Preventive or therapeutic agent for mild cognitive impairment

Abstract

This invention addresses the problem of providing a substance capable of preventing or treating mild cognitive impairment.　A substance capable of increasing the amount of mammalian YAP, the amount of a polynucleotide encoding mammalian YAP, or the amount of a mammalian YAP in the nucleus of brain neurons is used to prevent or treat mild cognitive impairment.

Description

[Technical field]

[0001] The present invention relates to a preparation for preventing and / or treating mild cognitive impairment (hereinafter, sometimes referred to as "MCI"), which contains mammalian YAP (Yes-associated protein), or a polynucleotide encoding mammalian YAP, or a substance capable of increasing the amount of mammalian YAP in the nuclei of brain neurons (hereinafter, these may be collectively referred to as "the YAP-related substance"). [Background technology]

[0002] Alzheimer's disease (hereinafter sometimes referred to as "AD") is one of the irreversible progressive central nervous system diseases, and is known to present symptoms such as cognitive impairment (dementia) accompanied by memory impairment and thinking disorder, behavioral disorder, personality change, etc. Alzheimer's dementia is the most common disease among dementia patients, accounting for approximately 60-80% of all dementia patients. Alzheimer's dementia generally develops in elderly people aged 65 years or older, but it can also develop in people aged 64 years or younger, in which case it is called early-onset Alzheimer's disease.

[0003] According to an announcement by the Cabinet Office, the number of dementia patients in Japan in 2012 was 4.62 million, equivalent to 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. If the number of dementia patients, such as Alzheimer's disease, 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.

[0004] Although MCI patients experience a decline in cognitive function, they do not meet the diagnostic criteria for dementia and do not have any problems with basic daily life or social life. Since some MCI patients are thought to develop Alzheimer's disease in the future, it is expected that treating MCI can prevent future onset of Alzheimer's disease.

[0005] It has been reported that a specific composition (ferulic acid, ginkgo leaf extract, α-glycerophosphocholine) has the effect of improving the symptoms of MCI (Patent Document 1). It has also been reported that hydrogen has the effect of preventing MCI (Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2019-6699 A [Patent Document 2] International Publication No. 2018 / 012596 Brochure Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a substance capable of preventing or treating mild cognitive impairment. [Means for solving the problem]

[0008] In the course of intensive research to solve the above problems, the present inventors have confirmed that 1) the concentration of HMGB1, which is known to be one of the damage-associated molecular patterns (DAMPs) released from necrotic cells, is higher in cerebrospinal fluid (CSF) samples from MCI patients than in CSF samples from individuals without MCI, and 2) Hippo pathway-dependent necrosis is increased in brain neurons of MCI patients. On the other hand, sphingosine 1-phosphate (S1P), a substance that promotes the nuclear translocation of YAP, and YAP and its isoforms are known to have the effect of suppressing Hippo pathway-dependent necrosis. Therefore, using S1P and YAP isoforms (specifically, rat YAPΔC-ins61), the researchers analyzed whether Hippo pathway-dependent necrosis caused by MCI could be suppressed. They found that the amount of YAP, which had decreased due to MCI, increased in the nuclei of brain neurons, suppressing Hippo pathway-dependent necrosis in those brain neurons, thereby making it possible to prevent or ameliorate cognitive decline, which led to the completion of the present invention.

[0009] That is, the present invention is as follows. [1] A preventive or therapeutic agent for mild cognitive impairment, comprising mammalian YAP, a polynucleotide encoding mammalian YAP, or a substance capable of increasing the amount of mammalian YAP in the nucleus of a brain neuron. [2] The preventive or therapeutic agent according to [1] above, characterized in that the mammalian YAP is a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the substance capable of increasing the amount of mammalian YAP in the nucleus of brain neurons is sphingosine 1-phosphate. [3] The preventive or therapeutic agent according to [1] or [2] above, wherein the mild cognitive impairment is characterized by an increased concentration of HMGB1 in cerebrospinal fluid compared to a control individual not suffering from mild cognitive impairment. [4] The preventive or therapeutic agent according to [3] above, wherein the control subject not suffering from mild cognitive impairment is a healthy subject or an Alzheimer's disease patient.

[0010] Further examples of embodiments of the present invention include a method for preventing or treating mild cognitive impairment, comprising the step of administering the YAP-related substance to a subject in need of such prevention or treatment; the YAP-related substance for use as a preventive or therapeutic agent for mild cognitive impairment; the YAP-related substance for use in the prevention or treatment of mild cognitive impairment; and use of the YAP-related substance for the manufacture of a preventive or therapeutic agent for mild cognitive impairment. Effect of the Invention

[0011] The YAP-related substance in question has the effect of suppressing Hippo pathway-dependent necrosis caused by mild cognitive impairment in brain neurons, preventing cognitive decline, and recovering impaired cognitive function. Therefore, the YAP-related substance in question is effective not only for preventing or treating mild cognitive impairment, but also for preventing dementia (e.g., Alzheimer's disease) that progresses from mild cognitive impairment. [Brief description of the drawings]

