Use of notch2 recombinant protein in preparation of medicine for improving social impairment and / or cognitive impairment of autism

Abstract

The application relates to application of a NOTCH2 recombinant protein in preparation of medicines for improving autism social disorder and / or cognitive disorder, and belongs to the technical field of biological medicines. In order to solve the problem that the prior art lacks medicines capable of effectively improving core autism social disorder and cognitive disorder, the application provides application of the NOTCH2 recombinant protein in preparation of medicines for improving autism social disorder and / or cognitive disorder, first discloses a key role mechanism of an S1P-NOTCH2-autophagy regulation axis in autism spectrum disorder, proves that the NOTCH2 recombinant protein can effectively improve core behavior phenotypes of the autism spectrum disorder by activating a NOTCH2 signal path and regulating autophagy function. Experiments prove that exogenous supplement of Notch2 can significantly improve anxiety-like behavior, social disorder and cognitive memory defects of model mice, and provide a brand-new drug target and intervention strategy for clinical treatment of the autism spectrum disorder.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of NOTCH2 recombinant protein in the preparation of drugs to improve social and / or cognitive impairments in autistic patients. Background Technology

[0002] Autism Spectrum Disorder (ASD), commonly known as autism, is a group of neurodevelopmental disorders characterized by impaired social communication, restricted interests, and repetitive and stereotyped behaviors. Cognitive-social impairment is a core symptom, manifesting as a lack of motivation for social interaction, difficulty understanding others' emotions, difficulties in verbal and nonverbal communication, and impaired learning and memory. This severely impacts individuals' ability to live independently and their social functioning, placing a heavy burden on families and society. In recent years, the prevalence of autism has been rising globally, becoming a major public health issue urgently needing to be addressed.

[0003] Currently, clinical interventions targeting the core symptoms of autism are mainly divided into two categories: behavioral / psychological interventions and pharmacological treatment. While behavioral interventions can improve some behavioral problems to a certain extent, they have limitations such as long treatment cycles, high costs, heavy reliance on professional educators, and difficulty in fundamentally improving central nervous system dysfunction. Regarding pharmacological treatment, there are currently no specific drugs that can reverse or improve the core cognitive and social impairments of autism. Existing drugs, such as risperidone and aripiprazole, are only approved by the U.S. Food and Drug Administration (FDA) for relieving accompanying symptoms such as irritability, aggression, or repetitive and stereotyped behaviors; however, there are still no effective treatments for the most fundamental social impairments and cognitive dysfunctions of autism.

[0004] Therefore, there is an urgent need to develop novel intervention strategies that target the core pathological mechanisms of autism and effectively improve cognitive and social impairments. In recent years, research has gradually shifted from relying solely on peripheral blood indicators to infer central nervous system pathology to integrating multidimensional data to explore real molecular events in the brain. Sphingosine-1-phosphate (S1P), an important bioactive lipid, has been found to be abnormally elevated in the peripheral blood of some autistic patients and participates in regulating key life processes such as autophagy. However, how abnormal S1P levels affect central nervous system function and lead to core autistic symptoms, and the molecular link between S1P and autophagy dysfunction, remains unclear. Currently, not only is a complete chain of evidence from "peripheral abnormalities" to "central nervous system impairments" lacking, but effective strategies for reversing cognitive and social deficits by regulating this pathway are also lacking. Summary of the Invention

[0005] To address the lack of drugs in the existing technology that can effectively improve the core social and cognitive impairments of autism, this invention provides the application of NOTCH2 recombinant protein in the preparation of drugs that improve social and / or cognitive impairments in autism.

[0006] The technical solution of the present invention:

[0007] The NOTCH2 recombinant protein is used in the preparation of drugs to improve social and / or cognitive impairment in autistic patients. The NOTCH2 recombinant protein contains amino acids 26 to 294 of the N-terminal region of the NOTCH2 protein, and the specific amino acid sequence is shown in SEQ ID No:1.

[0008] Furthermore, the drug is in the form of an injection, and the route of administration is intraventricular injection.

[0009] Furthermore, the drug comprises a nucleic acid molecule encoding the NOTCH2 recombinant protein.

[0010] Furthermore, the drug also contains pharmaceutically acceptable carriers and / or excipients.

[0011] Furthermore, the autism social impairment includes one or more of the following: reduced social interaction behavior, decreased social novelty preference, and increased repetitive and stereotyped behaviors.

[0012] Furthermore, the autistic cognitive impairment includes one or more of the following: decreased ability to recognize new objects, decreased object discrimination index, and decreased spatial learning and memory ability.

[0013] The beneficial effects of this invention are:

[0014] This invention, through bioinformatics analysis, reveals for the first time that NOTCH2 is a core differentially expressed gene shared by both brain tissue and peripheral blood of patients with autism spectrum disorder (ASD). A significant causal relationship exists between the NOTCH signaling pathway receptor and its ligand Delta and memory ability and social activity. Furthermore, a significant negative causal association exists between the NOTCH2-related activator protein ADAM19 and the risk of developing ASD. Building upon this, this invention further confirms that S1P regulates the expression of key autophagy molecules p62 and LC3 through the NOTCH2 signaling pathway, thus constructing a key mechanism of action of the S1P-NOTCH2-autophagy regulatory axis in the pathogenesis of ASD.

[0015] This invention directly upregulates Notch2 expression levels in the cortex of BTBR mice, a model of autism spectrum disorder, through stereotactic injection of recombinant Notch2 protein into the brain. Experiments have demonstrated that exogenous Notch2 supplementation significantly improves anxiety-like behavior, social impairment, and cognitive memory deficits in the model mice. This invention is the first to demonstrate that recombinant Notch2 protein can effectively improve the core behavioral phenotypes of autism spectrum disorder by activating the Notch2 signaling pathway and regulating autophagy, providing a novel drug target and intervention strategy for the clinical treatment of autism spectrum disorder, and possessing significant clinical application value and broad translational prospects. Attached Figure Description

[0016] Figure 1 This is a box plot of the corrected whole blood transcriptome data from ASD patients in Example 1;

[0017] Figure 2 This is a volcano diagram of differentially expressed genes in whole blood of ASD patients in Example 1;

[0018] Figure 3 The image shows the soft threshold screening diagram for the weighted gene co-expression network analysis in Example 1. A is the curve of the scale-free topology model fitting index changing with the soft threshold, and B is the curve of the average network connectivity changing with the soft threshold.

