KR102331240B1 - Diagnosis and therapy of brain neurological disease using SGK3 gene - Google Patents

Abstract

The present invention reveals the relationship between apoptosis caused by autophagy and serum and glucocorticoid-dependent kinase-3 (SGK3) that occurs in hippocampal neural stem cells by stress, and utilizes SGK3 to provide a variety of methods for the diagnosis and treatment of neurological diseases. Compositions and methods may be provided.

Description

Diagnosis and therapy of brain neurological disease using SGK3 gene

The present invention relates to a technique for using the SGK3 gene for diagnosis and treatment of neurological diseases.

Autophagy is one of the survival mechanisms of cells and is recognized as an essential phenomenon for normal cell survival, such as removing unnecessary cells and supplying nutrients to cells.

If autophagy does not normally occur, various diseases including cancer may occur, and there are diseases directly related to autophagy. When autophagy occurs excessively due to stress or external factors, cells may die by autophagy, which can cause very serious diseases.

When apoptosis by autophagy occurs in neural stem cells, various cells of the nervous system cannot differentiate or develop normally, and neurological diseases may occur. In particular, apoptosis caused by autophagy, which occurs in hippocampal neural stem cells (NSC) due to excessive stress, etc., inhibits the normal generation of neurons, and there is a risk of developing neurological diseases such as mental disorders or degenerative brain diseases. However, the relationship between hippocampal neural stem cells and apoptosis caused by autophagy has not been clearly elucidated, so it is difficult to diagnose or treat cranial nervous system diseases caused by autophagy of hippocampal neural stem cells.

Serum and glucocorticoid-inducible kinase (SGK) is known as one of the proteins acting on the function of nerve cells in the brain. However, it is known that there is no association between SGK1 and autophagy. Although Korean Patent Application Laid-Open No. 10-2006-0015467 discloses a technique for using SGK1 to control angiogenesis, it is shown to be a different technique from the treatment of diseases related to nerve cells in the brain.

In addition to SGK1, other genes included in the SGK family and proteins expressed therefrom are also not clearly known what function they have in the nerves of the brain. -inducible kinase-3) and the relationship between apoptosis by autophagy that occurs in hippocampal neurons has been revealed to complete the present invention.

Korean Patent Publication No. 10-2006-0015467

The present invention discloses the relationship between SGK3 (serum and glucocorticoid-inducible kinase-3) and apoptosis by autophagy occurring in adult hippocampal neural stem cells, and utilizes this to provide a technology for diagnosis, treatment and prevention of cranial nervous system diseases do.

In order to achieve the above object, the present invention comprises the steps of treating cells with a candidate therapeutic agent; measuring the SGK3 expression level of the cells or the degree of apoptosis of adult hippocampal neural stem cells; And when the level of SGK3 expression in cells treated with the candidate treatment material is suppressed compared to the untreated control or when the degree of apoptosis of adult hippocampal neural stem cells is reduced compared to the untreated control, the candidate material is selected as a therapeutic agent for brain and nervous system diseases It provides a screening method for a therapeutic agent for a brain nervous system disease, comprising the steps of:

In the method for screening a therapeutic agent for a brain nervous system disease according to an embodiment of the present invention, the cells may be derived from a mammalian model other than a stress-induced human or adult hippocampal neural stem cells treated with a stress hormone.

In one embodiment of the present invention, the brain nervous system disease is selected from the group consisting of panic disorder, memory loss, memory loss, depression, insomnia, nightmares, sleepwalking, cognitive impairment, Alzheimer's, Lou Gehrig's disease, stroke and Huntington's disease. .

The present invention provides a composition for diagnosing a brain nervous system disease, comprising an agent for measuring the level of SGK3 gene expression or SGK3 protein activity.

In one embodiment of the present invention, the agent may include a nucleic acid capable of specifically binding to the mRNA of the SGK3 gene or a derivative thereof.

In one embodiment of the present invention, the preparation may include an antibody capable of specifically binding to SGK3 protein.

The present invention provides a biomarker for diagnosing a neurological disease comprising the SGK3 gene or the SGK3 protein encoded therefrom.

The present invention provides a pharmaceutical composition for the treatment of neurological diseases comprising a substance that inhibits SGK3 gene expression or SGK3 protein activity.

In one embodiment of the present invention, the substance for inhibiting SGK3 gene expression may be a nucleic acid capable of complementary binding to SGK3 mRNA or a derivative thereof.

In one embodiment of the present invention, the SGK3 protein activity inhibitor may be an antibody capable of specifically binding to SGK3.

The present invention provides an animal model of cranial nervous system disease, except for humans, in which autophagic cell death of adult hippocampal neural stem cells is suppressed by deletion of the SGK3 gene in adult hippocampal neural stem cells.

The present invention provides a hippocampal neural stem cell in which apoptosis by autophagy is suppressed by deletion of the SGK3 gene.

According to the present invention, it is possible to prevent apoptosis caused by excessive autophagy in hippocampal neural stem cells by inhibiting SGK3 gene expression or SGK3 protein activity, and thus can be used for diagnosis of various neurological diseases.

In addition, the present invention provides a drug screening method for the treatment of neurological diseases by measuring the degree of inhibiting the death of neural stem cells through the inhibition of SGK3 gene expression or SGK3 protein activity.

In addition, the composition comprising a substance that inhibits the expression of SGK3 gene or the activity of SGK3 protein according to the present invention as an active ingredient inhibits the death of hippocampal neural stem cells, thereby preventing or treating diseases of the brain nervous system.

