Method for detecting neurodegenerative disease using short-chain RNA

Abstract

Disclosed is a method that enables the detection of the presence or absence of a neurodegenerative disease in a subject more simply and effectively than conventional methods. This method comprises: step (a) for preparing an extracellular vesicle fraction from a body fluid sample of a subject; step (b) for counting the extracellular vesicles contained in the extracellular vesicle fraction obtained in step (a) to obtain the number of extracellular vesicles; step (c) for measuring the total amount of short-chain RNA contained in all extracellular vesicles counted in step (b) to obtain the total amount of short-chain RNA per extracellular vesicle; and step (d) for, when the total amount of short-chain RNA per extracellular vesicle obtained in step (c) is greater than the total amount of short-chain RNA per extracellular vesicle obtained from body fluid samples from healthy subjects, then determining that the subject suffers from a neurodegenerative disease.

Description

Method for detecting neurodegenerative diseases using short RNA

[0001] The present invention is a method for detecting the presence or absence of a neurodegenerative disease using the total amount of short-chain RNA contained in extracellular vesicles in a body fluid sample from a subject.

[0002] It is known that extracellular vesicles released from cells in various organs and tissues exist in the body fluids of living organisms and circulate throughout the body.

[0003] Extracellular vesicles are small vesicles with diameters of approximately 10 nm to approximately 3000 nm secreted from various cells. Because they contain proteins derived from the releasing cell, mRNA, short-chain RNAs such as microRNA, and the like, their usefulness as biomarkers has been reported. Among these, exosomes are those with diameters of approximately 30 to 150 nm that are derived from endosomes. Exosomes are composed of a lipid membrane rich in ceramide, are produced in multivesicular endosomes, and are secreted extracellularly upon fusion of the multivesicular endosome with the cell membrane. Therefore, they contain membrane proteins, such as CD9 and CD63, which are endosome-specific markers, on their membranes (Non-Patent Document 1).

[0004] Neurodegenerative diseases are diseases that cause various neurological disorders due to the weakening, functional attenuation, or death of nerve cells, and are known to include Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease, amyotrophic lateral sclerosis (ALS), etc. If these diseases can be detected at an early stage, it may be possible to lead to early treatment, thereby improving the quality of life of patients.

[0005] To detect neurodegenerative diseases, various imaging tests such as MRI, FDG-PET, and DAT scans are performed, but they are highly invasive, expensive, and measurement facilities are limited. Therefore, blood tests and other tests are desirable as a less invasive and simple biochemical diagnosis.

[0006] Blood biomarkers for neurodegenerative diseases include amyloid beta (Non-Patent Document 2), phosphorylated tau (Non-Patent Document 3), and microRNA (Patent Document 1) for Alzheimer's disease, but each requires complex measurements and analysis algorithms using advanced equipment, making them difficult to widely use. There is a need for the development of diagnostic markers that can easily detect neurodegenerative diseases using blood samples.

[0007] International Publication No. 2019 / 159884

[0008] Lin J. et al. , The Scientific World Journal, Volume 2015, Article ID 657086 Nakamura A. et al. , Nature, 2018 Jan 31, vol. 554, p. 249-254 Tatebe H. et al. , Mol. Neurodegener. , 2017 Sep 4;12(1):63

[0009] The present invention provides a method for detecting the presence or absence of a neurodegenerative disease more simply and effectively, avoiding imaging tests that place a heavy burden on patients or complex analyses using advanced measuring equipment.

[0010] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors discovered that it is possible to detect the presence or absence of a neurodegenerative disease by measuring the total amount of short-chain RNA contained in extracellular vesicles per number of extracellular vesicles in a body fluid sample from a subject and comparing that amount with that of a healthy subject. Based on this finding, the present invention was completed.

[0011] That is, the present invention provides the following: (1) A method for detecting whether a subject is suffering from a neurodegenerative disease, comprising: (a) preparing an extracellular vesicle fraction from a body fluid sample from the subject; (b) counting extracellular vesicles contained in the extracellular vesicle fraction obtained in step (a) to obtain the number of extracellular vesicles; (c) measuring the total amount of short-stranded RNA contained in all the extracellular vesicles counted in step (b) to obtain the total amount of short-stranded RNA per number of extracellular vesicles; and (d) determining that the subject is suffering from a neurodegenerative disease if the total amount of short-stranded RNA per number of extracellular vesicles obtained in step (c) is greater than the total amount of short-stranded RNA per number of extracellular vesicles obtained from a body fluid sample from a healthy subject.

[0012] (2) The method of (1), wherein in the step (d), an average value of the total amount of short-stranded RNA per number of extracellular vesicles obtained from body fluid samples of multiple healthy subjects is calculated in advance, and the subject is determined to be suffering from a neurodegenerative disease if the total amount of short-stranded RNA per number of extracellular vesicles obtained from the subject's body fluid is 1.5 to less than 100 times the average value. (3) The method of (1) or (2), wherein in the step (a), the prepared extracellular vesicle fraction contains extracellular vesicles with a diameter of 30 to 200 nm. (4) The method of any of (1) to (3), wherein in the step (a), CD9, CD63, CD81, Tim4, or L1CAM protein is present on the surface of the prepared extracellular vesicles. (5) The method of any of (1) to (4), wherein in the previous step (b), the short-stranded RNA is 15 to 200 bases in length. (6) The method according to any one of (1) to (5), wherein the short-chain RNA is microRNA. (7) The method according to any one of (1) to (6), wherein the body fluid sample is blood, serum, plasma, or cerebrospinal fluid. (8) The method according to any one of (1) to (7), wherein in the step (a), the extracellular vesicle fraction is prepared by a method selected from the group consisting of centrifugation, immunoprecipitation, polymer precipitation, lipophilicity, liquid chromatography, size exclusion chromatography, ultrafiltration, and combinations thereof. (9) The method according to any one of (1) to (8), wherein in the step (b), the number of extracellular vesicles is counted by a tracking method, an antigen-antibody reaction method, or a flow cytometry method. (10) The method according to any one of (1) to (9), wherein in the step (c), the total amount of short-chain RNA contained in the extracellular vesicles is measured using a spectrophotometer, electrophoresis, a microarray, PCR, or a DNA sequencer. (11) The method according to any one of (1) to (10), wherein the neurodegenerative disease is Alzheimer's disease, dementia with Lewy bodies, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), or Parkinson's disease.

[0013] (12) A kit for detecting whether a subject is suffering from a neurodegenerative disease by the method described in (1), comprising: a means for preparing an extracellular vesicle fraction from a body fluid sample of the subject; a means for counting the extracellular vesicles contained in the prepared extracellular vesicle fraction to obtain the number of the extracellular vesicles; and a means for measuring the total amount of short-stranded RNA contained in all the counted extracellular vesicles to obtain the total amount of short-stranded RNA per number of extracellular vesicles. (13) The kit according to (12), wherein the means for preparing an extracellular vesicle fraction from a body fluid sample of a subject comprises a solid-phased antigen or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof specifically binds to a surface antigen of the extracellular vesicles of interest is solidified; the means for counting the extracellular vesicles contained in the prepared extracellular vesicle fraction to obtain the number of the extracellular vesicles comprises a labeled antibody or antigen-binding fragment thereof that specifically binds to a surface antigen of the extracellular vesicles of interest; and the means for measuring the total amount of short-chain RNA contained in all the counted extracellular vesicles to obtain the total amount of short-chain RNA per number of extracellular vesicles comprises a microarray equipped with nucleic acid probes that hybridize with multiple known microRNAs. (14) A system for detecting whether a subject is suffering from a neurodegenerative disease using the method of claim 1, comprising: a means for preparing an extracellular vesicle fraction from a body fluid sample of the subject; a means for counting extracellular vesicles contained in the prepared extracellular vesicle fraction to obtain the number of the extracellular vesicles; and a means for measuring the total amount of short-stranded RNA contained in all the counted extracellular vesicles to obtain the total amount of short-stranded RNA per number of extracellular vesicles.

