JP2011089982A - Method for screening therapeutic agent of affective disorder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for screening therapeutic agents of affective disorder. <P>SOLUTION: When a breakage of the JLP gene is found to cause affective disorder and furthermore the JLP gene knockout mouse is confirmed to force affective disorder, the method for screening therapeutic agent achieves the completion of affective disorder which utilizes the mouse concerned. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for screening a therapeutic agent for affective disorders, and more particularly to a method for screening a therapeutic agent for affective disorders using a JLP gene-deficient non-human animal that exhibits affective disorders compared to normal animals.

JLP {c-Jun NH ₂ -terminal Kinase (JNK) -associated leucine zipper protein} has been identified as a binding protein of the transcription factor Max and is thought to function as a scaffold protein for the JNK and p38 MAPK signaling modules ( Non-patent document 1).
JLP has also been reported to play an important role in myogenesis by interaction with the cell surface protein Cdo (Non-patent Document 2).
However, many of the biological functions of JLP in vivo were unknown.

The present inventors have analyzed the expression of JLP in the testis and epididymis of adult mice, and found that the expression of JLP is weak in spermatogonia and spermatocytes and strongly expressed in sperm cells. Furthermore, the present inventors conducted immunohistochemical analysis and biochemical analysis using macaque testis and epididymis, and as in mice, there was no expression in epididymal sperm, but in sperm cells. It has been reported to be highly expressed. In addition, according to the present invention, JLPKO mice were confirmed to be born in a normal state by analysis of JLP gene disruption (JLPKO) mice, but the pregnancy and birth rate of females after vaginal plug formation in natural mating was remarkable. It is reported that there is a tendency of male infertility (Non-patent Document 3).
However, there is no report that disruption of the JLP gene causes affective disorders.

On the other hand, screening of anxiolytic drugs, psychotropic drugs and the like using non-human animals in which various genes are disrupted has been reported.
Japanese Patent Application Laid-Open No. 10-127207 (Patent Document 1) discloses "Evaluation of psychotropic drug activity using a non-human animal in which a gene function encoding an angiotensin II type 2 receptor is deleted on the chromosome" Yes.
Japanese Patent Laid-Open No. 2006-238762 (Patent Document 2) discloses “screening a psychotropic drug using a non-human animal in which a part or all of the PKN1a gene is altered or deleted”.
Japanese Patent Application Laid-Open No. 2008-237072 (Patent Document 3) discloses “screening a psychotropic drug using a non-human animal in which a part or all of the motopsin gene is altered or deleted”.
In the above document, there is no disclosure or suggestion about the JLP gene.

In modern society, people have various stresses. Due to the stress, an increasing number of patients are mentally exhausted and complain of anxiety and depression.
Conventionally, benzodiazepine anxiolytics have been used to treat anxiety. However, these drugs have side effects such as addiction, and their treatment is performed under the strict management of specialists. The treatment of depression includes the administration of antidepressants such as noradrenaline, serotonin reuptake inhibitors, and monoamine oxidase inhibitors. These treatments are also performed by specialists in the same way as anxiety treatment. Yes.
In addition, long-term administration of these drugs has caused problems such as reduction of therapeutic effects and occurrence of side effects. Furthermore, it is known that there are individual differences in the effects of drugs.

  Based on the above situation, with the increase in anxiety and depression patients, the development of a new treatment for emotional disorders is desired.

Proc Natl Acad Sci USA (2002) 99: 14189-14194 J Cell Biol (2006) 175: 383-388 Transgenic Res (2008) 17: 1045-1058

JP 10-127207 A JP2006-238762 JP2008-237072

  This invention made it the subject which should solve solving the above-mentioned problem. More specifically, the present inventors made it a problem to be solved to provide a new screening method for a therapeutic agent for affective disorders.

  The inventors of the present invention have newly found that "disruption of the JLP gene causes affective disorders" during the process of analyzing JLP expression in the testis and epididymis using JLP gene knockout mice. The present invention was completed after confirming that the mouse had an affective disorder.

