Expression regulation of alpha-synuclein and its applications

Abstract

The application discloses expression regulation of alpha-synuclein and application thereof. The alpha-synuclein inhibitor is used for preparing a medicine, and the obtained medicine has the effects of preventing and treating anxiety caused by social isolation. Experiments prove that after the alpha-synuclein inhibitor is administered, the anxiety behavior of a model animal in a social isolation environment can be improved, and the alpha-synuclein inhibitor has a treatment effect on anxiety caused by social isolation. The alpha-synuclein inhibitor is selected from the following: 1) an ASO targeting SNCA mRNA coding alpha-synuclein; 2) a small-molecule compound targeting SNCA mRNA coding alpha-synuclein; or 3) an AAV interfering with the expression level of alpha-synuclein in a ventral hippocampal neuron.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the expression regulation of α-synuclein and its applications. Background Technology

[0002] General anxiety refers to a psychological reaction characterized by tension, worry, and unease when an individual faces potential threats, uncertainties, or stress. It may be accompanied by physiological or cognitive symptoms such as rapid heartbeat, insomnia, and difficulty concentrating. Its causes include a variety of factors such as genetics, environmental stress, cognitive biases, and physical illnesses. Symptoms can generalize to multiple areas of life and are not necessarily tied to a specific scenario.

[0003] As a subcategory of anxiety disorders, anxiety caused by social isolation refers to a targeted anxiety response directly triggered by a prolonged lack of sustained, effective, and meaningful interpersonal interaction and emotional connection (i.e., a state of social isolation). Prolonged social isolation is a key triggering factor for the exacerbation of anxiety disorders. The impact of social isolation is profound; it not only weakens an individual's social support system in real life but also triggers a series of cognitive impairments due to persistent environmental stress, specifically manifested as difficulty concentrating, memory loss, and decreased decision-making ability. Of particular concern is that groups in a prolonged state of isolation are more prone to developing negative cognitive schemas, such as catastrophic thinking and hypervigilance, which further exacerbate mood regulation disorders. This type of anxiety disorder stems from social isolation. Therefore, how to effectively prevent and treat anxiety caused by social isolation has become an urgent problem to be solved.

[0004] In clinical treatment, cognitive behavioral therapy (CBT) and serotonin reuptake inhibitors (SSRIs) are currently considered first-line treatments for anxiety disorders. However, these methods have significant limitations and are not specifically targeted when dealing with anxiety disorders caused by social isolation. CBT, as a widely accepted psychological intervention, is mostly designed for face-to-face consultations, making it difficult to implement remotely in isolated environments. Furthermore, its treatment cycle is long, and patient compliance is poor, leading some patients to abandon treatment midway. Traditional drug treatments for anxiety disorders mainly include benzodiazepines and serotonin reuptake inhibitors (SSRIs). SSRIs, as the most important and commonly used class of serotonin-containing drugs, exert their anti-anxiety effect by increasing the concentration of serotonin in the brain. However, SSRIs are prone to causing gastrointestinal reactions and sexual dysfunction, which reduces patient tolerance to some extent; while benzodiazepines, due to their high risk of addiction, also greatly limit their long-term use.

[0005] Current treatment methods suffer from significant gaps and low efficacy in targeting and regulating social isolation-specific neural circuits and molecular targets. Therefore, a thorough and systematic analysis of the molecular and neural mechanisms of anxiety disorders caused by social isolation, and the development of novel treatment technologies that are both highly effective and safe, is of paramount research value and practical significance, and is expected to pave new avenues for solving this problem. Summary of the Invention

[0006] The purpose of this invention is to disclose the expression regulation of α-synuclein and its application, so as to solve the technical problems of high risk of tolerance to SSRIs or addiction to benzodiazepines, and to provide at least one beneficial option or create conditions for the treatment of anxiety caused by social isolation.

[0007] The first aspect of this invention lies in providing the application of α-synuclein (α-Syn) inhibitors, specifically: using α-Syn inhibitors to prepare drugs, the resulting drugs having the efficacy of preventing and treating anxiety disorders caused by social isolation. Experiments have shown that administration of α-Syn inhibitors can improve anxiety behavior in model animals in a socially isolated environment, demonstrating its therapeutic effect on anxiety disorders caused by social isolation.

[0008] In some implementations of the first aspect of the present invention, the α-Syn inhibitor is selected from:

[0009] 1) Antisense oligonucleotides (ASOs) that target SNCA mRNA encoding α-Syn.

[0010] 2) Small molecule compounds that target SNCA mRNA encoding α-Syn; or,

[0011] 3) Adeno-associated virus (AAV) that targets and interferes with the expression level of α-Syn in ventral hippocampal neurons.

