Method for evaluating cognitive function, and apparatus for evaluating cognitive function

Abstract

To provide an evaluation method which enables quantitative and objective evaluation of cognitive functions.SOLUTION: A cognitive function evaluation method for evaluating cognitive functions of a subject is provided, the method involving evaluating cognitive functions of the subject on the basis of an expression level of phosphorylated TrkB in extracellular vesicles contained in a bodily fluid of the subject and an expression level of TrkB in the extracellular vehicles.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a cognitive function evaluation method or the like for evaluating the cognitive function of a subject. [Background Art]

[0002] Various compositions for improving human cognitive function have been conventionally known. Patent Document 1 describes a food composition for improving cognitive function, which contains L-ergothioneine as an active ingredient.

[0003] On the other hand, if the cognitive function of a human can be evaluated, it is highly useful for the treatment and prevention of dementia, and therefore various studies have been conducted on evaluation methods for cognitive function. Patent Document 2 describes a cognitive function evaluation apparatus that evaluates the cognitive function of a subject based on body motion data of the subject during walking.

[0004] In addition, Patent Document 3 describes, as a method for evaluating the effectiveness of a specific component on cognitive function, a method for evaluating the effectiveness of a green tea component on mild cognitive impairment. This method employs at least one test selected from a attention shift test, an emotion recognition test, and a 4-part continuous processing test in CogniTrax assessment as a predetermined evaluation test. [Prior Art Documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Unexamined Patent Publication No. 2019-180364 [Patent Document 2] Japanese Patent No. 6706809 [Patent Document 3] Japanese Unexamined Patent Publication No. 2020-38156 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] Incidentally, cognitive function tests such as the Cognitrax test primarily use questionnaires and observational methods, and are said to be subjective and easily influenced by psychological factors. Therefore, there is a need for evaluation methods that can quantitatively and objectively grasp the effect of improving cognitive function.

[0007] This invention has been made in view of these circumstances, and aims to realize an evaluation method that can quantitatively and objectively evaluate cognitive function. [Means for solving the problem]

[0008] The inventors of this application conducted intensive research into the mechanisms of cognitive function improvement and hypothesized that activation of TrkB (tropomyosin receptor kinase B: brain-derived neurotrophic factor receptor), which controls neurogenesis and neuronal maturation, could serve as a biomarker for cognitive function. Since it is not possible to directly evaluate TrkB activation in the brain, they focused on extracellular vesicles contained in human body fluids. Furthermore, human trials revealed that while the expression level of phosphorylated TrkB (an activated form of TrkB) in extracellular vesicles increased with cognitive function improvement, the expression level of TrkB in extracellular vesicles remained largely unchanged with cognitive function improvement.

[0009] The first invention is a method for evaluating cognitive function, which evaluates the cognitive function of a subject, based on the expression level of phosphorylated TrkB in extracellular vesicles contained in the subject's body fluid and the expression level of TrkB in extracellular vesicles.

[0010] The second invention, in the first invention, calculates an index value representing the cognitive function of a subject based on the expression level of phosphorylated TrkB and the expression level of TrkB.

[0011] The third invention is that, in the second invention, the index value is the ratio of the expression level of phosphorylated TrkB to the expression level of TrkB.

[0012] The fourth invention is a method in which, in any one of the first to third inventions, extracellular vesicles are recovered from the body fluid of a subject, and an expression level acquisition step is performed using the extracellular vesicles recovered in the recovery step to obtain information on the expression level of phosphorylated TrkB and the expression level of TrkB, wherein in the recovery step, extracellular vesicles are recovered using an immunoprecipitation method with brain-related antibodies.

