Disclosure of Invention
In view of the above, the present invention aims to provide an application of calmodulin gamma CaMKII as a target spot in screening or preparing a medicament for preventing and/or treating depression. As shown in FIG. 1, calmodulin gamma CaMKII refers to gamma CaMKII expressed in enrichment in hippocampal astrocytes, and the expression of gamma CaMKII is significantly down-regulated in a stress mouse model with depression-like behavior, and can be used as an index protein for reflecting depression to a certain extent. When gamma CaMKII protein is selectively over-expressed in hippocampal astrocytes or gamma CaMKII expression is targeted by utilizing glucocorticoid receptor inhibitor, the down-regulation of astrocyte activity caused by pressure stimulation can be timely inhibited, and the depression-like behavior of stressed mice can be relieved. Therefore, the regulatory mechanism of the gamma CaMKII protein discovered in the invention supplements the pathogenic mechanism of depression to a certain extent, and the process that the expression of the targeted gamma CaMKII protein can directly influence the depression-like behavior of a stressed mouse provides a new thought for developing and preparing anti-depression drugs.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect of the invention, there is provided the use of γcamkii as a target in the screening or preparation of a medicament for the prevention and/or treatment of depression, said γcamkii being located in hippocampal astrocytes. Knocking out gamma CaMKII in astrocytes significantly down-regulates its cell activity and directly results in mice exhibiting depression-like behavior, whereas overexpression of gamma CaMKII in astrocytes by viral injection or enhancement of gamma CaMKII protein levels by glucocorticoid receptor inhibitors can enhance astrocyte activity and suppress to some extent depression-like behavior in stressed mice. Based on this, the present invention provides a regimen for achieving anti-depressive behavior therapy with increased levels of gamma CaMKII protein. Therefore, in the process of preparing or screening the depression drugs, substances which can selectively up-regulate the level of gamma CaMKII protein in the astrocytes of the hippocampus, especially glucocorticoid signal pathway regulating drugs, can be selected by detecting the change of the gamma CaMKII expression level in the system to be used as candidate drugs for preventing and treating depression.
Preferably, the depression comprises stress and/or long-term stress stimulus induced depression.
In a second aspect of the present invention, there is provided the use of a gamma CaMKII expression promoter for promoting expression of gamma CaMKII in hippocampal astrocytes, comprising (1) preparing a product for enhancing astrocyte activity and function, (2) preparing a product for maintaining cerebral glutamate balance and/or repairing synaptic structures and functions, and (3) preparing a product for preventing and/or treating depression.
Preferably, the gamma CaMKII expression promoter comprises an expression vector with a Camk2g coding gene and/or a substance for targeted regulation of gamma CaMKII protein expression by modulating a signal pathway.
Preferably, the expression vector having the gene encoding Camk2g includes a lentiviral expression vector or an adeno-associated viral expression vector, which is packaged into a lentivirus or adeno-associated virus and then enters the cell of interest in the form of a viral infection.
Preferably, the agent that targeted modulates expression of γcamkii protein by modulating a signaling pathway comprises an agent that targeted modulates γcamkii protein levels by modulating a glucocorticoid signaling pathway and/or a specific modulating agent directed against a signaling pathway downstream of γcamkii protein itself.
In a third aspect of the present invention, there is provided a method for screening a drug for preventing and/or treating depression, comprising the steps of:
(1) Adding a substance to be detected into a detection system in a test group;
(2) Detecting the expression level of gamma CaMKII genes and/or proteins in a detection system of the test group, and comparing the gamma CaMKII genes and/or proteins with a negative control group;
(3) Comparing and analyzing the expression level of the gamma CaMKII gene and/or protein in the test group with the expression level of the gamma CaMKII gene and/or protein in the control group, wherein if the expression level of the gamma CaMKII gene and/or protein is obviously increased in the test group, the substance is a potential drug for preventing and/or treating depression;
wherein the detection system is hippocampal astrocytes.
In a fourth aspect of the invention, there is provided the use of γcamkii in the preparation of an animal model having a phenotype of depression-like behaviour by knocking out genes expressing γcamkii proteins in hippocampal astrocytes of an animal.
