WO2015023054A1 - Animal model with obsessive compulsive disorder using ninjurin 1 deficiency - Google Patents

Abstract

The present invention relates to an animal model with obsessive compulsive disorder, manufactured by deficiency of Ninjurin 1, and a method for searching candidate drugs for preventing or treating obsessive compulsive disorder using the same. According to the present invention, a Ninjurin 1 KO mouse can be provided as a new model for researching obsessive compulsive disorder, and memantine or a salt thereof, which is an NMDA receptor antagonist, can be provided as a new use for treating obsessive compulsive disorder

Description

OCD Animal Model Using Ｎｉｎｕｒｉｎ１ Deficiency

The present invention relates to an obsessive-compulsive animal model prepared by deficiency of ninjurin 1, and a method for searching for a candidate drug for preventing or treating OCD.

Ninjurin was first reported in 1996 by Toshiyuki Araki et al., Whose expression is increased in Schwann cells after injury by transection or crushing of the distal sciatic nerve. It was discovered in the process of finding a gene.

Phylogenetic trees have been reported using proteins known in GenBank to find proteins that have homology with Ninjurin. In vertebrates, two proteins are known, Ninjurin 1 and Ninjurin 2. Ninjurin 1 and Ninjurin 2 have been found in vertebrates such as humans, mice, and rats.

Human Ninjruin 1 is located on chromosome 9q22 and consists of 152 amino acids. Mouse Ninjurin 1 is located on chromosome 13 and consists of 152 amino acids. Two transmembrane domains can be predicted from the amino acid sequence of ninjurin 1. Through experiments, it is known that Ninjrurin 1 is a protein located in the cell membrane.

Ninjurin 1 is expressed in various tissues. For example, in the RNA stage, the heart, brain, placenta, lung, liver, skeletal muscle (sk.Muscle), kidneys, pancreas, spleen (spleen), thymus (prostate), prostate (testis), ovary (ovary), small intestine (sm.int.), large intestine (colon), blood (adrenal gland) and resection (Dorsal Root Ganglia, DRG), and protein levels have been reported in the liver, kidneys, thymus, uterus, adrenal gland, retina and dorsal root.

In addition, the functions of Ninjurin 1 so far known are related to cell adhesion, nerve outgrowth, cellular senescence and cancer.

Obsessive-Compulsive Spectrum Disorder (OCD), on the other hand, is an involuntary and persistent anxiety disease, and impulsive and repetitive behaviors are the main phenomena to solve this problem. OCD is a neurosis characterized by the presence of repetitive thoughts, illusions (obsessive) and repetitive urges or actions (forces) that the patient perceives as pathological or is strongly resistant within.

In the past, there have been many efforts to explain the causes of OCD based on psychological factors, but the results of recent drug studies and brain imaging studies show that biological factors are closely related to OCD. A representative example is the association with the serotonin system, a representative neurotransmitter of the brain. In clinical trials, drugs acting on the serotonin system have a pronounced effect on the treatment of OCD, and other clinical studies show that there is an association between serotonin and serotonin-related substances and OCD. In addition, many brain imaging studies have shown that certain neurocirculatory areas have problems in obsessive-compulsive disorder, and studies show that these areas are normalized after medication or behavioral therapy.

Recently, there is an increasing demand for research and treatment of various mental disorders such as OCD, depression, and autism, but there are no specific research models and methods yet, and the molecular mechanisms causing these mental diseases are well known. Not.

Accordingly, the present inventors first disclosed the relationship between OCD and Ninjurin 1, and suggests the possibility of using Ninjurin 1 KO mice as a new model for OCD studies and a target protein for treating OCD.

Specifically, an object of the present invention is to provide an obsessive-compulsive animal model prepared by deficiency of ninjurin 1 (Ninjurin 1).

In addition, an object of the present invention is to provide a method for searching for a candidate drug for preventing or treating OCD by using an OCD animal model prepared by deficient in ninjurin 1 (Ninjurin 1).