[0013] The fifth invention is a cognitive function evaluation device for evaluating the cognitive function of a subject, comprising an evaluation unit that evaluates the cognitive function of a subject based on the expression level of phosphorylated TrkB in extracellular vesicles contained in the subject's body fluid and the expression level of TrkB in extracellular vesicles. [Effects of the Invention]

[0014] In this invention, the cognitive function of a subject is evaluated based on the expression level of TrkB and phosphorylated TrkB in extracellular vesicles contained in the subject's body fluids, thus enabling a quantitative and objective evaluation of the subject's cognitive function. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 is a schematic diagram illustrating the procedure of the recovery step in the embodiment. [Figure 2] Figure 2 shows the target protein bands obtained by Western blotting using antibodies against CD63 and SNAP25. [Figure 3] Figure 3 is a chart showing the calculated values ​​for the ergonomic group and the placebo group regarding the ratio of the index value X in the example and comparative examples 1-4. [Figure 4] Figure 4 is a chart showing the correlation between the ratio of the index value X in the example and Comparative Example 1 and the blood ergothioneine concentration. [Modes for carrying out the invention]

[0016] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. The following embodiments are examples of the present invention, and are not intended to limit the scope of the present invention, its applications, or its uses.

[0017] The present embodiment is a cognitive function evaluation method (hereinafter referred to as "the present evaluation method") for evaluating the cognitive function of a subject by using a body fluid (for example, serum obtained from blood) of the subject who has ingested a composition (a food composition or a pharmaceutical composition) containing an ingredient effective for improving cognitive function. In the present evaluation method, in order to quantitatively evaluate the cognitive function of the subject, an index value X representing the cognitive function of the subject is calculated. The index value X is calculated using the expression level A of phosphorylated TrkB (p-TrkB) in extracellular vesicles obtained from the subject and the expression level B of TrkB in said extracellular vesicles. For example, the index value X can be calculated using Formula 1 below.

[0018] [Formula 1] X=A / B A: expression level of p-TrkB in extracellular vesicles obtained from the body fluid of the subject B: expression level of TrkB in extracellular vesicles obtained from the body fluid of the subject

[0019] In the present evaluation method, the cognitive function of the subject may be evaluated based on the expression level A and the expression level B. An index value representing the cognitive function of the subject may be calculated using a mathematical formula in which the numerator and denominator are reversed from those in Formula 1, or another mathematical formula into which the expression level A and the expression level B are substituted may be used to calculate the index value representing the cognitive function of the subject. Further, in the present evaluation method, the cognitive function of the subject may be evaluated by directly comparing the expression level A and the expression level B without calculating the index value X.

[0020] For example, when serum obtained from blood is used as the body fluid, the present evaluation method includes, in this order: a recovery step of recovering an extracellular vesicle fraction (human serum EVs fraction; hereinafter may be referred to as "EVs fraction") from the serum of the subject, and an expression level obtaining step of obtaining the expression level of p-TrkB and the expression level of TrkB in the EVs fraction obtained in the recovery step.

[0021] Extracellular vesicles are nano- to micro-sized particles surrounded by a lipid bilayer secreted by cells, and are present in various bodily fluids (blood, urine, saliva, etc.). In this evaluation method, extracellular vesicles derived from the brain are contained in serum obtained from human blood, so serum is used to collect the extracellular vesicles.

[0022] In the recovery step, methods such as ultracentrifugation, ultrafiltration, density gradient centrifugation, polymer precipitation, and immunoprecipitation can be used to recover extracellular vesicles from serum. Among these, when using immunoprecipitation, any antibody (protein) related to the brain is acceptable. For example, antibodies against SNAP25 (a marker protein for mature nerve cells) (anti-SNAP25 antibody), antibodies against CD271 (a marker protein for neural stem cells) (anti-CD271 antibody), and antibodies against PSA-NCAM (a marker protein for immature nerve cells) (anti-PSA-NCAM antibody) can be used. In this case, extracellular vesicles derived from the brain can be easily recovered, and highly accurate evaluation can be expected.

[0023] In the expression level acquisition step, methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), absolute quantification of protein amount using mass spectrometry, alphaLISA, and alphaScreen can be used to acquire the expression levels of p-TrkB and TrkB.

