRT-III structure subsequently stimulate neck constriction contributing towards detachment of ILVs by vesicle membrane scission.

Figure 8 :

 8 Figure 8 : Biogenesis and secretion of exosomes. Schematic representation of exosomes formation and release in the extracellular space. (1) Exosomes are produced as ILVs by inward budding of the endosomal membrane and accumulate in the lumen of the endosome. (2) Several mechanisms are involved in the biogenesis of exosomes such as ESCRT protein-, lipid-rafts-and tetraspanins microdomain-dependent pathways. Whether one or multiple pathways are required simultaneously on one population of MVB or if each pathway is specific to one population of MVB is still not clear. ESCRT-dependent pathways: A) the most described mechanism involve in the biogenesis of exosomes is the ESCRT-dependent pathway requiring protein of the ESCRT family. Specific transmembrane ubiquitinated cargo are recruited and clustered at the MVB membrane by ESCRT-0

  Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 50 molecules 278 . CD46 can shed from the tumor cells via exosomes and cleaved by metalloproteinases to produce soluble form in ovarian cancer cells 278 .

Table 8 :

 8 Summary ligandreceptor interaction in exosomes/cell communication. Known ligand/receptor interactions are listed and categorized according to the molecular origin of the exosomes' ligand. ICAM: InterCellular Adhesion Molecule, LFA-1: Lymphocyte Function-associated Antigen 1, MFG-E8: Milk Fat Globule-EGF factor 8 or lactadherin protein, SIRPα: SIgnal Regulatory Protein α, DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin C type lectin receptor.

  , thus affecting the exosome incorporation through the classic clathrin-and caveolin-dependent mechanisms. In this context, both phagocytosis and micropinocytosis pathways can form large vacuoles 301 and can engulf large exosomes clusters and aggregates. Despite their resemblance, phagocytosis and micropinocytosis occur through two distinct cellular machineries. The phagocytosis relies on the association between the receptor from plasma membrane and the vesicle's ligand, while the micropinocytosis occurs constitutively and requires the cytoskeleton actin protein-dependent extension formation 271 . Macropinocytosis is characterized by the formation of ruffled extension from the PM around the extracellular space including extracellular fluid and component that will be further internalized by the cell. The macropinocytosis machinery requires multiple mediators such as PAK1 kinase, rac1, ras and src, cholesterol, cytoskeleton actin protein and Na+/H+ exchanger 301 . Inhibition of this machinery leads to a decrease in exosome uptake, supporting the role of macropinocytosis in the uptake of exosomes 271 . The drugs used to inhibit the micropinocytosis include: 1) dynasore (inhibitor of dynamin), 2) NSC23766 (suppressor of rac1 activity, 3) cytochalasin D (disruption of the actin cytoskeleton function), 4) amiloride (blocking of the Na+/H+ exchanger and 5) alkalinising drugs such bafilomycin A, chloroquine or monensin to increase pH. Similarly to macropinocytosis, phagocytosis requires the formation of Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 53 membrane invagination around the targeted cargo to be internalized. Phagocytosis is a major pathway for exosome internalization 302 . It involves actin cytoskeleton, PI3K, dynamin and phosphatidylserine

  Lipid rafts are formed by cholesterol and sphingolipid-rich microdomain and are phospholipid clusters enriched in transmembrane proteins and sphingolipids like sphingomyelin297 . When classical endocytosis mechanisms such as clathrin-mediated endocytosis or macropinocytosis were not to part of the exosome uptake mechanisms, the colocalization of exosomes with a lipid raft marker pointed towards the role of lipid in the uptake of exosomes 273 . This non-clathrin dependent endocytosis of exosomes was confirmed by inhibiting vesicle uptake with cholesterol and specific lipid-raft dependent endocytosis inhibitor drugs 273 .

55 

 55 and paragraph IV. 4. 4. Recycling or degradation: fate of exosomes inside the targeted cells). As previously described exosomes are composed of various molecules cargo that can be functionally delivered to recipient cells, inducing a cell-to-cell cascade signalling to transduce the exosomes function. Physiological functions of exosomes are variable and still not entirely understood. In the next sections will be listed known functions and signal transduction activation mediated through exosomes.Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall Role in development biology and cancerThe internalization of exosomes by the targeted cell can, among others, activates the Wnt signalling pathway. This Wnt signalling pathway is a well-known signalling cascade involved in many processes during embryonic development, however less was known about the mechanisms involved in the transport of Wnt ligands to their targeted cells311 . Canonical and non-canonical Wnts molecules have been identified in secreted vesicles from mammalian cells and are functionally transferred to cells to induce Wnt signalling activity312 .Exosomes are also vehicle for pro-and anti-tumorigenic signalling molecules, acting as mediators of cellcell crosstalk in the tumor environment. Mesenchymal stem cells-derived exosomes can promote tumor growth by mediating angiogenesis in tumor cells via the activation of extracellular signal -regulated kinase 1/2 (ERK1/2) and p38 MAPK pathways313 . Promoting growth factor through improved vasculogenesis has been reported to be exosome-dependent in melanoma patients270 . Briefly, the receptor kinase MET was transferred via melanoma cell-derived exosomes and activated ERK phosphorylation and other downstream effectors to induce the tumor growth and enhance the metastatic activity270 . In endothelial cells, metastatic potential was stimulated through exosomal activation of MAPK/ERK signalling and stimulating the angiogenesis 314 . Moreover, the internalization of exosomes by monocytic cells induced the activation of NF-κB signalling, crucial step in IL6 production, activation of STAT3 and secretion of more cytokine contributing to the immune escape of tumor cells 281 . Contrary to a pro-tumorigenic potential of exosomes, other tumor cells-derived exosomes have been identified as mediators of tumor cell death through the activation of caspase pathways in an autocrine fashion. In this context, the caspases-3 and -9 are activated in tumor cells following the activation of GSK-3β neutralizing the PI3K/Akt survival signal transduction, and drive the tumor cells towards apoptosis 155 .

57 Chapter 3 :

 573 et al on B lymphocytes suggested a novel role of exosomes in antigen presentation 214 and stimulation of T cells activation. Finally, exosomes have also been identified as vehicle for infectious agents spreading such as HIV virus 150 . Altogether these results highlight the contribution of secreted exosomes in physiological and pathological conditions, suggesting the potential role of circulating exosomes in the communication between distant cells, but also their potential role in the propagation of toxic elements in different pathological context, including neurodegenerative diseases. Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall Neurotoxic communication in ALS I.

Figure 10 :

 10 Figure 10 : The neuromuscular junction and muscle contraction.Following an action potential that reaches the per-synaptic neuron, the neurotransmitter acetylcholine (ACh) is released in the synaptic cleft. Ach binds to its receptor on the post-synaptic nerve and triggers influx of Na + and 1) produce a synaptic potential that 2) activates voltage-gated sodium channels and produce an action potential that propagates along the membrane of the myofiber. 3) When it reaches the Tubule T invagination, calcium is released from the sarcoplasmic reticulum. 4) Finally calcium triggers muscle contraction through binding to troponin. 5) Myosin binds to the exposed binding site on the troponin protein and form a cross-bridge responsible for muscle contraction. Figure from Liu et al, 2018 340

  Figure 10 : The neuromuscular junction and muscle contraction.Following an action potential that reaches the per-synaptic neuron, the neurotransmitter acetylcholine (ACh) is released in the synaptic cleft. Ach binds to its receptor on the post-synaptic nerve and triggers influx of Na + and 1) produce a synaptic potential that 2) activates voltage-gated sodium channels and produce an action potential that propagates along the membrane of the myofiber. 3) When it reaches the Tubule T invagination, calcium is released from the sarcoplasmic reticulum. 4) Finally calcium triggers muscle contraction through binding to troponin. 5) Myosin binds to the exposed binding site on the troponin protein and form a cross-bridge responsible for muscle contraction. Figure from Liu et al, 2018 340

3 . 4 . 5 . 6 . 7 . 8 .

 345678 Leftover of skin and fat are gently removed using a pair of tweezers and scissors. The muscle biopsy is than roughly dissociated. The entire muscle biopsy is transferred in a watch glass containing serum and cut into small explants  It's important to use a glass receptacle in order to avoid damages to the muscles explants due to scratches in plastic containers. Using a small rubber pipette bulb and a glass pipette, the explants are moved to the 10cm petri dish prepared in step 1, while being careful not to create any scratches to the bottom of the dish. Using a plastic tip, explants are gently and evenly spread out. By gentle aspiration, the excess of serum is removed from around explants, and left to dry for approximatively 10-15mins. Once dried, 6-7ml of proliferative medium (see experimental procedure n°2 for instructions) are slowly added to avoid detachment of the muscle explants. The muscle biopsy is transferred to the 37°C incubator.Following the microdissection of the muscle biopsy, myoblast migrate and proliferate. Once enough myoblasts have migrate outside the muscle explant, they are transferred into new petri dishes/flasks for proliferation, sorting and further differentiation into myotubes. Other cell types such as fibroblasts will migrate into the petri dish, however we call easily enrich the human skeletal muscle stem cells by perfoming a CD56 cell sorting. The CD56 antigen (or N-CAM, the neutral cell adhesion molecule) was used as a marker of myogenic cells 372 .

13 . 14 .

 1314 Cells are resuspended in 1ml proliferative culture medium and counted using the cell counter moxi Z (ORFLO), and the number of divisions is evaluated as follows: Myoblasts are seeded in the appropriate flask for further proliferation.

  4.A new collection tube is used to directly flush out the magnetic labelled CD56 + cells (myogenic cells) with 1ml MACS buffer.5. The cell number is determined and enriched myoblasts cellpopulation are transferred into appropriate flask for further proliferation.

2 . 3 .

 23 Differentiation of myoblasts occurs in a serum-free medium, in order to get rid of most of the serum from the proliferative medium with minimum stress for the cells, cells are washed 5 times in DMEM The suitable volume of DMEM is added and cells are left for differentiation for 3 days at 37°C (see

Day 8 :

 8 N2B27 medium change n°21. Similarly, 75L of N2B27++ 2 medium is gently removed from each well 2. N2B27 ++ 3 medium is prepared as follows: RA, GDNF, DAPT and BDNT are added to N2B27 medium (1/500 dilution factor) 3. Carefully, 100 of fresh 100L N2B27++ 3 is added to each well and cells are incubated at 37°C for 2 days Day 10: N2B27 medium change n°3 1. The last medium change occurs at day 10 when iPSC-MN are fully differentiated, N2B27 ++ 4 medium is prepared as follows; GDNF and BDNF are added to the N2B27 medium (1/500 dilution factor) 2. Cells can stay in culture for further applications Secretion of neurotoxic vesicles by muscle cells of ALS patients Mycoplasma contamination screening have been performed regularly using the LookOut Mycoplasma PCR Detection kit from Sigma-Aldrich.

4 . 5 . 6 .Test Reaction tube rehydration 1 . 1 .