[0012] [Figure 1] This figure shows the results of measuring HMGB1 concentrations in CSF samples from three types of subjects (non-MCI / AD control subjects ["cc" in the figure], MCI patients ["MCI" in the figure], and AD patients ["AD" in the figure]). "***" in the figure indicates a statistically significant difference (p<0.001). ● in the figure indicates each individual subject. [Diagram 2] Figure 2A shows the results of constructing a receiver operating characteristic (ROC) curve based on the measurement results of HMGB1 concentrations in CSF samples from MCI patients and non-MCI / AD controls, and Figure 2B shows the results of constructing an ROC curve based on the measurement results of HMGB1 concentrations in CSF samples from MCI patients and AD patients. [Diagram 3]In FIG. 1, non-MCI / AD control subjects are divided into two groups: healthy subjects ("nc" in the figure) and disease patients ("dc" in the figure). [Figure 4] Figure 4A shows the results of an ROC curve created based on the results of measuring HMGB1 concentrations in CSF samples from MCI patients and healthy subjects, and Figure 4B shows the results of an ROC curve created based on the results of measuring HMGB1 concentrations in CSF samples from MCI patients and non-dementia neurological disease patients. [Diagram 5] The figures show the results of measuring the number of cells in which necrosis occurred (necrotic cell count) in the brain neurons of two types of AD model mice (5xFAD mice [Fig. 5A] and APP-KI mice [Fig. 5B], n=3 for each) and the three types of subjects. "*" and "**" in the figures indicate statistically significant differences (p<0.05 and p<0.01), respectively, based on Tukey's HSD test. [Figure 6] FIG. 6A shows the results of measuring endoplasmic reticulum (ER) volumes over time (1-3 hours) in brain neurons (n=9 each) of 1-month-old 5×FAD mice (left panel, n=3) and normal mice (right panel, n=3). FIG. 6B shows the results of measuring ER volumes over time (1-3 hours) in brain neurons (n=9 each) of 3-month-old 5×FAD mice (left panel, n=3) and normal mice (right panel, n=3). FIG. 6C shows the results of measuring ER volumes over time (1-3 hours) in brain neurons (n=9 each) of 6-month-old 5×FAD mice (left panel, n=3) and normal mice (right panel, n=3). [Figure 7] Figures 7A to 7C show the results of measuring the standard deviation (SD) (left panel) and quartile deviation (right panel) of the ER volume based on the results of Figures 6A to 6C, respectively. "**" in the figures indicates that there is a statistically significant difference (p<0.01) by Welch's test. [Figure 8]Figure 8A shows the YAP image, amyloid β (Aβ) image, and merge image (an image in which the YAP image, Aβ image, and DAPI image are superimposed) of the brain neurons of the three subjects. Figure 8B shows the original image of Figure 8A. [Figure 9] FIG. 9A is a diagram showing the results of measuring the nuclear YAP fluorescence level (i.e., nuclear YAP amount) for brain nerve cells (n=90 for each) of the above three types of subjects (n=3 for each) based on the YAP image of FIG. 8. FIG. 9B is a diagram showing the results of measuring the cytoplasmic pLATS1 level for brain nerve cells (n=90 for each) of the above three types of subjects (n=3 for each). FIG. 9C is a diagram showing the results of measuring the nuclear YAP amount for brain nerve cells (n=90 for each) of 3-month-old 5×FAD mice ("FAD" in the figure, n=3) and normal mice ("NC" in the figure, n=3). FIG. 9D is a diagram showing the results of measuring the nuclear YAP amount for brain nerve cells (n=90 for each) of 3-month-old APP-KI mice ("APP" in the figure, n=3) and normal mice ("NC" in the figure, n=3). In the figure, "*" and "**" indicate that there is a statistically significant difference (p<0.05 and p<0.01), respectively, by Tukey's HSD test. In the figure, "#" indicates that there is a statistically significant difference (p<0.05) by Student's t-test. [Figure 10]FIG. 10A is a diagram showing the results of measuring the amount of intranuclear YAP in brain neurons (n=60 each) of normal mice (indicated as "+S1P NC" and "-S1P NC" in the figure) that were continuously administered with S1P or aCSF from 1 to 6 months of age. FIG. 10B is a diagram showing the results of measuring the amount of intranuclear YAP in brain neurons (n=60 each) of normal mice (indicated as "+YAPΔC NC" and "-YAPΔC NC" in the figure) that were administered with AAV-YAPΔC-ins61 or AAV-NINS at the age of 1 month. FIG. 10C is a diagram showing the results of measuring the amount of intranuclear YAP in brain neurons (n=60 each) of 5×FAD mice (indicated as "+S1P FAD" and "-S1P FAD" in the figure) that were continuously administered with S1P or aCSF from 1 to 6 months of age. Figure 10D shows the results of measuring the amount of YAP in the nucleus of brain neurons (n ​​= 60 each) from 5xFAD mice administered AAV-YAPΔC-ins61 or AAV-NINS at 1 month of age (indicated as "+YAPΔC FAD" and "-YAPΔC FAD" in the figure, respectively). "**" in the figure indicates a statistically significant difference (p < 0.01) by Welch's test. [Figure 11]FIG. 11A is a diagram showing the results of measuring extracellular Aβ levels in brain tissues (n=30 each) of normal mice (indicated as "+S1P NC" and "-S1P NC" in the figure) that were continuously administered with S1P or aCSF from 1 to 6 months of age. FIG. 11B is a diagram showing the results of measuring extracellular Aβ levels in brain tissues (n=30 each) of normal mice (indicated as "+YAPΔC NC" and "-YAPΔC NC" in the figure) that were administered with AAV-YAPΔC-ins61 or AAV-NINS at the age of 1 month. FIG. 11C is a diagram showing the results of measuring extracellular Aβ levels in brain tissues (n=30 each) of 5×FAD mice (indicated as "+S1P FAD" and "-S1P FAD" in the figure) that were continuously administered with S1P or aCSF from 1 to 6 months of age. FIG. 11D shows the results of measuring extracellular Aβ levels in brain tissues (n=30 each) of 5×FAD mice administered AAV-YAPΔC-ins61 or AAV-NINS at 1 month of age (indicated as “+YAPΔC FAD” and “−YAPΔC FAD” in the figure, respectively). “**” and “ns” in the figure indicate that there is a statistically significant difference (p<0.01) or not (p≧0.05) by Welch's test, respectively. [Figure 12] FIG. 12A shows the results of measuring ER volume over time (1-3 hours) in brain neurons (n=3 each) of normal mice (indicated as "+S1P NC" and "-S1P NC" in the figure, n=3, respectively) that were continuously administered S1P or aCSF from the age of 1-6 months. FIGS. 12B and 12C show the results of measuring the standard deviation (FIG. 12B) and quartile deviation (FIG. 12C) of the ER volume based on the results of FIG. 12A. "ns" in the figure indicates that there is no statistically significant difference (p≧0.05) by Welch's test. [Figure 13]FIG. 13A shows the results of measuring ER volume over time (1-3 hours) in brain neurons (n=3 each) of 5×FAD mice (indicated as "+S1P FAD" and "-S1P FAD" in the figure, n=3, respectively) that were continuously administered with S1P or aCSF from the age of 1-6 months. FIGS. 13B and 13C show the results of measuring the standard deviation (FIG. 13B) and quartile deviation (FIG. 13C) of the ER volume based on the results of FIG. 13A. "**" in the figure indicates that there is a statistically significant difference (p<0.01) by Welch's test. [Figure 14] FIG. 14A shows the results of measuring ER volume over time (1 to 3 hours) in brain neurons (n=3 each) of normal mice (indicated as "+YAPΔC NC" and "-YAPΔC NC" in the figure, n=3, respectively) administered with AAV-YAPΔC-ins61 or AAV-NINS at the age of 1 month. FIGS. 14B and 14C show the results of measuring the standard deviation (FIG. 14B) and quartile deviation (FIG. 14C) of the ER volume based on the results of FIG. 14A. "ns" in the figure indicates that there is no statistically significant difference (p≧0.05) by Welch's test. [Figure 15] Figure 15A shows the results of measuring ER volume over time (1 to 3 hours) in brain neurons (n=3 each) of 5xFAD mice administered AAV-YAPΔC-ins61 or AAV-NINS at 1 month of age (indicated as "+YAPΔC FAD" and "-YAPΔC FAD" in the figure, respectively). Figures 15B and C show the results of measuring the standard deviation (Figure 15B) and quartile deviation (Figure 15C) of the ER volume based on the results in Figure 15A. "**" in the figure indicates that there is a statistically significant difference (p<0.01) by Welch's test. [Figure 16]FIG. 16A is a diagram showing the results of measuring pMARCK levels in brain neurons (n=30 each) of normal mice (indicated as "+S1P NC" and "-S1P NC" in the figure, respectively) that were continuously administered with S1P or aCSF from 1 to 6 months of age. FIG. 16B is a diagram showing the results of measuring pMARCK levels in brain neurons (n=30 each) of normal mice (indicated as "+YAPΔC NC" and "-YAPΔC NC" in the figure, respectively) that were administered with AAV-YAPΔC-ins61 or AAV-NINS at the age of 1 month. FIG. 16C is a diagram showing the results of measuring pMARCK levels in brain neurons (n=30 each) of 5×FAD mice (indicated as "+S1P FAD" and "-S1P FAD" in the figure, respectively) that were continuously administered with S1P or aCSF from 1 to 6 months of age. FIG. 16D shows the results of measuring pMARCK levels in brain neurons (n=30 each) of 5×FAD mice administered AAV-YAPΔC-ins61 or AAV-NINS at 1 month of age (indicated as "+YAPΔC FAD" and "-YAPΔC FAD" in the figure, respectively). "**" and "ns" in the figure indicate that there is a statistically significant difference (p<0.01) or not (p≧0.05) by Welch's test. [Figure 17]FIG. 17A shows the results of measuring the alternation behavior score for 5×FAD mice (indicated as “+S1P FAD” and “−S1P FAD”, n=6 and 9, respectively) and normal mice (indicated as “+S1P NC” and “−S1P NC”, n=7 and 9, respectively) that were continuously administered with S1P or aCSF from 1 to 6 months of age. FIG. 17B shows the results of measuring the alternation behavior score for 5×FAD mice (indicated as “+YAPΔC FAD” and “−YAPΔC FAD”, n=7 and 5, respectively) and normal mice (indicated as “+YAPΔC NC” and “−YAPΔC NC”, n=10 and 8, respectively) that were administered with AAV-YAPΔC-ins61 or AAV-NINS at the age of 1 month. FIG. 17C shows the results of measuring the alternation behavior score for 5×FAD mice (indicated as “+S1P FAD” and “−S1P FAD”, n=7 and 7, respectively) and normal mice (indicated as “+S1P NC” and “−S1P NC”, n=7 and 8, respectively) that were continuously administered with S1P or aCSF from the age of 5 to 6 months. FIG. 17D shows the results of measuring the alternation behavior score for 5×FAD mice (indicated as “+YAPΔC FAD” and “−YAPΔC FAD”, n=9 and 5, respectively) and normal mice (indicated as “+YAPΔC NC” and “−YAPΔC NC”, n=6 and 5, respectively) that were administered with AAV-YAPΔC-ins61 or AAV-NINS at the age of 5 months. In the figure, "*" and "**" indicate that there are statistically significant differences (p<0.05 and p<0.01), respectively, according to Tukey's HSD test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The preventive or therapeutic agent for mild cognitive impairment of the present invention is a preparation (hereinafter, sometimes referred to as the present preventive / therapeutic agent) containing at least one selected from mammalian YAP; a polynucleotide encoding mammalian YAP (hereinafter, sometimes referred to as a "mammalian YAP polynucleotide"); and a substance capable of increasing the amount of mammalian YAP in the nucleus of brain neurons (hereinafter, sometimes referred to as a "mammalian YAP enhancer"); preferably as an active ingredient, specified for the use of "preventing or treating mild cognitive impairment." Here, the prevention of mild cognitive impairment includes not only the prevention of onset of mild cognitive impairment, but also the prevention of the worsening of symptoms of mild cognitive impairment. The preventive / therapeutic agent may be used alone as a pharmaceutical (preparation), or may be used in the form of a composition (pharmaceutical composition) by further mixing with an additive.