[0012] In some embodiments of the first aspect of this invention, the drug is an injectable or nasal inhalation agent. Intravenous injection can increase the drug's cross-blood-brain barrier penetration rate through high blood drug concentration; intracerebral injection (such as stereotactic injection into the ventral hippocampus) can directly bypass the blood-brain barrier, precisely delivering the drug to the target area and avoiding systemic diffusion. The nasal mucosa is rich in blood vessels and olfactory nerve endings. Nasal inhalation agents can cross the blood-brain barrier after entering the bloodstream through the nasal mucosa vessels, or directly enter the central nervous system through the olfactory nerve, both of which can significantly increase the intracranial drug concentration without systemic metabolism.

[0013] A second aspect of the present invention is to provide a pharmaceutical combination for the prevention and treatment of anxiety disorder caused by social isolation. The pharmaceutical combination contains an α-Syn inhibitor.

[0014] In some implementations of the second aspect of the present invention, the α-Syn inhibitor is selected from:

[0015] 1) Targeting ASO that encodes SNCA mRNA of α-Syn;

[0016] 2) Small molecule compounds that target SNCA mRNA encoding α-Syn; or,

[0017] 3) AAV that interferes with the expression level of α-Syn in ventral hippocampal neurons.

[0018] In some embodiments of the second aspect of the present invention, the nucleotide sequence of the ASO is 5'-CUCCCTCCATGTCUUCU-3' (SEQ ID No:1). The nucleotide sequence of its control ASO is 5'-CCGTATCTGTAAGCATAC-3' (SEQ ID No:2). ASO has the advantages of not affecting animal cognition, memory, and behavior, has no obvious toxic side effects within appropriate doses, is easy to prepare, extract, and preserve, and has high stability.

[0019] In some embodiments of the second aspect of the present invention, the small molecule compound may be selected from Synucleozid. Synucleozid is a small molecule compound that targets SNCA mRNA encoding α-Syn. After entering the body, it is converted into a ribonuclease-targeting chimeric compound (RIBOTAC), namely Syn-RIBOTAC, which can selectively degrade SNCA mRNA, thereby significantly reducing the level of α-Syn.

[0020] In some application embodiments of the second aspect of the present invention, the AAV is recombinant adeno-associated virus type 5 (rAAV5), specifically constructed from the following elements: AAV5-CAG-DIO-Sncα-shRNA-EGFP-WPRE-hGH polyA. The sense strand oligo of the Sncα-shRNA is 5'-CACCGCAGAAATCGAAGCAGAGTTTCGAAAAACTCTGCTTCGATTTCTGC-3' (SEQ ID No:3); the antisense strand oligo is 5'-AAAAGCAGAAAYCGAAGCAGAGTTTTTCGAAACTCTGCTTCGATTTCTGC-3' (SEQ ID No:4). This oligo can specifically bind to and degrade SNCA mRNA through RNA interference (RNAi) mechanisms, thereby silencing SNCA gene expression and reducing α-Syn synthesis. Local application of shRNA technology to reduce α-Syn expression levels in the ventral hippocampus can effectively decrease α-Syn expression in the ventral hippocampus, improve abnormal neuronal cell metabolism in the ventral hippocampus of socially isolated animals, and specifically prevent or improve anxiety disorders induced by social isolation stress.

[0021] In some implementations of the second aspect of the present invention, the content of rAAV5 in the drug is (1~2.88)×10¹² μg / mL.

[0022] In some embodiments of the second aspect of the invention, the dosage form of the drug combination is selected from tablets, solutions, suspensions, emulsions, microcapsules, microspheres, injections, liposomes, or aerosols.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. This invention is the first to propose using multiple administration methods to interfere with α-Syn expression levels in the prevention and / or treatment of anxiety disorder caused by social isolation. Experiments have shown that in animal models of anxiety disorder caused by social isolation, the α-Syn expression level in the ventral hippocampus is significantly increased, and interfering with α-Syn expression levels using multiple administration methods can significantly improve the anxiety symptoms in these animal models.

[0025] 2. This invention provides the first in-depth explanation of the mechanism of anxiety disorder caused by social isolation from the perspective of interfering with α-Syn expression levels through multiple administration methods. It proposes intervention programs for key links in this mechanism and demonstrates the effectiveness and great application potential of the intervention programs, laying the foundation for further development of drugs for anxiety disorder caused by social isolation.

[0026] In summary, this invention focuses on drug innovation for the prevention and treatment of anxiety disorder caused by social isolation by interfering with α-Syn through multiple administration methods. It provides a variety of novel treatment strategies for the prevention and treatment of anxiety disorder caused by social isolation, and is expected to promote the innovation of treatment methods in this field, with broad application prospects. Attached Figure Description

[0027] Figure 1 These are the statistical results of open field experiments on mice in different treatment groups in Example 1 of this invention;

[0028] Figure 2 The above-ground cruciate maze test results of mice in different treatment groups in Example 1 of this invention are statistical results.

