KR20200012258A - Screening method of antidepressant by evaluation of the activity of mossy cells - Google Patents

Abstract

The present invention relates to a method for screening an antidepressant through evaluation of the activity of mossy cells. The screening method according to one aspect evaluates and compares a neural activity level of mossy cells based on a newly investigated mechanism of an antidepressant. Therefore, a new antidepressant can be simply and effectively discovered.

Description

Screening method of antidepressant by evaluating activity of hairy cells {Screening method of antidepressant by evaluation of the activity of mossy cells}

It relates to a method for screening antidepressants through evaluation of activity of hairy cells.

Depression is an emotional pathology that occurs irrespective of an objective situation, and the entire life of the patient is covered with a depressed mood, an interest decreases and becomes anhedonia, which leads to declining mental movements, feelings of depression, and despair. It is a mental illness. In addition, due to decreased appetite, insomnia, constipation, decreased libido, reduced immune function, the disease is more susceptible to the disease, and also shows various physical symptoms. The most promising hypothesis in the art is that the lack of monoamine neurotransmitters serotonin, norepinephrin, dopamine, etc., in the central nervous system synapse causes depression. It is considered.

Accordingly, most antidepressants have a pharmacological action that increases the concentration of neurotransmitters in serotonin or noradrenaline synapses. More specifically, antidepressants are largely tricyclic antidepressants (TCA), monoamine oxidase inhibitors (MAOIs), or selective serotonin reuptake inhibitors, depending on the mechanism of increasing neurotransmitter concentrations. inhibitors (SSRI) are commonly used. Monoamine oxidase inhibitors, such as phenelzine, have not been used recently because of the serious side effects of heart disease. Tricyclic antidepressants, such as imipramine, also have significant anticholinergic side effects, sedative effects, and cardiovascular side effects. It remains a problem. Therefore, the recent focus on the development of antidepressant drugs using selective serotonin (5-HT) reuptake inhibitors as an antidepressant with less such side effects, the representative drugs are fluoxetine (product name; Puroxak), paroxetine (paroxetine) , Product name; serozat), sertraline (product name; zoloput) and the like have been developed and widely recognized clinically.

However, these serotonin reuptake inhibitors are effective only in about 60% of patients in the entire dosing group, and do not show therapeutic efficacy in the remaining 30-40% of patients, and are spread throughout the central nervous system. Due to the widespread effects of serotonergic synapse, there are problems associated with unnecessary side effects in some patients. In addition, it takes less than an hour to increase serotonin levels in cerebral synapses in serotonin-based drug reactions, but at least three weeks of long-term drug until they show therapeutic efficacy leading to mood elevation. There is a limit in that it is necessary to take.

Under such technical background, there is a need for a study on the mechanism of occurrence of depression and the mechanism of action of antidepressants, which are therapeutic agents for depression, and the development of new antidepressants based on them (Korea Patent Publication 10-2018-0024527).

One aspect includes measuring neuronal activity levels of parental cells in contact with the test agent; And comparing the measured neuronal activity level of the parental cells with the neuronal activity level of the control group not in contact with the test substance.

One aspect includes measuring neuronal activity levels of parental cells in contact with the test agent; And comparing the measured neuronal activity level of the parental cells with the neuronal activity level of a control group not in contact with the test agent.

As used herein, the term "antidepressant" is a substance that treats or alleviates depressive symptoms such as depressed mood, motivation, interest, decreased mental activity, irritability, decreased appetite, insomnia, and persistent sadness and anxiety. Antidepressants, monoamine oxidase inhibitors, or selective serotonin reuptake inhibitors, and the like, and may include, for example, selective celltonin reuptake inhibitors of antidepressants, or therapeutic aids that enhance the therapeutic efficacy of the antidepressant. It may include all.

According to one embodiment, the change of neuronal activity in the dentate gyrus affects the neurodegenerative and behavioral responsiveness of antidepressant administration, and the neuronal activity of these parental cells is closely related to the p11 / AnxA2 / SMARCA3 complex formation. It was confirmed. Thus, increased neural activity of ramie cells, increased levels of p11 / AnxA2 / SMARCA3 complexes in ramie cells, or increased levels of binding between p11 / AnxA2 complexes and SMARCA3 in ramie cells assess the reactivity or therapeutic efficacy of antidepressant candidates. It can be used as an indicator.

In one embodiment, measuring the neuronal activity level of the parental cells is based on the detection of neuronal activity specific markers (eg, the number of BrdU + proliferating cells), electrophysiological analysis, and combinations thereof. It may be performed by, for example, by measuring the spontaneous firing rate (firing rate). Accordingly, the method may further comprise determining the test substance as an antidepressant when the neuronal activity level of the parental cells in contact with the test substance is increased compared to the control. Changes in these levels indicate that the activity level is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% and 1000% or more.

In another embodiment, measuring the neuronal activity level of the parental cells may be performed by measuring the level of p11 / AnxA2 / SMARCA3 complex in the parental cells or measuring the level of binding between the p11 / AnxA2 complex and SMARCA3. Measurements (eg, immunoprecipitation assays) can be made by methods well known in the art. Accordingly, the method comprises the steps of selecting a substance that promotes the formation of the p11 / AnxA2 / SMARCA3 complex in the parental cells, that is, the formation of the p11 / AnxA2 / SMARCA3 complex or the p11 / AnxA2 complex in the parental cells, compared to the control group. The method may further include determining an agent for increasing binding between SMARCA3 as an antidepressant. Changes in these levels indicate that the activity level is 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% and 1000% or more.

On the other hand, measuring the neuronal activity level of the parental cells may be assessed at the cellular level, or may be evaluated at the individual level in a depressed animal model.

In the screening method, the test substance is a group consisting of low molecular weight compounds, antibodies, antisense nucleotides, short interfering RNA, short hairpin RNA, nucleic acids, proteins, peptides, other extracts, and natural products. It may be any one selected from.

The test substance, hippocampal hairy cells, and the like may be attached with a detectable label. The label may be linked inside the nucleotide sequence using genetic recombination techniques. The binding level may be measured by two-hybrid method, co-immunoprecipitation assay, co-localization assay, scintillation proximity assay (SPA), depending on the type or method of labeling. ), UV or chemical crosslinking methods, bimolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), fluorescence polarization assays (FPA) and in vitro It can be performed by an in vitro pull-down assay or a combination thereof.

The screening method according to one aspect is based on the core mechanism of action of the newly identified antidepressant, and by evaluating and comparing the neuronal activity level of hairy cells, it is possible to find a new antidepressant more simply and effectively.

