Method of testing for risk of progression or onset of cognitive dysfunction in alzheimer's disease patient, and method of testing for alzheimer's disease

Abstract

To provide a technique for testing for the risk of progression or onset of cognitive dysfunction in an Alzheimer's disease patient.SOLUTION: A method of testing for the risk of future progression or onset of cognitive dysfunction in an Alzheimer's disease patient is provided, the method comprising measuring the amount of N-terminal fragment of SV2B in a bodily fluid collected from the Alzheimer's disease patient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to a method for examining a future progression risk or onset risk of cognitive impairment in an Alzheimer's disease patient, and also relates to a method for examining Alzheimer's disease, and also relates to a test kit used in these examination methods. [Background technology]

[0002] Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive cognitive decline and is characterized by amyloid-β (Aβ) deposition and neurofibrillary tangles in the cerebral cortex and subcutaneous gray matter. At present, AD can only be definitively diagnosed by autopsy after death, but if AD could be accurately and early diagnosed while alive, early therapeutic intervention would be possible and appropriate treatments for AD could be administered to patients.

[0003] Conventional AD diagnosis involves interviews, cognitive function tests, and imaging tests such as computed tomography (CT) and magnetic resonance imaging (MRI). In these diagnoses, it can be difficult to distinguish between AD and neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and dementia with Lewy bodies (DLB), and appropriate treatment cannot be provided to patients. Therefore, there is a demand for improved accuracy in determining whether or not a patient has AD in AD diagnosis.

[0004] Conventionally, cerebrospinal fluid testing has been known as a testing method for improving the accuracy of diagnosing AD. In the cerebrospinal fluid of AD patients, the concentrations of total tau protein (t-tau) and tau protein phosphorylated at threonine 181 (p-tau) are increased, and the concentration of Aβ42 is decreased. It is known that the accuracy of diagnosis for distinguishing between people with AD and people without AD can be improved by using these as biomarkers for AD (Non-Patent Documents 1 to 3).

[0005] On the other hand, AD progresses over a long period of 20 to 30 years from onset to the onset of cognitive impairment. Therefore, AD is classified into two types: those who do not develop cognitive impairment even if they are affected with AD, and those who will develop cognitive impairment in the near future. It is important to identify the latter type of AD patients early and provide appropriate treatment, but with the conventional diagnosis described above, it is still difficult to determine whether or not a patient falls into one of the above two types.

[0006] Furthermore, if the risk of developing cognitive impairment in AD patients and the risk of progression of cognitive impairment in AD patients could be distinguished using the same diagnostic method, it would be desirable to be able to continuously observe AD patients using the same diagnostic method. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Peskind ER. et al., Li G, Age and apolipoprotein E*4 allele effects on cerebrospinal fluid beta-amyloid 42 in adults with normal cognition. Arch Neurol. 2006;63:936-939. [Non-Patent Document 2] Shaw LM. et al., Cerebrospinal fluid biomarker signature in Alzheimer's disease neuroimaging initiative subjects. Ann Neurol. 2009;65:403-413. [Non-Patent Document 3] Tapiola T. et al., Cerebrospinal fluid [beta]-amyloid 42 and tau proteins as biomarkers of alzheimer-type pathologic changes in the brain. Arch Neurol. 2009;66:382-389. Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present disclosure is to provide a technique for examining the progression risk or onset risk of cognitive impairment in an AD patient. Another object of the present disclosure is to provide a technique for examining the presence or absence of AD. Yet another object of the present disclosure is to provide a technique for examining the presence or absence of severe AD. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems and have obtained the following findings. (1) Analysis of brain homogenates prepared from the supramarginal gyrus of the parietal lobe (hereafter referred to as brain tissue slices) of autopsy brains from non-AD, moderate AD, and severe AD patients revealed that the amount of synaptic vesicle protein 2B (SV2B) in the brain homogenates was significantly lower than other synaptic proteins not only in the severe AD group but also in the moderate AD group. (2) Analysis of synaptoneurosomes isolated from brain tissue slices from non-AD, moderate AD, and severe AD patients revealed that the ratio of SV2B N-terminal fragment (SV2B NTF) to full-length SV2B was significantly higher in the severe AD patients than in the non-AD and moderate AD patients. (3) Analysis of cerebrospinal fluid from non-AD and AD patients revealed that the amount of SV2B NTF in the cerebrospinal fluid was significantly higher in the AD patients than in the non-AD patients. (4) Patients with AD at the mild cognitive impairment (MCI) stage were followed up for one year, and after one year they were divided into two groups: those whose cognitive function had declined (progressive group) and those whose cognitive function had not declined (stable group). The cerebrospinal fluid was analyzed, and the amount of SV2B NTF in the cerebrospinal fluid of the progressive group at the start of the follow-up study was significantly higher than that of the stable group.

[0010] Based on these findings, the present disclosure has found that (i) the amount of SV2B NTF in a body fluid collected from an AD patient can be an index for predicting the risk of progression or onset of cognitive impairment in the future in an AD patient, (ii) the amount of SV2B NTF in a body fluid collected from a subject can be an index for predicting the presence or absence of AD, (iii) the amount of SV2B in a brain tissue slice collected from a subject can be an index for predicting the presence or absence of AD, and (iv) the ratio of SV2B NTF to full-length SV2B in synaptoneurosomes separated from a brain tissue slice collected from a subject can be an index for predicting the presence or absence of severe AD. The present disclosure has been completed based on these findings and through further investigation.

[0011] That is, the present disclosure provides inspection techniques of the following aspects. Item 1-1. A method for examining a risk of progression or onset of cognitive impairment in a patient with AD, comprising: A testing method comprising a step of measuring the amount of SV2B NTF in a body fluid collected from an AD patient. Item 1-2. The examination method according to Item 1-1, wherein the body fluid is cerebrospinal fluid. Item 1-3. The method according to Item 1-1 or 1-2, wherein the patient with AD is at a stage of mild cognitive impairment. Item 1-4. A kit for testing the risk of progression or onset of cognitive impairment in a patient with AD, A test kit comprising a reagent for measuring the amount of SV2B NTF in a body fluid. Item 1-5. The test kit according to Item 1-4, wherein the reagent is an antibody that exhibits binding to SV2B NTF.

[0012] The present disclosure also provides inspection techniques of the following aspects. Item 2-1. A method for testing for the presence or absence of AD, comprising: A testing method comprising a step of measuring the amount of SV2B NTF in a body fluid collected from a subject. Item 2-2. The examination method according to Item 2-1, wherein the body fluid is cerebrospinal fluid. Item 2-3. A kit for testing for the presence or absence of AD, comprising: A test kit comprising a reagent for measuring the amount of SV2B NTF in a body fluid. Item 2-4. The test kit according to Item 2-3, wherein the reagent is an antibody that exhibits binding to SV2B NTF.

