WO2015068075A2 - Detection of tau - Google Patents

Abstract

The present invention relates to an in vitro method for quantifying Tau protein, or a nucleic acid encoding Tau protein, in an individual, comprising the step of measuring an amount of Tau protein, or an amount of a nucleic acid encoding Tau protein, in an Ectosome-Enriched Fraction (EcEF) from said individual.

Description

DETECTION OF TAU

FIELD OF THE INVENTION

The invention relates to methods for detecting Tau protein or a nucleic acid encoding the same in an individual. The invention further relates to methods for detecting the occurrence of a Tau-related disease and for determining the efficacy of a treatment of a Tau-related disease in an individual.

BACKGROUND OF THE INVENTION

The abundant and abnormal accumulation of the hyperphosphorylated microtubule-associated protein, Tau, is a pathological feature of the neurodegenerative diseases collectively referred to as tauopathies (Buee et al, 2000).

From a pathological point of view, Tau proteins accumulate into distinguishes several intracellular inclusions, with the most common neuronal lesions being neurofibrillary tangles ( FTs), in which Tau aggregates into filaments. This neurofibrillary degeneration ( FD) is a slow process that lasts more than 20 years (Braak et al, 2011) and progresses from the pretangle to the ghost tangle stage in tauopathies such as Alzheimer disease (AD) (Augustinack et al., 2002), the most common sporadic tauopathy.

The sequence of pathological events leading to NFD follows reproducible loco- regional patterns of expression that differ between sporadic tauopathies such as AD (Braak et al., 1991; Delacourte, 1999), Progressive Supranuclear Palsy (PSP) (Verny et al, 1996) or Argyrophilic Grain Disease (AGD) (Saito et al, 2004). In AD for instance, Tau pathology first occurs in the locus cceruleus (Braak et al., 2011) and the trans-entorhinal cortex (Braak et al., 1991) followed with time by the successive involvement of other brain regions namely the hippocampus, polymodal and unimodal association cortical areas and finally secondary and primary cortical regions. Conversely, in rare hereditary Tauopathies such as FTDP-17 (Fronto-Temporal Dementia with parkinsonism linked to chromosome 17) mutations are found on the Tau gene. These mutant Tau forms rapidly aggregate and cause neurodegeneration. In these disorders, all neurons may have the mutant Tau form, which may then cause damage without spreading.

For many years, this sequence in the neurodegeneration process in sporadic tauopathies, or Tau-related disorders, was incriminated to a passive release of Tau in the extracellular space (ISF) during neuron death disrupting interneuronal connexions and leading to the degeneration of the neighboring neurons. There is now growing body of evidences that before cell death occurs, earlier events induce intracellular damages (Van der Jeugd et al, 2011; Mohamed et al, 2013). These data, generated from murine model of tauopathies, associated with the presence of Tau in human body fluids, have suggested a new concept of active Tau secretion.

It is now proposed that extracellular forms of Tau and mutant Tau (such as hTau46^{P 01L}) are responsible for autosomal dominant forms of frontotemporal lobar degeneration. To date, forty-six pathogenic mutations of the MAPT gene have been described, with a clinical phenotype of frontotemporal dementia, language disturbance or atypical parkinsonism (see also http://www.molgen.ua.ac.be/FTDmutations/) that could play a major role in the spatiotemporal evolution of FD (Clavaguera et al, 2009) and that could act on vulnerable neurons that form part of a circuit through a trans-synaptic mechanism (Liu et al, 2012; de Calignon et al., 2012). Mechanisms behind this process are subject of intense research but are still yet unknown. In particular, neither the function nor the mechanisms of Tau release into the interstitial fluid (ISF) or the cerebrospinal fluid (CSF) are yet understood.

WO2011/094645 teaches that extracellular forms of Tau may be detected in exosomes. This document further teaches a method for detecting Tau isoforms, which comprises a step of purification of the exosome fraction from a biological sample.

Exosomes relate to small vesicles, generally ranging from 50 to about 90 nm in diameter, which are secreted by eukaryotic cells, and which may contain a wide variety of proteins including, for example, proteins whose secretion correlates with various pathological states.

It is emphasized that the link between the presence of Tau in exosomes and the exosomal vesicular pathway is still a matter of debate, and the mechanism appears to be more complex, since non-vesicular "free" full-length Tau may also be found in the extracellular space (Karch et al, 2012; Chai et al., 2012). In line with this, other authors now even argue that exosome-related mechanisms may actually not be involved in the release of Tau (Santa-Maria et al, 2012).

Thus there remains a need for alternative methods for detecting Tau protein in an individual. There also remains a need for novel methods for detecting the occurrence of a Tau-related disease in an individual.

There also remains a need for novel methods for determining the efficacy of a treatment of a Tau-related disease in an individual.

SUMMARY OF THE INVENTION

The present invention has for purpose to meet these aforementioned needs.

Unexpectedly, the inventors have found that Tau is actively secreted in the extracellular media through non-exosomal pathways, and more particularly through ectosomal pathways. In particular, they have characterized the presence of Tau in those ectosomes (also known as microparticles), in cell lines, primary culture but also non- human primate cerebrospinal fluid and plasma. In particular, they have found that Tau may be detected on Ectosomes and/or an Ectosome-Enriched Fraction (EcEF).

Ectosomes should not be confused with Exosomes, as both extracellular compartments have distinct functional and structural properties.

Indeed, Ectosomes are vesicles which may range from 100 to 1000 nm in size, and which are formed directly by budding out of the plasma membrane, towards the extracellular compartment.

This result was completely unexpected for the following reasons: (i) because neither the function nor the mechanisms of Tau release into the intersitial fluid (ISF) or cerebrospinal fluid (CSF) are yet understood, and (ii) because the presence of extracellular Tau had been originally linked to the exosomes and the exosomal vesicular pathway, which relate to extracellular compartments and biological mechanisms which are completely distinct from ectosomes.

These results have allowed the inventors to design methods for detecting or for quantifying Tau protein, or a nucleic acid encoding Tau protein, from a biological sample containing ectosomes, which includes Ectosome-Enriched Fractions.

The invention relates to an in vitro method for quantifying Tau protein, or a nucleic acid encoding Tau protein, in an individual, comprising the step of measuring an amount of Tau protein, or an amount of a nucleic acid encoding Tau protein, in an Ectosome-Enriched Fraction (EcEF) from said individual. Thus, a first aspect of the invention is to provide an in vitro method for detecting Tau protein, or a nucleic acid encoding Tau protein, in an individual, comprising the steps of:

a) providing a biological sample from said individual, wherein said biological sample is susceptible to contain at least one Ectosome,

b) isolating from said biological sample an Ectosome-Enriched Fraction, c) detecting the presence of Tau protein, or of a nucleic acid encoding Tau protein, in said Ectosome-Enriched Fraction.

Another aspect of the invention is to provide an in vitro method for quantifying Tau protein, or a nucleic acid encoding Tau protein, in an individual, comprising the steps of:

a) providing a biological sample from said individual, wherein said biological sample is susceptible to contain at least one Ectosome,

b) isolating from said biological sample an Ectosome-Enriched Fraction, c) measuring an amount of Tau protein, or of a nucleic acid encoding Tau protein, in said Ectosome-Enriched Fraction.

Cerebrospinal Fluid (CSF) total-Tau and phosphorylated-Tau are now validated biomarkers of Alzheimer disease (AD). Their levels reflect the presence of neurofibrillary degenerative lesions in the brain from neuropathological studies.

Thus, the discovery of distinct secreted/non secreted Tau species opens new perspectives in diagnosis of neurodegenerative diseases. Deciphering the exact nature of Tau species associated to membrane vesicles involved in secretion mechanisms are new promising pathways to discover biomarkers of very early diagnosis of AD and other Tauopathies. Tauopathies may be referred herein as Tau-related diseases or Tau-related disorders.

Thus, the invention further relates to an in vitro method for detecting the occurrence of, or the risk of occurrence of, a Tau-related disease in an individual, comprising the steps of:

a) measuring an amount of Tau protein in an EcEF from said individual, b) comparing the amount of Tau protein measured at step a) with a reference value, c) determining from the comparison performed at step b) the occurrence of the Tau-related disease in said individual.

This invention also relates to an in vitro method for detecting the occurrence of, or the risk of occurrence of, a Tau-related disease in an individual, comprising the steps of:

a) measuring an amount of a nucleic acid encoding Tau protein in an EcEF from said individual,

b) comparing the amount of nucleic acid encoding Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the occurrence of, or the risk of occurrence of, the Tau-related disease in said individual. Targeting the clearance of specific Tau species that are rapidly secreted into the interstitial fluid during the pathological process should also be considered as new strategies to slow-down the spreading of Tau, and thus the appearance or prognosis of Tau-related diseases.

Thus, according to a third aspect, the invention further relates to an in vitro method for determining the efficacy of a treatment of a Tau-related disease in an individual, comprising the steps of:

a) measuring an amount of Tau protein in an EcEF from said individual, b) comparing the amount of Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the efficacy of said treatment in said individual.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Ectosomal and exosomal purification: Experimental procedures- Media coming from embryonic primary cultures (DIV 10, 5 flasks (25 cm²)) or hTau- overexpressing N1-E115 cell lines (48 hours post-differentiation, 4 flasks (75 cm²)) were processed by several centrifugations steps to isolate EcEF (ectosomes enriched fraction, pellet 1) or ExEF (exosomes enriched fraction, pellet 2). The exosomes were purified from the ExEF using a continous sucrose gradient. Figure 2: Characterisation of ectomal and exosomal fractions from rat primary embryonic cortical cells - Once purified from rat primary embryonic cortical cells (DIV 10), vesicles coming from EcEF (a, left panel) or ExEF (a, right panel) were observed by electron microscopy. Scale bar is indicated on the figure, (b) A semi-quantitative analyze was done to determine the percentage of vesicles in EcEF (size>100 nm) and ExEF (30 nm <size<70 nm). n=3 individual experience, at least 200 vesicles have been quantified per experience. Results are indicated at the top of the histogramm. (c) Exosomes were fractionated from ExEF. The association of two exosomal markers (Alix and Flotilin-1) was analyzed by western-blotting in cell lysate (CL) and fractions coming from a continuous sucrose gradient. The expected exosomal density is indicated on the figure (1.08<d<1.22) (Raposo et al., 1996).

Figure 3: Endogenous Tau is release from rat primary embryonic cortical cells in a vesicular but non-exosomal pathway: the ectosomes- (a) The presence of endogenous Tau in cell lysate (CL), EcEF and ExEF coming from primary embryonic cortical cells (10 DIV) were analyzed by western-blotting using a rabbit antibody against the C-terminal portion of Tau (Tau C-Ter). (b) EcEF (left panels) and ExEF (right panels) coming from primary embryonic cortical cells (10 DIV) were immunolabelled with antibodies against the C-terminal (C-Ter, lower panels) or the N-terminal part (M19G, upper panels) portion of Tau and the presence of Tau revealed using an 18 nm gold colloidal goat anti-rabbit antibody. Direct association of Tau to vesicles was observed by electron microscopy. Scale bar is indicated on the figure, (c) The percentage of positive vesicles was estimated by electron microscopy and is indicated on the top of the histogram. n=3 individual experience, at least 200 vesicles have been quantified per experience. Statistical analyzes were done using the chi² test (*** p<0.001).

