Method for determining synucleinopathy

Abstract

To provide a method for determining whether a subject suffers from synucleinopathy.SOLUTION: There is provided a method for determining whether a subject suffers from synucleinopathy, the method including comparing the level of ApoA1 bound to α-synuclein in a sample collected from the subject with a cutoff value.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to methods and kits for determining whether a subject is afflicted with a synucleinopathy. [Background technology]

[0002] Synucleinopathy is a general term for neurodegenerative diseases characterized by the accumulation of α-synuclein in the brain, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy (Non-Patent Documents 1 and 2). Drugs capable of curing these diseases are still under development, but symptomatic treatment using dopamine precursors and dopamine agonists has been established for the motor dysfunction of Parkinson's disease. However, the effectiveness of dopamine replacement therapy is relatively short-lived, and therefore, from the perspective of patient quality of life, it is important to diagnose Parkinson's disease patients as early as possible and intervene early in their treatment.

[0003] Parkinson's disease has traditionally been diagnosed based on its characteristic clinical symptoms, but diagnosis is difficult in early-stage patients with only mild motor dysfunction. Furthermore, a definitive diagnosis of Parkinson's disease requires nuclear medicine imaging (MIBG myocardial scintigraphy, DAT scan), which requires advanced measuring equipment. Therefore, the facilities that can perform this test are limited, the test takes a long time, and the test is expensive (Non-Patent Document 3).

[0004] Research into Parkinson's disease biomarkers has been conducted in body fluids (blood and cerebrospinal fluid). A study measuring the ratio of α-synuclein oligomers to α-synuclein in cerebrospinal fluid (Non-Patent Document 4) found that Parkinson's disease was significantly differentiated from healthy controls, but there was some overlap between the distributions of Parkinson's disease and healthy controls. Furthermore, collecting cerebrospinal fluid is highly invasive, placing a significant burden on patients and making it unsuitable for screening tests aimed at early diagnosis.

[0005] Previous studies measuring blood α-synuclein (Non-Patent Document 5) have reported that blood α-synuclein levels are significantly increased in Parkinson's disease patients, but the concentration distribution largely overlaps with that of healthy controls, making it difficult to use for diagnosing Parkinson's disease. Furthermore, research focusing on oxidized DJ-1 in red blood cells as a biomarker for Parkinson's disease has been reported, but its ability to distinguish between Parkinson's disease and healthy individuals is insufficient, making it difficult to use for diagnosis (Non-Patent Document 6).

[0006] It has been reported that apolipoprotein A1 (ApoA1) protein in the blood of Parkinson's disease patients is significantly lower than that of healthy individuals, but the ApoA1 concentration distributions in Parkinson's disease and healthy individuals overlap, and diagnostic accuracy sufficient to differentiate between the two cannot be expected (Non-Patent Document 7). Furthermore, although ApoA1 has been reported as one of the α-synuclein-binding proteins, there is no evidence that the binding state of α-synuclein and ApoA1 may change in Parkinson's disease (Non-Patent Document 8). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Clinical Neurology, Vol. 53, No. 8 (2013:8) Koichi Wakabayashi [Non-patent document 2] Non-patent document 2 Neurotherapy 37, 621-624 (2020) Kenjiro Ono [Non-patent document 3] Journal of the Japan Geriatrics Society, Vol. 53, No. 3 (2016:7) Tomoyuki Orimo [Non-patent document 4] Neurology 2010;75:1766-1772 T. Tokuda, et al. [Non-Patent Document 5] Annals of Clinical and Translational Neurology 2019; 6(3): 615-619 Adeline SL Ng, et al. [Non-patent document 6] Sci. Rep. 6, 30793; doi: 10.1038 / srep30793 (2016). Saito, Y., et al. [Non-Patent Document 7] Ann Neurol. 2013 July; 74(1): 119-127. doi:10.1002 / ana.23872. Judy K. Qiang, et al. [Non-patent document 8] J Mol Neurosci (2017) 63:165-172 DOI 10.1007 / s12031-017-0967-0 Fatemeh Nouri Emamzadeh & David Allsop Summary of the Invention [Problem to be solved by the invention]

[0008] One object of the present disclosure is to provide a method for determining whether a subject is suffering from a synucleinopathy. [Means for solving the problem]

[0009] The present inventors have found that the level of ApoA1 bound to α-synuclein is low in the blood of Parkinson's disease patients, and therefore, α-synuclein-bound ApoA1 can be used as a marker for synucleinopathies such as Parkinson's disease.