[0013] Furthermore, the present disclosure provides inspection techniques in the following aspects. Item 3-1. A method for testing for the presence or absence of AD, comprising: A testing method comprising a step of measuring the amount of SV2B in a brain tissue slice taken from a subject. Item 3-2. The testing method according to Item 3-1, in which the amount of SV2B in the brain tissue slice is measured using a brain homogenate or synaptoneurosome prepared from the brain tissue slice as a sample. Item 3-3. The amount of SV2B in the brain tissue slice is measured using a brain homogenate prepared from the brain tissue slice as a sample, and The testing method according to Item 3-1 or 3-2, which is performed to examine the presence or absence of AD whose pathological stage is moderate or higher. Item 3-4. A kit for testing for the presence or absence of AD, comprising: A test kit comprising a reagent for measuring SV2B in a brain tissue slice. Item 3-5. The test kit according to Item 3-4, wherein the reagent is an antibody that exhibits binding to SV2B.

[0014] Furthermore, the present disclosure provides inspection techniques in the following aspects. Item 4-1. A method for testing for the presence or absence of severe AD, comprising: A testing method comprising the steps of measuring the amount of full-length SV2B and the amount of SV2B NTF in synaptoneurosomes isolated from a brain tissue slice collected from a subject, and calculating the ratio of the amount of SV2B NTF to the amount of SV2B. Item 4-2. A kit for testing the presence or absence of severe AD, comprising: A test kit comprising a reagent for measuring full-length SV2B and SV2B NTF in synaptoneurosomes isolated from a brain tissue slice. Item 4-3. The test kit according to Item 4-2, wherein the reagent is an antibody that exhibits binding to SV2B NTF. Effect of the Invention

[0015] In one embodiment of the present disclosure, the amount of SV2B NTF in a body fluid collected from an AD patient can be used as a biomarker to examine the risk of progression or onset of cognitive impairment in the AD patient in the future. In this embodiment, AD patients who are likely to develop cognitive impairment in the future can be selected from AD patients at the stage of mild cognitive impairment. For example, when conducting a clinical trial of an AD therapeutic drug, the effectiveness of the AD therapeutic drug in the clinical trial can be efficiently evaluated by selecting AD patients who are likely to develop cognitive impairment in the future as clinical trial subjects using the test method 1 of the present disclosure.

[0016] In another embodiment of the present disclosure, the amount of SV2B NTF in a body fluid can be used as a biomarker to examine the presence or absence of AD.

[0017] Furthermore, in another embodiment of the present disclosure, by using the amount of SV2B NTF in a body fluid as a biomarker, it is possible to verify, by the same test method, the presence or absence of cognitive impairment in an AD patient in the case of AD, and the degree of progression of cognitive impairment. As a result, another embodiment of the present disclosure is excellent in that it makes it possible to observe a series of events from AD onset to the onset of cognitive impairment and the worsening of cognitive impairment over time using objective indicators.

[0018] In yet another embodiment of the present disclosure, the presence or absence of AD can be examined by using the amount of SV2B in a brain tissue slice as a biomarker. In this embodiment, when a brain homogenate is used as a sample for measuring the amount of SV2B in a brain tissue slice, not only severe AD but also moderate AD can be detected. [Brief description of the drawings]

[0019] [Figure 1] Brain homogenates (TH: Total homogenate) and synaptoneurosome fractions (SNS: Synaptoneurosomes) prepared from the supramarginal gyrus of the autopsy brain of 6 non-AD patients (Ctrl), 6 moderate AD patients (moderate), and 5 severe AD patients (severe) were subjected to Western blotting to measure the protein expression levels of SV2B, PSD95, SV2A, and Syt1. A shows the results for the brain homogenate, and B shows the results for the synaptoneurosome fraction. In A and B, the images shown on the left are images of immunostaining by Western blotting, and the bar graphs shown on the right are the results of correcting the expression level of each protein by setting the expression level of each protein in non-AD patients (Ctrl) to 1. In the figures, *, **, and ns indicate p<0.05, p<0.001, and p 0.05 or more by the Kruskal-Wallis test, respectively. [Diagram 2]Synaptoneurosome fractions (SNS: Synaptoneurosomes) prepared from the supramarginal gyrus of the autopsy brain parietal lobe of five non-AD patients (Ctrl), five moderate AD patients (moderate), and four severe AD patients (severe) were subjected to Western blotting to measure the protein expression levels of SV2B and SV2B NTF, and the ratio of the SV2B NTF expression level to the SV2B expression level was calculated. The image on the left is an image obtained by immunostaining using Western blotting, and the bar graph on the right is the result of correcting the ratio of the SV2B NTF expression level to the SV2B expression level in non-AD patients (Ctrl) to 1, with the corresponding ratio corrected for moderate AD patients and severe AD patients. In the figure, * indicates p<0.05 by Kruskal-Wallis test. [Diagram 3] The results are shown in the figure, in which the amount of SV2B and SV2B NTF was measured by Western blotting of concentrated cerebrospinal fluid from non-AD and AD patients, and brain homogenates (human, wild-type (WT) mice, and SV2B KO mice). The image shown in the upper left is an image of immunostaining of SV2B and SV2B NTF by Western blotting, and the image shown in the lower left is an image of Western blot albumin stained with Ponceau S staining solution. The bar graph on the right shows the relative value of the amount of SV2B NTF in concentrated cerebrospinal fluid from AD patients, with the amount of SV2B NTF in concentrated cerebrospinal fluid from non-AD patients set at 100. In the figure, * indicates p<0.05 by Mann-Whitney U test. [Figure 4]Patients with mild cognitive impairment were followed for one year, and were stratified into a stable group (12 patients) whose MMSE score change was 0 or more after one year, and a progressive group (13 patients) whose MMSE score change was less than -1 after one year. The amount of SV2B NTF in the cerebrospinal fluid at the start of the follow-up in each group was measured by Western blot. The image shown in the upper left shows an image of immunostaining of SV2B and SV2B NTF by Western blot, and an image of albumin stained with Ponceau S staining solution. The bar graph shown below shows the relative value of the amount of SV2B NTF in the cerebrospinal fluid of the stable group (stable) as 1, and the amount of SV2B NTF in the concentrated cerebrospinal fluid of the progressive group (progressive). In the figure, * indicates p<0.05 by Mann-Whitney U test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] 1. Terminology Terms used herein have the meanings generally understood by those skilled in the art of medicine, pharmacology, molecular biology, microbiology, organic chemistry, etc., unless otherwise specified. When a term defined in this specification does not have the same meaning as generally understood, the description in this specification takes precedence.