[0029] Figure 3 This is a bar chart showing the expression and protein levels of the α-Syn gene in the ventral hippocampus of a mouse model of anxiety disorder caused by social isolation in Example 1 of this invention.

[0030] Figure 4 This is a comparison of the correlation between the α-Syn expression level in the ventral hippocampus region of the brain and anxiety-related phenotypes in a mouse model of anxiety disorder caused by social isolation in Example 1 of this invention.

[0031] Figure 5 The above are the statistical results of the open field experiment of Sncα knockout mice in Example 2 of this invention;

[0032] Figure 6 The above-ground cross maze experiment results of Sncα knockout mice in Example 2 of this invention are statistical results.

[0033] Figure 7 This is a bar chart showing the protein levels administered to Synucleozid mice via implanted catheter in Example 3 of this invention;

[0034] Figure 8 This is the statistical result of the open field experiment administered to Synucleozid mice via implanted tube in Example 3 of the present invention;

[0035] Figure 9 The above-ground cruciate maze experiment results of Synucleozid mice administered via implanted tubes in Example 3 of this invention;

[0036] Figure 10 This is a bar chart showing the protein levels administered to ASO mice via implanted tubes in Example 3 of the present invention.

[0037] Figure 11 This is the statistical result of the open field experiment administered to ASO mice via implanted tube in Example 4 of the present invention;

[0038] Figure 12 The statistical results of the elevated cruciate maze experiment administered to ASO mice via implanted tubes in Example 4 of this invention;

[0039] Figure 13 The results of fluorescence microscopy detection in mice injected with rAAV5 in Example 5 of this invention;

[0040] Figure 14 This is a bar chart showing the protein levels in mice injected with rAAV5 in Example 5 of this invention.

[0041] Figure 15 This is the statistical result of the open field experiment in mice after injection of rAAV5 in Example 5 of the present invention;

[0042] Figure 16 The above-ground cruciate maze experiment results of mice injected with rAAV5 in Example 5 of this invention are statistical results.

[0043] Figure 17 The above are the statistical results of the open field experiment on mice treated with nasal administration in Example 6 of the present invention;

[0044] Figure 18 The above-ground maze experiment results are presented for mice treated with nasal administration in Example 6 of this invention.

[0045] In the figure, all those marked with "*" indicate that there is a significant difference between the two groups, where "*" means p < 0.05, "**" means p < 0.01, and "***" means p < 0.001. Detailed Implementation

[0046] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0047] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0048] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0049] Unless otherwise specified, all reagents and consumables used in the following experimental examples are commercially available.

[0050] The SPF-grade C57BL / 6J mice used in the following experiments were purchased from the Laboratory Animal Center of Southern Medical University and bred at South China University of Technology. The mice were housed in a standard animal facility with a temperature controlled at 23±2℃ and a relative humidity maintained at 50±10%, following a 12-hour light (lighting on at 8:00 AM) / 12-hour dark circadian rhythm. The mice had free access to food and drink. This experiment was conducted strictly in accordance with the "Guidelines for Laboratory Animal Care and Use" and has been approved by the Animal Ethics Committee of South China University of Technology.

[0051] In the following experimental examples, CαMK2α-Cre mice (strain number: 005359) and Sncα knockout(KO) mice (strain number: 016123) were obtained from the Jackson Laboratory and bred at South China University of Technology. The mice were housed in a standard animal room with a temperature of 23±2℃ and humidity of 50±10%, following a 12h / 12h (8:00 lighting) diurnal rhythm, with free access to water and food.

[0052] The primer pair for Sncα RT-qPCR includes: forward primer sequence: 5'-GAGGGCGTCCTCTATGTAGGT-3' (SEQ ID No: 5), and reverse primer sequence: 5'-CCCTCTTGTGGGTACCCCTTCT-3' (SEQ ID No: 6).

[0053] The antibody used for immunoblotting of α-Syn was Anti-Alphα-Synuclein [MJFR1] (1:1000, αb138501, purchased from Abcαm), and the internal control was αnti-GAPDH (1:2000, αb8245, purchased from Abcαm).

[0054] Synucleozid; acquired from MCE.

