Aβ biomarker in Alzheimer's disease model mice and its analytical method

Abstract

An AD model mouse-derived biological sample is subjected to detection of markers including mouse Aβ1-40 and mouse APP669-711, the respective measurement levels for mouse Aβ1-40 and mouse APP669-711 in the biological sample are obtained, and the ratio APP669-711 / Aβ1-40 of the mouse APP669-711 level to the mouse Aβ1-40 level is obtained. If the aforementioned ratio for the AD model mouse is higher than the aforementioned ratio for a reference mouse in which brain Aβ accumulation is absent, the brain Aβ accumulation amount in the AD model mouse is determined to be higher than the brain Aβ accumulation amount in the reference mouse.

Description

[Technical field]

[0001] The present invention relates to an Aβ biomarker for Alzheimer's disease (AD) model mice and an analytical method thereof. [Background technology]

[0002] AD is a neurodegenerative disease that is the main cause of dementia and accounts for more than half of all dementia cases. The number of dementia patients worldwide was estimated to be approximately 50 million in 2017, and is expected to reach approximately 82 million in 2030. In Japan, the number of dementia patients is also estimated to reach 7 million in 2025.

[0003] Amyloid beta (Aβ) is thought to be deeply involved in the onset of AD. Amyloid precursor protein (APP), which consists of 770 amino acid residues, is cleaved by various proteases to produce various molecular species of Aβ. The main Aβ are Aβ1-40, which consists of 40 amino acid residues, and Aβ1-42, which consists of 42 amino acid residues, and Aβ1-42 has strong aggregating properties. In AD, senile plaques appear in the brain due to aggregation accompanied by Aβ fibrillation, which is thought to be the earliest pathological change.

[0004] It is said that the accumulation of Aβ in the brain begins 20 years before the onset of AD, so it is important to determine the possibility of AD onset at an earlier stage. Testing methods used to diagnose AD include PET (Positron Emission Tomography) imaging (amyloid PET), which visualizes Aβ accumulation in the brain, and methods to detect Aβ in cerebrospinal fluid (CSF) as a biomarker (also simply called a "marker"). Of these, amyloid PET is a technology that visualizes the site and amount of Aβ amyloid accumulation in the brain with high accuracy, and is currently established as an imaging biomarker that can detect the early pathology of AD. However, amyloid PET is expensive, so it is realistic to use it only under limited conditions in clinical settings. On the other hand, CSF testing is highly invasive and therefore places a large physical burden. Under these circumstances, the development of a non-invasive and easy-to-collect biomarker was desired.

[0005] In recent years, it has been reported that the APP669-711 / Aβ1-42 ratio and Aβ1-40 / Aβ1-42 ratio in plasma, as well as a composite biomarker combining these, can estimate Aβ accumulation in the human brain with high accuracy by IP-MS (Immunoprecipitation-Mass Spectrometry) (Patent Document 1), which combines immunoprecipitation (Immuno Precipitation: IP) and MALDI-TOF MS (Matrix Assisted Laser Desorption / Ionization-Time of Flight Mass Spectrometry) for human plasma samples as a promising biomarker reflecting the state of Aβ accumulation in the brain (Patent Documents 2, 3, Non-Patent Documents 1, 2). This biomarker is useful as a risk factor for the differentiation of AD in humans and the onset of AD, and is expected to play an important role in determining intervention methods such as treatment and prevention, and therefore its clinical value is being emphasized.

[0006] On the other hand, there is no established method for fundamentally treating or preventing AD, so there is an urgent need to develop such methods. Experiments using AD model mice are necessary for research and development of intervention methods and for elucidating the mechanism of AD onset, and biomarkers can be used to understand the onset and progression of Aβ accumulation in the brains of AD model mice, to consider the timing of intervention, and to evaluate the effectiveness of intervention methods in the research stage, and are therefore valuable in basic research and non-clinical trials conducted by pharmaceutical companies and research institutions.