Figure 4: Tau secretion is an active mechanism that is not related to cell damage- Primary embryonic cultures were plated and maintained in culture 3, 10 or 15 days (3 DIV, 10 DIV or 15 DIV) (a). Differentiation process was initiated in stable N1-E115 overexpressing hTau46^WT of SEQ ID NO 3 or native N1-E115 cells using serum free media for 48 hours (b). The conditioned media were analyzed for LDH activity and expressed as percentage of LDH release. The statistical analyzes were done using a Mann and Whitney test (NS: non significant), (c). Western blot analysis of EcEF and ExEF coming from primary embryonic cultures, 3, 10 or 15 days after plating. Figure 5: Characterisation of ectomal and exosomal fractions from cell lines- Once purified from stable N1E-115 overexpressing hTau46^WT cells, vesicles coming from EcEF (a, left panels) or ExEF (a, right panels) were observed by EM. Scale bar is indicated on the figure, (b) A semi-quantitative analyze (n=100 vesicles) was done to determine the percentage of vesicles in EcEF (size>100 nm) and ExEF (30 nm <size<70 nm). Results are indicated at the top of the histogram, (c) Exosomes were fractionated from ExEF. The association of two exosomal markers (Alix and Flotilin-1) was analyzed by western- blotting in cell lysate (CL) and fractions coming from a continuous sucrose gradient. The expected exosomal density is indicated on the figure (1.08<d<1.22) (Raposo et al., 1996). Figure 6: A slight part of Tau is shifted to the classical secretory pathway when overexpressed in cells- (a) The presence of endogenous Tau in cell lysate (CL), EcEF and ExEF coming from differentiated stable N1E-115 overexpressing hTau46^WT cells were analyzed by western-blotting using a rabbit antibody against the total Tau (HT7), the N- terminal portion of Tau (Tau N-Ter) or the C-terminal portion of Tau (C-ter). Exosomes were purified from ExEF coming from conditioned media of differentiated stable NlE-115 overexpressing hTau46^WT (b, upper panel) or native N1-E115 (b, lower panel) cells using continuous sucrose gradient. The presence of Tau was analyzed using anti-total Tau (HT7). The expected exosomal density is indicated on the figure (1.08<d<1.22) ¹. EcEF (c, left panels) and ExEF (c, right panels) coming from stable N1E-115 overexpressing hTau46^WT cells were immunolabelled with antibodies against the C-terminal portion of Tau (N-Ter, upper panels), the C-terminal portion of Tau (C-Ter, middle panels) or a human specific total Tau (ADx215, lower panels). The presence of Tau was revealed using an 18 nm gold colloidal goat anti-mouse or anti rabbit antibody. Direct association of Tau to vesicles was then observed by electron microscopy. Scale bar is indicated on the figure (d) the percentage of positive vesicles was estimated by electron microscopy and is indicated on the histogram. n=l to 3 individual experience as indicated on the figure, at least 200 vesicles have been quantified per experience. Statistical analyzes were done using the chi² test (** pO.Ol, *** pO.001).

Figure 7: Tau is secreted in ISF-associated ectosomes & exosomes originating from lipid raft microdomains when overexpressed in the rat brain- Rats were stereotactically injected with LV-hTau46^WT (n=2) into the hippocampus. Five months p.L, ISF samples were collected by push pull microdialysis from the injection site to purify EcEF (left panels) and ExEF (right panels) and proceed for electron microscopy using an antibody against the N-terminal portion of Tau (N-Ter, upper panels), an antibody against the GMl (GMl, lower panels) or an antibody against the C-terminal portion of Tau (C-Ter, lower panels). The scale bar is indicated on the figure.

Figure 8: Tau is secreted in CSF and plasma-associated ectosomes & exosomes when overexpressed in the brain of non human primates- Primates were stereotactically injected with AAVs-hTau46^WT (n=l) or AAVs-hTau46^{P 01L} (n=l) into the hippocampus.

^~ry T P 301 L

Three months post-AAVs-hTau46 (upper panels) or AAVs-hTau46 (lower panels) delivery, CSF and plasma samples were collected to purify EcEFs (left panels) and ExEFs (right panels) from CSF (a) or plasma (b) and proceed for electron microscopy using antibody recognizing the C-terminal part of Tau. (c) The percentage of positive vesicles was estimated by electron microscopy and is indicated on the histogram. n=l individual experience, at least 200 vesicles have been quantified per experience.

Figure 9: Tau mRNAs are retrieved in ectosomes and exosomes-enriched fractions- After mRNA purification and reverse transcription, the presence of Tau mRNAs in EcEF and ExEF coming from hTau46^WT-overexpressing neuroblastoma cell lines (NlE-115) was analyzed by PCR. The upper panel shows human Tau PCR and lower panel shows the ribosomal 18s RNA control. The "Positive" lane refers to RNA extract from brain lysate of a rat expressing human Tau46^WT. The "Negative" lane refers to native NlE-115. Figure 10: Tau is inside the vesicles-

EcEF (upper panel - Figure 10A) and ExEF (lower panel - Figure 10B) obtained from stable N1E-115 overexpressing hlN4R cells (left part of the immunoblots) or from naive N1E-115 (right part of the immunoblots) were incubated with growing concentrations of NaCl (0.01 to 0.5M) before western blotting analyses using a total Tau N-ter antibody and a flotillin-1 antibody. EcEF and ExEF obtained from naive N1E-115 were previously incubated with recombinant hlN4R Tau. Cell lysates (CL) from both cell lines and recombinant hlN4R Tau were used as controls.

DETAILED DESCRIPTION OF THE INVENTION

Without wishing to be bound by any particular theory, the inventors are of the opinion that Tau is a soluble cytoplasmic protein that should not be addressed to the classical secretory endoplasmic reticulum-golgi secretory pathway. Indeed, the inventors are of the opinion that the association of Tau to the plasma membrane (Brandt et al, 1995; Kempf et al, 1996; Shea et al, 1996) is in favor of a direct vesicle shedding from specific microdomains that may be part of an active secretion process. The positioning of Tau in detergent-resistant membrane microdomains that is regulated by Fyn supports this hypothesis.

Indeed, Fyn associates to Tau into dendritic intracellular vesicles (Lee et al, 2012), influences its localization to the plasma membrane by phosphorylation at tyrosine 18 (Usardi et al, 2011). In this context, Ectosomes originating from lipid raft cell membrane (Davizon et al., 2010; Piccin et al, 2007) may drive the secretion of Tau from the neuron to the extracellular space.

Further, without wishing to be bound by any particular theory, the inventors believe that beyond containing Tau protein, ectosomes also contain nucleic acids encoding Tau protein, and more specifically messenger RNAs encoding Tau protein.

In view of the above, the invention relates to an in vitro method for detecting Tau protein, or a nucleic acid encoding Tau protein, comprising a step of measuring an amount of Tau protein, or an amount of nucleic acids encoding Tau protein, in a sample susceptible to contain one or more Ectosomes. To the knowledge of the inventors, Tau protein, including its isoforms, had never been reported or even measured specifically in Ectosomes, so far. Also, to the knowledge of the inventors, nucleic acids encoding Tau protein, especially mRNAs encoding Tau protein, have never been reported or even measured specifically in Ectosomes so far. The invention relates to an in vitro method for quantifying Tau protein, or a nucleic acid encoding Tau protein, in an individual, comprising the step of measuring an amount of Tau protein, or an amount of a nucleic acid encoding Tau protein, in an Ectosome-Enriched Fraction (EcEF) from said individual.

In view of the above, the invention further relates to an in vitro method for quantifying Tau protein in an individual, comprising the step of measuring an amount of Tau protein in an Ectosome-Enriched Fraction (EcEF) from said individual.

According to the invention, methods for « quantifying?) may, in some embodiments, encompass methods for « detecting ).

Thus, the invention also relates to an in vitro method for detecting Tau protein in an individual, comprising the steps of:

a) providing a biological sample from said individual, wherein said biological sample is susceptible to contain one or more Ectosomes,

b) obtaining an Ectosome-Enriched Fraction from said biological sample, c) detecting the presence of Tau protein in said Ectosome-Enriched Fraction. This invention also relates to an in vitro method for detecting Tau protein in an individual, comprising the steps of:

a) providing an Ectosome-Enriched Fraction from said individual, b) detecting the presence of Tau protein in said Ectosome-Enriched Fraction. This invention also relates to an in vitro method for detecting Tau protein in an individual, comprising the steps of:

a) providing one or more Ectosomes from said individual,

b) detecting the presence of Tau protein in the said one or more Ectosomes. This invention also relates to an in vitro method for detecting Tau protein in an individual, comprising the step of detecting the presence of Tau protein in an Ectosome- Enriched Fraction from said individual.

Thus, the invention relates to an in vitro method for quantifying Tau protein in an individual, comprising the steps of:

a) providing a biological sample from said individual, wherein said biological sample is susceptible to contain one or more Ectosomes,

b) obtaining an Ectosome-Enriched Fraction from said biological sample, c) measuring the amount of Tau protein in said Ectosome-Enriched Fraction.

This invention also relates to an in vitro method for quantifying Tau protein in an individual, comprising the steps of:

a) providing one or more Ectosomes from said individual,

b) measuring an amount of Tau protein in the said at least one Ectosome.

This invention also relates to an in vitro method for quantifying Tau protein in an individual, comprising the step of measuring the amount of Tau protein in an Ectosome- Enriched Fraction from said individual.

Thus, the invention relates to an in vitro method for detecting a nucleic acid encoding Tau protein in an individual, comprising the steps of:

a) providing a biological sample from said individual, wherein said biological sample is susceptible to contain one or more Ectosomes,

b) obtaining an Ectosome-Enriched Fraction from said biological sample, c) detecting the presence of a nucleic acid encoding Tau protein in said Ectosome-Enriched Fraction.

This invention also relates to an in vitro method for detecting a nucleic acid encoding Tau protein in an individual, comprising the steps of:

a) providing an Ectosome-Enriched Fraction from said individual, b) detecting the presence of a nucleic acid encoding Tau protein, in said Ectosome-Enriched Fraction.

This invention also relates to an in vitro method for detecting the presence of a nucleic acid encoding Tau protein in an individual, comprising the steps of:

a) providing one or more Ectosomes from said individual,

b) detecting the presence of a nucleic acid encoding Tau protein in the said one or more Ectosomes.

This invention also relates to an in vitro method for detecting a nucleic acid encoding Tau protein in an individual, comprising the step of detecting the presence of a nucleic acid encoding Tau protein in an Ectosome-Enriched Fraction from said individual.

Thus, the invention relates to an in vitro method for quantifying a nucleic acid encoding Tau protein in an individual, comprising the steps of:

a) providing a biological sample from said individual, wherein said biological sample is susceptible to contain one or more Ectosomes, b) obtaining an Ectosome-Enriched Fraction from said biological sample, c) measuring the amount of a nucleic acid encoding Tau protein in said Ectosome-Enriched Fraction.

This invention also relates to an in vitro method for quantifying a nucleic acid encoding Tau protein in an individual, comprising the steps of:

a) providing an Ectosome-Enriched Fraction from said individual, b) measuring the amount of a nucleic acid encoding Tau protein, in said Ectosome-Enriched Fraction.

This invention also relates to an in vitro method for quantifying a nucleic acid encoding Tau protein in an individual, comprising the steps of:

a) providing one or more Ectosomes from said individual,

b) measuring the amount of a nucleic acid encoding Tau protein in the said one or more Ectosomes.

This invention also relates to an in vitro method for quantifying a nucleic acid encoding Tau protein in an individual, comprising the step of measuring the amount of a nucleic acid encoding Tau protein in an Ectosome-Enriched Fraction from said individual.