[0014] Furthermore, the present invention described above encompasses the following preferred embodiments: (15) A method for detecting whether a subject is suffering from a neurodegenerative disease, comprising: a step (A) of measuring the number of extracellular vesicles contained in a body fluid sample from the subject; and a step (B) of measuring the amount of short-stranded RNA, preferably microRNA, contained in the extracellular vesicles whose number was measured in step (A), wherein a higher amount of short-stranded RNA per extracellular vesicle than that of a healthy subject indicates a high possibility that the subject is suffering from a neurodegenerative disease. (16) The method according to (15), wherein the extracellular vesicles express at least one neuronal cell surface marker selected from the group consisting of L1CAM, NCAM, Enolase 2, total tau protein (MAPT), Glutamate receptor 1 (GRIA1), and Proteolipid protein 1 (PLP1), preferably L1CAM protein. (17) The method according to (15) or (16), wherein the subject is determined to have a high likelihood of suffering from a neurodegenerative disease when the amount of short-stranded RNA per extracellular vesicle obtained from the subject's body fluid is 1.5 times or more but less than 100 times the average amount of short-stranded RNA per extracellular vesicle obtained from body fluid samples of multiple healthy subjects, calculated in advance. (18) The method according to any one of (15) to (17), wherein the extracellular vesicles further express at least one protein selected from the group consisting of CD63, CD9, CD81, and Tim4 on their surface. (19) The method according to (18), wherein step (A) is carried out by collecting extracellular vesicles using an immobilized antibody or antigen-binding fragment thereof that specifically reacts with the neuronal cell surface marker expressed on the surface of the extracellular vesicles, then reacting the extracellular vesicles with a labeled antibody or antigen-binding fragment thereof that specifically reacts with CD63, CD9, CD81, or Tim4, and measuring the label bound to the extracellular vesicles. (20) The method according to any one of (15) to (19), wherein step (B) is carried out by reacting short-stranded RNAs in exosomes with a microarray loaded with nucleic acid probes that hybridize with multiple known short-stranded RNAs, and measuring the short-stranded RNAs bound to the microarray.(21) A device included in the system described in (14) above, for preparing an extracellular vesicle fraction from a body fluid sample of a subject. (22) A device included in the system described in (14) above, for counting extracellular vesicles contained in a prepared extracellular vesicle fraction to obtain the number of extracellular vesicles. (23) A device included in the system described in (14) above, for measuring the total amount of short-chain RNA contained in all counted extracellular vesicles and obtaining the total amount of short-chain RNA per number of extracellular vesicles. (24) A program or a recording medium having a program written thereon, included in the system described in (14) above, for measuring the total amount of short-chain RNA contained in all counted extracellular vesicles and obtaining the total amount of short-chain RNA per number of extracellular vesicles.

[0015] According to the present invention, it becomes possible to determine whether or not a subject is suffering from neurodegeneration in a minimally invasive and simple manner.

[0016] The upper panel of Fig. 1 shows the results of counting the number of exosomes in the extracellular vesicle (containing exosomes, etc.) fraction prepared in Example 1. The lower panel shows the results of determining the relative ratio of the total amount of microRNA per number of exosomes in the exosome fraction prepared in Example 1 to a control. The upper panel of Fig. 2 shows the results of determining the relative ratio of the total amount of microRNA per number of exosomes in the extracellular vesicle (containing exosomes, etc.) fraction prepared in Example 2 to a control. The lower panel shows the results of determining the relative ratio of the total amount of microRNA per number of exosomes in the exosome fraction prepared in Example 2 to a control.

[0017] The present invention provides a method for testing whether a subject is suffering from a neurodegenerative disease, comprising the following steps (a) to (d): step (a): preparing an extracellular vesicle fraction from a body fluid sample of the subject; step (b): counting the extracellular vesicles contained in the extracellular vesicle fraction obtained in step (a) to obtain the number; step (c): measuring the total amount of short-stranded RNA contained in all the extracellular vesicles counted in step (b) to obtain the total amount of short-stranded RNA per number of extracellular vesicles; and step (d): determining that the subject is suffering from a neurodegenerative disease if the total amount of short-stranded RNA per number of extracellular vesicles obtained in step (c) is greater than the total amount of short-stranded RNA per number of extracellular vesicles obtained from a body fluid sample of a healthy subject.

[0018] The "subject" targeted in the method of the present invention refers to mammals such as humans, primates such as chimpanzees and gorillas, pet animals such as dogs and cats, livestock animals such as cows, horses, sheep and goats, rodents such as mice and rats, and animals kept in zoos. A preferred subject is a human. Note that, among subjects, a mammal, including a human, suffering from a neurodegenerative disease may be referred to as a "patient," and a preferred patient is a human. In the present invention, a "healthy subject" also refers to such a mammal, including a human, that is not suffering from the neurodegenerative disease to be detected. A preferred healthy subject is a human.

[0019] In the present invention, neurodegeneration refers to a state in which the structure and function of neurons, which are nerve cells, are lost. The most common examples of neurodegenerative diseases include Alzheimer's disease (Alzheimer's dementia), dementia with Lewy bodies, frontotemporal dementia, corticobasal degeneration, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS). Alzheimer's disease can cause cognitive impairment, Parkinson's disease can cause a decline in smooth motor function, and Huntington's disease can cause symptoms in both cognitive and motor function.

[0020] For example, dementias such as Alzheimer's disease, dementia with Lewy bodies, and frontotemporal dementia are diagnosed by measuring volumetric atrophy and reduced cerebral blood flow in specific brain regions using imaging tests such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Biomarkers of dementia, such as amyloid beta protein, tau protein, and phosphorylated tau protein in cerebrospinal fluid and blood, may be measured and used to aid in diagnosis. Dementia can also be diagnosed through screening tests, such as neuropsychological tests. One example of a screening test for dementia is the Mini-Mental State Examination (MMSE). A score of 23 or less out of a possible 30 points on the MMSE is considered to be a sign of suspected dementia.

[0021] In addition to the MRI and SPECT scans mentioned above, Parkinson's disease can be diagnosed using MIBG myocardial scintigraphy, which examines whether the sympathetic nervous system in the heart is functioning properly, and dopamine transporter scintigraphy (DatScan®), which evaluates the degree of degeneration and loss of dopaminergic neurons. There are also tests that measure alpha-synuclein protein aggregates in bodily fluids such as cerebrospinal fluid.