Figure 1 confirms the effect of the genetic deletion of p11 or Smarca3 on the behavioral response by chronic administration of SSRI in hippocampal ramie cells. (A) Schematic diagram showing the experimental procedure for identifying ramie cell specific Cre-recombination in hippocampus of D2-Cre transgenic mice; (B) Co-localization of Cre-dependent reporter (mTomato, red) and the cell line marker CRT (green, filled arrow); (C) Schematic diagram showing the process of genetically deleting p11 gene in ramie cells using the D2-Cre driver line; (D) NFS test on p11 knockout mice (hereinafter, herein, SAL refers to saline administration group, FLX refers to fluoxetine administration group); (E) the level of hunger was assessed by food intake (HCF) testing in breeding cages in the control and p11 cKO groups; (F) Schematic diagram showing the process of genetic deletion of Smarca3 gene in ramie cells using the D2-Cre driver line; (G) NFS testing of Smarca3 knockout mice; (H) Evaluation of hunger levels through food intake (HCF) testing in breeding cages in the control and Smarca3cKO groups; (I) Schematic diagram showing the experimental procedure for identifying ramie cell specific Cre-recombination in hippocampus of MC-Cre transgenic mice; (J) co-localization of Cre-dependent reporter (mTomato, red) and CRT (green, filled arrow), the ramie cell marker; (K) Schematic diagram showing the process of genetically deleting the Smarca3 gene in ramie cells using the MC-Cre driver line; (L) NFS testing of Smarca3 knockout mice; And (M) a result of evaluating the level of hunger through a food intake (HCF) test in a breeding cage for the same set of animals.
Figure 2 confirms the effect of the destruction of the cell line specific p11 / AnxA2 / SMARCA3 complex on the neurogenic and behavioral response by chronic treatment of SSRI. (A) Schematic diagram showing p11 / AnxA2 / SMARCA3 complex-inhibiting peptide (PASIP) -AcGFP1 fusion protein (PASIP-AcGFP1) and control construct (control AcGFP1); (B) Schematic diagram showing the process by which PASIP-AcGFP1 blocks the functional assembly of the p11 / AnxA2 / SMARCA3 heterohexamer complex; (C) the destruction of the p11 / AnxA2 / SMARCA3 complex by PASIP-AcGFP1 through in vitro pull-down analysis; (D) Schematic diagram showing stereotactic injection of recombinant AAV expressing the control AcGFP1 or PASIP-AcGFP1 into the tooth gyrus of MC-Cre transgenic mice; And (E) representative immune labeling images showing parental cell specific expression of control AcGFP1 or PASIP-AcGFP1 constructs.
Figure 3 confirms the ramie cell specific expression of the control AcGFP or PASIP-AcGFP1 constructs along the rostro-caudal axis of the hippocampus. (A) Schematic diagram showing double-flox Cre-dependent AAV vectors expressing control AcGFP1 and PASIP-AcGFP1 under EF-1α promoter control; (B) Schematic diagram of stereotactic injection of Cre-dependent AAV vectors expressing control AcGFP or PASIP-AcGFP1 constructs along the rostro-caudal axis of the hippocampus (Rostral: AP -2.1 mm, ML ± 1.4 mm, DV -1.95) mm.Caudal: AP -3.3mm, ML ± 2.7mm, DV -3.6mm); (C) representative images showing longitudinal expression of control AcGFP or PASIP-AcGFP1 along the rostro-caudal axis of the hippocampus of MC-Cre mice; (D) Moshi cell-specific expression of the control AcGFP or PASIP-AcGFP1 constructs was confirmed by immunostaining with GluR2 / 3 (moscle cell markers), PV (basket cell markers), NPY (HIPP cell markers).
4 confirms the effect of ramie cell-specific inhibition of p11 / AnxA2 / SMARCA3 complex on basal motility and anxiety-related behavior. (A) Schematic diagram schematically showing the experimental procedure; (B) representative images showing results immunostained with α-BrdU; (C) quantifying and comparing the number of BrdU-positive cells in the subgranular region (SGZ); (D) NFS test results; (E) Evaluation of the level of hunger through food intake (HCF) testing in breeding cages; (F) findings of anxiety-related behavior in light / dark box tests; And (G) findings of despair or depression-like behavior in the tail hanging test.
Figure 5 confirms the effect of the p11 / AnxA2 / SMAR complex on the excitability of the gynecological endothelial cells. (A) Schematic diagram showing the AcGFP1-labeling process of ramie cells for electrophysiological recordings; (B) representative fluorescence images of ramie cells labeled with AcGFP-1 in hippocampal sections in AAV-injected mice; (CF) Representative traces and minimal-stimulation of the spontaneous excitation rate of ramie cells by chronic FLX administration as a result of recording electrophysiological activity on AcGFP1-labeled ramie cells in saline or fluoxetine-treated tooth gyrus. Maximal plot (3 mice per n = 9 cells / group, scale bar: 1 sec, Student's t-test, ^* p <0.05); Representative traces and minimum-maximal plots (EF) showing spontaneous excitation rate of ramie cells by acute FLX administration (3 mice per n = 9 cells / group, scale bar: 1 second, Student's t-test, ^* p <0.05) ; (GH) Representative traces and minimum-maximal plots (n) showing spontaneous excitatory rates of ramie cells in the tooth gyrus of control and p11 cKO mice as a result of reduced electrophysiological activity in p11 cKO mice. = 3 mice for 10 cells / control, n = 3 mice for 6 cells / p11 cKO, scale bar: 200 ms, Student's t-test. ^* P <0.05); And (IJ) representative traces and minimum-maximal plots (n = 9) showing reduced electrophysiological activity of ramie cells in Smarca3 KO mice, showing spontaneous excitation rates of ramie cells in the tooth gyrus of control and Smarca3 cKO mice. 3 mice per cell / group, scale bar: 200 ms, Student's t-test, ^* p <0.05).
6 shows a series of recombinant constructs that inhibit the p11 / AnxA2 / SMARCA3 complex. (A) Schematic diagram showing a series of recombinant constructs, including control AcGFP1 (a), inhibitory recombinant construct (b, PASIP-AcGFP) and nuclear-targeting inhibitory recombinant construct (c, PASIP-AcGFP1-NLS); And (B) a double-flox AAV vector expressing control AcGFP1 (a), PASIP-AcGFP1 (b) or PASIP-AcGFP1-NLS (c) under EF-1α control.
Figure 7 confirms the effect of p11 / AnxA2 / SMAR complex on the excitability of the gynecological endothelial cells. (A) Immunofluorescence image showing Moshi cell specific expression in the tooth gyrus of the AAV-injected mouse of FIG. 6; (B) immunofluorescence images showing expression patterns of the AAV constructs in Hek 293 cells; And (CD) representative trace and minimum-maximal plots showing the spontaneous excitation rate of ramie cells expressing each construct, showing reduced electrophysiological activity of the ramie cells in which the p11 / AnxA2 / SMARCA3 complex was destroyed (n = 8 cells / 3 mice), scale bar: 200 ms ,. One-way ANOVA. F (2, 25) = 12.25, ^** P <0.01, ^*** p <0.005).
Figure 8 confirms the effect of Gi-DREADD-inhibition of ramie cells on the neurogenic and behavioral response by chronic treatment of SSRI. (A) Schematic diagram showing the process of silencing the activity of hairy cells through the Gi-DREADD system; (B) representative images showing ramie cell specific delivery through the Gi-DREADD system; (C) Representative traces showing spontaneous excitation rate of ramie cells expressing control mCherry or Gi-DREADD-mCherry (Gi-DREADD) before or after application of Gi-DREADD system (n = 4. Scale bar: 200 ms, Paired student's t-test. * P <0.05); (D) representative dot plot results; (E) Schematic diagram schematically showing the experimental procedure; (F) visualization of α-BrdU immunostained proliferating neural stem cells (BrdU, green); (G) Quantification of BrdU + cells (filled arrows) at subgranular site; (H) Immunostaining of immature neurons with α-doublecortin antibody after mitosis (DCX, green); And (I) quantifying the relative immunofluorescence intensity (Rel. DCX IF) of DCX subgranular and in granule regions using Image J software.
Figure 9 confirms the ramie cell specific expression of the control or Gi-DREADD constructs along the rostral-caudal axis of the hippocampus. (A) diagram showing a double-Flox Cre-dependent AAV vector expressing a control or Gi-DREADD construct under control of human Synapsin I (hSyn); (B) Schematic diagram showing the process of stereotactic injection of Cre-dependent AAV expressing control or Gi-DREADD along the rostral-caudal axis of hippocampus (Rostral: AP -2.1mm, ML ± 1.4mm, DV -1.95mm. Caudal: AP -3.3mm, ML ± 2.7mm, DV -3.6mm); And longitudinal expression of control or Gi-DREADD along the rostral-caudal axis of the hippocampus of D2-Cre mice.
Figure 10 confirms the effect of Gi-DREADD inhibition of ramie cells on the behavioral response by chronic administration of SSRI. (A) the results of NFS testing after inhibiting the activity of ramie cells via Gi-DREADD; (B) assessed the level of hunger through food intake (HCF) testing in breeding cages in the control and Gi-DREADD groups; And (C) findings of despair or depression-like behavior in the tail hanging test.
FIG. 11 is a diagram schematically illustrating the effect of changes in the activity of portal cells on neurodevelopmental and behavioral responses to chronic SSRI administration.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Experimental Materials and Experiment Preparation

