Method for detecting alzheimer's disease, and detection reagent

Abstract

The present invention relates to providing a method for easily detecting Alzheimer's disease (AD) and a method for easily stratifying AD pathology phases, as well as providing a reagent with that can be used in these methods. The present invention provides a method for easily detecting Alzheimer's disease (AD) and a method for easily stratifying AD pathology phases due to measurement of extracellular-secretion-vesicle-containing proteins making it possible to detect AD and stratify AD pathology phases, and a reagent that can be used in these methods.

Description

Method and reagent for detecting Alzheimer's disease

[0001] The present invention relates to a method and a reagent for detecting Alzheimer's disease, in which proteins contained in extracellular secretory vesicles are measured.

[0002] Alzheimer's disease (AD) is the most common form of dementia. The pathogenesis of AD begins before the onset of symptoms and progresses stepwise from the accumulation of amyloid beta protein (Aβ) in the initial pathology to neurofibrillary tangles in the tau pathology, followed by neurodegeneration, ultimately resulting in the onset of AD. Currently, cerebrospinal fluid tests and PET imaging using the concentrations of Aβ and tau protein as indicators are used as biomarkers to stratify this pathological phase. However, cerebrospinal fluid Aβ measurement is not covered by insurance, and there are many issues, such as the high invasiveness of cerebrospinal fluid collection and the limited number of expensive PET examination facilities. Therefore, there is a need for the development of biomarkers that can more easily stratify AD pathological phases in the living brain for use in clinical and basic research aimed at treating and preventing AD.

[0003] The present invention aims to provide a method for easily detecting Alzheimer's disease (AD) and a method for easily stratifying AD pathological phases in a living brain from before the onset of AD, as well as a reagent that can be used in the method.

[0004] The present inventors have conducted extensive research and found that a specific group of proteins in extracellular secretory vesicles changes between the molecular pathological phases of AD, using abnormal proteins (amyloid beta, tau) as indicators, and identified the proteins contained in extracellular secretory vesicles that change between the pathological phases. Based on this, they have conceived that measurement of proteins contained in extracellular secretory vesicles can be used to detect Alzheimer's disease and stratify the pathological phases of AD, and have completed the present invention.

[0005] That is, the present invention encompasses the following aspects: [1] A method for detecting Alzheimer's disease, comprising the step of measuring the amount of an extracellular secretory vesicle-containing protein in a sample collected from a subject. [2] The method of [1], wherein the amount of the extracellular secretory vesicle-containing protein in a subject with Alzheimer's disease varies from the amount of the protein in a normal subject. [3] The method of [2], wherein the variation is 2-fold or more or 0.5-fold or less. [4] Any of the methods of [1] to [3], comprising the steps of recovering an extracellular secretory vesicle fraction in a sample collected from a subject and measuring the extracellular secretory vesicle-containing protein in the extracellular secretory vesicle fraction.[5] The extracellular secretory vesicle-containing protein is selected from the group consisting of ectonucleotide pyrophosphatase / phosphodiesterase family member 3, α-1,3-fucosyltransferase 10, repulsive guidance molecule A, cAMP-dependent protein kinase type I-β regulatory subunit, DNA replication ATP-dependent helicase / nuclease DNA2, contactin 6, histone H4, exostosin 1, BCL6 corepressor-like protein 1, immunoglobulin μ heavy chain, Rho guanine nucleotide exchange factor 28, cathepsin B, keratin 87 putative protein, multi-splicing RNA-binding protein, serpin B3, β-galactoside α-2,6-sialyltransferase 1, calcium-binding protein 39, cytoplasmic isocitrate dehydrogenase [NADP], and melanoma-binding antibody. The method of any one of [1] to [4], wherein the extracellular secretory vesicle-containing protein is one or more proteins selected from the group consisting of B10, nodal modulator 1, nodal modulator 2, nodal modulator 3, phosphoribosyltransferase domain-containing protein 1, free homolog-like protein, myosin 10, helicase senataxin, FAM227B protein, serpin B4, cholinesterase, pericentrin, Unc13 homolog C protein, 26S proteosome non-ATPase regulatory subunit 1, neurexophilin 1, DNA-binding protein RFX5, pregnancy-specific β1 glycoprotein 8, amyloid βA4 precursor protein-binding family B member 2, kallikrein 11, cadherin 11, sodium channel protein type 4 subunit α, and transmembrane protein 62. [6] The method of any one of [1] to [5], wherein the extracellular secretory vesicle-containing protein comprises cathepsin B. [7] A method for detecting the amyloid accumulation stage of Alzheimer's disease, comprising the step of measuring extracellular secretory vesicle-containing proteins in a sample collected from a subject.[8] The extracellular secretory vesicle-containing protein is selected from the group consisting of ectonucleotide pyrophosphatase / phosphodiesterase family member 3, α-1,3-fucosyltransferase 10, repulsive guidance molecule A, cAMP-dependent protein kinase type I-β regulatory subunit, DNA replication ATP-dependent helicase / nuclease DNA2, contactin 6, histone H4, exostocin 1, BCL6 corepressor-like protein 1, immunoglobulin μ heavy chain, Rho guanine nucleotide exchange factor 28, cathepsin B, keratin 87 putative protein, and multi-splicing protein. [7] The method according to [7], wherein the extracellular secretory vesicle-containing protein is one or more proteins selected from the group consisting of phospho-RNA-binding protein, serpin B3, β-galactoside α-2,6-sialyltransferase 1, calcium-binding protein 39, cytoplasmic isocitrate dehydrogenase [NADP], melanoma-binding antibody B10, nodal modifier 1, nodal modifier 2, nodal modifier 3, phosphoribosyltransferase domain-containing protein 1, free homolog-like protein, myosin 10, helicase senataxin, FAM227B protein, and serpin B4. [9] The method according to [7] or [8], wherein the extracellular secretory vesicle-containing protein comprises cathepsin B.

[10] A method for detecting the tau pathogenesis and / or neurodegeneration stage of Alzheimer's disease, comprising the step of measuring the extracellular secretory vesicle-containing protein in a sample collected from a subject.

