JP2024064487A - Diagnostic markers for dementia - Google Patents

Abstract

To provide a method of accurately determining dementia using a relatively simple procedure that does not require a large-sized special device, a kit used for the determination, and the like.SOLUTION: A method disclosed herein comprises detecting an amyloid β-HMGB1 complex in one or more biological samples collected from a subject, and determining if the subject has dementia or not using concentration of the amyloid β-HMGB1 complex as an indicator.SELECTED DRAWING: None

Description

The present invention relates to a method for determining whether a subject suffers from dementia such as Alzheimer's disease using amyloid β-HMGB1 complex as an indicator, and a kit for detecting amyloid β-HMGB1 complex and determining whether a subject suffers from dementia.

Alzheimer's disease (hereinafter sometimes referred to as "AD") is an irreversible progressive central nervous system disease that is known to present symptoms such as cognitive impairment (dementia) accompanied by memory impairment and thinking disorders, behavioral disorders, and personality changes. Alzheimer's disease is the most common dementia disease, accounting for approximately 60-80% of all dementia patients. Alzheimer's disease generally develops in elderly people aged 65 or older, but it can also develop in people aged 64 or younger, in which case it is called early-onset Alzheimer's disease.

According to a Cabinet Office announcement, the number of dementia patients in Japan in 2012 was 4.62 million, or one in seven people aged 65 or older, but this number is expected to increase to approximately 7 million, or one in five, by 2025. As the number of dementia patients, such as AD, increases, it is expected that the economic and mental burden on the country and those related to patients will become more severe due to rising medical expenses and nursing care issues.

Dementia is mainly diagnosed by interview and questionnaire, and an objective diagnostic method has not yet been established. Interview and questionnaire methods have the disadvantage that they vary depending on the diagnostician and lack quantification. In order to solve this problem, research is being conducted to establish an objective diagnostic method. For example, in AD, cerebral atrophy is observed as the symptoms progress, so a method of performing image diagnosis using magnetic resonance imaging (MRI) has been devised. In addition, intracellular aggregation (neurofibrillary tangles) of excessively phosphorylated Tau (hereinafter sometimes referred to as "tau") is prominently observed in the brain along with extracellular accumulation (senile plaques) of amyloid beta (hereinafter sometimes referred to as "Aβ"). When Aβ accumulates, microtubules in nerve cells undergo structural changes and nerve cells die, and it is believed that tau leaks out of the cells and accumulates in cerebrospinal fluid (hereinafter sometimes referred to as "CSF") samples as the nerve cells die. For this reason, a diagnostic method using tau in a cerebrospinal fluid sample as a marker has also been devised. However, these methods require large, specialized equipment and are not sufficient in terms of convenience, economy, or versatility.

It has been reported that plasmalogens contained in red blood cells are useful as a diagnostic marker for dementia (Patent Document 1). The present inventor has also reported that HMGB1 (high mobility group box 1) is useful as a diagnostic marker for mild cognitive impairment (hereinafter sometimes referred to as "MCI") (Patent Document 2).

On the other hand, it has been reported that amyloid β and HMGB1 can form a complex in vitro (Non-Patent Document 1). However, it was not previously known that the amyloid β-HMGB1 complex was useful as a diagnostic marker for dementia.

International Publication No. 2012/090625 International Publication No. 2020/255938

Biochem Biophys Res Commun. 2003 Feb 14;301(3):699-703.

The objective of the present invention is to provide a method for accurately determining dementia in a relatively simple manner without the need for large specialized equipment, and to provide a kit or the like for use in said determination.

In the course of intensive research to solve the above problems, the present inventors have found that the concentration of Aβ-HMGB1 complex in biological samples from AD patients is increased using ELISA (Enzyme-Linked Immuno Sorbent Assay), and have found that the concentration of the Aβ-HMGB1 complex can be used as an index to distinguish between those suffering from dementia such as AD and those not suffering from dementia (MCI patients and healthy individuals not diagnosed with brain dysfunction). Furthermore, it has been confirmed that the accuracy of dementia determination can be improved by combining and analyzing the concentration of the Aβ-HMGB1 complex in the biological sample 1 or 2 and the concentration of HMGB1 in the biological sample. The aforementioned Patent Document 2 reports that the HMGB1 concentration is increased in MCI patients compared to healthy individuals, but is reduced to the level of healthy individuals in AD patients. For this reason, it was surprising that the Aβ-HMGB1 complex is useful as a diagnostic marker for dementia such as AD, and that the combination with HMGB1 improves the accuracy of dementia diagnosis. The present invention was completed based on these findings.

That is, the present invention is as follows.
[1] A method for diagnosing dementia, comprising the following steps (a) and (b):
(a) measuring the concentration of amyloid β-HMGB1 complex in one or more biological samples obtained from a subject;
(b) determining whether or not the subject is suffering from dementia using the concentration of amyloid β-HMGB1 complex in one or more biological samples collected from the subject as an index;
[2] The method of determining whether or not amyloid β-HMGB1 complex is likely to be affected by dementia, wherein in step (b), the concentration of the amyloid β-HMGB1 complex in one or more biological samples collected from the subject is compared with the concentration of the amyloid β-HMGB1 complex in one or more biological samples derived from a control individual not suffering from dementia, and if the concentration of the amyloid β-HMGB1 complex in the one or more biological samples collected from the subject is higher than the concentration of the amyloid β-HMGB1 complex in one or more biological samples derived from a control individual not suffering from dementia, the subject is determined to be likely to be affected by dementia, as described in [1] above.
[3] The method of determining the presence or absence of a biological sample according to [1] or [2] above, wherein the biological sample is a blood sample or a cerebrospinal fluid sample.
[4] In step (b),
comparing the concentration of amyloid β-HMGB1 complex in two or more biological samples taken from the subject with the concentration of amyloid β-HMGB1 complex in two or more biological samples taken from a control individual not suffering from dementia; and
comparing the concentration of HMGB1 in one or more biological samples taken from the subject with the concentration of HMGB1 in one or more biological samples from a control individual not suffering from dementia;
The method of any of the above-mentioned [1] to [3], wherein the subject is judged to have a high possibility of suffering from dementia if the concentration of the amyloid β-HMGB1 complex in two or more biological samples collected from the subject is higher than the concentration of the amyloid β-HMGB1 complex in two or more biological samples derived from a control individual not suffering from dementia, and the concentration of HMGB1 in one or more biological samples collected from the subject is higher than the concentration of HMGB1 in one or more biological samples derived from a control individual not suffering from dementia.
[5] The method of any one of [2] to [4] above, wherein the control person not suffering from dementia is a healthy person or a patient with mild cognitive impairment.
[6] The method for determining dementia according to any one of [1] to [5] above, wherein the dementia is Alzheimer's dementia.
[7] A kit for detecting amyloid β-HMGB1 complexes and diagnosing dementia, comprising an antibody that specifically binds to human amyloid β or a labeled version thereof, and an antibody that specifically binds to human HMGB1 or a labeled version thereof.
[8] The kit according to [7] above, further comprising: a kit for detecting HMGB1.

In another embodiment of the present invention,
A method for preparing data for diagnosing dementia, comprising the above step (a) and step (B) of preparing data for diagnosing whether or not a subject suffers from dementia, using the concentration of amyloid β-HMGB1 complex in one or more biological samples collected from the subject as an index; or
A method for diagnosing dementia, comprising the steps (a) and (b);
A method for treating dementia or preventing the worsening of dementia, comprising the above steps (a) and (b), and comprising a step of administering a treatment for treating dementia or a treatment for preventing the worsening of dementia (e.g., administering a dementia treatment drug) to a subject determined to have a high possibility of having dementia in step (b); or
A method for treating dementia or preventing the worsening of dementia, comprising a step of administering a treatment for treating dementia or a treatment for preventing the worsening of dementia (e.g., administering a dementia therapeutic drug) to a dementia patient having a higher concentration of amyloid β-HMGB1 complex in one or more biological samples than the concentration of amyloid β-HMGB1 complex in one or more biological samples derived from a control person not suffering from dementia; or
An antibody that specifically binds to human amyloid β, or a labeled product thereof, and an antibody that specifically binds to human HMGB1, or a labeled product thereof, for use in detecting an amyloid β-HMGB1 complex and in assessing (diagnosing) dementia;
Examples include:

The present invention does not require the use of large specialized equipment and can accurately diagnose dementia using a relatively simple method, which contributes to the treatment of dementia and the prevention of the progression of the disease by providing appropriate treatment to more dementia patients or by providing appropriate measures to prevent the deterioration of dementia.