[0010] In certain aspects, the present disclosure provides a method for determining whether a subject is affected by a synucleinopathy, comprising comparing the level of ApoA1 bound to alpha-synuclein in a sample obtained from the subject to a cutoff value.

[0011] In certain aspects, the present disclosure provides a kit for determining whether a subject is afflicted with a synucleinopathy, comprising a reagent that specifically binds to ApoA1 and a reagent that specifically binds to alpha-synuclein. [Effects of the Invention]

[0012] The present disclosure provides methods and kits for determining whether a subject is afflicted with a synucleinopathy. [Brief explanation of the drawings]

[0013] [Figure 1] 1 shows the results of measuring the amount of ApoA1 in the α-synuclein-bound fraction in the blood of healthy individuals and Parkinson's disease patients by mass spectrometry. [Figure 2] The results of measuring the blood ApoA1 levels in healthy subjects and Parkinson's disease patients by ELISA are shown. [Figure 3] 1 shows the results of measuring the amount of ApoA1 in the α-synuclein-bound fraction in the blood of healthy subjects and Parkinson's disease patients by ELISA. DETAILED DESCRIPTION OF THE INVENTION

[0014] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0015] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0016] In the present disclosure, the subject may be of any species, preferably a human. The sample may be blood, plasma, or serum collected from the subject. Blood samples may be collected by conventional methods, for example, from a vein or artery. Plasma and serum samples may be prepared by appropriately treating the blood using methods well known to those skilled in the art. This treatment is not particularly limited and may be any clinically acceptable treatment, such as the addition of an anticoagulant or centrifugation. The sample may also be other body fluids collected from the subject, such as cerebrospinal fluid, saliva, nasal discharge, sputum, pleural effusion, or ascites. The collected sample may be stored at a low temperature during or after its preparation prior to use, for example, frozen. The collected sample may also be appropriately concentrated or diluted as needed.

[0017] Alpha-synuclein is a protein expressed primarily in presynaptic terminals. Abnormal deposition of alpha-synuclein has been confirmed in the brains of patients with synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The amino acid sequences of alpha-synuclein from various species are readily available using publicly known databases. A representative amino acid sequence of human alpha-synuclein is registered in GenBank under accession number NP_000336.1 (SEQ ID NO: 1). In the present disclosure, alpha-synuclein includes its naturally occurring allelic products.

[0018] ApoA1 is known as a major component of HDL. The amino acid sequences of ApoA1 from various species are readily available from publicly known databases. A representative amino acid sequence of human ApoA1 is registered in GenBank under accession number NP_000030.1 (SEQ ID NO: 2). In the present disclosure, ApoA1 includes its naturally occurring allelic products.

[0019] In the present disclosure, α-synuclein and ApoA1 may contain sequences in which one or several amino acids have been deleted, substituted, or added to the original amino acid sequence (e.g., the amino acid sequence of SEQ ID NO: 1 for α-synuclein and the amino acid sequence of SEQ ID NO: 2 for ApoA1), so long as their functions are maintained. Note that "several" preferably means 2 to 7, more preferably 2 to 5, and most preferably 2 to 3 amino acids. Conservative substitutions between similar amino acid residues are preferred.

[0020] Furthermore, alpha-synuclein and ApoA1 may contain an amino acid sequence that, when calculated using BLAST or the like (e.g., using the default, i.e., initial, parameters of BLAST), has at least about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97%, about 98%, or about 99% or more identity with the original amino acid sequence, so long as the function is maintained.

