WO2018151135A1

WO2018151135A1 - Mental illness animal model and method for producing same

Info

Publication number: WO2018151135A1
Application number: PCT/JP2018/005019
Authority: WO
Inventors: 正啓岡; 哲嗣盛山; 悦啓米田; 洋一宮本; 聡辻井; 貴俊疋田; 真規子森田
Original assignee: 国立研究開発法人医薬基盤・健康・栄養研究所; 国立大学法人京都大学
Priority date: 2017-02-15
Filing date: 2018-02-14
Publication date: 2018-08-23
Also published as: JPWO2018151135A1; JP7012310B2

Abstract

The present invention relates to a method for producing a nonhuman mental illness animal model, wherein the method includes (a) a step for inducing a partial or complete loss of functionality of a karyopherin α (KPNA) 1 gene or homolog thereof in a nonhuman animal. The present invention also relates to a nonhuman mental illness animal model produced by this method, or to a progeny animal of this nonhuman mental illness animal model, and to a tissue or cell isolated from the animal. The present invention further relates to a method for screening, by using the animal, a drug for preventing or treating a mental illness; a method, by using the animal, for evaluating the efficacy and/or toxicity of a test substance for a mental illness; and a method for screening, by using the animal, a biomarker for mental illness.

Description

Psychiatric disease model animal and method for producing the same

The present invention relates to a mental disease model animal and a method for producing the same. The present invention also provides a method for screening a prophylactic or therapeutic agent for a mental illness using the animal model for mental illness, a method for evaluating the efficacy and / or harmfulness of the prophylactic or therapeutic agent, and a method for screening a biomarker for psychiatric disease. And so on.

In recent years, the number of mental illness patients in Japan has increased significantly, and in 2014 it was reported to be about 3.96 million. There are relatively many patients with schizophrenia, dementia, and depression. Schizophrenia is a frequent illness that takes nearly 1 in 100 people. The symptoms are classified into so-called “positive symptoms” such as delusions and hallucinations, and so-called “negative symptoms” such as expressionlessness, lethargy, decreased activity, attention deficit, speech dullness, cognitive impairment, and withdrawal. The cause of schizophrenia has not yet been elucidated. Dementia is a condition in which intellectual abilities such as memory, thought, understanding, calculation, learning, language, and judgment are impaired, and it impedes general life and social activities. Depression refers to a state in which depression continues, such as depression, which interferes with general life and social activities. Therefore, elucidation of the cause of psychiatric disorders such as schizophrenia, dementia, and depression, and establishment of effective treatment and prevention methods are desired. Some psychiatric disease model animals have been reported so far (Patent Documents 1 and 2). However, these psychiatric disorders are complex diseases that develop due to a combination of genetic factors and environmental factors such as stress, and a plurality of appropriate model animals are necessary for establishing effective treatments and prevention methods.

Drug addiction is one of the mental illnesses. When the effect of a drug is cut off, a strong desire (craving) for the drug is generated and the drug is used without controlling the craving. . Currently, abnormalities of the nervous system in the brain have been revealed as a cause of drug addiction. There are two types of drugs that cause drug dependence: drugs that cause only mental dependence and drugs that cause both mental and physical dependence. Alcohol, morphine, and heroin cause physical dependence as well as mental dependence. In contrast, nicotine, stimulants, and cocaine cause strong mental dependence but not physical dependence. Drug dependence is classified into three categories: drug abuse, drug dependence, and drug addiction. Representative examples of chronic poisoning include stimulant psychosis with hallucinations and delusions as the main symptom, and organic solvent psychosis characterized by “immobility syndrome”.

Caryopherin α (KPNA) is a nuclear transport factor and is also called importin α. So far, it has been reported that the expression of KPNA4 is decreased in the superior temporal gyrus of schizophrenic patients (Non-patent Document 1). It has also been reported that a single nucleotide polymorphism of KPNA4 is significantly seen in schizophrenic patients, and that the mutation is associated with a decrease in the KPNA4 gene (Non-patent Document 1). Similarly, it has been reported that the expression of KPNA3 tends to decrease in schizophrenic patients (Non-patent Document 1). In addition, it has been suggested that the single nucleotide polymorphism of KPNA3 is associated with schizophrenia, alcohol dependence, and opiate dependence (Non-Patent Documents 2 to 4). On the other hand, it has been reported that mice in which the KPNA1 gene is mutated do not exhibit abnormalities in the nervous system or abnormal behavior (Non-patent Document 5). In mice in which the KPNA1 gene was knocked out, abnormal development of female genital organs and abnormal gene regulation of estrogen receptor have been reported (Non-patent Document 6).

Japanese Patent No. 4106030 Japanese Patent No. 5277353

The problem to be solved by the present invention is to provide a model animal for mental disorders such as schizophrenia.

As a result of intensive studies to solve the above problems, the present inventors have found that a model animal for mental illness can be created by losing the function of the KPNA1 (importin α1) gene in the animal. Completed. The inventors have also found that a mental disease model animal can be created by applying stress to such an animal, and have completed the present invention.

That is, the present invention provides the following:
[1] A method for producing a non-human model animal of mental illness, comprising the step of (a) losing the function of all or part of the non-human animal caryopherin α (KPNA) 1 gene or a homologue thereof. Method;
[2] The method according to [1], further comprising the step of stressing the non-human animal obtained in step (b) (a);
[3] The method according to [2], wherein the stress is social isolation stress;
[4] The method according to [2], wherein the stress is drug stress;
[5] The method according to [4], wherein the drug is phencyclidine, dizocilpine (MK801), methamphetamine, amphetamine, cocaine, morphine, cannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC);
[6] Any of [1] to [5], wherein the mental illness is one or more diseases selected from schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, and depression A method according to claim 1;
[7] The method according to [4] or [5], wherein the mental illness is drug dependence;
[8] The method according to any one of [1] to [7], wherein step (a) is performed by knocking out the KPNA1 gene or a homolog thereof homologously;
[9] The method according to any one of [1] to [8], wherein the non-human animal is a non-human mammal;
[10] The non-human mammal is an animal selected from the group consisting of mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, goats, minipigs, pigs, sheep, cows, monkeys and chimpanzees. [9] The method described in;
[11] A non-human model animal of mental illness or a progeny thereof produced by the method according to any one of [1] to [10];
[12] A tissue or cell isolated from the animal according to [11];
[13] A screening method for a preventive or therapeutic agent for mental illness,
(A) administering a test substance to the animal according to [11],
(B) a method comprising imposing a behavioral task on the animal obtained in step (a), and (c) comparing the results of the behavioral task before and after administration of the test substance;
[14] A method for evaluating the efficacy and / or harmfulness of a test substance to mental illness,
(A) administering a test substance to the animal according to [11],
A method comprising: (b) imposing a behavioral task on the animal obtained in step (a); and (c) comparing the results of the behavioral task before and after administration of the test substance; and [15] mental illness A biomarker screening method for
(A) collecting a biological sample from each of the animal according to [11] and a wild-type animal of the same species as the animal,
(B) examining the nucleic acid, protein, metabolite, or lipid in the biological sample obtained in step (a) and comparing the transcription or expression pattern of the nucleic acid, protein, metabolite, or lipid in each animal; And (c) selecting a nucleic acid, protein, metabolite, or lipid that is indicative of mental illness based on the result of the comparison in step (b).