[0014] Examples of the above-mentioned mammals include rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees.

[0015] The mammalian YAP may be any mammalian YAP, that is, a polypeptide derived from a mammal that is localized in the nucleus of brain neurons and has the effect of suppressing Hippo pathway-dependent necrosis due to mild cognitive impairment, and examples thereof include a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of rat YAPΔC-ins61 (i.e., the amino acid sequence of SEQ ID NO: 1), a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of rat YAPΔC-ins13 (i.e., the amino acid sequence of SEQ ID NO: 3), a polypeptide comprising an amino acid sequence having at least 80% sequence identity with rat YAPΔCs-ins25 (i.e., the amino acid sequence of SEQ ID NO: 4), a polypeptide comprising an amino acid sequence having at least 80% sequence identity with rat YAP (i.e., the amino acid sequence of SEQ ID NO: 5), a polypeptide comprising an amino acid sequence having at least 80% sequence identity with mouse YAP (i.e., the amino acid sequence of SEQ ID NO: 6), an amino acid sequence having at least 80% sequence identity with human YAP2 (i.e., the amino acid sequence of SEQ ID NO: 7), and the like. a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1-2 delta (i.e., the amino acid sequence of SEQ ID NO: 8); a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1-2 alpha (i.e., the amino acid sequence of SEQ ID NO: 9); a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1-2 beta (i.e., the amino acid sequence of SEQ ID NO: 10); a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1-2 gamma (i.e., the amino acid sequence of SEQ ID NO: 11); a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1 (i.e., the amino acid sequence of SEQ ID NO: 12); a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1-1 gamma (i.e., the amino acid sequence of SEQ ID NO: 13); a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1-1 delta (i.e., the amino acid sequence of SEQ ID NO: 14);Examples of such polypeptides include a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of rat YAPΔC-ins61 (i.e., the amino acid sequence of SEQ ID NO: 15), a polypeptide comprising an amino acid sequence having at least 80% sequence identity with human YAP1-1 beta (i.e., the amino acid sequence of SEQ ID NO: 16), etc., and a preferred example is a polypeptide comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence of rat YAPΔC-ins61 (i.e., the amino acid sequence of SEQ ID NO: 1), as its effects have been demonstrated in the present Example described below. In addition, examples of polypeptides containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1 include rat YAPΔC-ins61 (a polypeptide consisting of the amino acid sequence of SEQ ID NO:1), as well as rat YAPΔC-ins13 (a polypeptide consisting of the amino acid sequence of SEQ ID NO:3), rat YAPΔC-ins25 (a polypeptide consisting of the amino acid sequence of SEQ ID NO:4), rat YAP (a polypeptide consisting of the amino acid sequence of SEQ ID NO:5), mouse YAP (a polypeptide consisting of the amino acid sequence of SEQ ID NO:6), human YAP2 (a polypeptide consisting of the amino acid sequence of SEQ ID NO:7), human YAP1-2 delta (a polypeptide consisting of the amino acid sequence of SEQ ID NO:8), human YAP1-2 alpha (a polypeptide consisting of the amino acid sequence of SEQ ID NO:9), human YAP1-2 beta (a polypeptide consisting of the amino acid sequence of SEQ ID NO:10), and human YAP1-2 gamma (a polypeptide consisting of the amino acid sequence of SEQ ID NO:11).