[0021] In the Examples described below, it was found that the level of ApoA1 bound to α-synuclein in the blood of Parkinson's disease patients is low. Parkinson's disease is a typical synucleinopathy. Therefore, ApoA1 bound to α-synuclein can be used as a marker to determine whether a subject suffers from a synucleinopathy.

[0022] In the present disclosure, synucleinopathies include Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, particularly Parkinson's disease. Multiple system atrophy includes olivopontocerebellar atrophy, striatonigral degeneration, and Shy-Drager syndrome.

[0023] For example, the amount of ApoA1 bound to alpha-synuclein can be measured by obtaining an alpha-synuclein fraction from a sample using a reagent that specifically binds to alpha-synuclein, contacting the obtained alpha-synuclein fraction with a reagent that specifically binds to ApoA1, and measuring the amount of bound reagent.

[0024] The reagents that specifically bind to alpha-synuclein or ApoA1 can be, for example, antibodies, RNA, DNA, polypeptides, or aptamers. These binding reagents can also be fragments, derivatives, or analogs that are capable of specifically binding to alpha-synuclein or ApoA1.

[0025] Reagents that specifically bind to alpha-synuclein or ApoA1 are typically antibodies. In the context of this disclosure, antibody refers to affinity ligands based on immunoglobulin scaffolds, including monoclonal and polyclonal antibodies of any origin, including murine, rat, rabbit, goat, human, and other antibodies, as well as chimeric antibodies containing sequences from multiple species, e.g., partially humanized antibodies, e.g., partially humanized mouse antibodies. Antibodies may also be fragments or derivatives thereof, provided they are capable of selectively interacting with alpha-synuclein or ApoA1.

[0026] Antibodies can be produced by conventional methods using α-synuclein or ApoA1, or a partial peptide thereof having antigenicity, as an immunogen. For example, polyclonal antibodies can be produced by immunizing animals with an antigen, and monoclonal antibodies can be produced using hybridoma technology. Alternatively, commercially available antibodies can be used.

[0027] The reagent that specifically binds to alpha-synuclein or ApoA1 may be labeled with a detectable substance. Examples of detectable substances include radioisotopes, fluorescent labels, luminescent labels, bioluminescent labels, enzyme labels, and biotin. Alternatively, the reagent may be unlabeled, and a labeled substance that recognizes the reagent, such as a secondary antibody, may be used.

[0028] The reagent that specifically binds to α-synuclein or ApoA1 may be bound to a suitable support. The support is not particularly limited as long as it can immobilize the binding reagent, and may be of any shape or material. Examples of supports include membranes such as nylon membranes, beads, glass, plastic, and metal.

[0029] Any known protein purification method may be used to obtain an α-synuclein fraction from a sample, such as affinity chromatography. Dialysis, ultrafiltration, ion exchange chromatography, gel filtration chromatography, and the like may also be combined as appropriate. For example, an α-synuclein fraction can be obtained by contacting a sample with a reagent that specifically binds to α-synuclein immobilized on a support and recovering the substance bound to the support. Alternatively, ApoA1 may be detected on the support without recovering the substance bound to the support.

[0030] ApoA1 can be detected by immunological techniques using reagents that specifically bind to ApoA1. Examples of immunological techniques include enzyme-linked immunosorbent assays (ELISAs, e.g., direct, indirect, sandwich, or competitive), immunochromatography, Western blotting, flow cytometry, and radioimmunoassays (RIAs), with sandwich ELISA being preferred. For example, an α-synuclein fraction can be purified from a sample, and ApoA1 can be detected by sandwich ELISA using a first ApoA1 antibody as a capture antibody and a second ApoA1 antibody as a detection antibody. Alternatively, ApoA1 bound to α-synuclein in a sample can be detected by sandwich ELISA using a capture antibody that binds to α-synuclein and a detection antibody that binds to ApoA1, without purifying the α-synuclein fraction from the sample.

[0031] Alternatively, ApoA1 can be measured by proteomic analysis of α-synuclein fractions. Relative quantification methods (SILAC, ICAT, ICPL, isobaric tag, etc.) or absolute quantification methods (SRM method) can be used. For example, ApoA1 can be measured by nanoLC-MS / MS analysis.

