Use of scamp5 in treatment of autism spectrum disorders

Abstract

The application discloses application of SCAMP5 in treatment of autism spectrum disorder. The application research proves that, compared with a CON group, VPA-induced ASD rats have significant neurotransmitter release disorder and synapse signal path abnormality in hippocampus, overexpression of SCAMP5 protein can improve abnormal release of Glu, abnormal expression of scaffold protein Homer1b / c and downstream protein IP3R, and meanwhile, can relieve autism-like behaviors, space working memory and synapse development abnormality of ASD rats. The application provides a new target and a new potential drug for treatment of autism spectrum disorder, and provides a new idea for target treatment of ASD by in-depth exploration of potential path in pathogenesis of autism.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of SCAMP5 in the treatment of autism spectrum disorder. Background Technology

[0002] Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder characterized by impaired social interaction, delayed language development, and stereotyped and repetitive behaviors. In recent years, epidemiological surveys have shown a significant increase in the global prevalence of ASD, transforming it from a rare disease into a major public health issue concerning childhood neurodevelopment. The pathogenesis of ASD is complex and diverse, with current research suggesting the interaction of genetic, epigenetic, and environmental factors. Among these, the imbalance between the excitation-inhibition (E / I) neurotransmitter system of excitatory glutamate (Glu) and inhibitory γ-aminobutyric acid (GABA) in the brain is considered one of the key pathological foundations of ASD. Glu, as the most important excitatory neurotransmitter in the central nervous system, not only participates in neuronal development and synaptic plasticity regulation, but its abnormal release is also closely related to various neuropsychiatric disorders. Defects in GABA function may lead to insufficient inhibitory function of neural networks, thereby triggering the characteristic behavioral abnormalities of ASD.

[0003] Recent studies have found that SCAMP5 (Secretory Carrier Membrane Protein 5), a member of the secretory carrier membrane protein family, significantly affects the release of glutamatergic neurotransmitters by regulating vesicle release mediated by the synaptic binding protein SYT1 (Synaptotagmin-1). Abnormal regulation of this process may lead to increased presynaptic glutamate release, thereby disrupting the E / I balance of neural networks. Meanwhile, the dynamic balance of Homer1, a key scaffold protein in the postsynaptic compact area, particularly its isoforms Homer1a and Homer1b / c, plays a crucial role in maintaining synaptic function and plasticity. Homer1 protein interacts with molecules such as metabolite glutamate receptors (mGluRs) and inositol triphosphate receptors (IP3Rs) to regulate downstream signaling pathways, affecting neuronal development and function. However, the specific molecular mechanisms by which SCAMP5 affects the pathogenesis of ASD by influencing glutamate release and causing abnormalities in the postsynaptic Homer1b / c scaffold protein remain unclear. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing an application of SCAMP5 in the treatment of autism spectrum disorder.

[0005] To achieve its objective, the present invention employs the following technical solution:

[0006] The first aspect of the present invention provides the use of SCAMP5 molecules as targets in screening drugs for the treatment of autism spectrum disorders, wherein the SCAMP5 molecule is the SCAMP5 protein or the SCAMP5 gene.

[0007] In the above-mentioned application technology solution, the drug promotes the expression of SCAMP5 molecules.

[0008] A second aspect of the invention provides the use of SCAMP5 expression promoters in the preparation of medicaments for treating autism spectrum disorders.

[0009] Preferably, the SCAMP5 expression promoter is a recombinant vector for expressing SCAMP5.

[0010] More preferably, the recombinant vector is a recombinant adeno-associated virus vector overexpressing SCAMP5.

[0011] A third aspect of the invention provides the use of the SCAMP5 protein or a gene expressing the SCAMP5 protein in the preparation of a medicament for treating autism spectrum disorder.

[0012] In any of the above application technologies, SCAMP5 improves the abnormal release of excitatory glutamate (Glu) from the presynaptic membrane.

[0013] In any of the above application technologies, SCAMP5 and SYT1 are used in conjunction with each other, and SCAMP5 affects the release of Glu from the presynaptic membrane through SYT1.

[0014] In any of the above application technologies, SCAMP5 improves abnormalities in the Homer1b / c scaffold and its downstream pathway IP3R protein in patients with autism spectrum disorder.

[0015] In any of the above application technologies, SCAMP5 improves spurious developmental abnormalities in patients with autism spectrum disorder.

