Application of TPP2 mutant zebrafish in ASD neurodevelopment-related disease model

Abstract

The application discloses application of a TPP2 mutant zebrafish in an ASD neurodevelopment-related disease model and belongs to the technical field of biological medicines. It is found for the first time that a TPP2 gene deletion type zebrafish has the following phenotypes: developmental retardation, developmental deformity, abnormal motor function, social dysfunction, stereotyped behavior, anxiety-like behavior and the like, has the core characteristics of ASD-related neurodevelopment-related diseases and can be used as an animal model for researching ASD neurodevelopment-related diseases, thereby providing an effective approach for diagnosing or treating ASD neurodevelopment-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of TPP2 mutant zebrafish as a model of ASD neurodevelopmental related diseases. Background Technology

[0002] TPP2 encodes tripeptidyl peptidase II (TPPI), a major exopeptidase in the cytoplasm responsible for degrading abnormal or ubiquitinated proteins and maintaining cellular protein homeostasis. It participates in antigen processing and regulates cytokine production, thereby influencing immune inflammatory responses. TPP2 also plays a role in lysosomal function; inactivated TPP2 leads to lysosomal accumulation, affecting intracellular protein degradation pathways. Studies have shown that TPP2 deficiency can lead to neurodegenerative diseases such as Alzheimer's disease (AD), with research primarily focused on mice.

[0003] Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder that differs fundamentally from neurodegenerative diseases. The two differ significantly in key dimensions such as onset time, core pathological mechanisms, clinical manifestations, and disease progression patterns. For example, their core pathological mechanisms differ. The core pathological mechanism of neurodegenerative diseases is the progressive and irreversible loss of neurons, often accompanied by abnormal protein aggregation (such as β-amyloid plaques and tau tangles in AD) and degenerative changes such as neurofibrillary degeneration. The core pathological mechanism of ASD, on the other hand, is abnormal network connectivity during neurodevelopment, including developmental disorders in neuronal migration, synapse formation and pruning, and neurotransmitter balance regulation. Its core is the "abnormal construction" of neural development, rather than the "degenerative loss" of mature neurons, and it lacks the progressive neuronal death pathological changes characteristic of neurodegenerative diseases.

[0004] Zebrafish have high reproductive capacity and low rearing costs, enabling large-scale experiments and meeting the needs of drug screening and molecular mechanism verification research. Furthermore, the behavioral characteristics of their juveniles and adults, such as locomotor ability, social behavior, and anxiety-like behavior, are mature and stable, effectively assessing neurodevelopmental and behavioral phenotypic abnormalities. This makes them an ideal model organism for studying ASD neurodevelopmental disorders mediated by TPP2 deletion. However, to date, no studies have confirmed whether TPP2 deletion leads to ASD neurodevelopmental disorders, and there are no reports on the use of TPP2 knockout zebrafish in the construction of ASD neurodevelopmental disease models, mechanism research, and the development of treatment plans. Summary of the Invention

[0005] This invention clarifies for the first time the key application value of TPP2 gene knockout zebrafish in the study of ASD neurodevelopmental related diseases, providing new technical tools and research ideas for the construction of models, exploration of pathogenesis and development of treatment plans for this type of disease.

[0006] The specific technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides the application of TPP2 mutant zebrafish in the preparation of ASD neurodevelopmental disease models, wherein the TPP2 gene in the TPP2 mutant zebrafish is deactivated.

[0008] The TPP2 mutant zebrafish exhibits a series of characteristics that closely match the core phenotypes of neurodevelopmental disorders: juveniles show significant developmental abnormalities, including delayed embryonic development, reduced embryonic survival rate, decreased embryonic hatching rate, and head deformities (primarily manifested as reduced body length, reduced head length, and reduced head area), accompanied by abnormal motor function (specifically, decreased basic motor ability under both light and dark conditions); adult fish show social dysfunction and typical behavioral abnormalities such as stereotyped behaviors and anxiety-like behaviors. These characteristics make this mutant zebrafish an ideal vehicle for constructing animal models of ASD-like neurodevelopmental disorders, providing a physiologically close experimental vehicle for in vivo research on the pathogenesis of ASD.

