Use of γCaMKII as a target in screening or preparing a drug for preventing and / or treating depression

Abstract

The present invention discloses the use of calmodulin γCaMKII as a target in hippocampal astrocytes in screening or preparing drugs for preventing and / or treating depression. The present invention reveals that γCaMKII is highly expressed in hippocampal astrocytes, and affects the hippocampal synaptic structure and brain emotion function by regulating the activity, morphology of astrocytes and the function of glutamate transporter GLT1. Deletion of γCaMKII expression in hippocampal astrocytes can lead to depressive-like behaviors in mice, and regulating the glucocorticoid signaling pathway can target and regulate the protein level of γCaMKII. In addition, the present invention discovers that overexpression of γCaMKII in hippocampal astrocytes can significantly improve the depressive-like behaviors of mice in the chronic stress restraint model. The present invention provides new insights into understanding the molecular mechanism of astrocyte-synapse interaction, and provides potential new targets for the discovery and screening of antidepressant drugs.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedicine and neuro-molecular biology, and particularly relates to the application of calmodulin γCaMKII as a target in screening or preparing drugs for preventing and / or treating depression. Background Art

[0002] Depression refers to a type of affective disorder disease mainly characterized by continuous and long-term low mood, decreased interest, and cognitive function impairment, which is caused by various factors. In modern society with increasing pressure, the incidence of depression shows an increasing trend year by year. Due to its difficult diagnosis and treatment, it has become one of the major mental diseases seriously affecting the quality of human life. Existing studies have found that there are abnormalities in the structure and neuronal discharge activities in multiple brain regions of depression patients, accompanied by changes in the number and morphology of astrocytes, but the specific pathogenic mechanism is still not fully understood. Based on the fact that the interaction between astrocytes and synapses is crucial for maintaining normal neural network activities, therefore, in-depth study of the regulatory mechanism of astrocytes on synapse function and its relationship with depressive-like behaviors is expected to provide possible drug targets for the treatment of depression, which has important scientific and social significance.

[0003] In widely used stress mouse models with depressive-like behaviors, such as chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) models, abnormal functions such as the morphology, activation degree, and glutamate transport of astrocytes in the brains of stressed mice have been detected. However, the reasons for the changes in astrocytes during this process and the relationship between such changes and synaptic dysfunction and depressive-like behaviors are still not fully understood. Considering that astrocytes can actively regulate neural network activities through calcium signal activation, and its abnormal function is closely related to depressive-like manifestations, it is very likely that the dysregulated calcium signal activity in astrocytes affects synaptic function by regulating processes such as glutamate transport and thus participates in the pathogenesis of depression. Therefore, further study of the decoding mechanism of calcium signal regulation of synaptic function in astrocytes and its physiological functions has important scientific and clinical significance.

[0004] In the nervous system, there is a class of calcium-binding protein CaMKII (calmodulin-dependent protein kinase II), which is a key molecule for decoding calcium signals. Its function has been widely studied in excitatory neurons, but the expression and function of CaMKII in various subtypes of astrocytes are still unclear. It is reported that there are four subtypes of CaMKII, namely α, β, γ, and δ. Among them, calmodulin kinase γ CaMKII is a key linker protein that mediates the calcium signal transduction and nuclear gene transcription process in nerve cells. Its abnormal expression will lead to defects in synaptic plasticity and learning ability. The results of single-cell RNA sequencing of the human brain found that γ CaMKII is not only expressed in neurons in the hippocampus, but also highly enriched in astrocytes, suggesting that γ CaMKII may be involved in the calcium signal transduction process in astrocytes. Based on the above research background, the inventors believe that γ CaMKII is very likely to be a key molecule for decoding calcium signals and regulating depressive-like behaviors in hippocampal astrocytes. Exploring and improving its mechanism involved in the pathogenesis of depression is of great significance for providing new drug targets and treatment regimens for the prevention and treatment of depression. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide the application of using calmodulin γ CaMKII as a target in screening or preparing drugs for preventing and / or treating depression. As Figure 1 shown, calmodulin γ CaMKII refers to γ CaMKII that is highly expressed and enriched in hippocampal astrocytes. Its expression is significantly down-regulated in a stress mouse model with depressive-like behaviors, and it can be used as an index protein reflecting the degree of depression to a certain extent. When γ CaMKII protein is selectively overexpressed in hippocampal astrocytes or the expression of γ CaMKII is targeted by a glucocorticoid receptor inhibitor, the down-regulation process of astrocyte activity caused by stress stimulation will be inhibited in time, and the depressive-like behaviors of stressed mice will also be alleviated. Therefore, the regulatory mechanism of γ CaMKII protein discovered in the present invention supplements the pathogenic mechanism of depression to a certain extent, and the process that targeting the expression of γ CaMKII protein can directly affect the depressive-like behaviors of stressed mice will provide new ideas for the development and preparation of anti-depressant drugs.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, there is provided the use of γCaMKII as a target in screening or preparing drugs for preventing and / or treating depression, wherein γCaMKII is located in hippocampal astrocytes. Knocking out γCaMKII in astrocytes significantly downregulates its cell activity and directly causes depressive-like behaviors in mice; while overexpressing γCaMKII in astrocytes by virus injection or using glucocorticoid receptor inhibitors to increase the γCaMKII protein level can enhance astrocyte activity and, to a certain extent, inhibit depressive-like behaviors in stressed mice. Based on this, the present invention provides a treatment plan for anti-depressive behaviors by increasing the γCaMKII protein level. Therefore, in the process of preparing or screening drugs for depression, substances that can selectively upregulate the γCaMKII protein level in hippocampal astrocytes, especially drugs that regulate the glucocorticoid signaling pathway, can be selected as candidate drugs for preventing and treating depression by detecting changes in the γCaMKII expression level in the detection system.

