WO2022163959A1 - Use of zbtb16 in degenerative brain disease - Google Patents
Use of zbtb16 in degenerative brain disease Download PDFInfo
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- WO2022163959A1 WO2022163959A1 PCT/KR2021/010056 KR2021010056W WO2022163959A1 WO 2022163959 A1 WO2022163959 A1 WO 2022163959A1 KR 2021010056 W KR2021010056 W KR 2021010056W WO 2022163959 A1 WO2022163959 A1 WO 2022163959A1
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- zbtb16
- protein
- degenerative brain
- brain disease
- dementia
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Definitions
- the present invention is capable of diagnosing degenerative brain disease through Zinc finger and BTB domain-containing protein 16 (ZBTB16). It relates to a kit, a method for providing information necessary for diagnosis of degenerative brain disease, a pharmaceutical composition for treating or preventing degenerative brain disease, and a method for screening a substance capable of preventing or treating degenerative brain disease.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- Degenerative brain disease is a disease that occurs in the brain as we age. Due to unknown causes, a specific group of brain cells in the brain gradually loses its function, death of cranial nerve cells, which is the most important for information transmission in the cranial nervous system, and problems with the shape or function of the synapse that transmits information between cranial nerve cells and cranial nerve cells Or it is known to be caused by an ideal increase or decrease in electrical activity of cranial nerves.
- Patent Document 1 Korean Patent Publication No. 10-2012-0004965
- the present invention has been devised to solve the above problems, and an object of the present invention is to provide a biomarker composition for diagnosing degenerative brain disease.
- Another object of the present invention is to provide a composition for diagnosing degenerative brain disease and a kit for diagnosing degenerative brain disease.
- Another object of the present invention is to provide a method for providing information necessary for diagnosing degenerative brain disease.
- Another object of the present invention is to provide a screening method for a therapeutic substance for degenerative brain disease.
- Another object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain disease.
- the present invention provides a biomarker composition for diagnosing degenerative brain disease, which includes a Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the ZBTB16 protein may be represented by the amino acid sequence of SEQ ID NO: 1.
- the present invention provides a composition for diagnosing degenerative brain disease comprising, as an active ingredient, an agent capable of measuring the expression level of ZBTB16 (Zinc finger and BTB domain-containing protein 16) protein or a gene encoding the same. do.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the agent for measuring the gene expression level is at least one selected from the group consisting of a probe, primer and antisense oligonucleotide that specifically binds to a gene encoding the ZBTB16 protein, and the agent for measuring the protein expression level is the ZBTB16 It may be any one or more selected from the group consisting of an antibody, peptide, aptamer, and compound that specifically binds to a protein.
- the ZBTB16 protein may be represented by the amino acid sequence of SEQ ID NO: 1.
- the present invention provides a kit for diagnosing degenerative brain disease comprising a composition in order to achieve the above another object.
- the present invention provides a method for providing information necessary for diagnosing degenerative brain disease, comprising the following steps.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the method may further include measuring the expression levels of LC3 and p62 proteins from the biological sample of the subject.
- the present invention comprises the steps of: a) treating a candidate material in a sample isolated from an animal or patient with degenerative brain disease; b) measuring the expression level of the ZBTB16 protein or a gene encoding the same in the candidate material treatment group; And c) when the expression level of the ZBTB16 protein or the gene encoding it measured in the above step is lower than that of the candidate material non-treated group (control group), selecting a degenerative brain disease treatment material; A screening method is provided.
- the ZBTB16 protein may be represented by the amino acid sequence of SEQ ID NO: 1.
- Measurement of the expression level is a polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), quantitative real-time polymerase reaction (qRT) -PCR), real time reverse transcription polymerase reaction (real time RT-PCR), RNase protection assay, Northern blot analysis (northern blot analysis), and measurement using any one method selected from the group consisting of DNA chip analysis. have.
- the method may further include measuring the expression levels of LC3 and p62 proteins in the serum of the candidate substance-treated group.
- the step of determining the candidate substance as a degenerative brain disease treatment material is more may include
- the present invention provides a pharmaceutical composition for preventing or treating degenerative brain disease, comprising as an active ingredient an agent capable of inhibiting the expression or activity of ZBTB16 (Zinc finger and BTB domain-containing protein 16) protein do.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the agent capable of inhibiting the expression is siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, which specifically binds to the mRNA of the gene encoding the ZBTB16 protein. It is characterized in that it contains any one or more selected from the group consisting of peptide nucleic acids (PNA) and antisense oligonucleotides, and the agent capable of inhibiting the activity is a compound capable of specifically binding to ZBTB16 protein, a peptide, It may include any one or more selected from the group consisting of aptamers, proteins and antibodies.
- PNA peptide nucleic acids
- ribozyme DNAzyme
- the shRNA is a recombinant adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposomes and niosomes in any one carrier selected from the group consisting of may be transmitted by
- the shRNA may be any one or more selected from the group consisting of a nucleotide sequence represented by SEQ ID NO: 7, a nucleotide sequence represented by SEQ ID NO: 8, a nucleotide sequence represented by SEQ ID NO: 9, and a nucleotide sequence represented by SEQ ID NO: 10.
- the degenerative brain disease includes Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment, It may be any one or more selected from the group consisting of Down's syndrome and Huntington's disease.
- the composition may improve, prevent or treat Behavioral and Psychological Symptoms of Dementia (BPSD) caused by degenerative brain diseases.
- BPSD Behavioral and Psychological Symptoms of Dementia
- BPSD Behavioral and Psychological Symptoms of Dementia
- delusions include delusions, hallucinations, agitation, aggression, activity disturbances, depression, and anxiety.
- diurnal rhythm disturbances include elation, irritability, lability, affective lability, defective self regulation, abnormal repetitive behavior ( aberrant motor behavior), anxiety and fear (anxiety and phobias), and may be any one or more selected from the group consisting of sleep disorders.
- a biomarker composition for diagnosing degenerative brain disease comprising Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient, wherein ZBTB16 in the brain striatum of an animal model of degenerative brain disease
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- FIG. 1 is a diagram showing the positions of the dorsal striatum and the nucleus accumbens (NAc) in the brain tissue (Brain) separated from the control group (WT) and the dementia animal model (APPPS1).
- Figure 2 is a control (WT) and dementia animal model (APPPS1) from the brain tissue (Brain) from the dorsal striatum (dorsal striatum) and the nucleus accumbens (nucleus accumbens, NAc) from the ZBTB16 protein expression was measured by immunohistochemistry. is the result
- FIG 3 is a graph showing the Y-maze test (YMT) results for the control group (WT+GFP), the dementia animal model (APPPS1+GFP), and the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16).
- FIG. 4 is a graph showing the results of a novel object recognition test (NOR) for a control group (WT+GFP), an animal model of dementia (APPPS1+GFP), and an animal model of ZBTB16 knockdown dementia (APPPS1+shZbtb16).
- NOR novel object recognition test
- FIG. 5 is a view showing the 3-chamber test (3CT) results of the animal model of dementia (APPPS1 + GFP), the animal model of ZBTB16 knockdown dementia (APPPS1 + shZbtb16) and the control (WT + GFP) prepared according to the present invention
- FIG. 5a is an inanimate object (0) and a living mouse (S1) of the animal model of dementia (APPPS1+GFP), ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and control (WT+GFP) prepared according to the present invention.
- Figure 2b is a familiar mouse (S1) and a new animal model (APPPS1 + GFP), ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and control (WT + GFP) prepared according to the present invention. It is a diagram showing the degree of interest in the mouse (S2).
- FIG. 6 is a diagram showing the results of the elevated cross maze experiment (EMP) of the dementia animal model (APPPS1 + GFP), the ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and the control group (WT + GFP) prepared according to the present invention.
- EMP elevated cross maze experiment
- LLB light-dark shift test
- FIG. 9 is a result of measuring the expression levels of LC3, p62 from the dorsal striatum in brain tissue isolated from the ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) prepared according to the present invention by immunohistochemistry.
- GFP green fluorescent protein
- 11 is a result of measuring the expression levels of ZBTB16 and p62 from dorsal striatum in brain tissue isolated from a 12-month-old normal animal model (WT+GFP (12mo)) by immunohistochemistry.
- FIG. 13 is a result of measuring the expression levels of ZBTB16 and p62 from dorsal striatum in brain tissue isolated from a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16(12mo)) by immunohistochemistry.
- Figure 14a is a 12-month-old dementia animal model (APPPS1 + GFP) prepared according to the present invention, a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16), and a 12-month-old control (WT + GFP) inanimate object (0) and living It is a diagram showing the degree of interest (3CT:sociability) for the mouse (S1).
- 14B shows familiar mice (S1) and new animals of a 12-month-old dementia animal model (APPPS1+GFP), a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and a 12-month-old control group (WT+GFP) prepared according to the present invention. It is a diagram showing the degree of interest (3CT: novelty) for the mouse (S2).
- FIG. 15 is a Y-maze of a 12-month-old normal animal model (control group (WT+GFP)) and a 12-month-old dementia animal model (APPPS1+GFP) and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) prepared according to the present invention.
- WT+GFP control group
- APPPS1+GFP 12-month-old dementia animal model
- APPPS1+shZbtb16 12-month-old ZBTB16 knockdown dementia animal model
- the present inventors have identified the interaction between degenerative brain disease and ZBTB16, and based on this, the present invention has been completed.
- One aspect of the present invention relates to a biomarker composition for diagnosing degenerative brain disease, which includes Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the biomarker composition may further include p62 and LC3, which are known autophagosome biomarkers.
- the LC3 protein is LC3 II (mitrotubule-associated protein light chain 3), and p62 is Sequestosome 1 and SQSTM1.
- 'Zinc finger and BTB domain-containing protein 16 (ZBTB16)' is a protein encoding the ZBTB16 gene.
- the gene is one of the Krueppel C2H2 type zinc-finger protein members, and encodes a zinc finger transcription factor including nine Kruppel-type zinc finger domains at the carbosil end.
- the protein is located in the nucleus, is involved in cell cycle progression, and is known to interact with Histone Deacetylase (HDAC).
- HDAC Histone Deacetylase
- amino acid sequence of ZBTB16 may be represented by SEQ ID NO: 1 registered on uniprot.org. In this case, it may include an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with the amino acid sequence represented by SEQ ID NO: 1. .
- nucleotide sequence of the gene encoding the ZBTB16 is registered at ncbi.nlm.nih.gov. More specifically, it can be found in In this case, it may include an amino acid sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence represented by SEQ ID NO: 2 .
- 'degenerative brain disease' is a disease that causes various symptoms as degenerative changes appear in nerve cells of the central nervous system.
- the disease continues to progress over many years or decades until death, and there is often a family history.
- Specific examples of the degenerative brain disease of the present invention are not limited thereto, but Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment, Down's syndrome, Huntington's disease, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's syndrome s Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia and Progressive Supranuclear Palsy ) may be any one or more selected from the group consisting of.
- the degenerative brain disease may be more preferably a degenerative brain disease induced by beta-amyloid accumulation, and even more preferably dementia.
- the dementia is not particularly limited, and for example, Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, Parkinson's disease ), mild cognitive impairment, Down's syndrome and Huntington's disease may be any one or more selected from the group consisting of.
- the present invention can diagnose early degenerative brain disease or dementia in which the core symptoms of dementia or degenerative brain disease such as cognitive impairment do not appear.
- the early degenerative brain disease or dementia may include delusions, hallucinations, agitation, aggression, activity disturbances, depression, anxiety, Diurnal rhythm disturbances, elation, irritability, lability, affective lability, defective self regulation, aberrant motor behavior , including those with only one or more mental and behavioral disorders selected from the group consisting of anxiety and phobias and sleep disorders.
- 'diagnosis' in a broad sense means judging the actual condition of a patient's disease in all aspects.
- the content of the judgment is the disease name, etiology, disease type, severity, detailed mode of the disease, presence or absence of complications, and prognosis.
- to determine the susceptibility of an object to a particular disease or condition to determine whether an object currently has a particular disease or disorder, or to determine the prognosis of an object with a particular disease or condition or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy).
- 'prognosis' means a prospect for future symptoms or progress determined by diagnosing a disease.
- the prognosis usually refers to the mode of degenerative brain disease within a certain period of time after the onset or treatment of the degenerative brain disease, the development of mental and behavioral symptoms, or the survival period.
- the prediction of prognosis is very important as it provides clues about the future direction of treatment for degenerative brain disease, especially for patients with degenerative brain disease.
- a 'biomarker' refers to an indicator that can detect changes in the body using proteins, DNA, RNA (Reebok nucleic acid), metabolites, and the like.
- ZBTB16 can be used as a biomarker for diagnosing or early diagnosis of degenerative brain disease. That is, if the expression level of the ZBTB16 protein or a gene encoding it is measured, it is possible to provide information necessary for the diagnosis of degenerative brain disease.
- "expression” means that a protein or nucleic acid is produced in a cell.
- 'Protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to, for example, a polymer of amino acid residues as commonly found in proteins in their natural state.
- 'Polynucleotide' or 'nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner analogous to naturally occurring nucleotides are also included.
- 'mRNA' refers to RNA that transfers genetic information (gene-specific nucleotide sequence) to ribosomes that specify an amino acid sequence from a specific gene during protein synthesis.
- Another aspect of the present invention relates to a composition for diagnosing degenerative brain disease, comprising as an active ingredient an agent capable of measuring the expression level of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the agent for measuring the gene expression level may be any one or more selected from the group consisting of a probe, primer, and antisense oligonucleotide that specifically binds to a gene encoding the ZBTB16 protein.
- the agent for measuring the protein expression level may be any one or more selected from the group consisting of an antibody, peptide, aptamer, and compound that specifically binds to the ZBTB16 protein.
- 'probe' refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases in length that can specifically bind to mRNA, and is labeled so that the presence or absence of a specific mRNA, the amount of expression can confirm.
- the probe may be manufactured in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. Suitable probe selection and hybridization conditions may be appropriately selected according to techniques known in the art.
- a 'primer' is a nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for template strand copying. say sequence.
- the primer is capable of initiating DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature.
- a reagent for polymerization ie, DNA polymerase or reverse transcriptase
- PCR conditions and lengths of sense and antisense primers may be appropriately selected according to techniques known in the art.
- the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods.
- the primer or probe can be variously modified according to methods known in the art within the range that does not interfere with hybridization with ZBTB16 mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and fluorescence or enzymatic binding of a labeling material.
- 'antibody' is a term known in the art and refers to a specific immunoglobulin directed to an antigenic site.
- the antibody in the present invention refers to an antibody that specifically binds to ZBTB16 of the present invention, and the antibody can be prepared according to a conventional method in the art.
- the form of the antibody includes polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included.
- the antibody refers to a complete form having two full-length light chains and two full-length heavy chains.
- the said antibody also includes special antibodies, such as a humanized antibody.
- the 'peptide' has an advantage of high binding strength to the target material, and no denaturation occurs even during heat/chemical treatment.
- the molecular size is small, it can be used as a fusion protein by attaching it to other proteins. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and drug delivery material.
- 'aptamer' refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample, Specifically, the presence of the target protein in the sample may be confirmed.
- the aptamer is synthesized by determining the sequence of an oligonucleotide having a selective and high binding affinity to the target protein (ZBTB16 protein in the present invention) to be confirmed, and then 5' of the oligonucleotide.
- -SH, -COOH, -OH or NH 2 It may be made by modifying it, but is not limited thereto.
- the gene of the present invention has a nucleic acid sequence registered in the gene bank, a person skilled in the art can design an antisense oligonucleotide, primer pair or probe that specifically amplifies a specific region of these genes based on the sequence.
- the diagnostic composition of the present invention comprising the ZBTB16 protein-specific agent may additionally include an agent necessary for a method for detecting a known protein, and the method for detecting a known protein using the composition can be used without limitation.
- the expression level of ZBTB16 protein in the sample can be measured.
- kits for diagnosing degenerative brain disease including Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the kit includes an antibody that specifically binds to a marker component, a secondary antibody conjugate to which a label that develops color by reaction with a substrate is conjugated, a color-developing substrate solution to react with the label, a washing solution and an enzyme reaction stop solution and the like, and may be manufactured as a plurality of separate packaging or compartments containing the reagent components used.
- the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction.
- the reverse transcription polymerase reaction kit includes each primer pair specific for a marker gene.
- the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of the control gene.
- reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.
- the DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescent probe.
- the substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.
- Samples used for analysis include blood, blood cells, brain tissue, nerve cells, cerebrospinal fluid, saliva, nasal fluid, sputum, capsular fluid, amniotic fluid, ascites, cervical or vaginal secretions, and urine degenerative brain diseases that can be distinguished from normal conditions. It includes a biological sample capable of identifying a specific protein associated with it. Preferably, it can be measured from a biological sample, for example, any one sample selected from the group consisting of blood, blood cells, brain tissue, nerve cells, and cerebrospinal fluid. The sample may be prepared to increase the detection sensitivity of the protein marker.
- a serum sample obtained from a patient may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, It may be pre-treated using a method such as sequential extraction or gel electrophoresis.
- Another aspect of the present invention relates to a method for providing information necessary for diagnosing degenerative brain disease, comprising the following steps.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- a biological sample is separated from a subject, and the expression level of a zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same is measured from the biological sample.
- ZBTB16 zinc finger and BTB domain-containing protein 16
- the term 'subject' refers to any single entity that is suspected of degenerative brain disease, including humans, cows, dogs, guinea pigs, rabbits, chickens, insects, mice, mice, and the like, and requires a diagnosis of degenerative brain disease. do. Also included in the subject are any subjects participating in a clinical study trial without any clinical findings of degenerative brain disease, or subjects participating in epidemiological studies or subjects used as controls. In an embodiment of the present invention, mice or mice were used.
- the 'subject suspected of degenerative brain disease' has the core symptoms of degenerative brain disease or Behavioral and Psychological Symptoms of Dementia (BPSD) caused by degenerative brain disease, and is expected to have degenerative brain disease may be a target.
- BPSD is a mental and behavioral disorder that occurs frequently as degenerative brain disease, especially dementia, progresses from moderate to severe. activity disturbances, depression, anxiety, diurnal rhythm disturbances, elation, irritability, lability, affective lability, deficits in self-regulation (defective self regulation), abnormal motor behavior (aberrant motor behavior), anxiety and fear (anxiety and phobias) and includes any one or more selected from the group consisting of sleep disorders.
- the 'biological sample' may be used without limitation as long as it is collected from a subject to be diagnosed with degenerative brain disease, for example, blood, blood cells, brain tissue, nerve cells, cerebrospinal fluid, saliva, nasal fluid, sputum, joint capsule fluid, It may be any one selected from the group consisting of amniotic fluid, ascites, cervical or vaginal secretions and urine, but is not particularly limited thereto, and is preferably selected from the group consisting of blood, blood cells, brain tissue, nerve cells, and cerebrospinal fluid. It may be any one sample.
- 'measurement' may preferably mean 'analysis', which means 'qualitative analysis', which means measuring and confirming the presence of a target substance, or the presence level (expression level) of a target substance. Or it may mean 'quantitative analysis' that measures and confirms the change in quantity.
- the measurement may be performed including both a qualitative method and a quantitative method, and preferably, a quantitative measurement may be performed.
- the sample may be pretreated prior to use for detection or diagnosis.
- it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.
- the protein expression level measurement is not particularly limited as long as it is by a protein expression measurement method known in the art, but for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immune diffusion method (Ouchterlony). immunodiffusion), rocket immunoelectrophoresis, immunostaining, immunoprecipitation, complement fixation, FACS, and protein chip.
