KR101978805B1 - X-Prolyl Aminopeptidase 1 knock-out Animal model - Google Patents

Abstract

The present invention relates to an animal model for at least one disease selected from the group consisting of hyperactivity, cognitive disorders, epilepsy, intellectual disabilities and attention deficit, wherein Xpnpep1 (X-Prolyl Aminopeptidase 1) gene is knocked out, And a screening method of the above-mentioned disease therapeutic agent. Using the animal model of the present invention, it is possible to stably carry out diseases or related studies related to hyperactivity, cognitive disorders, epilepsy, intellectual disorders and attention deficit. In addition, it can be usefully used for screening for a therapeutic agent related to the above-mentioned diseases.

Description

Xpnpep1 gene knockout animal model {X-Prolyl Aminopeptidase 1 knock-out Animal model}

The present invention relates to an animal model for at least one disease selected from the group consisting of hyperactivity, cognitive disorders, epilepsy, intellectual disabilities and attention deficit, wherein Xpnpep1 (X-Prolyl Aminopeptidase 1) gene is knocked out, And a screening method of the above-mentioned disease therapeutic agent.

Hyperactivity refers to excessive functional activation in the neurological aspect and developmental disorder characterized by excessive excitement, carelessness, and unstable behavior in the behavioral aspect. In addition, congitive impairment refers to a state in which memory, attention, language ability, time-space ability and judgment are degraded, and the degree varies from very mild to severe. Epilepsy is a disease in which seizures occur repeatedly. Causes of epilepsy include stroke, mental retardation such as mental retardation and cerebral palsy, head injury, tumors, metabolic diseases, and inflammatory diseases of the brain. In addition, intellectual disability refers to a state of severe limitation in adaptive behavior expressed by intellectual function and conceptual, social, practical, and adaptive skills. The causes of intellectual disability vary, but can be broadly categorized as biological, environmental or sociocultural causes, or a combination of the two preceding factors. Attention deficit also means distracted, hyperactivity, impulsive state due to lack of attention continuously.

In order to study mechanisms for at least one disease selected from the group consisting of hyperactivity, cognitive disorders, epilepsy, intellectual disabilities and attention deficits, and for the development of biological samples for therapeutic research, suitable animal models are indispensable and demanding .

It is an object of the present invention to provide an animal model for at least one disease selected from the group consisting of hyperactivity, cognitive disorders, epilepsy, intellectual disabilities and attention deficit, wherein Xpnpep1 (X-Prolyl Aminopeptidase 1) gene is knocked out.

It is also an object of the present invention to provide an animal model preparation method for at least one disease selected from the group consisting of hyperactivity, cognitive disorder, epilepsy, intellectual disability, and attention deficit including knocking out the Xpnpep1 gene in an animal other than a human .

It is also an object of the present invention to provide a screening method for one or more disease therapeutic agents selected from the group consisting of hyperactivity, cognitive disorders, epilepsy, intellectual disorders and attention deficit.

In order to solve the above-mentioned problems, the present invention provides an animal model for at least one disease selected from the group consisting of Xpnpep1 (X-Prolyl Aminopeptidase 1) knockout hyperactivity, cognitive disorder, epilepsy, Model.

Also, the present invention provides a method for preparing an animal model for at least one disease selected from the group consisting of hyperactivity, cognitive disorder, epilepsy, intellectual disorder, and attention deficit including knocking out the Xpnpep1 gene in an animal other than a human do.

(1) administering a test substance to the animal model as an experimental group; (2) measuring and comparing at least one selected from the group consisting of hyperactivity, cognitive disorder, epilepsy, intellectual disability, and attention deficit of the experimental group of the above (1) and the control animal model not treated with the test substance; And (3) selecting the subject substance in comparison with (2) the control group. The method for screening a therapeutic agent for one or more diseases selected from the group consisting of hyperactivity, cognitive disorder, epilepsy, to provide.

Using the animal model of the present invention, it is possible to stably carry out diseases or related studies related to hyperactivity, cognitive disorders, epilepsy, intellectual disorders and attention deficit. In addition, it can be usefully used for screening for a therapeutic agent related to the above-mentioned diseases.