[0008] Preferably, the depression includes depression induced by stress and / or long-term stress stimulation.

[0009] In the second aspect of the present invention, there is provided the use of a γCaMKII expression promoter in any one or more of the following, the γCaMKII expression promoter being used to promote the expression of γCaMKII in hippocampal astrocytes, and the use including: (1) preparing products for enhancing the activity and function of astrocytes; (2) preparing products for maintaining the balance of brain glutamate and / or repairing synaptic structure and function; (3) preparing products for preventing and / or treating depression.

[0010] Preferably, the γCaMKII expression promoter includes an expression vector having the Camk2g coding gene and / or a substance that targets and regulates the γCaMKII protein expression by regulating the signaling pathway.

[0011] Preferably, the expression vector having the Camk2g coding gene includes a lentiviral expression vector or an adeno-associated virus expression vector, which enters the target cells in the form of virus infection after being packaged into lentivirus or adeno-associated virus.

[0012] Preferably, the substance that targets and regulates the γCaMKII protein expression by regulating the signaling pathway includes a substance that targets and regulates the glucocorticoid signaling pathway to regulate the γCaMKII protein level and / or a specific regulatory substance for the upstream and downstream signaling pathways of the γCaMKII protein itself.

[0013] In the third aspect of the present invention, there is provided a method for screening drugs for preventing and / or treating depression, including the following steps:

[0014] (1) In the test group, a substance to be detected is added to the detection system;

[0015] (2) Detect the expression level of γCaMKII gene and / or protein in the detection system of the test group, and compare it with the negative control group;

[0016] (3) Compare and analyze the expression levels of γCaMKII gene and / or protein in the test group and the control group. If the expression level of γCaMKII gene and / or protein in the test group increases significantly, it indicates that the substance is a potential drug for preventing and / or treating depression;

[0017] Wherein, the detection system is hippocampal astrocytes.

[0018] In the fourth aspect of the present invention, there is provided the use of γCaMKII in the preparation of an animal model, the animal model having a phenotype of depressive-like behavior, which is achieved by knocking out the gene expressing γCaMKII protein in the hippocampal astrocytes of the animal.

[0019] Preferably, the animal is a mouse or a primate.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention first confirms that the γCaMKII signaling pathway in hippocampal astrocytes is involved in the regulation of astrocyte morphology and function, synaptic structure, and the process of depressive-like behavior.

[0022] (2) The present invention first targets the γCaMKII protein in astrocytes and regulates its expression by means of viral infection or inhibiting the glucocorticoid signaling pathway, and confirms its important beneficial effects in the treatment of depression.

[0023] (3) The present invention further supplements the pathogenesis of depression based on the expression changes of γCaMKII protein in mice with a depressive stress model, and also provides an important research basis for the prediction of depression and the development of clinical treatment drugs. In addition, based on the fact that γCaMKII protein actively participates in the interaction between brain astrocytes and neurons, this also provides new clinical ideas and social application values for the development of a series of neurodegenerative and psychiatric diseases targeting astrocytes and synaptic function impairment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0025] Figure 1It is a schematic diagram of the mechanism by which γCaMKII is involved in stress-induced depressive-like behaviors in mice.

