Differential diagnosis of Parkinson's disease and multiple system atrophy

Abstract

To provide a differential diagnosis method for Parkinson disease and multiple system atrophy.SOLUTION: A method for measuring abnormal α-synuclein associated with differential diagnosis of Parkinson disease and multiple system atrophy, comprises: introducing, into cultured cells that permanently express mutant α-synuclein, an RT-QuIC product against an anti-α-synuclein antibody reaction product in a blood sample derived from a subject as a seed; and measuring the density of aggregates in the cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for differential diagnosis of Parkinson's disease and multiple system atrophy. [Background technology]

[0002] Parkinson's disease (PD) and multiple system atrophy (MSA) are both progressive, intractable diseases accompanied by the deposition of α-synuclein (AS) aggregates. Diseases that share the common pathology of deposition of such AS aggregates are called α-synucleinopathy. PD progresses slowly, whereas MSA progresses rapidly, and it is often difficult to differentiate between the two diseases at the early stages. Delayed diagnosis of PD halves the effectiveness of symptomatic treatment and affects prognosis. In addition, as MSA progresses, various motor symptoms and autonomic nervous dysfunction appear, and if accurate diagnosis is not possible, this can lead to a significant decrease in quality of life.

[0003] Methods for early diagnosis of these α-synucleinopathies include genetic testing, myocardial MIBG scitigraphy, DAT scans, and RT-QuIC (Real-time quaking-induced conversion) of abnormal α-synuclein levels in cerebrospinal fluid. and HANABI (Handai Amyloid Burst Inducer: a fully automated protein aggregation detection device integrating a cross-type ultrasonic generator and a microplate reader) have been reported (Non-Patent Documents 1 and 2). The present inventor also reported a method for measuring the concentration of abnormal α-synuclein in test blood by reacting a test blood sample with an anti-α-synuclein antibody to effect immunoprecipitation, followed by RT-QuIC (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2020-34353 A [Non-patent literature]

[0005] [Non-Patent Document 1] Ann Clin Transl Neurol 2016;3:812-818 [Non-Patent Document 2] J Biol Chem. 2014;289:27290-27299. Summary of the Invention [Problem to be solved by the invention]

[0006] However, it remains difficult to distinguish between PD and MSA in the early stages with either test. Because PD and MSA differ in progression and severity, accurate diagnosis from the early stages is important for treatment. However, distinguishing between the two diseases is extremely difficult in the early stages, and there are no simple diagnostic biomarkers such as blood sampling, so even neurologists who perform detailed neurological examinations have difficulty. Neuroimaging is said to be helpful in diagnosis, but it is difficult for non-specialists to interpret, and no simple diagnostic method has been established. Delayed diagnosis of PD halves the effectiveness of symptomatic treatment and affects prognosis. In addition, MSA presents various motor symptoms and autonomic nervous dysfunction as it progresses, and if an accurate diagnosis cannot be made, it will result in a significant decrease in quality of life. Therefore, an object of the present invention is to provide a method that enables a simple and early differential diagnosis between PD and MSA. [Means for solving the problem]

[0007] Therefore, taking into consideration the use of blood samples as specimens and the ease of processing, the inventors conducted various studies and found that PD and MSA can be clearly differentiated and diagnosed by preparing cultured cells that constitutively express mutant AS, introducing the RT-QuIC product of the anti-AS antibody reactant in a blood sample from a subject into the cultured cells as a seed, and measuring the density of abnormal AS aggregates in the cells, thereby completing the present invention.

