WO2017032871A1

WO2017032871A1 - Method of differential diagnosis of dementia with lewy bodies and parkinson`s disease

Info

Publication number: WO2017032871A1
Application number: PCT/EP2016/070166
Authority: WO
Inventors: Brit Mollenhauer; Anja Schneider
Original assignee: Universitaetsmedizin Goettingen Georg August Universitaet
Current assignee: Universitaetsmedizin Goettingen Georg August Universitaet
Priority date: 2015-08-27
Filing date: 2016-08-26
Publication date: 2017-03-02
Anticipated expiration: 2018-02-27
Also published as: GB201515223D0

Abstract

The present invention is directed to methods of differential diagnosis of dementia with Lewy bodies and/or Parkinson's disease. Methods according to the present disclosure typically comprise a step of isolating exosomes from a sample of cerebrospinal fluid (CSF), and determining the number of exosomes and/or amount of exosomal α-Synuclein in a defined volume of CSF sample.

Description

Method of differential diagnosis of dementia with Lewy bodies

and Parkinson's disease

The present invention is directed to methods of differential diagnosis of dementia with Lewy bodies and/or Parkinson's disease, as defined in the claims. Methods according to the present disclosure typically comprise a step of isolating exosomes from a sample of cerebrospinal fluid (CSF), and determining the number of exosomes and/or amount of exosomal a-Synuclein in a defined volume of CSF sample.

BACKGROUND OF THE INVENTION

Parkinson's disease is a neurodegenerative disorder with motor symptoms which affects approximately 1 % of the population over 65 years. It is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and pathological aggregation of the intrinsically disordered protein a-Synuclein into Lewy bodies (Braak et al., 2003). Parkinson's disease and other neurodegenerative diseases with Lewy body pathology, such as Parkinson's disease dementia and dementia with Lewy bodies are considered as a continuum of a disease spectrum termed Lewy body disorders. Clinically, dementia with Lewy bodies is defined by dementia, visual hallucinations and fluctuating attention within 12 months of Parkinson syndrome onset. In contrast, the onset of dementia later than 12 months after initial motor symptoms specifies Parkinson's disease dementia (McKeith et al., 2005).

Atypical Parkinson syndrome arise generally from other neurodegenerative diseases. These include Multiple System Atrophy (MSA), Progressive supranuclear palsy (PSP), Dementia with Lewy bodies (DLB), Corticobasal degeneration (CBD). Atypical Parkinson syndromes are characterized by a non-responsiveness to dopaminergic therapies, as well as additional symptoms such as dementia, swallowing disorder, speaking disorder, falls, apraxia, and gaze palsy. Hence, a negative dopa test may exclude Parkinson's Disease but cannot differentiate between the atypical Parkinson syndromes.

The accurate distinction between Parkinson's disease, dementia with Lewy bodies and other non-a-Synuclein variants with Parkinson syndrome is challenging due to an overlap of clinical symptoms and neuropathological changes. Several comprehensive studies have identified imaging and fluid biomarkers, including dopamine transporter scans, serum peptide markers, and CSF α-Synuclein (Mollenhauer and Schlossmacher, 2010, Suzuki et al., 2015).

Dementia with Lewy bodies (DLB) differs from other idiopathic Parkinson's Diseases by an early onset of a dementia during course of disease. The distinction is made based on the definition that symptoms of a dementia prior to and within one year after onset of Parkinson syndrome are assigned to a DLB. In addition, DLB may be associated with optical hallucinations, and REM sleeping disorders with lively agitated dreams. Another symptom may be variations of vigilance during course of the day.

Progressive supranuclear palsy (PSP; also referred to as Steele-Richardson-Olszewski syndrome) is a degenerative disease which involves the gradual deterioration and death of specific brain volumes. Because of the slowed movements and gait difficulty, PSP is frequently misdiagnosed as Parkinson's Disease, or as Alzheimer's Disease due to the behavioral changes.

Polyneuropathy is a peripheral neuropathy (disease affecting peripheral nerves) in roughly the same areas on both sides of the body. Patients suffering from polyneuropathy often exhibit weakness, numbness, pins-and-needles, or even burning pain.

CT/MRT are not suggested in the Guidelines for diagnosing an idiopathic Parkinson disease, since it normally shows an age-corresponding normal result. Despite high specificity, the sensitivity of RT examinations is low for atypic Parkinson syndrome (ca. 60%), and dependent on the length of disease.

DAT-Scan/ B /PP-CIT-SPECT can be used to determine the degree of cell loss in the substantia nigra. A distinction between the different Parkinson syndromes is, however, not possible.

IBZM-SPECT can differentiate with moderate specificity and sensitivity between idiopathic Parkinson disease and atypic Parkinson syndrome. Differential diagnosing within atypic Parkinson syndrome is, however, not possible in a reliable way.

Although a-Synuclein does not contain a sorting signal for extracellular release, soluble and aggregated α-Synuclein was detected in tissue culture medium and body fluids, such as brain interstitial fluid, plasma and CSF (El-Agnaf et al., 2003, Lee et al., 2005, El-Agnaf et al., 2006, Tokuda et al., 2010, Emmanouilidou et al., 2011 , Hansson et al., 2014, Lee et al., 2014). Extracellular α-Synuclein was subsequently studied as a potential diagnostic biomarker, especially in the CSF, where the majority of α-Synuclein is derived from the CNS central nervous system rather than from peripheral blood (Mollenhauer et al., 2012). Most studies have shown a reduction of CSF α-Synuclein levels in Parkinson's disease and dementia with Lewy bodies (Tokuda et al., 2006, Hong et al., 2010, Mollenhauer et al., 201 1 ), however, conflicting results with either no differences compared to controls or even increased levels of extracellular α-Synuclein were reported (Noguchi-Shinohara et al., 2009, Ohrfelt et al., 2009, Reesink et al., 2010). In addition, the sensitivity and specificity of CSF α-Synuclein to distinguish Parkinson's disease or dementia with Lewy bodies from non a-Synuclein-related Parkinson syndrome and other neurological controls are low and up to date α-Synuclein has not been approved as a biomarker for clinical applications (Gao et al., 2014). Moreover, determination of a-synuclein in CSF (not exosomal CSF) is error-prone due to contamination with blood, which contains high levels of a-synuclein, which is introduced into the CSF during punctuation.

Recently, exosomes have been implicated in the dissemination of misfolded proteins in a variety of neurodegenerative disorders, including Parkinson's disease (Bellingham et al., 2012, Schneider and Simons, 2013). Exosomes are extracellular vesicles of 40-120 nm diameters which are released from various cells including neurons. However, it is not known whether exosomal a-Synuclein exists in the central nervous system in vivo.

Exosomal a-Synuclein was recently characterized in plasma samples from a large cohort of patients with Parkinson's disease and healthy controls. There, exosomes derived from the central nervous system were immune-captured by an antibody directed against the neural L1 cell adhesion molecule L1 CAM (Shi et al., 2014). Using this approach and in contrast to the findings shown in Comparative Example 4 herein, Shi et al. report increased amounts of exosomal α-Synuclein in Parkinson's disease compared to healthy controls.

In the context of Parkinson's Disease, WO 2010/056337 suggests the isolation of exosomes from blood, serum, CSF, and urine (Table 1 ), in order to conduct a mutational analysis of α-Synuclein (Figure 40a).

WO 2014/059052 acknowledges that there has not been a single effort attempted to analyse exosomes derived from Parkinson's Disease patients and controls, isolated from either CSF, urine, or plasma. The authors suggest to tackle urine exosomes first, and then move on to other types of samples which allow to investigate other proteins like tau and a- Synuclein.