FIG. 1 shows the results of Western blotting using an anti-Aβ antibody or an anti-HMGB1 antibody after mixing and incubating two types of Aβ (Aβ1-40 or Aβ1-42) with human HMGB1 including dsHMGB1 and atHMGB1 at a molar ratio of 1:1. FIG. 1 shows the results of a similar analysis performed in the experiment of FIG. 1a, except that the concentrations of Aβ (Aβ1-40 or Aβ1-42) and HMGB1 to be mixed were increased by 2-fold or 4-fold, respectively. FIG. 1 shows the results of Western blotting using anti-Aβ antibody or anti-HMGB1 antibody after mixing and incubating Aβ (Aβ1-40) and two types of HMGB1 (dsHMGB1 or atHMGB1) at a molar ratio of 1:1. FIG. 1 shows the results of Western blotting, performed with anti-Aβ antibody or anti-HMGB1 antibody, on cerebral cortex lysates derived from two types of AD model mice (5×FAD mice and APP-KI mice). FIG. 1 shows the results of immunoprecipitation using anti-Aβ antibody or anti-HMGB1 antibody for cerebral cortex lysates derived from different types of AD model mice (5×FAD mice and APP-KI mice), followed by Western blotting using anti-Aβ antibody or anti-HMGB1 antibody. FIG. 2a is a diagram showing the results of measuring the concentration of Aβ-HMGB1 complex in cerebrospinal fluid (CSF) samples in three groups (AD group [AD patients, n = 70], MCI group [MCI patients, n = 23], and control group [healthy subjects, n = 48]). "**" in the figure indicates that there is a statistically significant difference (p < 0.01) (same below). FIG. 2b is a diagram showing the results of measuring the concentration of Aβ-HMGB1 complex in plasma samples in three groups (AD group [AD patients, n = 31], MCI group [MCI patients, n = 3], and control group [healthy subjects, n = 30]). FIG. 2c is a diagram showing the relationship between the concentration of Aβ-HMGB1 complex and the concentration of HMGB1 in CSF samples in the AD group (AD patients, n = 70). Figure 2d is a diagram showing the results of a receiver operating characteristic (ROC) analysis between the AD group and the control group based on the results of Figure 2a. Figure 2e is a diagram showing the results of a receiver operating characteristic (ROC) analysis between the MCI group and the control group based on the results of Figure 2a. Figure 2f is a diagram showing the results of a receiver operating characteristic (ROC) analysis between the AD group and the control group based on the results of Figure 2b. Figure 2g is a diagram showing the results of a receiver operating characteristic (ROC) analysis between the MCI group and the control group based on the results of Figure 2b. Figure 2h shows the relationship between the plasma Aβ-HMGB1 complex concentration and the ADAS-cog score in the AD group and the control group. Figure 2i shows the relationship between the plasma Aβ-HMGB1 complex concentration and the MMSE score in the AD group and the control group. Figure 2j shows the relationship between the concentration of Aβ-HMGB1 complex in plasma and the annual change in ADAS-cog score in the AD group. Figure 2k shows the relationship between the concentration of Aβ-HMGB1 complex in plasma and the annual change in MMSE score in the AD group. Sagittal brain sections from two types of AD model mice (5xFAD mice [Fig. 3a] and APP-KI mice [Fig. 3b]) were analyzed by immunohistochemical staining using anti-Aβ and anti-HMGB1 antibodies. "***" in the figure indicates a statistically significant difference (p<0.001) (hereinafter the same). FIG. 1 shows the results of immunohistochemical staining analysis of sagittal brain sections from familial AD patients with M146L mutation (PS1-M146L) in the presenilin-1 (PS1) gene using anti-Aβ antibody and anti-HMGB1 antibody. FIG. 5 shows the results of immunohistochemical staining analysis of sagittal brain sections derived from two types of AD model mice (5×FAD mice [FIG. 5a] and APP-KI mice [FIG. 5b]) and a sagittal brain section derived from a familial AD patient with PS1-M146L (FIG. 5c) using anti-Aβ, anti-HMGB1, and anti-MAP2 antibodies. This figure shows the results of Western blotting using three types of antibodies (anti-pSer46-MARCKS antibody [Figure 6a], anti-phosphorylated tau antibody [Figure 6b], and anti-β-actin antibody [Figures 6a and 6b]) after incubating primary neurons in the presence of eight types of test substances (dsHMGB1, atHMGB1, Aβ1-40, Aβ1-42, Aβ1-40-dsHMGB1 complex, Aβ1-42-dsHMGB1 complex, and Aβ1-42-atHMGB1 complex). "##" in the figure indicates that there is a statistically significant difference (p<0.01) compared to "no treatment". Figure 6c shows the results of Western blotting using three types of antibodies (anti-pSer46-MARCKS antibody, anti-phosphorylated tau antibody, and anti-β-actin antibody) after incubating primary neurons in the presence of four types of test substances (dsHMGB1, Aβ1-40, Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex) and in the presence or absence of monoclonal antibody #129. FIG. 6d shows the results of Western blotting using four types of antibodies (anti-TLR4 antibody, anti-pSer46-MARCKS antibody, anti-phosphorylated tau antibody, and anti-β-actin antibody) after incubating primary neurons with TLR4 knockdown ("TLR4-siRNA(+)" in the figure) and primary neurons without TLR4 knockdown ("TLR4-siRNA(-)" in the figure) in the presence of four types of test substances (dsHMGB1, Aβ1-40, Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex). "##" in the figure indicates that there is a statistically significant difference (p<0.01) (same below). FIG. 6e shows the results of measuring the number of cells having ballooned endoplasmic reticulum (ER) after incubating primary neurons in the presence of eight types of test substances (dsHMGB1, atHMGB1, Aβ1-40, Aβ1-42, Aβ1-40-dsHMGB1 complex, Aβ1-40-atHMGB1 complex, Aβ1-42-dsHMGB1 complex, and Aβ1-42-atHMGB1 complex). FIG. 6f shows the results of measuring the number of cells having ballooned ER after incubating primary neurons in the presence of four types of test substances (dsHMGB1, Aβ1-40, Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex) and in the presence or absence of monoclonal antibody #129. FIG. 7a is a diagram showing the results of measuring the concentration of Aβ-HMGB1 complex in CSF samples in the AD group (n=31) and the MCI group (n=23) and performing ROC analysis between the two groups. FIG. 7b is a diagram showing the results of measuring the concentration of Aβ-HMGB1 complex in plasma samples in the AD group (n=30) and the MCI group (n=6) and performing ROC analysis between the two groups. FIG. 7c is a diagram showing the results of combining the concentration of Aβ-HMGB1 complex in the CSF sample in FIG. 7a and the concentration of HMGB1 in the CSF sample in both groups and performing ROC analysis between the two groups. FIG. 7d is a diagram showing the results of combining the concentration of Aβ-HMGB1 complex in the plasma sample in FIG. 7b and the concentration of HMGB1 in the CSF sample in both groups and performing ROC analysis between the two groups. Figure 7e shows the results of an ROC analysis between the two groups, combining the concentration of Aβ-HMGB1 complex in CSF samples in Figure 7a, the concentration of Aβ-HMGB1 complex in plasma samples in Figure 7b, and the concentration of HMGB1 in CSF samples in Figure 7c or 7d.

The method of the present invention is not particularly limited as long as it is a method of determining dementia (hereinafter, sometimes referred to as the "method of determination") that sequentially includes the steps of: (a) measuring (and, if necessary, quantifying) the concentration of the Aβ-HMGB1 complex (i.e., a complex of Aβ and HMGB1) in one or more biological samples collected from a subject; and (b) determining whether or not the subject is suffering from dementia (whether or not the subject has developed dementia) using the concentration of the Aβ-HMGB1 complex in one or more biological samples collected from the subject as an indicator. The method of determination of dementia is a method that assists a doctor in diagnosing the presence or absence of dementia, and does not include a diagnostic act by a doctor.

The kit of the present invention is not particularly limited as long as it is a kit (hereinafter sometimes referred to as "the present kit") that contains an antibody that specifically binds to human Aβ (i.e., an anti-human Aβ antibody) or a labeled anti-human Aβ antibody, and an antibody that specifically binds to human HMGB1 (i.e., an anti-human HMGB1 antibody) or a labeled anti-human HMGB1 antibody, and is specified for the uses of "detecting amyloid β-HMGB1 complexes" and "diagnosing dementia." The present kit is a use invention related to a kit for diagnosing dementia or a kit for assisting in the diagnosis of dementia, and these kits usually contain components generally used in this type of kit, such as a carrier, a pH buffer, and a stabilizer, as well as an instruction manual, instructions for diagnosing dementia, and other attached documents.

In the present invention, "dementia" refers to a state in which a decline in cognitive function (e.g., memory function, executive function, attention function, visuospatial cognition function, language comprehension function) is observed, the diagnostic criteria for dementia are met, and basic daily and social activities are impaired. In other words, "dementia" does not include a state in which a decline in cognitive function is observed but the diagnostic criteria for dementia are not met, and basic daily and social activities are not impaired (i.e., mild cognitive impairment). Here, "meeting the diagnostic criteria for dementia" means, for example, one or more selected from the following: a Mini Mental State Examination (MMSE) score of 20 points or less (e.g., 19 points or less, 18 points or less, 17 points or less, 16 points or less, 15 points or less, 14 points or less, 13 points or less, 12 points or less, 11 points or less, etc.); an ADAS-cog (Alzheimer's Disease Assessment Scale-cognitive subscale) score of 10 points or more (e.g., 11 points or more, 12 points or more, 13 points or more, 14 points or more, 15 points or more, 16 points or more, 17 points or more, 18 points or more, 19 points or more, 20 points or more, 21 points or more, 22 points or more, etc.); and a Clinical Dementia Rating (CDR) score of 1 point or more (e.g., 1.1 points or more, 1.2 points or more, 1.3 points or more, 1.4 points or more, etc.).