[0008] That is, the present invention provides the following inventions [1] to [4]. [1] A method for measuring abnormal α-synuclein relevant to the differential diagnosis of Parkinson's disease and multiple system atrophy, comprising: introducing, as a seed, the RT-QuIC product of an anti-α-synuclein antibody reactant in a blood sample from a subject into cultured cells that constitutively express mutant α-synuclein; and measuring the density of aggregates within the cells. [2] The method according to [1], wherein the cultured cells that constitutively express mutant α-synuclein are cultured cells that stably express mutant α-synuclein fused to a labeling protein. [3] The measurement method according to [1] or [2], wherein the RT-QuIC product is an α-synuclein aggregate obtained by performing RT-QuIC on an immunoprecipitate obtained by reacting a serum sample derived from a subject with an anti-α-synuclein antibody. [4] The method according to [2] or [3], wherein the labeled protein is a fluorescently labeled protein. Effect of the Invention

[0009] According to the present invention, since a blood sample is used as a specimen, the burden on the patient is reduced, and early differential diagnosis between PD and MSA can be performed accurately with simple operations. [Brief description of the drawings]

[0010] [Figure 1] The image shows a 0.33 μm-wide image of a cell with AS-GFP aggregates formed in a cultured cell, taken from the top to bottom in the Z direction, visualized using maximum intensity projection. [Diagram 2] FIG. 1 shows the difference in density of intracellular aggregates formed in PD and MSA. [Diagram 3] The mean intracellular aggregate density and its standard deviation obtained from each of the PD and MSA groups are plotted on the vertical and horizontal axes, respectively, to show the 95% prediction intervals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The method for measuring abnormal AS relevant to the differential diagnosis of PD and MSA of the present invention is characterized in that the RT-QuIC product of the anti-AS antibody reactant in a blood sample from a subject is introduced as a seed into cultured cells that constitutively express mutant AS, and the density of aggregates in the cells is measured.

[0012] "Constitutively expressing a mutant AS" refers to transiently or permanently expressing a mutant AS, and it is sufficient that the mutant AS is expressed when the seed is introduced. A mutant AS may be one in which a portion of the amino acid sequence of AS has been mutated, and is not particularly limited thereto, but examples thereof include AS having an A53T mutation, an A30P mutation, an E46K mutation, an H50Q mutation, a G51D mutation, or an A53E mutation.

[0013] In order to observe the aggregates finally formed in cultured cells, it is preferable to use the mutant AS as a fusion protein by fusing it with a labeling protein, such as a fluorescent protein or a luminescent protein. The fluorescent protein may be either natural or non-natural, so long as it emits fluorescence of a specific wavelength when irradiated with excitation light. Specific examples include GFP (including derivatives such as EGFP and 3xP3-EGFP), CFP, RFP, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, and the like. The luminescent protein may be a substrate protein that can emit light without the need for excitation light, or an enzyme that catalyzes the luminescence of the substrate protein. For example, luciferin as the luminescent substrate and luciferase as the enzyme are used.

[0014] As cultured cells that stably express mutant AS, human-derived or neural cultured cells are preferred, such as HEK293, HeLa, SHSY-5Y, Neuro2a, MEF, PC12, SK-MEL28, CHO, HepG2, and MRC-5.

[0015] As the cultured cells that constitutively express mutant AS, cultured cells that stably express mutant AS fused with a labeling protein are preferred, and cultured cells that stably express mutant AS fused with a fluorescent labeling protein are preferred, specifically, cultured cells of human origin that stably express mutant AS fused with GFP are preferred.

[0016] In the present invention, the RT-QuIC product corresponding to the anti-AS antibody reactant in a blood sample derived from a subject is introduced as a seed into the above-mentioned cultured cells. The subject is a patient with PD or MSA, or a patient suspected of having PD or MSA. The specimen used in the present invention may be any human body fluid, such as blood, cerebrospinal fluid, saliva, tears, or autopsy brain, and is preferably human blood, and more preferably human serum.

[0017] The anti-AS antibody reactant in a blood sample is a component obtained by reacting the blood sample with an anti-AS antibody, specifically, an immunoprecipitate obtained by reacting the blood sample with an anti-AS antibody. The anti-AS antibody may be a polyclonal or monoclonal antibody, and any commercially available antibody may be used, so long as it is specific to AS and recognizes all structures, including monomers, oligomers, and aggregates. Immunoprecipitation may be performed by a conventional immunoprecipitation method, for example, by reacting an anti-AS antibody with a blood sample, adding beads or magnetic beads to obtain a complex between the immune reaction mixture and the beads or magnetic beads, and then recovering the immune reaction mixture from the complex.