[0055] In the following experimental examples, recombinant adeno-associated virus type 5 (AAV5) was prepared by Priscilla Corporation. The AAV5 targeting Sncα was AAV5-CAG-DIO-Sncα-shRNA-EGFP-WPRE-hGH polyA, targeting the coding region of murine Snca (NCBI Gene ID: 20617) mRNA. This DIO strategy relies on the virus's ability to react with pyramidal cell-specific transgenic mice to produce cell-specific infection and Sncα knockout. AAV5-CAG-DIO-SC-shRNA-EGFP-WPRE-hGH polyA containing a negative control was also prepared as a control. The titer of all the aforementioned recombinant AAV5 types was 2.88 × 10⁻⁶.12 VG / mL. The above-mentioned AAV virus and control virus can be purchased from Priscilla Virus Company.

[0056] Specific steps for the elevated cross maze experiment: The experiment was conducted on the morning of the experiment, using an elevated cross maze with both closed walls and open arms measuring 65 cm in length. Before the experiment, the mice were gently soothed to alleviate their anxiety. During each experiment, a mouse was gently placed in the center of the maze and allowed to move freely for 5 minutes. During this time, a camera above the maze precisely recorded the mouse's movements. After each mouse completed its experiment, the apparatus was immediately thoroughly cleaned and disinfected with 75% (v / v) ethanol to ensure complete removal of any mouse odor and to avoid interference with subsequent experiments before placing the next mouse. When the mice entered the elevated cross maze, due to their innate fear of open, high-altitude environments, they instinctively preferred to remain within the closed walls. Normal mice typically exhibit strong exploratory traits and will generally explore the open arm areas a certain number of times and for a certain proportion of the time. However, the exploratory traits of the socially isolated model mice were significantly reduced, and their fear of open and high-altitude environments was significantly enhanced, thus showing a marked preference for the closed-wall environment. This experiment used the professional Smart 3.0 video-tracking software to accurately record and analyze the movement trajectory preferences of mice in the maze, thereby obtaining accurate experimental data.

[0057] Specific steps of the open field experiment:The open field experiment uses a rectangular box measuring 40 cm × 40 cm × 40 cm (length × width × height), with a rectangular area designated as the central zone at the bottom. Before the experiment, researchers gently soothe the mice to ensure the results are not affected by stress. At the start of each experiment, the mouse is gently placed in the rectangular box at the bottom center, allowing it to move freely within the open field. During the mouse's movement, the sensing device accurately records its path and automatically saves the data. After each mouse completes one open field experiment, the device is immediately and thoroughly cleaned and disinfected with 75% (v / v) ethanol to completely remove any odor left by the mouse and prevent interference with subsequent experiments before placing the next mouse. Once inside the open field device, mice, due to their instinctive fear of open, unprotected environments, tend to move along the four walls of the device. Normal mice, however, with their strong exploratory nature, will typically spend a certain percentage of their time exploring the central open area. The social isolation model mice exhibited significantly reduced exploration and novelty-seeking behaviors, and a markedly increased fear of open environments, thus clearly disliking being in the central open area. During the experiment, the mice's movement was continuously captured by a camera, and the data was recorded and analyzed using Smart 3.0 video-tracking software. The focus was on calculating the duration of the mice's stay in the central area to accurately reflect the animals' anxiety state.

[0058] Specific steps for catheter-based drug delivery: This invention employs a standardized stereotactic surgical procedure for targeted drug delivery: after scalp incision, a guide cannula (26 G) is implanted into the ventral hippocampus of the target brain region (vHip: -3.28 mm posterior to the anterior fontanelle, ±3.25 mm lateral to the midline, depth -4.50 mm) via a bilateral cannula system (RWD Life Science); the cannula is fixed with dental cement and incubated in a constant temperature and humidity environment (25±1℃, 55±5% humidity) for 7 days, during which time a closed cannula is left in place for protection and daily monitoring is performed. ASO was dissolved in artificial cerebrospinal fluid (ACSF, pH 7.4, 290–300 mOsm) and infused via a microprocessor-controlled pump (RWD R462) through a 33 G cannula (100 nL / min). Specific dosages followed pre-experimental or established protocols: Synucleozid (HY-135902A, 0.3 μg / 0.3 μL / side) and ASO (500 μg / 200 μL PBS / side, 0.5 μL / min flow rate) were delivered. All procedures were performed under aseptic conditions to ensure reproducibility and minimize experimental variability.

[0059] Nasal administration regimen:The α-Syn inhibitor Synucleozid and antisense oligonucleotides (ASO) were administered bilaterally daily between 09:00 and 11:00 AM (5 μL per nostril, 10 μL total) for 28 days according to a standardized protocol (30 μg / 10 μL PBS, containing 0.1% chitosan permeability enhancer). Nasal administration was performed as follows: Mice were fixed prone on a heated platform (37°C) with their necks extended at 15°. A calibrated micropipette (1 μL / second) was used to deliver the solution at a 45° nasal angle, forming an aspirable droplet. Freshly prepared solutions (stored at 4°C for <2 hours) were administered under laminar flow conditions with strict contamination control (gloves were changed between groups). Successful administration was confirmed when the droplet was completely cleared within 10 seconds (success rate >95%). Mice were then observed for 30 seconds before being returned to their individual ventilated cages.