[0016] The beneficial effects of this invention are as follows: Through multi-dimensional experiments, this study confirms that, compared to the CON group, valproic acid (VPA)-induced ASD rats exhibit significant neurotransmitter release disorders and synaptic signaling pathway abnormalities in the hippocampus. Overexpression of SCAMP5 protein can improve the abnormal release of Glu, the abnormal expression of scaffold protein Homer1b / c and downstream protein IP3R, and simultaneously alleviate autism-like behaviors, spatial working memory, and synaptic development abnormalities in ASD rats. This invention provides a new target and a new potential drug for the treatment of autism spectrum disorders. By deeply exploring the underlying pathways in the pathogenesis of autism, it offers new insights for targeted therapy of ASD. Attached Figure Description

[0017] Figure 1 This is the result of ELISA statistical analysis of SCAMP5 expression in the serum of children with ASD.

[0018] Figure 2 The expression level of SCAMP5 protein in the hippocampus of ASD rats is shown: A. Representative bands from Western blot; B. Statistical results of gray value analysis of bands in each group (n = 5, **P<0.01).

[0019] Figure 3 The following data shows the overexpression of SCAMP5 protein in the hippocampus of ASD rats: A. Representative bands from Western blot; B. Statistical results of gray value analysis of bands in each group (n = 5, *P<0.05, **P<0.01, ***P<0.001).

[0020] Figure 4 This indicates the localization of SCAMP5 protein in the hippocampus of ASD rats.

[0021] Figure 5 The effects of SCAMP5 overexpression on spatial cognitive ability in ASD rats were shown: A. Time statistics of entering new arms in the Y maze; B. Statistics of the percentage of people entering new arms in the Y maze; C. Statistics of the distance traveled in new arms in the Y maze (n = 10, *P<0.05, **P<0.01, ***P<0.001).

[0022] Figure 6 The effects of SCAMP5 overexpression on exploratory behavior, cognitive ability, adaptability, and mood changes in ASD rats were shown: A. Representational trajectory plot and heatmap of the open field; B. Statistical graph of dwell time in the central grid; C. Statistical graph of upright time; D. Statistical graph of defecation frequency; E. Statistical graph of urination frequency (n = 10, *P<0.05, **P<0.01, ***P<0.001).

[0023] Figure 7 The effects of SCAMP5 overexpression on social interaction and social preferences in ASD rats were shown: A. Phase 1 statistical plot of three boxes; B. Phase 2 statistical plot of three boxes; Chamber A: time spent in chamber A (chamber containing Stranger 2); Chamber B: time spent in chamber B (intermediate chamber); Chamber C: time spent in chamber C (chamber containing Stranger 1); Stranger 1: time spent in contact with stranger rat 1; Stranger 2: time spent in contact with stranger rat 2; (n = 10, *P<0.05, **P<0.01, ***P<0.001).

[0024] Figure 8 The effects of SCAMP5 overexpression on social interaction and repetitive stereotyped behaviors in ASD rats were shown: A. Chase time graph; B. Social time graph; C. Fur grooming time graph; D. Digging time graph (n = 10, *P < 0.05, **P < 0.01, ***P < 0.001).

[0025] Figure 9 The interaction between SCAMP5 and SYT1 is shown: A. Molecular docking diagram of SCAMP5 and SYT1; B. Co-IP result diagram of SCAMP5 and SYT1.

[0026] Figure 10 The expression of SYT1 after overexpression of SCAMP5 is shown: A. Representative bands of SYT1 in Western blot; B. Statistical results of gray value analysis of each group of bands (n = 5, *P<0.05, **P<0.01, ***P<0.001).

[0027] Figure 11 The results showed abnormal presynaptic membrane Glu release: A. Electron microscopy showing dense vesicle regions; B. Representative bands of vGlut1 Western blot; C. Statistical analysis of gray values ​​of each band; D. Statistical analysis of ELISA results (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001).

[0028] Figure 12 The density of dendritic spines in hippocampal cells of ASD rats reversibly induced by SCAMP5 overexpression is shown: A. Representative image with Golgi staining; B. Statistical results of dendritic spine density (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

[0029] Figure 13 The following data is presented: Homer1b / c stent and its downstream anomalies: A. Representative bands of Homer1b / c Western blot; B. Statistical results of grayscale value analysis of each group of bands; C. Representative bands of IP3R Western blot; D. Statistical results of grayscale value analysis of each group of bands (n = 5, *P<0.05, **P<0.01, ***P<0.001). Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0032] Example 1: Study on the effect of SCAMP5 on VPA-induced ASD behavior in rats

[0033] 1. Materials and Methods

[0034] 1.1 Materials

[0035] 1.1.1 Laboratory animals

[0036] Thirty Sprsgue-Dawley (SD) rats of reproductive age (20 females and 10 males) were purchased from the Experimental Animal Center of Chongqing Medical University (License No.: SYXK2022-0066). Females weighed ≥270 g and males weighed ≥300 g. Animal experiments were conducted under the guidance of the State Science and Technology Commission of China and in accordance with the regulations of the Experimental Animal Ethics Committee of Chongqing Medical University. The rats were housed at the Experimental Animal Center of Chongqing Medical University under a constant room temperature (22~25℃) and had free access to food and water.

