Method for diagnosis of TDP-43-related diseases via blood TDP-43 oligomer measurement

Abstract

The present invention relates to a method for diagnosis of TDP-43-related diseases through blood TDP-43 oligomer measurement. When the blood TDP-43 oligomer detection method developed in the present application is used, the blood TDP-43 oligomer can be effectively detected, enabling the diagnosis of TDP-43-related diseases with high specificity and reliability.

Description

Diagnosis of TDP-43-related diseases by measuring blood TDP-43 oligomers

[0001] The present invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the research project number 2021R1A6A1A03038996, the research management specialized organization of the project is the National Research Foundation of Korea, the research project name is "University Key Research Institute Support Project", the research project name is "Development of Diagnosis and Treatment Technology for Geriatric Diseases", the main organization is Gachon University, and the research period is 2021.06.01-2030.05.31.

[0002] The present invention was made under the support of the Ministry of Science and ICT of the Republic of Korea under the research project number RS-2023-00251396, the research management specialized organization of the project is the National Research Foundation of Korea, the research project name is "Individual Basic Research (MSIT)", the research project name is "Screening of Amyloid Beta Aggregation Inhibitor Compounds and Study of Mechanism of Action for Discovery of Alzheimer's Disease Treatment Candidates", the main organization is Gachon University, and the research period is 2023.06.01 - 2024.02.28.

[0003] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0117300, filed with the Korean Intellectual Property Office on September 4, 2023, the disclosure of which is incorporated herein by reference.

[0004] The present invention relates to a method for diagnosing a TDP-43-related disease by measuring blood TDP-43 oligomers.

[0005]

[0006] TDP-43 (TAR DNA-binding protein 43, transactive response DNA binding protein 43) is a protein that exists mainly in the nucleus. It binds to DNA or RNA to inhibit gene transcription and is involved in gene expression by regulating RNA splicing and translation processes.

[0007] Although the accumulation of insoluble aggregates of TDP-43 is a major pathological feature of various neurodegenerative diseases, some studies using cell models or animal models expressing TDP-43 have reported that overexpression of TDP-43 alone causes neurotoxicity without the formation of insoluble aggregates. This has led to the hypothesis that neurodegeneration is caused by the loss of normal TDP-43 function through post-translational modifications (PTMs), such as phosphorylation, acetylation, fragmentation, ubiquitination, oxidation of cysteine ​​residues, and sumoylation, or by the formation of toxic oligomers under cellular stress conditions. This has led to active research on TDP-43 oligomers.

[0008] Currently, the only way to diagnose diseases caused by the TDP-43 protein is postmortem staining of the brain. While expensive tests like MRI can aid in diagnosis, there are no clinically proven methods for making a definitive diagnosis. In particular, FTD and LATE share similar symptoms to Alzheimer's disease, making them highly susceptible to misdiagnosis. Furthermore, accurate treatment is impossible in the early stages. Therefore, the development of diagnostic technologies that can easily and reliably diagnose these neurodegenerative diseases is urgently needed.

[0009]

[0010] The present inventors developed a technique for measuring TDP-43 oligomers in blood to effectively diagnose TDP-43-related diseases, and diligently sought to confirm the diagnostic potential of TDP-43-related diseases through the measurement of TDP-43 oligomers. As a result, they completed the present invention by demonstrating that TDP-43 oligomer measurement can diagnose TDP-43-related diseases with high specificity and reliability.

[0011] Accordingly, an object of the present invention is to provide a method for providing information necessary for diagnosing a TDP-43-related disease, which comprises a step of measuring the concentration of TDP-43 oligomers in a biological sample isolated from a subject.

[0012] Another object of the present invention is to provide a composition for diagnosing a TDP-43-related disease, comprising a preparation for measuring the concentration of TDP-43 oligomers in a biological sample.

[0013] Another object of the present invention is to provide a diagnostic kit for diagnosing a TDP-43-related disease comprising the composition.

[0014]

[0015]

[0016] The present invention provides the following inventions 1 to 17.

[0017] 1. A method for providing information necessary for diagnosing TDP-43-related diseases, including the following steps:

[0018] (a) measuring the concentration of TDP-43 oligomers in a biological sample isolated from a subject; and

[0019] (b) comparing the measured concentration of TDP-43 oligomers with the measured value for a normal control group; if the concentration of TDP-43 oligomers in the subject is higher than that in the normal control group, the subject is determined to have a TDP-43-related disease.

[0020] 2. 1. A method for providing information necessary for diagnosing a TDP-43-related disease, comprising the following steps:

[0021] (a) measuring the concentration of TDP-43 oligomers and the concentration of total TDP-43 in a biological sample separated from a subject; and

[0022] (b) comparing the concentration of TDP-43 oligomers relative to the measured total TDP-43 concentration with the measured value for a normal control group; if the ratio of the concentration of TDP-43 oligomers relative to the total TDP-43 concentration of the subject is higher than that of the normal control group, the subject is determined to have a TDP-43-related disease.

[0023] 3. A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the TDP-43-related disease in 1 is at least one selected from the group consisting of LATE (Limbic-predominant Age-related TDP-43 Encephalopathy), semantic dementia (SD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).

[0024] 4.3 A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the semantic dementia is semantic dementia with frontotemporal dementia (SD-FTD) or semantic dementia with Alzheimer's disease (SD-AD).