[0060] Specific steps for the synthesis and purification of antisense oligonucleotides (ASO): This invention employs a chemically modified 18-mer ASO as a targeted degradation tool for α-synuclein mRNA. The gαpmer structure is designed to include a full-length thiophosphoryl (PS) backbone, with four locked nucleic acids (LNAs) modified at each of the 5' and 3' ends to form wing structures, and a 2'-deoxynucleotide (dN) gap in the center to activate RNαse H1-mediated mRNA cleavage. The designed ASO sequences include: (1) an active ASO targeting mouse SNCA gene exon 3: 5'-CUCCCTCCACTGTCUUCU-3' (SEQ ID No:1), with RNAhybrid prediction ΔG=-28.6 kcαl / mol, and no off-target sites in miRBαse v22; (2) a control ASO with <30% homology to the mouse transcriptome: 5'-CCGTATCTGTAAGCAGTAC-3' (SEQ ID No:2), verified by BLASTn GRCm39.

[0061] Synthesis process: ASO was synthesized by solid-phase phosphoramide method (Qiαgen), and after 2'-O-methylation modification at the end, it was purified by reversed-phase / ion-exchange dual-column chromatography system (XBridge OST C18 column + DNAPAc PA200 column) to obtain the product with a full-length purity of >98% (CE-LIF verification) and <0.01 EU / mg endotoxin (LAL detection). The product is the above-mentioned active ASO and control ASO.

[0062] Purification: For brain distribution studies, active ASO was site-directedly labeled with Alexα Fluor 594 via a 5'-hexylamino linker (DMT-on synthesis protocol), achieving a fluorescence labeling efficiency >95% (verified by MALDI-TOF MS). The target binding capacity was equivalent to unlabeled ASO (IC50 values ​​of primary neurons: labeled 12.3±1.7 nmol / L vs. unlabeled 11.8±2.1 nmol / L). The ASO stock solution was mixed with a lyophilization protectant in a specific ratio and placed in a container. The sample was rapidly cooled in a lyophilization chamber until completely frozen into a solid. It was then reconstituted with sterile PBS (pH 7.4) and equilibrated at 37°C for 2 hours to ensure proper folding. Based on the sequence-specific extinction coefficient (active ASO ε260 = 178500 M), the final sample was determined. -1 cm -1 Randomized control ε260=181200 M -1 cm -1 Spectrophotometric quantification was performed (NαnoDrop One), with A260 / A280 > 1.8 and A260 / A230 > 2.0; ASO working solution (0.1~10 mg / mL) was sterilized by passing it through a 0.22 μm PVDF membrane.

[0063] Administration routes: a) intranasal administration (30 μg / 10 μL PBS, containing 0.01 g chitosan permeation enhancer), or b) intraventricular infusion (500 μg / 200 μL PBS, 0.5 μL / min flow rate). This design integrates the triple advantages of nuclease resistance (PS backbone), high affinity binding (LNA modification), and catalytic degradation (gαpmer structure). The fluorescent labeling system supports intrabrain target imaging via confocal microscopy.

[0064] Example 1: Constructing an animal model of anxiety disorder caused by social isolation

[0065] To verify the association between social isolation-induced anxiety and α-Syn, a social isolation mouse model was constructed, and the expression of α-Syn in the ventral hippocampus and related anxiety behaviors were examined. The specific protocol is as follows:

[0066] 1) Construction of a social isolation mouse model

[0067] 1.1) Healthy 8-week-old male C57BL / 6J mice were selected and randomly divided into a social isolation group and a group (control) group. Each mouse in the social isolation group was housed alone in a cage, while 4 mice in the group were housed together in a cage. Both groups of mice had free access to water and food during the 28-day rearing period.

[0068] 1.2) After 28 days of rearing, anxiety behavioral tests, including the elevated cross maze test and the open field test, were conducted on both groups of mice. Before the experiments, it was ensured that both experimental setups were free of any special odors to avoid interfering with the mice's behavior. During the experiments, detailed experimental data were recorded for each group of mice. The results are as follows: Figure 1 and Figure 2 As shown.

[0069] Open field experiment results ( Figure 1 The results showed that mice in the socially isolated group spent significantly less time in the central region of the open field than mice in the social group. In the elevated cross maze experiment (…),… Figure 2 In the study, compared to the control group, the social isolation group mice spent significantly less time in the open arm and entered the open arm less frequently.

