Method of determining disease caused by, or accompanied by, synaptic dysfunction

Abstract

To provide a method of determining the presence or absence of onset or severity of a disease caused by or accompanied by synaptic dysfunction in an early and simple manner, and to provide a screening method for a therapeutic or preventive agent for diseases caused by or accompanied by synaptic dysfunction.SOLUTION: With the method of determining onset and severity of diseases caused by or accompanied by synaptic dysfunction using Drebrin A-related proteins (DARPs) as an indicator and the screening method for therapeutic and preventive agents for diseases caused by or accompanied by synaptic dysfunction using Drebrin A-related proteins (DARPs) as an indicator, in addition to providing a determination method that enables early and simple determination of these diseases, it can contribute to drug discovery research for these diseases.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for determining early and simply the presence or absence of onset and severity of a disease caused by or accompanied by synaptic dysfunction, a method for screening for preventive and / or therapeutic agents for a disease caused by or accompanied by synaptic dysfunction, antibodies for use in the determination method and screening method, and a kit for performing the determination method. [Background technology]

[0002] Alzheimer's disease, the main cause of dementia, is an irreversible progressive brain disease that is believed to occur when synapses in the brain become dysfunctional. The increase in the number of Alzheimer's disease patients has become a major social concern in recent years, and the establishment of an early diagnosis method and therapeutic drug is desired. Senile plaques have been observed in the postmortem brains of Alzheimer's disease patients, and these are known to be aggregates of "amyloid β protein" (amyloid plaques). It has been reported that the deposition of amyloid β protein is the earliest pathologically identifiable lesion, and that amyloid β protein aggregates and directly exhibits neuronal toxicity. It is now widely accepted that abnormalities in the production and accumulation of amyloid β protein are widely related to the onset of Alzheimer's disease based on genetic analysis of familial Alzheimer's disease patients. This is called the amyloid cascade hypothesis. Thus, it is widely accepted through numerous studies that amyloid β protein is the main cause of Alzheimer's disease (Non-Patent Document 1). It is also known that accumulation of amyloid beta protein in the brain begins more than 20 years before the onset of the disease.

[0003] In addition to Alzheimer's disease, it is known that synaptic dysfunction occurs due to decreased cerebral blood flow (depression, etc.), cerebral hemorrhage, cerebral infarction, etc. Furthermore, mitochondrial genetic diseases and dementia due to diabetes are also known. Synaptic dysfunction may also occur due to drug addiction. It is believed that cognitive function is reduced due to synaptic dysfunction in these diseases caused by or accompanied by synaptic dysfunction, but it is difficult to diagnose them. For example, Alzheimer's disease can only be diagnosed by performing a pathological autopsy after death, and diagnoses during life are performed by questionnaire tests and imaging tests, but both are diagnoses after the onset of subjective symptoms, and early treatment cannot be started. In addition, it has been reported that Alzheimer's disease can be diagnosed by a decrease in the concentration of amyloid β42 protein in cerebrospinal fluid (Non-Patent Document 2), but there is a problem that it is a highly invasive test and difficult to apply to elderly people. In addition, all of the drugs currently used to treat Alzheimer's disease are drugs that suppress symptoms, and there are no drugs that lead to fundamental treatment. In addition, for other diseases caused by or accompanied by synaptic dysfunction, we rely on questionnaire tests (depression, dementia, etc.) or imaging tests (cerebral hemorrhage, etc.), and there is no method for simple and early diagnosis.

[0004] The present inventors were the first in the world to discover drebrin, an actin-binding protein that is expressed in large amounts in developing nerve cells (see, for example, Non-Patent Documents 3 and 4). It has already been demonstrated that drebrin is involved in the morphogenesis of nerve cells, particularly process formation, by changing the properties of actin fibers (see, for example, Non-Patent Documents 5 to 7), and that in developing and migrating nerve cells, it is present in the cell body and the entire process, but in mature nerve cells, it is specifically present in the spine structure (dendritic spine) (see, for example, Non-Patent Documents 8 to 10). There are two isoforms of drebrin, embryonic type drebrin E and adult type drebrin A (see, for example, Non-Patent Document 4), and drebrin A, which is specifically found in the dendritic spine of mature nerve cells, is characterized by being expressed only in nerve cells (see, for example, Non-Patent Documents 9 and 10). The present inventors have further reported that drebrin in dendritic spines is widely lost in dementia diseases such as Alzheimer's disease (Non-Patent Document 11). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takashi Saito, Takaomi Saido, "Alzheimer's disease model mouse", Folia Pharmacol. Jpn. 144, 250-252, 2014 [Non-Patent Document 2] J. Neurol. Sci. 148, 41-45, 1997 [Non-Patent Document 3] J. Neurochem. 44, 1210-1216, 1985 [Non-Patent Document 4] J. Biochem. 117, 231-236, 1995 [Non-Patent Document 5] J. Neurosci. Res. 38, 149-159, 1994 [Non-Patent Document 6] Exp. Cell Res. 215, 145-153, 1994 [Non-Patent Document 7] J. Biol. Chem. 269, 29928-29933, 1994 [Non-Patent Document 8] J. Neurosci. 15, 7161-7170, 1996 [Non-Patent Document 9] Dev. Brain Res. 29, 233-244, 1986 [Non-Patent Document 10] Brain Res. 413, 374-378, 1987 [Non-Patent Document 11] J Neurosci. Res. 43(1), 87-92, 1996 Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, at present, it is not possible to easily and quickly determine diseases caused by or associated with synaptic dysfunction. Furthermore, it is not possible to directly treat synaptic dysfunction, and for example, in the treatment of Alzheimer's disease, a fundamental treatment has not been achieved, and symptomatic treatment is currently being performed. The present invention has been made in consideration of such circumstances, and aims to provide a method for easily and quickly determining the presence or absence of onset and severity of diseases caused by or associated with synaptic dysfunction, and to provide a method for screening therapeutic agents and preventive agents for diseases caused by or associated with synaptic dysfunction. [Means for solving the problem]