1-1. Animal breeding

Progeny for each line was produced using in vitro fertilization (IVF) and embryo transplantation (ET) techniques to produce a large number of co-age animal models sufficient for behavioral tests and other animal experiments. All animal experiments used littermates that matched age (10-15 weeks) and gender (male). BAC- [Drd2] -Cre Tg mice (GENSAT, Clone # ER44) and MC-Cre ([ Calcrl ] -Cre mice (JAX stock # 023014) were crossed with C57BL / 6 mice (Taconics) to form hemizygotes. P11- Flox mice and Smarca3- Flox mice were prepared by introducing the relevant genes into flanked loxP sites, as described in the previous study All experimental animals were subjected to room temperature conditions (22 ± 1 ° C.). , 12-hour light / dark cycles, were provided with a standard diet, water was freely consumed, and male mice were used in all experiments.

1-2. medication

For chronic drug administration, 2-5 mice per cage were housed. Fluoxetine hydrochloride (Spectrum Chemicals, USA) was administered by intraperitoneal injection (10 mg kg ⁻¹ day ⁻¹ ) daily for three weeks. The drug was prepared by dissolving in dimethyl sulfoxide (50%) and then diluting with saline. For the control group, the drug-free vehicle solution (0.9% saline) was administered daily. For Gi-DREADD mouse experiments, Clozapine-N-oxide (CNO); Sigma-Aldrich, USA) was dissolved directly in 0.9% saline solution. The dose of CNO was 0.3 mg / kg and two hours before each behavioral test, an intraperitoneal injection of a solution of volume of 100 μl / 10 g per body weight.

1-3. Construction of Plasmid Structures

The PASIP (p11 / AnxA2 / SMARCA3 complex inhibitory peptide) sequence was derived from the binding site between AHNAK1 (amino acid, 5654-5671) and p11 / AxnA2 complex. We then designed a series of PASIP constructs as follows: 1) Control AcGFP1 (pAAV-Ef1α-DIO-AcGFP1-myc.hGH), 2) PASIP-AcGFP1c (pAAV-Ef1α-DIO-PASIP-AcGFP1- myc.hGH) and 3) PASIP-AcGFP1-NLS targeting the nucleus (pAAV-Efla-DIO-PASIP-AcGFP1-myc-C3xNLS.hGH). The coding sequence of the PASIP construct was prepared using standard gene synthesis methods (GENEWIZ, USA) and the pAAV-Ef1α-DIO-EYFP vector (Addgene, # 29056) at the cleavage site recognized by two restriction enzymes (NheI, AscI). ) Was subcloned.

1-4. Immunoprecipitation of the p11 / AnxA2 / SMARCA3 Complex

HEK293 cells (CRL-1573, ATCC) were transfected with CMV-Cre plasmids and after 24 hours, the cells were infected with AAV carriers expressing the control AcGFP1 or PASIP-AcGFP1 constructs. Immunoprecipitation was performed with α-SMARCA3 antibodies (rabbit polyclonal antibodies made with immunized peptides). Immunoblotting was performed using the following antibodies according to standard protocols: α-p11 (mouse monoclonal, 1: 1000, BD bioscience), α-p11 (goat polyclonal, 1: 200, R & D systems ), α-SMARCA3 (goat polyclonal, 1: 200, NOVUS), α-AnxA2 (mouse monoclonal, Santa Cruz Biotech.), α-GFP (goat polyclonal, Santa Cruz Biotech.).

1-5. Stereotaxic surgery

Stereotaxic injection of AAV into mice was performed on an Angle Two ^™ stereotaxic frame (Leica, Buffalo Grove, IL, USA) (Leica, Buffalo Grove, IL, USA). Prior to stereotactic infusion of AAV, mice were anesthetized by intraperitoneal injection of avertin (100 mg / kg). Cre-dependent AAVs were injected bidirectionally stereotactically into the dorsal and ventral regions of the tooth gyrus using a 10 μl Hamilton syringe (33 gauge needle; Reno, NV, USA) (coordinates for dorsal: AP -2.1ML ± 1.4DV -1.95, coordinates for ventral: AP-3.3ML ± 2.7DV-3.6). Flow rate (0.2 μl / min) was controlled by nanopump controller (WPI, US). After infusion of the virus, the needle was left in place for 5 minutes, after which the incision site was closed. Mice were placed back into the recovery breeding cage. All animals were allowed to rest for at least 2 weeks before the next experimental phase.

1-6. Behavioral testing

For all behavioral tests, subject mice were transferred to the experimental site within one hour before the test. All tests were performed by the experimenter who was not informed about the treatment and genotype, and the tests were performed in the light / dark cycle cycle.

(1) Maze Finding Test (Elevated plus maze: EPM)

The test used a labyrinth placed 50 cm high from the bottom, with two open arms and two closed arms (30 cm long and 5 cm wide) as a plus-shaped maze structure. The mouse was placed in the center of the maze and allowed to move freely along the four arms for 10 minutes. The behavior of these mice was videoed and the total time spent on the open and closed arms was recorded in Ethovision? Recorded and measured using software (Noldus, USA).