Preferably, the animal is a mouse or primate.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention demonstrates for the first time that gamma CaMKII signaling pathways in hippocampal astrocytes are involved in the regulation of astrocyte morphology and function, synaptic structure and depression-like behavior.
(2) The invention carries out expression regulation and control in a virus infection mode or a glucocorticoid signal pathway inhibition mode through targeting gamma CaMKII protein in astrocytes for the first time, and proves the important beneficial effect in treating depression.
(3) The invention is further based on the expression change of gamma CaMKII protein in a depression stress model mouse, so that important supplement is made for the pathogenesis of depression, and an important research basis is provided for the prediction of depression and the research and development of clinical treatment medicaments. In addition, gamma CaMKII protein is based on the interaction between brain astrocytes and neurons, which also provides new clinical thought and social application value for developing a series of neurodegenerative and mental diseases with target astrocytes and impaired synaptic function.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a schematic diagram of gamma CaMKII involved in stress stimulation to induce depressive-like behavior in mice.
FIG. 2 shows that gamma CAMKII RNA and protein expression levels in hippocampal tissues are obviously down-regulated in astrocytes in a chronic constraint stress (CRS) -induced mouse depression model, and meanwhile, the gamma CAMKII RNA (Camk 2G) fluorescence in situ hybridization and GFAP protein immunostaining result graphs in a control group and a CRS mouse hippocampus are shown, dotted line circles mark the hippocampal brain astrocytes in the control group in the graph, dotted line boxes mark the hippocampal brain astrocytes in a depression model mouse, B) is a proportion statistical graph for expressing gamma CAMKII RNA (the fluorescent signal points in the hippocampal GFAP positive astrocytes are identified as expression), C is a cumulative frequency graph of gamma CAMKII RNA fluorescent in-situ hybridization signal points in the GFAP positive astrocytes, D) is a gamma CaMKII and GFAP protein immunostaining result graph in the control group and the CRS mouse hippocampus, E) is a gamma CaMKII and GFAP common positive signal occupation area, B) is a proportion statistical graph for expressing gamma CAMKII RNA (the fluorescent signal points in the hippocampal GFAP positive astrocytes is identified as expression in the hippocampal positive astrocytes, and a single-phase-contrast graph is shown in the hippocampal tissue of the graph, and the value of the gamma-type CAP positive signal points in the hippocampal positive tissue is shown in the graph is a statistical graph, and the value of the gamma-positive signal points in the hippocampal tissue is shown in the graph is a statistical graph and the comparison with the human tissue on the human tissue is shown in the graph, individual dots or squares represent statistics in individual brain tissue sections with p < 0.01, p < 0.001, n.s. without significant differences.
FIG. 3 shows a decrease in astrocyte activity in a gamma CaMKII conditional knockout mouse, while the mouse exhibits a pronounced depression-like phenotype, wherein (A) is a graph of results of gamma CaMKII and GFAP protein immunostaining in the hippocampus of wild-type (WT) and conditional knockout (cKO) mice, in which the dashed circle marks astrocytes in the hippocampus region of the WT mouse, the dashed square marks astrocytes in the hippocampus region of the cKO mice, (B) is a graph of the ratio of gamma CaMKII and GFAP co-positive signal areas in the hippocampus, (C) is a graph of the ratio of GFAP positive signal areas in the hippocampus, and (D) is a graph of relative stationary time statistics of the WT and cKO mice in a forced swimming test, (E) is a graph of stationary time statistics of the WT and cKO mice in a tail suspension experiment, in which dots represent statistics in a brain tissue section of the WT mouse, square dots represent statistics in a brain tissue section of the cKO mice, individual dots represent statistics in a brain tissue section of the individual or square dots represent statistics of individual behavior of the individual mice, and (D) is a dot represents individual behavior of the individual dots in 35. 0. Individual dot represents individual behavior of the mouse.