[0007] It has been found that not only Aβ1-42 but also Aβ1-40 (Aβ40) accumulates in the brains of AD model mice. For example, Non-Patent Document 3 shows that Aβ1-40 accumulates in the brains of AD model mice by staining with the Aβ1-40-specific antibody BA27, and Non-Patent Document 4 shows that Aβ1-40 in FA soluble Ab (fraction in which Aβ accumulation is dissolved) of mouse brain is increased in transgenic mice compared to wild type (WT). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 6424757 [Patent Document 2] Patent No. 6410810 [Patent Document 3] Patent No. 6467512 [Non-patent literature]

[0009] [Non-Patent Document 1] Kaneko N, Nakamura A, Washimi Y, Kato T, Sakurai T, Arahata Y, Bundo M, Takeda A, Niida S, Ito K, Toba K, Tanaka K, Yanagisawa K. : Novel plasma biomarker surrogating cerebral amyloid deposition. Proc Jpn Acad Ser B Phys Biol Sci. 2014;90(9):353-64

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[0010] The above-mentioned PET imaging is a method for evaluating the amount of Aβ accumulation in the brain of AD model mice, but it requires a PET device for experimental animals and requires the handling of radioactive materials, making it a potentially dangerous experiment (Non-Patent Document 5). In addition, since AD ​​model mice are small, it is difficult to collect CSF, and even if it is possible to collect it, the amount is small, and there is a risk of blood contamination. On the other hand, a relatively easy method for evaluating Aβ accumulation in the brain is to evaluate the state of amyloid accumulation in the brain by measuring the amyloid in the insoluble fraction after dissolving the brain of an AD model mouse, but continuous monitoring is impossible because the AD model mouse is killed (Non-Patent Documents 6 and 7).

[0011] An object of the present invention is to provide a biomarker for evaluating the state of Aβ accumulation in the brain of an AD model mouse, which is relatively easy to collect, safe, and allows for continuous observation, and an analytical method for the same. [Means for solving the problem]

[0012] A first aspect of the present invention relates to an analytical method for determining a state of Aβ accumulation in the brain, comprising the steps of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, determining the ratio of the mouse APP669-711 level to the mouse Aβ1-40 level: APP669-711 / Aβ1-40, and, when the ratio of the AD model mouse is higher than that of a reference mouse in which Aβ accumulation does not appear in the brain, determining that the amount of Aβ accumulation in the brain of the AD model mouse is greater than the amount of Aβ accumulation in the brain of the reference mouse.

[0013] A second aspect of the present invention relates to an analytical method for determining the effectiveness of an intervention with respect to the Aβ accumulation state in the brain, comprising the steps of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 before and after an intervention performed on the AD model mouse to obtain measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, determining the ratio of the mouse APP669-711 level to the mouse Aβ1-40 level: APP669-711 / Aβ1-40, and comparing the ratio of the AD model mouse before the intervention with the ratio of the AD model mouse after the intervention.

[0014] A third aspect of the present invention relates to an analytical method in which a biological sample derived from an AD model mouse is subjected to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain the measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, and a step of determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40 is performed multiple times over time on the same AD model mouse, and a change in the ratio in the AD model mouse is evaluated to determine whether the amount of Aβ accumulated in the brain is increasing or decreasing.

[0015] A fourth aspect of the present invention relates to an analytical method for screening a candidate substance for improving Aβ accumulation in the brain, comprising the steps of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 before and after providing the AD model mouse with a candidate substance for improving Aβ accumulation in the brain, to obtain measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, determining the ratio of the mouse APP669-711 level to the mouse Aβ1-40 level: APP669-711 / Aβ1-40, and comparing the ratio of the AD model mouse before providing the candidate substance with the ratio of the AD model mouse after providing the candidate substance.

[0016] A fifth aspect of the present invention relates to a marker for determining the state of Aβ accumulation in the brain, which comprises a combination of mouse Aβ1-40 and mouse APP669-711 in a biological sample derived from an AD model mouse. Effect of the Invention

[0017] The analytical method and marker of the present invention make it possible to provide an analytical method and biomarker for evaluating the state of Aβ accumulation in the brain of an AD model mouse, which is relatively easy to collect, safe, and allows for continuous observation. [Brief description of the drawings]