[0022] In the diagnosis of ALS, tests used to confirm the presence or absence of upper and lower motor neuron damage include head and spinal cord MRI, which are useful for ruling out neurogenic changes and denervation, needle electromyography (EMG), peripheral nerve conduction velocity testing (PNV), motor-evoked potential testing using central magnetic stimulation (CNS), and respiratory muscle assessment (FVC, overnight oxygen saturation measurement, and arterial blood gas analysis). Neuropsychological tests such as the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) and the ALS Cognitive Behavioral Screen (ALS-CBS) may also be used to screen for ALS. A score of 105 or less on the ECAS test out of a total of 136 points suggests possible functional impairment and raises suspicion of ALS. On the ALS-CBS test, a score of 36 or less suggests behavioral impairment, and a score of 32 or less suggests frontotemporal dementia.

[0023] Huntington's disease is diagnosed when head CT scans and MRI scans reveal atrophy of the caudate nucleus, a part of the basal ganglia, which leads to atrophy of the entire brain as the disease progresses, accompanied by enlargement of the anterior horn of the lateral ventricles. SPECT studies of cerebral blood flow often reveal decreased blood flow in the frontal and temporal lobes. Because Huntington's disease is caused by a genetic abnormality, a definitive diagnosis is made using genetic testing such as PCR. A CAG repeat count of 26 or fewer in the HTT gene is considered normal, a count of 36 or more indicates the possibility of developing Huntington's disease, and a count of 40 or more indicates a very high probability of developing the disease.

[0024] In the present invention, a subject "having a neurodegenerative disease" means that the subject has one of the above-mentioned neurodegenerative diseases. Furthermore, a subject "not suffering from a neurodegenerative disease" means that the subject does not have any of these diseases, and a "healthy subject" means a subject who does not have a neurodegenerative disease. More specifically, in the present invention, a healthy subject refers to a subject who shows no abnormalities in imaging tests such as the above-mentioned CT, MRI, PET, SPECT, MIBG myocardial scintigraphy, and dopamine transporter scintigraphy; a subject whose protein biomarkers in cerebrospinal fluid or blood, such as amyloid beta, tau, phosphorylated tau, and synuclein, have normal values; a subject whose HTT gene has 26 or fewer CAG repeats; a subject who scores more than 23 on the MMSE test; a subject who scores more than 105 on the ECAS test; or a subject who scores more than 36 on the ALS-CBS test.

[0025] In the present invention, the term "detection" can be replaced with the terms "test," "measurement," "detection," or supporting or assisting these. Furthermore, in the present invention, the terms "determination" or "evaluation" are used to mean supporting or assisting decision, diagnosis, or evaluation based on test results or measurement results.

[0026] As used herein, "P" or "P value" indicates the probability that a statistical value that is more extreme than the statistical value actually calculated from data under the null hypothesis will be observed in a statistical test. Therefore, the smaller the "P" or "P value," the more significant the difference between the compared subjects.

[0027] The "body fluid sample" to be detected in the present invention refers to a liquid biological material in which the amount of short-stranded RNA to be measured in the present invention may change depending on the presence or absence of neurodegeneration, the progression of neurodegeneration, and the effectiveness of a treatment for neurodegeneration. Specifically, the body fluid sample may be a body fluid such as cerebrospinal fluid, bone marrow fluid, blood, urine, saliva, sweat, lymph, tissue exudate or secretion, or serum or plasma prepared from blood. It also refers to a biological sample extracted from these, specifically a sample containing transcription products such as RNA or microRNA.

[0028] The present invention can be used directly or indirectly to detect the presence or absence of a neurodegenerative disease in a subject, or to diagnose the onset, prevalence, or degree of progression of a neurodegenerative disease, the presence or degree of improvement of a neurodegenerative disease, or the susceptibility to treatment of a neurodegenerative disease, or to screen for candidate substances useful for the prevention, amelioration, or treatment of a neurodegenerative disease.

[0029] The method for detecting a neurodegenerative disease of the present invention will be described below in detail for each step. 1. Step (a)

[0030] Step (a) of the present invention is a step of preparing an extracellular vesicle fraction from a body fluid sample of a subject.

[0031] Extracellular vesicles are small vesicles enclosed in a lipid bilayer membrane that are secreted from cells. They originate from the cells that secrete them, and when released into the extracellular environment, they may contain biological substances such as genes (e.g., RNA and DNA) and proteins. Extracellular vesicles are known to be contained in body fluids such as blood, serum, plasma, cerebrospinal fluid, and lymph.

[0032] Extracellular vesicles specifically include exosomes, microvesicles, and apoptotic vesicles. Exosomes, also called "exosomes" or "ectosomes," are approximately 30 to 150 nm in diameter and, because they are derived from endosomes, contain membrane proteins, such as CD9 and CD63, which are endosome-specific markers, on their membranes. Microvesicles, also called "microparticles" or "microvesicles," are approximately 100 to 1,000 nm in diameter and are released when the cell membrane buds and is constricted by the action of the ESCRT complex. Apoptotic vesicles are 1,000 to 3,000 nm in diameter and are formed when cells are rapidly fragmented during apoptosis.

[0033] The extracellular vesicle fraction prepared in step (a) is a fraction containing biological vesicles with a size range of 30 nm to 200 nm. The extracellular vesicle fraction preferably contains extracellular vesicles with a diameter of 20 nm to 1,000 nm, and more preferably contains biological vesicles with a diameter range of 10 nm to 3,000 nm, which is the size of typical extracellular vesicles. The extracellular vesicle fraction may be a fraction of larger cells (6 to 25 μm in diameter) and / or one obtained by purification to remove smaller cell fragments or debris (10 nm or less in diameter).

[0034] Methods for preparing or purifying extracellular vesicle fractions include those that utilize the size or density of extracellular vesicles or special proteins present on the surface of extracellular vesicles. Specific examples include centrifugation, immunoprecipitation, polymer precipitation, lipid affinity (affinity), liquid chromatography (e.g., high-performance liquid chromatography), size exclusion chromatography, and ultrafiltration. A combination of these methods may also be used. These methods are well known and can be easily performed using commercially available kits and devices, as described below.

[0035] Centrifugation is a method for separating or fractionating components that make up a sample by applying centrifugal force to the sample, and among these, ultracentrifugation is the most classic and gold standard. For example, in a method for obtaining an extracellular vesicle fraction by ultracentrifugation, a cell supernatant, serum, or plasma sample is centrifuged at 10,000 g for 30 minutes, the supernatant is extracted, and then centrifuged at 100,000 g for 70 minutes. The precipitate is washed with a phosphate buffer solution or the like, and centrifuged again at 100,000 g for 70 minutes, thereby obtaining an extracellular vesicle fraction as a precipitate (Clotilde Thery et al., Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids. Current Protocols in Cell Biology (2006)). 3.22.1-3.22.29) For the centrifugation method, a protocol can be applied in which the centrifugal force, centrifugation time, number of washings, etc. are optimized based on the relationship between the purity and yield of the extracellular vesicles to be obtained, and extracellular vesicle fractions can also be obtained appropriately using methods other than those described above.