[11] The method of

[10] , wherein the extracellular secretory vesicle-containing protein is one or more proteins selected from the group consisting of cholinesterase, pericentrin, Unc13 homolog C protein, 26S proteosomal non-ATPase regulatory subunit 1, α-1,3-fucosyltransferase 10, neurexophilin 1, DNA-binding protein RFX5, pregnancy-specific β1-glycoprotein 8, amyloid βA4 precursor protein-binding family B member 2, kallikrein 11, cadherin 11, β-galactoside α-2,6-sialyltransferase 1, sodium channel protein type 4 subunit α, FAM227B protein, and transmembrane protein 62.

[12] The method of

[10] or

[11] , wherein the extracellular secretory vesicle-containing protein includes cathepsin B.

[13] The method according to any one of [1] to

[12] , wherein the sample is cerebrospinal fluid or plasma.

[14] A diagnostic kit for Alzheimer's disease, comprising a reagent for measuring a protein contained in extracellular secretory vesicles.

[0006] The present invention provides a method for easily detecting Alzheimer's disease (AD), a method for easily stratifying AD pathological phases in a living brain from before the onset of AD, and reagents that can be used for these methods.

[0007] Graph showing cathepsin B concentrations in the extracellular vesicle fraction of cerebrospinal fluid in subjects with each ATN classification. ***: p<0.001. Graph showing cathepsin B concentrations in the extracellular vesicle fraction of cerebrospinal fluid in subjects with and without amyloid pathology (subjects in group A+ or A-). ***: p<0.001. Graph showing the correlation between cerebrospinal fluid Aβ42 concentrations and cathepsin B concentrations in the extracellular vesicle fraction of cerebrospinal fluid in each subject. r indicates the correlation coefficient. Graph showing cathepsin B concentrations in the extracellular vesicle fraction of plasma in subjects with and without amyloid pathology (subjects in group A+ or A-). ***: p<0.001. Graph showing the correlation between cerebrospinal fluid Aβ42 concentrations and cathepsin B concentrations in the extracellular vesicle fraction of plasma in subjects with and without amyloid pathology (subjects in group A+ or A-).

[0008] A first aspect of the present invention is a method for detecting Alzheimer's disease, comprising measuring the amount of extracellular secretory vesicle-containing protein in a sample collected from a subject. The subject is not particularly limited as long as it is an animal that may develop Alzheimer's disease, but is preferably a mammal, and more preferably a human. Furthermore, the detection method of the present invention is usually performed in vitro.

[0009] The sample includes body fluids, cells, tissues, etc. collected from a specimen. Body fluids include cerebrospinal fluid, blood, lymph, tissue fluid, etc., and blood includes plasma and blood cell components. Preferably, the sample is cerebrospinal fluid or plasma, and more preferably, cerebrospinal fluid.

[0010] The collection of a sample from a specimen may be performed by any appropriate method, including known methods, depending on the sample to be collected and the specimen to be collected. For example, cerebrospinal fluid can be collected by direct drip collection through lumbar puncture, and plasma can be collected from collected blood by centrifugation, but is not limited to these. Furthermore, the collected sample may be centrifuged or frozen for storage, if necessary.

[0011] Extracellular secretory vesicles are extracellular vesicles secreted from cells. Extracellular secretory vesicles typically contain cell membrane components of the producing cell on their surface and substances from the producing cell inside. The protein contained in extracellular secretory vesicles may be a protein contained inside the extracellular secretory vesicles, a protein present on the surface of the extracellular secretory vesicles, or a transmembrane protein of the extracellular secretory vesicles, but is not particularly limited as long as it is recovered together with the extracellular secretory vesicles. The protein contained in extracellular secretory vesicles is preferably a protein contained in the extracellular secretory vesicle fraction.

[0012] The measurement of proteins contained in extracellular secretory vesicles may be performed on the whole sample or on the extracellular secretory vesicle fraction of the sample. Thus, the present invention may include a step of recovering the extracellular secretory vesicle fraction from a sample collected from a subject.

[0013] An extracellular secretory vesicle fraction is a fraction containing a large amount of extracellular secretory vesicles in a sample, and can be fractionated using, for example, ultracentrifugal sedimentation, density gradient ultracentrifugal fractionation, size exclusion chromatography, antibodies against surface antigens, or techniques for capturing lipids specifically present on the membrane surface of extracellular secretory vesicles. The extracellular secretory vesicle fraction can be recovered by ultracentrifugation or column purification, or can also be recovered using commercially available products. Examples of such products include, but are not limited to, the size exclusion chromatography column EV-Second L70 (manufactured by GL Sciences).

[0014] A known protein measurement method can be used to measure the extracellular secretory vesicle-containing protein. Preferably, measurement using mass spectrometry with a liquid chromatography mass spectrometer (LC-MS) or an immunoassay using an antibody against the extracellular secretory vesicle-containing protein can be used. Examples of immunoassay methods include quantitative detection methods according to enzyme immunoassay, and measurement methods using fluorescent immunoassay, chemiluminescent immunoassay, etc. Enzyme immunoassay is a detection method among labeled immunoassay methods that uses an enzyme as a labeling substance. Another example of an enzyme immunoassay is ELISA.

[0015] Examples of ELISA include direct, indirect, and sandwich methods, and any of these methods can be used. The direct ELISA method may be a method well known in the art in which a labeled antibody is bound to a protein in a biological sample immobilized on a solid phase to detect the labeled substance. The indirect ELISA method may be a method well known in the art in which a primary antibody is bound to a protein in a biological sample immobilized on a solid phase to form an immune complex on the solid phase surface, and then a labeled secondary antibody that recognizes the primary antibody is used to detect the labeled substance. The sandwich ELISA method may be a method well known in the art in which a protein is captured by an immobilized antibody (primary antibody) immobilized on a solid phase, and then a labeled secondary antibody is bound to the captured protein to form an immune complex bound to the two antibodies on the solid phase surface, and then the labeled substance is detected.

[0016] LC-MS is a device that combines a high-performance liquid chromatograph (HPLC) with any mass spectrometer (MS). Measurements using mass spectrometry by LC-MS can identify and quantify proteins. Examples of MS used in LC-MS include, but are not limited to, Orbitrap Fusion Lumos (Thermo Scientific).

[0017] Proteins may be identified by any method suitable for each protein measurement method, and are not particularly limited thereto, but for example, in the case of measurement using mass spectrometry by LC-MS, the product ion spectrum data obtained by the LC-MS analysis can be searched against a protein database, such as the public protein database SwissProt.