Danzer et al. (2012) found that α-Synuclein oligomers can be found in different extracellular fractions in association with exosomes or as exosome free oligomers; and that α-Synuclein oligomers are more toxic to recipient cells compared to free a-synuclein oligomers.

There is currently no assay or imaging method available that provides security for clinical diagnosis of Parkinson's Disease and atypic Parkinson syndrome, respectively. As a consequence, atypic Parkinson's syndromes are often misdiagnosed as idiopathic Parkinson Disease, which may lead to long-lasting mistherapy.

There is still a need in the art for methods of differential diagnosis of dementia with Lewy bodies and/or Parkinson's disease.

SUMMARY OF THE INVENTION

The afore mentioned obstacles are overcome by isolation of exosomes from cerebrospinal fluid (CSF), which are then subjected to further analysis such as determination of the amount of α-Synuclein, as well as the concentration of exosomes (number of exosomes present in a defined volume). At the same time, the present disclosure addresses two major aspects: (1) the inventors provide the first comprehensive analysis of exosomal a- Synuclein in CSF from patient cohorts; and (2) the inventors show that CSF exosomes from patients with Parkinson's disease and dementia with Lewy bodies contain a pathogenic α-Synuclein species which serves as a seed to induce the oligomerisation of soluble α-Synuclein in recipient cells. Accordingly, CSF exosomal α-Synuclein may well be suited as a diagnostic marker.

More specifically, the inventors isolated exosomes from CSF from patients with Parkinson's disease, dementia with Lewy bodies, progressive supranuclear palsy as a non a-Synuclein-related disorder that clinically overlaps with Parkinson's disease, and neurological controls. CSF exosome numbers, a-Synuclein protein content of CSF exosomes and their potential to induce oligomerisation of α-Synuclein were analyzed. The quantification of CSF exosomai α-Synuclein showed distinct differences between patients with Parkinson's disease and dementia with Lewy bodies. It was also different compared to progressive supranuclear palsy patients and neurological controls, altogether with a sensitivity and specificity of up to 86%. In addition, exosomai α-Synuclein levels correlated with the severity of cognitive impairment in cross-sectional samples from patients with dementia with Lewy bodies. Importantly, CSF exosomes derived from Parkinson's disease and dementia with Lewy bodies induce oligomerisation of α-Synuclein in a reporter cell line in a dose-dependent manner. The present data in the examples suggest that CSF exosomes from patients with Parkinson's disease and dementia with Lewy bodies contain a pathogenic species of α-Synuclein which initiates oligomerisation of soluble a-Synuclein in target cells and reflect disease pathology.

The present disclosure provides a method of diagnosing dementia with Lewy bodies, comprising the steps of:

(a) isolating exosomes from a defined volume of a sample of cerebrospinal fluid (CSF), wherein the sample originates from a subject to be diagnosed; and

(b) determining the amount of exosomai α-Synuclein protein in the exosomes obtained in step (a) and/or

determining the number of exosomes obtained in step (a); and

(c) comparing the results obtained in step (b) with results obtained from appropriate controls;

wherein a significantly lower amount of exosomai α-Synuclein protein as compared to said appropriate controls is indicative for dementia with Lewy bodies, and wherein a significantly lower number of exosomes as compared to neurological controls is indicative for dementia with Lewy bodies.

Further provided is a method of differential diagnosis of dementia with Lewy bodies and Parkinson's disease, comprising the steps of:

(a) isolating exosomes from a defined volume of a sample of cerebrospinal fluid (CSF), wherein the sample originates from a subject to be diagnosed;

(b) determining the number of exosomes obtained in step (a); and

wherein

(i) a significantly lower number of exosomes as compared to said appropriate controls is indicative for dementia with Lewy bodies; and

(ii) a significantly higher number of exosomes as compared to said appropriate controls is indicative for Parkinson's disease. Finally, there is also disclosed a method of diagnosing Parkinson's disease, comprising the steps of:

(a) isolating exosomes from a defined volume of a sample of cerebrospinal fluid (CSF), wherein the sample originates from a subject to be diagnosed: and

(b) determining the number of exosomes obtained in step (a); and

wherein a significantly higher number of exosomes as compared to said appropriate controls is indicative for Parkinson's disease.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(b) determining the amount of exosomal S-synuclein protein in the exosomes obtained in step (a) and/or

determining the number of exosomes obtained in step (a); and

wherein a significantly lower amount of exosomal a-Synuclein protein as compared to said appropriate controls is indicative for dementia with Lewy bodies (preferably at least 0.1 - fold reduction, more preferably at least 0.2-fold reduction, more preferably at least 0.3-fold reduction, even more preferably at least 0.4-fold reduction, most preferably 0.5-fold or higher reduction), and wherein a significantly lower number of exosomes as compared to said appropriate controls is indicative for dementia with Lewy bodies (preferably at least 0.1 -fold reduction, more preferably at least 0.2-fold reduction, more preferably at least 0.3- fold reduction, even more preferably at least 0.4-fold reduction, most preferably 0.5-fold or higher reduction). Typical values indicative for DLB will be in the range of 3-12 pg/ml CSF, preferably 4-1 1 pg/ml CSF, more preferably 5-10 pg/ml CSF, and most preferably 6-9 pg/ml CSF.

Preferably, step (b) comprises both determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a) and determining the number of exosomes obtained in step (a).

The above method is not only suitable for diagnosing DLB, but also for differential diagnosing of DLB and Parkinson's disease. Therefore, the present disclosure also provides a method of differential diagnosis of dementia with Lewy bodies and Parkinson's disease, comprising the steps of:

(b) determining the number of exosomes obtained in step (a); and

wherein

(i) a significantly lower number of exosomes as compared to said appropriate controls is indicative for dementia with Lewy bodies (preferably at least 0.1 -fold reduction, more preferably at least 0.2-fold reduction, more preferably at least 0.3-fold reduction, even more preferably at least 0.4-fold reduction, most preferably 0.5-fold or higher reduction; typical values indicative for DLB are 0.5-1.5*10^A9/ml, in particular 0.6-1.4*10^A9/ml, more particularly 0.7-1.3^*10^A9/ml, such as 0.8- 1 .2*10^A9/ml); and

(ii) a significantly higher number of exosomes as compared to said appropriate controls is indicative for Parkinson's disease. Preferably, the number of exosomes is more than 0.5-fold higher, more preferably more than 1.0-fold higher, even more preferably more than 1.5-fold higher, and most preferably 2.0-fold or even higher as compared to said appropriate controls. Typical values indicative of PD are 2.5-

4.5*10^A9/ml, in particular 4.2-2.7*10^A9/ml, more particularly 4.0-3.0*10^A9/ml, such as 3.2-3.8^*10^A9/ml.

At the same time, the number of exosomes are at least two-fold increased, preferably 2.5- fold increased, more preferably 3.0-fold increased, even more preferably 3.5-fold increased, and in particular 4.0-fold or more increased as compared to the number of exosomes obtained from the same defined volume of a subject suffering from DLB.