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides an obsessive-compulsive animal model prepared by deficiency of ninjurin 1.

In one embodiment of the invention, the animal is characterized in that except for humans.

In another embodiment of the present invention, the ninjurin 1 deficiency is characterized by knock-out of the ninjurin 1 gene.

In addition, the present invention provides a method for searching for a candidate drug for preventing or treating OCD by using an OCD animal model prepared by deficient in ninjurin 1 (Ninjurin 1).

In one embodiment of the present invention, the detection method is characterized in that it comprises the step of determining whether the N-Methyl-D-aspartate receptor (NMDA receptor) is inhibited after administration of the candidate drug to the animal model.

In another embodiment of the present invention, the candidate drug is characterized in that the NMDA receptor antagonist.

In another embodiment of the invention, the NMDA receptor antagonist is characterized in that the memantine (Memantine).

According to the present invention, a Ninjurin 1 KO mouse can be provided as a new model for OCD.

In addition, the present invention can provide a novel target protein for the treatment of OCD.

In addition, the present invention can provide a method for searching for a drug candidate for OCD.

In addition, the present invention can provide a drug candidate for the treatment of OCD.

In addition, the present invention can provide a treatment for OCD as a novel use of NMDA receptor antagonists.

1 is a result of observing self-made injury, which is an OCD-like behavior of Ninjurin1 KO mice.

FIG. 2 shows the results of anxiety and repetitive behaviors, which are compulsive disorders of Ninjurin1 KO mice, through marble-burying test.

3 is a result of confirming the degree of anxiety of the Ninjurin1 KO mouse using Elevated-plus-maze.

4 shows the results of confirming the association between Ninjurin1 KO mice and autism using the Three-chamber test.

5 is a result of confirming the cortical-striatum circuit formation disorder of Ninjurin1 KO mouse using Golgi staining method.

6 is a result of confirming the change in the expression pattern of Ninjurin1 during brain development using Western blotting.

Figure 7 shows the results of confirming the expression of Ninjurin1 in synaptosomes of Puncta structure using immunocytochemical analysis.

8 shows the results of confirming the expression of Ninjurin1 in pre-synapse and post-synapse by double immunostaining.

9 is a result confirmed by comparing the expression of Fos B to increase the Neural activity of Ninjurin1 KO mice.

Figure 10 shows the results of confirming the expression of NMDAR and Src expression increase in Ninjurin 1 KO mice using Western blotting.

11 is a result confirming the inhibitory effect of memantine Ninjurin1 KO mouse OCD through the Marble-burying test.

The present inventors have predicted that OCD and Ninjurin 1 may be related to each other, and as a result of research, OCD-like behaviors such as self-made injury, repetitive behavior, and increased anxiety in Ninjurin 1-removed mice (Ninjurin 1 KO mice) And anatomical analysis confirmed abnormalities in the formation of cortical-stiatal circuits.

Furthermore, NMDA receptor expression and Src activity were increased in Ninjurin 1 deficient mice, and the treatment of obsessive compulsive behavior of Ninjurin1 KO mice was restored when Memantine treatment inhibited NMDA receptor activity. In other words, as a molecular mechanism of OCD, deficiency of Ninjurin 1 caused abnormalities in cortical-striatal circuit formation and induced NMDA receptor activity. As a result, it was first identified that OCD-like behavior occurred.

At the same time, we propose a method of searching for candidate drugs that can treat OCD caused by this mechanism, and the resulting candidate drugs.

NMDA receptors are neuroreceptors known to regulate cell death and signaling between normal cells. Direct interaction with the dopamine D1 receptor regulates cell death or induces communication between normal cells. When the NMDA receptor interacts with the D1 receptor, cell death and signaling regulation between cells are independently driven by different mechanisms. Conventionally, various studies on NMDA receptors have been made as part of treating various diseases such as stroke, osteoporosis, epilepsy and dementia.

