WO2021182900A2 - Method for preventing or treating mtor-associated diseases through regulation of vegfr-3 expression - Google Patents

Abstract

The present invention relates to a composition for preventing or treating mammalian target of rapamycin (mTOR)-mediated diseases and deficient mTOR signaling-associated diseases. The present invention provides an efficient therapeutic target for various disease caused by hyperactivity or deficient activity of mTOR, on the basis of the novel discovery that the regulation of VEGFR-3 expression leads to the regulation of mTOR signaling pathway activity. The present invention, especially through VEGFR-3 activation, blocks hyperexcitable states of epilepsy and protects nerves, and thus can be effectively used as a fundamental therapeutic agent for status epilepticus, which is an incurable disease.

Description

Method for preventing or treating mTOR-related diseases by regulating VEGFR-3 expression

The present invention relates to a method for treating mTOR-related diseases using an inhibitor or activator of VEGFR-3 based on the new discovery that the expression regulation of VEGFR-3 regulates the activity of the mTOR (mammalian target of rapamycin) signaling pathway will be.

Epilepsy is a disease that causes intermittent nervous system disorders due to episodic discharge of cranial nerve cells due to organic lesions or functional disorders, and exhibits symptoms such as neurological symptoms, loss of consciousness, convulsions, and sensory disturbances. It is the third most common neurological disease after Alzheimer's disease and stroke, affecting about 0.5 - 2% of the world's population with epilepsy. In addition, it is estimated that there are about 45 new cases per 100,000 people worldwide every year, and about 300,000 to 400,000 epilepsy patients in Korea. Looking at the age distribution of epilepsy patients, it is known that 70% of all epilepsy begins at the age of children and adolescents, and the incidence is particularly high in infancy. In addition, the incidence and prevalence rates were highest within the first year of life and then rapidly decreased, and showed a U-shape that rapidly increased again in the elderly over 60 years of age, and the prevalence of experiencing seizures during life reached 10 to 15%.

Epilepsy is a chronic disease in which epileptic seizures occur repeatedly, and the cause is very diverse and the exact etiology is unknown. However, with the recent development of neuroimaging technology, microscopic pathological changes in the brain that could not be observed in the past can be observed, and the identification of the cause of epilepsy is gradually expanding. According to the epilepsy epidemiologic survey published by the Korean Epilepsy Society in 2012, more than two-thirds of epilepsy patients are idiopathic or latent with no specific cause, and the rest are serious causes that can be found. In other words, if there is a past history of neuropathological changes or brain damage such as stroke, congenital anomaly, head trauma, encephalitis, brain tumor, degenerative encephalopathy, childbirth injury, central nervous system developmental disorder and genetic predisposition, epilepsy may be induced. it is known that there is

On the other hand, epilepsy treatment can be largely divided into drug treatment and non-drug treatment, that is, surgery, ketogenic diet, and vagus nerve stimulation. However, since non-drug treatment is being performed only for patients who are resistant to drugs, drug treatment has been used as a major method for epilepsy treatment. Conventional drugs include phenytoin (Dilantin®), valproate (Orfil®, Depakine®, Depakote®), carbamazepine (Tegretol®), phenobarbital (Luminal®), etosuccimide. (Ethosuximide: Zarontin®), and more recently topiramate (topamax®), lamotrigine (Lamictal®), vigabatrin (Sabril®), and oxcarbazepine (Oxcarbazepine: Trileptal®). developed and commercialized.

However, despite these drug treatments, more than 30% of epilepsy patients fall under drug refractory epilepsy, in which seizures are not controlled even with drugs with various mechanisms of action, and some drugs have immune hypersensitivity reactions. It shows side effects such as intractable skin rash, gastrointestinal irritation caused by drugs, and dizziness, and there have been reports of cases of mental disorder and suicide due to the use of conventional drugs. the current situation.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

The present inventors made intensive research efforts to discover effective and fundamental therapeutic compositions for various diseases caused by the lack of activity of the mTOR signaling pathway, including epilepsy. As a result, the expression of VEGFR-3 in astrocytes in the hippocampus and mTOR signaling activity are closely related, and when VEGFR-3 expression or activity is increased, the expression of glutamate transporter 1 (GLT-1) increases and mTOR The present invention has been completed by discovering that the signaling pathway is activated and effectively eliminates the etiology of diseases caused by mTOR inactivation.

Accordingly, an object of the present invention is to provide a composition for preventing or treating a disease related to mTOR signal deficiency and a screening method thereof.

Another object of the present invention is to provide a composition for preventing or treating mTOR-mediated diseases and a screening method thereof.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides an mTOR signal deficiency-related disease comprising at least one selected from the group consisting of VEGFR-3 protein, a nucleic acid molecule encoding the VEGFR-3 protein, and an activator of VEGFR-3 as an active ingredient It provides a composition for the prevention or treatment of.

The present inventors made intensive research efforts to discover effective and fundamental therapeutic compositions for various diseases caused by the lack of activity of the mTOR signaling pathway, including epilepsy. As a result, the expression of VEGFR-3 in astrocytes in the hippocampus and mTOR signaling activity are closely related, and when VEGFR-3 expression or activity is increased, the expression of glutamate transporter 1 (GLT-1) increases and mTOR It was found that the activation of the signaling pathway effectively eliminates the pathogenesis of diseases caused by mTOR inactivation.

mTOR (mammalian target of rapamycin) is a serine/threonine kinase belonging to the PIKK (PI3K-related kinase) family. Two proteins called mTOR C1 (mTOR Complex 1) and mTOR C2 (mTOR Complex 2) It forms a complex and regulates the metabolism of eukaryotic cells, such as cell growth and proliferation, autophagy, and protein synthesis, according to external environmental signals such as growth factors, nutrition, and stress stimuli. As used herein, the term “mTOR signal deficiency-related disease” is meant to encompass a series of diseases caused by the malfunction of the mTOR signaling pathway, which broadly regulates this comprehensive cellular metabolic process.

According to the present invention, as the expression of GLT-1 in astrocytes is increased by mTOR activation mediated by VEGFR-3, hyperexcitability in epilepsy is blocked and a neuroprotective effect is confirmed. In addition, it has been reported that activation of mTOR can promote BMP-induced muscle hypertrophy and prevent muscle atrophy, and promote protein synthesis in skeletal muscle (Gazzerro E, et al., Rev Endocr Metab Disord 2006; 7:51-65). . In addition, a decrease in mTOR signaling was observed in mood disorders including depression, and mTOR signaling causes synaptic neurotransmission and plasticity. (Yang C, Hu YM, et al., Ups J Med Sci 2013;118:3-8). In addition, mTOR signaling promotes limb skeletal growth by inducing the synthesis of factors necessary for growth in chondrocytes, and inhibits growth of limb mesenchymal cells by mTOR signal deficiency and reduces the growth of transcription factors involved in chondrogenesis. It has been reported that inhibition of expression can lead to skeletal developmental disorders (Jiang M, Fu X, et al., J Cell Biochem. 2017;118(4):748-753).

