Method and reagent for detecting alzheimer's disease

Abstract

To provide a method of easily detecting Alzheimer's disease (AD), and a method of easily stratifying AD pathological phases in a biological brain before AD outbreak.SOLUTION: The above challenge is cleared by providing a method of detecting Alzheimer's disease, comprising a step of measuring the level of protocadherin-1 (PCDH1) protein contained in extracellular vesicles in a sample collected from a specimen.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to a method and a detection reagent for Alzheimer's disease, in which extracellular secretory vesicle-containing protocadherin-1 (PCDH1) protein is measured. [Background technology]

[0002] Alzheimer's disease (AD) is the most common form of dementia. The pathogenesis of AD begins before the onset of the disease, and progresses stepwise from the accumulation of amyloid beta protein (Aβ) in the early pathology to neurofibrillary changes in tau pathology, to neurodegeneration, and finally to 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, but there are many problems, such as the fact that cerebrospinal fluid Aβ measurement is not covered by insurance and there are only a few facilities that perform expensive PET tests. Therefore, there is a need to develop biomarkers to 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 protocadherin-1 (PCDH1) gene is known to belong to the protocadherin subfamily of the cadherin superfamily, and is known to express a membrane protein that typically contains an extracellular region containing seven cadherin-like domains, a transmembrane region, and a C-terminal cytoplasmic region. However, the association of PCDH1 with Alzheimer's disease is unknown. Summary of the Invention [Problem to be solved by the invention]

[0004] 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, and also aims to provide a reagent that can be used in the method. [Means for solving the problem]

[0005] The present inventors have conducted extensive research and found that the amount of protocadherin-1 (PCDH1) protein in extracellular secretory vesicles changes between AD molecular pathology phases using abnormal proteins (amyloid beta, tau) as indicators, and between healthy subjects, amnestic mild cognitive impairment (MCI) patients, and Alzheimer's dementia patients. Based on this, the inventors have come to the conclusion that measurement of PCDH1 protein contained in extracellular secretory vesicles can detect Alzheimer's disease and stratify AD pathology phases, and have completed the present invention.

[0006] That is, the present invention includes the following aspects. [1] 1. A method for detecting Alzheimer's disease, comprising: A method comprising the step of measuring the amount of extracellular secretory vesicle-containing protocadherin-1 (PCDH1) protein in a sample taken from a subject. [2] 1. A method for detecting the amyloid accumulation, tau pathology, and / or neurodegeneration stages of Alzheimer's disease, comprising: A method comprising the step of measuring extracellular secretory vesicle-containing PCDH1 protein in a sample taken from a subject. [3] Methods [1] or [2] that do not detect amnestic mild cognitive impairment (MCI). [4] Any of the methods [1] to [3], comprising the steps of recovering an extracellular secretory vesicle fraction from a sample collected from a specimen, and measuring PCDH1 protein in the extracellular secretory vesicle fraction. [5] The method according to any one of [1] to [4], wherein the sample is cerebrospinal fluid. [6] A diagnostic kit for Alzheimer's disease, comprising a reagent for measuring extracellular secretory vesicle-containing PCDH1 protein. Effect of the Invention

[0007] The present invention provides a method for conveniently detecting Alzheimer's disease (AD), in particular a method for conveniently distinguishing between normal or MCI and AD, and a method for conveniently stratifying AD pathological phases, as well as reagents that can be used for these methods. [Brief description of the drawings]