1 shows changes in mRNA ((A)) and protein ((B)) levels of SGK1, SGK2 and SGK3 when corticosteroids are treated in adult hippocampal neural stem cells.
Figure 2 shows the protein expression confirmation results ((A), (C), (E)) in adult hippocampal neural stem cells in which SGK1, SGK2 and SGK3 are each deficient, and adult hippocampus in which SGK1, SGK2 and SGK3 are each deficient. Shows the degree of apoptosis when neural stem cells are treated with corticosteroids ((B), (D), (F)) , CORT: corticosteroid treatment].
3 shows changes in LC3 protein expression level in adult hippocampal neural stem cells deficient in SGK3 [sgCon: adult hippocampal neural stem cells without SGK3 gene, sgSGK3: adult hippocampal neural stem cells deficient in SGK3 gene, CORT: corticosteroid treatment , BafA1: Bafilomycin treatment].
4 shows the results of observing the LC3 protein expression level of SGK3 non-defective adult hippocampal neural stem cells and adult hippocampal neural stem cells with a fluorescence microscope [CORT: corticosteroid treatment, BafA1: Bafilomycin treatment] .
5 shows the results of observing the expression level of ZFYVE1 protein in adult hippocampal neural stem cells without SGK3 deletion and adult hippocampal neural stem cells without SGK3 deletion under a fluorescence microscope [sgCon: adult hippocampal neural stem cells without SGK3 gene deletion, sgSGK3: SGK3. Adult hippocampal neural stem cells in which the gene is deleted].
Figure 6 shows apoptosis changes by transfection of wild-type (SGK3-WT) and PX homology domain mutant (SGK3-R90A) in SGK3-deficient adult hippocampal neural stem cells (EV: empty vector) .
Figure 7 shows LC3 protein expression changes by transfection of wild-type (SGK3-WT) and PX homology domain mutant (SGK3-R90A) in SGK3-deficient adult hippocampal neural stem cells [CORT: corticosteroids. treatment, BafA1: treatment with Bafilomycin].
8 is a result of observing the expression level of LC3 protein by transfection of wild-type (SGK3-WT) and PX homology domain mutant (SGK3-R90A) in SGK3-deficient adult hippocampal neural stem cells under a fluorescence microscope. [CORT: corticosteroid treatment).
9 shows the results of analyzing SGK3 protein changes in blood cells after applying chronic restrain stress (CRS) for 7 days.

Hereinafter, the present invention will be described in detail. Unless otherwise defined, terms used in this specification should be interpreted as content commonly understood by those of ordinary skill in the art. The drawings and embodiments of the present specification are for those skilled in the art to easily understand and practice the present invention, and content that may obscure the gist of the present invention may be omitted from the drawings and embodiments, and the present invention is limited to the drawings and embodiments it's not going to be

Also, unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein is for the purpose of effectively describing particular embodiments only and is not intended to limit the invention.

Hereinafter, when it is said that any one component "includes" another component, it is not construed as being limited to only the component unless otherwise stated, and it is understood that other components may be further included. should be

The present invention examines the correlation between serum and glucocorticoid-inducible kinase (SGK3), one of the serum and glucocorticoid-inducible kinase family, and apoptosis by autophagy occurring in nerve cells. It is about the technology that reveals and utilizes it.

In particular, according to the present invention, SGK3 can be used to provide a pharmaceutical composition for the treatment and prevention of cranial nervous system diseases, can be used for drug screening for the development of therapeutic agents for cranial nervous system diseases, and can be used for diagnosis of cranial nervous system diseases. . In addition, it is possible to provide cells in which the SGK3 gene is deleted in the hippocampal neural stem cells of the brain and various animal models for research related to brain and nervous system diseases.

CNS diseases, such as mental disorders or degenerative brain diseases, are difficult to treat or prevent because the cause is unknown. When mammals, including humans, experience stress, various hormones are secreted to overcome stress according to signal transmission by the sympathetic nervous system.

In particular, when experiencing mental stress, corticosteroids (glucocorticoids and mineralcorticoids), such as Cortisol, Corticosterone, Aldosterone, Pregnenolone, Various stress hormones such as progesterone and catecholamines are secreted, and excessive or chronic secretion of these stress hormones can cause brain nervous system diseases.

According to the present invention, when exposed to stress, neurogenesis is reduced in the hippocampus of the brain. One of the main causes of this phenomenon is apoptosis caused by autophagy in adult neural stem cells of the hippocampus. Autophagy that occurs in adult hippocampal neural stem cells causes apoptosis of adult hippocampal neural stem cells, which reduces the number of neural stem cells, and can inhibit neuronal development and decrease in neuronal development.

According to the present invention, apoptosis by autophagy of adult hippocampal neural stem cells induced by stress is closely related to SGK3. According to an embodiment of the present invention, SGK3 is known to be involved in cellular ion channel regulation, but may also be involved in autophagy activity in adult neural stem cells of the hippocampus. According to another embodiment of the present invention, SGK3 contains phosphatidylinositol 3-phosphate, PI3P, Ptdins(3)P that regulates cell growth and survival through the PI3K pathway and is involved in autophagy activation. ) and a PX homology domain capable of binding, SGK3 can regulate autophagy that occurs in hippocampal adult neural stem cells.

More specifically, according to the present invention, when SGK3 gene expression or SGK3 protein activity is suppressed, it is possible to suppress apoptosis of adult hippocampal neural stem cells by autophagy, which may be induced by stress. According to an embodiment of the present invention related thereto, in the hippocampal adult neural stem cells deficient in SGK3, apoptosis by autophagy may not be induced even during stress hormone treatment induced by stress.

This function in which SGK3 is involved in autophagy is a characteristic function that distinguishes it from other SGK families. In the case of SGK1, it can regulate neurogenesis by inhibiting cell proliferation, but it is different from controlling apoptosis caused by autophagy like SGK3. can be clearly distinguished. According to an embodiment of the present invention, apoptosis of adult hippocampal neural stem cells by autophagy that occurs during stress induction can be suppressed only when SGK3 is deleted among SGK families, and stress can be suppressed even when SGK1 and SGK2, which are other SGK families, are deleted. The apoptosis of adult hippocampal neural stem cells by autophagy that occurs during induction is not inhibited.