Accordingly, according to a specific embodiment of the present invention, the disease related to mTOR signal deficiency that can be prevented or treated with the composition of the present invention is selected from the group consisting of epilepsy, muscular atrophy, depression and bone disease.

As used herein, the term “bone disease” refers to any disease in which degeneration or damage of bone or cartilage tissue leading to quantitative loss of bone or cartilage tissue is caused by pathological or physical causes, for example, osteoporosis, osteomalacia, It includes, but is not limited to, rickets, fibrous osteomyelitis, bone damage due to bone metastasis of cancer cells, aplastic bone disease, metabolic bone disease, and osteoarthritis.

According to another aspect of the present invention, the present invention provides a composition for preventing or treating mTOR-mediated diseases comprising an inhibitor of VEGFR-3 protein as an active ingredient.

As used herein, the term “inhibitor” refers to a substance that causes a decrease in the activity or expression of a target protein, whereby the activity or expression of the target protein becomes undetectable or exists at an insignificant level, as well as when the target protein is It refers to a substance that reduces the activity or expression to such an extent that the biological function can be significantly reduced.

Inhibitors of VEGFR-3 protein include, for example, shRNA, siRNA, miRNA, ribozyme, and peptide nucleic acids (PNA) that inhibit the expression of VEGFR-3, whose coding nucleotide sequence is known in the art at the gene level. Antisense oligonucleotides or CRISPR systems comprising a guide RNA for recognizing a target gene, and antibodies or aptamers that inhibit VEGFR-3 at the protein level, as well as compounds, peptides and natural products that inhibit their activity. All gene and protein level suppression means known in the art without limitation can be used.

As used herein, the term “shRNA (small hairpin RNA)” is a single strand of 50-70 nucleotides forming a stem-loop structure in vivo. An RNA sequence that creates a tight hairpin structure. Usually, a long RNA of 19-29 nucleotides is base-paired on both sides of a loop of 5-10 nucleotides to form a double-stranded stem, and in order to always be expressed, it is introduced into a cell through a vector containing a U6 promoter. It is transduced and is usually passed on to daughter cells so that suppression of the expression of the target gene is inherited.

As used herein, the term “siRNA” refers to a short double-stranded RNA capable of inducing an RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases, and if the expression of the target gene can be inhibited by the RNAi effect, blunt ends or cohesive ends Both ends are possible. The structure of the adhesive end can be both a structure in which the three-terminal protrudes and a structure in which the five-terminal side protrudes.

As used herein, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is not expressed in cells, has a short stem-loop structure, and inhibits the expression of a target gene through complementary binding to the mRNA of the target gene. do.

As used herein, the term “ribozyme” is a type of RNA and refers to an RNA molecule having the same function as an enzyme that recognizes the base sequence of a specific RNA and cuts it by itself. A ribozyme is a complementary nucleotide sequence of a target mRNA strand and consists of a region that binds with specificity and a region that cuts the target RNA.

As used herein, the term “peptide nucleic acid (PNA)” refers to a molecule capable of complementary binding to DNA or RNA while having both nucleic acid and protein properties. PNA is not found in nature but is artificially synthesized by a chemical method, and forms a double strand through hybridization with a natural nucleic acid of a complementary base sequence to control the expression of a target gene.

As used herein, the term “antisense oligonucleotide” refers to a nucleotide sequence complementary to a sequence of a specific mRNA, which binds to a complementary sequence in a target mRNA and translates its protein into a protein, translocation into the cytoplasm, maturation, or any other It refers to a nucleic acid molecule that inhibits an essential activity for an overall biological function. Antisense oligonucleotides may be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). . The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic sugarscholphonates, and the like.

As used herein, the term “gRNA (guideRNA)” refers to an RNA molecule used in a gene editing system that recognizes a target gene and specifically cuts the recognized loci by inducing a nuclease. A typical example of such a gene editing system is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.

According to the present invention, the expression inhibitor of the present invention may be a specific antibody that inhibits the activity of the protein encoded by the genes. The antibody specifically recognizing the target protein is a polyclonal or monoclonal antibody, preferably a monoclonal antibody.

Antibodies of the present invention can be prepared by methods routinely practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology , 6:511-519 (1976)), recombinant DNA methods (U.S. Patent No. 4,816,567). ) or phage antibody library methods (Clackson et al, Nature , 352:624-628 (1991) and Marks et al, J. Mol. Biol. , 222:58, 1-597 (1991)). . General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1999; and Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984.

The present invention may inhibit its activity by using an aptamer that specifically binds to a target protein instead of an antibody. As used herein, the term “aptamer” refers to a single-stranded nucleic acid (RNA or DNA) molecule or peptide molecule that binds to a specific target material with high affinity and specificity. For general information on aptamers, see Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):426-30(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24):14272-7 (1998).

As used herein, the term “mTOR-mediated disease” is meant to encompass a series of diseases caused by overactivation of the mTOR signaling pathway.

As shown in the Examples to be described later, the present inventors found that when a selective inhibitor of VEGFR-3 was administered, GLT-1 expression and reactivity of astrocytes were inhibited while the mTOR pathway was inhibited.

On the other hand, it was observed that the mTOR pathway is overactive in a number of cancer patients, and the major tumor suppressor genes TP53 and LKB1 not only inhibit TSC1 and TSC2, which are upstream regulators of mTORC1, but also mTOR signaling also causes apoptosis. It contributes to tumorigenesis by activating Akt, which induces proliferation processes such as glucose uptake and glycolysis while inhibiting Accordingly, inhibition of mTOR through inhibition of VEGFR-3 can be a significant anticancer target.

In addition, the mTOR pathway is a central mechanism of anabolic and catabolic pathways of lipid metabolism, and it has been reported that excessive activity of mTOR causes metabolic abnormalities such as obesity and insulin resistance (Zoncu R. , et al., Molecular Cell Biology 2011;12:21-35). In addition, in neurons, mTOR contributes to synaptic connections through the growth of axons and formation of dendrites. When the mTOR signal is excessively increased, it leads to a decrease in memory storage capacity and causes the progression of various neurodegenerative diseases (Bove J. , et al., NATURE REVIEWS 2011;12:437-52).

