Application of gamma CaMKII as target spot in screening or preparing medicine for preventing and / or treating depression

Abstract

The invention discloses application of calmodulin gamma CaMKII in hippocampal astrocyte as a target spot in screening or preparing a medicine for preventing and / or treating depression. According to the invention, gammaCaMKII is enriched and expressed in hippocampal astrocytes, and the activity and the form of the astrocytes and the function of a glutamate transporter GLT1 are regulated, so that the hippocampal synaptic structure and the brain emotional function are further influenced. The expression deletion of gamma CaMKII in hippocampal astrocytes can cause depression-like behaviors of mice, and the regulation of a glucocorticoid signal channel can regulate the gamma CaMKII protein level in a targeted manner. In addition, the depression-like behavior of a chronic stress constraint model mouse can be obviously improved by overexpressing the gamma CaMKII in the hippocampal astrocyte. The invention provides a new insight for understanding a molecular mechanism of interaction of astrocytes and synapses, and provides a potential new target for discovery and screening of antidepressant drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and neuromolecular biology, and specifically relates to the application of calmodulin gamma CaMKII 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 caused by multiple factors, with continuous and long-term low mood, loss of interest and cognitive impairment as the main clinical features. In the modern society with increasing stress, the incidence of depression has a tendency to increase year by year. Due to its difficulty in diagnosis and treatment, it has become one of the major mental illnesses that seriously affects the quality of human life. Studies have found that there are structural and neuronal discharge activity abnormalities in multiple brain regions of patients with depression, 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 to maintaining normal neural network activity, in-depth research on the regulatory mechanism of astrocytes on synaptic function and its relationship with depressive-like behavior 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 that show depressive-like behaviors, such as chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) models, abnormalities in the morphology, activation level, and glutamate transport of astrocytes in the brains of stressed mice were detected. However, why astrocytes change 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 activity through calcium signal activation, and their abnormal functions are closely related to depressive-like manifestations, it is very likely that the dysregulated calcium signal activity in astrocytes affects synaptic function through regulating glutamate transport and other processes, and thus participates in the pathogenesis of depression. Therefore, further research on the decoding mechanism of calcium signal regulation of synaptic function in astrocytes and its physiological functions is of great scientific and clinical significance.

[0004] In the nervous system, there is a class of calcium ion binding proteins 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 astrocyte subtypes are still unclear. It is reported that there are four subtypes of CaMKII, namely α, β, γ and δ. Among them, the calcium-regulated kinase γCaMKII is a key connecting protein that mediates the calcium signal transmission and nuclear gene transcription process of nerve cells. Its abnormal expression can lead to defects in synaptic plasticity and learning ability. The results of human brain single-cell RNA sequencing found that γCaMKII is not only expressed in neurons in the hippocampus, but also enriched in astrocytes, suggesting that γCaMKII may be involved in the calcium signal transduction process in astrocytes. Based on the above research background, the inventors believe that γCaMKII is very likely to be a key molecule in hippocampal astrocytes that decodes calcium signals and regulates depressive-like behaviors. It is of great significance to explore and improve its mechanism of involvement in the pathogenesis of depression and provide new drug targets and treatment plans for the prevention and treatment of depression. Summary of the invention

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

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

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

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

[0009] The second aspect of the present invention provides the use of a γCaMKII expression promoter in any one or more of the following applications, wherein the γCaMKII expression promoter is used to promote the expression of γCaMKII in hippocampal astrocytes, and the applications include: (1) preparing a product for improving the activity and function of astrocytes; (2) preparing a product for maintaining brain glutamate balance and / or repairing synaptic structure and function; and (3) preparing a product for preventing and / or treating depression.

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

[0011] Preferably, the expression vector having the Camk2g encoding gene comprises a lentivirus expression vector or an adeno-associated virus expression vector, which is packaged into a lentivirus or an adeno-associated virus and then enters the target cell in the form of viral infection.