[0024] In Western blotting, the primary antibodies used are an antibody against p-TrkB (anti-p-TrkB antibody) and an antibody against TrkB (anti-TrkB antibody). The expression level of p-TrkB corresponding to the band intensity is obtained by image analysis of the bands in the hydrophobic membrane obtained by reacting with the p-TrkB antibody as the primary antibody. The expression level of TrkB corresponding to the band intensity is also obtained by image analysis of the bands in the hydrophobic membrane obtained by reacting with the TrkB antibody as the primary antibody. The index value X is then calculated using Equation 1.

[0025] Unlike tests such as the Cognitrax test, this evaluation method is useful because it can quantitatively and objectively assess the cognitive function of subjects. In particular, the index value X can be considered a visualization of the phosphorylation of receptors in the brain, and is useful for evaluating cognitive function because it is an evaluation index that is close to the actual state of cognitive function.

[0026] This evaluation method can be used to assess the degree of cognitive function improvement in subjects (healthy individuals, those with mild cognitive impairment, etc.) who continuously ingest ingredients effective in improving cognitive function. In this case, the degree of cognitive function improvement is assessed based on the change in index value X over time. Furthermore, this evaluation method can be used to determine whether or not an ingredient whose effectiveness in improving human cognitive function is unknown can be improved by human intake of that ingredient. Therefore, this evaluation method is useful in the development of dementia treatments for Alzheimer's disease and other conditions, as well as supplements for improving cognitive function.

[0027] Furthermore, the following examples confirm that this evaluation method can be used to assess the cognitive function of subjects who have ingested (e.g., orally) ergothioneine (L-ergothioneine) as an effective component for improving cognitive function. However, the component ingested by the subject is not limited to ergothioneine; any component that can activate TrkB (for example, antidepressants such as imipramine and paroxetine, TrkB agonists such as 7,8-dihydroxyflavone, TrkB agonist antibodies such as AS86, and other components that can activate TrkB such as ketamine) is acceptable. It can also be used to determine whether a component whose TrkB activation effect is unknown can be improved by human ingestion of that component. In addition, it can be used to assess the cognitive function of subjects undergoing interventions effective for improving cognitive function (such as dementia rehabilitation) if TrkB is activated in those subjects.

[0028] Furthermore, to facilitate this evaluation method, a cognitive function evaluation device can be configured that includes an evaluation unit that evaluates the cognitive function of a subject based on the expression level A of phosphorylated TrkB in extracellular vesicles contained in the subject's body fluid and the expression level B of TrkB in extracellular vesicles. The evaluation unit may be configured, for example, by a computer, and when it receives input of expression levels A and B, it calculates an index value X using, for example, Equation 1. The cognitive function evaluation device may also have a function to automatically perform at least one of the above-described recovery step or expression level acquisition step. [Examples]

[0029] In this example, the index value X (p-TrkB / TrkB) of this evaluation method was obtained using blood samples collected from multiple subjects, including healthy individuals and those with mild cognitive impairment (MCI). In addition to the index value X, the ratio of expression levels of four types of proteins in extracellular vesicles was also obtained as a comparative example. Furthermore, since each subject underwent Cognitrax testing, the usefulness of index value X was verified based on its correlation with the index value X in this example and the ratios in the comparative example. It should be noted that this example is just one example of the invention, and the present invention is not limited by this example. <Subjects> A placebo-controlled, randomized, parallel-group, double-blind comparative study was employed, using two groups for consuming the test food: an ergothioneine group (test food group) and a placebo group (control group). Hereafter, the ergothioneine group will be referred to as the "ergo group," and the placebo group as the "placebo group."

[0030] For the selection of subjects, a screening survey was conducted on 70 individuals, and 52 individuals who met all of the eligibility criteria (1) to (11) and did not meet all of the exclusion criteria (A) to (D) were included as subjects.