In the method embodiments wherein one or more Ectosomes are provided for the purpose of detecting or quantifying Tau protein, or a nucleic acid encoding Tau protein, the said one or more Ectosomes may be provided either directly in a purified form, or as part of an Ectosome-Enriched Fraction.

The step of obtaining an Ectosome-Enriched Fraction from a biological sample may be achieved either directly on a "raw" biological sample from said individual, or after a first step of enrichment without departing from the teaching and scope of the invention.

A « biological sample » refers to any sample deriving from a biological material that is susceptible to contain one or more Ectosomes.

Thus, a biological sample that is used in a method according to the invention may be a complex sample that is susceptible to comprise one or more Ectosomes, and which may further comprise other vesicles such as Exosomes or Apoptotic bodies. Depending on the level of purity and/or the origin of the starting material, this biological sample may also comprise other contaminants, such as living or dead cells, in particular cell debris or cell « breakdown » products, without departing from the scope of the invention.

In some embodiments, a biological sample that is used for performing a method as described herein may be selected in a list comprising: a cell lysate, a cell culture supernatant, a cerebrospinal fluid sample, a blood sample or an interstitial fluid sample. According to the invention, a "blood sample" refers either to whole blood, or to a blood- derived fraction that is obtained following a fractionation step. Thus, a blood sample may include plasma or serum, and more particularly plasma.

According to the invention, the expression « an individual » includes a human or a non-human mammal, which also includes a primate or a rodent. As used herein, the expression "an individuaF preferably means a human mammal.

According to the invention, the expression « vesicle » includes any extracellular compartment that is derived from a cellular membrane, such as an Exosome, an Ectosome or an Apoptotic Body.

In a non-limitative manner, the expression « derived from a cellular membrane » may indicate that the extracellular compartment membrane is formed (i) directly by a budding-out mechanism directly at the plasma membrane (i.e. Ectosomes), (ii) indirectly by fusion of an endosomal compartment at the plasma membrane (i.e. Endosomes) or (iii) after shedding/fragmentation of the plasma membrane during apoptosis (i.e. Apoptotic bodies).

It should be understood from the above that the expression « vesicle » may thus encompass a wide variety of different structures, which are not assumed to be equivalent.

In particular, the origin of such vesicles, as well as their content, composition and/or biological origin may vary depending on sample preparations and tissues. Thus, the identification, or even the purification of one single kind of «vesicle» in a complex environment may be achieved using more than one characteristic. In view of such variability, it should be clear for the man skilled in the Art that the definitions given herebelow are provided only for reference, and may be considered either alone or in combination without departing from the spirit and scope of the invention.

It is proposed that vesicles should be characterized, and/or isolated based on:

- their size range, and/or - their biological origin, and/or

- their content, and/or

- their membrane composition.

Because of such variability, « vesicles » may have been incorrectly defined as « microvesicles » in earlier publications, in spite of the fact that the mean diameter of exosomes is generally smaller than 100 nm, and more often smaller than 90 nm.

In order to provide a clear framework, general definitions of the terms « Ectosome », « Exosome » and « Apoptotic body » will be provided herebelow.

According to the invention, the expressions « microvesicle » or « circulating microvesicle » or « microparticle » will only refer to Ectosomes.

According to the invention, an « Ectosome » is defined as a small vesicle ranging from 100 to 1000 nm in diameter that is formed by a budding-out mechanism at the plasma membrane. The membrane composition of an Ectosome is thus assumed to be similar to the said plasma membrane.

According to the invention, an « Exosome » is defined as a small vesicle smaller than 100 nm in diameter, for instance ranging from 30 to 100 nm in diameter, which includes 30 to 80 nm, 30 to 70 nm and 40 to 100 nm in diameter, in particular ranging from 50 to 90 nm in diameter, and is generally formed by a fusion/exocytosis mechanism of multivesicular bodies (or multivesicular endosomes) at the plasma membrane. Because such mechanism is indirect, the membrane composition of an Exosome is usually distinct from the said plasma membrane, which reflects its endocytic origin.

For recent reviews on microvesicles and exosomes, the man skilled in the Art may for instance refer to Schneider & Simons (Exosomes: vesicular carriers for intercellular communication in neurodegenerative disorders; Cell Tissue Res (2013) 352: 33-47) or Piccin et al, 2007.

According to the invention, an « Apoptotic Body » is defined as a large vesicle of 1 μπι or more in diameter, generally ranging from 1 to 5 μπι in diameter, that is formed after shedding/fragmentation of the plasma membrane during apoptosis. Thus, the membrane composition of an Apoptotic Body is assumed to be similar to the said plasma membrane. According to the invention, "Tau protein" may refer to any one of its known isoforms and/or peptides, and more particularly Tau isoforms of sequence SEQ ID NO 1 to 6, that is/are encoded by the MAPT gene (see for reference PI 0636 (TAU HUMAN), UniProtKB/Swiss-Prot). Unless specified, "total Tau" includes all known Tau isoforms and their truncated products, and more particularly brain Tau isoforms of sequence SEQ ID NO 1 to 6.

Of course, the expression "Tau protein" further applies to fragments derived from Tau, and/or mutant forms such as hTau46^{P 01L}.In particular, the Tau protein is selected in a list comprising: phosphorylated Tau, non-phosphorylated Tau, Total Tau, a Tau isoform, truncated Tau and/or Tau fragments, and/or a selection of Tau isoforms of sequence SEQ ID NO 1 to 6.

Thus the Tau protein can be selected in a list comprising: phosphorylated Tau, non-phosphorylated Tau, Total Tau, a Tau isoform, and/or a selection of Tau isoforms of sequence SEQ ID NO 1 to 6.

In view of the above, a Tau protein of the invention may be modified post- translationally, which includes modifications such as phosphorylation, acetylation and/or glycosylation.

Due to the diversity of known Tau isoforms and translational modifications, the one skilled in the Art will understand that the above-mentioned list is not exhaustive. For additional examples of epitopes of Tau, the one skilled in the Art may for instance refer to Hernandez et al. (Tissue-nonspecific Alkaline Phosphatase Promotes the Neurotoxicity Effect of Extracellular Tau; The Journal of Biological Chemistry; Vol.285, NO.42, pp. 32539-32548; 2010) and to Mohamed et al. (Mohamed et al., 2013).

According to a particular embodiment, a Tau protein of the invention is a non- phosphorylated, or de-phosphorylated Tau protein.

For a general review on known phosphorylation sites, the man skilled in the Art may refer to Sergeant & Buee (Sergeant et Buee 2001- Chapter 4: Tau pathophysiology. Vol. 13- Cytoskeleton in the Nerveous System (Ralph Nixon, David Yuan, eds). In: Handbook of Neurochemistry and Molecular Neurobiology. Lajtha A (Ed.). Spinger Publishers. ISBN: 978-l_4419-6786-2, pp. 83-132).

Goedert et al. (Goedert et al., 1989) determined the sequences of 6 tau isoforms of sequence SEQ ID NO 1 to 6 produced in adult human brain by alternative mRNA splicing. The proteins are composed of 352 to 441 amino acids. The isoforms differ from each other by the presence or absence of 29-amino acid or 58-amino acid inserts located in the N terminus and a 31 -amino repeat located in the C terminus. Inclusion of the latter, which is encoded by exon 10 of the MAPT Tau gene, gives rise to the 3 Tau isoforms with 4 repeats each; the other 3 isoforms have 3 repeats each. Normal human cerebral cortex contains similar levels of 3-repeat and 4-repeat tau isoforms.

As used herein, a "nucleic acid encoding Tau protein" encompasses mRNAs that are produced in the cell by expression of the Tau-encoding gene. Thus, a nucleic acid encoding Tau protein encompasses mRNAs encoding for at least one of the Tau isoforms, e.g. encompasses mRNAs encoding for at least one of the Tau isoforms comprising an amino acid sequence selected in a group comprising the amino acid sequences SEQ ID N° 1 to SEQ ID N° 6.

As used herein, an « amount » of Tau protein refers to a quantification value of a Tau protein in a sample and thus encompasses the amount or concentration of a Tau protein that is contained in the said sample as well as the amount or concentration of a Tau protein in the said sample. The amount value of a Tau protein may be expressed in a variety of quantification units, which encompasses arbitrary units and conventional units such as weight units, molar units or concentration units.

As used herein, an « amount » of a nucleic acid encoding Tau protein refers to a quantification value of the said nucleic acid in a sample and thus encompasses the amount or concentration of the said nucleic acid that is contained in the said sample as well as the amount or concentration of the said nucleic acid in the said sample. The amount value of a nucleic acid encoding Tau protein may be expressed in a variety of quantification units, which encompasses arbitrary units and conventional units such as weight units, molar units or concentration units.

Characterization and isolation of an Ectosome-enriched fraction

According to the invention, an « Ectosome-enriched » fraction (EcEF) refers to the product of at least one fractionation step of an individual's biological sample, wherein the amount of Ectosome is increased either (i) relatively to the total amount of vesicles in said sample before said fractionation step, or (ii) relatively to the initial amount of Ectosome in said sample before said at least one fractionation step. Thus, an « Ectosome-enriched » fraction (EcEF) may refer to the product of a fractionation step, wherein the amount of Ectosomes is increased relatively to the total amount of vesicles in said sample before said fractionation step.

An « Ectosome-enriched » fraction (EcEF) may also refer to the product of a fractionation step, wherein the amount of Ectosome is increased relatively to the initial amount of Ectosome in said sample before said fractionation step.

The total amount of vesicles includes (a) the total amount of Exosomes, (b) the total amount of Apoptotic Bodies, and (c) the total amount of Ectosomes.

Because the above-mentioned vesicles can be separated based on different criteria, the man skilled in the Art may thus purify each species in a different way and with various levels of purity, without departing from the teaching and scope of the invention.

Thus, an « Ectosome-enriched » fraction may refer in particular to the product of a fractionation step of an individual's biological sample, wherein the amount of Ectosome is increased relatively to either (a) the total amount of Exosomes, (b) the total amount of Apoptotic Bodies, (c) the total amount of Exosomes and Apoptotic Bodies or (d) the total amount of Exosomes and Apoptotic Bodies and Ectosomes in said sample before said fractionation step.

Preferably, an « Ectosome-enriched » fraction is obtained from a sample that is susceptible to contain at one or more Ectosomes, but which may further comprise other vesicles such as Exosomes or Apoptotic bodies.

More particularly, an « Ectosome-enriched » fraction (EcEF) may refer to the product of a fractionation step, wherein the amount of Ectosomes is increased relatively to the total amount of Exosomes in said sample before said fractionation step.

Alternatively, an « Ectosome-enriched » fraction (EcEF) may also refer to the product of a fractionation step, wherein the amount of Ectosomes is increased relatively to the total amount of Apoptotic bodies in said sample before said fractionation step.

According to the experimental protocol that is described in figure 1 and example 1, Apoptotic bodies should be discarded after the first centrifugation step. Thus, according to a preferred embodiment an EcEF should be depleted of Apoptotic Bodies.

Thus, an « Ectosome-enriched » fraction (EcEF) may refer to the product of a fractionation step, wherein the amount of Ectosomes is increased relatively to the total amount of Exosomes in said sample before said fractionation step, and that does not contain Apoptotic Bodies.

In a non-limitative manner, the step of « isolating an Ectosome-Enriched Fraction » may be achieved by flow cytometry, in particular Fluorescence-activated cell sorting (FACS), or centrifugation-based methods, in particular Differential centrifugation.