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

[0013] The third aspect of the present invention provides a method for screening drugs for preventing and / or treating depression, comprising the following steps:

[0014] (1) In the test group, add the substance to be tested into the test system;

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

[0016] (3) Comparative analysis of 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 is significantly increased, it indicates that the substance is a potential drug for preventing and / or treating depression;

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

[0018] A fourth aspect of the present invention provides the use of γCaMKII in preparing an animal model having a phenotype of depressive-like behavior, which is achieved by knocking out a gene expressing γCaMKII protein in 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) This invention is the first to demonstrate that the γCaMKII signaling pathway in hippocampal astrocytes is involved in regulating astrocyte morphology and function, synaptic structure, and depressive-like behavior.

[0022] (2) The present invention is the first to target the γCaMKII protein in astrocytes and regulate its expression by viral infection or inhibition of the glucocorticoid signaling pathway, confirming 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 depression stress model mice, and also provides an important research basis for the prediction of depression and the development of clinical treatment drugs. In addition, based on the active participation of γCaMKII protein in the interaction between brain astrocytes and neurons, it also provides new clinical ideas and social application value for the development of a series of neurodegenerative and psychiatric diseases targeting astrocytes and impaired synaptic function. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0025] Figure 1Diagram of the mechanism by which γCaMKII is involved in stress-induced depressive-like behavior in mice.

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

[0027] Figure 3The results show that the activity of astrocytes in γCaMKII conditional knockout mice is decreased, and the mice exhibit obvious depressive-like phenotypes: (A) is the immunostaining results 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 hippocampus of WT mice, and the dotted squares mark the astrocytes in the hippocampus of cKO mice; (B) is a statistical graph of the percentage of the area of ​​γCaMKII and GFAP co-positive signals in the hippocampus; (C) is a statistical graph of the percentage of the area of ​​GFAP positive signals in the hippocampus; (D) is a statistical graph of the relative immobility time of WT and cKO mice in the forced swimming test; (E) is a statistical graph of the immobility time of WT and cKO mice in the tail suspension test; in (B, C), dots represent the statistical values ​​in the brain tissue sections of WT mice, square dots represent the statistical values ​​in the brain tissue sections of cKO mice, and a single dot or square represents the statistical value in a single brain tissue section; (D, In E), dots represent the statistical values ​​of individual behaviors of WT mice, square dots represent the statistical values ​​of individual behaviors of cKO mice, and single dots or square dots represent the statistical values ​​of the behaviors of a single mouse; **p<0.01.

[0028] Figure 4 The richness of astrocyte branches was reduced in mice with conditional knockdown of γCaMKII: (A) is a concentric circle image of Sholl analysis of astrocytes in control and knockdown (sh-Camk2g) mice; (B) is a statistical graph of the number of astrocyte branches; (C) is a statistical graph of the total length of astrocyte branches; (D, E) are statistical graphs of the distribution of astrocyte branch intersections and the number of highest point intersections in Sholl analysis; in (B, C, D, E), dots represent the statistical values ​​in astrocytes of control mice, square dots represent the statistical values ​​in astrocytes of knockdown mice, and single dots or square dots represent the statistical values ​​in single astrocytes; *p<0.05, ***p<0.001.

[0029] Figure 5The results show that γCaMKII in astrocytes positively regulates glutamate transport function: (A) is a protein immunoblotting method to detect the interaction between γCaMKII and glutamate transporter 1 (GLT1) in hippocampal tissue; (B) tdTamato is specifically expressed in astrocytes after tamoxifen injection in Ai14 x Aldh1l1-CreERT2 mice, and the co-staining results of γCaMKII and GLT1 proteins show that they are co-labeled on the membranes of small branches of astrocytes; (C) is a real-time monitoring tracking diagram of the glutamate transport process of astrocytes in living brain slices of control group and γCaMKII conditional knockdown mice using the specific fluorescent probe iGluSnFR; (D) is a time series statistical diagram of the fluorescence changes of astrocyte glutamate probes in living brain slices of glutamate-treated control group and γCaMKII conditional knockdown mice; (E) is a graph showing the fluorescence changes of astrocytes in living brain slices at 15s, Statistical graph of glutamate probe fluorescence changes in the control group and γCaMKII knockdown astrocytes after 30s and 45s of glutamate drug action; *p<0.05, **p<0.01, ***p<0.001.