[0031] <<Eligibility Criteria>> (1) Individuals whose MMSE score from the screening test is 23 or higher (2) Age: Persons aged 20 or older and under 80 (3) Gender: Not specified (4) Those who do not smoke (5) Individuals who do not have a habit of consuming foods containing large amounts of ergothioneine (Foods containing large amounts of ergothioneine: mushrooms, liver, grains, legumes) (6) Persons who do not regularly use supplements or health foods (7) Persons who do not suffer from lifestyle-related diseases (such as hypertension or diabetes), rheumatism, liver disease, kidney disease, or other chronic diseases. (8) Persons with no history of treatment for malignant tumors, heart failure, or myocardial infarction. (9) Persons who have no history of allergies to mushrooms or pharmaceuticals containing large amounts of ergothioneine. (10) Persons who are not receiving outpatient treatment or medication for the purpose of treatment (11) Persons who fully understand the content of the clinical trial and have given written consent.

[0032] The Mini-Mental State Examination (MMSE), mentioned in eligibility criterion (1), is a test that evaluates the progression of dementia based on the total score out of a possible 30 points, with a score of 20 or less indicating suspected dementia. In this example, individuals with an MMSE score of 23-27 were classified as having MCI (Mild Cognitive Impairment), while those with a score of 28-30 were classified as healthy.

[0033] <<Exclusion criteria>> (A) Pregnant or breastfeeding women, or those who intend to become pregnant during the examination period. (B) Individuals currently participating in another clinical trial or study, or who have participated in another clinical trial or study within the past three months. (C) Persons who cannot follow the instructions of the attending physician and medical staff. (D) Any other person whom the examiner determines to have any problems.

[0034] To ensure that there was no bias in MMSE scores among the subjects, 52 subjects were randomly assigned to either the ergometer group (26 subjects) or the placebo group (26 subjects) using a substitution block method. The mean MMSE scores were similar, with the ergometer group at 27.2 points and the placebo group at 27.1 points.

[0035] Aside from MMSE scores, the gender of the subjects was as follows: the ergometer group consisted of 11 males and 15 females, while the placebo group consisted of 7 males and 19 females. Regarding the subject characteristics data, no significant differences were observed between the groups in height, weight, BMI, systolic blood pressure, diastolic blood pressure, pulse rate, or body temperature.

[0036] Of the 52 subjects, two individuals who contracted influenza midway through the study and were unable to undergo testing during the designated testing period, one individual whose food intake rate was 80% or higher but who had a two-week period during which they were unable to consume food, and one individual whose food intake rate was less than 80% were excluded from the analysis. Statistical analysis was then performed using the Per Protocol Set (PPS) cases, which were the target population that conformed to the study protocol (hereinafter referred to as "PPS case analysis").

[0037] Regarding the number of subjects in the PPS case analysis, there were 25 in the ergometer group (1 excluded) and 23 in the placebo group (3 excluded). In terms of gender, there were 11 males and 14 females in the ergometer group, and 5 males and 18 females in the placebo group. In addition, regarding whether the subjects were healthy or had MCI, there were 14 healthy subjects and 11 with MCI in the ergometer group, and 9 healthy subjects and 14 with MCI in the placebo group.

[0038] <Examination schedule, examination details> Each subject visited a medical institution (testing institution) according to the schedule in Table 1 and underwent examinations, diagnoses, and tests for the examination items in Table 1. Based on this schedule, examinations and tests were performed at 4 weeks (4 weeks: within 28 days ± 3 days from the first examination), 8 weeks (8 weeks: within 56 days ± 6 days from the first examination), and 12 weeks (12 weeks: within 84 days ± 6 days from the first examination), starting from week 0 of ingestion (0w: the first examination date).

[0039] [Table 1]

[0040] On each examination day, each subject woke up, ate breakfast, underwent a medical interview, physical measurements, blood pressure measurement, and blood test. The blood test measured the blood concentration of ergothioneine. Subsequently, each subject underwent the Cognitrax cognitive function test in accordance with the "Cognitrax Test Implementation Guide." In addition, a portion of the blood collected during the blood test was frozen and stored before being used in the examples and comparative examples.