[0026] Figure 2 It shows that in a mouse depression model induced by chronic restraint stress (CRS), the expression levels of γCaMKII RNA and protein in hippocampal tissues were significantly downregulated in astrocytes, accompanied by a decrease in astrocyte activity, while there was no significant difference in its expression in inhibitory neurons. Among them, (A) is the fluorescence in situ hybridization of γCaMKII RNA (Camk2g) and the immunostaining results of GFAP protein in the hippocampus of control group and CRS mice. The dotted circles in the figure mark the astrocytes in the hippocampal brain region of control group mice, and the dotted squares mark the astrocytes in the hippocampal brain region of the depression model mice; (B) is the statistical chart of the proportion of γCaMKII RNA-expressing (>2 fluorescent signal points are considered as expressing) in GFAP-positive astrocytes in the hippocampus; (C) is the cumulative frequency chart of the fluorescence in situ hybridization signal points of γCaMKII RNA in GFAP-positive astrocytes in the hippocampus; (D) is the immunostaining results of γCaMKII and GFAP proteins in the hippocampus of control group and CRS mice; (E) is the statistical chart of the proportion of the co-positive signal area of γCaMKII and GFAP in the hippocampus; (F) is the statistical chart of the proportion of the GFAP-positive signal area in the hippocampus; (G) is the correlation analysis chart of the proportion of the co-positive signal area of γCaMKII and GFAP and the proportion of the single GFAP-positive signal area in the hippocampus; (H) is the statistical chart of the fluorescence intensity of γCaMKII expression in PV-positive inhibitory neurons in the hippocampus. In (B, E, F, H), the dots represent the statistical values in the brain tissue sections of control group mice, the squares represent the statistical values in the brain tissue sections of the depression model mice, and a single dot or square represents the statistical value in a single brain tissue section; **p < 0.01, ***p < 0.001, n.s., no significant difference.

[0027] Figure 3It shows a decrease in astrocyte activity in γCaMKII conditional knockout mice, and these mice exhibit an obvious depressive-like phenotype: among them, (A) is the immunostaining result graph of γCaMKII and GFAP proteins in the hippocampus of wild-type (WT) and conditional knockout (cKO) mice. The dotted circles in the figure mark the astrocytes in the hippocampal brain region of WT mice, and the dotted squares mark the astrocytes in the hippocampal brain region of cKO mice; (B) is the statistical graph of the proportion of the co-positive signal area of γCaMKII and GFAP in the hippocampus; (C) is the statistical graph of the proportion of the GFAP positive signal area in the hippocampus; (D) is the statistical graph of the relative immobility time of WT and cKO mice in the forced swimming test; (E) is the statistical graph of the immobility time of WT and cKO mice in the tail suspension test; in (B, C), the dots represent the statistical values in the brain tissue sections of WT mice, the square dots represent the statistical values in the brain tissue sections of cKO mice, and a single dot or square dot represents the statistical value in a single brain tissue section; in (D, E), the dots represent the statistical values of the individual behaviors of WT mice, the square dots represent the statistical values of the individual behaviors of cKO mice, and a single dot or square dot represents the statistical value of the behavior of a single mouse; **p < 0.01.

[0028] Figure 4 It shows a decrease in the richness of astrocyte branches in γCaMKII conditional knockdown mice: among them, (A) is the concentric circle image of astrocyte Sholl analysis in control group and knockdown (sh-Camk2g) mice; (B) is the statistical graph of the number of astrocyte branches; (C) is the statistical graph of the total length of astrocyte branches; (D, E) are the statistical graphs of the distribution of astrocyte branch intersections and the number of the highest intersection points in Sholl analysis; in (B, C, D, E), the dots represent the statistical values in the astrocytes of control group mice, the square dots represent the statistical values in the astrocytes of knockdown mice, and a single dot or square dot represents the statistical value in a single astrocyte; *p < 0.05, ***p < 0.001.

[0029] Figure 5It shows that γCaMKII in astrocytes positively regulates glutamate transport function: among them, (A) is the detection of the interaction between γCaMKII and glutamate transporter 1 (GLT1) in the hippocampal tissue by Western blotting; (B) is the specific expression of tdTamato in astrocytes after tamoxifen injection in Ai14 x Aldh1l1-CreERT2 mice, and the co-staining results of γCaMKII and GLT1 proteins show their co-localization on the membranes of fine branches of astrocytes; (C) is the tracking diagram of the glutamate transport process in astrocytes in the living brain slices of control group and γCaMKII conditional knockdown mice monitored in real time by the specific fluorescent probe iGluSnFR; (D) is the time series statistical chart of the fluorescence changes of the glutamate probe in astrocytes in the living brain slices of the control group and γCaMKII conditional knockdown mice treated with glutamate; (E) is the statistical chart of the fluorescence changes of the glutamate probe in astrocytes of the control group and γCaMKII knockdown group after glutamate drug treatment at 15 s, 30 s and 45 s respectively; *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 6 It shows that knockdown of γCaMKII in astrocytes leads to changes in hippocampal synaptic structure: among them, (A) is the image of the secondary apical dendrites of CA1 pyramidal neurons labeled with GFP in the control group and knockdown mice; (B) is the statistical chart of the total dendritic spine density in CA1 in the control group and knockdown mice; (C) is the statistical chart of the density of mature dendritic spines in CA1 in the control group and knockdown mice; in (B, C), the dots represent the statistical values of the dendritic spine density on the dendrites in the control group mice, and the squares represent the statistical values of the dendritic spine density on the dendrites in the knockdown mice, and a single dot or square represents the statistical value on a single dendrite; **p<0.01, ***p<0.001.