FIG. 4 shows a drop in astrocyte branch abundance in gamma CaMKII conditional knockdown mice, wherein (A) is an image of concentric circles analyzed by astrocytes Sholl in control and knockdown (sh-Camk 2 g) mice, (B) is an astrocyte branch count plot, (C) is an astrocyte branch total length plot, (D, E) is a Sholl analysis astrocyte branch intersection distribution and peak intersection count plot, (B, C, D, E) wherein the dots represent statistics in control mouse astrocytes, the square dots represent statistics in knockdown mouse astrocytes, and individual dots or square dots represent statistics in individual astrocytes, p < 0.05, p < 0.001.
Fig. 5 shows that gamma CaMKII actively regulates glutamate transport function in astrocytes, wherein (a) is a trace graph of gamma CaMKII and glutamate transporter 1 (GLT 1) in hippocampal tissues detected by western immunoblotting, (B) is a time series statistical graph of astrocyte glutamate probe fluorescence change in Ai14 x Aldh1l1-CreERT2 mice injected with tamoxifen, tdTamato is specifically expressed in astrocytes, and co-staining results of gamma CaMKII and GLT1 protein show co-labeling on astrocyte fine branched membranes, (C) is a trace graph of astrocyte glutamate transport process in control group and gamma CaMKII conditional knockdown mice in vivo brain slices monitored in real time by using specific fluorescent probe iGluSnFR, (D) is a time series statistical graph of astrocyte glutamate probe fluorescence change in gamma CaMKII conditional knockdown mice in vivo brain slices, and (E) is a statistical graph of change of gamma CaMKII in astrocyte in 15s, 30s and 45s of glutamate drug effect, respectively, < 0.01.0.0.001.
Fig. 6 shows that knockdown γcamkii in astrocytes resulted in changes in hippocampal synaptic structure, wherein (a) is a secondary apical dendrite image of CA1 region pyramidal neurons labeled by GFP in control and knockdown mice, (B) is a statistical plot of total CA1 dendritic spine density in control and knockdown mice, (C) is a statistical plot of CA1 mature dendritic spine density in control and knockdown mice, (B, C) is a statistical value of dendritic spine density in control mice, square points are statistical values of dendritic spine density in knockdown mice, and individual dots or square points are statistical values on individual dendrites, where p < 0.01, p < 0.001.
Fig. 7 shows that overexpression of γcamkii in hippocampal astrocytes can increase astrocyte activity in CRS mice while inhibiting depression-like phenotype, wherein (a) is a graph of results of immunostaining of γcamkii and GFAP protein in control group and CRS model mice (CRS-Camk 2 g) overexpressing γcamkii, (B) is a graph of statistics of area of GFAP positive signal in hippocampus, dots in the graph represent statistics of brain tissue slices of control group mice, square dots represent statistics of brain tissue slices of overexpressing group mice, single dots or square dots represent statistics of single brain tissue slices, (C) is a graph of relative rest time of control group and mice overexpressing γcamkii in forced swimming test before and after molding, and triangular dots represent statistics of individual behaviors of mice before molding, single or triangular dots represent statistics of individual behaviors of single mice, and dots of 0.01 x < 0.n.
FIG. 8 shows that protein levels and cellular activity of gamma CaMKII in hippocampal astrocytes are regulated by stress hormones, wherein (A) is a graph showing the results of immunostaining of gamma CaMKII and GFAP proteins in hippocampus in mice pre-intraperitoneally injected with a control reagent (DMSO) or glucocorticoid receptor inhibitor RU486 (mifepristone) after undergoing Acute Restraint Stress (ARS) stimulation; (D) is a graph of results of gamma CaMKII and GFAP protein immunostaining in hippocampus after being subjected to plantar Electric Stimulation (ES) of mice pre-injected with a control reagent or RU486, (B, E) is a graph of statistics of gamma CaMKII and GFAP co-positive signal areas in hippocampus, (C, F) is a graph of statistics of GFAP positive signal areas in hippocampus, (B, C, E, F), wherein dots represent statistics in brain tissue sections of mice injected with the control reagent, square dots represent statistics in brain tissue sections of mice injected with the glucocorticoid receptor inhibitor RU486, and single dots or square dots represent statistics in single brain tissue sections, and p < 0.05 and p < 0.01.