NMDA receptor antagonists are substances that inhibit the function of the NMDA receptor, and (1) antagonistically bind to binding sites with promoters such as glutamic acid or NMDA (e.g., D-2-amino-5-phosphonoyl acetate) (2) antagonically binding to glycine binding sites essential for activation by NMDA receptor promoters (e.g., 7-chloroquinurene acid), (3) antagonistically binding to binding sites of polyamines, which are active enhancers (Eg, alkane), (4) binding to ion channel sites to block the flow of ions (eg MK-801, Mg2 +) is known.

Accordingly, the present invention provides a composition for the prevention or treatment of obsessive-compulsive disorder comprising memantine (Memantine) or a pharmaceutically acceptable salt thereof as an NMDA receptor antagonist.

Memantine (3,5-dimethyladamantan-1-amine) is one of the NMDA receptor antagonists that inhibits the excessive secretion of glutamate, a chemical in the brain. Glutamic acid plays a role in memory and learning when the amount is normal, but if the amount is excessive, it stimulates nerve cells to induce nerve cell destruction, which is related to stroke, epilepsy and dementia. It turned out. Memantine is one of the most commonly used treatments for Alzheimer's disease (dementia), but it is not known that it can be used to treat OCD.

The composition of the present invention may further comprise components such as existing therapeutically active ingredients, other adjuvants, pharmaceutically acceptable carriers and the like. Such pharmaceutically acceptable carriers include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and the like.

The present invention also provides a method of preventing or treating OCD by administering to a subject a pharmaceutical composition containing a pharmaceutically effective amount of an NMDA receptor antagonist.

As used herein, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle, etc. Mean mammal. Also, in the present invention, "pharmaceutically effective amount" means the type and severity of the disease to be administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route of administration and the rate of administration, the duration of treatment, the factors including the concurrent drug and other medicines It is determined according to factors well known in the art and can be easily determined by those skilled in the art in such an amount that the maximum effect can be obtained without side effects in consideration of all the above factors.

The composition of the present invention is not limited in the method of administration as long as it can reach the target tissue. Examples include oral administration, arterial injection, intravenous injection, transdermal injection, intranasal administration, coronary administration or intramuscular administration. The daily dosage is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, preferably administered once to several times a day.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 OCD-like Behavior of Ninjurin1 KO Mice: Self-made Injury

The differences between the wild-type (wt) and Ninjurin1 KO mice (5-7 months old) were observed and the behavioral patterns were compared. The Ninjurin1 KO mouse (C57BL / 6) used in the experiment was distributed at Ewha Womans University and kept aseptically in the animal laboratory at the College of Pharmacy, Seoul National University. All experiments were performed with the approval of the Seoul National University Laboratory Animal Management Committee (SNU-090316-). 9).

As a result, unlike wild type, damages such as barbering (FIG. 1A), self-made injury (FIG. 1B), and eye-scratching (FIG. 1C) were observed in Ninjurin1 KO mice, and the frequency was barbering (47, 83%). , self-made injury (7.25%) and eye-scratching (12,32%) were significantly increased compared to wild type (FIG. 1D). This phenotype is expected to be a self-made injury caused by excessive glooming behavior, and it is confirmed that the phenotype is a behavioral pattern similar to that of human OCD.

Example 2 OCD-like Behavior of Ninjurin1 KO Mice: Marble-burying Test

To confirm the association between Ninjurin1 KO mice (C57BL / 6) and OCD, a marble-burying test was performed as follows to investigate the degree of repetitive behavior and anxiety-related behaviors.

Mice of 3-5 months of age were placed in cages (25 x 20 x 20 cm) arranged regularly (5 x 4) of 20 marbles (size: 15 mm), and mouse behavior patterns were recorded as moving images. After 30 minutes the number of hidden marbles was analyzed and considered to be meaningful at least 2/3 hidden (FIGS. 2A and 2C). ****: p <0.0001; Comparison of Ninjurin1 KO mice to Ninjurin1 WT (FIG. 2B) or Hetero (FIG. 2D) mice, unpaired student's t-test.