[0009] Preferably, in the above applications, the TPP2 mutant zebrafish refers to the mutant strain formed by the deletion of 8 bases at positions 195-202 of exon 1 of the zebrafish TPP2 gene.

[0010] More preferably, in the above applications, the TPP2 mutant zebrafish is a homozygous mutant, i.e. TPP2 - / - Mutant zebrafish. Understandably, heterozygous mutants (i.e.,...) TPP2 + / - The mutant zebrafish exhibits a phenotype similar to that of the homozygous mutant and can also serve as a model of neurodevelopmental diseases related to ASD.

[0011] Secondly, this invention provides the application of TPP2 mutant zebrafish as a model of ASD (Acute Disorder of Development) in screening or evaluating candidate drugs for the treatment of neurodevelopmental disorders, wherein the TPP2 gene in the TPP2 mutant zebrafish is absent.

[0012] In the above applications, the candidate drugs can improve at least one of the following symptoms: developmental malformations, decreased motor function, social dysfunction, stereotyped behaviors, and anxiety-like behaviors.

[0013] Preferably, in the above application, the TPP2 mutant zebrafish refers to the mutant species formed by the deletion of 8 bases at positions 195-202 of exon 1 of the zebrafish TPP2 gene; more preferably, the TPP2 mutant zebrafish is a homozygous mutant.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The TPP2 gene-deleted zebrafish model provided by this invention can not only accurately simulate the core pathological features and behavioral manifestations of ASD neurodevelopmental related diseases, but also build a key technology platform for the discovery of disease diagnostic biomarkers and the development of therapeutic drugs, which has important basic research value and clinical translation prospects. Attached Figure Description

[0016] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0017] Figure 1 This is a clinical case analysis of an ASD patient with a TPP2 gene mutation. A is the Sanger sequencing result of the TPP2 gene in this patient, and B is the brain imaging (MRI) result of this patient.

[0018] Figure 2 The above are the statistical results of zebrafish embryo survival rate after TPP2 deletion in Example 1; data are expressed as Mean ± SD, and ns indicates no difference. p <0.05、 p <0.001 indicates a significant difference.

[0019] Figure 3 The above are the statistical results of zebrafish embryo hatching rate after TPP2 deletion in Example 1; data are expressed as Mean ± SD, and ns indicates no difference. p <0.05、 p <0.01、 p <0.005、 p <0.001 indicates a significant difference.

[0020] Figure 4 The figures show the morphological changes in zebrafish juveniles after TPP2 deletion in Example 1. A is a measurement image of the zebrafish juvenile's side view under a microscope after imaging (body length can be measured); B is a top view of the zebrafish juvenile under a microscope (head and body length can be measured); C is a top view of the zebrafish juvenile under a microscope (head area can be measured by framing the head region); D is the normalized statistical result of the zebrafish juvenile's body length after TPP2 deletion; E is the normalized statistical result of the zebrafish juvenile's head length after TPP2 deletion; and F is the normalized statistical result of the zebrafish juvenile's head area after TPP2 deletion. Data are expressed as Mean ± SD. p <0.005, p <0.001 indicates a significant difference.

[0021] Figure 5 The data presents the basal kinetic changes in zebrafish juveniles after TPP2 deficiency in Example 2. A represents the statistical results of the movement rate of zebrafish juveniles under light conditions after different degrees of TPP2 deficiency, and B represents the statistical results of the movement rate of zebrafish juveniles under dark conditions after different degrees of TPP2 deficiency. Data are expressed as Mean ± SD, and ns indicates no difference. p <0.05、 p <0.01、 p <0.001 indicates a significant difference.

[0022] Figure 6 The results show the changes in social behavior of adult zebrafish after TPP2 deletion in Example 3. A represents the pattern diagram and heatmap of social interaction in the three-box environment; B represents the statistical results of the dwell time of adult zebrafish in Zone I (Social zone) after different degrees of TPP2 deletion; C represents the statistical results of the dwell time of adult zebrafish in Zone II (Middle zone) after different degrees of TPP2 deletion; and D represents the statistical results of the dwell time of zebrafish in Zone III (Far zone) after different degrees of TPP2 deletion. Data are expressed as Mean ± SD, and ns indicates no difference. p<0.05、 p <0.01 indicates a significant difference.