Like in the method for diagnosing DLB, the method of differential diagnosis may further comprise the steps of

(b') determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a); and

(c') comparing the results obtained in step (b^:) with results obtained from said appropriate controls;

wherein a significantly lower amount of exosomal α-Synuclein protein as compared to said appropriate controls is indicative for dementia with Lewy bodies. For details, see above with regard to the method for diagnosing DLB. _,

As demonstrated in the Examples, it is possible to calculate a quotient from the amount of exosomal a-Synuciein protein in the exosomes, and the number of exosomes obtained from the defined volume of CSF sample in step (a). Said quotient is less prone to variation, and was shown to be indicative for Parkinson's disease. Hence, if both the amount of exosomal a-Synuclein protein in the exosomes, and the number of exosomes is determined, the method can comprise the additional steps of

(b") determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a), and calculating the quotient of [amount of exosomal α-Synuclein protein in the exosomes obtained in step (a) / number of exosomes obtained in step (a)]; and

(c") comparing the quotient obtained in step (b") with quotients obtained from said appropriate controls;

wherein a significantly lower quotient as compared to said appropriate controls is indicative for Parkinson's disease. For example, the quotient (also referred to as ratio in the examples) is reduced at least 0.5-fold, more preferably at least 1.0-fold, even more preferably at least 1.5-fold, still more preferably at least 2.0-fold, such as at least 2.5-fold, or most preferably at least 3.0-fold. Typical values indicative for Parkinson's disease are 0.1 -0.9 10^Λ-8 pg/number of exosomes per ml CSF, in particular 0.2-0.8 10^Λ-8 pg/number of exosomes per ml CSF, more particularly 0.3-0.7 10^Λ-8 pg/number of exosomes per ml CSF, such as 0.4-0.6 10^Λ-8 pg/number of exosomes per ml CSF. Typical values obtained from DLB patients are 1.0-2.0 10^Λ-8 pg/number of exosomes per ml CSF, in particular 1.1- 1.9 10^Λ-8 pg/number of exosomes per ml CSF, more particularly 1.2-1.8 10^Λ-8 pg/number of exosomes per ml CSF, such as 1.3-1.7 10^Λ-8 pg/number of exosomes per ml CSF.

In order to render the results comparable, it is necessary to know the volume of the CSF sample, i.e. the volume is defined. The defined volume of said sample of cerebrospinal fluid may be 0.5 ml to 10 ml (at least for humans it is not recommended to take higher sample volumes at a single puncture), and preferably 0.5 ml to 5 ml, more preferably 0.5 ml to 2 ml, and most preferably 0.5 ml to 1 ml. However, upon optimization of the assay, it may also be feasible to use smaller sample volumes such as 100 μΙ, 200 μΙ, 300μΙ or 400 pi. Preferably, the CSF sample is was centrifuged at 2000 x g for 10 minutes at room temperature following lumbar puncture, and the sample can be stored frozen at -80°C, in order to be thawed later on ice prior to step (a). In order to avoid false results due to blood contamination, CSF samples exhibiting erythrocyte counts < 50/mm³ shall not be used.

Generally, it is expected that the method is suitable for all kind of vertebrates. In preferred embodiments, the subject is a mammal, such as a human, mouse, rat, dog, monkey. Non- human transgenic or spontaneous or chemical or virus mediated animal disease models, such as Parkinson's disease animal models, are also contemplated. For small subjects, such as mice or rats, it may be necessary to combine more than one CSF sample in order to obtain a sufficient volume. The combined sample may origin from the same animal, or from the same (treatment) group of animals, if the method is applied in clinical animal studies. If the combined sample is from the same animal, the time between the two punctures should be kept as short as possible.

The appropriate controls may be selected from neurological controls and healthy controls. Unpublished data shows that the absolute numbers obtained for healthy controls are not too much different from neurological controls. However, healthy controls are usually not contained in the biobank collection for ethical reasons. Accordingly, another suitable control group are neurological controls. These controls include subjects with polyneuropathy and without indication of Parkinson's disease, and/or subjects with progressive supranuclear palsy.

Step (a) of the methods of the invention comprises at least one step of ultracentrif Ligation, in particular wherein said ultracentrifugation step comprises centrifuging at at least 100,000 x g for at least 50 minutes, in particular centrifuging at 100,000 x g for 60 minutes. Preferably, the ultracentrifugation step is preceded by at least two centrifugation steps at 3000-4000 x g, at least one centrifugation step at 4000-5000 x g, and at least one centrifugation step at 10000-20,000 x g. In a particular preferred embodiment, the ultracentrifugation step is preceded by one centrifugation step at 3500 x g, by two centrifugation steps at 4500 x g, and by one centrifugation step at 10,000 x g. The centrifugation steps at 3000-5000 x g are carried out preferably for 5-20 minutes, more preferably for 10-15 minutes. The centrifugation steps at 7000-20,000 x g, in particular 10,000-20,000 x g, are preferably carried out for 20-45 minutes, more preferably 25-40 minutes, such as for 30 minutes. In still another preferred embodiment, prior to preceding to step (b) the ultracentrifugation pellet is washed once, preferably using phosphate buffered saline (PBS), followed by centrifugation at 80,000-120,000 x g, preferably 90,000-1 10,000 x g, more preferably at 100,000 x g, for 50-70 minutes, preferably 55-65 minutes, more preferably 60 minutes. It is further advantageous to dissolve the ultracentrifugation pellet in PBS or HEPES buffer containing a surfactant which does not interfere with the integrity of the exosome membranes prior to any subsequent analysis. Preferably the surfactant is a non-ionic surfactant such as Tween 20, Tween 80, Triton-X 100, DDM or digitonin, preferably Tween 20 or Tween 80, more preferably Tween 20. A suitable concentration of the surfactant is 0.005-0.1%, preferably 0.01 -0.075%, more preferably 0.02-0.05%, e.g. 0.025%.

Alternatively, or in addition, it is also contemplated that step (a) comprises at least one step of size exclusion chromatography. To that end, the exosomes isolated in step (a) are characterized by a size of 20 nm to 170 nm, in particular 25 nm to 160 nm, more _

particularly 30 nm to 150 nm, more particularly 35 nm to 140 nm, more particularly 40 nm to 130 nm, such as 40 nm to 120 nm. Usually, step (a) is carried out at 4°C.

Quality and/or purity of the exosome preparation can be controlled by several assays as known in the art and further described in the experimental section herein. For example, the methods of the present disclosure may further comprising a step of testing the exosomes obtained in step (a) for one or more selected from (a) presence of flotillin-2, (b) absence of IgG heavy and/or light chains, and (c) absence of calnexin. Typically, such testing is carried out by Western Blotting.

Concerning step (b), (b') and (b") of the methods of the present disclosure, determining the number of exosomes obtained in step (a) can be made by using Nanoparticle tracking analysis (NTA). NTA, and suitable devices therefor are generally known in the art. Alternatively, the number of exosomes obtained in step (a) may also be determined using standard methods in the art such as Western Blotting, electron microscopy, ExoELISA, or ELISA.

Preferably, due to its high sensitivity, the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a) comprises electrochemiluminescence based ELISA using an a-Synuclein specific antibody or antibody fragment. More details with regard to this steps, such as suitable antibodies, can be found in the experimental section herein.

In still an alternative of the presently disclosed methods, the levels of exosomal a- Synuclein may be correlated with total exosomal protein content. In this case, the method may comprise the step of measuring the total protein content of the exosomes obtained in step (a), preferably by an assay selected from BCA or Bradford. However, any suitable assay for determining the total protein content can be applied in the presently disclosed methods.

The diagnostic methods of the present disclosure are particularly advantageous for backing-up a clinical diagnosis. Accordingly, the method may further comprise testing for McKeith consensus criteria indicative for dementia with Lewy bodies, as described in in McKeith et al. Neurology 2005; 6(12): 1863-1872; or determining the subject's Mini Mental State Examination (MMSE) scores. As shown in the experimental section, it could be shown that the results of the presently disclosed methods correlate well with MMSE scores. In another embodiment, the method may further comprise testing for UK-Brain Bank criteria indicative for Parkinson's disease.