[0030] Figure 6 The figure shows that knocking down γCaMKII in astrocytes leads to changes in hippocampal synaptic structure: (A) is an image of secondary apical dendrites of CA1 pyramidal neurons labeled by GFP in the control group and knockdown mice; (B) is a statistical graph of total dendritic spine density in CA1 in the control group and knockdown mice; (C) is a statistical graph of mature dendritic spine density in CA1 in the control group and knockdown mice; in (B, C), dots represent the statistical values ​​of dendritic spine density on dendrites in control mice, square dots represent the statistical values ​​of dendritic spine density on dendrites in knockdown mice, and single dots or square dots represent the statistical values ​​on single dendrites; **p<0.01, ***p<0.001.

[0031] Figure 7The results show that overexpression of γCaMKII in hippocampal astrocytes can increase astrocyte activity in CRS mice and inhibit the occurrence of depressive-like phenotypes: (A) is the immunostaining results of γCaMKII and GFAP proteins in the control group and CRS model mice overexpressing γCaMKII (CRS-Camk2g); (B) is a statistical graph of the percentage of GFAP positive signal area in the hippocampus, in which 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 a statistical graph of the relative immobility time in the forced swimming test of the control group and mice overexpressing γCaMKII before and after CRS modeling, in which the dots represent the statistical values ​​of the individual behavior of mice before modeling, the triangle dots represent the statistical values ​​of the individual behavior of mice after modeling, and a single dot or triangle dot represents the statistical value of the behavior of a single mouse; **p<0.01, ***p<0.001, ns, no significant difference.

[0032] Figure 8 The results show that the protein level of γCaMKII in hippocampal astrocytes and its cell activity are regulated by stress hormones: (A) is the immunostaining result of γCaMKII and GFAP proteins in the hippocampus of mice that were pre-injected with control reagent (DMSO) or glucocorticoid receptor inhibitor RU486 (mifepristone) intraperitoneally after acute restraint stress (ARS); (D) is the immunostaining result of γCaMKII and GFAP proteins in the hippocampus of mice that were pre-injected with control reagent or RU486 intraperitoneally after plantar electrical stimulation (ES); (B, E) are statistical graphs of the percentage of the area of ​​γCaMKII and GFAP co-positive signals in the hippocampus; (C, F) are statistical graphs of the percentage of the area of ​​GFAP positive signals in the hippocampus; in (B, C, E, F), the dots represent the statistical values ​​in the brain tissue sections of mice injected with the control reagent, the squares represent the statistical values ​​in the brain tissue sections of mice injected with the glucocorticoid receptor inhibitor RU486, and a single dot or square represents the statistical value in a single brain tissue section; *p<0 .05,**p<0 .01. DETAILED DESCRIPTION

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

[0034] Terminology explanation:

[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 of astrocyte activity and specificity.

[0036] Camk2g (Calcium / Calmodulin-dependent protein kinase II gamma): Gene encoding calmodulin-dependent protein kinase II gamma isoform (γCaMKII).

[0037] lxop-γCaMKII mouse: A transgenic mouse model constructed using the Cre / LoxP system, which achieves conditional knockout or regulation of the γCaMKII gene by specifically expressing Cre recombinase in specific neural 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 Cre recombinase and the ligand binding domain of the estrogen receptor (ERT2), and the activity of the Cre enzyme can only be activated by induction of tamoxifen or its active metabolite 4-hydroxytamoxifen (4-OHT).

[0039] Sholl analysis: A classic method for quantitatively analyzing neuronal morphological characteristics, originally proposed by Donald Sholl in 1953.

[0040] Ai14 mice: A genetically modified mouse strain commonly used in scientific research, carrying a tdTomato red fluorescent protein gene for reporter gene expression blocked by a STOP box sandwiched by loxP sites.

[0041] Depressive-like behavior refers to behavioral characteristics exhibited by animals that are similar to human depression symptoms. These behaviors usually reflect the core symptoms of depression such as low mood, loss of interest, and impaired cognitive function.

