KR102084767B1 - Pharmaceutical composition for preventing or treating stress disease comprising DISC1 protein or encoding gene thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a stressful disease, including a DISC1 protein or a gene encoding the same, and a method for screening a material for preventing or treating the stressful disease.
The present inventors studied the relationship between DISC1 and psychological stress. As a result, MAM binds to IP ₃ R1 (Inositol 1,4,5-trisphosphate (IP ₃ ) receptor type ₁ ) in MAM and competitively inhibits IP ₃ binding. DISC1's ability to regulate endoplasmic reticulum-mitochondrial calcium ion (Ca ²⁺ ) migration and its site of action by oxidative stress has been identified, which provides a model for intracellular calcium response to physiological stress. DISC1 and the model may be usefully used in related fields for the purpose of preventing and treating stressful diseases.

Description

Pharmaceutical composition for preventing or treating stress disease comprising DISC1 protein or encoding gene according to the present invention

The present invention relates to a pharmaceutical composition for preventing or treating stress disorders and a method for screening for preventing or treating stress disorders, comprising the DISC1 protein or a gene encoding the same.

Mitochondria-associated endoplasmic reticulum membrane (MAM) is a specialized lower compartment in which endoplasmic reticulum (ER) and mitochondria are formed in close proximity. Electron tomography analysis shows that there is a very close distance (10-25 nm) between the ER and the mitochondrial membrane, and that many chaperones and some key Ca ²⁺ channels involved in intracellular Ca ²⁺ homeostasis are present in the MAM. It is known to be concentrated. Inositol-1,4,5-trisphosphate receptors (IP ₃ Rs) and voltage-dependent anion channels (VDACs) are also abundant in MAM and regulate glucose Physically bound to protein 75 (glucose-regulated protein 75), as a result, Ca ²⁺ stored in the ER is transferred to the mitochondria quickly and efficiently through the MAM.

Neurons are highly polarized to suit their functions for intercellular communication. ER and mitochondria are widely distributed throughout the neuronal cell bodies and neurite end portions and serve as a major component of Ca ²⁺ signaling in neurons. ER Ca ²⁺ channels regulate a variety of neuron-specific processes, such as synaptic plasticity and neurotransmitter release, and because ER and mitochondria are regulated by the intracellular Ca ²⁺ levels, mitochondrial ATP production is tightly regulated. ^It is very closely related to the postsynaptic density (PSD), which supplies ATP in a manner that responds to ²⁺ , suggesting the potential importance of MAM in neurons. Indeed, some clues have been suggested in the neurological disorders such as Alzheimer's disease and atrophic axonal sclerosis, which are abnormal in ER-mitochondrial linkage and many related functions through MAM, which are common to mitochondrial dysfunction and disruption of intracellular Ca ²⁺ homeostasis. It is reported to show characteristic.

Oxidative stress causes Ca ²⁺ migration of ER-mitochondria in MAM. Hydrogen peroxide (H ₂ O ₂ ), superoxide anion (O- ² ) and C2-ceramide, which cause oxidative stress, cause Ca ²⁺ release from ER via IP ₃ R to mitochondria. Move it. Ca ²⁺ accumulation in mitochondria by oxidative stress has been reported to contribute to the depolarization of mitochondria and oxidative changes in oxidative phosphorylation, which are blocked by the addition of ER Ca ²⁺ channel blockers. Oxidative stress is attracting attention because it is a key mechanism underlying cellular and intracellular responses induced by various psychological stresses. In addition, short- and long-term treatment of physiological stress hormones cortisol and other glucocorticoids released in response to psychological stress impairs oxidative energy metabolism and inhibits antioxidant pathways, resulting in mitochondrial energy deficiency and free radicals in cells ( It is reported that a sharp rise in ROS) results in oxidative stress in the brain.

On the other hand, DISC1 (Disrupted-in-schizophrenia 1) has been studied in the analysis of various major mental disorders, including schizophrenia associated with chromosomal translocation affected by DISC1's open reading frame. Subsequent research suggests that functional disruption of the DISC1 protein will underlie the pathology of a wide range of major psychiatric disorders beyond the scope of individual diseases. For example, DISC1 mutant animal models exhibited a variety of behavioral patterns, including cognitive memory and social behavioral deficits associated with internal phenotypes of major mental disorders (Neuron 54, 387-402).

Accordingly, the present inventors assume that DISC1 may be involved in the interaction between psychological stress and environmental risk factors such as intracellular calcium cascade, and to verify this, ER under conditions that are physiological and pathologically related to the MAM location of DISC1 We investigated the effect of Ca ²⁺ transport between and mitochondria.

After a assumption such as the study of the association within the neurons of the DISC1 and psychological stress, the DISC1 binds to _{IP 3 R1 (Inositol 1,4,5-trisphosphate} (IP 3) receptor type1) in MAM IP ₃ Competitively inhibiting the binding to and down-regulated calcium ions (Ca ²⁺ ) migration from the endoplasmic reticulum by stress hormone-mediated oxidative stress to the mitochondria, the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating stress disorders, including DISC1 (Disrupted in schizophrenia 1) protein or a gene encoding the same as an active ingredient.

It is another object of the present invention to provide a method for screening a prophylactic or therapeutic substance for stress diseases.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a pharmaceutical composition for the prevention or treatment of stress diseases, including DISC1 (Disrupted in schizophrenia 1) protein or a gene encoding the same as an active ingredient.

In one embodiment of the present invention, the stress disorder may be any one selected from the group consisting of sleep disorders, depression, adaptation disorders, eating disorders, and anxiety disorders.

In another embodiment of the present invention, the gene may be inserted into a plasmid expression vector or a viral vector.

In another embodiment of the present invention, the DISC1 protein may regulate vesicle-mitochondrial calcium ion (Ca ²⁺ ) migration by stress hormone-mediated oxidative stress in MAM (Mitochondria-associated endoplasmic reticulum membrane).

In another embodiment of the present invention, the DISC1 protein binds to IP ₃ R1 (Inositol 1,4,5-trisphosphate (IP ₃ ) receptor type ₁ ) in MAM and competitively inhibits binding to IP ₃ to transfer calcium ions. Can be adjusted.

In another embodiment of the present invention, the stress hormone may be glucocorticoid.

In addition, the present invention provides a method for screening a material for preventing or treating a stressful disease, comprising the following steps.

(a) treating the candidate with cells expressing DISC1 (Disrupted in schizophrenia 1) protein or gene encoding the same in vitro ;

(b) measuring the expression level or activity of DISC1 protein in said cells; And

(c) selecting a substance that increases the expression level or activity of the DISC1 protein as a prophylactic or therapeutic substance for stress disorders as compared to the candidate non-treatment group.

In one embodiment of the present invention, the cell may be a neuron.

In another embodiment of the present invention, the candidate material may be selected from the group consisting of compounds, microbial cultures or extracts, natural product extracts, nucleic acids, and peptides.

In another embodiment of the present invention, the nucleic acid is composed of siRNA, shRNA, microRNA, antisense RNA, aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino It may be selected from the group.

In another embodiment of the present invention, the step (b) is the expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), Selected from the group consisting of immunoprecipitation, flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and protein chip It can be measured through one or more methods.

Another embodiment of the invention, the degree to which in step (b) the activity of DISC1 protein binds to the IP ₃ R1 in the MAM reduce the calcium ion transport in the ER to mitochondria by inhibiting the binding of IP ₃ competitively It may be to measure.

The present invention also provides a method for preventing or treating a stressful disease, comprising administering the pharmaceutical composition to a subject.

In addition, the present invention provides a use of the pharmaceutical composition for the prevention or treatment of stressful diseases.

The present inventors studied the relationship between DISC1 and psychological stress. As a result, MAM binds to IP ₃ R1 (Inositol 1,4,5-trisphosphate (IP ₃ ) receptor type ₁ ) in MAM and competitively inhibits IP ₃ binding. DISC1's ability to regulate down-regulated calcium ion (Ca ²⁺ ) migration from endoplasmic reticulum to oxidative stress by mitochondria and its site of action have been identified, providing a model of intracellular calcium response to physiological stress. The modulator DISC1 and the model may be usefully used in related fields for the purpose of preventing and treating stressful diseases.