[0021] In the present disclosure, Alzheimer's disease (AD) does not mean a clinical diagnosis based on dementia, but is defined by pathological changes that characterize the disease. That is, in the present disclosure, AD includes each stage of the preclinical stage, the mild cognitive impairment stage, and the dementia stage. In AD, the preclinical stage refers to a state in which the brain has pathological changes of AD, but the cognitive function is clinically normal. In AD, the mild cognitive impairment stage refers to a state in which the pathological changes that characterize AD are clinically exhibited as mild cognitive impairment. In AD, the dementia stage refers to a state in which the pathological changes of AD are clinically exhibited as a background. That is, the decline in cognitive function in AD patients does not occur in the preclinical stage, and becomes more severe in the order of the mild cognitive impairment stage and the dementia stage.

[0022] In the present disclosure, the risk of future cognitive decline in an AD patient refers to the risk of transitioning from the preclinical stage to the mild cognitive impairment stage or the dementia stage in the future, or the risk of transitioning from the mild cognitive impairment stage to the dementia stage in the future. The risk of future cognitive impairment in an AD patient refers to the risk of transitioning from the preclinical stage to the mild cognitive impairment stage or the dementia stage in the future in an AD patient. Cognitive function can be assessed by the Mini-Mental State Examination (MMSE).

[0023] In this disclosure, the pathological stage of AD is classified according to the CERAD score and the NFT Braak stage. The CERAD score is a pathological diagnostic criterion that uses the density of neuritic plaques (NP) as an index (Mirra SS, et al., Neurology 1991; 41: 479-486. ), and the density of NP is classified into four stages: 0: none, A: small amount, B: moderate amount, and C: large amount. The NFT Braak stage is a pathological diagnostic criterion that uses the progression stage of neurofibrillary tangles (NFTs) as an index (Braak H, et al., Acta Neuropathologica 1991; 82: 239-259.), and the degree of progression of NFTs is classified into seven stages: 0: none, I: only appears in the transitional entorhinal cortex, II: progresses to the entorhinal cortex, III: progresses to the hippocampus, IV: appears in large amounts in the hippocampus but small amounts in the neocortex, V: appears in large amounts in the neocortical association area, and VI: appears in large amounts in the neocortical primary area. In the present disclosure, moderate AD refers to AD with a CERAD score of B or higher and an NFT Braak stage of III or IV, and severe AD refers to AD with a CERAD score of C and an NFT Braak stage of V or VI. In the present disclosure, AD with a pathological stage of moderate or higher refers to AD with a pathological stage of moderate or severe.

[0024] In the present disclosure, synaptic vesicle protein 2B (SV2B) is a type of synaptic vesicle protein, and has been identified by the present inventors as a regulator of the Aβ-producing enzyme BACE1. Human SV2B has the amino acid sequence shown in SEQ ID NO: 1. In the present disclosure, when simply referred to as SV2B, it refers to full-length SV2B (a protein containing the amino acid sequence shown in SEQ ID NO: 1).

[0025] In the present disclosure, the N-terminal fragment of SV2B (SV2B NTF) refers to a fragment that is recognized by an anti-SV2B N-terminal antibody among fragments generated by cleavage of SV2B (full-length type). Here, the anti-SV2B N-terminal antibody is an anti-SV2B antibody that binds to the region of positions 2 to 17 from the N-terminus of SV2B, and an example of such an antibody is a commercially available anti-SV2 antibody (#119 102, Polyclonal rabbit antibody, Synaptic Systems). In addition, the molecular weight of SV2B NTF is more than 19 kDa and less than 39 kDa, or more than 19 kDa and less than 28 kDa, more specifically, about 25 kDa. For example, SV2B NTF can be detected as a band that appears in the region of molecular weight more than 19 kDa and less than 39 kDa by Western blotting using an anti-SV2B antibody that binds to the region of positions 2 to 17 from the N-terminus of V2B.

[0026] 2. Testing method 1 As shown in the Examples section below, the present inventors have found that the amount of SV2B NTF in a body fluid collected from an AD patient can be an index for predicting the risk of progression or onset of cognitive impairment in the future in an AD patient. Thus, in one embodiment of the present disclosure, a method for testing the risk of cognitive decline or onset of cognitive impairment in the future in an AD patient (hereinafter, sometimes referred to as "Testing Method 1") is provided, which includes a step of measuring the amount of SV2B NTF in a body fluid collected from an AD patient.

[0027] In the test method 1 of the present disclosure, the test subject is an AD patient for which prediction of the future risk of progression of cognitive impairment or the risk of developing cognitive impairment is required. The AD patient to be tested is a human or a non-human animal. Specific examples of non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats. Since the test method of the present disclosure is suitable for testing humans, the AD patient to be tested is preferably a human.

[0028] The subjects of the test may be those at any stage of AD, including preclinical, mild cognitive impairment, or dementia, but are preferably those at the preclinical or mild cognitive impairment stage of AD.

[0029] In the test method 1 of the present disclosure, the body fluid to be tested may be any of cerebrospinal fluid, blood, plasma, serum, saliva, etc., but from the viewpoint of predicting the progression risk or onset risk of cognitive impairment with high accuracy, cerebrospinal fluid is preferably used. Cerebrospinal fluid can be collected by lumbar puncture. In addition, the body fluid to be tested may be subjected to a concentration treatment as necessary to facilitate the quantification of SV2B NTF.

[0030] In the test method 1 of the present disclosure, the amount of SV2B NTF in a body fluid is measured in order to use the amount of SV2B NTF in the body fluid as a biomarker for predicting the risk of future cognitive decline or the risk of developing cognitive impairment. The amount of SV2B NTF in a body fluid can be measured by, for example, Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, liquid chromatography mass spectrometry (LC-MS / MS), etc. For example, in the Western blotting method, after separating proteins in the body fluid by electrophoresis, the proteins are transferred to a hydrophobic membrane, and SV2B NTF is colored or luminesced by an anti-SV2B antibody that exhibits binding affinity to SV2B NTF, and the signal intensity of the SV2B NTF band is measured by a densitometer. In addition, in the enzyme immunoassay, the total amount of SV2B NTF and full-length SV2B in the body fluid is measured using an anti-SV2B antibody that exhibits binding affinity to SV2B NTF, and the amount of full-length SV2B in the body fluid is measured using an anti-SV2B antibody that exhibits binding affinity to a region other than the NTF of SV2B, and the amount of SV2B NTF in the body fluid can be determined by subtracting the amount of full-length SV2B from the total amount of SV2B NTF and full-length SV2B. In the immunoprecipitation method, the SV2B protein (including SV2B NTF and full-length SV2B) in the sample is specifically separated by immunoprecipitation using an anti-SV2B antibody that exhibits binding affinity to SV2B NTF, and the presence or absence of SV2B NTF and the amount of SV2B NTF can be determined from the separated SV2B protein by Western blotting.