 45611 The column is placed into a new collection tube, and DNA is washed with 500µl of Wash Buffer II. Samples are centrifuged at maximum speed for 3mins and the collection tube is discarded. Elution of DNA is performed with 50µl Elution Buffer, incubated for 1min at room temperature. 7. Columns were centrifuged at maximum speed for 1min at room temperature, and extracted DNA is used to perform PCR. 𝐷𝑁𝐴 -𝑃𝑜𝑙/𝑅𝐵 = (23 × 𝑥 𝑠𝑎𝑚𝑝𝑙𝑒𝑠) + 23 (1 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) + 25 (𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) + 23 (1 𝑒𝑥𝑡𝑟𝑎 𝑣𝑜𝑙𝑢𝑚𝑒) 2. DNA Polymerase units determination  For JumpStart Taq Polymerase with 2,5units/l, a volume of 0,5l is required per reaction 𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐽𝑢𝑚𝑝 𝑆𝑡𝑎𝑟𝑡 𝑇𝑎𝑞 𝑃𝑜𝑙𝑦𝑚𝑒𝑟𝑎𝑠𝑒 = 0,5l × (x samples + 2 controls + 1 extra volume) 3. The DNA Polymerase/Rehydration buffer are carefully mixed by flicking tubes. Appropriate number of tubes are prepared as follows: (𝑥 𝑠𝑎𝑚𝑝𝑙𝑒𝑠) + (1 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙) 2. 23l of the prepared DNA Polymerase/rehydration Buffer is added to each tube Negative Control / Sample addition 1. 2l of DNA free water is added to the negative control tube 2. 2l of samples are added to the sample reaction tube Positive control preparation The positive tube contain positive mycoplasma DNA and are prepared last by adding 25l of the prepared DNA polymerase/Rehydration Buffer. Secretion of neurotoxic vesicles by muscle cells of ALS patients Each tube is mixed by flicking them and quickly centrifuged 2. Samples are incubated at room temperature for 5min and are immediately processed with the thermal cycling.

  1 cycle 94°C for 2 min  40 cycles 94°C for 30 sec 55°Cfor 30 sec 72°C for 40 sec  Cool down to 4-8°C Agarose gel migration 1. 1,2% agarose gel is used for PCR products migration. 2. 8l of PCR products are loaded into the gel and electrophoresis is run for approximatively 25 min at 100V. Results evaluation 1. The negative control shows a unique band at 481bp indicating successfully performed PCR. 2. The positive control shows 2 distinct bands, one at 481bp the internal PCR efficiency control and one at 259bp corresponding to the mycoplasma DNA. 3. Mycoplasma-positive samples show a band around 2608bp and depending on the contamination level a 481bp band for internal control.

1 .II. Exosomes extraction 1 . 2 . 3 . 4 . 3 . 4 . 5 .. 9 . 1 .

 1123434591 Cell debris elimination is performed by differential centrifugations: The culture medium is centrifuged at 300g for 10min The supernatant is centrifuged at 4000g for 20min at 4°C 2. Removal of microparticles is performed by filtration of the obtained supernatant using 0.22µm filters. The conditioned culture medium is ready for exosomes extraction. The required volume of culture medium is transferred in a new tube and mixed with 0.5 volumes of Total exosomes isolation kit (2:1, v:v) The culture medium/reagent is well mixed by pipetting up and down (or inverting the tube several times) until a homogeneous solution is obtained. The mixture is incubated at 4°C overnight. The next day exosomes are extracted by centrifuging the mixture at 10,000g for 1 hour at 4°C. 5. The exosome-free supernatant is removed and discarded, exosomes are in the pellet and are resuspended in 100µL PBS. dye that possess a long aliphatic tails allowing its anchoring to lipid regions of cell membranes 374 . PKH26 staining has been used in a variety of in vivo and in vitro cell tracking experiments and has also proven effective for exosomes uptake monitoring. Extracted muscle exosomes are stained with PKH26 label before applied to cell cultures (muscle and nerve cultures) for treatment. Procedure 1. A solution of 4µM PKH26 is prepared by mixing 3.2µL PKH26 with 800µL Diluent C. 2. 100µL of Diluent C are added to the 100µL exosomes suspension (see experimental protocol n°4 for exosomes extraction), immediately followed by 100µL of 4µM PKH26. The mixture should be immediately mixed to allow bright and uniform staining. Exosomes suspension are incubated for 5min at room temperature. The staining protocol of exosomes requires to wash the vesicles in concentrators to get rid of excess of PKH26 labelling reagent. The 100K concentrators are pre-rinsed with 500µL of PBS added to the sample chamber. Concentrators are centrifuged at 15,000rpm for 10min at 4°C and the filtrate is discarded. 6. PKH26 labelling is diluted in 250µL PBS 7. The exosomes sample is added to the sample chamber of the concentrator and centrifuged at 15,000rpm for 10min at 4°C. 8. The filtrate is discarded and exosomes are washed by adding 500µL PBS to the sample and centrifuge at 15,000rpm for 10min at 4°CThe filtrate is discarded and exosomes are washed again for a total of 3 PBS washings. 10. After the last centrifugation, exosomes can be resuspended in 100µL of PBS should kept in the sample chamber by gently scraping the column of the concentrator. 11. The labelled-exosomes are transferred to a new Eppendorf tube and immediately use for cell treatment. quantification from cell pellets sample Total proteins quantification of muscle cells sample was performed using the bicinchoninic acid (BCA) assay, allowing for a colorimetric quantification of proteins. The BCA assay is based on the reduction of Cu 2+ to Cu + and the sensitivity of bicinchoninic acid to detect Cu + . Protein extraction have been performed using different extraction buffer depending on further experiments. 1. Prepare appropriate extraction buffer:  RIPA buffer 1X  Urea 8M buffer  ½ RIPA 1X -½ Urea 8M buffer  Each extracting buffer supplemented with 10µL/mL proteases and phosphatases inhibitors cocktail 2. 30µL of extraction buffer is added to the cell pellet and samples are incubated 15 to 30 min at 4°C for complete cell lysis. 3. Lysed cell samples are centrifuged at 14,000g for 10 min at 4°C. 4. Soluble proteins contained in the supernatant are transferred to a new Eppendorf tube and stored at -80°C for further applications. BSA standards are prepared as follows using a 2mg/ml BSA stock solution: quantification from cell pellets sample 2. Standards are prepared in PBS and same extraction buffer concentration is added to each vial:  Cell samples are rich in proteins, thus extracted proteins are usually diluted 1/10 in PBS before quantification.  1/10 extraction buffer is thus added to each standards 3. Cell samples proteins are diluted in PBS (1/10 dilution factor) 4. BCA working reagent (WR) preparation:  Using the following formula, the required volume of WR is calculated (#𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑𝑠 + #𝑠𝑎𝑚𝑝𝑙𝑒𝑠) 𝑥 3 (𝑡𝑟𝑖𝑝𝑙𝑖𝑐𝑎𝑡𝑒𝑠) 𝑥 190 (𝑣𝑜𝑙 𝑜𝑓 𝑊𝑅 𝑝𝑒𝑟 𝑤𝑒𝑙𝑙) = 𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑊𝑅 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑  WR is prepared by mixing BCA reagent A with BCA reagent B (50:1, v:v) 5. 10µL of standards and samples and 190µL of WR are to the corresponding wells of the 96 wells plate.6. The plate is incubated at 37°C for 30 min before absorbance can be read at 562nm using a plate reader.

2 .

 2 Standards are prepared in PBS and same extraction buffer concentration is added to each vial (28% of final volume, i.e. for 30µl of standards 12µl of Urea 8M is added) 3. BCA working reagent (WR) preparation:  Using the following formula, the required volume of WR is calculated (#𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑𝑠 + #𝑠𝑎𝑚𝑝𝑙𝑒𝑠) 𝑥 3 (𝑡𝑟𝑖𝑝𝑙𝑖𝑐𝑎𝑡𝑒𝑠) 𝑥 190 (𝑣𝑜𝑙 𝑜𝑓 𝑊𝑅 𝑝𝑒𝑟 𝑤𝑒𝑙𝑙) = 𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑊𝑅 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑  WR is prepared by mixing BCA reagent A with BCA reagent B (50:1, v:v) 4. 10µL of standards and samples and 190µL of WR are to the corresponding wells of the 96 wells plate.

1 . 2 . 4 . 1 .

 1241 The 1X iBind Flex solution is prepared as follows:  500µL 100X additive  10mL iBind TM Flex 5X Buffer  39,5mL distilled water The PVDF membrane is incubated in the 1X iBind Flex solution for at least 10 mins, and continuously covered to avoid the membrane to dry out during preparation of antibodies solution.3. Primary and secondary antibodies are diluted in the 1X iBind Flex solution. The iBind Flex System is prepared for blotting by placing an iBind Flex Card in system and adding 10ml of 1X iBind Flex solution across the flow region as illustrated with picture below: The 1X iBind Flex Solution shouldn't be added to close to the stack of the iBind card as it may decrease the efficiency of the blotting. 5. 2ml (for midi membranes) or 1ml (for mini membranes) of 1X iBind Flex solution are added in the middle of the iBind Flex Card, and the pre-incubated membrane is places protein-side down and the low molecular weight region closest to the card stack, and air bubbles are removed. 6. The lid is closed and antibodies solutions are added to the appropriate rows: Row 4: iBind Flex solution Antibodies and appropriate dilutions used in western blotting are listed in Table from experimental procedure n°11) Amersham ECL Prime Western blotting Detection Reagent was used for detection of signal. Solution A containing luminol, and solution B containing peroxide are mixed in a 1:1 ratio.

4 .

 4 The cycling protocol includes:  Initial step: 94°C for 5 min  50 cycles of : 94°C for 10sec, 60°C for 5sec, and 70°C for 5sec Quantitative RT-PCR for FUS gene expression performed on muscles cells required the Light Cycler 480 Instrument Roche and Platinium® SYBR® Green qPCR SuperMix-UDG.

3 . 5 .

 35 The differentiation medium is replaced by 450µl of exosomes resuspensed in DMEM. 450µl of only DMEM is added to the nontreated wells. 4. Slowly, 50µl of Lipofectamine/siRNA mix (or DMEM) is added to the corresponding well. Cells are incubated for 3 days to allow integration of exosomes in the cells. On day 6 culture medium is replaced by fresh DMEM and cells are incubated for 3 more days before fixation. Secretion of neurotoxic vesicles by muscle cells of ALS patients ,000 healthy myoblasts are cultured in Ibidi 35mm µ-dishes 2. Exosomes are extracted from 200.000 ALS and healthy muscle cells cultures. 3. ALS and healthy exosomes are added to the culture of healthy myoblasts the same day the differentiation is induced. Exosomes absorption occurs for 3 days before fresh DMEM is added to the petri dishes. 4. Exosomes-treated myoblasts are fixed in 4% formaldehyde at day 3 or day 7 of differentiation.

2 .

 2 Apoptosis: apoptosis levels are measured with expression level of H2AX. Cells are stained with DAPI and H2AX and total number of nuclei and individual H2AX area are measured. The final H2AX are result from the individual H2AX sum normalized with the number of nuclei.

5 .

 5 Muscle cells are stained with DAPI and MF20 (myotube marker). Nuclei are counted in multinucleated myotubes and in mononucleated muscle cells and fusion index is determined using the following formula: Myonuclear domain: The myonuclear domain illustrating the atrophic level of myoblasts is calculated using the following formula:

2 . 1 . 1 .