[0007] Based on the finding that drebrin A in dendritic spines is widely lost in dementia diseases such as Alzheimer's disease, the present inventors considered that drebrin A leaks from neurons into the blood in Alzheimer's disease patients. Therefore, in order to detect drebrin A in the blood, protein A / G bead treatment was performed to remove antibodies in the blood according to a conventional method, and then Western blotting was performed using an anti-drebrin antibody that recognizes drebrin A and drebrin E, but no drebrin band was confirmed. As a precaution, the present inventors subjected the precipitated fraction of the blood sample using protein A / G beads to Western blotting using an anti-drebrin antibody, and contrary to expectations, a drebrin band was confirmed at a position of about 130 kDa. In order to confirm whether this drebrin band is drebrin A, Western blotting was performed with anti-drebrin A antibody on the precipitated fraction of the blood sample using protein A / G beads. Although the band of about 130 kDa was not full-length drebrin A but drebrin E, multiple bands with molecular weights smaller than full-length drebrin A that could be detected with anti-drebrin A antibody were detected. The band detected here was considered to be a fragment of drebrin A having a translated peptide of the Ins2 sequence characteristic of the drebrin A gene, or a splice variant. The present inventors further investigated and found that such drebrin A-related proteins (DARPs: Drebrin A Related Proteins) are present in large amounts in biological samples from patients suffering from diseases caused by or accompanied by synaptic dysfunction. Furthermore, the present inventors have found that DARPs bind to protein A / G beads via autoantibodies, that autoantibodies that recognize drebrin can damage nerve cells, and that diseases caused by or associated with synaptic dysfunction can be treated or prevented by suppressing damage to nerve cells by autoantibodies. The present invention was completed based on these findings.

[0008] That is, the present invention relates to the following. [1] A method for determining the risk of developing a disease caused by or accompanied by synaptic dysfunction, or the presence or absence of the disease, comprising the following steps (a-1) to (c-1): (a-1) measuring the concentration of drebrin A-related proteins (DARPs) in a biological sample collected from a subject; (b-1) comparing the concentration of DARPs measured in step (a-1) with the concentration of DARPs in a control subject who does not suffer from a disease caused by or associated with synaptic dysfunction; (c-1) a step of evaluating, when the concentration of DARPs measured in step (a-1) is higher than the concentration of DARPs in the control subject, that the subject is at high risk of developing a disease caused by or accompanied by synaptic dysfunction, or is likely to have developed a disease caused by or accompanied by synaptic dysfunction; [2] a method for determining the severity of a disease caused by or accompanied by synaptic dysfunction, characterized by comprising the following steps (a-2) to (c-2). (a-2) measuring the concentration of drebrin A-related proteins (DARPs) in a biological sample collected from a subject suffering from a disease caused by or accompanied by synaptic dysfunction; (b-2) comparing the concentration of DARPs measured in step (a-2) with the concentration of DARPs in a control subject suffering from a disease caused by or associated with synaptic dysfunction; (c-2) a step of evaluating that the subject is more likely to suffer from a more severe disease caused by or associated with synaptic dysfunction than the control, when the concentration of DARPs and / or DARPs autoantibodies measured in step (a-2) is higher than the concentration of DARPs and / or DARPs autoantibodies in the control; [3] A method for screening a preventive agent for a disease caused by or accompanied by synaptic dysfunction, or a therapeutic agent for a disease caused by or accompanied by synaptic dysfunction, comprising the following steps (a-3) to (c-3): (a-3) measuring the concentration of drebrin A-related proteins (DARPs) in a sample collected from a non-human animal model of synaptic dysfunction to which a test substance has been administered; (b-3) a step of comparing the concentration of drebrin A-related proteins (DARPs) measured in the step (a-3) with the concentration of drebrin A-related proteins (DARPs) in a non-human animal model of synapse dysfunction to which the test substance is not administered; (c-3) a step of evaluating that the test substance is effective for preventing a disease caused by or associated with synaptic dysfunction, or for treating a disease caused by or associated with synaptic dysfunction, when the concentration of drebrin A-related proteins (DARPs) measured in the step (a-3) is lower than the concentration of drebrin A-related proteins (DARPs) in a non-human animal model of synaptic dysfunction to which the test substance is not administered; [4] A method for screening a preventive agent for a disease caused by or accompanied by synaptic dysfunction, or a therapeutic agent for a disease caused by or accompanied by synaptic dysfunction, comprising the following steps (a-4) to (d-4): (a-4) administering a test substance to a cultured cell model of synapse dysfunction and culturing the cell; (b-4) measuring the concentration of drebrin A-related proteins (DARPs) in the culture medium of the synapse dysfunction model cultured cells; (c-4) comparing the concentration of DARPs measured in the step (b-4) with the concentration of DARPs in a culture medium of a cultured cell model of synapse dysfunction to which the test substance is not administered; (d-4) a step of evaluating, when the concentration of DARPs measured in step (b-4) is lower than the concentration of DARPs in a culture medium of a cultured cell model of synapse dysfunction to which the test substance is not administered, that the test substance is effective in preventing a disease caused by or associated with synapse dysfunction or in treating a disease caused by or associated with synapse dysfunction; [5] The method according to any one of [1] to [3] above, wherein the biological sample is a cerebrospinal fluid sample or a blood sample. [6] The method according to any one of [1] to [5] above, wherein the DARPs is one or more selected from DARP40, DARP60, DARP70, DARP90, and DARP100. [7] The method according to any one of [1] to [6] above, wherein the disease caused by or accompanied by synaptic dysfunction is a neurodegenerative disease. [8] The method according to [7] above, wherein the neurodegenerative disease is selected from Alzheimer's disease, corticobasal syndrome, Parkinson's disease, spinocerebellar degeneration and amyotrophic lateral sclerosis. [9] The method according to any one of [1] to [8] above, wherein the concentration of DARPs is measured using an antibody that recognizes a drebrin A-specific epitope.

[10] An antibody for use in the method according to [9] above, which recognizes a drebrin A-specific epitope.

[11] A kit for determining the risk of developing, the presence or absence of developing, or the severity of a disease caused by or associated with synaptic dysfunction, characterized by comprising the antibody described in

[10] above.

[12] A method for screening a preventive agent for a disease caused by or accompanied by synaptic dysfunction, or a therapeutic agent for a disease caused by or accompanied by synaptic dysfunction, comprising searching for a dominant negative peptide against an autoantibody against drebrin A-related proteins (DARPs).