[0032] In the Examples described below, it was found that the level of ApoA1 bound to α-synuclein is low in the blood of Parkinson's disease patients. Therefore, in this method, when the level of ApoA1 bound to α-synuclein in a sample collected from a subject is lower than the cutoff value, the subject is determined to have a synucleinopathy, and when the level is higher than the cutoff value, the subject is determined to not have a synucleinopathy.

[0033] When the level of ApoA1 bound to α-synuclein is equal to the cutoff value, the subject is determined to have or not have a synucleinopathy, and this can be set arbitrarily depending on the purpose of the determination. Thus, in one embodiment, when the level of ApoA1 bound to α-synuclein is below the cutoff value, the subject is determined to have a synucleinopathy, and when the level of ApoA1 bound to α-synuclein is equal to or greater than the cutoff value, the subject is determined not to have a synucleinopathy. In another embodiment, when the level of ApoA1 bound to α-synuclein is equal to or less than the cutoff value, the subject is determined to have a synucleinopathy, and when the level of ApoA1 bound to α-synuclein is higher than the cutoff value, the subject is determined not to have a synucleinopathy.

[0034] The cutoff value is a value that can statistically significantly separate a group of subjects with synucleinopathy from a group of subjects without the disease. The cutoff value can be set by known methods using various statistical analysis techniques. For example, the level of ApoA1 bound to α-synuclein in samples obtained from a group of subjects with synucleinopathy and the level of ApoA1 bound to α-synuclein in samples obtained from a group of subjects without the disease can be statistically analyzed to set the cutoff value. Statistical significance can be analyzed using known testing methods such as the chi-square test, generalized Wilcoxon test, Wilcoxon signed-rank test, Mann-Whitney test, log-rank test, and Cox proportional hazards. Statistical analysis software such as Prism can be used to set the cutoff value.

[0035] The cutoff value may be set based on sensitivity and / or specificity. Preferably, the cutoff value exhibits both high sensitivity and high specificity. Here, sensitivity refers to the true positive rate. Specificity refers to the true negative rate. For example, the level of ApoA1 bound to α-synuclein that exhibits a high positive rate in a group of subjects suffering from synucleinopathy and a high negative rate in a group of subjects not suffering from synucleinopathy may be set as the cutoff value.

[0036] For example, a cutoff value can be set using receiver operating characteristic (ROC) analysis, a commonly used method for assessing the usefulness of diagnostic tests. In ROC analysis, an ROC curve is created by plotting the sensitivity at each cutoff value on the vertical axis and the false positive rate (1 minus specificity) on the horizontal axis. For tests with no diagnostic ability, the ROC curve is a straight diagonal line. As diagnostic ability improves, the curve curve curves upward and to the left. The cutoff value that gives the point on the ROC curve with the smallest distance from the upper left corner is said to have excellent sensitivity and specificity. Alternatively, the cutoff value can be set based on the Youden index. Specifically, sensitivity and specificity are calculated from the levels of ApoA1 bound to α-synuclein in subjects with and without synucleinopathy, and an ROC curve is created using commercially available analysis software based on these values. The values ​​at which sensitivity and specificity are as close to 100% as possible are then determined and used as the cutoff value.

[0037] Furthermore, for example, the diagnostic efficiency (i.e., the ratio of the total number of cases in which subjects suffering from a synucleinopathy were correctly diagnosed as "afflicted" and the total number of cases in which subjects not suffering from a synucleinopathy were correctly diagnosed as "not afflicted") to the total number of cases can be calculated, and the level of ApoA1 bound to α-synuclein that gives the highest diagnostic efficiency can be used as the cutoff value.

[0038] Cutoff values ​​can also be set for patient subgroups according to characteristics, for example, cutoff values ​​may be set according to gender, age group, or race.

[0039] In some embodiments, the results of the present method can be provided as information for diagnosis. In some embodiments, the method includes obtaining a sample from a subject. In some embodiments, the method comprises purifying an alpha-synuclein fraction from the sample. In one embodiment, the method comprises measuring the level of ApoA1 bound to alpha-synuclein.