Finally, the present disclosure also provides a method of diagnosing Parkinson's disease, comprising the steps of:

(b) determining the number of exosomes obtained in step (a); and (c) comparing the results obtained in step (b) with results obtained from appropriate controls;

wherein a significantly higher number of exosomes as compared to said appropriate controls is indicative for Parkinson's disease. The method may further comprise the steps of:

(b") determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a), and calculating the quotient of [amount of exosomal a-Synuclein protein in the exosomes obtained in step (a) / number of exosomes obtained in step (a)]; and

(c") comparing the quotient obtained in step (b") with quotients obtained from said

appropriate controls;

wherein a significantly lower quotient as compared to said appropriate controls is indicative for Parkinson's disease. Further preferred embodiments are as described above. In particular, appropriate controls may be selected from neurological controls and healthy controls. Neurological controls can include subjects with polyneuropathy and without indication of Parkinson's disease, and/or subjects with progressive supranuclear palsy. Non-human transgenic or spontaneous or chemical or virus mediated animal disease models, such as Parkinson's disease animal models, are also contemplated. Finally, determining the amount of exosomal α-Synuclein protein in the exosomes obtained in step (a) preferably comprises electrochemiluminescence based ELISA using an α-Synuclein specific antibody or antibody fragment, as further detailed above and in the experimental section.

The invention is further described by the following embodiments.

1. A method of diagnosing dementia with Lewy bodies, comprising the steps of:

(a) isolating exosomes from a defined volume of a sample of cerebrospinal fluid

(CSF), wherein the sample originates from a subject to be diagnosed; and (b) determining the amount of exosomal α-Synuclein protein in the exosomes obtained in step (a) and/or

determining the number of exosomes obtained in step (a); and

(c) comparing the results obtained in step (b) with results obtained from

appropriate controls;

wherein a significantly lower amount of exosomal α-Synuclein protein as compared to said appropriate controls is indicative for dementia with Lewy bodies, and wherein a significantly lower number of exosomes as compared to said appropriate controls is indicative for dementia with Lewy bodies. - -

A method of differential diagnosis of dementia with Lewy bodies and Parkinson's disease, comprising the steps of:

(b) determining the number of exosomes obtained in step (a): and

(c) comparing the results obtained in step (b) with results obtained from

appropriate controls;

wherein

(ii) a significantly higher number of exosomes as compared to said appropriate controls is indicative for Parkinson's disease.

The method of embodiment 2, further comprising the steps of:

(c') comparing the results obtained in step (b') with results obtained from said appropriate controls;

wherein a significantly lower amount of exosomal α-Synuclein protein as compared to said appropriate controls is indicative for dementia with Lewy bodies.

The method of embodiment 2 or 3, further comprising the steps of:

(b") determining the amount of exosomal α-Synuclein protein in the exosomes obtained in step (a), and calculating the quotient of [amount of exosomal a- Synuclein protein in the exosomes obtained in step (a) / number of exosomes obtained in step (a)]; and

(c") comparing the quotient obtained in step (b ") with quotients obtained from said appropriate controls;

wherein a significantly lower quotient as compared to said appropriate controls is indicative for Parkinson's disease.

The method of any preceding embodiment, wherein the appropriate controls are selected from neurological controls and healthy controls, preferably wherein the appropriate controls are neurological controls, more preferably wherein said neurological controls are subjects with polyneuropathy and without indication of Parkinson's disease, and/or subjects with progressive supranuclear palsy.

The method of any preceding embodiment, wherein step (a) comprises at least one step of ultracentrifugation, in particular wherein said ultracentrifugation step - -

comprises centrifuging at at least 100,000 x g for at least 50 minutes, in particular centrifuging at 100,000 x g for 60 minutes.

. The method of embodiment 6, wherein the ultracentnfugation step is preceded by at least two centrifugation step at 3000-4000 x g, at least one centrifugation step at 4000-5000 x g, and at least one centrifugation step at 10000-20,000 x g.

. The method of embodiment 7, wherein the ultracentnfugation step is preceded by one centrifugation step at 3500 x g, by two centrifugation steps at 4500 x g, and by one centrifugation step at 10,000 x g.

. The method of any one of embodiments 7-8, wherein the centrifugation steps at 3000-5000 x g are carried out for 5-20 minutes, preferably 10-15 minutes.

0. The method of any one of embodiments 7-9, wherein the centrifugation steps at 7000-20,000 x g, in particular 10,000-20,000 x g are carried out for 20-45 minutes, preferably 25-40 minutes, such as for 30 minutes.

1. The method of any one of embodiments 6-10, wherein the ultracentrifugation pellet is washed once, followed by centrifugation at 80,000-120,000 x g, preferably

90,000-1 10,000 x g, more preferably at 100,000 x g, for 50-70 minutes, preferably 55-65 minutes, more preferably 60 minutes, most preferably using phosphate buffered saline (PBS).

12. The method of any one of embodiments 1-5, wherein step (a) comprises at least one step of size exclusion chromatography.

13. The method of any preceding embodiment, wherein step (a) is carried out at 4°C.

14. The method of any preceding embodiment, wherein the exosomes isolated in step

(a) are characterized by a size of 20 nm to 170nm, in particular 25 nm to 160 nm, more particularly 30 nm to 150 nm, more particularly 35 nm to 140 nm, more particularly 40 nm to 130 nm, such as 40 nm to 120 nm.

15. The method of any preceding embodiment, further comprising a step of testing the exosomes obtained in step for one or more selected from (a) presence of flotillin-2,

(b) absence of IgG heavy and/or light chains, and (c) absence of calnexin.

16. The method of embodiment 15, wherein said test comprises Western Blotting. 17. The method of any preceding embodiment, wherein the defined volume of said sample of cerebrospinal fluid is 0.5 ml to 10 ml, preferably 0.5 ml to 5 ml, more preferably 0.5 ml to 2 ml, and most preferably 0.5 ml to 1 ml.

18. The method of any preceding embodiment, wherein said CSF sample was

centrifuged at 2000 x g for 10 minutes at room temperature following lumbar puncture.

19. The method of embodiment 19, wherein the centrifuged sample is frozen at -80°C and later thawed on ice prior to step (a). The method of any preceding embodiment, wherein said CSF sample in step (a) exhibits erythrocyte counts < 507mm³.

The method of any preceding embodiment, wherein the subject is a vertebrate, in particular wherein the subject is a mammal, such as a human, mouse, rat, dog, monkey, or a non-human transgenic or spontaneous or chemical or virus mediated animal Parkinson's disease model.

The method of any preceding embodiment, wherein determining the number of exosomes obtained in step (a) comprises Nanoparticle tracking analysis (NTA). The method of any one of embodiments 1-21 , wherein determining the number of exosomes obtained in step (a) comprises Western Blotting, electron microscopy, ExoELISA, or ELISA.

The method of any preceding embodiment, wherein the method further comprises the step of measuring he total protein content of the exosomes obtained in step (a), preferably by an assay selected from BCA, Bradford.

The method of any one of embodiments 1 , and 3-24, wherein determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a) comprises electrochemiluminescence based ELISA using an a-Synuclein specific antibody or antibody fragment.