As a result, Ninjurin1 KO mice showed an increased marble-burying behavior pattern compared to wild-type (WT) and Hetero mice. Therefore, it was confirmed that Ninjurin1 KO mice had more repetitive behaviors and felt more anxiety than normal mice. Considering the marble burying test and the self-made injury test of Example 1, it can be seen that the Ninjurin1 KO mouse exhibits a similar behavior to that of the OCD.

Example 3. Comparison of Anxiety Degree in Ninjurin1 KO Mice: Elevated-plus-maze Test

The marble burying test of Example 2 confirmed that anxiety was increased in Ninjurin1 KO mice. To further confirm this, an elevated-plus-maze test was performed as follows to measure the degree of anxiety.

Ninjurin1 WT and KO mice (2 months old) were moved one hour before the experiment to adapt to the environment, and two open arms (25 x 5 x 0.5 cm) and two close arms (25 x 5 x 16 cm) in the vertical direction. A maze with a height of 50 cm was produced. The open arm has a low wall (0.5 cm) so that the mouse does not fall off, and the close arm has a high transparent plastic wall (16 cm) (Figure 3a). The test was conducted in a room with 100 UX lighting without ambient noise, and recorded video of mobility within the maze for 8 minutes and compared the time spent in the open and close arms.

The results are shown in FIGS. 3B and 3C, where the time of staying in each arm (FIG. 3B) and the number of times of entry (FIG. 3C) are indicative of the degree of anxiety. *: p <0.05; **: p <0.01 (Comparison of WT and KO) unpaired student's T-test, s.e.m

Example 4 Association with Autism in Ninjurin1 KO Mice: Three-chamber Test

To investigate the association of Ninjurin1 KO mice with autism, the degree of social communication, which is a major feature of autism, was compared with the nocturnal type through the three-chamber test as follows.

Target subjects (stranger 1, stranger 2, 6-8 weeks) Wild-type (WT) mice were placed in cages 3 days before the experiment and mice to be tested were placed 1 hour before. Three chambers were constructed as chambers with three transparent and removable chambers. The overall dimensions consist of 60 cm x 45 cm x 25 cm, with each chamber consisting of 20 cm x 45 cm x 25 cm. Each chamber is connected by one hole (5cm x 5cm) for easy opening and closing. The cage containing the Stranger mouse is made of steel cylindrical (height; 12cm, 9cm in diameter). In order to prevent the mice under test from rising above the cage, a transparent glass of water was put together (FIG. 4A).

Mice were allowed to move freely in three chambers for 5 minutes and then in contact with stranger 1 mice for 5 minutes. The new mouse (stranger 2) was placed in an empty cage and recorded as a video for 5 minutes. The time spent in each chamber (FIGS. 4C and 4F), the time of direct contact with the mice (FIGS. 4D and 4G), and the heat maps (FIGS. 4B and 4E) were analyzed using the Noldus Ethovision XT8.5 program. *: p <0.05; **: p <0.01; ****: p <0.0001 (comparison of WT and Ninjurin1 KO mice), s.e.m

As a result of the three chamber test, the social communication of Ninjurin1 KO mice was found to be similar to that of WT mice in the time of staying in each chamber and in direct contact with new mice. The interaction is shown. Therefore, it was confirmed that Ninjurin1 KO mice exhibited a different behavior from autism.

Example 5 Cotical-striatum Circuit Formation Disorders in Ninjurin1 KO Mice: Golgi Staining

Since the abnormalities of the Cortico-striatal circuit in the brain are known to be associated with the development of OCD, the cortex and striatum of Ninjurin1 KO mice via Golgi staining are as follows. Anatomical analysis of the structure was performed.