[0037] 1.1.2 Main Reagents

[0038]

[0039] 1.1.3 Related Antibodies

[0040]

[0041] 1.2 Experimental Methods

[0042] 1.2.1 Patient Information

[0043] Fasting blood samples were collected from 12 normal children and 13 children with ASD at Chongqing Women and Children's Hospital. The children with ASD included 9 males and 4 females (mean age ± SD = 5.7 ± 2.9 years), diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition. The 12 normal children included 7 males and 5 females (mean age ± SD = 5.8 ± 1.5 years). These healthy children did not exhibit any clinical findings suggestive of neuropsychiatric disorder. None of the participants had a recent history of infection or fever. The study protocol was approved by the Medical Research Ethics Committee of Chongqing Medical University (IACUC-CQMU-2023-0334). Written informed consent was obtained from all parents in accordance with the principles of the Declaration of Helsinki (2008).

[0044] 1.2.2 Establishment of a VPA-induced ASD rat model

[0045] After purchasing rats and placing them in the animal facility for two weeks to acclimatize, rats were randomly selected each evening between 7:00 PM and 8:00 PM and caged together at a female-to-male ratio of 1:1. The rats were then separated between 8:00 AM and 9:00 AM the following day, and vaginal plugs and vaginal smears were examined. Female rats with vaginal plugs and sperm detected in the vaginal smears were ear-tagged and marked as gestation day 0. In the VPA group, female rats were intraperitoneally injected with 600 mg / kg VPA solution (250 mg / ml dissolved in sterile 0.9% saline) at 12.5 days of gestation. The CON group received an equal volume of sterile saline. Pregnant rats were housed separately after intraperitoneal injection, and male offspring were selected as experimental subjects after birth.

[0046] 1.2.3 Adeno-associated virus (AAV) synthesis

[0047] AAV was designed and synthesized by Heyuan Biotechnology Co., Ltd. Cells were transfected with plasmid H33371. The AAV vector genome copy number was determined by qPCR to confirm the AAV viral particle concentration. pAAV-CMV-SCAMP5-EF1a-EGFP-tWPA was used as the vector, with primer sequences of 5'-TTACGCTATGTGGATACGC-3' (SEQ ID NO.1); 5'-ATCCTGGTTGCTGTCTCT-3' (SEQ ID NO.2). SCAMP5 overexpression was achieved using adeno-associated virus serotype 9 (AAV9).

[0048] 1.2.4 Stereoscopic injection of adenovirus into the hippocampus of ASD rats

[0049] Grouping: The offspring of pregnant rats were divided into VPA group and CON group. The offspring rats of VPA (PND9) were randomly divided into 3 groups: ①VPA group, ②VPA+OE-SCAMP5 group, and ③VPA+NC group.

[0050] Anesthesia: Weigh the patient and administer an intraperitoneal injection based on their weight.

[0051] Fixation: A quantitative amount of virus is drawn from a microsyringe and fixed onto the stereotaxic instrument. The anesthetized rat is then fixed onto the adapter. Both ear rods are fixed into the rat's external auditory canal. Fine adjustments are made to align the scales on the left and right ear rods and tightened to center the rat's head.

[0052] Location: Wipe with iodine solution, then use scissors and tweezers to cut the scalp along the midline of the skull to the appropriate size. Wipe with hydrogen peroxide to remove the fascia above the skull, and remove excess hydrogen peroxide with a cotton swab. Determine the anterior fontanelle (the junction of the coronal and sagittal sutures) as the origin, and balance left and right. Using bregma as the midpoint, move the needle 3 mm left and right and insert it until it just touches the skull surface. Read the DL values; if the error does not exceed 0.03 mm, the balancing is considered complete. Then determine the coordinates: ML = ± 2.5 mm, AP = - 3.45 mm, DV = - 3.0 mm, and mark these as the drilling locations.

[0053] Drilling: After positioning, use a skull drill to drill holes at the marked locations.

[0054] Injection: When the tip of the microinjector just touches the orifice, reset the DV coordinate value to zero, and then insert the microinjector to the predetermined DV coordinate value. Set the injection rate to 0.1 μL / min and the injection time to 10 min. After injection, stop the injection for 10-15 min before slowly withdrawing the microinjector to prevent fluid leakage. Suture the wound, place the rat on a warming blanket to prevent hypothermia, and continue until the rat regains consciousness.

[0055] 1.2.5 ELISA: Standard method.

[0056] 1.2.6 Brain tissue protein extraction: conventional method.

[0057] 1.2.7 BCA method for protein concentration determination: a conventional method.