Detailed Description
The invention is described in further detail below in connection with specific embodiments and with reference to the accompanying drawings. It should be understood that the examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way. The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated. In addition, unless specifically stated otherwise, the reagents involved in the examples of the present invention are commercially available products and are commercially available, and various processes and methods not described in detail in the examples of the present invention are conventional methods well known in the art.
Term interpretation:
GFAP (Glial Fibrillary Acidic Protein, glial acidic protein), an intermediate filament protein belonging to class III intermediate filaments, is specifically expressed in the Astrocyte (AS) cytoplasm of the central nervous system and can be used AS an astrocyte activity and specificity marker.
Camk2g (Calcium/Calmodulin-DEPENDENT PROTEIN KINASE II GAMMA) a gene encoding the calmodulin-dependent protein kinase II gamma subtype (gamma CaMKII).
Lxop-gamma CaMKII mice A transgenic mouse model constructed by using a Cre/LoxP system realizes conditional knockout or regulation of gamma CaMKII genes by specifically expressing Cre recombinase in specific nerve cells.
Aldh1l 1-Creet 2 mice, a transgenic mouse model, is mainly used for researching astrocytes in the central nervous system, and the specific expression of Cre recombinase in astrocytes is realized by inserting the Creet 2 gene into the stop codon of the Aldh1l1 gene, wherein Creet 2 is a fusion protein containing Cre recombinase and a ligand binding domain (ERT 2) of an estrogen receptor, and the activity of Cre recombinase can be activated by the induction of Tamoxifen (Tamoxifen) or an active metabolite 4-hydroxy Tamoxifen (4-OHT) thereof.
Sholl analysis A classical method for quantitative analysis of neuronal morphological characteristics, originally proposed by Donald Sholl in 1953.
Ai14 mice, a genetically modified mouse strain commonly used in scientific research, carry a tdTomato red fluorescent protein gene for reporter gene expression, which is blocked by a STOP box sandwiched by loxP sites.
Depression-like behavior (DEPRESSIVE-like behavior), which refers to behavior characteristics exhibited by animals that resemble the symptoms of human depression, typically reflecting the core symptoms of depression such as depressed mood, reduced interest, impaired cognitive function, etc.
EXAMPLE 1 detection of the expression level of gamma CaMKII and astrocyte Activity in hippocampal tissue of a Chronic stress model mouse
1. Test method
(1) Chronic restraint stress model mice were constructed by purchasing C57 male mice around 2 months, randomly dividing them into control group (Con) and experimental group (CRS) and placing them in cages with 12-hour light/12-hour dark cycle that can freely obtain food and water. After 1 week of adaptation to the environment, the mice were subjected to a pre-modelling forced swimming test to determine their baseline performance of depression-like behaviour. Thereafter, the control mice were not subjected to any treatment, whereas the experimental mice were placed daily in a50 mL centrifuge tube perforated to ensure that the mice were unable to escape, each tube was blocked with a card to ensure that each mouse was not seen its companion, and thereafter subjected to continuous 3-week restraint for 2-8 hours a day. Finally, whether the mice have obvious depression-like phenotypes is evaluated by comparing the data of the forced swimming test after modeling with the data of the relative resting time before modeling, so that the CRS mice successfully modeled are determined and used for researching the pathogenic mechanism of depression.
(2) Brain tissue sections were prepared by anesthetizing mice with sodium pentobarbital after behavioral testing, then perfusing with PBS to remove blood components from blood vessels, then taking out the brains of the mice and fixing them in 4% paraformaldehyde for 4 hours followed by an in situ hybridization experiment with RNA fluorescence for 20 hours. The fixed mouse brain was dehydrated with 30% sucrose at 4 ℃ for 24 hours. The dehydrated brain tissue was sectioned at-20 ℃ using a Leica CM1800 cryostat, and brain sections of 35 micron thickness were cut. If the RNA fluorescence in situ hybridization experiment is carried out subsequently, the cut sections are stuck on a glass slide without RNase, and are immediately put at-80 ℃ for preservation after air drying, and the brain slices which are subjected to the immunostaining subsequently are collected in a cryoprotection buffer solution, wherein the buffer solution consists of 40% glycerol, 30% 0.1M PBS and 30% glycol and are put at-20 ℃ for preservation for later use.