[0018] [Figure 1] 1 shows an immunostaining image of brain tissue from a 23-month-old APP / PS1 mouse in Example 1. [Diagram 2] 1 shows mass spectra of mouse Aβ-related peptides in plasma from 2-month-old (A) and 25-month-old (B) APP / PS1 mice in Example 1. [Diagram 3] 1 is a graph comparing the normalized intensity of plasma Aβ1-40 in young AD model mice (2-5 months old, N=11) with the normalized intensity of plasma Aβ1-40 in aged AD model mice (23-30 months old, N=13) in Example 1. [Figure 4] 1 is a graph comparing the normalized intensity of plasma APP669-711 in young AD model mice (2-5 months old, N=11) with the normalized intensity of plasma APP669-711 in aged AD model mice (23-30 months old, N=13) in Example 1. [Diagram 5] 1 is a graph comparing the plasma APP669-711 / Aβ1-40 ratio of young AD model mice (2-5 months old, N=11) with the plasma APP669-711 / Aβ1-40 ratio of aged AD model mice (23-30 months old, N=13) in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] <Analysis method> (First aspect) A first aspect of the present invention is an analytical method for determining the state of Aβ accumulation in the brain, comprising: A step of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample; determining the ratio of mouse APP669-711 levels to mouse Aβ1-40 levels: APP669-711 / Aβ1-40; and determining that the amount of Aβ accumulated in the brain of the AD model mouse is greater than the amount of Aβ accumulated in the brain of the reference mouse when the ratio of the AD model mouse is higher than the ratio of a reference mouse in which Aβ accumulation does not occur in the brain.

[0020] In the first aspect, the method includes a step of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain the respective measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample (this step is referred to as a "measurement step"). The AD model mouse is not particularly limited, and for example, an AD model mouse described in Non-Patent Document 8 can be appropriately used. Specifically, Tg(APPswe,PSEN1dE9)85Db o(Non-Patent Document 9), 5xFAD(B6SJL)(Non-Patent Document 10), 5xFAD(C57BL6)(Non-Patent Document 11), APP / PS1 / rTg21221(Non-Patent Document 12), APPSwe / PSEN1(A246E)(Non-Patent Document 13), APPSwe / PSEN1dE9(C3-3 x S-9)(Non-Patent Document 14), APPswe / PSEN1dE9(line 85)(Non-Patent Document 15), Tg2576 / Tau(P301L)(APPSwe-Tau)(Non-Patent Document 16), TREM2-BAC x 5xFAD(Non-Patent Document 17), 3xTg(Non-Patent Document 18), APP23(Non-Patent Document 19), APP Knock-in(Non-Patent Document 20), etc.

[0021] As an AD model mouse, an APP / PS1 mouse, which is a transgenic mouse in which human Swedish mutant APP and ΔExon mutant PS1 are overexpressed by the nervous system-specific prion promoter (PrP), is preferred. In Example 1 described below, Tg(APPswe,PSEN1dE9)85Db o It is known that Aβ accumulation appears in the brain of APP / PS1 mice from about 6 to 7 months of age (Non-Patent Document 21), and therefore, young and old APP / PS1 mice are used in the study to compare the presence or absence of Aβ accumulation in the brain.

[0022] In the measurement step, the "biological sample" can be selected from blood, urine, feces, bodily secretions (e.g., saliva, tears), and the like. Of these, the biological sample is preferably blood, for reasons such as ease of collection and ease of reflecting the state of Aβ in the brain. Blood is a sample that is directly subjected to the measurement step of the expression level of the marker, and includes whole blood, plasma, and serum. Blood can be prepared by appropriately processing whole blood collected from an AD model mouse. The processing performed when preparing a blood sample from the collected whole blood is not particularly limited, and any processing such as centrifugation may be performed. In addition, the blood to be subjected to the measurement step may be appropriately stored at a low temperature, such as by freezing, during the middle stage of the preparation step or after the preparation step.

[0023] The biological sample may be used as it is for measuring the concentration of the component, or may be pretreated as necessary before being used for measuring the concentration of the component. Examples of pretreatment include stopping the enzyme reaction in the biological sample, removing fat-soluble substances, removing proteins, etc. These pretreatments may be performed using known methods. In addition, the biological sample may be used after being appropriately diluted or concentrated.

[0024] In the measuring step, the respective measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample are obtained. Mouse Aβ1-40 and mouse APP669-711 have the following amino acid sequences.

[0025] Mouse Aβ1-40: DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 1) Mouse APP699-711: VKMDAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVV (SEQ ID NO: 2) In the present invention, the level of a marker basically means a concentration, but may also be other units that those skilled in the art use in a manner similar to concentration, such as detected ion intensity in mass spectrometry.