For instance, an Ectosome-enriched fraction (EcEF) and an Exosome-enriched fraction (ExEF) may be determined on the basis on their content in Ectosomes and Exosomes, and/or may be determined on the basis of the presence of specific biomarkers, respectively in Ectosomes and Exosomes. Such markers are known in the Art and may be used either alone or in combination.

Typically, several proteins are specifically enriched in exosomes, such as integrins and tetraspanins CD63, CD89, CD81, CD9 and CD82. Other markers which are specific of the endolysosomal pathway (such as the multivesicular endosome) may be further detected, such as the proteins Alix and TSGlOl, the endosomal and endosome maturation-related proteins Flotillin-1 and annexin and the heat shock proteins hsp70 and hsp90. The man skilled in the Art will understand that such markers are particularly useful for detecting, and isolating, Exosomes and/or Exosome-enriched fractions by labelling them with antibodies.

According to an exemplary embodiment, two exosomal markers are Alix and Flotilin-1. Alix is mainly an exosomal marker and is generally more expressed in the ExEF than in the EcEF.

Typically, those properties will be advantageously used for isolating Ectosome- enriched fractions using flow cytometry and/or FACS, as shown in Baron et al. for atherosclerosis lesions, liver and blood samples (Baron et al. ; PPARa activation differently affects microparticle content in atherosclerotic lesions and liver of a mouse model of atherosclerosis and NASH; Atherosclerosis (2011); 218: 69-76). FACS has the advantage of being a well-known method in the Art, which is suitable for combining both a fractionation step based on the content in specific biomarkers, and a size-exclusion criterium.

An EcEF is preferably obtained and characterized based on a size-exclusion criterium. For that purpose, differential centrifugation is a preferred method. In some embodiments, an EcEF contains at least 50% of Ectosomes, in particular at least 60% of Ectosomes, preferably at least 70% of Ectosomes, which includes at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of Ectosomes, relatively to the number of vesicles contained in the said EcEF.

Indeed, an EcEF comprises up to 100% of Ectosomes, relative to the number of vesicles contained in the said EcEF.

Such values correspond to a percentage relatively to the total amount of vesicles in the said EcEF.

According to another particular embodiment, an EcEF contains at least 50% of vesicles having a size of 100 nm or more, in particular at least 60% of said vesicles, preferably at least 70% of said vesicles, which includes at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of said vesicles, and up to 100% of said vesicles, relatively to the total amount of vesicles contained in the said EcEF.

In some embodiments, an EcEF contains at least 70% of vesicles having a size of 100 nm or more in diameter, in particular ranging from 100 to 1000 nm, and preferably ranging from 100 to 700 nm, relatively to the total amount of vesicles contained in the said EcEF.

In some embodiments, an EcEF contains at least 80% of vesicles having a size of 100 nm or more in diameter, in particular ranging from 100 to 1000 nm, and preferably ranging from 100 to 700 nm, relatively to the total amount of vesicles contained in the said EcEF.

Optionally, an EcEF may also be characterized by its particle-size distribution; a particle-size distribution may be measured for instance using Dynamic Light Scattering as described in Hupfeld et al. (Hupfeld et al, (2006) Liposome size analysis by dynamic/static light scattering upon size exclusion-/field flow- fractionation., J. Nanosci Nanotechnol.; 6(9-10): 3025-31).

In a non-limitative manner, an EcEF may be also characterized by electron microscopy analysis, as shown in the Material & Methods section and in the examples.

Because Exosomes and Ectosomes can be closely related in terms of size, it is preferable that the EcEF is substantially pure, and/or depleted of Exosomes. According to a particular embodiment, an EcEF is depleted of Exosomes, which includes an EcEF comprising less than 50% of Exosomes, in particular less than 40% of Exosomes, more particularly less than 30% of Exosomes, and preferably less than 20% of Exosomes, which includes less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of Exosomes, relatively to the total amount of vesicles contained in the said EcEF. In some embodiments, an EcEF is depleted of, or substantially depleted of, Exosomes.

According to this particular embodiment, an EcEF is depleted of vesicles having a size of less than 100 nm in diameter, which includes an EcEF comprising less than 50% of said vesicles, in particular less than 40% of said vesicles, more particularly less than 30% of said vesicles, and preferably less than 20% of said vesicles, which includes less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% of said vesicles, relatively to the total amount of vesicles contained in the said EcEF. In some embodiments, an EcEF is depleted of, or substantially depleted of, vesicles having a size of less than 100 nm in diameter.

According to a particular embodiment, the EcEF is isolated after at least one centrifugation step, and preferably at least two centrifugation steps.

According to a particular embodiment, the EcEF is isolated after a first centrifugation step and a second centrifugation step, wherein the first centrifugation step is suitable for recovering a first fraction depleted of cells and apoptotic bodies, and the second centrifugation step is suitable for recovering an Ectosome-Enriched Fraction.

Advantageously, the above-mentioned methods may be used for isolating both an EcEF and an ExEF. A complete protocol for isolating both an EcEF and an ExEF by centrifugation is described in the Material & Methods and in figure 1.

The man skilled in the Art will recognize that the parameters may be adjusted without departing from the scope and teaching of the invention. For instance, the duration of a centrifugation step may be adjusted, for instance increased or decreased, while at the same time adjusting the speed of centrifugation.

For convenience, centrifugation speed in preferably given in g or in rpm (round per minutes) units. The conversion for one unit the other is also well known in the art.

According to an exemplary embodiment, the EcEF is isolated after two centrifugation steps: a first centrifugation step of 2000xg for 10 minutes, after which the supernatant is recovered, followed by a second centrifugation step at 20000xg for 45 minutes, after which the pellet (also referred as "pellet 1" in figure 1) is recovered.

According to an exemplary embodiment, the ExEF is isolated after a third centrifugation step from the supernatant (also referred as "supernatant 2" in figure 1) recovered from the second centrifugation step. According to this embodiment, the ExEF corresponds to the second pellet (also referred as "pellet 2").

A third centrifugation step may be achieved at about lOOOOOxg for about 50 minutes.

Advantageously, the purity of the ExEF is advantageously improved after an additional step of sucrose gradient centrifugation, as described in figure 1. A sucrose gradient centrifugation may be achieved at about lOOOOOxg for 18 hours. Advantageously, small fractions may be collected and further rinsed once in imidazole at 150000xg for one hour.

This method is based on the well-established properties of Exosomes to "float" on sucrose gradients based on their density ranges. Indeed, Exosomes are vesicles that float on a sucrose gradient and their density ranges from 1.08 to 1.22 g ml^"1 depending on their cellular origin.

According to one embodiment, an ExEF may contain more than 50% of

Exosomes, in particular more than 60% of Exosomes, which includes more than 70%, more than 80% and more than 90% of Exosomes, which includes more than 80, 81, 82, 83,

84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of Exosomes, relatively to the total amount of vesicles contained in the said ExEF.

According to said embodiment, Exosomes may in particular be defined as vesicles with a size ranging from 30 to 70 nm in diameter.

According to one exemplary embodiment, an ExEF contains more than 95% of vesicles with a size between 30 and 70 nm, in particular at least 98% of vesicles with a size between 30 and 70 nm, relatively to the total amount of vesicles contained in the said

ExEF. Measuring the amount of Tau protein

The amount of Tau protein is measured in an Ectosome-Enriched Fraction (EcEF), and preferably an Ectosome. Such amount may include both the amount of Tau protein that is associated with Ectosomes, and the amount of Tau protein within Ectosomes. Preferably, the amount of Tau protein that is measured is the amount of Tau within Ectosomes.

Advantageously, prior to the measurement of an amount of Tau protein, the Ectosome-Enriched Fraction (EcEF) may be treated in a manner suitable for removing the amount of Tau which interacts with the extravesicular surface, such as a NaCl treatment as shown in figure 10.

In a non-limitative manner, the step of « measuring an amount of Tau protein » can be achieved by incubating said Ectosome-Enriched Fraction with a Tau-binding molecule.

A Tau-binding molecule may refer to any molecule which is able to bind to the Tau protein, which includes its known isoforms; and for which the presence may be further detected either alone or as a complex with said Tau protein. For instance, a Tau binding molecule may be an aptamer or an antibody, particularly an antibody, directly or indirectly labeled with a detectable molecule. When the Tau binding molecule is an antibody, it may be a monoclonal or a polyclonal antibody. Tau binding molecules and detectable molecules are well known in the Art.

According to a particular embodiment, the Tau-binding molecule may be directed against the C- or the N-terminal part of Tau.

A Tau-binding molecule targeting the N-terminal part of Tau can be directed against an epitope of sequence SEQ ID NO 7 or 8.

A Tau-binding molecule targeting the C-terminal part of Tau can be directed against an epitope of sequence SEQ ID NO 10.

According to another particular embodiment, the Tau-binding molecule may be directed against an epitope common to all Tau isoforms, which includes Tau isoforms of sequences SEQ ID NO 1 to 6.

According to a particular embodiment, a Tau-binding molecule can be an antibody directed against Tau protein, in particular an antibody directed against an epitope of sequence SEQ ID NO 7 to 10.

Advantageously, the amount of «Total Tau» may be determined using an antibody directed against the epitope of sequence SEQ ID NO 9, such as the mouse monoclonal antibody HT7 (Tau antibody (HT7) / Thermo Scientific). Of course the antibody HT7 is given here as a non-limitative example. Because Total Tau may include truncated forms, phosphorylated and non-phosphorylated forms of Tau, those proteins and/or peptides may be detectable by distinct antibodies, and/or have distinct molecular masses.

According to one particular embodiment, Tau protein may be detected by

Electron microscopy analysis and immuno-labelling of the EcEF with a Tau-binding antibody.

When the Tau-binding ligand is an antibody, it is preferably a monoclonal antibody.

Measuring the amount of a nucleic acid encoding Tau protein

In a non-limitative manner, the step of « measuring an amount of a nucleic acid encoding Tau protein » can be achieved by any method known to the one skilled in the art that allows quantification of gene-specific mRNAs in a sample, which encompasses any method for quantifying mRNAs produced by expression of a gene encoding Tau, preferably human Tau. Then, the step of measuring an amount of nucleic acid encoding Tau encompasses any method for quantifying mRNAs encoding the Tau isoforms of SEQ ID N° 1 to 6.

In a non-limitative manner, methods for quantifying nucleic acids in a sample include PCR (Polymerase Chain Reaction), RT-PCR, Q-PCR or real-time PCR, or in situ hybridation. More generally, all methods for quantifying nucleic acids are considered by the invention, including all methods which relate to the hybridization of at least one nucleic acid primer to the nucleic acid sequence which should be quantified.

Illustratively, measuring the amount of a nucleic acid encoding Tau protein may be performed by the well-known technique of real-time PCR with suitable pairs of nucleic acid primers. Quantitative PCR and semi-quantitative PCR are both considered by the invention. Examples of such primers are described in a non-limitative manner in example 6.

According to a particular embodiment, primers and pairs of primers suitable for the invention are primers of sequences SEQ ID NO 11 or 12 or 15 to 34, such as primers of sequences SEQ ID NO 11 and 12. Advantageously, the amplification can be compared to a positive control for calibration, as shown in example 7 or figure 9 with 18S ribosomal RNA, using primers of sequences SEQ ID NO 13 and 14.

The one skilled in the art may also refer to the articles of Wong et al. (2005, BioTechniques, Vol. 39: 75-85) or of Nolan et al. (2006, Nat Protoc, Vol. 1(3): 1559- 1582).

Since the mRNAs encoding the various Tau isoforms are known in the art, the one skilled in the art may design any suitable pair of nucleic acid primers on the basis of his general technical knowledge, and/or adapt or optimize hybridization conditions of pairs of primers, such as primers of sequences SEQ ID NO 11 to 34.