According to the present invention, a model animal for mental illness can be provided. In addition, according to a specific embodiment of the present invention, a model animal for mental illness whose onset is induced by stress can be provided.

FIG. 1 shows the result of a new object recognition test. Vertical axis: discrimination ratio (discrimination ratio,%), WT: wild type, Het: hetero knockout, KO: homo knockout. FIG. 2 shows the amount of self-issued movement for one hour in the open field test. Vertical axis: amount of action (total distance, cm / hour). A: Normal breeding (group housed), B: Isolated breeding (isolation housed). WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . FIG. 3 shows the result of the elevated plus maze test. Upper row: amount of action (total distance, cm / 15 minutes), middle row: stay time of open arm (Time in open arm,%), lower row: number of times to enter open arm (Entry to open arm). A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . FIG. 4 shows the results of the Y-shaped maze test. Upper row: amount of action (total distance, cm / 15 minutes), lower row: alternation (%). A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . FIG. 5 shows the results of the prepulse inhibition test. Upper row: startle response, lower row: prepulse inhibition (PPI,%). Lower horizontal axis: prepulse intensity (dB). A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . FIG. 6 shows the results of the passive avoidance test. Vertical axis: Time to enter the room that received the electric shock (latency to step through, seconds). A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . FIG. 7 shows the results of the forced swimming test. Vertical axis: immobility time (Immobility time, seconds). A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . FIG. 8 shows plasma corticosterone concentration (ng / ml). A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . FIG. 9-1 shows the monoamine concentration (% of WT) in the prefrontal cortex. A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . NE content: norepinephrine content, DOPAC content: 3,4-dihydroxyphenylacetic acid content, DA content: dopamine content. FIG. 9-2 shows the monoamine concentration (% of WT) in the prefrontal cortex. A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . HVA content: homovanillic acid content, 3-MT content: 3-methoxytyramine content, 5-HT content: serotonin content. FIG. 10-1 shows the monoamine concentration (% relative to WT) in the nucleus accumbens. A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . NE content: norepinephrine content, DOPAC content: 3,4-dihydroxyphenylacetic acid content, DA content: dopamine content. FIG. 10-2 shows the monoamine concentration (% relative to WT) in the nucleus accumbens. A: Normal breeding, B: Isolated breeding. WT: wild type, Het: hetero knockout, KO: homo knockout, isolation-WT: WT given social isolation stress, isolation-Het: Het given social isolation stress, isolation-KO: KO given social isolation stress . HVA content: homovanillic acid content, 3-MT content: 3-methoxytyramine content, 5-HT content: serotonin content. FIG. 11 shows the post-drug behavioral amount (total distance, cm / hour) on the seventh day of daily administration of physiological saline (saline) or phencyclidine hydrochloride (PCP). A: physiological saline administration group, B: PCP administration group. WT: wild type, Het: hetero knockout, KO: homo knockout, PCP-WT: WT given drug (PCP) stress, PCP-Het: Het given drug (PCP) stress, PCP-KO: drug (PCP) ) Stressed KO. FIG. 12 shows the results of an elevated plus maze test after daily administration of saline or PCP. Upper row: amount of action (total distance, cm / 15 minutes), middle row: stay time of open arm (Time in open arm,%), lower row: number of times to enter open arm (Entry to open arm). A: physiological saline administration group, B: PCP administration group. WT: wild type, Het: hetero knockout, KO: homo knockout, PCP-WT: WT given drug (PCP) stress, PCP-Het: Het given drug (PCP) stress, PCP-KO: drug (PCP) ) Stressed KO. FIG. 13 shows the results of a Y-shaped maze test after daily administration of physiological saline (saline) or phencyclidine hydrochloride (PCP). Upper row: amount of action (total distance, cm / 15 minutes), lower row: alternation (%). A: physiological saline administration group, B: PCP administration group. WT: wild type, Het: hetero knockout, KO: homo knockout, PCP-WT: WT given drug (PCP) stress, PCP-Het: Het given drug (PCP) stress, PCP-KO: drug (PCP) ) Stressed KO. FIG. 14 shows the results of a prepulse inhibition test after daily administration of physiological saline (saline) or phencyclidine hydrochloride (PCP). Upper row: startle response, lower row: prepulse inhibition (PPI,%). Lower horizontal axis: prepulse intensity (dB). A: physiological saline administration group, B: PCP administration group. WT: wild type, Het: hetero knockout, KO: homo knockout, PCP-WT: WT given drug (PCP) stress, PCP-Het: Het given drug (PCP) stress, PCP-KO: drug (PCP) ) Stressed KO. FIG. 15 shows the results of a passive avoidance test after daily administration of physiological saline (saline) or phencyclidine hydrochloride (PCP). Vertical axis: Time to enter the room that received the electric shock (latency to step through, seconds). A: physiological saline administration group, B: PCP administration group. WT: wild type, Het: hetero knockout, KO: homo knockout, PCP-WT: WT given drug (PCP) stress, PCP-Het: Het given drug (PCP) stress, PCP-KO: drug (PCP) ) Stressed KO.

In one embodiment, the present invention relates to a method for producing a non-human model animal of mental illness, comprising (a1) the function of all or part of the non-human animal caryopherin α (KPNA) 1 gene or a homologue thereof. Is provided (hereinafter, also referred to as “the method of the present invention”).

In this specification, “mental illness” refers to a disease caused by functional or organic disorders of the brain or heart. Mental illnesses include schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, depression, drug addiction and the like.

Caryoferrin alpha (KPNA) is widely found in various organisms such as mammals, birds, reptiles, amphibians and fish. In addition, it is known that there are 1 type of budding yeast, 3 types of nematodes and flies, 6 types of mice, and 7 types of humans. As shown in the following table, the abbreviations differ for each subtype between mouse and human.

The following table describes KPNA1 in other exemplary mammals.

In this specification, “homolog” refers to a group of highly similar genes having the same evolutionary origin. Homologs are classified into two types, “orthologs” and “paralogs”. An ortholog refers to a homolog that was the same gene at the time of species branching. Paralog refers to a homolog created by gene duplication.