Figure 1 shows that the DISC1 protein is present in MAM, the expression of DISC1 in the organelle fraction of the cerebral cortical neurons (MAM + Mito (DISC1 LI), MAM + Mito, MAM, ER, Mito, Whole lysate) Confirmed immune blotting results (FIG. 1A); Immunofluorescence assay to confirm the location of DISC1, ER, mitochondria (FIG. 1B); And immunofluorescence analysis and immunoblotting results showing that residues 1-201 of DISC1 are important for MAM localization (FIGS. 1C and 1D).
Figure 2 is the IP ₃ R1 as a result, make an important effect on the DISC1 positioned MAM, results (FIG. 2a to FIG. 2c) shows that the interaction IP ₃ R1 and MAM from DISC1; And the results showing the expression change of DISC1 according to the expression level of IP ₃ R1 in MAM (Figures 2d and 2e) is shown.
3 is a result confirming that DISC1 inhibits the binding of IP ₃ R1 and its ligand, immunoprecipitation results of analyzing the interaction of each functional domain of IP ₃ R1 with DISC1 protein (FIG. 3A); And Competitive IP ₃ binding analysis results (FIG. 3b) analyzing the effect of DISC1 on IP ₃ R1 and IP ₃ binding.
FIG. 4 shows that DISC1 in MAM is involved in ER-mitochondrial Ca ²⁺ migration. When IP ₃ is exposed to permeable neurons, mitochondrial Ca ²⁺ accumulation increases significantly when inhibition of DISC1 expression is observed. Showing results (FIG. 4A); And DISC1 overexpression or DISC1 (UBC6-DISC1) expression coupled with endoplasmic reticulum membrane proteins, showing a decrease in the increase in Ca ²⁺ accumulation of mitochondria (FIG. 4B).
FIG. 5 shows that ER-mitochondrial Ca ²⁺ migration regulated by DISC1 occurs mainly in MAM, mitochondrial Ca ²⁺ accumulation to control levels by simultaneously inhibiting the expression of MFN2 in neurons with DISC1 expression inhibition. Results showing decreasing (FIG. 5A); Results showing a significant increase in mitochondrial Ca ²⁺ accumulation upon induction of ER-mitochondrial contact enhancement by rapamycin-induced bridge forming modules in neurons overexpressing DISC1 (FIG. 5B); And Ca ²⁺ migration from ER to mitochondria according to the change in the level of DISC1 expression in crude MAM fraction derived from neuroblastoma CAD cells from IP ₃ (FIGS. 5C and 5D).
FIG. 6 shows that DISC1 regulates oxidative stress mediated ER-mitochondrial Ca ²⁺ migration. Hydrogen peroxide (H ₂ O ₂ ) or in control (CTL shRNA) and neurons (DISC1 shRNA) that inhibited expression of DISC1. Measurement results of mitochondrial Ca ²⁺ levels when treated with MS-treated glutathione peroxidase inhibitor (FIG. 6A); Measurement results of mitochondrial Ca ²⁺ levels when 2-APB was treated as an IP ₃ R blocker in each neuron under the same conditions as described above (FIG. 6B); Measurement results of mitochondrial Ca ²⁺ levels according to H ₂ O ₂ treatment in neurons overexpressing DISC1 or variant DISC1 (FIG. 6C); Measurement results of mitochondrial Ca ²⁺ levels according to H ₂ O ₂ treatment upon induction of ER-mitochondrial contact by rapamycin-induced bridge forming module in DISC1 overexpressed neurons (FIG. 6D); And mitochondrial Ca2 + level measurement results according to H ₂ O ₂ treatment in embryonic-derived cortical neurons (DISC1 LI) with damaged DISC1 locus (FIG. 6E).
FIG. 7 shows that DISC1 is closely related to dysfunction due to oxidative stress mediated mitochondrial Ca ²⁺ accumulation. When the expression of DISC1 is inhibited, mitochondrial membrane potential change according to time or concentration treatment of H ₂ O ₂ is shown. (FIG. 7A and FIG. 7B) and measurement of ROS generation degree (FIG. 7C and FIG. 7D); Measurement of the degree of ROS generation according to H ₂ O ₂ treatment in neurons expressing DISC1 overexpression or mutant DISC1 (DISC1 ^Δ1-201 ) expressed in ER-MAM (FIG. 7E); And mitochondrial membrane potential (FIG. 7f) and ROS generation degree (FIG. 7g) according to H ₂ O ₂ treatment in embryonic-derived cortical neurons (DISC1 LI) with damaged DISC1 locus.
8 is a result of confirming the increase of Ca ²⁺ migration from oxidative stress-dependent ER to mitochondria by corticosterone, the result of measuring the degree of ROS generation by corticosterone treatment in cortical neurons (FIG. 8A); And mitochondrial Ca ²⁺ level measurement results in APO treatment (FIG. 8B) as an antioxidant or 2-APB treatment (FIG. 8C) as an IP ₃ R blocker under the same conditions.
FIG. 9 shows that DISC1 down-regulates the increase of Ca ²⁺ migration from ER to mitochondria in corticosterone-dependent manner, and mitochondrial Ca ² according to changes in the expression level of DISC1 upon corticosterone (CORT) treatment in neurons. ⁺ Level measurement results (FIGS. 9A-9C); ROS generation degree measurement results (FIGS. 9D to 9F); And it shows the result of measuring the degree of ROS generation by corticosteroid treatment in embryo-derived cortical neurons (DISC1 LI) having a damaged DISC1 locus (FIG. 9H).
10 is a graphical illustration of the function of DISC1 identified in the present invention in Ca ²⁺ transport mechanism between ER and mitochondria by stress hormone mediated oxidative stress.

The inventors have identified a novel function of DISC1 in oxidative stress mediated calcium transport mechanism by stress hormone secretion, and based on this, the present invention has been completed.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating stress disorders comprising DISC1 (Disrupted in schizophrenia 1) protein or a gene encoding the same as an active ingredient.

As used herein, the term “stress” is a nonspecific biological response that occurs in the body to various injuries and stimuli applied to a living body, and was first named by Canadian endocrinologist H. celier. The stress response is a response of the adrenaline or other stress hormones to the body to protect our body. Stress causes physical symptoms such as fatigue, headaches, insomnia, myalgia, decreased concentration or memory, emptiness and confusion. Emotional symptoms such as mental symptoms, anxiety, depression, nervousness, and frustration, and behavioral symptoms such as restlessness and nervous behavior. Excessive or prolonged stress can cause stress-induced mental illness.

In the present invention, the stressful disease includes all diseases in which stress may act as a cause, and more preferably, may be a disease caused by physical effects due to stress hormone secretion, and more preferably sleep disorders and depression. , Adaptation disorders, eating disorders, and anxiety disorders may be any one selected from the group consisting of, but is not limited thereto.

In the present invention, the stress hormone may be a glucocorticoid, more preferably cortisone, cortisol, corticosterone, etc., but is not limited thereto.

As used herein, the term "prevention" means any action that inhibits or delays the development of a stressful disease by administration of a pharmaceutical composition according to the present invention.

As used herein, the term "treatment" means any action that improves or advantageously changes the symptoms for stressful disease by administration of the pharmaceutical composition according to the present invention.

The protein of DISC1 and the gene encoding the protein according to the present invention may be one or more selected from amino acid sequence information and nucleotide sequence information of human-derived DISC1 shown in Table 1, respectively, or more preferably from mouse. The protein may be composed of the amino acid sequence of SEQ ID NO: 1 (NCBI accession number: NP_061132.2) or SEQ ID NO: 2 (NP_777279.2), the gene encoding the protein is SEQ ID NO: 3 (NM_018662.2) or SEQ ID NO: It may consist of the base sequence of 4 (NM_174854.2), but is not limited thereto.

DISC1 isoform mRNA protein DISC1 isoform L NM_018662.2 NP_061132.2 DISC1 isoform Lv NM_001012957.1  NP_001012975.1 DISC1 isoform Es NM_001012958.1  NP_001012976.1 DISC1 isoform S NM_001012959.1  NP_001012977.1 DISC1 isoform a NM_001164537.1  NP_001158009.1 DISC1 isoform b NM_001164538.1 NP_001158010.1 DISC1 isoform c NM_001164539.1 NP_001158011.1 DISC1 isoform d NM_001164540.1 NP_001158012.1 DISC1 isoform e NM_001164541.1 NP_001158013.1 DISC1 isoform f NM_001164542.1  NP_001158014.1 DISC1 isoform g NM_001164544.1 NP_001158016.1 DISC1 isoform h NM_001164545.1 NP_001158017.1 DISC1 isoform i NM_001164546.1  NP_001158018.1 DISC1 isoform j NM_001164547.1  NP_001158019.1 DISC1 isoform k NM_001164548.1  NP_001158020.1 DISC1 isoform l NM_001164549.1 NP_001158021.1 DISC1 isoform m NM_001164550.1 NP_001158022.1 DISC1 isoform n NM_001164551.1 NP_001158023.1 DISC1 isoform o NM_001164552.1  NP_001158024.1 DISC1 isoform p NM_001164553.1 NP_001158025.1 DISC1 isoform q NM_001164554.1  NP_001158026.1 DISC1 isoform r NM_001164555.1 NP_001158027.1 DISC1 isoform t NM_001164556.1 NP_001158028.1 DISC1 isoform L NM_018662.2  NP_061132.2

The gene may be inserted into a plasmid expression vector or a viral vector, but is not limited thereto.

We have identified the DISC1 novel function related to stress through the examples.

In one embodiment of the present invention, by separating the brain of adult mice and analyzing the expression of DISC1 in the sequentially separated cell organelle fraction, DISC1 is located in the MAM, which is confirmed to be dependent on the 1-201 residue of DISC1 (See Example 2).

In another embodiment of the invention, the result of verifying the association of the IP ₃ known to abundantly present in the MAM R1 and DISC1, the level of the DISC1 change in MAM according to the level of expression of IP ₃ R1 and DISC1 the IP ₃ R1 It was confirmed that the binding to IP ₃ competitively inhibits binding to IP ₃ (see Example 3).

In another embodiment of the present invention, in consideration of the functional relationship between DISC1 and IP ₃ R1, analysis of whether MAM is involved in the ER-mitochondrial Ca ²⁺ migration, as a result of increasing the cellular permeability of neurons and exposure to IP ₃ intracellular Mitochondrial Ca ²⁺ level changes according to the expression level of DISC1, and it was also confirmed that DISC1 regulates Ca ²⁺ migration through IP ₃ R1 on the ER side. Moreover, further experiments confirmed that DISC1 regulates Ca ²⁺ migration in MAM (see Example 4).

In another embodiment of the present invention, it has been reported that oxidative stress induces Ca ²⁺ release from ER and migration to mitochondria in various cells, including neurons, analyzing whether DISC1 is involved in this process. It was. As a result, it was confirmed that ER-mitochondrial Ca ²⁺ migration changes according to expression level or variation of DISC1 in cells when neurons are exposed to H ₂ O ₂ (see Example 5).

In another embodiment of the present invention, excessive accumulation of Ca ²⁺ in the mitochondria due to oxidative stress may cause the dysfunction of the mitochondria. In this process, DISC1 was analyzed. As a result, when neurons were exposed to H ₂ O ₂ , mitochondrial membrane potential change and ROS production were changed according to the expression level or variation of DISC1 in cells, and the function of DISC1 in MAM was due to oxidative stress. It was confirmed that there is a close correlation with (see Example 6).

In another embodiment of the present invention, based on the results of previous studies that oxidative stress is induced in neurons by glucocorticoid stimulation, which is an excess of stress hormones, the correlation with DISC1 was verified. Corticosterone is one of the glucocorticoids. When the neurons were treated with ROS and mitochondrial Ca ²⁺ level was increased in proportion to the treatment concentration was observed (see Example 7). Furthermore, as a result of verifying the association between the phenomenon and DISC1, it was confirmed that ROS and mitochondrial Ca ²⁺ levels change according to the expression level or variation of DISC1 (see Example 8).

As shown in FIG. 10, the results of the above embodiments show that Ca ²⁺ migration through MAM is increased by oxidative stress due to stress hormone secretion, which may result in mitochondrial dysfunction. ER side of the MAM binds to IP ₃ R1 and down-regulates Ca ²⁺ migration, suggesting that DISC1 proteins or genes encoding them can be useful for the prevention or treatment of stress disorders. do.

