A method for constructing a mouse model of autism spectrum disorders and applications thereof

Abstract

The application belongs to the technical field of animal experiment models, and discloses a method for constructing a mouse model of autism spectrum disorder and application. Pregnant mice are orally exposed to different doses of 1-NP every day during the whole gestation period, the pregnant mice naturally give birth, and the offspring mice are evaluated for autism-like behaviors at PND28 after birth by using a three-chamber social experiment. The offspring mice are evaluated for autism-like behaviors at PND70 after birth by using a three-chamber social experiment. Maternal 1-NP exposure during pregnancy delays the migration of intermediate neurons in the forebrain of fetal mice, and the autism-like behaviors. 1-NP exposure during pregnancy down-regulates the expression of intermediate neuron migration-related genes by reducing DNA hydroxymethylation of the intermediate neuron migration-related genes. 1-NP exposure during pregnancy damages the synthesis of mitochondrial tricarboxylic acid cycle alpha-ketoglutarate (alpha-KG). Maternal alpha-KG supplementation during pregnancy improves the migration of intermediate neurons in 1-NP exposed fetal mice and the autism-like behaviors of offspring mice.

Description

Technical Field

[0001] This invention belongs to the field of animal experimental model technology, and particularly relates to a method for constructing a mouse model of autism spectrum disorder. Background Technology

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social impairments and stereotyped behaviors. In recent years, the prevalence of ASD in developed countries has increased from 0.76% to 1-2.5%. In my country, the prevalence is approximately 0.7%-1%. Numerous studies have shown that exposure to environmental toxins during pregnancy may be one of the reasons for the rapid increase in ASD prevalence. Several cohort studies have found that maternal exposure to motor vehicle exhaust during pregnancy increases the risk of ASD in children. 1-Nitropyrene (1-NP) is a representative nitropolycyclic aromatic hydrocarbon, mainly derived from diesel engine exhaust. Animal studies have found that exposure to 1-NP in late pregnancy is associated with reduced learning and memory abilities in offspring. Simultaneously, exposure to 1-NP throughout pregnancy induces anxiety-like behaviors in adult offspring. Although these neurological disorders are associated with maternal exposure to 1-NP during pregnancy, whether maternal exposure to 1-NP during pregnancy induces autism-like behaviors in offspring remains unclear. Mounting evidence suggests that interneurons in the medial prefrontal cortex (mPFC) play a crucial role in the regulation of social behavior. Population studies have found reduced interneurons in the mPFC of patients with ASD. In rodents, interneurons originate from ganglion eminences and migrate tangentially to the neocortex during late pregnancy. Two recent studies have shown that delayed interneuron migration induces autism-like behavior in mice. Multiple protein molecules, including neuregulin 1 (Nrg1) and semaphorin 3F (Sema3F), are involved in interneuron migration. Several studies have shown that hydroxymethylation modification is involved in the migration of placental trophoblast cells and neuronal stem cells. Our previous research found that the Nrg1 gene can regulate transcription through DNA hydroxymethylation modification. Therefore, we hypothesize that 1-NP exposure during pregnancy inhibits interneuron migration by reducing hydroxymethylation modification of genes related to interneuron migration.

[0003] Based on the above analysis, the existing technology has the following problems and shortcomings: It is necessary to construct a mouse model of autism spectrum disorder and reveal the role of DNA hypohydroxymethylation-induced interneuron migration delay in the induction of autism in offspring by environmental toxins during pregnancy. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a method for constructing a mouse model of autism spectrum disorder.

[0005] This invention is implemented as follows: a method for constructing a mouse model of autism spectrum disorder, wherein pregnant mice are orally exposed to different doses of 1-NP (0, 10, 100 μg / kg) daily throughout the entire pregnancy, the pregnant mice give birth naturally, and the pups are assessed for autism-like behavior during the weaning period using a three-box social test at 28 days postnatal day (PND); at PND70, the three-box social test is used to assess autism-like behavior in adult offspring;

[0006] In the 1-NP group, pregnant mice received different doses (0, 10, 100 μg / kg) of 1-NP; in the 1-NP group, pregnant mice were orally exposed to 100 μg / kg of 1-NP daily from the day of gestation GD0 to GD17.

[0007] The present invention also provides a method for constructing a mouse model of autism spectrum disorder, the method comprising:

[0008] a) Pregnant mice were randomly divided into different groups and administered different doses of 1-nitropyrene (1-NP) by gavage from day 0 of gestation (GD0) to day 17 of gestation (GD17); the control group was administered corn oil by gavage.

[0009] b) After the pregnant mice gave birth naturally, the pups were collected after weaning (PND28) and on day 70 after birth (PND70) for behavioral assessment. The three-box social test was used to assess the autism-like behaviors of the pups.

[0010] c) Extract tissue from the medial prefrontal cortex (mPFC) of pups for immunofluorescence, Western blotting, and whole-cell patch-clamp experiments to analyze the effect of gestational 1-NP exposure on inhibitory interneurons in pups' mPFCs.

[0011] d) Extract fetal rat forebrain or prefrontal cortex for immunofluorescence, Western blotting and RT-PCR to analyze the effect of gestational 1-NP exposure on fetal rat inhibitory interneurons;

[0012] e) Extract fetal rat forebrain for DNA hydroxymethylation level detection, high performance liquid chromatography-tandem mass spectrometry, transmission electron microscopy, TET enzyme activity detection and transcriptomics analysis to explore the mechanism of 1-NP exposure during pregnancy induced delayed migration of inhibitory interneurons in fetal rats.

[0013] f) Pregnant mice were randomly divided into a control group, an α-KG group, a 1-NP group, and a 1-NP+α-KG group. The control group was given corn oil by gavage, the α-KG group and the 1-NP+α-KG group were given 2 mg / kg of α-KG, and the 1-NP group and the 1-NP+α-KG group were given 100 μg / kg of 1-NP by gavage. All treatments were given from GD0 to GD17. After natural parturition, pups were collected after weaning (PND28) for autism-like behavior assessment to explore the protective effect of α-KG supplementation during pregnancy on autism-like behavior induced by 1-NP in pups.