Step (a1) may be performed by knocking out the KPNA1 gene or a homologous gene thereof. Gene knockout refers to deletion of a target gene by destroying a specific gene. Methods for knocking out a certain gene include, for example, a method of eliminating the expression of a functional protein by replacing the drug resistance gene with the whole or a part of the target gene using homologous recombination, or a site such as the Cre / Lox P system. Methods known in the art such as a method using a specific recombination reaction and a genome editing technology using TALEN or CRISPR-Cas9 can be used. For example, in the case of a knockout mouse, an ES cell having a target gene disruption is selected, a chimeric individual is prepared using the cell and a mouse embryo, and a cross of several generations is generated from a chimeric individual having germ cells derived from the ES cell. Will create an individual with the target gene disruption. In the case of livestock and the like, a knockout animal can be produced by a method that combines gene target disruption using somatic cells and a cloning technique. In the present invention, the “knockout” may be a homo knockout or a hetero knockout.

The knockout of the KPNA1 gene or its homologous gene may be carried out by dropping one or more exons of the gene. In this case, a part of an exon may be dropped off, or an entire exon may be dropped off. When dropping a plurality of exons, consecutive exons may be dropped, or separated exons may be dropped. One or more introns of the gene may be dropped together with one or more exons. In this case, a part of the intron may be dropped or the entire intron may be dropped. When knocking out the mouse KPNA1 gene, one or more exons selected from the first exon (exon 1) to the 14th exon (exon 14) may be dropped. For example, exon 2 in which the initiation codon of KPNA1 protein is present may be removed. Alternatively, exon 2 and exon 3 may be dropped.

Alternatively, step (a1) may be performed by knocking down the KPNA1 gene or its homologous gene. Gene knockdown refers to decreasing the transcription level of a specific gene or inhibiting translation from a specific gene. Unlike gene knockout, which destroys the gene itself, knockdown greatly diminishes the function of the gene but does not completely lose it. Methods for knocking down a gene include, for example, methods well known in the art, such as an antisense method for introducing RNA corresponding to the antisense strand of mRNA into a cell, and an RNAi method using siRNA, shRNA, microRNA, etc. Can be used. Knockdown efficiency can be confirmed by measuring changes in mRNA level or protein expression level. Methods for measuring mRNA levels include, but are not limited to, real-time quantitative PCR (qPCR) and Northern blotting. Examples of methods for measuring protein expression levels include, but are not limited to, Western blotting and ELISA. In the present invention, the knockdown efficiency is not particularly limited as long as it can be used as the mental disease model animal of the present invention by applying stress to the obtained knockdown animal (step (b1)). About 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95 %, About 96%, about 97%, about 98%, or about 99% efficiency.

The knockdown of the KPNA1 gene or its homologous gene may be performed by targeting one or more exons of the gene. In this case, a part of an exon may be targeted, or the entire exon may be targeted. When targeting multiple exons, successive exons may be targeted, or distant exons may be targeted. When knocking down the mouse KPNA1 gene, one or more exons selected from exons 1 to 14 may be targeted. For example, exon 2 may be targeted. Alternatively, exon 2 and exon 3 may be targeted.

In a particular embodiment, the method of the present invention further comprises the step of stressing the non-human animal obtained in step (b1) (a1).

In one embodiment, the stress applied in step (b1) is social isolation stress. In another embodiment, the stress imparted in step (b1) is drug stress. Examples of drugs include, but are not limited to, phencyclidine, dizocilpine (MK801), methamphetamine, amphetamine, cocaine, morphine, cannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC).

In another embodiment, the mental illness is one or more diseases selected from schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, and depression. In yet another embodiment, the stress applied in step (b1) is drug stress and the mental disorder is drug addiction.

In yet another embodiment, step (a1) is performed by knocking out the KPNA1 gene or a homolog thereof homologously.

As the “non-human animal” used in the present specification, a non-human animal having the KPNA1 gene or a homologue thereof can be used. In one embodiment, the non-human animal is a non-human mammal. Examples of non-human mammals include mice, rats, hamsters, guinea pigs, rabbits, ferrets, dogs, cats, goats, minipigs, pigs, sheep, cows, yaks, horses, donkeys, alpaca, common marmosets, monkeys, chimpanzees, Non-limiting examples include bonobos, orangutans, and gorillas. In certain embodiments, the non-human mammal is an animal selected from the group consisting of a mouse, rat, hamster, guinea pig, rabbit, dog, cat, goat, minipig, pig, sheep, cow, monkey and chimpanzee.

In another embodiment, the present invention is also referred to as a “non-human model animal of the mental illness or a progeny animal thereof” produced by the method of the present invention (hereinafter, “non-human model animal of the present invention or a progeny animal thereof”). )I will provide a. “Progeny animal” refers to a progeny animal obtained by further mating the non-human model animal of the present invention. The mating may be a mating between the non-human model animals of the present invention or a mating with an animal other than the non-human model animals of the present invention.

As will be described later (see the Examples section), in psychiatric disease model mice produced by the method of the present invention, plasma corticosterone concentration increased, cerebral prefrontal cortex 5-HT concentration decreased, And / or a decrease in the nucleus accumbens 5-HT concentration. Accordingly, in another embodiment, the present invention relates to (i) an increase in plasma corticosterone concentration, (ii) a decrease in pre-frontal cortex 5-HT concentration, and (iii) 5-HT in the nucleus accumbens. Non-human animals or their progeny animals that exhibit one or more of the conditions selected from the group consisting of decreasing concentrations are provided.

In another embodiment, the present invention provides a tissue or cell isolated from the above-described non-human model animal of the present invention or a progeny animal thereof. “Tissue” means any tissue obtained from the non-human model animals of the present invention or their progeny animals. For example, epithelial tissues (such as the epithelium, glandular epithelium, absorptive epithelium, sensory epithelium, and respiratory epithelium), connective tissues (such as the dermis, subcutaneous tissue, submucosa, periosteum, fascia, tendon, and vascular outer membrane) , Cartilage tissue, bone tissue, blood, lymph, muscle tissue (such as skeletal muscle tissue, smooth muscle tissue, and myocardial tissue), nerve tissue, and embryonic tissue, but are not limited thereto. “Cell” means any cell obtained from a non-human model animal of the present invention or a progeny animal thereof. Examples include epithelial cells, endothelial cells, fibroblasts, bone cells, muscle cells, nerve cells, blood cells, immune cells, adipocytes, mast cells, pigment cells, germ cells, progenitor cells, and stem cells. It is not limited to.

In yet another embodiment, the present invention provides a method for screening a preventive or therapeutic agent for mental illness, comprising the steps of (a2) administering a test substance to the non-human model animal of the present invention or a progeny animal thereof (b2 And (c2) comparing the results of the behavioral task before and after the administration of the test substance.

In yet another embodiment, the present invention relates to a method for evaluating the efficacy and / or harmfulness of a test substance on mental illness, comprising: (a3) a test substance in a non-human model animal of the present invention or a progeny animal thereof. (B3) A step of imposing a behavioral task on the animal obtained in step (a3), and (c3) comparing the results of the behavioral task before and after the administration of the test substance is provided. .