- the gene expression level measurement is not particularly limited as long as it is a gene expression measurement method known in the art, but preferably a polymerase reaction (PCR), a quantitative polymerase reaction (qPCR), a reverse transcription polymerase reaction (RT-PCR), competitive Reverse transcription polymerase reaction (competitive RT-PCR), quantitative real-time polymerase reaction (qRT-PCR), real time reverse transcription polymerase reaction (real time RT-PCR), RNase protection assay, northern blot analysis and DNA chip It may be any one method selected from the group consisting of analysis, and more preferably, quantitative polymerase reaction (qPCR) or northern blot analysis.
- the expression level of the Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding it in the subject sample measured by the method of step (1) described above was measured in the same manner as ZBTB16 in the normal control sample. It is compared with the expression level of the protein or the gene encoding it (step (2)).
- the sample is preferably a sample obtained by the same type or the same method between the subject and the normal control.
- step (3) when the expression level of the ZBTB16 protein or the gene encoding it is higher than that of the normal control sample, it can be determined or diagnosed as degenerative brain disease (step (3)). Specifically, when the ZBTB16 expression level of the subject sample measured by the above method is increased compared to the sample of a healthy normal control group, it may be determined or diagnosed as having a degenerative brain disease.
- the expression level of the biomarker (ZBTB16) of the present invention is measured in a biological sample obtained from a subject suspected of degenerative brain disease, and the present invention is obtained from a normal control biological sample obtained by the same type or method. After measuring the expression level of the biomarker (ZBTB16), the two are compared, but when the expression level of the subject is lower than the expression level of the normal control, it can be determined that the subject has degenerative brain disease.
- Another aspect of the present invention relates to a screening method for a therapeutic substance for degenerative brain disease, comprising the following steps.
- the term 'animal degenerative brain disease induced' refers to an animal in which various symptoms are induced while degenerative changes appear in nerve cells of the central nervous system, more preferably an animal in which dementia is induced by the accumulation of beta-amyloid, and a human It may be a mammal except for, preferably a mouse, a guinea pig, a pig, a bird, and the like.
- the 'isolated sample' may be a tissue or cell derived from an animal induced with degenerative brain disease.
- the sample includes a tissue or a cell thereof capable of detecting the expression level of ZBTB16 in an animal induced with a degenerative brain disease, and preferably a tissue or cell isolated from the brain of an animal induced with a degenerative brain disease.
- the present invention is not limited thereto.
- the 'candidate substance' means a substance to be tested for therapeutic activity for degenerative brain disease, and may include any molecule such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide range of compounds. have.
- the 'candidate material' may be a cell transfected with a liposome or a vector. The transfection may be performed using a microinjection method, a calcium phosphate co-precipitation method, an electroporation method, or a liposome method, but is not limited thereto.
- the 'candidate untreated group' is also referred to as a control group, which is an animal or a sample separated from degenerative brain disease induced without treatment with a candidate material, and is a degenerative disease belonging to the group treated with the candidate material and in a parallel relationship. It refers to an animal or a sample isolated from the induced brain disease.
- the 'screening method' may be performed by confirming the expression of ZBTB16, a biomarker of degenerative brain disease, and comparing the candidate substance with an untreated group (control group).
- the degenerative brain disease may specifically be a degenerative brain disease caused by the accumulation of beta-amyloid.
- the degenerative brain disease may be dementia, and the dementia includes Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, and Parkinson's. It may be any one or more selected from the group consisting of Parkinson's disease, mild cognitive impairment, Down's syndrome, and Huntington's disease.
- ZBTB16 expression was significantly higher in the brain tissue of the animal model of dementia compared with the normal animal model, and it was confirmed that there were mental and behavioral disorders in the animal model of dementia. However, it was confirmed that the expression of ZBTB16 was significantly reduced in the knockout animal model in which the expression of ZBTB16 was suppressed from the animal model of dementia, and the expression of mental and behavioral disorders was significantly lower than that of the normal animal model.
- 'prevention' includes all actions that suppress or delay the onset and symptoms of the degenerative brain disease, and includes prevention of disease recurrence after treatment as well as prevention before disease occurrence.
- 'treatment' includes all of the treatment of symptoms of the degenerative brain disease, improvement of symptoms, and inhibition of progression of symptoms.
- the present invention b) measures the expression level of the ZBTB16 protein or a gene encoding it in the candidate substance-treated group, and the expression level of the ZBTB16 protein or the gene encoding it measured in step b) is the candidate substance untreated group (control group) ), it can be selected as a therapeutic substance for degenerative brain disease (step c)).
- the present invention measures the expression level of ZBTB16 protein or a gene encoding the same in an animal or a sample isolated from degenerative brain disease induced in the absence of a candidate substance capable of preventing or treating the degenerative brain disease (candidate Substance-untreated group), after measuring the expression level of ZBTB16 protein or a gene encoding it in the presence of the candidate substance (candidate substance-treated group) and comparing both, ZBTB16 protein or coding for it when the candidate substance is present
- a substance that suppresses the expression level of a gene rather than the level in the absence of the candidate substance may be predicted or determined as a substance for preventing or treating degenerative brain disease.
- 'measurement of expression level' is a process of confirming the expression level of the ZBTB16 protein or a gene encoding the same in an animal or a sample isolated from degenerative brain disease induced in the absence and presence of a candidate substance.
- the expression level was measured by Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), quantitative real-time polymerase reaction (qRT- PCR), real-time RT-PCR, RNase protection assay, northern blot analysis, and DNA chip analysis may be any one method selected from the group consisting of.
- the screening method for a therapeutic substance for degenerative brain disease of the present invention may further include measuring the expression levels of LC3 and p62 proteins in the serum of the candidate substance-treated group.
- the step of determining the candidate substance as a degenerative brain disease treatment material is more may include
- 'LC3 and p62' are widely known as marker proteins for autophagy.
- ZBTB16 expression increases when degenerative brain disease occurs, autophagy is reduced, and autophagy is suppressed, so that the disease protein in the striatum cannot be reduced and accumulate, so it can be seen that degenerative brain disease is induced. Therefore, when ZBTB16 is inhibited in brain tissue during degenerative brain disease, autophagy is activated in the brain, so that the expression level of LC3 increases and the expression level of p62 decreases.
- ZBTB16 gene of the present invention is increased during degenerative brain disease.
- LC3 protein which is a biomarker related to autophagy
- p62 protein was significantly reduced. This means that ZBTB16 protein is not only involved in degenerative brain disease, but also the mechanism of action involved in degenerative brain disease, meaning that it can function as a diagnostic, therapeutic, and screening marker.
- the candidate material selected through the degenerative brain disease prevention or treatment material screening method may suppress or inactivate the expression level of the ZBTB16 gene when compared to the candidate material untreated group (control group) to which the candidate material is not administered.
- Another aspect of the present invention relates to a composition or kit for screening a substance for treating degenerative brain disease, comprising as an active ingredient an agent capable of inhibiting the expression or activity of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- ZBTB16 can function as a screening marker for a therapeutic substance for degenerative brain disease. That is, if the expression level of the ZBTB16 protein or a gene encoding it can be measured, a substance for preventing or treating degenerative brain disease can be screened.
- Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating degenerative brain disease, comprising an agent capable of inhibiting the expression or activity of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein as an active ingredient.
- ZBTB16 Zinc finger and BTB domain-containing protein 16
- the expression of ZBTB16 may include both mRNA expression of a gene, mRNA expression into protein, and mRNA degradation or protein degradation.
- the activity of ZBTB16 includes the biological function/activity of the ZBTB16 protein in a cell or tissue.
- 'activity' refers to a biological action that allows the expressed protein to perform its original function in a cell.
- 'expression' includes all steps of a process in which a gene is made into a protein in vitro or in a cell, and includes, for example, transcription from gene to mRNA and translation from mRNA to protein.
- the agent capable of inhibiting the expression of the Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein is an siRNA (small interference RNA) or shRNA (short hairpin RNA) that specifically binds to the mRNA of a gene encoding the ZBTB16 protein.
- siRNA small interference RNA
- shRNA short hairpin RNA
- miRNA miRNA
- ribozyme ribozyme
- DNAzyme peptide nucleic acids
- PNA peptide nucleic acids
- siRNA small interfering RNA
- shRNA small hairpin RNA
- miRNA miRNA
- RISC RNA Induced Silencing Complex
- An siRNA, shRNA or miRNA has a sequence that is highly complementary to its target sequence.
- highly complementary sequence is meant at least about 70% complementarity, at least about 80% complementarity, at least about 90% complementarity, or about 100% complementarity with a sequence of at least contiguous 15 bases in length of the target gene.
- Antisense oligonucleotides, siRNAs, shRNAs and/or miRNAs targeting the CTRP3 gene according to the present disclosure of various origins may be used as long as they bind to a target nucleic acid and function as a silencing, and bioequivalents, derivatives and analogs thereof will also include Antisense oligonucleotides are short synthetic nucleic acids that are known in the art and inhibit/reduce expression of the target protein, for example by binding to any coding sequence of the target protein.
- Antisense RNA may have an appropriate length depending on the target gene and delivery method, for example, about 6, 8 or 10 to 40, 60 or 100 nucleotides.
- shRNA Since the siRNA has to be prepared in vitro and the knockdown gene is typically delivered transiently for 6-10 days, it is preferable to use shRNA.
- the shRNA is a molecule of about 20 or more bases having a double-stranded structure in the molecule and having a hairpin-like structure in the molecule by including a partially palindromic nucleotide sequence in the single-stranded RNA.
- the shRNA for inhibiting ZBTB16 expression has a sequence complementary to a part of the ZBTB16 gene, and can degrade the mRNA of the ZBTB16 gene or inhibit translation.
- the complementarity is 80-90%, the translation of mRNA can be inhibited, and when the complementarity is 100%, the mRNA can be degraded.
- the shRNA that inhibits ZBTB16 expression is complementary to SEQ ID NO: 3 (GACTGGAGGATAGAGAAGACGTACCTCTA), SEQ ID NO: 4 (AATGGCTGTGGCAAGAAGTTCAGCCTCAA), SEQ ID NO: 5 (GCAAGCCAAGGCGGAGGACCTGGATGACC) and SEQ ID NO: 6 (ATCTCGAAGCATTCCCA complementary to mouse and human common mRNA) It is preferable to include a nucleotide sequence having 100% homology to the sequence.
- the shRNA relates to an shRNA that inhibits ZBTB16 expression, and may be any one or more of the nucleotide sequences shown in SEQ ID NOs: 7 to 10 below.
- the shRNA may be a sequence complementary to any one of SEQ ID NOs: 3 to 6 commonly found in mouse and human ZBTB16 mRNA, preferably SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 It may be any one sequence selected from the displayed nucleotide sequences.
- Each of the nucleotide sequences may be connected palindrome by a loop region of 4 to 10 bp to form a hairpin structure.
- SEQ ID NO: 7 TAGAGGTACGTCTTCTCTATCCTCCAGTC
- SEQ ID NO: 8 TTGAGGCTGAACTTCTTGCCACAGCCATT
- SEQ ID NO: 9 GGTCATCCAGGTCCTCCGCCTTGGCTTGC
- SEQ ID NO: 10 CCACTCTCCTCGCTGGAATGCTTCGAGAT
- shRNA short hairpin RNA
- Substances that inhibit ZBTB16 expression by RNAi may be artificially synthesized chemically, or hairpin-structured DNA in which the DNA sequences of the sense strand and the antisense strand are linked in the reverse direction using T7 RNA polymerase under laboratory conditions (in vitro). It may also be prepared by synthesizing RNA in When synthesizing under laboratory conditions, antisense and sense RNA can be synthesized from template DNA using T7 RNA polymerase and T7 promoter.
- introduction into cells induces RNAi and suppresses the expression of ZBTB16.
- the introduction into cells can be performed, for example, by a calcium phosphate method or a method using various transfection reagents (eg, oligofectamine, lipofectamine, lipofection, etc.).
- an expression vector containing shRNA or the DNA may be used, or cells containing the expression vector may be used.
- the type of the expression vector or cell is not particularly limited, but an expression vector or cell already used as a medicine is preferable.
- the vector refers to a means for expressing a target gene in a host cell.
- the vector includes elements for expression of a target gene, and may include an origin of replication, a promoter, an operator, a transcription termination sequence, and the like, into the genome of a host cell. Appropriate enzymatic sites (eg, restriction enzyme sites) for introduction of and/or ribosome binding sites (RBS) for translation into proteins and/or selectable markers to confirm successful introduction into host cells, IRES (Internal Ribosome Entry Site) and the like may be additionally included.
- the vector may further include a transcriptional control sequence (eg, enhancer, etc.) other than the promoter.
- the vector may be a plasmid DNA, a recombinant vector, or other medium known in the art, and specifically, a linear DNA plasmid DNA, a recombinant non-viral vector, a recombinant viral vector, or an inducible gene expression vector system), and the recombinant viral vector is a retrovirus, adenovirus, adeno associated virus, helper-dependent adenovirus, herpes simplex virus ( herpes simplex virus), lentivirus or vaccinia virus vector, but is not limited thereto.
- the method for introducing the expression vector into a cell is not particularly limited as long as it is a known method, but is preferably liposome-mediated transfection, calcium phosphate method, DEAE-dextran-mediated transfection, positively charged lipid-mediated transtransfection, electroporation, and phage system. It may include any one or more selected from transduction using a virus or an infection method using a virus.
- the present invention may include the recombinant expression vector for shRNA expression.
- shRNA having the nucleotide sequence represented by SEQ ID NO: 2 as the target sequence may be used.
- the recombinant vector is not particularly limited as long as it is a recombinant DNA method known in the art.
- the shRNA is delivered to any one carrier selected from the group consisting of recombinant adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposome and niosome. can be transmitted by any one carrier selected from the group consisting of recombinant adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposome and niosome. can be transmitted by AAV.
- AAV adeno-associated viruses
- the agent capable of inhibiting the activity of the ZBTB16 protein may include any one or more selected from the group consisting of compounds, peptides, aptamers, proteins and antibodies capable of specifically binding to the ZBTB16 protein.
- the substance capable of inhibiting the expression or activity of the ZBTB16 protein may also include a substance for treating degenerative brain disease screened according to the method for screening a substance for treating degenerative brain disease described above.
- ZBTB16 By inhibiting the expression or activity of ZBTB16 according to the embodiments described herein, it is possible to prevent or treat degenerative brain disease or mental and behavioral disorders resulting therefrom.
- the pharmaceutical composition may be for preventing or treating degenerative brain disease.
- the pharmaceutical composition may be provided in any dosage form suitable for topical application.
- it may be any one route selected from the group consisting of oral administration, transdermally administration, intravenous administration, intramuscular administration, subcutaneous injection and intra-brain administration.
- composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
- composition according to the present invention can be used as a suspending agent, solubilizer, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, if necessary depending on the administration method or formulation. , antioxidants and the like may be included as appropriate.
- pharmaceutically acceptable carriers and agents suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
- 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 Alternatively, it may be prepared by being introduced into a multi-dose container.
- the pharmaceutical composition may be administered by any device capable of moving the active ingredient to the target cell.
- an administration method using a non-invasive catheter may be used.
- the pharmaceutical composition of the present invention may be included in a therapeutically effective amount for the treatment of diseases.
- the term 'treatment' means reversing, alleviating, inhibiting the progression, or preventing one or more symptoms of a disease or disease to which the term is applied, unless otherwise stated.
- the term 'therapeutically effective amount' refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, doctor or other clinician, which is a therapeutic an amount that induces alleviation of the symptoms of the disease or disorder being used. It is apparent to those skilled in the art that the active ingredient included in the pharmaceutical composition of the present invention will change depending on the effect.
- the optimal content of the pharmaceutical composition can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, and the age, weight, general health status, sex and diet of the patient , administration time, administration route and secretion rate of the composition, treatment period, and drugs used at the same time may be adjusted according to various factors.
- the method is not particularly limited as long as shRNA or shRNA expression vector that suppresses ZBTB16 expression is expressed in the cell to which it is applied.
- a viral vector liposome
- animal viruses such as a retrovirus, a vaccinia virus, an adenovirus, and a synrinsemlici virus, are mentioned, for example.
- Substances that inhibit ZBTB16 expression by RNAi may be directly injected into cells.
- the dosage of the composition of the present invention is about 1x10 4 particles to 1x10 12 particles per kg of adult as an active ingredient.
- the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art In consideration of these factors, the dosage may be appropriately adjusted.
- the number of administration may be one time or two or more within the range of clinically acceptable side effects, and may be administered to one site or two or more sites for the administration site.
- the dose is the same as that of a human per kg, or the above dose is adjusted, for example, by the volume ratio (for example, the average value) of the ischemic organ (heart, etc.) between the target animal and the human.
- the converted amount can be administered.
- the animal to be treated according to the present invention include humans and other target mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, goats, horses, pigs, etc. This is included.
- the pharmaceutical composition of the present invention may contain various diseases or disorders related to degenerative brain diseases, such as Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia ( frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment, Down's syndrome, Huntington's disease, amyotrophic lateral sclerosis, spinal cerebellar Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, muscle tone It can be used for the treatment of Dystonia and Progressive Supranuclear Palsy.
- Alzheimer-type dementia Lewy body dementia
- frontotemporal dementia frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment
- Down's syndrome Huntington's disease
- amyotrophic lateral sclerosis spinal cerebellar Spinocerebellar Atrophy
- Tourette's Syndrome Friedrich's Ataxia
- treatment refers to (i) alleviating or suppressing symptoms of degenerative brain disease; (ii) alleviating or alleviating mental and behavioral disorders caused by degenerative brain disease; and (iii) inhibition of degenerative brain disease by promoting autophagy of disease proteins related to degenerative brain disease.
- therapeutically effective amount refers to an amount sufficient to achieve the aforementioned pharmacological effect.
- APP/PS1 mice were prepared for use as experimental animals.
- the APP/PS1 mouse is a representative Alzheimer's dementia animal model genetically mutated to overproduce beta amyloid (A ⁇ , ⁇ amyloid).
- a ⁇ beta amyloid
- This is APPswe/PSEN1dE9(line 85)(The Jackson Lab; available through the JAX MMRRC Stock# 034829(formerly Jackson Lab Stock #004462))(https://www.alzforum.org/research-models/appswepsen1de9-line-85) ) line, and was provided by the KIST Research Animal Resource Center, which maintains and sells it.
- control group WT+GFP
- a ⁇ , ⁇ amyloid a control group that was not genetically mutated to overproduce beta amyloid
- pGFP-C-shLenti-Zbtb16 shRNA vector (ORIGENE) expressing shRNA (SEQ ID NO: 7: 5'-TAGAGGTACGTCTTTCCTATCCTCCAGTC-3') against the gene (SEQ ID NO: 2) of Zbtb16 from ORIGENE (USA) and a specific portion as a negative control
- a pGFP-C-shLenti-non-targeting shRNA control vector TR30021 expressing shRNA with a random sequence was purchased.
- a lentivirus expressing Zbtb16 shRNA or control shRNA was prepared using the vector, third-generation packaging system (pMDLg/pRRE, pRSV-Rev, pMD2.G) and HEK293 T cell line according to the manufacturer's manual.
- HEK293T cell cultures containing lentivirus were concentrated by ultracentrifugation at 50,000 g at 4° C. for 90 minutes. Thereafter, the lentivirus concentrate was titrated using a Lenti-XTM p24 Rapid Titer Kit (clontech, USA).
- ZBTB16 protein knockdown dementia animal model (APPPS1 + shZtbt16) was prepared by breeding for 1 month (6 months old), control group (WT + GFP) for comparative experiments, and dementia animal model (APPPS1 + GFP) were also bred for 1 month under the same conditions. prepared (6 months old).
- the animal model was allowed to freely ingest drinking water and feed under conditions of 12 hours of light and 12 hours of shade in a kennel capable of constant temperature and constant humidity.
- a control group (WT+GFP) and an animal model of dementia (APPPS1+GFP) were prepared, and the experiment was performed at the age of 6 months by breeding for 1 month as in the ZBTB16 protein knockdown dementia animal model. They were randomly assigned to 8 subjects each.