In this specification, examples of dementia include Alzheimer's disease (Alzheimer's dementia), dementia caused by Parkinson's disease, dementia with Lewy bodies, frontotemporal dementia, dementia caused by progressive non-fluent aphasia, semantic dementia, and dementia caused by corticobasal degeneration. Since the effectiveness of this embodiment has been demonstrated in the examples described below, Alzheimer's dementia is a suitable example.

In the present invention, a "biological sample" may be any sample derived from a subject (living body), and examples thereof include blood samples, cerebrospinal fluid (CSF) samples, and urine samples. In the present embodiment described below, the effectiveness of such samples has been demonstrated, and therefore blood samples and CSF samples are preferred examples.

In the above step (a), the method for measuring and quantifying the concentration of the Aβ-HMGB1 complex in a biological sample may be any method capable of specifically detecting the Aβ-HMGB1 complex protein in the biological sample, and examples of such methods include immunological assays using an antibody that specifically recognizes the Aβ-HMGB1 complex using a biological sample or a processed product thereof (e.g., in the case of a blood sample, a serum sample or a plasma sample; in the case of a cerebrospinal fluid sample, cerebrospinal fluid from which impurities have been removed by centrifugation; in the case of a urine sample, urine from which impurities have been removed by centrifugation).

Examples of the immunological measurement method include immunohistochemical staining using an anti-human Aβ antibody or a labeled substance thereof and an anti-human HMGB1 antibody or a labeled substance thereof, sandwich ELISA, sandwich EIA, sandwich RIA, Western blotting, and a combination of immunoprecipitation and Western blotting.

In the present invention, the value of the Aβ-HMGB1 complex concentration may be an absolute value or a relative value. The absolute value of the Aβ-HMGB1 complex concentration can be calculated, for example, by creating a calibration curve showing the relationship between the amount of signal (e.g., amount of fluorescence, amount of dye) derived from the Aβ-HMGB1 complex and the Aβ-HMGB1 complex concentration using known concentrations of Aβ and/or HMGB1. The relative value of the Aβ-HMGB1 complex concentration can be calculated, for example, based on the amount of signal (e.g., amount of fluorescence, amount of dye) derived from the Aβ-HMGB1 complex derived from a control subject not suffering from dementia.

In the above step (b), the method for determining whether or not a subject has dementia, and in the above step (B), the method for creating data for determining whether or not a subject has dementia, can include, for example, a method for determining whether or not a subject has dementia based on a criterion of being higher than a certain threshold value (cut-off value). Examples of such cut-off values include the average value of Aβ-HMGB1 complex concentrations in biological samples from control subjects not suffering from dementia; the average value + standard deviation (SD); the average value + 2SD; the average value + 3SD; the median value of Aβ-HMGB1 complex concentrations in biological samples from control subjects not suffering from dementia; the third quartile of the Aβ-HMGB1 complex concentration; the maximum value of the Aβ-HMGB1 complex concentration; and the like. In addition, the cutoff value can be calculated using an ROC curve with statistical analysis software based on data on Aβ-HMGB1 complex concentrations in biological samples from dementia patients and data on Aβ-HMGB1 complex concentrations in biological samples from control subjects without dementia, so as to increase sensitivity (the proportion of people with dementia who can be correctly judged as positive) and specificity (the proportion of people without dementia who can be correctly judged as negative).

In the above step (b), the method for determining whether or not a subject suffers from dementia specifically includes:
A method for determining that a subject is highly likely to suffer from dementia, by comparing the concentration of the Aβ-HMGB1 complex in one or more biological samples collected from a subject with the concentration of the Aβ-HMGB1 complex in one or more biological samples from a control subject not suffering from dementia, when the concentration of the Aβ-HMGB1 complex in the one or more biological samples collected from the subject is higher than the concentration of the Aβ-HMGB1 complex in the one or more biological samples from the control subject not suffering from dementia; or
comparing the concentration of amyloid β-HMGB1 complex in two or more biological samples (preferably a blood sample and a cerebrospinal fluid sample) taken from the subject with the concentration of amyloid β-HMGB1 complex in two or more biological samples (preferably a blood sample and a cerebrospinal fluid sample) taken from a control individual not suffering from dementia; and
comparing the concentration of HMGB1 in one or more biological samples (preferably cerebrospinal fluid samples) taken from the subject with the concentration of HMGB1 in one or more biological samples (preferably cerebrospinal fluid samples) from a control individual not suffering from dementia;
A method for determining that a subject is likely to suffer from dementia, when the concentration of amyloid β-HMGB1 complex in two or more biological samples collected from the subject is higher than the concentration of amyloid β-HMGB1 complex in two or more biological samples derived from a control subject not suffering from dementia, and the concentration of HMGB1 in one or more biological samples collected from the subject is higher than the concentration of HMGB1 in one or more biological samples derived from a control subject not suffering from dementia;
Suitable examples include the following.

In addition, in the above step (B), a specific example of a method for creating data for determining whether or not a subject is suffering from dementia is a method in which the concentration of the Aβ-HMGB1 complex in one or more biological samples collected from the subject is compared with the concentration of the Aβ-HMGB1 complex in one or more biological samples from a control subject not suffering from dementia, and if the concentration of the Aβ-HMGB1 complex in one or more biological samples collected from the subject is higher than the concentration of the Aβ-HMGB1 complex in one or more biological samples from the control subject not suffering from dementia, data is created for determining that the subject is highly likely to be suffering from dementia.

In this specification, a "control subject not suffering from dementia" refers to a person not suffering from dementia (a control for the subject), that is, a person who satisfies at least one of the following criteria 1) to 3): 1) no decline in cognitive function; 2) not meeting the diagnostic criteria for dementia; and 3) no impairment in basic daily life or social life.Specific examples of such a person include healthy individuals and patients suffering from mild cognitive impairment, with healthy individuals and patients with mild cognitive impairment being preferred examples.

In the present invention, Aβ in the Aβ-HMGB1 complex, which is an index for determining dementia, is human-derived Aβ (Amyloid beta) protein or a peptide constituting the protein, and specific examples of the peptide constituting Aβ include one or more peptides selected from the following [Group B peptides]:
[B group peptides]
(1) A peptide (Aβ1-40) consisting of the amino acid sequence shown in SEQ ID NO: 1, or a peptide in the Aβ-HMGB1 complex consisting of the amino acid sequence shown in SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted and/or added, and the expression level in dementia patients is higher than that in controls not suffering from dementia;
(2) a peptide (Aβ1-42) consisting of the amino acid sequence shown in SEQ ID NO: 2, or a peptide in the Aβ-HMGB1 complex consisting of the amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted and/or added, and the expression level in dementia patients is higher than that in controls not suffering from dementia;

In the present invention, HMGB1 in the Aβ-HMGB1 complex, which is an indicator for diagnosing dementia, is human-derived HMGB1 (high mobility group box 1) protein or a peptide constituting the same, and specific examples of human-derived HMGB1 protein include one or more proteins selected from the following [Group A proteins]:
[Group A Protein]
(1) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 (HMGB1 isoform 1 [NCBI Reference Sequence: NP_001300821]), or a protein in the Aβ-HMGB1 complex consisting of the amino acid sequence shown in SEQ ID NO: 3 in which one or several amino acids have been deleted, substituted and/or added, and whose expression level in dementia patients is higher than that in controls not suffering from dementia;
(2) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 (HMGB1 isoform 4 [NCBI Reference Sequence: NP_001350590]), or a protein in the Aβ-HMGB1 complex consisting of the amino acid sequence shown in SEQ ID NO: 4 in which one or several amino acids have been deleted, substituted and/or added, and the expression level in dementia patients is higher than that in controls not suffering from dementia;

The above "amino acid sequence in which one or several amino acids have been deleted, substituted and/or added" generally means an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted and/or added, preferably 1 to 7 amino acids, more preferably 1 to 6 amino acids, even more preferably 1 to 5 amino acids, even more preferably 1 to 4 amino acids, particularly preferably 1 to 3 amino acids, especially preferably 1 to 2 amino acids, and most preferably 1 amino acid.

The anti-human Aβ antibody and anti-human HMGB1 antibody in the present kit are used to detect amyloid β-HMGB1 complexes using the immunological assay method and other methods described above. The present kit is preferably further specified for use as "for detecting HMGB1," and HMGB1 can be detected using the anti-human HMGB1 antibody in the present kit and the immunological assay method and other methods described above.

The anti-human Aβ antibody and anti-human HMGB1 antibody in the present kit may be an antibody such as a monoclonal antibody, a polyclonal antibody, a human antibody, a chimeric antibody, or a humanized antibody, and also includes antibody fragments consisting of a portion of an antibody, such as F(ab') ₂ , Fab, diabody, Fv, ScFv, and Sc(Fv) ₂ .

In the present kit, examples of the labeling substance in the labeling product of the anti-human Aβ antibody or anti-human HMGB1 antibody include enzymes such as peroxidase (horseradish peroxidase, etc.), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as FITC, Cy3, Cy5, rhodamine, and Alexa Fluor (registered trademark); fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and luciferase; ³ H, ¹⁴ C, ¹²⁵ Examples of the antibody include radioisotopes such as 131I or ^131I ; biotin; avidin; and the like.