[0025] 5. 1. A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the measurement of the TDP-43 concentration is performed through the following method:

[0026] Measurement of the concentration of TDP-43 oligomers or total TDP-43 is performed by an analytical method selected from the group consisting of Enzyme-Linked Immunosorbent Assay (ELISA), Western blot, Immunoassay, Affinity Probe Protein Identification (APPI), Immunofluorescence assay, and Surface Plasmon Resonance (SPR).

[0027] 6. A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, synovial fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine, cerebrospinal fluid, and combinations thereof.

[0028] 7. A composition for diagnosing a TDP-43-related disease, comprising a preparation for measuring the concentration of TDP-43 oligomers in a biological sample.

[0029] In 8.7, the agent for measuring the TDP-43 oligomer concentration is at least one agent selected from the group consisting of a compound that specifically reacts with TDP-43, a polypeptide that specifically binds, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer, a composition for diagnosing a TDP-43-related disease.

[0030] 9.7 A composition for diagnosing a TDP-43-related disease, wherein the composition further comprises a preparation for measuring the total TDP-43 concentration.

[0031] A diagnostic kit for diagnosing TDP-43 related diseases comprising a composition of 10.7.

[0032] 11. A method for diagnosing TDP-43-related diseases, comprising the following steps:

[0033] (a) measuring the concentration of TDP-43 oligomers in a biological sample isolated from a subject; and

[0034] (b) comparing the measured concentration of TDP-43 oligomers with the measured value for a normal control group; if the concentration of TDP-43 oligomers in the subject is higher than that in the normal control group, the subject is diagnosed as having a TDP-43-related disease.

[0035] 12. A method for diagnosing a TDP-43-related disease, comprising the following steps:

[0036] (a) measuring the concentration of TDP-43 oligomers and the concentration of total TDP-43 in a biological sample separated from a subject; and

[0037] (b) comparing the concentration of TDP-43 oligomers relative to the measured total TDP-43 concentration with the measured value for a normal control group; if the ratio of the concentration of TDP-43 oligomers relative to the total TDP-43 concentration of the subject is higher than that of the normal control group, the subject is diagnosed as having a TDP-43-related disease.

[0038] 13. A method for diagnosing a TDP-43-related disease, wherein the TDP-43-related disease is at least one selected from the group consisting of LATE (Limbic-predominant Age-related TDP-43 Encephalopathy), semantic dementia (SD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).

[0039] 14.13, a method for diagnosing a TDP-43-related disease, wherein the semantic dementia is semantic dementia with frontotemporal dementia (SD-FTD) or semantic dementia with Alzheimer's disease (SD-AD).

[0040] 15. In 11, the measurement of the TDP-43 concentration is performed through the following method: TDP-43 related disease diagnosis method:

[0041] Measurement of the concentration of TDP-43 oligomers or total TDP-43 is performed by an analytical method selected from the group consisting of Enzyme-Linked Immunosorbent Assay (ELISA), Western blot, Immunoassay, Affinity Probe Protein Identification (APPI), Immunofluorescence assay, and Surface Plasmon Resonance (SPR).

[0042] 16. A method for diagnosing a TDP-43-related disease, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, synovial fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine, cerebrospinal fluid, and combinations thereof.

[0043] 17. Use of a composition comprising a preparation for measuring the concentration of TDP-43 oligomers in a biological sample for diagnosing a TDP-43-related disease.

[0044]

[0045] According to one aspect of the present invention, the present invention provides a method for providing information necessary for diagnosing a TDP-43-related disease, comprising the following steps:

[0046] (a) measuring the concentration of TDP-43 oligomers in a biological sample isolated from a subject; and

[0047] (b) comparing the measured concentration of TDP-43 oligomers with the measured value for a normal control group; if the concentration of TDP-43 oligomers in the subject is higher than that in the normal control group, the subject is determined to have a TDP-43-related disease.

[0048] The present inventors developed a technique for measuring TDP-43 oligomers in blood to effectively diagnose TDP-43-related diseases and worked diligently to confirm the diagnostic potential of TDP-43-related diseases through the measurement of TDP-43 oligomers. As a result, they demonstrated that TDP-43 oligomer measurement can diagnose TDP-43-related diseases with high specificity and reliability.

[0049] The term TDP-43 (TAR DNA-binding protein of 43 kDa) in this specification refers to a protein with a molecular weight of 43 kDa, which has been identified through ongoing neuropathological research. TDP-43 plays a crucial role in the physiological and pathological biological processes of the central nervous system by participating in RNA degradation and binding. Known functions include RNA binding and processing, gene transcription regulation, and mRNA transport, and it is also known as a marker for degenerative diseases.

[0050] The term “TDP-43 oligomer” as used herein refers to a soluble or insoluble form of TDP-43 monomers aggregated together. The TDP-43 monomer is 43 kDa and consists of 414 amino acid sequences (SEQ ID NO: 1).

[0051] Oligomers can include dimers, which are two monomers joined together, trimers, tetramers, pentamers, hexamers, and more.

[0052] The term "diagnosis" as used herein includes determining an individual's susceptibility to a specific disease or condition, determining the prognosis of an individual with a specific disease or condition, or thermetrics (e.g., monitoring an individual's condition to provide information on the efficacy of treatment). In a broad sense, it refers to assessing all aspects of a patient's illness, such as the name of the disease, etiology, type of illness, severity, detailed aspects of the illness, and the presence or absence of complications.