[0026] The measurement of the marker is preferably carried out by a test based on biomolecule-specific affinity. The test based on biomolecule-specific affinity is a method well known to those skilled in the art, and is not particularly limited, but is preferably an immunoassay. Specifically, it includes immunoassays including competitive and non-competitive assay systems such as Western blot, radioimmunoassay, ELISA (Enzyme-Linked ImmunoSorbent Assay) (including sandwich immunoassay, competitive assay, and direct adsorption assay), immunoprecipitation, precipitation reaction, immunodiffusion, immunoagglutination, complement fixation analysis, immunoradiometric assay, fluorescent immunoassay, and protein A immunoassay. In the immunoassay, an antibody that binds to the above-mentioned marker in a biological sample is detected.

[0027] The measurement of the marker may be performed using an antibody-immobilized carrier prepared using an immunoglobulin having an antigen-binding site capable of recognizing a peptide derived from amyloid precursor protein (APP) or an immunoglobulin fragment having an antigen-binding site capable of recognizing a peptide derived from amyloid precursor protein (APP). The peptide in the sample can be detected by a mass spectrometer by immunoprecipitation using the antibody-immobilized carrier (IP-MS).

[0028] In the first embodiment, after the above-mentioned measurement step, a step of calculating the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40 (this step is referred to as a "calculation step"). The marker, which is the ratio APP669-711 / Aβ1-40, is determined by measuring the ratio of the APP669-711 / Aβ1-40 in a plasma sample from a reference AD ​​model mouse in which Aβ accumulation does not appear in the brain. ratio and in plasma samples from AD model mice with excessive accumulation of Aβ in the brain. ratio A significant difference was observed between the two.

[0029] In the analysis of AD in humans, etc., Aβ1-42 has been used as a suitable indicator (Patent Documents 2 and 3, Non-Patent Documents 1 and 2). Similarly, in mice, there exists mouse Aβ1-42 having the following amino acid sequence.

[0030] Mouse Aβ1-42: DAEFGHDSGFEVRHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 3) However, when AD model mice are used as subjects, it is difficult to measure mouse Aβ1-42, which has a low concentration, because the amount of blood collected is small, unlike in humans, etc. For this reason, the present inventors have found that mouse Aβ1-40 and mouse APP669-711 can be more suitable indicators than mouse Aβ1-42 when AD model mice are used as subjects.

[0031] In the first embodiment, after the above-mentioned calculation step, if the ratio of the AD model mouse is higher than that of a reference mouse in which Aβ accumulation does not occur in the brain, the step of determining that the amount of Aβ accumulated in the brain of the AD model mouse is greater than that of the reference mouse is included. In this step, the concentration of the marker in the biological sample is analyzed by comparing the measured value with the reference value. As the reference mouse, an AD model mouse or a wild-type mouse aged 2 to 5 months (young) can be suitably used.

[0032] According to the analytical method of the first aspect, it is possible to provide an analytical method for evaluating the state of Aβ accumulation in the brain of an AD model mouse, which is relatively easy to collect, safe, and allows for continuous observation.

[0033] (Second aspect) A second aspect of the present invention is an analytical method for determining the effectiveness of the intervention with respect to Aβ accumulation in the brain, comprising: Before and after the intervention in AD model mice, A step of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain the respective measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample (measurement step); A step of determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40 (calculation step); and comparing said ratio in the AD model mouse before the intervention with said ratio in the AD model mouse after the intervention.

[0034] The analysis method of the second embodiment is carried out before and after an intervention on an AD model mouse. Here, "intervention" includes administration of a therapeutic or preventive drug for AD, diet therapy, exercise therapy, learning therapy, surgery, etc. Note that exercise therapy, learning therapy, and surgery include both known and unknown therapies.

[0035] The measuring step and the calculating step in the second embodiment are similar to the measuring step and the calculating step in the first embodiment described above.

[0036] In the second embodiment, the measuring step and the calculating step are performed before and after the intervention performed on the AD model mouse, and then the ratio of the AD model mouse before the intervention is compared with the ratio of the AD model mouse after the intervention. According to the second embodiment, by applying the method before and after the intervention performed on the AD model mouse, it is possible to evaluate the efficacy of AD therapeutic drugs and preventive drugs, or to evaluate the effectiveness of other treatments.