[0018] Next, T-QuIC is performed on the anti-AS antibody reactants. RT-QuIC uses abnormally structured proteins as the seed of the amplification reaction and continuously performs agglutination reactions in a test tube to amplify and detect abnormally structured proteins in a sample. This amplification process is also a system that measures the presence or absence of abnormally structured proteins in real time by measuring the fluorescence intensity of thioflavin T (ThT), which specifically binds to β-sheet proteins and emits fluorescence. The amplification reaction is carried out through a step of (1) coaggregating normal AS and abnormal structural AS in a sample, and (2) promoting coaggregation to convert normal AS to abnormal structural AS, followed by detection with ThT. In order to promote coaggregation of abnormal structural AS, normal AS is preferably added to the reaction solution. The coaggregation and structural conversion reaction are preferably carried out under reaction solution conditions of pH 6 to 7.9. AS has a strong tendency to aggregate in an acidic state, and under strongly acidic conditions with a reaction solution of less than pH 6, healthy individuals also have endogenous AS, and undergo agglutination reactions in the same manner as PD and MSA patients. On the other hand, under alkaline conditions of pH 8.0, agglutination reactions are unlikely to occur in samples from PD, MSA, and healthy individuals. When the reaction solution is pH 6 to 7.9, the fluorescence intensity of PD and MSA samples increases to a maximum of 260,000 RFU, indicating agglutination reactions, but no agglutination reactions are observed in samples from healthy individuals, making it possible to distinguish between the two. The preferred reaction pH is 6.5 to 7.9, more preferably 6.5 to 7.8, and even more preferably 7.5.

[0019] T-QuIC yields abnormal AS aggregates that can be used as seeds. These abnormal AS aggregates are introduced into cultured cells that stably express the mutant AS, and labeled abnormal AS aggregates are formed within the cells, and their density is measured. The means for introducing the aggregate into the cultured cells include the lipofection method, the polycation method, the calcium phosphate method, the electroporation method, the microinjection method, etc. In the lipofection method, Lipofectamine3000, Multifectam, X-tremeGENE9, FuGENE6, Lipofectin, etc. can be used. Measurement of density within a cell can be performed by measuring the amount of labeled protein. MSA has significantly higher density than PD, allowing the differential diagnosis of the two with high sensitivity and specificity. EXAMPLES

[0020] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0021] Example 1 (Preparation of cultured cells constitutively expressing mutant AS) A plasmid in which AS with the A53T mutation, which has higher AS aggregation ability and GFP fused to the C-terminus of AS, was cloned under the CMV promoter was transfected into HEK293 cells, and a cell line (HEK293-AS-GFP) with stable expression was produced by selecting cells resistant to neomycin (G418). From among the stable expression lines, those with strong GFP fluorescence intensity were subcloned to produce a single clone-derived cell line with uniform expression of AS-GFP.

[0022] Example 2 (Formation of RT-QuIC products) (1) Immunoprecipitation was performed on human serum using magnetic beads. First, the magnetic beads and anti-AS antibodies are reacted overnight, and then the test blood sample is reacted for 3 hours. 0.1 M glycine (pH 2.5-3) is used to elute the immune reaction mixture. After reacting for a few seconds to elute, the pH is quickly adjusted to 7.5.

[0023] (2)RT-QuIC The amplification reaction involves (i) incubating the reaction solution with IP-serum to allow coaggregation of normal AS and structurally abnormal AS present in the reaction mixture, and (ii) promoting coaggregation to convert normal AS to structurally abnormal AS. For promotion of coaggregation and structural conversion, shaking was performed continuously for 120 hours at 200 rpm with 1 min shake and 14 min rest. The reaction solution consisted of 100 mmol / L phosphate buffer (pH 7.5) and 10 μmol / L thioflavin T. 10 μg of AS substrate was used. IP-serum was used as a seed. A BCA assay was performed, and 100 μL of serum with a protein concentration adjusted to 1 mg / mL was IPed, and 5 μL was used as a seed.