[0023] Figure 7 The results show the changes in stereotyped behavior of adult zebrafish after TPP2 deficiency in Example 3. A represents the normal and abnormal swimming trajectories of zebrafish; B represents the statistical results of the number of times small circling occurred after different degrees of TPP2 deficiency; and C represents the statistical results of the number of times walling occurred after different degrees of TPP2 deficiency. Data are expressed as Mean ± SD. p <0.05、 p <0.001 indicates a significant difference.

[0024] Figure 8 The results show the changes in anxiety behavior in adult zebrafish after TPP2 deficiency in Example 3. A represents the pattern and heatmap of diving in the new aquarium; B represents the statistical results of the time zebrafish spent in the upper zone after different degrees of TPP2 deficiency; C represents the statistical results of the time zebrafish spent in the bottom zone after different degrees of TPP2 deficiency; D represents the statistical results of the time zebrafish spent in the middle zone after different degrees of TPP2 deficiency; and E represents the statistical results of the average speed of zebrafish in the three zones after different degrees of TPP2 deficiency. Data are expressed as Mean ± SD, and ns indicates no difference. p <0.05、 p <0.01、 p <0.005 indicates a significant difference.

[0025] Figure 9 The table below shows the changes in aggression behavior of zebrafish after TPP2 deletion in Example 4. A represents the pattern and heatmap of mirror attack; B represents the statistical results of the swimming time of zebrafish in regions I and II after different degrees of TPP2 deletion; and C represents the statistical results of the swimming time of zebrafish in region III after different degrees of TPP2 deletion. Data are expressed as Mean ± SD, and ns indicates no difference. p <0.01、 p <0.005 indicates a significant difference. Detailed Implementation

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having” and any variations thereof are intended to cover non-exclusive inclusion.

[0027] In preliminary research, the inventors learned of a clinical case of an ASD patient with a TPP2 gene mutation from Wuhan Children's Hospital. With the consent of the patient's family, the inventors obtained the patient's genetic disease gene analysis report, Sanger sequencing results of the TPP2 gene, and MRI data. The patient's parents were phenotypically normal and had normal TPP2 genes, while the patient had a C→T mutation at position 3175 of exon 25 of the TPP2 gene on chromosome 13. Figure 1 A), causing premature termination of the protein at position 1059, is a pathogenic loss-of-function mutation; simultaneously, the patient's brain MRI ( Figure 1 B) Multiple small, patchy areas of long T1 and long T2 signal intensity were visible in the bilateral frontoparietal lobes, showing slightly high signal intensity on FLAIR sequences, suggesting demyelination. These results indicate that the TPP2 gene mutation in this ASD patient prematurely terminated the TPP2 protein function, and multiple demyelination changes were observed in the bilateral frontoparietal lobes of the brain. Therefore, the inventors hypothesize that the loss of TPP2 protein function due to TPP2 gene mutation may be related to neurodevelopmental disorders in ASD. Based on this hypothesis, this invention investigated the effects of constructing TPP2 mutant zebrafish (including homozygous and heterozygous TPP2-deficient zebrafish) on... TPP2 Different degrees of gene deletion are associated with neurodevelopmental disorders of ASD.

[0028] In the embodiments of this invention, the TPP2 mutant zebrafish used is a mutant strain formed by the deletion of 8 bases from positions 195 to 202 of exon 1 (nucleotide sequence shown in SEQ ID NO.1) of the zebrafish TPP2 gene; specifically, it can be targeted and knocked out using CRISPR-Cas9 technology, and the Cas9 target sequence is shown in SEQ ID NO.2. This base deletion directly causes the premature appearance of the stop codon during translation, forcing the translation process of the TPP2 protein to be interrupted, ultimately generating a truncated and nonfunctional abnormal TPP2 protein, thereby causing the complete loss of the normal biological function of the gene.