The pharmaceutical composition according to the present invention includes DISC1 (Disrupted in schizophrenia 1) protein or a gene encoding the same as an active ingredient, and may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in the preparation, and includes, but is not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, but preferably orally, Depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the time of day, and may be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and an effective dose level refers to a patient's disease type, severity, or drug. Can be determined according to the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical field. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary according to the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention by any method.

In another aspect of the present invention, the present invention provides a method for screening a material for preventing or treating a stressful disease, comprising the following steps.

(a) treating the candidate with cells expressing DISC1 (Disrupted in schizophrenia 1) protein or gene encoding the same in vitro ;

(b) measuring the expression level or activity of DISC1 protein in said cells; And

(c) selecting a substance that increases the expression level or activity of the DISC1 protein as a prophylactic or therapeutic substance for stress disorders as compared to the candidate non-treatment group.

In the present invention, the cells may be neurons, but are not limited thereto.

In the present invention, the candidate material may be selected from the group consisting of compounds, microbial cultures or extracts, natural product extracts, nucleic acids, and peptides, the nucleic acid is siRNA, shRNA, microRNA, antisense RNA, aptamer (aptamer) It may be selected from the group consisting of, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino (morpholino), but is not limited thereto.

In the step (b), the expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry flow cytometry, immunofluorescence, ouchterlony, complement fixation assays, and protein chips. However, the measurement method is not limited thereto.

In step (b), the activity may be to measure the extent to which the DISC1 protein binds to IP ₃ R1 in MAM and competitively inhibits IP ₃ binding to reduce calcium ion migration from the endoplasmic reticulum to the mitochondria, which is conventional The person skilled in the art can perform the method appropriately without limitation.

As another aspect of the present invention, the present invention comprises the step of administering to the subject a pharmaceutical composition comprising a DISC1 (Disrupted in schizophrenia 1) protein or a gene encoding the same as an active ingredient, preventing or treating a stressful disease Provide a method.

As another aspect of the present invention, the present invention provides a use of the pharmaceutical composition for the prevention or treatment of stressful diseases.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Experiment Preparation and Experiment Method

1-1. Laboratory animals

In an embodiment of the present invention, pregnant C57BL / 6 mice were purchased from Hyochang Science, and then cortical neurons were collected and cultured when the mouse embryos were in the E15-16 stage. On the other hand, wild-type male mice (C57BL / 6) and DISC1 locus-injured mice (DISC1 LI, C57BL / 6 strains) were randomly fed and reared for 10-12 weeks in light / dark cycles at 12 hour intervals. Was used for the experiment. All animal experiments in this example were performed according to the approved guidelines after approval by the Pohang University of Science Animal Experiment Management Committee.

1-2. Cell fraction

Brains were isolated from three adult mice, homogenized, centrifuged, and a portion of the supernatant was stored as a fraction of the total lysate and the rest was further centrifuged at 13,800 × g, 4 ° C. for 10 minutes. Thereafter, the pellet (crude MAM) was recovered, the supernatant was loaded onto a sucrose gradient, and then centrifuged again. The ER fraction, which appeared as a white band, was recovered from the centrifuged product. The remaining crude MAM pellets were again loaded into a sucrose gradient and centrifuged, the synaptosome fractions appearing as third white bands were collected, and the remaining pellets were loaded on top of the percoll gradient and centrifuged to separate the upper and lower bands, respectively. Recovered to MAM and mitochondrial fractions.

1-3. IP ₃  binding assay

Transfected HEK293FT cells were lysed in NP40 buffer and then proteins were immunoprecipitated with antibodies. Immunoprecipitated proteins were incubated with 3 nM [ ³ H] -IP ₃ for 1 hour at 4 ° C. and the concentration of cold IP ₃ was increased in CLM buffer without Ca ²⁺ . Meanwhile, brains were extracted from adult wild-type or DISC1 LI mice, solubilized in NP40 buffer by sonication, and immunoprecipitated with endogenous IP ₃ R1 and DISC1 proteins with specific antibodies. [ ³ H] -IP were cultured with ₃ increased the concentration of cold IP _3. The mixture was then filtered with a GF / B filter, washed three times with CLM buffer and then the filter was dried and radioactivity was measured using a scintillation counter.

1-4. Real-Time Ca Using GCaMP6s ²⁺  Imaging

Neurons transfected with Mito-GCaMP6 (DIV7-8) with each construct were subjected to ionogine without digitonin and Ca ²⁺ for 2 minutes at 37 ° C. in a modified EGTA-, Ca ²⁺ free buffer. The treatment increased permeability and then exposed to IP ₃ . To determine the effect of RiBFM on mitochondrial Ca ²⁺ responses, neurons were transfected with Mito GCaMP6, AKAP1-FKBP12, FRB-SAC1 with pFlag-cmv2 or Flag-DISC1 in DIV5-6 and rapamycin for 5 minutes. And precultured neurons were exposed to IP ₃ in the same manner as above.

1-5. Statistical analysis

Experimental data are presented as mean ± SEM after one or two-way ANOVA analysis by Student't-test or Bonferroni post-hoc tests, respectively, for comparison between two different groups or between groups. Differences between groups were considered significant when P values were less than 0.05 (<0.05). No statistical method was used to determine sample size and no randomization was used for analysis.

Example 2. Confirm DISC1 located in MAM

We attempted to determine the intracellular location of DISC1 in the brain of adult mice and in cortical neurons of mouse embryos using biochemical and immunofluorescent techniques. To this end, the brains of adult mice were first isolated and intracellular organelles were isolated by sequentially performing cell fractionation. Thereafter, immunoblotting was performed to confirm each fraction and its purity, and markers specific to organelles to be identified, namely, IP ₃ R1 (ER / MAM), VDAC1 (ER-related mitochondria), Tim17 (mitochondria), and PSD95 ( Synaptosome) expression level was measured. As a result, each fraction and its purity were confirmed through the expression of the markers as shown in Figure 1a.

In addition, as a result of observing the expression of endogenous DISC1 in each fraction, the DISC1 protein was observed in crude MAM fraction (MAM + Mito) to which mitochondria were attached to MAM, as shown in FIG. The fraction was observed but not after further fractionation. At this time, the DISC1 protein was not observed in the crude MAM fraction (MAM + Mito (DISC1 LI)) derived from a mouse having an injured DISC1 locus, thereby confirming the specificity of the DISC1 antibody. In addition, the endogenous DISC1 protein was observed in the endoplasmic reticulum (ER) fraction and was also included in the MAM fraction.

Based on the above results, immunofluorescence analysis was performed using the same DISC1 antibody as used in the immunoblotting, and as a result, endogenous DISC1 protein was partially dispersed in cortical neurons as shown in FIG. (ER-RFP) and mitochondria (Mito-Crimson) were found to be present at the same position.

Furthermore, as a result of the same experiment using the DISC1 protein (DISC1 ^Δ1-201 ), which ^lacks the 1-201 residue of DISC1, as shown in FIG. It was confirmed that the decrease significantly. These results show that the position of the DISC1 protein in MAM depends mainly on 1-201 residue of the protein. The results were also confirmed through the fact that almost no DISC1 ^Δ1-201 protein was detected in the crude MAM fraction (MAM + Mito) as compared to the wild type DISC1 protein.

Example 3. DISC1 and IP in MAM ₃ Interaction of R1 and IP by DISC1 ₃ Confirmation of Ligand Inhibition of R1

3-1. DISC1 and IP in MAM ₃ Confirm interaction of R1

IP ₃ R1 is mainly expressed in the brain and is known to be abundant in MAM. As a result, it was confirmed that DISC1 is present in MAM through Example 2, and the present inventors performed immunoprecipitation analysis on the two proteins to determine whether DISC1 is related to IP ₃ R1. As a result, as shown in FIG. 2A, IP ₃ R1 (GFP-IP ₃ R1) showed strong interaction with wild-type DISC1 (Flag-DISC1), whereas it did not show such interaction with DISC1 ^Δ1-201. . In addition, immunofluorescence analysis showed that endogenous DISC1 protein coexists with GFP-IP ₃ R1 in the contact region with mitochondria, denoted as Mito-Crimson, in cortical neurons as shown in FIG. 2B. In addition, as shown in the immunoblotting results of FIG. 2C, endogenous DISC1 and IP ₃ R1 proteins were expressed together in whole cell lysate and crude MAM fraction (MAM + Mito) derived from mature mouse brain. Confirmed.

Furthermore, to investigate the effect of IP ₃ R1 on DISC1 present in MAM, we analyzed the change of DISC1 protein in crude MAM fraction (MAM + Mito) according to the expression level of IP ₃ R1. As a result, as shown in Fig. 2d case that overexpression of _{IP 3 R1 (GFP-IP 3} R1) was in the fraction significantly increased expression of the DISC1 (Flag-DISC1), opposed to the expression by processing the IP ₃ R1 siRNA thereof When inhibited, as shown in Figures 2d and 2e it was confirmed that both Flag-DISC1 and endogenous DISC1 protein levels are significantly reduced. Contrary to the results in the crude MAM fraction, however, the amount of DISC1 protein in the whole cell lysate did not change.

These results demonstrate that IP ₃ R1 has a significant effect on the position of DISC1 in the MAM.

3-2. IP by DISC1 ₃ Confirmation of Ligand Inhibition of R1

On the basis of the above results, constructs expressing the functional domain of IP ₃ R1 as shown in FIG. 3A were constructed to identify the domain of IP ₃ R1 that binds to DISC1, and the immunoprecipitation analysis of each of the domains and DISC1 was performed. The interaction was analyzed. As a result, DISC1 interacts with the rest of the domains except the suppressor (SD), gate-keeping (GK), and transmembrane (TM) domains, namely the ligand-binding domain (LBD) and the regulatory domains (MD1, MD2, MD3). Appeared.

IP ₃ R1 in the ligand-binding domain, and since the control domain is a critical area on the binding of IP ₃ Rs present inventors investigated the DISC1 affect the binding of IP ₃ R1 and the IP ₃ ligand thereof. To this end, competitive IP ₃ binding assays were performed using the IP ₃ R1, DISC1 or DISC1 ^Δ1-201 protein isolated from immunoprecipitation using antibodies from HEK293FT cells according to the method of Examples 1-3. The resulting ^[3 H] was confirmed that a ratio of a cover for the IP ₃ IP ₃ binding capacity is decreased binding to IP ₃ R1 In contrast to DISC1 ^Δ1-201 DISC1 as shown in Figure 3b. In addition, the same experiments were performed with endogenous IP ₃ R1 and DISC1 protein isolated from wild type (WT) or DISC1 locus damaged mice (DISC1 LI). As a result, a sample derived from DISC1 LI compared with wild type (WT) IP ₃ R1. It was confirmed that the unlabeled IP ₃ which binds to is markedly increased.

