KR20190125098A - Animal model lacking social dominance, method for producing the same and uses thereof - Google Patents

Abstract

The present invention provides an animal model lacking social dominance, whose expression or activity of leucine rich repeat transmembrane neuronal 3 (LRRTM3) gene or protein is inhibited, a method of producing the animal model lacking social dominance and a method of screening a therapeutic agent for psychiatric disorder associated with the lack of social dominance using the same.

Description

Animal model lacking social dominance, method for producing the same and uses approximately}

It relates to an animal model lacking social hierarchies, a method of making and use thereof.

Many mental illnesses are characterized by a fundamental disruption of social behavior, and common examples include Autism spectrum disorder (ASD) and social anxiety disorder (SAD). In addition, secondary symptoms of various disorders include social atrophy, such as Schizophrenia and Major depressive disorder (MDD). There are currently no approved drugs that directly target social deficit, and conventional drug treatments for these diseases are known to have limited efficacy at best.

Recent preclinical studies have shown the neuropeptide Oxytocin as an excellent therapeutic agent in activating social behavior and reducing the self-administration of drugs and alcohol. However, oxytocin itself exhibits insufficient permeation to the blood brain barrier, minor oral bioavailability and short in vivo half-life. Because of these problems, clinical studies using oxytocin in the nasal cavity showed only minimal therapeutic efficacy in the relevant clinical group.

The mental illnesses mentioned above are a big social problem, and the nation has caused huge losses in the national economy such as medical expenses. A recent study by the Centers for Disease Control and Prevention (US) estimates that the incidence of autism in children reaches one in 110, and SAD is the second most common anxiety disorder, with the National Institutes of Health (NIH) 6.8%. It is estimated that the degree is suffering from the anxiety disorder.

According to the World Health Organization (WHO), schizophrenia occurs in about 24 million people worldwide and is one of the highest chronic diseases. The WHO also estimates that alcohol abuse contributed to more than 3 million deaths worldwide (5.9% of all deaths) in 2012. In developed countries, Psychoactive substance abuse accounts for 33.4% of men's total life-loss years.

The study of neural substrates on social behavior shows a paradigm shift from existing therapies for the disorder. For example, the use of antipsychotic drugs (eg Risperidone) against ASD aims to inhibit aggressive and challenging behavior rather than to activate prosocial behavior. The use of antidepressants for SAD (e.g., Paroxetine or Venlafaxine) is a low driver and generalized anxiety that is the primary driver of social atrophy in SAD. It is assumed that it affects social anxiety indirectly by improving mood and reducing global anxiety. Treatment of schizophrenia with antipsychotic drugs (for example, olanzapine or Aripiprazole) seeks to control benign symptoms and, to some extent, neurocognitive impairment, but the overall manifestations of chronic disease states. It does not deal directly with social atrophy.

Under these technical backgrounds, social deficiency symptoms are gradually being subdivided, and new animal models and treatments for the treatment of these symptoms are needed.

One aspect is to provide a social hierarchy lacking animal model in which the expression or activity of the LRRTM3 gene or protein is inhibited.

Another aspect is to provide a method of making a social hierarchy lacking animal model, comprising inhibiting the expression or activity of the LRRTM3 gene or protein.

Another aspect includes administering a candidate drug for the treatment of mental illness in the socially lacking animal model; And to provide a screening method of a psychological disorder therapeutic agent, comprising the step of identifying whether the symptoms of the mental disorders are alleviated.

One aspect provides a social hierarchy lacking animal model in which the expression or activity of the LRRTM3 gene or protein is inhibited.

As used herein, the term “social deficit” is a symptom found in mental illnesses such as Schizophrenia and Attention Deficit Hyperactivity Syndrome (ADHD), and as such, as the interest in mental illness has recently increased, Of segmentation, and tailored treatments based thereon, are drawing attention. For example, they may be classified into social interaction, social novelty memory, and social dominance according to behavioral classification. Here, the term "social hierarchical behavior or lack of social hierarchies" is a class of complex social behaviors that includes any behavior that falls outside the categories generally accepted in social interactions with others, including hierarchical relationships. Can be.

As used herein, the term "LRRTM3 (Leucine Rich Repeat Transmembrane Neuronal 3") is known to be able to modulate the structure and function of excitatory synapses at various sites in the brain hippocampus, the protein or gene sequence thereof can be identified through conventional methods Do.

