Differential diagnosis of tauopathies

Abstract

To provide a method for differentiating and easily diagnosing corticobasal degeneration and PSP among tauopathies.SOLUTION: A method for detecting tau fragments specific for corticobasal degeneration in a human-derived sample comprises reacting the human sample with an antibody which recognizes the N-terminal side of a C-terminal fragment starting from A 168 of human tau protein and an antibody which recognizes the C-terminal side of an N-terminal fragment truncated at N 368 of human tau protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for differential diagnosis of tauopathy. [Background Art]

[0002] Tauopathy is a group of neurodegenerative diseases accompanied by deposition of insoluble tau in neurons and glial cells of the human brain. Tauopathies include Alzheimer's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), etc. Among these, both CBD and PSP are typical tauopathies presenting with parkinsonism, but it is often clinically difficult to differentiate between the two. A definitive diagnosis is made pathologically based on the morphology of tau pathology in astrocytes, and biochemically based on the difference in the band pattern of C-terminal tau fragments of tau in the insoluble fraction of a patient's brain, but experience is required for the diagnosis. [Summary of Invention] [Problem to be Solved by the Invention]

[0003] An object of the present invention is to provide a method that can easily diagnose CBD and PSP by distinguishing between them among tauopathies. [Means for Solving the Problem]

[0004] Biochemically, CBD is characterized by the presence of a 37-40 kDa C-terminal tau fragment in the sarcosyl-insoluble fraction, but its generation mechanism had not been elucidated. Therefore, the inventors investigated the tau protein cleavage enzyme, specifically asparagine endopeptidase (regmine), and found that differences in the cleavage pattern of tau aggregates treated with legmine resulted in the generation of the 37-40 kDa C-terminal tau fragment. They also found that these differences in cleavage patterns stem from the structure of tau aggregates specific to CBD, and that by using an antibody that recognizes the legmine cleavage fragment, CBD and PSP can be differentiated by Western blotting. Furthermore, by using an antibody that specifically recognizes the legmine cleavage site, they found that it was possible to distinguish between CBD astrocyte plaques and PSP-specific tufted astrocytes, which had previously been difficult to distinguish immunohistochemically, thus completing the present invention.

[0005] In other words, the present invention provides the following inventions [1] to [7]. [1] A method for detecting CBD-specific tau fragments in a human sample, characterized by reacting a human-derived sample with an antibody that recognizes the N-terminal side of the C-terminal fragment starting at A168 of human tau protein and an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein. [2] The detection method according to [1], wherein a sample that reacts with an antibody that recognizes the N-terminal side of the C-terminal fragment starting at A168 of the human tau protein, and does not react with an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of the human tau protein, is determined to be a sample having a tau fragment specific to CBD. [3] The detection method according to [1] or [2], wherein the reaction of the antibody is immunohistochemical staining. [4] A method for detecting CBD-specific tau fragments in a human sample, characterized by performing a Western blot on a human-derived sample using one or more antibodies selected from an antibody that recognizes the N-terminal side of the C-terminal fragment of human tau protein starting at A168, an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein, and an antibody that recognizes the A168-N368 fragment of human tau protein. [5] The detection method according to any of [1] to [4], wherein the human-derived sample is a human-derived sample containing tau protein. [6] A CBD diagnostic agent containing an antibody that recognizes the N-terminal side of the C-terminal fragment of human tau protein starting at A168 and an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein. [7] A CBD diagnostic reagent for Western blotting, comprising one or more antibodies selected from an antibody that recognizes the N-terminal side of the C-terminal fragment of human tau protein starting at A168, an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein, and an antibody that recognizes the A168-N368 fragment of human tau protein. [Effects of the Invention]

[0006] According to the method of the present invention, CBD and PSP can be clearly distinguished and diagnosed by simple immunohistochemistry or Western blotting methods. [Brief explanation of the drawing]