According to another embodiment of the present invention, in cells deficient in SGK3, an increase in LC3 protein, known as a marker of autophagy, does not occur even after treatment with a stress hormone. According to another embodiment of the present invention, in cells deficient in SGK3, the expression of ZFYVE1, a functional group of PI3P involved in the initiation of autophagy, is hardly increased even after treatment with a stress hormone.

That is, unlike other SGK families, SGK3 can perform a special function that can mediate apoptosis by autophagy induced by stress in neural stem cells of the brain.

The above characteristics of the SGK3 gene can be utilized in the development of various compositions and methods for diagnosis, treatment, prevention, improvement, etc. of brain and nervous system diseases.

According to an embodiment of the present invention, it is possible to provide a screening method for a therapeutic agent for a brain nervous system disease in which the degree of inhibition of SGK3 expression or the degree of apoptosis of adult hippocampal neural stem cells is measured by treating a therapeutic agent candidate. Specifically, treating the cell with a candidate therapeutic agent; measuring the SGK3 expression level of the cells or the degree of apoptosis of adult hippocampal neural stem cells; And when the level of SGK3 expression in cells treated with the candidate treatment material is suppressed compared to the untreated control or when the degree of apoptosis of adult hippocampal neural stem cells is reduced compared to the untreated control, the candidate material is selected as a therapeutic agent for brain and nervous system diseases step; may include.

When SGK3 expression is suppressed in cells treated with the therapeutic candidate substance, it can be selected as a preventive or therapeutic agent for brain nervous system diseases caused by proliferation or growth disorders of nerve cells by inhibiting apoptosis caused by autophagy.

The therapeutic candidate may be a natural compound, a synthetic compound, DNA, RNA, a peptide, an enzyme, a ligand, a cell extract, a non-peptidic compound, a fermentation product, a plant extract, or plasma. Preferably, it can be obtained from a library of synthetic or natural compounds. Libraries of such compounds can be obtained by methods known in the art. Libraries of synthetic compounds are commercially available from Maybridge Chemical Co. (UK), Comgenex (USA), Brandon Associates (USA), Microsource (USA), and Sigma-Aldrich (USA), and libraries of natural compounds are available from Pan Laboratories (USA). ) and MycoSearch (USA).

The treatment target of the therapeutic candidate substance is a stress-induced human; non-human mammalian models; neural stem cells of the brain; Or hippocampal adult neural stem cells are preferred, but are not particularly limited thereto.

In the screening method for a therapeutic agent for a brain nervous system disease using SGK3, a method for confirming that the therapeutic agent is effective in treating, improving or preventing a brain nervous system disease is not particularly limited as long as it is a method generally used in the field to which the present invention belongs. Specifically, it is desirable to confirm the effect of a candidate therapeutic agent through the relationship between SGK3 and autophagy. For example, it is possible to measure the degree of death of adult hippocampal neural stem cells that are caused by stress or when treated with stress hormones. Alternatively, the level of a marker of autophagy such as LC3 protein may be measured, the level of an autophagy initiator such as ZFYVE1 may be checked, or the level of autophagosome formation may be confirmed. In addition, the level of SGK3 mRNA may be measured to determine the degree of SGK3 protein activity.

In one embodiment of the present invention, it is possible to provide a composition for diagnosing a neurological disease comprising an agent for measuring SGK3 gene expression or activity level of SGK3 protein.

According to the present invention, the expression of SGK3 together with SGK1 can be increased in animals exposed to stress environments such as direct treatment with stress hormones, brain neural stem cells, and blood. Since SGK1 is not related to autophagy, the onset or progression of neurological diseases can be diagnosed by directly measuring the level of SGK3 gene expression or SGK3 protein activity.

The agent used for measuring the expression level of the SGK3 gene in the composition for diagnosing a brain nervous system disease is not particularly limited as long as it is a nucleic acid capable of specifically binding to the mRNA of the SGK3 gene or a derivative thereof. For example, miRNA (microRNA), siRNA (small interference RNA), shRNA (short hairpin RNA), aptamer or PNA (peptide nucleic acid) designed to complementarily bind all or part of SGK3 mRNA. ) may be used as the formulation, but is not limited thereto.

In one embodiment of the present invention, "miRNA (microRNA)" refers to an untranslated RNA of approximately 22 nt that acts as a post-transcriptional repressor through base binding with the 3' untranslated region (UTR) region of mRNA. . The method for preparing miRNA is not particularly limited, and methods known in the art may be used.

"siRNA" refers to a small RNA fragment of 21-25 nucleotides in size produced by cleaving double-stranded RNA by Dicer, specifically binding to mRNA having a complementary sequence and inhibiting expression. In the present invention, the siRNA specifically binds to the mRNA of the SGK3 gene to inhibit the expression of the gene, and this siRNA may be chemically or enzymatically synthesized. The method for preparing siRNA is not particularly limited, and methods known in the art may be used.

The siRNA used in the present invention is a complete form having a polynucleotide pairing per se, that is, a form introduced into cells through two transformation processes in which siRNA is directly synthesized in vitro, or one form to have this form after administration in vivo A form in which the single-stranded oligonucleotide fragment and its reverse complement can be derived from a single-stranded polynucleotide separated by a spacer, for example, an siRNA expression vector prepared so that the siRNA is expressed in a cell or a PCR-derived siRNA The expression cassette may be in the form of being introduced into a cell through a transformation or infection process. The determination of a method for preparing an siRNA and introducing it into a cell or animal may vary depending on the purpose and the cellular biological function of the target gene product.

As an embodiment of the present invention, 5'-GAAGCGAATGGTTCGTCTTCAGG-3' (SEQ ID NO: 3) was used as an agent for inhibiting the expression of SGK3 or a gene encoding the shRNA, but is not particularly limited thereto.