[0029] Some embodiments of this invention demonstrate that the TPP2 mutant zebrafish exhibit a series of characteristics highly consistent with the core phenotypes of neurodevelopmental disorders: juveniles show obvious developmental abnormalities, specifically including developmental delay, reduced embryonic survival rate, and decreased hatching rate; head deformities are mainly manifested as reduced body length, reduced head length, and reduced head area, accompanied by weakened basic motor abilities under both light and dark conditions; adults exhibit typical behavioral abnormalities such as social dysfunction and stereotyped behaviors. These results indicate that adult TPP2 mutant zebrafish can serve as an ideal vehicle for constructing animal models of ASD-like neurodevelopmental disorders, and this model can be used for screening therapeutic drugs for ASD, evaluating the effects of candidate drugs on improving developmental abnormalities, restoring motor function, and correcting social behavior, providing an efficient screening tool for the development of clinical therapeutic drugs.

[0030] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] The following examples use WT wild-type zebrafish and TPP2 homozygous zebrafish (i.e., TPP2 - / - The mutant zebrafish was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. The TPP2 heterozygous zebrafish used (i.e., TPP2 + / - Mutant zebrafish) from adults TPP2 - / - The mutant zebrafish was obtained by crossing it with wild-type WT zebrafish.

[0032] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] Example 1

[0034] This example verifies the effect of TPP2 deficiency on zebrafish development. The specific experiment is as follows:

[0035] (1) The effect of TPP2 deficiency on the survival rate and hatching rate of zebrafish embryos and juveniles.

[0036] Wild-type zebrafish embryos, TPP2 heterozygous deletion zebrafish embryos, and TPP2 homozygous deletion zebrafish embryos were obtained and tested in six-well plates, with 20 zebrafish embryos per well. The number of surviving and hatching embryos was counted every 12 hours, and dead embryos were removed to maintain water quality. Zebrafish embryos coagulated to a milky white color after death, appearing black under a microscope. Healthy embryos began to hatch at 48 hours, indicated by the successful emergence of the embryonic membrane from the head or tail of the larvae. After death, the coagulated embryos turned white, and the cessation of heartbeat could be observed under a microscope. Based on this standard, the number of surviving and hatching zebrafish embryos was counted at 0h, 12h, 24h, 36h, 48h, 60h, 72h, and 84h. The survival rate (survival rate = current surviving number / total number of embryos × 100%) and hatching rate (hatching rate = number of hatched embryos / current surviving number × 100%) were calculated using the following formulas.

[0037] The results showed that, compared with the control group (WT), TPP2 heterozygous deletion (tpp2) was significantly reduced. + / - ) and TPP2 homozygous deletion (tpp2) - / - The TPP2 deletion group (p<0.0001) had a significant impact on the survival rate of zebrafish. At 12h and 24h, compared with the control group (99.4%), the survival rates of both the heterozygous TPP2 deletion group (88.3%) and the homozygous TPP2 deletion group (69.1%) changed, with the downregulation in the homozygous TPP2 deletion group (p<0.0001) being more significant than that in the heterozygous TPP2 deletion group (p=0.038). From 36h to 84h, only the homozygous TPP2 deletion group (p<0.0001) showed a significant decrease in survival rate. Figure 2 Hatching rate chart (); Figure 3The results showed that zebrafish in the control group began hatching at 36 hours, while hatching in the TPP2 homozygous deletion group began at 60 hours. From 48 hours onwards, the hatching rate of TPP2 heterozygous deletion (p < 0.0001) was significantly increased compared to the control group. However, from 60 hours onwards, the hatching rates of both TPP2 heterozygous deletion (p = 0.0269) and TPP2 homozygous deletion (p < 0.0001) zebrafish embryos were significantly decreased, with the TPP2 homozygous deletion group having a hatching rate of 3.8%. The TPP2 heterozygous deletion group (85%) also showed a significant decrease (p < 0.0001). At 72 h, compared with the control group, the hatching rate of zebrafish embryos with TPP2 heterozygous deletion (p = 0.0018) and TPP2 homozygous deletion (p < 0.0001) was significantly decreased. Similarly, at 84 h, the hatching rate of zebrafish embryos with TPP2 heterozygous deletion (p = 0.0018) and TPP2 homozygous deletion (p < 0.0001) was also significantly decreased compared with the control group. By 84 h, almost all zebrafish embryos in all groups had hatched. After 84 h, unhatched zebrafish embryos were observed under an inverted microscope, and it was found that all unhatched zebrafish embryos had stopped beating after 84 h.