[0014] Furthermore, a method for evaluating the effect of gestational 1-NP exposure on the migration of inhibitory interneurons in fetal rats was employed, the method comprising:

[0015] a) Pregnant mice were randomly divided into different groups and administered different doses of 1-NP by gavage from day 0 of gestation to day 17 of gestation (GD17) or day 13 of gestation.

[0016] b) Sacrifice pregnant mice at specific time points and collect forebrain or medial prefrontal cortex (mPFC) tissue from fetal mice;

[0017] c) Immunohistochemistry, Western blotting, immunofluorescence, transcriptome analysis, RT-PCR, DNA hydroxymethylation modification, and TETs enzyme activity detection were performed on the collected tissues to evaluate the effect of 1-NP exposure on the migration of fetal rat inhibitory interneurons.

[0018] Furthermore, a method for evaluating the protective effect of α-ketoglutarate (α-KG) supplementation during pregnancy on the reduction of inhibitory interneurons in the medial prefrontal cortex induced by 1-NP and on autism-like behaviors in offspring was employed. This method includes:

[0019] a) Pregnant mice were randomly divided into a control group, an α-KG group, a 1-NP group, and an α-KG+1-NP group. The pregnant mice in the 1-NP group and the α-KG+1-NP group were given 1-NP by gavage every day from day 0 of gestation (GD0) to a specific number of days. The pregnant mice in the α-KG group and the α-KG+1-NP group were given α-KG by gavage at the same time.

[0020] b) Collect medial prefrontal cortex (mPFC) tissue from offspring mice for Western blotting, immunofluorescence and whole-cell patch-clamp experiments to assess the effect of α-KG on the 1-NP-induced reduction in the number of inhibitory interneurons in offspring mPFC.

[0021] c) The three-box social experiment was used to assess autism-like behavior in offspring mice to evaluate the protective effect of α-KG against 1-NP-induced autism-like behavior in offspring.

[0022] The present invention also provides a method for constructing a mouse model of autism spectrum disorder, comprising the following steps:

[0023] a) Select pregnant mice and administer a specific dose of 1-NP orally daily throughout the pregnancy;

[0024] b) During the weaning period after birth (PND28), the three-box social test was used to assess the social behavior and autism-like behavior of the pups;

[0025] c) The three-box social test was used again to assess autism-like behaviors in mice at adulthood (PND70);

[0026] d) The 1-NP exposure dose and exposure time of the pregnant mice were determined according to the specific experimental design to ensure that the characteristics of autism-like behavior could be simulated.

[0027] Furthermore, a) all pregnant mice gave birth naturally to maintain the naturalness and validity of the experiment;

[0028] b) Pregnant mice were exposed to a specific dose of 1-NP by gavage daily during a specific time window during pregnancy;

[0029] c) Assess autism-like behaviors in mice at different life stages using a three-box social experiment to verify the long-term effects of 1-NP exposure on mouse behavior;

[0030] d) Mouse models constructed using this method can be used for basic research and drug testing of autism spectrum disorders, providing an important tool for the research and treatment of autism.

[0031] The present invention also provides a method for constructing a mouse model of autism spectrum disorder.

[0032] The present invention also provides the application of a mouse model of autism spectrum disorder in screening drugs for autism spectrum disorder.

[0033] The present invention also provides a method for assessing the effects of gestational 1-NP exposure on the medial prefrontal inhibitory synaptic transmission and number in offspring, the method comprising:

[0034] a) Pregnant mice were randomly divided into different groups and administered 100 μg / kg 1-NP by gavage daily from gestational day (GD0) to GD17.

[0035] b) Extracted mPFC tissue from rat pups for immunofluorescence, Western blotting, and whole-cell patch-clamp experiments to analyze the effects of 1-NP exposure on synaptic transmission and number of inhibitory interneurons in rat mPFC.

[0036] This invention also provides a method for evaluating the effect of gestational 1-NP exposure on the migration of inhibitory interneurons in the forebrain of fetal rats, the method comprising:

[0037] a) Pregnant mice were randomly divided into different groups and administered 100 μg / kg 1-NP by gavage daily from GD0 to GD17 or to GD13.

[0038] b) At specific time points (such as GD14 or GD18), pregnant mice are euthanized and fetal forebrain or mPFC tissue is collected.

[0039] c) Immunohistochemistry, Western blotting, immunofluorescence, transcriptome analysis, RT-PCR, DNA hydroxymethylation modification, and TETs enzyme activity detection were performed on the collected tissues to evaluate the effect of 1-NP exposure on the migration of fetal rat inhibitory interneurons.

[0040] This invention also provides a method for evaluating the protective effect of alpha-ketoglutarate (α-KG) supplementation during pregnancy against 1-NP-induced reduction in the number of inhibitory interneurons in the medial prefrontal cortex and autism-like behaviors in offspring, the method comprising:

[0041] a) Pregnant mice were randomly divided into a control group, an α-KG group, a 1-NP group, and an α-KG+1-NP group. The pregnant mice in the 1-NP group and the α-KG+1-NP group were given 1-NP by gavage daily from day 0 of gestation (GD0) to a specific number of days (such as GD13 or GD17). The pregnant mice in the α-KG group and the α-KG+1-NP group were given α-KG by gavage at the same time.

[0042] b) Medial prefrontal cortex tissues from offspring mice were collected for Western blotting, immunofluorescence, and whole-cell patch-clamp experiments to assess the effects of α-KG on 1-NP-induced synaptic transmission impairment and reduced number of inhibitory interneurons in offspring mPFC.

[0043] c) The three-box social experiment was used to assess autism-like behavior in offspring mice to evaluate the protective effect of α-KG against 1-NP-induced autism-like behavior in offspring.

[0044] Another object of the present invention is to provide a mouse model of autism spectrum disorder for use in screening drugs for autism spectrum disorder.

[0045] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0046] First, this invention provides a mouse model of autism spectrum disorder. Studies have shown that maternal exposure to 1-NP during pregnancy induces autism-like behavior in offspring by delaying the migration of interneurons in the forebrain of fetal mice. Simultaneously, studies have shown that 1-NP exposure during pregnancy delays interneuron migration through an epigenetic reprogramming mechanism that reduces DNA hydroxymethylation of genes related to interneuron migration, thereby inhibiting their expression.

