Methods of identifying subjects suffering from multiple system atrophy or parkinson's disease by alpha-synuclein seeded aggregation assays (SAA)

Abstract

The present invention relates to methods of identifying subjects suffering from Multiple System Atrophy (MSA) or Parkinson's Disease (PD). The method comprises subjecting a biological sample obtained from a subject to a seeded aggregation assay or a sequence of seeded aggregation assays. The invention also further provides methods of treating the identified MSA patient or the PD patient with compounds capable of inhibiting alpha synuclein aggregation.

Description

[0001] Methods of identifying subjects suffering from Multiple System Atrophy or Parkinson's disease

[0002] Field of the invention

[0003] The present invention relates to methods of identifying subjects suffering from Multiple System Atrophy (MSA) or Parkinson's Disease (PD). The method comprises subjecting a biological sample obtained from a subject to a seeded aggregation assay or a sequence of seeded aggregation assays. These seeded aggregation assays may be Real-Time Quaking Induced Conversion (RT-QuIC) assays. The invention also further provides methods of treating the identified MSA patient or the PD patient with compounds capable of inhibiting alpha synuclein aggregation.

[0004] Reference to sequence listing

[0005] This application includes Sequence Listings which are disclosed in file name: "1265-EP-EPA Seq list ST26.xml", created on 17 September 2024, which file is incorporated by reference herein in its entirety.

[0006] Background of the invention

[0007] Many neurodegenerative diseases are caused, at least in part, by the accumulation of specific misfolded proteins. These deposits are typically only identified upon post-mortem analysis of brain tissue, allowing diagnoses to be made based on specific neuropathological and molecular findings. Accordingly, prior to death of the patients it is challenging for clinicians to correctly diagnose and especially differentiate between neurodegenerative diseases. Currently, the diagnosis of such neurodegenerative disorders is often based on combinations of clinical evaluation of the signs and symptoms identified in the patient and biomarker levels.

[0008] One example of neurodegenerative disorders which can be challenging to correctly diagnose and especially differentiate are synucleinopathies. Synucleinopathies, refer to disorders characterized by the neural inclusion of pathologic alpha synuclein aggregates called Lewy bodies. Synucleinopathies include Parkinson's disease (PD) (including idiopathic and inherited forms of Parkinson's disease) and Diffuse Lewy Body (DLB) disease (also known as Dementia with Lewy Bodies (D LB) ), Lewy body variant of Alzheimer's disease (LBV), Combined Alzheimer's and Parkinson disease (CAPD), pure autonomic failure (PAF) and multiple system atrophy (MSA; e.g., Olivopontocerebellar Atrophy, Striatonigral Degeneration and Shy-Drager Syndrome). Real-Time Quaking Induced Conversion (RT-QuIC) assays exploits that fibrillar alpha synuclein (seeding material) can induce fibril formation of monomeric alpha synuclein substrates. When performing the RT-QuIC assay the seeding material is added to the monomeric alpha synuclein substrate, which causes the monomeric substrate to undergo fibril formation, and this reaction is followed and quantified over time. Fibril formation of the monomeric alpha synuclein substrate is evident of seeding material in a sample. The seeding material can be obtained from various sources such as from biological samples including plasma, brain homogenate, CSF etc., or from recombinant sources e.g. preformed fibrils (PFF). Different types of monomeric alpha synuclein substrate can be used in the RT- QuIC assay, including wild type monomeric alpha synuclein or recombinant mutant forms of monomeric alpha synuclein, such as mutant K23Q where the native lysine at position 23 of human alpha synuclein is changed to a glutamine.

[0009] Both wild type and mutant K23Q monomeric alpha synuclein substrate used in RT-QuIC assays has been shown to be useful in detecting alpha synuclein seeding in PD patient samples such as CSF or brain homogenate (see e.g. Groveman B. et al., 2018, Acta Neuropathol Commun. 2018; 6: 7). However, detection of MSA in patient samples using RT-QuIC assays has shown to be more challenging than for other synucleinopathies, which has been suggested to be related to the presence of a specific conformer of strain of alpha synuclein aggregates / fibrils in MSA patients (Srivastava A. et al., 2022, Biomolecules 12, 576).

[0010] Diagnosing multiple system atrophy (MSA) is particularly challenging as certain signs and symptoms of MSA, such as muscle rigidity and unsteady gait, also occur in other disorders, such as PD. Therefore, MSA may be difficult to distinguish clinically from other disorders, particularly in patients at the early stages of the disease. MSA patients often display parkinsonism (MSA parkinsonian type; MSA-P), and such patients may be misdiagnosed as being PD patients. The reverse also occurs; in a study it was found that approximately 20% of patients with a clinical diagnosis of MSA turned out to actually have PD or DLB at autopsy (Koga et al., 2015, Neurology 85:404-412). Patients presenting with the cerebellar phenotype of MSA (MSA-C) can mimic other adult-onset ataxias due to alcohol, chemotherapeutic agents, lead, lithium, and toluene, or vitamin E deficiency, as well as paraneoplastic, autoimmune, or genetic ataxias (e.g., spinocerebellar ataxias, fragile X-associated tremor ataxia syndrome, or late-onset Friedreich ataxia) (Klockgether, 2010, Lancet neurology 9:94- 104 and Lin et al., 2016, Cerebellum 15:663-679).

[0011] Currently there is a lack of methods for accurately separating patients with a diagnosis of multiple system atrophy (MSA) from Parkinson's Disease (PD), especially early in the disease progression. Many patients suffering from MSA are initially mis-diagnosed as Parkinson's patients and only after a few years the diagnosis of MSA is made based on clinical symptoms and a lack of response to standard parkinson's treatments such as L-DOPA. At that time the MSA disease has often progressed to a very severe state and the MSA patients' life-expectations are generally short. Such misdiagnosis of MSA and / or PD can have significant consequences for the patients as the result of such misdiagnosis often is an inappropriate treatment offered by the clinicians. Accordingly, there is a high unmet need for methods that enables correct diagnosis of MSA and separation from PD diagnosis earlier in the disease progression. Such diagnosis would enable more suitable interventional therapies to be offered to the patient earlier on in their disease progression, which therapies also potentially better target the MSA underlying pathology.

[0012] Summary of the invention

[0013] The inventors of the present invention have identified a method to differentiate PD from MSA patients. This method employs different alpha synuclein monomeric substrates, used in a specific sequential combination of RT-QuIC assays. When this method and the alpha synuclein monomeric substrates are applied to seeding material obtained from patient samples, the inventors have found that the results can be used to differentiate MSA patients from PD patients. This method has the potential to enable earlier and more accurate diagnosis of MSA and PD.

[0014] The inventors identified that if brain homogenates and CSF samples from MSA or PD patients are investigated in two separate RT-QuIC assays using wildtype alpha synuclein and alpha synuclein mutant K23Q, respectively, as the monomeric substrate, then samples from patients with MSA can be differentiated from samples from PD patients.

[0015] The RT-QuIC assay employing the wild type alpha synuclein monomeric substrate is useful in determining whether the patients are suffering from a synucleinopathy, such as MSA or PD. Surprisingly, the inventors found that if a seeded aggregation assay employing the alpha synuclein mutant K23Q. as the monomeric substrate is performed on these relevant patient samples, then this assay can be used to determine whether the patient are suffering from PD or MSA, as only the PD samples gives a positive signal in this type of assay.