- immunohistochemistry evaluation was performed.
- the control group (WT) and the dementia animal model (APPPS1) were anesthetized and perfused, respectively, and brains were removed and brain tissues were fixed with 4% PFA.
- the tissues of the striatum and the nucleus accumbens were obtained from the brain tissue, and a mouse anti-ZBTB16 antibody (D-9; sc-28319, Santa Cruz Biotechnology, Santa Cruz, CA) was attached to the tissues of the striatum and the nucleus accumbens, respectively ( 4°C, overnight). Then, the tissue was treated with a biotinylated anti-mouse secondary antibody, followed by staining for color development. At this time, the cell nucleus was stained with DAPI.
- Figure 2 is a control (WT) and dementia animal model (APPPS1) from the brain tissue (Brain) from the dorsal striatum (dorsal striatum) and the nucleus accumbens (nucleus accumbens, NAc) from the ZBTB16 protein expression was measured by immunohistochemistry. is the result
- the expression of ZBTB16 protein was significantly increased in the dorsal striatum of the dementia animal model.
- the expression level of ZBTB16 protein in the dementia animal model was significantly increased, so the ZBTB16 protein provides information for the diagnosis of degenerative brain diseases including dementia know you can do it.
- the novel object recognition test is a modification of the existing method and consists of an adaptor, a searcher, and a new material recognizer. After acclimatization by placing the mice in an open field for 10 minutes for two days, two identical objects were installed at regular intervals. The time spent in each object was measured by Ethovision while the experimental animals were allowed to freely explore for 3 minutes, and then returned to the cage for 30 minutes. Then, one of the installed objects was replaced with a new material and the movement of the experimental animal was measured for 3 minutes. The object's recognition index (%) was converted into a percentage of the time spent in each object among the total search time.
- FIG 3 is a graph showing the Y-maze test (YMT) results for the control group (WT+GFP), the dementia animal model (APPPS1+GFP), and the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16).
- the present invention aims to examine whether dementia can be diagnosed early from a dementia risk group without cognitive function symptoms, and the effect of improving, preventing or treating the prognostic symptoms (mental symptoms) that occur in the early stages of dementia, so the animal of the present invention
- 6-month-old mice which are 6 months younger than 12 months old, were used. Therefore, it can be seen that although dementia was clearly induced, there was no significant difference between the control group, the dementia animal model, and the ZBTB16 knockdown dementia animal model as a result of the YMT experiment. This result is consistent with what the inventors expected.
- N denotes a novel material
- F denotes an existing thing (familiar).
- the present invention aims to examine whether dementia can be diagnosed early from a dementia risk group without cognitive function symptoms, and the effect of improving, preventing or treating the prognostic symptoms (mental symptoms) that occur in the early stages of dementia, so the animal of the present invention
- 6-month-old mice which are 6 months younger than 12 months old, were used. Therefore, it can be seen that although early dementia was induced by the accumulation of beta-amyloid, there was no significant difference between the control group, the dementia animal model, and the ZBTB16 knockdown dementia animal model as a result of the YMT experiment, which is consistent with what the present inventor expected. is the result
- a measuring mouse (6 months old, male) was placed in an EthoVision XT 11.5 (manufactured by Noldus, Netherlands) in the middle of three consecutive rooms separated by a transparent wall, and a live mouse (S1) was placed in one of the two rooms. ), an inanimate object (O) was placed in the opposite room, or a familiar mouse (S1) and a new mouse (S2) were placed. Among the two rooms, the longer you stayed, the more red. The test was performed for 10 minutes, and a normal animal model (WT+GFP) was used as a control.
- WT+GFP normal animal model
- FIG. 5 is a view showing the 3-chamber test (3CT) results of the animal model of dementia (APPPS1 + GFP), the animal model of ZBTB16 knockdown dementia (APPPS1 + shZbtb16) and the control (WT + GFP) prepared according to the present invention
- FIG. 5a is an inanimate object (0) and a living mouse (S1) of the animal model of dementia (APPPS1+GFP), ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and control (WT+GFP) prepared according to the present invention.
- Figure 2b is a familiar mouse (S1) and a new animal model (APPPS1 + GFP), ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and control (WT + GFP) prepared according to the present invention. It is a diagram showing the degree of interest in the mouse (S2).
- the elevated cross maze experiment is conducted based on the premise that mice are innately dislike of bright and open spaces, and that they voluntarily explore new environments.
- the elevated cross maze apparatus consists of two open arms (30x5 cm) and two closed arms (5x30 cm, walls 15 cm high) extending from a common central platform (5x5 cm). became The structure was formed in the shape of a plus-sign with corresponding branches arranged to face each other. The device is raised 50 cm above floor level.
- mice belonging to the three groups were placed in the elevated cross maze device and observed for 7 minutes each. Each experimental animal was individually placed in the center of the open range, and the time spent on each open branch was measured. Behavioral observation images were taken using a video camera connected to the software (Smart Junior, Panlab SL, Barcelona, Spain). Thereafter, the time the mice stayed was measured in units of every second through video file analysis using video tracking software, and the results are shown in FIG. 6 .
- the contrast shift test is conducted based on the premise that mice naturally dislike bright light and voluntarily explore new environments.
- the experimental animals belonging to the three groups are placed in a dark room in a mouse cage in which a light box and a dark box are connected by a connecting passage, and while observing for 10 minutes, the delay time until the first exit to the light room , the time spent in a dark room, and the frequency of moving between each dark room and light room were measured. Its frequency is an index measuring the anti-anxiety function.
- FIG. 6 is a diagram showing the results of the elevated cross maze experiment (EMP) of the dementia animal model (APPPS1 + GFP), the ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and the control group (WT + GFP) prepared according to the present invention.
- EMP elevated cross maze experiment
- the dementia animal model (APPPS1+GFP) significantly reduced the dwell time in the open branch.
- the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) increased the dwell time in the open branch to a level similar to that of the control group (WT+GFP). That is, it was confirmed that when early dementia occurred due to the accumulation of beta-amyloid, the time spent in a bright and open space was significantly shortened. In other words, it can be seen that anxiety symptoms increase among mental and behavioral disorders despite the early dementia state in which memory and cognitive function are not significantly reduced by the accumulation of beta-amyloid. At this time, it can be seen that when ZBTB16 protein expression is suppressed, anxiety symptoms, which are mental and behavioral disorders of early dementia, are remarkably improved to a level similar to the normal level.
- LLB light-dark shift test
- the dementia animal model significantly decreased the time to stay in the light room compared to the control group (WT+GFP).
- the ZBTB16 knockdown dementia animal model APPPS1+shZbtb16 stayed in the light room for a long time at a level similar to that of the control group (WT+GFP), and the frequency of movement between the dark room and the light room was significantly increased.
- anxiety symptoms increase among mental and behavioral disorders despite the early dementia state in which memory and cognitive function are not significantly reduced by the accumulation of beta-amyloid.
- ZBTB16 protein expression is suppressed, anxiety symptoms, which are mental and behavioral disorders of early dementia, are remarkably improved to a degree similar to the normal level.
- Immunohistostaining was performed using p62 and LC3 as markers to determine the degree of p62 protein activity and autophagy activity according to the inhibition of the ZBTB16 protein according to the present invention.
- the section was treated with a primary antibody (anti-LC3; Novus Biologicals; NB100-2220, anti-p62; Abcam; ab91526), and a secondary antibody (Goat anti-Rabbit IgG; Alexa Fluor 488, Goat anti-Mouse IgG; Alexa Fluor 594).
- a primary antibody anti-LC3; Novus Biologicals; NB100-2220, anti-p62; Abcam; ab91526
- a secondary antibody Goat anti-Rabbit IgG; Alexa Fluor 488, Goat anti-Mouse IgG; Alexa Fluor 594.
- FIG. 8 is a result of measuring the expression levels of LC3 and p62 from dorsal striatum in brain tissue isolated from an animal model of dementia (APPPS1 + GFP) prepared according to the present invention by immunohistochemistry
- FIG. 9 is the present invention.
- LC3, p62 expression levels from the dorsal striatum in brain tissue isolated from the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) prepared according to the immunohistochemical staining method were measured.
- LC3 In order to confirm the autophagy phenomenon involved in the onset of dementia, LC3, an autophagy marker protein, and p62, an autophagy matrix protein, were identified.
- FIG. 11 is a result of measuring the expression levels of ZBTB16 and p62 from the dorsal striatum in brain tissue isolated from a 12-month-old control group (WT+GFP (12mo)) by immunohistochemistry
- FIG. 12 is a 12-month-old dementia animal model.
- ZBTB16, p62 expression levels from the dorsal striatum in brain tissue isolated from (APP/PS1+GFP(12mo)) were measured by immunohistochemistry
- FIG. 13 is a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1). + shZbtb16), ZBTB16, p62 expression levels from the dorsal striatum in the brain tissue isolated from the immunohistochemical staining method.
- a measurement animal model (12 months old, male) was placed in the EthoVision XT 11.5 (manufactured by Noldus, Netherlands) in the middle of three consecutive rooms separated by a transparent wall, and live mice ( S1), an inanimate object (O) was placed in the opposite room, or a familiar mouse (S1) and a new mouse (S2) were placed. Among the two rooms, the longer you stayed, the more red.
- the test was performed for 10 minutes, and the 12-month-old control group (WT+GFP) prepared in Experimental Example 8-1 was used as a control.
- 14 is a 12-month-old dementia animal model (APPPS1 + GFP) prepared according to the present invention, a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and a 12-month-old control group (WT + GFP) 3-chamber test (3CT) results 14A is an inanimate object of a 12-month-old dementia animal model (APPPS1+GFP), a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and a 12-month-old control group (WT+GFP) prepared according to the present invention.
- APPPS1 + GFP 12-month-old dementia animal model
- APPPS1 + shZbtb16 12-month-old ZBTB16 knockdown dementia animal model
- WT + GFP 12-month-old control group
- Figure 14b is a 12-month-old dementia animal model (APPPS1 + GFP) prepared according to the present invention, a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and a 12-month-old control group (WT+GFP) show the degree of interest (3CT: novelty) in familiar mice (S1) and new mice (S2).
- APPPS1 + GFP 12-month-old dementia animal model
- APPPS1+shZbtb16 a 12-month-old ZBTB16 knockdown dementia animal model
- WT+GFP 12-month-old control group
- ZBTB16 exhibits dementia prevention, treatment, and recovery effects not only in the dementia risk group without cognitive symptoms (6-month-old mice) but also in the dementia group with cognitive symptoms (12-month-old mice).
- the Y-maze experiment was conducted as a method to evaluate the short-term memory type and sequential behavioral ability. Experimental animals were placed at the tip of the branch of the Y-maze (42 ⁇ 3 ⁇ 12 cm) branched into three branches at a 120° angle and allowed to move freely for 8 minutes, and the movement was monitored using a computerized Ethovision system (Noldus IT b.v., Netherlands). was observed with After each branch was designated as A, B, and C, all four feet must completely enter one branch to be counted as the number of entry and exit. The case of entering three different branches one after another was defined as spontaneous alternation behavior. Alternation score (%) was calculated by the following formula.
- a 12-month-old normal animal model (control group (WT+GFP)) prepared by the method of Experimental Example 8-1, a 12-month-old dementia animal model (APPPS1+GFP), and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) ) was used, and the results were compared.
- the new substance discovery test is a modification of the existing method and consists of an adaptor, a searcher, and a new substance recognizer. After acclimatization by placing the mice in an open field for 10 minutes for two days, two identical objects were installed at regular intervals. The time spent in each object was measured by Ethovision while the experimental animals were allowed to freely explore for 3 minutes, and then returned to the cage for 30 minutes. Then, one of the installed objects was replaced with a new material and the movement of the experimental animal was measured for 3 minutes. The object preference was converted into a percentage of the time spent in each object among the total search time.
- a 12-month-old normal animal model (control group (WT+GFP)) prepared by the method of Experimental Example 8-1, a 12-month-old dementia animal model (APPPS1+GFP), and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) ) was used, and the results were compared.
- FIG. 15 is a Y-maze of a 12-month-old normal animal model (control group (WT+GFP)) and a 12-month-old dementia animal model (APPPS1+GFP) and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) prepared according to the present invention.
- WT+GFP control group
- APPPS1+GFP 12-month-old dementia animal model
- APPPS1+shZbtb16 12-month-old ZBTB16 knockdown dementia animal model
- the new substance discovery test can evaluate cognitive ability and memory.
- WT+GFP normal animal model
- APPPS1+GFP dementia animal model
- the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) was similar to the control group (WT+GFP), and it was confirmed that the retention time in the new substance significantly increased compared to the retention time in the familiar substance.
Abstract
In the present invention, it was found that degenerative brain disease can be diagnosed through zinc finger and BTB domain-containing protein 16 (ZBTB16) and the inhibition of expression of ZBTB16 leads to the amelioration, prevention, or treatment of the mental and behavioral symptoms of degenerative brain disease. Thus, the material can be utilized as a biomarker for degenerative brain disease as well as for prevention or treatment of degenerative brain disease.
Description
본 발명은 ZBTB16(Zinc finger and BTB domain-containing protein 16)을 통해 퇴행성 뇌질환을 진단할 수 있는 것으로, ZBTB16을 이용한 퇴행성 뇌질환을 진단하기 위한 바이오마커 조성물, 퇴행성 뇌질환 진단용 조성물, 이를 포함하는 키트, 퇴행성 뇌질환의 진단에 필요한 정보를 제공하기 위한 방법, 퇴행성 뇌질환 치료 또는 예방용 약학 조성물 및 퇴행성 뇌질환을 예방 또는 치료할 수 있는 물질을 스크리닝하는 방법에 관한 것이다.The present invention is capable of diagnosing degenerative brain disease through Zinc finger and BTB domain-containing protein 16 (ZBTB16). It relates to a kit, a method for providing information necessary for diagnosis of degenerative brain disease, a pharmaceutical composition for treating or preventing degenerative brain disease, and a method for screening a substance capable of preventing or treating degenerative brain disease.
퇴행성 뇌질환(degenerative brain disease)은 나이가 들어감에 따라 뇌에서 발생하는 질환을 뜻한다. 현재까지 잘 알려지지 않은 원인으로 뇌에서 특정 뇌세포 집단이 서서히 그 기능을 잃고 뇌신경계의 정보전달에 가장 중요한 뇌신경세포의 사멸, 뇌신경세포와 뇌신경세포 사이의 정보를 전달하는 시냅스의 형상이나 기능상의 문제 또는 뇌신경의 전기적 활동성의 이상적 증가나 감소로 인하여 야기되는 것으로 알려져 있다.Degenerative brain disease is a disease that occurs in the brain as we age. Due to unknown causes, a specific group of brain cells in the brain gradually loses its function, death of cranial nerve cells, which is the most important for information transmission in the cranial nervous system, and problems with the shape or function of the synapse that transmits information between cranial nerve cells and cranial nerve cells Or it is known to be caused by an ideal increase or decrease in electrical activity of cranial nerves.
이러한, 퇴행성 뇌질환 특히, 치매 환자들에서 망상 및 환각과 같은 정신병적 증상이 동반될 경우 병의 경과와 환자의 일상생활 기능에 부정적 영향을 미쳐 결국에 환자들의 조기 시설 입소 및 이로 인한 막대한 사회적, 경제적 비용의 증가를 초래한다. 치매에서 정신병적 증상의 신경병리적 기전을 이해하는 것이 조기 진단 및 치료를 위해서 매우 중요하나, 아직까지 명확히 밝혀진 바 없다. 따라서 퇴행성 뇌질환 진단뿐만 아니라 치료를 위한 새로운 치료제의 개발을 위한 연구가 절실한 실정이다.When psychotic symptoms such as delusions and hallucinations are accompanied by degenerative brain diseases, especially in dementia patients, it negatively affects the course of the disease and the patient's daily life functions, eventually leading to early hospitalization of patients and enormous social and social and It causes an increase in economic cost. Understanding the neuropathological mechanism of psychotic symptoms in dementia is very important for early diagnosis and treatment, but it has not yet been clearly elucidated. Therefore, there is an urgent need for research for the development of new therapeutic agents for the treatment as well as for the diagnosis of degenerative brain diseases.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
특허문헌 1. 대한민국공개특허공보 제10-2012-0004965호 Patent Document 1. Korean Patent Publication No. 10-2012-0004965
본 발명은 상기와 같은 문제점을 해결하기 위하여 안출된 것으로, 본 발명의 목적은 퇴행성 뇌질환 진단용 바이오마커 조성물을 제공하고자 하는 것이다.The present invention has been devised to solve the above problems, and an object of the present invention is to provide a biomarker composition for diagnosing degenerative brain disease.
본 발명의 다른 목적은 퇴행성 뇌질환 진단용 조성물 및 퇴행성 뇌질환 진단용 키트를 제공하고자 하는 것이다.Another object of the present invention is to provide a composition for diagnosing degenerative brain disease and a kit for diagnosing degenerative brain disease.
본 발명은 또 다른 목적은 퇴행성 뇌질환 진단에 필요한 정보를 제공하는 방법을 제공하고자 하는 것이다.Another object of the present invention is to provide a method for providing information necessary for diagnosing degenerative brain disease.
본 발명의 또 다른 목적은 퇴행성 뇌질환 치료물질의 스크리닝 방법을 제공하고자 하는 것이다.Another object of the present invention is to provide a screening method for a therapeutic substance for degenerative brain disease.
본 발명의 또 다른 목적은 퇴행성 뇌질환 예방 또는 치료용 약학 조성물을 제공하고자 하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain disease.
본 발명은 상기 목적을 이루기 위하여, ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 바이오마커 조성물을 제공한다.In order to achieve the above object, the present invention provides a biomarker composition for diagnosing degenerative brain disease, which includes a Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient.
상기 ZBTB16 단백질은 서열번호 1의 아미노산 서열로 표시되는 것일 수 있다.The ZBTB16 protein may be represented by the amino acid sequence of SEQ ID NO: 1.
본 발명은 상기 다른 목적을 이루기 위하여, ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 조성물을 제공한다.In order to achieve the above other object, the present invention provides a composition for diagnosing degenerative brain disease comprising, as an active ingredient, an agent capable of measuring the expression level of ZBTB16 (Zinc finger and BTB domain-containing protein 16) protein or a gene encoding the same. do.
상기 유전자 발현수준을 측정하는 제제는 ZBTB16 단백질을 코딩하는 유전자에 특이적으로 결합하는 프로브, 프라이머 및 안티센스 올리고뉴클레오타이드로 이루어진 군으로부터 선택되는 어느 하나 이상이고, 상기 단백질 발현수준을 측정하는 제제는 상기 ZBTB16 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 및 화합물로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The agent for measuring the gene expression level is at least one selected from the group consisting of a probe, primer and antisense oligonucleotide that specifically binds to a gene encoding the ZBTB16 protein, and the agent for measuring the protein expression level is the ZBTB16 It may be any one or more selected from the group consisting of an antibody, peptide, aptamer, and compound that specifically binds to a protein.
상기 ZBTB16 단백질은 서열번호 1의 아미노산 서열로 표시되는 것일 수 있다.The ZBTB16 protein may be represented by the amino acid sequence of SEQ ID NO: 1.
본 발명은 상기 또 다른 목적을 이루기 위하여, 조성물을 포함하는 퇴행성 뇌질환 진단용 키트를 제공한다.The present invention provides a kit for diagnosing degenerative brain disease comprising a composition in order to achieve the above another object.
본 발명은 상기 또 다른 목적을 이루기 위하여, 하기 단계를 포함하는 퇴행성 뇌질환 진단에 필요한 정보를 제공하는 방법을 제공한다.In order to achieve the above another object, the present invention provides a method for providing information necessary for diagnosing degenerative brain disease, comprising the following steps.