The method of any preceding embodiment, wherein the method further comprises testing for McKeith consensus criteria indicative for dementia with Lewy bodies, as described in in McKeith et al. Neurology 2005; 6(12): 1863-1872.

The method of any preceding embodiment, wherein the method further comprises determining the subject's Mini Mental State Examination (MMSE) scores.

The method of embodiments 2-27, wherein the method further comprises testing for UK-Brain Bank criteria indicative for Parkinson's disease.

A method of diagnosing Parkinson's disease, comprising the steps of:

(b) determining the number of exosomes obtained in step (a); and

(c) comparing the results obtained in step (b) with results obtained from

appropriate controls;

The method of embodiment 29, further comprising the steps of:

(b") determining the amount of exosomal α-Synuclein protein in the exosomes obtained in step (a), and calculating the quotient of [amount of exosomal a- Synuclein protein in the exosomes obtained in step (a) / number of exosomes obtained in step (a)]; and (c") comparing the quotient obtained in step (b") with quotients obtained from said appropriate controls;

31. The method of embodiment 29 or 30, wherein the appropriate controls are selected from neurological controls and healthy controls, preferably wherein the appropriate controls are neurological controls, more preferably wherein said neurological controls are subjects with polyneuropathy and without indication of Parkinson's disease, and/or subjects with progressive supranuclear palsy.

32. The method of any one of embodiments 29-31 , wherein the method is further

defined as in embodiments 6-24, and 28.

33. The method of embodiment 30, wherein determining the amount of exosomal a- Synuclein protein in the exosomes obtained in step (a) comprises

electrochemiluminescence based ELISA using an a-Synuclein specific antibody or antibody fragment.

In the following, the present invention as defined in the claims is further illustrated by the following figures and examples, which are not intended to limit the scope of the present invention. All references cited herein are explicitly incorporated by reference. DESCRIPTION OF THE FIGURES

Figure 1 : Isolation and characterization of exosomes from CSF. (A) Exosomes were isolated by subsequent centrifugation rounds including a final 100.000xg ultracentrifugation step from a CSF volume of 0.5 ml. Exosome numbers and a-Synuclein content were quantified in both, total CSF and exosome fractions, by nanoparticle tracking analysis and by electrochemoluminescence assay. (B) For Western blot (WB) analysis exosome pellets were prepared from 2.5 ml of CSF and resuspended in 20 μΙ of sample buffer. CSF was diluted 1 :5 in sample buffer and 20 μΙ of the exosome preparation and 20 μΙ of total CSF were probed with an antibody against the exosomal marker protein Flotillin- 2 (left panel). As a negative control, we probed exosome preparations and CSF with a secondary antibody against human IgG (middle panel). To rule out microsomal contamination of the exosome preparation, 20 μΙ of the exosome pellet, total CSF and of a cell lysate (C.L.) of mouse neuroblastoma N2a cells were blotted and incubated with an antibody against the endoplasmatic reticulum protein Calnexin (right panel). (C) Exosome numbers were determined by nanoparticle tracking analysis (NTA) in both, total CSF and the 100.000 x g exosome pellet derived from 0.5 ml CSF after resuspension in PBS. A representative plot depicting vesicle size and number of vesicles is shown. In contrast to the total CSF sample, the exosome preparation shows a peak between 20 nm and 170 nm peak (peak value: 102 nm). The values were adjusted for the respective dilution factors and calculated to represent the absolute vesicle numbers in 1 ml of CSF and in exosomes derived from the same CSF volume. (D) The coefficient of variance (CV) was determined for left: the number of exosomes in the exosome preparation (left column) and in total CSF (right column), for middle: the amount of exosomal a-Synuclein protein (left column) and total CSF α-Synuclein protein (right column), and right: for the ratio of exosomal α-Synuclein protein levels to the number of exosomes in the exosome preparation (left column) and total CSF (right column). CSF samples from 2 different patients were analyzed in replicates of n=3 and n=4.

Figure 2: Characterisation of exosomal α-Synuclein in dementia with Lewy bodies, Parkinson's disease and non a-Synuclein-related disease controls in the Kassel cohort. (A) Quantification of exosomal α-Synuclein protein content. Exosomes were prepared from of CSF samples from patients with dementia with Lewy bodies (DLB, very left bar), Parkinson's disease (PD, middle left bar), polyneuropathy (PNP, middle right bar) and progressive supranuclear palsy (PSP, very right bar). N=28 DLB, n=37 PD, n=15 PNP control, n=25 PSP control. ^* p<0.05, ** p<0.005, *** p<0.0005, 2-sided student's t-test. For ROC curve analysis please see Figure 3 and Table 2. (B) Correlation of CSF exosomal α-Synuclein with impaired cognitive function in patients with dementia with Lewy bodies (DLB). Cognitive state was measured by M SE (lower scores indicate a decrease in cognitive function) (n=17, Pearson's correlation coefficient r=-0.513, p=0.035, 2-tailed probability). (C) The number of exosomes per ml CSF was quantified by nanoparticle tracking analysis in CSF from patients with dementia with Lewy bodies (DLB) (very left bar), Parkinson's disease (PD) (middle left bar), polyneuropathy (PNP) (middle right bar) and progressive supranuclear palsy (PSP) (very right bar) (n=22 DLB, n=37 PD, n=15 PNP, n=24 PSP, * p<0.05, ** pO.005, *** p<0.0005, 2-sided student's t-test). (D) Ratio of exosomal α-Synuclein to number of exosomes in CSF from patients with DLB (very left bar), PD (left bar), PNP (middle right bar) and PSP (very right bar) (n=22 DLB, n=37 PD, n=15 PNP, n=24 PSP, * p<0.05, 2-sided student's t-test. For ROC curve analysis please see Figure 3 and Table 3.

Figure 3: Receiver operating characteristic (ROC) analysis, Kassel cohort (A) ROC curve analysis was performed to evaluate the diagnostic performance of CSF exosomal a- Synuclein to distinguish dementia with Lewy bodies from Parkinson's disease (Area under the curve AUC=0.883, p<0.001). (B) ROC curve of the ratio CSF exosomal α-Synuclein to number of exosomes to distinguish dementia with Lewy bodies from Parkinson's disease (AUC=0.665, p=0.025). (C) ROC curve of CSF exosomal α-Synuclein to distinguish dementia with Lewy bodies from polyneuropathy (AUC=0.804, p=0.001) and (D) to distinguish dementia with Lewy bodies from progressive supranuclear palsy (AUC=0.864, p=0.001 ). (E) ROC curve of CSF exosomal α-Synuclein to distinguish dementia with Lewy bodies from polyneuropathy (AUC=0.741 , p=0.007) and (F) from progressive supranuclear palsy (AUC=0.906, p<0.001 ). For sensitivity, specificity, positive and predictive values, please refer to Tables 2 and 3.

EXAMPLES

All used substances were Ph. Eur. Grade or of comparable quality. The buffers were prepared with purified and de-ionized water.

Material and methods

Reagents.

Primary antibodies were anti-Flotillin-2 (BD Biosciences), anti-Calnexin (StressGene), anti-a-Synuclein (Invitrogen), anti-a-Synuclein clone 42/a-Synuclein (BD Transduction Laboratories, Heidelberg, Germany). JF-1 clone 12.1 was kindly provided by Dr. Liyu Wu, Epitomics, Burlingame, USA. Secondary antibodies against mouse, rabbit and human IgG were obtained from Dako, Dianova and Invitrogen.

Plasmids

a-Synuclein hGLud (S1) and a-Synuclein hGLuc2 (S2) constructs were described previously (Outeiro et al., 2008).