[0008] [Figure 1] Graph showing the amount of extracellular secretory vesicle-containing PCDH1 in CSF in subjects with or without amyloid pathology (A+ or A- group subjects). [Diagram 2] Graph showing the amount of PCDH1 contained in extracellular secretory vesicles in cerebrospinal fluid in phosphorylated tau classification (subjects in the T+ or T- group). [Diagram 3] Graph showing the amount of PCDH1 contained in extracellular secretory vesicles in cerebrospinal fluid in total tau classification (subjects in N+ or N- groups). [Figure 4] Graph showing the amount of PCDH1 contained in extracellular secretory vesicles in cerebrospinal fluid in subjects with each ATN classification. *: p<0.05, **: p<0.01, ***: p<0.001. [Diagram 5] A graph showing the correlation between cerebrospinal fluid amyloid β1-42 (Aβ42) and the amount of PCDH1 contained in extracellular secretory vesicles in cerebrospinal fluid in each subject. r indicates the correlation coefficient. [Figure 6] Graph showing the amount of PCDH1 contained in extracellular secretory vesicles in cerebrospinal fluid in normal, amnestic mild cognitive impairment (MCI), or Alzheimer's disease (AD) subjects. **: p<0.01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] A first aspect of the present invention is a method for detecting Alzheimer's disease, comprising the steps of: The method includes measuring the amount of extracellular secretory vesicle-containing protocadherin-1 (PCDH1) protein in a sample taken from a subject. The specimen 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 according to the present invention is usually carried out in vitro.

[0010] The sample includes body fluids, cells, tissues, etc. collected from a specimen. Body fluids include cerebrospinal fluid, blood, lymphatic fluid, tissue fluid, etc., and blood includes plasma and blood cell components. Preferably, the sample is cerebrospinal fluid or plasma, and more preferably, cerebrospinal fluid.

[0011] The collection of the sample from the specimen may be performed by any suitable 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 performing lumbar puncture and directly dropping the sample, and plasma can be collected from the collected blood by centrifugation, but is not limited thereto. In addition, the collected sample may be centrifuged or frozen and stored as necessary.

[0012] 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 PCDH1 protein contained in extracellular secretory vesicles may be a PCDH1 protein contained inside the extracellular secretory vesicles, a PCDH1 protein present on the surface of the extracellular secretory vesicles, or a PCDH1 protein penetrating the membrane of the extracellular secretory vesicles, and is not particularly limited as long as it is a PCDH1 protein recovered together with the extracellular secretory vesicles. The PCDH1 protein contained in the extracellular secretory vesicles is preferably a PCDH1 protein contained in the extracellular secretory vesicle fraction.

[0013] The PCDH1 protein refers to a protein expressed from the protocadherin 1 (PCDH1) gene. The PCDH1 gene is known to belong to the protocadherin subfamily within the cadherin superfamily. The PCDH1 gene includes, but is not limited to, the human PCDH1 gene, and may be a corresponding homologue depending on the species to which the specimen belongs. For example, it is known that there are splicing variants in the human PCDH1 gene. Therefore, the PCDH1 protein may refer to all proteins of splicing variants among the proteins expressed from the PCDH1 gene, or may refer to a specific splicing variant. For example, the PCDH1 protein is known to include seven cadherin-like domains, a transmembrane region, and a C-terminal cytoplasmic region, and it is preferable that the protein expressed from the PCDH1 gene has one or more of these characteristics, and more preferably has all three of these characteristics.

[0014] The measurement of the extracellular secretory vesicle-containing protein may be performed in the whole sample or in the extracellular secretory vesicle fraction of the sample. Thus, the present invention may include a step of recovering the extracellular secretory vesicle fraction in the sample taken from the specimen.

[0015] An extracellular secretory vesicle fraction is a fraction that contains 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 a method for capturing lipids that are specifically present on the membrane surface of extracellular secretory vesicles. The extracellular secretory vesicle fraction can be collected by ultracentrifugation, column purification, or by using a commercially available product, such as, but not limited to, a size exclusion chromatography column, EV-Second L70 (GL Sciences).

[0016] A known protein measurement method can be used to measure the PCDH1 protein contained in extracellular secretory vesicles. Preferably, a measurement using mass spectrometry with a liquid chromatograph mass spectrometer (LC-MS) or an immunoassay using an antibody against the PCDH1 protein can be used. Examples of the immunoassay include a method of quantitative detection according to an enzyme immunoassay, a measurement method using a fluorescent immunoassay, a chemiluminescent immunoassay, etc. An enzyme immunoassay is a detection method among labeled immunoassay methods that uses an enzyme as a labeling substance. Another example of the enzyme immunoassay is an ELISA method.

