WO2017032871A1 - Procédé de diagnostic différentiel de la démence à corps de lewy et de la maladie de parkinson - Google Patents

Procédé de diagnostic différentiel de la démence à corps de lewy et de la maladie de parkinson Download PDF

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WO2017032871A1
WO2017032871A1 PCT/EP2016/070166 EP2016070166W WO2017032871A1 WO 2017032871 A1 WO2017032871 A1 WO 2017032871A1 EP 2016070166 W EP2016070166 W EP 2016070166W WO 2017032871 A1 WO2017032871 A1 WO 2017032871A1
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exosomes
synuclein
disease
parkinson
csf
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PCT/EP2016/070166
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Brit Mollenhauer
Anja Schneider
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Georg-August-Universitaet Goettingen Stiftung Oeffentlichen Rechts, Universitaetsmedizin
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • 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.
  • CSF cerebrospinal fluid
  • 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).
  • MSA Multiple System Atrophy
  • PSP Progressive supranuclear palsy
  • DLB Dementia with Lewy bodies
  • CBD Corticobasal degeneration
  • 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.
  • 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.
  • imaging and fluid biomarkers including dopamine transporter scans, serum peptide markers, and CSF ⁇ -Synuclein (Mollenhauer and Schlossmacher, 2010, Suzuki et al., 2015).
  • Dementia with Lewy bodies 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.
  • 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.
  • PSP Progressive supranuclear palsy
  • 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.
  • 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 ⁇ -Synuclein was subsequently studied as a potential diagnostic biomarker, especially in the CSF, where the majority of ⁇ -Synuclein is derived from the CNS central nervous system rather than from peripheral blood (Mollenhauer et al., 2012).
  • CSF ⁇ -Synuclein the sensitivity and specificity of CSF ⁇ -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 ⁇ -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.
  • 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 ⁇ -Synuclein in Parkinson's disease compared to healthy controls.
  • WO 2010/056337 suggests the isolation of exosomes from blood, serum, CSF, and urine (Table 1 ), in order to conduct a mutational analysis of ⁇ -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.
  • CSF cerebrospinal fluid
  • 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 ⁇ -Synuclein species which serves as a seed to induce the oligomerisation of soluble ⁇ -Synuclein in recipient cells. Accordingly, CSF exosomal ⁇ -Synuclein may well be suited as a diagnostic marker.
  • the present disclosure provides a method of diagnosing dementia with Lewy bodies, comprising the steps of:
  • step (c) comparing the results obtained in step (b) with results obtained from appropriate controls;
  • step (b) determining the number of exosomes obtained in step (a);
  • step (c) comparing the results obtained in step (b) with results obtained from appropriate controls;
  • step (b) determining the number of exosomes obtained in step (a);
  • step (c) comparing the results obtained in step (b) with results obtained from appropriate controls;
  • the present disclosure provides a method of diagnosing dementia with Lewy bodies, comprising the steps of:
  • step (b) determining the amount of exosomal S-synuclein protein in the exosomes obtained in step (a) and/or
  • step (c) comparing the results obtained in step (b) with results obtained from appropriate controls;
  • 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.
  • 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:
  • step (b) determining the number of exosomes obtained in step (a);
  • step (c) comparing the results obtained in step (b) with results obtained from appropriate controls;
  • 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 A 9/ml, in particular 0.6-1.4*10 A 9/ml, more particularly 0.7-1.3 * 10 A 9/ml, such as 0.8- 1 .2*10 A 9/ml); and
  • a significantly higher number of exosomes as compared to said appropriate controls is indicative for Parkinson's disease.
  • 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-
  • 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.
  • the method of differential diagnosis may further comprise the steps of
  • step (b') determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a);
  • step (c') comparing the results obtained in step (b : ) with results obtained from said appropriate controls;
  • the method can comprise the additional steps of
  • step (b) determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a), and calculating the quotient of [amount of exosomal ⁇ -Synuclein protein in the exosomes obtained in step (a) / number of exosomes obtained in step (a)];
  • step (c) comparing the quotient obtained in step (b") with quotients obtained from said appropriate controls;
  • a significantly lower quotient as compared to said appropriate controls is indicative for Parkinson's disease.
  • 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 ⁇ -8 pg/number of exosomes per ml CSF, in particular 0.2-0.8 10 ⁇ -8 pg/number of exosomes per ml CSF, more particularly 0.3-0.7 10 ⁇ -8 pg/number of exosomes per ml CSF, such as 0.4-0.6 10 ⁇ -8 pg/number of exosomes per ml CSF.
  • Typical values obtained from DLB patients are 1.0-2.0 10 ⁇ -8 pg/number of exosomes per ml CSF, in particular 1.1- 1.9 10 ⁇ -8 pg/number of exosomes per ml CSF, more particularly 1.2-1.8 10 ⁇ -8 pg/number of exosomes per ml CSF, such as 1.3-1.7 10 ⁇ -8 pg/number of exosomes per ml CSF.
  • 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.
  • sample volumes such as 100 ⁇ , 200 ⁇ , 300 ⁇ or 400 pi.
  • 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).
  • CSF samples exhibiting erythrocyte counts ⁇ 50/mm 3 shall not be used.
  • the method is suitable for all kind of vertebrates.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ultracentrifugation pellet prior to preceding to step (b) 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.
  • PBS phosphate buffered saline
  • 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%.
  • step (a) comprises at least one step of size exclusion chromatography.
  • 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 _
  • 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.
  • 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.
  • 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.
  • the levels of exosomal a- Synuclein may be correlated with total exosomal protein content.
  • 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.
  • any suitable assay for determining the total protein content can be applied in the presently disclosed methods.