In the present kit, the anti-human Aβ antibody or anti-human HMGB1 antibody can be produced using known techniques. For example, the anti-human Aβ polyclonal antibody or anti-human HMGB1 polyclonal antibody can be produced by a conventional method in which a non-human mammal (e.g., mouse, rat, rabbit, goat, or sheep) is immunized with a peptide constituting human Aβ (preferably Aβ1-40 or Aβ1-42) or a peptide constituting human HMGB1 as a sensitizing antigen, and the antiserum is collected and purified. Purification can be performed by appropriately combining general biochemical methods such as salting out, ion exchange chromatography, and affinity chromatography.

In addition, anti-human Aβ monoclonal antibodies and anti-human HMGB1 monoclonal antibodies can be produced, for example, by a known hybridoma method. First, an antibody-producing hybridoma is produced. A peptide constituting human Aβ (preferably Aβ1-40 or Aβ1-42) or a peptide constituting human HMGB1 is used as a sensitizing antigen, and a mammal is immunized with this according to a conventional immunization method. The resulting immune cells are fused with known parent cells by a conventional cell fusion method, and monoclonal antibody-producing cells are cloned by a conventional screening method. The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cells (myeloma cells) used for cell fusion, and rodents, such as mice, rats, and hamsters, are generally used. A known method can be used as a method for immunizing a mammal with the sensitizing antigen. Various known cell lines can be used as the myeloma cells to be fused with the above-mentioned immune cells. The cell fusion between immune cells and myeloma cells can be performed according to known methods, such as the method of Milstein et al. (Methods Enzymol. 73: 3-46 (1981)). The obtained fused cells can be selected by culturing in a conventional selection medium, such as HAT medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). The culture in this HAT medium is usually continued for several days to several weeks until cells other than the target hybridoma (non-fused cells) die. Next, a conventional limiting dilution method is performed to screen and clone hybridomas that produce antibodies that bind to human Aβ or hybridomas that produce antibodies that bind to human HMGB1. By purifying the culture supernatant of the hybridoma obtained in this way, a monoclonal antibody that binds to human Aβ or a monoclonal antibody that binds to human HMGB1 can be obtained. The purification can be performed by appropriately combining general biochemical methods, such as salting out, ion exchange chromatography, and affinity chromatography.

Anti-human Aβ monoclonal antibodies and anti-human HMGB1 monoclonal antibodies can also be produced by a host transformed with an expression vector containing an antibody gene by genetic engineering techniques. mRNA can be obtained from spleen cells, lymphocytes, or hybridomas producing monoclonal antibodies of a mammal immunized with a peptide constituting human Aβ (preferably Aβ1-40 or Aβ1-42) or a peptide constituting human HMGB1 as a sensitizing antigen, and a cDNA library can be prepared using this as a template. A clone producing an antibody that reacts with the sensitizing antigen is screened, the obtained clone is cultured, and the desired monoclonal antibody can be purified from the culture supernatant by an appropriate combination of general biochemical methods such as salting out, ion exchange chromatography, affinity chromatography, etc. Furthermore, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, single-chain antibodies, or antigen-binding fragments thereof can be produced, for example, by genetic engineering techniques using the variable regions or hypervariable regions of the above-mentioned monoclonal antibodies (e.g., Method in Enzymology 203: 99-121 (1991)).

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

1. Method [Analysis of Aβ-HMGB1 complex using sandwich ELISA]
A 96-well plate was coated with anti-HMGB1 monoclonal antibody (1 mg/L, clone 2D4, manufactured by Sinotest) diluted with phosphate-buffered saline (PBS) and incubated overnight at 4°C. After washing the wells with PBS containing 0.05% Tween 20 (washing solution), 400 μL of PBS containing 1% BSA was added to each well and incubated at 37°C for 2 hours to perform blocking treatment. After washing the wells with the above washing solution, human CSF samples, human plasma samples, or diluted calibrators (0, 175, 350, 700, 1400 pg/mL Aβ-HMGB1 complex-containing solutions) were each diluted to 1/4 concentration with PBS containing 1% BSA, and 100 μL of each was added to the wells and incubated at 37°C for 20 to 24 hours. After washing the wells with the above-mentioned washing solution, peroxidase-labeled anti-Aβ antibody (a mixture of clone 6E10 antibody [Biolegend] and clone 82E1 antibody [#10323, IBL]) was added and incubated at room temperature for 2 hours to carry out an antigen-antibody reaction. TMB (3,3',5,5'-tetra-methylbenzidine) (Dojindo Laboratories), a substrate for peroxidase, was added to each well, the reaction was stopped with 0.35 mol/L sodium sulfate, and the absorbance at 450 nm was measured using a microplate reader (model 680, Bio-Rad). It was confirmed that the concentration range of the detectable Aβ-HMGB1 complex was 30 to 2000 pg/mL. The anti-HMGB1 monoclonal antibody (clone 2D4) is an antibody that detects both dsHMGB1 and atHMGB1. Moreover, the above-mentioned anti-Aβ antibodies (clone 6E10 antibody and clone 82E1 antibody) are antibodies that detect two types of Aβ (Aβ1-40 or Aβ1-42).

[AD model mouse]
5xFAD transgenic B6/SJL mice (hereinafter referred to as "5xFAD mice"), i.e., AD model mice overexpressing human mutant APP (KM670/671NL Swedish mutation, I716V Florida mutation, and V717I London mutation) and human mutant PS1 (M146L mutation and L285V mutation) (see literature "J Neurosci 26, 10129-10140 (2006)"), were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). In addition, APP-KI mice, i.e., AD model mice having human mutant APP (KM670/671NL Swedish mutation, E693G Arctic mutation, I716F Beyreuther/Iberian mutation) (see literature "Nat Neurosci17, 661-663 (2014)"), were obtained from the Riken BioResource Research Center. Brain tissue samples were prepared from 5xFAD mice and APP-KI mice according to standard methods and cryopreserved.

[Human brain tissue samples]
Brain tissue samples were prepared from familial AD patients with PS1-M146L and non-demented controls (non-neurological disease controls) according to standard methods and cryopreserved. The use of human brain tissue samples was approved by the Ethics Committee of Tokyo Medical and Dental University, and informed consent was obtained.

[Immunohistochemical staining method using brain tissue samples from AD model mice]
The brain tissue samples of the AD model mice were fixed with 4% PFA (paraformaldehyde) solution, and then 5 μm-thick sagittal brain sections were prepared using a microtome (manufactured by Yamato Koki Kogyo Co., Ltd.). After deparaffinization with xylene and ethanol, the samples were boiled in 10 mM citric acid solution for 15 minutes for antigen retrieval. Sagittal brain sections were then incubated in PBS containing 10% FBS for 60 minutes and incubated in the presence of five primary antibodies (mouse anti-Aβ antibody [#10323, IBL, dilution ratio 1:1000], rabbit anti-HMGB1 antibody [ab18256, Abcam, dilution ratio 1:2000], Cy3-conjugated mouse anti-GFAP antibody [c9205, sigma-Aldrich, dilution ratio 1:5000], rabbit anti-MAP2 antibody [ab32454, Abcam, dilution ratio 1:1000], and goat anti-Iba1 antibody [011-27991, Fujifilm Wako Pure Chemical, dilution ratio 1:500]) at 4°C for 12 hours. Then, the sagittal brain sections were washed with PBS and incubated at room temperature for 1 hour in the presence of two types of secondary antibodies (Alexa Fluor 488-conjugated Donkey anti-mouse IgG [A21202, Thermo Fisher Scientific, dilution ratio 1:1000] and Cy3-conjugated Donkey anti-rabbit IgG [711-165-152, Jackson Laboratory, dilution ratio 1:1000]). The sagittal brain sections were washed with PBS and then stained with 0.2 μg/mL DAPI (4',6-diamidino-2-phenylindole) in PBS. Using a confocal laser scanning microscope (FV1200IX83, Olympus), images were obtained that detected fluorescent signals derived from various primary antibodies, images that detected DAPI fluorescent signals derived from cell nuclei (DAPI images), and images that were superimposed on these images (Merge images).

[Immunohistochemical staining method using human brain tissue samples]
Immunohistochemical staining of the above human brain tissue samples using anti-Aβ antibody, anti-HMGB1 antibody, and anti-MAP2 antibody was performed according to the method described in the above section [Immunohistochemical staining using brain tissue samples from AD model mice].

Preparation of CSF and Plasma Samples
CSF samples were collected by lumbar puncture from three types of fasting subjects (71 AD patients, 26 MCI patients, and 48 healthy subjects not diagnosed with brain dysfunction) (see Table 1), and then centrifuged (1000×g, 10 minutes, 4° C.) to remove impurities, and the supernatants were stored at −80° C. as CSF samples. Blood was also collected from the elbow vein of each of the three subjects, placed in a tube containing an anticoagulant (EDTA-2K), centrifuged (1500×g, 10 minutes, room temperature), and the supernatants were stored at −80° C. as plasma samples.

[Neuropsychological score]
The patient's physician administered the MMSE (Japanese version) and ADAS-cog.

Detection of Aβ and tau in CSF samples
Aβ1-40 and Aβ1-42 in the CSF samples were detected using a human Aβ(1-40) ELISA kit (292-62301, Fujifilm Wako Pure Chemical Industries, Ltd.) and a human Aβ(1-42) ELISA kit (298-62401, Fujifilm Wako Pure Chemical Industries, Ltd.). Phosphorylated tau (p-Tau) in the CSF samples was detected using INNOTEST Phospho-tau (181P) (Innogenetics).