[0037] (Third aspect) A third aspect of the present invention is an analytical method for determining whether an amount of Aβ accumulation in the brain is increased or decreased, comprising the steps of: A biological sample derived from an AD model mouse is subjected to detection of markers including mouse Aβ1-40 and mouse APP669-711, and a process (measurement method) for obtaining the measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample and a process (calculation process) for determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40 are performed multiple times over time on the same AD model mouse, and changes in the ratio in the AD model mouse are evaluated.

[0038] In the analysis method of the third embodiment, the measurement step and the calculation step are performed multiple times over time on the same AD model mouse. The measurement step and the calculation step in the third embodiment are also the same as the measurement step and the calculation step in the first embodiment described above.

[0039] In the third aspect, the measuring step and the calculating step are performed multiple times over time on the same AD model mouse to evaluate the change in the ratio of the AD model mouse. According to the third aspect, the change in Aβ accumulation in the brain of the AD model mouse can be evaluated over time, and it can be determined whether the amount of Aβ accumulation in the brain is increasing or decreasing.

[0040] (Fourth aspect) A fourth aspect of the present invention is an analytical method for screening a candidate substance for ameliorating Aβ accumulation in the brain, comprising: We administered a candidate substance to improve Aβ accumulation in the brain to AD model mice. Later, A step of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain the respective measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample (measurement step); A step of determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40 (calculation step); The method includes a step of comparing the ratio in the AD model mouse before administration of the candidate substance with the ratio in the AD model mouse after administration of the candidate substance.

[0041] The analysis method of the fourth embodiment is carried out before and after administering a candidate substance for improving Aβ accumulation in the brain to an AD model mouse. Here, the "candidate substance" refers to an unknown substance that may be a candidate for a therapeutic or preventive drug for AD to improve Aβ accumulation in the brain.

[0042] The measuring step and the calculating step in the fourth embodiment are similar to the measuring step and the calculating step in the first embodiment described above.

[0043] In the fourth embodiment, the ratio of the AD model mouse before the administration of the candidate substance is compared with the ratio of the AD model mouse after the administration of the candidate substance. According to the fourth embodiment, the effectiveness of the candidate substance for improving Aβ accumulation in the brain can be evaluated by administering the candidate substance to the AD model mouse before and after the administration.

[0044] <Markers for determining Aβ accumulation in the brain> (Fifth aspect) The present invention also provides a marker for determining the state of Aβ accumulation in the brain, which is composed of a combination of mouse Aβ1-40 and mouse APP669-711 in a biological sample derived from an AD model mouse. Since the blood APP669-711 / Aβ1-40 ratio has not been reported as a biomarker in humans, it can be said to be a biomarker specific to AD model mice. By using the marker of the present invention, Biological samples from AD model mice This will make it possible to determine the state of Aβ accumulation in the brain with high accuracy.

[0045] The biological sample and the AD model mouse for the marker of the present invention may be any of those described above in the first aspect, but similarly, the biological sample is preferably blood, and the AD model mouse is preferably an APP / PS1 mouse.

[0046] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0047] <Example 1> [1] AD model mouse APP / PS1 mice (Tg(APPswe,PSEN1dE9)85Dbo, Non-Patent Document 9), which are widely used as AD model mice, were used. APP / PS1 mice are transgenic mice that overexpress human Swedish mutant APP and ΔExon mutant PS1 driven by the nervous system-specific prion promoter (PrP). APP / PS1 mice aged 2 to 5 months (young) or 23 to 30 months (old) were used. It is known that Aβ accumulation appears in the brain of APP / PS1 mice from about 6 to 7 months of age (Non-Patent Document 21), and young and old APP / PS1 mice are used in the study to compare the presence or absence of Aβ accumulation in the brain.