[0038] The above results indicate that the absence of TPP2 reduces the survival rate of zebrafish and delays and reduces hatching.

[0039] (2) The effect of TPP2 deficiency on developmental malformations in zebrafish.

[0040] To evaluate the impact of TPP2 deficiency on zebrafish development, the body length, head length, and head area of ​​zebrafish juveniles obtained in each group in step (1) were measured. Specifically, zebrafish from each group were anesthetized with 0.004% tricaine and photographed. Side view images were taken under an inverted microscope with the same magnification, and the deformities, such as changes in body length, head length, and head area, were analyzed using ImageJ software.

[0041] The results are as follows Figure 4 As shown in the normalized statistical graph of zebrafish juvenile body length, it can be seen that with the absence of TPP2, compared with the control group, the body length of both heterozygous TPP2-deficient zebrafish juveniles (p < 0.0001) and homozygous TPP2-deficient zebrafish juveniles (p < 0.0001) was significantly reduced. Figure 4 D); As can be seen from the statistical graph of zebrafish head length after normalization, compared with the control group, the head length of zebrafish juveniles was significantly reduced after TPP2 heterozygous deletion (p < 0.0001) and homozygous deletion (p < 0.0001). Figure 4 E); As can be seen from the normalized statistical graph of zebrafish head area, compared with the control group, the head area of ​​zebrafish juveniles was significantly reduced after TPP2 heterozygous deletion (p=0.0006) and homozygous deletion (p<0.0001). Figure 4 F).

[0042] The above results indicate that the absence of TPP2 causes a reduction in body length, head length, and head area in zebrafish during embryonic development and juvenile development, resulting in developmental abnormalities.

[0043] Example 2

[0044] This example tested the effect of TPP2 deficiency on the basic movement of zebrafish juveniles. The specific experiment is as follows:

[0045] Embryos from wild-type zebrafish (WT) and TPP2 heterozygous deficient zebrafish (tpp2) were obtained. + / - Embryos and TPP2 homozygous deficient zebrafish (tpp2) - / - Embryos were cultured to 7 days post-fertilization (dpf) and then placed in 24-well plates, with one zebrafish in each well. 500 μL of E3 culture medium was added to each well. The 24-well plates were placed under the camera of a DanioVision behavioral tracking system. The aperture and camera focus were adjusted to ensure adequate light intake and accurate focusing. The experimental environment was set to 28°C using a temperature control system. In the light-conditioning exercise experiment, the lighting was turned on and the brightness was set to 100%, allowing the zebrafish larvae to acclimatize for 90 minutes. The tracking instrument then recorded the zebrafish larvae's movement trajectory over 10 minutes to analyze their movement speed under light conditions. In the dark-conditioning exercise experiment, the lighting was turned off and the brightness was set to 0%, allowing the zebrafish larvae to acclimatize for 90 minutes. The tracking instrument then recorded the zebrafish larvae's movement trajectory over 10 minutes to analyze their movement speed under dark conditions.

[0046] The results are as follows Figure 5 As shown: Compared with the control group, only the TPP2 homozygous deletion group showed a significant decrease in motility under light conditions (p=0.0058). Figure 5 A); Under dark conditions, compared with the control group, both the TPP2 heterozygous deletion group (p < 0.0001) and the TPP2 homozygous deletion group (p < 0.0001) were significantly reduced ( Figure 5 B).

[0047] Example 3

[0048] This example tested the effects of TPP2 deletion on the behavior of adult zebrafish, including the following experiments:

[0049] (1) Measurement of social behavior.