[0040] In some embodiments, a subject determined to have a synucleinopathy by this method is treated for the synucleinopathy. For example, if a therapeutic drug for synucleinopathy is approved in the future, the subject determined to have a synucleinopathy by this method is administered the therapeutic drug for synucleinopathy. For example, a therapeutic drug for Parkinson's disease may be administered to treat the parkinsonism symptoms of synucleinopathy.

[0041] A subject determined to have synucleinopathy by this method may be further clinically diagnosed and treated.For example, a subject determined to have synucleinopathy by this method and clinically diagnosed with Parkinson's disease is administered a therapeutic drug for Parkinson's disease.For example, a subject determined to have synucleinopathy by this method and clinically diagnosed with dementia with Lewy bodies is administered a therapeutic drug for dementia with Lewy bodies.For example, a subject determined to have synucleinopathy by this method and clinically diagnosed with multiple system atrophy is administered a therapeutic drug for multiple system atrophy.

[0042] Examples of drugs for treating Parkinson's disease include dopamine precursors (such as levodopa), dopamine agonists (such as pramipexole, ropinirole, apomorphine, and rotigotine), dopamine release enhancers (such as amantadine), dopamine degradation inhibitors (such as selegiline, rasagiline, safinamide, entacapone, opicapone, and carbidopa), non-dopamine drugs (such as zonisamide), adenosine receptor antagonists (such as istradefylline), anticholinergic agents, and serotonin 5-HT1A / 1B agonists (such as eltoprazine). Examples of drugs for treating dementia with Lewy bodies include drugs for treating Parkinson's disease, as well as drugs for treating dementia (such as donepezil), and drugs for treating sleep disorders. Examples of drugs for treating multiple system atrophy include drugs for treating Parkinson's disease, as well as drugs for treating cerebellar ataxia (such as taltirelin).

[0043] In another embodiment, a kit for determining whether a subject has a synucleinopathy is provided, comprising a reagent that specifically binds to ApoA1 and a reagent that specifically binds to α-synuclein. The reagents may be dissolved in water or a suitable buffer, such as phosphate-buffered saline (PBS), or lyophilized and provided in a suitable container. Suitable containers include bottles, vials, syringes, test tubes, plates, membranes, etc. The containers may be made of a variety of materials, such as glass or plastic. The kit may contain the reagent that specifically binds to ApoA1 and the reagent that specifically binds to α-synuclein as a single component or as separate components. The kit may further include other components or reagents necessary for detecting ApoA1 and / or α-synuclein. For example, the kit may further include a labeled secondary antibody, a chromogenic substrate, a blocking solution, a washing buffer, etc. The kit may further include other materials desirable from a commercial and user standpoint, such as a package insert containing instructions for use.

[0044] In one embodiment, a reagent that specifically binds to ApoA1 and a reagent that specifically binds to alpha-synuclein are provided for determining whether a subject is affected by a synucleinopathy. In one embodiment, there is provided the use of a reagent that specifically binds to ApoA1 and a reagent that specifically binds to alpha-synuclein to manufacture a kit for determining whether a subject is affected by a synucleinopathy.

[0045] For example, the following embodiments are provided: [1] A method for determining whether a subject is suffering from a synucleinopathy, comprising comparing the level of apolipoprotein A1 (ApoA1) bound to alpha-synuclein in a sample collected from the subject with a cutoff value. [2] The method according to item 1, wherein the subject is determined to be suffering from a synucleinopathy when the level of ApoA1 bound to α-synuclein is lower than the cutoff value. [3] The method according to item 1 or 2, wherein the subject is determined not to be suffering from a synucleinopathy when the level of ApoA1 bound to alpha-synuclein is higher than the cutoff value. [4] The method according to any one of items 1 to 3, comprising purifying an alpha-synuclein fraction from a sample collected from a subject. [5] The method according to any one of items 1 to 4, comprising measuring the amount of ApoA1 bound to α-synuclein in a sample collected from a subject. [6] The method according to any one of items 1 to 5, wherein the sample is blood, plasma, or serum. [7] The method according to any one of items 1 to 6, wherein the synucleinopathy is Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy. [8] The method according to any one of items 1 to 7, wherein the synucleinopathy is Parkinson's disease. [9] A kit for determining whether a subject is affected by a synucleinopathy, comprising a reagent that specifically binds to ApoA1 and a reagent that specifically binds to α-synuclein.