[0009] In another embodiment of the present invention, A method for determining the risk of developing a disease caused by or accompanied by synaptic dysfunction, or the presence or absence of the disease, comprising a step (a-1) of measuring the concentration of drebrin A-related proteins (DARPs) in a biological sample collected from a subject; or A method for determining the severity of a disease caused by or associated with synaptic dysfunction, comprising the step (a-2) of measuring the concentration of drebrin A-related proteins (DARPs) in a biological sample collected from a subject suffering from a disease caused by or associated with synaptic dysfunction; or A biomarker for determining the risk of developing a disease caused by or associated with synaptic dysfunction, or the presence or absence of development of the disease, or for determining the severity of a disease caused by or associated with synaptic dysfunction, comprising one or more drebrin A-related proteins (DARPs); or A method for screening a preventive agent for a disease caused by or associated with synaptic dysfunction, or a therapeutic agent for a disease caused by or associated with synaptic dysfunction, comprising a step (a-3) of measuring the concentration of drebrin A-related proteins (DARPs) in a sample collected from a non-human animal model of synaptic dysfunction to which a test substance has been administered; or A method for screening a preventive agent for a disease caused by or associated with synaptic dysfunction, or a therapeutic agent for a disease caused by or associated with synaptic dysfunction, comprising the above steps (a-4) and (b-4); or A method for collecting data for diagnosing the risk of developing a disease caused by or accompanied by synaptic dysfunction, or the presence or absence of the disease, comprising the steps (a-1) to (c-1); or A method for collecting data for diagnosing the severity of a disease caused by or accompanied by synaptic dysfunction, comprising the steps (a-2) to (c-2); or A method for determining the effectiveness of a preventive agent for a disease caused by or accompanied by synaptic dysfunction, or a therapeutic agent for a disease caused by or accompanied by synaptic dysfunction, comprising the above steps (a-3) to (c-3); or A method for determining the effectiveness of a preventive agent for a disease caused by or accompanied by synaptic dysfunction, or a therapeutic agent for a disease caused by or accompanied by synaptic dysfunction, comprising the above steps (a-4) to (d-4); or A preventive agent for a disease caused by or accompanied by synaptic dysfunction, or a therapeutic agent for a disease caused by or accompanied by synaptic dysfunction, comprising a dominant negative peptide against drebrin A-related proteins (DARPs) autoantibody; The following can be mentioned. Effect of the Invention

[0010] According to the determination method of the present invention, the presence or absence and severity of diseases caused by or accompanied by synaptic dysfunction, which have previously relied on question-based tests (depression, dementia, etc.) or imaging tests (cerebral hemorrhage, etc.), can be determined early and simply, leading to the prevention of the onset of these diseases and early treatment. Furthermore, DARPs and anti-DARPs autoantibodies can also serve as screening indicators for preventive and therapeutic agents, and can contribute to drug discovery research. [Brief description of the drawings]

[0011] [Figure 1] Upper panel: Results of Western blotting analysis of the beads absorbed fraction, plasma, and preclear plasma in Example 1. Lower panel: Results of Western blotting analysis of each IP fraction in Example 1. [Diagram 2] FIG. 1 shows the results of Western blotting performed on the bead-adsorbed fraction in Example 1 using various anti-drebrin antibodies (M2F6, 28D8, 3D9, 22G5, 17C3, 33A4: homemade) and anti-drebrin A antibodies (4C2: homemade; DAS2: Immunobiological Laboratories). [Diagram 3]FIG. 1 shows the results of Western blotting of blood samples obtained from two Alzheimer's disease patients (AD01, AD02), one corticobasal syndrome patient (CBS), one Parkinson's disease patient (PD01), and a healthy 65-year-old male (Healthy) in Example 2, using anti-drebrin A antibody DAS2 (Immunobiological Laboratories) on the adsorption fraction of protein A / G beads. [Figure 4] FIG. 1 shows the results of detection of DARPs by Western blotting using AD02 and control plasma (Ctrl) in Example 2. The concentrations of all of DARPs, DARP40, DARP60, DARP70, DARP90, and DARP100, were increased in AD02. [Diagram 5] This is a diagram showing the results of Western blotting of blood samples obtained from two Alzheimer's disease patients (AD01, AD02), one corticobasal syndrome patient (CBS), one Parkinson's disease patient (PD01), and a healthy 65-year-old male (Healthy) in Example 2, using anti-drebrin antibody 3D9 (homemade) for the adsorption fraction of protein A / G beads. Unlike Figure 3, no difference was found between the samples. [Figure 6] FIG. 13 shows the results of Western blotting of cerebrospinal fluid samples obtained from three Alzheimer's disease patients (GHAD01 to 03, all with mild dementia), one amyotrophic lateral sclerosis patient (ALS01), one spinocerebellar degeneration patient (SCD01), and one depression patient (Dep01) in Example 3, using anti-drebrin A antibody DAS2 (Immunobiological Laboratories) on the adsorption fraction of protein A / G beads. [Figure 7] FIG. 1 shows the results of detecting DARPs in cerebrospinal fluid samples by Western blotting for six Alzheimer's disease patients listed in Table 1 in Example 4. [Figure 8]FIG. 1 shows the results of subjecting GFP-tagged human drebrin A (GFP-hDA) in Example 5 to SDS-PAGE and detecting it by Western blotting using an anti-drebrin antibody (3D9: homemade) (left) and plasma derived from an Alzheimer's disease patient (ADplasma) (right) as primary antibodies. [Figure 9] This is a diagram showing the results of confirming whether an anti-drebrin antibody (22G5: homemade) can have toxicity to cultured rat hippocampal cells in Example 5. (A) shows cultured rat hippocampal cells treated with 22G5, and (B) shows cultured rat hippocampal cells treated with normal mouse IgG. [Figure 10] FIG. 13 shows the results of Western blotting in Example 6, which confirmed the presence of DARPs in cultured rat cerebral cortical cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention relates to a method for determining the risk of developing or the presence or absence of a disease caused by or associated with synaptic dysfunction, comprising the steps of: (a-1) measuring the concentration of drebrin A-related proteins (DARPs) in a biological sample collected from a subject; (b-1) comparing the concentration of DARPs measured in step (a-1) with the concentration of DARPs in a control subject not suffering from a disease caused by or associated with synaptic dysfunction; and (c-1) evaluating that, when the concentration of DARPs measured in step (a-1) is higher than the concentration of DARPs in the control subject, the subject is at high risk of developing a disease caused by or associated with synaptic dysfunction, or is highly likely to have developed a disease caused by or associated with synaptic dysfunction (hereinafter, sometimes referred to as "the present determination method 1"); The method comprises the steps of: (a-2) measuring the concentration of drebrin A-related proteins (DARPs) in a biological sample collected from a subject suffering from a disease caused by or associated with synaptic dysfunction; (b-2) comparing the concentration of DARPs measured in step (a-2) with the concentration of DARPs in a control subject suffering from a disease caused by or associated with synaptic dysfunction; and (c-2) determining whether the concentration of DARPs and / or DARPs autoantibodies measured in step (a-2) is higher than that of a control subject suffering from a disease caused by or associated with synaptic dysfunction. When the concentration of DARPs and / or DARPs autoantibodies in the subject is higher than that in the control subject, the subject is more likely to have a more severe disease caused by or associated with synaptic dysfunction than the control subject (hereinafter, this may be referred to as "the present determination method 2." Furthermore, the present determination method 1 and the present determination method 2 may be collectively referred to as "the present determination method" hereinafter). Here, "severity" includes an acute exacerbation phase in which synaptic damage progresses rapidly. In one aspect, the present determination method is a method for assisting a doctor in diagnosing the risk of onset of a disease caused by or associated with synaptic dysfunction, or diagnosing the presence or absence of onset or the severity of the disease, and may not include a diagnostic act by a doctor.