[0031] Figure 7It shows that overexpression of γCaMKII in hippocampal astrocytes can increase the activity of astrocytes in CRS mice and at the same time inhibit the emergence of depressive-like phenotypes: among them, (A) is the immunostaining result diagram of γCaMKII and GFAP proteins in control group and CRS model mice overexpressing γCaMKII (CRS-Camk2g); (B) is the statistical chart of the proportion of the area of GFAP positive signal in the hippocampus. In the figure, the dots represent the statistical values in the brain tissue sections of the control group mice, the square dots represent the statistical values in the brain tissue sections of the overexpression group mice, and a single dot or square dot represents the statistical value in a single brain tissue section; (C) is the statistical chart of the relative immobility time of control group and mice overexpressing γCaMKII in the forced swimming test before and after CRS modeling. In the figure, the dots represent the statistical values of the individual behaviors of the mice before modeling, the triangular dots represent the statistical values of the individual behaviors of the mice after modeling, and a single dot or triangular dot represents the statistical value of the behavior of a single mouse; **p<0.01, ***p<0.001, n.s., no significant difference.

[0032] Figure 8 It shows that the protein level of γCaMKII and its cell activity in hippocampal astrocytes are regulated by stress hormones: among them, (A) is the immunostaining result diagram of γCaMKII and GFAP proteins in the hippocampus of mice pre-injected intraperitoneally with a control reagent (DMSO) or a glucocorticoid receptor inhibitor RU486 (mifepristone) after acute restraint stress (ARS) stimulation; (D) is the immunostaining result diagram of γCaMKII and GFAP proteins in the hippocampus of mice pre-injected intraperitoneally with a control reagent or RU486 after foot shock (ES); (B, E) are the statistical charts of the proportion of the co-positive signal area of γCaMKII and GFAP in the hippocampus; (C, F) are the statistical charts of the proportion of the area of GFAP positive signal in the hippocampus; in (B, C, E, F), the dots represent the statistical values in the brain tissue sections of the mice in the control reagent injection group, the square dots represent the statistical values in the brain tissue sections of the mice in the glucocorticoid receptor inhibitor RU486 injection group, and a single dot or square dot represents the statistical value in a single brain tissue section; *p<0.05, **p<0.01. Detailed implementation manners

[0033] The present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that the embodiments are only for exemplifying the present invention and do not limit the scope of the present invention in any way. The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified. In addition, unless otherwise specified, the reagents involved in the embodiments of the present invention are commercially available products and can be obtained through commercial channels; the various processes and methods not described in detail in the embodiments of the present invention are conventional methods well known in the art.

[0034] Explanation of terms:

[0035] GFAP (Glial Fibrillary Acidic Protein): An intermediate filament protein belonging to class III intermediate filaments, specifically expressed in the cytoplasm of astrocytes (AS) in the central nervous system, and can be used as a marker for astrocyte activity and specificity.

[0036] Camk2g (Calcium / Calmodulin-dependent protein kinase II gamma): The gene encoding the gamma subtype of calcium / calmodulin-dependent protein kinase II (γCaMKII).

[0037] lxop-γCaMKII mouse: A transgenic mouse model constructed using the Cre / LoxP system, which can conditionally knockout or regulate the γCaMKII gene by specifically expressing Cre recombinase in specific nerve cells.

[0038] Aldh1l1-CreERT2 mouse: A transgenic mouse model mainly used to study astrocytes in the central nervous system. By inserting the CreERT2 gene into the stop codon of the Aldh1l1 gene, specific expression of Cre recombinase in astrocytes is achieved; CreERT2 is a fusion protein containing the Cre recombinase and the ligand-binding domain of the estrogen receptor (ERT2), and the activity of the Cre enzyme needs to be induced by tamoxifen or its active metabolite 4-hydroxytamoxifen (4-OHT).

[0039] Sholl analysis: A classic method initially proposed by Donald Sholl in 1953 for quantitatively analyzing the morphological characteristics of neurons.

[0040] Ai14 mouse: A genetically modified mouse strain commonly used in scientific research, carrying a tdTomato red fluorescent protein gene for reporting gene expression, which is blocked by a STOP cassette flanked by loxP sites.