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

[0043] 1. Test methods

[0044] (1) Construction of chronic restraint stress model mice: C57 male mice aged about 2 months were purchased and randomly divided into a control group (Con) and an experimental group (CRS) and placed in cages with a 12-hour light / 12-hour dark cycle with free access to food and water. After the mice adapted to the environment for 1 week, a forced swimming test was performed before modeling to determine the baseline performance of their depressive-like behavior. Thereafter, the control group mice were not treated in any way, while the experimental group mice were placed in a 50 mL centrifuge tube with a small hole every day to ensure that the mice could not escape. Each tube was separated by a card to ensure that each mouse could not see its companion. After that, they were restrained for 3 consecutive weeks, with an irregular time of 2-8 hours per day. Finally, the data of the forced swimming test after modeling and the relative immobility time before modeling were compared to evaluate whether the mice showed obvious depressive-like phenotypes, so as to determine the CRS mice that were successfully modeled for studying the pathogenic mechanism of depression.

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

[0046] (3) Brain slice RNA fluorescence in situ hybridization and data analysis: The slides with brain slices were taken out from -80°C and soaked in 50%, 70% and 100% ethanol for 5 minutes respectively. After baking at 60°C for 20 minutes, the slides were treated with pretreatment solution and digestive enzymes, washed with ultrapure water three times, and then incubated with Camk2g probe (purchased from Guangdong Pinbo Yishi Biotechnology Co., Ltd.) and corresponding channel type fluorescent labeling reaction solution, and finally GFAP immunostaining and sealing were performed. The slides were collected with 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) Immunohistochemical staining of brain slices and data analysis: After washing the tissue slices, the brain slices were blocked in 0.1% Triton X-100 and 4% donkey serum for 1 hour, then washed three times with PBS and incubated with primary antibodies for γCaMKII and GFAP (purchased from SYSY, 173011) at 4°C overnight. The next day, the slices were washed three times with PBS and incubated with appropriate fluorescently labeled Alexa Fluro secondary antibodies (purchased from Invitrogen) at room temperature for 3 hours in the dark. Finally, the stained slices were mounted on slides and covered with a medium containing 4',6-diamidino-2-phenylindole (DAPI). Slide images were collected using a Nikon A1 laser confocal microscope, and the fluorescence intensity and the ratio of the positive signal expression area of ​​γCaMKII and GFAP were quantitatively analyzed using ImageJ software.

[0048] (5) Statistical methods: All data were analyzed using GraphPad Prism and expressed as Mean ± SEM. The significance of the difference between two groups of data was analyzed using Student's t-test. One-way ANOVA was used for the analysis of multiple groups of data under single factor, and two-way ANOVA was used for the analysis of inter-group data under multi-factor. p < 0.05 was considered statistically significant.

[0049] 2. Experimental results

[0050] like Figure 2 As shown in Figures AC, in the chronic restraint stress (CRS)-induced mouse depression model, the activity of astrocytes marked by GFAP was significantly decreased, and the RNA level of γCaMKII in astrocytes was significantly decreased. Similar to the gene expression results, Figure 2 The immunostaining results of DF in the same study also confirmed that the γCaMKII protein level in astrocytes was downregulated 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 in the figure indicates that γCaMKII is an important molecule that regulates astrocyte activity and function. However, there is no significant difference in the expression of γCaMKII in inhibitory neurons between CRS and Con mice ( Figure 2 This indicates that the γCaMKII regulation process under CRS stress stimulation only occurs specifically in a specific neural cell population, astrocytes.

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

[0052] 1. Test methods

[0053] (1) Construction of astrocyte γCaMKII conditional knockout mice: lxop-γCaMKII and Aldh1l1-CreERT2 mice were hybridized, and tetrahydroxytamoxifen was injected intraperitoneally in the offspring to specifically activate the Cre recombinase in astrocytes, thereby achieving knockout of the γCaMKII gene in astrocytes throughout the brain.

[0054] (2) Depression model behavioral despair assessment paradigm - tail suspension test and forced swimming test: In the tail suspension test, the tail of each mouse is fixed in the tester, so that its head is suspended downward for 6 minutes, and each mouse is separated by a baffle; in the forced swimming test, the mouse is placed individually in a cylinder filled with water (water temperature 23-25℃) so that its legs cannot touch the bottom, and swims for 6 minutes. While conducting these two experiments, the animal behavior is recorded and filmed, and the time during the last 4 minutes of the test that the mouse remains relatively still, that is, the time without significant struggling movements, is analyzed. Finally, this parameter is used to evaluate the degree of depression of the experimental mouse. The longer the relative stillness time, the higher the degree of depression of the mouse.