[0053] As used herein, the terms "subject," "individual," or "patient" refer to any single individual requiring treatment. The term "subject" also includes any individual participating in a clinical research trial who does not exhibit any clinical manifestations of disease, a subject participating in an epidemiological study, or a subject used as a control.

[0054] The term “normal control group” as used herein refers to a healthy control group that does not show signs of a TDP-43-related disease, including, but not limited to, a group without abnormalities in cognitive ability or language ability, and a group without abnormalities in indicators used for diagnosing a TDP-43-related disease.

[0055] In the present specification, when the concentration of TDP-43 oligomers in the subject is higher than that in the normal control group, it means that the concentration of TDP-43 oligomers in the subject is significantly higher than that in the normal control group to the extent that the activity can be diagnosed as a TDP-43-related disease, and for example, it may be higher by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, or 30% or more, but is not limited thereto.

[0056] In one embodiment of the present invention, the method for providing information necessary for diagnosing a TDP-43-related disease comprises the following steps:

[0057] (a) measuring the concentration of TDP-43 oligomers and the concentration of total TDP-43 in a biological sample separated from a subject; and

[0058] (b) comparing the concentration of TDP-43 oligomers relative to the measured total TDP-43 concentration with the measured value for a normal control group; if the ratio of the concentration of TDP-43 oligomers relative to the total TDP-43 concentration of the subject is higher than that of the normal control group, the subject is determined to have a TDP-43-related disease.

[0059] In one embodiment of the present invention, the TDP-43 related disease is at least one selected from the group consisting of Limbic-predominant Age-related TDP-43 Encephalopathy (LATE), semantic dementia (SD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).

[0060] However, it is not limited to this and can include all diseases associated with the accumulation of TDP-43. For example, in addition to LATE (Limbic-predominant Age-related TDP-43 Encephalopathy), semantic dementia (SD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS), it includes Alzheimer's disease and Huntington's disease, which show TDP-43 lesions.

[0061] The term frontotemporal dementia (FTD) as used herein refers to a disease in which neurons in the areas of the brain, including the frontal and temporal lobes, are damaged. Lesions associated with the presence of the TDP-43 protein are found, and symptoms such as decline in cognitive function, social behavior, and language abilities are characterized. FTD has three clinical subtypes: (1) behavioral variant FTD (bvFTD), which is characterized by abnormal changes in behavior and personality related to cortical degeneration predominantly in the frontal lobe; (2) progressive nonfluent aphasia (PNFA), which is characterized by grammatical and motor language deficits associated with left perisylvian cortex atrophy; and (3) semantic dementia (SD), which is characterized by a progressive decline in word knowledge and word-object associations related to anterior temporal lobe neuronal loss. Neuropathologically, FTD is divided into FTD-TAU, which is characterized by tau pathology, and FTD-TDP, which is characterized by TDP-43 pathology. However, FTD-TAU and FTD-TDP are clinically phenotypically indistinguishable, especially in the PNFA type, and only semantic dementia (SD) presents with pure TDP-43 pathology.

[0062] Semantic dementia (SD) is a type of dementia characterized by impairments in the frontal lobe or parietal region, leading to a decline in speech, language comprehension, and semantic perception abilities, such as the ability to understand word meaning. In other words, brain functions related to the recognition of objects and people, as well as object recognition, are impaired, resulting in a decline in the ability to understand the meaning of objects, people, and places. SD dementia typically develops in people in their 50s to 70s and is characterized by impaired hypothetical language skills.

[0063] Amyotrophic lateral sclerosis (ALS) is a disease that causes movement-related muscle weakness and atrophy due to damage to motor neurons in the brain and spinal cord, and TDP-43 is known to be found in lesions associated with this disease.

[0064] Alzheimer's disease is the most common form of dementia, characterized by symptoms such as memory, thinking, and judgment decline. Although TDP-43 is not the primary cause of Alzheimer's disease, lesions associated with TDP-43 are found in some patients. Huntington's disease is a progressive, inherited disorder associated with damage to the central nervous system. Studies have also observed abnormalities in the localization of the TDP-43 protein in Huntington's disease. Limbic-predominant Age-related TDP-43 Encephalopathy (LATE) is a type of dementia caused by age-related brain cell damage. TDP-43 protein accumulates in the brain, and is found in brain regions associated with emotional functions such as memory and decision-making, such as the hippocampus, more than in other brain regions. For this reason, LATE is also called a subtype of hippocampal dementia.

[0065] In one embodiment of the present invention, the semantic dementia is semantic dementia with frontotemporal dementia (SD-FTD) or semantic dementia with Alzheimer's disease (SD-AD). In an embodiment of the present invention, it was confirmed that both semantic dementia with frontotemporal dementia (SD-FTD) and semantic dementia with Alzheimer's disease (SD-AD) can be effectively diagnosed based on the concentration of TDP-43 oligomers.