[0019] Figure 4 This is a heatmap of module-trait associations from the weighted gene co-expression network analysis in Example 1;

[0020] Figure 5 This is a Venn diagram showing the intersection of genes from key ASD cortical modules and differentially expressed genes in whole blood in Example 1.

[0021] Figure 6 This is a bar chart showing the GO functional enrichment analysis of common differentially expressed genes in Example 1;

[0022] Figure 7 This is a protein-protein interaction network diagram of the common differentially expressed genes in Example 1;

[0023] Figure 8 This is the key gene network diagram obtained by screening based on the CytoHubba plugin in Example 1;

[0024] Figure 9 The graphs show the Mendelian randomization analysis of NOTCH receptor and its ligand Delta with memory ability in Example 2. A is a scatter plot of MR analysis, and B is a forest plot of MR analysis.

[0025] Figure 10 The graphs show the Mendelian randomization analysis of NOTCH receptor and its ligand Delta with social activities in Example 2. A is a scatter plot of MR analysis, and B is a forest plot of MR analysis.

[0026] Figure 11 The figure shows the Mendelian randomization analysis of the NOTCH2 activator protein ADAM19 and the risk of ASD in Example 2. A is the scatter plot of MR analysis, and B is the forest plot of MR analysis.

[0027] Figure 12 This is a comparison of the relative expression levels of autophagy-related genes in the cortical tissues of BTBR mice and control mice in Example 3. A represents the relative expression level of P62 mRNA, and B represents the relative expression level of LC3 mRNA.

[0028] Figure 13 This is a comparison of the relative expression levels of autophagy-related genes in the cortical tissues of BTBR mice after SKI II intervention, uninterrupted BTBR mice, and control mice in Example 3. A represents the relative expression level of P62 mRNA, and B represents the relative expression level of LC3 mRNA.

[0029] Figure 14 This is a comparison of Notch2 expression levels in the cortical tissues of BTBR mice after SKI II intervention, uninterrupted BTBR mice, and control mice in Example 3. A is a Western Blot detection band, B is the relative expression level of Notch2 protein, and C is the relative expression level of Notch2 mRNA.

[0030] Figure 15 This is a comparison of Notch pathway ligand mRNA expression levels in the cortical tissues of BTBR mice after SKI II intervention, uninterrupted BTBR mice, and control mice in Example 3. A represents the relative expression level of Dll1 mRNA, B represents the relative expression level of Dll3 mRNA, C represents the relative expression level of Dll4 mRNA, and D represents the relative expression level of Jagged2 mRNA.

[0031] Figure 16 This is a comparison of the expression levels of downstream target genes mRNA of the Notch pathway in the cortical tissues of BTBR mice after SKI II intervention, uninterrupted BTBR mice, and control mice in Example 3. A represents the relative expression level of Hey1 mRNA, B represents the relative expression level of Hey2 mRNA, C represents the relative expression level of Hes1 mRNA, and D represents the relative expression level of Hes5 mRNA.

[0032] Figure 17 This is a comparison of Notch2 expression levels in the cortical tissues of BTBR mice injected with recombinant Notch2 protein and mice not injected with BTBR protein in Example 4. A is a Western Blot band of Notch2 protein expression, B is the relative expression level of Notch2 protein, and C is the relative expression level of Notch2 mRNA.

[0033] Figure 18 Example 4: Comparison of Notch pathway ligand mRNA expression levels in cortical tissues of BTBR mice injected with Notch2 recombinant protein and mice not injected with BTBR protein. A represents the relative expression level of Dll1 mRNA, B represents the relative expression level of Dll3 mRNA, C represents the relative expression level of Dll4 mRNA, and D represents the relative expression level of Jagged2 mRNA.

[0034] Figure 19 This is a comparison of the mRNA expression levels of downstream target genes of the Notch pathway in the cortical tissues of BTBR mice injected with recombinant Notch2 protein and mice not injected with BTBR protein in Example 4. A represents the relative expression level of Hey1 mRNA, B represents the relative expression level of Hey2 mRNA, C represents the relative expression level of Hes1 mRNA, and D represents the relative expression level of Hes5 mRNA.

[0035] Figure 20 This is a comparison of the mRNA expression levels of key autophagy molecules in the cortical tissues of BTBR mice injected with Notch2 recombinant protein and mice not injected with BTBR in Example 4. A represents the relative expression level of p62 mRNA, and B represents the relative expression level of LC3 mRNA.

[0036] Figure 21 This is a comparison chart of the discrimination index of the three groups of mice in the novel object recognition experiment in Example 5;

[0037] Figure 22 This is a schematic diagram of the movement trajectories of the three groups of mice in the open field experiment of Example 5;

[0038] Figure 23 This is a quantitative statistical comparison chart of the three groups of mice in the open field experiment in Example 5. A is the total activity distance, B is the activity time, and C is the average movement speed.

[0039] Figure 24 The image shows a comparison of the results of the three groups of mice in the three-box social experiment in Example 5. A represents the sociality index in the social preference stage, and B represents the social novelty preference index in the social novelty preference stage. Detailed Implementation

[0040] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0041] The experimental data from Examples 1-4 of this invention were analyzed using Prism 10. The normality of the data distribution was analyzed using the Shapiro-Wilk test. The homogeneity of variance between two groups was tested using a t-test, and the homogeneity of variance between three or more groups was tested using one-way ANOVA. If the data variances were unequal, Welch correction was used. If the data were not normally distributed, nonparametric tests were used for analysis. All data are described using mean ± SEM. A p-value < 0.05 was considered statistically significant. .

[0042] Example 1

[0043] This embodiment utilizes the Gene Expression Omnibus (GEO) database to perform differential gene screening, WGCNA analysis, and PPI network construction on transcriptome data from peripheral blood and cerebral cortex tissues of ASD patients.

[0044] (1) Screening of differentially expressed genes in whole blood of ASD patients

[0045] Download the GSE25507 dataset from the GEO database. This dataset is derived from a transcriptome analysis of peripheral blood from children with ASD and contains 64 healthy controls and 82 ASD patients.