Administration of the test substance in steps (a2) and (a3) is not particularly limited, and the test substance can be administered orally or parenterally. Parenteral routes of administration include, but are not limited to, nasal, ocular, otic, intravenous, intraarterial, ventricular, intraperitoneal, intramuscular, intradermal, and subcutaneous routes. The dose of the test substance can be appropriately set according to the type of active ingredient, the size of the molecule, the route of administration, the type of animal to be administered, the drug acceptability of the administration subject, body weight, age, and the like.

“Behavioral tasks” in steps (b2) and (b3) include open field test, elevated plus maze test, light / dark selection test, Y-shaped maze test, T-shaped maze test, new object recognition test, prepulse inhibition (PPI) test, latent inhibition test, passive avoidance test, forced swimming test, tail suspension test, but are not limited thereto. Conditions and means known in the art are used as specific conditions and means for these tests. For example, Proc Natl Acad Sci US A. 2007 Sep 4: 104 (36): 14501-6. Examples include the conditions and means described in the section of the embodiment.

In yet another embodiment, the present invention provides a method for screening a biomarker for mental illness, comprising: (a4) a non-human model animal of the present invention or a progeny animal thereof, and a wild-type animal of the same species as the animal. (B4) Step (b4) The nucleic acid, protein, metabolite, or lipid in the biological sample obtained in step (a4) is examined, and transcription of the nucleic acid, protein, metabolite, or lipid in each animal Alternatively, a method is provided comprising the steps of comparing expression patterns, and (c4) selecting nucleic acids, proteins, metabolites, or lipids that are indicative of mental illness based on the results of the comparison in step (b4).

Examples of the “biological sample” include cells, tissues (normal tissues and diseased tissues), whole blood, plasma, serum, lymph, cerebrospinal fluid, pleural effusion, ascites, gastric fluid, bile, pancreatic juice, intestinal fluid, joint fluid, tears, Examples include but are not limited to body fluids such as aqueous humor, saliva, sputum, nasal discharge, sweat, amniotic fluid, milk, and urine, and feces. The biological sample may be prepared into a state suitable for use in the above method using, for example, a buffer. The biological sample may be used fresh or may be used after a freezing treatment or a formalin fixing treatment. Alternatively, a biological sample stored at an appropriate temperature after collection may be used. As a method for measuring the transcription or expression pattern of nucleic acids, proteins, metabolites, or lipids, methods known in the art can be used. Examples include, but are not limited to, real-time quantitative PCR (qPCR), Northern blotting, Western blotting, and ELISA.

The present invention further provides the following:
[1 ′] A method for causing symptoms and / or disorders related to mental illness in a non-human animal, comprising: (a ′) all or part of the cariopherin α (KPNA) 1 gene of the non-human animal or a homolog thereof A method comprising the step of losing the function of
[2 ′] (b ′) The method according to [1 ′], further comprising applying stress to the non-human animal obtained in step (a ′);
[3 ′] The method according to [2 ′], wherein the stress is social isolation stress;
[4 ′] The method according to [2 ′], wherein the stress is drug stress;
[5 ′] The method according to [4 ′], wherein the drug is phencyclidine, dizocilpine (MK801), methamphetamine, amphetamine, cocaine, morphine, cannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC);
[6 ′] The mental illness is one or more diseases selected from schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, and depression, [1 ′] to [5 ′ ] The method according to any one of
[7 ′] The method according to [4 ′] or [5 ′], wherein the mental illness is drug dependence;
[8 ′] The method according to any one of [1 ′] to [7 ′], wherein step (a ′) is performed by knocking out the KPNA1 gene or a homologue thereof homologously;
[9 ′] The method according to any one of [1 ′] to [8 ′], wherein the non-human animal is a non-human mammal;
[10 ′] The non-human mammal is an animal selected from the group consisting of mouse, rat, hamster, guinea pig, rabbit, dog, cat, goat, minipig, pig, sheep, cow, monkey and chimpanzee. '] Method;
[11 ′] A non-human model animal of mental illness or a progeny thereof produced by the method according to any one of [1 ′] to [10 ′];
[12 ′] Tissue or cell isolated from the animal according to [11 ′];
[13 ′] A screening method for a preventive or therapeutic agent for mental illness,
(A ′) administering a test substance to the animal according to [11 ′],
A method comprising: (b ′) imposing a behavioral task on the animal obtained in step (a ′); and (c ′) comparing the results of the behavioral task before and after administration of the test substance;
[14 ′] A method for evaluating the efficacy and / or harmfulness of a test substance to mental illness,
(A ′) administering a test substance to the animal according to [11 ′],
A method comprising: (b ′) imposing a behavioral task on the animal obtained in step (a ′); and (c ′) comparing the results of the behavioral task before and after administration of the test substance; and [15 '] A screening method for biomarkers of mental illness,
(A ′) collecting a biological sample from each of the animal according to [11 ′] and a wild-type animal of the same species as the animal,
(B ′) Examine the nucleic acid, protein, metabolite, or lipid in the biological sample obtained in step (a ′) and compare the transcription or expression pattern of the nucleic acid, protein, metabolite, or lipid in each animal And (c ′) selecting a nucleic acid, protein, metabolite, or lipid that is indicative of mental illness based on the results of the comparison in step (b ′).

As used herein, “symptoms and / or disorders associated with mental disorders” refers to mental disorders (eg, schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, depression, drug dependence) ) Refers to the abnormal behavior or condition seen in Examples of such symptoms and / or disorders in non-human animals include decreased self-issued dynamics, restlessness, increased impulsivity, increased immobility time in forced swimming tests, decreased recognition of new objects, sensorimotor integration Examples include, but are not limited to, disability and disability learning.

