Application of adeno-associated virus vectors overexpressing the ATP5G1 gene in the preparation of drugs for treating depression

Abstract

This invention provides the application of an adeno-associated virus vector overexpressing the ATP5G1 gene in the preparation of drugs for the prevention, treatment, or improvement of depression. The adeno-associated virus vector comprises a neuron-specific promoter and a nucleotide sequence encoding the mouse ATP5G1 protein. Behavioral results consistently show that specific overexpression of ATP5G1 in the mPFC and hippocampus of CUMS mice significantly improves depressive-like manifestations such as weight loss, anhedonia, and behavioral hopelessness. Therefore, ATP5G1 may serve as a key regulator of chronic stress-induced depression, and its overexpression has potential antidepressant therapeutic value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for mental disorders, particularly to the intervention and treatment of depression, and especially to the use of adeno-associated virus vectors overexpressing the ATP5G1 gene in the preparation of drugs for the prevention, treatment or improvement of depression. Background Technology

[0002] The World Health Organization (WHO) predicts that depression (MDD) will become the leading cause of global disease burden by 2030. The monoamine hypothesis has long dominated the understanding of the pathogenesis and therapeutic targets of depression, but practice has shown that antidepressants based on this hypothesis are not very effective, with a remission rate of only about 40%, suggesting that this theory has certain limitations. The development of new technologies has promoted the emergence of many new biological theories related to depression, including the combination of genes and psychosocial stress, stress and inflammatory cytokines, abnormalities in the structure and function of the prefrontal cortex, hippocampal brain volume loss, neurotrophic factors and neural plasticity, and glutamatergic neurotransmission, among others. Mitochondria play a crucial role in these biological processes. Mitochondrial dysfunction interacts with monoamines, inflammation, and neural plasticity, and is considered the basis of MDD. Meanwhile, the incidence of depression and suicide rates gradually increase with age, and mitochondria play a central role in biological aging; mitochondrial homeostasis and function decline with age. Therefore, the "mitochondrial homeostasis imbalance hypothesis" has attracted increasing attention in the mechanism of depression. This hypothesis suggests that depression is a mitochondrial energy metabolism disorder that may be related to abnormal energy metabolism in the brain of depressed patients. Reversing mitochondrial dysfunction in the early stages may provide a new target for therapeutic intervention.

[0003] The ATP synthase membrane subunit c1 gene (ATP5G1, also known as ATP5MC1) is a key component of the oxidative phosphorylation chain complex V, encoding one of the c1 subunits of mitochondrial ATP synthase and catalyzing ATP synthesis. Cristofol et al. found that ATP5G1 plays a central role in encoding the c subunit. Knocking out the ATP5G1 c subunit isoform in HeLa cells via RNA interference resulted in significant ATP synthesis defects and disrupted the structure and function of the mitochondrial respiratory chain. However, the expression of its corresponding target peptide could rescue the oxidative phosphorylation defects induced by ATP5G1 protein (P1) silencing. Natera-Naranjo et al., using in situ hybridization and qPCR, discovered that ATP5G1 mRNA exists in the axons of rat primary sympathetic nerves and is translated within these axons. Downregulation of axonal ATP5G1 mRNA significantly reduced axonal ATP5G1 protein and ATP levels, with ATP5G1 protein levels decreasing by approximately 35-60% and axonal ATP levels decreasing by up to 60% compared to the control group. Local silencing of ATP5G1 expression increased total reactive oxygen species (ROS) levels in distal axons by 3-4 times, while ROS production in mitochondria increased by nearly 2 times, also leading to a significant decrease in axonal elongation rate. These cell experiments suggest that downregulation of ATP5G1 expression can significantly reduce oxidative phosphorylation capacity and disrupt mitochondrial homeostasis.

[0004] To date, research on ATP5G1 in depression and mouse models remains limited. In previous studies, the applicant first discovered a significant downregulation of ATP5G1 expression levels in the peripheral blood and cadaveric brain of MDD patients. This finding was published in *FrontGenet* in 2019 (Zeng Duan, first author) and has been cited multiple times by journals such as *JAMA Psychiatry* and *Molecular Psychiatry*. However, the relationship between depression and mitochondrial dysfunction is not a simple linear causal one. The abnormal expression of a mitochondrial-related gene in depressed patients, affecting mitochondrial function, does not reasonably imply that its reverse regulation will necessarily alter depressive behavior. Therefore, whether ATP5G1 is related to antidepressant efficacy remains unclear, and its potential role as a target for antidepressant treatment has not yet been reported in the literature. Therefore, this study focuses for the first time on the core scientific questions surrounding the relationship between the ATP5G1 gene and depressive-like behavior in mice, systematically conducting a series of experiments including animal model construction and functional validation. The aim is to elucidate the role of ATP5G1 in antidepressants, thereby providing new theoretical and experimental foundations for the study of the mechanisms of depression and the development of new drugs. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides the application of an adeno-associated virus vector overexpressing the ATP5G1 gene in the preparation of a medicament for the prevention, treatment, or improvement of depression.