[0070] In conclusion, social isolation stress can induce anxiety-like behaviors in mice, indicating that this experiment successfully constructed a social isolation mouse model.

[0071] 2) Determination of α-Syn expression in the ventral hippocampus of social isolation model mice

[0072] 2.1) α-Syn expression was measured using RT-qPCR and Western blotting. The RT-qPCR system consisted of 20 μL of 2×Master Mix, 0.4 μL each of forward and reverse primers, 2 μL of cDNA template, and 7.2 μL of RNase-free water. The reaction conditions followed a two-step procedure: pre-denaturation at 95℃ for 30 seconds; 40 cycles of amplification, each cycle including denaturation at 95℃ for 5–15 seconds and annealing / extension at 60℃ for 30–60 seconds; and finally, melting curve analysis was performed to verify amplification specificity.

[0073] The experiment was conducted using standard Western blotting methods. After the experiment, the obtained data were analyzed, and the results are as follows: Figure 3 As shown, social isolation leads to increased α-Syn expression in the ventral hippocampus of the mouse brain.

[0074] 2.2) Verify the correlation between α-Syn expression level and anxiety-like behaviors in socially isolated mice.

[0075] To further explore the potential link between α-Syn expression levels and anxiety-like behaviors in socially isolated mice, this invention employed linear regression analysis to analyze the correlation between α-Syn expression levels in the ventral hippocampus of socially isolated mice and the experimental results of anxiety-like behaviors. The analysis results are as follows: Figure 4 As shown. By Figure 4It was found that there was a significant negative correlation between α-Syn expression levels in the ventral hippocampus of the social isolation model mice and anxiety-like behavior. Data analysis showed that the correlation coefficient R between α-Syn expression levels and the time spent in the central region was [missing information]. 2 =0.8541, P value 0.0084, the correlation coefficient R between α-Syn expression level and open arm dwell time in social isolation model mice. 2 =0.7794, P=0.0198. This result indicates that increased α-Syn expression in the ventral hippocampus is closely associated with anxiety-like behavior in socially isolated mouse models. This finding provides important clues for further understanding the pathogenesis of socially isolated anxiety-like behavior, suggesting a complex interaction between α-Syn expression and socially isolated anxiety-like behavior, influencing the pathological process of anxiety disorders caused by social isolation.

[0076] To comprehensively verify the correlation between α-Syn deposition and anxiety disorder caused by social isolation, animal models of anxiety disorder caused by social isolation were treated with different methods, including Sncα knockout, small molecule inhibitors, ASO, and AAV, and the expression of α-Syn in the ventral hippocampus and related anxiety-like behaviors were detected.

[0077] Example 2: Correlation between Sncα knockout mice and social isolation-induced anxiety disorder

[0078] 1) Healthy 8-week-old male Sncα WT mice and Sncα knockout mice were randomly divided into a social isolation group and a group (control) group. The Sncα knockout mice are Sncα knockout (KO) mice (strain number: 016123). Each mouse in the social isolation group was housed alone in a cage, while four mice in the group were housed together in a cage. Both groups of mice had free access to water and food during the 28-day rearing period.

[0079] 2) After 28 days of rearing, anxiety behavioral tests, including the elevated cross maze and open field tests, were conducted on mice in both the social isolation and social groups. Before the experiments, it was ensured that both experimental setups were free of any unusual odors to avoid interfering with the mice's behavior. During the experiments, detailed data were recorded for each group of mice.

[0080] The results of the open field experiment are as follows Figure 5 As shown, the social isolation group of Sncα knockout mice spent significantly more time in the central region of the open field than the social isolation group of Sncα WT mice. The results of the elevated cruciate maze experiment are as follows: Figure 6As shown, compared to Sncα WT mice, Sncα knockout mice in the social isolation group exhibited significantly increased time spent in the open arm and a significantly increased number of entries into the open arm. These results demonstrate that Sncα knockout mice can alleviate anxiety-like behaviors, indicating a correlation between α-Syn deposition and anxiety-like behaviors induced by social isolation.

[0081] Example 3: The association between small molecule inhibitor treatment and social isolation-induced anxiety in mice

[0082] 1) Healthy 8-week-old male C57BL / 6J mice were selected for this experiment and randomly divided into a social isolation group and a group living (control) group. Each mouse in the social isolation group was housed alone in a cage, while 4 mice in the group living group were housed together in a cage.

[0083] 2) The socially isolated mice and the socially active mice were further divided into a Synucleozid group, which received a small molecule inhibitor, and a Vehicle group, which served as a control. The Synucleozid groups were administered Synucleozid at a dose of 0.3 μg / 0.3 μL / side according to the intubation administration protocol; the Vehicle groups received cerebrospinal fluid. All four groups of mice had free access to water and food for 28 days.