[0031] The amount of SV2B NTF in a body fluid may be determined as a relative amount to the amount of full-length SV2B in the body fluid. The amount of full-length SV2B in a body fluid can be measured by Western blotting, enzyme immunoassay, liquid chromatography mass spectrometry, etc. When measuring the amount of full-length SV2B by Western blotting or enzyme immunoassay, an anti-SV2B antibody that exhibits binding affinity to a region of SV2B other than the NTF may be used.

[0032] The amount of SV2B NTF in a body fluid is preferably determined relative to the amount of a loading control in the body fluid, which may be, for example, a housekeeping gene product such as albumin or actin.

[0033] The amount of SV2B NTF in body fluids is related to the risk of progression or onset of cognitive impairment in the future of AD patients, and AD patients with a high risk of progression or onset of cognitive impairment have a higher amount of SV2B NTF in body fluids than AD patients with a low risk. Therefore, in the test method 1 of the present disclosure, AD patients with a high amount of SV2B NTF in body fluids are predicted to have a high risk of progression of cognitive impairment or onset of cognitive impairment in the future. For example, when a preclinical AD patient is tested, if the amount of SV2B NTF in body fluids is high, it is predicted that the AD patient will progress in cognitive impairment in the future and may transition to a mild cognitive impairment stage or dementia stage. Also, for example, when a mild cognitive impairment stage AD patient is tested, if the amount of SV2B NTF in body fluids is high, it is predicted that the AD patient will progress in cognitive impairment in the future and may transition to a dementia stage.

[0034] According to the test method 1 of the present disclosure, the risk of progression or onset of cognitive impairment based on the amount of SV2B NTF in body fluids can be determined by comparing with a reference value previously obtained from AD patients with cognitive impairment or AD patients with no cognitive impairment. Here, the reference value is a value that serves as a criterion for determining the risk of progression or onset of cognitive impairment. Specifically, the reference value may be set to the average or median of the amount of SV2B NTF (baseline) in the body fluids of the progressive group or the stable group, or the cutoff value obtained from the amount of SV2B NTF (baseline) in each body fluid of the progressive group and the stable group, by performing a follow-up survey one year or several years after measuring the amount of SV2B NTF (baseline) in the body fluids of AD patients, and dividing the AD patients into a group with progressive cognitive impairment (progressive group) and a group with no progressive cognitive impairment (stable group).

[0035] For example, in the case of testing the risk of future progression of cognitive impairment in preclinical AD patients, a follow-up survey is conducted one or several years after measuring the amount of SV2B NTF in body fluids (baseline) of preclinical AD patients, and the patients are divided into a group of AD patients in which cognitive impairment progression or transition to mild cognitive impairment or dementia is observed (progressive group) and a group of AD patients in which cognitive impairment does not progress (stable group), and the average or median amount of SV2B NTF in body fluids (baseline) of the progressive group or stable group, or a cutoff value calculated from the amount of SV2B NTF in each body fluid (baseline) of the progressive group and the stable group may be used as the reference value. If the amount of SV2B NTF in the body fluids of a preclinical AD patient who is the subject of the test is equal to or higher than the average or median value of the progressive group, significantly higher than the average or median value of the stable group, or equal to or higher than the cutoff value, the AD patient is predicted to have a high risk of cognitive impairment progression within the period corresponding to the follow-up period.

[0036] For example, in the case of testing the risk of future cognitive impairment progression for AD patients in the mild cognitive impairment stage, a follow-up survey is conducted one or several years after measuring the amount of SV2B NTF in body fluids (baseline) of AD patients in the mild cognitive impairment stage, and the patients are divided into a group of AD patients in which cognitive impairment progression or transition to the dementia stage is observed (progressive group) and a group of AD patients in which cognitive impairment does not progress (stable group), and the average or median of the amount of SV2B NTF (baseline) in the body fluids of the progressive group or stable group, or a cutoff value calculated from the amount of SV2B NTF (baseline) in each body fluid of the progressive group and the stable group may be used as the reference value. If the amount of SV2B NTF in the body fluids of a subject AD patient in the mild cognitive impairment stage is equal to or greater than the average or median of the progressive group, significantly higher than the average or median of the stable group, or equal to or greater than the cutoff value, it is predicted that the AD patient has a high risk of cognitive impairment progression or transition to the dementia stage within a period corresponding to the follow-up period.

[0037] For AD patients who are predicted to have a high risk of progression or onset of cognitive impairment in the future by the testing method 1 disclosed herein, it is desirable to carefully monitor their progress so that early intervention and early treatment can be performed.

[0038] In addition, the test method 1 of the present disclosure can select AD patients who are likely to develop cognitive impairment in the future from among AD patients at the mild cognitive impairment stage. Therefore, when conducting a clinical trial of an AD therapeutic drug, by selecting AD patients who are likely to develop cognitive impairment in the future as clinical trial subjects using the test method 1 of the present disclosure, it becomes possible to efficiently evaluate the effectiveness of the AD therapeutic drug in the clinical trial. When a clinical trial is conducted using a group of clinical trial subjects of AD patients at the mild cognitive impairment stage, including patients who are not likely to develop cognitive impairment, when a subject does not develop cognitive impairment, it is not possible to determine whether this is due to the effect of the AD therapeutic drug or because the subject was originally not supposed to develop cognitive impairment. Therefore, in such a clinical trial, the effectiveness of the AD therapeutic drug will be erroneously evaluated for subjects who should not be affected by the efficacy of the therapeutic drug. On the other hand, in the testing method 1 disclosed herein, a clinical trial can be conducted using a group of clinical trial subjects consisting only of AD patients who are likely to develop cognitive impairment in the future. Therefore, if a subject does not develop cognitive impairment, it is highly likely that this is due to the effect of the AD therapeutic drug, and as a result, the effectiveness of the AD therapeutic drug can be appropriately evaluated in this clinical trial.

[0039] 3. Test kit 1 Another embodiment of the present disclosure is a kit for testing the risk of future cognitive decline or the risk of developing cognitive impairment in an AD patient, the test kit including a reagent for measuring the amount of SV2B NTF in a body fluid (hereinafter, sometimes referred to as "test kit 1"). Test kit 1 of the present disclosure is a test kit used to carry out test method 1 of the present disclosure, and the contents described in the above section "2. Test method 1" are also incorporated by reference in test kit 1 of the present disclosure.