(2) Open field test (OF)

Mice were placed in the center of the open field area (40 Х 40 Х 40 cm, Plexiglas chamber). Mice were allowed to move freely in an open field for 1 hour. Automated Superflex for duration in the center and surroundings Measurements were made using software (Accuscan Instruments, Columbus, OH, USA). The measurements were automated using two rows of infrared photocells located 31 mm apart and 20 mm and 50 mm from the bottom. Interruption of the photocell beam is Superflex? Recorded using software.

(3) Light and dark box test (LD box)

Mice were placed in the open field area (40Х40Х40 cm). A black box was inserted into the open field. Mice were placed in the light compartment at the beginning of the session and allowed to move freely between the compartments for 10 minutes. The time required in each compartment and the first time to enter the cancer compartment was measured using the automated Superflex ^™ software.

(4) Novelty suppressed feeding test (NSF) in a new environment

 The test was conducted for 15 minutes with some modifications to the previous method. Twenty four hours prior to conducting a behavioral test, all food provided to the breeding cage was removed. At the end of the experiment, a single 1.8 cm square food pellet was placed on a white filter paper located in the center of the test box. The mouse was positioned near the edge of the open field and the stopwatch was immediately activated. The time until the first bite of food was recorded. Immediately after this experiment, mice were placed in breeding cages and then food intake for 10 minutes was measured.

(5) Tail suspension test (TST)

The mouse was suspended for 6 minutes through the tail of the mouse. Test sessions were videoed and immobility was recorded using Clever system's automated TST analysis software (Reston, VA, USA). The dead time for the last 4 minutes (excluding the initial 2 minutes) was calculated.

1-7. Immune tissue chemistry

Immunostaining was performed using standard free-floating method. Sections were washed three times with PBS for 10 minutes each time and incubated with 2% normal donkey serum, 0.2% bovine serum albumin, and 0.3% Triton-X100. After the blocking step, the sections were incubated overnight at 4 ° C. with the primary antibody diluted in blocking buffer and the following primary antibody was used: α-doublecortin (goat polyclonal, 1: 200, Santa Cruz Biotech. ), α-calretinin (mouse monoclonal, 1: 500, SWANT), α-parvalbumin (rabbit polyclonal, 1: 1000, SWANT), α-neuropeptide Y (rabbit polyclonal, 1: 1000, Phoenix pharmaceutical ), α-GluR2 / 3 (rabbit polyclonal, 1: 100, Millipore). After incubation for 24 hours, the sections were washed three times with PBS containing 0.2% Triton-X100 (PBS-T) for 10 minutes, which were then AlexaTM-fluor-conjugated secondary antibody (1: 400, Life technologies , USA) for 3 hours at room temperature. Sections were washed three times for 10 minutes at room temperature with PBS-T and covered with Prolong ^™ Gold, anti-fading media (Life technologies, USA).

1-8. BrdU Labeling and Neurogenesis Analysis

Before sacrificing mice, BrdU solution (200 mg / kg) was administered intraperitoneally for 3 hours. After transcardial perfusion, brain tissue was fixed overnight with 4% PFA, and then 40-μm thick coronal sections were collected along the rostro-caudal axis of the hippocampus using Cryostat (CM3050S, Leica). Sections were processed using free-floating methods. BrdU immunohistochemical staining was performed for every sixth section spanning the hippocampus. The sections were then de-incubated with 1M HCl for 30 minutes at 45 ° C. to denature the DNA and neutralized by washing the sections three times with 0.1M PBS. Immunohistochemistry was performed using α-BrdU (let polyclonal, 1: 200, Abcam, Cambridge, MA, USA) and Alexa ^™ -fluor-conjugated secondary antibody (1: 400, Life technologies, USA) It became. The experimenter who was not informed of the slide code, in the total 12 sections from individual mice, the granule cell layer (GCL) and subgranular zone (SGZ) of the dental gyrus (DG) The number of BrdU-labeled cells located at was counted. The total number of BrdU-labeled cells per section was measured and multiplied by 6 to yield the total number of cells per tooth gyrus.

1-9. Electrophysiological Evaluation of Hippocampal Moshi Cells

(1) Fluorescent labeling of ramie cells

To label and visualize the hairy cells in the hippocampus slices, recombinant AAV vectors (AAV2.EF1α.DIO.AcGFP1-myc) were injected bilaterally into the ventral hippocampal dental gate of MC-Cre ([Calcrl] -Cre) Tg mice. AcGFP1 was selectively expressed in ramie cells. While recording the electrophysiological evaluation, changes in morphology, membrane capacitance, and electrophysiological properties for the hairy cells were further confirmed.

(2) preparation of slices

Male mice aged 12-15 weeks were euthanized with CO ₂ . The brains of the mice were then harvested and cutting solutions containing N-Methyl-D-glucamine on cold ice (93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH ₂ PO ₄ , 30 mM NaHCO ₃ , 25 mM glucose, 20 mM HEPES, 5 mM) sodium ascorbate, 3 mM sodium pyruvate, 2 mM thiourea, 0.5 mM CaCl ₂ , 10 mM MgSO 4, pH 7.4, 295-305 mM mOsm). Brain tissue slices (400 μm thick) containing ventral hippocampus were prepared using VT1000 S Vibratome (Leica Microsystems Inc., Buffalo Grove, IL, USA). After cutting, the slices were recovered in cutting solution at 37 ° C. for 15 minutes and then transferred to the recording solution at room temperature for a minimum of 1 hour before recording the electrophysiological evaluation.

(3) electrophysiological records

Electrophysiological recordings were performed as previously described. During recording, brain tissue slices were placed in a perfusion chamber attached to a fixed stage of an upright microscope BX51WI (Olympus, Tokyo, Japen), which was immersed in a continuously flowing, oxidized recording solution. The components contained in the solution are as follows: 125 mM NaCl, 25 mM NaHCO ₃ , 25 mM glucose, 2.5 mM KCl, 1.25 mM NaH ₂ PO ₄ , 2 mM CaCl ₂ and 1 mM MgCl ₂ , pH 7.4, 295-305 mM mOsm. Neurons were visualized on 40 × objective lenses using near infrared (IR). Electrophysiological recordings were made using a Multiclamp 700B / Digidata 1440A system (Molecular Devices, Sunnyvale, CA, USA). The patch electrode was filled with internal solution (126 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 10 mM creatine phosphate, 4 mM ATP and 0.3 mM GTP, pH 7.3, 290 mM mOsmol). Whole cell patch-clamp recordings were used to record the spontaneous action potentials of the hairy cells in the hippocampus slides, all recordings were performed at 37 ° C. In chemoelectric experiments, action potentials of ramie cells were recorded for 5 minutes according to criteria. CNO (1 mM) was also perfused onto brain tissue slices to confirm the effect of Gi-DREADD. Experimental data were analyzed by pClamp10 software (Molecular Devices), and GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA).

1-10. Statistical analysis

All experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism Version 7.0a. Comparison between the two groups was performed by a two-tailed, unpaired Student's t test. Comparisons between the multiple groups were evaluated using unidirectional or bidirectional ANOVA, followed by post hoc Bonferroni tests. Significant levels are as follows: ^* p <0.05, ^** p <0.01, ^*** p <0.001.