 211 ALS and healthy exosomes are extracted and exosomal proteins are quantified (see experimental protocol n°8) 3. 4µg or 8µg of exosomes are applied to the muscle cells cultures and differentiation is induced. 4. Cells were fixed at 6 days of differentiation. Secretion of neurotoxic vesicles by muscle cells of ALS patients Cell death and apoptosis levels: Similarly, cell death and apoptosis levels were assessed following directions explained above. 2. RNA localisation: Cells are stained with acridine orange (see experimental protocol n°11) and total nuclei is determined for each picture. The percentage of nuclear and cytoplasmic RNA accumulation is calculated. Muscle cell lines overexpressing wild type forms of FUS, SOD1 and TDP43 treatment: Dr J Dumoncaux and Dr V Mariot (NIHR Biomedical Research Centre, University College London, Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Trust, London, UK) generated the immortalized cell lines expressing FUSFLAG, SOD1 SOD1FLAG or TDP43FLAG. Healthy muscle cell lines are transduced with plasmid coding for a tagged (FLAG tag) of wild type FUS (FUSFLAG), SOD1 (SOD1FLAG) or TDP43 (TDP43FLAG), and selected with hygromycin.

  vesicles by muscle cells of ALS patientsLaura Le Gall 109

Figure 12 :

 12 Figure 12 : Similar myotubes differentiation after ALS and control exosomes treatment. At 3 days or 7 days of exosomes treatment the number of myonuclei per myotubes was measured as illustrated by cumulative rank plot showing a similar distribution in both conditions and time points. The fusion index was calculated as previously described, and was about 96% in both ALS-and healthy exosomes.

Figure 14 :

 14 Figure 14 : TDP43 granulation in ALS and healthy muscle cells. Similarly to Figure 4A from paper, TDP43 granulation was assessed in ALS and healthy muscle cells. Each circle represents one myonuclei, 100 nuclei were analysed per patient (x axis, n=3). A greater accumulation of TDP43 granules were measured in ALS muscle compared to. *, P<0,05 significantly different from healthy myonuclei.

Figure 1 :

 1 Figure 1: Accumulation of exosomal markers in muscle of ALS patients. Panel showing representative images of CD63 immunostaining performed on muscle biopsies from different subjects. Right insert: Quantification of exosomal marker CD63 in muscle biopsies (Pixel per µm 2 n= 6 ALS, 4 SBMA, 5 Healthy; ** significantly different from healthy subjects, P<0.01; TT significantly different from SBMA patients, P<0.01).

Figure 2 :

 2 Figure 2: Increased accumulation and secretion of exosomes by ALS myotubes. (A) PCA plot showing separation of ALS patients on the 1st component (x-axis), ALS: blue, SBMA: pink, SMA-III/IV: red, Healthy: green. The transcriptomic analysis was performed on primary human myoblasts differentiated for three days to form myotubes.

Figure 3 :

 3 Figure 3: ALS exosomes induce decreased neurite length and branching, and increased death, of human induced pluripotent stem cells derived motor neurons. (A) Equal quantities of ALS and healthy exosomes are taken up by motor neurons. (B) Neurite lengths of motor neurons are shortened when treated with ALS exosomes compared to healthy exosomes. Left panel: representative images of exosome-treated motor neurons. Right panel: quantification of neurite length (8 to 15 motor neurons analysed per well, with n=5 wells per treatment). ** and *** significantly different from ALS values P<0.01 and P<0.001, respectively. (C) Neurites of iPSC-MN cells have fewer neurite branch-points following treatment with ALS exosomes compared to treatment with healthy exosomes; two-sample Kolmogorov-Smirnov test confirmed the visual impression that the number of branches per neurite is decreased

Figure 4 :

 4 Figure 4: The toxicity of ALS muscle exosomes to healthy motor neurons involves the FUS protein and RNA processing. (A) ALS myonuclei present an increased granulation of FUS compared to healthy controls. Upper panel: representative images of FUS immunostaining in ALS and healthy myonuclei. Lower panel: quantification of granulation (100 myonuclei analysed per subject, with n=3 subjects per group). ***, P<0.001 significantly different from healthy myonuclei. (B) ALS myotubes present an accumulation of RNA in their nuclei. Left panel: representative images of RNA localisation in ALS and healthy myotubes. Right panel: percentage of myonuclei with high levels of RNA, assayed by acridine orange staining (50 myonuclei analysed per subject, with n=5 subjects per group). ***, P<0.001 significantly different from healthy myotubes. (C) RPL5, a protein involved in RNA transport and stress granules, is granulated in ALS myonuclei. Left panel: representative images of RPL5 localisation in ALS and healthy myotubes. Right panel: percentage of myonuclei with RPL5 granules (500 to 2,000 nuclei analysed per subject, with

Figure S2 :

 S2 Figure S2: ALS myoblasts form thinner myotubes with fewer myonuclei. (A) ALS myoblasts

Figure S3 :

 S3 Figure S3: ALS myotubes secrete exosomes. Denervated murine muscles do not show a global

  CD9 immunostaining was used to labelled murine exosomes on muscle sections. **, significantly different from innervated muscle, P<0.01. (C) No upregulation of exosomal markers was observed in cachectic murine muscles. *, significantly different from innervated muscle, P<0.05. (D) ALS and healthy exosomes are positive for CD63 and flotillin, and negative for calnexin. (E) NanoSight analysis showing that ALS and healthy exosomes sizes range between 90-200 nm. (F) SDS-PAGE analysis showing a greater quantity of protein in exosomally enriched fraction secreted by 800,000 ALS myotubes compared to healthy controls. Left panel: representative image of SDS-PAGE. Exosomes were extracted from the culture medium of 800,000 myoblasts differentiated into myotubes for 3 days. Right panel: quantification of protein levels per well. *, significantly different from healthy, P<0.05.

Figure S4 :

 S4 Figure S4: ALS exosomes are toxic toward healthy human iPSC-MN and myotubes. (A) ALS

Figure S5 :

 S5 Figure S5: RNA transport is affected in ALS myotubes, and in cells treated with ALS

Figure S6 :

 S6 Figure S6: The proteomic content of exosomes is enriched for FUS and TDP43 binding

  al, Amyotroph Lateral Scler Other Motor Neuron Disord 2000; 1: 293-299), who attended the Motor Neuron Diseases Center (Pitié Salpétrière, Paris). Genetic analyses were carried out on DNA extracted from blood samples for all ALS patients to screen several ALS related genes: the C9orf72 hexanucleotide repeat expansion (using gene scan and repeat primed PCR procedures described in(40)), ATXN2 repeat length (41) and the coding regions of SOD1, TARDBP, FUS, UBQLN2 and TBK1(sequences of the used primers are available upon request). An open deltoid biopsy was also obtained from 6 patients with SMN1-linked adult Spinal Muscular Atrophy (SMA type III/IV) and 10 patients with genetically confirmed Spinal and Bulbar Muscular Atrophy (SBMA, also known as Kennedy's Disease) recruited in the Paris MND Center or the Institute of Myology (Pitié Salpétrière, Paris). Two peroneal muscle biopsies were obtained from patients affected by sensorimotor polyneuropathy. Twenty-four deltoid muscle biopsies from healthy age and gendermatched subjects were obtained from the BTR (Bank of Tissues for Research, a partner in the EU network EuroBioBank) in accordance with European recommendations and French legislation. The main demographic, clinical, and genetic characteristics of the subjects are indicated in table

  the secondary antibodies used were goat anti-mouse IgG1 or anti-mouse IgG2a, or mouse IgG2b or anti-rabbit tagged with AlexaFluor 355 or AlexaFluor 488 or Alexa Fluor 555 or AlexaFluor 594 or AlexaFluor 647 (1:400, Invitrogen™). The slides were washed, counter-stained with 1µg.ml -1 DAPI for 1 min, rinsed 2 times and mounted with ibidi mounting medium (ibidi®).Ten to 20 non-overlapping pictures were acquired in a line along the diameter of the slide with an Olympus IX70, and an Olympus UPlan FI 10x/0.30 Ph1 and an Olympus BX60 objectives equipped with a Photomatics CoolSNAP™ HQ camera. For the human muscle sections, pictures were taken with an Olympus LCPlan FI 40x/0.60 Ph2 objective. Images were acquired using Zeiss software and analysed using either Fiji or ImageJ 1.37v.

  peptides were vacuum dried, resuspended and analyzed by data-dependent mass spectrometry on a Q Exactive HF (Thermo) with the following parameters: Positive Polarity, m/z 400-2000 MS Resolution 70,000, AGC 3e6, 100ms IT, MS/MS Resolution 17,500, AGC 5e5, 50ms IT, Isolation width 3 m/z, and NCE 30, cycle count 15.-Database Search and Quantification -Resulting mass spectral files were searched for protein identification using ProteomeDiscoverer (Thermo) against the Uniprot human database for semi tryptic peptides and filtered based on a false discovery rate of <1%. 718 proteins were detected in at least 1 sample Knockdown of FUS with siRNA 100,000 cells were plated in µ-Slide 4 Well ibiTreat (Ibibi®) one day before differentiation was induced. On day 2 of differentiation, cells were transfected using ) and treated with a low dose of PKH26-labelled ALS exosomes.

Figure 15 :

 15 Figure 15 : ALS muscle cells secrete neurotoxic exosomes. These vesicles disrupt the RNA-processing in MN. Potential role of muscle exosomes in body-wide propagation of disease.

  . Hence, muscles exosomes once secreted in the extracellular milieu could spread from LMN to UMN by retrograde Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 174 transport along axons, and the same way could be further transported to non-affected limb muscles through an anterograde transport manner.

Figure 16 :

 16 Figure 16 : Graphical abstract. Speculation on how muscle exosomes can have a role in the propagation of the disease.This project demonstrated that 1) ALS muscle stem cells share a specific ALS signature that is independent from any innervation/denervation process. 2) Multiple RNA processing dysregulation have been observed in ALS muscle cells such as nuclear accumulation of RNA, high FUS nuclear granulation, and aggregation of FUS partner, RPL5 in myonuclei. Here illustrated as brown nuclei from altered muscle fiber in dark red colour. 3) We showed a toxic secretion of exosomes by muscle cells of ALS patients that are toxic and induce cell death in 4) muscle cells and 5) motor neurons. 5) ALS muscle exosomes are toxic towards MN and could potentially be retrogradely transported towards to cell body of the affected MN (here in red dashed line versus green full line for healthy non-altered MN) and 6) induce an accumulation of RNA in MN nuclei. On the other hand, exosomes have been isolated from blood samples meaning they can 7) have access to the bloodstream and 8) pass the brain-blood-barrier. 9) Exosomes from motor neuron neighbouring cells such as astrocytes, microglia and oligodendrocytes also secrete exosomes that

  ISSN 2214-3599/15/$35.00 © 2015 -IOS Press and the authors. All rights reserved This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License.

Fig. 2 .

 2 Fig. 2. Aging changes the microenvironment of the satellite cell. Decreased muscle mass can be accompanied by a decrease in myokines and vesicles secreted into the microenvironment of the satellite cells. Aged myofibers produce more ROS and FGF-2, factors that can change epigenetic marking of the satellite cells and shut down their myogenic program and their capacity to re-quiesce. They also release less NO into their environment, stimulating vasoconstriction which may inhibit serum tissue perfusion. Aged fibroblasts present in the muscle can secrete more fibrous proteins, thickening the ECM. In turn, this decreases the diffusion of growth factors toward the satellite cells and thus their responsiveness to muscle repair cues. Increase in senescent cells with age can secrete factors that inhibit tissue regeneration. The microenvironment of the satellite cells is thus altered and affects their capacity to respond to any muscle damage.