[0040] Examples of reagents for measuring the amount of SV2B NTF include anti-SV2B antibodies that exhibit binding ability to SV2B NTF. The antibody may be either a polyclonal antibody or a monoclonal antibody. The antibody may also be an antibody fragment, so long as it exhibits binding ability to SV2B NTF. Examples of antibody fragments include Fab fragments, F(ab')2 fragments, and single-chain antibodies (scFv). The antibody may also be provided in a state where it is immobilized on a solid phase carrier such as a microtiter plate or particles.

[0041] Furthermore, the test kit 1 of the present disclosure may contain an antibody for measuring the amount of full-length SV2B in a body fluid (an anti-SV2B antibody that exhibits binding affinity to a region other than the NTF of SV2B) as needed. Furthermore, the test kit 1 of the present disclosure may contain a dilution or reaction buffer solution containing components necessary for the measurement, a washing solution, a coloring reagent, a reaction vessel, and the like as needed.

[0042] 4. Testing method 2 As shown in the Examples section below, the present inventors have found that the amount of SV2B NTF in a body fluid can be an index for predicting the presence or absence of AD. Thus, in one embodiment of the present disclosure, a method for testing the presence or absence of AD (hereinafter, sometimes referred to as "Testing Method 2") is provided, which includes a step of measuring the amount of SV2B NTF in a body fluid collected from a subject.

[0043] In the test method 2 of the present disclosure, the subject is a human or a non-human animal to be tested for the presence or absence of AD. Specific examples of non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats. Since the test method of the present disclosure is suitable for testing humans, the subject to be tested is preferably a human.

[0044] In the test method 2 of the present disclosure, the body fluid to be tested may be any of cerebrospinal fluid, blood, plasma, serum, saliva, etc., but from the viewpoint of predicting the presence or absence of AD with higher accuracy, cerebrospinal fluid is preferably used. Cerebrospinal fluid can be collected by lumbar puncture. In addition, the body fluid to be tested may be subjected to a concentration treatment, if necessary, to facilitate the quantification of SV2B NTF.

[0045] In the test method 2 of the present disclosure, the amount of SV2B NTF in a body fluid is measured in order to use the amount of SV2B NTF in a body fluid as a biomarker for predicting the presence or absence of AD. The method for measuring the amount of SV2B NTF in a body fluid is as described in the above section "2. Test method 1."

[0046] In the test method 2 of the present disclosure, the amount of SV2B NTF in a body fluid may be determined as a relative amount to the amount of full-length SV2B in the body fluid. The method for measuring the amount of full-length SV2B in a body fluid is as described in the above section "2. Test method 1."

[0047] In addition, in the test method 2 of the present disclosure, the amount of SV2B NTF in a body fluid is preferably determined as a relative amount to the amount of a loading control in the body fluid. As the loading control, for example, a housekeeping gene product such as albumin or actin can be used.

[0048] The amount of SV2B NTF in a body fluid correlates with the presence or absence of AD, and the amount of SV2B NTF in a body fluid of a person with AD is higher than that of a person without AD. Therefore, in the test method 2 of the present disclosure, a subject with a high amount of SV2B NTF in a body fluid is determined to be highly likely to have AD, and a subject with a low amount of SV2B NTF in a body fluid is determined to be highly likely to not have AD.

[0049] In the testing method 2 of the present disclosure, the presence or absence of AD can be determined based on the amount of SV2B NTF in a body fluid by comparing with a reference value previously determined from AD patients or non-AD patients. Here, the reference value is a value that serves as a standard for determining the presence or absence of AD, and specifically, is the average or median amount of SV2B NTF in the body fluid of AD patients or non-AD patients, or a cutoff value determined from the amount of SV2B NTF in the body fluid of AD patients and non-AD patients, etc.

[0050] For example, the average or median of the amount of SV2B NTF in the body fluid of non-AD patients is obtained in advance, and this is used as a reference value. If the amount of SV2B NTF in the body fluid of the subject is equal to or less than the reference value, it can be determined that the subject is highly likely to not have AD. Also, for example, the average or median of the amount of SV2B NTF in the body fluid of AD patients is obtained in advance, and this is used as a reference value. If the amount of SV2B NTF in the body fluid of the subject is equal to or more than the reference value, it can be determined that the subject is highly likely to have AD. Also, for example, the average or median of the amount of SV2B NTF in the body fluid of non-AD patients and the average or median of the amount of SV2B NTF in the body fluid of AD patients are obtained in advance, and a cutoff value for distinguishing the presence or absence of AD from these SV2B NTF amounts is determined in advance, and the cutoff value is used as a reference value. If the amount of SV2B NTF in the body fluid of the subject is equal to or more than the cutoff value, it can be determined that the subject is highly likely to have AD.

[0051] Since the testing method 2 disclosed herein can be performed as a test to assist in the diagnosis of AD, it is desirable to make a more accurate diagnosis of the presence or absence of AD for subjects who are determined to have a high probability of having AD by the testing method 2 disclosed herein based on the results of clinical symptoms, image findings, etc.

[0052] 5. Test Kit 2 Another embodiment of the present disclosure is a kit for testing the presence or absence of AD, the test kit including a reagent for measuring the amount of SV2B NTF in a body fluid (hereinafter, also referred to as "test kit 2"). Test kit 2 of the present disclosure is a test kit used to carry out test method 2 of the present disclosure, and the contents described in the above section "4. Test method 2" are also incorporated by reference in test kit 2 of the present disclosure.

[0053] The reagent for measuring the amount of SV2B NTF is as described in the section "3. Test Kit 1" above.

[0054] Furthermore, the test kit 2 of the present disclosure may contain an antibody for measuring the amount of full-length SV2B in a body fluid (an anti-SV2B antibody that exhibits binding affinity to a region other than the NTF of SV2B) as needed. Furthermore, the test kit 2 of the present disclosure may contain a dilution or reaction buffer solution containing components necessary for the measurement, a washing solution, a coloring reagent, a reaction vessel, and the like as needed.

[0055] 6. Testing method 3 As shown in the Examples section below, the present inventors have found that the amount of SV2B in brain tissue can be an index for predicting the presence or absence of AD. Thus, in one embodiment of the present disclosure, a method for testing the presence or absence of AD (hereinafter, sometimes referred to as "Testing Method 3") is provided, which includes a step of measuring the amount of SV2B in a brain tissue slice taken from a subject.

[0056] In the test method 3 of the present disclosure, the subject is a human or a non-human animal to be tested for the presence or absence of AD. Specific examples of non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats. Since the test method of the present disclosure is suitable for testing humans, the subject to be tested is preferably a human.