[0016] Accordingly, the present invention provides a method of analysing biological samples, such as CSF samples, from patients suffering from synucleinopathies and use the result of this analysis to determine if the patient suffers from MSA or PD. Definitions

[0017] In the present application amino acid residues are referred to throughout by their amino acid identity names or letter codes and position number in the polypeptide sequence. For example, with regard to SEQ ID NO: 1, "K23" refers to the lysine at position 23 of the protein described in SEQ ID NO:1 and so forth. Mutations to protein residues are referred to throughout by the amino acid letter code originally present in the protein and the position number of the amino acid in the protein, followed by the new amino acid letter code replacing the original amino acid in the mutated form of the protein. For example, "K23Q" indicates that the lysine (K) at position 23 has been changed to a glutamine (Q), see SEQ ID NO:2.

[0018] In the present application alpha synuclein is sometimes abbreviated as "aSyn" or "aSyn".

[0019] In the present context "truncated forms" of alpha synuclein are meant to describe versions of monomeric alpha synuclein which is shorter than the original full length WT alphasynuclein described in SEQ ID NO:1 or any mutated form thereof; the truncation consists of removal of a portion of the amino acid sequence described in SEQ ID NO:1 or any mutated form thereof, either from the C- terminal end, the N-terminal end or from both the C-terminal end and the N-terminal end.

[0020] In the present application the term "seeded aggregation assay" is meant to describe a type of in vitro assay which employs a biological sample and a seeding material. These two components are mixed together to form a reaction mixture which is allowed to incubate under conditions that may allow the seeding material and the monomeric alpha synuclein substrate present in a biological sample to coaggregate. This aggregation may or may not happen which is believed to depend on the compatibility between the seeding material and the sample material. The amount of alpha synuclein aggregation resulting from performing the seeded aggregation assay may be quantified in any suitable way, for example it may be quantified by using fluorescent methods. In some embodiments the seeded aggregation assay may be an RT-QuiC assay, a seed amplification assay (SAA) or a protein misfolding cyclic amplification assay (PMCA). In a preferred embodiment the seeded aggregation assay is a realtime quacking-induced conversion assay (RT-QuiC).

[0021] In the present context the term "agitation" is meant to indicate the introduction of any type of turbulence or motion into a reaction mix, for examples by sonication, stirring, or shaking. In some specific embodiments the agitation step includes shaking but not sonication. Description of drawings

[0022] Figure 1 shows the 96 well plate overview of RT-QuIC experiment described in example 1 testing brain homogenate samples from Parkinson's Disease (PD), Multiple System Atrophy (MSA) and Heathy Control (CTRL). In house preformed fibrils (PFF) in CSF were used as positive controls. The samples were tested in quadruplicates at each dilution. Controls were tested at four dilutions, while CSF from cases of PD or MSA were tested at five dilutions.

[0023] Figure 2 shows the K23Q. RT-QuIC results from brain homogenate obtained from either PD, MSA or Healthy control (HC). Each graph depicts results from all 5 replicates and show clear aggregation curves for PD brain homogenate whereas the curves for MSA and HC stayed close to the baseline.

[0024] Figure 3 shows the WT RT-QuIC results from brain homogenate obtained from either PD, MSA or Healthy control (HC). Each graph depicts results from all 5 replicates and under these conditions no clear differentiation was observed among PD, MSA or HC. They all started giving signals at a similar time point.

[0025] Figure 4 shows the 96 well plate overview of RT-QuIC experiment described in example 1 testing undiluted CSF samples from Parkinson's Disease (PD), Multiple System Atrophy (MSA) and Heathy Control (CTRL). The samples were tested in 5 replicates, and the individual samples are identified by the following numbers MSA1: 100140, MSA2: 100439, MSA3: 100460, MSA4: 100487, MSA5: 100527, PD1: 424777, PD2: 424779, CTRL1: 100554 and CTRL2: 100580.

[0026] In figure 4, the "MM" notation is master mix. Negative control is denoted (CTRL-) and is CSF from healthy donor, positive control is denoted (CTRL+) and is preformed fibrils (PFF) in CSF from healthy donor.

[0027] Figure 5A shows the K23Q. RT-QuIC results from undiluted CSF samples obtained from either PD, MSA or Healthy controls (HC). Each graph depicts results from all 5 replicates and show clear aggregation curves for PD brain homogenate whereas the curves for MSA and HC stayed close to the baseline. The individual samples are correlated to the plate setup of figure 4 by the following numbers MSA1= MSA:100140, MSA2= MSA:100439, MSA3= MSA:100460, MSA4= MSA:100487, MSA5= MSA:100527, PD1= PD:424777, PD2= PD:424779, CTRL1= HC:100554 and CTRL2= HC:100580.

[0028] Figure 5B shows the WT RT-QuIC results from brain homogenate obtained from either PD, MSA or Healthy control (HC). Each graph depicts results from all 5 replicates and show clear aggregation curves for 4 out of 5 MSA samples and 2 out of 2 PD samples, whereas the curves of HC stayed close to the baseline. The individual samples are correlated to the plate setup of figure 4 by the following numbers MSA1= MSA:100140, MSA2= MSA: 100439, MSA3= MSA:100460, MSA4= MSA:100487, MSA5= MSA:100527, PD1= PD:424777, PD2= PD:424779, CTRL1= HC:100554 and CTRL2= HC:100580. Figure 6A represents quantification of the K23Q RT-QuIC aggregation curves shown in figure 5A as bar graphs. The aggregation profiles that were above the pre-determined threshold was counted as positive, this threshold was based on a binary assessment where the decision time was selected as 40h and threshold for the K23Q RT-QuIC assay was set to 40000 a.u. The K23Q RT-QuIC result was only found to provide a positive signal when analysing the undiluted PD CSF samples, whereas the results were found not to provide a positive signal when analysing the undiluted CSF samples from MSA nor CSF samples form HC.

[0029] Figure 6B represents quantification of the WT RT-QuIC aggregation curves shown in figure 5B as bar graphs. The aggregation profiles that were above the pre-determined threshold was counted as positive, this threshold was based on a binary assessment where the decision time was selected as 40h and threshold for the WT RT-QuIC assay was set to 15000 a.u. The WT RT-QuIC shows high positive counts both with MSA (4 out of 5) and PD patient samples (2 out of 2), but not with samples from HC.

[0030] Detailed description of the invention

[0031] The present invention relates to the use of seeded aggregation assays to identify patients suffering from MSA and separate these patients from patient suffering from PD. This separation of MSA and PD is important in order to select the right cohorts for clinical trials and in order to offer the patients the therapies targeting the underlying disease pathology.

[0032] In some embodiments, methods employing seeded aggregation assays are disclosed for determining whether a subject suffers from a synucleinopathy. These methods include performing an aSyn seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeding assay includes: a) Providing a biological sample from a subject suspected of suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric wild type alpha synuclein (SEQ ID NO: 1) or a fragment thereof to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric wild type alpha synuclein with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from a synucleinopathy, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject not suffering from a synucleinopathy; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from a synucleinopathy.

[0033] In some embodiments, methods employing seeded aggregation assays are disclosed for determining whether a subject suffers from MSA or PD. These methods include performing an aSyn seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeding assay includes: a) Providing a biological sample from a subject suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from PD.