[0047] Secondly, the method for constructing a mouse model of autism spectrum disorder proposed in this invention aims to address the current lack of effective and reliable animal models in autism research. Through specific prenatal drug interventions and subsequent behavioral assessments, this method can simulate autism-like behaviors, thereby providing an animal model that more closely resembles the pathophysiological process of human autism.

[0048] From a technical perspective, this invention has the following significant features:

[0049] First, by exposing mice to the environmental toxin 1-NP during pregnancy, we simulated the environmental factors that contribute to the development of autism. This exposure method is highly reproducible and controllable, ensuring consistent conditions for each experiment, thereby improving the accuracy and reliability of the experimental results.

[0050] Secondly, this invention utilizes a three-box social test to assess autism-like behavior in mice. This testing method can quantify key indicators such as social interaction, exploratory behavior, and spatial memory in mice, thereby comprehensively evaluating their autism-like behavioral performance.

[0051] Next, this invention discovered that 1-NP exposure induces hypohydroxymethylation of genes related to interneuron migration in fetal mice. This method reveals the cause of autism-like behavior induced by 1-NP from the perspective of epigenetic reprogramming mechanisms of brain development.

[0052] Next, this study found that supplementation with the DNA hydroxymethyltransferase cofactor α-KG reversed autism-like behaviors in offspring induced by gestational 1-NP exposure. This approach has clinical and preventative significance, laying the theoretical foundation for the clinical application of α-KG in preventing developmental disorders caused by environmental toxins.

[0053] Furthermore, this invention emphasizes the natural parturition process of pregnant mice, which helps to eliminate the influence of the delivery method on the experimental results and makes the model closer to actual physiological conditions.

[0054] In terms of technical effectiveness, the autism mouse model constructed by this invention has the following advantages:

[0055] First, the model exhibits high stability. Due to the use of standardized methods for pregnancy drug intervention and behavioral assessment, the model demonstrates high consistency under different experimental conditions, which is beneficial for subsequent research and application.

[0056] Secondly, the model is highly reliable. It can simulate autism-like behaviors, including core symptoms such as social impairment, repetitive behaviors, and difficulties in language communication, thus providing an effective tool for studying the pathogenesis and treatment of autism.

[0057] Third, it has broad application prospects. This model can be used not only for basic research on autism, but also for research in drug screening, gene therapy, and environmental intervention, providing new ideas and directions for the clinical treatment of autism.

[0058] The method for constructing a mouse model of autism spectrum disorder proposed in this invention, through innovative prenatal drug intervention and behavioral assessment, has successfully constructed a stable, reliable, and promising animal model, providing a new and powerful tool for the research and treatment of autism. Attached Figure Description

[0059] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0060] Figure 1 This is a schematic diagram of the experimental protocol for animal 1-NP exposure and α-KG intervention provided in the embodiments of the present invention.

[0061] Figure 2 This invention relates to the effect of 1-NP exposure during pregnancy on autistic-like behaviors in offspring during weaning, as provided in this embodiment.

[0062] Figure 3 This invention relates to the effect of gestational 1-NP exposure on medial prefrontal cortex (mIPSC) in weaned offspring, as provided in this embodiment.

[0063] Figure 4 This invention provides an embodiment of the 1-NP exposure during pregnancy to GAD67 in the medial prefrontal cortex of weaned offspring. + The influence of interneurons.

[0064] Figure 5 This invention relates to the effect of gestational 1-NP exposure on the migration of interneurons in the forebrain of fetal rats, as provided in this embodiment.

[0065] Figure 6This invention relates to the effect of gestational 1-NP exposure on hydroxymethylation of genes related to interneuron migration in the forebrain of fetal rats, as provided in this embodiment.

[0066] Figure 7 This invention relates to the effect of gestational 1-NP exposure on mitochondrial function in the forebrain of fetal mice, as provided in this embodiment.

[0067] Figure 8 This invention relates to the effects of α-KG supplementation during pregnancy on hypohydroxymethylation of genes related to interneuron migration induced by 1-NP in fetal mice and the delayed migration of interneurons in the forebrain of fetal mice.

[0068] Figure 9 This invention relates to the effects of α-KG supplementation during pregnancy on interneurons in the medial prefrontal cortex of offspring during weaning, as provided in this embodiment.

[0069] Figure 10 This invention relates to the effects of α-KG supplementation during pregnancy on 1-NP-induced medial prefrontal cortex mIPSC transmission disorders and autism-like behaviors in offspring. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0071] The following are two specific embodiments provided by the present invention, describing how to construct a mouse model of autism spectrum disorder according to the above claims:

[0072] Example 1:

[0073] 1. Selection and treatment of pregnant mice:

[0074] Before the experiment began, a group of healthy pregnant mice were selected, and their environment and feeding conditions were ensured to be suitable during the experiment.

[0075] According to claims 3 and 4, these pregnant mice were exposed to 100 μg / kg of 1-NP treatment by gavage daily from pregnancy GD0 to GD17.

[0076] 2. Behavioral testing:

[0077] At the weaning period (PND28) after birth, the first three-box social test was performed on the weaned infants to assess social behavior and autism-like behavioral characteristics.

[0078] These mice were observed until adulthood (PND70), and the three-box social test was performed again to assess their autism-like behavior in adulthood.

[0079] 3. Analysis and Recording:

[0080] Record and analyze test results at two time points to determine the long-term effects of 1-NP exposure on social behavior in mice.

[0081] Example 2:

[0082] 1. Selection and treatment of pregnant mice:

[0083] Another group of healthy pregnant mice was selected, and all mice were raised in a suitable environment.

[0084] According to claims 3 and 5, these pregnant mice were exposed to 100 μg / kg of 1-NP by gavage daily from GD0 to GD13.

[0085] 2. Behavioral testing:

[0086] Similar to Example 1, the pups underwent their first three-box social test at PND 28 days to assess autism-like behaviors during the weaning period.