FIG. 1 shows activity patterns (left) and daily activity (right) for 24 hours in a groove-cage. FIG.
Figure 2 shows a sample of the open field activity of the wild type and Xpnpep1 - / - mice (left), the wild type and the open field activity of the Xpnpep1 - / - mice recorded.
Fig. 3 is a diagram showing results of observing hyperactivity (left) of wild-type and Xpnpep1 - / - mice and observation of presence or absence of anxiety disorder (right) in an open field test.
Figure 4 shows the results of tracing representative activities in an elevated plus maze test in wild-type (above) and Xpnpep1 - / - mice (below).
Figure 5 shows the time (left), the total number of open and closed arms (middle), the percentage of open and closed arm outs (right), the time spent on open arms during wild-type and Xpnpep1 - / - Fig.
FIG. 6 shows the result of performing Morris water-labyrinth analysis training in wild-type and Xpnpep1 - / - mice.
FIG. 7 shows the result of confirming the swimming path of each mouse during the probe test in the wild-type and Xpnpep1 - / - mice after the training period of 9 days and after 24 hours.
Figure 8 shows the time spent in each quadrant during a 60 second probe test in wild-type and Xpnpep1 - / - mice.
Fig. 9 shows the result of confirming swimming speed during probe test in wild-type and Xpnpep1 - / - mice.
Figure 10 shows the results for motor coordination and motor learning in rotarod analysis in wild-type and Xpnpep1 - / - mice.
Figure 11 shows an enlarged view of the epileptic discharge recorded in Xpnpep1 - / - mice (center, right, upper) and the diagram (left, upper) of the placement of epidural electrodes of Xpnpep1 - / - , Wild-type and Xpnpep1 - / - mice (below).
FIG. 12 is a graph showing an epileptic wave appearing in an Xpnpep1 - / - mouse according to frequency and duration.
FIG. 13 is a diagram showing the cumulative frequency distribution of epilepsy recorded in Xpnpep1 - / - mice.
FIG. 14 is a graph showing the behavioral change of Xpnpep1 - / - mice after intraperitoneal injection of pentylenetetrazol (PTZ, 30 mg / kg).
FIG. 15 is a graph showing the rate of reaching a kindling criterion (seizure, intermittent seizure, strap tail response, or jump) after treatment of pentylenetetrazole with wild type and Xpnpep1 - / - mice.
16 is a graph showing the degree of average seizure quantitated during the observation period of 30 minutes after treatment of pentylenetetrazole with wild-type and Xpnpep1 - / - mice.
FIG. 17 shows average seizure scores after treatment with pentylenetetrazole in wild type and Xpnpep1 +/- (heterotype) mice.
18 is a graph showing the proportion of kindled mice after treatment with pentylenetetrazole in wild type and Xpnpep1 +/- (heterotype) mice.
FIG. 19 shows the lack of a new object recognition in the Xpnpep1 - / - mouse, showing the results for the preference (left) and total observation time (right) for a novel object in the NOR test.
FIG. 20 is a graph showing the results of the quantitative determination of the movement behavior of the mouse before and after fear conditioning by the space (left) and the sound signal (middle) and the inhibition of activity during the fear conditioning experiment (right).

The present invention provides an animal model for one or more diseases selected from the group consisting of hyperactivity, cognitive disorders, epilepsy, intellectual disabilities and attention deficit, wherein Xpnpep1 (X-Prolyl Aminopeptidase 1) gene is knocked out.

In the present invention, " Xpnpep1 (X-Prolyl Aminopeptidase 1) " is a gene encoding aminopeptidase P1, which is a cytoplasmic form of metalloaminopeptidase that promotes cleavage of the N-terminal amino acid adjacent to the proline residue .

The animal is at least one selected from the group consisting of a mouse, a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a cattle, a sheep, a pig and chlorine, and is preferably a mouse.

Also, the present invention provides a method for preparing an animal model for at least one disease selected from the group consisting of hyperactivity, cognitive disorder, epilepsy, intellectual disorder, and attention deficit including knocking out the Xpnpep1 gene in an animal other than a human do.

(1) administering a test substance to the animal model as an experimental group; (2) measuring and comparing at least one selected from the group consisting of hyperactivity, cognitive disorder, epilepsy, intellectual disability, and attention deficit of the experimental group of the above (1) and the control animal model not treated with the test substance; And (3) selecting the subject substance in comparison with (2) the control group. The method for screening a therapeutic agent for one or more diseases selected from the group consisting of hyperactivity, cognitive disorder, epilepsy, to provide.

The animal model is at least one selected from the group consisting of a mouse, a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a cattle, a sheep, a pig, and chlorine.