[0046] First, the raw data is standardized and corrected to eliminate batch effects and systematic errors. For example... Figure 1 The box plot shown is the corrected sample expression level distribution. After correction, the batch effect interference is eliminated, and the comparability between groups is significantly improved.

[0047] Subsequently, using a p-value < 0.05 as the screening criterion, differentially expressed genes (DEGs) in the GSE25507 dataset were analyzed using the R package limma (R version 4.4.1). The results of the differential analysis were used to plot volcanoes using the R package ggplot2, as shown below. Figure 2 As shown, the horizontal axis (logFC) represents the fold change in gene expression (logarithm to base 2). The left side (logFC < 0) represents genes downregulated in the ASD group, and the right side (logFC > 0) represents genes upregulated. The vertical axis (-log...) 10 The p-value indicates the significance of the difference.

[0048] The differential expression analysis showed that, compared with the control group, 2709 differentially expressed genes were identified in the peripheral blood of ASD patients. Among them, the expression of some genes was significantly upregulated and the expression of some genes was significantly downregulated. Notch2 gene expression was significantly downregulated in the peripheral blood of ASD patients.

[0049] Unsupervised hierarchical clustering analysis of differential gene expression levels showed that the gene expression patterns of the ASD group and the control group could be clearly divided into two major clusters. The samples within the group showed good consistency, while the groups showed significant differences in expression profiles.

[0050] (2) Identification of cortical genes in ASD patients

[0051] The GSE113834 dataset was obtained from the GEO database. This dataset contains transcriptomic data from cortical tissue samples of 12 healthy controls and 15 children with ASD. To identify the genes most relevant to the disease, this embodiment uses the WGCNA package in R software to perform weighted gene co-expression network analysis on the GSE113834 dataset.

[0052] First, outlier samples were identified and removed through hierarchical clustering to ensure the reliability of subsequent data analysis. Then, soft thresholding was performed to construct a co-expression network that conforms to the characteristics of scale-free networks. The results are as follows: Figure 3 As shown. Figure 3 Figure A on the left shows the curve of the fit exponent of the scale-free topological model as a function of the soft threshold. The horizontal axis represents the soft threshold parameter, and the vertical axis represents the model fit R. 2 When R 2 When the preset threshold of 0.8 is reached for the first time, the corresponding soft threshold is the optimal parameter. This embodiment selects this parameter to ensure that the network conforms to the scale-free characteristic. Figure 3 Figure B on the right shows the curve of the average network connectivity as a function of the soft threshold. The horizontal axis represents the soft threshold parameter, and the vertical axis represents the average number of connections of nodes in the network. As the soft threshold increases, the average connectivity gradually decreases and tends to stabilize, indicating that the network connectivity distribution is reasonable under the selected parameters.

[0053] After constructing a weighted co-expression network based on a defined soft threshold, genes were divided into multiple co-expression modules labeled with different colors according to the similarity of gene expression patterns. Further analysis of the correlation between each module and ASD disease status or healthy control status yielded the following results: Figure 4 As shown. Figure 4This is a heatmap showing the association between modules and traits. The left vertical axis represents different co-expressed modules (named ME+module color), and the top horizontal axis represents the sample groups (healthy control group and ASD disease group). Each cell is labeled with the Pearson correlation coefficient and p-value between the module's characteristic genes and the corresponding trait. The color bars indicate the direction and strength of the correlation, with red representing a positive correlation and blue representing a negative correlation; the darker the color, the stronger the correlation. The MEbrown module shows a significant positive correlation with the ASD disease group (correlation coefficient r...). 2 =0.5, P<0.001), which is the module with the highest correlation to ASD among all modules, and therefore was identified as the key disease-related module for subsequent analysis.

[0054] (3) Identification of key genes in ASD patients

[0055] This embodiment first extracts 243 relevant genes from the MEbrown module, then takes the intersection of these genes with whole blood differentially expressed genes, and uses the online tool Evenn to construct a Venn diagram to obtain the common differentially expressed genes between the ASD cortex and whole blood. The results are as follows. Figure 5 As shown, the left circle represents 243 genes from the MEbrown module in the GSE113834 dataset, and the right circle represents 2709 differentially expressed genes in whole blood in the GSE25507 dataset. The overlapping part of the two circles represents the intersection genes between the two, totaling 22, which are the genes that are different in both the ASD cortex and whole blood.

[0056] To investigate the biological functions of common differentially expressed genes in ASD cortex and whole blood, Gene Ontology (GO) enrichment analysis was performed using the online gene enrichment analysis website Metascape. GO analysis is a method that encompasses biological processes, molecular functions, and cellular components and enables large-scale functional enrichment. The enrichment analysis thresholds were: Min Overlap = 3, P-Value Cutoff = 0.05, and Min Enrichment = 1.5. The results are as follows: Figure 6 As shown, the horizontal axis represents the negative logarithm of enrichment significance (-log10(P)), with a larger value indicating more significant enrichment; the vertical axis represents the enriched GO biological process entries. GO analysis results show that these genes are mainly enriched in biological processes such as humoral immune response and innate immune response, suggesting that their function is closely related to ASD-related neuroinflammatory responses.

[0057] Next, a protein-protein interaction (PPI) network of 22 commonly differentially expressed genes was constructed using the STRING database. The confidence level of the interaction pairs was set to ≥0.150. The results are as follows: Figure 7As shown, a PPI network with 22 nodes was constructed, in which the proteins corresponding to 14 genes interact with each other.

[0058] Genes closely linked to other nodes in a network are often considered to have important biological functions, i.e., key target genes, and are closely related to other nodes in the module. To screen for key target genes, we imported the PPI results into Cytoscape software (version 3.9.0) and calculated the connectivity of 14 connected gene nodes using the CytoHubba plugin. The results are as follows: Figure 8 As shown, NOTCH2 has the highest connectivity, suggesting that this gene may be a key gene with consistent differential expression in the cerebral cortex and peripheral blood of ASD patients.

[0059] The experimental results of this embodiment demonstrate that the NOTCH2 gene is downregulated in both the cerebral cortex and peripheral blood of ASD patients, and it is the core gene with the highest connectivity in the PPI network, making it a potential diagnostic and therapeutic target.

[0060] Example 2

[0061] To further verify the causal association between key molecules in the NOTCH signaling pathway and ASD and related behavioral phenotypes, this embodiment employs the two-sample Mendelian randomization (Two-sample MR) method and conducts analysis based on publicly available GWAS database data.