The present invention still further provides the following:
[16 ′] a non-human animal model of mental illness that has lost the function of all or part of the karyopherin α (KPNA) 1 gene or a homologue thereof;
[17 ′] The animal according to [16 ′], which is stressed;
[18 ′] The animal according to [17 ′], wherein the stress is social isolation stress;
[19 ′] The animal according to [17 ′], wherein the stress is drug stress;
[20 ′] The animal according to [19 ′], wherein the drug is phencyclidine, dizocilpine (MK801), methamphetamine, amphetamine, cocaine, morphine, cannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC);
[21 ′] The mental illness is one or more diseases selected from schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, and depression [16 ′] to [20 ′ ] The animal according to any one of
[22 ′] The animal according to [19 ′] or [20 ′], wherein the mental illness is drug dependence;
[23 ′] The animal according to any one of [16 ′] to [22 ′], wherein the KPNA1 gene or a homologue thereof is homozygously knocked out;
[24 ′] The animal according to any one of [16 ′] to [23 ′], which is a non-human mammal;
[25 ′] The animal according to [24 ′], which is an animal selected from the group consisting of a mouse, rat, hamster, guinea pig, rabbit, dog, cat, goat, minipig, pig, sheep, cow, monkey and chimpanzee ;
[26 ′] The progeny animal of the animal according to any one of [16 ′] to [25 ′];
[27 ′] Tissue or cell isolated from the animal according to any one of [16 ′] to [26 ′];
[28 ′] A screening method for a preventive or therapeutic agent for mental illness,
(A ′) administering a test substance to the animal according to any one of [16 ′] to [26 ′],
A method comprising: (b ′) imposing a behavioral task on the animal obtained in step (a ′); and (c ′) comparing the results of the behavioral task before and after administration of the test substance;
[29 ′] A method for evaluating the efficacy and / or harmfulness of a test substance to mental illness,
(A ′) administering a test substance to the animal according to any one of [16 ′] to [26 ′],
A method comprising: (b ′) imposing a behavioral task on the animal obtained in step (a ′); and (c ′) comparing the results of the behavioral task before and after administration of the test substance; and [30 '] A screening method for biomarkers of mental illness,
(A ′) collecting a biological sample from each of the animal according to any one of [16 ′] to [26 ′] and a wild-type animal of the same species as the animal,
(B ′) Examine the nucleic acid, protein, metabolite, or lipid in the biological sample obtained in step (a ′) and compare the transcription or expression pattern of the nucleic acid, protein, metabolite, or lipid in each animal And (c ′) selecting a nucleic acid, protein, metabolite, or lipid that is indicative of mental illness based on the results of the comparison in step (b ′).

The present invention still further provides the following:
[31 ′] Use of a non-human animal that has lost the function of all or part of the caryopherin α (KPNA) 1 gene or a homolog thereof as a non-human model animal for mental illness;
[32 ′] Use of a non-human animal that has lost the function of all or part of the caryopherin α (KPNA) 1 gene or a homolog thereof for the manufacture of a prophylactic or therapeutic drug for mental illness;
[33 ′] Use of a non-human animal that has lost the function of all or part of the caryopherin alpha (KPNA) 1 gene or a homolog thereof for screening for preventive or therapeutic agents for mental illness;
[34 ′] A non-human animal that has lost the function of all or part of the caryopherin α (KPNA) 1 gene or a homolog thereof for evaluating the efficacy and / or harmfulness of a test substance on mental illness use;
[35 ′] use of a non-human animal that has lost the function of all or part of the caryopherin alpha (KPNA) 1 gene or a homolog thereof for screening for biomarkers of mental illness;
[36 ′] The use according to any one of [31 ′] to [35 ′], wherein the non-human animal is stressed;
[37 ′] Use according to [36 ′], wherein the stress is social isolation stress;
[38 ′] Use according to [36 ′], wherein the stress is drug stress;
[39 ′] The use according to [38 ′], wherein the drug is phencyclidine, dizocilpine (MK801), methamphetamine, amphetamine, cocaine, morphine, cannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC);
[40 ′] The mental illness is one or more diseases selected from schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, and depression [31 ′] to [39 ′ ] Use according to any one of
[41 ′] Use according to [38 ′] or [39 ′], wherein the mental illness is drug dependence;
[42 ′] The use according to any one of [31 ′] to [41 ′], wherein the KPNA1 gene or a homolog thereof is homo knocked out in a non-human animal;
[43 ′] The use according to any one of [31 ′] to [42 ′], wherein the non-human animal is a non-human mammal; and [44 ′] the non-human mammal is a mouse, rat, hamster The use according to [43 ′], which is an animal selected from the group consisting of guinea pig, rabbit, dog, cat, goat, minipig, pig, sheep, cow, monkey and chimpanzee.

It should be understood that the terms used in this specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. “About” is typically ± 10%, more typically ± 5%, more typically ± 4%, more typically ± 3% of the numerical value to which the term is attached. It typically means a value in the range of ± 2%, and even more typically ± 1%.

Hereinafter, the present invention will be described in more detail and specifically with reference to examples. However, the examples do not limit the scope of the present invention.

1. Study Materials Kpna1-Het mice were prepared by the following method.
The mouse KPNA1 gene is on chromosome 16 and is composed of 14 exons. And the start codon of KPNA1 protein exists in the second exon. Accordingly, the second exon and the third exon were removed using the Cre / loxP system to obtain a mouse deficient in expression of the KPNA1 protein.

Specifically, first, a region consisting of the second and third exons of the mouse KPNA1 gene genomic region sandwiched between loxP recombination sequences, and a selection marker cassette (FRT recombination sequences are arranged on both sides). A targeting vector (SEQ ID NO: 4) having a neomycin resistance gene). Next, the prepared targeting vector was introduced into ES cells by electroporation. The ES cells after gene introduction were cultured in the presence of G418. Thereafter, screening of ES cells in which DNA recombination occurred as expected was performed by PCR and Southern blotting. The ES cells thus obtained were introduced into a mouse fertilized egg, returned to the mouse body, and implanted to obtain a chimeric mouse. Furthermore, the chimeric mouse and C57BL / 6J mouse were mated to obtain a transgenic mouse in which the region composed of the second exon and the third exon of the mouse KPNA1 gene was sandwiched between loxP recombination sequences. Next, the obtained transgenic mice were crossed with CAG-FLPe mice (transgenic mice expressing the recombinant enzyme FLPe systemically). As a result, the selection marker cassette sandwiched between the FRT recombination sequences was removed from the mouse genome. Subsequently, the obtained transgenic mice were crossed with CAG-Cre mice (transgenic mice expressing the recombinant enzyme Cre systemically). The resulting transgenic mouse was named Kpna1-Het mouse. A Kpna1-Het (NE10) mouse having a gene background of C57BL / 6J was obtained by crossing Kpna1-Het with C57BL / 6J for 10 generations or more. By mating Kpna1-Het (NE10) mice, sibling knockout mice (KO), hetero mice (Het), and wild type mice (WT) were obtained from their offspring.

Genotypes were confirmed by performing PCR on genomic DNA purified from a part of the mouse tail tissue using the primers shown in the following table. PCR was performed according to a conventional method.

After the PCR reaction, electrophoresis was performed on a 1% agarose gel. Those in which only a 622 bp PCR product was confirmed were determined to be wild type mice (WT). Those in which two PCR products of 622 bp and 420 bp were confirmed were judged as Kpna1-Het mice (Het). Those in which only the 420 bp PCR product was confirmed were determined to be knockout mice (KO). All experiments used siblings male KO, Het, and WT. The use of transgenic mice was approved by the Kyoto University Recombinant DNA Experiment Safety Committee and complied with the Kyoto University Recombinant DNA Experiment Safety Management Regulations.

1-1. Method of applying social isolation stress From the 5th to 8th week of life, mice continue to be reared in normal cages (21 x 32 x 13 cm) and small cages (12.5 x 20 x 11 cm) White paper was wrapped around each and divided into groups for rearing.