[0006] To achieve the above objectives, the present invention provides an adeno-associated virus vector overexpressing the ATP5G1 gene for use in the preparation of drugs for the prevention, treatment or improvement of depression. Its main feature is that the adeno-associated virus vector includes a neuron-specific promoter and a nucleotide sequence encoding the mouse ATP5G1 protein.

[0007] Preferably, the nucleotide sequence is as shown in SEQ ID NO:1.

[0008] Preferably, the adeno-associated virus vector includes a reporter gene.

[0009] Preferably, the reporter gene is eGFP.

[0010] Preferably, the adeno-associated virus vector is injected into the medial prefrontal cortex and / or hippocampus of the subject via stereotactic injection.

[0011] Preferably, the neuron-specific promoter is the hSyn promoter.

[0012] Preferably, the adeno-associated virus vector is the AAV2 / 9 serotype.

[0013] Preferably, the depression described is chronic, unpredictable, mild stress-induced depression. Attached Figure Description

[0014] Figure 1 shows the results of constructing the CUMS-induced depression mouse model in Example 1. Figure 1A shows the immobility time of mice in each group during the forced swimming test; Figure 1B shows the immobility time of mice in each group during the tail suspension test; Figure 1C shows the 3-hour sucrose preference test; and Figure 1D shows the 24-hour sucrose preference test. In Figure 1, CUMS represents chronic unpredictable stress; FST represents the forced swimming test; TST represents the tail suspension test; and SPT represents the sucrose preference test. p<0.05, p<0.01, p<0.001.

[0015] Figure 2 This is a flowchart of the experiment for Example 2. Figure 2As shown in Figure 2, C57BL / 6 mice were pretreated with CUMS for 2 weeks, then injected with AAV-hSyn-ATP5G1-eGFP or AAV-hSyn-eGFP (control virus) into the mPFC and vHip, and continued CUMS modeling for 6 weeks. Viral expression was verified by eGFP fluorescence, and depressive-like behaviors were evaluated using TST, FST, and SPT. In Figure 2, CUMS represents chronic unpredictable stress; FST represents the forced swimming test; TST represents the tail suspension test; and SPT represents the sucrose preference test. mPFC represents the medial prefrontal cortex; and vHip represents the ventral hippocampus.

[0016] Figure 3 shows the results of ATP5G1 overexpression reversing the depressive phenotype. Figures 3A and 3B show the baseline and mouse body weights 8 weeks after CUMS modeling; Figures 3C and 3D show the sucrose preference test at 3 hours and 24 hours; Figures 3E and 3F show the immobility time and number of movements in the tail suspension test; Figures 3G and 3H show the immobility time and number of movements in the forced swimming test. In Figure 3, CUMS represents chronic unpredictable stress; AAV-ATP5G1 is an adeno-associated virus vector for the ATP5G1 gene; SPT represents the sucrose preference test; TST represents the tail suspension test; and FST represents the forced swimming test. p<0.05, p<0.01, p<0.001, ns: no statistical difference. Detailed Implementation

[0017] To more clearly describe the technical content of the present invention, the following description is provided in conjunction with specific embodiments.

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0019] Example 1: Constructing an animal model of depression using CUMS SPF-grade C57BL / 6J mice were used in the experiment and were housed in a standard environment in accordance with animal experimental ethics.

[0020] Experimental grouping: C57BL / 6 mice were divided into two groups: control group (Ctrl) and CUMS model group. The CUMS model group was subjected to 8 weeks of chronic unpredictable mild stress method (CUMS) stimulation, while the Ctrl group received no stress stimulation treatment.