[0062] (1) Data sources and analysis methods

[0063] All GWAS (Genome-Wide Association Study) pooled data used in the analyses were obtained from the IEU OPEN GWAS PROJECT database. The dataset information for each phenotype is shown in Table 1.

[0064] Table 1

[0065]

[0066] Strict quality control was performed on the instrumental variable SNP before analysis: the threshold for GWAS p-value was relaxed to 1×10⁻⁶. -6 Screen for independent genetic variants that are significantly associated with the exposure factor, and exclude linkage disequilibrium (r) variants. 2 Overlapping SNPs (<0.001) ensure the independence of instrumental variables.

[0067] The inverse variance-weighted (IVW) method was used as the primary analytical method to assess the causal effect of exposure factors on outcome phenotypes. Robustness of the results was verified through various sensitivity analyses: 1. Weighted median cross-validation of association results; 2. The MR pleiotropic residual and outlier Delta test (MR-PRESSO) were introduced to identify and correct for potential levels of pleiotropic effects and outliers, and outliers were removed to calculate the corrected results; 3. Odds ratio (OR) and 95% confidence interval (CI) were used to describe the causal effect of exposure on the outcome. All analyses were performed using R software (version 4.4.1), TwoSampleMR, and MR-PRESSO packages. A p-value less than 0.05 was considered suggestive evidence of potential causality.

[0068] (2) Causal relationship between NOTCH receptor and its ligand Delta and memory ability

[0069] MR analysis was performed using genetic variations of NOTCH receptor and Delta ligand-related proteins as exposure factors and memory ability (maximum number of digits correctly memorized) as the outcome phenotype. A total of 14 independent SNPs were included as instrumental variables. The results are as follows: Figure 9 As shown, the scatter plot displays the effect size distribution of each SNP on NOTCH pathway protein (exposure) and memory ability (outcome), and the different colored lines correspond to the fitting trend lines of various analysis methods such as IVW and weighted median; the forest plot presents the effect sizes of individual SNPs and pooled analyses.

[0070] The results showed that IVW analysis indicated a significant negative causal association between NOTCH receptor and Delta ligand and the risk of decreased memory ability (OR=0.930, 95% CI: 0.886-0.977, P=0.004), suggesting that increased levels of NOTCH pathway proteins can significantly reduce the risk of memory decline, i.e., it has a protective effect on memory ability.

[0071] (3) Causal association between NOTCH receptor and its ligand Delta and social activities

[0072] MR analysis was performed using genetic variations of NOTCH receptor and Delta ligand-related proteins as exposure factors and social activity (leisure / social activity) as the outcome phenotype. Nine independent SNPs were included as instrumental variables. Results are as follows: Figure 10 As shown, the scatter plot shows the effect size distribution of each SNP on NOTCH pathway protein (exposure) and social activity (outcome) and the fitting trend lines of different analysis methods, while the forest plot presents individual DeltaSNPs and pooled effects.

[0073] The results showed that there was a significant negative causal association between NOTCH receptor and Delta ligand and the risk of reduced social activity ability (OR=0.755, 95% CI: 0.573-0.995, P=0.046), suggesting that increased levels of NOTCH pathway proteins can significantly reduce the risk of decreased social ability.

[0074] (4) Causal association between NOTCH2 activator protein ADAM19 and the risk of ASD.

[0075] MR analysis was performed using genetic variations of the NOTCH2 activator protein ADAM19 as the exposure factor and ASD as the outcome phenotype. A total of 7 independent SNPs were included as instrumental variables. The results are as follows: Figure 11 As shown, the scatter plot shows the effect size distribution of each SNP on the risk of ADAM19 (exposure) and ASD (outcome) and the fitting trend lines of different analysis methods, while the forest plot presents individual SNPs and the pooled effect.

[0076] The results showed that ADAM19 was significantly negatively associated with the risk of ASD, OR=0.904, 95% CI: 0.817-0.999, P=0.049, suggesting that elevated ADAM19 protein levels were associated with a reduced risk of ASD, which was consistent with the results of transcriptomic analysis showing that the NOTCH2 pathway was downregulated in ASD.

[0077] The experimental results of this embodiment demonstrate that moderate activation of the NOTCH signaling pathway has a direct causal protective effect on memory, social behavior, and ASD. Elevated levels of NOTCH receptor / ligand (OR=0.93 and 0.755) and elevated levels of ADAM19 (NOTCH2 activator protein) (OR=0.904) significantly reduced the risk of adverse outcomes (decreased memory / social abilities and the onset of ASD), providing genetic support for the feasibility of NOTCH2 as a therapeutic target.

[0078] Example 3

[0079] This embodiment verifies the molecular mechanism of "S1P abnormality → Notch2 signal suppression → autophagy dysfunction" in the ASD model (BTBR mouse).

[0080] (1) Laboratory animals

[0081] Four-week-old healthy, clean-grade male BTBR T+ Itpr3tf / J (BTBR) mice were purchased from the Model Animal Institute of Nanjing University. BTBR mice are recognized as an animal model of ASD because they stably express the core symptoms of ASD.

[0082] Healthy, clean-grade male C57BL / 6J (B6) mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and used as the control group for the ASD model mice. All mice were housed at the SPF-grade Animal Experiment Center of Harbin Medical University. All animal experiments were approved by the Animal Protection and Use Committee of Harbin Medical University, and the experimental procedures were strictly conducted in accordance with relevant regulations.

[0083] (2) Experimental methods

[0084] 1. Extraction of mouse cortical tissue: Using scissors, cut open the fur along the midline of the back of the mouse brain, then cut open the skull along the midline of the brain to expose the brain tissue. Carefully peel off the brain tissue with curved forceps and place it on an ice box with weighing paper attached. Using curved forceps, peel away the tissue along the anterior midline of the brain, removing the hippocampus, corpus callosum, olfactory bulb, and midbrain. The remaining brain tissue is the cerebral cortex. Separate the cerebral cortex into left and right sides along the midline and place them separately in EP tubes, storing at -80℃.