1-2. Method of applying drug stress To mice subjected to drug stress, phencyclidine hydrochloride [1- (1-phenylcyclohexane) piperidine hydrochloride: PCP] (10 mg / kg, 10 mL / kg, subcutaneous administration) was applied from 5 to 6 weeks after birth. It was administered once daily by subcutaneous injection for 7 days. In the control group, physiological saline was administered in the same schedule instead of PCP. On the seventh day, an open field test was conducted.

2. Behavioral experiments From the age of 8 weeks of age, new object recognition tests, open field tests, elevated plus maze tests, Y-shaped maze tests, prepulse inhibition (PPI) tests at the Animal Innovation Center Animal Facility, Kyoto University Graduate School of Medicine A passive avoidance test and a forced swimming test were conducted. All behavioral experiments were approved by the Kyoto University Animal Experiment Committee and followed the rules for conducting animal experiments at Kyoto University.

2-1. New object recognition test A box of width 40 cm x depth 40 cm x height 27 cm was used. Mice were habituated and acclimatized for 3 minutes in an empty box for 3 consecutive days. On the fourth day, two objects of the same shape, color and size were first placed in the box and trained for 5 minutes. Immediately after the training was finished, it was returned to the home cage. After 15 minutes, the mouse was placed in a box in which one of the two objects was replaced with a different new object and allowed to search for 5 minutes (retention). A video was taken of the training and retention for 5 minutes each. The behaviors taken for 5 minutes were followed and analyzed with EthoVision XT 8.5. The difference in time for searching for two objects was determined.

2-2. Open field test A mouse was placed in the center of a box 40 cm wide x 40 cm deep x 27 cm high. A 60-minute video was recorded immediately afterwards. The total moving distance (cm / 1 hour) for 60 minutes was analyzed with a video analysis system EthoVision XT 8.5 (Noldus), and the self-issued movement amount in a new environment was measured.

2-3. Elevated cross maze test Four arms measuring 30 cm in length and 7 cm in width were placed as a cross maze at a height of 30 cm from the floor. Of the four arms, two were open arms without walls and two were closed arms with walls 20 cm high. A mouse was placed in the center of this elevated cross maze. The video of the action for 15 minutes was recorded immediately after. 15 minutes of behavior in the cross maze was analyzed with EthoVision XT 8.5, total distance traveled (total distance, cm / 15 min), staying in open arm within the total time the mouse stayed in closed arm and open arm The time taken (Time in open arm,%) and the number of times the player entered the open arm (Entry to open arm) were measured.

2-4. Y-shaped maze test A Y-shaped maze in which three arms were 42 cm long, 15 cm deep, and the angle between the arms was 120 ° was installed. A mouse was placed in the center of the Y-shaped maze. The video of the action for 15 minutes was recorded immediately after. The captured 15-minute behavior was tracked and analyzed with EthoVision XT 8.5. The total travel distance (cm / 15 minutes) was measured. In addition, the number of times that the mouse entered all the arms and the number of times that the mouse entered three different arms (number of replacement actions) were counted, and the alternation (%) was calculated. Alternation (%) is represented by the following formula:
[Formula 1]
alternation (%) = number of alternations / (total number of intrusions -2) x 100

2-5. Prepulse Inhibition Test The startle response to sound stimuli was measured by The SR-LAB ™ Startle Response System (San Diego Instruments). The target animal was placed in an acrylic tube and conditioned for 30 minutes with 70 dB white noise. Then, after 40 milliseconds of white noise, one of six types of pre-pulses (white noise only, 74 dB, 78 dB, 82 dB, 86 dB, 90 dB) was given for 20 milliseconds. Subsequently, after a silence period of 100 milliseconds, a startle sound stimulus of 120 dB was given for 40 milliseconds, and the startle response at the startle sound was measured. Each combination of prepulse and startle sound stimulus was randomly presented 7 times each. The interval time after the startle sound stimulation was randomly given between 25 milliseconds and 60 milliseconds. Prepulse inhibition (PPI,%) was calculated from the measured data. PPI (%) is represented by the following formula:
[Formula 2]
{1- (magnitude of startle response with prepulse) / (magnitude of startle response without prepulse)} × 100

2-6. Passive avoidance test A device in which a bright room brightened by lighting and a dark room with a floor provided with a grid connected to an electric shock generator was connected. On the first day, the mouse was placed in the light room, and the time until it entered the dark room was measured. After entering the dark room, the door between the bright room and the dark room was closed, and an electric shock of 0.3 mA, 60 Hz was applied for 1 second. Then, it returned to the home cage. After 24 hours, it was put into a bright room and the time until entering the dark room was measured.

2-7. Forced swimming test Water at room temperature was put in a cylinder having a diameter of 13.5 cm and a height of 20 cm to a height of 14 cm, and a mouse was placed in the center. The video of the action for 6 minutes was recorded immediately after. The immobility time (second) for 6 minutes was measured with a stopwatch, and the immobility time in the underwater environment was measured.

3. Measurement of plasma corticosterone concentration Blood was collected from a mouse sufficiently anesthetized with isoflurane, and the corticosterone concentration in plasma was measured using an EIA kit (Cayman Chemical Company).

4). Measurement of the amount of monoamine in the brain The brain was quickly removed from a mouse sufficiently anesthetized with isoflurane, and a 1 mm thick coronal slice was prepared. The prefrontal cortex and nucleus accumbens were separated from the slice and stored frozen. The frozen sample was placed in a 0.2 M perchloric acid solution containing isoproterenol as an internal standard, and crushed in ice using an ultrasonic homogenizer (TAITEC). After homogenization for 30 minutes, the resulting solution was subjected to centrifugation at 20,000 g for 15 minutes at 4 ° C. The supernatant was filtered through 0.45 μm Cosmospin filter H (Nacalai Tesque). By high performance liquid chromatography, 3-methoxy-4-hydroxyphenyl glycol (MHPG), norepinephrine (NE), pinephrine (Epi), 3,4-dihydroxyphenylacetic acid (DOPAC), normetanephrine (NM), dopamine (DA) , 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 3-methoxytyramine (3-MT), and 5-hydroxytryptamine (5-HT) were measured.

5). Statistical processing All statistical processing was performed using Graph pad Prism 6.0 (Graph pad software). A t-test was performed as a 2-group comparison. For others, ANOVA analysis was performed, and significant differences were tested by Bonferroni multiple comparison.

6). Result The result of the test mentioned above is shown below.

6-1. The results of the new object recognition test are shown in FIG. In the WT that was normally bred, there was a significant difference in the discrimination rate between the two objects in Training and Retention. This indicates that a normally raised WT can recognize a new object. **, p <0.01. On the other hand, between Het and KO, no significant difference was found in the discrimination rate between the two objects in Training and Retention (ns: not significant). This indicates that normally raised Het and KO cannot recognize a new object, that is, the cognitive function is lowered. WT, n = 9; Het, n = 10; KO, n = 11. That is, Het and KO mice can be used as behavioral models for learning disabilities and dementia even in a state where no stress is applied.