  Fig.3. Increase of oxidative stress response in aged muscles. For this analysis we retrieved the gene expression matrix of old and young muscles of sedentary subjects (series GSE9103 from the Gene Expression Omnibus[139]). We discarded three samples as they did not pass the quality control threshold. To identify differentially expressed genes we used the characteristic direction method[140] followed by gene set enrichment with Enrichr[141]. The top figure depicts a bar graph from the top 10 up-regulated GO Biological Processes (combined score: p-value multiplied by z-score) and the bottom figure a network of the same Processes, where each node represents the enriched term and the edges represent the gene content similarity between the nodes.

  III. Accumulation of ALS exosomes in the culture medium II. Up-regulation of exosomal markers in ALS muscle cells V. Toxic autocrine and paracrine effects of ALS exosomes #4 Cell Death Blebbs containing labelled exosomes

  Secretion of toxic exosomes by muscle cells : role in ALS pathogenesis ALS Cont 2.4 M healthy cells 2.4 M ALS cells Human primary cells extracted from Deltoid muscle biopsies: -SBMA(n=12), ALS (n=18), SMA (n=12), Healthy subjects (n=15). -All Male, 20-76 years old. -Muscle cells sorted using CD56 MACS beads: >86% myogenic cells Material & methods -After removing cell debris and microparticles (centrifugation 200g,10 min RT; 2,000g 20 min 4C and filtration 0.22 mm), exosomes were extracted using Life Technologies kit (we validated the quality of the kit by electron microscopy and immunostaining and western blot) -Labelled ALS or control exosomes were added to healthy human muscle stem cells or healthy human iPSC-motor neuron n=6/supported by: • the Association pour la Recherche sur la Sclérose Latérale Amyotrophique (ARSLA), • INSERM &Direction Générale de l'Organisation des Soins (DGOS) • the Association Française contre les myopathies (AFM), • Target-ALS association and • European Union Regional Development Fund (ERDF) EU Sustainable Competitiveness Programme for N. Ireland; Northern Ireland Public Health Agency (HSC R&D) & Ulster University.

1 -

 1 th International Symposium on ALS/MND -Glasgow -UK "Alteration of FUS pathway leading to a communication breakdown in ALS" Laura Le Gall 1,2 , William J Duddy1 , Sylvain Roquevière 3 , Mariot Virginie 4 , Julie Dumonceaux 4 , TRANE group study, Olivier Lucas 5 , Cedric Raoul 35 , Susan Knoblach 6 , Cecile Martinat 3 , Jose-Luis Gonzales De Aguilar 7 , Pierre Francois Pradat 8* , Stephanie Duguez 1* *Co-last authors Affiliations: 1-Northern Ireland Center for Stratified Medicine, Biomedical Sciences Research Institute, Londonderry, UK. 2-Myologie Centre de Recherche, Université Sorbonne, UMRS 974 UPMC, INSERM, FRE 3617 CNRS, AIM, Paris, France. 3-I-Stem, INSERM/UEVE UMR 861, I-STEM, AFM, Evry, France. 4-NIHR Biomedical Research Centre, University College London, Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Trust, London, UK. 5-Équipe Pathologies du motoneurone : inflammation et thérapie, INSERM U1051 -INM, Montpellier, France. 6-Children's National Medical Center, George Washington University, Washington, US. 7-Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg, INSERM U1118, Strasbourg, France. 8-Département des Maladies du Système Nerveux, Hôpital de la Pitie-Salpetriere, Paris, France. Study group: Gillian Butler Brown 1 , Vincent Mouly 1 , Gisele Ouandaogo 1 , Alexandre XX 2 , Laura Robelin 2 , Franscesca Ratti 3 , Alexandre Mejat 3 , Pascal LeBlanc 3 , Salachas Francois 4 , Anne Cecile Durieux 5 , Loeffler jean Philippe 2 Myologie Centre de Recherche, Université Sorbonne, UMRS 974 UPMC, INSERM, FRE 3617 CNRS, AIM, Paris, France 2-Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg, INSERM U1118, Strasbourg, France 3-Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon, Lyon, France. 4-Département des Maladies du Système Nerveux, Hôpital de la Pitie-Salpetriere, Paris, France. 5-LPE, Université Jean Monnet de Saint Etienne, Faculté de Médecine Jacques Lisfranc, Saint Etienne, France.

••

  Exosomes contained FUS and FUS binding partners • ALS exosomes induce nuclear RNA accumulation • No post-mortem tissue • All primary cells • All sporadic ALS patients • 4 patients with mutations: 3x C9ORF72, 1x Sca2 : summarizing the subject samples used for each type of experiment. Cells with low expression level of FUS are less sensitive to ALS exosomes Secretion of neurotoxic vesicles by muscle cells of ALS patients For many years now, a vicious serial killer is attacking the nerves of ALS patients. What is ALS? ALS stands for Amyotrophic Lateral Sclerosis but I'm sure that some of you have already heard about this disease during summer 2014 when the ALS ice bucket challenge went viral. Today, many investigators are trying to solve the "ALS case" and here are the facts.

TABLE 1 :

 1 ALS AGE OF ONSET VARIABILITY AND THEIR CLINICAL FEATURES. ............................................................................

TABLE 2 :

 2 CLINICAL FEATURES FOR BEHAVIOURAL VARIANT FTD (BVFTD) DIAGNOSIS. ...........................................................

TABLE 3 :

 3 DIAGNOSTIC CRITERIA TABLE FOR ALS-BI AND ALS-CI VARIANT. ..........................................................................

TABLE 4 :

 4 EL ESCORIAL CRITERIA AND REVISIONS FOR ALS DIAGNOSIS. ..............................................................................

TABLE 5 :

 5 AMYOTROPHIC LATERAL SCLEROSIS FUNCTIONAL RATING SCALE, REVISED ALSFRS-R. ..............................................

TABLE 6 :

 6 CELL TYPES RELEASING EXOSOMES...............................................................................................................

TABLE 7 :

 7 EXOSOMES HETEROGENEITY EXPLAINED BY SIZE AND BUOYANT DENSITIES VARIABILITY IN EXTRACTED VESICLES POPULATION. ..............................................................................................................................................................

TABLE 8 :

 8 SUMMARY LIGAND -RECEPTOR INTERACTION IN EXOSOMES/CELL COMMUNICATION. ..............................................

TABLE 9 :

 9 TABLE OF ANTIBODIES AND THEIR DILUTIONS USED IN IMMUNOCYTOCHEMESTRY, IMMUNOHISTOCHEMISTRY OR IN WESTERN BLOTTING. .................................................................................................................................................

TABLE 10 :

 10 HOUSEKEEPING GENE PRIMER SEQUENCES. .................................................................................................

TABLE 11 :

 11 EXOSOMAL MARKER AND FUS PRIMER SEQUENCES. ......................................................................................

TABLE 12 :

 12 TABLE SUMMARIZING EXPERIMENTAL CONDITIONS AND ANALYSIS PERFORMED ON MN CULTURES TREATMENT...........

TABLE 13 :

 13 TABLE SUMMARIZING EXPERIMENTAL CONDITIONS AND ANALYSIS PERFORMED ON MUSCLES CELLS TREATMENT WITH EXOSOMES. .............................................................................................................................................

TABLE 14 :

 14 TABLE SUMMARIZING SAMPLES CHARACTERISTICS. ......................................................................................

TABLE 15 :

 15 NUMBER OF CELLS EXTRACTED PER BIOPSY AND PROPORTION OF MUSCLE CELLS. .................................................

Table 1 : ALS age of onset variability and their clinical features.

 1

	9

  ).

	Secretion of neurotoxic vesicles by muscle cells of ALS patients	Laura Le Gall
	Possible bvFTD	3 recurrent or persistent	Early behavioural disinhibition
		behavioural/cognition symptoms	Apathy or inertia
			Loss of sympathy or empathy
			Perseverative, stereotypes or
			compulsive/ritualistic behaviour
			Hyperorality and dietary changed
			Neurophysiological profile:
			executive/generation deficits with relative
			sparing of memory and visuospatial
			functions
	Probable bvFTD	Criteria for possible bvFTD and	Meets criteria for possible bvFTD
		functional disability
			Exhibits significant functional decline
			At least 1 Imaging results consistent with
			bvFTD:
			-Frontal/anterior temporal atrophy
			-Frontal/anterior hypofusion or
			hypermetabolism
	Definite bvFTD	Criteria for possible or probable bvFTD	Histopathological evidence of FTD on
		associated with supplemented with one	biopsy or at post-mortem
		of the following criterion
			Presence of a known pathogenic mutation
				12

Table 2 : Clinical features for behavioural variant FTD (bvFTD) diagnosis.

 2

Table 3 : Diagnostic criteria table for ALS-bi and ALS-ci variant.

 3

  5,6 do not include cognitive and behavioural deficits in ALS patients. However independent stratification tools have been developed to classify ALS patients with extra-motor involvement such as the Edinburgh Cognitive and Behavioural ALS Screen (ECAS), a cognitive test designed for patients with motor function impairment designed to minimise the impact of motor dysfunction on the classification of the patient35 .

	Secretion of neurotoxic vesicles by muscle cells of ALS patients	Laura Le Gall
	El Escorial criteria (1994)	Revised El Escorial criteria (2000)	Awaji-shima criteria (2008)
	Suspected ALS:
	-LMN signs only in 2 or more	_	_
	regions
	Possible ALS:	Clinically possible ALS:	Clinically possible ALS:
	-UMN + LMN signs in 1 region	-UMN + LMN signs in 1 region	Clinically and electrophysiological
	-UMN signs alone present in ≥2	-UMN signs alone present in ≥2	indication of :
	region	regions	-UMN + LMN signs in 1 region
	-LMN signs rostral to UMN signs	-LMN signs rostral to UMN signs	-UMN signs alone present ≥ 2
			regions
			LMN signs rostral on UMN signs
		Clinically probable ALS -
		laboratory supported:
		-UMN + LMN signs in 1 region
	_	only	_
		-UM signs in 1 region and LMN
		signs defined by EMG criteria
		present in ≥2 regions
	Probable ALS:	Clinically probable ALS:	Clinically probable ALS:
	UMN + LMN signs ≥ 2 regions	UMN + LMN signs ≥ 2 regions	Clinically and electrophysiological
			indication of UMN + LMN signs ≥
			2 regions with some UMN signs
			necessarily rostral to the LMN
			signs
	Definite ALS:	Clinically definite ALS:	Clinically definite ALS:
	-UMN + LMN signs in bulbar	-UMN + LMN signs in bulbar	Clinical and electrophysiological
	regions and ≥ 2 spinal regions	region and ≥2 spinal regions	indication of
	-UMN + LMN signs in 3 spinal	UMN + LMN signs in 3 spinal	-UMN + LMN signs in bulbar
	regions	regions	region and ≥ 2 spinal regions
			UMN + LMN signs in 3 spinal
			regions
			15

Differential diagnosis:

Among other motor neuron disease, spinal muscular atrophy type III and IV (SMAIII/IV) and spinal-bulbar muscular atrophy (SBMA or Kennedy's disease) are two motor neuron diseases mimicking early stages of ALSwith SBMA being one of the most frequently misdiagnosis with ALS

21

. Thus establishing a diagnosis at an early stage of ALS is challenging. The diagnosis is even more complexify by the existence of ALS variants with pure bulbar (pseudobulbar palsy and progressive bulbar palsy), pure UMN signs (PLS) or pure LMN dysfunctions (PMA and flail-arm/leg syndromes ALS variants) are part of ALS differential diagnosis

21

. However, ALS is defined as a motor neuron disease associated with progressive UMN and LMN dysfunction. Any symptoms that would not progress over months are signs of ALS mimic syndromes.