[0058] 1.2.8 Westren blot: a conventional method.

[0059] 1.2.9 Paraffin sectioning: conventional method.

[0060] 1.2.10 Immunohistochemical staining: standard method.

[0061] 1.2.11 Behavioral Testing

[0062] 1. Y-maze behavioral test (spatial recognition and memory ability)

[0063] Y-maze test: This test is conducted in a quiet, evenly lit room. Rats are allowed to acclimatize to the test room for 30 minutes beforehand to reduce stress. The Y-maze device is an opaque acrylic box with three arms of equal length at a 120° angle. The arm length × width × height = 40 × 10 × 20 cm, and the top is open for observation. Novelty arm preference test (assessing spatial memory) consists of two phases. In the training phase, one arm (novelty arm) is randomly closed, and rats are placed in from the middle area to freely explore the remaining two arms in 5 groups. After the test rats are removed, the device is cleaned of odors with 75% alcohol. One hour later, in the testing phase, the open arm is opened, and rats are placed in from the middle area. The time taken for each arm to explore by the rats in the 5 groups is recorded using Smart Behavioral Software.

[0064] 2. Autism-like behavioral tests

[0065] (1) Open field test: An open, opaque acrylic box (100 × 100 × 46.7 cm) was used to record the animals' movement and exploration. The box was divided into 25 uniformly sized square grids, with the nine central grids designated as the middle grids and the rest as the surrounding grids. Before the test, the rats were allowed to move freely in the box for 10 minutes to familiarize themselves with the environment. After the test began, the time spent in the middle and outer grids was recorded using Smart Behavioral Software. The time the rats spent standing upright in the box and the number of times they defecated throughout the test (anxiety level) were also recorded. After each test, the excrement was cleaned and the inside of the box was wiped with 75% alcohol to remove any residual odor.

[0066] (2) Three-chamber socialization test: In this test, a transparent rectangular acrylic box (120 × 45 × 40 cm) was divided into three equal-sized chambers, labeled from left to right as Chamber A, Chamber B, and Chamber C. Each chamber was separated by a doorway allowing rats to pass freely. Identical iron cages, labeled Circle a and Circle b, were placed in each end of the box. Smart behavioral software was used to record the rats' time spent in each chamber and circle, as well as their tracking patterns. The test consisted of three phases, each lasting 10 minutes.

[0067] Adaptation Phase: Under normal conditions of the three-chamber setup, the experimental rats were placed from chamber B into the adaptation environment and allowed to move freely for 10 minutes. Social Testing Phase: A stranger male rat of the same age (stranger 1) was placed in circle b as a social rat. Circle a was left empty. The experimental rats were then placed from chamber B, and the Smart software automatically timed the process for 10 minutes, recording their activity time and trajectory. Social Preference Testing Phase: After the first phase, the experimental rats were removed. Circle b remained unchanged (stranger 1), and the items in circle a were replaced with a new, stranger male rat of the same age (stranger 2). The experimental rats were then placed from chamber B, and the Smart software automatically timed the process for 10 minutes, recording their activity time and trajectory.

[0068] (3) Juvenile social play experiment: The experimental setup was a square acrylic box (50 × 50 cm). Before the experiment, a 5 cm layer of clean bedding was placed inside the box. During the adaptation phase, the rats were placed in the box and allowed to move freely for 10 minutes. After that, they were removed, and a strange male rat of the same age was placed in the box. The test rat was then placed in the box, and a 10-minute video recording was performed using the Smart video analysis system. During the test, the time spent chasing the strange rat, the social time, the attack time, as well as the time spent grooming (repetitive stereotyped behavior) and digging were recorded.

[0069] 1.2.12 Statistical Methods

[0070] Data analysis was performed using GraphPad Prism 10.0 statistical software (GraphPad Software, USA). For comparisons of three or more groups, one-way ANOVA combined with Tukey's multiple comparison test was used to analyze differences between groups. In experiments examining the interaction of different treatments and stimulus conditions, two-way ANOVA combined with Sidak's multiple comparison test was used. All experimental results are presented as mean ± standard error (mean ± SEM). The statistical significance level was set at a p-value less than 0.05.

[0071] 2. Results

[0072] 2.1 Expression of SCAMP5 in the serum of children with autism

[0073] To investigate the expression of SCAMP5 in children with autism, the expression levels of SCAMP5 in the serum of children with autism and normal children were detected using ELISA. The results showed that the expression of SCAMP5 in the serum of children with autism was significantly lower than that in normal children (**P < 0.01). Figure 1 This suggests that SCAMP5 is abnormally expressed in children with autism.