[0017] Examples of the ELISA method include a direct method, an indirect method, and a sandwich method, and any of these methods can be used. The direct method in the 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 a labeled substance. The indirect method in the 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 a labeled substance. The sandwich method in the ELISA method is 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 a labeled substance is detected. It may be.

[0018] 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).

[0019] Proteins may be identified by a method suitable for each protein measurement method, and are not particularly limited. 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. An example of the protein database is the public protein database SwissProt. The protein identification preferably includes the identification of PCDH1 protein.

[0020] 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 mass spectrometry by LC-MS, the amount may be calculated from the peak area of ​​precursor ion spectrum data obtained by LC-MS analysis. Protein identification and quantification may be performed using software. Examples of such software include, but are not limited to, Proteome Discoverer 2.4 software (manufactured by Thermo Fischer Scientific). Protein quantification preferably includes quantification of PCDH1 protein.

[0021] Proteins may be purified from a sample used for protein measurement. The protein purification method may be, for example, a purification method using polyacrylamide electrophoresis (SDS-PAGE), but is not limited thereto, and may be appropriately selected depending on the sample used, the protein measurement method, etc. It is preferable that the purified protein contains or may contain PCDH1 protein. Incidentally, "may contain PCDH1 protein" may refer to the fact that, regardless of whether or not the protein actually contains PCDH1 protein, for example, if the protein before purification contains PCDH1 protein, the protein is purified under conditions such that the protein contains PCDH1 protein even after purification. The protein may not be purified, and the sample may be used for protein measurement. The sample may also be appropriately diluted.

[0022] When the amount of PCDH1 protein contained in extracellular secretory vesicles of a specimen shows a certain or greater fluctuation in the measured value over a predetermined period, when the amount of PCDH1 protein contained in extracellular secretory vesicles of a normal specimen is lower, or when the amount is lower than a set threshold, Alzheimer's disease or the risk of developing Alzheimer's disease is detected. In this specification, the "risk of developing Alzheimer's disease" refers to the fact that the disease has not developed at the time of measurement, but there is a high probability that the disease will develop in the near future.

[0023] Specifically, for example, it is desirable to detect Alzheimer's disease or the risk of developing Alzheimer's disease when the amount of PCDH1 protein contained in extracellular secretory vesicles in a sample shows a downward fluctuation of more than a certain amount in measurements taken over a specified period of time, is at a lower level than the amount of PCDH1 protein contained in extracellular secretory vesicles in a normal sample, or shows a value below a set threshold.

[0024] More specific examples of the detection criteria include that the amount of PCDH1 protein contained in extracellular secretory vesicles in a specimen is 0.95 times or less, 0.75 times or less, 0.65 times or less, 0.5 times or less, or 0.4 times or less of the amount of PCDH1 protein contained in extracellular secretory vesicles in a normal specimen. It may be 0.3-fold or less, it may be 0.2-fold or less, and preferably it is 0.65-fold or less than the amount of PCDH1 protein contained in extracellular secretory vesicles in a normal specimen.

[0025] Alzheimer's disease is a type of dementia with symptoms such as disorientation, delayed recall disorder, visual-spatial cognitive dysfunction, and executive dysfunction. It is diagnosed when the causative gene or causative protein such as amyloid beta is identified, while Alzheimer's dementia is when the disease is diagnosed based only on clinical symptoms without gene or protein diagnosis. The precursor state of Alzheimer's disease (Alzheimer's dementia) is amnestic mild cognitive impairment (MCI).

[0026] In MCI, the amount of PCDH1 protein contained in extracellular secretory vesicles is not considered to fluctuate more than a certain amount in the measurement value for a certain period, not to exceed the amount of PCDH1 protein contained in extracellular secretory vesicles in normal specimens, or not to exceed a set threshold.Therefore, the method of the present invention may not detect MCI.In other words, the method of the present invention may be a method that detects Alzheimer's disease but does not detect MCI.