The results demonstrate that DISC1 inhibits the binding of IP ₃ R1 with its ligand.

Example 4 Reticulum-Mitochondrial Ca Via MAM ²⁺  Identify the role of DISC1 in transport

4-1. DISC1-mediated ER-mitochondrial Ca present in MAM ²⁺  Check movement adjustment

The present inventors tried to investigate whether DISC1 is involved in endoplasmic reticulum-mitochondrial Ca ²⁺ transport regulation in consideration of the functional relationship between IP ₃ R1 and DISC1 in MAM, which was described in the above embodiment. To this end, the target sequences for mitochondria or endoplasmic reticulum were combined to modify the expression construct of GCaMP6, a genetically encoded Ca ²⁺ marker. It was then confirmed that the organelle-specific GCaMP6 construct is expressed in cortical neurons. In the case of measuring the endoplasmic reticulum Ca ²⁺ , the results were verified using ER-GCaMP3 having a relatively low affinity for Ca ²⁺ .

To increase cell membrane permeabilization of cerebral cortical neurons, EGTA-, Ca ²⁺ -free buffers were pre-incubated for 2 minutes with digitonin and Ca ²⁺ -free ionomycin, followed by disruption of other organelle membranes. Washed to prevent. Since the process of increasing the membrane permeability was confirmed that the activation of L-type Ca ²⁺ channel in neurons did not affect the basic Ca ²⁺ level or general depolarization. After improving the neurons in cell permeability by the above-described method results of processing the IP _3, also in the neurons (DISC1 shRNA) expression of the DISC1 inhibition compared to control neurons (CTL shRNA), as shown in 4a IP ₃ dependent mitochondrial Ca ^{2 +} Increase was markedly increased and overexpression of human DISC1 protein resistant to shRNA (DISC1 shRNA + hDISC1) was confirmed that the Ca ²⁺ increase level recovered similarly to the control. In addition, it was confirmed that Ca ²⁺ levels stored in the endoplasmic reticulum in the neurons inhibited by the expression of DISC1 when IP ₃ is stimulated, which supports the above results. These results indicate that DISC1 regulates Ca ²⁺ secretion via IP ₃ R1 on the ER side before Ca ²⁺ migrates to the mitochondria. Contrary to the above results, as shown in FIG. 4B, in the case of overexpressing DISC1 in the permeable neurons (DISC1), the level of mitochondrial Ca ²⁺ induced by IP ₃ was significantly decreased compared to the control group (Vector). .

On the other hand, when the expression of DISC1 was inhibited, the mitochondrial Ca ²⁺ increase induced by ryanodine receptor agonist 4-chloro-m-cresol (4-cmc) did not change significantly. This indicates that it specifically acts on IP ₃ R mediated Ca ²⁺ migration.

Moreover, inhibition of expression of DISC1 did not affect mitochondrial capacity for Ca ²⁺ uptake in neurons pre-cultured with 2-aminoethyl diphenylborinate (2-APB), a selective IP ₃ R blocker. Turned out to be. To further investigate the intrinsic capacity of mitochondrial Ca ²⁺ uptake, in vitro mitochondrial Ca ²⁺ assays were performed using pure mitochondrial fractions derived from the brains of DISC1 LI mice with damaged wild-type or DISC1 loci. In response to Ca ²⁺ pulses in the extraocular muscles, the fluorescence signal of CaGreen-5N, ie intracellular permeable Ca ²⁺ staining was increased, but after reaching the peak they were reduced by mitochondrial Ca ²⁺ uptake. In addition, there was no significant difference between WT and DISC1LI for the rate of decrease indicating mitochondrial Ca ²⁺ uptake. These results suggest that the effect of DISC1 on endoplasmic reticulum-mitochondrial Ca ²⁺ migration is not due to a change in mitochondrial capacity for Ca ²⁺ uptake.

To further elucidate the association between the MAM location of DISC1 and the endoplasmic reticulum-mitochondrial Ca ²⁺ migration regulation, we present a DISC1 expression construct fused with the target sequence of yeast UBC6, a vesicle membrane protein that targets DISC1 on ER / MAM. (UBC6-DISC1) was prepared and confirmed localization of endoplasmic reticulum and MAM in neurons. As shown in FIG. 4B, neurons expressing the construct (UBC6-DISC1) significantly reduced IP ₃ -mediated Ca ²⁺ migration in a similar manner as overexpressing wild type DISC1, whereas Ca for DISC1 ^Δ1-201 It was confirmed that no significant change appeared in the ²⁺ migration. In addition, DISC1 was predominantly expressed in the outer mitochondrial membrane or in the mitochondrial inner space by recombining the anchoring sequence of the mouse AKAP1 or yeast MIA40 leader sequence, and did not alter the IP ₃ dependent mitochondrial Ca ²⁺ response.

4-2. ER-mitochondrial Ca of DISC1-mediated MAM ²⁺  Check movement adjustment

Next, to investigate whether vesicle-mitochondrial Ca ²⁺ transport regulated by DISC1 is regulated by changes in MAM formation, mitofusin 2 (MFN2), a protein that tethers endoplasmic reticulum to mitochondria in MAM, is used. It was. As shown in FIG. 5A, Ca ²⁺ accumulation of mitochondria was significantly increased in comparison with the control group (CTL shRNA + CTL siRNA) in the neurons (DISC1 shRNA + CTL siRNA) inhibited as shown in FIG. In neurons that inhibited the expression of MFN2 (DISC1 shRNA + MFN2 siRNA), the contact of MAM was decreased, and Ca ²⁺ accumulation in mitochondria was reduced to a level similar to that of the control group.

In addition, a rapamycin-inducible bridge-forming module (RibFM) was used to enhance endoplasmic reticulum-mitochondrial contact in response to rapamycin treatment. Specifically, the two rapamycin binding domains located in each of the ER and the mitochondria were dramatically merged after 5 minutes of rapamycin treatment, and after the rapamycin treatment, the merging continued for 1 hour even after the rapamycin was removed. It was. In addition, activation of the module by rapamycin was found to increase ER-mitochondrial Ca ²⁺ migration in neurons as previously reported. As a result, as shown in Figure 5b, the neurons (DISC1) overexpressing the DISC1 protein, while the increase rate of mitochondrial Ca ²⁺ accumulation following IP ₃ treatment decreased compared to the control group (Vector + Veh), the rapamycin treatment When ER-mitochondrial contact was significantly improved by the module, it was confirmed that mitochondrial Ca ²⁺ accumulation increased to a similar degree as the control. These results collectively show that ER-mitochondrial Ca ²⁺ migration regulated by DISC1 occurs mainly in MAM.

In addition, in vitro Ca ²⁺ analysis was performed to more directly verify Ca ²⁺ transport through MAM under the control of DISC1. To this end, crude MAM fractions (MAM with mitochondria attached) were isolated from neuroblastoma CAD cells transfected with GCaMP6s and shRNA. The experimental results, as shown in Fig. 5c crude MAM fraction isolated from the expression of the DISC1 inhibited cells was reduced by MAM Ca ²⁺ is greater width than the control group during treatment IP _3, mitochondrial Ca ²⁺ levels are opposed to the control group It was confirmed that the increase significantly compared to. Cortical neurons of DISC1 LI embryos with damaged DISC1 loci showed a significant increase in ER-mitochondrial Ca ²⁺ migration compared to the control group, as shown in FIG. 5D. These results confirmed that the expression of hDISC1 showed a significant level with the control.

Overall, these results indicate that loss of function of DISC1 may lead to abnormal ER-mitochondrial Ca ²⁺ migration in MAM.

Example 5 Oxidative Stress-Dependent ER-Mitochondrial Ca by DISC1 ²⁺  Shift adjustment

Recent studies suggest that sensitivity to oxidative stress is the basis of the neuronal environment associated with the pathophysiology of schizophrenia. Interestingly, oxidative stress induces progressive Ca ²⁺ release from ER and migration to mitochondria in various types of cells, including neurons. Based on these results, we tried to investigate whether DISC1 influences ER-mitochondrial Ca ²⁺ migration induced by oxidative stimulation.

To this end, treatment with H ₂ O ₂ in cerebral cortical neurons increased mitochondrial Ca ²⁺ in a smaller and slower manner than control neurons (CTL shRNA) as compared to IP ₃ treatment, as shown in FIG. 6A. Expression-inhibited neurons (DISC1 shRNA) were found to significantly increase mitochondrial Ca ²⁺ levels significantly. In addition, even when treated with MSC (mercaptosuccinic acid), an inhibitor of glutathione peroxidase, which inherently produces H ₂ O ₂ , mitochondrial Ca ²⁺ was significantly wider in neurons that inhibited DISC1 expression. Appeared to increase. Together with these results, it was confirmed that the level of ER Ca ²⁺ decreases in neurons (DISC1 shRNA) whose DISC1 expression is inhibited.

In view of the slow increase in mitochondrial Ca ²⁺ levels in response to oxidative stress, we measured mitochondrial Ca ²⁺ levels under oxidative stress for a long time. In order to measure Ca ²⁺ levels of mitochondria at specific time points during incubation of neurons with H ₂ O ₂ , mitochondrial-specific chemical Ca ²⁺ indicators are used instead of Mito-GCaMP6, which may cause differences due to various expression levels. Rhod2 / AM was used. As shown in FIG. 6B, mitochondrial Ca ²⁺ levels were increased in proportion to the incubation time after H ₂ O ₂ treatment. Moreover, Ca ²⁺ accumulation was significantly increased in neurons (DISC1 shRNA) which inhibited DISC1 expression. This result was not observed with pre-culture (DISC1 shRNA + 2-APB) with the selective IP ₃ R blocker 2-APB. This means that IP ₃ R is important for DISC1 to regulate ER-mitochondrial Ca ²⁺ migration by oxidative stress.