[0084] 3) After 28 days of rearing, the socially isolated mice underwent an immunoblotting experiment. Both the socially isolated and socially active mice underwent anxiety behavioral tests, including the elevated cross maze and open field tests. Before the experiments, it was ensured that both experimental setups were free of any unusual odors to avoid interfering with the mice's behavior. During the experiments, detailed data were recorded for each group of mice.

[0085] Immunoblotting assay, such as Figure 7 As shown, the expression level of α-Syn was significantly lower in the Synucleozid group compared to the Vehicle group. Open field experiment results are as follows... Figure 8 As shown, the Synucleozid group (socially isolated) mice spent significantly more time in the central region of the open field than the Vehicle group (socially isolated). The results of the elevated cruciate maze experiment are as follows: Figure 9 As shown, compared to the Vehicle group mice, the Synucleozid group mice in the social isolation group had a significantly increased time spent in the open arm and a significantly increased number of times they entered the open arm. These results demonstrate that Synucleozid can alleviate anxiety-like behaviors in the model animals, further indicating a correlation between α-Syn deposition and social isolation-induced anxiety disorder, and proving that small molecule inhibitors have a mitigating effect on social isolation-induced anxiety-like behaviors.

[0086] Example 4: The association between ASO treatment of mice targeting SNCA mRNA encoding α-Syn and social isolation-induced anxiety disorder.

[0087] 1) Healthy 8-week-old male C57BL / 6J mice were selected for this experiment and randomly divided into a social isolation group and a group living (control) group. Each mouse in the social isolation group was housed alone in a cage, while 4 mice in the group living group were housed together in a cage.

[0088] 2) The socially isolated mice and the socially active mice were further divided into an ASO group (SEQ ID No:1) and a Vehicle group (SEQ ID No:2) receiving ASO. Mice in all four groups were administered ASO (500 μg / 200 μL PBS / side, 0.5 μL / min flow rate) according to the intubation drug delivery procedure. All mice in all groups had free access to water and food for 28 days.

[0089] 3) After 28 days of rearing, the socially isolated mice underwent an immunoblotting experiment. Both the socially isolated and socially active mice underwent anxiety behavioral tests, including the elevated cross maze and open field tests. Before the experiments, it was ensured that both experimental setups were free of any unusual odors to avoid interfering with the mice's behavior. During the experiments, detailed data were recorded for each group of mice.

[0090] Immunoblotting assay, such as Figure 10 As shown, the expression level of α-Syn was significantly lower in the ASO group compared to the Vehicle group. The open field experiment results are as follows... Figure 11 As shown, the socially isolated ASO group mice spent significantly more time in the central region of the open field than the socially isolated Vehicle group mice. The results of the elevated cross maze experiment are as follows: Figure 12 As shown, compared to the Vehicle group mice, the ASO group mice in the social isolation group had a significantly increased time spent in the open arm and a significantly increased number of times they entered the open arm. These results demonstrate that ASO can alleviate anxiety-like behaviors in the model animals, suggesting that administering ASO targeting α-Syn mRNA via implanted catheters to degrade α-Syn can alleviate anxiety-like behaviors induced by social isolation.

[0091] Example 5: The association between Sncα-targeting AAV virus treatment in mice and social isolation-induced anxiety disorder.

[0092] 1) Healthy 8-week-old male C57BL / 6J mice were selected for this experiment and randomly divided into a social isolation group and a group living (control) group. Each mouse in the social isolation group was housed alone in a cage, while 4 mice in the group living group were housed together in a cage.

[0093] 2) CαMK2α-Cre mice (strain number: 005359) were divided into a social isolation group and a social group, and then further divided into a group receiving AAV targeting Sncα (Sncα-shRNA group) and a randomized control virus (SC-shRNA group). Stereotactic injection was performed on all four groups of mice according to the protocol, corresponding to the administration of AAV virus AAV5-CAG-DIO-Sncα-shRNA-EGFP-WPRE-hGH polyA and the randomized control (scramble control, SC) virus AAV5-CAG-DIO-SC-shRNA-EGFP-WPRE-hGH polyA, respectively. The titer of the aforementioned recombinant adeno-associated virus type 5 was 2.88 × 10⁻⁶. 12 VG / mL (300 μL / side, 0.3 μL / min flow rate), all four groups of mice had free access to water and food during the feeding period, which lasted for 28 days.