That is, the present invention is as follows.
“1. A screening method for a therapeutic agent for affective disorders, comprising a step of administering a test substance to a knockout non-human animal in which part or all of the JLP gene is altered or deleted.
2. The screening method according to item 1, further comprising a step of evaluating behavioral characteristics of the knockout non-human animal.
3. 3. The screening method according to item 1 or 2, wherein the knockout non-human animal is a mouse.
4). 4. The screening method according to any one of items 1 to 3, wherein the emotional disorder therapeutic drug is a psychotropic drug or an anxiolytic drug.
5). A screening method for a therapeutic agent for affective disorders, comprising a step of evaluating a test substance that activates or promotes expression of JLP.
6). A method for examining whether a patient has an affective disorder due to a JLP dysfunction by measuring the expression level of JLP in a sample derived from a patient suspected of having an affective disorder. "

  According to the present invention, it is possible to provide a screening method for a novel therapeutic agent for affective disorder based on the novel finding “disruption of JLP gene causes affective disorder”.

Explanation of the gene targeting method used to create JLP knockout mice Southern blotting analysis results for Jlp ⁺ / ⁺ , Jlp ⁺ / ^- , Jlp ^- / ^- mice Results of Western blotting with anti-JLP antibody Results of the number of seconds spent in each mouse in the elevated crossroad maze test Results of entry and recovery of each mouse in the elevated crossroad maze test Results of immobility time for each mouse in tail suspension test Mouse dorsal raphe nucleus diagram (the area surrounded by the broken circle in the figure is the dorsal raphe nucleus) Stained and fluorescently labeled figure of dorsal raphe nucleus of JLP knockout mouse Stained and fluorescently labeled figure of dorsal raphe nucleus of wild type mouse

The screening method of the present invention utilizes the fact that “disruption of JLP gene causes affective disorder” according to the following example.
Therefore, a therapeutic agent for affective disorders can be screened by administering a test substance to a JLP gene knockout non-human animal and confirming a change in behavioral characteristics of the knockout non-human animal.

(Emotional disorder)
“Emotional disorder” of the present invention refers to depression, generalized anxiety disorder, social anxiety disorder, post-traumatic stress disorder, obsessive compulsive disorder, panic disorder, panic attack, specific phobia, interpersonal phobia or agoraphobia Means failure including.

(Effective disorder treatment)
The “therapeutic agent for affective disorder” of the present invention means a drug for treating or ameliorating any of the above disorders. Examples include anxiolytic drugs (anti-anxiety drugs), psychotropic drugs (anti-autistic drugs, anti-coaddictive drugs, interpersonal behavior improving drugs, trauma improving drugs, anti-phobic drugs) and the like. .

(Test substance)
Any substance can be used as the test substance used in the present invention. The kind of the test substance is not particularly limited, and may be a low molecular synthetic compound, a compound present in a natural product extract, or a synthetic peptide.
Alternatively, the test substance may also be selected from a compound library, a phage display library, or a combinatorial library.

In the treatment of anxiety disorders, anxiolytics such as benzodiazepine anxiolytics, chenodiazepine anxiolytics, piperazine anxiolytics, serotonergic anxiolytics are used. In addition to anxiety disorders, anti-anxiety drugs are also associated with anxiety, such as prevention of somatic expression disorders (autonomic dysfunction), schizophrenia (schizophrenia), depression, and alcohol withdrawal (withdrawal). It can also be used to relieve anxiety.
For the treatment of depression, drugs that enhance the function of neurotransmitters are used, and mainly include tricyclic antidepressants, tetracyclic antidepressants, triazolopyridine antidepressants, selective serotonin reactivation. Uptake inhibitors (SSRI) and serotonin / noradrenaline reuptake inhibitors (SNRI) are known. SSRI and SNRI may be used to treat anxiety disorders.
Therefore, it is preferable to use a substituted compound, a pharmaceutically acceptable salt, or the like of a compound already known to have an affective affect as a candidate test substance.

(JLP)
JLP is a scaffold protein of the mitogen-activated protein kinase (MAPK) signal transduction pathway and is ubiquitous in non-human animals. Further, the base sequence of JLP can be obtained from a known database. For example, the Accession Number of the mouse JLP is AF327451.