[0016] The number of amino acid residues of the mammalian YAP is not particularly limited. Considering that the number of amino acid residues of the above-mentioned human, mouse, and rat YAP (i.e., the amino acid sequence of SEQ ID NO: 1 or 3 to 16) is within the range of 350 to 508, and the number of amino acid residues of rat YAPΔC-ins61 specifically used in the present Example described later is 366, the number of amino acid residues of the mammalian YAP is, for example, 280 to 700, 280 to 660, 280 to 630, 280 to 600, 280 to 560 pieces, 280~530 pieces, 280~510 pieces, 290~700 pieces, 300~700 pieces, 310~700 pieces, 320~700 pieces, 330~700 pieces, 340~700 pieces, 350~700 pieces, 366~700 pieces, 290~700 pieces, 290~660 pieces, 290~630 pieces, 290~600 pieces, 290~560 pieces, 290~530 pieces, 290~510 pieces, 300~700 pieces, 300~660 pieces, 300~630 pieces, 300~600 pieces, 300~560 pieces, 30 0~530 pieces, 300~510 pieces, 310~700 pieces, 310~660 pieces, 310~630 pieces, 310~600 pieces, 310~560 pieces, 310~530 pieces, 310~510 pieces, 320~700 pieces, 320~660 pieces, 320~ 630 pieces, 320~600 pieces, 320~560 pieces, 320~530 pieces, 320~510 pieces, 330~700 pieces, 330~660 pieces, 330~630 pieces, 330~600 pieces, 330~560 pieces, 330~530 pieces, 330~51 Examples of the amino acid residues of the mammalian YAP include 0, 340 to 700, 340 to 660, 340 to 630, 340 to 600, 340 to 560, 340 to 530, 340 to 510, 350 to 700, 350 to 660, 350 to 630, 350 to 600, 350 to 560, 350 to 530, 350 to 510, 360 to 700, 360 to 660, 360 to 630, 360 to 600, 360 to 560, 360 to 530, and 360 to 510. The amino acid residues of the mammalian YAP may be modified by glycosylation, acetylation, phosphorylation, lipid addition, stable isotope labeling, or the like.

[0017] With regard to the above mammalian YAP polynucleotide, by referring to the amino acid sequence of mammalian YAP and a known codon table, a person skilled in the art can specifically and clearly grasp the nucleotide sequence corresponding to the amino acid sequence. For example, when mammalian YAP is a polypeptide containing an amino acid sequence having at least 80% sequence identity with the amino acid sequence of rat YAPΔC-ins61 (i.e., the amino acid sequence of SEQ ID NO: 1), an example of the polynucleotide is a polynucleotide containing a nucleotide sequence having at least 80% sequence identity with a cDNA (polynucleotide) encoding rat YAPΔC-ins61 (i.e., a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2).

[0018] A suitable example of the mammalian YAP polynucleotide is a vector containing a polynucleotide in which a promoter is operably linked upstream of the mammalian YAP polynucleotide. Such a vector is not particularly limited as long as it can transcribe the mRNA encoded by the mammalian YAP polynucleotide, and can be appropriately selected according to the purpose, and examples thereof include non-viral vectors (e.g., episomal vectors, artificial chromosome vectors, and plasmid vectors) and viral vectors. The vector may be either circular or linear.

[0019] The promoter used in the above vector is not particularly limited as long as it is a region to which RNA polymerase (preferably RNA polymerase and a general transcription factor) binds and initiates transcription of the mRNA encoded by the mammalian YAP polynucleotide located downstream thereof, and examples thereof include the SRα promoter, the SV40 early promoter, the LTR of a retrovirus, the CMV (cytomegalovirus) promoter, the RSV (Rous sarcoma virus) promoter, the HSV-TK (herpes simplex virus thymidine kinase) promoter, the EF1α promoter, the metallothionein promoter, and the heat shock promoter. In addition, the enhancer of the IE gene of human CMV may be used together with the promoter. As an example, the CAG promoter (containing the cytomegalovirus enhancer, the chicken β-actin promoter, and the polyA signal site of the β-globin gene) can be used.

[0020] The episomal vector is a vector capable of autonomously replicating outside of a chromosome. A specific means of using an episomal vector is disclosed in Yu et al., Science, 324, 797-801 (2009). In a particularly preferred embodiment of the present invention, an episomal vector can be used in which loxP sequences are arranged in the same direction on the 5' and 3' sides of a vector element required for the replication of the episomal vector. Since an episomal vector can autonomously replicate outside of a chromosome, it can provide stable expression in a host cell even without being integrated into a genome.

[0021] Examples of the episomal vector include vectors that contain, as vector elements, sequences necessary for autonomous replication derived from EBV, SV40, etc. Specific examples of the vector elements necessary for autonomous replication include an origin of replication and a gene encoding a protein that binds to the origin of replication and controls replication, such as the origin of replication oriP and the EBNA-1 gene for EBV, and the origin of replication ori and the SV40LT gene for SV40.

[0022] Examples of the artificial chromosome vector include YAC (Yeast artificial chromosome) vectors, BAC (Bacterial artificial chromosome) vectors, and PAC (P1-derived artificial chromosome) vectors.

[0023] Examples of the above-mentioned plasmid vector include pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo.

[0024] The above-mentioned viral vector means a gene vector utilizing the infectivity and replicability of a virus, and more specifically means a viral particle (also called a "recombinant virus") that contains a viral vector plasmid (which may be DNA or RNA) in which a gene related to pathogenicity has been removed from the viral genome and a foreign gene (in the present application, a mammalian YAP polynucleotide) has been incorporated. Examples of the above-mentioned viral vector include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, Sendai virus vectors, herpes virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors. Adenoviral vectors and AAV vectors are preferred because they have a relatively high efficiency of introducing mammalian YAP polynucleotides into non-dividing cells such as brain nerve cells, and lentiviral vectors and AAV vectors are preferred because the expression of mammalian YAP can be sustained for a long time in brain nerve cells after the introduction of the mammalian YAP polynucleotide. AAV vectors are preferred because they are non-pathogenic and highly safe. Therefore, an AAV vector is a suitable example of the above-mentioned viral vector, because it has a relatively high efficiency of introducing mammalian YAP polynucleotides into non-dividing cells such as brain neurons, can sustain the expression of mammalian YAP for a long period of time in brain neurons after introduction of the mammalian YAP polynucleotide, and is non-pathogenic and highly safe.

[0025] An AAV vector containing a mammalian YAP polynucleotide can be produced in the nucleus by transfecting an AAV vector containing a mammalian YAP polynucleotide into HEK293 cells containing genes encoding AAV proteins required for AAV replication and AAV particle formation (rep gene, cap gene) and genes encoding proteins required for AAV proliferation (E2A gene, E4 gene, VARNA gene), and after freezing and thawing, the AAV vector can be recovered from the nucleus and separated and purified by methods such as density gradient centrifugation using cesium chloride or column method (see Li, XG et al. Mol Ther 13, 160-166 (2006); Matsushita, T. et al. Gene Ther 5: 938-945(1998); Urabe, M. et al. Mol Ther 13: 823-828(2006)).

[0026] In addition to the promoter, the vector may contain an enhancer, a polyA addition signal, a marker gene, a replication origin, a gene encoding a polypeptide that binds to the replication origin and controls replication, etc., if desired. The marker gene refers to a gene that enables cell selection or selection by introducing the marker gene into a cell. Specific examples of the marker gene include a drug resistance gene, a fluorescent protein gene, a luminescent enzyme gene, a chromogenic enzyme gene, etc. These may be used alone or in combination of two or more. Specific examples of the drug resistance gene include a neomycin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a zeocin resistance gene, a hygromycin resistance gene, etc. Specific examples of the fluorescent protein gene include a green fluorescent protein (GFP) gene, a yellow fluorescent protein (YFP) gene, a red fluorescent protein (RFP) gene, etc. Specific examples of the luminescent enzyme gene include a luciferase gene, etc. Specific examples of the chromogenic enzyme gene include a β-galactosidase gene, a β-glucuronidase gene, an alkaline phosphatase gene, etc.