[0018] Protein quantification may be performed by a method suitable for each protein measurement method, and is not particularly limited. For example, in the case of measurement using LC-MS mass spectrometry, protein quantification may be calculated from the peak area of ​​precursor ion spectrum data obtained by LC-MS analysis. Protein identification and quantification may also be performed using software. Examples of such software include, but are not limited to, Proteome Discoverer 2.4 software (manufactured by Thermo Fischer Scientific).

[0019] Proteins may be purified from a sample used for protein measurement. Examples of protein purification methods include, but are not limited to, purification methods using polyacrylamide gel electrophoresis (SDS-PAGE), and can be appropriately selected depending on the sample used, the protein measurement method, and the like. The sample may be used for protein measurement without purifying the protein. Alternatively, the sample may be diluted as appropriate.

[0020] Large-scale analysis of proteins contained in extracellular secretory vesicles is called extracellular secretory vesicle proteomics. Extracellular secretory vesicle proteomics may be a comprehensive analysis of proteins present in extracellular secretory vesicles. One aspect of extracellular secretory vesicle proteomics is, for example, comprehensive analysis of the concentrations of protein molecular species contained in extracellular secretory vesicles by mass spectrometry using LC-MS.

[0021] Extracellular secretory vesicle proteomics can be performed, for example, by subjecting an extracellular secretory vesicle fraction of a sample to mass spectrometry by LC-MS and identifying and quantifying proteins from the analytical data. Data obtained by proteomics analysis and listing quantitative data for each identified protein is also referred to as a protein profile. Therefore, data obtained by extracellular secretory vesicle proteomics and listing quantitative data for each identified protein contained in extracellular secretory vesicles is also referred to as an extracellular secretory vesicle-containing protein profile.

[0022] According to extracellular secretory vesicle proteomics, the protein profile of extracellular secretory vesicles in normal samples differs from that of Alzheimer's disease samples. Therefore, the extracellular secretory vesicle-containing protein measured in the present invention is preferably an extracellular secretory vesicle-containing protein whose amount in Alzheimer's disease samples varies from that in normal samples.

[0023] When the amount of extracellular secretory vesicle-containing protein in a sample shows a certain level of fluctuation in measurements over a predetermined period, when it exceeds or exceeds the amount of extracellular secretory vesicle-containing protein in a normal sample, or when it exceeds or exceeds a set threshold, Alzheimer's disease or the risk of developing Alzheimer's disease is detected. Note that, as used herein, "risk of developing Alzheimer's disease" refers to a high probability of developing Alzheimer's disease in the near future, even though the disease has not yet developed at the time of measurement.

[0024] With respect to an extracellular secretory vesicle-containing protein that is present at a higher level in the extracellular secretory vesicle-containing protein profile of an Alzheimer's disease specimen compared to the extracellular secretory vesicle-containing protein profile of a normal specimen, it is desirable to detect Alzheimer's disease or the risk of developing Alzheimer's disease when the amount of the extracellular secretory vesicle-containing protein in the specimen shows an upward fluctuation of a certain amount or more in measurements taken over a predetermined period of time, when the level is higher than the amount of the extracellular secretory vesicle-containing protein in a normal specimen, or when it shows a set threshold or higher.

[0025] Specific examples of detection criteria may include, for example, a detection level of 1.05 times or more, 1.5 times or more, 2.0 times or more, 3.0 times or more, or 5.0 times or more of the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen. Preferably, a detection level of 2.0 times or more of the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen.

[0026] With respect to an extracellular secretory vesicle-containing protein that is at a lower level in the extracellular secretory vesicle-containing protein profile of an Alzheimer's disease specimen compared to the extracellular secretory vesicle-containing protein profile of a normal specimen, it is desirable to detect Alzheimer's disease or the risk of developing Alzheimer's disease when the amount of the extracellular secretory vesicle-containing protein in the specimen shows a downward fluctuation of more than a certain amount in measurements taken over a predetermined period of time, when it is at a lower level than the amount of the extracellular secretory vesicle-containing protein in a normal specimen, or when it shows a value below a set threshold.

[0027] Specific examples of detection criteria include, for example, showing an amount of protein that is 0.95 times or less, 0.75 times or less, 0.5 times or less, 0.3 times or less, or 0.2 times or less than the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen. Preferably, showing an amount of protein that is 0.5 times or less than the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen.

[0028] Alzheimer's disease is a type of dementia characterized by symptoms such as disorientation, delayed recall disorder, visual-spatial cognitive impairment, and executive dysfunction, and is diagnosed after the causative genes and causative proteins such as amyloid beta are identified.Alzheimer's disease is diagnosed based on clinical symptoms alone, without genetic or protein diagnosis, and is called Alzheimer's dementia.The prodromal state of Alzheimer's disease (Alzheimer's dementia) is amnestic mild cognitive impairment (MCI).

[0029] Alzheimer's disease is typically characterized by the accumulation of amyloid beta outside neurons, in the cerebral cortex, and in cerebral blood vessels, the accumulation of phosphorylated tau in neurons, and neurodegenerative pathology.More typically, Alzheimer's disease first shows the accumulation of amyloid beta, then the accumulation of phosphorylated tau occurs, and then progresses to neurofibrillary tangles, neurodegeneration, cerebral atrophy, and cognitive dysfunction.Each of these representative stages of Alzheimer's disease is called Alzheimer's disease pathological phase (AD pathological phase).

[0030] Among the AD pathological phases, the stage where amyloid beta accumulation is observed is also called the amyloid accumulation stage. The stage where phosphorylated tau accumulation is observed is also called the tau pathogenesis stage. The stage where neurodegeneration is observed is also called the neurodegeneration stage.

[0031] A conventional method for biologically stratifying AD pathology phases is, for example, the ATN classification method, which classifies samples based on the presence or absence of amyloid accumulation, tau pathology, and neurodegeneration, and classifies the phases based on the abnormal proteins that primarily fluctuate so that they can be detected in the living brain. That is, in the ATN classification method, AD pathology phases are stratified into the amyloid accumulation stage, the tau pathology formation stage, and the neurodegeneration stage.