Example 2 Effects of Genetic Deletion of p11 or Smarca3 on Hippocampal Molecular Cells on Behavioral Responses by Chronic Administration of SSRI

In this example, the specific relevance of the p11 / AnxA2 / SMARCA3 complex to the action of antidepressants was determined. First, we prepared conditional KO mice that specifically lacked p11 or Smarca3 in ramie cells to specifically identify the role of three-dimensional complexes in specific cell types. In relation to the action of antidepressants, in order to investigate the specific functionality of p11 and / or Smarca3 against parental cells, the transgenic Cre driver line was used to target hippocampal ramie cells. That is, the dopamine D2 receptor promoter specific to the ramie cells and the D2-Cre ([ Drd2 ] -Cre) driver mouse line including the Cre recombinase and the mTomato reporter line were crossed to dopamine in a chylo cell-specific manner in the tooth gyrus. Expression of the D2 receptor promoter driven Cre recombinase was verified (see FIGS. 1A and 1B). Thereafter, they were crossed with p11 or Smarca3 flox conditional lines to induce specific deletion of p11 or Smarca3 in ramie cells (see FIGS. 1C and 1F). After chronic administration of SSRI to the p11 or Smarca3 conditional KO (cKO) lines, an NFS test was performed to investigate the presence of a depression-like condition. As a result, in the control group that did not knock out the related gene, the effect of the administration of the SSRI decreased the time to access to the food pellet (p11 ^{(f / f)} : SAL vs FLX, 351 ± 38 vs 165). ± 24, p <0.001; and Smarca3 ^{(f / f)} : SAL vs FLX, 257 ± 47 vs 104 ± 12, p <0.01). However, p11 conditional knockout mice (p11 ^{(f / f); D2-Cre} : SAL vs FLX, 364 ± 46 vs 296 ± 58; p = 0.37), or Smarca3 conditional knockout mice (Smarca3 ^{(f / f); D2-Cre} : SAL vs FLX, 192 ± 15 vs 232 ± 32, p = 0.24) did not show this reduction effect (see FIGS. 1D and 1G). In addition, after returning to the breeding cage, it was confirmed that there is no change in their food intake (see FIGS. 1E and 1H).

In addition, the above effects by SMARCA3 gene deletion in ramie cells were further validated using another kind of ramie cell specific Cre mouse line, MC-Cre ([ Calcrl ] -Cre). The MC-Cre line is very specific to the hippocampus, but exhibits a wide range of activity in both ramie cells and pyramidal cells in the CA3 region, which are present near the gates of the dental gyrus. We crossed the Smar3 flox conditional mouse (smarca3 ^{(f / f)} ) and MC-Cre mice to delete the Smarca3 gene (see FIG. 1K), and chronically administered SSRI, followed by NSF testing. As a result, in the control group that did not knock out related genes, the effect of the administration of the SSRI reduced the time to access to food pellets (Smarca3 ^{(f / f)} : SAL vs FLX, 338 ± 20 vs 229). ± 40; p <0.05). However, Smarca3 conditional knockout mice (Smarca3 ^{(f / f); MC-Cre} : SAL vs FLX, 405 ± 88 vs 404 ± 69, p = 0.99, or Smarca3 ^{(f / f); D2-Cre} : SAL vs FLX) , 192 ± 15 vs 232 ± 32, p = 0.24), this reduction effect was also not observed (see FIGS. 1L and 1M).

These experimental results indicate that selective deletion of p11 or Smarca3 in the parental cells of the tooth gyrus affects the antidepressant response. In other words, p11 and Smarca3 in parental cells are directly involved in the behavioral response to chronic antidepressant therapy.

Example 3 Effect of Cell-Specific Inhibition of p11 / AnxA2 / SMARCA3 Complex on Neurogenic and Behavioral Responses by Chronic Administration of SSRI

In Example 2, the effect of the genetic deletion of p11 or Smarca3 on the antidepressant response was specifically confirmed using a ramie cell-specific KO mouse. However, the transformative approach described above has the effect of p11 or Smarca3 in cKO mice. It cannot be excluded whether it is a behavioral change due to a developmental defect or off-target deletion of a gene. Therefore, we further investigated the effects of inhibition of p11 / AnxA2 / SMARCA3 complexes specific to ramie cells on adult neuronal and behavioral effects in adult mice. To this end, first, a recombinant construct that inhibits the assembly of the p11 / AnxA2 / SMARCA3 complex was derived. p11 / AnxA2 heterotetramers have been shown to form stable heterohexamers with SMARCA3 or AHNAK1 according to very similar recognition principles (not shown). In addition, short peptides from the binding sites of SMARCA3 or AHNAK1 are located in the hydrophobic binding pockets generated on the p11 / AnxA2 heterotetramer complex surface, thereby forming a stable complex with the p11 / AnxA2 heterotetramer. Therefore, based on the information on the simultaneous crystallization structure of the three-dimensional complex, the recombinant inhibitory structure of the p11 / AnxA2 / SMARCA3 complex (p11 / AnxA2 / SMARCA3 complex inhibitory peptide-AcGFP1 fusion construct; PASIP-AcGFP) was determined to be inactive. AcGFP1) (see Figure 2A). The inventors estimated that such recombinant inhibitory constructs exist competitively with endogenous SMARCA3 on the binding pocket, thereby blocking the functional assembly of the activated three-dimensional complex, p11 / AnxA2 / SMARCA3 heterohexamer (see FIG. 2B). In fact, in vitro immunoprecipitation analysis showed that the recombinant PASIP-AcGFP1 construct could block the assembly of the p11 / AnxA2 / SMARCA3 complex (see FIG. 2C).

In addition, Cre-dependent AAV was prepared to deliver the PASIP-AcGFP1 construct to hippocampal ramie cells. The Cre-dependent AAV was injected locally into the upper and posterior gate regions of MC-Cre transgenic mice (see FIGS. 2D, 3A, and 3B). Since MC-Cre mice display Cre-recombinant constructs in both dental ramie cells and some pyramid subpopulations in the CA3 region, the recombinant AAV solution was delivered to the center of the portal site to prevent unwanted diffusion of viral particles. . In histological analysis, AcGFP1 signal was found only in the gate and endothelial layer localized to neurons and axon fibers, not in the CA3 region where pyramidal cells are present (see FIG. 3C). In other words, these results show site specific delivery of the hippocampus. In addition, AcGFP1-fusion proteins (control AcGFP1, PASIP-AcGFP1) were specifically expressed in ramie cells, not in other GABA-like intermediate neurons, including PV + basket cells, and NPY + HIPP cells (FIG. 2E, and This result is indicative of cell specific expression in the tooth gyrus.

After blocking the formation of p11 / AnxA2 / SMARCA3 via moiety cell specific expression of the PASIP-AcGFP1 construct, we investigated the neurodegenerative and behavioral changes following chronic administration of SSRI (see FIG. 4A). In response to the treatment of chronic antidepressants, adult neurogenesis in the tooth gyrus is known. Thus, in control and PASIP-AcGFP1 mice, the proliferative activity of neural precursors induced by chronically administered fluoxetine was evaluated. Significant increases in BrdU + proliferating cells were observed in control mice chronically treated with fluoxetine (SAL vs FLX, 173 ± 16 vs 290 ± 43; p <0.05), but this effect was not observed in PASIP-AcGFP1 mice (SAL). vs FLX, 119 ± 27 vs 129 ± 13; p = 0.73) (see FIGS. 4B and 4C).