[0050] Adult zebrafish with a 3-month post-fertilization period (mpf) were used as experimental materials, and the grouping was the same as in Example 2. A transparent acrylic fish tank with dimensions of 37×15×15 cm was used as the experimental setup. Figure 6 A) The aquarium is divided into three sections by a transparent acrylic panel. Opaque, removable inserts and slots are located at the junctions of the center and the two sides. The central 20 cm section is used to observe and record the behavioral patterns of the zebrafish being measured. The other two sides have water on one side and a school of zebrafish on the other, simulating different social environments. An experimental light panel provides uniform illumination and maintains the background environment. During the experiment, the opaque partition is first inserted to allow the zebrafish to adapt without knowing the surroundings. After 5 minutes of adaptation, the partition is removed. At this point, the zebrafish are positioned with a school of fish on one side and water on the other. A side-mounted camera records their behavior and preferences, with each individual recorded for 10 minutes.

[0051] The results are as follows Figure 6 As shown: as TPP2 decreased from heterozygous deletion to homozygous deletion, the social tendency of zebrafish was significantly reduced. Figure 6 A); Compared with the control group, the TPP2 heterozygous deletion group (p=0.036) and the TPP2 homozygous deletion group (p=0.0064) both had significantly reduced time spent in region I. Figure 6 B); In region II, compared with the control group, both the TPP2 heterozygous deletion group (p=0.0229) and the TPP2 homozygous deletion group (p=0.0180) were significantly upregulated ( Figure 6 C); while in region III, only the TPP2 homozygous deletion group (p=0.0081) showed a significant upregulation compared to the control group (C). Figure 6 D).

[0052] (2) Measurement of stereotyped behaviors.

[0053] Adult zebrafish, aged 3 months, were used as experimental subjects, with the same grouping as above. A transparent acrylic fish tank measuring 40×40×20cm was used as the open field experimental setup. During the experiment, a zebrafish was placed in the open field experimental tank and allowed to adapt for 3 minutes. Then, a camera tracked and recorded the zebrafish's trajectory over 5 minutes.

[0054] The results are as follows Figure 7 As shown: Figure 7The three images at the top of panel A show the normal swimming trajectories of zebrafish, while the three images at the bottom show abnormal swimming trajectories: small circling and walling. The statistical analysis of the small circling trajectories shows that, compared to the control group, the number of small circling occurrences was significantly higher in both the TPP2 heterozygous deletion group (p=0.0185) and the homozygous deletion group (p=0.0169). Figure 7 B); The statistical chart of zebrafish wall-feeding trajectories shows that, compared with the control group, the number of wall-feeding trajectories was significantly increased in the TPP2 heterozygous deletion group (p < 0.0001) and the homozygous deletion group (p < 0.0001). Figure 7 C).

[0055] (3) Measurement of anxiety behavior.

[0056] Adult zebrafish with a 3 mpf size were used as experimental subjects, with the same grouping as above. A transparent acrylic aquarium measuring 28×20×5 cm was used as the experimental setup. To ensure consistent experimental conditions, square light panels were installed on the back and bottom of the aquarium to provide uniform illumination and maintain a constant background environment. During the experiment, zebrafish were placed in the aquarium, and their behavior was immediately recorded using a side-mounted camera. Recording time for each individual was 10 minutes, and the experiment was conducted daily from 9:00 AM to 5:00 PM.

[0057] To quantify the behavioral characteristics of zebrafish, the aquarium was vertically divided into three areas: upper, middle, and lower. Figure 8 A), and statistically analyze the following behavioral data: (1) dwell time in each area; (2) latency period when first entering the middle and upper areas; (3) zebrafish stillness time. Quantitative analysis of these behavioral indicators can be used to assess anxiety-like behavior in zebrafish. Anxious zebrafish are more likely to stay at the bottom of the tank when entering a new environment and are less likely to explore the top of the tank.