[0066] In one embodiment of the present invention, the measurement of the TDP-43 concentration is performed by the following method:

[0067] Measurement of the concentration of TDP-43 oligomers or total TDP-43 is performed by an analytical method selected from the group consisting of Enzyme-Linked Immunosorbent Assay (ELISA), Western blot, Immunoassay, Affinity Probe Protein Identification (APPI), Immunofluorescence assay, and Surface Plasmon Resonance (SPR). However, the measurement of the concentration of TDP-43 oligomers or total TDP-43 may be performed by any method capable of confirming the level of protein expression, such as protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDITOF (Sulface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation assay, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-Mass Spectrometry / Mass Spectrometry (LC-MS), Western blotting, and enzyme linked immunosorbent assay (ELISA). It can be done through this method.

[0068] The term “total TDP-43” as used herein is meant to include both TDP-43 monomers and oligomers.

[0069] In one embodiment of the present specification, the concentration of the TDP-43 oligomer or total TDP-43 can be measured by a detection method using an antibody or an antigen-binding fragment thereof. The detection method using the antibody or an antigen-binding fragment thereof can be an ELISA or a technique applied thereto. The measurement of the total TDP-43 concentration can be performed using an antibody that binds to a TDP-43 epitope. The measurement of the TDP-43 oligomer concentration can be performed using an antibody that binds to multiple epitopes of the TDP-43 oligomer. Specifically, the TDP-43 oligomer can be detected effectively through a multimer detection system (MDS) method that uses the same antibody that binds to multiple epitopes of the TDP-43 oligomer as a capture antibody and a detection antibody. The MDS refers to a method for discriminating and detecting a multimer form from a monomer form of a multimer-forming polypeptide.

[0070] In the present specification, the MDS method is a method for detecting a multimer form from a monomer form of a multimer-forming polypeptide of a sample, which may include the following steps: (a) contacting the sample with a capture antibody that recognizes an epitope on the multimer-forming polypeptide that captures the monomer form, the multimer form, or the monomer and the multimer form; (b) contacting the captured monomer form, the multimer form, or the monomer and the multimer form with a detection antibody that recognizes an epitope identical to or overlapping with the epitope of step (a); and (c) detecting a multimer form-detection antibody complex.

[0071] As used herein, the term "multimer-forming polypeptide" refers to a polypeptide capable of forming aggregates. The term "multimer-forming polypeptide" can be used interchangeably with the term "aggregate-forming polypeptide." For example, the multimer-forming polypeptide may be, but is not limited to, TDP-43.

[0072] The above MDS utilizes two types of antibodies: a capture antibody and a detection antibody. As used herein, the term "capture antibody" refers to an antibody capable of binding to a multimer-forming polypeptide to be detected in a sample. The term "detection antibody" refers to an antibody capable of binding to a multimer-forming polypeptide captured by the capture antibody. The most significant feature of the MDS method used in the present invention is that it utilizes a set of capture antibodies and detection antibodies that specifically recognize epitopes on a multimer-forming polypeptide, wherein the epitopes specifically recognized by the capture antibodies and detection antibodies are identical or overlap each other. The term "overlapped with" used when referring to the epitopes of the capture antibodies and detection antibodies encompasses epitopes that include completely or partially overlapping amino acid sequences. More specifically, the above MDS method may be a detection method disclosed in Republic of Korea Patent No. 10-0987639.

[0073] In one embodiment of the present invention, the biological sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, synovial fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine, cerebrospinal fluid, and combinations thereof.

[0074]

[0075] In one aspect of the present invention, the present invention provides a composition for diagnosing a TDP-43-related disease, comprising a preparation for measuring the concentration of TDP-43 oligomers in a biological sample.

[0076] Tools / reagents that may be included in the composition for determining whether the diagnosis or severity is alleviated include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, solubilizers, detergents, buffers, stabilizers, etc. Suitable carriers include, but are not limited to, soluble carriers, for example, physiologically acceptable buffers known in the art, for example, PBS, insoluble carriers, for example, polymers such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesins, cross-linked dextrans, polysaccharides, magnetic microparticles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

[0077] In one embodiment of the present invention, the agent for measuring the TDP-43 oligomer concentration is at least one agent selected from the group consisting of a compound that specifically reacts with TDP-43, a polypeptide that specifically binds, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer.

[0078] The term 'antibody' in the present invention is used in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting the desired biological activity. Specifically, the antibody used in MDS may bind to multiple epitopes of TDP-43.

[0079] In one embodiment of the present invention, the composition further comprises an agent for measuring the total TDP-43 concentration. The agent for measuring the total TDP-43 oligomer concentration is at least one agent selected from the group consisting of a compound that specifically reacts with TDP-43, a polypeptide that specifically binds, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer. Specifically, the antibody used for measuring the total TDP-43 concentration may bind to a single epitope of TDP-43.

[0080]

[0081] In one aspect of the present invention, the present invention provides a kit for diagnosing a TDP-43-related disease comprising the composition for diagnosing a TDP-43-related disease.

[0082] The kit for diagnosing TDP-43-related diseases of the present invention comprises one or more types of compositions, solutions, or devices suitable for an analysis method. For example, the kit is not particularly limited in the method as long as it can measure the concentration of TDP-43 oligomers, and preferably, a method for determining the concentration of TDP-43 oligomers using antibodies or reactive compounds can be applied.

[0083] The diagnostic kit of the present invention may include essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific for the TDP-43. The antibody is an antibody having high specificity and affinity for the marker protein and little cross-reactivity to other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. In addition, the ELISA kit may include an antibody specific for a control protein. In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.