  • 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.
  • the method may further comprise testing for UK-Brain Bank criteria indicative for Parkinson's disease.
  • the present disclosure also provides a method of diagnosing Parkinson's disease, comprising the steps of:
  • step (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 may further comprise the steps of:
  • step (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)];
  • step (c) comparing the quotient obtained in step (b") with quotients obtained from said
  • determining the amount of exosomal ⁇ -Synuclein protein in the exosomes obtained in step (a) preferably comprises electrochemiluminescence based ELISA using an ⁇ -Synuclein specific antibody or antibody fragment, as further detailed above and in the experimental section.
  • a method of diagnosing dementia with Lewy bodies comprising the steps of:
  • step (c) comparing the results obtained in step (b) with results obtained from
  • a method of differential diagnosis of dementia with Lewy bodies and Parkinson's disease comprising the steps of:
  • step (b) determining the number of exosomes obtained in step (a):
  • step (c) comparing the results obtained in step (b) with results obtained from
  • step (b') determining the amount of exosomal a-Synuclein protein in the exosomes obtained in step (a);
  • step (c') comparing the results obtained in step (b') with results obtained from said appropriate controls;
  • step (b) determining the amount of exosomal ⁇ -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)];
  • 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.
  • step (a) comprises at least one step of ultracentrifugation, in particular wherein said ultracentrifugation step - -
  • centrifuging at at least 100,000 x g for at least 50 minutes, in particular centrifuging at 100,000 x g for 60 minutes.
  • PBS phosphate buffered saline
  • step (a) comprises at least one step of size exclusion chromatography.
  • test comprises Western Blotting.
  • 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.
  • 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.
  • 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.
  • step (a) 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.
  • 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 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.
  • MMSE Mini Mental State Examination
  • a method of diagnosing Parkinson's disease comprising the steps of:
  • step (b) determining the number of exosomes obtained in step (a);
  • step (c) comparing the results obtained in step (b) with results obtained from
  • step (b) determining the amount of exosomal ⁇ -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;
  • 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.
  • step (a) comprises
  • 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 ⁇ of sample buffer.
  • CSF was diluted 1 :5 in sample buffer and 20 ⁇ of the exosome preparation and 20 ⁇ of total CSF were probed with an antibody against the exosomal marker protein Flotillin- 2 (left panel).
  • Flotillin- 2 As a negative control, we probed exosome preparations and CSF with a secondary antibody against human IgG (middle panel).
  • 20 ⁇ 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.
  • Figure 2 Characterisation of exosomal ⁇ -Synuclein in dementia with Lewy bodies, Parkinson's disease and non a-Synuclein-related disease controls in the Kassel cohort.
  • DLB Lewy bodies
  • PD Parkinson's disease
  • PNP polyneuropathy
  • PSP progressive supranuclear palsy
  • FIG. 3 Receiver operating characteristic (ROC) analysis, Kassel cohort
  • the buffers were prepared with purified and de-ionized water.
  • CSF specimens were collected at the Paracelsus-Elenagraphy 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/mm 3 were excluded. All samples were obtained in accordance with the ethical standards laid down in the1964 Declaration of Helsinki.
  • 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.
  • 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
  • Exosomes from human CSF for Western blot analysis were prepared from 2.5-5 ml of
  • NTA Nanoparticle tracking analysis
  • 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 ⁇ prior to analysis.
  • 100.000xg pellets derived from 0.5 ml total CSF were resuspended in 50 ⁇ 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.
  • NTA Nanoparticle Tracking Analysis
  • the reliability of the assay can be further improved by pipetting 500 ⁇ 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
  • 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).
  • ELISA enzyme-linked immunoabsorbent assay
  • ECL electrochemiluminescence
  • NTA nanoparticle tracking analysis
  • 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 ⁇ .
  • 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.
  • CV values were below 10 % for the number of exosomes (9.4 %) and for ⁇ -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.
  • 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 ⁇ -Synuclein quantificiation for differences in the preparation yields.
  • 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.
  • Example 2 CSF exosomal a-Synuclein distinguishes between Parkinson's disease, dementia with Lewy bodies and neurological controls and correlates with cognitive impairment
  • 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.
  • MMSE Mini Mental State Examination
  • SEM 3.37 x 10
  • a -9 pg a- Synuclein/number of exosomes, n 28
  • polyneuropathy mean 2.63 x 10 ⁇ -8 pg a- Synuclein/number of exosomes
  • progressive supranuclear palsy mean 4
  • exosomal ⁇ -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).
  • exosome numbers are significantly increased compared to dementia with Lewy bodies and neurological controls with comparable levels of total exosomal ⁇ -Synuclein and a lower ratio of ⁇ -Synuclein per exosomal particle (for a summary see Table 4 below).
  • Table 4 Summary table of exosomal a-Synuclein In different diseases
  • CNS-derived exosomal a- Synuclein isolated by this method comprised approximately 0.2-0.3% of total plasma a- Synuclein.
  • Braak H Del Tredici K, Rub U, de Vos RA, Jansen Steur EN. Braak E. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging. 2003;24(2): 197-211.
  • Heat-shock protein 70 modulates toxic extracellular alpha-synuclein oligomers and rescues trans- synaptic toxicity. FASEB J. 201 1 ;25(1):326-36.
  • Mollenhauer B Schlossmacher MG. CSF synuclein: adding to the biomarker footprint of dementia with Lewy bodies. J Neurol Neurosurg Psychiatry. 2010;81 (6):590-1. Mollenhauer B, Trautmann E, Otte B, Ng J, Spreer A, Lange P, et al. alpha-Synuclein in human cerebrospinal fluid is principally derived from neurons of the central nervous system. J Neural Transm. 2012;119(7):739-46.