[0034] In some embodiments, methods employing seeded aggregation assays are disclosed for identifying a subject suffering from MSA. These methods include performing an aSyn seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeding assay includes: a) Providing a biological sample from a subject suffering from a synucleinopathy, which is not dementia with lewy body; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA in the absence of a positive detection of alpha synuclein aggregation in step c) as this will be indicative of the subject suffering from MSA.

[0035] In some embodiments, methods employing seeded aggregation assays are disclosed for determining whether a subject suffers from a synucleinopathy and if so, whether this synucleinopathy is MSA or PD. These methods include performing two separate aSyn seeded aggregation assays on a biological sample from the subject, or a fraction thereof, wherein the first seeding assay includes: a) Providing a biological sample from a subject suspected of suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric wild type alpha synuclein (SEQ ID NO: 1) or a fragment thereof to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric wild type alpha synuclein with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from a synucleinopathy, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject not suffering from a synucleinopathy; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from a synucleinopathy; and wherein if the conclusion from the first assay is that the subject suffers from a synucleinopathy, then performing the second assay to determine whether the subject suffers from MSA or PD, which second assay includes: performing a second aSyn seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the second seeding assay includes: a) Providing a biological sample from the subject suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from PD.

[0036] In some embodiments, further methods employing seeded aggregation assays are disclosed for determining whether a subject suffers from a synucleinopathy and if so, whether this synucleinopathy is MSA or PD. These methods include performing two separate aSyn seeded aggregation assays on a biological sample from the subject, or a fraction thereof, wherein the first seeding assay includes: a) Providing a biological sample from a subject suspected of suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric wild type alpha synuclein (SEQ ID NO: 1) or a fragment thereof to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric wild type alpha synuclein with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from a synucleinopathy, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject not suffering from a synucleinopathy; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from a synucleinopathy; and wherein if the conclusion from the first assay is that the subject suffers from a synucleinopathy, then determine that dementia with lewy body is likely not the correct diagnosis; when the subjects potential diagnosis is expected to be limited to being either PD or MSA then performing the second assay to determine whether the subject suffers from MSA or PD, which second assay includes: performing a second aSyn seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the second seeding assay includes: a) Providing a biological sample from the subject suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from PD.

[0037] Some embodiments disclose the monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) for use in any of the methods or use in any of the assays described herein.

[0038] In some embodiments methods are disclosed herein for determining whether a subject has a synucleinopathy. In one embodiment the subject is a human. In one embodiment the subject is a human suspected of suffering from a synucleinopathy. In one embodiment the subject is a human suspected of suffering from a synucleinopathy but is not suspected of suffering from dementia with lewy body. In one embodiment the subject is a human suspected of suffering from a synucleinopathy which is not dementia with lewy body. In one embodiment the subject is a human diagnosed with a synucleinopathy, which may be either PD or MSA. In one embodiment the subject is a human diagnosed with a synucleinopathy, which is suspected to be either PD or MSA. In one embodiment the subject is a human diagnosed with either PD or MSA, wherein the diagnosis has not concluded which of these diseases are the correct diagnosis for the subject. The subject suffering from a synucleinopathy or suspected of suffering from a synucleinopathy may have been identified by any suitable diagnostic methods known in the art, e.g. by use of clinical scales, clinical questionnaire or other clinical tests, such as imaging techniques. Likewise, the subject suffering from a synucleinopathy or suspected of suffering from a synucleinopathy which is not dementia with lewy body may have been identified by any suitable diagnostic methods known in the art, e.g. by use of clinical scales, clinical questionnaire or other clinical tests, such as imaging techniques.

[0039] In one embodiment the subject determined to suffer from MSA or PD is treated with a compound capable of treating the disease and capable of inhibiting alpha synuclein aggregation.

[0040] In one embodiment the subject determined to suffer from MSA or PD is treated with a compound capable of treating the disease, such as an alphasynuclein antibody, a small molecule, an antisense oligonucleotide, or a peptide.

[0041] In one embodiment the subject determined to suffer from MSA or PD is treated with a compound capable of treating the disease, such as an anti-alphasynuclein monoclonal antibody

[0042] In one embodiment the subject determined to suffer from MSA or PD is treated with a compound capable of treating the disease selected from the list comprising prasinezumab, amlenetug, cinpanemab, ABBV-0805 / BAN0805, UCB7853, TAK-341 / M EDI 1341, Emrusolmin / TEV-56286, PBT434 / ATH434, BIIB101 / ION464 and UB-312.

[0043] In one embodiment the subject determined to suffer from MSA or PD is treated with a compound capable of treating the disease, wherein the treatment consists of slowing or delaying disease progression.

[0044] In one embodiment the subject determined to suffer from MSA or PD is treated with a compound capable of treating the disease, wherein the treatment consists of slowing or delaying clinical progression.

[0045] In some embodiments, the methods of the invention describe the use of seeded aggregation assays performed on a biological sample from the subject. These biological samples could for example be obtained from a nasal brushing, saliva, skin, whole blood, serum, plasma, cerebrospinal fluid, feces, urine or a tissue sample, such as, but not limited to, a brain tissue sample. In preferred embodiments the sample is a CSF sample or a plasma sample. Most preferred is CSF samples.

[0046] In some embodiments the biological sample of step a may be diluted, such as in a buffer before performing the remaining assay steps b though c. In some embodiments the buffer is a phosphate buffer, in some embodiments the buffer is CSF or artificial CSF. In some embodiments the biological sample can be diluted, for example 10-fold, 100-fold, l.OOO-fold or 10.000 fold.

[0047] In some embodiment the seeded aggregation assay is run multiple times on the same sample from the same subject to make a more accurate determination in step d. In certain embodiments the seeded aggregation assay step c is run in duplicates before making the determination in step d. In certain embodiments the seeded aggregation assay step c is run in triplicates before making the determination in step d. In certain embodiments the seeded aggregation assay step c is run in quadruplicates before making the determination in step d. In certain embodiments the seeded aggregation assay step c is run in quintuplicates before making the determination in step d. In certain embodiments the seeded aggregation assay step c is run in replicates of between 2 and 5 before making the determination in step d.

[0048] In some embodiment the seeded aggregation assay is run multiple times on the same sample from the same subject to make a more accurate determination in step d. In certain embodiments the seeded aggregation assay steps b through c is run in duplicates before making the determination in step d. In certain embodiments the seeded aggregation assay steps b through c is run in triplicates before making the determination in step d. In certain embodiments the seeded aggregation assay steps b through c is run in quadruplicates before making the determination in step d. In certain embodiments the seeded aggregation assay steps b through c is run in quintuplicates before making the determination in step d. In certain embodiments the seeded aggregation assay steps b through c is run in replicates of between 2 and 5 before making the determination in step d.

[0049] In some embodiment the determination of step d is a binary determination which is either positive or negative depending on how many of the replicates of step c were determined to be positive, this binary determination is decided based on a pre-determined threshold such as for example at least 2 out of 5 replicates of step c yielding a positive signal of aggregation, such as at least 3 out of 5 replicates of step c yielding a positive signal of aggregation, or such as at least 4 out of 5 replicates of step c yielding a positive signal of aggregation. In some embodiments this binary determination is decided based on a pre-determined threshold such as for example at least 40% of the replicates of step c yielding a positive signal of aggregation, such as at least 50 % of the replicates of step c yielding a positive signal of aggregation, such as at least 60 % of the replicates of step c yielding a positive signal of aggregation, such as at least 70 % of the replicates of step c yielding a positive signal of aggregation, such as at least 80 % of the replicates of step c yielding a positive signal of aggregation, or such as at least 90 % of the replicates of step c yielding a positive signal of aggregation.