Advantageously, primers and pairs of primers which have been described in example 7 may be used for detecting, for quantifying, or for discriminating between nucleic acids of the invention, such as Tau isoforms.

Even more advantageously, pairs of primers may be used for discriminating between different isoforms of Tau protein in a sample, which includes:

- Tau isoform (2+3+10+) of sequence SEQ ID NO 1,

- Tau isoform (2+3+10-) of sequence SEQ ID NO 2,

- Tau isoform (2+3-10+) of sequence SEQ ID NO 3,

- Tau isoform (2+3-10-) of sequence SEQ ID NO 4,

- Tau isoform (2-3-10-) of sequence SEQ ID NO 5,

- Tau isoform (2-3-10+) of sequence SEQ ID NO 6.

Because amplicons for each isoform will be different, they may be easily separated based on their size, as shown in example 6.

The one skilled in the Art will understand that, by selecting different pairs of primers and/or conditions of hybridization, the amplicon size may also vary depending on the nucleic acid that is amplified.

Methods for detecting the occurrence of a Tau-related disease

The invention further relates to an in vitro method for determining the occurrence of, or the risk of occurrence of, a Tau-related disease in an individual, comprising the steps of: a) measuring an amount of Tau protein in one or more Ectosomes or in an Ectosome-Enriched Fraction from said individual by a method described herein,

b) comparing the amount of Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the occurrence of, or the risk of occurrence of, the Tau-related disease in said individual.

In particular, the invention relates to an in vitro method for detecting the occurrence of a Tau-related disease in an individual, comprising the steps of:

a) measuring an amount of Tau protein in an Ectosome-Enriched Fraction from said individual as defined above,

b) comparing the amount of Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the occurrence of the

Tau-related disease in said individual.

According to a particular embodiment, the amount of extracellular Tau may be further quantified. Advantageously, a method for determining the occurrence of, or the risk of occurrence of, a Tau-related disease in an individual may comprise an additional step of determining the amount of extracellular Tau, such as the amount of extracellular Tau in a CSF sample from said individual.

As an illustration, the diagnosis of Alzheimer's disease may be established for a Tau concentration in the CSF beyond 400 pg/mL using ELISA assays (reference: Innogenetics: INNOTEST® hTAU Ag).

In a non-limitative manner, the Tau-related disease may be selected in a list comprising: Alzheimer's disease and/or other Tau-related diseases selected in a list comprising: Amyotrophic lateral sclerosis, Parkinson Dementia complex of Guam, Argyrophilic grain dementia, British type amyloid angiopathy, Corticobasal degeneration, Dementia pugilistica/autism with self-injury behaviour and other chronic traumatic injuries, Down's syndrome, FTDP-17, Gerstmann-Straussler-Scheinker disease, Hallenvorden-Spatz disease, Inclusion body myositis, Multisystem atrophy, Myotonic dystrophy type I and type II, Niemann-Pick disease type C, Hereditary inclusion body myopathy with Paget disease of bone and frontotemporal dementia, Parkinson with dementia of Guadeloupe, Pick's disease, Presenile dementia with tangles and calcifications, Prion protein cerebral amyloid angiopathy, Progressive supranuclear palsy, Post-encephalitic parkinsonism, Subacute sclerosing panencephalitis, Tangle only dementia.

Preferably, the one or more Ectosome(s) or the EcEF is obtained from either an interstitial fluid or a cerebrospinal fluid from the said individual.

According to a particular embodiment, an in vitro method for detecting the occurrence of a Tau-related disease in an individual may be used as a tool for determining the prognosis of the said Tau-related disease in the said individual.

Because Tau is a pathological feature of neurodegenerative diseases referred herein as Tauopathies" or "Tau-related diseases", the increase of an amount of Tau protein in Ectosomes (or EcEF) is predicted to be of poor prognosis. The man skilled in the Art may in particular refer to examples 4, 5 and 6 which provide support for active secretion of Tau in the interstitial fluids and cerebrospinal fluids, when overexpressed in the brain.

According to the invention, a « reference value » refers to the amount of Tau protein within one individual or group of individuals for which the presence or the absence of the occurrence of a Tau-related disease has been already determined.

When the above-mentioned method is used as a tool for prognosis, the reference value is advantageously determined from the same individual or group of individuals over time.

Methods for detecting the efficacy of a treatment of a Tau-related disease

The invention also relates to an in vitro method for determining the efficacy of a treatment of a Tau-related disease in an individual, comprising the steps of:

a) measuring an amount of Tau protein in one or more Ectosomes or in an

Ectosome-Enriched Fraction from said individual as defined above, b) comparing the amount of Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the efficacy of said treatment in said individual.

In particular, the invention relates to an in vitro method for determining the efficacy of a treatment of a Tau-related disease in an individual, comprising the steps of:

a) measuring an amount of Tau protein in an Ectosome-Enriched Fraction from said individual as defined above,

b) comparing the amount of Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the efficacy of said treatment in said individual. Preferably, the one or more Ectosomes or the EcEF is isolated from either an interstititial fluid or a cerebrospinal fluid from the said individual.

A treatment of a Tau-related disease is predicted to be useful if, after its administration, the amount of Tau proteins in Ectosomes (or EcEF) is decreased.

Advantageously, a « reference value » may refer to the amount of Tau protein in said individual or group of individuals prior to the administration of said treatment, or alternatively to the amount of Tau protein in another individual or group of individual which has been administered a placebo.

EXAMPLES

A. MATERIAL & METHODS

Antibodies- Mouse monoclonal antibody HT7 (Tau antibody (HT7) / Thermo Scientific; 1 :2000 for biochemistry) recognizes total Tau (epitope between AA159 to 163, of sequence SEQ ID NO 9). Polyclonal antibody against the C-terminal part of Tau recognizes the last 15 AA (Sergeant et al, 2001) (Home made, 1 : 10 000 for biochemistry and 1 : 1000 for electronic microscopy). Mouse monoclonal antibody is directed against Alix protein (Anti-PDC6I antibody [3A9]; Abeam 1 : 1000 for biochemistry) (Alvarez- Erviti et al, 2011). Mouse monoclonal antibody is directed against the Flotilin-1 protein (1 : 1000 for biochemistry) (de Gassart et al., 2003). The MAb ADx215 (1 :50 for EM) is a new human-specific monoclonal antibody (Caillierez et al; 2013) raised against the sequence SEQ ID NO 8 (GTYGLGDRK), which is part of the N-terminal end of Tau.

Viral vectors -

1) Plasmid construction- The packaging construct used was the pCMVAR8.92. To further decrease the risk of recombination and the production of replication-competent lentiviruses, the Rev gene was inserted into the pRSV-Rev plasmid. Viral particles were pseudotyped with the vesicular stomatitis virus G-protein encoded by the pMD2.G plasmid described previously (Hottinger et al, 2000). cDNAs encoding the 2+3-10+ 1N4R isoforms of human WT Tau and mutant P301L Tau were first cloned into the Gateway Entry pCR8/GW/TOPO vector (Invitrogen) using TOPO TA cloning methodology. The Gateway LR clonase (Invitrogen) catalyzed the in vitro recombination between the Gateway Entry pCR8/GW/TOPO vector (containing the Tau cDNA anked by attL sites) and the lentiviral destination vector (containing homologous attR sites).

2) Production and assay of recombinant LVs- LV vectors were amplified as previously described (Hottinger et al, 2000). Human 293T cells (4 x 10⁶) were plated onto 10 cm plates and transfected the following day with 13 μg of human Tau cDNA, 13 μg of pCMVAR8.92, 3 μg of pRSV-Rev and 3.75 μg of pMD.2G using the calcium phosphate DNA precipitation procedure. Four to six hours later, the medium was removed and replaced by fresh medium. Forty-eight hours later, the supernatant was collected and filtered. High-titer stocks were obtained by two successive ultracentrifugation steps at 19000 rpm (Beckman Coulter SW 32Ti and SW 60Ti rotors) and 4°C. The pellet was resuspended in PBS with 1% BSA and stored frozen at -80°C until used. Viral concentrations were determined by ELISA for the HIV-1 p24 antigen (Gentaur BVBA). The p24 is a lentiviral protein of the capsid that is commonly used in Elisa assay to determine the physical titre of lentiviral batches in quantity per mL. All viral batches were produced in appropriate areas in compliance with institutional protocols for genetically modified organism manipulation, as recommended by the 'Comite Scientifique du Haut Conseil des Biotechnologies' (Identification number 5258). 3) Production and assay of recombinant AAVs- Pseudotyped AAV9 vectors (CEA; MIRCen; Fontenay-aux-roses; Team of Dr Philippe Hantraye) were generated by packaging AAV2-based recombinant self-complementary (sc) genomes into the AAV9 capsid. Virions were produced by transfecting HEK293 cells with (i) the adenovirus helper plasmid (pXX6-80), (ii) the AAV packaging plasmid encoding the rep2 and cap9 genes (pXR9), and (iii) the AAV2 shuttle plasmid containing the respective transgenes under the transcriptional control of the CBA promoter sequence in a sc genome. Three days following transfection, cells were harvested and lysed by freeze/thaw cycles. AAV particles were then purified by ultracentrifugation on discontinuous iodixanol gradient. AAV-containing fractions were then desalted by tangential flow filtration and stored at 4°C in PBS until further use. Physical particles were quantified by real-time PCR. Vector titers were expressed as viral genomes per milliliter (vg/ml).

Animals (Primates)- 1) Animals and housing- All animal studies were conducted according European (EU Directive 2010/63) and French regulations (Decret 2013-118). The animal facility is authorized by local veterinarian authorities (authorization n° A 92-032-02) and complies with Standards for Humane Care and Use of Laboratory Animals of the Office of Laboratory Animal Welfare (OLAW - n°#A5826-01). All efforts were made to minimize animal suffering and animal care was supervised by veterinarians and animal technicians skilled in the healthcare and housing of non-human primates. All animals were housed in social groups under standard environmental conditions (12-hour light-dark cycle, temperature: 23 ± 1°C and humidity: 50%) with free access to food and water. Experiments were conducted on 1 male cynomolgus monkeys (Macaca fascicularis, supplied by Noveprim, Mauritius Island) of a mean age of 5 ± 1 years and a mean weight of 6 ± 2 kg.

2) Blood and CSF sampling- Primates were anesthetized with ketamine (lOmg/kg) and xylazine (0.5mg/kg) and the femoral sheath disinfected for a femoral blood draw of 10- 15ml using a vacuette blood collections system and EDTA tubes. For CSF sampling, the lumbar are shaved and disinfected and the animal's neck and knees are bent in full flexion to expose the vertebrae. A 20G Spinocan (B Braun) was then introduced between L3/L4 or L4/L5, the stylet was withdrawn and the CFS was collected into an eppendorf tube and immediately placed on ice.