In one embodiment, inhibition of expression of LRRTM3 specifically decreases the activation of neurons in the preliminary cortex, medial dorsal thalamus, and / or hippocampal CA1 region, thereby causing social hierarchy or social cognitive memory among various social deficits. Only symptoms could be reduced selectively, and among them, a decrease in social hierarchical behavior was observed. Therefore, it can be seen that the regulation of the expression or activity of the LRRTM3 gene or protein, in particular, is effective in selectively causing pathological symptoms associated with social hierarchical behavior.

As used herein, the term "inhibited expression or activity of or of the LRRTM3 gene" means that the function of the LRRTM3 gene or LRRTM3 protein is inhibited or disturbed. Such inhibition or interference may be achieved by disrupting the transcription of the LRRTM3 gene or by inhibiting the translation of the mRNA of the LRRTM3 gene. Or by specifically binding to the active site of the LRRTM3 protein or by modifying the protein structure, the activity of the LRRTM3 protein can be reduced or inactivated.

In one embodiment, the animal model may be a knock-down or knock-out of the LRRTM3 gene of the hippocampus teeth. Here, the term "knockout" means introducing a DNA from which a gene deletion is induced from the outside to induce a complete inactivation of a normal gene, and the term "knockdown" refers to, for example, using RNAi and the like. Degradation of expressing mRNA means reducing the amount of gene expression.

The animals are animals with various diseases that appear in humans, and may correspond to animal models used for identifying human diseases and developing treatment methods, such as long-term genetic studies requiring several generations or organ transplantation experiments that are difficult to directly target humans. Includes, but is not limited to, animals except humans. Specifically, it may be selected from the group consisting of mouse, rat, morphot, monkey, dog, cat, rabbit, cow, sheep, pig, and goat. Among the animals, the mouse has a good breeding ability, developmental ability, and environmental adaptability and a large number of offspring, thereby obtaining a large number of individuals belonging to the experimental group, thereby obtaining reliable results, and having an easy operation.

Another aspect provides a method of making a social hierarchy lacking animal model, comprising inhibiting the expression or activity of the LRRTM3 gene or protein.

The lack of sociality, social hierarchy, LRRTM3 gene or protein, the kind of animals and the like are as described above.

This step may be performed using a substance that inhibits the expression or activity of the LRRTM3 gene or protein. As the substance, any substance that reduces or interferes with the expression of the LRRTM3 gene or the activity of the LRRTM3 protein can be used without limitation, such as compounds, proteins, amino acids, peptides, viruses, carbohydrates, lipids, nucleic acids, and the like.

The substance that inhibits the expression of the LRRTM3 gene may be a substance for inhibiting the translation of the mRNA using a low molecular compound, the production of antisense nucleic acid sequences or RNA, siRNA or shRNA using RNAi techniques. In addition, the substance that inhibits the expression of the LRRTM3 gene may be to use a protein or compound that binds to a promoter, enhancer, or transcription regulator known to regulate the transcription of the LRRTM3 gene. Specifically, the substance includes antisense nucleotides complementarily binding to the LRRTM3 gene or its mRNA, small interfering RNA, short hairpin RNA, double strand RNA and microRNA. It may be one or more selected from the group consisting of In addition, specifically, nucleotide molecules that are antisense to nucleic acids encoding LRRTM3 proteins can be used as inhibitors. An 'antisense' nucleic acid may comprise a nucleic acid sequence that is complementary to a 'sense' nucleic acid encoding LRRTM3, eg complementary to the coding strand of a two stranded chain cDNA molecule or complementary to an mRNA sequence. The antisense nucleic acid may be complementary to the entire LRRTM3 coding strand or just a portion thereof (eg, coding region).

The agent that inhibits the activity of the LRRTM3 protein may be, for example, one or more selected from the group consisting of antibodies that specifically bind to LRRTM3 protein, antibody fragments having antigen binding properties thereof, and polypeptides having binding activity. The material may also include aptamers, compounds, peptides, or peptide mimetics that complementarily bind to the LRRTM3 protein.