[0027] The method of the present invention may include distinguishing between Alzheimer's disease and MCI. The distinction between Alzheimer's disease and MCI may be performed by detecting Alzheimer's disease or a risk of developing Alzheimer's disease, for example, when the amount of PCDH1 protein contained in extracellular secretory vesicles in a sample shows a certain or greater fluctuation in the measured value over a predetermined period, when the amount of PCDH1 protein contained in extracellular secretory vesicles in a sample with MCI exceeds a lower limit, or when the amount exceeds a set threshold.

[0028] Specifically, for example, Alzheimer's disease or the risk of developing Alzheimer's disease may be detected when the amount of extracellular secretory vesicle-containing PCDH1 protein in a sample shows a downward fluctuation of more than a certain amount in measurements taken over a specified period of time, is at a lower level than the amount of extracellular secretory vesicle-containing PCDH1 protein in an MCI sample, or shows a value below a set threshold.

[0029] As a more specific example of the detection criteria, for example, the amount of PCDH1 protein contained in extracellular secretory vesicles in a sample may be 0.95 times or less, 0.75 times or less, 0.65 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, or 0.2 times or less, and preferably, 0.65 times or less of the amount of PCDH1 protein contained in extracellular secretory vesicles in a sample with MCI.

[0030] In the methods of the present invention, detecting Alzheimer's disease may mean distinguishing between Alzheimer's disease and normal, or between Alzheimer's disease and normal and MCI, or between Alzheimer's disease and MCI.

[0031] Alzheimer's disease is typically characterized by the accumulation of amyloid-β extraneuronally, in the cerebral cortex, and in cerebrovascular tissue, the accumulation of phosphorylated tau intraneuronally, and neurodegenerative pathology. More typically, Alzheimer's disease begins with the accumulation of amyloid beta, followed by the accumulation of phosphorylated tau, which progresses to neurofibrillary tangles, neurodegeneration, cerebral atrophy, and cognitive impairment. Each stage of Alzheimer's disease, as represented by these, is called an Alzheimer's disease pathology phase (AD pathology phase).

[0032] 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 pathology formation stage. The stage where degeneration occurs is also called the neurodegenerative stage.

[0033] Conventional methods for biological stratification of AD pathology phases include, for example, the ATN classification method, which classifies samples according to the presence or absence of amyloid accumulation, tau pathology, and neurodegeneration, and classifies the phases according to abnormal proteins that mainly fluctuate so that they can be captured 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.

[0034] In the ATN classification method, amyloid beta, phosphorylated tau and total tau are measured in a living patient's sample. Amyloid beta, phosphorylated tau and total tau can be measured by known methods such as ELISA. Based on the measured value, the sample is classified into amyloid beta (A), phosphorylated tau (T) and total tau (N). The classification may be based on, for example, positive (+) or negative (-) obtained by comparing the measured value of A, T or N with a cutoff value. Specifically, for example, when amyloid beta is less than the cutoff value, it is determined as A+, when phosphorylated tau is greater than the cutoff value, it is determined as T+, when total tau is greater than the cutoff value, it is determined as N+, and when A, T and N are not as above, it is determined as negative (-). In the ATN classification, for example, ATN- is classified as normal, A+TN- 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.

[0035] In the present invention, it was found that the amount of PCDH1 protein contained in extracellular secretory vesicles was decreased in samples from the amyloid β-positive (A+) group, regardless of whether phosphorylated tau and total tau were positive or negative, respectively. Thus, a second aspect of the present invention provides a method for detecting the amyloid accumulation and / or tau pathogenesis / neurodegeneration stages of Alzheimer's disease, comprising the steps of: The method may include a step of measuring extracellular secretory vesicle-containing PCDH1 protein in a sample taken from a subject. The above-mentioned description can be used as a method for measuring the PCDH1 protein contained in extracellular secretory vesicles in a sample collected from a subject.