3) Surgery- Before surgery, all animals underwent an anatomical magnetic resonance imaging (MRI) session in a 7T Varian scanner in order to calculate bilateral stereotaxic coordinates for hippocampal targeting at -4, -6 and -11 from the anterior commissure. On the day of surgery, primates were induced with ketamine (lOmg/kg) and xylazine (0.5mg/kg) and kept under intravenous infusion of propofol (10-15mg/kg/h) for the duration of the procedure. They were placed on a stereotaxic frame (M2E, France) where the head was fixed by ear and mouth bars. Surgeries were performed in sterile conditions with constant monitoring of blood pressure, heart rate, expired C0₂, temperature and respiratory frequencies and rehydration by i.v. perfusion with 0.9% NaCl (lOml/kg/h). All primates were intracerebrally injected with a 26G needle attached to a Hamilton syringe, controlled by a KDS310 micropump (PHYMEP, France) delivering virus at a constant rate of Ι μΐ/min for a total volume of ΙΟμΙ per injection site and a total volume of 60μ1 per primate brain. After each injection, a 5-minute pause was respected before withdrawing the needle in order to minimize virus reflux along the needle track. A freshly sterilized needle was used for each animal while the same Hamilton syringe was used for each vector type (AAV9-CBA-Tau^WT or AAV9-CBA-Tau46^{P 01L}). Following surgery, the skull was cleaned and the skin sutured. An intradermic injection of bupivacaine and adrenaline was administered before suturing in order to prevent bleeding and provide local analgesia in the cutaneous wound area.

4) Euthanasia and tissue preparation- Animals were deeply anesthetized with 10: lmg/kg ketamine:xylazine and the thorax was opened to collect intra-cardiac blood samples (50ml) in EDTA-coated tubes. A lethal dose of pentobarbital was delivered before transcardial perfusion with room temperature 0.9% NaCl followed by ice-cold 4% paraformaldehyde. Brains were extracted and immediately placed in ice-cold 4% paraformaldehyde for 24 hours, and then in sucrose-containing phosphate buffer gradients for cryoprotection for 10 days before performing immunohistochemical analysis. Animals (Rats)-

1) Animals and housing- Two-month-old Wistar rats (Rattus norvegicus, 300 g) were included in this study. The animals were purchased from Janvier Laboratories and maintained in compliance with institutional protocols (Comite d'ethique en experimentation animale du Nord Pas-de-Calais n° 0508003). The animals were housed in a temperature-controlled room and maintained on a 12-hour day/night cycle with food and water ad libitum. All experiments on animals were performed in compliance with, and following the approval of, the local Animal Resources Committee (CEEA 342012 ) , standards for the care and use of laboratory animals and French and European Community rules.

2) Stereotaxic injections- Intracerebral injections of viral particles into the brains of anesthetized (ketamine 100 mg/kg, xylazine 10 mg/kg i.p.) 2-month-old Wistar rats were performed using standard stereotactic procedures at the following coordinates relative to the bregma: posterior: -5.3 mm; lateral: +/- 6.2 mm; depth: -7 mm and -6.2 mm. Injections were performed bilaterally. The standard injection procedure consisted of the delivery of 400 ng of p24 using a 10 μΙ_, glass syringe with a fixed needle (Hamilton). After injection at a rate of 0.25 μΙ_, per min, the needle was left in place for 1 min. A similar injection was then performed after 5 min at the second depth.

3) In vivo push-pull microdialysis- 6 months post-LVs delivery, a push- pull microdialysis probe was stereotaxically implanted at the injection site of anesthesized rats (Ketamine 100 mg/kg, Xylazine 10 mg/kg i.p.). Artificial CSF was then injected through the push-pull microdialysis probe at the rate of 1 μΕ/πήη. The same rate was applied to pull-out the interstitial fluid (ISF). The first collected fraction, corresponding to the 30 first minutes was discarded. Two aditional 30 minutes fractions per animal were retrieved, stored on ice before vesicle fractionation and electron microscopy analysis.

Cell Cultures- 1) Primary Embryonic Neuronal Cultures- Rat primary cortical neurons were prepared from 17-day-old Wistar rat embryos as previously described (Galas et al, 2006). Briefly, brain and meninges were removed. Cortex was carefully dissected out and mechanically dissociated in culture medium by triturating with a polished Pasteur pipette. Once dissociated and after blue trypan counting, cells were plated at a density of 2.5 ^χ 10⁴ cells/cm² in poly-d-lysine and laminin-coated T25. For dissociation, plating, and maintenance, we used Neurobasal medium supplemented with 2% B27 containing 500 μΜ glutamine and 1% antibiotic-antimycotic agent (GIBCO). Cells were maintained in a humidified incubator with 5% C0₂ for a defined period of differentiation (DIV, Days In Vitro). Extracellular media were collected from primary cultures at DIV3, 5, 10 and 12 and vesicles prepared as described after (Fig. 1).

2) Cell lines culture- Nl-El 15 (mouse neuroblastoma) cells were cultivated in Dulbecco's Modified Eagle's Medium with 10% fetal bovine serum (FBS), non-essential amino acids (1%), Penicillin/Streptomycin (1%) and L-Glutamine (1%). When indicated, Nl-El 15 were differentiated by total FBS starvation. Cells were maintained in a humidified incubator with 5% C0₂. All cell lines were passaged twice a week.

To generate the stable cell line (Nl-El 15-htau46^WT), N1E115 cells were seeded on 6-well plates and infected by LVs encoding hTau46^WT (400 ng p24 per well). Forty-eight hours later, cells were divided and a clonal selection performed using a limit dilution approach. To purify extracellular vesicles, Nl-El 15 and Nl-El 15-htau46^WT were differentiated 48 hours, the media were then collected from five T25 and vesicles prepared as described after (Fig. 1).

LDH assays- An LDH assay kit was used to control the LDH release from cells according to the manufacturer's instructions (Cytotoxicity Detection kit (LDH) / Roche applied Science). Briefly, culture media for each analyzed conditions were collected and centrifuged at 250xg for 10 minutes. The extracellular (EC) levels of LDH were estimated by incubating 10 μΕ of the supernatant in a 96-wells plate with 100 μΕ of a mixed substrate and dye from the kit. After one hour at room temperature incubation, absorbance was evaluated at 490 nm. The intracellular (IC) levels of LDH were also determined in cell lysate to normalize results between each condition. Cells were incubated with triton-XlOO IX diluted in culture medium for 1 hour to determine the 100 % of LDH release. Ratio Abs_Ec Absic were determined for each condition and results are presented as LDH release. The results were analyzed with the graphpad software. Statistical inter-groups comparisons were realized with an ANOVA and a Bonferonni post-test.

Ectosomes and exosomes isolation- For all procedures (Fig. 1), media were collected, placed on ice and proteases inhibitors added (Complete mini, Roche) before centrifuged 10 min at 2000 g at 4°C to remove cell debris. Supernatant is then centrifuged 45 min at 20 000 g. Pellets corresponding to ectosomes enriched fractions (EcEF) is either resuspended in RIPA buffer (NaCl 150 mM; NP40 1 %; Sodium Deoxycholate 0.5 %; SDS 0.1 %; Tris HC1 50 mM; pH=8.0) for biochemical assays or in PFA (paraformaldehyde) 2% (diluted in phosphate buffer 0.08 M Na₂HP0₄ and 0.02 M NaH₂P0₄) for electron analyses. The supernatant is centrifuged 50 min at 100 000 g to generate exosomes enriched fractions (ExEF). Pellets are processed as described for EcEF or resuspended in imidazole 3 mM, pH 7.4 to purified exosomes on linear sucrose gradient (2.25-0.25 M sucrose in imidazole 3 mM pH 7.4). Sample was centrifuged 18 h at 100 000 g at 4°C and 1 mL fractions collected from the top of the gradient. All the fractions were diluted in a final volume of 9 mL of imidazole 3 mM pH 7.4 and centrifuged once 1 h at 150 000 g at 4°C. The corresponding pellets were then resuspended in LDS buffer (Lithium Dodecyl Sulfate 2X containing 100 mM DTT, Invitrogen) for biochemical assays.

Electrophoresis and Immunoblotting- For cell lysates analyses, cells were rinced once in PBS, scrapped in RIPA buffer. For vesicles analyses, pellets were directly resuspended in RIPA buffer. Protein concentrations were determined (PIERCE 'BCA Protein Assay Kit') and samples diluted at lg/L in LDS containing 50 mM of DTT. 10 μg of proteins were denaturated at 100 °C during 10 min, loaded on 4-12% NuPAGE gels (Invitrogen), and transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline, pH 8, 0.05% Tween 20 with 5% skim milk or bovine serum albumin and incubated with the appropriate primary antibody overnight at 4°C. Membranes were then rinced and further incubated with horse-radish peroxidase-labeled secondary antibody (goat anti-rabbit or anti-mouse IgGs, Sigma), and bands were visualized by chemiluminescence (ECL, Amersham Biosciences). Electron microscopy- Vesicles pellets are resuspended in PFA (paraformaldehyde) 2% (diluted in phosphate buffer 0.08 M Na₂HP0₄ and 0.02 M NaH₂P0₄) over night at 4°C. Nickel grids (400 square mesh) were washed with ethanol 100 % and then placed on a Formvar film (2 % in chloroform). After air-drying, light-sensitive grids are conserved until used. Five of sample were deposited on a grid and incubated 20 min at room temperature. Fluid Excesses are removed using a blotting paper (Wattman). Grids were firstly then washed twice with PBS fixed in glutaraldehyde (1 % in PBS) and then seven washes were done in distilled water. Once incubated 5 min at room temperature in uranyl acetate 1 % pH 7.0, light-sensitive grids were incubated 10 min on ice in uranyl acetate 5 % pH 4.0-methylcellulose 2%. Fluid excesses are removed using a bloting-paper and grids are stored until observed under a transmission electron microscope (Zeiss EM902). When indicated, an immunolabelling was done. Grids were then rinsed once in PBS and incubated twice (3 min at RT) in a PBS-Glycin 50 mM before incubation in PBS-BSA 1 % 10 min at RT. Primary antibody diluted in PBS-BSA 1% was then applied 1 hour at RT and the grids before grids were rinsed 3 times in PBS-BSA 0.1 %. Primary antibody was revealed using appropriate secondary antibody diluted in PBS-BSA 1 % (18 nm gold colloidal goat anti-mouse or anti-rabbit 1 :20). After rinsed in PBS (3 times, 3 min at RT), grids were fixed 5 min at RT in PBS-Glutaraldehyde 1 %, rinsed in PBS (7 times, 2 minutes), and negatively stained with a 5 minutes incubation protected from light with 1% uranyl acetate (diluted into H₂0) and 10 minutes at 4°C protected from light with a solution of 1.2% uranyl acetate, 1.4% methyl cellulose. RNA extraction and RT-PCR/PCR- RT-PCR of human Tau mRNA was performed using total RNA. The brain slices were lysed, and total RNA was extracted using the RNeasy Lipid Tissue kit (Qiagen, France) according to the manufacturer's instructions. The RNA (1 μg) was denatured for 10 min at 68°C, and cDNA was generated using reverse transcription with 200 nmol/L of dNTPs, 1 ng/μΐ of random primers, 1 ng/μΐ of oligo dT, 5 mmol/L of dithiothreitol (DTT), 2 units/μΐ of RNase Out and 10 units/μΐ of M- MLV reverse transcriptase. The cDNAs were then amplified using oligonucleotides specific to human Tau of sequences SEQ ID NO 11 and 12 (forward: 5'-TGG-GGG-ACA- GGA-AAG-A-3' and reverse: 5'-CCT-CAG-ATC-CGT-CCT-CAG-TG-3'). The following primers of sequences SEQ ID NO 13 and 14 were used to amplify 18S for calibration: forward: 5'-AAA-CGG-CTA-CCA-CAT-CCA-AG-3' and reverse: 5'-CGC-TCC-CAA- GAT-CCA-ACT-AC-3'. PCR was performed using 2 μΐ of the previously obtained RT products, reverse and forward primers (0.5 μιηοΙ/L), dNTPs (1 μιηοΙ/L) and 0.02 unit/μΐ of DNA polymerase in a commercial reaction buffer (GoTaq Green Master Mix, Promega). The PCR products were electrophoresed on an 8 % acrylamide gel stained with 1 μg/ml ethidium bromide. Statistical analysis- The number of experiments and the statistical analysis performed are indicated in the figure legends.