For the purposes of the present invention, the Xpnpep1 gene knockdown animal model exhibits hyperactivity and cognitive impairment and exhibits intellectual and attention deficit behaviors. It is also an animal model in which epilepsy disease appears. Therefore, comparing the animal model in which the Xpnpep1 gene knocked down with the test substance was administered and the control group not administered, it was confirmed that the control group had a target substance which is low in hyperactivity, cognitive disorder, epilepsy, intellectual disorder and attention deficit, It is a selection.

Hereinafter, the present invention will be described in detail by way of examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention in any way to the scope of the invention as defined by the appended claims. It will be obvious.

Experimental Example 1. Experimental Preparation

1-1. Animal preparation

To prepare the Xpnpep1 (X-Prolyl Aminopeptidase 1) knockout mouse model, a mouse embryonic stem (ES) cell line (strain 129 Sv / Ev) containing the Xpnpep1 gene trap was used and this was provided by the Fred Hutchinson Cancer Research Center received. Embryonic stem cells were injected into blastocysts of C57BL / 6J mice to produce chimeric individuals, which were crossed with C57BL / 6J females to obtain a heterozygous Xpnpep1 mutation (N1). Gonadal metastases and mouse genotypes were monitored by PCR of genomic DNA using oligonucleotide primers: 5'-CCTTAGACGTGTCCCGAGGT-3 'and 5'-TGCAGCCTAGAGGAAGGACA-3' (wild type) or 5'-CCTGGACTACTGCGCCCTAC-3 ' area). Mice were backcrossed with C57BL / 6J and 129S4 / SvJae for 8-16 generations. All analyzes were performed on littermates of both genotypes generated by cross breeding between parental C57BL / 6J and 129S4 / SvJae heterozygotes. The mice were fed free of feed and water under a 12 hour light cycle and housed in a climate-controlled room animal facility. The maintenance of animal and all animal experiments was approved by Seoul National University's Institutional Animal Care and Use Committee (IACUC).

1-2. EEG (electroencephalograms) recording

Ten-week-old mice were used for EEG recording. Under avertin anesthesia (tribromoethanol, 20 mg / ml), four epidural electrodes were implanted in both the frontal and temporal regions of the reference electrode at the back of the skull. After 7 to 10 days of recovery, the EEG signal and video recording were simultaneously collected for 2 hours. The EEG signal was amplified (Grass model 9 polygraph, Grass Technologies) and digitized at a sampling rate of 500 Hz (Digidata 1320A, Molecular Devices).

1-3. Behavior analysis

All behavioral experiments were performed on male littermates. Data were analyzed using videotracking software from Noldus (Ethovision XT). To monitor the groove-cage activity, a specially designed transparent acrylic lid was attached to a standard mouse cage and the activity of the mouse was recorded with an infrared video recorder. The mice were each placed and adapted to the modified home cage for two weeks prior to recording. Open field activity was measured in a dark room with a white plastic chamber (40 x 40 x 40 cm) for 1 hour.

New object recognition tests were performed as described in the prior art (M. H. Kim et al., The Journal of the Society for Neuroscience 29, 1586-1595 (2009)). Specifically, the mice were individually adapted to an open field chamber (40 x 40 x 40 cm) for one hour before each training session. Two new objects were placed in the chamber during the training session and one of the objects was replaced with a new one during the recall session. In each session, the mouse was allowed to search for objects for 10 minutes. The interval between training and recall sessions was 24 hours. The preference index was calculated as the total time spent searching for new and familiar objects divided by the time spent searching for new objects.

The high-valued plus maze test was performed using a plus-shaped maze including open arms and closed arms surrounded by opaque walls. The maze is raised 50 cm above the floor. During the test, each mouse was placed in the middle of the maze and recorded as a video for 10 minutes. Open and closed arms, and the time spent in the cancer was analyzed using videotracking software (Ethovision XT, Noldus).

Fear conditioning is performed on the Coulbourn instruments with a sound of 18 seconds after a 300 second exploration and 2 seconds of foot shock (0.7 mA) for contextual and auditory cued A fear conditioning training session was conducted. Each mouse was then placed in the chamber for 60 seconds before being returned to the home cage. The fear conditioning test for space was performed in the same chamber (CS, contextual) after 24 hours monitoring mouse behavior for 5 minutes before training. For fear conditioning by sound signals, the mice were placed in separate chambers of different rooms, and the behavior of the mouse (pre-CS) was monitored for 180 seconds before the sound signal was presented (180 seconds, CS) mice. The degree of inhibition of activity can be expressed as: (travel distance during pre-CS-travel distance during CS) / (travel distance during pre-CS)

For the Morris water-maze analysis, a white circular pool (diameter 110 cm) was filled with 23-25 degrees of water. The two day pre-training session consisted of one test per day and each mouse was left on a visible platform (10 cm in diameter) for 30 seconds. During the hidden platform learning, each mouse was trained four times a day and each test interval was held for one minute. In each test, the mice were released to one of the four starting points to find the submerged platform. If the mouse can not find the platform within 90 seconds, it is manually guided to the platform. The mice were then allowed to remain on the platform for 30 seconds before returning to the test or home cage. The probe test was performed one day after the end of the training session.