[0041] Depressive-like behavior: Refers to the behavioral characteristics of animals that are similar to human depressive symptoms, and these behaviors usually reflect the core symptoms of depression such as low mood, reduced interest, and impaired cognitive function.

[0042] Example 1 Detection of the expression level of γCaMKII and astrocyte activity in the hippocampal tissue of mice in a chronic restraint stress model

[0043] 1. Experimental methods

[0044] (1)Construction of chronic restraint stress model mice: Male C57 mice around 2 months old were purchased and randomly divided into a control group (Con) and an experimental group (CRS), and were placed in a cage with a 12-hour light / 12-hour dark cycle where food and water were freely available. One week after the mice adapted to the environment, a pre-model forced swimming test was conducted to determine the baseline performance of their depressive-like behaviors. Thereafter, the control group mice were not treated with anything, while the experimental group mice were placed into 50 mL centrifuge tubes with small holes punched in them every day, ensuring that the mice could not escape. Each tube was separated by a card to ensure that each mouse could not see its companions. Thereafter, continuous restraint was carried out for 3 weeks, 2 - 8 hours per day at an indefinite time. Finally, by comparing the data of the relative immobility time in the forced swimming test after modeling with that before modeling, it was evaluated whether the mice showed obvious depressive-like phenotypes, and in this way, the successfully modeled CRS mice were determined for studying the pathogenic mechanism of depression.

[0045] (2)Preparation of brain tissue sections: After behavioral testing, the mice were anesthetized with sodium pentobarbital, and then perfused with PBS to remove the blood components in the blood vessels. Subsequently, the mouse brains were taken out and fixed in 4% paraformaldehyde. For subsequent RNA fluorescence in situ hybridization experiments, the fixation time was 4 hours, while for immunostaining, the fixation was 20 hours. The fixed mouse brains were dehydrated with 30% sucrose at 4°C for 24 hours. The dehydrated brain tissues were sectioned at -20°C using a Leica CM1800 cryostat, cutting out brain sections with a thickness of 35 microns. If subsequent RNA fluorescence in situ hybridization experiments were to be carried out, the cut sections were attached to RNase-free glass slides, air-dried and immediately stored at -80°C; while the brain sections for subsequent immunostaining were collected in a cryoprotectant buffer composed of 40% glycerol, 30% 0.1M PBS, and 30% ethylene glycol, and stored at -20°C for later use.

[0046] (3)Brain slice RNA fluorescence in situ hybridization and data analysis: The glass slides with brain slices attached were taken out from -80°C, soaked in 50%, 70%, and 100% ethanol for 5 minutes respectively. After baking the slices at 60°C for 20 minutes, the glass slides were treated with a pretreatment solution and digestive enzymes, washed 3 times with ultrapure water. Thereafter, they were incubated with a Camk2g probe (purchased from Guangdong Pinbo Yishi Biotechnology Co., Ltd.) and the corresponding channel type fluorescence-labeled reaction solution. Finally, GFAP immunostaining and mounting were carried out. Images of the glass slides were collected using a Nikon A1 laser confocal microscope, and the number of Camk2g RNA fluorescence in situ hybridization signal points in each GFAP-positive cell was quantitatively counted using ImageJ software.

[0047] (4)Brain slice immunohistochemical staining and data analysis: After washing the tissue sections, the brain slices were placed in 0.1% Triton X-100 and 4% donkey serum for blocking for 1 hour. Then the slices were washed three times with PBS, and then the slices were incubated overnight with primary antibodies against γCaMKII and GFAP (purchased from SYSY, 173011) at 4°C. The next day, after washing the slices three times with PBS, they were incubated with appropriate fluorescently labeled Alexa Fluro secondary antibodies (purchased from Invitrogen) for 3 hours in the dark at room temperature. Finally, the stained slices were mounted on glass slides and sealed with a medium containing 4',6-diamidino-2-phenylindole (DAPI). Images of the glass slides were collected using a Nikon A1 laser confocal microscope, and the fluorescence intensity and the ratio of the expression area of γCaMKII and GFAP positive signals were quantitatively analyzed using ImageJ software.

[0048] (5)Statistical methods: All data were statistically analyzed using GraphPad Prism and are expressed as Mean±SEM. The significance analysis of the differences between two groups of data was tested by Student’s t-test, the analysis of multiple groups of data under a single factor was performed using one-way ANOVA, and the analysis of the data between groups under multiple factors was performed using two-way ANOVA. p<0.05 was considered statistically significant.