6-2. The open field test results are shown in FIG. There was no difference in the amount of self-issued WT, Het, and KO reared normally in the new environment (A). One way ANOVA p = 0.8419. WT, n = 9; Het, n = 10; KO, n = 11. KO mice subjected to social isolation stress (isolation-KO) showed a significant decrease in spontaneous movement (B). One way ANOVA p = 0.0004. *, P <0.05. WT, n = 10; Het, n = 14; KO, n = 9.

6-3. The results of the elevated plus maze test are shown in FIG. WT, Het, and KO bred normally did not differ in the amount of behavior in the elevated plus maze test, the stay time of the open arm, and the number of times they entered the open arm (A). WT, n = 9; Het, n = 10; KO, n = 11. KO mice subjected to social isolation stress (isolation-KO) significantly increased the amount of behavior in the elevated plus maze test (B upper row, One way ANOVA p = 0.0383). *, P <0.05. Moreover, when social isolation stress was given, the stay time (%) of open arm increased significantly in the order of WT, Het, and KO (B middle stage, One way ANOVA p = 0.0003). Furthermore, when social isolation stress was applied, the number of times the arm entered the open arm increased in the order of WT, Het, and KO (lower B, One way ANOVA p <0.0001). *, P <0.05, ***, p <0.001, ***, p <0.0001. WT, n = 10; Het, n = 14; KO, n = 9. This indicates that the Het mice (isolation-Het) and KO mice (isolation-KO) subjected to social isolation stress have increased restlessness and impulsivity in the maze test. That is, Het mice and KO mice subjected to social isolation stress can be used as behavior models for schizophrenia, bipolar disorder, and hyperactivity disorder.

6-4. The results of the Y-shaped maze test are shown in FIG. In WT, Het, and KO bred normally, there was no difference in the amount of behavior and alternation (%) in the Y-shaped maze test (A). WT, n = 9; Het, n = 10; KO, n = 11. KO mice subjected to social isolation stress (isolation-KO) significantly increased the amount of behavior in the Y-shaped maze test (B upper row, One way ANOVA p = 0.0347). *, P <0.05. There was no difference in alternation (%) (lower B, One way ANOVA p = 0.7173). WT, n = 10; Het, n = 14; KO, n = 9. This shows that there is no difference in short-term memory in KO mice (isolation-KO) subjected to social isolation stress, but there is no calmness in the maze test.

6-5. The results of the prepulse inhibition test are shown in FIG. There was no difference in startle response and PPI (%) in the prepulse inhibition test in WT, Het, and KO bred normally. WT, n = 9; Het, n = 10; KO, n = 11. There was no difference in startle response in the prepulse inhibition test in KO mice subjected to social isolation stress (B upper row, One way ANOVA p = 0.8297). On the other hand, when social isolation stress was applied, the PPI (%) decreased in the order of WT, Het, and KO (lower B, Two way ANOVA p = 0.007). *, P <0.05. WT, n = 10; Het, n = 14; KO, n = 9. This indicates that there is a disturbance of sensorimotor integration in Het mice (isolation-Het) and KO mice (isolation-KO) subjected to social isolation stress. That is, Het mice (isolation-Het) and KO mice (isolation-KO) subjected to social isolation stress can be used as schizophrenia models.

6-6. The results of the passive avoidance test are shown in FIG. WT, Het, and KO bred normally had significantly increased time (vertical axis) until they entered the room that received the electric shock (Day 2) the day after receiving the electric shock (Day 2). ***, p <0.0001. This indicates that repellent learning is possible. WT, n = 10; Het, n = 6; KO, n = 9. WT (isolation-WT) and Het (isolation-Het) that applied social isolation stress significantly increased the time (vertical axis) to enter the room that received the electric shock the day after receiving the electric shock (Day 2). Increased (B). **, p <0.01, ***, p <0.0001. On the other hand, no significant difference was observed in KO (isolation-KO) applied with social isolation stress (ns: not significant), and obstacles were observed in repelling learning (B). WT, n = 10; Het, n = 14; KO, n = 9. That is, KO mice subjected to social isolation stress (isolation-KO) can be used as behavioral models for schizophrenia, learning disorders, and dementia.

6-7. The results of the forced swimming test are shown in FIG. There was no difference in the immobility time in the forced swimming test in WT, Het, and KO bred normally (A). WT, n = 11; Het, n = 10; KO, n = 9. KO mice subjected to social isolation stress (isolation-KO) showed a significant increase in immobility time in the forced swimming test (B). One way ANOVA p = 0.0003. *, P <0.05, **, p <0.01. WT, n = 10; Het, n = 14; KO, n = 9. This indicates that KO mice subjected to social isolation stress (isolation-KO) have a strong tendency to depression. That is, KO mice subjected to social isolation stress (isolation-KO) can be used as behavioral models for depression and schizophrenia.

6-8. Plasma corticosterone concentration The results are shown in FIG. There was no difference in plasma corticosterone concentration among WT, Het, and KO bred normally (A). WT, n = 10; Het, n = 10; KO, n = 9. In KO mice subjected to social isolation stress (isolation-KO), a significant increase in plasma corticosterone concentration was observed (B). One way ANOVA p = 0.0088. *, P <0.05. WT, n = 10; Het, n = 14; KO, n = 9.

6-9. Preamine cortex monoamine concentration The results are shown in FIGS. 9-1 and 9-2. In WT, Het, and KO bred normally, the DA concentration in the prefrontal cortex was significantly increased in order (A). *, P <0.05. WT, n = 8; Het, n = 5; KO, n = 6. In WT (isolation-WT), Het (isolation-Het), and KO (isolation-KO) that applied social isolation stress, there was a tendency for the 5-HT concentration in the prefrontal cortex to decrease in turn (B). . WT, n = 6; Het, n = 7; KO, n = 5.

6-10. Results of monoamine concentration in nucleus accumbens are shown in FIGS. 10-1 and 10-2. There was no difference in the monoamine concentration in the nucleus accumbens in WT, Het, and KO reared normally (A). *, P <0.05. WT, n = 8; Het, n = 5; KO, n = 6. In WT (isolation-WT), Het (isolation-Het), and KO (isolation-KO) applied with social isolation stress, the 5-HT concentration in the nucleus accumbens tended to decrease in turn (B). WT, n = 6; Het, n = 7; KO, n = 5.