[0024] Example 3 (Seed introduction and observation) HEK293-AS-GFP cell line DMEM high glucose+10 1x10 cells were cultured in 10% FBS medium and seeded into 24-well plates the day before transfection. 4 100 cells were seeded. 2 μL of seed amplified from patient serum by RT-QuIC was added to 50 μL of AS (1 mg / mL; 30 mM TrisHCl pH8.0 + 150 mM NaCl) purified from E. coli using His6-tag, and amplified at 37°C for 1 week. After ultrasonic disruption (about 1 minute with a bath sonicator), 5 μL was diluted with 20 μL of OPTI-MEM, 1 μL of P3000 Reagent was added, and the mixture was mixed with 25 μL of OPTI-MEM plus 1 μL of Lipofectamine 3000 Reagent, and added to the cells after 10 minutes of standing. 48 hours after lipofection, the cells were fixed with 10% neutral formalin (formalin content 4%), nuclear stained with 50 mM TrisHCl pH8.0 + DAPI (0.1 μg / mL), and mounted on slides using ProLong-Diamond.

[0025] In the observation, a super-resolution microscope using structured illumination (Zeiss Elyra) was used to capture images of cells with intracellular AS-GFP aggregates in the Z direction with a width of 0.1 μm from the top to the bottom, and the images were visualized by maximum intensity projection (Figure 1). In addition, cells with more than 100 aggregates were randomly observed using a Keyence BZ-H4XF / Sectioning Module, and the density of intracellular AS-GFP aggregates was analyzed by hybrid cell counting. As a result, cells with high density were derived from MSA, and those with low density were derived from PD. A total of 100 cells were analyzed for PD and MSA origin (sensitivity 94%, specificity 92%, cutoff value 45.5, AUC: 0.96). The average density of aggregates was calculated for 12-13 cells per patient, and it was possible to distinguish 8 cases of PD and 8 cases of MSA with sensitivity and specificity of 100% (cutoff value 46.1) (Figure 2). The measured values ​​of intracellular aggregate density in PD and MSA have a potential distribution of deviations from the mean. By evaluating the variability of the mean intracellular aggregate density in PD and MSA, i.e., the standard deviation, it is possible to determine whether the mean intracellular aggregate density in newly obtained cases is reliable enough to distinguish between PD and MSA. The sample standard deviation of the mean intracellular aggregate density in the PD group was 7.031 (orange in the lower part of Figure 3), whereas the sample standard deviation of the mean intracellular aggregate density in the MSA group was 2.767 (blue in the upper part of Figure 3; significance level 5%, p=0.025). Figure 3 shows the 95% prediction intervals obtained by plotting the mean intracellular aggregate density and its standard deviation obtained from each of the PD and MSA groups on the vertical and horizontal axes, respectively. The 95% prediction interval means the interval within which the newly obtained sample can exist with a 95% probability. From Figure 3, it can be said that it is possible to distinguish between PD and MSA with high accuracy if the standard deviation of the mean intracellular aggregate density of newly obtained cases is within the 95% prediction interval of 9.021 or less.

Claims

1. A method for measuring abnormal α-synuclein relevant to the differential diagnosis of Parkinson's disease and multiple system atrophy, comprising: introducing, as a seed, an RT-QuIC product of an anti-α-synuclein antibody reactant in a blood sample from a subject into cultured cells that constitutively express mutant α-synuclein; measuring the density of aggregates in the cells; and using as an indicator whether the density is higher in multiple system atrophy than in Parkinson's disease.

2. 2. The method according to claim 1, wherein the cultured cells constitutively expressing mutant α-synuclein are cultured cells stably expressing mutant α-synuclein fused with a labeling protein.

3. The measurement method according to claim 1 or 2, wherein the RT-QuIC product is an α-synuclein aggregate obtained by performing RT-QuIC on an immunoprecipitate obtained by reacting a serum sample derived from a subject with an anti-α-synuclein antibody.

4. The method according to claim 2, wherein the labeled protein is a fluorescently labeled protein.

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