[0021] A mouse depression model was constructed using CUMS (Cumulative Stress Methods) over an 8-week period. Mice were housed individually throughout the modeling process to eliminate interference from group living. Nine different mild stimuli were used, with one stress method randomly selected daily to prevent stress tolerance in the mice. The parameters for each stimulus are as follows: Fasting and water restriction: depriving someone of food or water for 24 hours; Moist bedding: Add water to the cage to wet the bedding and keep the cage in a moist environment for 12 hours; Restraint stress: Mice were restricted from free movement using restraint tubes for 4 hours; Cage rotation: Place the cage in a rotating device and shake it at a uniform speed for 30 minutes at a time; Noise stimulation: Continuous environmental noise interference, lasting 4-6 hours; Reversed day and night cycle: 24-hour full light feeding to reverse the day and night rhythm; Cage tilt: The rearing cage is placed at a 45° angle and reared for 6 hours; Tail clamping stimulation: Non-invasively clamp the base of the mouse's tail and stimulate it once for 1-2 minutes; Cold water swimming: Forced swimming for 6 minutes in a 4 ℃ low-temperature clean water environment.

[0022] After 8 weeks of CUMS stimulation, behavioral assessments were conducted. Classical behavioral paradigms were used to evaluate depressive-like behaviors in the animal model of depression: the sucrose preference test detected anhedonia, while the tail suspension test and forced swimming test detected hopeless behavior. Environmental conditions were strictly controlled throughout the experiment, standardized operating procedures were followed, and an animal behavioral analysis system was used to automatically record behavioral indicators, including key parameters such as immobility time, activity time, and sucrose intake, ensuring the objectivity and accuracy of the data. Specific behavioral experiments are as follows: Forced swimming test (FST): This test assesses the depressive state of mice by measuring their cumulative immobility time. Mice are placed alone in a transparent glass container and allowed to swim freely. A state of despair and helplessness is indicated when the mouse floats with its head above water without struggling or making slight movements. The test lasts for a total of 6 minutes, and the cumulative immobility time of the mouse is monitored within the first 4 minutes.

[0023] Sugar Water Preference Test (SPT): The sucrose preference test lasted for 4 days. For the first 2 days (acclimation phase), each mouse had access to two water bottles (50ml each) containing pure water and a 1% sucrose solution, respectively, for free drinking. To prevent positional preference, the positions of the two water bottles were changed every 12 hours. On day 3, the mice were deprived of water for 18 hours. On day 4, the mice were given one bottle of pure water and one bottle of sugar water, ensuring free access to water, with the bottle positions changed every 2 hours. Sugar water preference index (%) = Sugar water consumption / (Sugar water consumption + Pure water consumption) 100% of the sugar water preference index was calculated for a short period of 3 hours and a daily period of 24 hours. In this embodiment, 3 hours specifically refers to the three hours from 4 PM to 7 PM, and 24 hours specifically refers to the two hours from 4 PM to 4 PM the next day.

[0024] Tail Suspension Test (TST): Mice were suspended 60 cm above the ground and secured with tape. The test lasted for 6 minutes, and the total time the mice remained still was recorded for the last 4 minutes.

[0025] Statistical analysis was performed using GraphPad Prism software. Independent samples t-tests were used for comparisons between two groups; one-way ANOVA was used for comparisons among multiple groups, and Tukey's multiple comparison test was used post-hoc to determine differences between groups. p < 0.05 was considered statistically significant.

[0026] The results are shown in Figure 1. After 8 weeks of CUMS stimulation, as shown in Figure 1A, the immobility time of the model mice (CUMS group) in the forced swimming test was significantly longer than that of the normal control group (Ctrl group) (P<0.001). As shown in Figure 1B, the immobility time in the tail suspension test was also significantly prolonged (P<0.01), indicating that chronic unpredictable mild stress can effectively induce behavioral despair phenotypes in mice. Meanwhile, as shown in Figures 1C and 1D, the results of the sucrose preference test showed that the CUMS group mice had significantly lower sucrose intake than the control group within a short-term detection window of 3 hours (P<0.001), and also significantly lower intake than the control group in a long-term detection window of 24 hours (P<0.05), indicating that the model mice exhibited a persistent and stable loss of pleasure. These behavioral results consistently confirm the successful establishment of the CUMS depressed mouse model.

[0027] Example 2: ATP5G1 overexpression improves depressive-like behavior See the experimental procedure. Figure 2 Adeno-associated virus vector (AAV-hSyn-ATP5G1-eGFP) overexpressing the ATP5G1 gene was constructed. Figure 2 As shown, C57BL / 6 mice were pretreated with CUMS for 2 weeks, and then injected with AAV-hSyn-ATP5G1-eGFP or AAV-hSyn-eGFP (control virus) into the mPFC and vHip using stereotactic injection technique, followed by CUMS modeling for another 6 weeks. After modeling, viral expression was verified by eGFP fluorescence, and depressive-like behavior was evaluated using behavioral experiments such as TST, FST, and SPT to observe whether overexpression of ATP5G1 could improve CUMS-induced depressive-like behavior in mice. The specific steps are as follows.