 Next, we investigated the functional role of the p11 / AnxA2 / SMARCA3 complex in ramie cells related to the behavioral changes induced by SSRI administration. After chronic administration of fluoxetine, the time to food intake decreased in control AcGFP1 mice (SAL vs FLX, 296 ± 48 vs 147 ± 22; p <0.01), but not in PASIP-AcGFP1 mice (SAL vs FLX, 304 ± 51 vs 293 ± 54; p = 0.88) (see FIG. 4D). Fluoxetine treatment or inhibition of the p11 / AnxA2 / SMARCA3 complex did not have a significant effect on food intake in the breeding cages, thus indicating that these behavioral changes were not the result of differences in levels of hunger among the comparison groups. (See Figure 4E).

 In addition, control AcGFP1 mice and PASIP-AcGFP1 showed significant differences in the light / dark box test (Control AcGFP1 vs PASIP-AcGFP1, 154 ± 16 vs 94 ± 13; p <0.01) (see FIG. 4F), NSF test As with the antidepressant-induced behavioral changes observed in, the inhibition of the p11 / AnxA2 / SMARCA3 complex also reduced the effects of fluoxetine administration in the tail suspension test (TST) (see FIG. 4G).

Taken together, these results indicate that the p11 / AnxA2 / SMARCA3 complex formed in hairy cells plays an important role in behavioral changes after chronic treatment of SSRIs.

Example 4 Effect of Cell Specific Inhibition of p11 / AnxA2 / SMARCA3 Complex on Neural Activity of Mosh Cells

In this example, the effect of the administration of antidepressant on the neuronal activity of hairy cells was investigated. Moshi cells are neurons that spontaneously regulate neuronal excitability at the gate, and their activity regulates the neural circuits and functions of the tooth gyrus. For electrophysiological recordings of ramie cells, Cre-dependent AcGFP1 AAVs were injected into ramie cell-specific Cre (MC-Cre) mice to label the ramie cells with the fluorescent protein AcGFP1 (see FIG. 5A). As a result, it was confirmed that AcGFP1 is limited to the expression of hairy cells in the dentate gyrus (see FIG. 5B). In addition, changes in morphology, tonic excitation pattern, and membrane capacity (50-100 pF) for these ramie cells were further confirmed. In electrophysiological records, chronic treatment of fluoxetine significantly improved the spontaneous firing rate of ramie cells (control: 2.36 ± 0.41 Hz, fluoxetine: 4.19 ± 0.92 Hz) (FIGS. 5C and 5D). There was no effect on acute treatment with the drug (control: ^*** Hz, Fluoxetine: ^** Hz) (see FIGS. 5E, 5F).

In addition, by measuring the spontaneous action potential frequency in ramie cells, it was confirmed whether p11 and SMARCA3 regulated ramie cell activity. As a result, the rate of spontaneous excitatory regulation of ramie cells was significantly reduced in p11 cKO mice (control: 3.37 ± 0.63 Hz, p11 cKO: 0.65 ± 0.10 Hz) (FIG. 3G and 3H), and the resting membrane of ramie cells. The translocation was also more hyperpolarized in p11 cKO mice (control: -47.70 ± 2.11 mV, p11 cKO: 56.01 ± 0.63 mV). Likewise, the spontaneous excitatory regulation rate of ramie cells was significantly reduced in Smarca3 cKO mice (control: 3.67 ± 0.48 Hz, Smarca3 cKO: 1.75 ± 0.33 Hz) (see FIGS. 5I and 5J). These experimental results suggest that p11 and SMARCA3 play an important role in regulating neuronal activity of hairy cells.

In addition, the effect of cell-specific inhibition of p11 / AnxA2 / SMARCA3 complex by expression of PASIP-AcGFP1 construct on neuronal activity of hairy cells was further investigated. According to previous studies, the p11 / AnxA2 / SMARCA3 complex targets nuclear substrates to regulate gene transcription. Thus, PASIP-AcGFP1-NLS constructs were specifically targeted to the nuclei of ramie cells via additional nuclear localization signals (NLS) (see FIG. 6A). Cre-dependent viral vectors were prepared comprising the controls AcGFP1, PASIP-AcGFP1, and PASIP-AcGFP1-NLS (see FIG. 6B). Cells were then co-transfected into cultured Hek cells and subjected to stereotyped injection into parental cell-specific Cre (MC-Cre) transgenic mice, to cell-type specific expression of PASIP-AcGFP1 construct and PASIP-AcGFP1-. Nuclear specific expression of NLS constructs was verified (see FIGS. 7A and 7B). In addition, the excitatory regulation rate of ramie cells was significantly reduced compared to the control AcGFP1 by PASIP-AcGFP1 or PASIP-AcGFP1-NLS expression (control: 3.58 ± 0.56 Hz, PASIP-AcGFP1: 0.64 ± 0.10 Hz, PASIP- AcGFP1-NLS: 1.39 ± 0.36 Hz) (see FIGS. 7C and 7D), as above, resting membrane translocation was more polarized by PASIP-AcGFP1 or PASIP-AcGFP1-NLS in control cells (control: -47.33 ± 1.77). mV, PASIP-AcGFP1: -54.33 ± 1.32 mV, PASIP-AcGFP1-NLS: -56.30 ± 1.20 mV).

Taken together, the p11 / AnxA2 / SMARCA3 complex is responsible for regulating the activity of ramie cells, thus indicating that altered activity of ramie cells may affect the reactivity or action of antidepressants.

Example 5 Effect of Selective Inhibition of Ramie Cells on Antidepressant Action of Hippocampus

In this example, the effects of selective inhibition of hairy cells on other neurodegenerative and behavioral effects on chronic treatment of antidepressants were investigated. In order to modulate the activity of parental cells, we applied the DREADD technique (Designer Receptors Exclusively Activated by Designer Drug). To introduce the Gi-DREADD system into ramie cells, viral vectors expressing the control mcherry or hM4D-Gi-mcherry (Gi-DREADD) were injected into the rostral and caudal gate regions of D2-Cre transgenic mice (FIG. 8A, 9A, and 9B). Then, longitudinal expression of the control group or Gi-DREADD was determined according to the rostral-caudal axis of the hippocampus of D2-Cre mice (see FIG. 9C). In addition, electrophysiological recordings of mCherry-labeled ramie cells have been used to confirm that the activity of ramie cells is silenced by treatment of clozapine-N-oxide (CNO), an artificial agent of the DREADD system. Only observed in Gi-DREADD mice, not in control mice (see FIGS. 8C and 8D).