[0027] The mammalian YAP polynucleotide may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA from the viewpoint of stability. In addition, the polynucleotide may be partially or entirely substituted with an artificial nucleotide such as PNA (polyamide nucleic acid), LNA (registered trademark, locked nucleic acid, bridged nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), GNA (glycerol nucleic acid), or TNA (threose nucleic acid). In addition, the mammalian YAP polynucleotide may be a single-stranded (sense strand) polynucleotide encoding mammalian YAP, or a double-stranded polynucleotide consisting of the sense strand and an antisense strand of its complementary sequence, but a double-stranded polynucleotide is preferred.

[0028] In the present specification, "at least 80% sequence identity with an amino acid sequence" means that the ratio of amino acids identical to the amino acid sequence to be compared is 80% or more, preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, even more preferably 93% or more, particularly preferably 95% or more, especially more preferably 98% or more, and most preferably 99% sequence identity. In addition, in the present specification, "at least 80% sequence identity with a nucleotide sequence" means that the ratio of nucleotides identical to the nucleotide sequence to be compared is 80% or more, preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, even more preferably 93% or more, particularly preferably 95% or more, especially more preferably 98% or more, and most preferably 99% sequence identity. The identity of amino acid sequences or nucleotide sequences can be determined using known programs such as ClustalW, GENETYX, and BLAST.

[0029] The mammalian YAP enhancer is not particularly limited as long as it is a substance (e.g., polypeptides such as antibodies; polynucleotides; sugars; lipids; organic compounds; inorganic compounds) that can increase the amount of mammalian YAP in the nuclei of brain neurons by promoting the migration of mammalian YAP into the nuclei of brain neurons, enhancing the expression of mammalian YAP in the nuclei of brain neurons, or the like. Examples of the mammalian YAP enhancer include lysophospholipids (e.g., lysophosphatidic acid [LPA], sphingosine 1 phosphate [S1P], lysophosphatidylserine [LPS], lysophosphatidylinositol [LPI], lysophosphatidylethanolamine [LPE]), S1P receptor agonists (e.g., KRP-203 [2-Amino-2-[2-[2-chloro-4-[[3-(phenylmethoxy)phenyl]thio]phenyl]ethyl]-1,3-propanediol, CS- Examples of such compounds include 0777 [1-[5-[(3R)-3-amino-4-hydroxy-3-methylbutyl]-1-methylpyrrol-2-yl]-4-(4-methylphenyl)butan-1-one], ponesimod [(2Z,5Z)-5-(3-chloro-4-((R)-2,3-dihydroxypropoxy)benzylidene)-2-(propyliMino)-3-(o-tolyl)thiazolidin-4-one], and siponimod [1-(4-[1-[(E)-4-Cyclohexyl-3-trifluoromethyl-benzyloxyimino]-ethyl]-2-ethyl-benzyl)-azetidine-3-carboxylic acid]), and since the effect of such compounds has been demonstrated in the present embodiment described later, S1P can be preferably exemplified.

[0030] The present preventive / therapeutic agent is administered to a subject who needs to prevent or treat mild cognitive impairment. The method of administering the present preventive / therapeutic agent to the subject can be appropriately selected in consideration of the form of the present YAP-related substance. For example, when the present YAP-related substance is a mammalian YAP or a mammalian YAP polynucleotide, parenteral administration (e.g., intravenous administration, intracerebrospinal fluid administration by lumbar puncture, etc.) can be mentioned, and when the present YAP-related substance is a mammalian YAP enhancer (preferably a low molecular weight substance), parenteral administration (e.g., intravenous administration, intracerebrospinal fluid administration by lumbar puncture, etc., subcutaneous administration, intramuscular administration, intranasal administration) and oral administration can be mentioned.

[0031] In the present invention, "mild cognitive impairment" means a state in which a decline in cognitive function (e.g., memory function, executive function, attention function, visuospatial cognitive function, language comprehension function) is observed but the diagnostic criteria for dementia are not met (in other words, the person is not suffering from dementia) and there is no impairment in basic daily life or social life; and / or a state in which the concentration of HMGB1 in cerebrospinal fluid is elevated compared to control individuals not suffering from mild cognitive impairment. Here, "cognitive decline is observed but the diagnostic criteria for dementia are not met" means, for example, a Mini Mental State Examination (MMSE) score within the range of 19-27 points (e.g., 20-27 points, 21-27 points, 22-27 points, 23-27 points, 24-27 points, 19-26 points, 19-25 points, 19-24 points, 19-23 points, 21-26 points, 21-25 points, 21-24 points, 21-23 points); and / or a Clinical Dementia Rating (CDR) score of 0.5 points.

[0032] 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.

[0033] In this specification, a "control subject not suffering from mild cognitive impairment" refers to a person not suffering from mild cognitive impairment (a control subject), that is, a person who satisfies at least one of criteria 1) to 3): 1) no decline in cognitive function; 2) meeting the diagnostic criteria for dementia; and 3) having difficulty in basic daily life or social life. Specifically, the control subject may include healthy individuals and patients suffering from diseases other than mild cognitive impairment (e.g., one or more diseases selected from Alzheimer's disease, conus medullaris syndrome, neuromyelitis optica, paraneoplastic syndrome, idiopathic normal pressure hydrocephalus, alcoholism, peripheral neuropathy, multiple sclerosis, systemic lupus erythematosus, lung cancer, and herpes zoster meningitis), and preferable examples thereof include healthy individuals and Alzheimer's disease patients.

[0034] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0035] 1. Materials and Methods [Preparation of CSF samples] Of the three types of subjects shown in Table 1 (those not suffering from either MCI or AD [non-MCI / AD control subjects], MCI patients, and AD patients), 30 non-MCI / AD control subjects (19 healthy subjects, 11 disease patients), 21 MCI patients, and 56 AD patients were given CSF samples by lumbar puncture, and then centrifuged (1000×g, 10 minutes, 4°C) to remove impurities, and the supernatant was stored at -80°C as a CSF sample. Whether or not a subject suffered from MCI or AD was diagnosed based on the criteria defined in the International Classification of Diseases, 10th Edition (ICD-10) by the World Health Organization. Note that subjects diagnosed as suffering from MCI are subjects who, although they have a decline in cognitive function, do not meet the diagnostic criteria for dementia and have no problems with basic daily life.

[0036] [Measurement of Aβ and pTau in CSF samples] The concentrations of two types of Aβ (Aβ40 and Aβ42) in the CSF samples were measured using an ELISA kit for detecting human Aβ40 (292-62301, Wako Pure Chemical Industries, Ltd.) and an ELISA kit for detecting human Aβ42 (298-62401, Wako Pure Chemical Industries, Ltd.). The pTau concentration in the CSF samples was measured using INNOTEST PHOSPHO-TAU(181P) (Innogenetics).