Table 4 : El Escorial criteria and revisions for ALS diagnosis

 4

.

El Escorial diagnosis criteria created in 1990 and revised in 2000 to help with diagnosis of ALS patients. In the original El Escorial criteria patients were classified into 4 categories (suspected ALS, possible ALS, probable ALS or definite ALS). In 2000, the revised criteria or Airlie House criteria disease burden ranges clinically possible to clinically definite ALS. The Awaji-shima criteria further simplified the revised El Escorial criteria into 3 subgroups: clinically possible, clinically probable and clinically definite. UMN: upper motor neuron. LMN: lower motor neuron.

Table evaluating

 evaluating Secretion of neurotoxic vesicles by muscle cells of ALS patientsLaura Le Gall 16 gross motor task, fine motor task, bulbar function and respiratory function, including dyspnea, orthopnea and the need for ventilator support, see Table5). Shortly after establishment of the revised scale, ALSFRS-R was validated in 2 studies38 as ALSFRS scores powerfully correlated with level of muscle weakness and survival. Lower ALSFRS scores were assigned to ALS patients who died during the course of the study.

	Speech	Turning in bed
	4 Normal	4 Normal
	3 Detectable disturbance	3 Slow and clumsy, no help required
	2 Intelligible with repeating	2 Can turn alone and adjust sheets but with great difficulty
	1 Combined with non-vocal communication	1 Can initiate, but unable to turn and adjust sheets alone
	0 Loss of speech	0 Helpless
	Salivation	Walking
	4 Normal	4 Normal
	3 Slight, but definite excess of saliva	3 Early ambulation difficulties
	2 Moderate excessive saliva	2 Walks with assistance
	1 Marked excess of saliva, and drooling	1 Non-ambulatory functional movement
	0 Marked drooling	0 No purposeful leg movement
	Swallowing	Climbing stairs
	4 Normal	4 Normal
	3 Early eating problems	3 Slow
	2 Dietary consistency changes	2 Mild unsteadiness or fatigue
	1 Supplemental tube feeding needed	1 Needs assistance
	0 Exclusively parental or enteral feeding	0 Cannot do
	Handwriting	Dyspnoea*
	4 Normal	4 None
	3 Slow and legible	3 Occurs when walking
	2 Not all words are legible	2 Occurs with 1 or more of the following: eating,
		bathing, dressing
	1 Able to grip a pen but, unable to write	1 Occurs at rest, difficulty breathing when either sitting
		or lying
	0 Unable to grip a pen	0 Significant difficulty, considering using mechanical
		respiratory support
	Cutting food and handling utensils	Orthopnoea*
	4 Normal	4 None
	3 Slow and clumsy, no help required	3 Some difficulty sleeping sur to shortness of breath
	2 Can cut foods, but clumsy and needs some help	Not more than 2 pillows routinely required
	1 Food must be cut by someone else	2 Extra pillows needed to be able to sleep (>2)
	0 Needs to be fed	1 Sleeping only in sitting up position
		0 Unable to sleep
	Dressing and hygiene	Respiratory insufficiency*
	4 Normal	4 None
	3 Independent, although decreased efficiency for	3 Intermittent use of BiPAP
	self-care

2 Intermittent assistance or substitute methods 2 Continuous use of BiPAP at night 1 Needs attendant for self-care 1 Continuous use of BiPAP all day 0 Total dependence 0 Invasive mechanical ventilation by intubation or tracheostomy

Table 5 : Amyotrophic lateral sclerosis functional rating scale, revised ALSFRS-R.

 5 In 1996, a simple questionnaire measuring physical function carried out daily by ALS patients was validated. A revision of the ALSFRS aimed to better assess respiratory function of ALS patients (added questions are marked by an *) resulting in the above improved 10 questions questionnaire. The ALSFRS is used by clinicians in practice and in clinical trials, the ALSFRS allows to establish a rate of decline in ALS patients based on 4 domains: gross and fine motor tasks, bulbar function and respiratory function. BiPAP: bi-level intermittent positive air pressure (non-invasive ventilator assistance for ALS patients).

  Vesicle associated membrane protein-associated protein B or VAPB mutations are first observed in ALS patients in 1960's 40 . VAPA and VAPB are part of the VAP protein family which are well known components of vesicular regulation and trafficking. VAPB plays a role in degradation of misfolded proteins

. Similarly to TDP43, FUS is nuclear RNA-binding protein Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 21 possessing multiple functions in RNA and miRNA metabolism. It also may play a key role in neuronal integrity and plasticty

40

. Mutations in FUS, and more particularly in its nuclear localization signal (NLS) domain, causes a dominant cytoplasmic mislocalisation probably associated with both toxic loss-and gainfunction

55

.

 Other gene mutations associated with ALS VAPB:

morphology References Non cancerous cells Neuronal cells

		Secretion of neurotoxic vesicles by muscle cells of ALS patients			Laura Le Gall
						T-cells			x	x
		Stem cells	Mesenchymal stem cells Neural stem cells	Human mesenchymal stem cells Human neural stem cells Neural fetal stem cells		x		x	x x x
		Epithelial	cells		Intestinal epithelial cells Mammary epithelial cell Salivary gland epithelial cells	x		x	x	x x
						Adipocytes				x	x
				Endothelial cells	Human umbilical vein endothelial cells	x	x		x
					Granulosa cells	x		x	x
		Other			Hepatocytes Keratinocytes				x	x	x x
				Exosomes origin Macrophages Monocyte Monocyte-derived macrophages Aortic adenocarcinoma	Alix x	CD9	CD63 x	CD81 x	Flotillin	Hsp70 x	Tsg101	General	x
				Basophilic leukemia cells				x
					Rat primary cortical neurons	x			x	x	x
					Bladder cancer cells		x	x	x	x	x
							Microglial cell		x	x	x
		Brain cancer	Neuroglial D54MG / SMA560 brain cancer Murine neuroglial cells cell line				x	x	x	x
			cells		cells Primary cell cultures from pediatric Murine oligodendrocytes Rat schwann cells high grade gliomas	x	x	x	x	x	x	x x	x x
					Breast cancer	Rat astrocytes	x x			x x	x
	Cancerous cells	Rat hippocampal neurons Cardiac muscle cells Cardiomyocytes progenitors cells Skeletal muscle cells Human satellite cells derived muscle cells Murine colon adenocarcinoma cells HCT116 and COLO205 cells lines RKO and Caco-2 colon cell lines Glioblastoma multiforme Lung carcinoma Smooth muscle cells Human aortic vascular Lymphoma smooth muscle cell Mammary adenocarcinoma cell carcinoma Colon Muscle cells	x	x x x x x	x x x x x x x	x x x x	x x	x x x	x x x	x x x x x x x x
		Hematopoietic cells	B-cells Dendritic cells (DC) Mast cells mouse bone marrow-derived mast cells Natural killer cells Mastocytoma cell Myeloma Nasopharyngeal carcinoma Ovarian carcinoma Pancreatic adenocarcinoma Platelets Prostate cancer cell line		x x	x x x x x x	x	x	x x x
											35

Table 6 : Cell types releasing exosomes

 6

	Non exhaustive list of different cell types secreting exosomes including (1) non-cancerous like cells
	neuronal cells (cortical neurons and neuroglial cells), muscle cells (skeletal muscle cells) and hematopoietic
	cells (B cells and dendritic cells) and (2) cancerous cells. Exosomes are also secreted in-vivo and isolated
	from various body fluids such as plasma, urine and saliva. Listed are the classic positive exosomal markers
	used to identify and characterize the extracted vesicles (Alix, CD9, CD63, CD81, Flotillin, Hsp70, and
	Tsg101). General morphology column indicate the analysis of physical properties of exosomes including
	shape (electron microscopy analysis) and size (nanoparticle tracking analysis method).

Density (g/ml) Exosome origin Ref Exosome marker 1,07 1,08 1,09 1,1 1,11 1,12 1,13 1,14 1,15 1,16 1,17 1,18 1,19 1,2 Rat

	3. 3.
	Exosomes secretions: MVB and PM fusion below), and lead to the secretion of specific exosome
	subpopulations 227 . Even if heterogeneous exosome populations are secreted by cells, most of the studies
	analyse exosomes as bulk isolates masking vesicle subpopulations. It is still challenging to specifically
	target a distinct group of exosomes, but solving this technical challenge would allow researchers to target
	the exosome subpopulation of interest whether it is for general exosome biogenesis and secretion
	understanding or for therapeutic purposes.

Table 7 : Exosomes heterogeneity explained by size and buoyant densities variability in extracted vesicles population.

 7

  role in the propagation of neurotoxic proteins. In ALS patients, neuronal exosomes have been reported to contain ALS-associated proteins such as SOD1354,355 , FUS 356 , TDP43 357,358 and C9orf72mediated DPRs 359 . Interestingly, misfolded SOD1 seeds were identified in exosomes and are transmitted to recipient cells inducing propagation of the prion-like properties of SOD1 360 . In addition, astrocytes-derived exosomes contain mutant SOD1 and contribute to the death of motor neurons 355 . Likewise, TDP43 and

. Exosomes are secreted by many cells types including neurons 315 , astrocytes

165

, oligodendrocytes 147 and microglia 162 and may participate in the maintenance of physiological conditions in the brain

146,147,161,162,165,353

. However, increasing number of studies are reporting a pathological role of exosomes in neurodegenerative diseases such as Alzheimer's and Parkinson's disease

316,317

as Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall 62 exosomes have a

Muscle stem cells extraction and culture (2) -Myoblasts proliferation, sorting and differentiation

  table below). Human iPSCs-derived motor neurons (iPSC MN) are obtained following Maury et al 373 protocol. iPSC MN progenitors and supplements were obtained from Dr C Martinat (I-Stem, INSERM/UEVE UMR 861, I-STEM, AFM, Corbeil-Essones, France).