In contrast, in the case of overexpressing wild type DISC1 and UBC6-DISC1, as shown in FIG. 6C, the level of mitochondrial Ca ²⁺ was significantly decreased compared to the control group at 1 hour after H ₂ O ₂ culture. , DISC1 ^Δ1-201 did not show this reduction. In addition, as shown in FIG. 6D, when ER-mitochondrial contact by RiBFM was enhanced (DISC1 + Rapa), mitochondrial Ca ²⁺ accumulation in response to oxidative stress was increased, whereby DISC1 overexpression was induced by oxidative stress. It was found to counteract the effects on mitochondrial Ca ²⁺ accumulation. Consistent with these results, as shown in FIG. 6E, cerebral cortical neurons (DISC1 LI) derived from mouse embryos with damaged DISC1 loci were inhibited in the expression of DISC1 in culture with H ₂ O ₂ compared to wild-type neurons (WT). Similar to the case, the increase in the level of mitochondrial Ca ²⁺ was confirmed to increase significantly.

Example 6 Control of Oxidative Stress Mediated Dysfunction by DISC1 in Mitochondria

Excessive Ca ²⁺ accumulation in mitochondria has been reported to lead to uncontrolled activity of the mitochondrial electron transport chain, leading to mitochondrial membrane potential disruption (depolarization) and promotion of ROS production. These mitochondrial dysfunctions have been observed in schizophrenic patients and animal models representing the phenotype of schizophrenia, suggesting that deterioration of mitochondrial activity may be a component of schizophrenia pathophysiology. Therefore, we investigated the effect of DISC1 on oxidative stress mediated mitochondria dysfunction in cerebral cortical neurons. More specifically, neurons were preincubated with TMRM, a chemical indicator of mitochondrial potential, and exposed to H ₂ O ₂ to measure changes in mitochondrial membrane potential in response to oxidative stress.

As a result, as shown in FIGS. 7A and 7B, neurons whose DISC1 expression was inhibited (DISC1 shRNA), compared to control neurons (CTL shRNA), exhibited the exposure time and treatment of disruption of mitochondrial membrane potential induced by H ₂ O ₂ . It was observed to accelerate depending on the concentration. According to the same experiment, as a result of measuring the degree of ROS generation using DHR-123, a mitochondrial ROS generation index, as shown in FIGS. 7C and 7D, the ROS generation time was increased in DISC1-expressed neurons (DISC1 shRNA). And dose dependently. In addition, pre-depletion of Ca ²⁺ stored in ER was dependent on H ₂ O ₂ in both control (CTL shRNA + ER Ca ²⁺ depletion) and DISC1-inhibited neurons (DISC1 shRNA + ER Ca ²⁺ depletion). Phosphorus ROS production was reduced and it was confirmed that there was no difference in ROS production between the control and DISC1 knockdown neurons. Moreover, when overexpressing DISC1, UBC6-DISC1 significantly reduced ROS generation in response to H ₂ O ₂ , but in the case of DISC1 ^Δ1-201 , there was no significant difference compared to the control vector. It was confirmed that it was not. Consistent with these results, as shown in FIG. 7F and FIG. 7G, neurons derived from DISC1 LI embryos showed greater changes in mitochondrial membrane potential and ROS production when cultured with H ₂ O ₂ compared to wild type neurons.

Overall, these results demonstrate that the function of DISC1 in MAM is closely related to mitochondrial function in oxidative stress through ER-mitochondrial Ca ²⁺ migration.

Example 7 Mitochondrial Ca Dependent on Oxidative Stress by Corticosterone ²⁺  Accumulation Induction

Previous studies have shown that acute and chronic treatment with excess glucocorticoids results in impaired oxidative phosphorylation, resulting in a deficiency in mitochondrial ATP production, and ROS in the brain region where there are many glococorticoid receptors, including the hippocampus and cerebral cortex. It has been reported that the rapid rise of leads to oxidative stress. We therefore assumed that excessive glucocorticoids could induce ER-mitochondrial Ca ²⁺ migration by inducing oxidative stress.

To test this hypothesis, changes in ROS and mitochondrial Ca ²⁺ levels in cerebral cortical neurons were treated after glucocorticoid stress hormone corticosterone (CORT). As a result, a significant increase in ROS and mitochondrial Ca ²⁺ levels was observed in proportion to treatment concentration when treated with corticosterone for 1 hour as shown in FIGS. 8A and 8B. An antioxidant and a ROS scavenger, APO (apocynin), were used to verify whether this increase in glucocorticoid-dependent mitochondrial Ca ²⁺ levels is dependent on oxidative stress induction. As a result, as shown in Figure 8b it was confirmed that the pretreatment with APO significantly reduced the increase in mitochondrial Ca ²⁺ level by the corticosteroid as described above. Furthermore, as shown in FIG. 8C, when neurons were preincubated with 2-APB, an IP ₃ R blocker, there was no increase in mitochondrial Ca ²⁺ levels by corticosterone. In contrast, when corticosteroids were treated with neurons for 1 hour, ER's ability to store Ca ²⁺ or produce IP ₃ did not change.

Overall, the results show that ER-mitochondrial Ca ²⁺ migration can be regulated by corticosterone interconnected with the induction of oxidative stress.

Example 8 Corticosterone-Dependent ER-Mitochondrial Ca by DISC1 ²⁺  Check movement adjustment

Since the above examples confirm that DISC1 regulates ER-mitochondrial Ca ²⁺ migration induced by oxidative stress, the present inventors further investigated whether DISC1 influences mitochondrial Ca ²⁺ accumulation by corticosterone. . As a result, as shown in FIG. 9A, the degree of increase of mitochondrial Ca ²⁺ levels was significantly increased in the neurons whose DISC1 expression was inhibited compared to the control neurons (CTL shRNA) when treated with corticosterone for 1 hour. And increased significantly by the antioxidant and ROS scavenger APO treatment (CTL shRNA + APO and DISC1 shRNA + APO).

In addition, the inventors further investigated whether the MAM position of DISC1 affects the increase in corticosterone-induced mitochondrial Ca ²⁺ levels. As a result, as shown in FIG. 9B, incubation of UBC6-DISC1 overexpressed neurons (UBC6-DISC1) with corticosteroid resulted in decreased Ca ²⁺ accumulation in mitochondria compared to control neurons (Vector), whereas DISC1 ^{Δ1 In} the case of ^-201, the same reduction effect was not observed. Furthermore, as shown in FIG. 9C, when rapamycin was treated in a neuron (DISC1) overexpressing DISC1 as described above, corticosteroid-induced mitochondrial Ca ²⁺ accumulation was increased again. These results indicate that DISC1 plays an essential role in ER-mitochondrial Ca ²⁺ migration induced by glucocorticoids in MAM.

Next, we investigated whether excessive Ca ²⁺ accumulation of mitochondria by corticosterone causes ROS overproduction in neurons with DISC1 expression inhibition. As a result, as shown in FIG. 9D, the degree of ROS increase was significantly increased in DISC1-inhibited neurons (DISC1 shRNA) compared to control neurons (CTL shRNA), and these results showed that neurons deficient in ER Ca ²⁺ beforehand. (CTL shRNA + ER Ca ²⁺ depletion and DISC1 shRNA + ER Ca ²⁺ depletion). Furthermore, as shown in FIG. 9E, neurons overexpressing wild-type DISC1 and UBC6-DISC1 were found to incubate with corticosteroids to reduce increased ROS levels, whereas DISC1 ^Δ1-201 did not reduce increased ROS levels. It was. In addition, as shown in FIG. 9F, the degree of ROS generation in DISC1 overexpressing neurons increased again upon induction of MAM formation by RiBFM. In this experimental environment, rapamycin itself did not affect the increase of mitochondrial Ca ²⁺ levels and ROS production induced by oxidative stress and glucocorticoids. Finally, neurons derived from DISC1 LI mouse embryos exhibited excessive ROS production and mitochondria compared to control (WT) in response to corticosterone similarly to neurons that inhibited DISC1 expression as shown in FIGS. 9G and 9H. Ca ²⁺ accumulation was seen. However, APO treatment significantly reduced the difference in mitochondrial Ca ²⁺ accumulation between wild-type and DISC1 LI neurons consistent with the results of FIG. 9A.