[0074] 2.2 VPA-induced decrease in hippocampal SCAMP5 in ASD rats

[0075] To investigate the difference in SCAMP5 protein expression in the hippocampus of VPA-induced autistic rats and CON-induced rats, Western blot was used to detect the SCAMP5 protein expression level in the hippocampus of both groups of rats at 35 days after birth. The results showed that the expression level of SCAMP5 in the VPA group was significantly lower than that in the CON group (**P<0.01). Figure 2 The results suggest that SCAMP5 protein levels in the hippocampus of ASD rats are significantly decreased.

[0076] 2.3 Intrahippocampal injection of r-AAV overexpressing SCAMP5

[0077] To investigate the role of SCAMP5 in VPA-induced ASD rats, we overexpressed SCAMP5 and found that the SCAMP5 expression level was increased in the VPA+OE-SCAMP5 group compared with the VPA+NC group. The results indicate that SCAMP5 was successfully overexpressed in the hippocampus of VPA-induced ASD rats (*P<0.05). Figure 3 ).

[0078] 2.4 Localization of SCAMP5 in cells

[0079] To investigate the localization of SCAMP5 in VPA-induced ASD rats, IHC results showed that SCAMP5 was mainly expressed in the cytoplasm and cell membrane. Figure 4 ).

[0080] 2.5 Overexpression of SCAMP5 in the hippocampus improves spatial cognitive ability in ASD rats

[0081] The Y-maze test primarily assesses the spatial cognitive ability of ASD rats. The experimental results shown in the figure indicate that all indicators in the VPA+OE-SCAMP5 group were significantly improved compared to the VPA group, with an increased time to enter the new heteroarm in the second stage (**P<0.01). Figure 5 -A), the proportion of new heteroarms entered increased significantly (***P<0.001) Figure 5 -B), the distance traveled by the new arm increased or decreased significantly (***P<0.001) Figure 5 -C).

[0082] 2.6 Hippocampal overexpression of SCAMP5 improves autism-like behavior in ASD rats

[0083] The results of autism-like behavior detection showed that overexpression of SCAMP5 in animals could improve autism-like behavior in ASD rats.

[0084] 2.6.1 Open Field Test

[0085] Open field test results showed that the VPA+OE-SCAMP5 group rats had a significantly increased time spent in the central grid (*P<0.05). Figure 6 AB), upright time increased (**P<0.01) Figure 6 -C), while there was no difference in the frequency of urination and defecation (-C). Figure 6 DE). Experimental results showed that rats in the VPA+OE-SCAMP5 group exhibited increased exploration behavior and cognitive abilities in their surroundings, and enhanced curiosity, but showed no significant anxiety.

[0086] 2.6.2 Three-Box Social Experiment

[0087] Figure 7 The results showed that in the first phase of the experiment (0-10 min), compared with the CON group, the VPA group spent significantly less time in chamber C where the unfamiliar mouse 1 was located (***P<0.001), while the VPA+OE-SCAMP5 group rats were more inclined to be in chamber C where the unfamiliar mouse 1 was located (***P<0.01); in the second phase (10-20 min), compared with the CON group, the VPA group spent significantly more time in chamber C where the unfamiliar mouse 1 was located (**P<0.01), while the VPA+OE-SCAMP5 group rats were more inclined to be in chamber A where the unfamiliar mouse 2 was located (***P<0.001); this indicates that compared with the VPA group rats, the VPA+OE-SCAMP5 group rats showed a more obvious bias towards the new social mouse.

[0088] 2.6.3 Youth Play Experiment

[0089] The adolescent play test is mainly used to reflect the social interaction behavior and repetitive stereotyped behaviors in rats. Figure 8 The results showed that, compared with the VPA group, the VPA+OE-SCAMP5 group rats had significantly increased chase time (***P<0.001) and social time (***P<0.001) with the tool rats, while there was no significant difference in digging time. This indicates that the VPA+OE-SCAMP5 group rats had stronger social abilities. The fur-stroking time of the VPA+OE-SCAMP5 group rats was lower than that of the VPA group (***P<0.001), indicating that the VPA+OE-SCAMP5 group rats exhibited reduced repetitive and stereotyped behaviors.

[0090] 3. Summary and Analysis

[0091] Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social impairment, delayed language development, and repetitive behaviors. According to the latest epidemiological surveys, the global prevalence of ASD has reached 1-2%, posing a serious public health problem. The etiology of ASD is complex and diverse, but synaptic dysfunction is considered one of its core pathological mechanisms. Secretory carrier membrane proteins (SCAMPs), as key molecules regulating vesicle transport, are closely associated with the development and progression of ASD through abnormal expression of SCAMP5, a member of the secretory carrier membrane protein family, in the nervous system.