[0087] At PND70, adult mice underwent a second three-box social test to further assess autism-like behaviors.

[0088] 3. Observation and recording:

[0089] The results of the three-compartment tests at PND28 and PND70 were recorded in detail, and the effects of 1-NP treatment on mouse behavior were compared and analyzed.

[0090] Through the above embodiments, researchers can effectively construct and evaluate mouse models of autism spectrum disorder, which can contribute to further biomedical research and the development of potential treatment strategies.

[0091] The material method used in this invention:

[0092] Anti-GAD67 (198013) was purchased from Synaptic Systems, Germany. Anti-NeuN (ab177487), anti-TET1 (ab191698), anti-TET2 (ab213369), anti-TET3 (ab139311), anti-SDHB (ab175225), and anti-vATP5A (ab110413) were purchased from Abcam, USA. 1-NP (N22959) and α-ketoglutarate (α-KG, No. K1128) were purchased from Sigma Aldrich, Germany. Nucleoprotein extraction reagent (78833) was purchased from Thermo Fisher Scientific, USA. TET enzyme activity assay kit (P-3086-96) was purchased from Epigentek, USA. All other reagents were purchased from Sigma Aldrich, Germany.

[0093] Animal experiments of this invention:

[0094] In this study, the animal experiments were divided into five parts. The animals used in the study complied with the regulations of the Biomedical Ethics Committee of Anhui Medical University (ethics approval number: LLSC20170498).

[0095] Experiment 1: To investigate the effects of 1-NP exposure throughout pregnancy on autistic-like behaviors in offspring, 30 pregnant mice were randomly divided into three groups. From GD0 to GD17, mice were administered different doses of 1-NP (0, 10, and 100 μg / kg) daily via gavage. The 1-NP exposure doses were based on previous studies conducted by our research group. All pregnant mice were delivered naturally. Some pups were assessed for autistic-like behaviors in weaned offspring using a three-box social test at PND28.

[0096] Furthermore, some pups were assessed for autism-like behaviors in adult offspring using a three-box social experiment at PND 70 days after birth.

[0097] Experiment 2: To investigate the effects of 1-NP exposure throughout pregnancy on inhibitory interneurons in offspring mPFC, 20 pregnant mice were randomly divided into two groups. From GD0 to GD17, the pregnant mice were administered different doses of 1-NP (0, 100 μg / kg) via gavage daily. All pregnant mice were delivered naturally. At PND28 after birth, some weaned pups were euthanized, and the medial prefrontal cortex was collected for whole-cell patch-clamp, immunofluorescence, and Western blotting experiments. Furthermore, at PND70 after birth, adult pups were euthanized, and the medial prefrontal cortex was collected for whole-cell patch-clamp experiments.

[0098] Experiment 3: To investigate the effect of 1-NP exposure during pregnancy on the migration of inhibitory interneurons in fetal rats, 10 pregnant rats were randomly divided into two groups. From GD0 to GD17, the pregnant rats were administered different doses of 1-NP (0, 100 μg / kg) via gavage daily. On GD18, all pregnant rats were sacrificed, and fetal mPFCs were collected for immunohistochemistry and western blotting experiments. GD14 is a critical period for the tangential migration of inhibitory interneurons from the ganglion eminence to the cortex (Sun et al., 2021). Therefore, another 20 pregnant rats were randomly divided into two groups, and from GD0 to GD13, the pregnant rats were administered different doses of 1-NP (0, 100 μg / kg) via gavage daily. Fetal rat forebrain was collected for western blotting, immunofluorescence, transcriptome analysis, RT-PCR, DNA hydroxymethylation modification, and TETs enzyme activity detection.

[0099] Experiment 4: To investigate the protective effect of α-KG supplementation during pregnancy on the delayed migration of inhibitory interneurons in the fetal rat forebrain induced by 1-NP. Twenty pregnant rats were randomly divided into four groups: control group (Ctrl), α-KG group, 1-NP group, and α-KG+1-NP group. In the 1-NP and 1-NP+α-KG treatment groups, pregnant rats were administered 100 μg / kg 1-NP by gavage daily from GD0 to GD13. In the α-KG and 1-NP+α-KG groups, pregnant rats were administered 2 g / kg α-KG by gavage daily from GD0 to GD13 (Mehra et al., 2016). On GD14, all pregnant rats were sacrificed, and the fetal forebrain was collected for DNA hydroxymethylation modification, RT-PCR, and immunofluorescence experiments.

[0100] Experiment 5: To investigate the protective effect of α-KG supplementation during pregnancy against the reduction in the number of inhibitory interneurons in the mPFC of offspring induced by 1-NP and autism-like behavior, 40 pregnant mice were randomly divided into four groups: control group (Ctrl), α-KG group, 1-NP group, and 1-NP+α-KG group. In the 1-NP and 1-NP+α-KG treatment groups, pregnant mice were administered 100 μg / kg 1-NP by gavage daily from GD0 to GD17. In the α-KG and 1-NP+α-KG groups, pregnant mice were administered 2 g / kg α-KG by gavage daily from GD0 to GD17. All pregnant mice gave birth spontaneously. The mPFCs of offspring mice were collected for Western blotting, immunofluorescence, whole-cell patch-clamp, and three-box social behavior experiments.

[0101] Behavioral evaluation of this invention:

[0102] The three-chamber socialization experiment assesses autism-like behaviors in mice. The apparatus consists of three identical chambers connected by doors, allowing mice free movement between them. The assessment of autism-like behaviors comprises three phases. First, a test mouse is placed in the central chamber and allowed to freely explore and adapt to the three chambers for 10 minutes. Next, two cages are placed in fixed positions in the side chambers. One cage contains a strange mouse (strange 1, S1) of the same age and sex as the test mouse, while the other cage is empty (E). The test mouse is allowed to move freely in the three chambers for 10 minutes, and the sniffing time in S1 and E is recorded using behavioral software. Finally, another strange mouse (strange 2, S2) is placed in the empty cage. The test mouse is allowed to move freely in the three chambers for 10 minutes, and the sniffing time of the test mouse towards mice S1 and S2 is recorded using behavioral software.