CSF collection

CSF specimens were collected at the Paracelsus-Elena Klinik in Kassel and the Goettingen University Memory Clinic, Department of Psychiatry, Germany (IRB approval by the local board of Hessen, Germany, IRB 09/07/04 and 26/07/02 and by the Ethics committee of the University Medical Center, Gottingen, IRB 02/05/09) between 2009 and 2012 by lumbar puncture between 9 and 12 a.m. Specimens were collected in polypropylene tubes and centrifuged at 2,000xg for 10 minutes at room temperature, aliquoted and frozen at -80°C within 30 min of the procedure's completion (Mollenhauer et al., 201 1). Samples with erythrocyte counts > 50/mm³ were excluded. All samples were obtained in accordance with the ethical standards laid down in the1964 Declaration of Helsinki.

The cross-sectional Kassel cohort

The cross-sectional Kassel cohort is described in detail in (Mollenhauer et al., 201 1 ). All Parkinson's disease patients fulfilled UK-Brain Bank criteria. Dementia with Lewy body patients were diagnosed according to McKeith consensus criteria (McKeith et al., 2005). Progressive supranuclear palsy patients fulfilled the National Institute of Neurological Disorders and Stroke-Society for Progressive Supranuclear Palsy (NINDS-SPSP) criteria - - for possible or probable disease (Litvan et a!., 1996). None of the neurological control patients suffered from Parkinson's disease or dementia.

Purification of exosomes from CSF

Exosomes were isolated as described before (Strauss et al., 2010). CSF was thawed on ice and subjected to subsequent centrifugation steps at 4°C: 3,500xg 10 minutes, 2 times

4,500xg for 10 minutes, 10,000xg for 30 minutes and 100,000xg for 60 minutes (see Fig.

1A). The 100,000xg pellet was washed once with PBS before resuspension of the pellet.

Exosomes from human CSF for Western blot analysis were prepared from 2.5-5 ml of

CSF and resuspended in Lammli buffer. Exosomes for electrochemiluminescence measurements were dissolved in 1 % CHAPS, 5 mM EDTA, 50 mM Tris-HCI, pH 8.0 lysis buffer. Total CSF was diluted in 2x lysis buffer.

Nanoparticle tracking analysis (NTA)

Exosomes in CSF or in the ultracentrifugation pellet were analysed by nanotracking analysis with a NanoSight LM14 instrument equipped with a 532 nm laser (NanoSight Ltd., Amesbury, United Kingdom). Total CSF samples were diluted 1 :40 in PBS (Gibco) to a final volume of 400 μΙ prior to analysis. 100.000xg pellets derived from 0.5 ml total CSF were resuspended in 50 μΙ PBS and diluted 1 :40 in PBS. Samples were measured in triplicates for 30 s. Particle numbers were then analysed with the Nanoparticle Tracking Analysis (NTA) 2.3 software.

The reliability of the assay can be further improved by pipetting 500 μΙ PBS or HEPES buffer containing 0,025% Tween-20 onto the ultra centrifugation pellet after ultra centrifugation, and mixing at room temperature for at least 1 min until the pellet is fully suspended. Afterwards, particle content is measured at room temperature using Nanosight LM-10. Suspension times of 1 h and 16h were also tested and revealed that for 16h incubation time a slight reduction of particle counts has been observed.

ElectroGhemiluminescence assay fo g-Synuelein quantification

Quantification of a-Synuclein protein in CSF and in exosomes prepared from CSF was performed as described (Kruse et al., 2012). Standard 96-well plates (Meso Scale Discovery, Gaithersburg, USA) were coated overnight at 4°C with 3 g/ml antibody MJF-1 clone 12.1 in PBS. After three times washing with 150 μΙ PBS + 0.05% Tween-20 plates were blocked with 150 μΙ 1 % BSA (Meso Scale Discovery) for one hour under shaking at 300 rpm at room temperature. After three times washing recombinant a-Synuclein standards (kindly provided by Dr. Omar el-Agnaf, United Arab Emirates University, Al Ain, United Arab Emirates) and samples were applied in duplicates for 1 hour at room- temperature and under shaking at 700 rpm. After three times washing, Sutfo-TAG-labelied anti-a-Synuclein clone 42 (1 g/ml) was added for 1 hour at room temperature and 700 rpm shaking. After three times washing 2x Read Buffer T (Meso Scale Discovery TM) was applied to each well and plates were measured in a Sector Imager 6000 (Meso Scale Discovery TM). Data analysis was performed with the MSD Discovery Workbench 3.0 Data Analysis Toolbox.

Data and Statistical Analysis

Data were analyzed blinded to the diagnosis. Statistical analysis was performed using SPSS statistics software (SPSS-Statistics 17.0, IBM GmbH, Munich, Germany) and Microsoft Excel software. Correlation analysis was performed by using Pearson's correlation. Receiver operating characteristic curves were used to evaluate sensitivity and specificity relationships to determine the diagnostic performance of exosomal a-Synuclein as a diagnostic test.

Example 1 : Isolation of exosomes from CSF

Exosomes can be prepared from CSF by subsequent centrifugation rounds followed by a final 100,000xg ultracentrifugation step, as also described previously ((Kunadt et al., 2015) and Fig. 1A+B). The ultracentrifugation pellet contained vesicles of 40 to 120 nm diameters with the typical cup-shaped morphology of exosomes (data not shown). The CSF exosome fraction was immunoreactive for the exosomal marker protein Flotillin-2 (Fig. 1 B) whereas in total CSF, Flotillin-2 was only detectable at a much higher exposure time (data not shown). In contrast, the heavy and light chain of immunoglobuline G (IgG) was abundant in total CSF but absent from the CSF exosome fraction (Fig. 1 B). In addition, the endoplasmatic reticulum protein Calnexin was not detected in the CSF exosome preparation, indicating the absence of microsomal contamination (Fig. 1 B). The inventors have already demonstrated by Western blot that CSF exosome preparations from 5 ml starting volume contain α-Synuclein (Kunadt et al., 2015). However, for quantification of exosomal α-Synuclein from smaller CSF volumes as low as 500 μΙ a more sensitive detection method is required. The inventors have previously described an optimized enzyme-linked immunoabsorbent assay (ELISA) which is based on an electrochemiluminescence (ECL) platform and allows for CSF α-Synuclein quantification with a large dynamic range and high sensitivity (Kruse et al., 2012). Exosomes were prepared from 1 ml CSF and the number and size distribution of vesicles in total CSF and in the CSF exosome preparations were measured by nanoparticle tracking analysis (NTA) (Sokolova et al., 2011 , Oosthuyzen et al., 2013, van der Pol et al.. 2014). A representative - - graph is depicted in Fig. 1C and shows vesicles in the range from 0 to 550 nm in total CSF. In contrast, the exosome preparation predominantly consists of vesicles in the size range of exosomes. In addition, we quantified ct-Synuclein protein levels in CSF and CSF exosome preparations by an electrochemiluminescence assay using MJF-1 clone 12.1 as a capture and Sulfo-TAG-labelled anti-a-Synuclein clone 42 as a detection antibody (Kruse et al., 2012). This assay has an average lower limit of detection of 5 pg/ml a- Synuclein which allows the quantification of exosomal a-Synuclein levels from a CSF starting volume as low as 500 μΙ. The present inventors next verified the reproducibility of these quantification methods (Fig. 1 D). Exosomes were prepared from several aiiquots of the same sample and exosome numbers as well as a-Synuclein protein levels were measured in total CSF and in the exosome preparation. This was repeated with a second series of aiiquots from an independent CSF sample. Coefficient of variance (CV) values were below 10 % for the number of exosomes (9.4 %) and for α-Synuclein protein (8.2 %) in total CSF. CV values for the same measurements carried out in the exosome preparations were 22.3 % for both, the number of exosomes and for exosomal a- Synuclein (Fig. 1 D). CV values were also calculated for the ratio of exosomal a-Synuclein protein levels to the number of exosomes (9.9 % for the determination of exosome numbers in the exosome pellet and 8.3% for the quantification of exosome numbers in the starting volume of total CSF).