[0057] In the testing method 3 of the present disclosure, the brain tissue specimen to be tested can be obtained by brain biopsy.

[0058] In the test method 3 of the present disclosure, the amount of SV2B in the brain tissue slice is measured in order to use the amount of SV2B in the brain tissue slice as a biomarker for predicting the presence or absence of AD. To measure the amount of SV2B contained in the brain tissue slice, the brain tissue slice is added to a liquid medium such as physiological saline or buffer solution, suspended to dissolve proteins, and then the brain homogenate (liquid fraction) recovered by solid-liquid separation such as filtration and centrifugation may be used as a sample. Synaptoneurosomes separated from the brain homogenate may also be used as a sample. Separation of synaptoneurosomes from the brain homogenate can be performed by stepwise centrifugation. The method for measuring the amount of SV2B in these samples is as described in the section "2. Test Method 1" above.

[0059] In addition, in the test method 3 of the present disclosure, the amount of SV2B in the brain tissue slice is preferably determined as a relative amount to the amount of a loading control in the brain tissue slice. As the loading control, for example, a housekeeping gene product such as albumin or actin can be used.

[0060] The amount of SV2B in brain tissue slices correlates with the presence or absence of AD, and subjects with AD have lower amounts of SV2B in brain tissue slices than subjects without AD. Therefore, in the test method 3 disclosed herein, subjects with low amounts of SV2B in brain tissue slices are determined to be highly likely to be suffering from AD.

[0061] In addition, with conventional testing techniques using biomarkers, it was difficult to predict whether or not a patient had moderate pathological stage AD. However, in the testing method 3 disclosed herein, when a brain homogenate is used as a sample for measuring the amount of SV2B in a brain tissue slice, not only severe AD but also moderate AD can be detected.

[0062] In the test method 3 of the present disclosure, the presence or absence of AD can be determined based on the amount of SV2B in the brain tissue slice by comparing it with a reference value previously determined from AD patients or non-AD patients. Here, the reference value is a standard value for determining the presence or absence of AD, and specifically, it is the average or median amount of SV2B in brain tissue slices from AD patients or non-AD patients, or a cutoff value determined from the amount of SV2B in brain tissue slices from AD patients and non-AD patients, etc.

[0063] For example, the average or median amount of SV2B in the brain tissue slice of a non-AD patient is obtained in advance, and this is used as a reference value. If the amount of SV2B in the brain tissue slice of the subject is equal to or greater than the reference value, it can be determined that the subject is highly likely to not have AD. Also, for example, the average or median amount of SV2B in the brain tissue slice of an AD patient is obtained in advance, and this is used as a reference value. If the amount of SV2B in the brain tissue slice of the subject is equal to or less than the reference value, it can be determined that the subject is highly likely to have AD. Also, for example, the average or median amount of SV2B in the brain tissue slice of a non-AD patient and the average or median amount of SV2B in the brain tissue slice of an AD patient are obtained in advance, and a cutoff value for distinguishing the presence or absence of AD from these SV2B amounts is determined, and if the amount of SV2B in the brain tissue slice of the subject is equal to or less than the cutoff value, it can be determined that the subject is highly likely to have AD.

[0064] Since the testing method 3 of the present disclosure can be performed as a test to assist in the diagnosis of AD, it is desirable to make a more accurate diagnosis of the presence or absence of AD for a subject determined to be highly likely to have AD by the testing method 3 of the present disclosure based on the results of clinical symptoms, image findings, etc.

[0065] 7. Test Kit 3 Another embodiment of the present disclosure is a kit for testing the presence or absence of AD, the test kit including a reagent for measuring the amount of SV2B in a brain tissue slice (hereinafter, sometimes referred to as "Test Kit 3"). Test Kit 3 of the present disclosure is a test kit used to carry out Test Method 3 of the present disclosure, and the contents described in the above section "6. Test Method 3" are also incorporated by reference in Test Kit 3 of the present disclosure.

[0066] Examples of reagents for measuring the amount of SV2B include anti-SV2B antibodies that exhibit binding affinity to a region of SV2B other than the NTF. The antibody may be either a polyclonal antibody or a monoclonal antibody. The antibody may also be an antibody fragment such as a Fab fragment, a F(ab')2 fragment, or a single-chain antibody (scFv). The antibody may also be provided in a state where it is immobilized on a solid phase carrier such as a microtiter plate or particles.

[0067] Furthermore, the test kit 3 of the present disclosure may contain, as necessary, a reagent for preparing a brain homogenate and / or a synaptoneurosome from a brain tissue slice. Furthermore, the test kit 3 of the present disclosure may contain, as necessary, a dilution or reaction buffer solution containing components necessary for measurement, a washing solution, a coloring reagent, a reaction vessel, and the like.

[0068] 8. Testing method 4 As shown in the Examples section below, the present inventors have found that the ratio of the amount of SV2B NTF to the amount of full-length SV2B in synaptoneurosomes isolated from brain tissue slices can be an index for predicting the presence or absence of severe AD. Therefore, in one embodiment of the present disclosure, a method for testing the presence or absence of severe AD is provided, which includes a step of measuring the amount of full-length SV2B and the amount of SV2B NTF in synaptoneurosomes isolated from a brain tissue slice taken from a subject, and determining the ratio of the amount of SV2B NTF to the amount of full-length SV2B (hereinafter, also referred to as "Test Method 4").

[0069] In the test method 4 of the present disclosure, the subject is a human or non-human animal to be tested for the presence or absence of severe AD, including a person suspected of having AD, an AD patient whose pathological stage has not been determined, etc. Specific examples of non-human animals include non-human mammals such as primates, rats, mice, gerbils, guinea pigs, hamsters, ferrets, rabbits, cows, horses, pigs, goats, dogs, and cats. Since the test method of the present disclosure is suitable for testing humans, the subject to be tested is preferably a human.

[0070] In the test method 4 of the present disclosure, the synaptoneurosomes to be tested can be isolated from a brain tissue slice collected by brain biopsy. The method for isolating synaptoneurosomes from a brain tissue slice is as described in the above section "6. Test method 3." The method for measuring the amount of full-length SV2B and the amount of SV2B NTF contained in synaptoneurosomes is as described in the above section "2. Test method 1."

[0071] In addition, in the test method 4 of the present disclosure, the amount of full-length SV2B and the amount of SV2B NTF in synaptoneurosomes are preferably determined as relative amounts to the amount of a loading control in a body fluid. As the loading control, for example, a housekeeping gene product such as albumin or actin can be used.