The accelerated Ugo Basile test was performed by training the mice once a day for 4 days. The mice were placed on a stationary rod and allowed to accelerate for 5 minutes at 5-40 rpm. The waiting time to fall from the rotating rod was measured.

Seizures and seizure scores were performed with reference to the conventional literature (Y. S. Bae et al., Biochemical and biophysical research communications 433, 175-180 (2013)). Specifically, a score of 0 indicates non-seizure, a score of 1 indicates nodding of head, a score of 2 indicates intermittent seizures or sporadic shaking, a score of 3 indicates full seizure, Straub tail tail, hind legs, , Run, fall, score of 5 means intense seizure, death.

Example  One. Aminopeptidase  For the deficiency of P1 Xpnpep1 - / - Effects of mouse hyperactivity, anxiety level, motor function, learning disorder and memory

The effects of aminopeptidase P1 deficiency on mouse hyperactivity, anxiety level, motor function, learning disability and memory were examined.

Specifically, according to the method shown in Experimental Example 1-3, the behavior of Xpnpep1 ^{- / -} mice was observed in a groove-cage for 3 consecutive days. (9-11 weeks old) adult and Xpnpep1 ^{- / -} mice (wild type, WT), which are littermates. In addition, the presence and activity of anxiety behaviors of Xpnpep1 - / - mice were observed in open field test and high - cost plus maze test. In addition, probe testing and rotarod analysis were performed on each mouse. The results are shown in Figs. 1 to 10. Fig.

As shown in FIG. 1, the Xpnpep1 ^{- / -} mice showed a significantly higher moving distance (m) at night than in the daytime compared to the wild type (WT), indicating that hyperactivity was high. In addition, as shown in Fig. 2, in the groove-cage activity test, it was confirmed that Xpnpep1 ^{- / -} mice had a significantly higher moving distance (m) than the wild type (WT) .

Also, as shown in FIG. 3, an open field test was performed to confirm that Xpnpep1 - / - mice showed hyperactivity and no anxiety behavior. Specifically, Xpnpep1 - / - mice were confirmed to have increased motility activity during 1 hour of test as a result of confirming the movement distance measurement. In addition, the anxiety level due to the deficiency of aminopeptidase P1 was changed by confirming that the contact time (thigmotax (%)), which is the time spent close to the wall in the open field box, is similar to that of Xpnpep1 ^{- / -} Respectively.

In addition, as shown in Figs. 4 and 5, in the high-valued plus maze test, the Xpnpep1 ^{- / -} mice had no difference in the time taken to open the arm compared to the wild type (WT) And the number of close - arm accesses was significantly higher.

Also, as shown in FIG. 6, in the Morris water-lab analysis training, it was confirmed that Xpnpep1 - / - mice had a significantly higher escape latency time than the wild type (WT). As shown in FIG. 7, when swimming was conducted for 24 hours after 9 days of training, the swimming path of each mouse was checked. As a result, compared to the wild type (WT), the Xpnpep1 - / - I have confirmed that you are passing the non-part. As shown in FIG. 8, the time spent in each quadrant during the 60-second probe test was confirmed, and it was confirmed that the time to stay in the quadrant where the target was located was reduced in the Xpnpep1 - / - mouse as compared with the wild type (WT).

As shown in FIG. 9, the swimming speed was checked during the probe test. As a result, it was confirmed that the swimming speed of the Xpnpep1 - / - mouse was not significantly different from that of the wild type (WT).

Further, as shown in Fig. 10, in the rotarod analysis for the detection of movement disorder, the time taken from the accelerated rotation rod to fall was confirmed by confirming that the wild type (WT) and Xpnpep1 - / - mice were similar It was confirmed that there was no abnormality in exercise coordination and exercise learning.

From the above results, it was confirmed that the Xpnpep1 - / - mice of the present invention exhibited hyperactivity, and the motor function and anxiety level were normal.