[0013] The present invention also relates to the following steps: (a-3) measuring the concentration of drebrin A-related proteins (DARPs) in a sample collected from a non-human animal model of synapse dysfunction to which a test substance has been administered; (b-3) comparing the concentration of drebrin A-related proteins (DARPs) measured in step (a-3) with the concentration of drebrin A-related proteins (DARPs) in a non-human animal model of synapse dysfunction to which the test substance has not been administered; and (c-3) determining whether the concentration of drebrin A-related proteins (DARPs) measured in step (a-3) is higher than the concentration of drebrin A-related proteins (DARPs) in a sample collected from a non-human animal model of synapse dysfunction to which the test substance has not been administered. a step of evaluating that the test substance is effective for preventing a disease caused by or associated with synaptic dysfunction or for treating a disease caused by or associated with synaptic dysfunction when the concentration of drebrin A-related proteins (DARPs) in the test substance is lower than that in a non-human animal model of synaptic dysfunction (hereinafter, referred to as "the present invention"). The present invention also relates to a screening method for synapse dysfunction model cells, which includes the steps of: (a-4) administering a test substance to a cultured cell of a synapse dysfunction model and culturing the cell; (b-4) measuring the concentration of drebrin A-related proteins (DARPs) in a culture medium of the cultured cell of the synapse dysfunction model; (c-4) comparing the concentration of DARPs measured in step (b-4) with the concentration of DARPs in a culture medium of the cultured cell of the synapse dysfunction model to which the test substance is not administered; and (d-4) determining whether the concentration of DARPs measured in step (b-4) is higher than the concentration of DARPs in a culture medium of the cultured cell of the synapse dysfunction model to which the test substance is not administered. a step of evaluating that the test substance is effective in preventing a disease caused by or accompanied by synaptic dysfunction, or in treating a disease caused by or accompanied by synaptic dysfunction, when the concentration of DARPs in the culture medium of a synaptic dysfunction model cultured cell is lower than the concentration of DARPs in the culture medium of a synaptic dysfunction model cultured cell;Furthermore, there are no particular limitations as long as the present screening method 1 and the present screening method 2 are collectively referred to as "the present screening method" hereinafter.

[0014] The present invention was completed by the first discovery that drebrin A-related proteins (DARPs), which are fragments or splice variants of human drebrin A (SEQ ID NO: 1) localized in the brain, are abundant in biological samples from patients suffering from diseases caused by or accompanied by synaptic dysfunction. In the present invention, DARPs may be fragments or splice variants of drebrin A having at least a part of the Ins2 translation sequence RPYCPFIKASDSGPSSSSSSSSSPPRTPFPYITCHRTPNLSSSLPC (SEQ ID NO: 2) characteristic of drebrin A, and preferred examples of DARPs include DARP100 with a molecular weight of about 100 kDa, DARP90 with a molecular weight of about 90 kDa, DARP70 with a molecular weight of about 70 kDa, DARP60 with a molecular weight of about 60 kDa, and DARP40 with a molecular weight of about 40 kDa. DARPs can be used alone in the present assessment method or screening method, or one or more DARPs can be used in combination in the present assessment method or screening method. In addition, the total concentration of DARPs can be measured and used in the present assessment method or screening method.

[0015] In the present invention, the DARPs concentration can be measured by any known method. In a preferred embodiment, the DARPs concentration is measured by Western blotting, ELISA (Enzyme-Linked Immunosorbent Assay), immunoprecipitation, or the like, using an antibody that recognizes DARPs (hereinafter sometimes referred to as "anti-DARPs antibody"). The anti-DARPs antibody may be either a monoclonal antibody or a polyclonal antibody, as long as it has a part of DARPs as an epitope, and may be used alone or in combination of two or more types. In addition, in order to distinguish it from drebrin E present throughout the body, it is preferable to measure the DARPs concentration using an antibody that recognizes a drebrin A-specific epitope (hereinafter sometimes referred to as "the present antibody") for use in the present determination method and / or the present screening method. Here, the term "drebrin A-specific epitope" means an epitope that is not present in drebrin E but is present in drebrin A, and is preferably a region including at least a part of the Ins2 translation sequence RPYCPFIKASDSGPSSSSSSSSSPPRTPFPYITCHRTPNLSSSLPC (SEQ ID NO: 2), and / or a specific three-dimensional structure formed because drebrin A includes the Ins2 translation sequence.

[0016] When performing Western blotting or ELISA, a method in which an anti-DARPs antibody is used as a primary antibody and detection is performed using a labeled secondary antibody that recognizes the primary antibody can be preferably used. For example, when the primary antibody is a rabbit antibody, a labeled anti-rabbit IgG antibody can be used as the secondary antibody, and when the primary antibody is a mouse antibody, a labeled anti-mouse IgG antibody can be used as the secondary antibody.

[0017] Examples of the labeling substance in the labeled secondary antibody include enzymes, radioisotopes, fluorescent substances, luminescent substances, gold colloids, and the like. Among these, enzymes are preferred from the viewpoint of sensitivity and ease of operation, and horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase (GOD), and the like are more preferred. When HRP is used as the labeling substance, TMB (3,3',5,5'-tetramethylbenzidine) and the like can be used as substrates, and when AP is used, AMPPD (3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane disodium salt), 9-(4-chlorophenylthiophosphoryloxymethylidene)-10-methylacridan disodium salt, and the like can be used as substrates. In addition, fluorescent dyes such as FITC (fluorescein isothiocyanate) and rhodamine can also be used as labeling substances.