[0037] [Measurement of HMGB1 in CSF samples] Each well of a polystyrene microtiter plate (manufactured by Nunc) was coated with 100 μL of PBS containing 1 mg / L of anti-human HMGB1 monoclonal antibody (2D4, manufactured by Shino Test Co., Ltd.) and incubated overnight at 2 to 8 ° C. Then, each well was washed three times with PBS containing 0.05% Tween 20, blocked with 400 μL / well of PBS containing 1% BSA, washed three times with PBS containing 0.05% Tween 20, and then 100 μL of CSF sample was added to each well and incubated at 37 ° C. for 24 hours. Then, each well was washed three times with PBS containing 0.05% Tween 20, and 100 μL of PBS containing 0.5 mg / L of anti-human HMGB1, 2 monoclonal antibody conjugated with peroxidase (R04, manufactured by Shino Test Co., Ltd.) was added to each well and incubated at room temperature for 2 hours. Then, each well was washed three times with PBS containing 0.05% Tween 20, and TMB (3,3',5,5'-tetra-methyl benzidine) (manufactured by Dojindo Laboratories) was added to each well as a color-developing substrate. The TMB reaction was stopped by adding 0.35 M sodium sulfate to each well, and the absorbance at the maximum absorption wavelength of 450 nm was measured using a Model 680 microplate reader (manufactured by Bio-Rad). The standard curve for the absorbance was prepared using purified porcine thymus-derived HMGB1 (manufactured by Sino-Test).

[0038] [Preparation of brain tissue samples] Brain tissues were isolated from five non-MCI / AD control subjects, eight MCI patients, and eight AD patients among the three types of subjects shown in Table 1, and paraffin sections of the brain tissues were prepared according to standard methods. The use of human brain tissue samples from the above patients was approved by the Ethics Committee of Tokyo Medical and Dental University.

[0039] [Table 1] "Age," "MMSE score," and "CDR score" in the table are shown as mean values ​​± standard deviation (SD). Specifically, the 14 patients with diseases mentioned above were one patient with spinal conus syndrome, one patient with neuromyelitis optica, one patient with paraneoplastic syndrome, four patients with idiopathic normal pressure hydrocephalus, one patient with brain stem tumor, one patient with idiopathic normal pressure hydrocephalus and alcoholism, one patient with peripheral neuropathy, one patient with multiple sclerosis, one patient with systemic lupus erythematosus, one patient with lung cancer and idiopathic normal pressure hydrocephalus, and one patient with Herpes zoster meningitis.

[0040] [AD model mouse] 5×FAD transgenic B6 / SJL mice (hereinafter referred to as "5×FAD 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 reference "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 carrying human mutant APP (KM670 / 671NL Swedish mutation, E693G Arctic mutation, I716FBeyreuther / Iberian mutation) (see reference "Nat Neurosci 17, 661-663 (2014)"), were obtained from the RIKEN BioResource Research Center.

[0041] [Immunohistochemical staining of brain tissue] Brain tissues were excised from the subjects or AD model mice and fixed with a 4% PFA (paraformaldehyde) solution, and then paraffin sections 5 μm thick were prepared using a microtome (manufactured by Yamato Optical Instruments Co., Ltd.). Four types of primary antibodies (rabbit anti-pSer46-MARCKS antibody [Biologica, dilution ratio 1:2000], mouse anti-amyloid β antibody [clone 82E1, #10323, Immuno-Biological Laboratories, dilution ratio 1:5000 or 1:1000], rabbit anti-pSer909-LATS1 antibody [#9157, Cell Signaling Technology, dilution ratio 1:100], and rabbit anti-YAP antibody [sc-15407, Santa Cruz, dilution ratio 1:100]) and two types of secondary antibodies (Alexa488-labeled Donkey anti-mouse IgG [A-21202, Molecular Probes, dilution ratio 1:1000] and Alexa568-labeled Donkey anti-rabbit IgG [#A-10042, Molecular Probes, dilution ratio 1:1000]) were used. Immunohistochemical staining using 0.2 μg / mL DAPI (4',6-diamidino-2-phenylindole) was performed according to standard methods. Cell nuclei were stained in PBS containing 0.2 μg / mL DAPI (4',6-diamidino-2-phenylindole). Images of the fluorescent signals derived from various primary antibodies were obtained using a fluorescence microscope (IX70, Olympus) or a confocal laser scanning microscope (FV1200, Olympus). Specifically, YAP images, images of phosphorylation of serine 909 of LATS1 [pLATS1], images of the DAPI fluorescent signals derived from cell nuclei (DAPI images), and images of these images superimposed (Merge images) were obtained. In the DAPI images, cells with deformed and / or shrunken cell nuclei were evaluated as necrotic cells, and the number of necrotic cells in 30 fields of view (143 μm × 143 μm) was counted to calculate the number of necrotic cells per field of view (see the vertical axis of Figures 5A to C).

[0042] [In vivo imaging of ER and amyloid beta in brain neurons] The skull bone on the RSD surface of normal mice (non-transgenic B6 / SJL mice) or 5×FAD mice was removed using a high-speed microdrill. 1 μL of a solution containing 100 nM BTA1 (2-(4'-(methylamino)phenyl) benzothiazole, Sigma-Aldrich), an amyloid-binding substance, and 100 nM ER-Tracker Red (E34250, Thermo Fisher Scientific) was injected into the RSD in the presence of 1% isoflurane (anesthetic). After 4 hours, ER-Tracker Red was imaged using an upright microscope (BX61WI, Olympus), a water immersion objective lens (XLPlanN25xW, numerical aperture: 1.05), and a pulsed laser (MaiTaiHP DeepSee, Spectra-Physics) equipped with a scanning laser microscope system FV1000MPE2 (Olympus). The images showing the BTA1 fluorescent signal (BTA1 image) and the ER-Tracker Red image (E34250, Thermo Fisher Scientific) were recorded. An image in which the Red fluorescent signal was detected (ER-Tracker image) was obtained, and the volume of the ER-Tracker Red fluorescent signal (i.e., the volume of the ER) was measured.

[0043] [Construction of recombinant AAV] A recombinant AAV expressing rat YAPΔC-ins61 (GenBank: ABA33617) (i.e., a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1) (hereinafter, sometimes referred to as "AAV-YAPΔC-ins61") was produced by introducing a cDNA encoding rat YAPΔC-ins61 (GenBank: DQ186898) (i.e., a cDNA consisting of the nucleotide sequence of SEQ ID NO: 2) into an AAV vector, according to the method described in the literature "Li, XG et al. Mol Ther 13, 160-166 (2006)". The produced rAAV was purified by cesium chloride density gradient centrifugation, and the virus titer was measured using the quantitative PCR method. As a control, a recombinant AAV (hereinafter, sometimes referred to as "AAV-NINS") produced using an AAV vector not containing the above cDNA was produced.

[0044] [Administration of rAAV and sphingosine 1-phosphate (S1P) to the retrosplenial cortex] AAV-YAPΔC-ins61 or AAV-NINS (each with a titer of 1 × 10 10 The injection of S1P (40 nM, 0.15 μL / hr; Sigma-Aldrich) or control artificial cerebrospinal fluid (aCSF) (0.15 μL / hr; Alzet) into the mouse retrosplenial cortex (RSD) (-2.0 mm from bregma, 0.6 mm mediolateral, depth) and the injection of S1P (40 nM, 0.15 μL / hr; Sigma-Aldrich) into the mouse subarachnoid space were performed according to the method described in the literature "Yang, G. et al. Nat Protoc 5, 201-208 (2010)". The injection of AAV-YAPΔC-ins61 or AAV-NINS into mice was performed at 1 month or 5 months of age. The injection of S1P into mice was performed continuously from 1 to 6 months of age or from 5 to 6 months of age.