On the other hand, inhibitors of mTOR, including rapamycin, have a strong immunosuppressive function by inhibiting the proliferation of T cells induced by growth factors. It can be a therapeutic target for immune diseases.

Accordingly, the mTOR-mediated disease that can be prevented or treated with the composition of the present invention is selected from the group consisting of cancer, neurodegenerative disease, metabolic disease, inflammatory disease and actrocytosis.

More specifically, the cancer is selected from the group consisting of kidney cancer, breast cancer, lung cancer, stomach cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, lymphoma, leukemia and myelodysplastic syndrome.

More specifically, the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, myasthenia gravis and Pick's disease ( Pick's disease).

More specifically, the metabolic disease is selected from the group consisting of obesity, diabetes, dyslipidemia, fatty liver and insulin resistance syndrome.

As used herein, the term “metabolic disease” conceptualizes the phenomenon in which the risk factors of various cardiovascular diseases and type 2 diabetes, which are caused by metabolic abnormalities, cluster with each other as one disease group, and insulin resistance and related complex and It is a concept that encompasses various metabolic abnormalities and clinical aspects.

As used herein, the term “obesity” refers to a state in which the amount of energy intake exceeds energy consumption over a long period of time and the excess energy is stored as fat, resulting in an excess of adipose tissue in the body. Usually, if the body mass index (Body mass index: weight (kg)/[height (m)] ² ) is 25 or more, it is defined as fertilely obese.

As used herein, the term “diabetes” refers to a chronic disease characterized by a relative or absolute lack of insulin resulting in glucose-intolerance. Diabetes treated or prevented by the composition of the present invention includes all types of diabetes, for example, type 1 diabetes, type 2 diabetes, and hereditary diabetes. Type 1 diabetes is insulin-dependent diabetes mellitus, mainly caused by destruction of β-cells. Type 2 diabetes is non-insulin-dependent diabetes mellitus, caused by insufficient insulin secretion after a meal or by insulin resistance.

As used herein, the term “dyslipidemia” refers to a pathologic condition in which the level of fat concentration in the blood is outside the normal range, for example, hypercholesterolemia, hypertriglyceridemia, low-HDL-cholesterol. In addition to hyperlipidemia and hyper-LDL-cholesterolemia, it includes all abnormal lipid states caused by abnormal lipoprotein metabolism.

As used herein, the term “fatty liver” refers to a state in which fat is accumulated in hepatocytes in an excessive amount due to a disorder of fat metabolism in the liver, which causes various diseases such as angina pectoris, myocardial infarction, stroke, arteriosclerosis, fatty liver and pancreatitis.

As used herein, the term “insulin resistance” refers to a state in which cells cannot effectively burn glucose because the function of insulin to lower blood sugar is low. When insulin resistance is high, the body makes too much insulin, which can lead to high blood pressure or dyslipidemia, as well as heart disease and diabetes. In particular, in type 2 diabetes mellitus, an increase in insulin in muscle and adipose tissue is not noticed, and the action of insulin does not occur. The term “insulin resistance syndrome” is a concept that collectively refers to diseases induced by insulin resistance. Cell resistance to insulin action, hyperinsulinemia, and increase in very low density lipoprotein (VLDL) and triglycerides, high density It refers to a disease characterized by a decrease in high density lipoprotein (HDL) and hypertension, and is a concept recognized as a risk factor for cardiovascular disease and type 2 diabetes (Reaven GM, Diabetes, 37: 1595-607, (1988)).

According to a specific embodiment of the present invention, the dyslipidemia prevented or treated with the composition of the present invention is hyperlipidemia.

As used herein, the term “hyperlipidemia” refers to a disease caused by maintaining a high lipid concentration in the blood because fat metabolism such as triglycerides and cholesterol is not properly performed. More specifically, hyperlipidemia includes hypercholesterolemia or hypertriglyceridemia with a high incidence in a state in which lipid components such as triglycerides, LDL cholesterol, phospholipids and free fatty acids in the blood are increased.

According to a specific embodiment of the present invention, the fatty liver to be prevented or treated by the composition of the present invention is non-alcoholic fatty liver.

As used herein, the term “non-alcoholic fatty liver (NAFL)” refers to a disease in which an excessive amount of fat is accumulated in liver cells regardless of alcohol absorption, and includes simple fatty liver (steatosis) and non-alcoholic non-alcoholic steatohepatitis (NASH). Although simple fatty liver has a good clinical prognosis, NASH with inflammation or fibrosis is a progressive liver disease and is recognized as a progenitor disease that causes cirrhosis or liver cancer. Obesity and insulin resistance are major risk factors for nonalcoholic fatty liver disease. Risk factors for the progression of liver fibrosis include obesity (BMI > 30), blood liver function index ratio (AST/ALT > 1), and diabetes. can 69-100% of nonalcoholic fatty liver patients are obese, and 20-40% of obese patients have nonalcoholic fatty liver. This is because obesity is the most important risk factor for nonalcoholic liver disease.

According to a specific embodiment of the present invention, the inflammatory disease (or autoimmune disease) that can be prevented or treated with the composition of the present invention is rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes mellitus, systemic lupus erythematosus, multiple sclerosis , idiopathic fibroal alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis or Crohn's disease.

As used herein, the term “astrocytosis” refers to an abnormal increase in astrocytes due to damage to adjacent neurons, such as trauma of the central nervous system, tissue damage caused by tumors, infection, ischemia, stroke, neurodegenerative disease, etc. Or it is meant to encompass all pathological conditions that cause activation. Astrocytes that are activated or proliferated in the lesion area where brain tissue is damaged by various causes have some protective effect on the regeneration process of brain tissue, but the increase in astrocytes appearing after injury inhibits the regeneration process by secreting various inflammatory substances. .

More specifically, the astrocytosis is glioma, glioblastoma, astrocytoma, stroke, traumatic brain injury, and amyotrophic lateral sclerosis. Lateral Sclerosis: ALS).

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or disease in a subject who has never been diagnosed with a disease or disease, but is likely to have the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of the disease, condition or condition; or (c) eliminating the disease, condition or symptom. When the composition of the present invention is administered to a subject, it regulates the mTOR signaling pathway by inhibiting or increasing the activity or expression level of VEGFR-3, inhibits the development of symptoms of various diseases caused by deficiency or excessive mTOR activity, or removes it or act as a mitigator. Accordingly, the composition of the present invention may be a composition for treating these diseases by itself, or may be administered together with other pharmacological ingredients and applied as a therapeutic adjuvant for the above diseases. Accordingly, as used herein, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant”.

As used herein, the term “administration” or “administering” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.

In the present invention, the term “therapeutically effective amount” refers to the content of the composition in which the pharmacological component in the composition is sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered. prophylactically effective amount”.