[0050] In some embodiment the determination of step d is made using a pre-determined threshold for what is indicative of a positive detection of alpha synuclein aggregation. It is well within the capabilities of the skilled person to set this threshold to match the aggregation curves detected and the apparatus used to perform step c. In some embodiment this threshold may be about 15.000 or about 40.000 arbitrary intensity units. In some embodiment the determination of step d is a binary determination which is either positive or negative depending on whether the signal is above or below the predetermined threshold for what is indicative of a positive detection of alpha synuclein aggregation.

[0051] In some embodiments "step c)" comprises quantifying the alpha synuclein fibril formation in the reaction mixture; and "step d)" comprises determining if the alpha synuclein fibril formation quantified in step c) is indicative of a positive detection of alpha synuclein fibril formation; and "step e)" comprises identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein fibril formation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein fibril formation in step c) will be indicative of the subject suffering from PD.

[0052] In some embodiments the methods employing seeded aggregation assays for determining whether a subject suffers from MSA or PD utilize a mutated form of monomeric alpha synuclein as substrate. Such mutated form of monomeric alpha synuclein is selected from the group consisting of monomeric alpha synuclein containing between one and eight point mutations in SEQ ID NO: 1, wherein the point mutations are at residues K23, D2, K21, K45, T59, G67, V77, and / or A78. In certain embodiments the mutation is at residue K23, in even further embodiments the mutation is K23Q.

[0053] In some embodiments the seeded aggregation assays of the invention employs truncated forms of monomeric alpha synuclein as substrate, which truncated forms contains a deletion of up to 30 amino acids of SEQ ID NO: 1, such as up to 25 amino acids deletion, such as up to 20 amino acids deletion, such as up to 15 amino acids deletion, such as up to 10 amino acids deletion, such as up to 5 amino acids deletion.

[0054] In further embodiments the seeded aggregation assays of the invention employs monomeric alpha synuclein as substrate, which is both truncated and mutated, i.e. such mutated form of monomeric alpha synuclein is selected from the group consisting of monomeric alpha synuclein containing between one and eight point mutations in SEQ ID NO: 1, wherein the point mutations are at residues K23, D2, K21, K45, T59, G67, V77, and / or A78, such as K23Q; and which further contains a deletion of up to 30 amino acids of SEQ. ID NO: 1, such as up to 25 amino acids deletion, such as up to 20 amino acids deletion, such as up to 15 amino acids deletion, such as up to 10 amino acids deletion, such as up to 5 amino acids deletion.

[0055] In some embodiments the monomeric alpha synuclein as substrate is for use in the assays or the methods of the invention.

[0056] In further embodiments the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) in the seeded aggregation assays of the invention employs the use of a fluorescent dye. The fluorescent dye may be any suitable dye to quantify the alphasynuclein aggregation, such as thioflavin T (ThT) or thioflavin S (ThS). In further embodiments the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) is performed within 24 - 48 hours after the reaction mixture in step b) i. was first formed. In further embodiments the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) is performed within 24 - 48 hours after the incubation and agitation of the reaction mixture in step b) ii. was initiated.

[0057] In some embodiments the seeded aggregation assays of the invention employs a step b) i. comprising contacting the biological sample with a monomeric alpha synuclein substrate to form a reaction mixture. In some embodiment the biological sample to be tested in the seeded aggregation assay is an undiluted sample. In some embodiment the volume of the biological sample to be tested in the seeded aggregation assay is about 15 uL. In some embodiments the monomeric alpha synuclein substrate is provided in a master mix composition which is then added to the biological sample to form the reaction mixture. In some embodiments this master mix composition comprises the monomeric alpha synuclein substrate in a concentration of about 0.04 to 1.00 mg / mL, such as about 0.06 to 0.08 mg / mL or such as about 0.08 mg / mL. In further embodiments the master mix composition further comprises a 20 mM phosphate buffer. In further embodiments the master mix composition added to form the reaction mixture of step b) i. has a pH of about 7.5. In certain embodiments the reaction mixture in step b) i. also contain sodium dodecyl sulfate (SDS), in certain embodiments the SDS is present in a concentration of 0.001 %-0.005%. In certain embodiments the reaction mixture in step b) i. contain SDS in a concentration of 0.0015%.

[0058] In certain embodiments the reaction mixture in step b) i. also contain a fluorescent dye which is suitable to detect alpha synuclein aggregation, such as such as thioflavin T (ThT) or thioflavin S (ThS). In certain embodiments the reaction mixture in step b) i. contain the fluorescent dye in a concentration of about 10 uM. In certain embodiments the reaction mixture in step b) i. contain thioflavin T (ThT) in a concentration of about 10 uM.

[0059] In some embodiments the seeded aggregation assays of the invention employs incubation and agitation conditions in step b) ii. which permits and / or promotes coaggregation of misfolded aSyn aggregates present in the biological sample, or fraction thereof, with the aSyn substrate. Such conditions permits and / or promotes coaggregation of the aSyn substrate with the misfolded aSyn aggregates to result in a conversion of the soluble aSyn to aSyn aggregates while inhibiting spontaneous aggregation of soluble aSyn. In certain embodiments the incubation and agitation in step b) ii. comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking. In further embodiments the shaking cycles are repeated throughout the incubation and agitation step, such as repeating shaking cycles of about 20 to 180 seconds shake and about 20 to 180 seconds rest throughout this step. In further embodiments the shaking cycles are repeated throughout the incubation and agitation step, such as repeating shaking cycles of about 30 to 70 seconds shake and about 30 to 70 seconds rest throughout this step. In further embodiments the shaking cycles are repeated throughout the incubation and agitation step, such as repeating shaking cycles of about 50-70 seconds shake and about 50-70 seconds rest throughout this step. In further embodiments the shaking cycles are repeated throughout the incubation and agitation step, such as repeating shaking cycles of about 1 minute shake and about 1 minute rest throughout this step. In further embodiment the shaking is performed at about 500 rpm. In some embodiments the incubation and agitation in step b) ii. are performed at about 30-45 degrees Celsius. In some embodiments the incubation and agitation in step b) ii. are performed at about 35-40 degrees Celsius. In some embodiments the incubation and agitation in step b) ii. are performed at about 37 degrees Celsius. In some embodiments the incubation and agitation in step b) ii. takes about 40 hours. In some embodiments the incubation and agitation in step b) ii. Is performed in the presence of silica beads, and in specific embodiments these silica beads are about 0.1 mm in size.

[0060] In some embodiments the incubation and agitation in step b) ii. does not comprise sonication, i.e in some embodiment the incubation and agitation is performed in the absence of sonication.

[0061] In some embodiments the seeded aggregation assay os the invention are in vitro assays.

[0062] In some embodiments the methods of the invention are in vitro methods.

[0063] Numbered embodiments In the following specific embodiments of the invention are disclosed. The first embodiment is denoted El, the second embodiment is denoted E2 and so forth.

[0064] El. A method for identifying a subject suffering from a synucleinopathy, which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suspected of suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric wild type alpha synuclein (SEQ ID NO: 1) or a fragment thereof to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric wild type alpha synuclein with the aSyn aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from a synucleinopathy, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject not suffering from a synucleinopathy; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from a synucleinopathy.