The antibody can be used both prepared by injecting LRRTM3 or purchased commercially. In addition, the antibody may include polyclonal antibodies, monoclonal antibodies, fragments capable of binding epitopes, and the like. Polyclonal antibodies can be produced by conventional methods of injecting the LRRTM3 into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be purified by any method known in the art and can be made from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs and the like. In addition, all of the fragments capable of binding to the monoclonal antibody or epitope can be produced by methods known in the art.

In one embodiment, the step may be to knock down or knock out the LRRTM3 gene of the hippocampus. In this step, if it is possible to reduce the total inactivation or expression of the LRRTM3 gene, various techniques or methods already known in the art may be applied.

Another aspect includes administering a candidate drug for the treatment of mental illness in the socially lacking animal model; And it provides a screening method of a mental disorder treatment, comprising the step of identifying whether the symptoms of the mental disorders are alleviated.

The "candidate drug" is a newly synthesized or known compound, and may include, without limitation, a substance expected to be effective in preventing or treating a mental disorder.

In addition, if the candidate drug is administered to alleviate the symptoms of mental illness, the candidate drug may be determined to be effective in preventing or treating the mental disorder. In this case, the mental disorder may be a symptom of social hierarchical deficiency, and for example, a wide range of brain and mental disorders and brain development disorders associated with social behavioral abnormalities such as schizophrenia or attention deficit syndrome, hyperactivity syndrome, etc. fall into this category. Can be.

By “administration” of the candidate drug is meant introducing the substance into the subject in a suitable manner. The route of administration of the candidate drug can be administered via any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto.

In the screening method, the step of confirming whether symptoms of mental disorders are alleviated may be a method known in the art, such as measuring the concentration of a mental disease related label in blood of an animal model, analyzing an electroencephalogram (EEG), Three- Chamber test, social dominance tube test, or a combination thereof.

In the lack of social hierarchy according to one aspect, the animal model selectively lowers social hierarchy behavior by inhibiting the expression or activity of the LRRTM3 gene or protein. Therefore, it can be usefully used for the screening of candidate substances for the alleviation or treatment of the symptoms of lack of social hierarchy.

Figure 1 confirms the behavioral change of the animal model by inhibiting the expression of LRRTM3 through the three chamber test. Experimental results are expressed as mean ± SEM (control; n = 10 mice, LRRTM3 KO; n = 16 mice, * p: <0.05, ** p: <0.01, *** p: <0.001, ns: significant) One-way ANOVA and Student's t test).
Figure 2 is a result of confirming the behavioral change of the animal model by inhibiting the expression of LRRTM3 through a social dominance tube test. Experimental results are expressed as mean ± SEM (control; n = 10 mice, LRRTM3 KO; n = 16 mice, * p: <0.05, ** p: <0.01, *** p: <0.001, ns: significant) One-way ANOVA and Student's t test).
FIG. 3 shows a change in c-fos, which is an indicator of activation of nerve cells, in each region of the brain after a social dominance tube test is performed on an animal model in which LRRTM3 expression is suppressed. 3A shows the results of confirming the expression of c-fos by immunofluorescence (cg1: cingulate cortex, PrL: prelimbic cortex, IL: infralimbic cortex, CA1: hippocampus) CA1 (Hippocampus CA1)). 3B is a result of confirming the expression of c-fos by immunofluorescence (MDT: Medial dosal thalamus). 3C is a result of quantitative analysis of changes in c-fos for each region of the brain. Experimental results are expressed as mean ± SEM (control; n = 5 mice, LRRTM3 KO; n = 5 mice, * p: <0.05, ** p: <0.01, *** p: <0.001, Student's t test) .
Figure 4 shows the quantitative correlation between the Win percentage (%) and the number of neurons expressing c-Fos in the preliminary cortex after the social dominance tube test in the animal model in which LRRTM3 expression is suppressed. .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Preparation of LRRTM3 Knockout Animal Model

LRRTM3 knockout animal models were prepared as described in the literature (Laakso et al., 2012; Um et al 2016 Cell Rep.). In other words, by using homologous recombination method, a targeting construct was prepared by replacing the start coding exon of mouse LRRTM3 with an IRES-tauGFP-LNL cassette. Thereafter, germline chimeras were produced by morula aggregation, and several backcrosses were performed to prepare mice in which LRRTM3 knocked out of the hippocampus.

Example 2 Analysis of Behavioral Changes in LRRTM3 Knockout Animal Models

In this example, we tried to identify the social-related behavioral changes of the prepared LRRTM3 knockout animal model (LRRTM3 KO). To this end, according to the methods commonly performed in behavioral analysis, the sociability measures the degree of interest in other homologous mice and the social novelty measures the degree of interest in new mice. Three chamber tests were conducted to observe intimacy and curiosity between the individuals. In addition, a social dominance test was conducted to confirm the degree of social hierarchy through aggression seen when encountering an unfamiliar mouse in a confined space. Meanwhile, normal mice were used as the control group (WT) in this example, a total of 10 controls and a total of 16 LRRTM3 knockout animal models.

As a result, as shown in FIGS. 1 and 2, in the LRRTM3 knockout animal model, social interactions were similar to those of the control group (see Sy in FIG. 1), whereas social cognitive memory (SN, FIG. 1, 2), and social hierarchical behavior (see FIG. 2) were significantly lower than the control group. In other words, these experimental results indicate that inhibition of LRRTM3 expression can selectively reduce only social hierarchical behavior and / or social cognitive memory symptoms.

Example 3 Analysis of Neuronal Activity in LRRTM3 Knockout Animal Models

In the present embodiment, after analyzing the social hierarchical behavior, it was intended to specifically check the profile of the activated neuron. To this end, changes in c-fos, which are indicators of activation of neurons in each region of the brain, were analyzed by immunofluorescence and quantified (cg1: cingulate cortex, PrL: prelimbic cortex, IL: infralimbic cortex, CA1: hippocampus CA1, MDT: medial dosal thalamu). In addition, the quantitative correlation between the Win percentage (%) and the number of c-Fos-expressing neurons in the preliminary cortex among these regions was confirmed. Meanwhile, normal mice were used as the control group (WT) in this example, and 5 control and LRRTM3 knockout animal models were used.

As a result, as shown in Figs. 3 and 4, in the LRRTM3 knockout animal model, the number of activated neurons in the preliminary cortical region, the hippocampal CA1 region, and the medial dorsal sagittal region was selectively reduced (Fig. 3). ), A significant correlation between the number of c-Fos-expressing neurons and Win percent (%) was confirmed (see FIG. 4). In other words, these results indicate that inhibition of LRRTM3 expression reduces the activation of neurons in specific regions of the brain, thereby selectively lowering only social hierarchical behavior and / or social cognitive memory symptoms among various social deficiency symptoms. .

Claims (12)

Leucine Rich Repeat Transmembrane Neuronal 3 (LRRTM3) An animal model that lacks social hierarchies with suppressed expression or activity of genes or proteins.
The animal model of claim 1, wherein the animal is selected from the group consisting of mouse, hamster, rat, morphot, monkey, dog, cat, rabbit, cow, sheep, pig, and goat.
The animal model of claim 1, wherein the animal model is knocked down or knocked out of the LRRTM3 gene of the hippocampus teeth.
The animal model of claim 1, wherein the animal model further exhibits symptoms of social novelty memory.
The method of claim 1, wherein the animal model is selected from the group consisting of prelimbic cortex, medial dosal thalamu, hippocampus CA1 (Hippocampus CA1), and combinations thereof, wherein the activation of neurons is selective Animal model.
A method of making an animal model lacking social hierarchy, comprising the step of inhibiting the expression or activity of a Leucine Rich Repeat Transmembrane Neuronal 3 (LRRTM3) gene or protein.
The method of claim 6, wherein the animal is selected from the group consisting of mouse, hamster, rat, morphot, monkey, dog, cat, rabbit, cow, sheep, pig, and goat.
The method of claim 6, wherein the step is knocking down or knocking out the LRRTM3 gene of the hippocampus teeth.
The method of claim 6, wherein the animal model further exhibits symptoms of social novelty memory.
Administering a candidate drug for the treatment of mental illness in the socially devoid of animal model of claim 1; And
Checking whether the symptoms of the mental illness is alleviated, the screening method of the psychological disease agent.
The screening method of claim 10, wherein the mental disorder has a lower social hierarchy.
The method of claim 10, wherein the identifying step is performed by measuring a concentration of a mental disease related label in blood of the animal model, analyzing an electroencephalogram (EEG), a three-chamber test, a social dominance tube test, or a combination thereof. Phosphorus, screening method.

Images