[0032] In the ATN classification method, amyloid beta, phosphorylated tau, and total tau are measured in samples from living patients. Measurements of amyloid beta, phosphorylated tau, and total tau can be performed by known methods, such as ELISA. Based on the measured values, the patient is classified as amyloid beta (A), phosphorylated tau (T), or total tau (N). Classification may be based on a comparison of the measured values ​​for A, T, or N with a cutoff value, resulting in a positive (+) or negative (-) result. Specifically, for example, amyloid beta below the cutoff value is considered A+; phosphorylated tau greater than the cutoff value is considered T+; and total tau greater than the cutoff value is considered N+. Anything other than the above for A, T, or N may be considered negative (-). In the ATN classification, for example, A-T-N- is classified as normal, A+T-N- is classified as amyloid accumulation stage, and A+T+N+ is classified as tau pathogenesis / neurodegeneration stage. The cutoff value for ATN classification is not limited to this, but may be set, for example, by performing ROC analysis between the normal and MCI groups and the AD group based on clinical diagnosis.

[0033] Combining conventional methods for stratifying AD pathology phases with extracellular secretory vesicle proteomics can reveal the protein profiles of extracellular secretory vesicles in each AD pathology phase. For example, in samples classified by the ATN classification system, the protein profiles of extracellular secretory vesicles in A-T-N-, A+T-N-, or A+T+N+ samples reflect the protein profiles of extracellular secretory vesicles in the normal, amyloid accumulation, or tau pathogenesis / neurodegeneration stages, respectively. Samples from surviving patients corresponding to each AD pathology phase of Alzheimer's disease show different protein profiles in extracellular secretory vesicle proteomics.

[0034] Therefore, each AD pathology phase may be detected when the amount of extracellular secretory vesicle-containing protein in a sample shows a certain degree of variation in measurements over a predetermined period of time, exceeds above or below the amount of extracellular secretory vesicle-containing protein in a normal sample, or exceeds above or below a set threshold.

[0035] Amyloid accumulation stage marker proteins are proteins whose measured values ​​vary in the extracellular secretory vesicle-containing protein profile of a specimen in the amyloid accumulation stage of Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of a normal specimen. Preferably, the amyloid accumulation stage marker proteins are proteins whose measured values ​​vary by 2-fold or more, or 0.5-fold or less, in the extracellular secretory vesicle-containing protein profile of a specimen in the amyloid accumulation stage of Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of a normal specimen. More preferably, the amyloid accumulation stage marker proteins are ectonucleotide pyrophosphatase / phosphodiesterase family member 3, α-1,3-fucosyltransferase 10 (Alpha-(1,3)-fucosyltransferase 10), repulsive guidance molecule A, cAMP-dependent protein kinase type I-beta regulatory subunit, DNA replication ATP-dependent helicase / nuclease DNA2, contactin-6, histone H4, exostosin-1, BCL-6 corepressor-like protein 1 (BCL-6) corepressor-like protein 1), immunoglobulin mu heavy chain, Rho guanine nucleotide exchange factor 28, cathepsin B, putative keratin-87 protein, RNA-binding protein with multiple splicing, serpin B3, beta-galactoside α-2,6-sialyltransferase 1, and alpha-2,6-sialyltransferase 1, calcium-binding protein 39, cytoplasmic isocitrate dehydrogenase [NADP], melanoma-associated antigen B10, nodal modulator 1, nodal modulator 2, nodal modulator 3, phosphoribosyltransferase domain-containing protein 1 The protein is selected from the group consisting of domain-containing protein 1, free homolog-like protein, myosin-10, helicase senataxin, FAM227B protein, and serpin B4.

[0036] Even more preferably, the amyloid accumulation stage marker protein includes cathepsin B. Cathepsin B may be both an amyloid accumulation stage marker protein and a tau pathogenesis / neurodegeneration stage marker protein.

[0037] The amyloid accumulation stage may be detected when the amount of amyloid accumulation stage marker protein in a sample shows a certain level of fluctuation in measurements over a predetermined period of time, when the amount of amyloid accumulation stage marker protein exceeds or exceeds above or below the amount of amyloid accumulation stage marker protein in a normal sample, or when the amount exceeds or exceeds a set threshold value.

[0038] Specifically, with regard to an amyloid accumulation stage marker protein that is present at a higher level in the extracellular secretory vesicle-containing protein profile of a specimen at the amyloid accumulation stage of Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of a normal specimen, it is desirable to detect the amyloid accumulation stage of Alzheimer's disease, Alzheimer's disease, MCI, or the risk of developing Alzheimer's disease when the amount of the amyloid accumulation stage marker protein in the specimen shows an upward fluctuation of at least a certain amount in measurements taken over a predetermined period of time, when the level is higher than the amount of the amyloid accumulation stage marker protein in a normal specimen, or when the level is above a set threshold.

[0039] More specific examples of the detection criteria may include, for example, showing 1.05 times or more, 1.5 times or more, 2.0 times or more, 3.0 times or more, or 5.0 times or more of the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen. Preferably, showing 2.0 times or more of the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen.

[0040] As mentioned above, examples of amyloid accumulation stage marker proteins that are present at higher levels in the extracellular secretory vesicle-containing protein profile of a specimen in the amyloid accumulation stage of Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of a normal specimen include, but are not limited to, cathepsin B.

[0041] With regard to an amyloid accumulation stage marker protein that is present at a lower level in the extracellular secretory vesicle-containing protein profile of a specimen of amyloid accumulation stage Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of a normal specimen, it is desirable to detect the amyloid accumulation stage of Alzheimer's disease, Alzheimer's disease, MCI, or the risk of developing Alzheimer's disease when the amount of the amyloid accumulation stage marker protein in the specimen shows a downward fluctuation of more than a certain amount in measurements over a predetermined period of time, when the level is lower than the amount of the amyloid accumulation stage marker protein in a normal specimen, or when the level is below a set threshold.

[0042] More specific examples of the detection criteria may include, for example, showing an amount of protein that is 0.95 times or less, 0.75 times or less, 0.5 times or less, 0.3 times or less, or 0.2 times or less than the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen. Preferably, showing an amount of protein that is 0.5 times or less than the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen.