In one embodiment of the present invention, "shRNA" is a single-stranded RNA having a length of 45 to 70 nucleotides, and an oligo connecting a 3-10 base linker between the sense of the target gene siRNA base sequence and the complementary nonsense sequence. After synthesizing DNA, cloning it into a plasmid vector or inserting shRNA into retroviruses, lentivirus and adenovirus, and expressing shRNA with a looped hairpin structure is produced and intracellularly It means that it is converted to siRNA by Dicer and shows RNAi effect. Expression constructs/vectors including small hairpin RNA (shRNA) can be prepared by methods known in the art.

The agent for measuring the activity level of the SGK3 protein is not particularly limited as long as it is a material capable of binding to the SGK3 protein, and may preferably be an antibody that specifically binds to the SGK3 protein. In order to measure the expression level of the SGK3 gene or the activity level of the SGK3 protein, various known markers may be used as needed.

As another embodiment for diagnosing cranial nervous system disease, the SGK3 gene or SGK3 protein encoded therefrom may be provided as a biomarker for diagnosing cranial nervous system disease.

Alternatively, the reliability or accuracy of the prediction of the onset and progression of a brain nervous system disease may be improved through the combination of the composition for diagnosis or the biomarker. For example, it may be a combination of an siRNA capable of complementary binding to mRNA of SGK3 as an agent inhibiting the expression of SGK3 gene and an antibody for measuring the level of SGK3 activity.

In one embodiment of the present invention, it is possible to provide a pharmaceutical composition for treating a neurological disease comprising a substance that inhibits SGK3 gene expression or SGK3 protein activity.

According to the present invention, since SGK3 is involved in apoptosis caused by autophagy occurring in adult hippocampal neural stem cells, deletion of SGK3 can inhibit apoptosis of adult hippocampal neural stem cells by autophagy, thereby inhibiting SGK3 gene expression or SGK3 In the case of an agent that inhibits the activity of a protein, it can be used as a composition for the treatment of diseases of the brain nervous system.

The agent for inhibiting SGK3 gene expression is not particularly limited as long as it is a nucleic acid capable of complementary binding to SGK3 mRNA or a derivative thereof. Nucleic acids or derivatives thereof that bind to the mRNA of the SGK3 gene block translation, which is a protein synthesis process, thereby preventing the normal functioning of the mRNA. As an example of a nucleic acid binding to mRNA or a derivative thereof, miRNA, siRNA, aptamer or PNA (peptide nucleic acid) designed to complementarily bind to part or all of SGK3 mRNA may be used.

The agent for inhibiting the activity level of the SGK3 protein is not particularly limited as long as it is a substance capable of inhibiting the function of the SGK3 protein, and may preferably be an antibody that specifically binds to the SGK3 protein. In order to measure the expression of SGK3 gene or inhibition of SGK3 protein activity, various well-known markers may be used as needed.

In one embodiment of the present invention, the antibody specifically binding to SGK3 protein is selected by domain-searching a region with high specificity in the peptide sequence of SGK3, and binding a carrier protein to the selected peptide as an antigen. Can be used. The rabbit is immunized with the carrier-peptide complex as an antigen, and subcutaneously injected through the first, second and third steps to measure the antigen immune response. At each stage, blood is collected and serum can be obtained to check whether the antibody is produced. The immune response induced by antigen injection can determine the level of antibody present in the rabbit's serum through ELISA. For purification of the antibody, the antigen is attached to an affinity gel, a column is made to separate the antibody, and protein A It can be purified through a column method using

In one embodiment of the present invention, the SGK3 gene is deleted in adult hippocampal neural stem cells to suppress autophagic cell death of adult hippocampal neural stem cells, and it is possible to provide an animal model of cranial nervous system disease except for humans. Specifically, for example, in one embodiment of the present invention, SGK3 gene-deficient mice are produced using modified embryonic stem cells. The modified embryonic stem cells are made in a form in which loxP sites are inserted on both sides of exons 4 and 5 of the SGK3 gene, and the modified embryonic stem cells can be injected into a pseudo-pregnant mouse to produce a genetically modified mouse.

Examples of experimental mice that can be used for the preparation of the animal model according to the present invention include ICR or DDY belonging to a closed colony according to phylogenetic classification; BALB/cA, C57BL/6N, C3H/HeN, DBA/2N or CBA/N belonging to Inbred; BDF1 (C57BL/6 x DBA/2), CDF1 (CBA/N x DBA/2) or B6C3F1 (C57BL/6 x C3H/HeN) belonging to the hybrid; Any one used as an experimental mouse, such as BALB/c-nu or C,B-17SCID belonging to the Mutant family, can be used.

In a preferred embodiment of the present invention, the SGK3 gene-deficient mouse is crossed with a Nestin-Cre/ERT2 mouse with a Nestin promoter expressed in hippocampal neural stem cells to confirm the role of SGK3 in autophagy apoptosis and brain disease. In this crossbred mouse, Cre gene expression is induced by injection of tamoxifen, and the SGK3 gene deletion is selectively performed at a desired time, thereby conducting a study on the function of SGK3 in adult hippocampal neural stem cells.

Specifically, an animal model in which the adult hippocampal neural stem cell SGK3 gene is deficient has resistance to cranial nervous system diseases that can be induced by stress, so it can be used for research related to cranial nervous system diseases.

In addition, as an embodiment of the present invention, adult hippocampal neural stem cells in which apoptosis by autophagy is suppressed by deletion of the SGK3 gene, not in an animal model, may be used as a research model.

In the present invention, the cranial nervous system disease may include diseases that may be caused by abnormalities occurring in nerve cells, such as damage to nerve cells in the brain, disorder in the development of nerve cells, disorder in growth of nerve cells, or disorder in proliferation of nerve cells. Preferably, it is a mental disease or degenerative brain disease among stress-related diseases caused by stress or stress hormones. Stress-related diseases refer to all diseases induced by stress that change or affect the emotions, behavioral patterns, thinking patterns, and cognitive levels of animals including humans. More specifically, panic disorder, memory loss, memory loss, depression, insomnia, nightmares, sleep disorders such as sleepwalking, cognitive disorders, Alzheimer's disease, Lou Gehrig's disease, stroke and Huntington's disease, etc., but are not limited thereto.