Golgi staining was performed using standard Golgi-Cox impregnation method using FD Rapid GolgiStain Kit (Neurodigitech). WT and Ninjurin 1 KO mice (8-week-old male) brain tissue were separated and fixed, and Gogi-Cox solution was perfused at 25 ° C. for 18 days. Using a microtome (HM 525, Thermo Scientific), the cortical filigree was cut to 150 um in thickness and observed using an optical microscope (Leica, CTR 5000). Scale bar = 100 um (FIGS. 5A and 5C), 50 um (FIG. 5B).

As a result, in the striatum (FIG. 5A), Medium Spiny Neurons (MSN) (FIG. 5B), and cortical-striatum neurons (FIG. 5C) in Ninjurin1 KO mice, dendritic branching complex ( Significant decreases in dendritic arbor complexity and dendritic branching were observed. Therefore, it was confirmed that Ninjurin1 induced the resin complex of neurons, and in the absence of Ninjurin1, abnormality occurred in the formation of cortical line.

Example 6 Expression Pattern Change of Ninjurin1 during Brain Development

Since ninjurin1 influences the neural cell network formation, Western blotting was used to investigate the expression of ninjurin1 in the developing brain.

Example 6-1. sample preparation

In order to investigate the expression of Ninjurin1 in each part of the mouse brain, males at 2 weeks (FIG. 6A) and 8 weeks (FIG. 6B) were perfused using PBS and brains were extracted. After washing twice with PBS, olfactory bulb (OB), cerebral cortex (Ctx), cerebellum (CB), medulla oblongata (MO), and spinal cord (SC) were isolated. Each separated portion was extracted protein using lysis buffer. In order to investigate the expression of Ninurin1 in each developmental stage, brains of 2, 4, 6, 8, 10 and 14 day old mice were extracted (FIG. 6C). In addition, the mouse cortical neurons (cortical neuron) was first cultured to prepare the cells by differentiation time (3, 5, 7, 12, 16 days) and then extracted the protein (Fig. 6d).

Example 6-2. Western blotting

After the extracted protein was isolated via SDS-PAGE, each primary antibody was incubated overnight at 4 ℃. The next day, HRP-labeled anti-mouse IgG was treated as a secondary antibody at room temperature for 1 hour, washed with PBST (PBS, 1% Tween-20), and then detected using ECL solution (Intron, Westzol). The primary antibodies used were PSD-95 (Millipore, mouse) and Synaptophysin (Millipore, mouse). Both antibodies were used as markers of synaptogenesis. Β-actin (sigma, rabbit) and α-tubulin (sigma, mouse) were used as controls.

As a result, as shown in Figure 6, as the development progresses in the mouse brain it was confirmed that the expression of Ninjurin1 increases. In other words, the expression of Ninjurin1 is evenly expressed in each part of the brain, and the expression of Ninjurin1 is increased similarly to the time of neural junction formation during developmental stage.

Example 7 Identification of Ninjurin1 Distribution in Synaptosomes of Puncta Structure

To investigate intracellular expression of ninjurin1, synaptosomes of mice were isolated as follows.

Example 7-1. sample preparation

The brains of 8-week-old female mice were extracted and ground with scissors and then ground in cold HEPES-Sucrose buffer (Hom, homogenate). The pellet was centrifuged (1000 g, 5 min) to obtain a pellet (P1, nuclear fraction), and the supernatant (S1, synaptosome) was centrifuged again (10000 g, 15 min) to pellet (P2, crude synaptosomal fraction) and supernatant ( S2, light membrane & cytosolic fraction). P2 was then resuspended in cold HEPES buffer, and then centrifuged again (10000 g, 15 min) to obtain pellet (P2 ', washed synaptosomal fraction). After dissolving P2 pellet by hypo-osmotic shock using 9 times volume of cold distilled water, 4mM HEPES was added and centrifuged again (21000g, 20min) to obtain pellet (P3, lysed synaptosomal membrane fraction). . Then, the supernatant (S3, crude synaptic vesicle fraction) was centrifuged again (55000 rpm) to obtain pellets (S3 ', synaptic vesicle fraction) (Fig. 7a).