	Secretion of neurotoxic vesicles by muscle cells of ALS patients	Laura Le Gall
			Experimental protocol n°2 Experimental protocol n°3
		Human iPSCs motor neurons culture
	Myotubes retrieval:		Products:
	1. The 3 days differentiation medium is harvested and stored for further applications (see Exosomes extraction -experimental Procedure	M199 (Gibco #41150-020)
	I.	procedure n°5) Poly-L-lysine / laminin coating (Day 1 and 2):	Insulin (91077C-5G)
	2. Adherent myotubes are washed once in PBS and appropriate The experimental procedure from Maury et al. 373 was adapted to Ibidi® 8 well volume of Trypsin is added (see Table above). Cells are incubated plates.	Fetuin (Millipore #341506-1GM)
	5min at 37°C. Day 1: Poly-L-ornithine (PLO) coating	bFGF (Millipore #GF407)
		3. To inhibit the trypsin 5% FBS-PBS is added and cells are 1. PLO is diluted in PBS (final concentration 20g/ml) and 150L is added
	centrifuged at 200g -5min. Following volumes are used for 1.10 6 cells. per well.
	IV.	Proliferative medium
		1. DMEM/M199 medium is prepared by adding 125ml of M199 to
		500ml of DMEM
		2. 100ml of FBS is mixed with 400ml of DMEM/M199 and
		supplements are added to complete the proliferative medium:
		 125µl Fetuin (final concentration of 25µg/ml)
		 25µl bFGF (final concentration of 0.5ng/ml)
		 25µl EGF (final concentration of 5ng/ml)
		 250µl insulin (final concentration 5µg/ml)
		3. The proliferative medium is filtered with a 0.22µm filter
		Petri dish/Flask	Number of cells	Differentiation culture medium (DMEM)
		6cm petri dish	0,8.10 6 cells	4ml
		Flask 175cm²	5.10 6 cells	16ml
		Flask 225cm²	7,5.10 6 cells	22ml
				70

4. Harvested myotubes are resuspended in 100µl PBS and stored in eppendorf tubes at -80°C for further applications. 2. Plates are incubated at 37°C overnight.

Day 2: Laminin coating 1. PLO solution is removed and each well is rinsed 3 times with sterile water and once with PBS. 2. Laminin solution is prepared in PBS (final concentration 5g/ml) and 150L is added per well. 3. Plates are incubated at 37°C overnight. II.

iPSC MN culture (Day 3 to day 10)

					Experimental protocol n°3
				Human iPSCs motor neurons culture
	2. Additives including RI, RA, SAG, SAPT, GDNF and BDNF are	Products:
	diluted (1/1000) to the N2B27 medium. The supplemented N2B27
	medium also contains laminin to promote cell adhesion of iPSCs-	GDNF (Life Technologies #PHC7044)
	MN progenitors.
							BDNF (Life Technologies Products: #10908010)
	The N2B27 ++	1 is prepared as described below:	µ-Slide 8 Well ibidi treat
	N2B27 ++ 1				Dilution Volume	Stock [X] Final [X]	(Thistle Scientific #80826)
						(L)
	Rock Inhibitor (RI)	1/1000	6,4	100M	100nM	Laminin (Gibco # 23017-015)
	Retinoic Acid (RA) SAG (Smoothened Agonist) 1/1000 1/1000	6,4 6,4	100M 500M	100nM 500nM	PLO (Sigma #P4957-50ml)
	DAPT				1/1000	6,4	10mM	10M	Neurobasal (Life Technologies
	GDNF				1/1000	6,4	10g/mL 10ng/mL	#21103049)
	BDNF				1/1000	6,4	10g/mL 10ng/mL
	Laminin				1/1000	6,4	5g/mL	5ng/mL	DMEM/F12-Glutamax (Life
							Technologies # 31331028)
	iPSC-MN culture:				N2 (Life Technologies #
	1. Progenitor are defrosted in N2B27 medium without additives and	17502-048)
	centrifuged at 260g for 5min.		B27 (Life Technologies #
	2. Cells are resuspended in 1ml N2B27 ++ 1 and cell count is	A18956)
	determined.			Penicilin/Streptamicin (Gibco
	3. 6,000 progenitors are seeded per well in a final volume of 200L	#15070-063)
	iPSCs-derived MN are cultured in N2B27 differentiation medium for 7 N2B27 ++ 1 and incubated at 37°C for 2 days.	Technologies # 31350010) 2-Mercaptoethanol (Life
	days until reaching full differentiated state.
	Day 5: N2B27 medium change n°1 Day 3: iPSCs-MN progenitor's seeding N2B27 culture medium preparation, N2B27 ++ 1: N2B27 medium needs to be replaced regularly and different combination	Glutamax (Life Technologies #35050061
	1. N2B27 medium is prepared as described below: of additives are required depending on the differentiation step.
	500mL 50mL 1. iPSCs-MN are very sensitive to any harsh medium flow so 75L
	of N2B27 ++	Neurobasal 1 medium is gently removed to avoid cells to detach. 250mL 25mL
	2. N2B27 ++	DMEM/F12-Glutamax 250mL 25mL N2 5mL 500L 2 medium is prepared as follows: RA, SAG, DAPT and
	B27 BDNT additives are added to N2B27 medium (1/500 dilution 10mL 1mL
	factor)		Penicilin/Streptamicin 5mL	500L
				2-Mercaptoethanol	1,25mL 125L
				Glutamax		2,5mL	250L

n°9 Western blotting -Cell lysate samples

	Procedure
	I.	Sample preparation
		1. Proteins from cell lysates are extracted and quantified as needed
		(see experimental procedure n°7).
		2. If samples are running under reducing conditions, a NuPAGE mix
		composed of 1 volume of NuPAGE reducing agent (10X) and 2.5
		volumes of NuPAGE LDS sample buffer (4X) (1:2,5, v:v) is
		prepared, if non reducing conditions are required only NuPAGE
		LDS sample buffer (4X) is mixed with the samples.
		The required volume of NuPage to be added per sample is
		calculated as follows:
		𝑁𝑢𝑃𝐴𝐺𝐸 𝑣𝑜𝑙𝑢𝑚𝑒 𝑓𝑜𝑟 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋 =	𝑉𝑜𝑙𝑢𝑚𝑒 𝑠𝑎𝑚𝑝𝑙𝑒 𝑋	2.85 ⁄
		3. Samples are boiled at 70°C for 10min and quickly centrifuged.

Table 9 : Table of antibodies and their dilutions used in immunocytochemestry, immunohistochemistry or in western blotting.

 9

		Mounting medium
	(Ibidi #50001)
	Acridine	Orange	(Sigma
	#158550)

Experimental protocol n°12 RT-qPCR and relative gene expression analysis

	1. RT-qPCR was performed in 384 wells plate, and each well
	contained 9µl	Products:
	2. A SYBR Green mix was set-up for each gene, SYBR Green mix	M-MLV-RT	(Invitrogen
	for 1 well is prepared as follows:	#28025-013)
	 0.14µl distiller water	RNase	OUT	(Invitrogen
	 4.5µl SYBR Green	#10777-019)
	 0.36µl reverse and forward primers (20pmol/µl) for the	10mM dNTP Mix (Invitrogen
	gene of interest (see Table below).	#18427-013)
	3. 5µl of mix and 4µl of cDNA were added per well, in triplicate, and	Oligo(dT)		(Invitrogen
	the plate was quickly centrifuged.	#18418012)
		LightCycler 480 DNA SYBR
		Green Master 1 (Roche
		#04707516001)
		Instrument:
		LightCycler 480 Instrument
		Roche

qPCR and relative gene expression analysis Figure 11 : Housekeeping gene expression level measurement

	Secretion of neurotoxic vesicles by muscle cells of ALS patients	Laura Le Gall
			Experimental protocol n°12
	RT-Primers	Sequence (5' → 3')
	β2M	Fw Primer	CTCTCTTTCTGGCCTGGAGG
		Rev Primer	TGCTGGATGACGTGAGTAAACC
	GAPDH	Fw Primer	AAGGTGAAGGTCGGAGTCAACGG
		Rev Primer	TGACAAGCTTCCCGTTCTCAGCC
	GUS	Fw Primer	CTCATTTGGAATTTTGCCGATT
		Rev Primer	CCGAGTGAAGATCCCCTTTTTA
	HPRT1	Fw Primer	TGATAGATCCATTCCTATGACTGTAGA
		Rev Primer	CAAGACATTCTTTCCAGTTAAAGTTG
	P0 (RPL0)	Fw Primer	TCCAGGCTTTAGGTATCACCAC
		Rev Primer	GCTCCCACTTTGTCTCCAGTC
	PPIA	Fw Primer	CCTAAAGCATACGGGTCCTG
		Rev Primer	TTTCACTTTGCCAAACACCA
	PSMA2	𝐵2𝑀 Fw Primer 𝐶𝑡 𝐵2𝑀 ⁄ 𝐸 𝑔𝑒𝑛𝑒 𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡 CATTCAGCCCGTCTGGTAAA 𝐶𝑡 𝑔𝑒𝑛𝑒 𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡 Rev Primer GTTTTTTCTCAGTTGCTAATACCACA
				96

.

RT-qPCR was performed on healthy, ALS SMA and KD muscle cells in order to test 7 housekeeping genes. The comparison of the different genes expression levels has led us to use B2M as housekeeping gene because of its consistent expression in all experimental condition.

Table 10 : Housekeeping gene primer sequences.

 10

	unit)	40	Healthy ALS	SMA KD
	mRNA levels (arbitrary	0 10 20 30
		B 2 M	G A P D H	G U S	P 0	P P I A	P S M A 2

qPCR and relative gene expression analysis

	Primers		Sequence (5' → 3')
	Lamp1	Forward Primer CAGATGTGTTAGTGGCACCCA Reverse Primer TTGGAAAGGTACGCCTGGATG
	Lamp2	Fw Primer Rev Primer	GAAAATGCCACTTGCCTTTATGC AGGAAAAGCCAGGTCCGAAC
	CD81	Fw Primer Rev Primer	TTCCACGAGACGCTTGACTG CCCGAGGGACACAAATTGTTC
	CD63	Fw Primer Rev Primer	CAGTGGTCATCATCGCAGTG ATCGAAGCAGTGTGGTTGTTT
	CD82	Fw Primer Rev Primer	GCTCATTCGAGACTACAACAGC GTGACCTCAGGGCGATTCA
	SCARB2	Fw Primer Rev Primer	GGCCGATGCTGCTTCTACA GGTCTCCCCTCTGAGGATCTC
	EPDR1	Fw Primer Rev Primer	GTCCAGGAGTGGTCGGACA ACACCGAGGGGTCTTTAATACC
	VPS29	Fw Primer Rev Primer	TGCAACAGTTTGCCAGCTAAA CCTCTGCAACAGGGCTAAGC
	RAB11FIP1	Fw Primer Rev Primer	GGACAAGGAGCGAGGAGAAAT GTCGTGCTAGGGATGATGGC
	CD44	Fw Primer Rev Primer	CTGCCGCTTTGCAGGTGTA CATTGTGGGCAAGGTGCTATT
	RAB5b	Fw Primer Rev Primer	TCACAGCTTAGCCCCCATGTA CTTCACCCATGTCTTTGCTCG
	HLA-G	Fw Primer Rev Primer	GAGGAGACACGGAACACCAAG GTCGCAGCCAATCATCCACT
	TSG101	Fw Primer Rev Primer	GAGAGCCAGCTCAAGAAAATGG TGAGGTTCATTAGTTCCCTGGA
	FUS	Fw Primer Rev Primer	TGGTCTGGCTGGGTTACTTT TAACTGGTTGGCAGGTACGT

Table 11 : Exosomal marker and FUS primer sequences.

 11

Table 12 : Table summarizing experimental conditions and analysis performed on MN cultures treatment. Table 13 : Table summarizing experimental conditions and analysis performed on muscles cells treatment with exosomes.

 12

	See following page.

  , with some overlap between different experiments (See Supplementary Table2in manuscript). For each experiments conducted, samples were randomly picked-as we did not know at the time, the genetic results for the ALS patients.