(3) Taking out slide glass stuck with brain slice from-80 deg.C, soaking in 50%, 70% and 100% alcohol for 5 min, baking at 60 deg.C for 20min, treating slide glass with pretreatment liquid and digestive enzyme, washing with ultrapure water for 3 times, incubating with Camk2g probe (purchased from Guangdong Boyi Biotechnology Co., ltd.) and corresponding channel type fluorescent labeling reaction liquid, and GFAP immunostaining and sealing. Slide pictures were collected with a Nikon A1 laser confocal microscope and the number of cam 2g RNA fluorescent in situ hybridization signal spots in each GFAP positive cell was counted quantitatively with ImageJ software.
(4) Brain slice immunohistochemical staining and data analysis after tissue section washing, brain slices were blocked in 0.1% Triton X-100,4% donkey serum for 1 hour, then sections were washed three times with PBS, and after sections were incubated overnight with gamma CaMKII and GFAP (commercially available from SYSY, 173011) primary antibodies at4 ℃. The following day, the sections were washed three times with PBS and incubated for 3 hours in a dark environment at room temperature with an appropriate fluorescent labelled Alexa Fluro secondary antibody (purchased from Invitrogen). Finally, the stained sections were mounted on slides and blocked using a medium containing 4', 6-diamidino-2-phenylindole (DAPI). Slide pictures were taken with a Nikon A1 laser confocal microscope and fluorescence intensity and the ratio of gamma CaMKII to GFAP positive signal expression area were quantitatively analyzed with ImageJ software.
(5) Statistical methods all data were statistically analyzed using GRAPHPAD PRISM, expressed as mean±sem. The significance of the differences between the two sets of data was analyzed by Student's ' s t-test (Student's t-test), one-way ANOVA was used for analysis between the multiple sets of data in one factor, and two-way ANOVA was used for analysis between the multiple sets of data in multiple factors. p <0.05 is considered statistically significant.
2. Experimental results
As shown in a-C of fig. 2, GFAP-labeled astrocytes were significantly reduced in the chronic stress-binding (CRS) -induced mouse depression model, while gamma CaMKII was significantly reduced in RNA levels in astrocytes. Similar to the gene expression results, the immunostaining results of D-F in fig. 2 also confirm that the protein level of γcamkii in astrocytes was down-regulated in the hippocampus of CRS mice, and that the protein expression level of γcamkii in the hippocampus was positively correlated with the activity of astrocytes (G in fig. 2), indicating that γcamkii is an important molecule for regulating astrocyte activity and function. However, there was no significant difference in expression of γcamkii in inhibitory neurons of CRS and Con mice (H in fig. 2), indicating that the γcamkii modulation process under CRS stress stimulation only specifically occurred in this particular neural cell population of astrocytes.
EXAMPLE 2 construction of astrocyte gamma CaMKII conditional knockout mice and evaluation of their depression-like phenotype
1. Test method
(1) The method comprises the steps of hybridizing lxop-gamma CaMKII with an Aldh1l 1-Creet 2 mouse, and specifically activating Cre recombinase in astrocytes by intraperitoneal injection of tetrahydroxy tamoxifen in offspring of the mice, so as to realize the knockout of gamma CaMKII genes in whole brain astrocytes.
(2) In the model behavior desperate evaluation model-tail suspension experiment and forced swimming test, the tail of each mouse is fixed in the tester, the head of each mouse is suspended downwards by 6 min, the mice are separated by baffle plates, and in the forced swimming test, the mice are independently placed in a cylinder filled with water (the water temperature is 23-25 ℃) so that the legs of the mice cannot touch the bottom, and swimming is performed by 6 min. Animal behavior was recorded and photographed while the two experiments were being conducted, and the time during which the mice remained relatively stationary during the last 4 min tests, i.e. without significant struggling action, was analyzed, and finally the experimental mice were evaluated for their extent of depression with this parameter, the longer the time of relative rest, the higher the extent of depression of the mice was indicated.