[0018] The detection and quantification method of DARPs varies depending on the labeling method, and can be performed by a method well known and conventional to those skilled in the art. For example, when HRP, AP, GOD, etc. are used as the labeling substance, a chromogenic substrate or a luminescent substrate is added, and the absorbance or luminescence intensity is measured to quantify the substance to be measured. When a fluorescent substance is used as the labeling substance, the substance to be measured can be quantified by measuring its fluorescence intensity. When a radioisotope is used as the labeling substance, the substance to be measured can be quantified by measuring the radioactivity. When gold colloid is used as the labeling substance, the substance to be measured can be quantified by measuring the absorbance. When quantifying, for example, a calibration curve (standard curve) is prepared in advance using a sample with a known DARPs concentration, and the measured value is compared with the calibration curve to calculate the DARPs concentration in the sample, which may be used for comparison in the present determination method or the present screening method, or the absorbance, radioactivity, and luminescence intensity may be directly used for comparison in the present determination method or the present screening method without calculating the DARPs concentration. Here, in the present determination method, in order to evaluate (determine) whether the DARPs concentration in the subject (the DARPs concentration, absorbance, radioactivity, and luminescence intensity calculated above) is higher or lower than the DARPs concentration in the control subject (the DARPs concentration, absorbance, radioactivity, and luminescence intensity calculated above), any threshold (cut-off value) can be set, and examples of such thresholds include the mean value, mean value + standard deviation (SD), mean value + 2SD, mean value + 3SD, mean value -SD, mean value -2SD, mean value -3SD, median, median + SD, median + 2SD, median + 3SD, median -SD, median -2SD, and median -3SD of the DARPs concentration in the control subjects.Furthermore, in the present screening method, in order to evaluate (determine) whether the DARPs concentration (the DARPs concentration, absorbance, radioactivity, and luminescence intensity calculated above) in a non-human synapse dysfunction model animal or cultured synapse dysfunction model cell administered with a test substance is higher or lower than the DARPs concentration (the DARPs concentration, absorbance, radioactivity, and luminescence intensity calculated above) in a non-human synapse dysfunction model animal or cultured synapse dysfunction model cell not administered with the test substance, any threshold (cut-off value) can be set. Examples of such thresholds include the mean value, mean value + standard deviation (SD), mean value + 2SD, mean value + 3SD, mean value -SD, mean value -2SD, mean value -3SD, median, median + SD, median + 2SD, median + 3SD, median -SD, median -2SD, and median -3SD of the DARPs concentration in a non-human synapse dysfunction model animal or cultured synapse dysfunction model cell not administered with a test substance.

[0019] In the present invention, a biological sample means a body fluid collected from a subject or a control. Here, examples of the body fluid include blood, lymph, tissue fluid, body cavity fluid, cerebrospinal fluid, and the like, and among these, cerebrospinal fluid and blood are preferred. Examples of blood include serum and plasma. The biological sample may be directly subjected to the measurement of the DARPs concentration, but is preferably pretreated before the measurement to enhance the detection sensitivity of DARPs. The pretreatment method may be any method that enhances the detection sensitivity of the protein to be measured in the biological sample, and may be, for example, immunoprecipitation using an anti-DARPs antibody. In addition, the present inventors have succeeded in enhancing the detection sensitivity of DARPs by treating the biological sample with protein A / G beads and subjecting the fraction adsorbed to the beads to the present determination method or the present screening method. The protein A / G bead treatment is performed prior to immunoprecipitation with the aim of reducing the background caused by autoantibodies, and the target protein is originally detected from the flow-through fraction after the protein A / G bead treatment. The discovery by the present inventors that DARPs are adsorbed to protein A / G beads is not only an unexpected discovery, but also suggests the presence of anti-DARPs autoantibodies in biological samples. The present inventors confirmed the presence of anti-DARPs autoantibodies in biological samples through further investigations, and further found that anti-DARPs autoantibodies can damage brain nerve cells. These findings indicate that dominant-negative peptides against anti-DARPs autoantibodies can suppress damage to brain nerve cells caused by anti-DARPs autoantibodies and treat and / or prevent diseases caused by or associated with synaptic dysfunction. Therefore, in one aspect, the present invention relates to a method for screening a preventive agent for a disease caused by or associated with synaptic dysfunction, or a therapeutic agent for a disease caused by or associated with synaptic dysfunction, characterized by searching for a dominant-negative peptide against anti-DARPs autoantibody.

[0020] In the present invention, a disease caused by or accompanied by synaptic dysfunction refers to a disease caused by synaptic dysfunction, i.e., a disorder in information transmission at synapses (Alzheimer's disease, vascular dementia, dementia with Lewy bodies, Parkinson's disease, corticobasal degeneration, amyotrophic lateral sclerosis, spinocerebellar degeneration, etc.), or a disease in which synaptic dysfunction occurs after the onset of a disease (cerebral hemorrhage, cerebral infarction, brain tumor, etc.). Depression caused by reduced cerebral blood flow, drug addiction, mitochondrial genetic disease, diabetes, etc. are also known to be accompanied by synaptic dysfunction and to cause cognitive function to decrease, and are included in diseases caused by or accompanied by synaptic dysfunction. Furthermore, the present determination method can also be used to evaluate sequelae in the event of trauma to the brain. Preferably, the disease caused by or associated with synaptic dysfunction refers to a neurodegenerative disease, and more preferably a neurodegenerative disease selected from Alzheimer's disease, corticobasal syndrome, Parkinson's disease, spinocerebellar degeneration and amyotrophic lateral sclerosis.

[0021] In the present determination method 1, when determining the risk of developing a disease caused by or associated with synaptic dysfunction, the subject may be a subject who has not developed a disease caused by or associated with synaptic dysfunction and is not known whether or not he or she will develop a disease caused by or associated with synaptic dysfunction in the future. In addition, when determining whether or not he or she will develop a disease caused by or associated with synaptic dysfunction, the subject may be a subject who has not developed a disease caused by or associated with synaptic dysfunction in the future. Preferably, the subject is evaluated (determined) as having a high risk of developing a disease caused by or associated with synaptic dysfunction by the method for determining the risk of developing the present invention. In the present determination method 1, the control refers to a healthy person who has a low or almost no risk of developing a disease caused by or associated with synaptic dysfunction and has not developed a disease caused by or associated with synaptic dysfunction. In addition, when setting the above threshold, at least one of the following can be used as controls: (i) all healthy subjects whose DARPs concentrations have been measured, or healthy subjects selected at random, (ii) healthy subjects of the same age or age range as the subject (the amount of drebrin in the brain decreases with age), and (iii) subjects before the onset of disease. The threshold set based on the DARPs concentrations of these controls can be used to distinguish the acute exacerbation phase of synaptic dysfunction.