(1) 피검체로부터 분리된 생물학적 시료로부터 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하는 단계;(1) measuring the expression level of a Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same from a biological sample isolated from a subject;
(2) 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 정상 대조군의 시료와 비교하는 단계; 및(2) comparing the expression level of the ZBTB16 protein or a gene encoding it with a sample of a normal control; and
(3) 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준이 정상 대조군의 시료보다 높을 경우 퇴행성 뇌질환이라고 판단하는 단계.(3) When the expression level of the ZBTB16 protein or the gene encoding it is higher than that of the normal control sample, determining that the disease is degenerative brain disease.
상기 피검체의 생물학적 시료로부터 LC3 및 p62 단백질 발현량을 측정하는 단계를 더 포함할 수 있다.The method may further include measuring the expression levels of LC3 and p62 proteins from the biological sample of the subject.
본 발명은 상기 목적을 이루기 위하여, a) 퇴행성 뇌질환 동물 또는 환자에서 분리된 시료에 후보물질을 처리하는 단계; b) 상기 후보물질 처리군에서 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하는 단계; 및 c) 상기 단계에서 측정된 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현 수준이 후보물질 비처리군(대조군)보다 낮아지면, 퇴행성 뇌질환 치료물질로 선정하는 단계;를 포함하는 퇴행성 뇌질환 치료물질의 스크리닝 방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of: a) treating a candidate material in a sample isolated from an animal or patient with degenerative brain disease; b) measuring the expression level of the ZBTB16 protein or a gene encoding the same in the candidate material treatment group; And c) when the expression level of the ZBTB16 protein or the gene encoding it measured in the above step is lower than that of the candidate material non-treated group (control group), selecting a degenerative brain disease treatment material; A screening method is provided.
상기 ZBTB16 단백질은 서열번호 1의 아미노산 서열로 표시되는 것일 수 있다.The ZBTB16 protein may be represented by the amino acid sequence of SEQ ID NO: 1.
상기 발현수준의 측정은 중합효소반응(PCR), 정량적 중합효소반응(qPCR), 역전사 중합효소 반응(RT-PCR), 경쟁적 역전사 중합효소 반응(competitive RT-PCR), 정량적 실시간 중합효소반응(qRT-PCR), 실시간 역전사 중합효소반응(real time RT-PCR), RNase 보호 분석법, 노던 블롯 분석법(northern blot analysis) 및 DNA 칩 분석으로 이루어진 군으로부터 선택되는 어느 하나의 방법을 이용하여 측정하는 것일 수 있다.Measurement of the expression level is a polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), quantitative real-time polymerase reaction (qRT) -PCR), real time reverse transcription polymerase reaction (real time RT-PCR), RNase protection assay, Northern blot analysis (northern blot analysis), and measurement using any one method selected from the group consisting of DNA chip analysis. have.
상기 후보물질 처리군의 혈청에서 LC3 및 p62 단백질 발현량을 측정하는 단계를 더 포함할 수 있다.The method may further include measuring the expression levels of LC3 and p62 proteins in the serum of the candidate substance-treated group.
상기 LC3 단백질 발현량이 후보물질 비처리군(대조군)보다 증가하고, 상기 p62 단백질 발현량은 후보물질 비처리군(대조군)보다 감소한 경우, 상기 후보물질을 퇴행성 뇌질환 치료물질로 판단하는 단계를 더 포함할 수 있다.When the LC3 protein expression level increases than the candidate substance untreated group (control group), and the p62 protein expression level decreases than the candidate substance untreated group (control group), the step of determining the candidate substance as a degenerative brain disease treatment material is more may include
본 발명은 상기 다른 목적을 이루기 위하여, ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질의 발현 또는 활성을 억제할 수 있는 제제를 유효성분으로 포함하는 퇴행성 뇌질환 예방 또는 치료용 약학 조성물을 제공한다.In order to achieve the above other object, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain disease, comprising as an active ingredient an agent capable of inhibiting the expression or activity of ZBTB16 (Zinc finger and BTB domain-containing protein 16) protein do.
상기 발현을 억제할 수 있는 제제는 ZBTB16 단백질을 코딩하는 유전자의 mRNA에 특이적으로 결합하는 siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids) 및 안티센스 올리고뉴클레오타이드으로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것을 특징으로 하며, 상기 활성을 억제할 수 있는 제제는 ZBTB16 단백질에 특이적으로 결합할 수 있는 화합물, 펩티드, 앱타머, 단백질 및 항체로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것일 수 있다.The agent capable of inhibiting the expression is siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, which specifically binds to the mRNA of the gene encoding the ZBTB16 protein. It is characterized in that it contains any one or more selected from the group consisting of peptide nucleic acids (PNA) and antisense oligonucleotides, and the agent capable of inhibiting the activity is a compound capable of specifically binding to ZBTB16 protein, a peptide, It may include any one or more selected from the group consisting of aptamers, proteins and antibodies.
상기 shRNA는 재조합 아데노바이러스, 아데노-관련 바이러스(Adeno-associated viruses: AAV), 레트로바이러스, 렌티바이러스, 헤르페스 심플렉스 바이러스, 배시니아 바이러스, 리포좀 및 니오좀로 이루어진 군으로부터 선택되는 어느 하나의 전달체에 의해 전달되는 것일 수 있다.The shRNA is a recombinant adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposomes and niosomes in any one carrier selected from the group consisting of may be transmitted by
상기 shRNA는 서열번호 7로 표시되는 뉴클레오티드 서열, 서열번호 8로 표시되는 뉴클레오티드 서열, 서열번호 9로 표시되는 뉴클레오티드 서열 및 서열번호 10으로 표시되는 뉴클레오티드 서열로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The shRNA may be any one or more selected from the group consisting of a nucleotide sequence represented by SEQ ID NO: 7, a nucleotide sequence represented by SEQ ID NO: 8, a nucleotide sequence represented by SEQ ID NO: 9, and a nucleotide sequence represented by SEQ ID NO: 10.
상기 퇴행성 뇌질환은 알츠하이머형 치매(Alzheimer-type dementia), 루이체 치매(Lewy body dementia), 전측두엽 치매(frontotemporal dementia), 뇌혈관 치매(cerebrovascular dementia), 파킨슨병(Parkinson's disease), 경도인지장애, 다운증후군(Down's syndrome) 및 헌팅톤병(Huntington's disease)로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The degenerative brain disease includes Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment, It may be any one or more selected from the group consisting of Down's syndrome and Huntington's disease.
상기 조성물은 퇴행성 뇌질환에서 유발되는 정신 및 행동장애(Behavioral and Psychological Symptoms of Dementia, BPSD)를 개선, 예방 또는 치료할 수 있다.The composition may improve, prevent or treat Behavioral and Psychological Symptoms of Dementia (BPSD) caused by degenerative brain diseases.
상기 정신 및 행동장애(Behavioral and Psychological Symptoms of Dementia, BPSD)는 망상(delusion), 환각(hallucination), 초조(agitation), 공격적 행동(aggression), 이상 행동(activity disturbances), 우울(depression), 불안(anxiety), 일중주기장애(diurnal rhythm disturbances), 기분의 들뜸(elation), 과민(irritability), 불안정(lability), 정서적 불안정성(affective lability), 자기조절 결손(defective self regulation), 비정상적인 반복행동(aberrant motor behavior), 불안과 공포(anxiety and phobias) 및 수면 장애로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The Behavioral and Psychological Symptoms of Dementia (BPSD) include delusions, hallucinations, agitation, aggression, activity disturbances, depression, and anxiety. (anxiety), diurnal rhythm disturbances, elation, irritability, lability, affective lability, defective self regulation, abnormal repetitive behavior ( aberrant motor behavior), anxiety and fear (anxiety and phobias), and may be any one or more selected from the group consisting of sleep disorders.
본 발명에 따르면, ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 바이오마커 조성물에 관한 것으로서, 퇴행성 뇌질환 동물모델의 뇌 선조체에서 ZBTB16의 발현이 증가하는 것을 규명하고, 퇴행성 뇌질환 동물모델의 뇌 선조체에 ZBTB16 발현을 억제시킬 경우, 동물의 정신 및 행동증상이 정상수준으로 회복되는 것을 검증하였는 바, 퇴행성 뇌질환을 진단하기 위한 바이오마커로 활용할 수 있다.According to the present invention, there is provided a biomarker composition for diagnosing degenerative brain disease comprising Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient, wherein ZBTB16 in the brain striatum of an animal model of degenerative brain disease In the case of suppressing ZBTB16 expression in the brain striatum of an animal model of degenerative brain disease, it was verified that the mental and behavioral symptoms of the animal were restored to normal levels. It can be used as a marker.
본 발명에 따르면 ZBTB16의 발현을 억제시키는 경우, 퇴행성 뇌질환의 정신 및 행동 증상이 개선, 예방 또는 치료되는 것을 확인하였는 바, 상기의 물질은 퇴행성 뇌질환의 바이오마커뿐만 아니라, 퇴행성 뇌질환의 예방 또는 치료에도 활용할 수 있다.According to the present invention, when the expression of ZBTB16 is suppressed, it has been confirmed that the mental and behavioral symptoms of degenerative brain disease are improved, prevented, or treated. Or it can be used for treatment.
도 1은 대조군(WT)과 치매 동물모델(APPPS1)에서 분리된 뇌조직(Brain)에서 배측선조체(dorsal striatum)와 측좌핵(nucleus accumbens, NAc)의 위치를 도시한 도면이다.1 is a diagram showing the positions of the dorsal striatum and the nucleus accumbens (NAc) in the brain tissue (Brain) separated from the control group (WT) and the dementia animal model (APPPS1).
도 2는 대조군(WT)과 치매 동물모델(APPPS1)에서 분리된 뇌조직(Brain)에서 배측선조체(dorsal striatum)과 측좌핵(nucleus accumbens, NAc)으로부터 ZBTB16 단백질 발현량을 면역조직염색법으로 측정한 결과이다. Figure 2 is a control (WT) and dementia animal model (APPPS1) from the brain tissue (Brain) from the dorsal striatum (dorsal striatum) and the nucleus accumbens (nucleus accumbens, NAc) from the ZBTB16 protein expression was measured by immunohistochemistry. is the result
도 3은 대조군(WT+GFP)과 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에 대한 Y-maze 시험(YMT) 결과를 나타낸 그래프이다.3 is a graph showing the Y-maze test (YMT) results for the control group (WT+GFP), the dementia animal model (APPPS1+GFP), and the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16).
도 4는 대조군(WT+GFP)과 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에 대한 신물질탐색 시험(novel object recognition test, NOR) 결과를 나타낸 그래프이다.4 is a graph showing the results of a novel object recognition test (NOR) for a control group (WT+GFP), an animal model of dementia (APPPS1+GFP), and an animal model of ZBTB16 knockdown dementia (APPPS1+shZbtb16).
도 5는 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 3-chamber test(3CT) 결과를 나타낸 도면으로, 도 5a는 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 무생물인 물체(0)와 살아있는 생쥐(S1)에 대한 관심 정도를 나타낸 도면이고, 도 2b는 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 낯익은 생쥐(S1)와 새로운 생쥐(S2)에 대한 관심 정도를 나타낸 도면이다.5 is a view showing the 3-chamber test (3CT) results of the animal model of dementia (APPPS1 + GFP), the animal model of ZBTB16 knockdown dementia (APPPS1 + shZbtb16) and the control (WT + GFP) prepared according to the present invention; FIG. 5a is an inanimate object (0) and a living mouse (S1) of the animal model of dementia (APPPS1+GFP), ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and control (WT+GFP) prepared according to the present invention. It is a view showing the degree, and Figure 2b is a familiar mouse (S1) and a new animal model (APPPS1 + GFP), ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and control (WT + GFP) prepared according to the present invention. It is a diagram showing the degree of interest in the mouse (S2).
도 6은 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 고가식 십자 미로 실험(EMP) 결과를 나타낸 도면이다.6 is a diagram showing the results of the elevated cross maze experiment (EMP) of the dementia animal model (APPPS1 + GFP), the ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and the control group (WT + GFP) prepared according to the present invention.
도 7은 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 명암 이동 시험(LDB) 결과를 나타낸 도면이다.7 is a diagram showing the results of a light-dark shift test (LDB) of an animal model of dementia (APPPS1+GFP), an animal model of ZBTB16 knockdown dementia (APPPS1+shZbtb16), and a control group (WT+GFP).
도 8은 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP)에서 분리된 뇌조직에서 배측선조체(dorsal striatum)으로부터 LC3, p62 발현량을 면역조직염색법으로 측정한 결과이다.8 is a result of measuring the expression levels of LC3 and p62 from the dorsal striatum in brain tissue isolated from the dementia animal model (APPPS1 + GFP) prepared according to the present invention by immunohistochemistry.
도 9는 본 발명에 따라 제조된 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에서 분리된 뇌조직에서 배측선조체(dorsal striatum)으로부터 LC3, p62 발현량을 면역조직염색법으로 측정한 결과이다.9 is a result of measuring the expression levels of LC3, p62 from the dorsal striatum in brain tissue isolated from the ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) prepared according to the present invention by immunohistochemistry.
도 10은 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP, APPPS1+shZbtb16)에서 분리된 뇌조직에서 배측선조체(dorsal striatum)으로부터 녹다운 바이러스 전달체에 포함되어 함께 발현되는 녹색형광단백질(GFP) 발현량과 발현 영역을 면역조직염색법으로 측정한 결과이다.10 is a green fluorescent protein (GFP) expression included in the knockdown virus delivery system from the dorsal striatum in brain tissue isolated from the dementia animal model (APPPS1 + GFP, APPPS1 + shZbtb16) prepared according to the present invention. These are the results of measuring the amount and expression region by immunohistochemistry.
도 11은 12 개월령 정상 동물모델(WT+GFP(12mo))에서 분리된 뇌조직에서 배측선조체(dorsal striatum)로부터 ZBTB16, p62 발현량을 면역조직염색법으로 측정한 결과이다.11 is a result of measuring the expression levels of ZBTB16 and p62 from dorsal striatum in brain tissue isolated from a 12-month-old normal animal model (WT+GFP (12mo)) by immunohistochemistry.
도 12는 12 개월령 치매 동물모델(APP/PS1+GFP(12mo))에서 분리된 뇌조직에서 배측선조체(dorsal striatum)로부터 ZBTB16, p62 발현량을 면역조직염색법으로 측정한 결과이다.12 is a result of measuring the expression levels of ZBTB16 and p62 from dorsal striatum in brain tissue isolated from a 12-month-old dementia animal model (APP/PS1+GFP (12mo)) by immunohistochemistry.
도 13은 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16(12mo))에서 분리된 뇌조직에서 배측선조체(dorsal striatum)으로부터 ZBTB16, p62 발현량을 면역조직염색법으로 측정한 결과이다.13 is a result of measuring the expression levels of ZBTB16 and p62 from dorsal striatum in brain tissue isolated from a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16(12mo)) by immunohistochemistry.
도 14a는 본 발명에 따라 제조된 12 개월령 치매 동물모델(APPPS1+GFP), 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 12 개월령 대조군(WT+GFP)의 무생물인 물체(0)와 살아있는 생쥐(S1)에 대한 관심 정도(3CT:sociability)를 나타낸 도면이다.Figure 14a is a 12-month-old dementia animal model (APPPS1 + GFP) prepared according to the present invention, a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16), and a 12-month-old control (WT + GFP) inanimate object (0) and living It is a diagram showing the degree of interest (3CT:sociability) for the mouse (S1).
도 14b는 본 발명에 따라 제조된 12 개월령 치매 동물모델(APPPS1+GFP), 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 12 개월령 대조군(WT+GFP)의 낯익은 생쥐(S1)와 새로운 생쥐(S2)에 대한 관심 정도(3CT:novelty)를 나타낸 도면이다.14B shows familiar mice (S1) and new animals of a 12-month-old dementia animal model (APPPS1+GFP), a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and a 12-month-old control group (WT+GFP) prepared according to the present invention. It is a diagram showing the degree of interest (3CT: novelty) for the mouse (S2).
도 15는 본 발명에 따라 제조된 12 개월령 정상 동물모델(대조군(WT+GFP))과 12 개월령 치매 동물모델(APPPS1+GFP) 및 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)의 Y-maze 시험(YMT) 결과를 나타낸 그래프(좌측)와 신물질탐색 시험(novel object recognition test, NOR) 결과를 나타낸 그래프(우측)이다.15 is a Y-maze of a 12-month-old normal animal model (control group (WT+GFP)) and a 12-month-old dementia animal model (APPPS1+GFP) and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) prepared according to the present invention. A graph showing the test (YMT) results (left) and a graph showing the novel object recognition test (NOR) results (right).
이하에서, 본 발명의 여러 측면 및 다양한 구현예에 대해 더욱 구체적으로 살펴보도록 한다. Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.
본 발명자들은 퇴행성 뇌질환을 진단할 수 있는 물질을 찾을 수 있는 방법을 얻고자 예의 노력한 결과, 퇴행성 뇌질환과 ZBTB16의 상호작용을 규명하였고, 이에 기초하여 본 발명을 완성하게 되었다.As a result of earnest efforts to obtain a method for finding a substance capable of diagnosing degenerative brain disease, the present inventors have identified the interaction between degenerative brain disease and ZBTB16, and based on this, the present invention has been completed.
본 발명의 일 측면은 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 바이오마커 조성물에 관한 것이다.One aspect of the present invention relates to a biomarker composition for diagnosing degenerative brain disease, which includes Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient.
상기 바이오마커 조성물은 기존에 알려진 자가포식 바이오마커(autophagosome marker)인 p62 및 LC3을 추가적으로 더 포함할 수 있다. 상기 LC3 단백질은 LC3 II(mitrotubule-associated protein light chain 3)이고, p62는 Sequestosome 1, SQSTM1이다.The biomarker composition may further include p62 and LC3, which are known autophagosome biomarkers. The LC3 protein is LC3 II (mitrotubule-associated protein light chain 3), and p62 is Sequestosome 1 and SQSTM1.
본 발명에서 'ZBTB16(Zinc finger and BTB domain-containing protein 16)'은 ZBTB16 유전자를 암호화하는 단백질이다. 상기 유전자는 Krueppel C2H2 type zinc-finger 단백질 구성원 중 하나로, 카르보실 말단에서 9개의 Kruppel-type zinc finger 도메인을 포함하는 Zinc finger 전사인자를 암호화한다. 상기 단백질은 핵 내에 위치하며, 세포주기 진행에 관여하고, Histone Deacetylase (HDAC)와 상호작용을 하는 것으로 알려져 있다.In the present invention, 'Zinc finger and BTB domain-containing protein 16 (ZBTB16)' is a protein encoding the ZBTB16 gene. The gene is one of the Krueppel C2H2 type zinc-finger protein members, and encodes a zinc finger transcription factor including nine Kruppel-type zinc finger domains at the carbosil end. The protein is located in the nucleus, is involved in cell cycle progression, and is known to interact with Histone Deacetylase (HDAC).
또한, 상기 ZBTB16의 아미노산 서열은 uniprot.org에 등록되어 있는 서열번호 1로 표시될 수 있다. 이때, 상기 서열번호 1로 표시되는 아미노산 서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 갖는 아미노산 서열을 포함할 수 있다.In addition, the amino acid sequence of ZBTB16 may be represented by SEQ ID NO: 1 registered on uniprot.org. In this case, it may include an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with the amino acid sequence represented by SEQ ID NO: 1. .
또한, 상기 ZBTB16을 암호화하는 유전자의 염기서열은 ncbi.nlm.nih.gov에 등록되어 있다. 보다 구체적으로에서 확인할 수 있다. 이때, 상기 서열번호 2로 표시되는 염기서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 갖는 아미노산 서열을 포함할 수 있다.In addition, the nucleotide sequence of the gene encoding the ZBTB16 is registered at ncbi.nlm.nih.gov. More specifically, it can be found in In this case, it may include an amino acid sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more to the nucleotide sequence represented by SEQ ID NO: 2 .