In the case of preparing the pharmaceutical composition in one embodiment of the present invention, the form or type is not particularly limited, and may be prepared in various formulations according to the administration or intake method. Examples of the dosage form may include various dosage forms such as external preparations or injections in addition to oral forms such as tablets, syrups, capsules, and powders.

The composition of the present invention may be administered orally or parenterally according to a desired method, and when administered parenterally, external or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection It is preferable to select The dosage varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

In addition, excipients, carriers, or diluents commonly used in the art when preparing the pharmaceutical composition may be suitably further included. Examples of carriers or excipients include various compounds including sorbitol, sugar alcohols such as xylitol or malthiol, monosaccharides, polysaccharides, gelatin, water, alcohol, starch, edible rubbers, mineral oil, and the like, and mixtures thereof. The types of wetting agents, surfactants, excipients, fillers, binders, etc. generally used during formulation are not particularly limited.

As another embodiment of the present invention, a health functional food, feed, or food additive for improving or preventing brain and nervous system diseases may also be provided, and in addition, it may be implemented in various other forms or methods.

In the case of manufacturing a health functional food or food additive according to an embodiment of the present invention, the form or type is not particularly limited and may be prepared by mixing with other ingredients capable of increasing anti-inflammatory activity. There is no particular limitation on the type of food, for example, drinks, vitamins, various beverages, processed meat, processed sausages, processed noodles, processed oils and fats, candy, gum, etc. Foods manufactured and sold in general may be included.

Preferably, it is preferable to prepare it as a drink or other beverage type composition, and various flavoring agents such as sweeteners may be included as additional ingredients to help ingestion. It may include carbohydrates of various flavors, such as simple sugar, fructose, and glucose, and may also include sugar alcohols such as sorbitol or xylitol. Examples of the sweetener include not only natural sweeteners but also synthetic sweeteners such as saccharin.

Examples for carrying out the present invention will be described below. The examples correspond to one example for carrying out the present invention, and the present invention should not be construed as being limited by the examples.

1-1. Preparation of SGK family-deficient (SGK3 knock-out) hippocampal neural stem cells including SGK3

All procedures in the management and use of laboratory animals were approved by the Animal Care and Use Committee of DGIST, and all animals were bred and used in DGIST animal facilities in the absence of specific pathogens.

Adult hippocampal neural stem cells (HCN cells, hereinafter may be referred to as 'HCN') collected from 8-week-old female SD rats (Sprague-Dawley rats) were prepared and N ₂ and basic fibroblast growth factor (bFGF); Pepro tech) added DMEM (Dulbecco's modified Eagle's medium) / serum-free medium containing F-12, cultured in a 37 °C incubator. Cells were subcultured every 3-4 days.

Deletion of ULK1, SGK1, SGK2, and SGK3 in HCN cells is caused by pRGEN_cas9_Hyg/EGFP CMV, dRGEN-Ulk1 (GATTGGACACGGCGCTTTCGCGG), -SGK1 (GCTTCTGAATAAAATCGTTCAGG), -SGK2 (GTCCATCATGAATAAAGG guide by RNAs GG), -SGK2 (GTCCATCATGAATAAGGGGAGG). Transfection was accomplished by a CRISPR/Cas-9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated genes-9) system. After performing each gene deletion, culture was performed for 24 hours, and transformation was confirmed and selected for 72 hours using 300 μg/ml of hygromycin.

1-2. Confirmation of mRNA and protein levels of SGK1, SGK2 and SGK3 by stress hormone in adult hippocampal neural stem cells

HCN cells were treated with corticosteroids (hereinafter, may be referred to as 'CORT') according to the method below, and changes in the mRNA and protein levels of SGK1, SGK2, and SGK3 were confirmed.

For the mRNA level, RNA extraction was performed using QIAZol lysis reagent for each sample. After the extracted RNA was annealed with OligodT at 65 ° C, it was mixed with reverse transcriptase and dNTP, and then reverse transcribed from 25 ° C to 42 ° C using a PCR machine for 30 minutes for stable cDNA synthesis. After that, heat inactivation was performed at 85°C. After adding SGK1, SGK3, SGK3 primers and SYBR-GREEN dye to the DNA for each condition, the mRNA level was checked by measuring the amplification and amount of the SGK family through real-time polymerase chain reaction (RT-PCR).

Protein levels were confirmed by western blotting. Cells were lysed with RIPA buffer (radioimmunoprecipitation assay) (Sigma-Aldrich) containing 1X protease and a phosphatase inhibitor cocktail (Thermo) on ice for 15 min. After centrifugation (12,000 rpm, 15 minutes), the supernatant was collected and the protein concentration of the lysate was measured using BCA protein assay reagent (Thermo). For analysis, 10-20 μg of total protein was added per sample. The protein was transferred to a polyvinylidene fluoride membrane, and the membrane was blocked with TBS containing 5% skim milk powder and 0.1% Tween 20 at room temperature for 1 hour. Thereafter, the membrane was treated with a suitable primary antibody and incubated overnight, followed by treatment with a peroxidase-conjugated secondary antibody and incubated for 1 hour. Next, protein levels were analyzed using a detection kit (supersignal chemiluminescence; Thermo).

Referring to FIG. 1 , it can be seen that the level of mRNA and protein of SGK1 and SGK3 increased during CORT treatment, but the level of SGK2 hardly changed compared to before treatment.

1-3. Confirmation of apoptosis by stress hormone in SGK1, SGK2 and SGK3 deficient adult juvenile neural stem cells

SGK1, SGK2 and SGK3 deficient HCN cells were treated with stress hormones and the degree of apoptosis was confirmed.