[0043] A protein whose measured value varies in an extracellular secretory vesicle-containing protein profile of a specimen at the tau pathogenesis / neurodegeneration stage of Alzheimer's disease compared to an extracellular secretory vesicle-containing protein profile of a normal specimen is defined as a tau pathogenesis / neurodegeneration stage marker protein. Preferably, the tau pathogenesis / neurodegeneration stage marker protein is a protein whose measured value varies by 2-fold or more, or 0.5-fold or less, in an extracellular secretory vesicle-containing protein profile of a specimen at the tau pathogenesis / neurodegeneration stage of Alzheimer's disease compared to an extracellular secretory vesicle-containing protein profile of a normal specimen. More preferably, the tau pathogenesis / neurodegeneration stage marker protein is cholinesterase, pericentrin, Protein unc-13 homolog C, 26S proteasome non-ATPase regulatory subunit 1, α-1,3-fucosyltransferase 10, neurexophilin-1, DNA-binding protein RFX5, pregnancy-specific beta-1-glycoprotein 8, or the like. 8), amyloid-beta A4 precursor protein-binding family B member 2, kallikrein-11, cadherin-11, β-galactoside α-2,6-sialyltransferase 1, sodium channel protein type 4 subunit alpha, FAM227B protein, and transmembrane protein 62.

[0044] Furthermore, the tau pathogenesis / neurodegeneration stage marker protein may include cathepsin B. In this case, the tau pathogenesis / neurodegeneration stage marker protein may be cathepsin B alone, or may be a combination of cathepsin B and another tau pathogenesis / neurodegeneration stage marker protein. Note that cathepsin B may be a tau pathogenesis / neurodegeneration stage marker protein and an amyloid accumulation stage marker protein at the same time.

[0045] Tau pathogenesis / neurodegeneration may be detected when the amount of tau pathogenesis / neurodegeneration marker protein in a sample shows a certain level of variation in measurements over a predetermined period of time, exceeds or exceeds above or below the amount of tau pathogenesis / neurodegeneration marker protein in a normal sample, or exceeds or exceeds a set threshold.

[0046] Specifically, with regard to a tau pathogenesis / neurodegeneration stage marker protein that is present at a higher level in the extracellular secretory vesicle-containing protein profile of a specimen at the tau pathogenesis / neurodegeneration stage of Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of a normal specimen, it is desirable to detect the tau pathogenesis / neurodegeneration stage of Alzheimer's disease or Alzheimer's disease when the amount of the tau pathogenesis / neurodegeneration stage marker protein in the specimen shows a certain amount of upward fluctuation in measurements over a predetermined period of time, when it is at a higher level than the amount of the tau pathogenesis / neurodegeneration stage marker protein in a normal specimen, or when it is above a set threshold.

[0047] Specific examples of detection criteria may include, for example, a detection level of 1.05 times or more, 1.5 times or more, 2.0 times or more, 3.0 times or more, or 5.0 times or more of the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen. Preferably, a detection level of 2.0 times or more of the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen.

[0048] As described above, examples of amyloid accumulation stage marker proteins that are present at higher levels in the extracellular secretory vesicle-containing protein profile of specimens at the tau pathogenesis / neurodegeneration stage of Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of normal specimens include, but are not limited to, cathepsin B.

[0049] With regard to a tau pathogenesis / neurodegeneration stage marker protein that is present at a lower level in the extracellular secretory vesicle-containing protein profile of a specimen in the tau pathogenesis / neurodegeneration stage of Alzheimer's disease compared to the extracellular secretory vesicle-containing protein profile of a normal specimen, it is desirable to detect the tau pathogenesis / neurodegeneration stage of Alzheimer's disease or Alzheimer's when the amount of the tau pathogenesis / neurodegeneration stage marker protein in the specimen shows a certain downward fluctuation in measurements over a predetermined period of time, when the level is lower than the amount of the tau pathogenesis / neurodegeneration stage marker protein in a normal specimen, or when the level is below a set threshold.

[0050] Specific examples of detection criteria include, for example, showing an amount of protein that is 0.95 times or less, 0.75 times or less, 0.5 times or less, 0.3 times or less, or 0.2 times or less than the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen. Preferably, showing an amount of protein that is 0.5 times or less than the amount of protein in the extracellular secretory vesicle-containing protein profile of a normal specimen.

[0051] The method according to the first aspect of the present invention includes the step of detecting Alzheimer's disease, but does not include the final determination of the diagnosis of Alzheimer's disease. A physician can diagnose Alzheimer's disease and determine a treatment plan by referring to the detection results obtained by the method of the present invention.

[0052] A second aspect of the present invention is a diagnostic kit for Alzheimer's disease, which comprises a reagent for measuring proteins contained in extracellular secretory vesicles.

[0053] The above description can be applied to the measurement of extracellular secretory vesicle-containing proteins using a reagent for measuring extracellular secretory vesicle-containing proteins. The reagent for measuring extracellular secretory vesicle-containing proteins is not particularly limited as long as it can detect and / or quantify extracellular secretory vesicle-containing proteins, but it is preferable that it can quantify extracellular secretory vesicle-containing proteins.

[0054] The kit may also include reagents, instruments, etc. used in the means for isolating a sample from a specimen. This means may include means for isolating cerebrospinal fluid or blood from a specimen, and may further include means for isolating plasma from blood. The kit may also include means for recovering an extracellular secretory vesicle fraction from a specimen.

[0055] The diagnostic kit of the present invention can be produced using a reagent for measuring proteins contained in extracellular secretory vesicles. That is, another aspect of the present invention includes use of a reagent for measuring proteins contained in extracellular secretory vesicles for the production of a diagnostic kit for Alzheimer's disease.

[0056] The reagent for measuring the protein contained in extracellular secretory vesicles can be as described above, and examples thereof include, but are not limited to, antibodies that recognize the protein contained in extracellular secretory vesicles. The reagent for measuring the protein contained in extracellular secretory vesicles preferably includes, but is not limited to, an antibody that recognizes an amyloid accumulation stage marker protein or a tau pathogenesis / neurodegeneration stage marker protein, and more preferably includes an antibody that recognizes cathepsin B.

[0057] A third aspect of the present invention is a method for treating Alzheimer's disease in a patient, comprising: (i) identifying the patient as having a marker whose measured value exceeds a predetermined reference value; and (ii) administering treatment to the identified patient. In the identification step (i), the marker may be measured using an antibody that specifically recognizes the marker, or may be measured using mass spectrometry. Exceeding the reference value refers to exceeding the reference value by a value above or below the reference value, and the reference value is preferably set depending on the marker.

[0058] The marker is preferably an extracellular secretory vesicle-containing protein, more preferably an amyloid accumulation stage marker protein or a tau pathogenesis / neurodegeneration stage marker protein, and even more preferably cathepsin B.