[0084]

[0085] The features and advantages of the present invention are summarized as follows:

[0086] (a) The present invention provides a method for providing information necessary for diagnosing a TDP-43-related disease, comprising the step of measuring the concentration of TDP-43 oligomers in a biological sample separated from a subject.

[0087] (b) The present invention provides a composition for diagnosing a TDP-43-related disease, comprising a preparation for measuring the concentration of TDP-43 oligomers in a biological sample.

[0088] (c) Another object of the present invention is to provide a diagnostic kit for diagnosing a TDP-43-related disease comprising the composition.

[0089] (d) When using the blood TDP-43 oligomer detection method developed by this invention, blood TDP-43 oligomer can be effectively detected, and TDP-43-related diseases can be diagnosed with high specificity and reliability.

[0090]

[0091] Figure 1 illustrates a schematic diagram of a multimer detection system (MDS) for detecting TDP-43 oligomers. In (A), the protein monomer has only a single epitope bound by the capture antibody. This prevents the biotinylated detection antibody from binding to the protein, resulting in no signal generation. Conversely, in (B), the protein oligomer has multiple epitopes that remain exposed when the protein is immobilized. The biotinylated detection antibody is free to bind to the epitopes, resulting in signal generation.

[0092] Figure 2 shows the plasma concentrations of oligomeric TDP-43 in Korean patients with various neurodegenerative diseases. Tukey's test showed statistically significant differences in the plasma concentrations of oligomeric TDP-43 (o-TDP-43) in patients with semantic dementia (SD) compared with all other neurodegenerative diseases, including the normal control group. No significant differences were found for other neurodegenerative diseases compared with the normal control group. Oligomer TDP-43 concentrations were measured in duplicate. Disease groups included the normal control group, mild cognitive impairment (MCI), early-onset Alzheimer's disease (EOAD), late-onset Alzheimer's disease (LOAD), idiopathic Parkinson's disease (IPD), and semantic dementia (SD).

[0093] Figure 3 shows total TDP-43 plasma concentrations in Korean patients with various neurodegenerative diseases. Analysis of variance (ANOVA) revealed no significant differences in total TDP-43 (t-TDP-43) concentrations across all neurodegenerative diseases. t-TDP-43 concentrations were measured in duplicate.

[0094] Figure 4 shows the ratio of oligomeric TDP-43 (o-TDP-43) to total TDP-43 (t-TDP-43) concentrations. Tukey's test showed a statistically significant difference in the ratio of o-TDP-43 to t-TDP-43 concentrations in semantic dementia (SD) samples compared to other neurodegenerative diseases and healthy controls. No significant differences were found in any other group compared to all other groups except SD.

[0095] Figure 5 shows the results of the analysis of oligomeric TDP-43 (o-TDP-43) concentrations in stratified semantic dementia (SD) samples compared to other neurodegenerative diseases. The o-TDP-43 concentrations in SD-FTD (frontotemporal dementia) samples were significantly increased compared to all other groups except SD-AD (Alzheimer's disease) (p < 0.05). The o-TDP-43 concentrations in SD-AD samples were also significantly greater than those in normal controls, mild cognitive impairment (MCI), early-onset Alzheimer's disease (EOAD), and late-onset Alzheimer's disease (LOAD).

[0096] Figure 6 shows the results of ROC analysis of the concentrations and ratios of oligomeric TDP-43 (o-TDP-43) and total TDP-43 (t-TDP-43). Only the ratios of o:t-TDP-43 and o-TDP-43 showed superior diagnostic performance in identifying semantic dementia (SD) samples among other groups, including normal controls.

[0097]

[0098]

[0099] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0100]

[0101] Example

[0102] Example 1: Materials and Methods

[0103] 1-1. Anti-TDP-43 antibodies and recombinant TDP-43

[0104] Anti-human TDP-43 antibodies were obtained from Proteintech (10782-2-AP and 12892-1-AP, N- and C-terminal detection, respectively). Samples extracted from both antibodies were diluted to 100 μg / mL in phosphate-buffered saline (PBS) and biotinylated for 2 h at room temperature. Both antibodies were then dialyzed for 2 h at room temperature using a Slide-A-Lyzer Dialysis cassette and incubated overnight against fresh PBS at 4°C. Recombinant TDP-43 (rTDP-43) was purchased from Abcam and stored at -80°C until needed.

[0105]