[0036] Immunoprecipitation is an immunochemical technique that uses antibodies that recognize specific antigens to selectively separate and analyze target antigens or molecules that exhibit affinity for antigens from a mixture. In the present invention, specific antigens or markers present on the surface of extracellular vesicles can be used. Common antigens or markers for such extracellular vesicles include CD9, CD63, CD81, HSP70, ALIX, ANXA5, TSG101, FLOT-1, ICAM1, calnexin (CANX), CXCR4, EpCAM, Vimentin, and Tim4. Neuronal surface markers such as L1CAM, NCAM, Enolase 2, total tau protein (MAPT), Glutamate receptor 1 (GRIA1), and proteolipid protein 1 (PLP1) can also be used. Extracellular vesicles can be bound to magnetic beads bound with antibodies to these markers, and then separated and purified by immunoprecipitation. For example, the "Exo-Flow Exosome IP Kit" from System Biosciences or the ExoCap™ Streptavidin Kit (MBL) can be used. It is preferable to collect extracellular vesicles that express both at least one of the above-mentioned extracellular vesicle markers and at least one of the above-mentioned neuronal surface markers.

[0037] The polymer precipitation method is a method that uses polymers to precipitate extracellular vesicles by a single centrifugation step due to their relative specific gravity, and for example, "ExoQuick" from System Biosciences can be used.

[0038] The lipid affinity method, also called the binding affinity method or affinity method, is a method for separating and purifying a target substance or its complex by utilizing the reaction of a molecule (ligand) that reversibly binds to the target substance. For example, separation can be performed using a substance that binds to phosphatidylserine present on the surface of the lipid bilayer membrane of extracellular vesicles in a calcium-dependent manner, and the binding can be released using a chelating agent. For example, "MagCapture" from Fujifilm Wako Pure Chemical Industries, Ltd. ＴＭ The "Exosome Isolation Kit PS" can be used.

[0039] Liquid chromatography is a method in which a sample dissolved in a solvent is passed through a tube, and components are separated based on differences in migration speed due to differences in molecular size and charge. In particular, high performance liquid chromatography is a method that uses a highly pressurized liquid as the mobile phase. For example, Shimadzu's "Shim-pack Scepter Chemistry" can be used.

[0040] Size exclusion chromatography, also known as gel filtration chromatography, is a method that uses a composite matrix packed in a column to separate and purify extracellular vesicles from other impurities such as proteins, and has the advantage of being able to obtain vesicles without affecting their shape or function. For example, a "PURE-EV column" from HansaBioMed Life Sciences or an "EVSecond L70" from GL Sciences Inc. can be used.

[0041] Ultrafiltration is a technique in which pressure is applied to a solution on one side of a dialysis membrane to push extremely small molecules such as water and salts in the solution to the other side of the membrane, separating and purifying the remaining material. In the present invention, this method can be applied to the separation of extracellular vesicles. This method is excellent at removing biomolecules smaller than extracellular vesicles, and is suitable for separating and purifying extracellular vesicles contained in, for example, solutions from which polymers have been removed in advance from serum or plasma, or the supernatant of cells cultured without serum. In this case, for example, Merck's "Amicon (registered trademark) Ultra-15" can be used. 2. Step (b)

[0042] Step (b) of the present invention is a step of counting the extracellular vesicles contained in the extracellular vesicle fraction obtained in step (a) to obtain the number thereof.

[0043] The tracking method uses a high-sensitivity camera to analyze the trajectory of each particle due to Brownian motion, calculates the movement speed of each particle in the sample suspension, and analyzes particle size based on this. Specifically, the "NanoSight" by Malvern Panalytical, a Spectris company, can be used.

[0044] The antigen-antibody reaction method is a method for measuring the relative quantity of extracellular vesicles by sandwich ELISA using an antibody against a surface marker specific to extracellular vesicles. ＴＭ A "Ready to use ELISA kit" can be used. Alternatively, an "ExoCounter" from JVC Kenwood Corporation can be used, which enables absolute quantification by capturing extracellular vesicles through an antibody-antigen reaction and then digitally measuring them using nanobeads. Furthermore, the number of extracellular vesicles can be roughly quantified by utilizing enzymes contained in extracellular vesicles, such as acetyl-CoA acetylcholinesterase activity. Specifically, an "EXOCET Exosome Quantitation Kit" from System Biosciences can be used.

[0045] Flow cytometry is a method in which extracellular vesicles are stained with a fluorescent dye, bound to a carrier such as beads using an antibody-antibody interaction, and measured using a fluorescence microscope. ＴＭ The "Exosome Flow Cytometry Kit" can be used. 3. Step (c)

[0046] Step (c) of the present invention is a step of measuring the total amount of short-chain RNA contained in all extracellular vesicles counted in step (b) to obtain the total amount of short-chain RNA per number of extracellular vesicles.

[0047] As used herein, "RNA" includes total RNA, mRNA, rRNA, microRNA, siRNA, shRNA, piRNA, snoRNA, snRNA, and non-coding RNA.

[0048] In the present invention, short RNA refers to RNA having a length of 15 to 200 bases. Examples include microRNA, siRNA, shRNA, piRNA, snoRNA, snRNA, etc. Among these, microRNA is preferred.

[0049] Unless otherwise specified, microRNAs, among short-stranded RNAs, refer to RNAs transcribed as hairpin-like microRNA precursors or RNAs cleaved after transcription by dsRNA cleaving enzymes with RNase III cleavage activity, and are incorporated into a protein complex called RISC. MicroRNAs are involved in the translational repression of mRNA. MicroRNAs are typically non-coding RNAs of 15 to 30 bases.

[0050] As used herein, microRNAs include "immature microRNAs" and "mature microRNAs." Immature microRNAs refer to microRNAs that do not yet become mature microRNAs, as described below, and include "microRNA precursors."

[0051] As used herein, the term "mature microRNA" refers to a microRNA that can be incorporated into RISC after being cleaved by the dsRNA cleaving enzyme.

[0052] Furthermore, the microRNA of the present invention encompasses not only the microRNA itself represented by a specific base sequence, but also precursors of the microRNA (pre-microRNA, pri-microRNA), microRNAs with biological functions equivalent to the microRNA represented by the specific base sequence, such as its homologs (i.e., homologs or orthologs), variants such as genetic polymorphisms, or derivatives. Furthermore, when mature microRNAs are excised from RNA precursors with hairpin-like structures as mature microRNAs, one to several bases before or after the sequence may be excised shorter or longer, or base substitutions may occur, resulting in mutants called isomiRs (Morin RD. et al., 2008, Genome Res., Vol. 18, pp. 610-621). Specific microRNAs that are such precursors, homologs, variants, derivatives, or isomiRs can be identified, for example, using "miRBase" (version 22). It should be noted that "miRBase" (version 22) is an online database that provides microRNA base sequences, annotations, and predicted target genes. All microRNAs registered in "miRBase" (http: / / www.mirbase.org / ) are limited to those that have been cloned or that have been shown to be expressed and processed in vivo. Furthermore, the microRNAs of the present invention may be gene products of miR genes, and such gene products include mature microRNAs (e.g., non-coding RNAs of 15 to 30 bases involved in translational repression of mRNAs as described above) or microRNA precursors (e.g., pre-microRNAs or pri-microRNAs as described above).