[Preparation of Aβ-HMGB1 complex]
Two types of Aβ peptide powders (Aβ1-40 [Amyloid β-Protein [Human, 1-40], 4307-v, Peptide Institute]; and Aβ1-42 [Amyloid β-Protein [Human, 1-42], 4349-v, Peptide Institute]) were dissolved in DMSO at a concentration of 1 mM to prepare an Aβ1-40-containing solution and an Aβ1-42-containing solution. Disulfide-type human HMGB1 (dsHMGB1) (HM-120, HMGBiotech) was dissolved in PBS at a concentration of 140 μM to prepare a dsHMGB1-containing solution. In addition, 100 μM dsHMGB1 was incubated with 20 mM DTT at 37 ° C. for 24 hours, and then dialyzed with PBS at 4 ° C. for 3 hours to prepare a fully reduced HMGB1 (atHMGB1)-containing solution. The dsHMGB1-containing solution or the atHMGB1-containing solution and the Aβ-containing solution were each diluted to 60 μM with PBS, mixed in an equimolar ratio, and incubated at 37 ° C. for 48 hours in a PCR thermal cycler with a heated lid to prepare four types of Aβ-HMGB1 complexes (Aβ1-40-dsHMGB1 complex, Aβ1-40-atHMGB1 complex, Aβ1-42-dsHMGB1 complex, or Aβ1-42-atHMGB1 complex)-containing solutions, which were then stored at −80 ° C. When used in an experiment, the Aβ-HMGB1 complex-containing solution was thawed on ice and diluted with PBS to the working concentration.

[Tricin-SDS-PAGE and Western blotting]
To detect the Aβ-HMGB1 complex, Tricin-SDS-PAGE was performed according to the method described in the literature "Nat. Protoc.1, 16-22 (2006)". First, a gel (upper gel: 4%, spacer gel: 10%, lower gel: 16%) was cast 6 hours before electrophoresis. Next, an equal amount of sample buffer (a solution containing 1% SDS, 3% mercaptoethanol, 15% glycerol, 65 mM Tris/HCl [pH 7.0], and 0.05% BPB [bromophenol blue]) was added to the sample (the Aβ-HMGB1 complex-containing solution, or the cerebral cortex lysate prepared according to a standard method from an AD model mouse), incubated at 37°C for 15 minutes, and electrophoresis was performed at 30V for 30 minutes in the presence of an electrophoresis buffer (anode buffer [100 mM Tris, 22.5 mM HCl [pH 8.9]], cathode buffer [100 mM Tris, 100 mM Tricin, 0.1% SDS [pH 8.25 or less]]). Then, electrophoresis was switched to 150V and stopped before the sample finished flowing. The gel was transferred to an Immobilon®-P polyvinylidene fluoride membrane (Millipore) by semi-dry transfer in the presence of electrode buffer (300 mM Tris, 100 mM acetic acid [pH 8.6]) at 0.4 mA/ ^cm2 for 24 hours. The membrane was then blocked for 1 hour in TBST solution (10 mM Tris-HCl [pH 8.0], 150 mM sodium chloride, 0.05% Tween-20) containing 5% skim milk, and then incubated with three types of primary antibodies (rabbit anti-HMGB1 antibody [ab18256, Abcam, dilution ratio 1:2000], rabbit anti-Aβ antibody [#2454S, CellSignaling Technology, dilution ratio 1:1000], and mouse anti-Aβ antibody [#10323, IBL, dilution ratio 1:200]) and two types of secondary antibodies (HRP-labeled anti-rabbit IgG [NA934, GE Healthcare, dilution ratio 1:3000] and HRP-labeled anti-mouse IgG [NA931, GE Healthcare, dilution ratio 1:3000]). Western blotting was performed using ECL Select Western Blotting Detection Reagent (RPN2235, GE Healthcare, dilution ratio 1:3000) according to a standard method. Proteins bound to various primary antibodies were detected using ECL Select Western Blotting Detection Reagent (RPN2235, GE Healthcare) and a luminescence image analyzer (ImageQuant LAS500, GE Healthcare).

[Culture of primary neurons]
Four to six cerebral cortices were dissected from E15 mouse embryos and incubated in 4 mL of PBS (#25200056, Thermo Fisher Scientific) containing 0.05% trypsin for 15 minutes at 37°C, after which the cells were dissociated by pipetting. The cells were passed through a 70 μm cell strainer (#22-363-548, Thermo Fisher Scientific), collected by centrifugation, and cultured in neurobasal medium (Thermo Fisher Scientific) containing 2% B27, 0.5 mM L-glutamine, 1% penicillin/streptomycin, and 0.5 μM Ara-C (arabinofuranosyl cytosine) to obtain mouse primary cerebral cortical neurons. Seven days after the start of culture, 0.56 nM of eight types of test substances (dsHMGB1, atHMGB1, Aβ1-40, Aβ1-42, Aβ1-40-dsHMGB1 complex, Aβ1-40-atHMGB1 complex, Aβ1-42-dsHMGB1 complex, and Aβ1-42-atHMGB1 complex)-containing solution was added and incubated at room temperature for 3 hours. The concentration of the test substance was determined with reference to the HMGB1 concentration in CSF from MCI/AD patients (see the literature "Nat. Commun.11, 1-22 (2020)"). In addition, in order to confirm the effect of human anti-HMGB1 antibody, an experiment was also performed in which 0.56 nM of human anti-HMGB1 antibody was added together with the test substance and incubated in the same manner. Thereafter, neurite degeneration was analyzed according to the method described in the following [Western blotting] section. ER ballooning was analyzed by time-lapse photography of live cells using a BZ-X700 (Keyence). TLR4 knockdown was performed by transfecting cells with TLR4 siRNA (Santa Cruz Biotechnology) using lipofectamineRNAiMAX reagent (#13778030, Thermo Fisher Scientific). Cells were cultured for 36 hours after TLR4 knockdown before the addition of test substances.

[Western blotting]
The incubated cells were scraped with PBS and collected. After centrifugation (10,000×g, 1 min, 4° C.), the cell pellet was dissolved in extraction buffer (containing 2% SDS, 1 mM DTT, and 10 mM Tris-HCl [pH 7.5]). After heating at 100° C., the cell lysate was centrifuged (16,000 g×10 min, 4° C.), and the supernatant was added to an equal amount of sample buffer (containing 125 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 12% mercaptoethanol, and 0.05% BPB) to prepare a sample. The sample was separated by SDS-PAGE according to a standard method, and transferred to an Immobilon-P polyvinylidene fluoride membrane (Millipore) by the semi-dry method. The membrane was then blocked for 1 hour in TBST solution (10 mM Tris-HCl [pH 8.0], 150 mM sodium chloride, 0.05% Tween-20) containing 5% skim milk, and then incubated with four types of primary antibodies (rabbit anti-pSer46-MARCKS antibody [GL Biochem, dilution ratio 1:10,000, reaction conditions: 1 hour at room temperature], mouse anti-phosphorylated tau antibody [AT-8] [#90206, Innogenetics, dilution ratio 1:2000, reaction conditions: 3 hours at room temperature], mouse anti-β-actin antibody [#sc-47778, Santa Cruz Biosciences, dilution ratio 1:2000, reaction conditions: 3 hours at room temperature], and mouse anti-β-actin antibody [#sc-47778, Santa Cruz Biosciences, dilution ratio 1:2000, reaction conditions: 3 hours at room temperature]). Western blotting was performed according to a standard method using Can GetSignal Solution (Toyobo) containing Can 10000 IgG (Novus Biotechnology, dilution ratio 1:1000, reaction conditions: 12 hours at 4 ° C.) and mouse anti-TLR4 antibody [#NB100-56566, Novus, dilution ratio 1:5000, reaction conditions: 3 hours at room temperature] and Can Get Signal Solution (Toyobo) containing two types of secondary antibodies (HRP-labeled anti-rabbit IgG [NA934, GE Healthcare, dilution ratio 1:3000] and HRP-labeled anti-mouse IgG [NA931, GE Healthcare, dilution ratio 1:3000]). For detection of proteins bound by various primary antibodies, ECL Select Western Blotting Detection Reagent (RPN2235, GE Healthcare) and a luminescence image analyzer (ImageQuant LAS500, GE Healthcare) were used.