The description of the present invention set forth above is for illustrative purposes, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Pharmaceutical composition for preventing or treating stress          disease comprising DISC1 protein or encoding gene <130> PD17-173-P1 <150> KR 10-2017-0164209 <151> 2017-12-01 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 854 <212> PRT <213> Homo sapiens DISC1 protein <400> 1 Met Pro Gly Gly Gly Pro Gln Gly Ala Pro Ala Ala Ala Gly Gly Gly   1 5 10 15 Gly Val Ser His Arg Ala Gly Ser Arg Asp Cys Leu Pro Pro Ala Ala              20 25 30 Cys Phe Arg Arg Arg Arg Leu Ala Arg Arg Pro Gly Tyr Met Arg Ser          35 40 45 Ser Thr Gly Pro Gly Ile Gly Phe Leu Ser Pro Ala Val Gly Thr Leu      50 55 60 Phe Arg Phe Pro Gly Gly Val Ser Gly Glu Glu Ser His His Ser Glu  65 70 75 80 Ser Arg Ala Arg Gln Cys Gly Leu Asp Ser Arg Gly Leu Leu Val Arg                  85 90 95 Ser Pro Val Ser Lys Ser Ala Ala Ala Pro Thr Val Thr Ser Val Arg             100 105 110 Gly Thr Ser Ala His Phe Gly Ile Gln Leu Arg Gly Gly Thr Arg Leu         115 120 125 Pro Asp Arg Leu Ser Trp Pro Cys Gly Pro Gly Ser Ala Gly Trp Gln     130 135 140 Gln Glu Phe Ala Ala Met Asp Ser Ser Glu Thr Leu Asp Ala Ser Trp 145 150 155 160 Glu Ala Ala Cys Ser Asp Gly Ala Arg Arg Val Arg Ala Ala Gly Ser                 165 170 175 Leu Pro Ser Ala Glu Leu Ser Ser Asn Ser Cys Ser Pro Gly Cys Gly             180 185 190 Pro Glu Val Pro Pro Thr Pro Pro Gly Ser His Ser Ala Phe Thr Ser         195 200 205 Ser Phe Ser Phe Ile Arg Leu Ser Leu Gly Ser Ala Gly Glu Arg Gly     210 215 220 Glu Ala Glu Gly Cys Pro Pro Ser Arg Glu Ala Glu Ser His Cys Gln 225 230 235 240 Ser Pro Gln Glu Met Gly Ala Lys Ala Ala Ser Leu Asp Gly Pro His                 245 250 255 Glu Asp Pro Arg Cys Leu Ser Arg Pro Phe Ser Leu Leu Ala Thr Arg             260 265 270 Val Ser Ala Asp Leu Ala Gln Ala Ala Arg Asn Ser Ser Arg Pro Glu         275 280 285 Arg Asp Met His Ser Leu Pro Asp Met Asp Pro Gly Ser Ser Ser Ser     290 295 300 Leu Asp Pro Ser Leu Ala Gly Cys Gly Gly Asp Gly Ser Ser Gly Ser 305 310 315 320 Gly Asp Ala His Ser Trp Asp Thr Leu Leu Arg Lys Trp Glu Pro Val                 325 330 335 Leu Arg Asp Cys Leu Leu Arg Asn Arg Arg Gln Met Glu Val Ile Ser             340 345 350 Leu Arg Leu Lys Leu Gln Lys Leu Gln Glu Asp Ala Val Glu Asn Asp         355 360 365 Asp Tyr Asp Lys Ala Glu Thr Leu Gln Gln Arg Leu Glu Asp Leu Glu     370 375 380 Gln Glu Lys Ile Ser Leu His Phe Gln Leu Pro Ser Arg Gln Pro Ala 385 390 395 400 Leu Ser Ser Phe Leu Gly His Leu Ala Ala Gln Val Gln Ala Ala Leu                 405 410 415 Arg Arg Gly Ala Thr Gln Gln Ala Ser Gly Asp Asp Thr His Thr Pro             420 425 430 Leu Arg Met Glu Pro Arg Leu Leu Glu Pro Thr Ala Gln Asp Ser Leu         435 440 445 His Val Ser Ile Thr Arg Arg Asp Trp Leu Leu Gln Glu Lys Gln Gln     450 455 460 Leu Gln Lys Glu Ile Glu Ala Leu Gln Ala Arg Met Phe Val Leu Glu 465 470 475 480 Ala Lys Asp Gln Gln Leu Arg Arg Glu Ile Glu Glu Gln Glu Gln Gln                 485 490 495 Leu Gln Trp Gln Gly Cys Asp Leu Thr Pro Leu Val Gly Gln Leu Ser             500 505 510 Leu Gly Gln Leu Gln Glu Val Ser Lys Ala Leu Gln Asp Thr Leu Ala         515 520 525 Ser Ala Gly Gln Ile Pro Phe His Ala Glu Pro Pro Glu Thr Ile Arg     530 535 540 Ser Leu Gln Glu Arg Ile Lys Ser Leu Asn Leu Ser Leu Lys Glu Ile 545 550 555 560 Thr Thr Lys Val Cys Met Ser Glu Lys Phe Cys Ser Thr Leu Arg Lys                 565 570 575 Lys Val Asn Asp Ile Glu Thr Gln Leu Pro Ala Leu Leu Glu Ala Lys             580 585 590 Met His Ala Ile Ser Gly Asn His Phe Trp Thr Ala Lys Asp Leu Thr         595 600 605 Glu Glu Ile Arg Ser Leu Thr Ser Glu Arg Glu Gly Leu Glu Gly Leu     610 615 620 Leu Ser Lys Leu Leu Val Leu Ser Ser Arg Asn Val Lys Lys Leu Gly 625 630 635 640 Ser Val Lys Glu Asp Tyr Asn Arg Leu Arg Arg Glu Val Glu His Gln                 645 650 655 Glu Thr Ala Tyr Glu Thr Ser Val Lys Glu Asn Thr Met Lys Tyr Met             660 665 670 Glu Thr Leu Lys Asn Lys Leu Cys Ser Cys Lys Cys Pro Leu Leu Gly         675 680 685 Lys Val Trp Glu Ala Asp Leu Glu Ala Cys Arg Leu Leu Ile Gln Ser     690 695 700 Leu Gln Leu Gln Glu Ala Arg Gly Ser Leu Ser Val Glu Asp Glu Arg 705 710 715 720 Gln Met Asp Asp Leu Glu Gly Ala Ala Pro Pro Ile Pro Pro Arg Leu                 725 730 735 His Ser Glu Asp Lys Arg Lys Thr Pro Leu Lys Val Leu Glu Glu Trp             740 745 750 Lys Thr His Leu Ile Pro Ser Leu His Cys Ala Gly Gly Glu Gln Lys         755 760 765 Glu Glu Ser Tyr Ile Leu Ser Ala Glu Leu Gly Glu Lys Cys Glu Asp     770 775 780 Ile Gly Lys Lys Leu Leu Tyr Leu Glu Asp Gln Leu His Thr Ala Ile 785 790 795 800 His Ser His Asp Glu Asp Leu Ile Gln Ser Leu Arg Arg Glu Leu Gln                 805 810 815 Met Val Lys Glu Thr Leu Gln Ala Met Ile Leu Gln Leu Gln Pro Ala             820 825 830 Lys Glu Ala Gly Glu Arg Glu Ala Ala Ala Ser Cys Met Thr Ala Gly         835 840 845 Val His Glu Ala Gln Ala     850 <210> 2 <211> 852 <212> PRT <213> Mus musculus DISC1 protein <400> 2 Met Gln Gly Gly Gly Pro Arg Gly Ala Pro Ile His Ser Pro Ser His   1 5 10 15 Gly Ala Asp Ser Gly His Gly Leu Pro Pro Ala Val Ala Pro Gln Arg              20 25 30 Arg Arg Leu Thr Arg Arg Pro Gly Tyr Met Arg Ser Thr Ala Gly Ser          35 40 45 Gly Ile Gly Phe Leu Ser Pro Ala Val Gly Met Pro His Pro Ser Ser      50 55 60 Ala Gly Leu Thr Gly Gln Gln Ser Gln His Ser Gln Ser Lys Ala Gly  65 70 75 80 Gln Cys Gly Leu Asp Pro Gly Ser His Cys Gln Ala Ser Leu Val Gly                  85 90 95 Lys Pro Phe Leu Lys Ser Ser Leu Val Pro Ala Val Ala Ser Glu Gly             100 105 110 His Leu His Pro Ala Gln Arg Ser Met Arg Lys Arg Pro Val His Phe         115 120 125 Ala Val His Ser Lys Asn Asp Ser Arg Gln Ser Glu Arg Leu Thr Gly     130 135 140 Ser Phe Lys Pro Gly Asp Ser Gly Phe Trp Gln Glu Leu Leu Ser Ser 145 150 155 160 Asp Ser Phe Lys Ser Leu Ala Pro Ser Leu Asp Ala Pro Trp Asn Lys                 165 170 175 Gly Ser Arg Gly Leu Lys Thr Val Lys Pro Leu Ala Ser Pro Ala Leu             180 185 190 Asn Gly Pro Ala Asp Ile Ala Ser Leu Pro Gly Phe Gln Asp Thr Phe         195 200 205 Thr Ser Ser Phe Ser Phe Ile Gln Leu Ser Leu Gly Ala Ala Gly Glu     210 215 220 Arg Gly Glu Ala Glu Gly Cys Leu Pro Ser Arg Glu Ala Glu Pro Leu 225 230 235 240 His Gln Arg Pro Gln Glu Met Ala Ala Glu Ala Ser Ser Ser Asp Arg                 245 250 255 Pro His Gly Asp Pro Arg His Leu Trp Thr Phe Ser Leu His Ala Ala             260 265 270 Pro Gly Leu Ala Asp Leu Ala Gln Val Thr Arg Ser Ser Ser Arg Gln         275 280 285 Ser Glu Cys Gly Thr Val Ser Ser Ser Ser Ser Asp Thr Gly Phe Ser     290 295 300 Ser Gln Asp Ala Ser Ser Ala Gly Gly Arg Gly Asp Gln Gly Gly Gly 305 310 315 320 Trp Ala Asp Ala His Gly Trp His Thr Leu Leu Arg Glu Trp Glu Pro                 325 330 335 Met Leu Gln Asp Tyr Leu Leu Ser Asn Arg Arg Gln Leu Glu Val Thr             340 345 350 Ser Leu Ile Leu Lys Leu Gln Lys Cys Gln Glu Lys Val Val Glu Asp         355 360 365 Gly Asp Tyr Asp Thr Ala Glu Thr Leu Arg Gln Arg Leu Glu Glu Leu     370 375 380 Glu Gln Glu Lys Gly Arg Leu Ser Trp Ala Leu Pro Ser Gln Gln Pro 385 390 395 400 Ala Leu Arg Ser Phe Leu Gly Tyr Leu Ala Ala Gln Ile Gln Val Ala                 405 410 415 Leu His Gly Ala Thr Gln Arg Ala Gly Ser Asp Asp Pro Glu Ala Pro             420 425 430 Leu Glu Gly Gln Leu Arg Thr Thr Ala Gln Asp Ser Leu Pro Ala Ser         435 440 445 Ile Thr Arg Arg Asp Trp Leu Ile Arg Glu Lys Gln Arg Leu Gln Lys     450 455 