[0053] In the present invention, the "total amount of short-stranded RNA" refers to the number of molecules or copies of the short-stranded RNA. The amount of short-stranded RNA can be measured, for example, by spectrophotometer, electrophoresis, microarray, PCR, or DNA sequencer, and refers to the total amount of short-stranded RNA that can be detected, regardless of the individual RNA sequence. The "total amount of short-stranded RNA" refers to the total amount of short-stranded RNA detected by the measurement method used. Therefore, for example, when "3D-Gene" (registered trademark), a type of microarray, is used as the measurement method, multiple microRNAs are simultaneously detected, and the sum of the measured values ​​can be used as the total amount of short-stranded RNA of the present invention.

[0054] Methods for obtaining RNA from extracellular vesicles include, for example, the common acid phenol method (Acid Guanidinium-Phenol-Chloroform (AGPC) method), or an RNA extraction reagent containing acid phenol such as "Trizol" (registered trademark) (Life Technologies) or "Isogen" (Nippon Gene Co., Ltd.), or a kit such as "miRNeasy Mini Kit" (Qiagen) may be used, or the RNA extraction reagent in "3D-Gene (registered trademark) RNA extraction reagent from liquid sample kit" (Toray Industries, Inc.) may be used, but are not limited to these.

[0055] The amount of short-chain RNA can be measured, for example, by using the entire obtained RNA as a sample and separating and measuring the short-chain RNA, such as by measuring using electrophoresis, microarray, PCR, or a DNA sequencer, or by measuring the short-chain RNA from the RNA sample in advance using an ultrafiltration column or the like, such as by using a spectrophotometer.

[0056] Electrophoresis is a technique in which an electric field is applied to a gel matrix through which the objects to be measured (such as nucleic acids or proteins) pass, separating them based on differences in mobility due to differences in size, charge, and structure. This method is particularly useful for analyzing the base sequence of nucleic acids. The amount of short-chain RNA can be measured using polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions using a surfactant such as SDS, or capillary electrophoresis for measuring smaller amounts of sample. The latter method, in particular, can be used to measure the amount of short-chain RNA using, for example, Agilent Technologies' "2100 Bioanalyzer."

[0057] A microarray is a relatively small device capable of simultaneously measuring multiple minute target substances, particularly nucleic acids (DNA, RNA), and is also referred to as a nucleic acid array, nucleic acid chip, or DNA chip. In the present invention, the total amount of short-stranded RNAs, such as microRNA and piRNA, can be measured by using an array specialized for these substances. For example, the amount of short-stranded RNA can be calculated from the sum of the microRNA and piRNA expression levels. The amount of short-stranded RNA can be obtained by using such microarrays, such as Toray Industries, Inc.'s "3D-Gene" (registered trademark) or Agilent Technologies' microRNA microarray. In the following examples, the total amount of microRNA is measured using Toray Industries, Inc.'s "3D-Gene" (registered trademark). "3D-Gene" (registered trademark) is a microarray equipped with DNA complementary to 2,632 types of microRNA, covering most of the known human microRNAs, and is preferably used in the present invention. When using a microarray, it is preferable to use one that is equipped with DNA complementary to as many known human microRNAs as possible, preferably 1,000 or more, more preferably 2,000 or more, and even more preferably 2,600 or more. The versions of the microRNAs complementary to the DNA installed in "3D-Gene" (registered trademark) are listed at https: / / www.3d-gene.com / products / dna / , and the version details are registered in miRBase release 22 (http: / / www.mirbase.org / ). The amount of microRNA bound to the microarray can be measured by fluorescently labeling the microRNA in advance, hybridizing it with the DNA on the microarray, and then quantifying the fluorescent label immobilized on the microarray. This can be easily performed using commercially available kits and devices, as specifically described in the Examples below.

[0058] PCR (Polymerase Chain Reaction), particularly multiplex PCR, which simultaneously detects multiple target short RNAs, is useful in the present invention. Kits capable of simultaneously detecting multiple microRNAs are available, such as Qiagen's "miRCURY LNA miRNA PCR Assays" and "miScript miRNA PCR Arrays" and Thermo Fisher Scientific's "TaqMan ＴＭ MicroRNA Assay, etc. can be used. After simultaneously detecting multiple microRNAs, the amount of short-chain RNA can be obtained by summing up the measured values.

[0059] A DNA sequencer is a method for determining the sequence of a DNA fragment, that is, the sequence of each base. In the present invention, a classical method such as the Sanger method or a next-generation sequencer may be used.

[0060] When using next-generation sequencers such as "Miseq, Hiseq, NexSeq" (Illumina), "Ion Proton, Ion PGM, Ion S5 / S5 XL" (Thermo Fisher Scientific), "PacBio RS II, Sequel" (Pacific Bioscience), or "Nanopore Sequencer," a commercially available measurement kit specially designed for measuring microRNAs may be used, such as "MinION" (Oxford Nanopore Technologies).

[0061] Next-generation sequencing is a sequencing technology using a next-generation sequencer, and is characterized by the ability to perform a vast number of sequencing reactions simultaneously in parallel compared to the Sanger method (see, for example, Rick Kamps et al., Int. J. Mol. Sci., 2017, 18(2), p. 308 and Int. Neurourol. J., 2016, 20(Suppl. 2), S76-83). An example of next-generation sequencing for short-stranded RNA includes a step of adding adapter sequences having a predetermined base sequence to both ends of a sample-derived short-stranded RNA or its cDNA, and a step of reverse transcribing the total RNA (total RNA) derived from the sample into cDNA before or after the addition of the adapter sequence. After reverse transcription and before the sequencing step, the cDNA derived from a specific target short-stranded RNA may be amplified by a nucleic acid amplification method such as PCR or concentrated using a probe or the like. The details of the subsequent sequencing process vary depending on the type of next-generation sequencer, but typically, cDNA is linked to a substrate via an adapter sequence, and a sequencing reaction is carried out using the adapter sequence as a priming site for a sequencing primer to determine and analyze the base sequence. For details on the sequencing reaction, see, for example, Rick Kamps et al. (cited above). Finally, data is output. This provides a collection of sequence information (reads) obtained in the sequencing reaction and analysis data for the information. For example, next-generation sequencing can identify target microRNAs based on the obtained sequence information, and determine their expression levels based on the number of reads containing the target microRNA's base sequence.

[0062] Unlike the above-mentioned measurement methods, measurement using a spectrophotometer is a method in which RNA length cannot be specified, and therefore RNA samples are first fractionated using an ultrafiltration column or the like to obtain short-chain RNA, and then measurement is performed. Measurement can be performed using absorbance or fluorescence, but a measurement method using a fluorescent dye is preferable from the viewpoint of high sensitivity. For example, short-chain RNA is fluorescently labeled using Thermo Fisher Scientific's "Quant-iT RiboGreen RNA Kit" and then transferred to the "NanoDrop" of the same company. ＴＭ " can be measured.

[0063] Although the amount of short-stranded RNA can be obtained by the above-mentioned measurement method, the "total amount" of short-stranded RNA is used in the present invention. The "total amount" does not need to be limited to a specific RNA sequence, but refers to the amount of a collection of multiple types of short-stranded RNA sequences.