[0041] <Test food, method of consumption of test food> The ergometer group received a food product containing 5 mg of ergothioneine derived from Tamogitake mushroom extract (per 4 tablets), while the placebo group received a food product containing 0 mg of ergothioneine (per 4 tablets). The test foods for each group were packaged in aluminum pouches that could be stored at room temperature. The test foods were blinded and distributed to subjects based on the identification number assigned to each food product. Blinding applied to all individuals involved in the study (subjects, intervention providers, evaluators, etc.), and the allocation sheets were not opened until the subjects for analysis were finalized.

[0042] The test food for the ergo group was manufactured using a freeze-drying method. Specifically, the test food for the ergo group was obtained by sequentially performing the following steps: a preparation step in which dextrin is dissolved in Tamogitake mushroom extract to create a material solution; a pre-freezing step in which the material solution obtained in the preparation step is frozen; and a freeze-drying step in which the material solution after the pre-freezing step is freeze-dried under low temperature and low pressure conditions.

[0043] Each participant was instructed to take two tablets with breakfast and two tablets with dinner, each with water, starting the day after the initial test (i.e., the ergo group's daily intake of ergothioneine was 5 mg). No specific dietary restrictions were given, but participants were advised to avoid consuming large amounts of mushrooms during the intake period. If a participant forgot to take a tablet after breakfast, they were instructed to take it as soon as possible, and if they forgot to take a tablet after dinner, they were instructed to take it before going to bed. As a general rule, only cases with a food intake rate of 80% or higher were included in the analysis. In addition, to accurately measure cognitive function, each participant was instructed to avoid overeating and excessive drinking the day before each test.

[0044] <Cognitrax Test> The Cognitrax test is a cognitive function testing service designed for Japan by Health Solution Co., Ltd., based on cognitive function testing technology developed by CNS Vital Signs, Inc. in the United States. The Cognitrax test includes verbal memory tests, visual memory tests, finger tapping tests, coding tests (SDC tests), Stroop tests, sustained processing tests, and 4-part sustained processing tests. The test results are quantified for 13 items (cognition-related test items) including overall memory, working memory, verbal memory, visual memory, cognitive speed, reaction speed, overall attention, cognitive flexibility, processing speed, executive function, sustained attention, simple attention, and motor speed. The test results for each item in this example are shown in Table 2. In Table 2, numbers in parentheses indicate negative changes.

[0045] [Table 2]

[0046] <Assessment of cognitive function using this evaluation method, etc.> As described above, the index value X for this evaluation method was obtained using blood collected from each subject, and the ratio of the expression levels of two types of proteins in extracellular vesicles was obtained as comparative examples 1-4. Blood samples were used at 0 weeks (0w), 4 weeks (4w), 8 weeks (8w), and 12 weeks (12w) of intake to obtain the index value X and the ratios for each comparative example. A detailed explanation follows below.

[0047] First, serum was obtained from the blood of each subject at each time of ingestion. Serum was obtained by allowing each subject's blood to stand in a test tube, and then collecting the supernatant portion of the test tube.

[0048] Next, the EVs fraction was recovered from the serum using ultracentrifugation. Specifically, the EVs fraction was recovered using five treatments. In the first treatment, the serum was centrifuged at a temperature of 4°C, a centrifugal force of 1200g, and a centrifuge operating time of 20 minutes to obtain a primary treatment solution from which the pellet had been removed. In the second treatment, the primary treatment solution was centrifuged at a temperature of 4°C, a centrifugal force of 10000g, and a centrifuge operating time of 20 minutes to obtain a secondary treatment solution from which the pellet had been removed. In the third treatment, the secondary treatment solution was filtered through a 0.22 μm filter to obtain a tertiary treatment solution. The tertiary treatment solution contained extracellular vesicles ranging in size from 50 nm to 200 nm. In the fourth treatment, the third treatment solution was centrifuged at a temperature of 4°C, a centrifugal force of 100000g, and a centrifuge operating time of 90 minutes, and the supernatant was removed. As a result, a pellet of extracellular vesicles remained at the bottom of the test tube. In the fifth treatment, the pellet of extracellular vesicles remaining at the bottom of the test tube in the fourth treatment was resuspended in 50 μL of phosphate-buffered saline (PBS). This resuspended material was used as a sample for Western blotting.