460 Glu Ile Glu Ala Leu Gln Ala Arg Met Ser Ala Leu Glu Ala Lys Glu 465 470 475 480 Lys Arg Leu Ser Gln Glu Leu Glu Glu Gln Glu Val Leu Leu Arg Trp                 485 490 495 Pro Gly Cys Asp Leu Met Ala Leu Val Ala Gln Met Ser Pro Gly Gln             500 505 510 Leu Gln Glu Val Ser Lys Ala Leu Gly Glu Thr Leu Thr Ser Ala Asn         515 520 525 Gln Ala Pro Phe Gln Val Glu Pro Pro Glu Thr Leu Arg Ser Leu Arg     530 535 540 Glu Arg Thr Lys Ser Leu Asn Leu Ala Val Arg Glu Leu Thr Ala Gln 545 550 555 560 Val Cys Ser Gly Glu Lys Leu Cys Ser Ser Leu Arg Arg Arg Leu Ser                 565 570 575 Asp Leu Asp Thr Arg Leu Pro Ala Leu Leu Glu Ala Lys Met Leu Ala             580 585 590 Leu Ser Gly Ser Cys Phe Ser Thr Ala Lys Glu Leu Thr Glu Glu Ile         595 600 605 Trp Ala Leu Ser Ser Glu Arg Glu Gly Leu Glu Met Phe Leu Gly Arg     610 615 620 Leu Leu Ala Leu Ser Ser Arg Asn Ser Arg Arg Leu Gly Ile Val Lys 625 630 635 640 Glu Asp His Leu Arg Cys Arg Gln Asp Leu Ala Leu Gln Asp Ala Ala                 645 650 655 His Lys Thr Arg Met Lys Ala Asn Thr Val Lys Cys Met Glu Val Leu             660 665 670 Glu Gly Gln Leu Ser Ser Cys Arg Cys Pro Leu Leu Gly Arg Val Trp         675 680 685 Lys Ala Asp Leu Glu Thr Cys Gln Leu Leu Met Gln Ser Leu Gln Leu     690 695 700 Gln Glu Ala Gly Ser Ser Pro His Ala Glu Asp Glu Glu Gln Val His 705 710 715 720 Ser Thr Gly Glu Ala Ala Gln Thr Ala Ala Leu Ala Val Pro Arg Thr                 725 730 735 Pro His Pro Glu Glu Glu Lys Ser Pro Leu Gln Val Leu Gln Glu Trp             740 745 750 Asp Thr His Ser Ala Leu Ser Pro His Cys Ala Ala Gly Pro Trp Lys         755 760 765 Glu Asp Ser His Ile Val Ser Ala Glu Val Gly Glu Lys Cys Glu Ala     770 775 780 Ile Gly Val Lys Leu Leu His Leu Glu Asp Gln Leu Leu Gly Ala Met 785 790 795 800 Tyr Ser His Asp Glu Ala Leu Phe Gln Ser Leu Gln Gly Glu Leu Gln                 805 810 815 Thr Val Lys Glu Thr Leu Gln Ala Met Ile Leu Gln Leu Gln Pro Thr             820 825 830 Lys Glu Ala Gly Glu Ala Ser Ala Ser Tyr Pro Thr Ala Gly Ala Gln         835 840 845 Glu Thr Glu Ala     850 <210> 3 <211> 7069 <212> DNA <213> Homo sapiens DISC1 mRNA <400> 3 ggaaggagca ggaggcagcc caggcggagc gggaggagct ggcagcgggg cgcatgccag 60 gcgggggtcc tcagggcgcc ccagccgccg ccggcggcgg cggcgtgagc caccgcgcag 120 gcagccggga ttgcttacca cctgcagcgt gctttcggag gcggcggctg gcacggaggc 180 cgggctacat gagaagctcg acagggcctg ggatcgggtt cctttcccca gcagtgggca 240 cactgttccg gttcccagga ggggtgtctg gcgaggagtc ccaccactcg gagtccaggg 300 ccagacagtg tggccttgac tcgagaggcc tcttggtccg gagccctgtt tccaagagtg 360 cagcagcccc tactgtgacc tctgtgagag gaacctcggc gcactttggg attcagctca 420 gaggtggcac cagattgcct gacaggctta gctggccgtg tggccctggg agtgctgggt 480 ggcagcaaga gtttgcagcc atggatagtt ctgagaccct ggacgccagc tgggaggcag 540 cctgcagcga tggagcaagg cgtgtccggg cagcaggctc tctgccatca gcagagttga 600 gtagcaacag ctgcagccct ggctgtggcc ctgaggtccc cccaacccct cctggctctc 660 acagtgcctt tacctcaagc tttagcttta ttcggctctc gcttggctct gccggggaac 720 gtggagaagc agaaggctgc ccaccatcca gagaggctga gtcccattgc cagagccccc 780 aggagatggg agccaaagct gccagcttgg acgggcctca cgaggacccg cgatgtctct 840 ctcggccctt cagtctcttg gctacacggg tctctgcaga cttggcccag gccgcaagga 900 acagctccag gccagagcgt gacatgcatt ctttaccaga catggaccct ggctcctcca 960 gttctctgga tccctcactg gctggctgtg gtggtgatgg gagcagcggc tcaggggatg 1020 cccactcttg ggacaccctg ctcaggaaat gggagccagt gctgcgggac tgcctgctga 1080 gaaaccggag gcagatggag gtaatatcct taagattaaa acttcagaaa cttcaggaag 1140 atgcagttga gaatgatgat tatgataaag ctgagacgtt acaacaaaga ttagaagacc 1200 tggaacaaga gaaaatcagc ctgcactttc aacttccttc aaggcagcca gctcttagca 1260 gtttcctggg tcacctggca gcacaagtcc aggctgcctt gcgccgtggg gccactcagc 1320 aggccagcgg agatgacacc cacaccccac tgagaatgga gccgaggctg ttggaaccca 1380 ctgctcagga cagcttgcac gtgtccatca cgagacgaga ctggcttctt caggaaaagc 1440 agcagctaca gaaagaaatc gaagctctcc aagcaaggat gtttgtgctg gaagccaaag 1500 atcaacagct gagaagggaa atagaggagc aagagcagca actccagtgg cagggctgcg 1560 acctgacccc actggtgggc cagctgtccc tgggtcagct gcaggaggtc agcaaggcct 1620 tgcaggacac cctggcctca gccggtcaga ttcccttcca tgcagagcca ccggaaacca 1680 taaggagcct ccaggaaaga ataaaatccc tcaacttgtc acttaaagaa atcactacta 1740 aggtgtgtat gagtgagaaa ttctgcagca ccctgaggaa gaaagttaac gatattgaaa 1800 cccaactacc agccttgctt gaagccaaaa tgcatgccat atcaggaaac catttctgga 1860 cggctaaaga cctcaccgag gagattagat cattaacatc agagagagaa gggctggagg 1920 gactcctcag caagctgttg gtgttgagtt ccaggaatgt caaaaagctg ggaagtgtta 1980 aagaagatta caacagactg agaagagaag tggagcacca ggagactgcc tatgaaacaa 2040 gtgtgaagga aaatactatg aagtacatgg aaacacttaa gaataaactg tgcagctgca 2100 agtgtccact gcttgggaaa gtgtgggaag ctgacttgga agcttgtcga ttgcttatcc 2160 agagcctaca gctccaggaa gccaggggaa gcctgtctgt agaagatgag aggcagatgg 2220 atgacttaga gggagctgct cctcctattc cccccaggct ccactccgag gataaaagga 2280 agaccccttt gaaggtattg gaagaatgga agactcacct catcccctct ctgcactgtg 2340 ctggaggtga acagaaagag gaatcttaca tcctttctgc agaacttgga gaaaagtgtg 2400 aagacatagg caagaagcta ttgtacttgg aagatcaact tcacacagca atccacagtc 2460 atgatgaaga tctcattcag tctctcagga gggagctcca gatggtgaag gaaactctgc 2520 aggccatgat cctgcagctc cagccagcaa aggaggcggg agaaagagaa gctgcagctt 2580 cctgcatgac agctggtgtc cacgaagcac aagcctgagg agtgacggga tgggggaggg 2640 aggtgggcca ccatgtttgg acccgggggg ctgctcttcc ctcccccgcc atagctaaga 2700 tgcctgaatc aattacggag atacagagcc ttgaggtctt tcagtggaaa ggtggttcat 2760 gttcattctc atcagtgtga aactgaggag tctgcaattt ggaatatgga gagagagact 2820 gatttgctga atttccttct aaatgtcact caaaaatttc ttttccatgt cattcttggg 2880 aatgtcttcc acaggatttg agaatagttt catctcagcc cccattagag agaagttggg 2940 gtgaattctg gaaaaatgtc tctttttcct gtgccatttg ccttctgctg caacgaaaat 3000 atttcctgat tcaagattct ataaaaagga aaccaagcat aagactctgt catcatacct 3060 gttacacgtt cctacaggtg cacaatctaa gagagctaat taacctcaga gtctggagtt 3120 aacagctttt caccttactt ctcctgtgat ctaatattat cttagaaaaa ttaatatgca 3180 atttccaaaa gatattttgg taagacaaca acctcccagt gatatgccac ctttcaattt 3240 tccttttgtg gcaatgattg catctgaaga aaggatccct gagagtctct gtttcatcag 3300 gacattctga aatttaccca cagtgaggct gtggatggat caggggacct gtataaaatg 3360 tttgagcctg ttccattttc ccgtggaacc tgtttcactc aatgccaggc agtgcagcat 3420 ttaggaaagc agtgcagtac tcagtaaggc agtgcagtac tcagtaacac aatacagtac 3480 tcaggcagtg cagtactcag taagacagtg cagtgctcag taaggcagtg caatactcag 3540 taacagtgta gtactcagta acagtgaagt actcagtaat acagtacagt attcagtaag 3600 gcagtgaagt actcagtaat acaatacagt actcagttag gcagtgcagt actcaggaat 3660 gcagcacagt actcaggcag tgcaatactc agtgcggtac tcagtaacac agtgcagtac 3720 tcagtaacag tgcagtactc agtaacagtg cagtactcag taaggcagtg cagtactcag 3780 taacacagtg cagtactcag taatacagta cagtactcag taaggcagta tggtactcag 3840 taaagcaatg caatgctcag taacacagtg cagtgctcaa taaggcagtg cagtgctcag 3900 taaggcagtg aattgcttag taacacagtg tagtgctcag taggacagca tagtactcag 3960 taacacaggg cagctagtac tcagtactat aagtactgag tacttatata ggcaatgtag 4020 tactcagtaa atcagtgcag tactcagtaa tgcaagggca tttcaggctc ctgctgggct 4080 gcttctttgg cccagctggg actcctattg agacagctgc aaaacaggct gatttcaatt 4140 aggcagcact tcccaaagtg cactgaggaa ggtggcccca agagaagctc tctaaacaaa 4200 ggagtaccct ctctggtcaa gtacctttgg taaatacacc ataccataat atctgcttgg 4260 agaaccacaa tgcacattag catattagtc tgagagagaa cttatagtaa ggaaactcac 4320 ttgattttat ctaacctcaa actttccaag tttaatggat cgtgaatttt tttcatgtaa 4380 ctcctattca tatcccatag atctagtatt gtacagcact gcattctctg aggaagtccc 4440 agtccaaact ctgatttaca tcactttaga aaccacactc acacttttgc agagtgttga 