[0036] When the amount of PCDH1 protein contained in extracellular secretory vesicles in a sample shows a certain degree of variation in measurements over a specified period of time, when it exceeds the amount of PCDH1 protein contained in extracellular secretory vesicles in a normal sample, or when it exceeds a set threshold, the amyloid accumulation stage and / or the tau pathogenesis / neurodegeneration stage may be detected, preferably the amyloid accumulation stage and the tau pathogenesis / neurodegeneration stage are detected.

[0037] Specifically, for example, it is desirable to detect the amyloid accumulation stage and / or tau pathogenesis / neurodegeneration stage of Alzheimer's disease, Alzheimer's disease, or the risk of developing Alzheimer's disease when the amount of extracellular secretory vesicle-containing PCDH1 protein in a sample shows a downward fluctuation of more than a certain amount in measurements taken over a specified period of time, when it is at a lower level than the extracellular secretory vesicle-containing PCDH1 protein in a normal sample, or when it shows a set threshold or below.

[0038] More specific examples of detection criteria may include, for example, an amount of PCDH1 protein contained in extracellular secretory vesicles that is 0.95 times or less than that of a normal specimen, an amount of PCDH1 protein contained in extracellular secretory vesicles that is 0.75 times or less, an amount of PCDH1 protein contained in extracellular secretory vesicles that is 0.65 times or less, an amount of PCDH1 protein contained in extracellular secretory vesicles that is 0. ...4 times or less, an amount of PCDH1 protein contained in extracellular secretory vesicles that is 0.3 times or less, or an amount of PCDH1 protein contained in extracellular secretory vesicles that is 0.2 times or less, and preferably an amount of PCDH1 protein contained in extracellular secretory vesicles that is 0.75 times or less than that of a normal specimen.

[0039] As described above, the method of the present invention may not detect MCI, i.e., the method of the present invention may detect the amyloid accumulation stage and / or tau pathogenesis / neurodegeneration stage of Alzheimer's disease, but not MCI.

[0040] The methods according to the first and second aspects of the present invention include a step of detecting Alzheimer's disease and / or its pathological phase, but do not include a final decision regarding the diagnosis of Alzheimer's disease. A doctor can diagnose Alzheimer's disease and determine a treatment plan by referring to the detection results by the method of the present invention.

[0041] A third aspect of the present invention is a diagnostic kit for Alzheimer's disease, comprising a reagent for measuring extracellular secretory vesicle-containing PCDH1 protein.

[0042] The above description can be applied to the measurement of PCDH1 protein contained in extracellular secretory vesicles using a reagent for measuring PCDH1 protein contained in extracellular secretory vesicles. The reagent for measuring PCDH1 protein contained in extracellular secretory vesicles is not particularly limited as long as it is a reagent capable of detecting and / or quantifying PCDH1 protein contained in extracellular secretory vesicles, but it is preferable that the reagent be capable of quantifying PCDH1 protein contained in extracellular secretory vesicles.

[0043] The kit may include reagents, instruments, etc., used in the means for isolating a sample from a specimen. The means may include a means for isolating cerebrospinal fluid from the specimen. The kit may also include a means for recovering an extracellular secretory vesicle fraction from the sample.

[0044] The diagnostic kit of the present invention can be produced using a reagent for measuring PCDH1 protein contained in extracellular secretory vesicles. That is, another embodiment of the present invention includes the use of a reagent for measuring PCDH1 protein contained in extracellular secretory vesicles for the production of a diagnostic kit for Alzheimer's disease.

[0045] The above-mentioned description can be applied to the reagent for measuring the PCDH1 protein contained in the extracellular secretory vesicles, and examples thereof include, but are not limited to, an antibody that recognizes the PCDH1 protein. The antibody that recognizes the PCDH1 protein preferably specifically binds to the PCDH1 protein.