Using the Gi-DREADD system, we investigated the role of ramie cells in the neurogenic and behavioral changes induced by chronic fluoxetine administration (see FIG. 8E). Adult neurogenic activity was measured using BrdU labeling and histological analysis of control and Gi-DREADD mice. Chronic administration of SSRI significantly increased the number of BrdU + proliferating cells in the subgranular region of the dental groin of control mice (SAL vs FLX, 177 ± 18 vs 337 ± 37; p <0.01), whereas in Gi-DREADD mice The effect of treatment with this fluoxetine was lost (SAL vs FLX, 172 ± 27 vs 232 ± 20; p = 0.89) (see Figures 8F and 8G). In addition, the expression level of double cortin (DCX) was analyzed as a marker of immature neurons indicating a specific snapshot of cells after neonatal mitosis during neuronal maturation and differentiation. Chronic administration of fluoxetine improved the survival of neoplastic precursors and promoted differentiation into mature neurons, but significantly decreased survival and / or differentiation of neoplastic cells after mitosis when parental cell activity was inhibited (FIG. 8H and 8I). That is, these experimental results show a similar tendency to the effects of the inhibition of the p11 / AnxA2 / SMARCA3 complex of Example 2, wherein the activity of the parental cells is induced by the administration of fluoxetine to the proliferation of neural precursors and the neoplastic granule cells. It can be seen that it is directly involved in controlling differentiation and maturation.

In addition, various behavioral tests were carried out to monitor the general motility activity and emotional behavior change to investigate the effect of ramie cell activity on antidepressant action. As a result of conducting a food intake suppression test (NSF) in a new environment, behavioral changes induced by chronic administration of antidepressants are affected by the inhibition of CNO-induced activity of parental cells, thereby effecting fluoxetine. Was offset in Gi-DREADD mice (control: SAL vs FLX, 484 ± 85 vs 238 ± 40; p <0.05, Gi-DREADD: SAL vs FLX, 450 ± 36 vs 365 ± 40; p = 14) (See Figure 10A). In addition, since the food intake level in the cage was not affected by the introduction of the Gi-DREADD system, it could be seen that this behavioral change was not due to the difference in the level of hunger among the comparison groups (see FIG. 10B). In addition, in the tail hanging test (TST), the effect of fluoxetine on inactivity was significantly reduced in control mice (SAL vs FLX, 141 ± 9 vs 110 ± 9; p <0.05), but as above, Gi- This effect was not observed in DREADD mice (SAL vs FLX, 128 ± 9 vs 129 ± 10, p = 0.94) (see FIG. 10C). Taken together, it can be seen that the cell line activity in the tooth gyrus plays an important role in the action and / or response of the antidepressant.