[0049] 2. Experimental results

[0050] As Figure 2 shown in A-C below, in the mouse depression model induced by chronic restraint stress (CRS), the activity of astrocytes labeled with GFAP decreased significantly, and at the same time, the RNA level of γCaMKII in astrocytes decreased significantly. Similar to the gene expression results, Figure 2 the immunostaining results in D-F below also confirmed the downregulation of the protein level of γCaMKII in astrocytes in the hippocampus of CRS mice, and the protein expression level of γCaMKII in the hippocampus was positively correlated with the activity of astrocytes ( Figure 2 G below), indicating that γCaMKII is an important molecule regulating the activity and function of astrocytes. However, there was no significant difference in the expression of γCaMKII in inhibitory neurons between CRS and Con mice ( Figure 2 H below), which indicates that the regulation process of γCaMKII under CRS stress stimulation only occurs specifically in this specific population of astrocytes.

[0051] Example 2 Construction of astrocyte γCaMKII conditional knockout mice and evaluation of their depressive-like phenotypes

[0052] 1. Experimental methods

[0053] (1) Construction of astrocyte γCaMKII conditional knockout mice: Cross lxop-γCaMKII with Aldh1l1-CreERT2 mice. In their offspring, specifically activate the Cre recombinase in astrocytes by intraperitoneal injection of tamoxifen, thereby achieving knockout of the γCaMKII gene in astrocytes throughout the brain.

[0054] (2) Assessment paradigms for behavioral despair in the depression model - tail suspension test and forced swimming test: During the tail suspension test, each mouse's tail is fixed in the tester, making its head hang downwards for 6 min, separated from each other by baffles; during the forced swimming test, the mouse is placed alone in a water-filled cylinder (water temperature 23 - 25 °C) so that its legs cannot touch the bottom and swim for 6 min. Record and photograph the animal behavior during these two experiments, and analyze the time when the mouse remains relatively stationary during the last 4 min of the test, that is, the time without large struggling movements. Finally, evaluate the depression level of the experimental mice based on this parameter. The longer the relatively stationary time, the higher the depression level of the mouse.

[0055] 2. Experimental results

[0056] As Figure 3 shown in A - C, the expression of γCaMKII protein in astrocytes in conditional knockout mice decreased significantly, and at the same time, the activity of hippocampal astrocytes was also significantly downregulated. As Figure 3 shown in D - E, when compared with WT mice, the relative stationary time of cKO mice increased significantly in the tail suspension test and forced swimming test, indicating that specific knockout of γCaMKII in astrocytes directly increases the probability of mice showing depressive-like behaviors.

[0057] Example 3 Specific knockdown of γCaMKII expression in the mouse hippocampus not only impairs the morphology and glutamate transport function of astrocytes, but also directly affects synaptic structure

[0058] 1. Experimental methods

[0059] (1) Stereotactic virus injection: After the mouse is anesthetized with isoflurane, the mouse brain is fixed with a stereotaxic device, the mouse head skin is cut with sterile scissors to expose the skull, and a 0.5 mm hole is drilled on the skull surface with an electric drill according to the specific coordinates of the mouse hippocampus. 200-400 nL of virus is injected into the bilateral hippocampus at a rate of 50-100 nL / min via a syringe. After the injection, the needle remains in place for 5 min to allow the virus to spread. After that, the incision is sutured with sterile sutures, and the mouse is placed in a specific incubator. After it wakes up, it is returned to the cage. Finally, wait for 3-4 weeks for the virus to be fully expressed before conducting relevant experiments. In the experiment of specifically knocking down the expression of γCaMKII in hippocampal astrocytes, AAV2 / 8-GfaABC1D-shcamk2g-P2A-mCherry virus produced by Shanghai Shengbo Biotechnology Co., Ltd. will be injected into the bilateral hippocampal CA1 brain region, where GfaABC1D is an astrocyte-specific promoter and shcamk2g is a shRNA targeting γCaMKII to interfere with the gene expression of γCaMKII in the hippocampus. In addition, when detecting astrocyte or synaptic morphology in mice with knocked down γCaMKII expression, GFP virus driven by GfaABC1D or αCaMKII promoter produced by Wuhan Shumi Brain Science Technology Co., Ltd. will be injected into the bilateral hippocampal CA1 brain region at the same time.

[0060] (2) Sholl analysis of astrocytes and analysis of dendritic spine density of pyramidal neurons: After the virus-injected mice were sliced, GFP immunostaining was performed, and the number of astrocytes and dendritic spines was analyzed using ultra-high resolution imaging technology and morphological methods. When randomly selecting GFP-labeled astrocytes in the hippocampus for Sholl analysis using ImageJ, Figure 4 As shown in A, concentric circles are drawn outward with the cell body as the center, and then concentric circles are drawn with the cell body as the center to count the number of intersections between its branches and the concentric circles. The data point on each Sholl diagram corresponds to the average Sholl intersection, which represents the average value of the corresponding group. After labeling CA1 pyramidal neurons with GFP, their secondary apical dendrites were photographed with a high-resolution microscope, and the density of total dendritic spines and mature dendritic spines (i.e., mushroom-shaped dendritic spines) was counted using ImageJ software.