2. Experimental results
As shown in a-C of fig. 3, expression of gamma CaMKII protein in astrocytes was significantly reduced in conditional knockout mice, while hippocampal astrocyte activity was also significantly down-regulated. As shown by D-E in fig. 3, cKO mice significantly increased relative resting time in tail suspension experiments and forced swim tests when compared to WT mice, demonstrating that specific knockout of γcamkii in astrocytes directly increases the probability of mice developing depression-like behavior.
Example 3 specific knock down of γCaMKII expression in the hippocampus of mice not only resulted in impaired astrocyte morphology and glutamate transport function, but also directly affected synaptic structures
1. Test method
(1) Stereotactic virus injection after gas anesthesia of mice with isoflurane, fixing the brains of the mice with a stereotactic device, shearing the skin of the heads of the mice with sterile scissors to expose the skull positions, and drilling holes of 0.5 mm on the surface of the skull with electric drills according to specific coordinates of the hippocampus of the mice. 200-400 nL virus was injected into bilateral hippocampus by syringe at a rate of 50-100 nL/min, and the needle was kept in place 5min after injection was completed to allow virus to spread. And then, suturing the incision by using a sterile suture line, putting the mice into a specific heat preservation box, putting the mice back into a rearing cage after the mice are awake, and finally, carrying out related experiments after waiting for 3-4 weeks for complete expression of viruses. In experiments to specifically knock down hippocampal astrocyte γcamkii expression, AAV2/8-GfaABC1D-shcamk g-P2A-mCherry virus produced by Shanghai boy corporation would be injected into the bilateral hippocampal CA1 brain region, where GfaABC D is an astrocyte-specific promoter and shcamk g is shRNA targeting γcamkii for gene expression interference of γcamkii in hippocampus. In addition, when astrocytes or synaptic morphology are detected in mice knocked down by γcamkii expression, GFP virus driven by the GfaABC D or αcamkii promoter produced by the marsupium brain science and technology limited will be injected simultaneously into the bilateral hippocampal CA1 brain region.
(2) Astrocyte Sholl analysis and dendritic spine density analysis of vertebro neurons mice injected with virus will be GFP immunostained after taking sections, and the number of astrocytes and dendritic spine will be analyzed by ultra-high resolution imaging technique and morphological means. When GFP-labeled astrocytes were randomly selected from the hippocampus and analyzed using ImageJ Sholl, concentric circles were made outwards with the cell bodies as the center, and the number of intersections between the branches and the concentric circles was counted with the cell bodies as the center, as shown in A in FIG. 4, and the data points on each Sholl chart correspond to the average Sholl intersections, representing the average of the corresponding group. After labeling CA1 cone neurons with GFP, their secondary apical dendrites were photographed with a high resolution microscope and the densities of total and mature dendrite (i.e., mushroom-like dendrite) were counted by ImageJ software.
(3) Western immunoblotting and immunoprecipitation by taking hippocampal tissue of mice on ice, adding a lysate containing protease and phosphatase inhibitors for disruption, centrifuging after sufficient lysis to obtain supernatant, measuring Protein concentration by BCA Protein quantification, adjusting the above tissue lysate to a consistent concentration according to BCA Protein quantification results, adding antibody (1-2 μg) of specific target Protein therein, incubating overnight at 4 ℃, then adding Protein G-Sepharose beads (G Protein modified agarose beads) at 4 ℃ for 1 hour to allow the beads and antibody to fully bind, then rinsing the beads with the lysate sufficiently to leave only other proteins interacting with the target Protein on the beads, detecting the interacted proteins by SDS-PAGE gel electrophoresis, blocking 1 hour at room temperature with 5% skim milk after Protein transfer, incubating overnight at 4 ℃ for 1 hour at secondary room temperature, and finally detecting the expression of the target Protein by a chemiluminescent imager.