[0046] A fourth aspect of the invention is a method of treating Alzheimer's disease in a patient, comprising the steps of: (i) identifying a patient as having a measured value of PCDH1 protein below a predetermined reference value; and (ii) administering treatment to the identified patient. In the identification step (i) above, the PCDH1 protein may be measured using an antibody that specifically recognizes the PCDH1 protein, or may be measured using mass spectrometry.

[0047] The method for treating Alzheimer's disease of the present invention can be applied to the method for detecting Alzheimer's disease of the present invention. That is, the method according to the fourth aspect of the present invention can be provided by the first and / or second aspects of the present invention. Specifically, for example, in the identification in the step (i), a patient may be identified by detecting Alzheimer's disease by the method according to the first and / or second aspects of the present invention, a patient may be identified by detecting the amyloid accumulation stage of Alzheimer's disease by the method according to the second aspect of the present invention, or a patient may be identified by detecting the tau pathogenesis stage and / or neurodegeneration stage of Alzheimer's disease by the method according to the second aspect of the present invention.

[0048] The treatment is not particularly limited and may be appropriately selected at the discretion of the physician. EXAMPLES

[0049] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following embodiments.

[0050] Collection of cerebrospinal fluid, collection method of cerebrospinal fluid The subjects for lumbar puncture were 41-80 years old, not taking antiplatelet or anticoagulant drugs, and without blood disorders. Participants had finished dinner by 9 p.m. the previous night, had not consumed alcohol, and had slept for at least 6 hours. After fasting overnight, lumbar puncture was performed between 9:00 a.m. and 10:00 a.m. CSF was collected using a steel needle and a 23G-25G top spinal needle with a polypropylene base. CSF samples for Aβ1-42 / p-Tau181 / t-Tau ELISA measurement were then collected by following step 1 below, and CSF samples for extracellular secretory vesicle analysis were collected by following step 2 below. All samples were collected by a single experienced physician using the same method and equipment at the same time. 1. Cerebrospinal fluid samples for Aβ1-42 / p-Tau181 / t-Tau ELISA measurement The cerebrospinal fluid dripped from the spinal needle during the above collection process was collected in a sterile 10cc polypropylene tube. It was then centrifuged at 3500 RPM (2200g) for 7 minutes at room temperature to precipitate cells and other insoluble materials. The supernatant was then dispensed into polypropylene microtubes (1.5ml) in 500μl portions using polypropylene dispensing 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 extracellular secretory vesicle analysis 500 μl of the collected sample was dropped directly into a polypropylene microtube (1.5 ml) from the spinal needle, and the sample was frozen at -80°C within 1 hour after collection and stored until the next analysis. Only one freeze-thaw cycle was performed.

[0051] ATN classification Amyloid beta (Aβ1-42), phosphorylated tau (p-Tau181), and total tau (t-Tau) were measured by ELISA in cerebrospinal fluid samples taken from subjects, and subjects were classified into ATN classifications, i.e., amyloid beta (A), phosphorylated tau (T), and neurodegeneration (N), based on the measured values. The cutoff values ​​were set based on the clinical diagnosis of all subjects by performing receiver operating characteristic (ROC) analysis between normal and MCI vs. AD groups, and were set as follows: amyloid beta: <680pg / mL, phosphorylated tau: ≥76.5pg / mL, and total tau: ≥598pg / mL.

[0052] Extracellular secretory vesicle fraction Extracellular secretory vesicle fractions were prepared from 500 μL of CSF samples for extracellular secretory vesicle analysis by ultracentrifugation, which consisted of three cycles of 2,000 g for 10 min, 10,000 g for 30 min, and then 100,000 g for 70 min.

[0053] ELISA measurement of protocadherin-1 (PCDH1) The collected extracellular secretory vesicle fraction was dissolved in 0.1% SDS / PBS, and the concentration of PCDH1 was measured using the Human Protocadherin-1 (PCDH1) ELISA Kit (MyBioSource #MBS269386) according to the recommended protocol.