4500 gcttaataac tacctgccac agattggtaa atttaatcca gtggttgttc tgtttgtgct 4560 tctgttctca tttatgtgtt tagggatagt gaggttcctg ccttcactag gatccacgga 4620 tatgagacca tttttgtcat ttcctgaagt cacactggcg tttccagaag gcatctggtg 4680 ctttgctcag ccttccatgc tgtgcagcac ttctgtcctc agtcaaggag atggccatgc 4740 ttaagccagc aattggctgg ggtccaggaa acaaagcaaa agcacaatat gtgaatgtgc 4800 tgattgtgtt ccctatggct ttatctcgag caaaatacac tctacatatt ttaataataa 4860 gtataattag cttgttcctg gacttcattt tcaatgatga accaaattcc tgaattattt 4920 ataattgtgt ctaaagaaaa ttatgaactg gtcacatggc acttggaatc cttgagttaa 4980 ttccagtgaa gcaaaacttg ggaagagtca ggattggcca cattgccaat aacaaattcc 5040 tacttcgaca tatgtctttt caaaaagcct cccagacaca agacatctta accgtcacta 5100 gcccaagtgt tttgtattac tcagacacca tcatgaaata attctgtgag gtcatgatgt 5160 atttgaaaat tctgcaagtt aataactgcc ttgaattgtt tgaacccgaa ataagggttc 5220 tttggtacct ctagtagata gtgtgttcat ttccctgctg caaattttga agtatttggg 5280 caggtgagtc atgttttaac cacaagccat aactcatctg ttgtctttgc ttggtcttag 5340 agtatcattc agaaagtccg ctaagggcca gcgtgcttct tctggctaca caaccttctc 5400 aggacaagcc cactgtctta agccactttg accctgggag acacaggact gtgtatcctc 5460 aatcatacta tacagcagtt tttgtcaggg gaacataaaa atatccaaga gaggttaggg 5520 cttagattta aaagcatcaa aacaacaaca atggaaattt atgttggcga tagccaagac 5580 cacaagcaaa agcacatact ggaaatgatg agttagaatc tgatttgact gggatgtttt 5640 atgagaatgt aagtgtgata ttatactgtc tgccttgctg gaatgctggc tttcaaatgg 5700 tcacccattt ttctttcact ggcctgagtt aggacatgct atcagtaata gtcccagttc 5760 catccaactt tctgaaattt catttttttt tttgagatgg agtctctctc tgtcacccaa 5820 gttggagtgc agtggccccg caatctcggc tcactgcaac ctctacctcc caggttcaag 5880 ctattctact gcctcagcct cccaagtagc tggggttaca ggcatttgcc accgggccct 5940 gatgattttt gtatttttag tagagacagg gcttcaccat gttggctagg agggtctcaa 6000 actcctgacc tcaggcgatc cacccccacc tcggcttccc aaagtgctga gattacaggt 6060 gtgagccacc gcacccggcc aactttctga aatttcaaaa ctgaattgat ccttctccaa 6120 attagtatat actattggaa acttgtcttt ccctgcagta aggctggttt ccccacccca 6180 gaaacatgta acggttggta ccatgctaag cccttgccat gctaagccct ttacagtcat 6240 atcctataat ccccatatca accttataag gaaggtgttt gtagatgatg caactgagcc 6300 ttaagaggac taattccctt tttctaaggc acagagctgg taaaatgtga agtaatagtg 6360 aacctaacag tcagagacag gcagcatgct cttaactagt gctcttccta aagttccttt 6420 aatgtccttt tgagattttg agccatggaa cttacttgtt cacctggcta agaactcatg 6480 gccactgtgg aaatcttggt tagggagtca aagaaactga gcctggggca aacgaggctt 6540 cccacactgc caggggagcc tcactgtgaa gtctaggctc agacaggcat caacaaacct 6600 attcacccca ccatcatcct gatctaacca ttccccagtc atcccaggaa aaccactcac 6660 agcctgacac tgggctgact ttcttgaaga tcctcatcca attggtgttt ttcagaagtg 6720 ttccaatatt atgaattctg tgttgtggag aaaagcaacc atgcatttac tggtcaatgc 6780 cttcttgtat atgtaattca atacttttac ttttaatatc ctcaccttat ctaatctttg 6840 aattttgtca tgtaatttat tgcttcatta aggttacttt ttgttataca aaataaaagc 6900 tgatatccaa ggcatggtgc atcttgatga ttttttgtcc tttgaagtat ggatgataga 6960 aaaatgtatc aggtttattc atctcatctt tctgttacag gatgattaat tgtacagtta 7020 catcacacga aacatttata ataaagtcat gctttagaaa aaaaaaaaa 7069 <210> 4 <211> 2597 <212> DNA <213> Mus musculus DISC1 mRNA <400> 4 cagtcaccgc cgtccggggc gcagccggcc gcacgatgca gggcgggggt ccccggggcg 60 ctccgatcca cagtccgagc cacggcgcag acagtgggca tggcttaccg cctgcagtag 120 cccctcagag gcggcggctg acacggagac caggctacat gagaagcaca gcgggttctg 180 ggatcgggtt cctctctcca gcagtgggca tgccacaccc gagctcagca gggctgacag 240 gccagcagtc ccaacactca cagtccaagg ctggacagtg cggacttgac cctgggagcc 300 actgccaagc ctcactggtg ggcaagcctt ttctcaagag ctcccttgtc cctgctgtgg 360 cctctgaggg ccacctgcac ccagcccagc gctctatgag aaaaagacca gtgcactttg 420 cggttcattc caagaatgac agcagacaat ctgagaggct gactgggtca tttaagcctg 480 gggacagtgg gttttggcaa gaattattat cttcagacag ctttaagtct ctggctccta 540 gccttgatgc accctggaac aagggatcaa ggggcctgaa gactgtgaaa cctctggcat 600 caccggcgtt gaatggcccc gctgatatcg catcccttcc cggcttccaa gacaccttca 660 cttccagctt cagcttcatc caactctccc ttggtgctgc tggagaacgc ggagaagcag 720 aaggttgcct gccatccaga gaggccgaac ctctgcatca gaggccccaa gagatggcag 780 ctgaagcatc tagctcagac aggccccacg gggatcctcg gcatctctgg accttcagtc 840 ttcacgctgc tccaggcttg gcggacttgg ctcaggtgac aaggagcagc agcaggcaat 900 cagaatgtgg cacggtctcc tcctcctcct cggacactgg cttctcttcc caggatgcat 960 cctccgctgg tgggcggggc gaccagggcg gcggctgggc cgatgcccat ggatggcata 1020 cattgctcag ggaatgggag cccatgctgc aggactacct actgagcaac cgcaggcagc 1080 tggaggtcac ttccttaatt ttaaagcttc agaaatgtca agaaaaagtg gtcgaggatg 1140 gcgattacga tactgcagag acattgagac agaggttgga agaactggaa caggagaaag 1200 gccgcctgtc ctgggctctg ccttcacagc aacctgctct tcgcagcttc ttgggttacc 1260 tggcagcaca gatacaggtg gccttgcatg gagccaccca aagggccggc agcgatgatc 1320 cagaagcccc acttgaagga cagctgagga ctaccgccca ggatagcctg cctgcatcca 1380 tcaccaggag ggactggctt attcgagaga aacagcgatt gcagaaggaa atcgaagctc 1440 tccaagcacg gatgtctgcg ctggaggcaa aggaaaaacg gctgagccaa gagttggagg 1500 agcaggaggt gctgctccgg tggccgggct gtgacctgat ggcactggtg gcccagatgt 1560 ccccaggcca gctgcaggag gtcagcaagg ccttgggaga gaccctgacc tctgccaacc 1620 aggctccctt ccaggtggag ccacctgaga ccctcaggag cctccgggaa aggacaaaat 1680 cactgaacct ggctgtcaga gaactcactg ctcaggtgtg ctcaggtgag aagctgtgca 1740 gctctctgag gaggagactc agtgacctcg acaccaggct gcctgccttg ctggaagcca 1800 agatgctggc cctatcagga agctgcttct ccacagccaa ggagctcacg gaggagattt 1860 gggccttgtc gtcagagcgg gaagggctag agatgttcct gggcaggctg ttggcactca 1920 gctccaggaa cagcagaagg ctgggcatcg tcaaagagga tcacctcagg tgcaggcagg 1980 acctggcact ccaggacgcc gcccacaaaa cacgcatgaa ggcaaacact gtgaagtgca 2040 tggaagtgtt ggaaggtcag ctgagcagct gcaggtgccc gctgcttggg agagtgtgga 2100 aagcagactt ggagacttgt cagttgctaa tgcagagcct gcagcttcag gaagcaggca 2160 gcagcccaca cgcagaggac gaggagcagg tgcatagcac aggagaggcc gcccagacag 2220 ctgctctggc tgtccctcga acaccccacc ccgaagaaga aaagtccccc ttgcaggtgc 2280 tccaggagtg ggacacccac tcagctcttt caccacactg tgctgcaggc ccatggaaag 2340 aggattctca catcgtttct gctgaagttg gagaaaagtg cgaagccata ggcgtgaagc 2400 tcctacacct ggaagaccag cttctcggag ccatgtacag tcacgatgag gctctctttc 2460 agtctctcca gggggagctc cagacggtga aggaaacact gcaggccatg atcctgcagc 2520 tccagccaac aaaggaggca ggagaggcct cagcttccta tccgacagct ggtgctcagg 2580 aaaccgaggc ctgaggt 2597

Claims (12)

delete delete delete delete delete delete (a) treating the candidate with cells expressing DISC1 (Disrupted in schizophrenia 1) protein or gene encoding the same in vitro ;
(b) measuring the activity of the DISC1 protein in the cell; And
(c) selecting a substance that increases the activity of the DISC1 protein as a prophylactic or therapeutic substance for stress disorders, compared to a candidate non-treatment group;
Activity in the step (b) is characterized in that the DISC1 protein binds to IP ₃ R1 in MAM competitively inhibits the binding of IP ₃ by measuring the extent to reduce the calcium ion migration from the endoplasmic reticulum to mitochondria, .
The method of claim 7, wherein
The cell is characterized in that the neurons, the screening method.
The method of claim 7, wherein
The candidate material is selected from the group consisting of compounds, microbial cultures or extracts, natural product extracts, nucleic acids, and peptides, the screening method.
The method of claim 9,
The nucleic acid is selected from the group consisting of siRNA, shRNA, microRNA, antisense RNA, aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino, Screening method.
delete delete

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