본 발명에서 '퇴행성 뇌질환'은 중추신경계의 신경세포에 퇴행성 변화가 나타나면서 여러가지 증상을 유발하는 질환으로 대부분 질병의 발병이 서서히 시작하며 출생 후 오랜 기간 정상적인 기능을 하다가 증상이 나타난다. 또한 일단 발병하면 사망 시까지 수년 또는 수십 년에 걸쳐 지속적으로 병이 진행하며, 가족력이 있는 경우가 많다. 본 발명의 퇴행성 뇌질환의 구체적인 예는 이에 한정되지 아니하나, 알츠하이머형 치매(Alzheimer-type dementia), 루이체 치매(Lewy body dementia), 전측두엽 치매(frontotemporal dementia), 뇌혈관 치매(cerebrovascular dementia), 파킨슨병(Parkinson's disease), 경도인지장애, 다운증후군(Down's syndrome), 헌팅톤병(Huntington's disease), 근위축성 측삭경화증(amyotrophic lateral sclerosis), 척수소뇌성 운동실조증(Spinocerebellar Atrophy), 뚜렛 증후군(Tourette`s Syndrome), 프리드리히 보행실조(Friedrich`s Ataxia), 마차도-조셉 병(Machado-Joseph`s disease), 루이 소체 치매(Lewy Body Dementia), 근육긴장이상(Dystonia) 및 진행성 핵상 마비(Progressive Supranuclear Palsy)로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.In the present invention, 'degenerative brain disease' is a disease that causes various symptoms as degenerative changes appear in nerve cells of the central nervous system. In addition, once onset, the disease continues to progress over many years or decades until death, and there is often a family history. Specific examples of the degenerative brain disease of the present invention are not limited thereto, but Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment, Down's syndrome, Huntington's disease, amyotrophic lateral sclerosis, Spinocerebellar Atrophy, Tourette's syndrome s Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, Dystonia and Progressive Supranuclear Palsy ) may be any one or more selected from the group consisting of.
본 발명에서 상기 퇴행성 뇌질환은 보다 바람직하게 베타 아밀로이드 축적에 의해 유도되는 퇴행성 뇌질환일 수 있고, 보다 더 바람직하게는 치매일 수 있다. 상기 치매는 특별히 제한되지 않으며, 예를 들어 알츠하이머형 치매(Alzheimer-type dementia), 루이체 치매(Lewy body dementia), 전측두엽 치매(frontotemporal dementia), 뇌혈관 치매(cerebrovascular dementia), 파킨슨병(Parkinson's disease), 경도인지장애, 다운증후군(Down's syndrome) 및 헌팅톤병(Huntington's disease)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.In the present invention, the degenerative brain disease may be more preferably a degenerative brain disease induced by beta-amyloid accumulation, and even more preferably dementia. The dementia is not particularly limited, and for example, Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, Parkinson's disease ), mild cognitive impairment, Down's syndrome and Huntington's disease may be any one or more selected from the group consisting of.
게다가, 본 발명은 인지장애와 같은 퇴행성 뇌질환 또는 치매의 핵심 증상은 나타나지 않은 초기 퇴행성 뇌질환 또는 치매를 진단할 수 있다. In addition, the present invention can diagnose early degenerative brain disease or dementia in which the core symptoms of dementia or degenerative brain disease such as cognitive impairment do not appear.
상기 초기 퇴행성 뇌질환 또는 치매는 인지기능 저하 없이 망상(delusion), 환각(hallucination), 초조(agitation), 공격적 행동(aggression), 이상 행동(activity disturbances), 우울(depression), 불안(anxiety), 일중주기장애(diurnal rhythm disturbances), 기분의 들뜸(elation), 과민(irritability), 불안정(lability), 정서적 불안정성(affective lability), 자기조절 결손(defective self regulation), 비정상적인 반복행동(aberrant motor behavior), 불안과 공포(anxiety and phobias) 및 수면 장애로 이루어진 군으로부터 선택되는 어느 하나 이상의 정신 및 행동 장애만을 갖고 있는 것을 포함한다.The early degenerative brain disease or dementia may include delusions, hallucinations, agitation, aggression, activity disturbances, depression, anxiety, Diurnal rhythm disturbances, elation, irritability, lability, affective lability, defective self regulation, aberrant motor behavior , including those with only one or more mental and behavioral disorders selected from the group consisting of anxiety and phobias and sleep disorders.
본 발명에서 '진단'은 넓은 의미로는 환자의 병의 실태를 모든 면에 걸쳐서 판단하는 것을 의미한다. 판단의 내용은 병명, 병인, 병형, 경중, 병상의 상세한 양태, 합병증의 유무 및 예후 등이다. 구체적으로 특정 질병 또는 질환에 대한 한 객체의 감수성(susceptibility)을 판정하는 것, 한 객체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 객체의 예후(prognosis)를 판정하는 것 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 객체의 상태를 모니터링하는 것)을 포함한다.In the present invention, 'diagnosis' in a broad sense means judging the actual condition of a patient's disease in all aspects. The content of the judgment is the disease name, etiology, disease type, severity, detailed mode of the disease, presence or absence of complications, and prognosis. Specifically, to determine the susceptibility of an object to a particular disease or condition, to determine whether an object currently has a particular disease or disorder, or to determine the prognosis of an object with a particular disease or condition or therametrics (eg, monitoring the condition of an object to provide information about treatment efficacy).
본 발명에서 '예후(prognosis)'란 질병을 진단하여 판단된 장래의 증세 또는 경과에 대한 전망을 의미한다. 특히 퇴행성 뇌질환 환자에 있어서 예후는 통상적으로 퇴행성 뇌질환 발병 또는 치료 후 일정기간 내의 퇴행성 뇌질환의 양태, 정신적 및 행동적 증상 발전 여부 또는 생존기간을 뜻한다. 예후의 예측은 특히 퇴행성 뇌질환 환자의 향후 퇴행성 뇌질환 치료의 방향에 대한 단서를 제시하므로 매우 중요하다.In the present invention, 'prognosis' means a prospect for future symptoms or progress determined by diagnosing a disease. In particular, in patients with degenerative brain disease, the prognosis usually refers to the mode of degenerative brain disease within a certain period of time after the onset or treatment of the degenerative brain disease, the development of mental and behavioral symptoms, or the survival period. The prediction of prognosis is very important as it provides clues about the future direction of treatment for degenerative brain disease, especially for patients with degenerative brain disease.
본 발명에서 '바이오마커'란 단백질이나 DNA, RNA(리복핵산), 대사 물질 등을 이용해 몸 안의 변화를 알아낼 수 있는 지표를 의미한다. 바이오마커를 활용하면 생명체의 정상 또는 병리적인 상태, 약물에 대한 반응 정도 등을 객관적으로 측정할 수 있으며 퇴행성 뇌질환을 비롯해 뇌졸중, 암 등 각종 난치병을 진단하기 위한 효과적 방식이다. 또한, 신약개발과정에 반영할 수 있어 안전성확보는 물론 비용절감 효과까지 바라볼 수 있다.In the present invention, a 'biomarker' refers to an indicator that can detect changes in the body using proteins, DNA, RNA (Reebok nucleic acid), metabolites, and the like. By using biomarkers, it is possible to objectively measure the normal or pathological state of an organism, the degree of response to drugs, etc. In addition, since it can be reflected in the new drug development process, it is possible to see not only safety but also cost reduction effect.
본 발명의 ZBTB16과 퇴행성 뇌질환의 관계는 아직까지 알려진바 없었으나, 본 발명을 통해 베타 아밀로이드 축적에 의해, 상기 ZBTB16의 발현이 증가함을 밝혀냈다. 또한, 동물에서 ZBTB16을 억제시킬 경우, 퇴행성 뇌질환에 의해 유도되는 정신 및 행동 장애를 정상수준으로 회복시킬 수 있음을 확인하였다. 이는 ZBTB16이 퇴행성 뇌질환을 진단 또는 조기 진단할 수 있는 바이오마커로서 활용할 수 있음을 의미하는 것이다. 즉, 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현 수준을 측정한다면 퇴행성 뇌질환의 진단에 필요한 정보를 제공할 수 있는 것이다.Although the relationship between ZBTB16 of the present invention and degenerative brain disease has not been known yet, the present invention revealed that the expression of ZBTB16 is increased by the accumulation of beta-amyloid. In addition, it was confirmed that when ZBTB16 is inhibited in animals, mental and behavioral disorders induced by degenerative brain diseases can be restored to normal levels. This means that ZBTB16 can be used as a biomarker for diagnosing or early diagnosis of degenerative brain disease. That is, if the expression level of the ZBTB16 protein or a gene encoding it is measured, it is possible to provide information necessary for the diagnosis of degenerative brain disease.
본 발명에서, "발현(expression)"은 세포에서 단백질 또는 핵산이 생성되는 것을 의미한다. '단백질'은 '폴리펩타이드(polypeptide)' 또는 '펩타이드(peptide)'와 호환성 있게 사용되며, 예컨대, 자연 상태의 단백질에서 일반적으로 발견되는 바와 같이 아미노산 잔기의 중합체를 말한다. '폴리뉴클레오티드(polynucleotide)' 또는 '핵산'은 단일-또는 이중-가닥의 형태로 된 데옥시리보뉴클레오티드(DNA) 또는 리보뉴클레오티드(RNA)를 말한다. 다른 제한이 없는 한, 자연적으로 생성되는 뉴클레오티드와 비슷한 방법으로 핵산에 혼성화되는 자연적 뉴클레오티드의 공지된 아날로그도 포함된다. 'mRNA'는 단백질 합성 과정에서 특정 유전자로부터 아미노산 서열을 특정하게 되는 리보솜으로 유전 정보(유전자 특이적 염기 서열)를 전달하는 RNA를 의미한다.In the present invention, "expression" means that a protein or nucleic acid is produced in a cell. 'Protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to, for example, a polymer of amino acid residues as commonly found in proteins in their natural state. 'Polynucleotide' or 'nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single- or double-stranded form. Unless otherwise limited, known analogs of natural nucleotides that hybridize to nucleic acids in a manner analogous to naturally occurring nucleotides are also included. 'mRNA' refers to RNA that transfers genetic information (gene-specific nucleotide sequence) to ribosomes that specify an amino acid sequence from a specific gene during protein synthesis.
본 발명의 다른 측면은 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 조성물에 관한 것이다.Another aspect of the present invention relates to a composition for diagnosing degenerative brain disease, comprising as an active ingredient an agent capable of measuring the expression level of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same.
상기 유전자 발현수준을 측정하는 제제는 ZBTB16 단백질을 코딩하는 유전자에 특이적으로 결합하는 프로브, 프라이머 및 안티센스 올리고뉴클레오타이드로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The agent for measuring the gene expression level may be any one or more selected from the group consisting of a probe, primer, and antisense oligonucleotide that specifically binds to a gene encoding the ZBTB16 protein.
상기 단백질 발현수준을 측정하는 제제는 상기 ZBTB16 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 및 화합물로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.The agent for measuring the protein expression level may be any one or more selected from the group consisting of an antibody, peptide, aptamer, and compound that specifically binds to the ZBTB16 protein.
본 발명에서 '프로브'는 mRNA와 특이적으로 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있어서 특정 mRNA의 존재 유무, 발현양을 확인할 수 있다. 프로브는 올리고뉴클레오타이드(oligonucleotide) 프로브, 단쇄 DNA(single strand DNA) 프로브, 이중쇄DNA(double strand DNA)프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적절한 프로브의 선택 및 혼성화 조건은 당해 기술 분야에 공지된 기술에 따라 적절히 선택할 수 있다. In the present invention, 'probe' refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases in length that can specifically bind to mRNA, and is labeled so that the presence or absence of a specific mRNA, the amount of expression can confirm. The probe may be manufactured in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, or the like. Suitable probe selection and hybridization conditions may be appropriately selected according to techniques known in the art.
본 발명에서 '프라이머'는 짧은 자유 3-말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로서 작용하는 짧은 핵산 서열을 말한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(즉, DNA 폴리머라제 또는 역전사효소) 및 상이한 4 가지의 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성을 개시할 수 있다. PCR 조건, 센스 및 안티센스 프라이머의 길이는 당업계에 공지된 기술에 따라 적절히 선택될 수 있다.In the present invention, a 'primer' is a nucleic acid sequence having a short free 3' hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for template strand copying. say sequence. The primer is capable of initiating DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers may be appropriately selected according to techniques known in the art.
본 발명에서 상기 프라이머 또는 프로브는 포스포아미다이트(phosphoramidite) 고체지지체 합성법이나 기타 널리 공지된 방법을 이용하여 화학적으로 합성할 수 있다. 또한 프라이머 또는 프로브는 ZBTB16 mRNA와의 혼성화를 방해하지 않는 범위에서 당해 기술 분야에 공지된 방법에 따라 다양하게 변형시킬 수 있다. 이러한 변형의 예로는 메틸화, 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환 및 뉴클레오티드 간의 변형, 예를 들면 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등), 그리고 형광 또는 효소를 이용한 표지물질(labeling material)의 결합 등이 있다.In the present invention, the primer or probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, the primer or probe can be variously modified according to methods known in the art within the range that does not interfere with hybridization with ZBTB16 mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and fluorescence or enzymatic binding of a labeling material.
본 발명에서 '항체'는 당해 기술분야에 공지된 용어로서 항원성 부위에 대하여 지시되는 특이적인 면역 글로불린을 의미한다. 본 발명에서의 항체는 본 발명의 ZBTB16에 대해 특이적으로 결합하는 항체를 의미하며, 당해 기술분야의 통상적인 방법에 따라 항체를 제조할 수 있다. 상기 항체의 형태는 폴리클로날 항체 또는 모노클로날 항체를 포함하며, 모든 면역글로불린 항체가 포함된다. 상기 항체는 2개의 전체 길이의 경쇄 및 2 개의 전체 길이의 중쇄를 갖는 완전한 형태를 의미한다. 또한, 상기 항체는 인간화 항체 등의 특수 항체도 포함된다.In the present invention, 'antibody' is a term known in the art and refers to a specific immunoglobulin directed to an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to ZBTB16 of the present invention, and the antibody can be prepared according to a conventional method in the art. The form of the antibody includes polyclonal antibodies or monoclonal antibodies, and all immunoglobulin antibodies are included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. Moreover, the said antibody also includes special antibodies, such as a humanized antibody.
본 발명에서 '펩타이드'는 표적 물질에 대한 결합력 높은 장점이 있으며, 열/화학 처리시에도 변성이 일어나지 않는다. 또한 분자 크기가 작기 때문에 다른 단백질에 붙여서 융합 단백질로의 이용이 가능하다. 구체적으로 고분자 단백질 체인에 붙여서 이용이 가능하므로 진단 키트 및 약물전달 물질로 이용될 수 있다.In the present invention, the 'peptide' has an advantage of high binding strength to the target material, and no denaturation occurs even during heat/chemical treatment. In addition, since the molecular size is small, it can be used as a fusion protein by attaching it to other proteins. Specifically, since it can be used by attaching it to a polymer protein chain, it can be used as a diagnostic kit and drug delivery material.
본 발명에서 '앱타머'는 시료 내의 검출하고자 하는 분석물질과 특이적으로 결합할 수 있는 물질로 그 자체로 안정된 삼차 구조를 가지는 단일 가닥 핵산(DNA, RNA, 또는 변형 핵산)을 의미하는 것으로, 특이적으로 시료 내의 표적 단백질의 존재를 확인할 수 있다. 앱타머의 제조는 일반적인 앱타머의 제조 방법에 따라, 확인하고자 하는 표적 단백질(본 발명에서는 ZBTB16 단백질)에 대해 선택적이고 높은 결합력을 가지는 올리고뉴클레오티드의 서열을 결정하여 합성한 후, 올리고뉴클레오티드의 5' 말단이나 3' 말단을 앱타머 칩의 관능기에 결합할 수 있도록, -SH, -COOH, -OH 또는 NH2로 변형을 시킴으로써 이루어질 수 있으나, 이에 제한되지 않는다.In the present invention, 'aptamer' refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure as a substance capable of specifically binding to an analyte to be detected in a sample, Specifically, the presence of the target protein in the sample may be confirmed. According to the general aptamer preparation method, the aptamer is synthesized by determining the sequence of an oligonucleotide having a selective and high binding affinity to the target protein (ZBTB16 protein in the present invention) to be confirmed, and then 5' of the oligonucleotide. To be able to bind the terminal or 3' end to the functional group of the aptamer chip, -SH, -COOH, -OH or NH 2 It may be made by modifying it, but is not limited thereto.
본 발명의 유전자는 핵산 서열이 유전자 은행에 등록되어 있으므로 당업자는 상기 서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 디자인할 수 있다.Since the gene of the present invention has a nucleic acid sequence registered in the gene bank, a person skilled in the art can design an antisense oligonucleotide, primer pair or probe that specifically amplifies a specific region of these genes based on the sequence.
상기 ZBTB16 단백질 특이적인 제제를 포함하는 본 발명의 진단용 조성물은 공지된 단백질을 감지하는 방법에 필요한 제제를 추가적으로 포함할 수 있으며, 본 조성물을 이용하여 공지된 단백질을 감지하는 방법을 제한없이 사용하여 피검체에서 ZBTB16 단백질의 발현 수준을 측정할 수 있다.The diagnostic composition of the present invention comprising the ZBTB16 protein-specific agent may additionally include an agent necessary for a method for detecting a known protein, and the method for detecting a known protein using the composition can be used without limitation. The expression level of ZBTB16 protein in the sample can be measured.
또한, 본 발명의 또 다른 측면은 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 키트에 관한 것이다.In addition, another aspect of the present invention relates to a kit for diagnosing degenerative brain disease including Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient.
상기 키트는 마커 성분에 특이적으로 결합하는 항체, 기질과의 반응에 의해서 발색하는 표지체가 접합된 2차 항체 접합체(conjugate), 상기 표지체와 발색 반응할 발색 기질 용액, 세척액 및 효소반응 정지용액 등을 포함할 수 있으며, 사용되는 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다.The kit includes an antibody that specifically binds to a marker component, a secondary antibody conjugate to which a label that develops color by reaction with a substrate is conjugated, a color-developing substrate solution to react with the label, a washing solution and an enzyme reaction stop solution and the like, and may be manufactured as a plurality of separate packaging or compartments containing the reagent components used.
구체적인 양태로서 상기 진단 키트는 역전사 중합효소반응을 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 진단용 키트일 수 있다. 역전사 중합효소반응 키트는 마커 유전자에 대한 특이적인 각각의 프라이머 쌍을 포함한다. 프라이머는 각 마커 유전자의 핵산서열에 특이적인 서열을 가지는 뉴클레오타이드로서, 약 7bp 내지 50bp의 길이, 보다 바람직하게는 약 10bp 내지 30bp의 길이이다. 또한 대조군 유전자의 핵산 서열에 특이적인 프라이머를 포함할 수 있다. 그 외 역전사 중합효소반응 키트는 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNAse, RNAse 억제제 DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다.In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer pair specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.
또 다른 양태로는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 진단 키트일 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드(oligonucleotide)가 부착되어 있는 기판, 및 형광표식 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한 기판은 대조군 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드를 포함할 수 있다.In another aspect, it may be a diagnostic kit comprising essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescent probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.