[0007] [Figure 1] This figure shows the full-length tau protein, the legmine cleavage site, the A168 C-terminal fragment, the N368 N-terminal fragment, and the recognition sites of the anti-A168 antibody and anti-N368 antibody. [Figure 2] This is a Western blot image of the 1% sarcosyl-insoluble fraction of a patient's brain. Black arrowheads indicate the specific 37-40 kDa C-terminal fragment of CBD. A commercially available tau monoclonal antibody, T46, was used as the tau C-terminal antibody. [Figure 3] This is a Western blot showing the time course of the reaction between PSP and CBD-insoluble tau and regmine. Black arrowheads indicate full-length tau, and white arrowheads indicate 37-40 kDa C-final fragments. [Figure 4] This image shows double immunofluorescence staining of tufted astrocytes from PSP and astrocyte plaques from CBD using anti-phosphorylated tau antibody and anti-A168 antibody. The commercially available monoclonal antibody AT8 was used as the anti-phosphorylated tau antibody. [Figure 5]This figure shows double immunofluorescence staining of PSP tufted astrocytes and CBD astrocyte plaques using antiphosphorylated tau antibody and anti-N368 antibody. [Figure 6] This is a Western blot showing the changes over time after the three-dimensional structure of insoluble tau was broken down with guanidine hydrochloride and then reacted with regmine. Black arrowheads indicate full-length tau, and white arrowheads indicate 37-40 kDa C-terminal fragments. [Modes for carrying out the invention]

[0008] One embodiment of the present invention is a method for detecting CBD-specific tau fragments in a human sample, characterized by reacting a human-derived sample with an antibody that recognizes the N-terminal side of the C-terminal fragment starting at A168 of human tau protein and an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein.

[0009] Examples of specimens used in the present invention include tissues containing cells suspected of containing human-derived tau protein, and bodily fluids such as cerebrospinal fluid. Examples of such cells include nerve cells, tissues containing astrocytes, bodily fluids, and cultured cells infected with tau protein derived from patient bodily fluids.

[0010] The antibodies used in this invention are an antibody that recognizes the N-terminal side of the C-terminal fragment of human tau protein starting at A168, and an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein. The C-terminal fragment of human tau protein starting at A168 is the A168C-terminal fragment shown in Figure 1, that is, the C-terminal fragment of tau protein starting at the 168th alanine (A). Therefore, the antibody that recognizes the N-terminal side of the C-terminal fragment of human tau protein starting at A168 is the antibody that recognizes the N-terminal side of the A168C-terminal fragment shown in Figure 1 (anti-A168 antibody). Such anti-A168 antibodies can be produced using 6 to 20 peptides, preferably 6 to 16 peptides, starting with A168 as antigens, according to the usual methods for producing monoclonal or polyclonal antibodies. From the obtained antibodies, it is preferable to isolate the antibody that recognizes the N-terminal side of the A168 C-terminal fragment by affinity purification.

[0011] The N-terminal fragment of human tau protein cleaved at N368 is the N368N-terminal fragment shown in Figure 1, i.e., the N-terminal fragment cleaved at the 368th asparagine (N) group of the tau protein. Therefore, the antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein is the antibody that recognizes the C-terminal side of the N368N-terminal fragment shown in Figure 1 (anti-N368 antibody). Such anti-N368 antibodies can be produced using, for example, 6 to 20 peptides, preferably 6 to 16 peptides, with N368 at the N-terminus as the antigen, according to the usual methods for producing monoclonal or polyclonal antibodies. From the obtained antibodies, it is preferable to isolate the antibody that recognizes the C-terminal side of the N368 N-terminal fragment by affinity purification. Alternatively, commercially available antibodies that recognize the C-terminal side of the N368 N-terminal fragment can also be used.

[0012] Western blotting and immunohistochemistry are methods for reacting human-derived samples with antibodies that recognize the N-terminal side of the C-terminal fragment starting with human tau protein 168 and antibodies that recognize the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein, but immunohistochemistry is preferred.

[0013] Immunohistochemical staining may be performed using either fluorescent staining or dye staining. Fluorescent staining may use fluorescent dyes such as FITC, Alexa, Fluor dyes, or Cy dyes, or fluorescent proteins such as PE and APC. Dye staining may use labeled enzymes, peroxidases, alkaline phosphatases, etc. Furthermore, the staining method may be direct, indirect, or amplified. Here, the procedure for immunohistostaining using an anti-A168 antibody and an anti-N368 antibody is as follows: a patient brain specimen thinly sliced on a glass slide is inactivated by a heating method, and then reacted with a primary antibody and a secondary antibody. Microscopic observation can be performed by performing a treatment to eliminate autofluorescence and mounting the specimen with a cover glass.