The reliability of the method could even be further improved by dissolving the exosome pellet in HEPES or PBS buffer containing 0.025 % Tween, which does not interfere with the integrity of exosome membranes while it resolves exosome aggregates induced during UC. Using biological replicates, this method further decreases the coefficient of variation (CV) for direct quantification of exosome concentrations in the UC pellet from 16 to 6 % as compared to the UC method without Tween, which was performed in parallel with the same pool of CSF. The ratio of exosome numbers in the obtained UC pellet to total CSF exosome numbers also allows to correct exosomal α-Synuclein quantificiation for differences in the preparation yields. Thus, the CV of exosomal a-Synuclein quantification improves greatly from 22% to 9% using PBS or HEPES buffer containing 0,025% Tween-20 for dissolving the UC pellet prior to measuring the particle content using Nanosight LM-10.

This data shows that the obtained exosome preparation is robust and suitable for quantitative analysis in clinical samples.

Example 2: CSF exosomal a-Synuclein distinguishes between Parkinson's disease, dementia with Lewy bodies and neurological controls and correlates with cognitive impairment In order to examine CSF exosomal a-Synuclein, the inventors prepared CSF exosomes from 37 clinically diagnosed patients with Parkinson's disease with different grades of motor symptoms as assessed by Hoehn&Yahr staging (mean H&Y stage 3.9, SEM=0.20). All samples were randomly selected from the "Kassel cohort" and complemented with two different neurological control groups (Table 1) (Mollenhauer et al. , 201 1 ).

Table 1 Demographics of analyzed CSF samples of the Kassel cohort

Tomlinson 2010: MDS Systematic review of levodopa dose equivalency reporting in Parkinson's disease

One group consisted of 15 patients with polyneuropathy and with no indication of Parkinson's disease. A second group contained 25 patients with progressive supranuclear palsy which is characterized by a Parkinson syndrome without underlying a-Synuclein pathology. When absolute amounts of α-Synuclein protein in the exosome fraction of equal CSF starting volumes were quantified, no significant differences between Parkinson's disease, polyneuropathy and progressive supranuclear palsy groups was found (Parkinson's disease mean=17.20 pg in exosomes derived from 1 ml CSF, SEM=1 .50 pg, n=37; polyneuropathy control mean=17.20 pg in exosomes derived from 1 ml CSF, SEM=2.97 pg, n=15, progressive supranuclear palsy control mean=20.60 pg in exosomes derived from 1 ml CSF, SEM=4.56 pg, n=25, no significant differences, student's 2-tailed t-test) (Fig. 2A). Additionally, exosomal α-Synuclein levels in CSF from altogether 35 dementia with Lewy bodies samples from both, the Kassel and the Goettingen cohort, was analysed as another disease with a-Synuclein-related Parkinson syndrome. As shown in Fig. 2A we found significantly lower exosomal α-Synuclein CSF from dementia with Lewy bodies patients as compared to Parkinson s disease, polyneuropathy and progressive supranuclear palsy groups (dementia with Lewy bodies mean=7.92 pg in exosomes derived from 1 ml, SEM=0.78 pg, n=35,*<0.05, * p<0.05, *** p<0.0005, student's 2-tailed t-test). Importantly, exosomal a-Synuclein levels correlated inversely with the dementia with Lewy bodies patients' Mini Mental State Examination (MMSE) scores (Pearson's coefficienct, r=-0.382, n=33 for whom MMSE data were available, p=0.028, 2-tailed probability) (Fig. 2B). This data indicates that higher amounts of exosomal a-Synuclein in CSF are associated with a lower MMSE performance and therefore with cognitive impairment.

Next a preliminary evaluation of the performance of exosomal α-Synuclein as a potential diagnostic biomarker was performed by receiver operating characteristic (ROC) curve analysis (Fig. 3 and Table 2). The sensitivity and specificity of CSF exosomal a-Synuclein to distinguish dementia with Lewy bodies from Parkinson's disease were 85.7% and 80.6% with a positive and negative predictive value of 81.1% and 85.3%. The sensitivity and specificity for the distinction of dementia with Lewy bodies from polyneuropathy were 85.7% and 66.7%; that for dementia with Lewy bodies versus progressive supranuclear palsy 71.4% and 92.0% (Fig. 3 and Table 2).

Table 2: Summary of ROC results: Exosomal α-Synuclein [pg] in 1 ml CSF

As depicted in Fig. 2C, the number of exosomes in CSF differs highly between the various diagnostic groups, with nearly 4 fold higher values in Parkinson's disease as compared to dementia with Lewy bodies (Parkinson's disease mean=3.69 x 0^Λ9 exosomes/ml CSF, SEM=1.83 x 10^Λ8 exosomes/ml CSF, n=37; dementia with Lewy bodies mean=1.08 x 10^Λ9 exosomes/ml CSF, SEM=1.83 x 10^Λ8 exosomes/ml CSF, n=30, p=5.67 10^Λ-8, student's 2-tailed t-test) and approximately 2 times higher numbers as compared to polyneuropathy and progressive supranuclear palsy (polyneuropathy mean=2.03 x 10^Λ9 exosomes/ml CSF, SEM= 4.09 x 10^Λ8 exosomes/ml CSF, n=15; progressive supranuclear palsy mean=1.39 x 10^Λ9 exosomes/ml CSF, SEM= 5.23 x 10^Λ9 exosomes/ml CSF, n=24, *p<0.05, **p<0.005, ***p<0.0005 2-tailed student's t-test). In contrast, no significant differences were observed between the polyneuropathy and progressive supranuclear palsy groups (Fig. 2C). We next calculated the ratio of exosomal α-Synuclein per number of exosomes in the different diagnostic groups (Fig. 2D). Interestingly, an approximately 2 to 3 times lower ratio was detected in Parkinson's disease patients compared to all other groups (Parkinson's disease mean=6.74 x 10^Λ-9 pg a-Synuclein/number of exosomes, SEM=1.04 x 10^Λ-9 pg a-Synuclein/number of exosomes, n=35; dementia with Lewy bodies mean=1.50 x 10Λ-8 pg α-Synuclein/number of exosomes, SEM=3.37 x 10^A-9 pg a- Synuclein/number of exosomes, n=28; polyneuropathy mean=2.63 x 10^Λ-8 pg a- Synuclein/number of exosomes, SE =1.03x 10^Λ-8 pg a-Synuclein/number of exosomes, n=15; progressive supranuclear palsy mean=4.99 x 10^Λ-8 pg α-Synuclein/number of exosomes, SEM=1.27 x 10^Λ-8 pg a-Synuclein/number of exosomes, n=24, *p<0.05, **p<0.005, 2-tailed student's t-test). In contrast, no significant differences were found between polyneuropathy and progressive supranuclear palsy groups. ROC curve analysis of this marker for the distinction of Parkinson's disease versus dementia with Lewy bodies, polyneuropathy and progressive supranuclear palsy is shown in Fig. 3 and in Table 3. The ratio of CSF exosomal α-Synuclein to CSF exosome numbers distinguishes Parkinson's disease from dementia with Lewy bodies with a sensitivity of 74.3% and a specificity of 60.7% (Table 3).