분석을 위해 사용되는 시료는 혈액, 혈구, 뇌 조직, 신경세포, 뇌척수액, 타액, 비액, 객담, 관절낭액, 양수, 복수, 자궁경부 또는 질 분비물 및 소변 등 정상적인 상태와 구별될 수 있는 퇴행성 뇌질환과 관련된 특이적 단백질을 확인할 수 있는 생체 시료를 포함한다. 바람직하게는 생물학적 시료, 예를 들어 혈액, 혈구, 뇌 조직, 신경세포, 뇌척수액으로 이루어진 군에서 선택되는 어느 하나의 시료로부터 측정될 수 있다. 상기 시료는 단백질 마커의 탐지 감도를 증가시키도록 준비될 수 있는데 예를 들어 환자로부터 수득한 혈청 시료는 음이온 교환 크로마토그래피, 친화도 크로마토그래피, 크기별 배제 크로마토그래피(size exclusion chromatography), 액체 크로마토그래피, 연속추출(sequential extraction) 또는 젤 전기영동 등의 방법을 이용하여 전처리될 수 있다.Samples used for analysis include blood, blood cells, brain tissue, nerve cells, cerebrospinal fluid, saliva, nasal fluid, sputum, capsular fluid, amniotic fluid, ascites, cervical or vaginal secretions, and urine degenerative brain diseases that can be distinguished from normal conditions. It includes a biological sample capable of identifying a specific protein associated with it. Preferably, it can be measured from a biological sample, for example, any one sample selected from the group consisting of blood, blood cells, brain tissue, nerve cells, and cerebrospinal fluid. The sample may be prepared to increase the detection sensitivity of the protein marker. For example, a serum sample obtained from a patient may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, It may be pre-treated using a method such as sequential extraction or gel electrophoresis.
본 발명의 또 다른 측면은 하기 단계를 포함하는 퇴행성 뇌질환 진단에 필요한 정보를 제공하는 방법에 관한 것이다.Another aspect of the present invention relates to a method for providing information necessary for diagnosing degenerative brain disease, comprising the following steps.
(1) 피검체로부터 분리된 생물학적 시료로부터 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하는 단계;(1) measuring the expression level of a Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same from a biological sample isolated from a subject;
(2) 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 정상 대조군의 시료와 비교하는 단계; 및(2) comparing the expression level of the ZBTB16 protein or a gene encoding it with a sample of a normal control; and
(3) 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준이 정상 대조군의 시료보다 높을 경우 퇴행성 뇌질환이라고 판단하는 단계;를 포함한다.(3) when the expression level of the ZBTB16 protein or a gene encoding it is higher than that of a normal control sample, determining that it is a degenerative brain disease; includes.
우선, (1) 피검체로부터 생물학적 시료를 분리하고, 상기 생물학적 시료로부터 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현 수준을 측정한다.First, (1) a biological sample is separated from a subject, and the expression level of a zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same is measured from the biological sample.
본 발명에서 '피검체'란 인간, 소, 개, 기니아 피그, 토끼, 닭, 곤충, 마우스, 생쥐 등을 포함하여 퇴행성 뇌질환이 의심되는, 퇴행성 뇌질환 진단이 요구되는 임의의 단일 개체를 의미한다. 또한, 임의의 퇴행성 뇌질환 질병 임상 소견을 보이지 않는 임상 연구 시험에 참여한 임의의 대상 또는 역학 연구에 참여한 대상 또는 대조군으로 사용된 대상이 대상에 포함된다. 본 발명의 일 실시예에서는 마우스 또는 생쥐를 대상으로 하였다.In the present invention, the term 'subject' refers to any single entity that is suspected of degenerative brain disease, including humans, cows, dogs, guinea pigs, rabbits, chickens, insects, mice, mice, and the like, and requires a diagnosis of degenerative brain disease. do. Also included in the subject are any subjects participating in a clinical study trial without any clinical findings of degenerative brain disease, or subjects participating in epidemiological studies or subjects used as controls. In an embodiment of the present invention, mice or mice were used.
본 발명에서 '퇴행성 뇌질환이 의심되는 대상'은 퇴행성 뇌질환의 핵심 증상 또는 퇴행성 뇌질환에서 유발되는 정신 및 행동장애(Behavioral and Psychological Symptoms of Dementia, BPSD)를 가져, 퇴행성 뇌질환을 가질 것으로 예상되는 대상일 수 있다. BPSD는 퇴행성 뇌질환 특히 치매가 보통으로부터 중증으로 진행함에 따라 빈번히 발생되는 정신 및 행동장애로, BPSD는 망상(delusion), 환각(hallucination), 초조(agitation), 공격적 행동(aggression), 이상 행동(activity disturbances), 우울(depression), 불안(anxiety), 일중주기장애(diurnal rhythm disturbances), 기분의 들뜸(elation), 과민(irritability), 불안정(lability), 정서적 불안정성(affective lability), 자기조절 결손(defective self regulation), 비정상적인 반복행동(aberrant motor behavior), 불안과 공포(anxiety and phobias) 및 수면 장애로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함한다.In the present invention, the 'subject suspected of degenerative brain disease' has the core symptoms of degenerative brain disease or Behavioral and Psychological Symptoms of Dementia (BPSD) caused by degenerative brain disease, and is expected to have degenerative brain disease may be a target. BPSD is a mental and behavioral disorder that occurs frequently as degenerative brain disease, especially dementia, progresses from moderate to severe. activity disturbances, depression, anxiety, diurnal rhythm disturbances, elation, irritability, lability, affective lability, deficits in self-regulation (defective self regulation), abnormal motor behavior (aberrant motor behavior), anxiety and fear (anxiety and phobias) and includes any one or more selected from the group consisting of sleep disorders.
상기 '생물학적 시료'는 퇴행성 뇌질환 여부를 진단하고자 하는 피검체에서 채취된 것이면 제한없이 사용될 수 있으며, 예를 들어 혈액, 혈구, 뇌 조직, 신경세포, 뇌척수액, 타액, 비액, 객담, 관절낭액, 양수, 복수, 자궁경부 또는 질 분비물 및 소변으로 이루어진 군으로부터 선택되는 어느 하나일 수 있으나, 특별히 이에 제한되는 것은 아니며, 바람직하게는 혈액, 혈구, 뇌 조직, 신경세포, 뇌척수액으로 이루어진 군에서 선택되는 어느 하나의 시료일 수 있다.The 'biological sample' may be used without limitation as long as it is collected from a subject to be diagnosed with degenerative brain disease, for example, blood, blood cells, brain tissue, nerve cells, cerebrospinal fluid, saliva, nasal fluid, sputum, joint capsule fluid, It may be any one selected from the group consisting of amniotic fluid, ascites, cervical or vaginal secretions and urine, but is not particularly limited thereto, and is preferably selected from the group consisting of blood, blood cells, brain tissue, nerve cells, and cerebrospinal fluid. It may be any one sample.
본 발명에서 '측정'은, 바람직하게 '분석'을 의미하는 것일 수 있고, 이는 목적하는 물질의 존재 여부를 측정 및 확인하는 것을 의미하는 '정성분석' 혹은 목적하는 물질의 존재 수준(발현 수준) 또는 양의 변화를 측정 및 확인하는 '정량분석'을 의미하는 것일 수 있다. 본 발명에서 측정은 정성적인 방법과 정량적인 방법을 모두 포함하여 수행될 수 있으며, 바람직하게는 정량적인 측정이 수행될 수 있다.In the present invention, 'measurement' may preferably mean 'analysis', which means 'qualitative analysis', which means measuring and confirming the presence of a target substance, or the presence level (expression level) of a target substance. Or it may mean 'quantitative analysis' that measures and confirms the change in quantity. In the present invention, the measurement may be performed including both a qualitative method and a quantitative method, and preferably, a quantitative measurement may be performed.
상기 시료는 검출 또는 진단에 사용하기 전에 전처리할 수 있다. 예를 들어, 균질화(homogenization), 여과, 증류, 추출, 농축, 방해 성분의 불활성화, 시약의 첨가 등을 포함할 수 있다.The sample may be pretreated prior to use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like.
상기 단백질 발현 수준 측정은 당업계에 공지된 단백질 발현 측정 방법에 의한 것이라면 측정 방법이 특별히 제한되지 않으나, 예를 들어 웨스턴 블랏, ELISA, 방사선면역분석, 방사선 면역 확산법, 오우크테로니 면역 확산법(Ouchterlony immunodiffusion), 로케트 면역전기영동(Rocket immunoelectrophoresis), 면역염색법, 면역침전 분석법, 보체 고정 분석법, FACS 및 단백질칩으로 이루어진 군으로부터 선택된 하나 이상일 수 있다.The protein expression level measurement is not particularly limited as long as it is by a protein expression measurement method known in the art, but for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immune diffusion method (Ouchterlony). immunodiffusion), rocket immunoelectrophoresis, immunostaining, immunoprecipitation, complement fixation, FACS, and protein chip.
상기 유전자 발현 수준 측정은 당업계에 공지된 유전자 발현 측정 방법이라면 특별히 제한되지 않으나, 바람직하게는 중합효소반응(PCR), 정량적 중합효소반응(qPCR), 역전사 중합효소 반응(RT-PCR), 경쟁적 역전사 중합효소 반응(competitive RT-PCR), 정량적 실시간 중합효소반응(qRT-PCR), 실시간 역전사 중합효소반응(real time RT-PCR), RNase 보호 분석법, 노던 블롯 분석법(northern blot analysis) 및 DNA 칩 분석으로 이루어진 군으로부터 선택되는 어느 하나의 방법일 수 있고, 보다 바람직하게는 정량적 중합효소반응(qPCR) 또는 노던 블롯 분석법(northern blot analysis)일 수 있다.The gene expression level measurement is not particularly limited as long as it is a gene expression measurement method known in the art, but preferably a polymerase reaction (PCR), a quantitative polymerase reaction (qPCR), a reverse transcription polymerase reaction (RT-PCR), competitive Reverse transcription polymerase reaction (competitive RT-PCR), quantitative real-time polymerase reaction (qRT-PCR), real time reverse transcription polymerase reaction (real time RT-PCR), RNase protection assay, northern blot analysis and DNA chip It may be any one method selected from the group consisting of analysis, and more preferably, quantitative polymerase reaction (qPCR) or northern blot analysis.
다음, 상술한 (1) 단계의 방법으로 측정한 피검체 시료의 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현 수준을, 동일한 방법으로 측정한 정상 대조군 시료의 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준과 비교한다((2) 단계). 이때 상기 시료는 피검체와 정상 대조군 간에 동일한 종류 또는 동일한 방법에 의해 수득된 시료인 것이 바람직하다.Next, the expression level of the Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding it in the subject sample measured by the method of step (1) described above was measured in the same manner as ZBTB16 in the normal control sample. It is compared with the expression level of the protein or the gene encoding it (step (2)). In this case, the sample is preferably a sample obtained by the same type or the same method between the subject and the normal control.
최종적으로 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준이 정상 대조군의 시료보다 높을 경우 퇴행성 뇌질환이라고 판단 또는 진단할 수 있다((3) 단계). 구체적으로 상기 방법으로 측정한 피검체 시료의 ZBTB16 발현수준이 건강한 정상 대조군의 시료에 비하여 증가한 경우, 퇴행성 뇌질환인 것으로 판단 또는 진단할 수 있다.Finally, when the expression level of the ZBTB16 protein or the gene encoding it is higher than that of the normal control sample, it can be determined or diagnosed as degenerative brain disease (step (3)). Specifically, when the ZBTB16 expression level of the subject sample measured by the above method is increased compared to the sample of a healthy normal control group, it may be determined or diagnosed as having a degenerative brain disease.
즉, 퇴행성 뇌질환으로 의심되는 피검체로부터 수득한 생물학적 시료에서 본 발명의 바이오마커(ZBTB16)의 발현수준을 측정하고, 이와 동일한 종류 또는 동일한 방법에 의해 수득된 정상 대조군의 생물학적 시료로부터 본 발명의 바이오마커(ZBTB16)의 발현 수준을 측정한 후, 양자를 비교하되, 피검체의 발현 수준이 정상 대조군의 발현 수준보다 낮을 경우 피검체는 퇴행성 뇌질환인 것으로 판단할 수 있다.That is, the expression level of the biomarker (ZBTB16) of the present invention is measured in a biological sample obtained from a subject suspected of degenerative brain disease, and the present invention is obtained from a normal control biological sample obtained by the same type or method. After measuring the expression level of the biomarker (ZBTB16), the two are compared, but when the expression level of the subject is lower than the expression level of the normal control, it can be determined that the subject has degenerative brain disease.
본 발명의 또 다른 측면은 하기 단계를 포함하는 퇴행성 뇌질환 치료물질의 스크리닝 방법에 관한 것이다.Another aspect of the present invention relates to a screening method for a therapeutic substance for degenerative brain disease, comprising the following steps.
a) 퇴행성 뇌질환 동물 또는 환자에서 분리된 시료에 후보물질을 처리하는 단계;a) treating a candidate material in a sample isolated from an animal or patient with degenerative brain disease;
b) 상기 후보물질 처리군에서 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하는 단계; 및b) measuring the expression level of the ZBTB16 protein or a gene encoding the same in the candidate material treatment group; and
c) 상기 단계에서 측정된 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현 수준이 후보물질 비처리군(대조군)보다 낮아지면, 퇴행성 뇌질환 치료물질로 선정하는 단계.c) If the expression level of the ZBTB16 protein or a gene encoding it measured in the above step is lower than that of the candidate material untreated group (control group), selecting a degenerative brain disease treatment material.
본 발명에서 '퇴행성 뇌질환이 유도된 동물'은 중추신경계의 신경세포에 퇴행성 변화가 나타나면서 여러가지 증상을 유발된 동물로, 보다 바람직하게 베타 아밀로이드의 축적에 의해 치매가 유도된 동물로, 인간을 제외한 포유동물일 수 있고, 바람직하게는 마우스, 기니아 피크, 돼지, 조류 등일 수 있다.In the present invention, the term 'animal degenerative brain disease induced' refers to an animal in which various symptoms are induced while degenerative changes appear in nerve cells of the central nervous system, more preferably an animal in which dementia is induced by the accumulation of beta-amyloid, and a human It may be a mammal except for, preferably a mouse, a guinea pig, a pig, a bird, and the like.
상기 '분리된 시료'는 퇴행성 뇌질환이 유도된 동물로부터 유래된 조직 또는 세포일 수 있다. 상기 시료는 퇴행성 뇌질환이 유도된 동물에서 ZBTB16 발현수준을 검출할 수 있는 조직 또는 이의 세포를 포함하며, 바람직하게는 퇴행성 뇌질환이 유도된 동물의 뇌로부터 분리된 조직 또는 세포일 수 있으나, 특별히 이에 제한되는 것은 아니다.The 'isolated sample' may be a tissue or cell derived from an animal induced with degenerative brain disease. The sample includes a tissue or a cell thereof capable of detecting the expression level of ZBTB16 in an animal induced with a degenerative brain disease, and preferably a tissue or cell isolated from the brain of an animal induced with a degenerative brain disease. However, the present invention is not limited thereto.
본 발명에서, 상기 '후보물질'은 퇴행성 뇌질환 치료 활성을 테스트할 물질을 의미하며, 예컨대 추출물, 단백질, 올리고펩티드, 소형 유기 분자, 다당류, 폴리뉴클레오티드 및 광범위한 화합물 등의 임의 분자를 포함할 수 있다. 또한, 상기 '후보물질'은 리포좀(liposome) 또는 백터(vector)에 트렌스펙션시킨 세포일 수도 있다. 상기 트랜스펙션은 미세 주입법, 칼슘 포스페이트 공동-침전법, 전기 천공법 또는 리포좀 이용법 등을 이용하여 실시할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the 'candidate substance' means a substance to be tested for therapeutic activity for degenerative brain disease, and may include any molecule such as extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide range of compounds. have. In addition, the 'candidate material' may be a cell transfected with a liposome or a vector. The transfection may be performed using a microinjection method, a calcium phosphate co-precipitation method, an electroporation method, or a liposome method, but is not limited thereto.
본 발명에서 '후보물질 비처리군'은 대조군이라고도 하며, 이는 후보물질을 처리하지 않은 퇴행성 뇌질환이 유도된 동물 또는 이로부터 분리된 시료로써, 상기 후보물질을 처리한 군과 병렬 관계에 속하는 퇴행성 뇌질환이 유도된 동물 또는 이로부터 분리된 시료를 의미한다.In the present invention, the 'candidate untreated group' is also referred to as a control group, which is an animal or a sample separated from degenerative brain disease induced without treatment with a candidate material, and is a degenerative disease belonging to the group treated with the candidate material and in a parallel relationship. It refers to an animal or a sample isolated from the induced brain disease.
본 발명에서 '스크리닝 방법'은, 퇴행성 뇌질환의 바이오마커인 ZBTB16 발현을 확인함으로써, 후보물질을 처리하지 않은 비처리군(대조군)과 비교하는 방식으로 이루어지는 것일 수 있다.In the present invention, the 'screening method' may be performed by confirming the expression of ZBTB16, a biomarker of degenerative brain disease, and comparing the candidate substance with an untreated group (control group).
본 발명에서는 퇴행성 뇌질환 실험을 위해, 베타 아밀로이드(Aβ, β amyloid)를 과생성하도록 유전적으로 변이하여 알츠하이머 치매를 유도하였다. 따라서 상기 퇴행성 뇌질환은 구체적으로 베타 아밀로이드 축적에 의한 퇴행성 뇌질환일 수 있다. 특히, 상기 퇴행성 뇌질환은 치매일 수 있으며, 상기 치매는 알츠하이머형 치매(Alzheimer-type dementia), 루이체 치매(Lewy body dementia), 전측두엽 치매(frontotemporal dementia), 뇌혈관 치매(cerebrovascular dementia), 파킨슨병(Parkinson's disease), 경도인지장애, 다운증후군(Down's syndrome) 및 헌팅톤병(Huntington's disease)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다.In the present invention, for the degenerative brain disease experiment, Alzheimer's dementia was induced by genetically mutating to overproduce beta amyloid (Aβ, β amyloid). Therefore, the degenerative brain disease may specifically be a degenerative brain disease caused by the accumulation of beta-amyloid. In particular, the degenerative brain disease may be dementia, and the dementia includes Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, and Parkinson's. It may be any one or more selected from the group consisting of Parkinson's disease, mild cognitive impairment, Down's syndrome, and Huntington's disease.
본 발명의 실시예에서 치매 동물모델의 뇌조직에서 ZBTB16 발현이 정상 동물모델과 비교하여 현저히 많이 발현되었고, 이외 치매 동물모델의 정신 및 행동장애가 있는 것을 확인하였다. 그러나 치매 동물모델로부터 ZBTB16 발현을 억제시킨, 녹아웃 동물모델에서는 ZBTB16 발현이 현저히 감소하였으며, 정신 및 행동장애가 정상 동물모델과 비교하여 현저히 적게 발현되는 것을 확인하였다. In the examples of the present invention, ZBTB16 expression was significantly higher in the brain tissue of the animal model of dementia compared with the normal animal model, and it was confirmed that there were mental and behavioral disorders in the animal model of dementia. However, it was confirmed that the expression of ZBTB16 was significantly reduced in the knockout animal model in which the expression of ZBTB16 was suppressed from the animal model of dementia, and the expression of mental and behavioral disorders was significantly lower than that of the normal animal model.
또한, ZBTB16 발현과 자가포식 작용의 상호관련성을 살펴본 결과, 치매 동물모델로부터 ZBTB16 발현을 녹아웃 시킬 경우, 자가포식 작용이 증가되어(LC3, p62, 자가포식 작용의 마커 단백질 발현량이 각각 증가, 감소함), 선조체 내 질환 단백질 발생을 감소시키며, 알츠하이머와 같은 퇴행성 뇌질환에 의해 유발되는 정신 및 행동 장애를 해소할 수 있음을 확인하였다.In addition, as a result of examining the correlation between ZBTB16 expression and autophagy, when ZBTB16 expression was knocked out from an animal model of dementia, autophagy increased (LC3, p62, and the expression levels of marker proteins of autophagy increased and decreased, respectively) ), it was confirmed that it can reduce the generation of disease proteins in the striatum and resolve mental and behavioral disorders caused by degenerative brain diseases such as Alzheimer's.