[0022] The subject in the present determination method 2 may be, for example, a subject who has developed a disease caused by or accompanied by synaptic dysfunction, but the severity of the disease caused by or accompanied by synaptic dysfunction is unknown. The control in the present determination method 2 means, for example, a patient who has developed a disease caused by or accompanied by synaptic dysfunction, and whose severity has been determined. Here, the "patient whose severity has been determined" may be a patient whose severity has been determined based on a known treatment guideline. As the known treatment guideline, it is preferable to use a treatment guideline for the target disease, and examples of such treatment guidelines include treatment guidelines for dementia, and treatment guidelines for cerebral hemorrhage and cerebral infarction. For example, in dementia due to Alzheimer's disease, dementia is divided into early, middle, and late stages by interview, etc., and DARPs in plasma are thought to be maximum from the early to middle stages of onset. In fact, it has been shown that DARPs are rather reduced after widespread neuronal death occurs in the later stages (severe dementia patients) (AD01 patient in Example 2). In addition, in cerebral hemorrhage, DARPs are thought to be high for a while after the hemorrhage occurs, and then, over time, in the chronic phase, they decrease and reach a constant value. As such, the relationship between changes in DARPs in control subjects and severity differs depending on the target disease, so it is preferable to judge the severity according to appropriate clinical guidelines.

[0023] The non-human animal with synaptic dysfunction in the present screening method 1 may be a non-human animal that naturally develops synaptic dysfunction, or a non-human animal in which the onset of synaptic dysfunction has been induced. Specifically, the non-human animal in which synaptic dysfunction has been induced may be a model non-human animal for a disease caused by or accompanied by the above-mentioned synaptic dysfunction. Examples of the above-mentioned non-human animal include non-human mammals such as mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats, and dogs.

[0024] The cultured cells as a model of synaptic dysfunction in the present screening method 2 may be cultured cells in which synaptic dysfunction naturally occurs, or cultured cells in which synaptic dysfunction has been induced. Specifically, the cultured cells in which the onset of synaptic dysfunction has been induced may be model cultured cells of a disease caused by or accompanied by the above-mentioned synaptic dysfunction. As the cultured cells, preferred examples include cultured nerve cells, and examples thereof include cultured cells derived from humans, as well as cultured cells derived from non-human mammals such as mice, rats, hamsters, guinea pigs, monkeys, cows, pigs, horses, rabbits, sheep, goats, cats, and dogs.

[0025] In one aspect, the present invention relates to a kit (hereinafter, sometimes referred to as "the present kit") for determining the risk of developing, the presence or absence of developing, or the severity of a disease caused by or accompanied by synaptic dysfunction, comprising the present antibody. In a preferred aspect, the present kit is a kit for use in the present determination method. In addition to the present antibody, the present kit may also include a means for obtaining a biological sample from a subject, a labeled secondary antibody that recognizes the present antibody, and an accompanying document describing the procedure and diagnostic criteria.

[0026] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0027] 1. Detection of DARPs in blood samples The present inventors assumed that drebrin exists in the plasma of Alzheimer's disease patients, and performed Western blotting using anti-drebrin antibody on the plasma, but could not detect drebrin. Therefore, in order to remove the antibody components contaminating the plasma, the plasma was treated with protein A / G beads and subjected to Western blotting.

[0028] The experimental procedure is as follows. (1) 1.8 ml of plasma was suspended in 200 μl of Protein A / G beads (Protein A / G PLUS-Agarose: manufactured by Santa Cruz) and reacted (pre-cleared) at 4° C. for 1 hour on a rotator. (2) Centrifuge to prepare the supernatant (preclear plasma) and sediment (Beads Absorbed fraction). (3) 30 μl of SDS sample buffer was added to the bead-adsorbed fraction, which was then heat-denatured and eluted. (4) A portion of the centrifuged supernatant (preclear plasma) is saved for input. The remaining precleared plasma was mixed with four types of antibodies (anti-drebrin antiserum DEp: homemade; anti-drebrin monoclonal antibodies 28D8 and 3D9 that recognize drebrin A and E: homemade; control IgG: Fujifilm Wako Pure Chemical Industries, Ltd.) and reacted overnight (IP: immunoprecipitation). (5) 30 μl of protein A / G beads were further suspended in the IP tube (4) above that had been reacted overnight, and the mixture was reacted on a rotator at 4°C for 1 hour. The beads were then washed, and 30 μl of SDS sample buffer was added to the bead fraction, which was then heat-denatured and eluted (IP, immunoprecipitation fraction). (6) Each sample (top row: Beads absorbed, Plasma, Preclear Plasma; bottom row: each IP image) (min) were analyzed by Western blot (a mixture of monoclonal antibodies 28D8 and 3D9 was used for detection).

[0029] The results are shown in Figure 1. Drebrin could not be detected in the western blot of the lower panel after immunoprecipitation, whereas a drebrin band was confirmed, contrary to expectations, in the protein A / G beads adsorbed fraction used for preclearing (Beads absorbed) (upper panel, arrow). Therefore, to confirm whether such band is drebrin E present throughout the body or drebrin A localized in brain synapses, western blotting was performed on the protein A / G beads adsorbed fraction using various anti-drebrin antibodies (M2F6, 28D8, 3D9, 22G5, 17C3, 33A4: homemade) that recognize both drebrin A and E, and an anti-drebrin A antibody (4C2, homemade; DAS2: Immunobiology Laboratories) that recognizes a region of drebrin A that includes at least a part of Ins2. As a result, the full-length drebrin band of approximately 130 kDa was not stained with drebrin A-specific antibodies 4C2 and DAS2, and was found to be a drebrin E band (Figure 2). This drebrin E was thought to be derived from a renal tumor. On the other hand, several bands thought to be fragments or splice variants of drebrin A, which were also stained with anti-drebrin A antibodies 4C2 and DAS2, were found on the lower molecular weight side of the drebrin E band. These were named drebrin A related proteins (DARPs). In FIG. 2, drebrin E and DARP40, which has a molecular weight of about 40 kDa among DARPs, are indicated by arrows. DARP40 was not clearly stained with control IgG or M2F6, a general antibody against drebrin, and was discovered for the first time by the present inventors. Since drebrin A is localized in dendritic spines in the brain, it was suggested that DARPs leak from damaged dendritic spines in Alzheimer's disease patients and migrate into the blood through the blood-brain barrier. EXAMPLES

[0030] 2. Use of DARPs in blood samples as diagnostic biomarkers From Example 1, it was considered that DARPs leaked from damaged dendritic spines in Alzheimer's disease patients. In other diseases caused by or accompanied by synaptic dysfunction, DARPs may leak from damaged dendritic spines and migrate into the blood. Therefore, in order to confirm whether DARPs in blood samples can be used as a diagnostic biomarker for diseases caused by or accompanied by synaptic dysfunction, the following experiment was performed.