[0092] SCAMPs are an evolutionarily conserved family of transmembrane proteins that act as carriers in the post-Golgi circulation pathway. Five members of the SCAMP family (SCAMP1 to SCAMP5) have been identified. SCAMPs are widely distributed in the post-Golgi membrane, synaptic vesicles, secretory granules, and transporter vesicles, and are involved in various membrane transport functions. SCAMP1 has been reported to be associated with various solid tumors, such as ovarian cancer, pancreatic cancer, gallbladder cancer, breast cancer, and colorectal cancer. Scamp5 consists of an N-terminal tail, four TMDs, and a C-terminal tail; the TMDs contain a 2 / 3 loop domain. SCAMP5 is associated with various neurodegenerative diseases. SCAMP5-mediated exosome secretion is a mechanism for the clearance of neurotoxic proteins; aberrant clearance of α-synuclein may affect the progression of PD; two patients with heterozygous SCAMP5 G180R mutations have been reported to present with ASD, ID, and EP episodes.

[0093] This experiment first used ELISA to detect changes in SCAMP5 expression in the serum of children with autism and normal individuals. Then, by establishing a VPA-induced ASD rat model, SCAMP5 was overexpressed in the hippocampus of the rats to explore its effect on the core symptoms of ASD. The results showed that SCAMP5 expression was decreased in the serum of children with autism. After VPA-induced overexpression of SCAMP5 in the hippocampus of ASD rats, social impairment, repetitive and stereotyped behaviors, and spatial exploration abilities were significantly improved in the ASD rats.

[0094] In conclusion, SCAMP5 significantly improved spatial cognition and autism-like behavior induced by VPA in ASD rats. However, its mechanism of affecting Glu release in ASD remains unclear.

[0095] Example 2: Study on the development mechanism of SCAMP5 in VPA-induced ASD rats.

[0096] 1. Materials and Methods

[0097] 1.1 Materials

[0098] 1.1.1 Laboratory Animals

[0099] See Example 1 for details.

[0100] 1.1.2 Main Reagents

[0101]

[0102] The rest is the same as in Example 1.

[0103] 1.1.3 Related Antibodies

[0104]

[0105] The rest is the same as in Example 1.

[0106] 1.2 Experimental Methods

[0107] 1.2.1 Constructing a VPA-induced ASD rat model

[0108] Same as Example 1.

[0109] 1.2.2 Brain tissue sampling

[0110] Same as Example 1.

[0111] 1.2.3 Protein concentration determination by BCA method

[0112] Same as Example 1.

[0113] 1.2.4 Western bolt

[0114] Same as Example 1.

[0115] 1.2.5 Molecular docking

[0116] Molecular docking and visualization analysis were performed using PyMOL software and the HDOCK platform.

[0117] 1.2.6 Co-IP

[0118] (1) Pretreatment of magnetic beads: Take 50 μL of A / G magnetic beads and wash them three times with 400 μL of IP lysis buffer. Magnetic separation is performed, and the supernatant is discarded.

[0119] (2) Antibody-magnetic bead binding: Dilute 3 μL of antibody with 400 μL IP lysis buffer, add the diluted antibody to the magnetic beads prepared in step 1, suspend thoroughly, and incubate in a reverse mixer at 4℃ for 2 h.

[0120] (3) Washing: Add 400 μL of IP lysis buffer, fully suspend the magnetic beads, separate them magnetically, discard the supernatant, and repeat the washing 4 times.

[0121] (4) Weigh and record the hippocampal tissue. Prepare RIPA:PMSF = 100:1 according to the instructions, using freshly prepared solution. Add 7 μL of RIPA lysis buffer to 1 μg of fresh tissue. Add 3 grinding beads to each sample and set 5 grinding cycles in a tissue homogenizer (4.5 m / s speed, 1 min per cycle, 30 s interval between cycles). After complete lysis, centrifuge at 12000 rpm for 20 min in a 4°C centrifuge. Transfer the supernatant to a new EP tube and label it.

[0122] (5) Take 100 μL as input, add 25 μL of 5 × loading buffer, and heat at 95℃ for 5 min to denature.

[0123] (6) Antigen-antibody-magnetic bead complex binding: Add 400 μL of the antigen sample prepared in step 4, fully suspend, and incubate in a reverse mixer at 4°C overnight.

[0124] (7) Magnetic separation of antigen-antibody-magnetic bead complex, discarding the supernatant, and washing five times with 400 μL IP lysis buffer (for the fourth wash, transfer the resuspended and mixed liquid to a new EP tube and then magnetically separate and discard the supernatant).

[0125] (8) Add 40 μL of 1 × loading buffer (SDS-DTT), boil at 99℃ for 5 min to wash out the protein.

[0126] (9) Magnetic separation, transfer the supernatant to the new EP tube.

[0127] (10) Perform immunoblotting experiments.