As used herein, the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the subject of the present invention is a human.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and specifically may be administered orally, intravenously, subcutaneously or intraperitoneally.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient. can be A preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg/kg for adults.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating a disease related to mTOR signal deficiency, comprising the following steps:

(a) contacting a test substance with a biological sample containing a VEGFR-3 protein or a gene encoding the same; and

(2) measuring the expression level or activity of the VEGFR-3 protein or a gene encoding the same in the sample;

When the expression level or activity of the VEGFR-3 protein or a gene encoding the same is increased, the test substance is determined as a composition for preventing or treating mTOR signal deficiency related diseases.

Since the mTOR signal deficiency-related disease to be prevented or treated by the composition screened in the present invention has already been described above, the description thereof will be omitted to avoid excessive overlap.

As used herein, the term “biological sample” refers to any sample containing VEGFR-3 expressing cells obtained from mammals including humans, including, but not limited to, tissue, organ, cell or cell culture medium.

The term “test substance” used while referring to the screening method of the present invention is added to a sample containing cells expressing VEGFR-3 and used in screening to test whether it affects the activity or expression level of VEGFR-3 It means an unknown substance. The test substance includes, but is not limited to, compounds, nucleotides, peptides and natural extracts. The step of measuring the expression level or activity of VEGFR-3 in the biological sample treated with the test substance may be performed by various methods for measuring the expression level and activity known in the art. As a result of the measurement, when the expression level or activity of VEGFR-3 is increased, the test substance may be determined as a composition for preventing or treating mTOR signal deficiency related diseases.

As used herein, the term “increase in expression or activity” refers to an increase in the expression level of VEGFR-3 or intrinsic function of VEGFR-3 in vivo to such an extent that mTOR activation mediated by VEGFR-3 is increased to a measurable level. it means. Specifically, it may refer to a state in which the activity or expression level is increased by 20% or more, more specifically, a state increased by 40% or more, more specifically, a state increased by 60% or more compared to the control group.

According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating an mTOR-mediated disease, comprising the steps of:

(a) contacting a test substance with a biological sample containing a VEGFR-3 protein or a gene encoding the same; and

(2) measuring the expression level or activity of the VEGFR-3 protein or a gene encoding the same in the sample;

When the expression level or activity of the VEGFR-3 protein or a gene encoding the same is decreased, the test substance is determined as a composition for preventing or treating mTOR-mediated diseases.

Since the mTOR-mediated disease to be prevented or treated by the composition screened in the present invention has already been described above, the description thereof will be omitted to avoid excessive overlap.

As used herein, the term “reduction of expression or activity” refers to inhibiting the expression level of VEGFR-3 or the intrinsic function of VEGFR-3 in vivo to such an extent that mTOR activation mediated by VEGFR-3 is reduced to a measurable level. it means. Specifically, it may refer to a state in which the activity or expression level is reduced by 20% or more, more specifically, a state in which the activity or expression is reduced by more than 40%, more specifically, a state in which the activity or expression level is reduced by more than 60% compared to the control.

According to a specific embodiment of the present invention, the biological sample is a biological sample including astrocytes.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising at least one selected from the group consisting of VEGFR-3 protein, a nucleic acid molecule encoding the VEGFR-3 protein, and an activator of VEGFR-3 as an active ingredient. It provides a method for preventing or treating a disease related to mTOR signal deficiency, comprising administering to a subject.

According to another aspect of the present invention, the present invention provides a method for preventing or treating mTOR-mediated diseases, comprising administering to a subject a pharmaceutical composition comprising an inhibitor of VEGFR-3 protein as an active ingredient.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for preventing or treating mTOR signaling deficiency-related diseases and mTOR (mammalian target of rapamycin)-mediated diseases, and a screening method thereof.

(b) The present invention provides an effective therapeutic target for various diseases caused by excessive activity or lack of activity of mTOR based on the new discovery that regulation of VEGFR-3 expression leads to regulation of mTOR signaling pathway activity

(c) The present invention can be usefully used as a fundamental therapeutic agent for overlapping epilepsy, an intractable disease, by blocking the hyperexcited state in epilepsy and protecting nerves through VEGFR-3 activation, in particular.

1 is a diagram showing the time-dependent expression patterns of pS6 and VEGFR-3 in the hippocampus after epilepsy overlap is induced with pilocarpine. panel A, E shows that p6S-positive neuron-like cells are expressed in the hippocampus of the normal control panel. Panels B, F show that pS6-positive signal is slightly increased compared to that of the normal control group on the 1st day after epilepsy superposition induction. The figure shows that pS6-positive cells increased remarkably on the 4th day after induction of epilepsy superposition. Panels D and H show that pS6 positivity is reduced on the 7th day of the epilepsy superposition compared to the previous time. Scale bars = 100 μm, 20 μm. Figure 1b shows the results of confirming that the protein expression changes appear the same as the immunohistochemical staining results through western blot analysis of pS6, S6, VEGFR-3, and GFAP. * P < 0.05, ** P < 0.01, and *** P < 0.001 were considered to be significantly different from the control group and the group (Tukey's post-hoc test after one-way ANOVA, n = 5 for each group). All values are expressed as mean ± standard error.

2 is a diagram showing the results of confirming the expression of pS6 in VEGFR-3 positive reactive astrocytes after induction of epilepsy overlap. Panels A-C show that in normal controls, most pS6 expression (green) was not observed in GFAP-positive astrocytes (blue), but some VEGFR-3 expression (red) was observed on the surface of GFAP-positive astrocytes. Panels D-F are diagrams confirming that pS6 expression in activated astrocytes on day 4 after epilepsy induction coincides with VEGFR-3 expression. Scale bar = 20 μm

3 is a diagram showing that VEGFR-3 expression in astrocytes is reduced by inhibiting mTOR activation. Figure 3a is the result of double immunofluorescence staining of VEGFR-3 (red) and NeuN (green), confirming that the expression of astrocyte VEGFR-3 in the CA1 region of the hippocampus on the 4th day after induction of epilepsy overlap is higher than that of the normal control group. shows However, in the hippocampus of an animal model of epilepsy administered with rapamycin, an mTOR inhibitor, cells expressing VEGFR-3 were significantly reduced. Scale bar = 50 μm. Figure 3b is a schematic diagram of the hippocampal sub-pyramid region subjected to quantitative analysis of the number of VEGFR-3 expressing cells. Figure 3c is a picture showing the results of quantitative analysis of VEGFR-3 expression in the indicated hippocampal sub-pyramid region, it was confirmed that the number of VEGFR-3 positive astrocytes increased by induction of epilepsy overlap was significantly reduced by the administration of rapamycin. . Figure 3d is a result of analyzing the VEGFR-3 protein expression pattern through Western blot, it shows that the expression of VEGFR-3 increased by the induction of epilepsy overlap is again reduced by the administration of rapamycin. Figure 3e is a diagram showing the density-based quantitative analysis result as a histogram of the VEGFR-3 protein band (normalized to β-actin). Also, the increase in VEGFR-3 expression due to epilepsy overlap is significant by rapamycin administration. shows a significant decrease. * P < 0.05 and *** P < 0.001 was considered to be significantly different from the SE 4d-Veh group (Tukey's post-hoc test after one-way ANOVA, n = 5 for each group). All values are expressed as mean ± standard error.