[0049] Next, the aforementioned resuspension was used as the sample for Western blotting. First, polyacrylamide electrophoresis (SDS-PAGE) was performed. In this electrophoresis, the sample (protein material) treated with SDS was electrophoresed in a polyacrylamide gel. This separated the proteins according to their peptide chain length (molecular weight). Next, the proteins separated by polyacrylamide electrophoresis were transferred to a hydrophobic membrane. A polyvinylidene fluoride (PVDF) membrane was used as the hydrophobic membrane. After blocking, the hydrophobic membrane was reacted with a primary antibody against the target protein, and after washing, a secondary antibody (HRP-labeled secondary antibody) that specifically binds to the primary antibody was reacted, causing the hydrophobic membrane to develop color by chemiluminescence. Then, by image analysis of the bands (target protein bands) in the hydrophobic membrane, the expression level of the target protein was obtained from the intensity of the bands.

[0050] Specifically, an equal volume of buffer (composition: 100 mM Tris-HCl (pH 7.5), 300 mM NaCl, 2% NP-40, 2% sodium deoxycholate, 0.4% sodium dodecylsulfate, 2 mM EDTA, 20 mM sodium furoride, 20 mM β-glycerophosphate disodium salt hydrate, 20 mM sodium pyrophosphate decahydrate, 2 mM sodium orthovanadate, 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 2 μg / mL leupeptin, 2 μg / mL antipain, 1 mM benzamidine hydrochloride hydrate) was added to 25 μL of the above-mentioned resuspension, and the solution was solubilized using a tip sonicator (manufactured by Tommy Seiko Co., Ltd.). The resulting solution was then prepared by Pierce TM After measuring the protein content using the BCA Protein Assay Kit, the protein was denatured by adding 1 / 4 the volume of buffer (composition: 10 mM Tris-HCl buffer (pH 6.8), 10% glycerol, 2% sodium dodecylsulfate, 0.01% bromophenol blue, 5% mercaptoethanol) and incubating at 95°C for 5 minutes. The sample was then applied to a 12.5% ​​polyacrylamide gel so that the protein content was 20 μg / lane, and electrophoresis was performed for approximately 30 minutes using a Compact PAGE-Twin (manufactured by ATTO Corporation).

[0051] Next, the proteins that had been electrophoresed on the gel were transferred to a PVDF membrane immersed in 100% methanol for 20 seconds. After transfer, blocking was performed at room temperature for 1 hour. For blocking with anti-p-TrkB antibody (anti-phospho-TrkB antibody), anti-TrkB antibody, and anti-NT-5 antibody, 2% BSA TBST (Tris-buffered saline containing 0.1% Tween 20) was used; for anti-SNAP25 antibody, 1% skim milk TBST was used; and for anti-CD63 antibody, 3% skim milk TBST was used. For reactions with primary antibodies, the following primary antibody solutions diluted with Can Get Signal Solution 1 (manufactured by Toyobo Co., Ltd.) were reacted overnight at 4°C. Note that rabbit-derived antibodies were used for anti-p-TrkB antibody, anti-TrkB antibody, anti-NT-5 antibody, and anti-CD63 antibody, while mouse-derived antibody was used for anti-SNAP25 antibody. ·Rabbit anti-phospho-TrkB antibody (Merck Millipore, 1:100 dilution) • Rabbit anti-TrkB antibody (Cell Signaling Technology, 1:1,000 dilution) • Rabbit anti-NT-5 antibody (Merck Millipore, 1:250 dilution) • Mouse anti-SNAP25 antibody (Santa Cruz, 1:50 dilution) • Rabbit anti-CD63 antibody (Santa Cruz, 1:100 dilution)