[0059] The method for detecting Alzheimer's disease of the present invention can be applied to the method for treating Alzheimer's disease of the present invention. That is, the first aspect of the present invention described above can provide a method according to the third aspect of the present invention. Specifically, for example, in the identification step (i), a patient may be identified by detecting Alzheimer's disease using the method according to the first aspect of the present invention, or by detecting the amyloid accumulation stage of Alzheimer's disease using the method according to the first aspect of the present invention, or by detecting the tau pathogenesis stage and / or neurodegeneration stage of Alzheimer's disease using the method according to the first aspect of the present invention.

[0060] The treatment is not particularly limited and may be appropriately selected at the discretion of the physician.

[0061] Example 1: Cerebrospinal fluid extracellular secretory vesicle proteomics

[0062] Breakdown of subjects The following subject samples (n=16) were used for cerebrospinal fluid extracellular secretory vesicle proteomics (Table 1).

[0063]

[0064] Cerebrospinal fluid collection Patients eligible for cerebrospinal fluid testing had to meet the following conditions: - Between the ages of 40 and 80, not taking antiplatelet or anticoagulant drugs - The patient had eaten dinner by 9 PM the night before collection, had slept for at least six hours without consuming alcohol - The patient was allowed to drink water, but had no tea or coffee, had no breakfast, and could have cerebrospinal fluid collected on an empty stomach between 9:00 AM and 10:00 AM

[0065] Cerebrospinal fluid collection method: With the patient in the left lateral position, a lumbar puncture is performed at the 3rd and 4th lumbar intervertebral space or the 4th and 5th lumbar intervertebral space. The spinal needle used for the puncture is a 22G top spinal needle with a stainless steel needle tube and a polypropylene needle hub. The usual polystyrene spinal needle, pressure rod, three-way stopcock, and extension tube are not used; the cerebrospinal fluid sample is collected directly from the spinal needle hub.

[0066] First, approximately 500 μl of cerebrospinal fluid is directly dripped into a 15 cc sterile polypropylene dispenser. After that, the container is changed and 500 μl of cerebrospinal fluid is dripped directly from the needle hub into a polypropylene microtube (1.5 ml). After that, drip the fluid directly into the same 15 cc sterile dispenser again, and then drip 500 μl of cerebrospinal fluid into another 1.5 ml polypropylene microtube, and this process is repeated. Continue until one 15 cc sterile dispenser (approximately 4-5 cc) and a total of 10-12 microtubes (500 μl each) have been collected. If macroscopic bleeding is observed during the process, the procedure is discontinued. The 500 μl of cerebrospinal fluid in each microtube is frozen and stored at -80°C within one hour of the start of collection (specimen for extracellular secretory vesicle analysis).

[0067] The cerebrospinal fluid samples collected in the polypropylene microtubes were centrifuged at 3,500 rpm (2,200 x g) for 7 minutes, and the supernatant was dispensed into 500 μl aliquots into polypropylene microtubes (1.5 ml) using polypropylene dispensing tips, and immediately frozen and stored at -80°C (Aβ40, Aβ42, pTau, tTau ELISA samples). All of the above steps were performed by the same surgeon at the same time using instruments made of the same material.

[0068] ATN Classification: Aβ (Aβ1-42), phosphorylated tau (p-Tau181), and total tau (t-Tau) were measured by ELISA in cerebrospinal fluid samples collected from subjects. Based on the measured values, subjects were classified into ATN classifications of Aβ (A), phosphorylated tau (T), and neurodegeneration (N). Cutoff values ​​were determined based on the clinical diagnosis of all subjects, using ROC analysis between normal and MCI vs. AD groups. In this example, the cutoff values ​​were Aβ: 680 pg / mL, phosphorylated tau: 76.5 pg / mL, and total tau: 598 pg / mL. Note that even when recalculated using the cutoff values ​​set in Example 2 below, Aβ: 747.2 pg / mL, phosphorylated tau: 58.4 pg / mL, and total tau: 756.6 pg / mL, the ATN classification of the subjects in this example remained unchanged from that listed in Table 1.

[0069] Preparation of Extracellular Secretory Vesicle Fraction An extracellular secretory vesicle fraction was prepared from 0.5 mL of cerebrospinal fluid using an extracellular secretory vesicle purification column (EV-Second L70, GL Sciences).

[0070] Proteomics analysis (LC-MS) Comprehensive identification and quantification of proteins in the extracellular secretory vesicle fraction was carried out by LC-MS analysis. Specifically, this was carried out as follows.

[0071] (1) Sample preparation: The extracellular secretory vesicle fraction was dissolved in 30 μl of Laemmli's sample buffer, reduced with 10 mM TCEP at 100°C for 10 minutes, and then alkylated with 50 mM iodoacetamide. SDS-PAGE was performed, and the protein bands were excised from the gel, digested with trypsin / Lys-C-Mix, and then subjected to LC-MS analysis.

[0072] (2) LC-MS Analysis Analysis was performed using an Orbitrap Fusion Lumos mass spectrometer (ThermoScientific) equipped with an Ultimate 3000 RSLC nano-flow HPLC system (DIONEX, USA). Proteins were identified by searching the SwissProt human protein database using Proteome Discoverer 2.4 software (Thermo Scientific).

[0073] (3) Label-Free Quantitative Analysis The LC-MS data set was subjected to label-free quantitative analysis using Proteome Discoverer 2.4 software (Thermo Scientific) to perform relative quantification of all detected proteins.

[0074] (4) Results Quantitative data for 1,758 proteins were obtained from the cerebrospinal fluid extracellular secretory vesicle fraction. Proteins whose average amount in the extracellular secretory vesicle fraction from Group 3 (A-T-N- group) was 2-fold or more or 0.5-fold or less than the average amount in the extracellular secretory vesicle fraction from Group 2 (A+T-N- group), and proteins whose expression was confirmed only in either Group 1 or Group 2, were determined to have a certain degree of effectiveness as biomarkers for detecting the amyloid accumulation stage, and the following molecular species were selected (Table 2). Ectonucleotide pyrophosphatase / phosphodiesterase family member 3, alpha-(1,3)-fucosyltransferase 10, repulsive guidance molecule A, cAMP-dependent protein kinase type I-beta regulatory subunit, DNA replication ATP-dependent helicase / nuclease DNA2 ATP-dependent helicase / nuclease DNA2), contactin-6, histone H4, exostosin-1, BCL-6 corepressor-like protein 1, immunoglobulin mu heavy chain, Rho guanine nucleotide exchange factor 28, cathepsin B, putative keratin-87 protein protein), RNA-binding protein with multiple splicing, Serpin B3, β-galactoside α-2,Beta-galactosid alpha-2,6-sialyltransferase 1, calcium-binding protein 39, isocitrate dehydrogenase [NADP] cytoplasmic, melanoma-associated antigen B10, nodal modulator 1, nodal modulator 2, nodal modulator 3 3), phosphoribosyltransferase domain-containing protein 1, protein furry homolog-like protein, myosin-10, helicase senataxin, FAM227B protein, and serpin B4. These molecular species may also be potential targets for therapeutic and preventive drugs.