[10] The kit according to item 9, wherein the reagent is an antibody.

[0046] All documents cited herein are hereby incorporated by reference. The above description is non-limiting, and the present invention is defined in the appended claims, and various modifications are possible within the scope of the technical idea. The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0047] [method] Samples used for mass spectrometry Mass spectrometry was performed using plasma samples from healthy volunteers (n = 5) and Parkinson's disease patients (n = 5). The Parkinson's disease patients included four with moderate motor dysfunction and one with severe motor dysfunction. The patient background of each sample is shown in Table 1. [Table 1]

[0048] Samples used for ELISA measurement For ELISA measurements, plasma samples from healthy volunteers (n = 5) and Parkinson's disease patients (n = 5) were used, which were different from the samples used for mass spectrometry. The Parkinson's disease patients used samples were from four patients with moderate motor dysfunction and one patient with severe motor dysfunction. The patient background for each sample is shown in Table 2. [Table 2]

[0049] Preparation of α-synuclein antibody beads 300 μg of anti-α-synuclein monoclonal antibody (Becton Dickinson Japan, 610787) was covalently coupled to magnetic beads (Dynabeads M-270 Epoxy, Thermo Fisher Scientific) by reacting with 2 mL (40 mg of beads) at room temperature for 30 minutes. Finally, 50 mM Tris pH 8.0 buffer was added to block any remaining amino groups. The antibody-immobilized magnetic beads were stored at 4°C until use.

[0050] Purification of α-synuclein-binding proteins 10 μL of a protease inhibitor (Halt protease inhibitor cocktail SIGMA P8340) was added to 0.5 mL of plasma derived from a healthy subject or a Parkinson's disease patient, and the mixture was centrifuged at 1500 g at 4°C for 5 minutes to obtain the supernatant. 0.5 mL of binding buffer (1% BSA, PBS, 0.05% Tween 20) was added to the supernatant, and 0.1 mL of the prepared α-synuclein antibody-immobilized magnetic beads was added. The mixture was incubated overnight at 4°C. After recovery, the sample was immobilized on a magnetic plate, and the magnetic beads were recovered. The recovered magnetic beads were washed three times with 1 mL of washing buffer (PBS, 0.05% Tween 20). 60 μL of elution buffer (0.1 M citrate, pH 2.8) was added to the washed magnetic beads, and the beads were then immobilized on a magnetic plate. The eluted fraction was recovered. The recovered eluted fraction was neutralized by adding 10 μL of 1 M Tris pH 8.0. The prepared sample was stored at -80°C until mass spectrometry and ELISA analysis.

[0051] Proteomic analysis of α-synuclein-binding proteins Proteomic analysis of α-synuclein-binding proteins was performed at the National Institutes of Biomedical Innovation, Health, and Nutrition (6-8, Asagi 7-chome, Saito, Ibaraki, Osaka). 10 μL of α-synuclein-binding protein sample was purified using a StageTip, dried in a SpeedVac, and redissolved in 10 μL of buffer (0.1% formic acid, 2% acetonitrile). The prepared sample was subjected to nanoLC-MS / MS analysis using an ExactivePlus mass spectrometer (ThermoFisherScientific), an UltiMate 3000 RSLC nanoHPLC (ThermoFisherScientific), and an HTC-PAL autosampler (CTC Analytics). The peptide fragment data obtained were used to calculate the area values ​​of each peptide using Xcalibur 2.4. Binding proteins were identified using MaxQuant 1.6.14.0.

[0052] ELISA measurement ApoA1 protein concentrations in blood and α-synuclein-binding protein fraction samples were measured using a Human APOA1 sandwich ELISA kit (Proteintech, KE00157) and an Apolipoprotein A1 (APOA1) Human SimpleStep ELISA Kit (Abcam, ab189576).