(4) Glutamic acid probe imaging in Aldh1l1-CreERT2 mice hippocampus was injected with rAAV-EF1 alpha-DIO-iGluSnFR produced by Brinkel Biotechnology Co., ltd.) and AAV2/8-GfaABC1D-shcamk g-P2A-mCherry virus produced by Shanghai-Shengbo BioCo., ltd., while tamoxifen was intraperitoneally injected to ensure that the specificity was obtained for expressing glutamic acid probe iGluSNFR in astrocytes. After waiting for adequate virus expression, mice were harvested and subjected to acute brain slice sections under oxygenated conditions, the resuscitated brain slice was first centered over the selected area of sh-RNA expression (i.e., mCherry labeling), after which 2mM glutamate drug treatment was performed and live photography was performed simultaneously.
2. Experimental results
(1) As shown in a in fig. 4, when analyzed by Sholl after labeling astrocyte morphology with GFP, it was found that specific knock-down γcamkii not only affects astrocyte activity, but also significantly down-regulates the number and total length of its branches, and the number of branch junctions (B-E in fig. 4) in Sholl analysis, indicating that γcamkii actively regulates the abundance of its branches in astrocytes, and that it is highly likely to affect astrocyte function and interactions with synapses.
(2) As shown in a in fig. 5, the co-immunoprecipitation experiments revealed that there was a significant interaction between the gamma CaMKII protein and glutamate transporter 1 (GLT 1) in adult mouse hippocampal tissue. When the hippocampal brain slices of Ai14 x Aldh1l1-CreERT2 mice were further immunostained imaged by high resolution microscopy (B in fig. 5), the results showed that γcamkii showed a distinct co-localization phenomenon with GLT1 on the tiny branches at the ends of tdmamato-labeled astrocytes. This finding suggests that there is an interaction between γcamkii and GLT1 in astrocytes, especially in its branching portion, suggesting that γcamkii is highly likely to play an important role in regulating glutamate transport function of astrocytes, thereby affecting the function of the nerve synapse. When the glutamate transport function was continuously detected in the brain of the knock-down mice expressing the glutamate probe virus, the real-time tracking imaging result found that the acute brain sections of the mice were treated with the glutamate drug, and the fluorescence value of the glutamate probe was increased and then decreased, which means that the glutamate drug treatment successfully activated the glutamate transporter on the astrocyte membrane, and the fluorescence value was correspondingly changed as glutamate was transported from extracellular to intracellular (C in fig. 5). Compared with the control results, the astrocyte glutamate transport process in the mouse living brain tablet is obviously slowed down by the gamma CaMKII conditional knockdown (C-E in figure 5), which indicates that the glutamate transporter function is inhibited to a certain extent. Based on the obvious interaction between gamma CaMKII and GLT1, gamma CaMKII is highly likely to regulate the glutamate transport function of astrocytes through the process and influence the interaction process between astrocytes and neurons to a certain extent.
(3) As shown in fig. 6, when GFP was used to label hippocampal CA1 pyramidal neurons, this example found that specific knockdown of γcamkii resulted in a significant decrease in the total and mature dendritic spine density of its secondary apical dendrites in mice. Given that glutamate transport function of astrocytes is impaired in gamma CaMKII knockout 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 abundance of its branches, but also affects the structure and function of its peripheral nerve synapses by altering glutamate transport function.
Example 4 overexpression of γCaMKII in mouse hippocampal astrocytes improves depressive-like behavior
1. Test method
Overexpression of gamma CaMKII in the astrocytes of the hippocampus of CRS mice AAV2/8-GfaABC D-mCherry-P2A-Camk2g produced by Shanghai Bobio Co or control virus was injected into the bilateral CA1 brain regions of the hippocampus of adult mice, behavioural tests before CRS modeling were performed at the same time as the time point when the virus began to express after 3 weeks recovery of the mice, after which the control and over-expression mice were subjected to a chronic tethering process for three weeks, and after modeling was completed, the extent of change in the mouse depression-like behaviour was analyzed by forced swimming behavioural test.