[0061] (3) Protein immunoblotting and immunoprecipitation: The hippocampal tissue of mice was taken on ice, and a lysis buffer containing protease and phosphatase inhibitors was added to disrupt the tissue. After sufficient lysis, the supernatant was centrifuged and the protein concentration was measured by BCA protein quantification. The above tissue lysate was adjusted to a consistent concentration according to the BCA protein quantification result, and the antibody of the specific target protein (1-2 μg) was added thereto. The tissue was incubated at 4°C overnight, and then Protein G-Sepharose beads (G protein modified agarose beads) were added and incubated at 4°C for 1 hour to allow the beads and the antibody to fully bind. The beads were then rinsed thoroughly with lysis buffer to allow other proteins that only interacted with the target protein to remain on the beads. The interacting proteins were then detected by SDS-PAGE gel electrophoresis. After protein transfer, the tissue was blocked with 5% skim milk at room temperature for 1 hour, and the primary antibody was incubated at 4°C overnight, and the secondary antibody was incubated at room temperature for 1 hour. Finally, the expression of the target protein was detected by chemiluminescence imaging.

[0062] (4) Glutamate probe imaging: rAAV-EF1α-DIO-iGluSnFR produced by Brinkase Biotechnology Co., Ltd. and AAV2 / 8-GfaABC1D-shcamk2g-P2A-mCherry produced by Shanghai Biobio Biotechnology Co., Ltd. were injected into the hippocampus of Aldh1l1-CreERT2 mice, and tamoxifen was injected intraperitoneally to ensure the specific expression of the glutamate probe iGluSNFR in astrocytes. After waiting for the virus to be fully expressed, the mice were sampled and acute brain slices were made under oxygenation. After the brain slices were revived, the area of ​​sh-RNA expression (i.e., mCherry labeling) was first centered, and then 2mM glutamate drug was treated and real-time imaging was performed at the same time.

[0063] 2. Experimental results

[0064] (1) If Figure 4 As shown in A, when the astrocyte morphology was marked with GFP and Sholl analysis was performed, it was found that specific knockdown of γCaMKII not only affected the activity of astrocytes, but also significantly downregulated the number and total length of their branches, as well as the number of branch intersections in the Sholl analysis ( Figure 4 BE in ), saying that γCaMKII in astrocytes actively regulates the abundance of their branches, and it is very likely that this will affect the function of astrocytes and their interaction with neural synapses.

[0065] (2) If Figure 5As shown in A of Figure 5 , co-immunoprecipitation experiments revealed a significant interaction between γCaMKII protein and glutamate transporter 1 (GLT1) in the hippocampal tissues of adult mice. When further performing immunostaining imaging on hippocampal brain slices of Ai14 x Aldh1l1-CreERT2 mice using a high-resolution microscope ( Figure 5 B), the results showed obvious co-localization of γCaMKII and GLT1 on the fine branches at the ends of tdTomato-labeled astrocytes. This finding indicates that there is an interaction between γCaMKII and GLT1 in astrocytes, especially in their branched parts, suggesting that γCaMKII is very likely to play an important role in regulating the glutamate transport function of astrocytes, thereby affecting synaptic function. When continuing to detect the glutamate transport function in the brains of knockdown mice expressing glutamate probe virus, real-time tracking imaging results found that when treating acute brain slices of mice with glutamate drugs, the fluorescence value of the glutamate probe first increased and then decreased, indicating that the glutamate drug treatment successfully activated the glutamate transporter on the astrocyte membrane, and as glutamate was transported from the extracellular to the intracellular space, the fluorescence value changed accordingly ( Figure 5 C). Compared with the results of the control group, the glutamate transport process in astrocytes in the in vivo brain slices of γCaMKII conditional knockdown mice was significantly slower (

[0066] C-E), indicating that its glutamate transporter function was inhibited to a certain extent. Based on the obvious interaction between γCaMKII and GLT1, γCaMKII is very likely to regulate the glutamate transport function of astrocytes through this process and affect the interaction process between astrocytes and neurons to a certain extent. Figure 6 As shown in

[0067] Example 4 Overexpression of γCaMKII in mouse hippocampal astrocytes improves depressive-like behaviors

[0068] 1. Experimental method

[0069] Overexpression of γCaMKII in hippocampal astrocytes of CRS mice: Bilateral CA1 brain regions of the hippocampus in adult mice were injected with AAV2 / 8-GfaABC1D-mCherry-P2A-Camk2g or control virus produced by Shanghai Shengbo Biotechnology Company. Three weeks after the injection, which was also the time point when the virus began to express, behavioral tests were performed before CRS modeling. Subsequently, control group and overexpression group mice underwent a three-week chronic restraint process. After the modeling, forced swimming behavioral tests were used to analyze the degree of change in depressive-like behavior of the mice.