[0103] Immunofluorescence of this invention:

[0104] After anesthetizing, pups were perfused with 4% paraformaldehyde. Whole brain tissue was fixed in 4% paraformaldehyde for 24 h and dehydrated in 30% sucrose solution for 48 h. Brain tissue was embedded in OCT and cut into 30 µm thick slices in the coronal region. Approximately three regions of mPFC brain slices from each pup were randomly selected for immunofluorescence experiments. First, the slices were blocked with goat serum at room temperature for 1 hour, then incubated with GAD67 (1:1000) and NeuN (1:1000) antibodies at 4°C for 24 hours. Then, the slices were stained with the corresponding secondary antibodies. Finally, the nuclei were stained with Choechst 33342 and mounted with neutral resin. For fetal rats, after tissue collection, the brain was cut into 40 µm thick slices in the coronal region. Approximately three regions of ganglion elevation were randomly selected and incubated with GAD67 (1:1000) antibody as the primary antibody. Cell nuclei were stained with Hoechst 33342. GAD67 was analyzed using the panoramic tissue cell quantitative analysis system (TissueFAXS Plus S). + Neuron and NeuN + The number of neurons.

[0105] Mitochondrial morphology detection in this invention:

[0106] The ganglion ridges of fresh fetal rat forebrains were harvested and cut into 1 mm thick slices. The slices were dehydrated in acetone at room temperature and fixed with 1% osmium tetroxide at 4°C. After infiltration and embedding in embedding medium and propylene oxide, the slices were further sectioned and stained with uranium acetate. Transmission electron microscopy (Talos L120CG2) was used to photograph the mitochondria of nerve cells. The mitochondrial circumference and percentage of abnormal mitochondria were calculated using ImageJ.

[0107] Whole-cell electrophysiology in mouse mPFC of this invention:

[0108] Fresh brain tissue was prepared into 300 μm thick coronal sections in a 6% cold sucrose solution. Sections containing mPFCs were transferred to artificial cerebrospinal fluid filled with 95% O2 and 5% CO2 for 1 hour. Tetrodotoxin was used to block action potentials. 6-Cyano-7-nitroquinoxaline-2,3-dione was used to block AMPA receptors. Amino-5-phosphatidylvaleric acid was used to block N-methyl-D-aspartate receptors. mIPSCs of mPFC pyramidal neurons were detected in voltage-clamp mode. The frequency and amplitude of mPFC pyramidal neurons were analyzed using Mini60 software.

[0109] Measurement of 5hmC in this invention:

[0110] APOBEC-coupled epigenetic sequencing (ACE-Seq) is a novel technique for detecting 5hmC. First, 5hmC is converted to 5ghmC using T4 phage β-glucosyltransferase. Second, APOBEC3A (A3A) specifically replaces the amino group of "C" and "5mC" with "U". Since 5ghmC cannot be recognized by A3A and is detected as "C", this distinguishes "5hmC" in the target gene from "C" and "5mC" (Song et al. 2022). The multivariate PCR sequence of the target gene is shown in Table 1.

[0111] Table 1 Primers for Multiplex PCR in measurement of 5hmC

[0112]

[0113] This invention relates to the detection of TET enzyme activity:

[0114] After necropsy of pregnant mice, fetal forebrain tissue was collected and placed in pre-cooled PBS. The forebrain tissue was minced, and nuclear proteins were extracted using the Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, USA). After extraction, all samples were adjusted to the same concentration for later use. The activity of TET enzyme was detected using the Epigenase™ 5hmCHydroxylase TET activity / inhibition enzyme kit. TET enzyme activity = (sample OD value - blank well OD value) / (sample nuclear protein amount × enzymatic reaction time) (Lv et al. 2022).

[0115] The detection of α-KG levels in this invention:

[0116] After necropsy of pregnant mice, fetal forebrain tissue was collected. The forebrain tissue was lysed using a lysis buffer (methanol:acetonitrile:water mixture = 2:2:1), and the supernatant was collected by centrifugation. The supernatant was concentrated under nitrogen and then analyzed by LC-MS / MS. LC-MS / MS mobile phase A: ultrapure water containing 0.2% formic acid; mobile phase B: acetonitrile. The elution conditions for mobile phase B were: 0–3 min: 0–10% B, 6–8 min: 50% B, 8–10 min: 30% B, 10–12 min: 3% B (Lv et al. 2022).

[0117] RNAseq of this invention:

[0118] Total RNA was extracted from the samples using TRIzol reagent. RNA-seq libraries were constructed using the total RNA from the VAHTS mRNA-seq V3 library. Quality assessment was performed using BioAnalyzer and the Qubit database. All libraries were sequenced on an Illumina NovaSeq 6000 system. After quality control, sequence data were processed with STAR to generate read alignments. Genes with P < 0.05 and a Fold change of ≥ 1.5 were considered differentially expressed genes by DESeq2 analysis. To understand the potential roles of differentially expressed genes, GO, KEGG, and Reactome enrichment analyses were performed using the clusterProfiler R package.

[0119] RNA extraction and RT-PCR:

[0120] Total RNA was extracted from brain tissue using the Trizol method. The Promega reverse transcription kit (A3500) was used to reverse the total RNA into cDNA. The LightCycler 480 SYBR Green I kit (Roche) was used for RT-PCR detection. Primer sequences for the target gene were synthesized by Shanghai Sangon Biotech. A list of primers targeting specific genes is shown in Table 2.

[0121] Table 2 Primers for RT-PCR

[0122]

[0123] Western blotting of this invention:

[0124] RIPA lysis buffer was used to lyse fetal rat forebrain or adult male rat hippocampus tissue, and the supernatant was collected by centrifugation. Protein was extracted using the BCA method, and different samples were quantified to the same concentration for later use. Immediately before use, 20-30 μg of total protein was pipetted, and proteins of different molecular weights were separated by SDS-PAGE. The separated proteins were transferred to a PVDF membrane. Specific antibodies against the target protein and their corresponding secondary antibodies were used to bind the target protein in the PVDF membrane. An ECL detection kit was used to detect the target bands. ImageJ was used to detect the grayscale values ​​of the protein bands.