[0075]

[0076] Furthermore, proteins whose average amount in the extracellular secretory vesicle fraction of Group 2 (A-T-N- group) was more than 2 times or 0.5 times lower than the average amount in the extracellular secretory vesicle fraction of Group 1 (A+T+N+ group), and proteins whose expression was confirmed only in either Group 1 or Group 2, were judged to have a certain degree of effectiveness as biomarkers for detecting tau pathogenesis and the neurodegenerative stage, and the following molecular species were selected (Table 3). Cholinesterase, Pericentrin, Protein unc-13 homolog C, 26S proteasome non-ATPase regulatory subunit 1, α-1,3-fucosyltransferase 10, Neuroxophilin-1, DNA-binding protein RFX5, pregnancy-specific beta-1-glycoprotein 8 8), amyloid-beta A4 precursor protein-binding family B member 2, kallikrein-11, cadherin-11, β-galactoside α-2,6-sialyltransferase 1, sodium channel protein type 4 subunit alpha, FAM227B protein, and transmembrane protein 62. These molecular species may also be potential targets for therapeutic and preventive drugs.

[0077]

[0078] Example 2: Measurement of Cathepsin B

[0079] Of the molecular species selected as the amyloid accumulation stage markers, the concentrations of cathepsin B in the extracellular secretory vesicle fractions of the cerebrospinal fluid and plasma samples were measured by ELISA.

[0080] Breakdown of subjects: The following subject samples were used for cathepsin B measurement (Table 4). Cerebrospinal fluid and plasma samples were collected from each subject. All subjects were also classified into normal, MCI, or AD groups based on their clinical diagnosis (data not shown). Based on the values ​​of Aβ, phosphorylated tau, and total tau measured by ELISA using the cerebrospinal fluid samples for Aβ1-42 / p-Tau181 / t-Tau ELISA measurement described below from each subject's cerebrospinal fluid sample, ROC analysis was performed between the normal and MCI vs. AD groups based on the clinical diagnosis of all subjects to set cutoff values ​​(Aβ: 747.2 pg / mL, phosphorylated tau: 58.4 pg / mL, total tau: 756.6 pg / mL) for ATN classification.

[0081]

[0082] Cerebrospinal fluid collection and collection method: Subjects for lumbar puncture were 40-80 years old, not taking antiplatelet or anticoagulant drugs, and without hematological disorders. Participants had eaten dinner by 9:00 PM the night before, had not consumed alcohol, and had slept for at least six hours. After fasting overnight, lumbar puncture was performed between 9:00 AM and 10:00 AM. Cerebrospinal fluid was collected using a 22G top-spinal needle with a steel needle and a polypropylene base. Cerebrospinal fluid samples for Aβ1-42 / p-Tau181 / t-Tau ELISA measurement were then collected using step 1 below, and cerebrospinal fluid samples for EV analysis were collected using step 2 below. All samples were collected at the same time by a single experienced physician using the same method and equipment. 1. CSF samples for Aβ1-42 / p-Tau181 / t-Tau ELISA assay: CSF samples dispensed from the spinal needle during the collection process were collected in a sterile 15cc polypropylene tube. The samples were then centrifuged at 3500 RPM (2200g) for 7 minutes at room temperature to precipitate cells and other insoluble material. The supernatant was then dispensed in 500µl aliquots into 1.5ml polypropylene microtubes using polypropylene dispenser tips for ELISA assays. The samples were frozen at -80°C within 1 hour of collection and stored until further analysis. Only one freeze-thaw cycle was performed. 2. CSF samples for EV analysis: CSF samples dispensed from the spinal needle during the collection process were dispensed in 500µl aliquots into 1.5ml polypropylene microtubes. The samples were frozen at -80°C within 1 hour of collection and stored until further analysis. Only one freeze-thaw cycle was performed.

[0083] After collecting cerebrospinal fluid using the method described above, whole blood was collected from the left upper arm of the same patient into a vacuum tube containing EDTA2Na. Plasma was then collected by centrifugation at 2200 g and 4°C for 15 minutes and dispensed into sterile 15cc polypropylene tubes in approximately 0.5 ml aliquots. All samples were frozen at -80°C within 1 hour of collection and stored until further analysis. Only one freeze-thaw cycle was performed.

[0084] Extracellular secretory vesicle fractions were prepared from 0.1 mL of cerebrospinal fluid samples or 0.4 mL of plasma samples for EV analysis. The extracellular secretory vesicle fractions were prepared by ultracentrifugation. The ultracentrifugation consisted of three cycles: 10 min at 2,000 g, 30 min at 10,000 g, and 70 min at 100,000 g.

[0085] Cathepsin B ELISA Measurement The collected extracellular secretory vesicle fraction was dissolved in 0.1% SDS / PBS, and the concentration of cathepsin B was measured using a Human Cathepsin B ELISA Kit (Abcam: #ab119584) according to the recommended protocol.

[0086] Results: The results of ELISA assays of cathepsin B in the extracellular vesicle fraction of CSF samples are shown in Figures 1 and 2. The results of each ATN classification and cathepsin B concentration shown in Figure 1 indicate that cathepsin B concentrations were significantly elevated in the A+T-N- group compared to the A-T-N- group. This is consistent with the results of the CSF extracellular vesicle proteomics analysis described above, confirming the effectiveness of cathepsin B as a biomarker for detecting the amyloid accumulation stage. Furthermore, cathepsin B concentrations were significantly elevated in the A+T+N+, A+T-N+, and A+T-N- groups, respectively, compared with the A-T+N+, A-T-N+, and A-T-N- groups. This suggests that cathepsin B may also be useful as a marker for detecting the tau pathogenesis / neurodegeneration stage.