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Abstract

La présente invention a pour objet des procédés de diagnostic différentiel de la démence à corps de Lewy et/ou de la maladie de Parkinson. Des procédés selon la présente invention comprennent en règle générale une étape consistant à isoler des exosomes d'un échantillon de liquide céphalo-rachidien (CSF pour CerebroSpinal Fluid) et à déterminer le nombre d'exosomes et/ou la quantité d'α-synucléine exosomale dans un volume défini d'échantillon de liquide céphalo-rachidien.
PCT/EP2016/070166 2015-08-27 2016-08-26 Procédé de diagnostic différentiel de la démence à corps de lewy et de la maladie de parkinson WO2017032871A1 (fr)

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US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
US11827695B2 (en) 2017-02-17 2023-11-28 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
US20200400687A1 (en) * 2017-12-19 2020-12-24 Chase Therapeutics Corporation Methods for developing pharmaceuticals for treating neurodegenerative conditions
EP4273882A3 (fr) * 2017-12-19 2024-01-03 Chase Therapeutics Corporation Méthode d'évaluation d'une synucléinopathie
EP3728567A4 (fr) * 2017-12-19 2021-09-22 Chase Therapeutics Corporation Procédés pour le développement de produits pharmaceutiques pour le traitement d'affections neurodégénératives
WO2019126395A1 (fr) 2017-12-19 2019-06-27 Chase Therapeutics Corporation Procédés pour le développement de produits pharmaceutiques pour le traitement d'affections neurodégénératives
WO2019171035A1 (fr) * 2018-03-06 2019-09-12 University Of Newcastle Upon Tyne Détection d'agrégation de protéines pathologiques
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WO2020223523A1 (fr) 2019-04-30 2020-11-05 Chase Therapeutics Corporation Dosages d'alpha-synucléine
JP7348604B2 (ja) 2019-11-29 2023-09-21 富士フイルム和光純薬株式会社 パーキンソン病の診断を補助する方法、バイオマーカー、試薬キット及び装置
JPWO2021107155A1 (fr) * 2019-11-29 2021-06-03
WO2021107155A1 (fr) * 2019-11-29 2021-06-03 富士フイルム和光純薬株式会社 Procédé d'aide au diagnostic de la maladie de parkinson, biomarqueur, kit de réactifs et dispositif
GB2601997B (en) * 2020-12-09 2023-04-26 Mursla Ltd Extracellular vesicle characterization systems
GB2601997A (en) * 2020-12-09 2022-06-22 Mursla Ltd Extracellular vesicle characterization systems
WO2022122768A1 (fr) 2020-12-09 2022-06-16 Mursla Limited Systèmes de caractérisation de vésicule extracellulaire

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