[0058] Test results as follows Figure 8 As shown: Figure 8 The three images on the right (A) are heatmaps of the swimming trajectories of zebrafish in different groups. Compared with the control group, zebrafish in the TPP2 heterozygous deletion group and the TPP2 homozygous deletion group were more inclined to explore the bottom area of ​​the new aquarium. Statistical results of the zebrafish's dwell time in the three areas show that, compared with the control group, the dwell time in the upper areas of the TPP2 heterozygous deletion group (p=0.0013) and the TPP2 homozygous deletion group (p=0.0486) was significantly reduced. Figure 8 B); Compared with the control group, the TPP2 heterozygous deletion group (p=0.0046) and the TPP2 homozygous deletion group (p=0.0355) both showed a significantly increased time spent in the bottom region. Figure 8C); Compared with the control group, there was no difference in the time spent in the central region between the TPP2 heterozygous deletion group (p>0.9999) and the TPP2 homozygous deletion group (p=0.2241). Figure 8 D); Statistical results of the average speed of zebrafish in the three regions show that, compared with the control group, the TPP2 heterozygous deletion group (p=0.0077) and the TPP2 homozygous deletion group (p=0.0006) both showed a significant decrease ( Figure 8 E).

[0059] (4) Measurement of aggressive behavior.

[0060] Adult zebrafish that had reached 3 mpf were placed in a transparent acrylic aquarium measuring 18×4.5×15 cm as the experimental setup (grouping as above). The zebrafish were allowed 5 minutes to acclimatize (during which time the mirror was covered). After acclimatization, the cover was removed, and their behavior was recorded using a side-mounted camera for 10 minutes per individual. The aquarium was divided into three zones: the area within 1 cm of the mirror was the interaction / aggression zone, the area 1-4 cm from the mirror was the approach zone, and the area greater than 4 cm was the distance zone. The time the zebrafish spent in each zone was recorded.

[0061] Test results as follows Figure 9 As shown: Figure 9 A. The left side shows a schematic diagram of a mirror mold. A mirror is placed on the far left. The mold is divided into three areas: Area I, Area II, and Area III. Figure 9 The three images on the right are heatmaps of zebrafish swimming trajectories, representing the WT group, the TPP2 heterozygous deletion group, and the TPP2 homozygous deletion group, respectively. This indicates that, compared to the control group, zebrafish in the TPP2 heterozygous deletion group and the TPP2 homozygous deletion group tended to move away from the mirror. Statistical results of the zebrafish's swimming time in the three regions show that, compared to the control group, the time spent in regions I and II by the TPP2 heterozygous deletion group (p=0.0012) and the TPP2 homozygous deletion group (p=0.0007) was significantly reduced. Figure 9 B), but there was no significant difference in the swimming time in region III between the TPP2 heterozygous deletion group (p=0.7627) and the TPP2 homozygous deletion group (p=0.3888). Figure 9 C).

[0062] The above results indicate that TPP2 gene knockout can cause adult zebrafish to exhibit the ASD phenotype.

[0063] In summary, this invention is the first to discover that TPP2 deletion can cause developmental abnormalities and developmental delays in zebrafish, as well as basic motor defects in juvenile zebrafish and social defects, increased stereotyped behaviors, increased anxiety-like behaviors, and decreased aggression in adult zebrafish. This indicates that the TPP2 gene knockout zebrafish model can serve as a new ideal vector for studying animal models of ASD-like neurodevelopmental diseases.

[0064] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Application of TPP2 mutant zebrafish in the preparation of ASD disease model, wherein TPP2 mutant zebrafish refers to a homozygous mutant species formed by the deletion of 8 bases at positions 195-202 of exon 1 of the zebrafish TPP2 gene, and the nucleotide sequence of exon 1 is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The TPP2 mutant zebrafish exhibited developmental abnormalities, including at least one of delayed embryonic development, decreased survival rate, decreased embryonic hatching rate, and head malformation.

3. The application according to claim 1, characterized in that, The TPP2 mutant zebrafish exhibited decreased locomotor function.

4. The application according to claim 1, characterized in that, The TPP2 mutant zebrafish exhibited social dysfunction as well as stereotyped, anxiety-like, and aggressive behaviors.

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