[0128] 1.2.7 Transmission Electron Microscopy

[0129] (1) Weigh the patient and, after anesthesia, infuse the apex of the heart with normal saline and 20% glutaraldehyde for internal fixation until the outflowing fluid is clear.

[0130] (2) The rat's neck was severed, the scalp was cut open, the skull was dissected, the entire brain was dissected, and the hippocampus was trimmed to approximately 1 mm. 3 The tissues of both sizes were externally fixed in 20% glutaraldehyde and stored at 4°C.

[0131] (3) Slicing + photography: magnification 30000 × .

[0132] 1.2.8 Golgi staining: standard method.

[0133] 1.2.9 Analysis of Dendritic Thorn Quantity

[0134] ImageJ opens and processes the captured image, overlays the image, adjusts the brightness, and sets the ruler.

[0135] The counting function of Neuron J is used to count the number of dendritic spines within a certain distance.

[0136] 1.2.10 Statistical Methods

[0137] Data analysis was performed using GraphPad Prism 10.0 statistical software (GraphPad Software, USA). For comparisons of three or more groups, one-way ANOVA combined with Tukey's multiple comparison test was used to analyze differences between groups. In experiments examining the interaction of different treatments and stimulus conditions, two-way ANOVA combined with Sidak's multiple comparison test was used. All experimental results are presented as mean ± standard error (mean ± SEM). The statistical significance level was set at a p-value less than 0.05.

[0138] 2 Results

[0139] 2.1 Interaction between SCAMP5 and SYT1

[0140] Synaptic vesicle exocytosis is a key step in neurotransmitter release, and SYT1, a calcium-sensitive synaptic vesicle membrane protein, plays a central role in the fusion of synaptic vesicles with the presynaptic membrane. To detect the interaction between SCAMP5 and SYT1, we performed molecular docking using PyMOL software and the HDOCK platform, performed PyMOL visualization analysis on model1, and used Co-IP to detect the presence of an interaction between SCAMP5 and SYT1. The results showed that the confidence score for SCAMP5 and SYT1 was higher than 0.7, indicating good binding (e.g., ...). Figure 9 -A), Visual analysis such as Figure 9 -B, Co-IP suggests that SCAMP5 and SYT1 may interact (e.g., Figure 9 -B).

[0141] 2.2 SYT1 expression after SCAMP5 overexpression

[0142] SYT1 senses calcium ion influx through its C2 domain, triggering the assembly of the SNARE complex and thus promoting neurotransmitter release. To examine the changes in SYT1 expression after SCAMP5 overexpression, we used Western blot to detect SYT1 expression levels. The results showed that SYT1 expression in the hippocampus of rats in the VPA group was significantly higher than that in the CON group. After SCAMP5 overexpression, the SYT1 expression level in the VPA+OE-SCAMP5 group was lower than that in the VPA group (e.g., ...). Figure 10 ).

[0143] 2.3 Abnormal release of Glu from the presynaptic membrane

[0144] Based on the previously discovered abnormal SYT1 expression, we further systematically investigated the changes in presynaptic membrane Glu release function. Transmission electron microscopy was used to detect differentially expressed neurotransmitters, and gas chromatography was used to detect the dense region of presynaptic membrane vesicles. Figure 11 -A), Western blot was used to detect glutamate transporter 1 (vGlut1), and ELISA was used to detect Glu release. Results indicated that Glu and GABA were differentially expressed proteins in the VPA model. vGlut1 expression in the hippocampus of rats in the VPA group was significantly higher than that in the CON group, and the expression level of vGlut1 decreased after SCAMP5 overexpression compared to the VPA group. Figure 11 BC), ELISA results showed that the Glu content was significantly increased in the VPA group, and the Glu expression level decreased after SCAMP5 overexpression. Figure 11 -D). In summary, increased Glu release in the VPA model suggests that overexpression of SCAMP5 may reduce VPA-induced Glu abnormalities.

[0145] 2.4 Overexpression of SCAMP5 in the hippocampus improves dendritic density of hippocampal neurons in ASD rats

[0146] Neuronal dendritic density is related to synaptic plasticity and neuronal development. We performed high-magnification observation of dendritic spines in rat hippocampal cells using Golgi staining and counted the density of dendritic spines. The results showed that compared with the CON group ( Figure 12 In the VPA group, dendritic spine density was significantly decreased, while overexpression of SCAMP5 increased dendritic spine density. This suggests that SCAMP5 may affect synaptic plasticity and neuronal development.