[0031] Blood samples were obtained from two Alzheimer's disease patients (AD01 (5 years since onset, with renal tumor), AD02 (3 years since onset, MMSE score 21)), one corticobasal syndrome patient (CBS), one Parkinson's disease patient (PD01), and a healthy 65-year-old male (Healthy), and the protein A / G bead adsorption fraction was subjected to Western blotting in the same manner as in Example 1. Detection was performed using anti-drebrin A antibody DAS2 (Immunobiological Laboratories).

[0032] The results are shown in Figure 3. Many DARPs bands were found in all blood samples. The confirmed DARPs were named DARP40, DARP60, DARP70, DARP90, and DARP100 according to their molecular weights. The DARPs concentrations were higher in blood samples from disease patients, except AD01, than in a healthy 65-year-old male (Healthy). Furthermore, as shown in Figure 4, when DARPs were detected using the same procedure using AD02 and control plasma (Ctrl: Kohjin Bio (KJB), normal human plasma pool EDTA-2Na, lot: HMN389081), the concentrations of all DARPs, DARP40, DARP60, DARP70, DARP90, and DARP100, were increased in AD02. Therefore, it was shown that these DARPs can be used as diagnostic biomarkers for diseases caused by or accompanied by synaptic dysfunction. On the other hand, the DARPs concentration was lower in AD01 than in Healthy (Figure 3). This suggests that in chronic AD patients who have progressed to the final stage of dementia (high tau and poor MMSE scores) due to neuronal cell death, the rapid leakage of DARPs that occurs in the acute exacerbation stage does not occur, resulting in a lower DARPs concentration.

[0033] Furthermore, when the same sample as in Figure 3 was stained with anti-drebrin antibody 3D9 (homemade) that recognizes both drebrin A and E, no difference in the intensity of the band was observed between the disease patient and a healthy 65-year-old male (Healthy) (Figure 5). The results here show that drebrin-related proteins derived from drebrin E cannot be used as diagnostic biomarkers, and that antibodies (DAS2, etc.) that recognize a region in drebrin A that includes at least a part of Ins2 can be used to diagnose diseases. EXAMPLES

[0034] 3. Use of DARPs in cerebrospinal fluid samples as diagnostic biomarkers From Example 2, it was confirmed that DARPs in blood samples can be used as a diagnostic biomarker for diseases caused by or associated with synaptic dysfunction. Since DARPs are related to drebrin A localized in the brain, it is considered that DARPs are also present in large amounts in cerebrospinal fluid in patients with diseases caused by or associated with synaptic dysfunction. Therefore, in order to confirm that DARPs in cerebrospinal fluid samples can be used as a diagnostic biomarker for diseases caused by or associated with synaptic dysfunction, the following experiment was performed.

[0035] Cerebrospinal fluid samples were obtained from three Alzheimer's disease patients (GHAD01-03, all mild dementia), one amyotrophic lateral sclerosis patient (ALS01), one spinocerebellar degeneration patient (SCD01), and one depression patient (Dep01), and the protein A / G bead adsorption fraction was subjected to Western blotting in the same manner as in Example 2, except that the cerebrospinal fluid sample was used instead of the blood sample. Detection was performed using anti-drebrin A antibody DAS2 (Immunobiological Laboratories).

[0036] The results are shown in Figure 6. Many DARPs bands were found in all cerebrospinal fluid samples, and DARP40, DARP60, DARP70, DARP90, and DARP100 were confirmed, as in the blood samples. The DARPs concentrations were higher in two patients with Alzheimer's disease (GHAD01 and 03), one patient with amyotrophic lateral sclerosis (ALS01), and one patient with spinocerebellar degeneration (SCD01), which are neurodegenerative diseases, than in one patient with depression (Dep01). In addition, even in patients with the same Alzheimer's disease, there was a difference in DARPs concentration as shown by the comparison between GHAD01 and 03, suggesting that the DARPs concentration may reflect the severity of the disease (degree of damage to dendritic spines). On the other hand, GHAD02 had a DARPs concentration similar to that of one patient with depression (Dep01). In GHAD02, the progression of AD has shifted to the chronic phase, and rapid leakage of DARPs, as in the acute exacerbation phase, has not occurred, and it is considered that the DARPs concentration is low. In addition, the DARPs concentration in patients with depression was lower than that in other neurodegenerative diseases, but considering that depression can sometimes progress to cognitive impairment, it is considered that the transition to cognitive impairment can also be evaluated by the increase in DARPs concentration in patients with depression. EXAMPLES

[0037] 4. Relationship between DARPs levels and other indices in Alzheimer's disease patients Example 3 shows that DARPs in cerebrospinal fluid samples can be used as a diagnostic biomarker for Alzheimer's disease. On the other hand, in patients whose Alzheimer's disease has progressed to the chronic phase, rapid leakage of DARPs like that in the acute exacerbation phase does not occur, and it is considered that the DARPs concentration is low. Therefore, for six Alzheimer's disease patients listed in Table 1 below, DARPs in cerebrospinal fluid samples were detected by Western blotting in the same manner as in Example 3. In the table, "TAU↑" means that the concentration of tau is high and has led to tau accumulation, and "Aβ↑" means that the concentration of amyloid β is high and has led to amyloid β accumulation.

[0038] [Table 1]

[0039] The results are shown in Figure 7. DARP100, DARP70, and DARP40 were confirmed in cerebrospinal fluid samples from all patients. The DARPs concentration was high in patients in the exacerbation stage (case numbers 1107 to 1110) who had not yet accumulated tau, but the DARPs concentration was low in patient with case registration number 1111, who had the highest tau concentration. These results also showed that the DARPs concentration was low in patients whose Alzheimer's disease had progressed to the chronic stage. EXAMPLES

[0040] 5. Confirmation of the presence of DARPs autoantibodies and verification of neuronal toxicity Example 1 showed that DARPs were adsorbed to Protein A / G beads having the ability to bind to antibodies (IgG, IgA). In order to confirm whether such adsorption was caused by direct adsorption of DARPs to Protein A / G beads or via DARPs autoantibodies in blood samples, human drebrin A tagged with GFP (GFP-hDA: a sample expressed in HEK293 and homogenized in SDS buffer) was subjected to SDS-PAGE, and anti-drebrin antibody (3D9: homemade) and plasma (ADplasma) derived from an Alzheimer's disease patient were used as primary antibodies for detection.