[0106] 1-2. Participants and Samples

[0107] The demographics of the participants enrolled in the Alzheimer's Disease All Markers (ADAM) study at Seoul National University Bundang Hospital, including patients with Semantic Dementia (SD; n = 16), Mild Cognitive Impairment (MCI; n = 18), Early-Onset Alzheimer's Disease (EOAD; n = 32), Late-Onset Alzheimer's Disease (LOAD; n = 16), and Parkinson's Disease (PD; n = 12), are summarized in Table 1 . A clinical diagnosis of AD was made according to the following criteria: (1) probable AD criteria proposed by the National Institute on Aging-Alzheimer's Association Working Group (NIAAA, 2011), (2) male or female patients aged 50 to 80 years, (3) at least 6 years of education, and (4) at least 6 months of follow-up by a board-certified neurologist to determine the clinical course of AD. AD was classified into two groups, EOAD or LOAD, based on the age of onset set at 65 years. MCI was diagnosed based on impairment in one or more cognitive domains but preserved functional ability and not meeting the National Institutes of Health and Aging (NIA-AA) criteria for dementia due to AD. PD was diagnosed based on a positive positron emission tomography (PET) scan with 18F radiolabeled N-(3-fluoropropyl)-2β-carbomethoxy-3β-(4-liodophenyl) nortrophan (FPCIT). Healthy controls (n = 79) included the following: (1) from a community-based population, (2) no abnormalities on a health screening questionnaire, (3) no complaints of memory abnormalities, (4) a Korean Dementia Screening Questionnaire score of 6 or less, (5) normal general cognition (within 1 standard deviation [SD] of the age- and education-adjusted Mini-Mental State Examination [MMSE] score of 26 or more), and (6) full activities of daily living (ADL) on the Korean version of the Activities of Daily Living Scale (K-IADL ≤0.05).42), (7) no depression (Medium-Scale Geriatric Depression Scale ≤7), (8) no history of thyroid dysfunction, vitamin B12 deficiency, or folate deficiency. Exclusion criteria consisted of any cognitive impairment except Alzheimer's disease, stroke, and delirium.

[0108]

[0109] Demographics of the trial participants Patient characteristics Normal MCIADPDSDEOADLOADSD-ADSD-FTD Age 40-49 50 10000 50-59 20 21 9 0 20 26 0-69 35 31 0 32 1 4 7 0-79 1 1 9 0 9 6 4 3 8 0-89 1 3 0 4 2 1 1 Gender Male 25 6 12 6 8 3 3 Female 47 1 1 8 10 4 3 7 MMSE score 0-9 0 9 2 2 3 10-19 21 0 7 2 2 0-24 1 7 9 7 1 1 2 5-30 7 1 8 2 0 1 2 Race: East Asian (Korean)

[0110] 1-3. Plasma collection

[0111] Approximately 10 mL of whole blood was collected from each patient using a vacutainer holder and a vacutainer ethylenediaminetetraacetic acid (EDTA) collection tube. Blood samples were centrifuged at 850 x g for 30 minutes at room temperature within 2 hours of collection. 40 to 120 μL of plasma were diluted in purple-capped polypropylene cryovials (Axygen by Corning), appropriately labeled, and stored at -80°C until further use.

[0112]

[0113] 1-4. Synthesis of oligomers mimicking standard proteins

[0114] Maleimide-activated ovalbumin (OVA) was dissolved in ddH2O to a final concentration of 10 mg / mL. The TDP-43 peptide corresponding to the target epitope was dissolved in ddH2O to a final concentration of 5 mg / mL. Then, 50 μL of OVA was mixed with 50 μL of the target peptide and up to 300 μL of binding buffer (0.1 M EDTA + PBS). The mixed solution was incubated with rotation at room temperature for 2 hours. The OVA in the solution was then blocked with 1 μL of 120 mg / mL cysteine ​​dissolved in acidic PBS (pH 4.5) and incubated with rotation at room temperature for 1 hour. The solution was then transferred to a dialysis cassette and dialyzed against PBS for 2 hours at room temperature, then transferred to 4°C and incubated overnight in fresh PBS. The target peptide was attached to the OVA carrier protein, resulting in an oligomeric mimic standard protein (OMSP), which was collected in a microcentrifuge tube and stored at 4°C until use.

[0115]

[0116] 1-5. ELISA and MDS (multimer detection system) for measuring TDP-43

[0117] 100 μL of 1 μg / mL 10782-2-AP antibody (MDS) or 12892-1-AP (total ELISA) was coated onto black Nunc Maxisorp microwells overnight at 4°C. The microwells were then washed three times with 300 μL of PBS containing Tween (PBST; Thermo Scientific) and blocked with 200 μL of BlockAce (Bio-Rad) diluted 25% in PBS for 1 h at room temperature. After washing the microwells once more with PBST, 100 μL of standards in the form of OMSP (50, 10, 2, 0 ng / mL) serially diluted in PBST (MDS) or recombinant TDP-43 (50, 10, 2, 0 ng / mL) in PBST was added (total ELISA). For patient or control samples, 50 μL of plasma sample was added to the microwell in 150 μL of PBST and incubated at 37°C for 1 hour. After washing the analyte three times with PBST, 100 μL of 0.5 μg / mL biotinylated anti-TDP-43 (10782-1-AP) antibody in PBST containing 5% BlockAce was added and incubated at 37°C for 1 hour to perform MDS (multimer detection system) and ELISA. The microwell was then washed again and 100 μL of streptavidin was conjugated with horseradish peroxidase (streptavidin-poly-HRP, Pierce Thermo Scientific, USA) diluted 10,000-fold in PBST containing 5% BlockAce and incubated at room temperature for 30 minutes. After a final wash of the microwells, 100 μL of SuperSignal ELISA Pico chemiluminescent substrate (Thermo Scientific) was added, and the resulting luminescence was immediately measured using a PerkinElmer Victor 3 multilabel microplate reader. A schematic of the MDS method for measuring TDP-43 oligomers is shown in Figure 1.

[0118]

[0119] 1-6. Statistical Analysis

[0120] Statistical analyses were performed using SPSS 25 (SPSS Inc., Chicago, IL). For all tests, p <0.05 was considered statistically significant. The normality of the distribution was assessed using the Kolmogorov-Smirnov test, and skewed distributions were transformed using the Box-Cox method. A one-way analysis of variance (ANOVA) was then used to assess significant differences between the disease groups and the healthy control group. Post hoc analysis (Tukey's test) was used to identify pairs of groups with significantly different concentrations. Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic accuracy of the ELISA for phenotypes with statistically higher parameters compared to the healthy control group.