B. RESULTS

EXAMPLE 1. Characterisation of ectosomal and exosomal fractions from rat primary embryonic cortical cells.

The ability of rat primary embryonic cortical cells to secrete exosomes and ectosomes was analyzed. Cell culture supernatants were processed as described in figure 1 to isolate membrane vesicles. Ectosomes Enriched Fractions (EcEF, pellet 1) or Exosomes Enriched Fractions (ExEF, pellet 2) were analyzed by electron microscopy (Fig. 2a). A semi-quantitative analysis allowed us to determine the percentage of vesicles (ectosomes or exosomes) in these fractions based on size exclusion criteria: EcEF contains 71 % of vesicles having a size larger than 100 nm whereas ExEF contains 98 % of vesicles with a size between 30 and 70 nm (Fig. 2b). Exosomes are vesicles that float on a sucrose gradient and their density ranges from 1.08 to 1.22 g ml^"1 depending on their cellular origin (Raposo et al, 1996). We fractionated ExEF on continuous sucrose gradient and validated the presence of exosomes by biochemical assays using two specific markers, Alix (Thery et al, 2001) and Flotillin-1 (de Gassart et al., 2003) (Fig. 2c). Conclusion: Ectosomes and Exosomes can be purified and fractionated based on size-exclusion criteria and density. They can be further identified using biochemical assays, which validates the efficiency of the protocol. In view of the above, Ectosomes- enriched Fractions with purity levels of more than 70% can be obtained reproducibly. EXAMPLE 2. Murine Endogenous Tau is released from rat primary embryonic cortical cells in a vesicular but non-exosomal pathway: the ectosomes-

The association of murine endogenous Tau protein into vesicles isolated from rat primary embryonic cortical cells was analyzed. Conditioned media coming from E17 rat primary cortical cells cultured for 10 days (10 DIV) were collected. EcEF and ExEF pelleted from these media were suspended in RIPA buffer and analyzed by western- blotting to reveal the presence of total Tau using an antibody directed against the C- or the N-terminal parts of Tau (Sergeant et al., 2001) . Whereas the ExEF is devoid of Tau, a signal corresponding to the endogenous Tau (50 kDa) and truncated species, is observed in EcEF, using an antibody directed against the C-terminal part of Tau (Fig. 3a).

In order to avoid an artefactual signal due to the slight contamination of EcEF by exosomes (Fig. 2b), the association of Tau to ectosomes was validated by EM using antibodies that both recognized the total Tau either at its N-terminal (Fig. 3b, upper panels) or at its C-terminal portion (Fig. 3b, lower panels). The number of vesicles (30 to 70 nm for exosomes and larger than 100 nm for ectosomes) immunopositive for both antibodies was quantified and confirmed that whereas only 3 % of exosomes are immunoreactive, Tau is associated to 22 (N-Ter) or 12 (C-Ter) % of ectosomes (Fig. 3c).

The presence of Tau in the extracellular medium may reflect either active secretion or neuronal death. To exclude the latter one, we controlled cell damage induces by the neuronal differentiation in our culture conditions. Media from 3, 10 and 15 DIV were collected and the presence of a cytoplasmic protein, the LDH (lactate dehydrogenase) quantified from an aliquot. The differentiation process does not induce cell death (Fig 4a) suggesting that the presence of Tau in the vesicles fractionated from media is related to an active secretion (with DIV) (Fig 4c) rather than cell damage.

Conclusion: From these results, we conclude that in physiological conditions, in rat primary embryonic cortical cells, Tau is actively secreted preferably in ectosomes rather than in exosomes. Thus, the differential distribution of secreted Tau into particular fractions may be considered as a sensor of the physiological state of neurons. When this latter is deregulated protein accumulation occurs; specific mechanisms of proteolysis, such as macroautophagy, are activated and result in Tau targeting to MVBs. EXAMPLE 3. Characterisation of ectomal and exosomal fractions from cell lines-

As already done for rat primary embryonic cortical cells (as shown above and in Fig. 2), the ability of neuroblastoma cell lines N1-E115 overexpressing human Tau (hTau46^WT, 2+3-10+) to secrete exosomes and ectosomes was analyzed. EcEF and ExEF pelleted from conditioned media were analyzed by EM (Fig. 5a). EcEF contains 80 % of vesicles having a size larger than 100 nm whereas ExEF contains 97 % of vesicles with size between 30 and 70 nm (Fig. 5b). The presence of exosomes in a continuous gradient was also confirmed by biochemical detection of Alix and flotilin (Fig. 5c).

Conclusion: EcEF and ExEF fractions can be obtained reproducibly from neuroblastoma cell lines with high levels of purity (more than 80%).

EXAMPLE 4. A minor part of Tau is shifted to the classical secretory pathway when overexpressed in cells-

The mechanism supporting cell-to-cell Tau transfer in vivo is not yet understood but a few studies identified exosomes isolated from cell lines as potential transfer vehicles (Simon et al, 2012; Saman et al. 2012). In the context of protein over- expression, cells may activate their own degradation cellular processes. Among them, the multivesicular body (MVB) pathway allowed degradation of proteins into lysosomal vesicles. Two distinct populations of MVBs co-exist in cells, the first one target proteins to lysosomes and the second one, a cholesterol-rich population, don't fuse with lysosomes but drive exosome outside the cells (Raposo et al. 2013). A slight leakage from the MVB may then shuttle Tau outside the cells in exosomal vesicles.

We then wondered if this particular trafficking pathway might be involved in Tau secretion in pathological conditions where Tau accumulated in neurons. To investigate this, we generated a sable cell line overexpressing the full-length 4R human Tau isoform (2+3-10+, hTau46^WT) in N1E-115 cells using the lentiviral technology as described in the material and methods section. After differentiation, extracellular vesicles were isolated from media as described above (Fig. 1) to generate EcEF & ExEF. Exosomes were fractionated from the ExEF using a continuous sucrose gradient and immunoblotted with a human specific antibody against the total Tau (HT7), in order to selectively detect the over-expressed human species from the endogenous Tau.

As shown in rat primary embryonic cortical cells (Fig. 3), exogenous human WT Tau is mainly associated to ectosomes (Fig. 6a). Although degraded and/or dephosphorylated Tau fragments are detected using a total Tau antibody HT7 or an antibody directed against the N-Terminal part of Tau, no signal has been found with an antibody directed against the C-terminal part of Tau. In this context of Tau over- expression, a minor part of human Tau is also found in the exosomal fractions (Fig. 6b, upper panel). We didn't find endogenous murine Tau in fractionated exosomes in the native N1-E115 cells (Fig. 6b, lower panel). The presence of Tau in EcEF and ExEF is not related to cell death as confirmed using a cytotoxic assay (Fig. 4b). On the other hand, the presence of Tau in EcEF suggests that there is a growing secretion of Tau over time (Fig. 4c). EM confirmed a direct association of Tau to vesicles (Fig. 6d).

Conclusion: Thus, in cells over-expressing WT-Tau, Tau is mainly found in ectosomes even though a low percentage of exosome is associated to Tau (Fig. 6e).What is more, those results show that Tau is progressively secreted, and that this secretion is not due to neuronal death.

EXAMPLE 5. Tau is secreted in ISF-associated ectosomes and exosomes when over-expressed in the rat brain.

Taking advantage of our recently lentiviral-mediated rat model of sporadic tauopathy (Caillierez et al, 2013), we demonstrated the transport of Tau into vesicles in vivo. Rats were stereotactically injected with LV-hTau46^WT (n=2) into the hippocampus. Five months p.L, interstitial fluids (ISF) samples were collected by push pull microdialysis from the injection site in order to purify EcEF and ExEF (Fig. 7a). These fractions were analyzed with electron microscopy using an antibody against the N-terminal portion of Tau (N-Ter, upper panels), an antibody against the GMl (GMl, middle panels) or an antibody against the C-terminal portion of Tau (C-Ter, lower panels).

Conclusion: This analysis confirms that in vivo, and in a context of Tau over- expression, Tau is transported into the ISF and can be detected in vesicles. Moreover, the presence of GMl, a specific marker of lipid raft, at the surface of ectosomes and exosomes shows that both vesicles are originating from those specific membrane domains (Fig. 7b).

EXAMPLE 6. Tau is secreted in CSF-associated ectosomes&exosomes when over-expressed in non human primate brain- In this context, we further investigated if vesicular-Tau could be purified from non-human primates CSF samples. In that way, AAVs vectors, encoding either a human WT form of Tau (hTau46^WT, 2+3-10+) or its mutated counterpart (hTau46^{P 01L}, 2+3-10+) were bilaterally injected into the hippocampal formation of one male cynomolgus monkeys (Macaca fascicularis).

Four months post-AAVs delivery, the presence of pathological Tau epitopes was confirmed at the injection sites using AT8, MCI and AT 100 antibodies (data not shown). Indeed, immunological tools are available to distinguish among the different biochemical forms of Tau that characterize pathological Tau stages: the antibody AT8 labels hyperphosphorylated Tau (Mercken et al, 1992), whereas MCI labels conformational changes (Jeganathan et al, 2008) and AT 100 labels aggregated Tau (Allen et al, 2002). Those antibodies were previously validated in our rat model of neurodegenerative Tau pathology (Caillierez et al, 2013).

Three months post-AAVs delivery, CSF and plasma samples were collected to purify EcEFs (Fig8a and b, left panels) and ExEFs (Fig. 8a and b, right panel) and analyzed by electron microscopy using an antibody that recognized the C-terminal part of Tau. In this non-human primate model of Tau over-expression, Tau is mainly found associated to ectosomes (59 to 83 %) regardless the Tau form (WT or mutant Tau) but a minor part of the protein is also detected in exosomes (10 to 16 %) (Fig. 8c).

Thus, the experiment has been successfully reproduced with EcEFs obtained from CSF and plasma samples. Conclusion: Ectosomes can be purified and collected in EcEFs from primates

CSF and plasma samples. The detection of WT Tau or mutant Tau in this compartment opens up the possibility to follow the amount of Tau associated with ectosomes as a novel method for the detection and/or prognosis of Tau-related diseases. EXAMPLE 7. Tau mRNAs are retrieved in ectosomes and exosomes- enriched fractions.

As shown in figure 9 with hTau46^WT, Tau mRNa can be detected both in EcEFs and ExEFs after steps of mRNA purification and reverse transcription.