 In primary cortical neurons (DIV 16) incubated for 16 days, using the Syn-PER synaptic protein extraction reagent (Pierce, Thermo scientific), the nuclear fraction, homogenate, synaptosome The cytosolic fraction was obtained. After dissolving primary cortical neurons (DIV 16) with Syn-PER buffer, centrifugation (1000g, 10min) to obtain the nuclear fraction and supernatant, and the supernatant again centrifuged (10000g, 20min) to synaptosomes and cytoplasmic fraction Got. These were analyzed for proteins using conventional western blotting methods (FIG. 7B).

Example 7-2. Immunocytochemistry

In order to further confirm the expression distribution of ninjurin1 in cells, immunocytochemical analysis was performed. GFP-Ninj1 and Flag-mNinj1 were transfected (Lipofectamine 2000, Invitrogen) into primary cortical neurons DIV4 and DIV8 cultured for 4 days and 8 days, respectively. After 24 hours, fixed and stored using PFA (4%, 10 min) (PBS, 4 ° C). After washing twice with cold PBS for immunocytochemical analysis, the reaction of PFA was inhibited with 20 mM glycine / PBS. After permeabilization with 1% Triton X-100, nonspecific binding was inhibited via blocking solution (1% BSA (w / v), 5% FBS (v / v)). Each primary antibody was treated overnight at 4 ° C. After washing twice with PBS, a secondary antibody (Alexa 488 / 546-conjugated donkey anti-rabbit / mouse IgG) was treated for 1 hour at room temperature followed by confocal microscopy (LSM 700, Carl Zeiss). It was observed using (Fig. 7c). Primary antibodies used were Anti-Flag (sigma), anti-GFP (Abcam), Ninjurin1 (custom-made, Ab1-15). Scale bar: 50um.

As a result, Ninjurin1 is mainly present in the synaptosome fractions (P2, P2 ', P3), but not in the synaptic vesicle fractions (S3, S3') (Fig. 7a), and synapses in primary cultured cortical neurons. It was confirmed that it was present in the tosome (FIG. 7B). In addition, as a result of immunocytochemical analysis, it was confirmed that exogenously overexpressed Ninjurin1 is expressed in puncta-like structures (FIG. 7C).

Example 8. Expression of Ninjurin1 in pre-synapse and post-synapse

Since ninjurin1 is expressed in the synaptosome region with puncta structure, the distribution of Ninjurin1 expression in synapses was examined in detail through several synaptic markers and dual immunostaining.

Primary antibodies used for dual immunostaining are as follows.

Pre-synapse markers: Synaptophysin (Millipore, mouse), vGlut1 (Abcam, sheep),

Post-synapse marker: Homer1 (Synaptic systems, mouse)

As a result of the experiment, as shown in Figure 8, it was confirmed that Ninjurin1 is partially co-expressed with each marker. However, the presynapse markers Synaptophysin and vGlut1 were overlaid in various parts, but the postsynapse markers Homer1 were overlaid. Therefore, Ninjurin1 is expressed in both pre-synapse and post-synapse, but it can be seen that it is present in pre-synapse more than post-synapse.

Example 9 Confirmation of Increased Neural Activity of Ninjurin1 KO Mice

Since Ninjurin1 KO mice showed OCD-like behavior and Ninjurin1 was expressed at synapses in Examples 1 to 8, nerve activity was compared by immunostaining Fos B and comparing expression levels as follows.

Brain tissue and lysate were prepared from wild-type (WT) and Ninjurin1 KO mice (3 months old male), and the expression levels of Fos B (Santa cruz) were compared by Western blotting (40ug, 12% gel). . For immunohistochemistry, mice were perfused with PBS and fixed at 4 ° C. overnight with 4% PFA. After dehydration using 10%, 20% and 30% sucrose, OCT blocks were made. The microtomes (HM 525, Thermo Scientific) were cut to 20 μm and placed in free-floating solution and stored at −70 ° C. until use. The cut brain tissue was washed with PBS (5 min, 3 times) and then blocked with blocking solution (0.03% Triton X-100, 10% BSA, 5% FBS) and Fos B antibody (Santa Cruz) overnight at 4 ° C. Was treated to tissue. The following day, after washing three times with PBS for 10 minutes, the secondary antibody was treated for 1 hour at room temperature. Nuclei were stained with Hechst33442 (Molecular probe, Invitrogen) and observed under confocal microscopy.