[0048] [2] Immunostaining of brain tissue In the 23-month-old APP / PS1 mouse used in this example, the appearance of intracerebral Aβ accumulation was confirmed by immunostaining of the brain tissue by the following procedure. The half brain removed from the mouse was infiltrated with 4% PFA (paraformaldehyde) / PBS (phosphate buffer solution) (pH 6.7) for 24 hours, and the brain was cut into pieces. The brain was then dehydrated and embedded in 70% ethanol for 1 hour, 80% ethanol for 2 hours, 90% ethanol for 2 hours, 99% ethanol for 3 hours x 3 times, xylene for 3 hours, xylene for 2 hours, and paraffin for 3 hours x 3 times. The prepared block was thinly sliced ​​to a thickness of 4 mm using a MICROM HM 430 (Thermo SCIENTIFIC), stretched on a PLL (poly-L-lysine)-coated slide glass, and dried at 37 ° C for 2 days.

[0049] To deparaffinize the prepared brain sections, they were immersed in xylene for 5 minutes three times. Then, they were immersed in 100% ethanol for 2.5 minutes twice, 90% ethanol for 2.5 minutes, 80% ethanol for 2.5 minutes, 70% ethanol for 2.5 minutes, and running water for 10 minutes. To activate the antigen, they were autoclaved in 0.01M citric acid (pH 6.0) / DW (distilled water) at 121°C for 10 minutes, and immersed in running water for 10 minutes in TS (Tris buffer). Then, they were blocked with inactivated 10% calf serum / PBS for 30 minutes at room temperature, and various primary antibodies diluted 1000-fold were applied at room temperature overnight. Anti-human Aβ mouse monoclonal antibody (82E1, IBL) and anti-mouse Aβ rabbit polyclonal antibody (APP597, IBL) were used as primary antibodies. Next, the sections were infiltrated with various 500-fold diluted biotinylated secondary antibodies at room temperature for 2 hours, ABC elite (VECTOR) for 1 hour, ImmPACT SG (VECTOR) for 5 minutes, 0.1% hematoxylin solution for 10 seconds, and running water for 5 minutes. Finally, for dehydration, the sections were infiltrated with 70% ethanol for 2.5 minutes, 80% ethanol for 2.5 minutes, 90% ethanol for 2.5 minutes, 100% ethanol for 2.5 minutes x 2 times, and xylene for 5 minutes x 3 times for clearing. The sections were then mounted using mounting medium HSR solution (Sysmex), dried, and observed with EVOS FL Auto2 (Invitrogen). Immunostaining of the brain tissue of 23-month-old APP / PS1 mice confirmed the appearance of Aβ accumulation in the brain (Figure 1).

[0050] [3] Blood collection from AD model mice The facial vein near the jawbone of an AD model mouse was punctured with a 21G injection needle, and the blood that came to the surface was collected. To prevent blood clotting during collection, EDTA-2Na (final concentration: 0.15%) was added to each blood collection tube in advance. The collected blood was immediately centrifuged at 4°C, 3,500 rpm, and 5 minutes, and the supernatant, plasma, was collected. The above blood components obtained were each frozen and stored at -80°C.

[0051] [4] Measurement of plasma Aβ-related peptides Aβ-related peptides in plasma were measured using IP-MS, a combination of immunoprecipitation (IP) and mass spectrometry (MS). Anti-mouse Aβ rabbit polyclonal antibody APP597 (IBL) was purified with Protein G (Thermo Fisher Scientific) to remove BSA, and then antibody beads covalently bound to Dynabeads Epoxy (Thermo Fisher Scientific) were used for IP. Frozen mouse plasma (40-100 μL) was thawed and mixed with equal amounts of the internal standard peptide solution and plasma. The mixture was mixed with the antibody beads and allowed to react with the antigen at 4°C for 1 hour. The internal standard peptide used was 40 pM stable isotope-labeled mouse Aβ1-42 (SIL-Aβ1-42, theoretical mass m / z 4471.30). After the antigen-antibody reaction, the antibody beads were washed, and the Aβ-related peptides were eluted with glycine buffer (pH 2.8) containing DDM (n-dodecyl-β-D-maltoside). After returning the solution to neutral with Tris buffer, the antibody beads were washed again. of After the antigen-antibody reaction with the Aβ-related peptides and washing, the Aβ-related peptides were eluted with an elution solution (5 mM HCl, 0.1 mM methionine, 70% (v / v) acetonitrile). The elution solution after IP was dropped onto 4 wells of a μFocus MALDI plate (trademark) 900 μm (Hudson Surface Technology, Inc., Fort Lee, NJ) on which 0.5 mg / mL CHCA (α-cyano-4-hydroxycinnamic acid) / 0.2% (w / v) MDPNA (methylenediphosphonic acid) had been dropped and dried in advance, and the solution was then dropped onto the 4 wells of the plate and dried.