Table 3: Summary of ROC results: Exosomal α-Synuclein [pg]/number of exosomes, 1 ml CSF

Taken together, exosomal α-Synuclein is significantly decreased in CSF from dementia with Lewy bodies patients compared to neurological controls, mainly due to a lower absolute number of CSF exosomes. Disease progression in dementia with Lewy bodie, defined by the severity of cognitive dysfunction, is paralleled by higher exosomal a- Synuclein levels (Fig. 2B). In Parkinson's disease, exosome numbers are significantly increased compared to dementia with Lewy bodies and neurological controls with comparable levels of total exosomal α-Synuclein and a lower ratio of α-Synuclein per exosomal particle (for a summary see Table 4 below). Table 4: Summary table of exosomal a-Synuclein In different diseases

It was demonstrated that exosomal α-Synuclein is present in CSF where it is accessible for quantification as a potential biomarker to detect a-Synuclein-related pathology (Table 4). ROC curve analysis revealed a high sensitivity and specificity for the discrimination of dementia with Lewy bodies, Parkinson's disease and neurological controls. Moreover, a highly significant correlation of exosomal α-Synuclein with cognition in dementia with Lewy bodies was detected. Thus, CSF exosomal α-Synuclein could serve as a surrogate marker to monitor disease progression. Such markers have not been available in the past and could aid future interventional studies in dementia with Lewy bodies.

Comparative Example 3: Analysis of plasma exosomal a-Synuclein

Recently, plasma exosomal α-Synuclein was suggested as a potential diagnostic marker in PD. Shi et al. specifically immune-isolated exosomes derived from the central nervous system (CNS) with an antibody directed against the neuronal L1 cell adhesion molecule. The authors found that the quantity of α-Synuclein in these CNS-derived plasma exosomes was significantly higher in PD compared to controls. CNS-derived exosomal a- Synuclein isolated by this method comprised approximately 0.2-0.3% of total plasma a- Synuclein. By ultracentrifugation and without further selection for CNS-derived exosomes the inventors prepared the total amount of exosomes present in plasma. When the a- Synuclein in plasma exosome preparations to α-Synuclein detected in total plasma from the Kassel cohort was quantified, we found that approximately 0.2-0.3% of plasma a- Synuclein is associated with exosomes (data not shown). This is comparable to the percentage reported for CNS-derived plasma exosomal α-Synuclein and surprisingly indicates that the majority of exosomal α-Synuclein in plasma might be CNS derived. In contrast to the findings by Shi et al, the present inventors detected no significant differences for plasma exosomal a-Synuclein levels between PD, DLB and neurological controls in our cohort. The number of exosomes within 1 ml plasma did not differ between DLB, PD and controls.

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Claims

1. A method of differential diagnosis of dementia with Lewy bodies and

Parkinson's disease, comprising the steps of:

(b) determining the number of exosomes obtained in step (a); and

(c) comparing the results obtained in step (b) with results obtained from

appropriate controls selected from neurological controls and healthy controls;

wherein

(ii) a significantly higher number of exosomes as compared to said

appropriate controls is indicative for Parkinson's disease.

2. The method of claim 1, further comprising the steps of:

wherein a significantly lower amount of exosomal a-synuclein protein as compared to said appropriate controls is indicative for dementia with Lewy bodies.

3. The method of claim 1 or 2, further comprising the steps of:

(b") determining the amount of exosomal α-synuclein protein in the exosomes obtained in step (a), and calculating the quotient of [amount of exosomal α-synuclein protein in the exosomes obtained in step (a) / number of exosomes obtained in step (a)]; and

4. A method of diagnosing dementia with Lewy bodies, comprising the steps of:

(b) determining the amount of exosomal a-synuclein protein in the exosomes obtained in step (a) and/or

determining the number of exosomes obtained in step (a); and

(c) comparing the results obtained in step (b) with results obtained from

appropriate controls selected from neurological controls and healthy controls;

wherein a significantly lower amount of exosomal a-synuclein protein as compared to said appropriate controls is indicative for dementia with Lewy bodies, and wherein a significantly lower number of exosomes as compared to said appropriate controls is indicative for dementia with Lewy bodies.

5. The method of any preceding claim, wherein the appropriate controls are

neurological controls, preferably wherein said neurological controls are subjects with polyneuropathy and without indication of Parkinson's disease, and/or subjects with progressive supranuclear palsy.

6. The method of any preceding claim, wherein step (a) comprises at least one step of ultracentrifugation, in particular wherein said ultracentrifugation step comprises centrifuging at at least 100,000 x g for at least 50 minutes, in particular centrifuging at 100,000 x g for 60 minutes,

wherein the ultracentrifugation step is preceded by at least two centrifugation steps at 3000-4000 x g, at least one centrifugation step at 4000-5000 x g, and at least one centrifugation step at 10000-20,000 x g;

in particular wherein the ultracentrifugation step is preceded by one

centrifugation step at 3500 x g, by two centrifugation steps at 4500 x g, and by one centrifugation step at 10,000 x g, and/or

7. The method of claim 6, wherein the centrifugation steps at 3000-5000 x g are carried out for 5-20 minutes, preferably 10-15 minutes; wherein the

centrifugation steps at 10000-20,000 x g are carried out for 20-45 minutes, preferably 25-40 minutes, such as for 30 minutes; and/or wherein the ultracentrifugation pellet is washed once, followed by centrifugation at 80,000- 120,000 x g, preferably 90,000-110,000 x g, more preferably at 100,000 x g, for 50-70 minutes, preferably 55-65 minutes, more preferably 60 minutes, most preferably using phosphate buffered saline (PBS).

8. The method of any preceding claim, wherein the exosomes isolated in step (a) are characterized by a size of 20 nm to 170nm, in particular 25 nm to 160 nm, more particularly 30 nm to 150 nm, more particularly 35 nm to 140 nm, more particularly 40 nm to 130 nm, such as 40 nm to 120 nm; and/or

wherein the defined volume of said sample of cerebrospinal fluid is 0.5 ml to 10 ml, preferably 0.5 ml to 5 ml, more preferably 0.5 ml to 2 ml, and most preferably 0.5 ml to 1 ml.

9. The method of any preceding claim, wherein determining the number of

exosomes obtained in step (a) comprises Nanoparticle tracking analysis (NTA).

10. The method of any one of claims 1, and 3-9, wherein determining the amount of exosomal a-synuclein protein in the exosomes obtained in step (a) comprises electrochemiluminescence based ELISA using an a-synuclein specific antibody or antibody fragment.

11. A method of diagnosing Parkinson's disease, comprising the steps of:

(b) determining the number of exosomes obtained in step (a); and

(c) comparing the results obtained in step (b) with results obtained from

appropriate controls;

12. The method of claim 11, further comprising the steps of:

13. The method of claim 11 or 12, wherein the appropriate controls are selected from neurological controls and healthy controls, preferably wherein the appropriate controls are neurological controls, more preferably wherein said neurological controls are subjects with polyneuropathy and without indication of Parkinson's disease, and/or subjects with progressive supranuclear palsy.

14. The method of any one of claims 11-13, wherein the method is further defined as in claims 6-9.

15. The method of claim 12, wherein determining the amount of exosomal a- synuclein protein in the exosomes obtained in step (a) comprises