[0045] [Cognitive function analysis] Exploratory behavior of 6-month-old normal mice (non-transgenic B6 / SJL mice) and 5×FAD mice administered with AAV-YAPΔC-ins61, AAV-NINS, or S1P was tested using a Y-shaped maze (Ohara Medical Industries Co., Ltd.) in which three arms of the same size were connected at 120°C. Specifically, 2-month-old mice were placed in the corner of a certain arm and allowed to move freely around the Y-shaped maze for 8 minutes. The number of times the mouse moved from the previous arm to an arm different from the previous arm was divided by the number of times the mouse moved between arms was calculated as the rate of spontaneous alteration (alternation score, see Figure 14).

[0046] [Generation of human ADiPS cells] Human iPS cells with the KM670 / 671NL Swedish mutation (AD mutation) were produced using a CRISPR / Cas9 knockout system by Applied StemCell. Specifically, a plasmid containing DNA (DNA consisting of the nucleotide sequence of SEQ ID NO: 17) encoding a single-stranded guide (g)RNA, a plasmid expressing a Cas9 gene fused with a 2A peptide gene and a GFP gene, and a single-stranded donor DNA (single-stranded DNA consisting of the nucleotide sequence of SEQ ID NO: 18) for introducing the KM670 / 671NL Swedish mutation in human APP were transfected into human normal iPS cells (ASE-9203, Applied Stem Cell) by electroporation (1200V, 30ms, 1 pulse) using a Neon transfection system (Thermo Fisher Scientific). After culturing the cells in the presence of 0.4 μg / mL puromycin (selection drug) for 24 to 48 hours, the formed colonies (cell masses) were picked up, and GFP-positive colonies were transferred to a 96-well plate and cultured for 7 to 10 days. Human iPS cells with human APP KM670 / 671NL Swedish mutation in a heterozygous (AD / -) or homozygous (AD / AD) form (referred to as "human AD / -iPS cells" and "human AD / ADiPS cells", respectively, hereinafter collectively referred to as "human ADiPS cells") were subjected to PCR using a primer set (a forward primer consisting of the nucleotide sequence of SEQ ID NO: 19 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 20) and confirmed by Sanger sequencing.

[0047] 2.Results [HMGB1 concentrations in CSF samples from MCI patients compared with controls without MCI] The median HMGB1 concentration in CSF samples from MCI patients was 1548 pg / mL, which was higher than the median HMGB1 concentration in CSF samples from non-MCI / AD control subjects (229 pg / mL) and the median HMGB1 concentration in CSF samples from AD patients (327 pg / mL) (see Figure 1). Next, the non-MCI / AD control subjects were divided into two groups, healthy subjects and disease patients, and compared with the MCI patients. As a result, the median HMGB1 concentration in CSF samples from MCI patients (1548 pg / mL) was higher than the median HMGB1 concentration in CSF samples from healthy subjects (338 pg / mL) and the median HMGB1 concentration in CSF samples from disease patients (133 pg / mL) (see Figure 3).

[0048] These results indicate that the HMGB1 concentration in biological samples from MCI patients is higher than that in biological samples from individuals other than MCI patients (e.g., healthy individuals; dementia patients such as AD). The interquartile ranges in the box plots of "cc", "MCI", and "AD" in Figure 1 are 20-680.75 pg / mL, 205-2701 pg / mL, and 3-961.75 pg / mL, respectively. The interquartile ranges in the box plots of "nc", "dc", "MCI", and "AD" in Figure 3 are 56-796.5 pg / mL, 20-298 pg / mL, 205-2701 pg / mL, and 3-961.75 pg / mL, respectively.

[0049] Next, based on the measurement results of HMGB1 concentrations in CSF samples from non-MCI / AD control subjects (specifically, healthy subjects and disease patients), MCI patients, and AD patients, ROC curves for discriminating between MCI patients and non-MCI / AD control subjects or AD patients were created (see Figures 2 and 4), and cutoff values ​​were calculated. As a result, when 754 was set as the cutoff value for discriminating between MCI patients and non-MCI / AD control subjects, the sensitivity (proportion of test-positive subjects among MCI patients) and specificity (proportion of test-negative subjects among non-MCI / AD control subjects) for MCI patients were 86% and 80%, respectively. In addition, when 754 was set as the cutoff value for discriminating between MCI patients and AD patients, the sensitivity (proportion of test-positive subjects among MCI patients) and specificity (proportion of test-negative subjects among AD patients) for MCI patients were 86% and 70%, respectively. When the cutoff value for distinguishing between MCI patients and healthy subjects was set at 992, the sensitivity (proportion of MCI patients who tested positive) and specificity (proportion of healthy subjects who tested negative) for MCI patients were 76% and 84%, respectively.When the cutoff value for distinguishing between MCI patients and disease patients was set at 754, the sensitivity (proportion of MCI patients who tested positive) and specificity (proportion of disease patients who tested negative) for MCI patients were 86% and 91%, respectively.

[0050] These results indicate that by measuring the HMGB1 concentration in a biological sample from a subject and setting an appropriate cutoff value, it is possible to accurately determine whether or not a subject has MCI using the HMGB1 concentration as an index. The AUC (area under the curve) in the ROC curve for discriminating between MCI patients and non-MCI / AD control subjects (see Figure 2A) is 0.887, the AUC in the ROC curve for discriminating between MCI patients and AD patients (see Figure 2B) is 0.809, the AUC in the ROC curve for discriminating between MCI patients and healthy subjects (see Figure 4A) is 0.861, and the AUC in the ROC curve for discriminating between MCI patients and disease patients (see Figure 4B) is 0.931, indicating that the ability to discriminate between MCI patients and disease patients is high.

[0051] [AD model mice around 6 months of age reflect the state of MCI] HMGB1 is known to be one of the DAMPs released from necrotic cells (see Nature 418, 191-195 (2002) and Nat Rev Immunol 5, 331-342 (2005)). We then measured the number of necrotic cells in brain neurons from three types of subjects (non-MCI / AD patients, MCI patients, and AD patients). We found that necrotic cells were hardly detected in non-MCI / AD patients, whereas the number of necrotic cells increased in MCI patients (see Figure 5C). On the other hand, the number of necrotic cells in AD patients was decreased rather than that in MCI patients (see Figure 5C). These results indicate that necrosis occurs in the brain neurons of MCI patients.

[0052] Furthermore, when the number of necrotic cells was measured in the brain neurons of two types of AD model mice (5×FAD mice and APP-KI mice), it was shown that the number of necrotic cells increased in both mice from 1 to 6 months, but rather decreased after 6 months (see Figures 5A and B). Considering these results together with the human results described above, it is suggested that AD model mice around 6 months old reflect the state (symptoms) of MCI, AD model mice before 6 months old (specifically, 1-month-old AD model mice) reflect the state before the onset of MCI or the very early stage after the onset of MCI, and AD model mice over 6 months old (specifically, 12- to 18-month-old AD model mice) reflect the state (symptoms) of AD patients.