[ELISA regarding affinity of Aβ-HMGB1 complex to TLR4]
100μL of PBS containing 1nM of two types of Aβ-HMGB1 complexes (Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex) and human anti-dsHMGB1 antibody (monoclonal antibody #129) (International Publication No. WO 2020/059847) were incubated (pre-incubated) at room temperature for 30 minutes, and then the pre-incubated sample and 100μL of PBS containing 1nM of four types of HMGB1 (atHMGB1, dsHMGB1, Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex) were added to a 96-well plate pre-coated with human anti-dsHMGB1 antibody (HMGB1 ELISA kit Exp, 326078738, manufactured by Shino Test Co., Ltd.), and incubated at 37 ° C. for 24 hours. Each well was washed three times with a washing solution (HMGB1 ELISA KIT Exp, 326078738, manufactured by Chinotestu Co., Ltd.), and then 100 μL of various concentrations (0, 0.25, 0.5, 1, 2, or 4 nM) of peroxidase-labeled (Peroxidase Labeling Kit-NH2, LK11, manufactured by Dojindo Laboratories Co., Ltd.) human TLR4 (1478-TR-050, manufactured by R&D Co., Ltd.) was added to each well and incubated for 24 hours at 25° C. After washing each well with the above washing solution, 100 μL of a fluorescent reagent (HMGB1 ELISA KIT Exp, 326078738, manufactured by Chinotestu Co., Ltd.) was added and incubated for 30 minutes at 25° C. The reaction was stopped with 100 μL of stopping solution (HMGB1 ELISA KIT Exp, 326078738, manufactured by Chinotestu), and the absorbance at 450 nm was measured using a plate reader (SPARK 10M, manufactured by Tecan).

[Statistical analysis]
Statistical analysis was performed using R version 3.6.2. Box plots were used to depict data distribution. Box plots show median, quartiles, and whiskers, with data outside the 25-75% range. p-values were calculated using Wilcoxon rank sum test with post-hoc Bonferroni correction. Student's t test was used for other between-group comparisons. Tukey's HSD test was used for multiple between-group comparisons. Correlation analysis was performed with Microsoft Excel for Microsoft 365, and p-values for Pearson's correlation coefficient were calculated with R. ROC (Receiver Operating Characteristic) analysis was performed with the ROCR package.

2. Results [Aβ-HMGB1 complexes are formed in vitro and are also present in the brains of AD model mice (in vivo)]
First, two types of Aβ (Aβ1-40 or Aβ1-42) and human HMGB1 containing dsHMGB1 and atHMGB1 (1690-HMB-050, R&D) were mixed and incubated at a 1:1 molar ratio, and Western blotting was performed using an anti-Aβ antibody or an anti-HMGB1 antibody. As a result, when either antibody was used, a band was detected at a position of approximately 35 kDa, which is the predicted molecular weight of the 1:1 complex of Aβ (Aβ1-40 or Aβ1-42) and HMGB1 (Figure 1a, see arrow).

When Aβ (Aβ1-40 or Aβ1-42) was used alone, bands corresponding to Aβ oligomers were detected in addition to bands corresponding to Aβ monomers, whereas the addition of human HMGB1 prevented the detection of bands corresponding to Aβ oligomers (see arrowhead in Figure 1a).

Furthermore, when the concentrations of Aβ (Aβ1-40 or Aβ1-42) and human HMGB1 were increased, bands derived from the higher molecular weight Aβ-HMGB1 complex were detected (see Figure 1b). For example, a band derived from the Aβ:HMGB1 complex with a ratio of 2:1 was detected at the molecular weight position of 65 kDa (see arrow in Figure 1b), and a band derived from the Aβ:HMGB1 complex with a ratio of 2:3 was detected at the molecular weight position of 100 kDa (see arrow in Figure 1b).

In addition, when Aβ (Aβ1-40) and dsHMGB1 were mixed and incubated, the band derived from the Aβ-HMGB1 complex was detected more strongly than when Aβ (Aβ1-40) and atHMGB1 were mixed and incubated (see Figure 1c).

These results confirmed that a complex of Aβ and HMGB1 is formed in vitro.

Next, we analyzed whether Aβ-HMGB1 complexes existed in the brains of AD model mice (see Figures 1d and 1e). Western blotting was performed using anti-Aβ or anti-HMGB1 antibodies on cerebral cortex lysates prepared according to standard methods from two types of AD model mice (5xFAD mice and APP-KI mice) and a control mouse (B6/SJL mouse). As a result, the bands with molecular weights of 45 kDa, 50 kDa, and 75 kDa detected by anti-Aβ and anti-HMGB1 antibodies were more strongly detected in the cerebral cortex lysates of AD model mice than in the cerebral cortex lysates of control mice (see Figure 1d). In addition, when the cerebral cortex lysates of AD model mice were immunoprecipitated according to standard methods using anti-Aβ or anti-HMGB1 antibodies, the bands detected by anti-Aβ and anti-HMGB1 antibodies were also detected by anti-HMGB1 and anti-Aβ antibodies (see Figure 1e).

These results indicate the presence of Aβ-HMGB1 complexes in the brains of AD model mice, and that the amount of Aβ-HMGB1 complexes is increased in AD model mice compared to normal mice. HMGB1 has been reported to undergo multiple modifications, including acetylation (EMBO J. 22,5551-5560 (2003)), phosphorylation (J Immunol. 182,5800 (2009), Lab. Invest. 89,948-959 (2009)), and methylation (J Biol. Chem.282,16336-16344 (2007)). Interestingly, in two-dimensional electrophoresis analysis of HMGB1 purified from thymus, HMGB1 protein spots with molecular weights of 30 kDa, 35 kDa, and 60 kDa were detected (EMBO J. 22, 5551-5560 (2003)). This suggests that the bands with molecular weights of 45 kDa, 50 kDa, and 75 kDa detected in this example with anti-Aβ and anti-HMGB1 antibodies are bands derived from complexes between these modified HMGB1 proteins and Aβ.

[The concentration of Aβ-HMGB1 complexes is increased in biological samples from AD patients]
The results of Figures 1d and e suggest the usefulness of the Aβ-HMGB1 complex as a biomarker for AD. Therefore, in order to analyze whether AD can be diagnosed using the Aβ-HMGB1 complex as an indicator, the concentrations of the Aβ-HMGB1 complex in the CSF and plasma samples in three groups (AD group [AD patients], MCI group [MCI patients], and control group [healthy subjects]) were measured by ELISA. As a result, in many AD groups, the concentrations of the Aβ-HMGB1 complex in the CSF and plasma samples increased (see Figures 2a and 2b). On the other hand, no such increase was observed in the MCI group, and in the control group, only some (three cases [NC11, NC15, and W232] in the CSF samples; two cases [NC05 and NC17] in the plasma samples) showed an increase (see Figures 2a and 2b). Of these, one patient (W232) was found to have a subnormal MMSE score (24.0) and elevated p-Tau level (181 pg/mL), returned to the hospital, and was diagnosed with early AD within 4 years.

These results suggest that it may be possible to distinguish between patients with dementia caused by AD and other conditions and those without dementia (patients with mild cognitive impairment and healthy individuals who have not been diagnosed with brain dysfunction) by using increased concentrations of Aβ-HMGB1 complexes in biological samples as an indicator.

In contrast to the results in Figure 2a, which show that the concentration of Aβ-HMGB1 complexes was elevated in CSF samples from the AD group but not in the MCI group, we have previously reported that the expression level of HMGB1 is higher in CSF samples from MCI patients than in CSF samples from AD patients (see Nat. Commun.11, 1-22 (2020)). Therefore, we compared the concentration of HMGB1 and the concentration of Aβ-HMGB1 complexes in CSF samples taken at the same time from the same AD patients. Interestingly, the concentration of HMGB1 in CSF samples and the concentration of Aβ-HMGB1 complexes showed a negative correlation (see Figure 2c). This negative correlation indicates that by measuring both the concentration of HMGB1 and the concentration of Aβ-HMGB1 complexes in CSF samples, it is possible to distinguish between MCI and AD and evaluate the level of cognitive decline in patients.

Furthermore, based on the results of Figures 2a and 2b, ROC analysis was performed between the AD group and the control group, and the AUC (Area Under the Curve) values were 0.5314 and 0.7742, respectively, both of which exceeded 0.5 (see Figures 2d and 2f). On the other hand, based on the results of measuring the concentrations of Aβ (Aβ1-40) in CSF samples in the AD group (n=8) and the control group (n=6), ROC analysis was performed between the AD group and the control group, and the AUC value was 0.333, which was below 0.5.

These results indicate that the concentration of the Aβ-HMGB1 complex (rather than Aβ) can be used as an indicator to distinguish between patients with dementia caused by AD, etc., and those without dementia. Based on the results of Figures 2a and 2b, ROC analysis was performed between the MCI group and the control group, and the AUC values were 0.4688 and 0.4667, respectively, both of which were below 0.5, confirming that the concentration of the Aβ-HMGB1 complex cannot be used to distinguish between patients with mild cognitive impairment and those without mild cognitive impairment (see Figures 2e and 2g).

In addition, when the relationship between the concentration of Aβ-HMGB1 complex and two types of cognitive function scores (ADAS-cog and MMSE) in the AD and control groups was analyzed, a positive correlation was observed between the concentration of Aβ-HMGB1 complex and the ADAS-cog score in the AD group, whereas no such correlation was observed in the control group (see FIG. 2h). In addition, a negative correlation was observed between the concentration of Aβ-HMGB1 complex and the MMSE score in the AD group, whereas no such correlation was observed in the control group (see FIG. 2i).
The results indicate that there is a positive correlation between increased concentrations of Aβ-HMGB1 complexes and decline in cognitive function.

Furthermore, when the relationship between the concentration of Aβ-HMGB1 complex in the AD group and the annual changes in two types of cognitive function scores (ADAS-cog and MMSE) was analyzed, a positive correlation was found between the concentration of Aβ-HMGB1 complex and the annual changes in ADAS-cog scores (see Figure 2j), and a negative correlation was found between the concentration of Aβ-HMGB1 complex and the MMSE scores (see Figure 2k).
The results indicate that there is a positive correlation between the concentration of Aβ-HMGB1 complexes and the decline in cognitive function.