[0085] 2. Real-time quantitative polymerase chain reaction (qRT-PCR):

[0086] This invention uses qRT-PCR to detect the mRNA expression levels of relevant genes in mouse cerebral cortex tissue. The specific steps and parameters are as follows:

[0087] (i) Primer design and synthesis

[0088] Specific primers were designed based on gene sequences from the NCBI database. All primers were synthesized by Beijing Ruikexing Biotechnology Co., Ltd., and their nucleotide sequences are shown in SEQ ID No:3-26. Specific sequences are listed in Table 2.

[0089] Table 2

[0090]

[0091] (ii) Total RNA extraction and reverse transcription

[0092] Cortical tissue was homogenized using a homogenizer (conditions: 4°C, 60Hz, 60 seconds, repeated 3 times). Total RNA was extracted using the standard TRIzol method, and RNA purity was determined using a NanoDrop-2000 spectrophotometer (OD260 / 280 between 1.9 and 2.1). Reverse transcription was performed using Novizan's HiScript III RT SuperMix for qPCR (+gDNA wiper) kit: 4 μL of 5×All-in-one qRT SuperMix and 1 μL of Enzyme Mix were added to every 20 μL reaction mixture, along with 1 μg of total RNA template, and the volume was brought to 20 μL with enzyme-free water. The reaction program was: 50°C for 15 minutes (reverse transcription), 85°C for 5 seconds (enzyme inactivation). The resulting cDNA was stored at -20°C for later use.

[0093] (iii) Real-time quantitative PCR

[0094] qPCR was performed using the Novizan ChamQ Universal SYBR qPCR Master Mix kit. The reaction volume (20 μL) consisted of: 10 μL 2×Mix, 0.4 μL each of forward and reverse primers (10 μM), 1 μL cDNA template, and enzyme-free water to a final volume of 20 μL. The following program was run on a Roche LightCycler 480 instrument: 95°C pre-denaturation for 30 seconds; 95°C denaturation for 3–10 seconds, 60°C annealing / extension for 30 seconds, for a total of 40 cycles; finally, melting curve analysis was performed (95°C for 15 seconds, 60°C for 60 seconds, 95°C for 15 seconds). Each sample was tested in triplicate.

[0095] (iv) Data Analysis

[0096] Using 2- ΔΔCt The relative expression level of the target gene was calculated using a method that... Gapdh was used as an internal reference gene, and samples from C57BL / 6J control mice were used as calibration samples. The calculation formulas are: ΔCt = Ct (target gene) - Ct (Gapdh); ΔCt = ΔCt (experimental group) - ΔCt (control group); relative expression level = 2 - ... ΔΔCt Experimental data are expressed as mean ± standard error (SEM).

[0097] 3. Western blotting detection technology

[0098] The cortical tissue was removed from -80°C and washed with PBS to remove blood. A mixture of RIPA lysis buffer, protease inhibitor, and phosphatase inhibitor was prepared in a 100:1:1 ratio. Two grinding beads and 500 μL of the lysis mixture were added to each tissue tube, and the tissue was homogenized at 4°C and 60 Hz for 60 seconds, repeated three times. The homogenate was lysed on ice for 30 minutes, sonicated for 10 seconds, and then incubated on ice for 30 minutes. The homogenate was centrifuged at 4°C and 12000 rpm for 15 minutes, and the supernatant was collected as the total protein extract.

[0099] Protein concentration was determined using the BCA method. 20 μL of standard or test sample (1 μL sample per well, brought to 20 μL with PBS) was added to each 96-well plate, with three replicates per plate. 200 μL of BCA working solution was prepared at a 1:1 ratio and added to each well. The plate was incubated at 37°C for 30 minutes, and the absorbance was measured at 562 nm. The protein concentration was calculated based on the standard curve and adjusted to an isostatic ratio. The protein sample was then mixed with 5×SDS-PAGE loading buffer at a 4:1 ratio, denatured at 100°C for 10 minutes, cooled, and stored at -20°C for later use.

[0100] After preparing the separating and stacking gels, place them in an electrophoresis tank and add 1× electrophoresis buffer. Add protein markers and an equal volume of denatured protein sample sequentially, and perform electrophoresis at a constant voltage of 220V for 30 minutes. After electrophoresis, cut the gel region according to the target molecular weight. Activate the PVDF membrane with methanol, then place it in a transfer clamp along with the gel and filter paper. Transfer the membrane at a constant current of 0.3A for 60 minutes (on ice). After transfer, wash once with TBST and block in 5% skim milk blocking buffer at room temperature for 1 hour. After washing the membrane three times with TBST, incubate it overnight in primary antibody working solution at 4°C. The next day, recover the primary antibody, wash the membrane three times with TBST, and then incubate it in secondary antibody working solution (1:5000) at room temperature in the dark for 1 hour. After washing the membrane three times with TBST, add ECL chemiluminescent substrate, develop and acquire images in a fully automated chemiluminescence image analysis system. Calculate the relative expression level of the target protein using GAPDH as an internal control.

[0101] (3) Verify autophagy dysfunction in ASD model mice

[0102] The mRNA expression levels of p62 and LC3, key markers of autophagy, in the cerebral cortex of BTBR mice and control C57BL / 6J mice were detected, and the results are as follows: Figure 12As shown, compared with the C57BL / 6J group, the expression of p62 mRNA in the cerebral cortex of the BTBR group showed an increasing trend (P < 0.05), while the expression of LC3 mRNA was significantly decreased (P < 0.001), and the difference was statistically significant. This indicates that autophagy dysfunction exists in the cerebral cortex of BTBR mice, which is consistent with the neuropathological characteristics of ASD.

[0103] (4) Confirm the regulatory role of S1P on autophagy and Notch2 expression levels.

[0104] To verify the effect of S1P levels on the expression levels of key autophagy molecules in the cortex of BTBR mice, BTBR mice were treated with a sphingosine kinase inhibitor (SKI II) to reduce their S1P levels. Four-week-old male BTBR mice were selected, and the pre-dissolved sphingosine kinase inhibitor SKI II stock solution was extracted using a syringe.

[0105] SKI II solution was prepared using a gradient dilution method: 100 mg of the active pharmaceutical ingredient was dissolved in 1 ml of DMSO (dimethyl sulfoxide) by vortexing to prepare a stock solution of 100 mg / ml. The working solution preparation system consisted of 5% stock solution, 40% PEG300, 5% Tween-80, and 50% ultrapure water, yielding a final concentration of 5 mg / ml. The working solution was administered intraperitoneally to mice at a concentration of 50 mg / kg. Injections were given every other day for a total of 14 days. BTBR mice and C57BL / 6J mice injected with the same solvent but without SKI-II served as controls.