[0121]

[0122] Example 2: Analysis of blood TDP-43 concentration

[0123] By analyzing the concentration of TDP-43 in the blood of patients with neurodegenerative diseases, we verified the possibility of effective diagnosis of neurodegenerative diseases through measurement of blood TDP-43 oligomers.

[0124] The results are shown in Figures 2, 3, 4, 5 and 6.

[0125] As shown in Figure 2, plasma TDP-43 oligomer (o-TDP-43) concentrations were significantly higher in SD patients compared to other neurodegenerative disease groups and normal controls (p <0.05). Plasma o-TDP-43 concentrations ranged from 7.43 to 37.45 (mean 23.86±4.24) ng / mL in SD, 6.86 to 15.09 (mean 9.97±0.79) ng / mL in PD, 0 to 12.35 (mean 4.89±0. 90) ng / mL in LOAD, 0 to 18.15 (mean 7.03±0.92) ng / mL in EOAD, and 0 to 11.92 (mean 3.26±0.46) ng / mL in healthy controls. There was a significant difference between groups in o-TDP-43 plasma concentrations (ANOVA; p <0.05). Post hoc analysis (Tukey test) on o-TDP-43 concentrations showed that SD patients had significantly higher o-TDP-43 concentrations compared to other disease groups and normal controls (p <0.05).

[0126] However, as shown in Figure 3, no statistically significant difference was found in the blood total TDP-43 (t-TDP-43) concentrations in all groups.

[0127] As shown in Fig. 4, when o-TDP-43 concentrations were plotted as a ratio to t-TDP-43 concentrations, significant differences between the groups were also observed. Tukey's test showed that the ratio of o-TDP-43 to t-TDP-43 was significantly higher in SD patients than in all other groups. Plasma o-TDP-43 concentrations were significantly higher in SD-FTD patients than in other disease groups and in the normal control group (p <0.005). When SD patients were classified into the comorbid FTD group (SD-FTD) and the comorbid AD group (SD-AD), the plasma o-TDP-43 concentrations in SD-FTD patients showed a significant difference compared to the other disease groups (excluding SD-AD) and the normal control group.

[0128] As shown in Figure 5, o-TDP-43 concentrations in SD-AD patients were also significantly higher than in other disease groups and the normal control group, but not as significantly as in SD-FTD. No significant differences were observed in plasma o-TDP-43 concentrations between SD-FTD and SD-AD patients. No significant differences in t-TDP-43 plasma concentrations were observed across all groups.

[0129] As shown in Fig. 6, ROC analysis for o-TDP-43 showed excellent diagnostic accuracy in diagnosing SD among other AD pathologies and normal controls. ROC analysis for o-TDP-43 concentration was performed to compare SD with all other groups. The area under the curve (AUC) for the ratio of o:t-TDP-43 was the highest at 0.916 (sensitivity = 84%, specificity = 89%, positive predictive value [PPV] = 46%, negative predictive value [NPV] = 98%), followed by o-TDP-43 at 0.904 at 95% confidence level (sensitivity = 89%, specificity = 86%, PPV = 39%, NPV = 99%). Total TDP-43 performed poorly as a SD marker, with an AUC of 0.351 at the 95% confidence level.

[0130] In this study, SD plasma samples showed significantly higher o-TDP-43 concentrations through MDS compared to plasma collected from other disease groups (p <0.05). However, no significant differences in o-TDP-43 concentrations were found in MCI, EOAD, LOAD, and PD plasma samples compared to the normal control group. Furthermore, no significant differences in t-TDP-43 concentrations were found in plasma from any disease group compared to the normal control group using a conventional ELISA. This is in contrast to a previous study by Foulds et al., which reported significantly increased t-TDP-43 concentrations in the plasma of 46% of FTD patients (n=16) and 22% of AD patients (n=23) in their cohort (n=137). The ELISA used in this study consisted of a monoclonal capture antibody that binds to the RRM-2 (residues 205–222) of TDP-43 and a polyclonal detection antibody that binds to the NTD (residues 1–19) and RRM-2 regions but does not overlap with the capture antibody epitope (residues 194–213). In this study, the same polyclonal antibody (10782-2-AP) used by Foulds et al. to measure o-TDP-43 levels in plasma was used as both capture and detection antibodies. In contrast, a different set of polyclonal antibodies was used to capture and detect t-TDP-43. The capture antibody used (12892-1-AP) is specific for the first 19 residues (272-290) of the low-complexity domain (LCD) of TDP-43, whereas the detection antibody was an NTD and RRM-2-specific polyclonal antibody (10782-2-AP).