Immunohistochemical staining of FIG. 9A and Western blot of FIG. 9B confirmed that the expression of Fos B was increased in the brain of Ninjurin1 KO mouse. Therefore, it can be seen that the neuronal activity is enhanced in Ninjurin1 KO mice.

Example 10 Confirmation of Increased Expression of NMDAR and Src in Ninjurin1 KO Mice

Western blotting was used to examine the expression of NMDA receptors in the brain and cortex of Ninjurin1 KO mice. Brain, cortex, and synaptosme were prepared as tissues, followed by Western blotting. The primary antibodies used were as follows.

1) Millipore: NMDAR2A (Rabbit), NMDAR2B (Rabbit), NMDAR1 (Rabbit), PSD-95 (Mouse), Synaptophysin (Mouse), Synapsin (Mouse), GABAR _A (Mouse)

2) Cell signaling: Src, p-Src (Y416)

3) Sigma: β-actin, α-tubulin

As a result, the expression of glutamate subunits (NMDAR2A, NMDAR2B, NMDAR1) was increased in Ninjurin1 KO mice, but no change was observed in synapsin and synaptophysin, factors regulating synapse formation (FIGs. 10A and 10B). The amount of phosphorylated Src was also increased in Ninjurin1 KO mouse synaptosomes (FIG. 10C). Therefore, it is expected that the obsessive-like behavior of Ninjurin1 KO mice is regulated by the glutamate pathway through activation of Src.

Example 11 Confirmation of Inhibitory Effect of Memantine on Ninjurin1 KO Mouse OCD

Obsessive-compulsive behavior of Ninjurin1 KO mice was associated with an increase in NMDAR. Therefore, the effect of inhibiting OCD against memantine hydrochloride, an antagonist of NMDAR, was treated for Alzheimer's disease.

Example 11-1. Barbering vs. skin damage

Memantine hydrochloride (sigma, 5 mg / kg) was intraperitoneally injected daily for 10 days in Barbering and skin injury Ninjurin1 KO mice (5 months old). Two weeks later, Barbering and the degree of skin damage were compared with before injection (FIG. 11A).

Example 11-2. Marble-burying test

Ninjurin1 KO mice were intraperitoneally injected with 5 mg / kg of memantine hydrochloride. After 1 hour, a marble-burying test was conducted in the same manner as in Example 2 (FIG. 11D).

As a result, the injection of memantine hydrochloride significantly reduced OCD-like behaviors such as barbering (FIG. 11B), self-made injury (FIG. 11C), and marble-burying (FIG. 11D) of Ninjurin1 KO mice. In conclusion, NMDAR mediates OCD-like behavior in Ninjurin1 KO mice, and memantine hydrochloride, which inhibits its activity, is expected to be a possible candidate for treating OCD behavior.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (7)

1. Obsessive-compulsive animal model produced by deficient in ninjurin 1 (Ninjurin 1).
2. The animal model of claim 1, wherein the animal is not human.
3. The animal model of claim 1, wherein the ninjurin 1 deficiency is caused by knock-out of the ninjurin 1 gene.
4. A method of searching for a candidate drug for preventing or treating OCD using the animal model of claim 1.
5. The method of claim 4, wherein the detection method comprises measuring whether the N-Methyl-D-aspartate receptor (NMDA) receptor is inhibited after administering the candidate drug to the animal model.
6. The method of claim 5, wherein the candidate drug is an NMDA receptor antagonist.
7. 7. The method of claim 6, wherein said NMDA receptor antagonist is memantine.

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