[0064] The total amount of short-chain RNA contained in all extracellular vesicles obtained by the above measurement is divided by the number of extracellular vesicles obtained in step (b), thereby obtaining the total amount of short-chain RNA per number of extracellular vesicles. In this case, instead of the "total amount of short-chain RNA contained in all extracellular vesicles," a value obtained by extracting a portion of the extracellular vesicle fraction obtained in step (a) and measuring the amount of short-chain RNA contained therein may be used, or the "total amount of short-chain RNA contained in all extracellular vesicles" may be calculated based on the amount of short-chain RNA. 4. Step (d)

[0065] Step (d) of the present invention is a step of determining that a subject is suffering from a neurodegenerative disease if the total amount of short-stranded RNA per extracellular vesicle obtained in step (c) is greater than the total amount of short-stranded RNA per extracellular vesicle obtained in the same manner from a body fluid sample of a control healthy subject. Here, the healthy subject refers to a subject not suffering from a neurodegenerative disease, and the total amount of short-stranded RNA per extracellular vesicle obtained from a body fluid sample of a subject not suffering from a neurodegenerative disease is used as the control.

[0066] For example, a body fluid sample from a subject who is clearly not suffering from a neurodegenerative disease is used as a control, and an extracellular vesicle fraction is obtained in the same manner as in step (a). The extracellular vesicles are counted simultaneously with the body fluid sample from the subject to be detected (step (b)). The total amount of short-chain RNA contained in all the counted extracellular vesicles is measured to obtain the total amount of short-chain RNA per number of extracellular vesicles (step (c)). Using this as a control, if the total amount of short-chain RNA per number of extracellular vesicles in the subject is high, the subject can be determined to be suffering from a neurodegenerative disease. Furthermore, the total amount of short-chain RNA per number of extracellular vesicles in the control is set to 1, and the ratio of the total amount of short-chain RNA per number of extracellular vesicles in the subject to this is calculated. If this ratio exceeds 1, the subject can be determined to be suffering from a neurodegenerative disease.

[0067] Furthermore, by using body fluid samples from multiple subjects who do not suffer from neurodegenerative diseases as controls, the standard error of the control measurement value (total amount of short-stranded RNA) can be calculated. When comparing with the subject measurement value, thresholds can be set to reliability standards such as 70%, 80%, 90%, and 100%.

[0068] It is also possible to measure a control in advance, set the measured value as a threshold, and use it repeatedly. The threshold can be set appropriately for each detection system as a value that can distinguish between a control and a subject based on the total amount of short-stranded RNA per number of extracellular vesicles in a body fluid sample. For example, when the total amount of short-stranded RNA is measured using "3D-Gene" (registered trademark) and the number of extracellular vesicles is counted using the nanotracking method, if a control consisting of a single healthy subject is used, the threshold can be set at, for example, 1.1-fold, 1.5-fold, preferably 2-fold, more preferably 2.5-fold, or 5-fold the total amount of short-stranded RNA per number of extracellular vesicles in the control. Alternatively, if a control consisting of multiple healthy subjects is used, the threshold can be set at, for example, 1.1-fold, 1.5-fold, preferably 2-fold, more preferably 2.5-fold, or 5-fold the average total amount of short-stranded RNA per number of extracellular vesicles. Alternatively, the threshold can be set as the maximum total amount of short-stranded RNA per extracellular vesicle count in a control sample of multiple healthy individuals, or 1.1 times, 1.5 times, preferably 2 times, more preferably 2.5 times, or 5 times that value. Since the average value for the control is the value for healthy individuals, for example, once the initial measurement is performed, the measured value can be used from the next time onward, and the threshold can be set in advance using this average value. Such a threshold can be described, for example, in the instructions (instructions for use) for the kit for performing the method of the present invention.

[0069] If the total amount of short-stranded RNA per extracellular vesicle count obtained from a subject's body fluid is significantly higher than the average value, it is possible that the total amount has not been measured correctly, leading to the risk of erroneously determining that a subject without a neurodegenerative disease has the disease. To prevent this problem, an upper limit may be set, and a subject may be determined to have a neurodegenerative disease if the total amount of short-stranded RNA is below the upper limit. For example, when measuring the total amount of short-stranded RNA using "3D-Gene" (registered trademark) and counting the number of extracellular vesicles using the nanotracking method, if a control consisting of a single healthy subject is used, the upper limit can be set at, for example, 500-fold, 300-fold, preferably 200-fold, or more preferably 100-fold the total amount of short-stranded RNA per extracellular vesicle count in the control. Alternatively, if a control consisting of multiple healthy subjects is used, the upper limit can be set at 500-fold, 300-fold, preferably 200-fold, or more preferably 100-fold the average total amount of short-stranded RNA per extracellular vesicle count. Alternatively, the upper limit can be set at 500 times, 300 times, preferably 200 times, and more preferably 100 times the maximum total amount of short-chain RNA per extracellular vesicle in a control consisting of multiple healthy individuals.

[0070] When the total amount of short-stranded RNA per vesicle count obtained from the subject's body fluid is a multiplier within a specific range relative to the total amount of short-stranded RNA when a single control is used, or relative to the average or maximum value when multiple controls are used, the subject can be determined to be suffering from a neurodegenerative disease. The range may be 1.1 to less than 500 times, preferably 1.5 to less than 300 times, and most preferably 1.5 to less than 100 times.

[0071] The method of the present invention for determining whether a subject is suffering from a neurodegenerative disease can also be used in combination with imaging tests such as CT, MRI, PET, SPECT, MIBG myocardial scintigraphy, or dopamine transporter scintigraphy, protein biomarker tests such as amyloid beta, tau, phosphorylated tau, and synuclein in cerebrospinal fluid or blood, genetic tests using PCR, and neuropsychological tests such as the MMSE test, ECAS test, and ALS-CBS test.

[0072] The present invention will be explained in more detail by the following examples, but the scope of the present invention is not limited to these examples.

[0073] [Example 1] <Preparation of extracellular vesicle fraction> (Step (a)) A fraction equivalent to an extracellular vesicle (including exosomes, etc.) fraction derived from a subject suffering from a neurodegenerative disease was prepared as follows.

[0074] As nerve cells, SH-SY5Y cell line (ATCC) was plated in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque) / Ham's F-12 medium (Nacalai Tesque) containing 10% FBS (Gibco), and retinoic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the medium to a final concentration of 10 μM. The cells were incubated at 37°C and 5% CO. ２、 The cells were cultured for 5 days under dark conditions. After that, 1 × 10 cells were added to each well of a 6-well plate. ５The cells were plated, and after 24 hours, the culture supernatant was removed. After washing twice with 1 mL of phosphate-buffered saline (PBS) (Nacalai Tesque), 3 mL of DMEM (Nacalai Tesque) / Ham's F-12 medium (Nacalai Tesque) was added to give a final concentration of 10 μM amyloid β oligomers, and the cells were cultured at 37°C for 48 hours. This treatment caused neurodegeneration in the neurons, resulting in Alzheimer's disease-like morphology. Amyloid β oligomers were prepared by dissolving amyloid β peptide (1-42) (Abcam) in DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 25 mM, adding hexafluoropropanol (Nacalai Tesque) to this solution to a final concentration of 1 mM, drying the solution in a concentrator centrifuge, redissolving it in DMSO (Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 5 mM, adding Ham's F-12 medium (Nacalai Tesque) to a final concentration of 100 μM, and incubating the solution at 4°C for 24 hours. After culturing, the cell supernatant was collected and centrifuged at 10,000×G at 4°C for 30 minutes to recover the supernatant.