4 is a diagram showing that mTOR activation in astrocytes is reduced when VEGFR-3 expression in the hippocampus is suppressed after induction of epilepsy overlap. Figure 4a shows that the astrocyte pS6 immune response (red) is significantly increased in the hippocampal CA1 region on the 4th day after epilepsy induction, but pS6 expression is decreased by administration of the VEGFR-3 inhibitor, SAR131675 (SAR). Scale bar = 50 μm. Figure 4b is a diagram showing that the quantitative analysis result confirms that the number of astrocytes pS6-positive cells increased by epilepsy overlap is significantly reduced by SAR administration. FIG. 4c is a Western blot analysis result showing changes in pS6 expression by SAR administration, and it was confirmed that pS6 protein expression increased by epilepsy overlap was significantly inhibited by SAR administration. * When P < 0.05, it was considered that there was a significant difference with the SE 4d-Veh group (Tukey's post-hoc test after one-way ANOVA, n = 5 for each group). All values are expressed as mean ± standard error.

5 is a diagram showing that RA treatment reduces GLT-1 expression in the hippocampus after SE induction. 5a is Representative figure showing the increase of glial GLT-1-positive cells by SE in the subpyramid region (SE 4d-Veh); However, RA treatment attenuated glial GLT-1 immunoreactivity in the hippocampus after SE induction (SE 4d-RA). Scale bar = 50 μm; The magnification of the rest of the images is the same. 5b is a quantitative analysis result showing that the number of glial GLT-1-positive cells is significantly increased in the sub-pyramid region of SE 4d-veh and SE 4d-RA groups compared to Sham-Veh. *** P <0.001, statistically significant (Tukey's post-hoc test after one-way ANOVA, n = 5 for each group). All values are expressed as mean ± standard error.

6 is a diagram showing that inhibition of VEGFR-3 attenuates GLT-1 expression and activation of astrocytes in the hippocampus after induction of epilepsy overlap. As shown in FIG. 6a , GLT-1 expression (red) was confirmed in the paramid neuron layer of the CA1 region of the hippocampus of the normal control group, whereas an increase in the expression of astrocyte GLT-1 was observed in the hippocampus on the 4th day after epilepsy induction. became Scale bar = 50 μm. Figure 6b shows that, as a result of quantitative analysis, the number of GLT-1 expressing cells in the hippocampus after induction of epilepsy superimposition is significantly increased compared to the normal control group, but when VEGFR-3 is suppressed due to SAR administration, GLT-1 expression of astrocytes is decreased. It is a picture that shows Figure 6c shows the results confirmed through Western blot that SAR administration reduces the expression of GLT-1 protein. Figure 6d shows immunofluorescence staining results showing that GFAP-positive astrocytes (green) are remarkably activated by pilocarpine-induced epilepsy overlap, and that GFAP-positive astrocyte expression is reduced by administration of SAR, a VEGFR-3 inhibitor. show Scale bar = 50 μm. 6e is a diagram showing the result of verifying that GFAP expression is significantly reduced by SAR administration in Western blot analysis, identical to the immunofluorescence staining result. * When P < 0.05, it was considered that there was a significant difference with the SE 4d-Veh group (Tukey's post-hoc test after one-way ANOVA, n = 5 for each group). All values are expressed as mean ± standard error.

7 is a diagram showing the effect of an increase in VEGFR-3 in reactive astrocytes after SE induction. Elevated VEGFR-3 affects mTOR activation in reactive astrocytes following the same prolonged seizure behavior. Furthermore, VEGFR-3-mediated mTOR activation may reduce SE-induced hyperexcitability during interstitial subacute phase (gray arrow) by increasing GLT-1 expression in astrocytes. Taken together, the increase in VEGFR-3 in reactive astrocytes appears to play an important role in calming hyperexcitability in the hippocampus.

8 is a diagram showing the effect of RA treatment on Akt/mTOR activation in the hippocampus in normal and epilepsy conditions. 8A is a Western blot analysis result showing the change in the expression level of phosphorylated Akt (pAkt) after RA treatment. At steady state, RA treatment significantly increased the expression level of pAkt in the hippocampus in sham-manipulated mice compared to the Sham-Veh group. Under epilepsy conditions, the expression level of pAkt in the hippocampus of SE 4d-Veh and SE 4d-RA groups was significantly increased compared to Sham-Veh group. The results of quantitative analysis of each sample based on the density of the pAkt band were shown as histograms (normalized to β-actin). * P <0.05 and *** P < 0.001 had a statistically significant difference with Sham-Veh animals (Tukey's post-hoc test after one-way ANOVA, n = 5 for each group). 8B is a Western blot result showing the change in the expression level of RA-treated pS6. Although there was no significant difference between Sham-Veh and Sham-Veh groups, pS6 expression in the hippocampus of mice (SE 4d-RA) 4 days after epilepsy induction by RA treatment was significantly suppressed compared to the SE 4d-Veh group. became The results of quantitative analysis of each sample based on the density of the pS6 band were shown as histograms (normalized to β-actin). *** Statistically significant difference from Sham-Veh animals when P < 0.001. ^# Statistically significant difference with SE 4d-Veh animals when P <0.05 (Tukey's post-hoc test after one-way ANOVA, n = 5 for each group). All values are expressed as mean ± standard error.

9 is a diagram showing the effect of VEGFR-3 inhibition on acute epilepsy induced by pilocarpine injection. One hour before pilocarpine injection, mice were pretreated with SAR (50 mg/kg; ip) or vehicle solution, and seizure behavior was recorded after pilocarpine injection until the end of the seizure. Figure 9a is a diagram showing that the total number of convulsive seizures (stages 3-5) is significantly increased in SAR-pretreated mice compared to Veh-pretreated group. ** Statistically significant difference from Veh-pretreatment group when P < 0.01 (Student's t -test, n=10 for each group). Figures 9b and 9c show that there was no significant difference between the SAR- and Veh-pretreatment groups (n = 10 for each group) in the onset times of convulsive seizures and SE. All values are expressed as mean ± standard error.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

laboratory animal

Male C57BL/6 mice (8 weeks old, Orient Bio, Gyeonggi, Korea) were treated in a light-controlled environment (from 8:00 am to 8:00 pm) under standard temperature (22 ± 1 °C) and humidity (50 ± 1%). ) was bred with free access to food and drinking water. All experiments were conducted in accordance with the guidelines of the Animal Experimentation Ethics Committee of Yonsei University, and the number and suffering of experimental animals were minimized.