[0014] As shown in Fig. 4, tufted astrocytes in progressive supranuclear palsy (PSP) reacted with both of the two types of antibodies. On the other hand, as shown in Fig. 5, astrocytic plaques in corticobasal degeneration (CBD) reacted with the anti-A168 antibody but did not react with the anti-N368 antibody. It was found that in CBD, tau protein is cleaved at N167 but is resistant to cleavage at N368.

[0015] From the results of the immunostaining, a specimen that reacts with the antibody recognizing the N-terminal side of the C-terminal fragment starting at A168 of human tau protein and does not react with the antibody recognizing the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein can be determined as a specimen having a tau fragment specific to CBD.

[0016] One aspect of the present invention is a diagnostic agent for CBD comprising an antibody that recognizes the N-terminal side of the C-terminal fragment starting at A168 of human tau protein and an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein. The antibody used in the diagnostic agent of the present invention may be the antibody itself described above, or may be an antibody labeled with various enzymes, dyes, or the like. Further, the diagnostic agent of the present invention may contain buffers, inactivating agents, protocols, and the like.

[0017] Another aspect of the present invention is a method for detecting a CBD-specific tau fragment in a human specimen, characterized by performing Western blotting on a human-derived specimen using one or more antibodies selected from the group consisting of: an antibody that recognizes the N-terminal side of the C-terminal fragment starting at A168 of human tau protein, an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein, and an antibody that recognizes the A168-N368 fragment of human tau protein.

[0018] Examples of specimens used in the present invention include cell suspensions derived from tissues containing cells suspected of containing human-derived tau protein, and body fluids such as cerebrospinal fluid. Examples of such cells include nerve cells, tissues containing astrocytes, body fluids, and cultured cells infected with tau protein derived from a patient's body fluid.

[0019] For the antibody that recognizes the N-terminal side of the C-terminal fragment starting at A168 of human tau protein and the antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein used in the present invention, those described above can be used. Further, as the antibody that recognizes the A168-N368 fragment of human tau protein, a commercially available antibody that recognizes this fragment can be used.

[0020] In Western blotting, for example, tau protein in the insoluble fraction of a patient's brain is reacted with an anti-A168 antibody. Alternatively, tau protein in the insoluble fraction of a patient's brain is reacted with regmaine, and then reacted with, for example, an anti-A168 antibody. As shown in Figure 2, when tau protein in the insoluble brain fraction of a CBD patient is reacted with an anti-A168 antibody, a clear band of 37-40 kDa is obtained, whereas no clear band is observed in PSP. Further, as shown in Figure 3, when tau protein in the insoluble brain fraction of a patient is reacted with regmaine and then reacted with an anti-A168 antibody, a 37-40 kDa band resistant to cleavage by regmaine is obtained in CBD, whereas in PSP, cleavage by regmaine proceeds, so it was found that the 37-40 kDa band disappears over time.

[0021] Differences in the structure of tau aggregates observed in CBD based on differences in the cleavage pattern by regmaine can also be distinguished by Western blotting. In this method, in addition to using an anti-A168 antibody, a commercially available anti-tau antibody can also be used. When an anti-A168 antibody is used, distinction can be made based on whether or not a band recognized by the anti-A168 antibody is generated. When an anti-tau antibody is used, distinction can be made based on the difference in molecular weight (the band pattern in Western blotting).

[0022] Another aspect of the present invention is a CBD diagnostic agent for Western blotting, comprising one or more antibodies selected from an antibody that recognizes the N-terminal side of the C-terminal fragment of human tau protein starting at A168, an antibody that recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein, and an antibody that recognizes the A168-N368 fragment of human tau protein. The antibody used in the diagnostic agent of the present invention may be the antibody itself, or it may be an antibody labeled with various enzymes, dyes, etc. Furthermore, the diagnostic agent of the present invention may contain buffers, inactivators, protocols, etc.