[0041] The results are shown in Figure 8. A band was confirmed at the same position (arrow in the figure) in the case of detection with 3D9 (left) and in the case of detection with plasma from an Alzheimer's disease patient (AD plasma) (right). These results demonstrated that plasma derived from Alzheimer's disease patients contained autoantibodies capable of recognizing drebrin A. It is believed that DARPs were adsorbed to protein A / G beads via the autoantibodies.

[0042] Since DARPs in cerebrospinal fluid are also adsorbed to protein A / G beads, it is believed that anti-DARPs autoantibodies are also present in cerebrospinal fluid. Therefore, in order to confirm whether anti-DARPs autoantibodies can be toxic to brain neurons, anti-drebrin antibody (22G5: homemade) or commercially available normal mouse IgG (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was administered to rat hippocampal cultured cells (homemade) at DIV23 (in vitro culture days), incubated for 1 hour, washed with PBS, fixed, and reacted with anti-MAP2 antibody (manufactured by Abcam), and 22G5 and normal mouse IgG were visualized with anti-mouse IgG-Alexa568 (manufactured by Thermo Fisher Scientific Co., Ltd.), and MAP2 was visualized with anti-rabbit IgG-Alexa488 (manufactured by Thermo Fisher Scientific Co., Ltd.) to verify the effect of anti-drebrin antibody on neurons.

[0043] The results are shown in Figure 9. In rat hippocampal cultured cells that were administered with commercially available normal mouse IgG (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) for 1 hour, washed, and fixed, mouse IgG (red, not detected) was not taken up into the cells, and MAP2 staining (green) similar to that in normal cells was observed (Figure 9B). On the other hand, in cells that were administered with anti-drebrin antibody (22G5) for 1 hour, washed, and fixed, In cultured neurons, 22G5 was observed to have penetrated into the cell body and some dendrites (red). Furthermore, in the area where 22G5 was incorporated, abnormalities occurred in microtubules and normal MAP2 staining (green) disappeared (Figure 9A). These results suggest that when DARPs autoantibodies are produced in large quantities in the body due to synaptic dysfunction or due to the onset of a disease accompanied by synaptic dysfunction, they damage brain neurons and further progress the disease. Therefore, dominant-negative peptides that suppress the brain neuron-damaging action of DARPs autoantibodies are thought to be candidate compounds for therapeutic and / or preventive agents for the above-mentioned diseases that can suppress brain neuron damage. EXAMPLES

[0044] 6. Confirmation of the presence of DARPs in rat neurons To confirm whether disease model animals and cultured nerve cells derived from disease model animals can be used for screening of therapeutic and preventive agents for diseases, the presence of DARPs in rat nerve cells was verified by the following procedure. (1) Two dishes (60 mm dishes) of cultured rat cerebral cortical cells on DIV8 (days of in vitro culture) were harvested in PBS and disrupted by sonication. (2) After centrifugation at 15,000 rpm for 20 minutes, the supernatant was collected (Figure 10, left lane, Input). (3) For immunoprecipitation, anti-drebrin antibody (28D8: homemade) and 30 μl of protein A / G beads were added to the supernatant and rotated (1 hour). After that, the beads were washed three times with PBS (add PBS, mix, centrifuge at 1500g for 10 seconds, and remove the supernatant), and the bound substance was eluted with 50 μl of SDS buffer (center lane in Figure 10, 28D8 IP). (4) As a negative control, 30 μl of protein A / G beads were added to the supernatant and rotated (for 1 hour). The beads were then washed three times with PBS (add PBS, mix, centrifuge at 1500 g for 10 seconds, and remove the supernatant), and the bound substances were eluted with 50 μl of SDS buffer (right lane in Figure 10, A / G beads). (5) Each of the obtained fractions was subjected to Western blotting using anti-drebrin A antibody DAS2 (Immunobiological Laboratories, Inc.).

[0045] The results are shown in Figure 10. The bands of DARPs could be confirmed in the fraction immunoprecipitated with anti-drebrin antibody, and it was revealed that DARPs are also present in animals having drebrin other than humans and in their cultured cells. The results here show that the change in concentration of DARPs leaking out of the cells (into the culture medium) due to synaptic dysfunction can be used as an index for screening preventive agents for diseases caused by or accompanied by synaptic dysfunction, or therapeutic agents for diseases caused by or accompanied by synaptic dysfunction. EXAMPLES

[0046] 7. Confirmation of the presence of DARPs in Alzheimer's disease model mice The present inventors crossed 5xFAD mice (mice with five copies of the gene for familial Alzheimer's disease) with drebrin knockout mice (homozygotes) to generate mice with a heterozygous 5xFAD gene and a heterozygous drebrin gene (+ / -) (5xFAD / DXKO + / - ) has been reported (Patent Application No. 2019-185245). Blood samples were obtained from these mice, and the protein A / G bead adsorption fraction was subjected to Western blotting in the same manner as in Example 1. Detection was performed using anti-drebrin A antibody DAS2 (Immunobiology Laboratories), and 5xFAD / DXKO + / - It was confirmed that the DARP100 concentration in the rats was higher than that in wild-type mice. [Industrial Applicability]

[0047] According to the determination method of the present invention, the presence or absence and severity of diseases caused by or accompanied by synaptic dysfunction, which have previously relied on question-based tests (depression, dementia, etc.) or imaging tests (cerebral hemorrhage, etc.), can be determined early and simply, leading to the prevention of the onset of these diseases and early treatment. Furthermore, DARPs and anti-DARPs autoantibodies can also serve as screening indicators for preventive and therapeutic agents, and can contribute to drug discovery research. Therefore, the present invention has extremely high industrial applicability in the medical and pharmaceutical fields.

Claims

1. An agent comprising an antibody recognizing a drebrin A-specific epitope for measuring a drebrin A-related protein, which is a fragment of drebrin A or a splice variant of drebrin A, in a biological sample collected from a subject, said measurement being for determining synaptic dysfunction, and said drebrin A-specific epitope being a region including the amino acid sequence shown in SEQ ID NO:

2.

2. A method for determining synaptic dysfunction, comprising the step of measuring the concentration of a drebrin A-related protein, which is a fragment of drebrin A or a splice variant of drebrin A, in a biological sample collected from a subject.

Images