[0065] E2. A method for identifying a subject suffering from MSA or PD, which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i) contacting the biological sample with a mutated form of monomeric alpha synuclein to form a reaction mixture; and ii) incubating and agitating the reaction mixture of i) to induce coaggregation of the mutated form of monomeric alpha synuclein with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from PD.

[0066] E3. A method for identifying a subject suffering from MSA or PD, which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i) contacting the biological sample with a mutated form of monomeric alpha synuclein to form a reaction mixture; and ii) incubating and agitating the reaction mixture of i) to induce coaggregation of the mutated form of monomeric alpha synuclein with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation, and where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from MSA and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from PD.

[0067] E4. A method for identifying a subject suffering from MSA or PD, which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from PD.

[0068] E5. A method for identifying a subject suffering from MSA or PD, which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suffering from a synucleinopathy, which is not dementia with lewy body; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA in the absence of a positive detection of alpha synuclein aggregation in step c).

[0069] E6. A method for identifying a subject suffering from MSA, which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suffering from MSA or PD; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA in the absence of a positive detection of alpha synuclein aggregation in step c) as this will be indicative of the subject suffering from MSA.

[0070] E7. A method for identifying a subject suffering from PD, which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suffering from MSA or PD; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from PD if a positive detection of alpha synuclein aggregation in step c) is observed as this will be indicative of the subject suffering from PD.

[0071] E8. A method for identifying a subject suffering from MSA or PD, which method comprises performing two separate or independent alpha synuclein seeded aggregation assays on a biological sample from the subject, or a fraction thereof, wherein the first seeded aggregation assay includes: a) Providing a biological sample from a subject suspected of suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric wild type alpha synuclein (SEQ ID NO: 1) or a fragment thereof to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric wild type alpha synuclein with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from a synucleinopathy, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject not suffering from a synucleinopathy; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from a synucleinopathy; and wherein if the conclusion from the first assay is that the subject suffers from a synucleinopathy, then performing the second assay to determine whether the subject suffers from MSA or PD, which second assay includes: aa) Providing a biological sample from the subject suffering from a synucleinopathy; bb) Performing on the biological sample provided in step aa) a seeded aggregation assay, wherein the assay comprises: il. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and iil. incubating and agitating the reaction mixture of il) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the alpha synuclein aggregates present in the biological sample; cc) Quantifying the alpha synuclein aggregation in the reaction mixture; dd) Determining if the alpha synuclein aggregation quantified in step cc) is indicative of a positive detection of alpha synuclein aggregation; and ee) Identifying if the sample from step aa) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step cc) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step cc) will be indicative of the subject suffering from PD. E9. A method for identifying a subject suffering from MSA or PD, which method comprises performing two separate or independent alpha synuclein seeded aggregation assays on a biological sample from the subject, or a fraction thereof, wherein the first seeded aggregation assay includes: a) Providing a biological sample from a subject suspected of suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric wild type alpha synuclein (SEQ ID NO: 1) or a fragment thereof to form a reaction mixture; and ii. incubating and agitating the reaction mixture of i) to induce coaggregation of the monomeric wild type alpha synuclein with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from a synucleinopathy, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject not suffering from a synucleinopathy; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from a synucleinopathy; and wherein if the conclusion from the first assay is that the subject suffers from a synucleinopathy, then determine that dementia with lewy body is not the correct diagnosis; whereby the subjects potential diagnosis is limited to being either PD or MSA then performing the second assay to determine whether the subject suffers from MSA or PD, which second assay includes: aa) Providing a biological sample from the subject suffering from a synucleinopathy; bb) Performing on the biological sample provided in step aa) a seeded aggregation assay, wherein the assay comprises: il. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and iil. incubating and agitating the reaction mixture of il) to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the alpha synuclein aggregates present in the biological sample; cc) Quantifying the alpha synuclein aggregation in the reaction mixture; dd) Determining if the alpha synuclein aggregation quantified in step cc) is indicative of a positive detection of alpha synuclein aggregation; and ee) Identifying if the sample from step aa) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step cc) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step cc) will be indicative of the subject suffering from PD.

[0072] E10. The method of embodiments E1-E9 wherein the subject is a human.

[0073] Ell. The method of embodiments E1-E1O wherein the subject is a human suffering from a synucleinopathy.

[0074] E12. The method of embodiments El-Ell wherein the subject suffers from a synucleinopathy, which is not dementia with lewy body.

[0075] E13. The method of embodiments E1-E12 wherein the subject suffers from either PD or MSA.

[0076] E14. The method of embodiments E1-E13 wherein the subject suffers from either PD or MSA, wherein the diagnosis has not concluded which of these diseases are the correct diagnosis for the subject.

[0077] E15. The method of embodiments E1-E14 wherein the biological sample is obtained from a nasal brushing, saliva, skin, whole blood, serum, plasma, cerebrospinal fluid (CSF), feces, urine or a tissue sample, such as, a brain tissue sample.

[0078] E16. The method of embodiments E1-E15 wherein the biological sample is a plasma sample or a CSF sample.

[0079] E17. The method of embodiments E1-E16 wherein the biological sample is a CSF sample.

[0080] E18. The method of embodiments E1-E17 wherein the biological sample of step a is diluted before performing the remaining assay steps b though c. E19. The method of embodiments E1-E18 wherein the biological sample of step a is diluted in a phosphate buffer or artificial CSF before performing the remaining assay steps b though c.

[0081] E20. The method of embodiments E1-E19 wherein the biological sample of step a is diluted 10-fold, 100-fold, l.OOO-fold or 10.000 fold before performing the remaining assay steps b though c.

[0082] E21. The method of embodiments E1-E20 wherein the seeded aggregation assay step c is run in replicates of between 2 and 5 before making the determination in step d.

[0083] E22. The method of embodiments E1-E21 wherein the seeded aggregation assay step c is run in duplicates before making the determination in step d.

[0084] E23. The method of embodiments E1-E21 wherein the seeded aggregation assay step c is run in triplicates before making the determination in step d.

[0085] E24. The method of embodiments E1-E21 wherein the seeded aggregation assay step c is run in quadruplicates before making the determination in step d.

[0086] E25. The method of embodiments E1-E21 wherein the seeded aggregation assay step c is run in quintuplicates before making the determination in step d.

[0087] E26. The method of embodiments E1-E20 wherein the seeded aggregation assay steps b through c is run in replicates of between 2 and 5 before making the determination in step d.

[0088] E27. The method of embodiments E1-E26 wherein the seeded aggregation assay steps b through c is run in duplicates before making the determination in step d.

[0089] E28. The method of embodiments E1-E26 wherein the seeded aggregation assay steps b through c is run in triplicates before making the determination in step d.

[0090] E29. The method of embodiments E1-E26 wherein the seeded aggregation assay steps b through c is run in quadruplicates before making the determination in step d. E30. The method of embodiments E1-E26 wherein the seeded aggregation assay steps b through c is run in quintuplicates before making the determination in step d.

[0091] E31. The method of embodiments E1-E30 wherein the determination of step d is a binary determination which is either positive or negative.

[0092] E32. The method of embodiments E1-E31 wherein the determination of step d is a binary determination which is either positive or negative depending on how many of the replicates of assay steps b through c or assay step c were determined to be positive, such as at least 2 replicates yielding a positive signal of aggregation, such as at least 3 replicates yielding a positive signal of aggregation, or such as at least 4 replicates yielding a positive signal of aggregation.