SE induction with pilocarpine

A pilocarpine-induced status epilepticus (SE) mouse model was established by a previously reported method (Cho et al., 2019; Jeong, Lee, Kim, & Cho, 2011). Briefly, 30 min after treatment with atropine methyl nitrate (1 mg/kg; ip; Sigma-Aldrich), mice were treated with pilocarpine hydrogen chloride (280-300 mg/kg; Sigma-Aldrich; St. Louis, MO, USA) or Or saline (normal control) was intraperitoneally injected. After pilocarpine treatment, the seizure stage was determined according to the Racine scale (Racine, 1972). Animals that have reached stage 4 (standing on hind legs, rearing) and stage 5 (rearing and falling) and exhibiting general tonic-clonic seizures are considered to have superposition epilepsy (SE). and selected for further study. To monitor motor seizures after pilocarpine injection, seizure frequency and latency were recorded from immediately after pilocarpine injection to the end of seizures. The number of seizures refers to the total number of convulsive seizures (stages 3-5) after pilocarpine injection. The onset times of seizures and SE were measured as the time elapsed from pilocarpine injection to the first convulsive seizure (stage 3) and SE, respectively. Two hours after SE induction, the seizure was terminated by administration of diazepam (10 mg/kg; i.p.). To promote recovery, a 5% glucose solution was intraperitoneally injected into all experimental animals, and food soaked in water was supplied, and then placed in an incubator (30 ± 1 °C) to maintain physiological body temperature. All experimental mice were sacrificed 1, 4 or 7 days after treatment with pilocarpine or saline.

Rapamycin and SAR131675 treatment

To suppress the mTOR signal, rapamycin (RA; LC laboratories; Woburn, MA, USA) was first administered (6 mg/kg; ip) 1 hour before pilocarpine treatment and 3 days from the 1st day after SE induction. It was administered daily (3 mg/kg; ip). According to a previously reported method (L.-H. Zeng, Rensing, & Wong, 2009), rapamycin was dissolved in 100% ethanol and immediately before injection 5% Tween 80, 5% polyethylene glycol 400 (Sigma-Aldrich) and Dilute in vehicle solution containing 4% ethanol. SAR131675 (SAR; Selleck Chemicals; Munich, Germany), a VEGFR-3 inhibitor, was dissolved in 5% DMSO with saline according to a previously reported method (Bhuiyan, Kim, Hwang, Lee, & Kim, 2015). As with the RA administration method, SAR was administered 1 hour before pilocarpine treatment (50 mg/kg; ip), and after induction of SE with pilocarpine, daily injections were performed for 3 days after 1 day (25 mg/kg; ip). ).

Immunohistochemistry and immunofluorescence staining

For immunohistochemical staining, mice were anesthetized with 15% chloral hydrate and cardiac perfused with saline followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB; pH 7.4). Thereafter, brains were rapidly excised as previously reported (Cho et al., 2019) and cryopreserved with 30% sucrose solution for 3 days. Next, the samples were rapidly frozen in liquid nitrogen. Serial sections (20 μm thick) were cut coronal at 120 μm intervals (720 μm in total, between -1.58 and -2.30 from the grand sac in every 7th slice) (Franklin & Paxinos, 2008). All sections were washed with 0.01 M PBS (pH 7.4). For immunohistochemistry, sections were incubated with 3% H ₂ O ₂ and 10% methanol in 0.01M PBS to remove endogenous peroxidase activity. Next, the sections were fixed with 10% goat serum (VectorLaboratories; Burlingame, CA, USA) dissolved in 0.01M PBS for 1 hour at 4°C and anti-phospho S6 ribosomal protein antibody (pS6; 1:200; Cell Signaling Technology). ; Beverly, MA, USA) and incubated overnight. The next day, the sections were incubated with biotinylated anti-rabbit IgG (1:200; Vector Laboratories) at room temperature for 2 hours and placed in an avidin-biotin peroxidase complex solution (Vector Laboratories) for 1 hour. Finally, the sections were stained with 0.1% diaminobenzidine tetrahydrochloride and 0.005% H ₂ O _{2 dissolved in 0.05M Tris-HCl (pH7.4).} The stained sections were observed with an optical microscope (BX51; Olympus; Tokyo, Japan).

For multi-immunofluorescence analysis, sections were prepared from anti-neuronal nuclei (NeuN; 1:100; Millipore; Temecula, CA, USA), anti-glial fibrillary acidic protein (GFAP; 1:400; Millipore), anti-VEGFR-3 (1:500; Abnova; Taipei, Taiwan, China), anti-VEGFR-3 (1:100; Abcam; Cambridge, A, USA), anti-pS6 (1:200; Cell) Signaling Technology), and anti-GLT-1 (1:200; Thermo Scientific; Rockford, IL, USA) antibodies, followed by Cy3- (1:500; Jackson ImmunoResearch; West Grove, PA, USA), Cy5- (1:500; Jackson ImmunoResearch) and Alexa fluor 488-conjugated IgG (1:300; Invitrogen; Carlsbad, CA, USA) were incubated overnight at 4°C. Sections were mounted and observed under a confocal microscope (LSM 700; Carl Zeiss; Thornwood, NY, USA).

Quantitative analysis of glia-like VEGFR-3, pS6 and GLT-1-expressing cells

To analyze the expression changes of VEGFR-3, pS6 and GLT-1-positive cells in the hippocampus of the normal control group and 4 days after seizure induction, 6 coronal sections were obtained. The number of cells was measured according to a previously reported method (Cho et al., 2019; Jeong et al., 2011). The number of VEGFR-3, pS6 and GLT-1-positive cells was measured in the region including the stratum radiatum, lacunosum moleculare, and dentate gyrus molecular layers ( FIG. 3B ). The number of VEGFR-3, pS6 and GLT-1-labeled cells in each section was measured using ZEN image inspection software (Carl Zeiss).