“The JLP gene was disrupted (knockout mouse)” means that the JLP gene region was artificially altered, thereby inhibiting normal expression of the JLP gene. It means that JLP is not functioning normally in the living body (especially brain).
In addition, “all” in “a part or all of the JLP gene is altered or deleted” refers to a region from the 5 ′ end of exon 1 to the 3 ′ end of the last exon of JLP genomic DNA.
In addition, “part” refers to a part of this region and a length necessary to inhibit the normal expression of the JLP gene.
Furthermore, “modification” refers to modifying the base sequence of a target region in genomic DNA to another base sequence by substituting, deleting, inserting, and / or transposing single or multiple nucleotides. .

The “knockout animal in which part or all of the JLP gene is altered or deleted” of the present invention can be prepared by a known method.
For example, it can be prepared using a gene targeting method as described in the Examples below. In this method, normal expression of JLP can be inhibited by substituting at least the initiation codon region of exon 1 of the JLP gene with another base sequence by homologous recombination.
Moreover, the knockout animal used for the screening method of the present invention includes not only the knockout animal produced by the same method but also its offspring.

(Knockout animal)
The animal that is the subject of the knockout animal of the present invention is a non-human animal and is not particularly limited. For example, mammals such as cows, pigs, sheep, goats, rabbits, dogs, cats, guinea pigs, hamsters, mice, rats and the like are exemplified. Of these, rabbits, dogs, cats, guinea pigs, hamsters, mice, and rats are preferred for use as experimental animals, of which rodents are more preferred, and many inbred strains have been produced. Considering techniques such as in vitro fertilization, mice and rats are particularly preferable.

(Construction of targeting vector)
To construct the targeting vector, a part of the JLP gene of the target animal is isolated. For example, when a knockout mouse is prepared, a JLP gene is screened from a mouse genomic DNA library.
A targeting vector for homologous recombination is constructed using the genomic DNA clone obtained by the above screening. The genomic DNA clone need not be the full length of the gene, but only the region necessary for disrupting the JLP gene and suppressing the expression of JLP.
The targeting vector can be prepared by a known method. For example, each of the above-described genomic DNA clone, positive selection marker, negative selection marker (DT-A gene, etc.) using a commercially available plasmid as a backbone. Fragments can be generated by appropriate ligation (see: FIG. 1).

(Generation of JLP gene knockout animals)
The targeting vector prepared by the above method is introduced into a cell having an individual forming ability (differentiating totipotency) such as a fertilized egg, an early embryo, or an embryonic stem cell (ES cell) by electroporation, etc. Cells that have undergone homologous recombination are selected. Sorting can be performed using a drug by a positive-negative selection method.
After selection, the cells in which the desired homologous recombination has occurred are confirmed by Southern blotting or PCR. The cells finally confirmed to have the desired homologous recombination are introduced into 8-cell embryos or blastocysts (blast cysts) collected from the oviduct or uterus during pregnancy.

  The 8-cell embryo or blastocyst is transplanted to a temporary parent according to a conventional method. By crossing a germline chimeric animal (preferably male) born from a foster parent with a wild-type animal (preferably female) having a wild-type JLP gene homozygously, as a first generation (F1), A heterozygote in which one JLP gene is disrupted by homologous recombination can be obtained. Furthermore, by mating these heterozygotes, a homozygote in which both JLP genes on the homologous chromosome are disrupted, that is, the JLP knockout animal of the present invention can be obtained as the second generation (F2). The homozygote can be identified by cutting a part of the body (eg, tail), extracting the DNA, and examining the genotype by Southern blotting or PCR.

(Screening method for therapeutic agents for affective disorders using JLP gene knockout animals)
As shown in the examples below, JLP knockout mice did not show clear morphological abnormalities. However, JLP knockout mice showed anxiety behavior, which is a kind of emotional disorder in the elevated crossroad maze test, and depression, which is a kind of emotional disorder, in the tail suspension test.
From the results of these examples, it is considered that the affective disorder was caused by the functional suppression of JLP.
That is, the behavioral characteristics of a JLP knockout non-human animal administered with a test substance (emotional disorder treatment candidate compound), the behavioral characteristics of a normal non-human animal and / or a JLP knockout non-human not administered with the test substance By comparing with the behavioral characteristics of animals, it is possible to select a therapeutic agent for affective disorders including psychotropic drugs such as psychotropic drugs and anxiolytic drugs.