[0093] E33. The method of embodiments E1-E31 wherein the determination of step d is a binary determination which is either positive or negative depending on how many of the replicates of assay steps b through c or assay step c were determined to be positive, such as for example at least 40% of the replicates of step c yielding a positive signal of aggregation, such as at least 50 % of the replicates of step c yielding a positive signal of aggregation, such as at least 60 % of the replicates of step c yielding a positive signal of aggregation, such as at least 70 % of the replicates of step c yielding a positive signal of aggregation, such as at least 80 % of the replicates of step c yielding a positive signal of aggregation, or such as at least 90 % of the replicates of step c yielding a positive signal of aggregation.

[0094] E34. The method of embodiments E1-E32 wherein the determination of step d is a binary determination which is either positive or negative, wherein at least 2 replicates of assay steps b through c or assay step c yielding a positive signal of aggregation is determined to be positive signal for aggregation.

[0095] E35. The method of embodiments E1-E32 wherein the determination of step d is a binary determination which is either positive or negative, wherein at least 3 replicates of assay steps b through c or assay step c yielding a positive signal of aggregation is determined to be positive signal for aggregation. E36. The method of embodiments E1-E32 wherein the determination of step d is a binary determination which is either positive or negative, wherein at least 4 replicates of assay steps b through c or assay step c yielding a positive signal of aggregation is determined to be positive signal for aggregation.

[0096] E37. The method of embodiments E1-E36 wherein the determination of step d is a binary determination which is either positive or negative decided based on a pre-determined threshold for what is indicative of a positive detection of alpha synuclein aggregation.

[0097] E38. The method of embodiments E37 wherein the quantification in step c is done by fluorescent methods and the pre-determined threshold is set to about 15.000 or about 40.000 arbitrary intensity units.

[0098] E39. The method of embodiments E1-E38 wherein step c) comprises quantifying the alpha synuclein fibril formation in the reaction mixture; and step d) comprises determining if the alpha synuclein fibril formation quantified in step c) is indicative of a positive detection of alpha synuclein fibril formation; and step e) comprises identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein fibril formation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein fibril formation in step c) will be indicative of the subject suffering from PD.

[0099] E40. The method of embodiments E1-E39 wherein the seeded aggregation assay utilizes a truncated form of monomeric alpha synuclein as substrate.

[0100] E41. The method of embodiments E1-E39 wherein the seeded aggregation assay utilizes a mutated form of monomeric alpha synuclein as substrate.

[0101] E42. The method of embodiments E1-E39 wherein the seeded aggregation assay utilizes a mutated and truncated form of monomeric alpha synuclein as substrate.

[0102] E43. The method of embodiments E39-E42 wherein the mutated form of monomeric alpha synuclein as substrate contains between one and eight point mutations in SEQ. ID NO: 1. E44. The method of embodiments E43 wherein the point mutations are at residues K23, D2, K21, K45, T59, G67, V77, and / or A78.

[0103] E45. The method of embodiments E44 wherein the point mutation is at residue K23, such as a K23Q. mutation.

[0104] E46. The method of embodiments E40 and E42 wherein the truncated forms of monomeric alpha synuclein contains a deletion of up to 30 amino acids of SEQ. ID NO: 1, such as up to 25 amino acids deletion, such as up to 20 amino acids deletion, such as up to 15 amino acids deletion, such as up to 10 amino acids deletion, such as up to 5 amino acids deletion.

[0105] E47. The method of embodiments E1-E46 wherein the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) employs the use of a fluorescent dye.

[0106] E48. The method of embodiments E1-E47 wherein the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) employs the use of a fluorescent dye which is suitable to quantify the alphasynuclein aggregation.

[0107] E49. The method of embodiments E1-E48 wherein the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) employs the use of a fluorescent dye selected from as thioflavin T (ThT) or thioflavin S (ThS).

[0108] E50. The method of embodiments E1-E48 wherein the fluorescent dye is thioflavin T (ThT).

[0109] E51. The method of embodiments E47-E49 wherein the fluorescent dye is added to the reaction mixture of step b i.

[0110] E52. The method of embodiments E1-E50 wherein the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) is performed within 24 - 48 hours after the reaction mixture in step b) i. was first formed.

[0111] E53. The method of embodiments E1-E52 wherein the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) is performed within 24 - 48 hours after the incubation and agitation of the reaction mixture in step b) ii. was initiated. E54. The method of embodiments E1-E53 wherein the biological sample to be tested in the seeded aggregation assay is an undiluted sample.

[0112] E55. The method of embodiments E1-E54 wherein the biological sample to be tested in the seeded aggregation assay is about 15 uL in volume.

[0113] E56. The method of embodiments E1-E55 wherein the monomeric alpha synuclein substrate is provided in a composition which is then added to the biological sample to form the reaction mixture of step b i.

[0114] E57. The method of embodiments E56 wherein the monomeric alpha synuclein substrate is added in a composition in a concentration of about 0.04 to 1.00 mg / mL, such as about 0.06 to 0.08 mg / mL or such as about 0.08 mg / mL.

[0115] E58. The method of embodiments E56-E57 wherein the monomeric alpha synuclein substrate is added in a composition which further comprises a 20 mM phosphate buffer.

[0116] E59. The method of embodiments E56-E58 wherein the monomeric alpha synuclein substrate is added in a composition which has a pH of about 6-8, such as about 7-8, such as about 7.5.

[0117] E60. The method of embodiments E56-E59 wherein the monomeric alpha synuclein substrate is added in a composition which has a pH of about 7.5.

[0118] E61. The method of embodiments E56-E60 wherein the monomeric alpha synuclein substrate is added in a composition which further comprises sodium dodecyl sulfate (SDS).

[0119] E62. The method of embodiments E61 wherein the sodium dodecyl sulfate (SDS) is present in a concentration of about 0.001 % - 0.005%.

[0120] E63. The method of embodiments E61-E62 wherein the sodium dodecyl sulfate (SDS) is present in a concentration of about 0.0015%. E64. The method of embodiments E1-E63 wherein the monomeric alpha synuclein substrate is added in a composition which further comprises a fluorescent dye in a concentration of about 10 uM.

[0121] E65. The method of embodiments E1-E64 wherein the monomeric alpha synuclein substrate is added in a composition which further comprises thioflavin T (ThT) in a concentration of about 10 uM.

[0122] E66. The method of embodiments E1-E65 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which permits and / or promotes coaggregation of alpha synuclein aggregates present in the biological sample, or fraction thereof, with the monomeric alpha synuclein substrate.

[0123] E67. The method of embodiments E1-E66 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which permits and / or promotes coaggregation of alpha synuclein aggregates present in the biological sample, or fraction thereof, with the monomeric alpha synuclein substrate, while inhibiting spontaneous aggregation of the monomeric alpha synuclein substrate.

[0124] E68. The method of embodiments E1-E67 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which comprise shaking the reaction mixture in a shaking cycle, wherein each shaking cycle comprises a period of rest and a period of shaking.

[0125] E69. The method of embodiments E1-E68 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which comprise shaking the reaction mixture in a shaking cycle, wherein the shaking cycles are repeated throughout the incubation and agitation step.