[0052] After washing the PVDF membrane with TBST, it was reacted with the following secondary antibody solutions diluted with Can Get Signal Solution 2 (manufactured by Toyobo Co., Ltd.) at room temperature for 1 hour. ·HRP-labeled anti-Mouse-IgG antibody (1:20,000 dilution) • HRP-labeled anti-Rabbit-IgG antibodies (anti-p-TrkB antibody and anti-TrkB antibody are diluted 1:5,000 times; anti-NT-5 antibody is diluted 1:10,000 times; anti-CD63 antibody is diluted 1:100,000 times)

[0053] Proteins reacted with these secondary antibodies were color-developed using Pierce ECL Plus Substrate and detected using a lumino image analyzer LAS-4000 (Fujifilm Corporation). Band intensity was quantified using the image processing software ImageJ. This allowed us to obtain the expression levels of p-TrkB, TrkB, NT-5, CD63, and SNAP25.

[0054] Figure 2(a) shows the results using an antibody against CD63, an extracellular vesicle marker derived from all tissues, as the primary antibody (marker size: 40-50 kDa), and Figure 2(b) shows the results using an antibody against SNAP25, an extracellular vesicle marker derived from the brain, as the primary antibody (marker size: 25 kDa). As shown in Figures 2(a) and 2(b), CD63 and SNAP25 were detected from the hydrophobic membrane after transcription. This confirmed that extracellular vesicles derived from the brain were present in the serum.

[0055] Next, as an example, the mean value of the index value X(p-TrkB / TrkB), which is the ratio of p-TrkB expression level to TrkB expression level, was calculated for all subjects in the placebo group and all subjects in the ergometer group. In Figure 3(a), the index value X(p-TrkB / TrkB) is plotted on the vertical axis and the test dates (weeks 0, 4, 8, and 12) are plotted on the horizontal axis, showing the calculated values ​​for each group. In Figures 3(a) to (e), open bars represent the calculated values ​​for the placebo group, and filled bars represent the calculated values ​​for the ergometer group.

[0056] As Comparative Example 1, the average value of the ratio of p-TrkB expression level to CD63 expression level (p-TrkB / CD63) was calculated for all subjects in the placebo group and all subjects in the ergometer group. Figure 3(b) shows the calculated values ​​for each group, with the ratio (p-TrkB / CD63) on the vertical axis and the test date on the horizontal axis.

[0057] As a comparative example 2, the mean ratio of TrkB expression level to CD63 expression level (TrkB / CD63) was calculated for all subjects in the placebo group and all subjects in the ergometer group. Figure 3(c) shows the calculated values ​​for each group, with the ratio (TrkB / CD63) on the vertical axis and the test date on the horizontal axis.

[0058] As Comparative Example 3, the mean ratio of NT-5 expression level to CD63 expression level (NT-5 / CD63) was calculated for all subjects in the placebo group and all subjects in the ergometer group. Figure 3(d) shows the calculated values ​​for each group, with the ratio (NT-5 / CD63) on the vertical axis and the test date on the horizontal axis.

[0059] As Comparative Example 4, the mean ratio of SNAP25 expression level to CD63 expression level (SNAP25 / CD63) was calculated for all subjects in the placebo group and all subjects in the ergometer group. Figure 3(e) shows the calculated values ​​for each group, with the ratio (SNAP25 / CD63) on the vertical axis and the test date on the horizontal axis.

[0060] As shown in Figure 3(a), the index value X (p-TrkB / TrkB) of the example showed significant differences (p<0.05) between the 12w placebo group and the 12w ergometer group, between the 0w ergometer group and the 8w ergometer group, and between the 0w ergometer group and the 12w ergometer group. Furthermore, as shown in Figure 3(b), the ratio (p-TrkB / CD63) of Comparative Example 1 showed significant differences (p<0.05) between the 12w placebo group and the 12w ergometer group, and between the 0w ergometer group and the 12w ergometer group.