  A test substance or a mixture containing the test substance is prepared according to the characteristics thereof, and subcutaneously or intraperitoneally injected, intravenously injected, orally administered, etc. are selected as appropriate and administered to a JLP knockout non-human animal.

(Evaluation method of behavioral characteristics)
As a method for evaluating the behavioral characteristics of a JLP knockout non-human animal, a method known per se can be used. For example, the following method can be used.
To check anxiety, you can use the elevated plus maze test (Neurosci. Methods 14, 525-529, 1986) and the light-dark box method (Psychopharmacology, 94, 392-396, 1988). The forced swimming method (Arch. Int. Pharmacodyn. 229, 327-336, 1977) or the tail suspension test can be used, and the open-field method (Life Sciences 58, 701-709, 1996) can be used. Also, Food Neophobia (Physiol. Behav., 51, 979-985, 1992), Vogel-type conflict Staircase test (Psychopharmacol., 92, 106-109, 1987) or Learning helplessness test (Psychopharmacol., 90, 90-94) , 1986) can also be used.

(Elevated cross maze test)
The elevated plus maze test can be performed according to the test procedure described in the paper by S. Pellow et al. {J. Neurosci. Methods, vol.14 (1985), p-149-167}.
As a test device, two open arms and two closed arms are connected to the central part (common central platform) so that they are located on the same line. These devices are installed at a certain height (for example, 30 to 100 cm, preferably 40 to 50 cm) from the floor. Around the open arm (end in the width direction), there is a lip with a certain height (for example, 0.1 to 1.0 cm, preferably 0.3 to 0.5 cm) so that the test animal does not fall easily. Has been.
The illuminance of the test chamber is set appropriately (for example, 5 to 200 lux, preferably 5 to 20 lux). The subject animal has a relatively bright illuminance (for example, 10 to 1000 lux, preferably 20 to 500) in a separate room from the start of the test (for example, 1 hour or more, preferably 3 hours or more) to the start of the test (except during administration). (Lux). When the test substance is administered before the test, the administration is performed, for example, 180 minutes before the start of the test, and preferably 15 to 30 minutes before the test.
The test animal is allowed to move freely for a certain period of time from the start of the test, and the situation is recorded on a video tape.
After the test, the total time spent on the open arm for each test animal is measured, and the result is expressed, for example, as “average value + SEM”. In addition, the time when all the limbs of the test animal are on the open arm is defined as the time when the test animal is on the open arm, and is measured.

The test animals used in this test are animals that have been accustomed to a relatively bright illuminance until immediately before the test, and under the relatively dark illuminance test conditions thereafter, they are on open arms with no side walls. It seems to have anxiety. Therefore, in the elevated plus maze test, as a result, the test animal that has been on the open arm for a longer time can be evaluated as an animal with less anxiety, that is, an animal with reduced or eliminated anxiety.
In addition, if multiple test animals with different test substance administration concentrations are prepared and tested, the test substance will improve affective disorder (has an anxiolytic effect), and what dose (concentration) It is possible to analyze and evaluate whether or not the effect is exhibited (whether a significant difference is observed) or whether a dose (concentration) -dependent tendency is observed.

(Light / dark exploration test)
The light and dark exploration test can be performed using a test apparatus composed of two compartments, a dark compartment and a light compartment. The dark compartment is surrounded by a black material such as plastic, and has a structure with a lid on top. The bright compartment is surrounded by a white material such as plastic, and the top is It has an open structure. The light and dark compartments are connected by a tunnel made of black material such as plastic and can be moved back and forth.
The illuminance of the test chamber is set appropriately (for example, 10 to 1000 lux, preferably 150 lux). The test animal should have a relatively dark illuminance (eg, 50 lux or less, preferably 10 lux) in a separate room from the start of the test (eg, 1 hour or more, preferably 3 hours or more) to the start of the test (except during administration) Get used to it. When the test substance is administered before the test, the administration is performed, for example, 180 minutes before the start of the test, preferably 15 to 30 minutes before the test.