[0126] E70. The method of embodiments E1-E69 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which comprise shaking the reaction mixture in a shaking cycle, wherein the shaking cycles comprise repeating shaking cycles of about 20 to 180 seconds shake and about 20 to 180 seconds rest throughout this step. E71. The method of embodiments E1-E69 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which comprise shaking the reaction mixture in a shaking cycle, wherein the shaking cycles comprise repeating shaking cycles of about 30 to 70 seconds shake and about 30 to 70 seconds rest throughout this step.

[0127] E72. The method of embodiments E1-E69 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which comprise shaking the reaction mixture in a shaking cycle, wherein the shaking cycles comprise repeating shaking cycles of about 50 to 70 seconds shake and about 50 to 70 seconds rest throughout this step.

[0128] E73. The method of embodiments E1-E69 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which comprise shaking the reaction mixture in a shaking cycle, wherein the shaking cycles comprise repeating shaking cycles of about 1 minute shake and about 1 minute rest throughout this step.

[0129] E74. The method of embodiments E1-E73 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which comprise shaking the reaction mixture in a shaking cycle, wherein the shaking cycles is performed at about 500 rpm.

[0130] E75. The method of embodiments E1-E74 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. which are performed at about 30-45 degrees Celsius.

[0131] E76. The method of embodiments E1-E75 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. which are performed at about 30-40 degrees Celsius.

[0132] E77. The method of embodiments E1-E76 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. which are performed at about 37 degrees Celsius.

[0133] E78. The method of embodiments E1-E77 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. for about 24-48 hours, such as about 40 hours.

[0134] E79. The method of embodiments E1-E78 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. wherein the reaction mixture comprises silica beads. E80. The method of embodiments E1-E79 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. which is performed in the presence of silica beads, which beads are about 0.1 mm in size.

[0135] E81. The method of embodiments E1-E80 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. which is performed in the absence of sonication.

[0136] E82. The method of embodiments E1-E81 wherein the seeded aggregation assay is an in vitro assay.

[0137] E83. The method of embodiments E1-E82 wherein the seeded aggregation assay is a realtime quacking-induced conversion (RT-QuIC) assay.

[0138] E84. The method of embodiments E1-E83 wherein the seeded aggregation assay is a diagnostic assay.

[0139] E85. A method of treating MSA or PD, which method comprises administering to an MSA or PD patient an effective amount of a compound capable of treating the disease, wherein the MSA or PD patient is identified via any of the methods described in E1-E84.

[0140] E86. The method of embodiment E1-E84, which method further comprises step f), wherein step f) comprises administering to the MSA or PD patient an effective amount of a compound capable of treating the disease.

[0141] E87. The method of embodiment E1-E84, which method further comprises step f), wherein step f) comprises administering to the MSA or PD patient an effective amount of a compound capable of inhibiting alphasynuclein aggregation.

[0142] E88. A method of treating PD, which method comprises administering to a PD patient an effective amount of a compound capable of treating the disease, wherein PD patient is identified via any of the methods described in E1-E84. E89. A method of treating MSA, which method comprises administering to an MSA patient an effective amount of a compound capable of treating the disease, wherein the MSA patient is identified via any of the methods described in E1-E84.

[0143] E90. A method of treating MSA or PD according to any of E85-E87, wherein the compound capable of treating the disease is selected from monoclonal antibodies, small molecules, antisense oligonucleotides or peptides.

[0144] E91. A method of treating MSA or PD according to any of E85-E88, wherein the compound capable of treating the disease is a monoclonal antibody, such as an anti-alphasynuclein monoclonal antibody.

[0145] E92. A method of treating MSA or PD according to any of E85-E88, wherein the compound capable of treating the disease is a small molecule.

[0146] E93. A method of treating MSA or PD according to any of E85-E88, wherein the compound capable of treating the disease is an antisense oligonucleotides.

[0147] E94. A method of treating MSA or PD according to any of E85-E88, wherein the compound capable of treating the disease is a peptide capable of inducing active immunotherapy.

[0148] E95. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is selected from the list consisting of prasinezumab, amlenetug, cinpanemab, ABBV-0805 / BAN0805, UCB7853, TAK-341 / MEDI1341,

[0149] Emrusolmin / TEV-56286, PBT434 / ATH434, BIIB101 / ION464 and UB-312.

[0150] E96. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is selected from the list consisting of prasinezumab, amlenetug, cinpanemab, ABBV-0805 / BAN0805, UCB7853 and TAK-341 / M EDI 1341.

[0151] E97. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is Emrusolmin / TEV-56286 or PBT434 / ATH434. E98. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is BIIB101 / ION464 or UB-312.

[0152] E99. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is UB-312.

[0153] E100. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is selected from prasinezumab, amlenetug or TAK- 341 / MEDI1341.

[0154] E101. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is prasinezumab.

[0155] E102. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is amlenetug.

[0156] E103. A method of treating MSA or PD according to any of E85-E92, wherein the compound capable of treating the disease is TAK-341 / MEDI1341.

[0157] E104. A method of treating MSA or PD according to any of E85-E103, wherein the treatment comprises slowing or delaying disease progression.

[0158] E105. A method of treating MSA or PD according to any of E85-E103, wherein the treatment comprises slowing or delaying clinical progression.

[0159] E106. A monomeric alpha synuclein K23Q. mutant substrate for use in any of the methods of embodiment E1-E105.

[0160] Experimental section

[0161] Example 1 - RT-QuIC assays for identifying MSA patients In this example the inventors of the present invention demonstrate that a specific sequential combination of RT-QuIC assays can be used to differentiate patients with Multiple System Atrophy (MSA) from patients with Parkinson's Disease (PD).

[0162] The RT-QuIC assay:

[0163] The basis for the assay is that if a sample containing alpha synuclein fibrils (seeding material) is added to an alpha synuclein monomeric substrate, this will in general causes the alpha synuclein monomeric substrate to undergo fibril formation and this reaction was followed and quantified over time. The alpha synuclein seeding material can be obtained from various sources such as from biological samples. In this example brain homogenate and CSF from patients suffering from Parkinson's Disease (PD) or Multiple System Atrophy (MSA) as well as brain homogenate and CSF from Heathy Control (CTRL) were used as seeding material.

[0164] These samples were analysed in two separate RT-QuIC assays using different alpha synuclein monomeric substrates. One assay, denoted K23Q RT-QuIC, is using the alpha synuclein monomeric mutant K23Q (SEQ ID NO: 2) as the substrate and the other assay, denoted WT RT-QuIC, are using wild type alpha synuclein (SEQ ID NO: 1) monomeric substrate.

[0165] A positive control was also included in each assay run to verify the ability to quantify fibril formation over time. This positive control employed recombinant preformed fibrils (PFF) of wildtype alpha synuclein in CSF as the seeding material (data not shown).

[0166] RT-QuIC on human brain homogenates

[0167] The RT-QuIC assay was performed using 3 different samples i.e. Parkinson's Disease (PD), Multiple System Atrophy (MSA) and Heathy Control (HC) brain homogenates.

[0168] Each sample was tested at 4-5 different dilutions and each of the diluted samples was tested in 4 replicates in the RT-QuIC assays. The 96 well plate setup for this assay is depicted in figure 1. The K23Q RT-QuIC results from brain homogenate is depicted in figure 2 and the WT RT-QuIC results are depicted in figure 3.