[Aβ-HMGB1 complexes are present in neurons surrounding Aβ plaques]
We previously classified the phenotype of cell death pathology into three patterns (active necrosis, secondary necrosis, and ghost of cell death) (see reference "Nat. Commun.11, 1-22 (2020)"). To investigate the pathological significance of increased concentrations of Aβ-HMGB1 complexes, sagittal sections of brains from two types of AD model mice (5xFAD mice and APP-KI mice) were analyzed by immunohistochemical staining using anti-Aβ and anti-HMGB1 antibodies. As a result, in cells in which secondary necrosis occurred around extracellular Aβ aggregates (Aβ plaques), Aβ-derived signals and HMGB1-derived signals were colocalized (see microscopic images in Figures 3a and b), indicating that Aβ-HMGB1 complexes are present in cells around Aβ plaques. In addition, these overlapping signals, together with weak DAPI signals, were present in the cytoplasm of dying cells, suggesting that Aβ-HMGB1 complexes are formed in neurons that have suffered secondary damage around the formation of senile plaques. Furthermore, the colocalization of Aβ-derived signals and HMGB1-derived signals was quantified, and it was confirmed that such areas were significantly increased in AD model mice compared to normal mice (see graphs in Figures 3a and b).

Furthermore, sagittal sections of brains from familial AD patients with PS1-M146L were analyzed by immunohistochemical staining using anti-Aβ and anti-HMGB1 antibodies. As a result, Aβ-derived signals and HMGB1-derived signals were colocalized with weak DAPI signals in the cytoplasm of cells surrounding extracellular Aβ aggregates (senile plaques) (see Figure 4). Furthermore, whereas HMGB1-derived signals were detected in the nuclei of normal cells without intracellular Aβ (see arrows in Figure 4), they were detected in the cytoplasm as well as the nuclei of abnormal cells, where they colocalized with Aβ-derived signals (see boxed area in Figure 4). These abnormal neuronal morphological changes are consistent with typical features of TRIAD (Transcriptional Repression-Induced Atypical cell Death) necrosis associated with impaired YAP function identified in HD and AD pathology (see references Nat.Commun. 11, 1-22 (2020), J Cell Biol. 172, 589-604 (2006), Hum. Mol.Genet. 25, 4749-4770 (2016), and Acta Neuropathol. Commun. 5, 19 (2017)). Furthermore, quantification of areas where Aβ-derived signals and HMGB1-derived signals colocalized confirmed that such areas were significantly increased in familial AD patients with PS1-M146L compared to non-neurological controls (see graph in Figure 4).

[Aβ-HMGB1 complexes are produced in dying neurons and cleared by microglia]
To identify the cell type involved in the generation of the Aβ-HMGB1 complex, sagittal sections of brains from two types of AD model mice (5×FAD mice and APP-KI mice) and sagittal sections of brains from familial AD patients with PS1-M146L were analyzed by immunohistochemical staining using anti-Aβ and anti-HMGB1 antibodies, anti-MAP2 antibodies, anti-Iba1 antibodies, or anti-GFAP antibodies. First, as a result of analysis using anti-MAP2 antibodies, i.e., antibodies against MAP2, a neuronal cell-specific marker, in brain tissues from AD model mice (5×FAD mice and APP-KI mice), it was confirmed that Aβ-derived signals and HMGB1-derived signals colocalized in dying neurons surrounding Aβ plaques (see Figures 5a and 5b). In addition, in brain tissue from familial AD patients with PS1-M146L, Aβ-derived signals and HMGB1-derived signals were confirmed to colocalize in dying neurons with intracellular Aβ (primary necrosis [see Nat. Commun. 11, 1-22 (2020)] and in dying neurons around or near senile plaques (secondary necrosis [see Commun. Biol.4, 1175 (2021) and Nat. Commun.11, 1-22 (2020)]) (see Figure 5c).
These results indicate that Aβ-HMGB1 complexes are generated in dying neurons.

In addition, analysis using anti-Iba1 antibodies, i.e., antibodies against Iba1, a microglia-specific marker, confirmed that Aβ-derived signals and HMGB1-derived signals colocalized at the boundaries of extracellular Aβ aggregates in brain tissue from AD model mice (5xFAD mice and APP-KI mice) and brain tissue from familial AD patients with PS1-M146L. However, considering that microglia themselves do not produce Aβ by cleaving APP (Aβ precursor) and that microglia function as phagocytes, it is reasonable to consider that these microglia degraded by phagocytosis the Aβ-HMGB1 complexes generated from clusters of necrotic neurons that later form Aβ plaques (see references "Commun. Biol. 4, 1175 (2021)" and "Nat. Commun.11, 1-22 (2020)").

Furthermore, analysis using anti-GFAP antibodies, i.e., antibodies against GFAP, an astrocyte-specific marker, showed no clear colocalization of Aβ-derived signals and HMGB1-derived signals in brain tissue from AD model mice (5xFAD mice and APP-KI mice) or in brain tissue from familial AD patients with PS1-M146L.

Taken together, these results indicate that intracellular amyloid beta induced by the HMGB1-TLR4-PKC-Ku70 pathway accumulates, and Aβ-HMGB1 complexes are generated in the cytoplasm of neurons undergoing the process of TRIAD necrosis (see Commun. Biol. 4, 1175 (2021) and Nat. Commun.11, 1-22 (2020)). As the AD pathology progresses, the number of neurons that have fallen into TRIAD necrosis increases exponentially through a mechanism known as "necrosis-to-necrosis amplification" (see Commun. Biol.4, 1175 (2021) and Nat.Commun. 11, 1-22 (2020)). As the AD pathology progresses, the Aβ load also increases, so it is thought that the production of the Aβ-HMGB1 complex increases in the later stages of AD. The localization of the Aβ-HMGB1 complex in the secondary TRIAD neurons, where it is produced, explains why the expression level of the Aβ-HMGB1 complex is low in the MCI stage and high in the advanced AD stage.

[Aβ-dsHMGB1 complex induces neurite degeneration]
To examine whether the Aβ-HMGB1 complex induces neurite degeneration in the same way as HMGB1, primary neurons were incubated in the presence of eight types of test substances (dsHMGB1, atHMGB1, Aβ1-40, Aβ1-42, Aβ1-40-dsHMGB1 complex, Aβ1-40-atHMGB1 complex, Aβ1-42-dsHMGB1 complex, and Aβ1-42-atHMGB1 complex), and then Western blotting was performed using an antibody that detects phosphorylation of Ser46 in MARCKS, a marker of neurite/synaptic degeneration (anti-pSer46-MARCKS antibody), and an antibody that detects phosphorylation of Ser202 and Thr205 in tau, which has been established as a marker of synaptic degeneration (anti-phosphorylated tau antibody). As a result, the levels of pSer46-MARCKS and phosphorylated tau did not change when primary neurons were incubated in the presence of atHMGB1 compared to when they were incubated in the absence of the test substance ("no treatment" in the figure), whereas the levels of pSer46-MARCKS and phosphorylated tau increased when they were incubated in the presence of dsHMGB1 (see Figures 6a and 6b). On the other hand, the levels of pSer46-MARCKS and phosphorylated tau did not change when primary neurons were incubated in the presence of amyloid β (Aβ1-40 and Aβ1-42) compared to when they were incubated in the absence of the test substance ("no treatment" in the figure) (see Figures 6a and 6b). Furthermore, when primary neurons were incubated in the presence of Aβ-atHMGB1 complexes (Aβ1-40-atHMGB1 complexes and Aβ1-42-atHMGB1 complexes), the levels of pSer46-MARCKS and phosphorylated tau did not change compared to when the cells were incubated in the absence of the test substance ("no treatment" in the figure), whereas when the primary neurons were incubated in the presence of Aβ-dsHMGB1 complexes (Aβ1-40-dsHMGB1 complexes and Aβ1-42-dsHMGB1 complexes), the levels of pSer46-MARCKS and phosphorylated tau increased (see Figures 6a and 6b).

These results indicate that the Aβ-dsHMGB1 complex (rather than the Aβ-atHMGB1 complex) has a neurite degenerative effect.

Next, to examine whether human anti-dsHMGB1 antibody (monoclonal antibody #129) affects the neurite degeneration caused by the Aβ-dsHMGB1 complex, primary neurons were incubated in the presence of four test substances (dsHMGB1, Aβ1-40, Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex) and in the presence or absence of monoclonal antibody #129, and then Western blotting was performed using anti-pSer46-MARCKS antibody and anti-phosphorylated tau antibody. As a result, when primary neurons were incubated in the presence of Aβ-dsHMGB1 complexes (Aβ1-40-dsHMGB1 complexes and Aβ1-42-dsHMGB1 complexes), the levels of pSer46-MARCKS and phosphorylated tau increased compared to when incubated in the absence of the test substance ("no treatment" in the figure), whereas when incubated with the Aβ-dsHMGB1 complexes in the presence of monoclonal antibody #129, the increase in the levels of pSer46-MARCKS and phosphorylated tau was suppressed (see Figure 6c).