SGK family deficient cells were placed in a 96-well plate at 1.5X10 ⁵ cells/ml, CORT was treated with 200 uM, and 48 hours later, it was confirmed.

For cell death assay, cells were stained with Hoechst 33342 (Invitrogen) and PI (propidium iodide; Sigma) and imaged using a fluorescence microscope (Axiovert 40 CFL; Carl Zeiss), the percentage of cell death was Cell death (%) was calculated according to the formula below.

Cell death (%) = (PI [red] positive cells / Hoechst [blue] positive cells) × 100

As a result of apoptosis analysis through FIG. 2 , it can be confirmed that, unlike SGK1 and SGK2, cell death by CORT, a stress hormone, is inhibited when SGK3 is absent.

1-4. Confirmation of changes in the level of autophagy markers in SGK3-deficient adult hippocampal neural stem cells

SGK3-deficient HCN cells were treated with a stress hormone and the level of LC3 protein, known as an autophagy marker, was checked. LC3 (MAP1LC3B) is an autophagy marker protein, and the increase in LC3-II and the degree of autophagy are proportional.

To confirm this, Control HCN cells and SGK3-deficient cells were cultured on a ^{35 mm plate at 2×10 5} cell/ml, respectively, under CORT-treated and non-CORT-treated conditions for 48 hours, and then the cells were collected and Western blotting was performed. Quantitative increase of LC3-II, a lipidation form of LC3, was confirmed as an autophagy marker.

3, as a result of confirming the level of LC3-II protein in the control group, the level of LC3-II increased when the stress hormone was treated, but the level of LC3-II was hardly increased in the SGK3-deficient cells. (sgCon) In HCN cells, LC3 puncta formation was increased during stress hormone treatment, but LC3 puncta formation was decreased in SGK3-deficient cells. Fluorescence-labeled LC3 with RFP is observed in the form of puncta in autophagosomes, and the increase in fluorescently-labeled LC3 puncta and the degree of autophagy are proportional.

1-5. Identification of proteins involved in autophagy initiation in SGK3-deficient adult hippocampal neural stem cells.

The ZFYVE1 protein was observed as a marker protein for the initial structure of the autophagosome, which is known to be involved in the initiation of autophagy and treatment of SGK3-deficient HCN cells with stress hormones.

To confirm this, a vector expressing mCherry-ZFYVE1 in SGK3-deficient HCN cells was introduced into the cells using Lipofectamine, incubated for 24 hours, and after CORT treatment, the number of ZFYVE1 puncta fluorescence expression was confirmed using a Confocal 780 microscope (Zeiss). .

As a result of the observation of ZFYVE1 through FIG. 5 , it can be confirmed that, in control (sgCon) HCN cells, the formation of ZFYVE1 puncta was increased during stress hormone treatment, but in SGK3-deficient cells, the number of ZFYVE1 puncta decreased compared to the control group. The increase in the fluorescently labeled ZFYVE1 puncta is proportional to the degree of autophagy induction.

1-6. Confirmation of changes in apoptosis by transfection of SGK3-WT and PX homology domain mutant SGK3-R90A in SGK3-deficient adult hippocampal neural stem cells.

To confirm this, SGK3-WT vector and SGK3-R90A expression vector were introduced into SGK3-deficient cells using lipofectamine. After culturing for 24 hours, CORT was treated, and ^{after seeding at 1.5×10 5} cell/ml in 96 well plates, 48 hours later, the degree of apoptosis was confirmed using Hoechst/PI technique.

As a result of confirming changes in apoptosis by transfection of SGK3-WT and SGK3-R90A through FIG. 6 , the degree of apoptosis increased only during transfection of SGK3-WT, so that the PX homology domain contributed to apoptosis induced by stress hormone. involvement can be confirmed.

1-7. Confirmation of changes in autophagy level by transfection of SGK3-WT and PX homology domain mutant SGK3-R90A in SGK3-deficient adult hippocampal neural stem cells.

To confirm this, after gene injection was carried out in the same way as apoptosis, the cells were picked up 48 hours after CORT treatment on a 35 mm plate, and the difference in LC3-II protein expression, which can confirm the level of autophagy, was measured using Western blotting technique. to confirm.

Referring to FIG. 7 , as a result of confirming changes in apoptosis by transfection of SGK3-WT and SGK3-R90A, the level of LC3-II was increased only during transfection of SGK3-WT, and LC3-R90A transfection through FIG. 8 . It can be confirmed by fluorescence microscopy that puncta does not appear, and it can be confirmed that the PX homology domain is involved in apoptosis by autophagy induced by stress hormone.

1-8. Confirmation of SGK3 protein expression change for chronic restraint stress (CBS).

A 4-week-old mouse was subjected to chronic restraint stress by restraining it in a PVC (Polyvinylchloride) tube blocked on both sides for 6 hours every day for 7 days. . 9 , it can be confirmed that the expression of SGK3 protein was increased in the stressed experimental group compared to the control group that was not subjected to chronic restraint stress. This means that the change in SGK3 expression can be confirmed through blood, which means that it can be easily utilized for the diagnosis of neurological diseases according to the present invention.

Measurement of the degree of death of SGK3 inhibitors on hippocampal neural stem cells

By treating adult hippocampal neural stem cells with the SGK3 inhibitor library, the degree of apoptosis was confirmed, and those that inhibit apoptosis were selected. As the SGK3 inhibitor selected in this Example, shRNA (GAAGCGAATGGTTCGTCTTCAGG; SEQ ID NO: 3) (Toolzen) was used to knock-down the expression of SGK3. Accordingly, it was confirmed that apoptosis was inhibited compared to the control due to SGK3 siRNA treatment.