[0072] The ratio of the amount of SV2B NTF to the amount of full-length SV2B in synaptoneurosomes (ratio SV2B NTF / full-length SV2B) correlates with the presence or absence of severe AD, and the ratio is higher in subjects with severe AD than in subjects without AD and subjects with mild AD. Therefore, in the test method 4 of the present disclosure, subjects with a high ratio in synaptoneurosomes are determined to be likely to have severe AD, and subjects with a low ratio in synaptoneurosomes are determined to be likely not to have severe AD.

[0073] In the test method 4 of the present disclosure, the presence or absence of severe AD based on the ratio SV2B NTF / SV2B in synaptoneurosomes can be determined by comparing with a reference value previously obtained from patients with severe AD, patients with mild AD, or patients without AD. Here, the reference value is a value that serves as a standard for determining the presence or absence of severe AD, and specifically, is the average or median value of the ratio SV2B NTF / full-length SV2B in synaptoneurosomes of patients with severe AD, patients with mild AD, or patients without AD, or a cutoff value obtained from the ratio in synaptoneurosomes of patients with severe AD and patients with mild AD or patients without AD, or the like.

[0074] For example, the average or median of the ratio SV2B NTF / full-length SV2B in the synaptoneurosomes of mild AD patients or non-AD patients is obtained in advance and used as a reference value. If the ratio SV2B NTF / full-length SV2B in the synaptoneurosomes of the subject is equal to or less than the reference value, it can be determined that the subject is highly likely to not have severe AD. Also, for example, the average or median of the ratio SV2B NTF / full-length SV2B in the synaptoneurosomes of severe AD patients is obtained in advance and used as a reference value. If the ratio SV2B NTF / full-length SV2B in the synaptoneurosomes of the subject is equal to or more than the reference value, it can be determined that the subject is highly likely to have severe AD. Furthermore, for example, the average or median ratio SV2B NTF / full-length SV2B in synaptoneurosomes of subjects with mild AD or not with AD, and the average or median ratio SV2B NTF / full-length SV2B in synaptoneurosomes of subjects with severe AD are determined in advance, and a cutoff value is determined based on these ratios SV2B NTF / full-length SV2B to distinguish the presence or absence of severe AD.If the ratio SV2B NTF / full-length SV2B in the synaptoneurosomes of a subject is equal to or greater than the cutoff value, it can be determined that the subject is highly likely to be suffering from severe AD.

[0075] Since the testing method 4 disclosed herein can be performed as a test to assist in the diagnosis of severe AD, it is desirable to make a more accurate diagnosis of the presence or absence of severe AD for a subject determined to be highly likely to have severe AD by the testing method 4 disclosed herein based on the results of clinical symptoms, image findings, etc.

[0076] 9. Test Kit 4 Another embodiment of the present disclosure is a kit for testing the presence or absence of severe AD, which includes a reagent for measuring full-length SV2B and SV2B NTF in synaptoneurosomes isolated from a brain tissue slice (hereinafter, also referred to as "Test Kit 4"). Test Kit 4 of the present disclosure is a test kit used to carry out Test Method 2 of the present disclosure, and the contents described in the above section "8. Test Method 4" are also incorporated by reference in Test Kit 4 of the present disclosure.

[0077] The reagents for measuring full-length SV2B and SV2B NTF are as described above in sections "3. Test kit 1" and "7. Test kit 3."

[0078] In addition, the test kit 4 of the present disclosure may contain a reagent for isolating synaptoneurosomes from a brain tissue slice, if necessary. Furthermore, the test kit 4 of the present disclosure may contain a dilution or reaction buffer solution containing components necessary for measurement, a washing solution, a coloring reagent, a reaction vessel, and the like, if necessary. EXAMPLES

[0079] The present disclosure is not limited in any way to the above-described embodiment of the invention or the following description of the examples. Various modifications that do not deviate from the scope of the claims and that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of the documents and the like shown in this specification are hereby incorporated by reference in their entirety.

[0080] Test Example 1: Verification that the amount of SV2B in brain tissue slices is a biomarker for AD (1) Sample Human autopsy brain samples were provided from six non-AD patients, six moderate AD patients, and five severe AD patients from the brain bank managed by the Sawarabi Medical Corporation Fukushimura Hospital. The pathological stages of the AD patients were classified according to the CERAD score and NFT Braak stage. The characteristics of each sample are shown in Table 1.

[0081] [Table 1]

[0082] (2) Method for measuring full-length SV2B, SV2B NTF, PSD95, SV2A, and Syt1 in brain homogenates and synaptoneurosomes The supramarginal gyrus of each human autopsy brain was mixed with homogenate buffer and homogenized, then filtered through an 80 μm filter to obtain a total homogenate, which was then further filtered through a 5 μm filter to obtain a homogenate rich in synaptoneurosomes (SNS). The SNS-rich homogenate was centrifuged at 1,000 g for 10 minutes to obtain an SNS pellet. The total homogenate and the SNS pellet were dissolved in a tissue lysis solution containing a final 1% SDS (sodium dodecyl sulfate), and the SDS-solubilized fractions, the total lysate and the SNS fraction, were collected and subjected to Western blotting. In Western blotting, the tissue was stained with labeled antibodies that bind to full-length SV2B, SV2B NTF, PSD95, SV2A, and Syt1. Staining of full-length SV2B and SV2B NTF was performed using a labeled anti-SV2B antibody (#119 102, Polyclonal rabbit antibody, Synaptic Systems) that binds to the region 2-17 from the N-terminus of SV2B. The signal intensity of each protein band was quantified by densitometry, and the expression level of each protein was normalized to the expression level of actin, which was used as a loading control.

[0083] (3) Results The results of measuring SV2B, PSD95, SV2A, and Syt1 in the total lysate and SNS fraction using human autopsy brains from 6 non-AD, 6 moderate AD, and 5 severe AD patients are shown in Figure 1. The expression levels of SV2A and Syt1 in the total lysate and SNS fraction were not significantly different between non-AD and AD patients. The expression level of PSD95 in the total lysate was also not significantly different between non-AD and AD patients, and the expression level of PSD95 in the SNS fraction was significantly lower in severe AD patients than in non-AD patients. On the other hand, the expression level of SV2B in the total lysate was significantly lower in AD patients than in non-AD patients. The expression level of SV2B in the SNS fraction was also significantly lower in severe AD patients than in non-AD patients. These results indicate that the expression level of SV2B in AD brains decreases earlier than that of other major synaptic proteins, making it possible for SV2B to be an indicator of whether or not the patient has AD.