[0125] Statistical analysis in this invention: In the two-group experiments, t-tests were used for normally distributed data and data with homogeneous variance. Corrected t-tests were used for normally distributed data and data with heterogeneous variance. In the four-group experiments, two-way ANOVA and the Bonferroni test were used to analyze differences. A 2-1 -ΔΔCt The RT-PCR results were analyzed using the method.

[0126] Results of the Invention

[0127] (1) Effects of 1-NP exposure during pregnancy on autistic-like behaviors in offspring

[0128] In social behavior tests, experimental mice typically exhibited a preference for interacting with the unfamiliar mouse S1, spending more time sniffing S1 than in an empty cage (Fig. 2A and G). In social novelty preference tests, experimental mice typically exhibited a preference for interacting with the new unfamiliar mouse S2, spending more time sniffing S2 than with S1 (Fig. 2D and J). This study found that while male weaning mice in the high-dose group showed a preference for interacting with S1, this was not observed in the low-dose group (Fig. 2B). On the other hand, female weaning mice in the high-dose group did not show a preference for interacting with the unfamiliar S1 mouse (Fig. 2C). Social novelty preference tests were performed on weaning offspring. The results showed that male weaning mice from the low-dose group showed a preference for interacting with S2, while male weaning mice from the high-dose group did not show a preference for interacting with the unfamiliar S2 mouse. Figure 2 E). Weaned female mice exhibited a phenotype similar to that of male mice. Figure 2 F). Next, the social behavior and social novelty preference behavior of adult offspring were further evaluated. In the social behavior test, neither of the two 1-NP exposure groups of adult male mice showed a preference for interaction with S1 mice (Fig. 2H). On the other hand, the high-dose group of adult female mice showed a preference for interaction with S1 mice, but this was not observed in the low-dose group ( Figure 2I). In the social novelty preference behavior test, neither of the two 1-NP exposure groups showed a social preference for S2 mice (Fig. 2K). Furthermore, the low-dose group of 20-year-old female mice showed a preference for interacting with S2 mice, but not in the high-dose group. Figure 2 L).

[0129] (2) Effects of maternal 1-NP exposure on mIPSC in offspring mPFC

[0130] To assess the effect of gestational 1-N exposure on miniature inhibitory postsynaptic currents (mIPSC) in offspring mPFC neurons, whole-cell recordings were performed on mPFC neurons. Results showed that although there was no significant difference in mIPSC amplitude between the control group and the 1-NP treatment group (…),… Figure 3 In A and C, the frequency of mIPSC in male weaned offspring was significantly reduced in the 1-NP treatment group. Figure 3 A and B). Although there is no difference in mIPSC frequency ( Figure 3 D and E), gestational 1-NP exposure significantly reduced female offspring at weaning ( Figure 3 The mIPSC amplitude of D and F was measured. Next, mIPSC was detected in the mPFC of adult offspring. The results showed that both the frequency and amplitude of mIPSC were reduced in adult male offspring exposed to 1-NP (D and F). Figure 3 GI). Although the mIPSC frequency did not change ( Figure 3 J and K), the mIPSC amplitude in the mPFC of female adult offspring exposed to 1-NP was significantly reduced ( Figure 3 J and L).

[0131] (3) Effects of maternal 1-NP exposure on GAD67+ interneurons in apoptotic mPFC

[0132] To investigate the effects of 1-NP exposure during pregnancy on interneurons in the mPFC of weaned offspring, this study examined the expression and distribution of GAD67, a marker of inhibitory interneurons in the mPFC. The results showed that GAD67 protein expression in the mPFC of male offspring at weaning was significantly reduced in the 1-NP exposure group. Figure 4 A and B). The expression levels of the neuron-specific marker NeuN in the mPFC of the control group and the 1-NP exposure group were not different ( Figure 4 C and D). Further analysis of NeuN + Neuron and GAD67 +The percentage of neurons in each subregion of the mPFC (Cg1: cingulate cortex; PrL: prelimbic cortex; IL: sublimbic cortex) was analyzed, and the results showed that in the 1-NP exposure group, the GAD67 value in each subregion of the mPFC of weaned male offspring was significantly increased. + With NeuN + The proportion of neurons decreased significantly ( Figure 4 E and F), while NeuN in each subregion of mPFC + The percentage of neurons did not differ significantly between the 1-NP and control groups. Figure 4 E and G). Meanwhile, the level of GAD67 protein in the mPFC of weaned female offspring was significantly reduced in the 1-NP exposure group (E and G). Figure 4 H and I), while there was no significant difference in NeuN protein between the two groups ( Figure 4 J and K). Figure 4 L and M show the levels of GAD67 in different subregions of the mPFC (Cg1, PrL, IL) in female mice in the 1-NP exposure group. + With NeuN + The proportion of neurons is reduced. Despite exposure to the PrL subregion of the mPFC in female mice with 1-NP, NeuN... + The percentage of neurons increased, but there were no significant differences between the two groups in the remaining subregions of the mPFC (Figure 4L and N).

[0133] (4) Effects of 1-NP exposure during pregnancy on the migration of interneurons in the fetal forebrain