Western blot analysis

Brain tissue samples for Western blot analysis were prepared by a previously reported method (Jeong, Lee, & Kim, 2015). The mouse hippocampal tissue was homogenized and centrifuged at 14,000 g for 15 minutes at 4°C. The supernatant was transferred to a new tube, and the concentration was measured using a bicinchoninic acid assay kit (Thermo Scientific). Proteins were separated by gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore) using an electrophoretic transfer system (Bio-Rad Laboratories; Hercules, CA, USA). Membranes were incubated overnight at 4°C with the following primary antibodies: anti-VEGFR-3 (Abcam), anti-phosphorylated Akt (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-pS6 (Cell Signaling Technology) ), anti-S6 (Cell Signaling Technology), anti-GFAP (Millipore), anti-GLT-1 (Thermo Scientific), and anti-β-actin (Santa Cruz Biotechnology; Dallas, TX, USA). After washing, the membrane was again incubated with a secondary antibody (Enzo Life Science; Farmingdale, NY, USA), and blotting was performed using ECL western blotting detection reagent (Amersham Biosciences; Piscataway, NJ, USA). The density of the band was measured using computer imaging equipment and related software (Fuji Film; Tokyo, Japan).

statistical analysis

All statistical analyzes were performed using IBM SPSS statistics Version 25 (SPSS; Chicago, IL, USA). Data are expressed as mean ± standard error (SEM) and Tukey post-hoc test was performed after Student's t -test or one-way ANOVA. When P < 0.05, it was considered to have statistical significance.

Experiment result

Changes in pS6 and VEGFR-3 expression in the hippocampus after SE

Expression of the phosphorylated ribosomal protein S6 (pS6) is often utilized as a marker of mTOR activation (Deli et al., 2012; Macias et al., 2013; L.-H. Zeng et al., 2009). To confirm the change in mTOR activation in the hippocampus after SE induced by pilocarpine, immunohistochemistry for pS6 expression was performed. As a result, pS6 immunoreactivity in normal control animals was mainly observed in the pyramidal neurons and some dentate granule neurons of the CA1 and CA3 subfields (Panel A of Fig. It was also detected in the interspersed cells of the layer (panel E of Figure 1a). Glue-like pS6 expression was slightly increased in the emissive layer and the subfield CA1 subfields 1 day after SE induction (panels B and F of FIG. 1A ). The expression of glial-like pS6 reached a maximum level on day 4 after SE induction (panels C and G of FIG. 1A ) and gradually decreased from day 7 after SE induction (panels D and H of FIG. 1A ). As with the immunostaining results, as a result of western blot analysis, the expression level of pS6 was significantly increased in the hippocampus from 4 days after SE induction ( P = 0.001 compared to the normal control group (sham); FIG. 1b ). Similar to the results of previous studies by the present inventors (Cho et al., 2019), it was confirmed that the protein level of VEGFR-3 was significantly increased in the hippocampus from 4 days after SE induction as compared to normal control mice as a result of immunoblotting (Cho et al., 2019) ( P = 0.04 compared to normal control (sham); FIG. 1B ). GFAP protein levels were significantly increased at 4 days (P =0.003 compared to normal control; FIG. 1B) and 7 days ( P=0.001 compared to normal control; FIG. 1B) after SE induction.

After confirming the expression change of pS6 and VEGFR-3 after SE induction, it was investigated whether pS6 and VEGFR-3 coexist in astrocytes by triple-immunofluorescence staining (FIG. 2). In normal control animals, pS6 expression in GFAP-expressing astrocytes was not observed, but VEGFR-3 expression was more or less observed on the surface of GFAP-expressing astrocytes (FIG. 2 panels A-C). However, SE-induced pS6 expression was significantly increased in hippocampal VEGFR-3-expressing reactive astrocytes 4 days after SE induction (FIG. 2 panels D-F). These results suggest that mTOR activation is associated with increased VEGFR-3 expression during astrocyte activation after SE induction.

Positive correlation between mTOR activation and VEGFR-3 expression in reactive astrocytes after SE induction

Next, it was investigated whether inhibition of mTOR activation by rapamycin (RA) treatment induces changes in VEGFR-3 expression in astrocytes in the hippocampus. As a result of immunofluorescence staining, the expression of VEGFR-3 in astrocytes was remarkably increased in the subpyramid region of the hippocampus 4 days after SE induction with pilocarpine compared to the sham-Veh and sham-RA groups. VEGFR-3 expression of astrocytes was significantly attenuated in the subpyramid region of (Fig. 3a). When we quantified the number of VEGFR-3-positive cells in the hippocampal subpyramid region (Fig. 3b), it was confirmed that the number of VEGFR-3-expressing cells was significantly increased in the SE4d-Veh group compared to the sham-Veh group (sham-Veh). Contrast P <0.001; Fig. 3c). However, RA treatment induced seizures day 4 as compared to animals which were VEGFR-3- significantly reduced the number of immunoreactive cells in the hippocampal pyramid sub-region (SE 4d-Veh contrast P <0.001; Fig. 3c). Similar to the immunostaining results, the Western blot results showed that the protein level of VEGFR-3 significantly increased 4 days after SE induction compared to the vehicle-treated normal control group ( P < 0.001 compared to Sham-Veh; FIGS. 3D and 3F) ); However, VEGFR-3 levels were significantly attenuated by RA treatment (SE 4d-Veh vs. P = 0.02; FIGS. 3D and 3F). In sham-manipulated mice, VEGFR-3 levels were significantly increased by RA treatment compared to the vehicle-treated sham group ( P = 0.012 versus Sham-Veh; FIGS. 3D and 3F). Furthermore, phosphorylated Akt levels by RA treatment were significantly increased compared to the vehicle-treated sham group ( P = 0.025 versus Sham-Veh; FIG. 8 ).

To further confirm the correlation between VEGFR-3 expression and mTOR activation in reactive astrocytes after SE induction, inhibition of VEGFR-3 by a VEGFR-3 specific inhibitor, SAR131675(SAR) (Alam et al., 2012) It was investigated by immunofluorescence staining and western blotting to determine whether or not induced changes in pS6 expression of astrocytes in the hippocampus of animals on the 4th day of seizure induction. As a result of immunofluorescence staining, it was confirmed that pS6 expression was increased in hippocampal astrocytes, whereas pS6 expression of SE-induced glial cells was significantly decreased in the hippocampus of SAR-treated animals (SE 4d-SAR) on the 4th day of seizure induction (SE 4d-SAR) ( Fig. 4a). Quantitative analysis also showed that SAR treatment significantly reduced the number of pS6-expressing glial cells in the hippocampus 4 days after SE induction ( P < 0.001 compared to SE 4d-Veh; FIG. 4b ). Similarly, Western blot results showed that pS6 protein expression induced by SE was significantly attenuated by SAR treatment ( P = 0.032 versus SE 4d-Veh; FIG. 4C). Through these results, it was found that the increase in the expression of VEGFR-3 in astrocytes is related to the activation of mTOR in the hippocampus after SE induction.