[0084] The ratio of SV2B NTF to full-length SV2B in the SNS was measured using human autopsy brains from five non-AD, five moderate AD, and four severe AD patients. The ratio of SV2B NTF to full-length SV2B in the SNS was not significantly different between non-AD and moderate AD patients, but was significantly higher in severe AD patients than in non-AD and moderate AD patients. In other words, it was revealed that the ratio of SV2B NTF to full-length SV2B in the SNS can be an indicator of the presence or absence of severe AD.

[0085] Test Example 2: Verification that SV2B NTF in cerebrospinal fluid is a biomarker for AD (1) Sample Human cerebrospinal fluid samples from 13 AD patients and 13 non-AD patients stored at Kyoto University Hospital were used. The presence or absence of AD was diagnosed by the ratio of Aβ40 / 42, which are AD core biomarkers, and the concentration of p-tau. Table 2 shows the characteristics of each sample.

[0086] [Table 2]

[0087] In addition, the frontal lobe of a human autopsy brain from a non-AD patient provided by the brain bank managed by the Sawarabikai Welfare Village Hospital was used as a positive control.

[0088] In addition, cerebral cortices were collected from wild-type mice and SV2B knockout mice (SV2B KO mice) (both 32-week-old females of the 129P2 / OlaHsd strain) and used as positive and negative controls, respectively.

[0089] (2) Measurement method of SV2B and SV2B NTF in cerebrospinal fluid and brain homogenate Human cerebrospinal fluid was filtered through a centrifugal ultrafiltration filter (Amicon R The tissue was concentrated using Ultra Centrifugal Filter Devices 10 kDa (#UFC501096). Brain homogenates were prepared under the same conditions as in Test Example 1 using the frontal lobe of a human autopsy brain and mouse cerebral cortex (live-type mouse and SV2B KO mouse).

[0090] The obtained cerebrospinal fluid concentrate and brain homogenates (human, wild-type mouse, and SV2B KO mouse) were dissolved in Tris-HCl buffer containing 1% SDS (sodium dodecyl sulfate) and subjected to Western blotting. In Western blotting, both full-length SV2B and SV2B NTF were stained using a labeled anti-SV2B antibody (#119 102, Polyclonal rabbit antibody, Synaptic Systems) that binds to the region 2-17 from the N-terminus of SV2B. In addition, albumin was stained using Ponceau S staining solution in Western blotting. The amount of full-length SV2B and SV2B NTF was quantified by densitometry and normalized to the expression level of albumin.

[0091] (3) Results The results are shown in Figure 3. The amount of SV2B NTF in the cerebrospinal fluid was significantly higher in AD patients than in non-AD patients. In other words, this result demonstrated that the amount of SV2B NTF in the cerebrospinal fluid can be an indicator of the presence or absence of AD.

[0092] Test Example 3: Verification that SV2B NTF in cerebrospinal fluid is a biomarker for predicting the risk of cognitive decline or the risk of developing cognitive impairment in patients with AD (1) Method A one-year follow-up study was conducted on 25 AD patients (humans) in the mild cognitive impairment stage to examine the degree of cognitive decline. First, cerebrospinal fluid was collected from each AD patient at the start of the follow-up study (baseline), and the concentrations of Aβ40, Aβ42, and p-tau in the cerebrospinal fluid were measured. Furthermore, the amount of SV2B NTF in the cerebrospinal fluid was measured under the same conditions as in Test Example 2. In addition, at the start of the follow-up study (baseline) and one year after the start of the follow-up study, each AD patient was administered the Mini Mental State Examination (MMSE) to examine cognitive function.

[0093] (2) Stratification according to the degree of cognitive decline Patients with AD whose MMSE score change after one year was 0 or more were classified as the stable group (12 cases), and those whose MMSE score change after one year was less than -1 were classified as the progressive group (13 cases). Table 3 shows the characteristics of each stratified group.

[0094] [Table 3]

[0095] (3) Comparison of SV2B NTF levels in cerebrospinal fluid between stable and progressive disease The results of comparing the amount of SV2B NTF in the cerebrospinal fluid between the stable group and the progressive group are shown in Figure 4. As a result, it was confirmed that the amount of SV2B NTF in the cerebrospinal fluid of the progressive group was significantly higher than that of the stable group. In other words, it was revealed that the amount of SV2B NTF in the cerebrospinal fluid can be an indicator for predicting the future risk of cognitive decline or the risk of developing cognitive impairment in AD patients.

Claims

1. A method for examining a future risk of progression or onset of cognitive impairment in an Alzheimer's disease patient, comprising: A testing method comprising a step of measuring the amount of N-terminal fragment of synaptic vesicle protein 2B in body fluid collected from a patient with Alzheimer's disease.

2. The method according to claim 1 , wherein the body fluid is cerebrospinal fluid.

3. The method of claim 1 or 2, wherein the patient with Alzheimer's disease is at a stage of mild cognitive impairment.

4. A kit for examining a future risk of progression or onset of cognitive impairment in an Alzheimer's disease patient, comprising: A test kit comprising a reagent for measuring the amount of N-terminal fragment of synaptic vesicle protein 2B in a body fluid.

5. The test kit according to claim 4, wherein the reagent is an antibody that exhibits binding to the N-terminal fragment of synaptic vesicle protein 2B.

6. A method for testing for the presence or absence of Alzheimer's disease, comprising: A testing method comprising a step of measuring the amount of N-terminal fragment of synaptic vesicle protein 2B in a body fluid collected from a subject.

7. The method according to claim 6 , wherein the body fluid is cerebrospinal fluid.

8. A kit for testing for the presence or absence of Alzheimer's disease, comprising: A test kit comprising a reagent for measuring the amount of N-terminal fragment of synaptic vesicle protein 2B in a body fluid.

9. The test kit according to claim 8, wherein the reagent is an antibody that exhibits binding to an N-terminal fragment of synaptic vesicle protein 2B.

10. A method for testing for the presence or absence of Alzheimer's disease, comprising: A testing method comprising a step of measuring the amount of synaptic vesicle protein 2B in a brain tissue slice taken from a subject.

11. The examination method according to claim 10, wherein the amount of synaptic vesicle protein 2B in the brain tissue slice is measured using a brain homogenate or synaptoneurosome prepared from the brain tissue slice as a sample.

12. The amount of synaptic vesicle protein 2B in the brain tissue slice is measured using a brain homogenate prepared from the brain tissue slice as a sample; and The method according to claim 10 or 11, which is performed to examine the presence or absence of Alzheimer's disease, whose pathological stage is moderate to severe.

13. A kit for testing for the presence or absence of Alzheimer's disease, comprising: A test kit containing a reagent for measuring synaptic vesicle protein 2B in brain tissue slices.

14. The test kit according to claim 13, wherein the reagent is an antibody that exhibits binding to synaptic vesicle protein 2B.

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