[0169] RT-QuIC on undiluted human CSF samples

[0170] The RT-QuIC assay was performed using CSF from PD patients, MSA patients and healthy controls (HC). The CSF samples were obtained from Bispebjerg Hospital and BioIVT. CSF samples from 5 MSA patients, 2 PD patients and 2 healthy controls were used. Each sample was tested in 5 replicates. The 96 well plate setup for this assay is depicted in figure 4 - the individual samples are identified by the following numbers MSA1: 100140, MSA2: 100439, MSA3: 100460, MSA4: 100487, MSA5: 100527, PD1: 424777, PD2: 424779, CTL1: 100554 and CTL2: 100580. The K23Q RT-QuIC results from CSF samples is depicted in figure 5A and the WT RT-QuIC results are depicted in figure 5B. These results are also presented in figure 6A (K23Q. RT-QuIC) and figure 6B (WT RT-QuIC) as bar graphs representing quantification of the RT-QuIC aggregation curves shown in figure 5A and 5B.

[0171] Table 1: Assay conditions for the RT-QuIC used in example 1

[0172] Instrument used to quantify fibril formation over time: BMG ClarioStar, head-sealed plate, top read, 500 rpm, shake rest cycle of 1 min on-1 min off, for 40 h.

[0173] Assay reaction was done in a 100 uL volume in a heat-sealed, flat-bottom microwell plate containing 40 mg of 0.1 mm silica / zirconium beads per well. 15 uL of test sample (CSF) was added into 85 uL master mix (MM, containing 20 mM phosphate buffer pH 7.5, 85 mM NaCI, 10 uM ThT and 0.08 mg / ml K23Q / WT alpha synuclein).

[0174] The plate was incubated in a BMG ClarioStar at 37 degrees and fluorescence readout (ex 440 nm, em 480 nm, gain set at 800) was performed after cycles of 1 min shake (500 rpm), 1 min rest. Total run time was 40 hours.

[0175] Results of the RT-QuIC on human brain homogenates: The WT RT-QuIC results from brain homogenates are depicted in figure 3 and show that under these conditions no clear differentiation could be observed between the PD, MSA or HC. All samples started giving signals at a similar time point.

[0176] The K23Q. RT-QuIC results from brain homogenate are depicted in figure 2 and here clear aggregation curves were observed for PD brain homogenate samples whereas the curves of MSA and HC homogenate samples stayed close to the baseline.

[0177] Results of the RT-QuIC on undiluted human CSF samples

[0178] The WT RT-QuIC results from undiluted CSF samples are depicted in figure 5B and 6B and show clear aggregation curves for 4 out of 5 MSA samples and 2 out of 2 PD samples, whereas the curves of HC stayed close to the baseline. In figure 6B the aggregation profiles presented in figure 5B that were above the pre-determined threshold were counted as positive; as each sample was run in 5 replicates the amount of positive aggregation curves for each sample was between 0 and 5. This threshold was based on a binary assessment where the decision time was selected as 40 hours (h) and threshold for the WT RT-QuIC assay was set to 15000 arbitrary intensity units (a.u.).

[0179] The K23Q. RT-QuIC results from undiluted CSF samples are depicted in figure 5A and 6A and show clear aggregation curves for PD samples whereas the curves for MSA and HC stayed close to the baseline. In figure 6A the aggregation profiles of figure 5A that were above the pre-determined threshold were counted as positive; as each sample was run in 5 replicates the amount of positive aggregation curves for each sample was between 0 and 5. This threshold was based on a binary assessment where the decision time was selected as 40 hours (h) and threshold for the K23Q. RT-QuIC assay was set to 40000 arbitrary intensity units (a.u.).

Claims

Claims1. A method for identifying a subject suffering from Multiple System Atrophy (MSA) or Parkinson's Disease (PD), which method comprises performing an alpha synuclein seeded aggregation assay on a biological sample from the subject, or a fraction thereof, wherein the seeded aggregation assay includes: a) Providing a biological sample from a subject suffering from a synucleinopathy; b) Performing on the biological sample provided in step a) a seeded aggregation assay, wherein the assay comprises: i. contacting the biological sample with a monomeric alpha synuclein K23Q mutant (SEQ ID NO: 2) to form a reaction mixture; and ii. incubating and agitating the reaction mixture of step i. to induce coaggregation of the monomeric alpha synuclein K23Q mutant with the alpha synuclein aggregates present in the biological sample; c) Quantifying the alpha synuclein aggregation in the reaction mixture; d) Determining if the alpha synuclein aggregation quantified in step c) is indicative of a positive detection of alpha synuclein aggregation; and e) Identifying if the sample from step a) is from a subject suffering from MSA or PD, where an absence of a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from MSA; and a positive detection of alpha synuclein aggregation in step c) will be indicative of the subject suffering from PD.

2. The method of claim 1, wherein step a) comprises: a) Providing a biological sample from a subject suffering from a synucleinopathy, which is not dementia with lewy body.

3. The method of claim 1-2 for identifying a subject suffering from MSA, wherein step a) comprises: a) Providing a biological sample from a subject suffering from MSA or PD; and step e) comprises: e) Identifying if the sample from step a) is from a subject suffering from MSA in the absence of a positive detection of alpha synuclein aggregation in step c) as this will be indicative of the subject suffering from MSA.

4. The method of claim 1-2 for identifying a subject suffering from PD, wherein step a) comprises: a) Providing a biological sample from a subject suffering from MSA or PD; and step e) comprises: e) Identifying if the sample from step a) is from a subject suffering from PD if a positive detectionof alpha synuclein aggregation in step c) is observed as this will be indicative of the subject suffering from PD.

5. The method of claims 1-4 wherein the subject is a human.

6. The method of claims 1-5 wherein the biological sample is a CSF sample.

7. The method of claims 1-6 wherein the seeded aggregation assay step c is run in replicates of at least 2 before making the determination in step d.

8. The method of claims 1-6 wherein the seeded aggregation assay steps b through c is run in replicates of at least 2 before making the determination in step d.

9. The method of claims 1-8 wherein the determination of step d is a binary determination which is either positive or negative depending on how many of the replicates of assay step c were determined to be positive, such as at least 60 % of the replicates of step c yielding a positive signal of aggregation, such as at least 70 % of the replicates of step c yielding a positive signal of aggregation, such as at least 80 % of the replicates of step c yielding a positive signal of aggregation, or such as at least 90 % of the replicates of step c yielding a positive signal of aggregation.

10. The method of claims 1-9 wherein the determination of step d is a binary determination which is either positive or negative decided based on a pre-determined threshold for what is indicative of a positive detection of alpha synuclein aggregation.

11. The method of claims 1-10 wherein the quantification of the alpha synuclein aggregation in the reaction mixture performed in step c) employs the use of a fluorescent dye selected from as thioflavin T (ThT) or thioflavin S (ThS).

12. The method of claims 1-11 wherein the seeded aggregation assay employs incubation and agitation conditions in step b) ii. which permits and / or promotes coaggregation of alpha synuclein aggregates present in the biological sample, or fraction thereof, with the monomeric alpha synuclein substrate, while inhibiting spontaneous aggregation of the monomeric alpha synuclein substrate.

13. The method of claims 1-12 wherein the seeded aggregation assay employs incubation and agitation in step b) ii. which is performed in the absence of sonication.

14. The method of claims 1-13 wherein the seeded aggregation assay is a real-time quacking-induced conversion (RT-QuIC) assay.

15. A compound capable of treating MSA or PD for use in the treatment of MSA or PD, wherein the MSA or PD patient to be treated is identified by any of the methods described in claims 1-14.

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