즉, ZBTB16의 억제를 통해 베타 아밀로이드 축적에 의해 유도되는 퇴행성 뇌질환의 정신 및 행동 장애를 대조군과 비교하여 정상화시킴을 확인하였다. 퇴행성 뇌질환의 핵심 증상 뿐만 아니라, 이의 BPSD까지 유용하게 적용될 수 있음을 알 수 있다.That is, it was confirmed that the inhibition of ZBTB16 normalized the mental and behavioral disorders of degenerative brain disease induced by beta-amyloid accumulation compared with the control group. It can be seen that not only the core symptoms of degenerative brain disease, but also its BPSD can be usefully applied.
본 발명에서 '예방'은, 상기 퇴행성 뇌질환의 발병과 증상을 억제 또는 지연시키는 모든 행위를 포함하며, 질병 발생 전의 예방 뿐만 아니라 치료 후 질병 재발의 예방을 포함한다. 본 발명에서 '치료'란, 상기 퇴행성 뇌질환의 증상의 치유, 증상의 개선 및 증상의 진행 억제를 모두 포함한다.In the present invention, 'prevention' includes all actions that suppress or delay the onset and symptoms of the degenerative brain disease, and includes prevention of disease recurrence after treatment as well as prevention before disease occurrence. In the present invention, 'treatment' includes all of the treatment of symptoms of the degenerative brain disease, improvement of symptoms, and inhibition of progression of symptoms.
본 발명은 b) 상기 후보물질 처리군에서 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하고, 상기 b) 단계에서 측정된 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현 수준이 후보물질 비처리군(대조군)보다 낮아지면, 퇴행성 뇌질환 치료물질로 선정할 수 있다(단계 c)).The present invention b) measures the expression level of the ZBTB16 protein or a gene encoding it in the candidate substance-treated group, and the expression level of the ZBTB16 protein or the gene encoding it measured in step b) is the candidate substance untreated group (control group) ), it can be selected as a therapeutic substance for degenerative brain disease (step c)).
구체적으로 본 발명은 상기 퇴행성 뇌질환을 예방 또는 치료할 수 있는 후보물질의 부재 하에, 퇴행성 뇌질환이 유도된 동물 또는 이로부터 분리된 시료에서 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하고(후보물질 비처리군), 상기 후보물질의 존재 하에서 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하여(후보물질 처리군) 양자를 비교한 후, 상기 후보물질이 존재할 때의 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 상기 후보물질의 부존재 하에서의 수준보다 억제시키는 물질을 퇴행성 뇌질환 예방 또는 치료용 물질로 예측 또는 판단할 수 있다.Specifically, the present invention measures the expression level of ZBTB16 protein or a gene encoding the same in an animal or a sample isolated from degenerative brain disease induced in the absence of a candidate substance capable of preventing or treating the degenerative brain disease (candidate Substance-untreated group), after measuring the expression level of ZBTB16 protein or a gene encoding it in the presence of the candidate substance (candidate substance-treated group) and comparing both, ZBTB16 protein or coding for it when the candidate substance is present A substance that suppresses the expression level of a gene rather than the level in the absence of the candidate substance may be predicted or determined as a substance for preventing or treating degenerative brain disease.
본 발명에서 '발현수준 측정'이란 후보물질 부재 그리고 존재 하에 퇴행성 뇌질환이 유도된 동물 또는 이로부터 분리된 시료에서 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 확인하는 과정이다.In the present invention, 'measurement of expression level' is a process of confirming the expression level of the ZBTB16 protein or a gene encoding the same in an animal or a sample isolated from degenerative brain disease induced in the absence and presence of a candidate substance.
상기 발현수준의 측정은 웨스턴 블랏, ELISA, 방사선면역분석, 방사선 면역 확산법, 오우크테로니 면역 확산법(Ouchterlony immunodiffusion), 로케트 면역전기영동(Rocket immunoelectrophoresis), 면역염색법, 면역침전 분석법, 보체 고정 분석법, FACS, 단백질칩, 중합효소반응(PCR), 정량적 중합효소반응(qPCR), 역전사 중합효소 반응(RT-PCR), 경쟁적 역전사 중합효소 반응(competitive RT-PCR), 정량적 실시간 중합효소반응(qRT-PCR), 실시간 역전사 중합효소반응(real time RT-PCR), RNase 보호 분석법, 노던 블롯 분석법(northern blot analysis) 및 DNA 칩 분석으로 이루어진 군으로부터 선택되는 어느 하나의 방법일 수 있다.The expression level was measured by Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), quantitative real-time polymerase reaction (qRT- PCR), real-time RT-PCR, RNase protection assay, northern blot analysis, and DNA chip analysis may be any one method selected from the group consisting of.
본 발명의 퇴행성 뇌질환 치료물질의 스크리닝 방법은 상기 후보물질 처리군의 혈청에서 LC3 및 p62 단백질 발현량을 측정하는 단계를 더 포함할 수 있다. 상기 LC3 단백질 발현량이 후보물질 비처리군(대조군)보다 증가하고, 상기 p62 단백질 발현량은 후보물질 비처리군(대조군)보다 감소한 경우, 상기 후보물질을 퇴행성 뇌질환 치료물질로 판단하는 단계를 더 포함할 수 있다.The screening method for a therapeutic substance for degenerative brain disease of the present invention may further include measuring the expression levels of LC3 and p62 proteins in the serum of the candidate substance-treated group. When the LC3 protein expression level increases than the candidate substance untreated group (control group), and the p62 protein expression level decreases than the candidate substance untreated group (control group), the step of determining the candidate substance as a degenerative brain disease treatment material is more may include
본 발명에서 'LC3 및 p62'은 자가포식 작용의 마커 단백질로 널리 알려져 있다. 본 발명에서 퇴행성 뇌질환 발생시 ZBTB16 발현이 증가하면서 자가포식 작용을 감소시키고, 자가포식 현상을 억제하여 선조체 내 질환 단백질이 감소되지 못하고 축적됨에 따라 퇴행성 뇌질환이 유도됨을 알 수 있다. 따라서 퇴행성 뇌질환 발생시, 뇌조직에서 ZBTB16을 억제하면 뇌내에서 자가포식 작용이 활성화되어 LC3 발현량이 증가하고, p62 발현량은 감소함을 확인할 수 있다.In the present invention, 'LC3 and p62' are widely known as marker proteins for autophagy. In the present invention, when ZBTB16 expression increases when degenerative brain disease occurs, autophagy is reduced, and autophagy is suppressed, so that the disease protein in the striatum cannot be reduced and accumulate, so it can be seen that degenerative brain disease is induced. Therefore, when ZBTB16 is inhibited in brain tissue during degenerative brain disease, autophagy is activated in the brain, so that the expression level of LC3 increases and the expression level of p62 decreases.
본 발명의 ZBTB16 유전자와 퇴행성 뇌질환 및 ZBTB16과 LC3 및 p62의 관계는 아직까지 알려진바 없었으나, 본 발명을 통해 퇴행성 뇌질환시, 상기 ZBTB16 유전자의 발현이 증가함을 밝혀냈다. 또한, 동물에서 ZBTB16 억제시킬 경우, 자가포식 작용 관련 바이오마커인 LC3 단백질 발현은 증가하고, p62 단백질 발현은 현저히 감소함을 확인하였다. 이는 ZBTB16 단백질이 퇴행성 뇌질환에 관여하고 있다는 것뿐만 아니라, 퇴행성 뇌질환에 관여하게 되는 작용기작까지 밝힌 것으로써, 진단, 치료, 스크리닝 마커로서 모두 기능할 수 있음을 의미하는 것이다.Although the relationship between the ZBTB16 gene of the present invention and degenerative brain disease and between ZBTB16 and LC3 and p62 has not been known, it was found through the present invention that the expression of the ZBTB16 gene is increased during degenerative brain disease. In addition, it was confirmed that when ZBTB16 was inhibited in animals, the expression of LC3 protein, which is a biomarker related to autophagy, increased, and the expression of p62 protein was significantly reduced. This means that ZBTB16 protein is not only involved in degenerative brain disease, but also the mechanism of action involved in degenerative brain disease, meaning that it can function as a diagnostic, therapeutic, and screening marker.
상기 퇴행성 뇌질환 예방 또는 치료물질 스크리닝 방법을 통해 선별된 후보물질은 후보물질을 투여하지 않은 후보물질 비처리군(대조군)과 비교하였을 때, ZBTB16 유전자의 발현량을 억제 또는 비활성 시킬 수 있다.The candidate material selected through the degenerative brain disease prevention or treatment material screening method may suppress or inactivate the expression level of the ZBTB16 gene when compared to the candidate material untreated group (control group) to which the candidate material is not administered.
본 발명의 다른 측면은 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질의 발현 또는 활성을 억제할 수 있는 제제를 유효성분으로 포함하는 퇴행성 뇌질환 치료물질의 스크리닝용 조성물 또는 키트에 관한 것이다.Another aspect of the present invention relates to a composition or kit for screening a substance for treating degenerative brain disease, comprising as an active ingredient an agent capable of inhibiting the expression or activity of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein.
본 발명의 ZBTB16과 퇴행성 뇌질환 및 ZBTB16과 LC3 및 p62 단백질의 관계는 아직까지 알려진바 없었으나, 본 발명을 통해 퇴행성 뇌질환시, 상기 ZBTB16의 발현이 증가함을 밝혀냈다. 또한, 동물에서 ZBTB16 억제시킬 경우, 자가포식 작용 관련 바이오마커인 LC3 단백질 발현은 증가하고, p62 단백질 발현은 현저히 감소함을 확인하였다. 이는 ZBTB16가 퇴행성 뇌질환 치료물질 스크리닝 마커로서 기능할 수 있음을 의미하는 것이다. 즉, 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정할 수 있다면 퇴행성 뇌질환 예방 또는 치료물질을 스크리닝할 수 있다.Although the relationship between ZBTB16 and degenerative brain disease and ZBTB16 and LC3 and p62 proteins of the present invention has not been known yet, the present invention revealed that the expression of ZBTB16 is increased in degenerative brain disease. In addition, it was confirmed that when ZBTB16 was inhibited in animals, the expression of LC3 protein, which is a biomarker related to autophagy, increased, and the expression of p62 protein was significantly reduced. This means that ZBTB16 can function as a screening marker for a therapeutic substance for degenerative brain disease. That is, if the expression level of the ZBTB16 protein or a gene encoding it can be measured, a substance for preventing or treating degenerative brain disease can be screened.
상기 발현수준을 측정할 수 있는 제제는 앞서 상세히 서술한 바 있으므로, 이를 참고로 하고, 반복되는 설명은 생략하도록 한다.Since the agent capable of measuring the expression level has been described in detail above, it will be referred to and repeated description will be omitted.
본 발명의 다른 측면은 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질의 발현 또는 활성을 억제할 수 있는 제제를 유효성분으로 포함하는 퇴행성 뇌질환 예방 또는 치료용 약학 조성물에 관한 것이다.Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating degenerative brain disease, comprising an agent capable of inhibiting the expression or activity of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein as an active ingredient.
상기 ZBTB16의 발현은 유전자의 mRNA으로의 발현, mRNA의 단백질로의 발현, 및 mRNA 분해 또는 단백질 분해를 모두 포함하는 것일 수 있다. ZBTB16의 활성은 ZBTB16 단백질의 세포 또는 조직에서의 생물학적 기능/활성을 포함하는 것이다.The expression of ZBTB16 may include both mRNA expression of a gene, mRNA expression into protein, and mRNA degradation or protein degradation. The activity of ZBTB16 includes the biological function/activity of the ZBTB16 protein in a cell or tissue.
본 발명에서 '활성'이란 발현된 단백질이 세포 내에서 본래의 기능을 할 수 있도록 하는 생물학적 작용을 의미한다.In the present invention, 'activity' refers to a biological action that allows the expressed protein to perform its original function in a cell.
본 발명에서 '발현'이란 인비트로 또는 세포내에서 유전자가 단백질로 만들어지는 과정의 모든 단계를 포함하는 것으로, 예를 들면 유전자에서 mRNA로의 전사, mRNA에서 단백질로의 번역을 포함하는 것이다.In the present invention, 'expression' includes all steps of a process in which a gene is made into a protein in vitro or in a cell, and includes, for example, transcription from gene to mRNA and translation from mRNA to protein.
상기 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질의 발현을 억제할 수 있는 제제는 ZBTB16 단백질을 코딩하는 유전자의 mRNA에 특이적으로 결합하는 siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids) 및 안티센스 올리고뉴클레오타이드으로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함할 수 있다.The agent capable of inhibiting the expression of the Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein is an siRNA (small interference RNA) or shRNA (short hairpin RNA) that specifically binds to the mRNA of a gene encoding the ZBTB16 protein. , miRNA (microRNA), ribozyme, DNAzyme, PNA (peptide nucleic acids) and may include any one or more selected from the group consisting of antisense oligonucleotides.
상기 siRNA(small interfereing RNA) 또는 shRNA (small hairpin RNA) 또는 miRNA(microRNA)는 RNA 간섭(interference) 작용을 통해, 예를 들면 짧은 길이의 간섭 RNA (siRNA)가 서열 특이적으로 전사체에 결합하여, RISC(RNA Induced Silencing Complex)를 형성하여, 전사된 RNA가 세포내에 단백질로 발현될 수 없도록(silencing) 한다. siRNA, shRNA 또는 miRNA는 그 표적 서열에 상당히 상보적인 서열을 갖는다. 상당히 상보적 서열이란, 표적 유전자의 적어도 연속적인 15 베이스 길이의 서열과 적어도 약 70% 상보성, 적어도 약 80% 상보성, 적어도 약 90% 상보성, 또는 약 100% 상보성을 의미한다. 표적 핵산에 결합하여 사일런싱의 기능을 하는 한 다양한 유래의, 본원에 따른 CTRP3 유전자를 표적으로 하는 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및/또는 miRNA가 사용될 수 있으며, 이들의 생물학적 동등체, 유도체 및 유사체도 포함하는 것이다. 안티센스 올리고뉴클레오타이드는 당업계에 공지된 바와 같으며, 예를 들면 표적 단백질의 임의의 코딩 서열에 결합하여, 표적 단백질의 발현을 억제/감소시키는 짧은 합성 핵산이다. 안티센스 RNA는 표적 유전자 및 전달 방법에 따라 적절한 길이를 가질 수 있으며, 예를 들면 약 6, 8 또는 10 내지 40, 60 또는 100 뉴클레오타이드이다.The siRNA (small interfering RNA) or shRNA (small hairpin RNA) or miRNA (microRNA) is sequence-specifically bound to a transcript through RNA interference, for example, a short interfering RNA (siRNA). , to form a RISC (RNA Induced Silencing Complex), so that the transcribed RNA cannot be expressed as a protein in the cell (silencing). An siRNA, shRNA or miRNA has a sequence that is highly complementary to its target sequence. By highly complementary sequence is meant at least about 70% complementarity, at least about 80% complementarity, at least about 90% complementarity, or about 100% complementarity with a sequence of at least contiguous 15 bases in length of the target gene. Antisense oligonucleotides, siRNAs, shRNAs and/or miRNAs targeting the CTRP3 gene according to the present disclosure of various origins may be used as long as they bind to a target nucleic acid and function as a silencing, and bioequivalents, derivatives and analogs thereof will also include Antisense oligonucleotides are short synthetic nucleic acids that are known in the art and inhibit/reduce expression of the target protein, for example by binding to any coding sequence of the target protein. Antisense RNA may have an appropriate length depending on the target gene and delivery method, for example, about 6, 8 or 10 to 40, 60 or 100 nucleotides.
상기 siRNA는 시험관 내에서 제조되어야 하고 녹다운 유전자를 통상적으로 6-10일 동안 일시적으로 전달된다는 단점이 있으므로, shRNA를 사용하는 것이 바람직하다.Since the siRNA has to be prepared in vitro and the knockdown gene is typically delivered transiently for 6-10 days, it is preferable to use shRNA.
상기 shRNA는 1본쇄 RNA에서 부분적으로 회문상의 염기서열을 포함함으로써, 분자 내에서 2본쇄 구조를 가지고 헤어핀과 같은 구조가 되는 약 20염기 이상의 분자이다.The shRNA is a molecule of about 20 or more bases having a double-stranded structure in the molecule and having a hairpin-like structure in the molecule by including a partially palindromic nucleotide sequence in the single-stranded RNA.
본 발명에서 ZBTB16 발현을 억제하는 shRNA는 ZBTB16 유전자의 일부에 상보적인 서열을 가지고, ZBTB16 유전자의 mRNA를 분해하거나, 번역을 억제할 수 있다. 상보성이 80-90%인 경우에는 mRNA의 번역을 억제할 수 있고, 100%인 경우에는 mRNA를 분해시킬 수 있다. 따라서, 본 발명에서 ZBTB16 발현을 억제하는 shRNA는 마우스와 인간의 공통 mRNA의 서열번호 3(GACTGGAGGATAGAGAAGACGTACCTCTA), 서열번호 4(AATGGCTGTGGCAAGAAGTTCAGCCTCAA), 서열번호 5(GCAAGCCAAGGCGGAGGACCTGGATGACC) 및 서열번호 6(ATCTCGAAGCATTCCAGCGAGGAGAGTGG)에 대한 상보적인 서열에 대하여 100% 상동성을 갖는 염기서열을 포함하는 것이 바람직하다. In the present invention, the shRNA for inhibiting ZBTB16 expression has a sequence complementary to a part of the ZBTB16 gene, and can degrade the mRNA of the ZBTB16 gene or inhibit translation. When the complementarity is 80-90%, the translation of mRNA can be inhibited, and when the complementarity is 100%, the mRNA can be degraded. Accordingly, in the present invention, the shRNA that inhibits ZBTB16 expression is complementary to SEQ ID NO: 3 (GACTGGAGGATAGAGAAGACGTACCTCTA), SEQ ID NO: 4 (AATGGCTGTGGCAAGAAGTTCAGCCTCAA), SEQ ID NO: 5 (GCAAGCCAAGGCGGAGGACCTGGATGACC) and SEQ ID NO: 6 (ATCTCGAAGCATTCCCA complementary to mouse and human common mRNA) It is preferable to include a nucleotide sequence having 100% homology to the sequence.
상기 shRNA는 ZBTB16 발현을 억제하는 shRNA에 관한 것으로, 하기 서열번호 7 내지 10으로 표시되는 염기서열 중에서 어느 하나 이상일 수 있다.The shRNA relates to an shRNA that inhibits ZBTB16 expression, and may be any one or more of the nucleotide sequences shown in SEQ ID NOs: 7 to 10 below.
구체적으로 상기 shRNA는 마우스와 인간의 ZBTB16 mRNA에서 공통적으로 나타나는 서열번호 3 내지 6 중에서 어느 하나에, 상보적인 서열일 수 있는데, 바람직하게 서열번호 7, 서열번호 8, 서열번호 9 및 서열번호 10으로 표시되는 염기서열 중에서 선택되는 어느 하나의 서열일 수 있다. 상기 각각의 염기서열은 4 내지 10 bp의 루프 영역에 의해 회문적으로(palindrome) 연결되어 헤어핀 구조를 형성하는 것일 수 있다.Specifically, the shRNA may be a sequence complementary to any one of SEQ ID NOs: 3 to 6 commonly found in mouse and human ZBTB16 mRNA, preferably SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 It may be any one sequence selected from the displayed nucleotide sequences. Each of the nucleotide sequences may be connected palindrome by a loop region of 4 to 10 bp to form a hairpin structure.
