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. Tauopathy includes Alzheimer's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and the like. Among these, both CBD and PSP are representative tauopathies presenting parkinsonism, but it is often clinically difficult to distinguish between the two. A definitive diagnosis is made pathologically based on the morphology of astrocytic tau pathology and biochemically based on the difference in the band pattern of tau C-terminal fragments in the insoluble fraction of the patient's brain, but the diagnosis requires experience.

Summary of the Invention

Problems to be Solved by the Invention

[0003] An object of the present invention is to provide a method capable of easily diagnosing by distinguishing between CBD and PSP among tauopathies.

Means for Solving the Problems

[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, as a procedure for immunohistochemical staining using anti-A168 antibody and anti-N368 antibody, a thinly sliced patient's brain specimen on a slide glass is inactivated by a heating method, and a primary antibody and a secondary antibody are reacted. A treatment for eliminating autofluorescence is performed, and the specimen is sealed with a cover glass, so that microscopic observation can be performed.

[0014] As shown in FIG. 4, the atrial astrocytes in PSP reacted with both of the two kinds of antibodies. On the other hand, as shown in FIG. 5, in the astrocytic plaques in CBD, the anti-A168 antibody reacted, but did not react with the anti-N368 antibody. It was found that in CBD, it is cleaved at N167 but has cleavage resistance at N368.

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

[0016] One aspect of the present invention is a CBD diagnostic agent containing an antibody recognizing the N-terminal side of the C-terminal fragment starting from A168 of the human tau protein and an antibody recognizing the C-terminal side of the N-terminal fragment cleaved at N368 of the human tau protein. The antibody used in the diagnostic agent of the present invention may be the above-mentioned antibody itself, or may be an antibody labeled with various enzymes, dyes, etc. Further, the diagnostic agent of the present invention may contain a buffer, an inactivator, a protocol, etc.

[0017] Another aspect of the present invention is a method for detecting a tau fragment specific to CBD in a human specimen, which comprises performing a Western blot method on a human-derived specimen using one or more antibodies selected from an antibody recognizing the N-terminal side of the C-terminal fragment starting from A168 of the human tau protein, an antibody recognizing the C-terminal side of the N-terminal fragment cleaved at N368 of the human tau protein, and an antibody recognizing the A168-N368 fragment of the human tau protein.

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

[0019] Antibodies that recognize the N-terminal side of the C-terminal fragment starting from A168 of human tau protein and antibodies that recognize the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein can be those described above. In addition, as an antibody that recognizes the A168-N368 fragment of human tau protein, a commercially available antibody that recognizes this fragment can be used.

[0020] In the Western blotting method, for example, tau protein in the insoluble fraction of a patient's brain is reacted with an anti-A168 antibody. Alternatively, after reacting tau protein in the insolubilized patient's brain with legumain, it is reacted with, for example, an anti-A168 antibody. As shown in Figure 2, when tau protein in the insoluble fraction of a CBD patient's brain is reacted with an anti-A168 antibody, a distinct band of 37-40 kDa is obtained, but no distinct band is observed in PSP. Also, as shown in Figure 3, when tau protein in the insoluble fraction of a patient's brain is reacted with legumain and then with an anti-A168 antibody, a 37-40 kDa band resistant to cleavage by legumain is obtained in CBD, but it was found that the 37-40 kDa band disappears over time in PSP because cleavage by legumain progresses.

[0021] Differences in the structure of tau aggregates observed in CBD due to differences in the cleavage pattern of legumain can also be distinguished by Western blotting. In this method, in addition to the use of an anti-A168 antibody, commercially available anti-tau antibodies can also be used. When using an anti-A168 antibody, it can be distinguished by whether a band recognized by the anti-A168 antibody appears. In the case of an anti-tau antibody, it can be distinguished by the difference in molecular weight (the pattern of bands 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 specimen, characterized by reacting a human-derived specimen 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 claim 1, wherein a specimen that reacts with an antibody that recognizes the N-terminal side of the C-terminal fragment starting at A168 of the human tau protein but 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 specimen containing a tau fragment specific to corticobasal degeneration.

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

4. A method for detecting a tau fragment specific to corticobasal degeneration in a human specimen, comprising carrying out a Western blotting method on a human-derived specimen using one or more antibodies selected from an antibody recognizing the N-terminal side of a C-terminal fragment starting from A168 of human tau protein, an antibody recognizing the C-terminal side of an N-terminal fragment cleaved at N368 of human tau protein, and an antibody recognizing the A168-N368 fragment of human tau protein.

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

6. A diagnostic agent for corticobasal degeneration, comprising an antibody which recognizes the N-terminal side of the C-terminal fragment starting from A168 of human tau protein and an antibody which recognizes the C-terminal side of the N-terminal fragment cleaved at N368 of human tau protein.

7. A diagnostic agent for corticobasal degeneration for western blotting, comprising one or more antibodies selected from an antibody recognizing the N-terminal side of a C-terminal fragment starting from A168 of human tau protein, an antibody recognizing the C-terminal side of an N-terminal fragment cleaved at N368 of human tau protein, and an antibody recognizing the A168-N368 fragment of human tau protein.

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