[0052] Mass spectrum data were acquired using an AXIMA Performance (Shimadzu / KRATOS, Manchester, UK) with a Linear TOF in positive ion mode. The m / z values ​​of the Linear TOF were expressed as the average mass of the peaks. The m / z values ​​and signal intensities were calibrated using standard peptides. Human angiotensin II, human ACTH fragment 18-39, bovine insulin oxidized beta-chain, and bovine insulin were used as external standards to calibrate the m / z values. The internal standard peptide SIL-Aβ1-42 was used to normalize the signal intensities of each Aβ-related peptide in plasma. The normalized intensity of the four mass spectra obtained from one sample was then averaged, and this was evaluated as the amount of Aβ-related peptide. If there were two or more data points that did not reach the lower limit of detection (S / N<3), they were deemed not detectable.

[0053] This measurement method uses an antibody that specifically reacts with mouse Aβ sequences, and analyzes the peak detected at the theoretical mass of mouse Aβ sequences in the mass spectrum. Therefore, this analysis data is targeted at mouse Aβ-related peptides, and does not target Aβ-related peptides derived from overexpressed human Swedish mutant APP.

[0054] [5] Analysis of plasma Aβ-related peptides Mass spectra of mouse Aβ-related peptides obtained by analyzing plasma from 2-month-old and 25-month-old APP / PS1 mice by IP-MS are shown in Figure 2. Data from one mouse, aged 2 months and 25 months, is shown here. In these mass spectra, peaks for Aβ1-40, Aβ1-42, and APP669-711 were detected at the theoretical mass positions shown in Table 1.

[0055] Next, the subjects were divided into a young group (2-5 months old) without Aβ accumulation in the brain and an elderly group (23-30 months old) with Aβ accumulation in the brain, and the standardized intensities of Aβ1-40 and APP669-711 were compared between the groups. Comparisons between the two groups were performed using Student's t-test with a significance level of 5%. As a result, no significant differences were confirmed between the young and elderly groups for Aβ1-40 and APP669-711 (Figures 3 and 4). On the other hand, when the ratio of the standardized intensity of APP669-711 to Aβ1-40 (APP669-711 / Aβ1-40) was calculated and compared between the two groups, the result was p=0.0012, and a significant difference was confirmed between the young and elderly groups. In other words, APP669-711 / Aβ1-40 in the elderly group was increased more than in the young group (Figure 5). This suggests that the blood APP669-711 / Aβ1-40 ratio reflects Aβ accumulation in the brain of AD model mice. Since the blood APP669-711 / Aβ1-40 ratio has not been reported as a biomarker in humans, it can be said to be a biomarker specific to AD model mice.

[0056] [Aspects] It will be understood by those skilled in the art that the exemplary embodiments and experimental examples described above are examples of the following aspects.

[0057] (Section 1) An analytical method according to one embodiment is an analytical method for determining the state of Aβ accumulation in the brain, and includes the steps of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40, and, when the ratio of the AD model mouse is higher than that of a reference mouse in which Aβ accumulation does not occur in the brain, determining that the amount of Aβ accumulated in the brain of the AD model mouse is greater than the amount of Aβ accumulated in the brain of the reference mouse.

[0058] According to the analytical method described in paragraph 1, it is possible to provide an analytical method for evaluating the state of Aβ accumulation in the brain of an AD model mouse, which is relatively easy to collect, safe, and allows for continuous observation.

[0059] (Section 2) An analytical method according to one embodiment is an analytical method for determining the effectiveness of an intervention with respect to the Aβ accumulation state in the brain, and includes the steps of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 before and after an intervention performed on the AD model mouse, thereby obtaining the respective measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40, and comparing said ratio of the AD model mouse before the intervention with said ratio of the AD model mouse after the intervention.

[0060] According to the analytical method described in Section 2, by applying it to AD model mice before and after an intervention, it is possible to evaluate the efficacy of AD therapeutic drugs, preventive drugs, or other treatments.

[0061] (Section 3) One embodiment of the analytical method is an analytical method for determining whether the amount of Aβ accumulated in the brain is increasing or decreasing, and involves subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain the measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, and determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40, multiple times over time in the same AD model mouse, and evaluating changes in the ratio in the AD model mouse.