	Secretion of neurotoxic vesicles by muscle cells of ALS patients MN cultures)Secretion of neurotoxic vesicles by muscle cells of ALS patients 66 Subjects Age at the time of the biopsie Gender ALS/FRS Genetic Muscle testing Site of onset 0=spinal; 1=Upper limb; 2=Lower limb; 3=bulbar; 4=respiratory	Duration of disease (months)	Histology	Electron microscopy	Cell culture	Transcriptome	Laura Le Gall Laura Le Gall Immunostaining cell culture RT-qPCR exosome treatment MN exosome treatment myotubes
		58	F	38	130	1	13
		67 M 45	146	2	25,2
		56 M 41	140	2	26,7
		59	F	39 C9orf72	146	3	16,5
		53	F	28 C9orf72	119	3	6
		61 M 38 ATXN2	130	2	11,3
		64 M 40	130	1	39,3
		70 M 37	126	3	48,3
		68 M 33	123	1	53,4
		76 M 32	104	1	28
		59	F	23	103	3	33
	24 ALS	61 M 40	97	1	11
	(35-76y: 58.54±8.28; 7F, 17M)	61 M 38	88	1	52
		53 M 31	94	2	40
		52 H	37	No data	0	101
		35	F	32	No data	0	85
		52	F	43	135	1	33,6
		49 M 41	111	2	44.1
		53 M 40	130	2	17,4
		57 M 37 C9orf72	145	3	20,6
		66 M 41	147	2	28.7
		51 M 36	139	2	12
		66 M 43	149	1	12
		58	F	40	98	3	14
		61 M
		75 M
		56	F
		64	F
		51 M
		52	F
		47	F
		59 M
		19 M
		59 M
		54 M
	24 Healthy	59	F
	(21-75y: 48.29 ± 17.56; 7F, 17M)	49	F
		53 M
		70 M
		21 M
		21 M
		53 M
		69	F
		66 M
		21 M
		33 M
		22 M
		25 M
	2 Polyneuritis	70 M				13
	(70 & 45 y; 1F, 1M)	45	F				8
		60 M 42 47 repeats	132	2	420
		43 M 44 48 repeats	148	2	60
		57 M 44 44 repeats	148	2	72
		71 M 35 45 repeats	136	2	324
	10 SBMA	45 M 39 50 repeats	134	2	168
	(43-71y: 58.18 ± 9.65; 10M)	71 M 40 41 repeats	149	1	372
		66 M 40 49 repeats	142	2	144
		56 M 36 46 repeats	121	2	324
		62 M 38 47 repeats	143	2	156
		64 M 37 45 repeats	138	2	108
		48 M 42	140	2	156
		36 M 41	106	2	276
	6 SMA-III/IV	61 M 40	122	2	588
	(30-61y: 42.5 ± 10.27; 1F, 5M)	45 M 41	124	2	324
		30 M 43	139	2	84
		35	F	44	142	2	384
								110 111

Table 14 : Table summarizing samples characteristics.

 14 Muscle biopsies were isolated from deltoid muscles. The table summarizes the age of onset, ALSFRS-R score, muscle testing results and genetics when applicable. Each sample is associated to every experimental procedure it has been used in.

Table 15 : Number of cells extracted per biopsy and proportion of muscle cells.

 15 Table summarizing total number of cells after each amplification step. Muscles cells represents approximatively 10.91% and are enriched by CD56 MACS sorting. exosomes are co-purified 384 . Moreover exosomes can easily aggregates and block the filtrate and may cause loss of vesicles 385 . For many years virus extraction are successfully performed using polymer-based isolation techniques 221,384,386,387 . Polymer-based exosomes extraction kit are now commercially available, but still raise controversy in the exosomes field. It has been demonstrated that polymer-based exosomes extraction from blood samples co purify contaminants such as immunoglobulins or larger proteins such as albumin 385 . Culture media is not as rich in contaminants as blood samples, and precipitated exosomes are labelled and washed in PBS multiple time before further application allowing withdrawal of any possible contaminants as illustrated on polyacrylamide gel from healthy and ALS exosomes extracted using LifeTechnologies extraction kit (See Figure supplemental 3F in paper). Important critics about the use of polymer-based extraction technique is based on the observation of residual gel matrix in exosomes pellet 388 . It is important to note that this study, contrary to ours is conducted on blood samples richer in contaminants to start with.

	Absolute purification of exosomes is not achievable, however several procedures allows concentration of
	exosomes, such as ultracentrifugation that is the used separation technique in exosomes field. If it is assume
	that low protein contaminant are isolated with exosomes during ultracentrifugation, the high speed
	(100,000g 213 to 140,000g 217,218 ) during ultracentrifugation can induce mechanical damage. During cell
	treatment it is primordial to keep the integrity of exosomes by avoiding damaging them. Importantly
	ultracentrifugation requires large volume of starting material and working with primary cells doesn't suit
	this requirement. Density-gradient separation is another well-known exosomes isolation procedure however
	only a sub-population of vesicles at a particular buoyant density is studied and in order to illustrate the
	heterogeneity of secreted vesicles in ALS patients we want to study the whole muscle exosome population.

It also require a first step to isolate total exosomes by ultracentrifugation, and again requires a large amount of cell culture media as described above. Size-exclusion chromatography or ultrafiltration allows the separation of exosomes depending on their size, exosomes as well as non-vesicular component sharing Authors declared that the extraction kit influences the uptake of exosomes, however approximatively same amount of vesicles were monitored in recipient cells treated either with exosomes extracted with the polymer-based kit or by ultracentrifugation, in accordance with other studies 389 . Importantly authors demonstrated that washing exosomes with PBS allows the removal of residual polymer 389 , and during our exosomes staining procedure, exosomes samples are washed several times in PBS suggesting that the extraction kit is washed away and can't interfere with the further applications.

  Table summarizing the number of genes dysregulated in different secretory compartments in ALS muscles, by re-analysis of data of Bakay et al (2006) and Pradat et al (2012) -referred to in the table as Ref 1 and Ref 2 respectively. The GEO repository accession number for Bakay et al. is GDS2855, and the ArrayExpress repository number for Pradata at al. is E-MEXP.3260The column '% expected' shows the percentage expected by chance based on the proportion of genes of that study's microarray chip that are annotated to the respective cell compartment. A Fisher's exact test was performed for each compartment to determine if it was significantly enriched (Fisher p value is given).

	Supplemental Table. S2
	66 Subjects	Ref 1 Gender ALS/FRS 58 F Age at the time of the biopsie 38	Ref Genetic n e g	As % Ref 1 Ref Muscle testing Site of onset 0=spinal; 1=Upper limb; 2=Lower limb; 3=bulbar; 4=respiratory 130 1	Duration of disease (months) 13	% expected Ref 1 Ref 2 Ref 1 Enrichment (Fisher test) Histology Electron microscopy Cell culture Transcriptome Immunostaining cell culture RT-qPCR exosome treatment MN exosome treatment myotubes Ref 2 x
		67 M 45 56 M 41	n e g n e g	2	146 140		2 2	2	25.2 26.7			x x	x x	x x
		59 F	39 C9orf72	146		3		16.5			x		x
		53 F	28 C9orf72	119		3		6		x		x		x	x
	Secreted	41 61 M 38 ATXN2 16 64 M 40 n e g	130 130	5.0	2 1	8.3 10.3 10.0 11.3 x 39.3 x	x x	-x x	x	x	-
		70 M 37	n e g		126		3		48.3			x	x	x	x
		68 M 33	n e g		123		1		53.4	x		x	x	x	x
	Secreted (not TM)	31 76 M 32 59 F 23	n e g n e g	13	104 103	3.8	1 3	6.6	28 33		9.0		8.8 x x	x x	-	x	-
		61 M 40	n e g		97		1		11				x	x
		61 M 38	n e g		88		1		52				x	x
	Golgi + secreted	3 53 M 31 52 H 37	n e g n e g	2	0.4 No data 94	2 0	1.0	40 101	0.2	x	0.2 x		-	x	x	0.078
		35 F	32	n e g		No data	0		85			x
		52 F	43	n e g		135		1		33.6	x
	Clathrin	0 49 M 41 53 M 40	n e g n e g	0	111 130	0.0	2 2	0.0	44.1 17.4	0.3		0.3 x x	x x	-x x	x	-
		57 M 37 C9orf72	145		3		20.6			x	x	x
		66 M 41	n e g		147		2		28.7			x	x	x
	Endosome	27 51 M 36 66 M 43	n e g n e g	8	139 149	3.3	2 1	4.0	12 12		2.0 x x		2.0	x	0.012	x	0.057
		58 F	40	n e g		98		3		14
	Lysosome		11		8		1.3		4.0			1.2		1.2		-	0.003
	Autophagosome		4		1		0.5		0.5			0.2		0.2		0.074	-
	All secretion-related
			75		28		9.6 15.5 14		13.6		-	-
	(union of above)

  Analysis of gene expression data -Expression data were uploaded to the GEO

	III/IV, and Healthy, were identified, and the top 50 ranked by fold-change in ALS -Housekeeping genes: Out of the 7 housekeeping genes listed in the table Rev
		v Healthy were uploaded to CellWhere (43). CellWhere was used to identify below and tested, Beta-2-microglobulin (B2M) showed a constant expression Primer GTTTTTTCTCAGTTGCTAATACCACA
		secretory-related subcellular localization annotations of proteins encoded by level in all samples, and was used to normalize the gene expression levels of
	these genes, and to visualize these proteins and their localizations on a schema exosomal markers. -Exosomal markers and FUS expression level: Quantitative PCR was
		of performed on LightCycler® 480 Instrument (Roche) using LightCycler® 480 the cell. For enrichment mapping, the GSEA tool Primers Sequence (5' → 3')
		(http://software.broadinstitute.org/gsea/index.jsp) was used to assess the Fw Primer CTCTCTTTCTGGCCTGGAGG DNA SYBR Green I Master (Roche). Primers used are listed in table below.
		distribution of gene sets across the differential expression profile of ALS β2M Rev
		compared to Healthy myotubes. 6,349 gene sets were tested, including all of Primer TGCTGGATGACGTGAGTAAACC
		the Gene Ontology Biological Process and Cellular Component collections, and Fw Primer AAGGTGAAGGTCGGAGTCAACGG
		repository at accession number GSE122261. Raw data (.CEL Intensity files) all of the Canonical Pathways from MSigDB. In addition, custom gene sets were GAPDH Rev
		were processed using R/Bioconductor packages. Briefly, sample quality was created listing genes encoding the known protein binding partners of FUS (31) Primer TGACAAGCTTCCCGTTCTCAGCC
		verified by assessment of MA plots, normalized unscaled standard error and TDP43 (32). Cytoscape v3 and the enrichment map plugin were used to Fw Primer CTCATTTGGAATTTTGCCGATT
		(NUSE; all samples had median <1.1), and relative log expression (RLE; all create a graph representing as nodes the gene sets identified by GSEA to be GUS Rev
		sample had divergence <0.2), using the oligo package. Background-corrected significantly enriched with FDR < 0.05, with edges shown for those pairs of Primer CCGAGTGAAGATCCCCTTTTTA
		normalized log2-transformed probe set signal intensities were obtained by gene sets having overlap coefficient >0.5. Cytoscape's selection features were Fw Primer TGATAGATCCATTCCTATGACTGTAGA
		Robust Multi-array Averaging (RMA) using default settings. Affycoretools and used to isolate a sub-graph of the enrichment map showing only the FUS and HPRT1 Rev
		huex10stprobeset.db were used to annotate probeset IDs. For exon-level Primer TDP43 binding gene sets and those gene ontology or canonical pathway gene CAAGACATTCTTTCCAGTTAAAGTTG
		analysis, probesets were retained if oligo's paCalls function declared them sets with which they shared genes (overlap coefficient >0.5). Fw Primer TCCAGGCTTTAGGTATCACCAC
	-	present with p<0.05 in all samples of at least one experimental group. For gene-P0 (RPL0) Rev
	level analysis, probesets were retained if they had expression level >log250 in Quantitative RT-qPCR Primer GCTCCCACTTTGTCTCCAGTC
	at least 4 of the 23 samples. A design matrix (~0 + condition; condition = ALS, -cDNA synthesis: RNA from 3 days differentiated muscle cells was extracted Fw Primer CCTAAAGCATACGGGTCCTG
		SBMA, SMA, or Healthy) was created, to which Limma was used to fit a linear as described above. 1 ug of RNA was used to synthesise cDNA using M-MLV PPIA Rev
		model to the normalized expression values, and differentially expressed genes Primer Reverse Transcriptase (LifeTechologies™). Quantitative PCR was performed TTTCACTTTGCCAAACACCA
		were identified using Limma's empirical Bayes method. Genes that were on LightCycler® 480 Instrument (Roche) using LightCycler® 480 DNA SYBR PSMA2 Fw Primer CATTCAGCCCGTCTGGTAAA
		dysregulated in the same direction in all comparisons of ALS v SBMA, SMA-Green I Master (Roche).