[0087] Furthermore, focusing on amyloid pathology, each sample was classified into two groups, A+ group (A+T+N+ group, A+T-N+ group, and A+T-N- group) and A- group (A-T+N+ group, A-T-N+ group, and A-T-N- group), and the results (Figure 2A) confirmed that the concentration of cathepsin B was significantly elevated in the A+ group. It was also confirmed that the concentration of Aβ42 in the cerebrospinal fluid, which is an indicator of amyloid pathology, is inversely correlated with the concentration of cathepsin B in the extracellular secretory vesicle fraction of the cerebrospinal fluid sample (Figure 2B). These findings further confirm the potential of cathepsin B as a biomarker for detecting the amyloid accumulation stage.

[0088] The results of ELISA measurement of cathepsin B in the extracellular secretory vesicle fraction of plasma samples are shown in Figure 3. From the results shown in Figure 3A, it was confirmed that, in plasma samples as well as in cerebrospinal fluid samples, the concentration of cathepsin B was significantly elevated in the A+ group compared to the A- group. Furthermore, as with the results of cerebrospinal fluid samples, it was confirmed that the concentration of cathepsin B in the extracellular secretory vesicle fraction of plasma samples also showed an inverse correlation with cerebrospinal fluid Aβ42 concentration (Figure 3B). From these results, it can be said that cathepsin B is effective as a biomarker for detecting the amyloid accumulation stage, even when plasma is used as a sample.

[0089] From the above, it has become clear that cathepsin B contained in the extracellular secretory vesicle fraction is a powerful cerebrospinal fluid / blood biomarker for Alzheimer's disease, which can also be used to evaluate the preclinical / early stage of Alzheimer's disease, including the amyloid accumulation stage.

Claims

1. 1. A method for detecting Alzheimer's disease, comprising: measuring the amount of extracellular secretory vesicle-containing protein in a sample taken from the subject; The method, wherein the extracellular secretory vesicle-containing protein comprises cathepsin B.

2. The method of claim 1, wherein the extracellular secretory vesicle-containing protein is an extracellular secretory vesicle-containing protein whose amount in an Alzheimer's disease specimen varies from the amount of the extracellular secretory vesicle-containing protein in a normal specimen.

3. The method of claim 2, wherein the variation is greater than or equal to 2-fold or less than or equal to 0.5-fold.

4. The method of claim 1, comprising the steps of recovering an extracellular secretory vesicle fraction from a sample collected from a subject and measuring proteins contained in the extracellular secretory vesicle fraction.

5. The extracellular secretory vesicles containing proteins include cathepsin B, ectonucleotide pyrophosphatase / phosphodiesterase family member 3, α-1,3-fucosyltransferase 10, repulsive guidance molecule A, cAMP-dependent protein kinase type I-β regulatory subunit, DNA replication ATP-dependent helicase / nuclease DNA2, contactin 6, histone H4, exostosin 1, BCL6 corepressor-like protein 1, immunoglobulin μ heavy chain, Rho guanine nucleotide exchange factor 28, keratin 87 putative protein, multi-splicing RNA-binding protein, serpin B3, β-galactoside α-2,6-sialyltransferase 1, calcium-binding protein 39, cytoplasmic isocitrate dehydrogenase [NADP], and melanoma binding protein.

2. The method of claim 1, wherein the antibody is one or more proteins selected from the group consisting of antibody B10, nodal modulator 1, nodal modulator 2, nodal modulator 3, phosphoribosyltransferase domain-containing protein 1, free homolog-like protein, myosin 10, helicase senataxin, FAM227B protein, serpin B4, cholinesterase, pericentrin, Unc13 homolog C protein, 26S proteosome non-ATPase regulatory subunit 1, neurexophilin 1, DNA binding protein RFX5, pregnancy-specific beta 1 glycoprotein 8, amyloid beta A4 precursor protein binding family B member 2, kallikrein 11, cadherin 11, sodium channel protein type 4 subunit alpha, and transmembrane protein 62.

6. 1. A method for detecting the amyloid accumulation stage of Alzheimer's disease, comprising: measuring extracellular secretory vesicle-containing proteins in a sample collected from the subject; The method, wherein the extracellular secretory vesicle-containing protein comprises cathepsin B.

7. The extracellular secretory vesicle-containing proteins include cathepsin B, ectonucleotide pyrophosphatase / phosphodiesterase family member 3, α-1,3-fucosyltransferase 10, repulsive guidance molecule A, cAMP-dependent protein kinase type I-β regulatory subunit, DNA replication ATP-dependent helicase / nuclease DNA2, contactin 6, histone H4, exostosin 1, BCL6 corepressor-like protein 1, immunoglobulin μ heavy chain, Rho guanine nucleotide exchange factor 28, keratin 87 putative protein, and multi-splicing R and one or more proteins selected from the group consisting of NA-binding protein, serpin B3, β-galactoside α-2,6-sialyltransferase 1, calcium-binding protein 39, cytoplasmic isocitrate dehydrogenase [NADP], melanoma-binding antibody B10, nodal modifier 1, nodal modifier 2, nodal modifier 3, phosphoribosyltransferase domain-containing protein 1, free homolog-like protein, myosin 10, helicase senataxin, FAM227B protein, and serpin B4.

8. 1. A method for detecting tau pathogenesis and / or neurodegeneration in Alzheimer's disease, comprising: measuring extracellular secretory vesicle-containing proteins in a sample collected from the subject; The method, wherein the extracellular secretory vesicle-containing protein comprises cathepsin B.

9. 9. The method of claim 8, wherein the extracellular secretory vesicle-containing protein is cathepsin B and one or more proteins selected from the group consisting of cholinesterase, pericentrin, Unc13 homolog C protein, 26S proteosomal non-ATPase regulatory subunit 1, α-1,3-fucosyltransferase 10, neurexophilin 1, DNA-binding protein RFX5, pregnancy-specific β1-glycoprotein 8, amyloid βA4 precursor protein-binding family B member 2, kallikrein 11, cadherin 11, β-galactoside α-2,6-sialyltransferase 1, sodium channel protein type 4 subunit α, FAM227B protein, and transmembrane protein 62.

10. The method according to any one of claims 1 to 9, wherein the sample is cerebrospinal fluid or plasma.

11. A diagnostic kit for Alzheimer's disease, comprising a reagent for measuring cathepsin B.

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