[0062] According to the analytical method described in item 3, it is possible to evaluate changes in Aβ accumulation in the brain of an AD model mouse over time and determine whether the amount of Aβ accumulation in the brain is increasing or decreasing.

[0063] (Section 4) An analytical method according to one embodiment is an analytical method for screening candidate substances for improving Aβ accumulation in the brain, and includes the steps of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 before and after providing the AD model mouse with a candidate substance for improving Aβ accumulation in the brain, thereby obtaining the respective measurement levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample, determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40, and comparing said ratio in the AD model mouse before providing the candidate substance with said ratio in the AD model mouse after providing the candidate substance.

[0064] According to the analytical method described in paragraph 4, the effectiveness of a candidate substance for improving Aβ accumulation in the brain can be evaluated by applying the method to AD model mice before and after administering the candidate substance for improving Aβ accumulation in the brain.

[0065] (Section 5) In the analytical method according to any one of items 1 to 4, the biological sample is blood.

[0066] According to the analytical method described in item 5, biological samples can be collected more easily than biological samples other than blood.

[0067] (Section 6) In the analysis method according to any one of items 1 to 5, the AD model mouse is an APP / PS1 mouse.

[0068] According to the analysis method described in item 6, the AD model mouse is widely used and can be easily obtained.

[0069] (Section 7) A marker for determining the state of Aβ accumulation in the brain according to one embodiment comprises a combination of mouse Aβ1-40 and mouse APP669-711 in a biological sample derived from an AD model mouse.

[0070] According to the markers described in paragraph 7, Biological samples from AD model mice This will make it possible to determine the state of Aβ accumulation in the brain with high accuracy.

[0071] (Section 8) In the marker described in item 7, the biological sample is blood.

[0072] The marker described in item 8 can be collected more easily than biological samples other than blood.

[0073] (Section 9) In the marker according to item 7 or 8, the AD model mouse is an APP / PS1 mouse.

[0074] The markers described in item 9 are widely used in AD model mice and are easily available.

Claims

1. A step of subjecting a biological sample derived from an Alzheimer's disease (AD) model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample; determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40; and determining that the amount of Aβ accumulated in the brain of the AD model mouse is greater than the amount of Aβ accumulated in the brain of the reference mouse when the ratio of the AD model mouse is higher than the ratio of a reference mouse not exhibiting Aβ accumulation in the brain, The analytical method, wherein the biological sample is blood.

2. Before and after the intervention performed on AD model mice, A step of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample; determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40; A method for determining the effectiveness of the intervention with respect to Aβ accumulation in the brain, comprising a step of comparing the ratio of the AD model mouse before the intervention with the ratio of the AD model mouse after the intervention, The analytical method, wherein the biological sample is blood.

3. A method for analyzing the effect of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample and determining a ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40, the steps being repeated multiple times over time for the same AD model mouse, and evaluating changes in the ratio in the AD model mouse to determine whether the amount of Aβ accumulated in the brain is increased or decreased, The analytical method, wherein the biological sample is blood.

4. Before and after administering a candidate substance for improving Aβ accumulation in the brain to AD model mice, A step of subjecting a biological sample derived from an AD model mouse to detection of markers including mouse Aβ1-40 and mouse APP669-711 to obtain measured levels of mouse Aβ1-40 and mouse APP669-711 in the biological sample; determining the ratio of mouse APP669-711 level to mouse Aβ1-40 level: APP669-711 / Aβ1-40; An analytical method for screening a candidate substance for improving Aβ accumulation in the brain, comprising a step of comparing the ratio of an AD model mouse before administration of a candidate substance with the ratio of an AD model mouse after administration of the candidate substance, The analytical method, wherein the biological sample is blood.

5. The method according to any one of claims 1 to 4, wherein the AD model mouse is an APP / PS1 mouse.

6. A marker for determining the state of Aβ accumulation in the brain, which comprises a ratio of mouse APP669-711 level to mouse Aβ1-40 level in a biological sample derived from an AD model mouse: APP669-711 / Aβ1-40, wherein the biological sample is blood.

7. The marker according to claim 6 , wherein the AD model mouse is an APP / PS1 mouse.

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