  Smirnov test was used to compare the distribution of number of vesicles per MVBs in ALS and healthy myotubes, the distribution of neurites branching in ALS and healthy exosomes-treated MN, the distribution of H2AX expression levels in myonuclei treated with different doses of exosomes, and the distribution of myotube nuclear numbers in control myotubes treated with ALS and control exosomes. One-way ANOVA followed by a Dunnet's multiple comparison test was used to evaluate the differences in the in silico secretome, in the immunostaining for exosomal markers, RT-qPCR, neurites length, and MN treatments. Two-way ANOVA was used to evaluate changes in bleb counts, and cell death in different cells lines (Cont, FUSFLAG, SOD1FLAG, TDP43FLAG).Differences were considered to be statistically different at P < 0.05.

	TM ) and resuspended in lysis buffer (8M urea; 2% SDS; 10 µL/mL protease inhibitor cocktail). Extracted proteins were loaded into NuPage polyacrylamide 4-12% BisTris gels for electrophoresis under reducing conditions (Calnexin, Flotillin) and non-reducing conditions (CD63). Transfer on polyvinylidene difluoride (PVDF) membrane was performed using the iBlot® Dry Blotting System (Life Technologies TM ) and upon transfer polyacrylamide gels were stained with Blue Coomassie Gel Code Blue Stain Reagent (LifeTechnologies TM ) to visualize proteins. Immunoblotting was carried out using the iBind TM Flex Western System and primary antibodies (Calnexin, CD63, Flotillin and RPL5 see Table S3) with respective secondary antibodies (Goat anti Mouse HRP, Donkey anti Goat HRP, Goat anti Rabbit HRP). The signal was detected using the Amersham ECL TM Prime Western blotting Detection Reagent and the UVP ChemiDoc-It 2 Imager. Statistics All values are presented as means ± SEM. Student's T-Test was used to compare differences between ALS and control samples for all the protein quantifications, electron microscopy quantifications, exosome integration, immunostaining (FUS, Acridine Orange, RPL5, Caprin1), FUS expression level, exosome-treated MN and myotube death, myotube atrophy. A Kolmogorov-Secretion of neurotoxic vesicles by muscle cells of ALS patients Laura Le Gall
	172

  formation of D-loops, essential for homologous recombination and DNA damage response 410 . Interestingly, the treatment of healthy muscle cells with ALS exosomes have a high level of H2AX (see Figure supplemental 4C & 4E). Altogether these results suggests that the treatment with ALS exosomes might influence the DNA damage response, contributing to the dysregulation of RNA processing and transport observed in ALS patients. It is noteworthy that ALS exosome treatment allows the reproduction of ALS pathogenesis observed in patients and might represent a good ALS model. Importantly this PhD study was based on sporadic ALS

patients samples (with known and unknown gene mutations) suggesting a common toxic pathway in ALS muscle stem cells.

  Laura Le Gall1,2 , William J Duddy1 , Sylvain Roquevière 3 , Mariot Virginie4 , Blandine Madji Hounoum5 , Jeanne Lainé 2 , Romain Joubert 6 , Julie Dumonceaux 4 , Pascal Leblanc 7 , Gisele Ouandaogo 2 , Laura Robelin 8 , Franscesca Ratti 7 , Alexandre Mejat 7 , Gillian Butler Browne 2 , Jean Philippe Loeffler8 , Anne Cecile Durieux 9 , Jose-Luis Gonzales De Aguilar 8 , Helene Blasco5 , Cecile Martinat 3 , Pierre Francois Pradat 10* , Stephanie Duguez 1* Northern Ireland Center for Stratified Medicine, Biomedical Sciences Research Institute, Londonderry, UK. 2-Myologie Centre de Recherche, Université Sorbonne, UMRS 974 UPMC, INSERM, FRE 3617 CNRS, AIM, Paris, France. 3-I-Stem, INSERM/UEVE UMR 861, I-STEM, AFM, Evry, France. 4-NIHR Biomedical Research Centre, University College London, Great Ormond Street Institute of Child Health and Great Ormond Street Hospital NHS Trust, London, UK. 5-Université François-Rabelais, INSERM U930 "Imagerie et Cerveau", CHRU de Tours, 10 Bv Tonnellé, 37044 Tours, France. 6-Unité Physiologie et Pathologie Moléculaires des Rétrovirus Endogènes et Infectieux, CNRS UMR 9196, Institut Gustave Roussy, Villejuif, France. 7-Laboratory of Molecular Biology of the Cell, Ecole Normale Supérieure de Lyon, Lyon, France. 8-Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg, INSERM U1118, Strasbourg, France. 9-LPE, Université Jean Monnet de Saint Etienne, Faculté de Médecine Jacques Lisfranc, Saint Etienne, France. 10-Département des Maladies du Système Nerveux, Hôpital de la Pitie-Salpetriere, Paris, France.

*Co-last authors 1-

Material and methods

Acknowledgements

I would like to express my deepest gratitude to my supervisor Stéphanie Duguez. You've made this PhD journey so much better by always being patient and supportive, no matter the circumstances. I can't thank you enough for sharing your passion about science and research, but above all to trust me to complete this project. I could not have asked for anything else during all this time, and for that I'll be forever grateful. Alors Stéphanie, merci infiniment pour tout ce que tu as fait pour moi depuis le début de cette aventure en 2015. Merci de m'avoir transmis toutes ces connaissances, et merci pour tout le

Acknowledgments:

We thank the Human Cell Culture Platform of The Institute of Myology, and the "Plateforme Biopuces et Séquençage de l'IGBMC". This work was financed by Target-ALS (ViTAL consortium, PI: S Duguez), ARsLA (TEAM consortium, PI: S Duguez ; PULSE, PI: D Devos), European Union Regional Development Fund (ERDF) EU Sustainable Competitiveness Programme for N. Ireland, Northern Ireland Public Health Agency (HSC R&D) & Ulster University (PI : T Bjourson). LLG is a recipient from ArSLA. It was also partially supported by European Community's Health Seventh Framework Programme under grant agreement No. 21 259867 (Euro-MOTOR), INSERM, Sorbonne University, and the AFM. The study was sponsored by APHP.

The authors declare that they have no conflict of interest. SD, LLG, SR, VM, and UV performed the myoblast and motor neuron cell cultures and measurements, SK performed the nanosight and proteomic analysis on exosomes. JLGDA generated the transcriptome data on differentiated myoblasts. WJD performed the computational analysis, JL performed the electron microscopy analysis, SM performed genetic analysis. PB, AB, HB, GB, MDMA, DD, ACD, DFr, AH, AdHes, LL, PL, TL, PLeb, NLF, TM, VM, AM, GO, FR, LR, FS, TS, GQ and PFP generated the samples, and had some input into data generation. SD with the help of WJD took the lead in interpretation of results and writing the manuscript. CM, SK, JD, JLGDA, CR, GBB, OL discussed the results and provided critical feedback on the manuscript. SD and PFP conceived the original idea. SD supervised the project.

ACKNOWLEDGMENTS

This work was financed by the EU FP7 Programme project MYOAGE (contract HEALTH-F2-2009-223576), the ANR Genopath-INAFIB, the AFLD, the CNRS, INSERM, the University Pierre and Marie Curie Paris 6 and the AFM (Association Franc ¸aise contre les Myopathies).

Supplementary figures:

. Each circle represents a compartment related to a secretory pathway. The size of each circle is proportional to the number of genes related to each compartment. Red colour indicates that the gene expression of the compartment is altered in ALS muscle compared to healthy (* and **, P<0.05 and P<0.01, respectively). See Table S1A for more details.

CONFLICTS OF INTEREST

All authors declare no conflicts of interest. Methods:

Cont

To explore this in a way that is independent and upstream of muscle denervation we used primary muscle stem cells extracted from deltoid muscles biopsies from sporadic ALS patients (n=18) and aged-matched healthy subjects (n=21). After differentiating the cells for 3 days, exosomes were extracted from the culture medium. Exosomal proteins were extracted using 8M Urea buffer and quantified using the BCA Protein Assay. Equal amounts of PKH26-labelled ALS and healthy exosomes were added to the culture medium of healthy muscle cells and IPS-derived motor neurons. We created healthy muscle cell lines expressing tagged form of wild type FUS (FLAG-FUS), TDP43 (FLAG-TDP43) or SOD1 (FLAG-SOD1).

Results:

ALS exosomes induce an increased cell death when added to the culture medium of healthy human muscles and IPS-derived motor neurons. A consistently greater cell death was observed when muscle cells were treated with ALS exosomes -no matter the amounts of exosomes loadedsuggesting that both quantity and quality of exosomes are important for the toxicity of ALS exosomes.

Recently, we detected the presence of FUS in exosomes. To further investigate the role of exosomes on the FUS pathway, all FLAG-FUS/TDP43/SOD1 cell lines were treated with equal amounts of exosomes. ALS exosomes induced greater cell stress and cell death than healthy exosomes, in all cell lines, but the effect was markedly greater in FLAG-FUS cell line. Interestingly, FUS distribution is also altered in ALS muscle cells.

Discussion:

Together, these data demonstrate that the muscle cells from sporadic patients secrete toxic agents -for muscle cells and motor neurons -through their exosomes. The increased toxicity of ALS exosomes with FLAG-FUS cell line is suggesting that ALS exosomes toxicity is exacerbated in the presence of FUS overexpression. Moreover, results are showing that FUS function is impacted in ALS muscle cells. We are now further exploring the impact of ALS muscle exosomes with the FUS pathway.

Subject samples table

• INSERM &Direction Générale de l'Organisation des Soins (DGOS)

• the Association Française contre les myopathies (AFM),

• Target-ALS association and