Example  2. Aminopeptidase  For the deficiency of P1 Xpnpep1 - / - mouse EEGs (electroencephalograms) analysis and Pentylenetetrazole ( Pentylenetetrazole ) Identify the degree of seizure following injection

EEGs analysis of Xpnpep1 - / - mice against deficiency of aminopeptidase P1 and seizure severity following pentylenetetrazole injection were determined.

Specifically, in the wild-type (WT) and Xpnpep1 - / - mice, the spontaneous epileptic EEG rhythm was measured by the method shown in Example 1-2 above. Specifically, epileptic discharges were measured via epidural electrodes of each mouse. In addition, intraperitoneal injection of pentylenetetrazol (PTZ, 30 mg / kg) followed by a kindling criterion of Xpnpep1 - / - mice (convulsions, intermittent convulsions, strap tail responses, or jumps) The percentage achieved, the seizure score, the percentage of kindled mice, and the average seizure score. The results are shown in Fig. 11 to Fig.

As shown in FIGS. 11 to 13, it was confirmed that spontaneous epileptic activity appeared in the brain of Xpnpep1 - / - mice as compared with the wild type (WT). However, it was confirmed that the spontaneous epilepsy of Xpnpep1 - / - mice of the present invention is a seizure epileptic with no seizure.

14, behavioral changes of Xpnpep1 - / - mice after intraperitoneal injection of pentylenetetrazol (PTZ, 30 mg / kg) inducing epileptic seizure were examined. As a result, Xpnpep1- / - convulsant-induced seizures were observed in mice. As a result of seizure scoring, it was confirmed that seizure scores were significantly higher in Xpnpep1 - / - mice as compared to wild type (WT), as shown in Figs. 15 and 16.

In addition, as shown in Figs. 17 and 18, it was confirmed that the average seizure score was significantly higher in the Xpnpep1 +/- (heterotype) mouse than in the wild type (WT), and the proportion of the Kindled mouse was also higher.

From the above results, it was confirmed that the Xpnpep1 - / - mouse of the present invention is a model in which spontaneous epileptic activity occurs and that the sensitivity to seizure inducer is higher than that of wild type.

Example  3. Aminopeptidase  For the deficiency of P1 Xpnpep1 - / - Cognitive dysfunction and fear conditioning test in mice

The cognitive dysfunction of Xpnpep1 - / - mice against deficiency of aminopeptidase P1 was confirmed. In addition, a fear conditioning experiment was performed by contextual or cued signals.

Specifically, the cognitive function was tested in the same manner as in Example 1-3. In addition, panic conditioning tests by contextual or cued measures the movement distance and analyzed activity inhibition. The results are shown in Fig. 19 and Fig.

As shown in Fig. 19, it was confirmed that novel object recognition appears at a significantly lower rate in Xpnpep1 - / - mice as compared to wild type (WT). In addition, it was confirmed that the search time did not show a significant difference between the two groups.

In addition, as shown in Fig. 20, the fear conditioning by contextual means that there is no change in the activity of Xpnpep1 - / - mice after pre-CS (conditioned stimulus) and stimulation (CS) It was confirmed that the fear conditioning function due to space was significantly reduced. On the other hand, fear conditioning by auditory cue showed similar level of activity inhibition as wild type (WT).

As a result, it was confirmed that the Xpnpep1 - / - mice of the present invention had significantly lowered cognition, learning and memory functions as compared with the wild type (WT).

Claims (5)

Xpnpep1 (X-Prolyl Aminopeptidase 1) gene is knocked out and susceptibility to seizure inducer is increased,
An animal model for the screening of prophylactic or therapeutic substances against seizures not accompanied by seizures,
Wherein the animal model maintains motor coordination and motor learning capabilities and does not exhibit symptoms of anxiety behavior.
delete Knocking out an Xpnpep1 gene in an animal other than a human, comprising the steps of: (a)
Wherein said animal model is characterized by increased sensitivity to seizure inducing agents, ability to maintain motor coordination and motor learning, and no symptoms of anxiety behavior.
(1) administering a test substance to the animal model of claim 1 as an experimental group;
(2) measuring and comparing non-convulsions with the experimental group of (1) and the control animal model not treated with the test substance; And
(3) selecting the target substance in comparison with (2) the control group. 5. The method of claim 4,
Wherein the animal model is at least one selected from the group consisting of a mouse, a monkey, a dog, a cat, a rabbit, a guinea pig, a rat, a cattle, a sheep, a pig, and chlorine.

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