[0055] 2. Experimental results

[0056] like Figure 3 As shown in Figures AC, the expression of γCaMKII protein in astrocytes was significantly decreased in conditional knockout mice, and the activity of hippocampal astrocytes was also significantly downregulated. Figure 3 As shown in DE in Figure 3, when compared with WT mice, the relative immobility time of cKO mice in the tail suspension test and forced swim test was significantly increased, indicating that specific knockout of γCaMKII in astrocytes directly increases the probability of mice exhibiting depressive-like behaviors.

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

[0058] 1. Test 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 Figure A, co-immunoprecipitation experiments revealed a significant interaction between γCaMKII protein and glutamate transporter 1 (GLT1) in adult mouse hippocampal tissue. When further immunostained hippocampal slices from Ai14 x Aldh1l1-CreERT2 mice were imaged by high-resolution microscopy ( Figure 5 B), the results showed that γCaMKII and GLT1 showed obvious co-localization on the tiny 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 branches, which suggests that γCaMKII is very likely to play an important role in regulating the glutamate transport function of astrocytes, thereby affecting the function of synapses. When the glutamate transport function was continued to be detected in the brains of knockdown mice expressing the glutamate probe virus, the real-time tracking imaging results showed that when acute brain slices of mice were treated with glutamate drugs, the fluorescence value of the glutamate probe would first increase and then decrease, indicating that glutamate drug treatment successfully activated the glutamate transporter on the astrocyte membrane, and as glutamate was transported from the extracellular to the intracellular, the fluorescence value also changed accordingly ( Figure 5 C). Compared with the control group, the glutamate transport process of astrocytes in the living brain slices of γCaMKII conditional knockdown mice was significantly slowed down ( Figure 5 CE in the glial cells), indicating that its glutamate transporter function is 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 between astrocytes and neurons to a certain extent.

[0066] (3) If Figure 6 As shown, when GFP is used to mark hippocampal CA1 pyramidal neurons, this example found that specific knockdown of γCaMKII in mice resulted in a significant decrease in the total dendritic spines and mature dendritic spine density of its secondary apical dendrites. Given that the glutamate transport function of astrocytes is impaired in γCaMKII knockdown mice, this change in neuronal synaptic structure is likely due to excitotoxicity caused by extracellular glutamate accumulation. These results indicate that γCaMKII in astrocytes not only actively regulates the richness of its branches, but also affects the structure and function of its peripheral nerve synapses by changing the glutamate transport function.

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

[0068] 1. Test methods

[0069] Overexpression of γCaMKII in hippocampal astrocytes of CRS mice: AAV2 / 8-GfaABC1D-mCherry-P2A-Camk2g or control virus produced by Shanghai Shengbo Biotechnology Co., Ltd. was injected into the CA1 brain region of the hippocampus of adult mice on both sides. Behavioral tests were performed before CRS modeling 3 weeks after the mice recovered, which was also the time when the virus began to express. Thereafter, the 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 changes in the depressive-like behavior of the mice.

[0070] 2. Experimental results

[0071] like Figure 7 As shown in AB in Figure 1, overexpression of γCaMKII in hippocampal astrocytes can significantly increase the activity of astrocytes in CRS mice. Further analysis of the behavioral test results before and after modeling revealed that the control group mice showed obvious depressive-like phenotypes after 3 weeks of restraint stress, while the overexpression group mice showed no significant changes in depressive-like behavior before and after modeling ( Figure 7 C in the figure). Given that astrocyte function is closely related to depressive-like behavior, this suggests that overexpression of γCaMKII in hippocampal tissue can inhibit depression in mice during the stress restraint model by enhancing astrocyte function. This finding provides a potential target for the development of drugs that specifically prevent depression.