Effect of VEGFR-3 specific inhibitors on reactive astrocytosis after SE induction

The PI3K/Akt/mTOR pathway has been reported to affect GLT-1 regulation in primary cultures of astrocytes (Wu, Kihara, Akaike, Niidome, & Sugimoto, 2010). Therefore, we aimed to investigate whether mTOR inhibition by RA treatment regulates GLT-1 expression in the hippocampus after SE. As a result, it was confirmed that the number of glial-like GLT-1-positive cells increased in the hippocampus 4 days after SE induction (SE 4d-Veh; FIG. 5a ). On the other hand, SE-induced glial GLT-1 expression was found to be decreased by RA treatment (SE 4d-RA; FIG. 5A ). Similarly, the quantitative analysis results vehicle - were significantly increased in the sub-pyramid region of the hippocampus compared to the glue in the treated SE induced 4 days later mouse GLT-1- positive cells sham-Veh group (Sham-veh contrast P <0.001; Figure 5b), the increased number of glial GLT-1-positive cells was found to be significantly reduced by RA treatment ( P = 0.029 versus SE 4d-Veh; Figure 5b). As a result of Western blot, there was no significant difference in total GLT-1 protein level between the sham-Veh and SE 4d-Veh groups ( FIG. 5c ). However, RA treatment significantly reduced total GLT-1 protein levels compared to the SE 4d-Veh group ( P = 0.011 versus SE 4d-Veh; FIG. 5c ).

Since the study results of the present invention suggested an interaction between VEGFR-3 induction and mTOR activation in the activation of astrocytes ( FIGS. 3 and 4 ), the present inventors found that inhibition of VEGFR-3 is GLT-1 expression in the hippocampus after SE induction. And whether it can change the activation of astrocytes was investigated. Pretreatment with SAR significantly increased the number of seizures compared to the vehicle group ( P = 0.002; FIG. 9), whereas there was no significant difference between convulsive seizures and SE onset times. As a result of immunofluorescence staining, it was confirmed that the glial-like GLT-1 immunoreactivity in the hippocampus 4 days after SE induction was significantly increased compared to the sham-Veh and sham-SAR groups (FIG. 6a). However, the increased glia-like GLT-1 expression was again reduced by SAR treatment (SE4d-SAR; FIG. 6A ). Consistent with these results, as a result of quantitative analysis, the number of glial GLT-1-positive cells was significantly increased compared to the Sham-Veh group in the hippocampal subpyramid region 4 days after SE induction ( P < 0.001 compared to Sham-Veh; FIG. 6b ) ), this increase was again attenuated by SAR treatment in the hippocampus (SE 4d-Veh vs. P = 0.005; Fig. 6b). As a result of Western blot for GLT-1, there was no significant difference between the vehicle-treated normal control group (sham-manipulated) and vehicle-treated mice on the 4th day of seizure induction, but the level of total GLT-1 protein was significant by SAR treatment. decreased significantly (P = 0.023 compared to SE 4d-Veh; FIG. 6c ). In addition, as a result of immunofluorescence staining, it was confirmed that GFAP-expressing reactive astrocytes increased in the hippocampus after SE induction and decreased again by SAR treatment ( FIG. 6d ). Similar to the double-immunofluorescence staining results, Western blot analysis results show that the SE-induced GFAP expression level was significantly reduced by SAR treatment compared to the SE 4d-Veh group ( P = 0.042 for SE 4d-Veh; 6e). These results suggest that VEGFR-3 expression in astrocytes after SE may be involved in mTOR-mediated GLT-1 expression and activation of astrocytes in reactive astrocytes in the hippocampus.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

1. VEGFR-3 protein, a nucleic acid molecule encoding the VEGFR-3 protein, and a composition for preventing or treating mTOR signal deficiency-related diseases comprising at least one selected from the group consisting of an activator of VEGFR-3 as an active ingredient.
2. The composition of claim 1, wherein the mTOR signal deficiency-related disease is selected from the group consisting of epilepsy, muscular atrophy, depression, and bone disease.
3. The composition according to claim 2, wherein the bone disease is selected from the group consisting of osteoporosis, osteomalacia, rickets, fibrous osteitis, bone damage due to bone metastasis of cancer cells, aplastic bone disease, metabolic bone disease, and degenerative arthritis.
4. A composition for preventing or treating mTOR-mediated diseases, comprising an inhibitor of VEGFR-3 protein as an active ingredient.
5. 5. The composition of claim 4, wherein the mTOR-mediated disease is selected from the group consisting of cancer, neurodegenerative disease, metabolic disorder, inflammatory disease, and actrocytosis.
6. The method of claim 5, wherein the cancer is selected from the group consisting of kidney cancer, breast cancer, lung cancer, stomach cancer, bladder cancer, prostate cancer, ovarian cancer, cervical cancer, lymphoma, leukemia and myelodysplastic syndrome. composition to do.
7. The method of claim 5, wherein the neurodegenerative disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, myasthenia gravis and Pick's disease. (Pick's disease) composition, characterized in that selected from the group consisting of.
8. The composition according to claim 5, wherein the metabolic disease is selected from the group consisting of obesity, diabetes, dyslipidemia, fatty liver and insulin resistance syndrome.
9. The method of claim 5, wherein the astrocytosis is glioma, glioblastoma, astrocytoma, stroke, traumatic brain injury, amyotrophic lateral sclerosis ( Amyotrophic Lateral Sclerosis (ALS)).
10. A method for screening a composition for preventing or treating a disease related to mTOR signal deficiency, comprising the steps of:

    (a) contacting a test substance with a biological sample containing a VEGFR-3 protein or a gene encoding the same; and

    (2) measuring the expression level or activity of the VEGFR-3 protein or a gene encoding the same in the sample;

    When the expression level or activity of the VEGFR-3 protein or a gene encoding the same is increased, the test substance is determined as a composition for preventing or treating mTOR signal deficiency related diseases.
11. A method for screening a composition for preventing or treating an mTOR-mediated disease, comprising the steps of:

    (a) contacting a test substance with a biological sample containing a VEGFR-3 protein or a gene encoding the same; and

    (2) measuring the expression level or activity of the VEGFR-3 protein or a gene encoding the same in the sample;

    When the expression level or activity of the VEGFR-3 protein or a gene encoding it is reduced, the test substance is determined as a composition for preventing or treating mTOR-mediated diseases.
12. The method according to claim 10 or 11, wherein the biological sample is a biological sample comprising astrocytes.

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