[0106] The expression levels of p62 and LC3, key molecules in cortical autophagy, were detected in three groups of mice. The results are as follows: Figure 13 As shown, after SKI II intervention, the expression levels of p62 and LC3 mRNAs, molecules related to the autophagy pathway, were significantly altered in the cortical tissue of BTBR mice. p62 mRNA expression decreased, while LC3 mRNA expression increased, with statistically significant differences (P < 0.05). This indicates that after SKI II intervention, p62 levels decreased and LC3 levels significantly increased in the cortical tissue of BTBR mice, suggesting that reduced S1P levels can improve autophagy impairment in the ASD model.

[0107] The expression levels of Notch2 molecules in the cortex of three groups of mice were detected, and the results are as follows: Figure 14As shown, compared with normal C57BL / 6J mice, the expression levels of Notch2 protein and mRNA in BTBR mice were significantly reduced. This is completely consistent with the results of the previous transcriptomic and MR analyses showing "downregulation of the Notch2 pathway in ASD patients," verifying the inhibitory state of the Notch2 pathway in the BTBR mouse model. After SKI II intervention, the expression levels of Notch2 protein and mRNA in the cortex of BTBR mice significantly rebounded, indicating that SKI II intervention can effectively reverse the downregulation of the Notch2 pathway in the ASD model, providing direct in vivo experimental evidence for targeted therapy of this target.

[0108] (5) Confirm that S1P regulates the expression level of ligands in the Notch2 signaling pathway.

[0109] During neural development, Dll1, Dll3, Dll4, and Jagged2 act as key ligands in the Notch signaling pathway. Through the Notch signaling pathway, they jointly regulate the proliferation, differentiation, and apoptosis of nerve cells, thereby shaping the structure and function of the nervous system.

[0110] The expression levels of Dll1, Dll3, Dll4, and Jagged2 in the cortex of three groups of mice were detected, and the results are as follows: Figure 15 As shown, compared with normal C57BL / 6J mice, the mRNA expression levels of Notch pathway ligands Dll1, Dll3, Dll4, and Jagged2 in the cortex of BTBR mice were significantly upregulated. This is consistent with the results of transcriptomic analysis showing abnormal activation of Notch pathway-related ligands in ASD, further verifying the disordered expression of Notch pathway ligands in the ASD model. After SKI II intervention, although the mRNA expression levels of Dll1, Dll3, and Dll4 in the cortex of BTBR mice showed a decreasing trend, there was no statistically significant difference (ns); however, the mRNA expression level of Jagged2 was significantly downregulated. This indicates that SKI II intervention can specifically reverse the abnormally high expression of Jagged2 in the ASD model, while its regulatory effect on Dll1 / 3 / 4 is not significant, suggesting that SKI II's regulation of the Notch pathway may mainly be achieved by targeting Jagged2, thereby affecting the activation of the Notch2 receptor.

[0111] (6) Confirm that S1P regulates the expression levels of target genes in the Notch2 signaling pathway.

[0112] During neural development, target genes such as Hes1, Hes5, Hey1, and Hey2 regulate the maintenance and differentiation of neural stem cells through the Notch signaling pathway, affecting neuronal generation and synaptic plasticity. Changes in the expression levels of these genes may be closely related to the occurrence and development of neural developmental disorders.

[0113] The expression levels of downstream target genes of the Notch pathway, Hey1, Hey2, Hes1, and Hes5, in the cortex of three groups of mice were detected. The results are as follows: Figure 16 As shown, compared with normal C57BL / 6J mice, the mRNA expression levels of Hey1 and Hes1, downstream target genes of the Notch pathway, were significantly downregulated in the cortex of BTBR mice, while the mRNA expression level of Hey2 was significantly upregulated, and the expression of Hes5 showed no significant difference. This corroborates the results of Notch2 receptor downregulation, indicating that the transcriptional activity of the Notch pathway is generally suppressed in the ASD model, and the expression pattern of downstream target genes is disordered. After SKI II intervention, the mRNA expression levels of Hey1 and Hes1 in the cortex of BTBR mice significantly recovered; the mRNA expression level of Hey2 did not change significantly; and the mRNA expression level of Hes5 showed no significant difference among the three groups. This indicates that SKI II intervention can specifically restore the expression of Hey1 and Hes1, key downstream target genes of the Notch pathway, and reverse the transcriptional repression state of the pathway, suggesting that it may affect the neurodevelopment and behavioral characteristics of BTBR mice by regulating the Notch signaling pathway.

[0114] The experimental results in this embodiment demonstrate that abnormal S1P levels inhibit the Notch2 signaling pathway and autophagy function. Reducing S1P levels with drugs restores Notch2 expression and autophagy homeostasis, revealing that S1P is a key upstream molecule regulating the Notch2 / autophagy axis.

[0115] Example 4

[0116] This embodiment verifies the upregulation of Notch2 expression levels in the cortex of BTBR mice and its regulatory effect on the expression levels of Notch2 signaling pathway-related molecules (ligands, target genes) and key autophagy molecules, further clarifying the core regulatory function of Notch2 in ASD models and providing direct in vivo experimental evidence for Notch2 as a targeted therapeutic target for ASD.

[0117] (1) Stereoscopic injection of Notch2 recombinant protein into the brain

[0118] The Notch2 recombinant protein used in this embodiment was purchased from Wuhan Yunclone Technology Co., Ltd. The mouse protein has the catalog number RPL148Mu01. This recombinant protein contains the N-terminal region of the Notch2 protein (amino acids 26 to 294), and the specific amino acid sequence is shown in SEQ ID No:2.