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

[0073] 1. Test methods

[0074] (1) Acute stress stimulation: During acute restraint stress stimulation (ARS), adult mice will be placed in a 50 mL centrifuge tube with a small hole to ensure that the mice cannot escape. Each tube will be separated by a card to ensure that each mouse cannot see its companion. The restraint time is 2 hours. During the plantar electrical stimulation (ES) experiment, the mice will be placed in the conditioning room for 2 minutes to familiarize themselves with the environment, and then given a 2-second foot shock with a current intensity of 0.7 mA. After the shock, the mice will continue to stay in the conditioning room for 2 minutes. After that, the mice will be returned to the cage. Both acute stress stimulations started 3 hours after the injection of mifepristone. After the stimulation, the mice will be sampled 2 hours later for subsequent brain slice sectioning and immunostaining experiments.

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

[0076] 2. Experimental results

[0077] like Figure 8 As shown in Figures AC, compared with the control group, which showed lower γCaMKII protein levels after acute restraint (ARS), after the glucocorticoid signaling pathway was inhibited by RU486 in advance, the γCaMKII protein level and cell activity in hippocampal astrocytes were limited by ARS and remained in a relatively normal state. In addition, when mice experienced another stress stimulus, electrical stimulation (ES) in the plantar, mice that were pre-injected with RU486 intraperitoneally also maintained higher γCaMKII and GFAP protein levels ( Figure 8 DF in the figure). These results indicate that the downregulation of γCaMKII protein levels and cell activity in hippocampal astrocytes in mice with depressive-like behaviors is most likely due to the regulation of stress hormones. Based on the fact that patients with depression often have dysregulated hypothalamic-pituitary-adrenal axis responses and abnormal glucocorticoid release, and that mice can have a certain degree of antidepressant effect after being treated with mifepristone (RU486), it is very likely that the regulation of γCaMKII protein in astrocytes is an important target of RU486 in the treatment of depression. Therefore, as a target protein for depression, the regulation of γCaMKII protein by glucocorticoids is an important supplementary mechanism in the pathogenesis of depression.

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

[0079] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of γCaMKII as a target in screening or preparing drugs for preventing and / or treating depression, characterized in that: The γCaMKII is localized in hippocampal astrocytes.

2. The use according to claim 1, characterized in that: The depression includes depression induced by stress and / or long-term stress stimulation.

3. Use of a γCaMKII expression promoter in any one or more of the following, characterized in that: The γCaMKII expression promoter is used to promote the expression of γCaMKII in hippocampal astrocytes, and the applications include: (1) preparing products for improving the activity and function of astrocytes; (2) preparing products for maintaining brain glutamate balance and / or repairing synaptic structure and function; and (3) preparing products for preventing and / or treating depression.

4. The use according to claim 3, characterized in that: The γCaMKII expression promoter includes an expression vector having a Camk2g coding gene and / or a substance that targets and regulates the expression of γCaMKII protein by regulating a signal pathway.

5. The use according to claim 4, characterized in that: The expression vector having the Camk2g coding gene includes a lentivirus expression vector or an adeno-associated virus expression vector, which is packaged into a lentivirus or an adeno-associated virus and then enters the target cell in the form of virus infection.

6. The use according to claim 4, characterized in that: The substance that regulates the expression of γCaMKII protein by regulating the signal pathway includes a substance that regulates the level of γCaMKII protein by regulating the glucocorticoid signal pathway and / or a specific regulating substance for the upstream and downstream signal pathways of the γCaMKII protein itself.

7. A method for screening drugs for preventing and / or treating depression, characterized in that: The steps include: (1) In the test group, add the substance to be tested into the test system; (2) detecting the expression level of γCaMKII gene and / or protein in the detection system of the test group, and comparing it with that of the negative control group; (3) Comparative analysis of 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 is significantly increased, it indicates that the substance is a potential drug for preventing and / or treating depression; Wherein, the detection system is hippocampal astrocytes.

8. The use of γCaMKII in preparing an animal model, characterized in that: The animal model has a phenotype of depressive-like behavior, which is achieved by knocking out the gene expressing γCaMKII protein in the hippocampal astrocytes of the animal.

9. The use according to claim 8, characterized in that: The animal is a mouse or a primate.

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