[0134] This study examined the level of GAD67 in the mPFC of GD18 fetal rats. As shown in Figures 5A and 5B, GAD67 protein levels in the mPFC were significantly reduced in male fetal rats exposed to 1-NP. Further analysis of GAD67... + The number of interneurons in each subregion (SVZ: subventricular zone; VZ: ventricular zone; IZ: intermediate zone; CP: cortical lamina; MZ: limbic zone). Results showed that in the 1-NP exposure group, GAD67... + The number of interneurons in GAD67 in CP and MZ layers + The number of interneurons was significantly reduced ( Figure 5 C and D), while the number of interneurons in the SVZ / VZ layer was significantly increased in the 1-NP exposure group ( Figure 5 C and D). Meanwhile, GAD67 levels were detected in the mPFC of female fetal mice. Results showed that GAD67 protein levels in the mPFC of female fetal mice were significantly downregulated in the 1-NP exposure group (C and D). Figure 5 (E and F). Although there was no difference in GAD67+ interneurons in the IZ layer between the two groups, GAD67 in the CP and MZ layers... + Interneurons were significantly reduced in the 1-NP exposure group ( Figure 5 G and H). In contrast, GAD67 in SVZ / VZ layers. + Interneurons were significantly increased in the 1-NP exposure group ( Figure 5 G and H). Previous studies have shown that in mice, starting from day 14 of GD, interneurons migrate tangentially to the frontal cortex. This study found that although there was no difference in GAD67 protein levels between the two groups ( Figure 5 In groups I and J, the number of GAD67+ interneurons in the distal mid-distal forebrain cortex (areas 2 and 3) of fetal rats exposed to 1-NP was reduced. Figure 5 K and L). Finally, the expression levels of 12 interneuron migration-related genes were examined in the control group and the 1-NP exposure group. The results showed no difference in Cxcl12, Cxcr4, Cxcr7, Slit1, Efna5, Arx, Nrp1, Nrp2, and Sema3A. Figure 5 MU), Nrg1, Erbb4 and Sema3F gene expression levels were significantly reduced in the forebrain of fetal mice in the 1-NP exposure group ( Figure 5 VX).

[0135] (5) Effects of 1-NP exposure during pregnancy on hydroxymethylation of genes related to migration of interneurons in the fetal forebrain

[0136] This study investigated the effects of 1-NP exposure during pregnancy on TET activity and hydroxymethylation modification of genes related to interneuron migration in the forebrain of fetal rats. Hydroxymethylation sites in the Nrg1, Erbb4, and Sema3F genes are shown in Figure 6A. Although the 5hmC levels of CpG-rich fragments in the Nrg1 and Erbb4 genes did not differ significantly between the control and 1-NP treatment groups, the 5hmC level of a CpG-rich fragment in the Sema3F gene was significantly reduced in the forebrain of 1-NP-exposed fetal rats. Figure 6 B). The content of 5hmC at the CpG sites of the Nrg1, Erbb4, and Sema3F genes was further examined. (e.g.) Figure 6 As shown in Figure C, the 5hmC content at one CpG site of the Nrg1 gene was significantly reduced in the fetal rat forebrain in the 1-NP treatment group. The 5hmC content at two CpG sites of the Erbb4 gene was significantly reduced in the fetal rat forebrain in the 1-NP treatment group. The 5hmC content at all three CpG sites of the Sema3F gene was significantly reduced in the fetal rat forebrain in the 1-NP treatment group. Although 1-NP had no effect on the expression levels of TET1, TET2, and TET3 (Figures 6D-F), the activity of TETs enzymes in the forebrain of fetal rats exposed to 1-NP was significantly reduced. Figure 6 G).

[0137] (6) Effects of 1-NP exposure during pregnancy on mitochondrial function in the forebrain of fetal rats

[0138] KEGG and GO analyses revealed that 1-NP induces mitochondrial-related metabolic dysfunction in the fetal rat forebrain, affecting nicotinic acid / nicotinamide metabolism, steroid biosynthesis, and long-chain fatty acid metabolism (Figures 7A and 7B). Further gene set enrichment analysis (GSEA) of KEGG showed a significant negative enrichment of the oxidative phosphorylation pathway in the 1-NP-exposed fetal rat forebrain (Figure 7C). GSEA of GO revealed significant negative enrichment of the electron transport chain and oxidative phosphorylation pathway in the 1-NP-exposed fetal rat forebrain. Figure 7 D and E). Reactome's GSEA results showed negative enrichment of the TCA cycle and electron transport chain in the forebrain of fetal rats exposed to 1-NP (Fig. 7F). Further studies found no difference in mitochondrial area between the control and 1-NP groups, but in the forebrain of fetal rats exposed to 1-NP, the mitochondrial cristae structure in the ganglion ridges at the origin of interneurons disappeared (D and E). Figure 7 GI). SDHB and vATP5A, two proteins associated with oxidative phosphorylation, were significantly reduced in the forebrain of fetal rats in the 1-NP exposure group. Figure 7 JL). Finally, this study found that the mitochondrial α-KG synthase IDH2 was significantly reduced in the 1-NP-induced fetal rat forebrain (Fig. 7M and N). The TET enzyme cofactor α-KG was also reduced in the 1-NP-induced fetal rat forebrain (Fig. 7O).

[0139] (7) Effects of α-KG supplementation during pregnancy on hypohydroxymethylation of genes related to interneuron migration induced by 1-NP

[0140] To investigate the role of TET enzyme inhibition in 1-NP-induced hypohydroxymethylation of genes related to interneuron migration in the fetal rat forebrain and the resulting delay in interneuron migration, this study conducted an intervention experiment involving the supplementation of the TET enzyme cofactor α-KG. The results showed that α-KG supplementation during pregnancy inhibited the 1-NP-induced reduction of 5hmC at the CpG site in the Nrg1 gene. Figure 8 A). Simultaneously, α-KG supplementation during pregnancy suppressed the 1-NP-induced decrease in 5hmC at one CpG site (chr9: 107710238) in the Sema3F gene. Figure 8 F). Prenatal α-KG supplementation has a protective trend against 1-NP-induced reductions in 5hmC at Erbb4 or other Sema3F CpG sites (Figures 8B-E). More importantly, prenatal α-KG supplementation can alleviate 1-NP-induced reductions in Sema3F, Nrg1, and Erbb4 genes in the fetal rat forebrain (F). Figure 8 GI).

[0141] (8) Effect of α-KG supplementation during pregnancy on 1-NP-induced delay in interneuron migration

[0142] like Figure 8 As shown in J and K, α-KG supplementation during pregnancy reverses 1-NP-induced GAD67. + Interneurons were reduced in the mid and distal parts of the forebrain cortex. Further investigation was conducted to explore the effect of α-KG supplementation during pregnancy on interneurons in the mPFC of male offspring at weaning. Results are shown in the figure. Figure 9 A and B, α-KG supplementation during pregnancy can prevent the downregulation of GAD67 protein in mPFC caused by 1-NP. Although α-KG supplementation during pregnancy has no effect on NeuN + Neurons were not affected. Figure 9 (C and E), α-KG supplementation during pregnancy reversed GAD67 in 1-NP-induced mPFC. + With NeuN + The proportion of neurons decreased ( Figure 9 (C and D) The effects of α-KG supplementation during pregnancy on interneurons in the mPFC of female offspring at weaning were consistent with the results in male offspring. Figure 9 F and J).