[0119] The sequence shown in SEQ ID NO:1 in this invention is the amino acid sequence from position 26 to 294 of the N-terminal region of the human NOTCH2 protein. Although the mouse Notch2 recombinant protein (provided by Wuhan Yunclone Technology Co., Ltd., corresponding to the Leu26~Phe294 region of the mouse Notch2 protein) was used as an example for verification in this embodiment, the technical solution has cross-species universality and is also applicable to the human NOTCH2 recombinant protein. According to sequence alignment analysis from public databases such as UniProt and NCBI, the NOTCH2 protein is highly conserved in mammals. The amino acid sequence homology between human NOTCH2 protein and mouse Notch2 protein exceeds 90%, and the structure and biological function of its key functional domains (such as EGF-like repeat domains, RAM domains, and ankyrin repeat sequences) are highly conserved in evolution. The recombinant protein intervention experiment involved in this invention mainly relies on the binding ability of the extracellular segment of the NOTCH2 protein to the receptor and its regulatory function on downstream signaling pathways (such as Hes / Hey family genes). These functions have predictable similarities between humans and mice. Therefore, the dosage, intervention time, and effects on downstream signaling pathways validated on murine Notch2 recombinant protein can be reasonably extrapolated to the application scenarios of human Notch2 recombinant protein, providing experimental evidence for clinical translation.

[0120] Five-week-old male BTBR mice were randomly divided into two groups:

[0121] BTBR+Notch2 group: Ten 5-week-old male BTBR mice were used. Prior to injection, the mice were anesthetized with isoflurane gas, and their head hair was removed. The mice's heads were then fixed using a stereotaxic apparatus, and a 0.5 mm drill bit was used to drill a hole. The recombinant protein was injected into the mouse cortex using a microinjector (ML: ±1.00, AP: 3.10, DV: 1.60). The titer was 1×10⁻⁶. 13 The dose was administered at a rate of 100 nmol / min, totaling 1 μl. The syringe was kept in place for 10 minutes after injection to prevent extravasation, and then the scalp was sutured. Behavioral testing was performed 3 days after protein injection, followed by extraction of cortical tissue to detect the expression levels of relevant molecules.

[0122] BTBR group: BTBR mice injected with an equal volume of physiological saline served as the control group.

[0123] (2) Verify the intervention effect of exogenous Notch2 recombinant protein

[0124] The mRNA and protein expression levels of Notch2 in the cortical tissues of two groups of mice were detected by qRT-PCR and Western Blot experiments. The results are as follows: Figure 17As shown, compared with the untreated BTBR model group, the protein expression level and mRNA expression level of Notch2 in the cortex of mice injected with recombinant Notch2 protein were significantly increased, and the differences were statistically significant. This indicates that exogenous Notch2 recombinant protein can effectively increase the expression level of endogenous Notch2.

[0125] (3) Verify the regulatory effect of upregulation of Notch2 on ligands in the Notch2 signaling pathway.

[0126] To investigate the effect of recombinant Notch2 protein on the expression levels of Notch2 signaling pathway-related ligands in the cortical tissue of BTBR mice, the expression levels of Dll1, Dll3, Dll4, and Jagged2 in the cortical tissue of two groups of mice were detected by qRT-PCR. The results are as follows: Figure 18 As shown, when Notch2 levels were upregulated, the expression levels of Dll1, Dll3, and Dll4 in the cortex were significantly reduced (P<0.05), while the expression level of Jagged2 showed no significant difference (P>0.05). This indicates that injection of recombinant Notch2 protein can regulate the activation state of the Notch signaling pathway by downregulating the expression of Dll1, Dll3, and Dll4 ligands.

[0127] (4) Verify the regulatory effect of upregulating Notch2 on target genes of the Notch2 signaling pathway.

[0128] To investigate the effect of recombinant Notch2 protein on the expression levels of Notch2 signaling pathway-related target genes in the cortical tissue of BTBR mice, the expression levels of downstream Notch pathway target genes Hey1, Hey2, Hes1, and Hes5 in the cortical tissue of two groups of mice were detected by qRT-PCR. The results are as follows: Figure 19 As shown, when Notch2 levels were upregulated, the expression levels of Hey1 and Hes1 mRNA in the cortex of BTBR mice increased significantly (P<0.05), the expression level of Hey2 mRNA decreased significantly (P<0.0001), while the expression level of Hes5 showed no significant difference (P>0.05).

[0129] (5) Verify the regulatory effect of upregulating Notch2 on cortical autophagy function in BTBR mice.

[0130] To clarify the effect of Notch2 upregulation on the expression levels of autophagy pathway-related molecules in the cortical tissue of BTBR mice, the mRNA expression levels of p62 and LC3 in the cortical tissue of two groups of mice were detected by qRT-PCR. The results are as follows: Figure 20As shown, the expression levels of p62 mRNA and LC3 mRNA in the cortex of BTBR mice were decreased and significantly increased (P < 0.05). This indicates that upregulation of Notch2 can effectively improve autophagy dysfunction in the cortex of BTBR mice, confirming the regulatory role of Notch2 in autophagy homeostasis.

[0131] The experimental results of this embodiment demonstrate that exogenous supplementation of recombinant Notch2 protein can effectively activate its downstream signaling pathways and significantly restore autophagy function in the cerebral cortex of BTBR mice. Combined with the results of Example 3, this further confirms the regulatory mechanism of S1P abnormality → Notch2 signal inhibition → autophagy dysfunction, providing complete in vivo experimental evidence for NOTCH2 as a targeted therapeutic target for ASD.

[0132] Example 5

[0133] This embodiment verifies, from a behavioral perspective, the effect of upregulating Notch2 expression levels on improving autism-like behavior in BTBR autism model mice.

[0134] (1) New object recognition experiment

[0135] The Novel Object Recognition Test (NORT) is used to assess the short-term memory ability of mice. The experimental procedure consists of three stages:

[0136] Adaptation phase: The mice were placed in an empty test box (40cm×40cm×50cm) and allowed to explore freely for 10 minutes to eliminate environmental unfamiliarity.

[0137] Familiarization phase: Two objects of the same material (A1 / A2) were placed symmetrically in the test box. The cumulative sniffing time of the mouse on the two objects was recorded by a camera tracking system for 10 minutes.

[0138] Testing phase (1 hour interval): Replace one of the objects with a new object (B) of the same material but different shape, and keep the other familiar object (A). Record the sniffing time of the mouse for the new and old objects.

[0139] The Discrimination Index (DI) is calculated, with a value ranging from ±1, to quantify the mouse's preference for new and old objects and reflect its memory retention ability.