Through the above results, it can be seen that the SGK3 protein is reduced by changing the SGK3 gene in the active state to the inactive state by binding to shRNA in the neural stem cells, and suppressing the expression of SGK3 mRNA as a protein, which is immediately treated with the SGK3 antibody or When SGK3 was knocked down to suppress expression, it was confirmed that it was effective in preventing or treating degenerative brain diseases caused by neuronal proliferation disorders, etc. by suppressing the death of hippocampal neural stem cells due to autophagy activation.

Construction of SGK3 gene-deficient mice

In order to generate SGK3 deletion specifically in hippocampal neural stem cells, the modified embryonic stem cells in which loxP sites are inserted on both sides of exons 4 and 5 of the SGK3 gene were injected into pseudo-pregnant mice, and the transgenic mouse SGK3 ^flox/flox was obtained. Among SGK3 heterozygous knockout mice obtained in the first generation by crossing this with Nestin-Cre/ERT2 mice that specifically express Cre in hippocampal neural stem cells, SGK3 ^flox/+ ; Nestin-Cre / by breeding a mouse with SGK3 ^{flox / flox} mouse with mouse genotype ERT2 from the second generation born SGK3 ^{flox / flox;} SGK3 homozygous knockout mice (-/-) having the genotype of Nestin-Cre/ERT2 were obtained. In SGK3 homozygous knockout mice, Cre gene expression was induced by tamoxifen injection, and it can be used to time-selectively study the effects of SGK3 gene deletion on adult hippocampal neural stem cells.

Preparation Example 1. Preparation of a pharmaceutical composition comprising an SGK3 inhibitor

It is prepared according to a conventional manufacturing method by mixing the following components including an inhibitor of SGK3. Components according to the formulation form are shown in Tables 1 to 5 below.

A powder can be prepared by mixing the above ingredients according to a conventional manufacturing method and filling the airtight cloth.

According to a conventional manufacturing method, the above ingredients may be mixed and tableted to prepare a tablet.

The capsules can be prepared by mixing the above ingredients according to a conventional manufacturing method and filling the gelatin capsules.

According to a conventional manufacturing method, it can be prepared in the above content per ampoule.

After mixing each component according to a conventional liquid preparation method, dissolving it in purified water and adding a predetermined flavor, the volume of the entire composition is adjusted to 100 ml. A sterilized solution can be prepared by filling in a brown bottle.

In the above, an embodiment of the present invention has been described, but those of ordinary skill in the art may add, change or delete components, etc. without departing from the scope of the technical idea of the present invention described in the claims. Accordingly, the present invention can be variously modified.

<110> DAEGU GYEONGBUK INSTITUTE OF SCIENCE & TE <120> Diagnosis and therapy of brain neurological disease using SGK3 gene <130> P20030390223 <150> KR 10-2019-0032275 <151> 2019-03-21 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 2391 <212> DNA <213> Homo sapiens <400> 1 ggtgtgctct tgagggatta aatgcaaaga gatcacacca tggactacaa ggaaagctgc 60 ccaagtgtaa gcattcccag ctccgatgaa cacaagagaga aaaagaagag gtttactgtt 120 tataaagttc tggtttcagt gggaagaagt gaatggtttg tcttcaggag atatgcagag 180 tttgataaac tttataacac tttaaaaaaa cagtttcctg ctatggccct gaagatcct 240 gccaagagaa tatttggtga taattttgat ccagatttta ttaaacaaag acgagcagga 300 ctaaacgaat tcattcagaa cctagttagg tatccagaac tttataacca tccagatgtc 360 agagcattcc ttcaaatgga cagtccaaaa caccagtcag atccatctga agatgaggat 420 gaaagaagtt ctcagaagct acactctacc tcacagaaca tcaacctggg accgtctgga 480 aatcctcatg ccaaaccaac tgactttgat ttcttaaaag ttattggaaa aggcagcttt 540 ggcaaggttc ttcttgcaaa acggaaactg gatggaaaat tttatgctgt caaagtgtta 600 cagaaaaaaa tagttctcaa cagaaaagag caaaaacata ttatggctga acgtaatgtg 660 ctcttgaaaa atgtgaaaca tccgtttttg gttggattgc attattcctt ccaaacaact 720 gaaaagcttt attttgttct ggattttgtt aatggagggg agcttttttt ccacttacaa 780 agagaacggt cctttcctga gcacagagct aggttttacg ctgctgaaat tgctagtgca 840 ttgggttact tacattccat caaaatagta tacagagact tgaaaccaga aaatattctt 900 ttggattcag taggacatgt tgtcttaaca gattttgggc tttgtaaaga aggaattgct 960 atttctgaca ccactaccac attttgtggg acaccagagt atcttgcacc tgaagtaatt 1020 agaaaacagc cctatgacaa tactgtagat tggtggtgcc ttggggctgt tctgtatgaa 1080 atgctgtatg gattgcctcc tttttattgc cgagatgttg ctgaaatgta tgacaatatc 1140 cttcacaaac ccctaagttt gaggccagga gtgagtctta cagcctggtc cattctggaa 1200 gaactcctag aaaaagacag gcaaaatcga cttggtgcca aggaagactt tcttgaaatt 1260 cagaatcatc ctttttttga atcactcagc tgggctgacc ttgtacaaaa gaagatcca 1320 ccaccattta atcctaatgt ggctggacca gatgatatca gaaactttga cacagcattt 1380 acagaagaaa cagttccata ttctgtgtgt gtatcttctg actattctat agtgaatgcc 1440 agtgtattgg aggcagatga tgcattcgtt ggtttctctt atgcacctcc ttcagaagac 1500 ttaatttttgt gagcagtttg ccattcagaa accattgagc aaaataagtc tatagatggg 1560 actgaaactt ctatttgtgt gaatatattc aaatatgtat aactagtgcc tcatttttat 1620 atgtaatgat gaaaactatg aaaaaatgta ttttcttcta tgtgcaagaa aaatagggca 1680 tttcaaagag ctgttttgat taaaatttat attcttgttt aataagctta tttttaaaca 1740 atttaaaagc tattattctt agcattaacc tatttttaaa gaaacctttt ttgctattga 1800 ctgttttttc cctctaagtt tacactaaca tctacccaag atagactgtt ttttaacagt 1860 caatttcagt tcagctaaca tatattaata cctttgtaac tctttgctat ggcttttgtt 1920 atcacaccaa aactatgcaa ttggtacat gttgtttaag aagaaaccgt atttttccat 1980 gataaatcac tgtttgaaat atttggttca tggtatgatc gaaatgtaaa agcataatta 2040 acacatggc tgctagttaa caattggaat aactttattc tgcagatcat ttaagaagta 2100 acaggccggg cgcggtggct cacgcctgta atcccagcac tttgggaggc tgaggcgggc 2160 agatcacctg aggtcaggag ttggagacca gcctgaccaa catggacaaa ccccgtctct 2220 actaaaaata caaaattggc agggtgtggt ggcacatgcc tataatccca gctacttggg 2280 aggctaaggc aggagaatcg cttgaacccg ggaggcggag 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Asp Gly Lys Phe Tyr Ala Val Lys Val 180 185 190 Leu Gln Lys Lys Ile Val Leu Asn Arg Lys Glu Gln Lys His Ile Met 195 200 205 Ala Glu Arg Asn Val Leu Leu Lys Asn Val Lys His Pro Phe Leu Val 210 215 220 Gly Leu His Tyr Ser Phe Gln Thr Thr Glu Lys Leu Tyr Phe Val Leu 225 230 235 240 Asp Phe Val Asn Gly Gly Glu Leu Phe Phe His Leu Gln Arg Glu Arg 245 250 255 Ser Phe Pro Glu His Arg Ala Arg Phe Tyr Ala Ala Glu Ile Ala Ser 260 265 270 Ala Leu Gly Tyr Leu His Ser Ile Lys Ile Val Tyr Arg Asp Leu Lys 275 280 285 Pro Glu Asn Ile Leu Leu Asp Ser Val Gly His Val Val Leu Thr Asp 290 295 300 Phe Gly Leu Cys Lys Glu Gly Ile Ala Ile Ser Asp Thr Thr Thr Thr 305 310 315 320 Phe Cys Gly Thr Pro Glu Tyr Leu Ala Pro Glu Val Ile Arg Lys Gln 325 330 335 Pro Tyr Asp Asn Thr Val Asp Trp Trp Cys Leu Gly Ala Val Leu Tyr 340 345 350 Glu Met Leu Tyr Gly Leu Pro Pro Phe Tyr Cys Arg Asp Val Ala Glu 355 360 365 Met Tyr Asp Asn Ile Leu His Lys Pro Leu Ser Leu Arg Pro Gly Val 370 375 380 Ser Leu Thr Ala Trp Ser Ile Leu 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Claims (14)