2. Experimental results
As shown in a-B in fig. 7, overexpression of γcamkii in hippocampal astrocytes significantly increased astrocyte activity in CRS mice. Further analysis of the behavioral test results before and after modeling revealed that the control mice exhibited a pronounced depression-like phenotype after 3 weeks of binding stress, whereas the overexpressed mice did not significantly change depression-like behavior before and after modeling (C in fig. 7). Given that astrocyte function is closely related to depression-like behavior, this suggests that overexpression of γcamkii in hippocampal tissue can inhibit depressed mood in mice during stress-binding modeling by enhancing astrocyte function. This finding provides a potential target for developing drugs specifically preventing depression.
Example 5 inhibition of the stress hormone-glucocorticoid signalling pathway target modulation of gamma CaMKII protein levels in hippocampal astrocytes
1. Test method
(1) Acute stress stimulus when acute restraint stress stimulus (ARS) is carried out, adult mice are placed in a 50mL centrifuge tube with small holes, so that the mice cannot escape, each tube is separated by a card, and each mouse cannot see a companion, and restraint time is 2 hours. In conducting plantar Electrical Stimulation (ES) experiments, mice will be placed in a conditioned reflex chamber for 2 minutes for environmental familiarity, followed by a2 second foot shock with a current intensity of 0.7 mA, and allowed to continue to reside in the conditioned reflex chamber for 2 minutes after the shock. The mice were then returned to the feeder cages. Both acute stress stimulations were started 3 hours after mifepristone drug injection, mice were harvested 2 hours after stimulation and subsequent brain slice sections and immunostaining experiments were performed.
(2) Mifepristone (RU 486) pharmaceutical effect powder of glucocorticoid receptor inhibitor mifepristone (RU 486), purchased from Shanghai aladine Biotechnology, inc., will be dissolved in 5% DMSO corn oil and RU486 will be introduced into mice (20 mg/Kg) in intraperitoneal injection to target modulation of glucocorticoid signaling pathway prior to initiation of acute stress stimulation.
2. Experimental results
As shown in a-C in fig. 8, the protein level of γcamkii in hippocampal astrocytes and its cellular activity was more limited by ARS after pre-inhibiting glucocorticoid signaling with RU486, which remained in a relatively normal state, compared to the control group which showed lower protein levels of γcamkii after acute tethering (ARS). In addition, mice pre-intraperitoneally injected with RU486 also maintained higher levels of gamma CaMKII and GFAP protein (D-F in fig. 8) when they underwent another stress stimulus, plantar Electrical Stimulation (ES). These results demonstrate that the down-regulation of protein levels of γcamkii and its cellular activity in hippocampal astrocytes in mice exhibiting depression-like behavior is most likely due to the regulation by stress hormones. Based on the frequent observation of hypothalamic-pituitary-adrenal axis imbalance and abnormal glucocorticoid release in depressed patients, mice can have some degree of antidepressant effect when they are treated with mifepristone (RU 486), which suggests that the gamma CaMKII protein regulation process in astrocytes is highly likely an important target for RU486 to treat depression. Thus, gamma CaMKII protein, which is the target protein for depression, is an important complementary mechanism in the pathogenesis of depression by the regulatory process of glucocorticoids.
Through the animal experiments, the overexpression of gamma CaMKII protein in hippocampal astrogliosis can inhibit the depression-like phenotype in mice with chronic constraint stress model. The discovery provides a new thought for exploring and improving the cerebral depression progress and provides support for the health and social functions of depression patients. In conclusion, the invention further discusses the potential application of the interaction between astrocytes and nerve synapses based on gamma CaMKII protein in improving the brain emotion function, and provides a certain theoretical and experimental basis for developing related medicaments or treatment means for preventing depression in the future.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Those skilled in the art will appreciate that many modifications, adaptations and variations of the present invention can be made using the techniques disclosed herein without departing from the spirit and scope of the invention, and that many modifications, adaptations and variations of the present invention are within the scope of the invention as defined by the appended claims.