[0053] [MCI induces Hippo pathway-dependent necrosis in brain neurons] Next, to investigate necrotic cells in brain neurons in more detail, we performed live imaging analysis of ER and amyloid beta in neurons using ER-TrackerRed and BTA1 as indicators, respectively. As a result, in the brain neurons of normal control mice, BTA1 (i.e., amyloid beta) was not detected and the ER was normal, whereas in the brain neurons of 1-6 month old 5×FAD mice, amyloid beta was detected and ER expansion and instability were observed (see Figures 6 and 7).

[0054] It has been reported that ER expansion and instability are associated with transcriptional repression induced cell death (TRIAD), i.e., Hippo pathway-dependent necrosis (see J Cell Biol 172, 589-604 (2006)), CellDeath Dis 7, e2207 (2016), and HumMol Genet 25, 4749-4770 (2016)). Therefore, we analyzed the expression of YAP, a major factor of the Hippo pathway, in brain neurons from the above three types of subjects. As a result, YAP was detected in the nucleus of brain neurons from control non-MCI / AD disease patients, whereas the amount of YAP in the nucleus was reduced in brain neurons from AD patients, and furthermore, the amount of YAP in the nucleus was further reduced in brain neurons from MCI patients than in brain neurons from AD patients (see Figures 8 and 9A). In addition, YAP was shown to co-localize with amyloid beta in the cytoplasm in brain neurons derived from AD and MCI patients (see Figure 8A). Furthermore, since it is known that LATS1 is activated when serine 909 is phosphorylated and inhibits the nuclear translocation of YAP (see Genes Dev 30, 1-17 (2016)), the level of phosphorylated LATS1 (pLATS1) was analyzed. The pLATS1 level in brain neurons derived from AD patients was higher than that in brain neurons derived from non-MCI / AD disease patients, and the pLATS1 level was even higher in brain neurons derived from MCI patients than in brain neurons derived from AD patients (see Figure 9B). The decrease in the amount of YAP in the nucleus was also observed in two types of AD model mice (5×FAD mice and APP-KI mice) at 3 months (see Figures 9C and D). These results indicate that Hippo pathway-dependent necrosis (TRIAD) occurs in human and mouse brain neurons at the time of the onset of MCI.

[0055] [Nuclear YAP-increasing substances and YAP have the effect of suppressing Hippo pathway-dependent necrosis caused by MCI in brain neurons] S1P promotes YAP nuclear translocation by inhibiting phosphorylation of LATS1 via the S1P receptor. In addition, S1P, YAP and its isoforms are known to have the effect of suppressing Hippo pathway-dependent necrosis (see references "J Cell Biol 172, 589-604 (2006)", "CellDeath Dis 7, e2207 (2016)", and "HumMol Genet 25, 4749-4770 (2016)"). Therefore, we examined whether S1P and rat YAPΔC-ins61 can suppress Hippo pathway-dependent necrosis caused by MCI.

[0056] First, when S1P or AAV-YAPΔC-ins61 was administered to 5×FAD mice at 1 month of age (i.e., before the onset of MCI or at the very early stage after the onset of MCI), it was confirmed that the amount of YAP in the nucleus of brain neurons increased and recovered to the same level as in normal mice at 6 months of age (i.e., at the onset of MCI) compared to untreated 5×FAD mice (see Figure 10). Next, analysis of extracellular amyloid-β levels in brain tissue showed that 5×FAD mice administered S1P or AAV-YAPΔC-ins61 at 1 month of age had lower amyloid-β levels at 6 months of age compared to untreated 5×FAD mice (see Figure 11). Furthermore, analysis of ER in brain neurons showed that administration of S1P or AAV-YAPΔC-ins61 to 5×FAD mice at 1 month of age significantly alleviated ER dilation and instability at 6 months of age (see Figures 13 and 15), restoring the ER to levels comparable to that of normal mice (see Figures 12 and 14). These results indicate that nuclear YAP-increasing substances such as S1P and YAP such as AAV-YAPΔC-ins61 have the effect of suppressing Hippo pathway-dependent necrosis caused by MCI in brain neurons.

[0057] [Nuclear YAP-increasing substances and YAP have the effect of restoring cognitive function impaired by MCI] It is known that HMGB1 leaks from necrotic cells in brain neurons and phosphorylates the 46th serine (Ser46) in MARCKS (pSer46-MARCKS) (see SCIENTIFIC REPORT 25, 31895 (2016)). We analyzed the pSer46-MARCKS level in brain neurons and found that 5×FAD mice administered S1P or AAV-YAPΔC-ins61 at 1 month of age had reduced pSer46-MARCKS levels to the same level as normal mice at 6 months of age, compared to untreated 5×FAD mice (see Figure 16). These results indicate that S1P and AAV-YAPΔC-ins61 have the effect of suppressing an increase in the number of necrotic cells.

[0058] Furthermore, we analyzed the cognitive function of mice using a Y-maze with alternation scores as an index. First, we confirmed that the cognitive function of 5×FAD mice was lower than that of normal mice at 6 months of age (i.e., at the onset of MCI) (see Figure 17). Next, 5×FAD mice administered S1P or AAV-YAPΔC-ins61 at 1 month of age (i.e., before the onset of MCI or at the very early stage after the onset of MCI) showed cognitive function comparable to that of normal mice at 6 months of age compared to untreated 5×FAD mice (see Figures 17A and B). These results indicate that substances that increase YAP in the nucleus, such as S1P, and YAP, such as AAV-YAPΔC-ins61, have the effect of preventing cognitive decline due to MCI.

[0059] Furthermore, 5×FAD mice administered S1P or AAV-YAPΔC-ins61 at the age of 5 months (i.e., when MCI developed) showed cognitive function comparable to that of normal mice at the age of 6 months, compared with untreated 5×FAD mice (see Figures 17C and D). These results indicate that substances that increase YAP in the nucleus, such as S1P, and YAP, such as AAV-YAPΔC-ins61, have the effect of restoring cognitive function impaired by MCI. [Industrial Applicability]

[0060] The present invention contributes to the prevention or treatment of mild cognitive impairment, and the prevention of dementia (e.g., Alzheimer's disease) that progresses from mild cognitive impairment.

Claims

1. A preventive or therapeutic agent for mild cognitive impairment, comprising a mammalian YAP, a polynucleotide encoding a mammalian YAP, or a substance capable of increasing the amount of mammalian YAP in the nucleus of a brain neuron, the mammalian YAP is a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; The substance capable of increasing the amount of mammalian YAP in the nucleus of the brain nerve cell is sphingosine 1 phosphate. The preventive or therapeutic agent.

2. The preventive or therapeutic agent according to claim 1, characterized in that the mild cognitive impairment is a state in which the concentration of HMGB1 in cerebrospinal fluid is elevated compared to a control individual not suffering from mild cognitive impairment.

3. The method according to claim 2, wherein the control subject not suffering from mild cognitive impairment is a healthy subject or an Alzheimer's disease patient.

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