treating the cells with a therapeutic candidate;
Measuring the expression level of SGK3 in the cells; And
When the expression level of SGK3 in cells treated with the candidate therapeutic substance is suppressed compared to the untreated control, selecting the candidate substance as a therapeutic agent for a brain nervous system disease; The method of claim 1,
The cell is a stress hormone-treated adult hippocampal neural stem cells derived from a mammalian model other than a stress-induced human, a screening method for a therapeutic agent for a brain nervous system disease. The method of claim 1,
The cranial nervous system disease is selected from the group consisting of panic disorder, memory loss, memory loss, depression, insomnia, nightmares, sleepwalking, cognitive impairment, Alzheimer's, Lou Gehrig, stroke and Huntington's disease. The method of claim 1,
Measuring the SGK3 expression level of the cells further comprises measuring the degree of apoptosis of adult hippocampal neural stem cells,
When the level of SGK3 expression in the cells treated with the candidate treatment material is suppressed compared to the untreated control group, the step of selecting the candidate material as a treatment for brain nervous system disease is when the degree of apoptosis of adult hippocampal neural stem cells is reduced compared to the untreated control group , The screening method for a therapeutic agent for a brain nervous system disease, further comprising the step of selecting the candidate substance as a therapeutic agent for a brain nervous system disease. A composition for diagnosing brain and nervous system diseases, comprising an agent for measuring SGK3 gene expression or activity level of SGK3 protein. 6. The method of claim 5,
The composition for diagnosing a brain nervous system disease, wherein the agent comprises a nucleic acid capable of specifically binding to the mRNA of the SGK3 gene or a derivative thereof. 6. The method of claim 5,
The agent is a composition for diagnosing a brain nervous system disease comprising an antibody capable of specifically binding to the SGK3 protein. 6. The method of claim 5,
The cranial nervous system disease is selected from the group consisting of panic disorder, memory loss, memory loss, depression, insomnia, nightmares, sleepwalking, cognitive impairment, Alzheimer's, Lou Gehrig, stroke and Huntington's disease. A biomarker for diagnosing a neurological disease comprising the SGK3 gene or the SGK3 protein encoded therefrom. a substance that inhibits SGK3 gene expression or SGK3 protein activity, wherein the SGK3 gene expression inhibitory substance is a nucleic acid capable of complementary binding to SGK3 mRNA or a derivative thereof, and the SGK3 protein activity inhibitory substance is specific for SGK3 A pharmaceutical composition for treating brain and nervous system diseases, which is an antibody that can be physically bound. 11. The method of claim 10,
The cranial nervous system disease is selected from the group consisting of panic disorder, memory loss, memory loss, depression, insomnia, nightmares, sleepwalking, cognitive impairment, Alzheimer's, Lou Gehrig, stroke and Huntington's disease. An animal model of cranial nervous system disease, except for humans, in which autophagic cell death of adult hippocampal neural stem cells is suppressed by deletion of the SGK3 gene in adult hippocampal neural stem cells. Hippocampal neural stem cells in which apoptosis by autophagy is suppressed by deletion of the SGK3 gene. delete

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