Furthermore, to examine whether TLR4 affects the neurite degeneration action of the Aβ-dsHMGB1 complex, primary neurons with TLR4 knockdown and primary neurons without TLR4 knockdown were incubated in the presence of four types of test substances (dsHMGB1, Aβ1-40, Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex), and then Western blotting was performed using anti-TLR4 antibody, anti-pSer46-MARCKS antibody, and anti-phosphorylated tau antibody. As a result, when primary neurons without TLR4 knockdown were incubated in the presence of dsHMGB1 or Aβ-dsHMGB1 complexes (Aβ1-40-dsHMGB1 complexes and Aβ1-42-dsHMGB1 complexes), the levels of pSer46-MARCKS and phosphorylated tau increased compared to when incubated in the absence of the test substance ("no treatment TLR4-siRNA(-)" in the figure), whereas when primary neurons with TLR4 knockdown were incubated in the presence of dsHMGB1 or Aβ-dsHMGB1 complexes (Aβ1-40-dsHMGB1 complexes and Aβ1-42-dsHMGB1 complexes), the increase in the levels of pSer46-MARCKS and phosphorylated tau was suppressed (see FIG. 6d).
Furthermore, ELISA was performed regarding the affinity of the Aβ-HMGB1 complexes (Aβ1-40-dsHMGB1 complex and Aβ1-42-dsHMGB1 complex) to TLR4, and the results confirmed that the Aβ-dsHMGB1 complexes have high binding affinity to TLR4, similar to dsHMGB1.

These results indicate that the Aβ-dsHMGB1 complex binds to TLR4 and induces neurite degeneration, and also suggest that the binding domain (surface) in the Aβ-dsHMGB1 complex is present in the outer region of dsHMGB1 and that Aβ (Aβ1-40 and Aβ1-42) stabilizes the shape of the binding domain.

[Aβ-dsHMGB1 complex induces neuronal cell death]
Next, to examine whether the Aβ-dsHMGB1 complex, which has a neurite degenerative effect, induces cell death, primary neurons were incubated in the presence of eight types of test substances (dsHMGB1, atHMGB1, Aβ1-40, Aβ1-42, Aβ1-40-dsHMGB1 complex, Aβ1-40-atHMGB1 complex, Aβ1-42-dsHMGB1 complex, and Aβ1-42-atHMGB1 complex), and then the number of cells with ballooned ER, which is one form of cell death, was measured. As a result, when primary neurons were incubated in the presence of atHMGB1, amyloid beta (Aβ1-40 and Aβ1-42), or Aβ-atHMGB1 complexes (Aβ1-40-atHMGB1 complex and Aβ1-40-atHMGB1 complex), the number of cells with ballooned ER was almost unchanged compared to when incubated in the absence of the test substance ("no treatment" in the figure), whereas when incubated in the presence of dsHMGB1 or Aβ-dsHMGB1 complexes (Aβ1-40-dsHMGB1 complex and Aβ1-42-dsHMGB1 complex), the number of cells with ballooned ER increased (see FIG. 6e).
This result indicates that the Aβ-dsHMGB1 complex has the effect of inducing cell death.

In addition, primary neurons were incubated in the presence of four types of test substances (dsHMGB1, Aβ1-40, Aβ1-40-dsHMGB1 complex, and Aβ1-42-dsHMGB1 complex) and in the presence or absence of monoclonal antibody #129, and the number of cells having ballooned ER was then measured. As a result, when primary neurons were incubated in the presence of Aβ-dsHMGB1 complexes (Aβ1-40-dsHMGB1 complexes and Aβ1-42-dsHMGB1 complexes), the number of cells having ballooned ER increased compared to when incubated in the absence of the test substances ("no treatment" in the figure), whereas when incubated in the presence of the Aβ-dsHMGB1 complexes and monoclonal antibody #129, the increase in the number of cells having ballooned ER was suppressed (see FIG. 6f).
These results confirmed that monoclonal antibody #129 inhibits cell death caused by the Aβ-dsHMGB1 complex.

These results indicate that dsHMGB1 and Aβ form an Aβ-dsHMGB1 complex in dying neurons during the process of secondary necrosis. In the later stages of AD pathology, the amount of extracellular HMGB1 is thought to decrease due to the inhibition of HMGB1 release caused by the formation of the Aβ-dsHMGB1 complex in neurons and the fixation of HMGB1 to the Aβ-dsHMGB1 complex after the uptake of extracellular HMGB1 in microglia. On the other hand, the amount of extracellular Aβ-dsHMGB1 complex increases due to its release from necrotic neurons. It has been revealed that the Aβ-dsHMGB1 complex, like HMGB1, induces synaptic damage and secondary necrosis. It is expected that the increase in the amount of extracellular Aβ-dsHMGB1 complex in the later stages of AD will lead to the progression of AD pathology.

[Analysis of the combination of Aβ-HMGB1 complex and HMGB1 improves the accuracy of dementia diagnosis]
The results shown in Figures 2d and 2f indicate that dementia can be diagnosed using the concentration of Aβ-HMGB1 complexes in biological samples from the AD and control groups as an index. Next, we investigated whether the accuracy of dementia diagnosis could be improved by combining factors other than the concentration of Aβ-HMGB1 complexes in biological samples.

First, the concentrations of Aβ-HMGB1 complexes in CSF and plasma samples from the AD and MCI groups were measured, and ROC analysis was performed between the two groups. As with the AD and control groups, the AUC values were 0.600 and 0.725, respectively, both exceeding 0.5 (see Figures 7a and 7b). On the other hand, ROC analysis was performed between the AD and control groups based on the results of measuring the concentrations of Aβ (Aβ1-40) in CSF samples from the AD (n=8) and MCI (n=2) groups, resulting in an AUC value of 0.438, below 0.5.

These results confirmed that the concentration of the Aβ-HMGB1 complex (rather than Aβ) can be used as an indicator to distinguish between patients with dementia caused by AD etc. and patients with mild dementia (i.e., those without dementia).

Next, the concentrations of the Aβ-HMGB1 complex in the CSF or plasma samples were combined with the concentrations of HMGB1 in the CSF samples to perform ROC analysis between the two groups, and the AUC values improved to 0.644 and 0.758, respectively (see Figures 7c and 7d). Furthermore, the concentrations of the Aβ-HMGB1 complex in the CSF samples, the Aβ-HMGB1 complex in the plasma samples, and the HMGB1 concentration in the CSF samples were combined to perform ROC analysis between the two groups, and the AUC value further improved to 0.817 (see Figure 7e).

These results indicate that the accuracy of dementia diagnosis is improved by analyzing the concentration of Aβ-HMGB1 complexes in CSF samples or plasma samples in combination with the concentration of HMGB1 in CSF samples, or by analyzing the concentration of Aβ-HMGB1 complexes in CSF samples and plasma samples in combination with the concentration of HMGB1 in CSF samples, compared to analyzing the concentration of Aβ-HMGB1 complexes in CSF samples or plasma samples alone.

The present invention contributes to the early detection of dementia, the treatment of dementia, and the prevention of the progression of dementia.

Claims (8)

A method for diagnosing dementia, comprising the following steps (a) and (b).
(a) measuring the concentration of amyloid β-HMGB1 complex in one or more biological samples obtained from a subject;
(b) determining whether or not the subject is suffering from dementia using the concentration of amyloid β-HMGB1 complex in one or more biological samples collected from the subject as an index; The method according to claim 1, wherein in step (b), the concentration of the amyloid β-HMGB1 complex in one or more biological samples collected from the subject is compared with the concentration of the amyloid β-HMGB1 complex in one or more biological samples from a control individual not suffering from dementia, and if the concentration of the amyloid β-HMGB1 complex in one or more biological samples collected from the subject is higher than the concentration of the amyloid β-HMGB1 complex in one or more biological samples from a control individual not suffering from dementia, the subject is determined to be highly likely to suffer from dementia. The method according to claim 1, wherein the biological sample is a blood sample or a cerebrospinal fluid sample. In step (b),
comparing the concentration of amyloid β-HMGB1 complex in two or more biological samples taken from the subject with the concentration of amyloid β-HMGB1 complex in two or more biological samples from a control individual not suffering from dementia; and
comparing the concentration of HMGB1 in one or more biological samples taken from the subject with the concentration of HMGB1 in one or more biological samples from a control individual not suffering from dementia;
The method of claim 1, wherein the subject is determined to be highly likely to be suffering from dementia if the concentration of the amyloid β-HMGB1 complex in two or more biological samples collected from the subject is higher than the concentration of the amyloid β-HMGB1 complex in two or more biological samples derived from a control subject not suffering from dementia, and the concentration of HMGB1 in one or more biological samples collected from the subject is higher than the concentration of HMGB1 in one or more biological samples derived from a control subject not suffering from dementia. The method of claim 2 or 4, wherein the control subject not suffering from dementia is a healthy subject or a patient with mild cognitive impairment. The method for determining dementia according to any one of claims 1 to 4, wherein the dementia is Alzheimer's dementia. A kit for detecting amyloid β-HMGB1 complexes and diagnosing dementia, comprising an antibody or a labeled substance thereof that specifically binds to human amyloid β, and an antibody or a labeled substance thereof that specifically binds to human HMGB1. The kit according to claim 7, further comprising a kit for detecting HMGB1.