The test begins with the animal in the dark compartment. The test animal is allowed to move freely for a certain period of time (for example, 5 minutes or more, preferably 5 minutes) from the start of the test, and the state is recorded on a video tape.
After the test, the total time spent in the light compartment for each test animal is measured, and the result is expressed, for example, as “average value + SEM”. In addition, the time when all the limbs of the test animal are in the light compartment is defined as the time when the test animal is in the light compartment.

  The test animal used in the test is an animal accustomed to a relatively dark illuminance until immediately before the test as described above, and thus is considered to have anxiety about the subsequent relatively bright illuminance region. Therefore, in this light / dark exploration test, it can be evaluated that the test animals that have been in the light compartment for a longer time have less anxiety, that is, animals with reduced or eliminated anxiety.

(Tail suspension test)
In the tail suspension test (TST), the mouse tail is fixed and hung. The hung mouse rampages as it tries to escape, but after a while it abandons it and becomes immobile. It is believed that the time of immobility (immobility time) reflects the degree of depression in humans.
Therefore, screening for therapeutic agents for candidate affective disorders can be performed using the decrease in immobility time as an index.

(Screening method for therapeutic agents for affective disorders focusing on the function of JLP gene)
From the results of the following examples, it has been clarified that inhibition of the function of JLP causes affective disorder. Therefore, the present invention also provides a screening method for a therapeutic agent for affective disorders, which comprises a step of evaluating a substance that activates or promotes JLP expression.
That is, it is considered that the affective disorder can be improved or completely cured by administering a substance that activates or promotes the expression of JLP to a patient who exhibits affective disorder by suppressing the expression of JLP.

The evaluation method can be performed by a method known per se.
For example, the expression level of JLP mRNA or JLP in vitro or in vivo is measured as one index for confirming whether the functional activity or promotion of JLP has been performed by administration of a test substance.
JLP is a scaffold protein for the mitogen-activated protein kinase (MAPK) signaling pathway. A scaffold protein is a protein that allows a phosphorylation reaction of a continuous MAPK cascade to proceed efficiently by forming a complex of kinase molecules related to the MAPK cascade. That is, phosphorylation of the MAPK cascade is measured as one index for confirming whether JLP functional activity or promotion has been performed.

  The therapeutic drug for behavioral disorder obtained by the above screening is a powder, granule, tablet, capsule, intestinal solvent, liquid, injection (liquid, suspension) or according to the purpose such as prevention, improvement or treatment. Various forms such as those used for gene therapy can be prepared according to conventional methods. The therapeutic agent of the present invention is usually preferably prepared as a pharmaceutical composition using one or more pharmaceutical carriers.

  The dose or intake of the therapeutic agent for behavioral disorders is not particularly limited, and the effectiveness of the contained components, the mode of administration, the route of administration, the type of disease, the nature of the subject (weight, age, medical condition and It is appropriately selected according to the presence or absence of use of other medicines, etc.) and the judgment of the doctor in charge. The medicament or food of the present invention can be administered or ingested in 1 to several times a day, and may be intermittently administered or ingested once every several days or weeks.

(Examination method for affective disorder focusing on the expression level of JLP in biological samples)
From the results of the following examples, it has been clarified that inhibition of the function of JLP causes affective disorder. Therefore, the present invention also provides an emotional disorder test method including a step of measuring the expression level of JLP in a biological sample.
That is, a sample is obtained from a patient suspected of emotional disorder. Then, by comparing the expression level of JLP in the sample with the expression level of JLP in the sample derived from a healthy person, it is examined whether it is an emotional disorder due to a functional disorder of JLP.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

(Preparation of JLP knockout mice)
A JLP knockout mouse was prepared by the following method (see Non-Patent Document 3).
The following operations were performed in accordance with the basic policy regarding the implementation of animal experiments at research institutions established by the Ministry of Education, Culture, Sports, Science and Technology, and the guidelines for the management and use of laboratory animals established by the National Institutes of Health in 1985. .

  FIG. 1 is a diagram for explaining the gene targeting method used for the preparation of the JLP knockout mouse of this example. In the figure, in order from the top, normal JLP gene (Wild type allele), targeting vector for creating JLP knockout mice, and the structure of the JLP gene disruption (mutation) allele that has undergone homologous recombination (Targeted allele) Is schematically shown.