[0070] 2. Experimental results

[0071] As Figure 7 shown in A - B of Figure 7 , overexpression of γCaMKII in hippocampal astrocytes could significantly increase the activity of astrocytes in CRS mice. Further analysis of the behavioral test results before and after modeling found that control group mice showed obvious depressive-like phenotypes after three weeks of restraint stress, while there were no significant changes in depressive-like behavior in overexpression group mice before and after modeling (

[0072] Example 5 Inhibition of the stress hormone - glucocorticoid signaling pathway can target and regulate the protein level of γCaMKII in hippocampal astrocytes

[0073] 1. Experimental methods

[0074] (1) Acute stress stimulation: During acute restraint stress stimulation (ARS), adult mice were placed in a 50 mL centrifuge tube with small holes punched, ensuring that the mice could not escape. Each tube was separated by a card to ensure that each mouse could not see its companions. The restraint time was 2 hours. During the plantar electric shock (ES) experiment, mice were placed in a conditioned reflex chamber for 2 minutes for environmental familiarization, and then given a 2-second foot shock with a current intensity of 0.7 mA. After the shock, the mice were allowed to stay in the conditioned reflex chamber for another 2 minutes. Subsequently, the mice were returned to the breeding cage. Both of these acute stress stimulations started 3 hours after the injection of mifepristone. After the stimulation ended, the mice were sacrificed 2 hours later and subsequent brain slice sectioning and immunostaining experiments were carried out.

[0075] (2)Drug action of mifepristone (RU486): The glucocorticoid receptor inhibitor mifepristone (RU486) powder purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. will be dissolved in corn oil containing 5% DMSO. Before the start of acute stress stimulation, RU486 will be intraperitoneally injected into mice (20 mg / Kg) to target and regulate the glucocorticoid signaling pathway.

[0076] 2. Experimental results

[0077] As Figure 8 shown in A - C, compared with the control group which showed lower protein levels of γCaMKII after acute restraint stress (ARS), after pre - inhibiting the glucocorticoid signaling pathway with RU486, the protein levels of γCaMKII and its cell activity in hippocampal astrocytes were less affected by ARS and remained in a relatively normal state. In addition, when mice experienced another stress stimulation - foot - shock stress (ES), mice pre - intraperitoneally injected with RU486 also maintained relatively high protein levels of γCaMKII and GFAP ( Figure 8 shown in D - F). These results indicate that the down - regulation of the protein levels of γCaMKII and its cell activity in hippocampal astrocytes in mice with depressive - like behaviors is most likely regulated by stress hormones. Based on the frequently observed dysregulation of the hypothalamic - pituitary - adrenal axis response and abnormal glucocorticoid release in patients with depression, and the fact that mice can have a certain degree of antidepressant effect after being treated with mifepristone (RU486), it indicates that the regulation process of γCaMKII protein in astrocytes is most likely an important target for the drug action of RU486 in treating depression. Therefore, as a target protein for depression, the regulation process of γCaMKII protein by glucocorticoids is an important supplementary mechanism in the pathogenesis of depression.

[0078] Through the above - mentioned animal experiments, it was found that overexpressing the γCaMKII protein in hippocampal astrocytes can inhibit the depressive - like phenotype in mice of the chronic restraint stress model. This finding provides a new idea for exploring ways to improve the process of depression in the brain and supports the health and social functions of patients with depression. In summary, this invention deeply explores the potential application of the interaction between astrocytes and synapses based on the γCaMKII protein in improving brain emotional functions, providing a certain theoretical and experimental basis for the future development of drugs or treatment methods for preventing depression.

[0079] The above are only the preferred embodiments of the present invention, and do not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art of this technology, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as within the protection scope of the present invention. Any equivalent changes in the form of slight modifications, decorations and evolutions that can be made by those who are familiar with the technology in this field without departing from the spirit and scope of the present invention by using the technical content disclosed above are equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments according to the substantial technology of the present invention still fall within the scope of the technical solutions of the present invention.

Claims

1. Use of a γCaMKII expression promoter in the preparation of a product for preventing and / or treating depression, characterized in that, The γCaMKII expression promoter is used to promote the expression of γCaMKII in hippocampal astrocytes, and the γCaMKII expression promoter includes an expression vector having Camk2g a coding gene.

2. The application according to claim 1, wherein The said one having Camk2g The expression vector with the coding gene includes a lentiviral expression vector or an adeno-associated virus expression vector, which, after being packaged into lentivirus or adeno-associated virus, enters the target cells in the form of virus infection.

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