[0131] We have previously demonstrated the broad utility of MDS in quantifying oligomers in other proteinopathies. This is the first time this technique has been used in FTD plasma. In another study, postmortem analysis of brain pathology from FTD patients revealed that patients diagnosed with semantic dementia exhibited only ubiquitin and TDP-43-positive inclusions, without tau pathology, which may explain the greater prominence of o-TDP-43 in SD samples. In contrast, the presence of tau pathology leads to the development of PNFA or bvFTD. PNFA is characterized by grammatical and motor language deficits, with tau pathology being more prominent than TDP-43 pathology. Conversely, bvFTD manifests as behavioral and personality abnormalities due to fronto-predominant cortical degeneration, which carries an equal burden of both tau and TDP-43 pathology. When SD patients were stratified by comorbidity with FTD or AD, it was interesting to note that o-TDP-43 concentrations in SD-FTD samples were significantly higher than in the other disease groups (p<0.05; Figure 5). The o-TDP-43 concentrations in SD-AD samples were also significantly higher compared to the other disease groups and healthy controls (p<0.05; Figure 5). However, no significant differences in o-TDP-43 concentrations were observed between the SD-FTD and SD-AD groups. The decreased plasma o-TDP-43 concentrations in the MCI and AD groups may be due to the presence of other pathological proteins, such as Aβ42 and tau neurofibrillary tangles (NFTs), in AD subtypes and MCI, which sequester o-TDP-43 into large fibrils deposited within neurons. Recent studies in a Drosophila model have shown that the presence of TDP-43 specifically enhances tau but not Aβ42 neurotoxicity, thereby increasing tau accumulation. Another study using a tau-induced cell model (iHEK) showed accumulation of cytoplasmic TDP-43.Tau's key role in cytosolic sequestration of other proteins may explain why free TDP-43 levels are unclear and not readily detectable in the plasma of patients with Alzheimer's disease. Another study used 1D and 2D Western immunoblotting and quantitative mass spectrometry in cerebrospinal fluid (CSF), serum samples, blood lymphocytes, brain component fractions, and purified exosomes from patients with amyotrophic lateral sclerosis (ALS), FTD, and non-neurodegenerative diseases. The TDP-43 CSF-to-blood concentration ratio was found to be 1:200, suggesting that CSF TDP-43 can be secreted into the blood. Therefore, in this study, TDP-43 concentrations were measured only in plasma samples.

[0132] Immunohistochemistry of brain tissue still considers total TDP-43 protein pathology the gold standard for identifying neurodegenerative diseases. However, as reported in this study, the ratio of o-TDP-43 and t-TDP-43 concentrations in plasma of SD patients performed well as a diagnostic marker for differentiating TDP-43 protein pathology in SD-FTD patients, with an AUC value of 0.916, demonstrating its potential as an excellent diagnostic marker alongside o-TDP-43 concentration alone (AUC 0.904). In contrast, t-TDP-43 concentration performed poorly as a diagnostic marker, with an AUC value of 0.351. Therefore, as reported in this study, it is expected that o-TDP-43 or the ratio of o-TDP-43 to t-TDP-43 concentrations can effectively diagnose neurodegenerative diseases.

Claims

1. A method for providing information necessary for diagnosing TDP-43-related diseases, including the following steps: (a) measuring the concentration of TDP-43 oligomers in a biological sample isolated from a subject; and (b) comparing the measured concentration of TDP-43 oligomers with the measured value for a normal control group; if the concentration of TDP-43 oligomers in the subject is higher than that in the normal control group, the subject is determined to have a TDP-43-related disease.

2. A method for providing information necessary for diagnosing a TDP-43-related disease, comprising the following steps: (a) measuring the concentration of TDP-43 oligomers and the concentration of total TDP-43 in a biological sample separated from a subject; and (b) comparing the concentration of TDP-43 oligomers relative to the measured total TDP-43 concentration with the measured value for a normal control group; if the ratio of the concentration of TDP-43 oligomers relative to the total TDP-43 concentration of the subject is higher than that of the normal control group, the subject is determined to have a TDP-43-related disease.

3. A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the TDP-43-related disease is at least one selected from the group consisting of LATE (Limbic-predominant Age-related TDP-43 Encephalopathy), semantic dementia (SD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).

4. A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the semantic dementia in the third paragraph is semantic dementia with frontotemporal dementia (SD-FTD) or semantic dementia with Alzheimer's disease (SD-AD).

5. A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the measurement of the TDP-43 concentration in paragraph 1 is performed through the following method: Measurement of the concentration of TDP-43 oligomers or total TDP-43 is performed by an analytical method selected from the group consisting of Enzyme-Linked Immunosorbent Assay (ELISA), Western blot, Immunoassay, Affinity Probe Protein Identification (APPI), Immunofluorescence assay, and Surface Plasmon Resonance (SPR).

6. A method for providing information necessary for diagnosing a TDP-43-related disease, wherein the biological sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, synovial fluid, amniotic fluid, ascites, cervical secretion, vaginal secretion, urine, cerebrospinal fluid, and combinations thereof of the subject in the first paragraph.

7. A composition for diagnosing a TDP-43-related disease, comprising a preparation for measuring the concentration of TDP-43 oligomers in a biological sample. In paragraph 8.7, the agent for measuring the TDP-43 oligomer concentration is at least one agent selected from the group consisting of a compound that specifically reacts with TDP-43, a polypeptide that specifically binds to TDP-43, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), or an aptamer, a composition for diagnosing a TDP-43-related disease.

9. A composition for diagnosing a TDP-43-related disease, wherein the composition further comprises a preparation for measuring the total TDP-43 concentration in accordance with paragraph 7.

10. A diagnostic kit for diagnosing a TDP-43-related disease comprising the composition of Article 7.

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