[0053] statistical processing The statistical significance test between the Parkinson's disease group and the healthy control group was performed using the Mann-Whitney U test, a non-parametric test method.

[0054] [result] Proteomic analysis of α-synuclein-binding proteins was performed using α-synuclein antibody magnetic beads on plasma samples from five healthy individuals and five Parkinson's disease patients. The 15 types of α-synuclein-binding proteins shown in Table 3 were obtained. [Table 3]

[0055] The abundance ratio of identified α-synuclein-binding proteins between healthy and Parkinson's disease (PD) subjects (average of five samples) was calculated, and apolipoprotein A1 (ApoA1) was found to be approximately 43-fold lower in the PD group compared to healthy subjects. The α-synuclein protein itself was only approximately half lower in the PD group compared to healthy subjects. These results indicate that, although circulating α-synuclein is lower in PD patients than in healthy subjects, the amount of ApoA1 bound to α-synuclein is even more significantly reduced.

[0056] The amount of ApoA1 in the α-synuclein-bound fraction of each of the five samples varied among healthy individuals, but a significant decrease was observed in all five Parkinson's disease samples, revealing a significant difference between the healthy and Parkinson's disease groups (Figure 1). While the amount of α-synuclein-bound ApoA1 protein in the blood showed individual differences among healthy individuals, it was uniformly decreased in all samples from Parkinson's disease patients.

[0057] Next, we analyzed the relationship between blood ApoA1 protein concentration and the amount of ApoA1 in the α-synuclein-bound fraction using an ELISA assay system for the same samples. As shown in Figure 2, blood ApoA1 protein concentration was significantly lower in the Parkinson's disease (PD) group compared to the healthy control group (HC), but there was overlap in the concentration ranges. This trend was confirmed by the measurement results using two different commercially available ApoA1 ELISA kits.

[0058] The ApoA1 concentration in the α-synuclein-bound fraction was also analyzed by ELISA. As shown in Figure 3, the ApoA1 content in the α-synuclein-bound fraction was significantly lower in the Parkinson's disease (PD) group compared to the healthy control (HC) group, with no overlap in the concentration ranges. In the Parkinson's disease group, a uniformly significant decrease was observed in all samples measured, as in the mass spectrometry analysis. This suggests that the decrease in ApoA1 content in the α-synuclein-bound fraction may be indicative of some pathological condition in Parkinson's disease.

[0059] These results suggest that the ApoA1 protein in the α-synuclein-bound fraction can be used as a marker to adequately distinguish between healthy individuals and patients with synucleinopathy. [Industrial Applicability]

[0060] According to the present disclosure, it is possible to determine whether or not a person is suffering from a synucleinopathy, which is useful in the medical field.

Claims

1. A method for determining whether a subject is suffering from a synucleinopathy, comprising comparing the level of apolipoprotein A1 (ApoA1) bound to alpha-synuclein in a sample taken from the subject with a cutoff value.

2. The method of claim 1, wherein the subject is determined to be suffering from a synucleinopathy when the level of ApoA1 bound to alpha-synuclein is lower than the cutoff value.

3. The method of claim 1, wherein the subject is determined not to be suffering from a synucleinopathy when the level of ApoA1 bound to alpha-synuclein is higher than the cutoff value.

4. 10. The method of claim 1, comprising purifying an alpha-synuclein fraction from a sample taken from a subject.

5. The method of claim 1, comprising measuring the amount of ApoA1 bound to alpha-synuclein in a sample taken from the subject.

6. The method of claim 1 , wherein the sample is blood, plasma, or serum.

7. 2. The method of claim 1, wherein the synucleinopathy is Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy.

8. 2. The method of claim 1, wherein the synucleinopathy is Parkinson's disease.

9. A kit for determining whether a subject is suffering from a synucleinopathy, comprising a reagent that specifically binds to ApoA1 and a reagent that specifically binds to α-synuclein.

10. The kit of claim 9 , wherein the reagent is an antibody.

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