[0075] As an affinity method, "MagCapture ＴＭ Using the "Exosome Isolation Kit PS" (Fujifilm Wako Pure Chemical Corporation), an extracellular vesicle (including exosomes, etc.) fraction was obtained from 1 mL of the cell culture supernatant after the above-mentioned centrifugation according to the specified protocol.

[0076] On the other hand, as a control corresponding to an extracellular vesicle fraction derived from a subject not suffering from a neurodegenerative disease, an extracellular vesicle fraction was prepared in the same manner as above, but without the addition of amyloid β oligomers.

[0077] <Counting of extracellular vesicles> (Step (b)) The number of extracellular vesicles contained in the extracellular vesicle fraction prepared in step (a) above was counted by the nanotracking method. Using a nanoparticle tracking analyzer "NanoSight NS 300" (NanoSight Ltd.), the number of extracellular vesicles was counted using 1 / 5 of the amount of the extracellular vesicle fraction obtained in step (a), and the analysis was performed using NTA3.2 (NanoSight Ltd.). The results are shown in the upper part of Figure 1. n = 3 was performed, and the vertical axis of the graph represents the number of exosome (diameter 80-120 nm) particles ( / mL), and the bar graph represents the mean ± standard deviation.

[0078] The average number of exosomes prepared with the addition of amyloid β oligomers was 6.68 × 10 ６ On the other hand, the average number of exosomes prepared without adding amyloid β oligomers as a control was 2.61 × 10 ６ The total amount of short-chain RNA in extracellular vesicles was measured (step (c)).

[0079] The total amount of short-chain RNA contained in all extracellular vesicles contained in the extracellular vesicle fraction prepared in step (a) above was obtained as follows. Total RNA was obtained from 4 / 5 of the extracellular vesicle fraction obtained in step (a) using the RNA extraction reagent in the "3D-Gene (registered trademark) RNA extraction reagent from liquid sample kit" (Toray Industries, Inc.) according to the protocol specified by the company. MicroRNA was fluorescently labeled from the obtained total RNA using the "3D-Gene (registered trademark) miRNA Labeling kit" (Toray Industries, Inc.) according to the protocol specified by the company. The oligo DNA chip used was the "3D-Gene® Human miRNA Oligo Chip" (Toray Industries, Inc.), which is equipped with probes with sequences complementary to 2,632 types of microRNAs registered in miRBase release 22 (http: / / www.mirbase.org / ). Hybridization and post-hybridization washing were performed under highly stringent conditions according to the protocol established by the company. The DNA chip was scanned using a "3D-Gene® Scanner" (Toray Industries, Inc.), and the images were acquired. The fluorescence intensity was then quantified using "3D-Gene® Extraction" (Toray Industries, Inc.) to obtain comprehensive microRNA gene expression levels. The sum of the detected microRNA gene expression levels (linear values) was calculated as the total microRNA amount.

[0080] The total amount of microRNA contained in exosomes prepared with the addition of amyloid β oligomers was 2.46 × 10 ５ On the other hand, the total amount of microRNA contained in the control exosomes prepared without adding amyloid β oligomers was 3.10 × 10 ５ It was.

[0081] The total amount of microRNA obtained was divided by the number of exosomes obtained in step (b) above to calculate the amount of microRNA per number of exosomes. The total amount of microRNA per number of exosomes in exosomes prepared with the addition of amyloid beta oligomers was 0.037. On the other hand, the total amount of microRNA per number of exosomes in control exosomes prepared without the addition of amyloid beta oligomers was 0.119.

[0082] <Determination of the Presence or Absence of Neurodegeneration> (Step (d)) The total amount of microRNA per number of exosomes obtained in step (c) above was used to determine the relative ratio of the total amount of microRNA in the presence of amyloid β oligomers, when the total amount of microRNA in the absence of amyloid β oligomers was set to 1. The results are shown in the lower part of Figure 1. The experiment was performed with n = 3, and the vertical axis of the graph shows the relative ratio of the total amount of microRNA per number of exosomes.

[0083] As a result, the total amount of microRNA per exosome was significantly increased when amyloid beta oligomers were added (Figure 1, lower right (+)) compared to when amyloid beta oligomers were not added (Figure 1, lower left (-)) (P value < 0.005).

[0084] These results demonstrated that it is possible to determine whether a subject suffers from neurodegeneration by comparing the total amount of microRNA per exosome count with a control.

[0085] [Example 2] <Preparation of extracellular vesicle fraction> (Step (a)) A fraction equivalent to an extracellular vesicle (including exosomes, etc.) fraction derived from a subject suffering from a neurodegenerative disease was prepared as follows.

[0086] The serum samples were purchased from Busicom Japan and were obtained from three patients with informed consent: a healthy subject, an Alzheimer's disease patient, and an ALS patient (Table 1).

[0087] CONTROL: Healthy individuals AD: Alzheimer's disease ALS: Amyotrophic lateral sclerosis MMSE: Mini-Mental state examination

[0088] For immunoprecipitation, biotin-labeled anti-L1CAM antibody (Invitrogen) and the ExoCap™ Streptavidin Kit (MBL) were used, and 1 mL of the above serum was centrifuged at 100,000 × G for 10 minutes at 4°C according to the protocol specified by the ExoCap™ Streptavidin Kit. An extracellular vesicle (including exosomes, etc.) fraction was obtained from the supernatant.

[0089] <Counting Extracellular Vesicles> (Step (b)) The number of extracellular vesicles contained in the extracellular vesicle fraction prepared in step (a) above was detected by antigen-antibody reaction. A 500-fold diluted solution of HRP-labeled anti-CD63 antibody (Santa Cruz Biotechnology) was added to 1 / 5 of the extracellular vesicle fraction obtained in step (a) and stirred at 4°C for 4 hours. The extracellular vesicle value (luminescence intensity) was then detected using a microplate reader (MOLECULAR DEVICES) according to the protocol specified for the ExoCap™ Streptavidin Kit. The extracellular vesicle value prepared from the serum of an Alzheimer's disease patient was 7.51 x 10 ５ The value of extracellular vesicles prepared from serum of ALS patients was 7.11 × 10 ５ On the other hand, the value of extracellular vesicles prepared from serum of healthy individuals was 9.42 × 10 ５ The results of the relative values ​​are shown in the upper panel of Figure 2. The experiment was performed with n = 3, and the vertical axis of the graph shows the relative number of exosomes to the average value of healthy subjects, while the bar graph shows the relative value when the average value of healthy subjects was set to 1. The average number of exosomes prepared from Alzheimer's disease patients (relative to healthy subjects) was 0.798 (top middle of Figure 2 (AD)). The average number of exosomes prepared from ALS patients (relative to healthy subjects) was 0.755 (top right of Figure 2 (ALS)).