In addition, primers of sequences SEQ ID NO 15 to 34 are provided with their corresponding %GC contents, melting temperatures (or Tm), as well as amplicon size for each isoform. A. Quantitative PCR (or QPCR)

1) Primer QPCR for the 6 human Tau isoforms

Sense primer: AGGGGGC TGATGGT A A A AC G Tm: 60.03°C %GC: 55 Antisense primer: AGAGCTGGGTGGTGTCTTTG Tm: 59.89°C %GC: 55 Amplicon size: 128bp

2) Pairs of primers for differentiating the 6 human Tau isoforms

3-1 -Isoforms 2+3+

Sense primer: GGAAGATGTGACAGCACCCT Tm: 59.67°C %GC: 55 Antisense primer. TCCTTCTGGGATCTCCGTGT Tm: 59.96°C %GC. 55 Amplicon size for isoforms 2+3+: 85bp

3-2- Isoforms 2+3-

Sense primer: GGCTACACCATGCACCAAGA Tm: 60.32°C %GC: 55 Antisense primer: CTTCAGCTTCCGCTGTTGGA Tm: 60.60°C %GC: 55 Amplicon size for isoforms 2+ 3-: 145 nucleotides

3-3- Isoforms 2-3-

Sense primer: GGCCTGAAAGCTGAAGAAGC Tm: 59.47°C %GC: 55 Antisense primer: CTTCCAGTCCCGTCTTTGCT Tm: 59.96°C %GC: 55 Amplicon size for isoforms 2- 3-: 113 nucleotides

3-4- Isoforms 10+

Sense primer: GGCGGGAAGGTGCAGATAAT Tm: 60.18°C %GC: 55 Antisense primer: ACCTTGCTCAGGTCAACTGG Tm: 59.89°C %GC: 55 Amplicon size for isoforms 10+ : 137 nucleotides

3-5- Isoformes 10-

Sense primer: GC GGGA AGGTGC A A AT AGTC Tm: 59.27 °C %GC: 55 Antisense primer: TTTACTTCCACCTGGCCACC Tm: 59.89 °C %GC: 55 Amplicon size for isoforms 10- : 112 nucleotides B. Semi-quantitative PCR

1) Primers for differentiating between the 6 Tau isoforms

Sense primer: ATGCACCAAGACCAAGAGGG Tm: 59.96°C %GC: 55%

Antisense primer: GAGGTCACCTTGCTCAGGTC Tm: 60.04°C %GC: 60%

Amplicon size for isoforms:

Htau40wt 441AA NM0Q5910 (2+3+10+) : 869bp

Htau39wt 410AA M001203252 (2+3+10-) : 776bp

Htau46 wt 412AA M001123067 (2+3-10+) : 782bp

Htau37 wt 381AA M001203251 (2+3-10-) : 689bp

Htau44wt 352AA M016841 (2-3-10-) : 602bp

Htaif 3wt 383AA NM0i6834 (2-3-10+) : 695bp

2) Primers for differentiating between two or more Tau isoforms

2-1 Tau E2-E10 (ex 2-10): isoforms 2+3-10+ et 2+3+10+

Sense primer: GGCTCTGAAACCTCTGATGC Tm: 60°C

Antisense primer: TTTGAGCCACACTTGGACTG

2-2- Tau 5 ' (ex 1-4): isoforms 2+3+, 2+3- et 2-3- Sense primer: TACGGGTTGGGGGACAGGAAAGAT Tm: 65°C

Antisense primer: GGGGTGTCTCCAATGCCTGCTTCT

2-3- Tau 3 ' (ex 9-13): isoforms 10+ et 10-

Sense primer: CATGCCAGACCTGAAGAATGTCAAG Tm: 65°C

Antisense primer: TCACAAACCCTGCTTGGCCA

EXAMPLE 8. Tau is inside the vesicles.

One may ask if Tau is really inside the vesicles and not stuck on the vesicular surface. In order to demonstrate that Tau is intravesicular, ExEF and EcEF fractionated from N1E-115 cells overexpressing the full-length 4R human Tau isoform (2+3-10+, hlN4R) were treated with increasing NaCl concentrations (from 0.01 to 0.5 M) to remove Tau interacting with the extravesicular surface. Recombinant Tau (hlN4R) was added to extravesicular fractions from naive N1-E115 cells as a control to validate the effect of NaCl treatment. As expected, extravesicular recombinant Tau was removed from plain/naive vesicles whereas NaCl treatment did not affect Tau-immunoreactivity from EcEF/ExEF demonstrating that Tau is indeed inside vesicles or anchored to the internal vesicles membrane (see Figure 10).

Conclusion: This analysis confirms that the measured amount of Tau corresponds to the amount inside vesicles, and not stuck on the vesicular surface.

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SEQUENCE LISTING

SEP ID NO 1 (Tau isoform)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTE DGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIG DTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQK GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTR EPKK V A VVRTPPK SP S S AK SRLQT AP VPMPDLKN VK SKIGS TE LKHQPGGGK VQI INKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPG GGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGG KKIETHKLTFRENAKAKTDH GAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

SEP ID NO 2 (Tau isoform)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTE DGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIG DTPSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQK GQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTR EPKK V A VVRTPPK SP S SAK SRLQT APVPMPDLKNVK SKIGS TENLKHQPGGGK VQI VYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHV PGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDM VDSPQLATLADEVSASLAKQGL

SEP ID NP 3 (Tau isoform)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTE DGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGS DDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGD RSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMP DLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVPGGGSV QIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITH VPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSID MVDSPQLATLADEVSASLAKQGL SEP ID NO 4 (Tau isoform)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTE DGSEEPGSETSDAKSTPTAEAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGS DDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGD RSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPMP DLKNVKSKIGSTE LKHQPGGGKVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQV EVKSEKLDFKDRVQSKIGSLDNITHVPGGG KKIETHKLTFRENAKAKTDHGAEIV YKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL SEP ID NO 5 (Tau isoform)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDT PSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQ ANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPK K V A VVRTPPK SP S S AK SRLQT AP VPMPDLKNVK SKIGS TE LKHQPGGGK VQI V Y KPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPG GG KKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVD SPQLATLADEVSASLAKQGL

SEP ID NP 6 (Tau isoform)

MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKAEEAGIGDT PSLEDEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQ ANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPK KV A VVRTPPK SP S SAK SRLQT AP VPMPDLKNVK SKIGS TENLKHQPGGGK VQIINK KLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGG QVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGA EIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL

SEP ID NP 7 (Tau-epitope N-terminal)

MAEPRQEFEVMEDHAGTYG

SEP ID NP 8 (Tau-epitope N-terminal)

GTYGLGDRK SEP ID NO 9 (Exon 7)

PPGQK SEP ID NP 10 (Tau-epitope C-terminal)

TLADEVSASLAKQGL

SEP ID NP 11

TGGGGGACAGGAAAGA

SEP ID NP 12

CCTCAGATCCGTCCTCAGTG

SEP ID NP 13

AAACGGCTACCACATCCAAG

SEP ID NP 14

CGCTCCCAAGATCCAACTAC SEP ID NP 15

AGGGGGCTGATGGTAAAACG

SEP ID NP 16

AGAGCTGGGTGGTGTCTTTG

SEP ID NP 17

GGAAGATGTGACAGCACCCT

SEP ID NP 18

TCCTTCTGGGATCTCCGTGT SEP ID NO 19

GGCTACACCATGCACCAAGA

SEP ID NO 20

CTTCAGCTTCCGCTGTTGGA

SEP ID NP 21

GGCCTGAAAGCTGAAGAAGC SEP ID NP 22

CTTCCAGTCCCGTCTTTGCT

SEP ID NP 23

GGCGGGAAGGTGCAGATAAT

SEP ID NP 24

ACCTTGCTCAGGTCAACTGG

SEP ID NP 25

GCGGGAAGGTGCAAATAGTC

SEP ID NP 26

TTTACTTCCACCTGGCCACC SEP ID NP 27

ATGCACCAAGACCAAGAGGG

SEP ID NP 28

GAGGTCACCTTGCTCAGGTC

SEP ID NP 29

GGCTCTGAAACCTCTGATGC SEP ID NO 30

TTTGAGCCACACTTGGACTG SEP ID NO 31

TACGGGTTGGGGGACAGGAAAGAT

SEP ID NP 32

GGGGTGTCTCCAATGCCTGCTTCT SEP ID NP 33

CATGCCAGACCTGAAGAATGTCAAG

SEP ID NP 34

TCACAAACCCTGCTTGGCCA

Claims

1. An in vitro method for quantifying Tau protein, or a nucleic acid encoding Tau protein, in an individual, comprising the step of measuring an amount of Tau protein, or an amount of a nucleic acid encoding Tau protein, in an Ectosome-Enriched Fraction (EcEF) from said individual.

2. The in vitro method according to claim 1, comprising a step of measuring an amount of Tau protein in said Ectosome-Enriched Fraction.

3. The in vitro method according to any one of claims 1 and 2, wherein the EcEF is obtained from an individual's biological sample that is selected in a list comprising: a cell lysate, a cell culture supernatant, a cerebrospinal fluid sample, a blood sample, an interstitial fluid sample.

4. The in vitro method according to any one of claims 1 to 3, wherein the individual is a human or a non-human mammal, preferably a human mammal.

5. The in vitro method according to any one of claims 1 to 4, wherein the

EcEF comprises at least 70% of vesicles having a size ranging of 100 nm or more in diameter, relatively to the total amount of vesicles in the said EcEF.

6. The in vitro method according to any one of claims 1 to 5, wherein the Ectosome-Enriched Fraction is depleted of Exosomes.

7. The in vitro method according to any one of claims 1 to 6, wherein the

EcEF comprises less than 30% of vesicles having a size of less than 100 nm in diameter, relatively to the total amount of vesicles contained in the said EcEF.

8. The in vitro method according to claim 1, wherein said Ectosome-Enriched Fraction is obtained by using a method selected in a list comprising Fluorescence- Activated Cell Sorting (FACS) or differential centrifugation.

9. The in vitro method according to claim 1, wherein the Ectosome-Enriched Fraction is obtained by a method comprising a step of submitting the biological sample to a first centrifugation step, so as to obtain a first fraction depleted of cells and apoptotic bodies, and wherein the said first fraction is submitted to a second centrifugation step, so as to obtain the said Ectosome-Enriched Fraction.

10. The in vitro method according to any one of claims 1 to 8, wherein the step of measuring an amount of Tau protein is achieved by incubating said Ectosome-Enriched Fraction with a Tau-binding molecule.

11. The in vitro method according to claim 10, wherein said Tau-binding molecule is an antibody directed against Tau protein, in particular an antibody directed against an epitope of sequence SEQ ID NO 7 to 10.

12. The in vitro method according to any one of claims 1-11, wherein the Tau protein is selected in a list comprising: phosphorylated Tau, non-phosphorylated Tau, Total Tau, a Tau isoform, and/or a selection of Tau isoforms of sequence SEQ ID NO 1 to 6.

13. An in vitro method for detecting the occurrence of a Tau-related disease in an individual, comprising the steps of:

a) measuring an amount of Tau protein in an EcEF from said individual, b) comparing the amount of Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the risk of occurrence of the Tau-related disease in said individual.

14. The in vitro method according to claim 13, wherein the Tau-related disease is selected in a list comprising: Alzheimer's disease, Amyotrophic lateral sclerosis, Parkinson Dementia complex of Guam, Argyrophilic grain dementia, British type amyloid angiopathy, Corticobasal degeneration, Dementia pugilistica/autism with self-injury behaviour and other chronic traumatic injuries, Down's syndrome, FTDP-17, Gerstmann- Straussler-Scheinker disease, Hallenvorden-Spatz disease, Inclusion body myositis, Multisystem atrophy, Myotonic dystrophy type I and type II, Niemann-Pick disease type C, Hereditary inclusion body myopathy with Paget disease of bone and frontotemporal dementia, Parkinson with dementia of Guadeloupe, Pick's disease, Presenile dementia with tangles and calcifications, Prion protein cerebral amyloid angiopathy, Progressive supranuclear palsy, Post-encephalitic parkinsonism, Subacute sclerosing panencephalitis, Tangle only dementia.

15. An in vitro method for determining the efficacy of a treatment of a Tau- related disease in an individual, comprising the steps of:

a) measuring an amount of Tau protein in an EcEF from said individual, b) comparing the amount of Tau protein measured at step a) with a reference value,

c) determining from the comparison performed at step b) the efficacy of said treatment in said individual.