[0054] Example 1 Comparison of PCDH1 content in extracellular secretory vesicles according to ATN classification

[0055] Subject samples (80 in total) were classified as ATN as shown in Table 1.

[0056] [Table 1]

[0057] For subjects with each ATN classification, PCDH1 ELISA was performed on the extracellular secretory vesicle fraction prepared from the cerebrospinal fluid specimen. The amount of PCDH1 contained in the extracellular secretory vesicles measured from the cerebrospinal fluid is compared by amyloid beta classification (A+ and A-) in Figure 1, by phosphorylated tau classification (T+ and T-) in Figure 2, and by total tau (neurodegeneration) classification (N+ and N-) in Figure 3.

[0058] The amount of PCDH1 contained in extracellular secretory vesicles derived from CSF was significantly decreased in the amyloid beta positive (A+) group, whereas no significant difference was observed in the phosphorylated tau positive (T+) group and total tau (neurodegeneration) positive (N+) group compared with the respective negative groups.

[0059] Furthermore, the results for each ATN classification are shown in Figure 4. Regardless of the presence or absence of changes in phosphorylated tau or neurodegeneration, the CSF-derived extracellular secretory vesicle-containing PCDH1 was decreased in amyloid beta-positive (A+) specimens. In particular, the A+TN- group showed a significant decrease in CSF-derived extracellular secretory vesicle-containing PCDH1 compared to all A- groups.

[0060] Next, the correlation between the amount of amyloid β (Aβ1-42) in cerebrospinal fluid samples and the amount of PCDH1 contained in extracellular secretory vesicles was analyzed, and the results are shown in Figure 5. When the amount of amyloid β decreased, the amount of PCDH1 contained in extracellular secretory vesicles also decreased, and a correlation was observed between the changes in the two.

[0061] These findings demonstrate that measuring the amount of PCDH1 contained in extracellular secretory vesicles is useful for detecting the amyloid accumulation stage, which is thought to be the early stage of Alzheimer's disease.

[0062] Example 2 Comparison of PCDH1 levels in extracellular secretory vesicles by diagnostic classification

[0063] Next, the subject samples (a total of 80 samples) were classified according to the results of the diagnosis based on clinical symptoms as normal, amnestic mild cognitive impairment (MCI), or Alzheimer's disease (AD), as shown in Table 2.

[0064] [Table 2]

[0065] For subjects in each diagnostic category, ELISA measurement of PCDH1 was performed on the extracellular secretory vesicle fraction prepared from the cerebrospinal fluid specimen. The results are shown in Figure 6. The amount of PCDH1 contained in the extracellular secretory vesicles derived from the cerebrospinal fluid of subjects diagnosed with AD was significantly reduced compared to that of subjects diagnosed with normal or MCI. Therefore, it is believed that by measuring the amount of PCDH1 contained in the extracellular secretory vesicles, it is possible to distinguish AD from normal and MCI, and to detect AD.

Claims

1. 1. A method for detecting Alzheimer's disease, comprising: A method comprising the step of measuring the amount of extracellular secretory vesicle-containing protocadherin-1 (PCDH1) protein in a sample taken from a subject.

2. 1. A method for detecting the amyloid accumulation, tau pathology, and / or neurodegeneration stages of Alzheimer's disease, comprising: A method comprising the step of measuring extracellular secretory vesicle-containing protocadherin-1 (PCDH1) protein in a sample taken from a subject.

3. The method according to claim 1 or 2, which does not detect amnestic mild cognitive impairment (MCI).

4. The method according to claim 1 or 2, comprising the steps of recovering an extracellular secretory vesicle fraction from a sample taken from a specimen, and measuring PCDH1 protein in the extracellular secretory vesicle fraction.

5. The method of claim 1 or 2, wherein the sample is cerebrospinal fluid.

6. A diagnostic kit for Alzheimer's disease, comprising a reagent for measuring extracellular secretory vesicle-containing PCDH1 protein.

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