The 8.7-kb gene region that is between 4.5-kb upstream of exon 1 and 4.2-kb downstream of exon 1 of the mouse JLP gene was used for the construction of the targeting vector.
Gene fragments of 3.1-kb (4.5-kb to 1.4-kb upstream of exon 1) and 3.4-kb (0.8-kb to 4.2-kb downstream of exon 1) were amplified by PCR, respectively, and the left arm of the targeting vector and Used for light arm. A Neo resistance gene was inserted between the left arm and the right arm to completely delete exon 1 of the JLP gene. The DT-A gene was introduced for negative selection.

   The above targeting vector was linearized with NotI and then introduced into 129 / Ola E14-1 ES cells {Reference: EMBO J (1997) 16: 1850-1857, Neurosci Lett (2007) 429: 43-48}. A clone having an allele in which the desired homologous recombination occurred was selected by PCR and Southern blotting using a 5′-flanking probe and a neo probe.

The above-mentioned ES cell clone in which homologous recombination occurred was introduced into a mouse embryo according to a known method (reference: EMBO J 16: 1850-1857) to obtain a chimeric mouse.
Furthermore, this chimeric mouse was backcrossed 3 times with wild type C57BL / 6J mice. The JLP knockout mouse (Jlp ⁻ / ⁻ ) used in Example 2 below was obtained by crossing a hetero mouse (Jlp ⁺ / ⁻ ).

(Confirmation of JLP knockout mouse)
It was confirmed that targeting of the JLP gene was successful by Southern blotting (FIG. 2).
Also, hetero mice (Jlp ⁺ / ^-) were obtained by crossing homomice (Jlp ^- / ^-) was born according expected mendelian frequency, there are viable, and appearance was normal.
In addition, Western blot analysis using an anti-JLP antibody revealed that the JLP band detected in wild-type mice was not detected in homozygous mice (FIG. 3).
From the above results, it was confirmed that a JLP knockout mouse could be produced.

(Confirmation of emotional behavior of JLP knockout mice)
In order to confirm the emotional behavior of JLP knockout mice, especially the anxiety behavior, an elevated crossroad maze test was conducted. Details are as follows.

The emotion of the mouse was confirmed using an elevated plus maze (EP-3002, Ohara Medical Industry) and image analysis software (Image J EP, manufactured by Ohara Medical Industry).
The device consists of an open arm without walls (length 50 cm, width 5 cm), a closed arm with walls (15 cm) (length 50 cm, width 5 cm), and a platform that is the intersection of both runways. The apparatus was set at a height of 50 cm from the floor.
Above Examples JLP knockout mice prepared in ^{1 (Jlp - / -, n} = 4) and hetero mice ^{(Jlp + / -, n =} 5) is placed on the platform at the start of the experiment, the ceiling moving path of the subsequent 10 minutes Taken with a CCD camera attached to.
After taking the captured image data into an analysis notebook computer, the time spent in each arm, the time spent in the platform (central section), and the number of times of entry into each arm were calculated by image analysis software. Using the preference for each arm of the mouse as an index, the approach and stay to the open arm is evaluated as “low emotion”, and the approach and stay to the closed arm is evaluated as “high emotion”. It was evaluated that it had behavioral characteristics.

(Confirmation results of emotional behavior of JLP knockout mice)
In the measurement of the number of seconds the JLP knockout mouse stayed for 10 minutes, the stay time of the central section, the open arm and the closed arm was an average of 107.5 seconds, an average of 1.0 seconds, and an average of 491.5 seconds (see: FIG. 4). ).
On the other hand, in the measurement of the number of seconds the hetero mouse stayed for 10 minutes, the stay time of the central section, the open arm, and the closed arm was 82.5 seconds, 37.0 seconds, and 480.5 seconds on average, respectively (see: FIG. 4). .
Moreover, in the measurement of the number of times of entry of the JLP knockout mouse, the number of times of entry of the open arm and the closed arm was 0.75 times on average and 22.25 times on average (see: FIG. 5).
On the other hand, in the measurement of the number of times of entry of the hetero mice, the number of times of entry of the open arm and the closed arm was 4.0 times on average and 25.4 times on average (see: FIG. 5).
From the above results, JLP knockout mice clearly showed anxiety behavior.