[0140] A novel object recognition experiment was conducted using BTBR+Notch2 group, BTBR group mice, and C57BL / 6J mice from Example 4 as test subjects. The results are as follows: Figure 21As shown, compared with the C57BL / 6J group, the discrimination index of the BTBR group was significantly lower, with a statistically significant difference (P < 0.01), indicating that BTBR mice have obvious cognitive memory deficits. Compared with the BTBR group, the discrimination index of the BTBR+Notch2 group rebounded, but did not reach the level of statistical difference (ns, P > 0.05). This suggests that upregulating Notch2 has a certain improving trend on short-term memory impairment in BTBR mice.

[0141] (2) Open field experiment

[0142] The Open Field Test (OFT) was conducted using the SMART 3.0 behavioral analysis system to assess anxiety-like behaviors in mice. The experimental setup was constructed of black polycarbonate, with dimensions of 45×45×40 cm and an open top to simulate a natural exploration environment. An infrared camera was mounted on top of the chamber to track mouse behavior in real time. During the experiment, mice were gently placed in the center of the chamber and allowed to explore freely for 10 minutes without disturbance. The system simultaneously recorded behavioral parameters such as movement trajectory, activity duration, and distance traveled, with a focus on anxiety-related indicators such as activity distance and duration. All data were automatically collected and analyzed using the SMART 3.0 system. By comparing differences in activity distance and time, the anxiety-like behavioral characteristics of the mice were quantitatively assessed.

[0143] Open field experiments were conducted using mice from the BTBR+Notch2 group, the BTBR group, and the C57BL / 6J group as test subjects, as shown in Example 4. The mouse movement trajectories are as follows: Figure 22 As shown, the movement trajectories of mice in the C57BL / 6J group were evenly distributed, with a moderate range of activity and no signs of hyperactivity. The movement trajectories of mice in the BTBR group were significantly denser and covered a wider area, indicating that the frequency and intensity of activity were significantly higher than those of normal mice, exhibiting typical hyperactive behavior. The movement trajectories of mice in the BTBR+Notch2 intervention group were significantly sparser than those in the BTBR group, with fewer activity paths, suggesting that hyperactivity symptoms were alleviated.

[0144] like Figure 23 As shown, compared with the C57BL / 6J group, the BTBR group had significantly increased activity distance, activity time, and average speed (P < 0.01); compared with the BTBR group, the BTBR+Notch2 group had significantly decreased activity distance, activity time, and average speed (P < 0.05). This indicates that upregulating Notch2 can effectively improve the hyperactive behavior of BTBR mice and alleviate their hyperactivity symptoms.

[0145] (3) Three-box social experiment

[0146] The Three-Chamber Social Test is used to systematically assess the social abilities and novelty preferences of mice. The experimental setup consists of three interconnected compartments (20cm × 40cm / compartment), with a transparent cylindrical container placed in each of the left and right compartments.

[0147] The experimental procedure was divided into three stages: environmental adaptation, social preference, and social novelty. First, in the environmental adaptation stage, mice were placed in the central compartment, and the partition was opened, allowing them to freely explore the entire setup for 10 minutes to eliminate unfamiliarity. Next, in the social preference test stage, a new unfamiliar mouse (Stranger 1) was introduced into the right-hand container, while the left-hand container remained empty. A camera system tracked the mice's time spent in the three compartments in real time, quantifying the duration of their active sniffing of the new unfamiliar mouse to determine their natural inclination towards social stimuli. Finally, in the social novelty test, the previously encountered Stranger 1 mouse was moved to the left-hand container, while a new unfamiliar mouse (Stranger 2) was introduced into the right-hand container. The mice's ability to recognize social novelty was assessed by comparing their exploratory behaviors towards the known social object (Stranger 1) and the new social object (Stranger 2). All behavioral parameters were standardized using intelligent analysis software to ensure the objectivity and reproducibility of the experimental results.

[0148] A three-box social experiment was conducted using BTBR+Notch2 group, BTBR group mice and C57BL / 6J mice as test subjects in Example 4. The results are as follows: Figure 24 As shown, during the social preference test, C57BL / 6J mice showed a significantly greater preference for sniffing unfamiliar mice, indicating normal social willingness. BTBR mice exhibited a significant decrease in sniffing time with unfamiliar mice, almost matching the interaction time with empty cages, a typical social avoidance / social motivation deficit. Compared to the BTBR group, the BTBR+Notch2 group showed a significant increase in sniffing time with unfamiliar mice (P<0.01), converging with the behavioral pattern of normal mice, indicating a significant improvement in basic social willingness.

[0149] During the social novelty test, C57BL / 6J mice actively explored new, unfamiliar mice instead of staying with familiar mice, which is a normal social novelty preference. BTBR mice showed a significant increase in sniffing time with familiar mice and almost no exploration desire with new, unfamiliar mice, exhibiting a lack of social novelty preference, a typical behavioral characteristic of ASD. Compared to the BTBR group, the BTBR+Notch2 group showed a significant reduction in over-reliance on familiar mice (P<0.001) and a slight recovery in exploration time with unfamiliar mice, indicating a partial recovery of social novelty preference. This demonstrates that upregulating Notch2 expression can effectively reverse the social motivation deficit and lack of social novelty preference in BTBR mice, providing direct behavioral evidence for NOTCH2 as a key target for improving social symptoms of ASD.

[0150] The experimental results of this embodiment demonstrate that supplementing the brain with recombinant Notch2 protein can significantly improve the core symptoms of autism, such as repetitive stereotyped behaviors, social impairment, and cognitive memory deficits, in ASD model mice.

Claims

1. The use of NOTCH2 recombinant protein in the preparation of drugs to improve social and / or cognitive impairments in autistic patients, characterized in that, The amino acid sequence of the NOTCH2 recombinant protein is shown in SEQ ID No:

1.

2. The application according to claim 1, characterized in that, The drug is in the form of an injection and is administered via intraventricular injection.

3. The application according to claim 2, characterized in that, The drug contains a nucleic acid molecule encoding the NOTCH2 recombinant protein.

4. The application according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

5. The application according to any one of claims 1-4, characterized in that, The autism social impairments include one or more of the following: reduced social interaction, decreased social novelty preference, and increased repetitive and stereotyped behaviors.

6. The application according to any one of claims 1-4, characterized in that, The cognitive impairments associated with autism include one or more of the following: decreased ability to recognize new objects, reduced object discrimination index, and decreased spatial learning and memory abilities.

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