[0028] The CUMS modeling method and behavioral experimental methods such as FST are described in Example 1.

[0029] Experimental Groups C57BL / 6 mice were divided into three groups: blank control group (Ctrl), CUMS model group, and AAV-ATP5G1 intervention group.

[0030] Ctrl control group: AAV-hSyn-eGFP (control virus) was injected into mPFC and vHip at the end of week 2, with no CUMS stress throughout the process; CUMS model group: CUMS pretreatment for 2 weeks, followed by injection of AAV-hSyn-eGFP (control virus) at the end of the second week, and then CUMS modeling for 6 weeks. AAV-ATP5G1 intervention group: CUMS pretreatment for 2 weeks, followed by injection of AAV-hSyn-ATP5G1-eGFP at the end of week 2, and then CUMS modeling for 6 weeks.

[0031] Weight monitoring Before the modeling process began (baseline), the initial body weight of mice in each group was measured to ensure that the baseline body weights of the groups matched. After 8 weeks of CUMS modeling, the body weight of mice was measured again, and the differences in body weight between groups were statistically analyzed.

[0032] Viral vector construction This study commissioned Wuhan BrainVTA to construct an adeno-associated virus vector (AAV-hSyn-ATP5G1-eGFP) that overexpresses ATP5G1 based on the AAV2 / 9 serotype and the neuron-specific promoter hSyn. Simultaneously, the company custom-constructed a control virus, AAV-hSyn-eGFP. The specific design is as follows: AAV2 / 9: This indicates a chimeric serotype, which is the ITR of AAV2 + the capsid (cap protein) of AAV9. AAV9 has the ability to cross the blood-brain barrier and the ability to efficiently transduce cells such as myocardium and neurons.

[0033] hSyn: The name of a neuron promoter, which has neuron-specific expression characteristics.

[0034] ATP5G1: The target gene (specific sequence shown in SEQ ID NO:1) encodes the mitochondrial ATP synthase subunit c1, which is involved in ATP synthesis.

[0035] eGFP: Enhanced green fluorescent protein, used as a reporter gene to visualize target gene expression.

[0036] The ATP5G1 gene (also known as ATP5MC1) sequence was obtained from the Gene database https: / / www.ncbi.nlm.nih.gov / gene / 11951. Mus musculus strain C57BL / 6J chromosome 11,GRCm39. NCBI Reference Sequence: NC_000077.7.

[0037] >NC_000077.7:c95966520-95963619 Mus musculus strain C57BL / 6Jchromosome 11, GRCm39, with the specific sequence shown in SEQ ID NO:1.

[0038] Brain region localization injection For CUMS stimulation, C57BL / 6 mice were pretreated with CUMS for 2 weeks. On the last day of the second week, stereotactic injection of AAV-hSyn-ATP5G1-eGFP (AAV-ATP5G1 group) or AAV-hSyn-eGFP (CUMS group) was performed into the mPFC and vHip brain regions using stereotactic injection technique, followed by 6 weeks of CUMS modeling. For the Ctrl group, AAV-hSyn-eGFP was injected into the mPFC and vHip brain regions at the end of the second week, but no CUMS stress stimulation was administered. All surgical procedures followed standardized protocols to ensure experimental reproducibility. Details are as follows: Bilateral intracranial catheterization and microinjection: After anesthetizing C57BL / 6J mice (0.5% sodium pentobarbital, 50 mg / kg), they were fixed on a stereotaxic instrument, the scalp was cut, and the skull was carefully wiped with 1% hydrogen peroxide solution (H2O2) to fully expose the skull. AAV virus was injected into the bilateral hippocampus and PFC brain tissue using precise stereotaxic positioning based on the coordinates of the PFC (X= ±0.4mm, Y=+1.96mm, Z=-2.04) and bilateral hippocampus (X= ±2.90mm, Y=-3.25mm, Z=-3.95). After the operation, the mice were allowed to recover carefully in a room with a suitable temperature for 5 days before subsequent experiments. Using a 5μl micro-adjustment syringe, 0.6μl of adeno-associated virus overexpressing ATP5G1 was injected into each ventral hippocampus at a rate of 0.1μl / min, and 0.2μl of overexpressing virus was injected into each PFC brain tissue. After injection, the syringe was not removed immediately, but left in place for 6 minutes before slowly withdrawing the syringe for about 10 minutes.