<110> Daegu Gyeongbuk Institute of Science and Technology <120> Screening method of antidepressant by evaluation of the activity          of mossy cells <130> PN122566KR <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> PASIP <400> 1 Gly Asn Gly Asn Gly Lys Val Thr Phe Pro Lys Met Lys Ile Pro Lys   1 5 10 15 Phe Ser Gly Arg              20 <210> 2 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> C3xNLS <400> 2 Pro Lys Lys Lys Arg Lys Val Asp Pro Lys Lys Lys Ars Lyg Val Asp   1 5 10 15 Pro Lys Lys Lys Arg Lys Val              20 <210> 3 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> AHNAK1 <400> 3 Gly Lys Val Thr Phe Pro Lys Met Lys Ile Pro Lys Phe Thr Phe Ser   1 5 10 15 Gly arg         <210> 4 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> AcGFP1-myc control <400> 4 Met Val Ser Lys Gly Ala Glu Leu Phe Thr Gly Ile Val Pro Ile Leu   1 5 10 15 Ile Glu Leu Asn Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Glu Asp Asp Gly Asn Tyr Lys Ser Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Thr Gly         115 120 125 Thr Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly Asn Lys Met Glu Tyr     130 135 140 Asn Tyr Asn Ala His Asn Val Tyr Ile Met Thr Asp Lys Ala Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser                 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly             180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu         195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Tyr Phe Gly Phe     210 215 220 Val Thr Ala Ala Ala Ile Thr His Gly Met Asp Glu Leu Tyr Lys Glu 225 230 235 240 Gln Lys Leu Ile Ser Glu Glu Asp Leu                 245 <210> 5 <211> 279 <212> PRT <213> Artificial Sequence <220> <223> AcGFP1-myc C-AHNAK1 <400> 5 Met Val Ser Lys Gly Ala Glu Leu Phe Thr Gly Ile Val Pro Ile Leu   1 5 10 15 Ile Glu Leu Asn Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly              20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile          35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr      50 55 60 Leu Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys  65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu                  85 90 95 Arg Thr Ile Phe Phe Glu Asp Asp Gly Asn Tyr Lys Ser Arg Ala Glu             100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Thr Gly         115 120 125 Thr Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly Asn Lys Met Glu Tyr     130 135 140 Asn Tyr Asn Ala His Asn Val Tyr Ile Met Thr Asp Lys Ala Lys Asn 145 150 155 160 Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser                 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly             180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu         195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Ile Tyr Phe Gly Phe     210 215 220 Val Thr Ala Ala Ala Ile Thr His Gly Met Asp Glu Leu Tyr Lys Glu 225 230 235 240 Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Asn Gly Asn Gly Asn Gly                 245 250 255 Asn Gly Asn Gly Ser Gly Lys Val Thr Phe Pro Lys Met Lys Ile Pro             260 265 270 Lys Phe Thr Phe Ser Gly Arg         275 <210> 6 <211> 296 <212> PRT <213> Artificial Sequence <220> <223> N-AHNAK1-AcGFP1-myc C3xNLS <400> 6 Gly Lys Val Thr Phe Pro Lys Met Lys Ile Pro Lys Phe Thr Phe Ser   1 5 10 15 Gly Arg Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Ser Lys Gly Ala              20 25 30 Glu Leu Phe Thr Gly Ile Val Pro Ile Leu Ile Glu Leu Asn Gly Asp          35 40 45 Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala      50 55 60 Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu  65 70 75 80 Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Ser Tyr Gly Val Gln                  85 90 95 Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys             100 105 110 Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu Arg Thr Ile Phe Phe Glu         115 120 125 Asp Asp Gly Asn Tyr Lys Ser Arg Ala Glu Val Lys Phe Glu Gly Asp     130 135 140 Thr Leu Val Asn Arg Ile Glu Leu Thr Gly Thr Asp Phe Lys Glu Asp 145 150 155 160 Gly Asn Ile Leu Gly Asn Lys Met Glu Tyr Asn Tyr Asn Ala His Asn                 165 170 175 Val Tyr Ile Met Thr Asp Lys Ala Lys Asn Gly Ile Lys Val Asn Phe             180 185 190 Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His         195 200 205 Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp     210 215 220 Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu 225 230 235 240 Lys Arg Asp His Met Ile Tyr Phe Gly Phe Val Thr Ala Ala Ala Ile                 245 250 255 Thr His Gly Met Asp Glu Leu Tyr Lys Ser Gly Leu Arg Ser Arg Ala             260 265 270 Asp Pro Lys Lys Lys Arg Lys Val Asp Pro Lys Lys Lys Lys Arg Lys Val         275 280 285 Asp Pro Lys Lys Lys Arg Lys Val     290 295 <210> 7 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> C3xNLS <400> 7 ccaaaaaaga agagaaaggt agatccaaaa aagaagagaa aggtagatcc aaaaaagaag 60 agaaaggta 69 <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> AHNAK1 <400> 8 ggaaaagtaa cattccctaa aatgaagatc cccaaattta ccttctctgg ccgt 54 <210> 9 <211> 768 <212> DNA <213> Artificial Sequence <220> <223> AcGFP1-myc control <400> 9 gctagccacc atggtgagca agggcgccga gctgttcacc ggcatcgtgc ccatcctgat 60 cgagctgaat ggcgatgtga atggccacaa gttcagcgtg agcggcgagg gcgagggcga 120 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcctgtgcc 180 ctggcccacc ctggtgacca ccctgagcta cggcgtgcag tgcttctcac gctaccccga 240 tcacatgaag cagcacgact tcttcaagag cgccatgcct gagggctaca tccaggagcg 300 caccatcttc ttcgaggatg acggcaacta caagtcgcgc gccgaggtga agttcgaggg 360 cgataccctg gtgaatcgca tcgagctgac cggcaccgat ttcaaggagg atggcaacat 420 cctgggcaat aagatggagt acaactacaa cgcccacaat gtgtacatca tgaccgacaa 480 ggccaagaat ggcatcaagg tgaacttcaa gatccgccac aacatcgagg atggcagcgt 540 gcagctggcc gaccactacc agcagaatac ccccatcggc gatggccctg tgctgctgcc 600 cgataaccac tacctgtcca cccagagcgc cctgtccaag gaccccaacg agaagcgcga 660 tcacatgatc tacttcggct tcgtgaccgc cgccgccatc acccacggca tggatgagct 720 gtacaaggag cagaaactca tctctgaaga ggatctgtga ggcgcgcc 768 <210> 10 <211> 857 <212> DNA <213> Artificial Sequence <220> <223> AcGFP1-myc C-AHNAK1 <400> 10 gctagccacc atggtgagca agggcgccga gctgttcacc ggcatcgtgc ccatcctgat 60 cgagctgaat ggcgatgtga atggccacaa gttcagcgtg agcggcgagg gcgagggcga 120 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcctgtgcc 180 ctggcccacc ctggtgacca ccctgagcta cggcgtgcag tgcttctcac gctaccccga 240 tcacatgaag cagcacgact tcttcaagag cgccatgcct gagggctaca tccaggagcg 300 caccatcttc ttcgaggatg acggcaacta caagtcgcgc gccgaggtga agttcgaggg 360 cgataccctg gtgaatcgca tcgagctgac cggcaccgat ttcaaggagg atggcaacat 420 cctgggcaat aagatggagt acaactacaa cgcccacaat gtgtacatca tgaccgacaa 480 ggccaagaat ggcatcaagg tgaacttcaa gatccgccac aacatcgagg atggcagcgt 540 gcagctggcc gaccactacc agcagaatac ccccatcggc gatggccctg tgctgctgcc 600 cgataaccac tacctgtcca cccagagcgc cctgtccaag gaccccaacg agaagcgcga 660 tcacatgatc tacttcggct tcgtgaccgc cgccgccatc acccacggca tggatgagct 720 gtacaaggag cagaaactca tctctgaaga ggatctgggt aacggaaatg gcaacgggaa 780 tggtaacgga tctggaaaag taacattccc taaaatgaag atccccaaat ttaccttctc 840 tggccgttga gcgcgcc 857 <210> 11 <211> 951 <212> DNA <213> Artificial Sequence <220> <223> N-AHNAK1-AcGFP1-myc C3xNLS <400> 11 gctagccacc atggtgggat ctggaaaagt aacattccct aaaatgaaga tccccaaatt 60 taccttctct ggccgtggta acggaaatgg caacgggaat ggtaacgagc agaaactcat 120 ctctgaagag gatctgagca agggcgccga gctgttcacc ggcatcgtgc ccatcctgat 180 cgagctgaat ggcgatgtga atggccacaa gttcagcgtg agcggcgagg gcgagggcga 240 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcctgtgcc 300 ctggcccacc ctggtgacca ccctgagcta cggcgtgcag tgcttctcac gctaccccga 360 tcacatgaag cagcacgact tcttcaagag cgccatgcct gagggctaca tccaggagcg 420 caccatcttc ttcgaggatg acggcaacta caagtcgcgc gccgaggtga agttcgaggg 480 cgataccctg gtgaatcgca tcgagctgac cggcaccgat ttcaaggagg atggcaacat 540 cctgggcaat aagatggagt acaactacaa cgcccacaat gtgtacatca tgaccgacaa 600 ggccaagaat ggcatcaagg tgaacttcaa gatccgccac aacatcgagg atggcagcgt 660 gcagctggcc gaccactacc agcagaatac ccccatcggc gatggccctg tgctgctgcc 720 cgataaccac tacctgtcca cccagagcgc cctgtccaag gaccccaacg agaagcgcga 780 tcacatgatc tacttcggct tcgtgaccgc cgccgccatc acccacggca tggatgagct 840 gtacaagtcc ggactcagat ctcgagctga tccaaaaaag aagagaaagg tagatccaaa 900 aaagaagaga aaggtagatc caaaaaagaa gagaaaggta tgaggcgcgc c 951

Claims (10)

Measuring the neuronal activity level of the parental cells in contact with the test substance; And
And comparing the measured neuronal activity level of the parental cells with the neuronal activity level of a control group that is not in contact with the test agent.
The method of claim 1, wherein measuring the neuronal activity level of the ramie cells is performed by detection of neuronal activity specific markers of the ramie cells, electrophysiological analysis, and combinations thereof.
The method of claim 1, further comprising determining the test substance as an antidepressant when the neuronal activity level of the parental cells in contact with the test substance is increased relative to the control.
The method of claim 1, wherein measuring the neuronal activity level of the ramie cells is performed by measuring the level of p11 / AnxA2 / SMARCA3 complex in the ramie cells.
The method of claim 4, further comprising determining the test substance as an antidepressant when the level of p11 / AnxA2 / SMARCA3 complex in the parental cells in contact with the test substance is increased relative to the control.
The method of claim 4, wherein the step is measuring the level of binding between the p11 / AnxA2 complex and SMARCA3 in the parental cells.
The method of claim 6, wherein the binding level between the p11 / AnxA2 complex and SMARCA3 is determined by a two-hybrid method, a co-immunoprecipitation assay, a co-localization assay, a scintillation proximity assay. : SPA), UV or chemical crosslinking methods, bimolecular interaction analysis (BIA), mass spectrometry (MS), nuclear magnetic resonance (NMR), fluorescence polarization assays (FPA), and And is measured by any one or more methods selected from the group consisting of in vitro pull-down assays.
The method of claim 6, further comprising determining the test substance as an antidepressant when the level of binding between p11 / AnxA2 complex and SMARCA3 in the hairy cells in contact with the test substance is increased compared to the control.
The method according to claim 1, wherein the antidepressant is a Serotoin Reuptake Inhibitor family antidepressant.
The method of claim 9, wherein the antidepressant enhances the therapeutic efficacy of the serotonin reuptake inhibitor family antidepressant.

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