6-11. FIG. 11 shows the behavioral amount after drug administration on day 7 of daily administration of physiological saline or PCP. When physiological saline was administered every day, there was no difference in the amount of behavior after administration between WT, Het, and KO (A). WT, n = 7; Het, n = 6; KO, n = 5. When PCP was administered every day, the amount of behavior after drug administration on day 7 significantly increased in the order of WT, Het, and KO. One way ANOVA p = 0.0034. *, P <0.05. WT, n = 5; Het, n = 6; KO, n = 5. This indicates that KO mice subjected to drug stress (PCP-KO) have increased sensitivity to PCP. That is, KO mice subjected to drug stress (PCP-KO) can be used as behavioral models for drug addiction and schizophrenia.

6-12. FIG. 12 shows the results of the elevated plus maze test after daily administration of physiological saline or PCP. There was no difference in the amount of behavior in the elevated plus maze test between WT, Het, and KO even when physiological saline was administered every day or when PCP was administered every day (A upper stage, B upper stage). WT, n = 8; Het, n = 8; KO, n = 5; PCP-WT, n = 5; PCP-Het, n = 7; PCP-KO, n = 6. When PCP was administered every day, the open arm stay time (%) increased significantly in the order of WT, Het, and KO (B middle row, One way ANOVA p = 0.004). In addition, when PCP was administered every day, the number of times to enter open arm significantly increased in the order of WT, Het, and KO (B lower row, One way ANOVA p = 0.154). *, P <0.05. This indicates that there is an increase in impulsivity in KO mice subjected to drug stress (PCP-KO). That is, KO mice given drug stress can be used as behavioral models for schizophrenia, bipolar disorder, and hyperactivity disorder.

6-13. FIG. 13 shows the results of a Y-shaped maze test after daily administration of physiological saline or PCP. When physiological saline was administered every day, there was no difference in the amount of behavior and alternation (%) in the Y-shaped maze test in WT, Het, and KO (A). WT, n = 8; Het, n = 8; KO, n = 5. When PCP was administered every day, the amount of behavior in the Y-shaped maze test increased significantly in the order of WT, Het, and KO (B upper row, One way ANOVA p = 0.0014). *, P <0.05, **, p <0.01. There was no difference in Alternation (%) (B lower row, One way ANOVA p = 0.2351). WT, n = 5; Het, n = 7; KO, n = 6. This indicates that there is no difference in short-term memory in KO mice subjected to drug stress (PCP-KO), but there is no rest in the maze test.

6-14. FIG. 14 shows the results of the prepulse inhibition test after daily administration of physiological saline or PCP. There was no difference in startle response in the prepulse inhibition test between WT, Het, and KO even when physiological saline was administered daily or when PCP was administered daily (A upper stage, B upper stage). WT, n = 9; Het, n = 8; KO, n = 6; PCP-WT, n = 5; PCP-Het, n = 7; PCP-KO, n = 8. When PCP was administered daily, a decrease in PPI (%) was observed in the order of WT, Het, and KO (lower B). PCP-WT vs. PCP-KO, *, p <0.05. This indicates that there is a disorder of sensorimotor integration in KO mice subjected to drug stress (PCP-KO). That is, KO mice given drug stress can be used as behavioral models for schizophrenia.

6-15. The results of the passive avoidance test after daily administration of physiological saline or PCP are shown in FIG. When physiological saline was administered every day, WT, Het, and KO significantly increased the time (vertical axis) to enter the room that received the electric shock on the day after receiving the electric shock (Day 2) (A) . ***, p <0.0001, **, p <0.01. WT, n = 8; Het, n = 8; KO, n = 5. When PCP was administered daily, WT (PCP-WT) and Het (PCP-Het) had significant time (vertical axis) to enter the room that received the electric shock on the day after receiving the electric shock (Day 2). (B). ***, p <0.0001, **, p <0.01. In contrast, KO (PCP-KO) showed no significant difference (ns: not significant), and obstacles were observed in repellent learning (B). WT, n = 5; Het, n = 7; KO, n = 6. That is, KO mice subjected to drug stress (PCP-KO) can be used as behavioral models for schizophrenia, learning disorders, and dementia.

The psychiatric disease model animal of the present invention can be used as an animal model corresponding to psychiatric diseases such as schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, depression, and drug dependence. Therefore, it is useful as a model animal for developing a therapeutic drug for mental illness. In addition, biomarkers for mental illness can be screened by using the psychiatric disease model animal of the present invention. Therefore, the present invention is useful in the medical field and drug development.

SEQ ID NO: 1: primer KPNA1-F1
SEQ ID NO: 2: primer KPNA1-R1
SEQ ID NO: 3: primer KPNA1-R2
SEQ ID NO: 4: KPNA1 targeting vector

Claims

A method for producing a non-human animal model of mental illness, comprising the step of (a) losing the function of all or part of the non-human animal caryopherin α (KPNA) 1 gene or a homologue thereof.
The method according to claim 1, further comprising the step of: (b) applying stress to the non-human animal obtained in step (a).
3. The method according to claim 2, wherein the stress is social isolation stress.
The method according to claim 2, wherein the stress is stress caused by a drug.
5. The method according to claim 4, wherein the drug is phencyclidine, dizocilpine (MK801), methamphetamine, amphetamine, cocaine, morphine, cannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC).
The psychiatric disorder is one or more diseases selected from schizophrenia, bipolar disorder, hyperactivity disorder, learning disorder, dementia, and depression. the method of.
The method according to claim 4 or 5, wherein the mental illness is drug dependence.
The method according to any one of claims 1 to 7, wherein step (a) is carried out by knocking out the KPNA1 gene or a homologue thereof homologously.
The method according to any one of claims 1 to 8, wherein the non-human animal is a non-human mammal.
The non-human mammal is an animal selected from the group consisting of mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, goats, minipigs, pigs, sheep, cows, monkeys and chimpanzees. Method.
A non-human model animal of mental illness or a progeny thereof produced by the method according to any one of claims 1 to 10.
A tissue or cell isolated from the animal according to claim 11.
A method for screening a preventive or therapeutic agent for mental illness,
(A) administering a test substance to the animal according to claim 11;
(B) A method comprising the steps of imposing a behavioral task on the animal obtained in step (a), and (c) comparing the results of the behavioral task before and after administration of the test substance.
A method for evaluating the efficacy and / or harmfulness of a test substance to mental illness, comprising:
(A) administering a test substance to the animal according to claim 11;
(B) A method comprising the steps of imposing a behavioral task on the animal obtained in step (a), and (c) comparing the results of the behavioral task before and after administration of the test substance.
A method for screening a biomarker for mental illness,
(A) collecting a biological sample from each of the animal according to claim 11 and a wild-type animal of the same species as the animal,
(B) examining the nucleic acid, protein, metabolite, or lipid in the biological sample obtained in step (a) and comparing the transcription or expression pattern of the nucleic acid, protein, metabolite, or lipid in each animal; And (c) selecting a nucleic acid, protein, metabolite, or lipid that is indicative of mental illness based on the result of the comparison in step (b).