[0039] In vivo validation of adeno-associated virus (AAV): C57BL / 6J mice that had previously received bilateral hippocampal and PFC AAV injections were anesthetized by intraperitoneal injection of sodium pentobarbital. The blood was washed away with physiological saline, followed by perfusion with 4% paraformaldehyde solution. After this, the mice were decapitated, and the brains were immediately placed in 4% paraformaldehyde solution for post-fixation. The brain tissue was then dehydrated successively with 20% and 30% sucrose solutions. After settling, the tissue was embedded in embedding medium and coronally sectioned using a cryostat to achieve a 50 μm thickness. The brain slices were then transferred to glass slides for observation and photography under an inverted fluorescence microscope to examine AAV infection status.

[0040] As shown in Figure 2, a large number of green eGFP fluorescent signals can be seen in the mPFC and vHip brain region slices, suggesting that the AAV virus carrying ATP5G1 can achieve specific overexpression in neurons. eGFP can be used as a reporter gene to trace and monitor the expression efficiency of the target gene.

[0041] Monitor weight changes, results as follows Figure 3 A and Figure 3 As shown in Figure 3A, there was no significant difference in body weight among the groups at baseline (P>0.05). However, as shown in Figure 3B, after 8 weeks of CUMS stimulation, the body weight of the CUMS group was significantly lower than that of the control group (Ctrl group) (P<0.001). Figure 3 As shown in B, after overexpression of ATP5G1 in the mPFC and hippocampus (AAV-ATP5G1 group), the body weight of mice increased significantly compared with the CUMS group (P<0.05), suggesting that ATP5G1 may reverse stress-induced weight loss by improving energy metabolism.

[0042] As shown in Figures 3C and 3D, in the sucrose preference experiment, the CUMS group mice had significantly lower sucrose intake at both 3 and 24 hours compared to the control group (P<0.001), indicating a significant loss of pleasure. Overexpression of ATP5G1 significantly increased sucrose intake at both time points (P<0.001), demonstrating that ATP5G1 can effectively reverse CUMS-induced loss of pleasure.

[0043] As shown in Figures 3E and 3F, in the tail suspension test, the immobility time of mice in the CUMS group was significantly longer than that in the control group (P<0.05), while the activity time was significantly reduced (P<0.05), exhibiting behavioral despair. After overexpression of ATP5G1, the immobility time was significantly shortened (P<0.01), and the activity time was significantly increased (P<0.01), indicating that ATP5G1 can alleviate despair behavior and enhance proactive coping ability.

[0044] As shown in Figures 3G and 3F, in the forced swimming experiment, the immobility time of mice in the CUMS group was significantly longer than that in the control group (P<0.001); after overexpression of ATP5G1, the immobility time was significantly reduced (P<0.01). There was no statistically significant difference in activity time among the groups, but the significant change in immobility time was sufficient to support the antidepressant effect of ATP5G1.

[0045] The above behavioral results consistently demonstrate that specific overexpression of ATP5G1 in the mPFC and hippocampus of CUMS mice can significantly improve depressive-like symptoms such as weight loss, anhedonia, and behavioral despair.

[0046] Therefore, ATP5G1 may be a key regulator of depression caused by chronic stress, and its overexpression has potential antidepressant therapeutic value.

[0047] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. The use of an adeno-associated virus vector overexpressing the ATP5G1 gene in the preparation of drugs for the prevention, treatment, or improvement of depression, characterized in that, The adeno-associated virus vector includes a neuron-specific promoter and a nucleotide sequence encoding the ATP5G1 protein.

2. The application according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The adeno-associated virus vector includes a reporter gene.

4. The application according to claim 3, characterized in that, The reporter gene is eGFP.

5. The application according to claim 1, characterized in that, The adeno-associated virus vector was injected into the medial prefrontal cortex and / or hippocampus of the subject via stereotactic injection into the brain.

6. The application according to claim 1, characterized in that, The neuron-specific promoter is the hSyn promoter.

7. The application according to claim 1, characterized in that, The adeno-associated virus vector is of serotype AAV2 / 9.

8. The application according to claim 1, characterized in that, The depression described is chronic, unpredictable, mildly stress-induced depression.

Images