[0061] Furthermore, the index value X in the example and the ratio in Comparative Example 1 showed a high correlation with the blood ergothioneine concentration, as shown in Figures 4(a) and 4(b).

[0062] Next, for the ergometer group, Table 3 shows the correlation coefficients for each Cognitrax test item: index value X(p-TrkB / TrkB), ratio (p-TrkB / CD63), ratio (TrkB / CD63), ratio (NT-5 / CD63), ratio (SNAP25 / CD63), blood ergothioneine concentration, serum ergothioneine concentration, and change in serum ergothioneine concentration exposure (ΔAUC). Among the correlation coefficients in Table 3, those enclosed in thick borders indicate statistically significant correlations (*: p<0.05, **: p<0.01).

[0063] [Table 3]

[0064] Table 4 shows the correlation coefficients for each Cognitrax test item in the placebo group, including the index value X(p-TrkB / TrkB), ratio (p-TrkB / CD63), ratio (TrkB / CD63), ratio (NT-5 / CD63), ratio (SNAP25 / CD63), blood ergothioneine concentration, serum ergothioneine concentration, and change in serum ergothioneine concentration exposure (ΔAUC). In Table 4, the correlation coefficients enclosed in bold boxes indicate statistically significant correlations (*: p<0.05, **: p<0.01).

[0065] [Table 4]

[0066] Tables 3 and 4 show that the index value X(p-TrkB / TrkB) in the ergometer group showed a significant positive correlation with the results of the Cognitrax test (overall memory, verbal memory, reaction time, working memory, reaction time, overall attention, cognitive flexibility, processing speed, and executive function). In particular, overall memory, verbal memory, and processing speed also showed a significant positive correlation with the change in serum ergothioneine exposure (ΔAUC). On the other hand, the ratios (p-TrkB / CD63), (TrkB / CD63), (NT-5 / CD63), and (SNAP25 / CD63) did not show a significant correlation with the results of the Cognitrax test. Therefore, it was found that the index value X(p-TrkB / TrkB) is useful for evaluating cognitive function. Furthermore, the significant positive correlation observed, particularly with working memory, suggests that ergothioneine intake activates p-TrkB in the hippocampus, which is crucial for working memory, and that this leads to neurogenesis. Therefore, the index value X can be considered useful for evaluating neurogenesis in the hippocampus. [Industrial applicability]

[0067] This invention can be applied to methods for evaluating cognitive function, such as those used to evaluate the cognitive function of a subject.

Claims

1. A method for assisting in the assessment of a subject's cognitive function, A method for presenting information for evaluating the cognitive function of a subject based on the expression level of phosphorylated TrkB in extracellular vesicles contained in the subject's body fluid and the expression level of TrkB in the extracellular vesicles.

2. The method according to claim 1, wherein an index value representing the cognitive function of the subject is calculated based on the expression level of phosphorylated TrkB and the expression level of TrkB.

3. The method according to claim 2, wherein the index value is the ratio of the expression level of phosphorylated TrkB to the expression level of TrkB.

4. A recovery step of recovering extracellular vesicles from the body fluid of the subject, Using the extracellular vesicles recovered in the above recovery step, an expression level acquisition step is performed to obtain information on the expression level of phosphorylated TrkB and the expression level of TrkB. The method according to any one of claims 1 to 3, wherein the recovery step involves recovering extracellular vesicles using an immunoprecipitation method with brain-related antibodies.

5. A cognitive function evaluation device for evaluating the cognitive function of a subject, A cognitive function evaluation device comprising an evaluation unit that evaluates the cognitive function of the subject based on the expression level of phosphorylated TrkB in extracellular vesicles contained in the subject's body fluid and the expression level of TrkB in the extracellular vesicles.

Images