(Confirmation of depression in JLP knockout mice)
A tail suspension test was conducted to confirm depression in JLP knockout mice. Details are as follows.

The JLP knockout mice 7 months of age that were prepared in Example 1 (Jlp ^- / ^-) and hetero mice as a control (Jlp ⁺ / ^-) each of the tail tip of the fixed with vinyl tape, both mouse suspended state did.
After fixing both mice in a suspended state, a 300-second movie was recorded using a digital camera (Panasonic; DMC-FS25) fixed with a tripod. In addition, the immobility time of the mouse | mouth was measured manually using the timer (minimum unit; second). The immobility time was measured 3 times for each mouse.

(Confirmation result of depression in JLP knockout mice)
The average immobility time of JLP knockout mice during 300 seconds was 220.7 seconds (see FIG. 6).
On the other hand, the average immobility time of the hetero mice during 300 seconds was 87.3 seconds (see FIG. 6).
Apparently, JLP knockout mice had a longer immobility time compared to heterozygous mice.
From these results, JLP knockout mice clearly showed depression.

(Confirmation of serotonin nervous system expression in the dorsal raphe nucleus of JLP knockout mice)
The serotonergic nervous system with the origin nucleus in the dorsal raphe nucleus (Fig. 7) projects to the forebrain region, especially the hippocampus, amygdala, hypothalamus, etc., and activates the serotonin receptor, thereby causing anxiety and depression. It is known to suppress emotionality. Moreover, it is known that inactivation of the serotonin nervous system causes anxiety, depression and the like. There are many anti-anxiety drugs and antidepressants currently used and developed for the purpose of activating the serotonin nervous system.
Therefore, the expression of the serotonin nervous system in the dorsal raphe nucleus of the JLP knockout mouse prepared in Example 1 was confirmed. Details are as follows.

  The dorsal raphe nucleus of the JLP knockout mouse prepared in Example 1 and the dorsal raphe nucleus of the wild-type mouse were stained with Hoechst and at the same time using an anti-serotonin (also known as 5-hydroxytryptamine, 5-HT) antibody. Fluorescently labeled.

FIG. 8 is a diagram showing staining and fluorescent labeling of the dorsal raphe nucleus of a JLP knockout mouse, and FIG. 9 is a diagram showing staining and fluorescence labeling of the dorsal raphe nucleus of a wild type mouse. 8 and 9, the expression of the serotonin nervous system in the dorsal raphe nucleus of knockout mice is decreased compared to the expression of the serotonin nervous system in the dorsal raphe nucleus of wild type mice. It was.
From the above results, inactivation of the serotonin nervous system is considered as one cause of affective disorder in JLP knockout mice.

(General)
From the results of Example 2 and Example 3, the JLP knockout mouse of this Example showed anxiety / depression-like behavioral characteristics. In addition, from the results of Example 4, in the mice, a decrease in serotonin nerves in the dorsal raphe nucleus was observed.
Therefore, by administering drugs acting on the serotonin nervous system to JLP knockout mice and analyzing behavioral changes, it is possible to know the effects and mechanism of action of anxiolytics and antidepressants related to the serotonin nervous system New drug screening can be performed.

  A compound that can improve or treat the behavioral characteristics of a JLP knockout non-human animal or a compound that activates or promotes the expression of JLP can be a therapeutic agent for affective disorders.

Claims (5)

  A screening method for a therapeutic agent for affective disorders, comprising a step of administering a test substance to a knockout non-human animal in which part or all of the JLP gene is altered or deleted.   Furthermore, the screening method of Claim 1 including the process of evaluating the behavior characteristic of the said knockout non-human animal.   The screening method according to claim 1 or 2, wherein the knockout non-human animal is a mouse.   The screening method according to any one of claims 1 to 3, wherein the therapeutic agent for affective disorder is a psychotropic drug or an anxiolytic drug.   A screening method for a therapeutic agent for affective disorders, comprising a step of evaluating a test substance that activates or promotes expression of JLP protein.