서열번호 7 : TAGAGGTACGTCTTCTCTATCCTCCAGTCSEQ ID NO: 7: TAGAGGTACGTCTTCTCTATCCTCCAGTC
서열번호 8 : TTGAGGCTGAACTTCTTGCCACAGCCATTSEQ ID NO: 8: TTGAGGCTGAACTTCTTGCCACAGCCATT
서열번호 9 : GGTCATCCAGGTCCTCCGCCTTGGCTTGCSEQ ID NO: 9: GGTCATCCAGGTCCTCCGCCTTGGCTTGC
서열번호 10 : CCACTCTCCTCGCTGGAATGCTTCGAGATSEQ ID NO: 10: CCACTCTCCTCGCTGGAATGCTTCGAGAT
RNAi에 의해 ZBTB16의 발현을 억제하는 물질로서는, 3'말단에 돌출부를 가지는 짧은 헤어핀 구조로 구성된 shRNA(short hairpin RNA)를 사용할 수도 있다. RNAi에 의해 ZBTB16의 발현을 억제하는 물질은, 인공적으로 화학 합성하여도 좋고, 센스가닥 및 안티센스 가닥의 DNA 서열을 역방향으로 연결한 헤어핀 구조의 DNA를 T7 RNA 폴리머라제에 의해 실험실 조건(in vitro)에서 RNA를 합성하여 제작하여도 무방하다. 실험실 조건에서 합성하는 경우, T7 RNA 폴리머라제 및 T7 프로모터를 이용하여, 주형 DNA로부터 안티센스 및 센스 RNA를 합성할 수 있다. 이들을 실험실 조건에서 어닐링한 후, 세포에 도입하면 RNAi가 유발되어, ZBTB16의 발현이 억제된다. 세포에의 도입은 예를 들면, 인산칼슘법, 또는 각종 트랜스펙션 시약(예를 들면, oligofectamine, lipofectamine, 및 lipofection 등)을 이용한 방법에 의해 행할 수 있다.As a substance inhibiting the expression of ZBTB16 by RNAi, shRNA (short hairpin RNA) having a short hairpin structure having a protrusion at the 3' end may be used. Substances that inhibit ZBTB16 expression by RNAi may be artificially synthesized chemically, or hairpin-structured DNA in which the DNA sequences of the sense strand and the antisense strand are linked in the reverse direction using T7 RNA polymerase under laboratory conditions (in vitro). It may also be prepared by synthesizing RNA in When synthesizing under laboratory conditions, antisense and sense RNA can be synthesized from template DNA using T7 RNA polymerase and T7 promoter. After annealing them under laboratory conditions, introduction into cells induces RNAi and suppresses the expression of ZBTB16. The introduction into cells can be performed, for example, by a calcium phosphate method or a method using various transfection reagents (eg, oligofectamine, lipofectamine, lipofection, etc.).
RNAi에 의해 ZBTB16의 발현을 억제하는 물질로서는, shRNA 또는 상기 DNA을 포함하는 발현벡터를 이용하여도 좋고, 상기 발현벡터를 함유하는 세포를 이용하여도 좋다. 상기 발현벡터나 세포의 종류는 특별히 한정되지 않으나, 이미 의약으로서 사용되고 있는 발현벡터나 세포가 바람직하다.As a substance that inhibits the expression of ZBTB16 by RNAi, an expression vector containing shRNA or the DNA may be used, or cells containing the expression vector may be used. The type of the expression vector or cell is not particularly limited, but an expression vector or cell already used as a medicine is preferable.
상기 벡터는 숙주 세포에서 목적 유전자를 발현시키기 위한 수단을 의미한다. 상기 벡터는 목적 유전자 발현을 위한 요소(elements)를 포함하는 것으로, 복제원점(replication origin), 프로모터, 작동 유전자(operator), 전사 종결 서열(terminator) 등을 포함할 수 있고, 숙주 세포의 게놈 내로의 도입을 위한 적절한 효소 부위(예컨대, 제한 효소 부위) 및/또는 숙주 세포 내로의 성공적인 도입을 확인하기 위한 선별 마커 및/또는 단백질로의 번역을 위한 리보좀 결합 부위(ribosome binding site; RBS), IRES(Internal Ribosome Entry Site) 등을 추가로 포함할 수 있다. 상기 벡터는 상기 프로모터 이외의 전사 조절 서열(예를 들어, 인핸서 등)을 추가로 포함할 수 있다.The vector refers to a means for expressing a target gene in a host cell. The vector includes elements for expression of a target gene, and may include an origin of replication, a promoter, an operator, a transcription termination sequence, and the like, into the genome of a host cell. Appropriate enzymatic sites (eg, restriction enzyme sites) for introduction of and/or ribosome binding sites (RBS) for translation into proteins and/or selectable markers to confirm successful introduction into host cells, IRES (Internal Ribosome Entry Site) and the like may be additionally included. The vector may further include a transcriptional control sequence (eg, enhancer, etc.) other than the promoter.
또한, 상기 벡터는 당업계에 공지된 플라스미드 DNA, 재조합 벡터 또는 기타 매개체일 수 있고, 구체적으로 선형 DNA 플라스미드 DNA, 재조합 비바이러스성 벡터, 재조합 바이러스성 벡터 또는 유전자 발현 유도 벡터 시스템(inducible gene expression vector system)일 수 있으며, 상기 재조합 바이러스성 벡터는 레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 아데노 부속 바이러스(adeno associated virus), 헬퍼-의존형 아데노바이러스(helper-dependent adenovirus), 헤르페스 심플렉스 바이러스(herpes simplex virus), 렌티바이러스(lentivirus) 또는 백시니아바이러스(vaccinia virus) 벡터일 수 있으나, 이에 제한하는 것은 아니다.In addition, the vector may be a plasmid DNA, a recombinant vector, or other medium known in the art, and specifically, a linear DNA plasmid DNA, a recombinant non-viral vector, a recombinant viral vector, or an inducible gene expression vector system), and the recombinant viral vector is a retrovirus, adenovirus, adeno associated virus, helper-dependent adenovirus, herpes simplex virus ( herpes simplex virus), lentivirus or vaccinia virus vector, but is not limited thereto.
상기 발현벡터를 세포에 도입하는 방법은 공지된 방법이라면 특별히 제한되지 않으나, 바람직하게 리포좀 매개 전달이입, 칼슘포스페이트법, DEAE-덱스트란 매개 전달이입, 양하전 지질 매개 전달이입법, 전기천공, 파아지 시스템을 이용한 형질도입(transduction) 또는 바이러스를 이용한 감염 방법 중에서 선택되는 어느 하나 이상을 포함할 수 있다.The method for introducing the expression vector into a cell is not particularly limited as long as it is a known method, but is preferably liposome-mediated transfection, calcium phosphate method, DEAE-dextran-mediated transfection, positively charged lipid-mediated transtransfection, electroporation, and phage system. It may include any one or more selected from transduction using a virus or an infection method using a virus.
따라서, 본 발명은 상기 shRNA 발현용 재조합 발현벡터를 포함할 수 있다. 이때, 서열번호 2로 표시되는 염기서열을 표적서열로 하는 shRNA를 이용할 수 있다. 재조합 벡터는 당해 분야에 공지된 재조합 DNA 방법이라면 특별히 이에 제한되지 않는다.Accordingly, the present invention may include the recombinant expression vector for shRNA expression. In this case, shRNA having the nucleotide sequence represented by SEQ ID NO: 2 as the target sequence may be used. The recombinant vector is not particularly limited as long as it is a recombinant DNA method known in the art.
상기 shRNA는 재조합 아데노바이러스, 아데노-관련 바이러스(Adeno-associated viruses: AAV), 레트로바이러스, 렌티바이러스, 헤르페스 심플렉스 바이러스, 배시니아 바이러스, 리포좀 및 니오좀로 이루어진 군으로부터 선택되는 어느 하나의 전달체에 의해 전달될 수 있다.The shRNA is delivered to any one carrier selected from the group consisting of recombinant adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposome and niosome. can be transmitted by
상기 ZBTB16 단백질의 활성을 억제할 수 있는 제제는 ZBTB16 단백질에 특이적으로 결합할 수 있는 화합물, 펩티드, 앱타머, 단백질 및 항체로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함할 수 있다.The agent capable of inhibiting the activity of the ZBTB16 protein may include any one or more selected from the group consisting of compounds, peptides, aptamers, proteins and antibodies capable of specifically binding to the ZBTB16 protein.
본 발명의 일 실시예에 있어서, 상기 ZBTB16단백질의 발현 또는 활성을 억제할 수 있는 물질은 상술한 퇴행성 뇌질환 치료물질 스크리닝 방법에 따라 스크리닝된 퇴행성 뇌질환 치료물질도 포함될 수 있다.In one embodiment of the present invention, the substance capable of inhibiting the expression or activity of the ZBTB16 protein may also include a substance for treating degenerative brain disease screened according to the method for screening a substance for treating degenerative brain disease described above.
본 명세서에 기재된 상기 일 실시예들에 따른 ZBTB16의 발현 또는 활성을 억제함으로써 퇴행성 뇌질환 또는 이로 인한 정신 및 행동 장애를 예방 또는 치료하도록 할 수 있다. By inhibiting the expression or activity of ZBTB16 according to the embodiments described herein, it is possible to prevent or treat degenerative brain disease or mental and behavioral disorders resulting therefrom.
상기 약학 조성물은 퇴행성 뇌질환의 예방 또는 치료용일 수 있다. 또한, 상기 약학 조성물은 국소 적용에 적합한 모든 제형으로 제공될 수 있다. 예를 들면, 경구 투여, 경피(trandermally) 투여, 정맥 투여, 근육 투여, 피하주사 및 뇌(Brain)내 투여로 이루어진 군으로부터 선택되는 어느 하나의 경로일 수 있다.The pharmaceutical composition may be for preventing or treating degenerative brain disease. In addition, the pharmaceutical composition may be provided in any dosage form suitable for topical application. For example, it may be any one route selected from the group consisting of oral administration, transdermally administration, intravenous administration, intramuscular administration, subcutaneous injection and intra-brain administration.
본 발명에 따른 약학 조성물은 일반적으로 사용되는 약학적으로 허용가능한 담체와 함께 적합한 형태로 제형화될 수 있다. 약학적으로 허용되는 담체로는 예를 들면, 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등과 같은 비경구 투여용 담체 등이 있으며 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 또한 본 발명에 따른 약학 조성물은 그 투여방법이나 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 산화방지제 등을 적절히 포함할 수 있다. 상기에 예시된 것들을 비롯하여 본 발명에 적합한 약학적으로 허용되는 담체 및 제제는 문헌[Remington's Pharmaceutical Sciences, 최신판]에 상세히 기재되어 있다.The pharmaceutical composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the pharmaceutical composition according to the present invention can be used as a suspending agent, solubilizer, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, if necessary depending on the administration method or formulation. , antioxidants and the like may be included as appropriate. Pharmaceutically acceptable carriers and agents suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.
본 발명의 약학 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다.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 Alternatively, it may be prepared by being introduced into a multi-dose container.
또한 상기 약학 조성물은 유효성분이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수도 있다. 구체적으로 비침습적인 카테터를 이용하는 투여방법을 사용하여도 좋다. 본 발명의 약학 조성물은 질환의 치료를 위하여 치료학적으로 유효한 양으로 포함할 수 있다. 본 발명에서 용어 '치료'란, 달리 언급되지 않는 한, 상기 본 용어가 적용되는 질환 또는 질병의 하나 이상의 증상을 역전시키거나, 완화시키거나, 그 진행을 억제하거나, 또는 예방하는 것을 의미한다. 또한 상기 용어 '치료학적으로 유효한 양'은 연구자, 수의사, 의사 또는 기타임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다. 본 발명의 약학 조성물에 포함되는 유효 성분은 효과에 따라 변화될 것임은 당업자에게 자명하다.In addition, the pharmaceutical composition may be administered by any device capable of moving the active ingredient to the target cell. Specifically, an administration method using a non-invasive catheter may be used. The pharmaceutical composition of the present invention may be included in a therapeutically effective amount for the treatment of diseases. In the present invention, the term 'treatment' means reversing, alleviating, inhibiting the progression, or preventing one or more symptoms of a disease or disease to which the term is applied, unless otherwise stated. In addition, the term 'therapeutically effective amount' refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, which is considered by a researcher, veterinarian, doctor or other clinician, which is a therapeutic an amount that induces alleviation of the symptoms of the disease or disorder being used. It is apparent to those skilled in the art that the active ingredient included in the pharmaceutical composition of the present invention will change depending on the effect.
그러므로 최적의 약학 조성물 함량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.Therefore, the optimal content of the pharmaceutical composition can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of other components contained in the composition, the type of formulation, and the age, weight, general health status, sex and diet of the patient , administration time, administration route and secretion rate of the composition, treatment period, and drugs used at the same time may be adjusted according to various factors.
ZBTB16 발현을 억제하는 shRNA 발현벡터 등 유전자 송달에 관해서는, 적용되는 세포 내에서 ZBTB16 발현을 억제하는 shRNA 또는 shRNA 발현벡터를 발현시키는 한, 특별히 방법은 한정되지 않으나, 예를 들면, 바이러스 벡터, 리포좀을 이용한 유전자 도입을 이용하는 것이 가능하다. 바이러스 벡터로서는, 예를 들면, 레트로바이러스, 백시니아 바이러스, 아데노바이러스, 신린셈리키 바이러스 등의 동물바이러스를 들 수 있다. RNAi에 의해 ZBTB16 발현을 억제하는 물질은 세포에 직접 주입하여도 무방하다.Regarding gene delivery, such as an shRNA expression vector that suppresses ZBTB16 expression, the method is not particularly limited as long as shRNA or shRNA expression vector that suppresses ZBTB16 expression is expressed in the cell to which it is applied. For example, a viral vector, liposome It is possible to use gene introduction using As a viral vector, animal viruses, such as a retrovirus, a vaccinia virus, an adenovirus, and a synrinsemlici virus, are mentioned, for example. Substances that inhibit ZBTB16 expression by RNAi may be directly injected into cells.
상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 포함하는 것이 중요하다. 예컨대, 본 발명의 조성물의 투여량은 유효성분으로서 성인 1 kg 당 약 1x104 particles 내지 1x1012 particles이다. 다만, 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있고, 당업자라면 이러한 요인들을 고려하여 투여량을 적절히 조절할 수 있다. 투여 횟수는 1회 또는 임상적으로 용인가능한 부작용의 범위 내에서 2회 이상이 가능하고, 투여 부위에 대해서도 1개 부위 또는 2개 부위 이상에 투여할 수 있다. 인간 이외의 동물에 대해서도, kg당 인간과 동일한 투여량으로 하거나, 또는 예를 들면 목적의 동물과 인간과의 허혈기관(심장 등)의 용적비(예를 들면, 평균값) 등으로 상기의 투여량을 환산한 양을 투여할 수 있다. 본 발명에 따른 치료의 대상동물로서는, 인간 및 그 밖의 목적으로 하는 포유동물을 예로 들 수 있고, 구체적으로는 인간, 원숭이, 마우스, 래트, 토끼, 양, 소, 개, 염소, 말, 돼지 등이 포함된다.In consideration of all of the above factors, it is important to include an amount that can obtain the maximum effect with a minimum amount without side effects. For example, the dosage of the composition of the present invention is about 1x10 4 particles to 1x10 12 particles per kg of adult as an active ingredient. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity, and those skilled in the art In consideration of these factors, the dosage may be appropriately adjusted. The number of administration may be one time or two or more within the range of clinically acceptable side effects, and may be administered to one site or two or more sites for the administration site. For animals other than humans, the dose is the same as that of a human per kg, or the above dose is adjusted, for example, by the volume ratio (for example, the average value) of the ischemic organ (heart, etc.) between the target animal and the human. The converted amount can be administered. Examples of the animal to be treated according to the present invention include humans and other target mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, goats, horses, pigs, etc. This is included.
본 발명의 shRNA는 ZBTB16 발현을 억제하므로, 본 발명의 약학 조성물은 퇴행성 뇌질환과 관련된 다양한 질병 또는 질환, 예컨대 알츠하이머형 치매(Alzheimer-type dementia), 루이체 치매(Lewy body dementia), 전측두엽 치매(frontotemporal dementia), 뇌혈관 치매(cerebrovascular dementia), 파킨슨병(Parkinson's disease), 경도인지장애, 다운증후군(Down's syndrome), 헌팅톤병(Huntington's disease), 근위축성 측삭경화증(amyotrophic lateral sclerosis), 척수소뇌성 운동실조증(Spinocerebellar Atrophy), 뚜렛 증후군(Tourette`s Syndrome), 프리드리히 보행실조(Friedrich`s Ataxia), 마차도-조셉 병(Machado-Joseph`s disease), 루이 소체 치매(Lewy Body Dementia), 근육긴장이상(Dystonia) 및 진행성 핵상 마비(Progressive Supranuclear Palsy) 등의 치료에 이용될 수 있다. 본 명세서에서 용어 "치료"는 (i) 퇴행성 뇌질환의 증상 완화 또는 억제 ; (ii) 퇴행성 뇌질환에 의한 정신 및 행동 장애 경감 또는 완화; 및 (iii) 퇴행성 뇌질환과 관련된 질환 단백질의 자가포식 작용 촉진에 따른 퇴행성 뇌질환 억제;를 의미한다. 따라서, 본 명세서에서 용어 "치료학적 유효량"은 상기한 약리학적 효과를 달성하는 데 충분한 양을 의미한다.Since the shRNA of the present invention inhibits ZBTB16 expression, the pharmaceutical composition of the present invention may contain various diseases or disorders related to degenerative brain diseases, such as Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia ( frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment, Down's syndrome, Huntington's disease, amyotrophic lateral sclerosis, spinal cerebellar Spinocerebellar Atrophy, Tourette's Syndrome, Friedrich's Ataxia, Machado-Joseph's disease, Lewy Body Dementia, muscle tone It can be used for the treatment of Dystonia and Progressive Supranuclear Palsy. As used herein, the term “treatment” refers to (i) alleviating or suppressing symptoms of degenerative brain disease; (ii) alleviating or alleviating mental and behavioral disorders caused by degenerative brain disease; and (iii) inhibition of degenerative brain disease by promoting autophagy of disease proteins related to degenerative brain disease. Accordingly, as used herein, the term “therapeutically effective amount” refers to an amount sufficient to achieve the aforementioned pharmacological effect.
이하에서 실시예 등을 통해 본 발명을 더욱 상세히 설명하고자 하며, 다만 이하에 실시예 등에 의해 본 발명의 범위와 내용이 축소되거나 제한되어 해석될 수 없다. 또한, 이하의 실시예를 포함한 본 발명의 개시 내용에 기초한다면, 구체적으로 실험 결과가 제시되지 않은 본 발명을 통상의 기술자가 용이하게 실시할 수 있음은 명백한 것이며, 이러한 변형 및 수정이 첨부된 특허청구범위에 속하는 것도 당연하다.Hereinafter, the present invention will be described in more detail by way of Examples and the like, but the scope and content of the present invention may not be construed as being reduced or limited by the Examples below. In addition, based on the disclosure of the present invention including the following examples, it is clear that a person skilled in the art can easily practice the present invention for which no specific experimental results are presented, and such modifications and variations are included in the attached patent. It goes without saying that it falls within the scope of the claims.
또한 이하에서 제시되는 실험 결과는 상기 실시예 및 비교예의 대표적인 실험 결과만을 기재한 것이며, 아래에서 명시적으로 제시하지 않은 본 발명의 여러 구현예의 각각의 효과는 해당 부분에서 구체적으로 기재하도록 한다.In addition, the experimental results presented below describe only the representative experimental results of the Examples and Comparative Examples, and the effects of each of the various embodiments of the present invention that are not explicitly presented below will be described in detail in the corresponding part.
실험예 1. 치매 동물모델(APPPS1)Experimental Example 1. