CN115517226B - Application of DMWD as target point in construction of social disorder model and screening of drugs - Google Patents

Abstract

The invention provides applications of DMWD as a target point in constructing a social disorder model and screening drugs. Specifically, the invention provides application of DMWD as a target in constructing a social disorder model animal, application of DMWD as a target in screening a medicament for treating social disorders, and application of an agent for over-expressing DMWD in preparing a medicament for treating social disorders. The invention proves that DMWD has high feasibility as a treatment target of social disorder, and the intervention effect is evaluated by a behavioristics means, thereby providing a reliable theoretical basis for the clinical application of DMWD.

Description

Application of DMWD as target point in construction of social disorder model and screening of drugs

Technical Field

The invention relates to a novel social disorder target, in particular to a social disorder model constructed by taking WD (fibroblast repeat binding, DMWD) of a dystonia site as a target, and application of the DMWD as the target in screening of drugs for treating the social disorder.

Background

Social dysfunction is a group of heterogeneous disorders with common abnormal social functions in the development process, and is mainly reflected in the defect of social interaction behaviors. This social deficit is one of the core symptoms in patients with Autism Spectrum Disorder (ASD). However, the neural mechanisms behind this behavioral disorder are still not well-explained, largely due to their inherent complexity and the heterogeneity of autism. Furthermore, since there are few animal models that address social deficits only, the study of this obstacle also appears to be redundant and inadequate. Therefore, finding a suitable animal model is of great significance for studying the social deficits and even the pathogenic mechanism of autism.

Similarly, social deficits are also present among the major or secondary symptoms of many psychiatric disorders, such as schizophrenia, but due to the complexity of the nervous system and heterogeneity of psychiatric disorders, the mechanism remains unexplained and clinically ineffective treatments are not available. Most of the current anti-mental disease drugs used in clinic are targeted drugs of neurotransmitter systems. However, in the application process, the medicines have a plurality of problems which exceed the scope of the existing theory, including: 1) The drug has low applicability, and only takes effect in 40-50% of patients; 2) The onset time is long, and the curative effect can be observed for at least weeks after the use; 3) The clinical application has short timeliness, and the same medicine can not be used for the same patient for a long time; 4) Serious toxic and side effects, which cause systemic toxic and side effects such as weakness, nausea, increased muscle stress and the like; 5) And not solely for a symptom, such as social deficit. Furthermore, the molecular mechanisms of these drugs remain unclear and cannot be reasonably explained by existing theories. Therefore, researching pathogenic mechanisms according to a single target point and developing a medicine with better curative effect, smaller side effect and more pertinence to overcome social defects is an important scientific problem to be solved urgently at present.

Disclosure of Invention

It is an object of the present invention to provide social disorder-related targets.

Another object of the invention is to provide the use of said social disorder-related target.

In particular, the invention provides a social disorder-associated target, which is dyenophia mycoonica, WD repeat association (DMWD).

DMWD is the WD repeat at the site of dystonia, located at 19q13.32. It is currently believed to be ubiquitously expressed in a variety of structures, the highest of which are in brain and testis, and the translated protein is predicted to be present in cells in dendrites or in or around the nucleus, but the DMWD protein is largely an unidentified protein, demonstrated to bind USP12 and USP46 by direct co-immunoprecipitation of epitope-tagged proteins. Furthermore, DMWD is thought to be involved in the multisystem manifestation of type 1 muscular dystrophy (DM 1) caused by the reduction in multigene doses resulting from abnormal amplification of CTG repeats in DMPK.

Experimental research shows that the behavioral indexes of the DMWD homozygous and heterozygous knockout mice in a three-compartment social and social interaction test are remarkably changed compared with those of a normal wild type control group, but the behavioral indexes are not remarkably different in self-organizing and mining experiments, which indicates that the DMWD gene deletion causes the social defects of the mice, but does not generate all symptoms of self-closing. Behaviours in 12-month-old mice also demonstrated that deletion of the DMWD gene resulted in social deficits in the mice, but did not produce all of the self-closing symptoms. A plurality of ethological experimental results show that the DMWD homozygotic knockout mice and the DMWD heterozygotic knockout mice have no obvious difference from wild mice in the aspects of learning, memory, emotion, violent behavior and the like, and the specificity of the DMWD gene knockout mice on the ethological phenotype defect is proved. The DMWD gene knockout mouse can be used as a social disorder model animal. Furthermore, the invention over-expresses DMWD in the brain of the mouse, so that the social defects of homozygotic and heterozygotic mice can be recovered, and the social ability of wild mice can not be influenced. Experiments show that DMWD can be used as a therapeutic target for social disorder.

Therefore, the invention provides application of DMWD as a target point in constructing social disorder model animals.

The invention also provides application of the DMWD as a target in screening medicines for treating social disorders.

The invention also provides the use of an agent that overexpresses DMWD in the preparation of a medicament for treating a social disorder. DMWD may be overexpressed by any method available in the art, for example, by injecting DMWD-loaded vectors (e.g., AAV viral vectors) into the frontal lobe of the brain.

According to a particular embodiment of the present invention, preferably, the social disorder model animal is a DMWD knockout animal. The DMWD gene can be knocked out by any method available in the art.

According to a particular embodiment of the invention, preferably, the animal is a mouse.

According to a particular embodiment of the invention, preferably, the social disorder is a social dysfunction.

According to a particular embodiment of the invention, preferably, said social disorder model animal has a behavioral phenotypic deficiency specificity.

According to a particular embodiment of the invention, preferably, the DMWD is a DMWD gene and/or a DMWD protein.

In conclusion, the invention discovers that a mouse with the DMWD gene knockout has social function defects, and further provides an animal model using the DMWD knockout as social disorder.

Drawings

Figure 1 shows the behavioural results of the homozygous, heterozygous, wild-type three group mice in a teenage period (6 weeks old) three-compartmental social interaction (panel a, panel B), (panel C) social interaction test, (panel D) self-organisation and (panel E) mining experiments in a specific embodiment of the invention (× × p < 0.001).

Fig. 2 shows the behavioural results of the homozygous, heterozygous, wild-type three mice age (12 months) in the three-compartment socialization (panel a, panel B), (panel C) social interaction test, (panel D) self-grooming and (panel E) mining experiments in a specific example of the invention (. P <0.05,. P < 0.01).

Fig. 3 shows the behavioral results of the homozygous, heterozygous and wild type three groups of mice in the present embodiment during their adolescent age (6 weeks old) (panel a) open field experiment, (panel B) scene fear experiment, (panel C) tail overhang test, (panel D) Y maze test, (panel E) startle reflex experiment, (panel F) water maze experiment and (panel G) violent behavior experiment.

Fig. 4 shows the behavioral results of the homozygous, heterozygous and wild type three mice in the old age period (12 months old) in the present embodiment of the invention in the open field experiment (picture a), (picture B) the scene fear experiment, (picture C) the tail overhang test, (picture D) the Y maze test, (E) the startle reflex experiment, (picture F) the water maze experiment and (picture G) the violent behavior experiment.

Fig. 5 shows the behavioral results of the social interaction test (panel C) in three-compartment (panel a, panel B) of mice from different treatment groups in a specific example of the invention ([ p ] p <0.05, [ p ] p <0.01, [ p ] p < 0.001).

Detailed Description

For a clearer understanding of the technical features, objects, and advantages of the present invention, reference will now be made in detail to the present technical solutions with reference to specific embodiments, and it should be understood that these examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art.

Example 1 establishment of mouse social Defect model

The gene knockout heterozygous mouse (the knockout mouse is purchased from the science and technology company of the race industry) is hybridized and bred to obtain mice of three genotypes, namely, homozygous (MUT), heterozygous (HET) and Wild (WT). After three groups of mice were bred under the same conditions for 6 weeks, the behavioral phenotype of the mice was evaluated. And performing the ethology after 12 months to verify the same social defect of the model mouse at different ages.

The experimental method of the behavioural test comprises the following steps: the experimental system comprises a three-chamber social experiment, a social interaction experiment, a mining experiment, a self-organizing experiment, an open field experiment, a scene fear experiment, a tail suspension experiment, a Y maze experiment, a shock frightening reflex experiment, a water maze experiment and a violent behavior experiment.

Three-compartment social experiments: the experimental device for the three-chamber social contact of the mouse also comprises three rectangular boxes, wherein the specification of each box is 19 multiplied by 45cm, a partition plate between every two boxes is made of transparent resin glass, and a channel is arranged in the middle to enable the three boxes to be communicated; one metal cage of consistent gauge was placed in each of the left and right boxes, sufficient to hold one mouse. Laboratory mice were placed in the behavioural testing room for more than half an hour prior to the start of the experiment. The whole process is divided into three stages:

the first stage is as follows: the experimental mice were placed from the middle box and the partition channel was opened so that the mice could explore freely for 5 minutes in the three boxes.

And a second stage: the experimental mice were introduced into the central box, the passages of the left and right boxes were closed with a plexiglas partition, and then a strange mouse was placed in the metal cage on one of the left and right sides, the metal cage on the other side was empty, and the left and right box passages were opened, so that the mouse was freely explored in the three boxes for 10 minutes. Shooting and recording related parameters: the duration of direct contact (sniffing, fast round-robin exploration and climbing of the metal cage belonging to direct contact) between the experimental mice and the stranger mice (here mice which have never been contacted before) or the empty metal cage, 3-5cm around the metal cage being defined as the contact range.

And a third stage: similarly, the experimental mouse is led to the central box, the resin glass partition plate closes the channels of the left box and the right box, then a second strange mouse is placed in the hollow metal cage in the second stage, the channels of the left box and the right box are opened, the mouse is freely explored for 10 minutes in the three boxes, relevant parameters are shot and recorded, and the duration of direct contact (sniffing, fast surrounding exploration and metal cage climbing) between the experimental mouse and the metal cages of the two strange mice respectively is prolonged.

Social interaction experiments: the method mainly comprises the step of enabling an experimental mouse and a strange mouse to freely socialize so as to detect the social ability of the experimental mouse and the strange mouse. Laboratory mice were placed in the behavioural testing room for more than half an hour prior to the start of the experiment. The experimental mice were placed in a new padded mouse cage and allowed to move freely for 15 minutes under low light conditions, which is the acclimation period. Then, one strange mouse (the mouse should be similar to or slightly smaller than the experimental mouse in age and shape) was placed, and both mice were allowed to move freely for 10 minutes. Video recording is carried out in the whole course, and the time of direct contact behavior of the experimental mouse to the strange mouse is observed. Direct contact here includes sniffing of the experimental mice over the whole body of the strange mouse, following and climbing actions.

And (3) excavating action: the experimental mice were placed in a squirrel cage filled with new padding and allowed to move freely for 20 minutes. The whole course of the experiment process is recorded by video, the first 10 minutes is an adaptation period, and the time of the excavation behavior of the mouse is recorded after the last 10 minutes. Excavation here refers to the act of spreading the padding with the forelimbs and head of the mouse.

Self-organizing behavior: the experimental mice were placed in an open field without padding and allowed to explore freely for 20 minutes. The whole course of the experiment process is recorded by video, the first 10 minutes is an adaptation period, and the time of the self-organizing behavior of the mouse is recorded after the last 10 minutes. Self-grooming as used herein refers to the act of the mouse stroking or scratching the body or face, or licking parts of the body.

Open field experiment: is an experiment for researching the spontaneous activity and exploration behavior of the mouse. The experimental animals are observed and researched for neuropsychiatric changes and various behaviors after entering the open environment, such as fear of the animals to the new open environment, so that the animals mainly move in the peripheral area and less move in the central area. The open field experimental device is a big iron box with a square bottom surface and an upward opening, wherein the square bottom surface is 81cm multiplied by 28 cm. Mice were acclimated in the behavioural laboratory for 30 minutes before testing, placed into one of 4 corners facing the wall under appropriate lighting and allowed to explore the environment freely for 5min. And then carrying out relevant parameter statistics (total movement distance, central region activity time and distance and the like) on the video by using Taisui open field analysis software. After each mouse test, the experimental set was cleaned with 75% alcohol and kept clean.

Scene fear experiment: is a common experimental method for testing the condition memory ability of experimental animals. In a special animal ethology laboratory, an independent isolation box is used as an isolation large environment for ethology test, a 2W incandescent lamp is installed in the isolation large environment, a small exhaust fan is used for ventilation, a stainless steel fence is arranged at the bottom of the isolation large environment and used for foot electrical stimulation, and a loudspeaker is installed at the top of the isolation large environment and used for sound stimulation. Each mouse was wiped with alcohol after completion of the test to eliminate odor interference with the results. On day 1, the animals were trained and exposed to an isolation box. After 60s acclimation, white noise (75 dB) stimulation lasting for 30s is respectively given at 110s, 160s, 210s, 260s, 310s, 360s, 410s, 460s, and a foot shock (30 mA) lasting for 2s is given at the last 2s of each white noise stimulation. The whole experiment lasted 490s, and the animals were observed for catalepsy by a camera. Day 2 is a short-term fear memory test and the animals are placed in the experimental box for 120s and given only sound stimuli. The dead time of the animal is recorded by camera observation. Day 6 is a long-term fear memory test, and animals were placed in the experimental box for 120s and given only sound stimuli. The dead time of the animal is recorded by camera observation.

Tail suspension test: for characterizing the degree of despair and helplessness in animal behavior. Before the experiment, the tail of the mouse is fixed on a bracket by using an adhesive tape, and the mouse is hung upside down in a hanging cabin. After 1min of adaptation, immobility time of mice within the following 5min was recorded and analyzed with tail-overhang software (activity threshold = 30%).

Y maze experiment: because the animal needs to remember the direction explored at the previous time when switching the exploration direction in the Y maze every time, the Y maze experiment can effectively measure the cognitive memory of the animal, and completely utilizes the curiosity of the animal on a new environment. The Y-shaped maze consists of three arms with equal length (50 cm multiplied by 18cm multiplied by 35 cm), the included angle between every two arms is 120 degrees, a movable clapboard is respectively arranged at the center, and the inner arm and the bottom of the maze are coated with black. Mice were acclimated in the behavioural laboratory for 30 minutes prior to testing. One of the three arms was blocked with a plate and the mouse was placed from either of the remaining two arms and allowed to explore freely for 5 minutes. After 30 minutes a second experiment was performed, in which the third arm had to be opened and the mouse was still placed in the Y maze from either of the previous arms, allowed to explore freely for 5 minutes, and the number of times and the time the mouse entered the new arm was recorded. After each mouse test, the device floor was wiped with 75% alcohol.

Startle reflex test: the background noise (59 dB) is opened in the whole course of the experimental stage, and the experimental stage is divided into an adaptation period, a Block I stage and a Block II stage. The duration of the adaptation period is 3min. Phase I (BlockI) was set to 10 tails with no pre-pulse stimulation per test (tail), startle stimulation was set to white noise,120dB: stimulation duration 20ms, and the average takeoff amplitude was recorded. The intervals between Trail vary randomly between 10-30 s. Stage II (Block II) was set to 30 trails with the spacing between each trail varying randomly between 10-30 s. The stimulation types in Block II are divided into 3. Each stimulation type occurred 10 times in 30 trails, with a random order of occurrence. The pre-stimulation gradients were 59dB,64dB (+ 5), 69dB (+ 10), 80dB (+ 21), duration 10ms. The pre-stimulus-startle stimulus interval was set to 90ms. Startle was set at 120dB, stimulation duration 20ms, and mean takeoff amplitude was recorded.

Water maze experiment: is a classical experimental method for detecting the learning and memory abilities of experimental animals. The experimental field is divided into quadrants, one quadrant is provided with a transparent platform for the mouse to stand, the platform position cannot be changed once the platform is arranged in the experimental process, and simultaneously, the periphery of the experimental pool can be marked with a graph respectively for the reference memory of the experimental animal. Before the experiment begins, diluted milk is added into a water pool to enable the water surface to be turbid, and the water surface height needs to exceed the distance of one finger joint of a standing platform. The temperature of the water is required to be constant in the experimental process, and is generally 18 to 22 ℃. The experiment comprises two stages of positioning navigation and space exploration. The design days of the positioning navigation experiment are 6 days, the single training time is 60s, each mouse is respectively put into water from 4 quadrants until a platform in the water is found, the time used in the process is called the latency period, and if the mouse does not find the platform within the specified time, the mouse is manually guided to arrive at the platform and then stays for 10s to deepen the learning. The platform was removed on day 7, and the mice were launched from the contraposition quadrant of the platform and their movements recorded over 60 s.

Violent behavior experiment: the resident male mice and the female mice with the same week age are bred in a matched mode for one week until the resident male mice are not interfered in the subsequent adaptation period and the experimental period, and padding is not changed. The test was started one week later, the female mice were taken out from the cages of the residents one hour before the test, the male mice having a volume slightly smaller than that of the residents, i.e., the invaders were placed in the cages, and the animal behavior was recorded for 10 minutes to count the attack time of the residents.

The ethological evaluation results of the 6-week-old mice in fig. 1 show that the ethological indexes of the DMWD homozygous and heterozygous knockout mice in the three-compartment social and social interaction test are significantly changed compared with the normal wild-type control group, but the ethological indexes are not significantly different in the self-organizing and mining experiments, which indicates that the DMWD gene deletion causes the social deficiency of the mice, but does not produce all symptoms of the self-imposed type.

The behaviours of 12-month old mice in figure 2 also demonstrated that deletion of the DMWD gene resulted in social deficits in the mice, but did not produce all of the self-closing-like symptoms.

The results of various ethological experiments in fig. 3 and fig. 4 show that the DMWD homozygotic and heterozygotic knockout mice have no significant difference from wild mice in the aspects of learning, memory, emotion, violent behavior and the like, and the specificity of the DMWD gene knockout mice on the ethological phenotype defect is proved.

In conclusion, the DMWD knockout mouse can be used as a social disorder model animal.

Example 2 DMWD as a therapeutic target for social deficits

The homozygous mice are divided into two groups, one group (MUT-M +) is injected with AAV virus (pAAV-ITR-hsyn promoter-DMWD-EGFP-3 flag-WPRE-SV40_ polyA-ITR) which overexpresses DMWD in the forehead lobe of the brain of the mice at a single time, and the other group (MUT-EGFP) is injected with control virus (pAAV-ITR-hsyn promoter-EGFP-3 flag-WPRE-SV40_ polyA-ITR) (the virus is purchased from synbiotic biotechnology company); the heterozygote (HET-M +/HET-EGFP) and wild-type (WT-M +/WT-EGFP) mice were the same, and six groups were used in the experiment. Three weeks after virus injection, the three-compartment social and social interaction ethology method of example 1 was used to evaluate DMWD for virus treatment.

The results in FIG. 5 show that the overexpression of DMWD in mouse brain can restore the social deficiency of homozygotic and heterozygotic mice, and has no influence on the social ability of wild type mice.

In conclusion, the experiment of the invention shows that DMWD can be used as a target for treating social disorder.

Claims (9)

1. The application of DMWD as a target in constructing a social disorder model animal with specificity of behavioral phenotypic deficiency.

2. The use according to claim 1, wherein the social disorder model animal is a DMWD knockout animal.

3. The use of claim 1 or 2, wherein the animal is a mouse.

4. The use of claim 1, wherein the social disorder is a social dysfunction.

5. The use according to claim 1, wherein the DMWD is a DMWD gene and/or a DMWD protein.

6. Use of DMWD as a target for screening a medicament for the treatment of social disorders specific for behavioral phenotypic defects.

7. Use of an agent that overexpresses DMWD in the preparation of a medicament for treating a social disorder specific for a behavioral phenotypic defect.

8. Use according to claim 6 or 7, wherein the social disorder is a social dysfunction.

9. The use according to claim 6, wherein the DMWD is a DMWD gene and/or a DMWD protein.

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