[0023] In CBD, there were no abnormalities in regmine function compared to Alzheimer's disease, PSP, and the control group. Resistance to cleavage at the N368 site disappeared by disrupting the structure of the tau aggregate, suggesting that the difference in cleavage patterns is due to differences in the structure of the tau aggregate. Furthermore, it was revealed that by using an antibody that specifically recognizes the regmine cleavage site, it is possible to immunohistochemically distinguish between astrocyte plaques and the clustered astrocytes seen in PSP. [Examples]

[0024] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0025] Example 1 (Preparation of anti-A168 antibody) Two rabbits were immunized a total of four times using a peptide antigen (SEQ ID NO: 1) consisting of 11 amino acids, starting with A168, a 10-amino acid cleavage fragment of tau protein, with a cysteine ​​residue added to the C-terminus. Total blood collection was performed on day 49 after the start of immunization. Affinity purification was performed on serum showing a strong reaction to recombinant tau starting with the A168-C-terminal fragment. Affinity purification was performed using columns immobilized with recombinant full-length tau and recombinant A168-C-terminal fragment, respectively, to purify antibodies that react with the A168-C-terminal fragment but not with full-length tau.

[0026] Example 2 (Reaction between sample and antibody) (material) Frozen brain tissue from a patient, or formalin-fixed paraffin-filled tissue sections from a patient's brain.

[0027] (method) In immunohistochemistry, patient brain tissue, fixed in formalin and embedded in paraffin, is sectioned and spread on a glass slide. After deparaffinization, thermal activation is performed with a pH 9 buffer. After blocking, the primary antibody is reacted. The primary antibody is used at approximately a 200-fold dilution. After reacting with Alexa fluor dye as a secondary antibody, the autofluorescence is eliminated, and the sample is mounted on a coverslip. Observation is performed using a confocal microscope. In the Western blotting method, a cell suspension is prepared from frozen patient brain tissue, reacted with sarcosyl, and then ultracentrifuged to obtain a pellet (sarcosyl-insoluble fraction). The obtained pellet is divided into two groups: one treated with guanidine hydrochloride to break down the three-dimensional structure of tau aggregates, and the other not treated. Both groups are then treated with regmine, followed by Western blotting and reaction with anti-A168 antibody.

[0028] (result) Immunohistochemical staining showed no staining of astrocyte plaques with antibodies that recognize the N368 cleavage site, but staining was observed in the tufted astrocytes of PSP. Western blotting revealed that the anti-168 antibody recognized the 37-40 kDa C-terminal tau fragment of CBD, but showed little reaction to PSP. Legmine cleaved the N167 and N368 sites of tau aggregates, but resistance to cleavage at N368 was observed only in CBD tau aggregates. The 37-40 kDa C-terminal tau fragment was formed from tau fragments cleaved at N167 but not at N368. As shown in Figure 6, the resistance to cleavage at the N368 site disappeared by breaking the structure of the tau aggregate.

Claims

1. A method for detecting tau fragments specific to corticobasal degeneration in a human sample, characterized by reacting a human-derived sample with an antibody that recognizes the N-terminus containing A168 in the C-terminal fragment of human tau protein starting at A168 and an antibody that recognizes the C-terminus containing N368 in the N-terminal fragment of human tau protein cleaved at N368.

2. The detection method according to claim 1, wherein a sample is determined to have a tau fragment specific to corticobasal degeneration if it reacts with an antibody that recognizes the N-terminus containing A168 in the C-terminal fragment of the human tau protein starting at A168, but does not react with an antibody that recognizes the C-terminus containing N368 in the N-terminal fragment cleaved at N368 of the human tau protein.

3. The detection method according to claim 1, wherein the reaction of the antibody is immunohistochemical staining.

4. Western blotting is performed on human samples using one or more antibodies selected from the following: an antibody that recognizes the N-terminus containing A168 in the C-terminal fragment starting with A168 of human tau protein, an antibody that recognizes the C-terminus containing N368 in the N-terminal fragment cleaved at N368 of human tau protein, and an antibody that recognizes the A168-N368 fragment of human tau protein. A method for detecting tau fragments specific to [a certain substance].

5. The detection method according to any one of claims 1 to 4, wherein the human-derived sample is a human-derived sample containing tau protein.

6. A diagnostic agent for corticobasal degeneration containing an antibody that recognizes the N-terminus containing A168 in the C-terminal fragment of human tau protein starting at A168, and an antibody that recognizes the C-terminus containing N368 in the N-terminal fragment of human tau protein cleaved at N368.

7. A diagnostic agent for Western blotting of corticobasal degeneration containing one or more antibodies selected from an antibody that recognizes the N-terminus containing A168 in the C-terminal fragment of human tau protein starting at A168, an antibody that recognizes the C-terminus containing N368 in the N-terminal fragment cleaved at N368 of human tau protein, and an antibody that recognizes the A168-N368 fragment of human tau protein.

Images