[0094] 3) After 28 days of rearing, the socially isolated mice underwent an immunoblotting experiment. Both the socially isolated and socially active mice underwent anxiety behavioral tests, including the elevated cross maze and open field tests. Before the experiments, it was ensured that both experimental setups were free of any unusual odors to avoid interfering with the mice's behavior. During the experiments, detailed data were recorded for each group of mice.

[0095] Fluorescence image as follows Figure 13 As shown, this indicates that the virus has infected pyramidal neurons. The results of the Western blot experiment are as follows: Figure 14 As shown, the expression level of α-Syn was significantly decreased in the Sncα-shRNA group compared to the SC-shRNA group. The open field experiment results are as follows... Figure 15 As shown, the socially isolated Sncα-shRNA group mice spent significantly more time in the central region of the open field than the socially isolated SC-shRNA group mice. The results of the elevated cruciate maze experiment are as follows: Figure 16 As shown, compared to the SC-shRNA group mice, the social isolation group Sncα-shRNA group mice exhibited significantly increased time spent in the open arm and a significantly increased number of entries into the open arm. These results demonstrate that the Sncα-shRNA group mice can alleviate anxiety-like behaviors, suggesting that viral injection to degrade α-Syn can also alleviate anxiety-like behaviors induced by social isolation, and that pyramidal cells play a crucial role in this process.

[0096] Example 6

[0097] To further verify the effect of α-Syn deposition in the ventral hippocampus of socially isolated mice, intervention was performed by intranasal administration of ASO, which targets α-Syn, and Synucleozid, an α-Syn inhibitor. Behavioral tests were conducted on mice after intranasal administration of ASO (SEQ ID No:1) and Synucleozid, as provided in Example 4.

[0098] 1) Healthy 8-week-old male C57BL / 6J mice were randomly divided into four groups: I: social group (GL group); II: social isolation group (SI group); III: social isolation and ASO administration (SI+ASO group); IV: social isolation and Synucleozid administration (SI+Synucleozid group). 5 μL was administered to each nostril, for a total of 10 μL. Each mouse in the social isolation group was housed individually in a cage, while four mice in each social group were housed together in a cage.

[0099] 2) The mice in the above four groups were administered the drugs according to the steps of intranasal administration. All four groups of mice had free access to water and food during the feeding period, which lasted for 28 days.

[0100] 3) After 28 days of rearing, anxiety behavioral tests, including the elevated cross maze test and the open field test, were conducted on all four groups of mice. Before the experiments, it was ensured that both experimental setups were free of any special odors to avoid interfering with the mice's behavior. During the experiments, detailed experimental data were recorded for each group of mice.

[0101] The results of the open field experiment are as follows Figure 17 As shown, the SI+ASO group and the SI+Synucleozid group spent significantly more time in the central region of the open field than the SI group. The results of the elevated cruciate maze experiment are as follows: Figure 18 As shown, compared to the SI group mice, the SI+ASO and SI+Synucleozid groups exhibited significantly increased dwell time in the open arm and a significantly increased number of entries into the open arm. These results demonstrate that anxiety-like behaviors were alleviated in the SI+ASO and SI+Synucleozid groups, suggesting that intranasal administration of α-Syn can alleviate anxiety-like behaviors induced by social isolation.

[0102] In summary, this experiment strongly demonstrates that social isolation stress leads to increased α-Syn expression levels in the ventral hippocampus of animal brains, and that this increased α-Syn expression is associated with anxiety-like behaviors. Furthermore, the experiment also confirmed that targeting α-Syn with ASO and Synucleozid via implanted catheters and intranasal administration, as well as using recombinant adeno-associated virus to interfere with α-Syn expression, all have significant therapeutic and preventative effects on anxiety disorders induced by social isolation stress, and hold promise for widespread application in the development of preventative and / or therapeutic drugs.

[0103] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A pharmaceutical composition for treating anxiety disorder caused by social isolation, characterized in that, Contains an α-synuclein inhibitor, wherein the α-synuclein inhibitor is selected from: 1) An antisense oligonucleotide targeting SNCA mRNA encoding α-synuclein, wherein the nucleotide sequence of the antisense oligonucleotide is shown in SEQ ID No:1; or, 2) An adeno-associated virus that interferes with the expression level of α-synuclein in ventral hippocampal neurons, wherein the adeno-associated virus is a recombinant adeno-associated virus type 5 containing shRNA with nucleotide sequences as shown in SEQ ID No:3 and SEQ ID No:

4.

2. The pharmaceutical composition according to claim 1, characterized in that, The content of the recombinant adeno-associated virus type 5 in the drug is (1~2.88)×10. 12 μg / mL.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The dosage form of the pharmaceutical composition is selected from tablets, solutions, suspensions, emulsions, microcapsules, microspheres, injections, liposomes, or aerosols.

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