[0090] <Measurement of the total amount of short-chain RNA in extracellular vesicles> (Step (c)) The total amount of short-chain RNA contained in all extracellular vesicles contained in the extracellular vesicle fraction prepared in step (a) above was obtained as follows. Total RNA was obtained from 4 / 5 of the extracellular vesicle fraction obtained in step (a) using the RNA extraction reagent in the "3D-Gene (registered trademark) RNA extraction reagent from liquid sample kit" (Toray Industries, Inc.) according to the protocol specified by the company. MicroRNA was fluorescently labeled from the obtained total RNA using the "3D-Gene (registered trademark) miRNA Labeling kit" (Toray Industries, Inc.) according to the protocol specified by the company. The oligo DNA chip used was the "3D-Gene® Human miRNA Oligo Chip" (Toray Industries, Inc.), which is equipped with probes with sequences complementary to 2,632 types of microRNAs registered in miRBase release 22 (http: / / www.mirbase.org / ). Hybridization and post-hybridization washing were performed under highly stringent conditions according to the protocol established by the company. The DNA chip was scanned using a "3D-Gene® Scanner" (Toray Industries, Inc.), and the images were acquired. The fluorescence intensity was then quantified using "3D-Gene® Extraction" (Toray Industries, Inc.) to obtain comprehensive microRNA gene expression levels. The sum of the detected microRNA gene expression levels (linear values) was calculated as the total microRNA amount.

[0091] The total amount of microRNA contained in exosomes prepared from serum of Alzheimer's disease patients was 9.56 × 10 ３ The total amount of microRNA contained in exosomes prepared from the serum of ALS patients was 7.74 × 10 ３ On the other hand, the total amount of microRNA contained in exosomes prepared from serum of healthy individuals was 8.13 × 10 ３ It was.

[0092] The total amount of microRNA obtained was divided by the value obtained in step (b) above to calculate the amount of microRNA per exosome. The total amount of microRNA per exosome in serum exosomes from healthy individuals was 0.011. On the other hand, the total amount of microRNA per exosome in serum exosomes from Alzheimer's disease patients was 0.019. Furthermore, the total amount of microRNA per exosome in serum exosomes from ALS patients was 0.075.

[0093] <Determining the Presence or Absence of Neurodegeneration> (Step (d)) The total amount of microRNA per exosome obtained in step (c) above was calculated relative to the total amount of microRNA in Alzheimer's disease and ALS patients, with the healthy controls set at 1. The results are shown in the lower panel of Figure 2. The experiment was performed in triplicate. The vertical axis of the graph represents the total amount of microRNA per exosome (relative to the average value for healthy controls), and the bar graph represents the relative value when the healthy controls are set at 1. The total amount of microRNA per exosome (relative to the healthy controls) prepared from Alzheimer's disease patients was 1.705 (Figure 2, lower middle (AD)). The total amount of microRNA per exosome (relative to the healthy controls) prepared from ALS patients was 6.641 (Figure 2, lower right (ALS)). The total amount of microRNA per exosome was more than 1.5-fold higher in Alzheimer's disease patients and more than 5-fold higher in ALS patients.

[0094] These results demonstrated that it is possible to determine whether a subject suffers from neurodegeneration by comparing the total amount of microRNA per exosome count with that of a healthy individual as a control.

Claims

1. 1. A method for detecting whether a subject is suffering from amyotrophic lateral sclerosis, comprising: (a) preparing an extracellular vesicle fraction from a body fluid sample of a subject; (b) counting the extracellular vesicles contained in the extracellular vesicle fraction obtained in step (a) to obtain the number of the extracellular vesicles; Step (c) of measuring the total amount of short-chain RNA contained in all the extracellular vesicles counted in step (b) to obtain the total amount of short-chain RNA per number of extracellular vesicles; and a step (d) of determining that the subject is suffering from amyotrophic lateral sclerosis if the total amount of short-stranded RNA per extracellular vesicle obtained in step (c) is greater than the total amount of short-stranded RNA per extracellular vesicle obtained from a body fluid sample of a healthy subject; A method comprising:

2. The method of claim 1, wherein in step (d), an average value of the total amount of short-stranded RNA per number of extracellular vesicles obtained in advance from body fluid samples of multiple healthy subjects is calculated, and the subject is determined to have amyotrophic lateral sclerosis if the total amount of short-stranded RNA per number of extracellular vesicles obtained from the subject's body fluid is 1.5 to less than 100 times the average value.

3. 3. The method according to claim 1 or 2, wherein the extracellular vesicle fraction prepared in step (a) contains extracellular vesicles having a diameter of 30 nm to 200 nm.

4. The method of claim 1 or 2, wherein in step (a), CD9, CD63, CD81, Tim4 or L1CAM protein is present on the surface of the prepared extracellular vesicles.

5. 3. The method according to claim 1, wherein in the step (b), the short RNA has a length of 15 to 200 bases.

6. The method of claim 1 or 2, wherein the short RNA is a microRNA.

7. The method of claim 1 or 2, wherein the body fluid sample is blood, serum, plasma, or cerebrospinal fluid.

8. The method of claim 1 or 2, wherein in step (a), the extracellular vesicle fraction is prepared by a method selected from the group consisting of centrifugation, immunoprecipitation, polymer precipitation, lipid affinity, liquid chromatography, size exclusion chromatography, ultrafiltration, and combinations thereof.

9. The method according to claim 1 or 2, wherein in step (b), the number of extracellular vesicles is counted by a tracking method, an antigen-antibody reaction method, or a flow cytometry method.

10. The method according to claim 1 or 2, wherein in step (c), the total amount of short-chain RNA contained in the extracellular vesicles is measured using a spectrophotometer, electrophoresis, a microarray, PCR, or a DNA sequencer.

11. A means for preparing an extracellular vesicle fraction from a body fluid sample of a subject; A means for counting the extracellular vesicles contained in the prepared extracellular vesicle fraction to obtain the number of the extracellular vesicles; and a means for measuring the total amount of short-chain RNA contained in all counted extracellular vesicles and obtaining the total amount of short-chain RNA per number of extracellular vesicles. A kit for detecting whether a subject has amyotrophic lateral sclerosis using the method of claim 1.

12. The means for preparing an extracellular vesicle fraction from a body fluid sample of a subject includes an immobilized antibody or antigen-binding fragment thereof that specifically binds to a surface antigen of the extracellular vesicles of interest, and the means for counting the extracellular vesicles contained in the prepared extracellular vesicle fraction to obtain the number of the extracellular vesicles comprises a labeled antibody or an antigen-binding fragment thereof that specifically binds to a surface antigen of the extracellular vesicle of interest; The kit of claim 11, wherein the means for measuring the total amount of short-chain RNA contained in all counted extracellular vesicles and obtaining the total amount of short-chain RNA per number of extracellular vesicles comprises a chip equipped with nucleic acid probes that hybridize with multiple known microRNAs.

13. A means for preparing an extracellular vesicle fraction from a body fluid sample of a subject; A means for counting the extracellular vesicles contained in the prepared extracellular vesicle fraction to obtain the number of the extracellular vesicles; and a means for measuring the total amount of short-chain RNA contained in all the counted extracellular vesicles and obtaining the total amount of short-chain RNA per number of extracellular vesicles. A system for detecting whether a subject is affected by amyotrophic lateral sclerosis using the method of claim 1.