[0147] 2.5 Homer1b / c bracket malfunction

[0148] Homer1b / c is a scaffold protein located on the postsynaptic membrane and is associated with various neurological disorders. Some studies have indicated that Homer1b / c interacts with mGluR1 / 5, and changes in Glu may alter this interaction. To detect changes in Homer1b / c expression, we used Western blot to examine changes in Homer1b / c expression and the protein activity of its downstream pathway, IP3R. The results showed that in the VPA model, Homer1b / c protein expression decreased, and overexpression of SCAMP5 increased Homer1b / c expression levels compared to the VPA group. Figure 13 In the VPA model, compared with the CON group, IP3R protein expression decreased, and the expression level of IP3R increased after hippocampal overexpression of SCAMP5 compared with the VPA group. Figure 13 CD).

[0149] 3. Summary and Analysis

[0150] Brain information processing depends on a balance between excitatory (primarily glutamate-mediated) and inhibitory (primarily GABA-mediated) neurotransmission. An imbalance between excitation and inhibition can lead to dysfunction of neural circuits, resulting in various neuropsychiatric disorders, including Drawe syndrome, schizophrenia, Fragile X syndrome, and depression. The E / I imbalance hypothesis has become one of the important theories governing the pathological mechanisms of ASD.

[0151] Synaptotagmins (SYTs) are a family of membrane transport proteins, composed of an N-terminal transmembrane domain (TMD), a variable linker region, and two cytoplasmic C2 domains (C2A and C2B domains) at the C-terminus of the protein. Genetic and functional studies have shown that SYT1 plays a crucial role in mediating the release of calcium-triggered neurotransmitters (NTs) and is associated with various neuropsychiatric disorders. Some studies have indicated that SYT1 acts as a calcium-sensitive neurotransmitter... 2+ Receptors can influence vesicle release via SNARE. Homer proteins are located in the postsynaptic compact area of ​​mammalian neurons and function as adaptors to many postsynaptic compact proteins, including three isoforms: Homer1, Homer2, and Homer3. Homer plays an important role in secondary brain injury and cerebral hemorrhage, and is also associated with the pathogenesis of various neurological diseases. Recent articles have indicated that Homer1a is controlled by a bimodal response to circadian rhythms and CREB in the mouse brain, and that Homer1a has significant implications for synaptic remodeling and antidepressant mechanisms.

[0152] This study revealed the interaction between SCAMP5 and SYT1 and its regulatory role in a VPA-induced autism spectrum disorder (ASD) rat model. Western blot analysis showed a significantly increased expression level of SYT1, a key protein in presynaptic vesicle release, in the ASD model group. Overexpression of SCAMP5 reversed this phenomenon, suggesting that SCAMP5 may influence presynaptic vesicle release by regulating SYT1. Further analysis revealed a significant increase in Glu content in the ASD model group, which decreased after SCAMP5 overexpression, indicating that SCAMP5 may regulate synaptic Glu release through a SYT1-dependent pathway. Golgi staining revealed significantly lower dendritic density in the hippocampus of VPA model rats compared to the control group; overexpression of SCAMP5 improved dendritic density, demonstrating that SCAMP5 can improve synaptic development abnormalities in ASD rats. Furthermore, analysis of the postsynaptic dense scaffold protein Homer1b / c and its downstream signaling molecule IP3R showed that abnormal changes in Glu may lead to assembly or functional disorders of Homer1b / c, thereby disrupting postsynaptic signal transduction and ultimately causing abnormal synaptic development.

Claims

1. The application of SCAMP5 molecules as targets in screening drugs for the treatment of autism spectrum disorders, wherein the SCAMP5 molecule is the SCAMP5 protein or the SCAMP5 gene.

2. The application according to claim 1, characterized in that: The drug promotes the expression of SCAMP5 molecules.

3. Application of SCAMP5 expression promoters in the preparation of drugs for treating autism spectrum disorders.

4. The application according to claim 3, characterized in that: The SCAMP5 expression promoter is a recombinant vector that expresses SCAMP5.

5. The application according to claim 4, characterized in that: The recombinant vector is a recombinant adeno-associated virus vector that overexpresses SCAMP5.

6. The application of SCAMP5 protein or the gene expressing SCAMP5 protein in the preparation of drugs for the treatment of autism spectrum disorder.

7. The application according to any one of claims 1 to 6, characterized in that: SCAMP5 improves the abnormal release of excitatory glutamate (Glu) from the presynaptic membrane.

8. The application according to claim 7, characterized in that: SCAMP5 and SYT1 interact with each other; SCAMP5 affects the release of Glu from the presynaptic membrane through SYT1.

9. The application according to any one of claims 1 to 6, characterized in that: SCAMP5 improves abnormalities in the Homer1b / c scaffold and its downstream pathway IP3R protein in patients with autism spectrum disorder.

10. The application according to any one of claims 1 to 6, characterized in that: SCAMP5 improves spurious developmental abnormalities in patients with autism spectrum disorder.

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