[0143] (9) Effects of α-KG supplementation during pregnancy on 1-NP-induced mIPSC transmission disorder.

[0144] Although α-KG supplementation during pregnancy has no effect on mIPSC amplitude ( Figure 10 A and C), α-KG supplementation during pregnancy can salvage the 1-NP-induced decrease in mIPSC frequency in male offspring ( Figure 10 A and B). Meanwhile, although α-KG supplementation during pregnancy has no effect on mIPSC frequency ( Figure 10 D and E), α-KG supplementation during pregnancy can rescue 1-NP-induced reduction in mIPSC amplitude in female offspring ( Figure 10 D and F).

[0145] (10) Effects of α-KG supplementation during pregnancy on autistic-like behaviors induced by 1-NP

[0146] Prenatal alpha-kJ supplementation can prevent social behavioral disorders in male offspring caused by 1-NP. Figure 10 G) and α-KG supplementation during pregnancy alleviated 1-NP-induced social novelty preference behavior disorder in male offspring. Figure 10 H). Simultaneously, α-KG supplementation during pregnancy can protect against 1-NP-induced social behavioral disorders in female offspring (H). Figure 10 I). Furthermore, α-KG supplementation during pregnancy protects against 1-NP-induced social novelty preference behavior disorder in female offspring ( Figure 10 J).

[0147] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model of autism spectrum disorder, characterized in that, Pregnant mice were orally exposed to different doses of 1-NP daily throughout their pregnancy; the three-box social test was used on day 28 after birth to assess autism-like behavior in weaned pups; and the three-box social test was used on day 70 after birth to assess autism-like behavior in adult offspring. In the 1-NP group, pregnant mice received different doses of 1-NP; in the 1-NP group, pregnant mice were orally exposed to 100 μg / kg 1-NP daily from day 0 of gestation to day 17 of gestation. The method for constructing the mouse model of autism spectrum disorder, the method comprising: a) Pregnant mice were randomly divided into different groups and administered different doses of 1-NP by gavage from day 0 of gestation to day 17 of gestation; the control group was administered corn oil by gavage. b) After the pregnant mice gave birth naturally, the pups were collected on day 28 and day 70 after birth for behavioral assessment. The three-box social test was used to assess the autism-like behaviors of the pups. c) Extracted medial prefrontal lobe tissue from pups for immunofluorescence, Western blotting, and whole-cell patch-clamp experiments to analyze the effect of gestational 1-NP exposure on inhibitory interneurons in the medial prefrontal lobe of pups. d) Extract fetal rat forebrain or prefrontal cortex for immunofluorescence, Western blotting and RT-PCR to analyze the effect of gestational 1-NP exposure on fetal rat inhibitory interneurons; e) Extract fetal rat forebrain for DNA hydroxymethylation level detection, high performance liquid chromatography-tandem mass spectrometry, transmission electron microscopy, TET enzyme activity detection and transcriptomics analysis to study the mechanism of gestational 1-NP exposure induced delayed migration of fetal rat inhibitory interneurons. f) Pregnant mice were randomly divided into a control group, an α-KG group, a 1-NP group, and a 1-NP+α-KG group. The control group was given corn oil by gavage, the α-KG group and the 1-NP+α-KG group were given 2 mg / kg of α-KG, and the 1-NP group and the 1-NP+α-KG group were given 100 μg / kg of 1-NP by gavage. All treatments were given from day 0 of pregnancy to day 17 of pregnancy. After the pregnant mice gave birth naturally, the pups were collected on day 28 after birth for autism-like behavior assessment to study the protective effect of α-KG supplementation during pregnancy on autism-like behavior induced by 1-NP in pups.

2. The method for constructing a mouse model of autism spectrum disorder as described in claim 1, characterized in that, A method for assessing the effects of gestational 1-NP exposure on the migration of inhibitory interneurons in fetal rats is employed, the method comprising: a) Pregnant mice were randomly divided into different groups and administered different doses of 1-NP by gavage from day 0 of gestation to day 17 of gestation or day 13 of gestation. b) Sacrifice pregnant mice at specific time points and collect the forebrain or medial prefrontal lobe tissue of the fetal mice; c) Immunohistochemistry, Western blotting, immunofluorescence, transcriptome analysis, RT-PCR, DNA hydroxymethylation modification, and TETs enzyme activity assays were performed on the collected tissues to evaluate the effect of 1-NP exposure on the migration of inhibitory interneurons in fetal rats.

3. The method for constructing a mouse model of autism spectrum disorder as described in claim 1, characterized in that, A method for evaluating the protective effect of α-KG supplementation during pregnancy on the reduction of inhibitory interneurons in the medial prefrontal cortex induced by 1-NP and on autism-like behaviors in offspring was employed. This method includes: a) Pregnant mice were randomly divided into a control group, an α-KG group, a 1-NP group, and a 1-NP+α-KG group. The pregnant mice in the 1-NP group and the α-KG+1-NP group were given 1-NP by gavage daily from day 0 of gestation to day 17 or day 13 of gestation. The pregnant mice in the α-KG group and the α-KG+1-NP group were given α-KG by gavage during the same period. b) Collect medial prefrontal cortex tissue from offspring mice for Western blotting, immunofluorescence, and whole-cell patch-clamp experiments to assess the effect of α-KG on the 1-NP-induced reduction in the number of inhibitory interneurons in the medial prefrontal cortex of offspring. c) The three-box social experiment was used to assess autism-like behavior in offspring mice to evaluate the protective effect of α-KG against 1-NP-induced autism-like behavior in offspring.

4. A mouse model of autism spectrum disorder constructed by a method for constructing a mouse model of autism spectrum disorder as described in any one of claims 1 to 3.

5. The use of a mouse model of autism spectrum disorder as described in claim 4 in screening drugs for autism spectrum disorder.

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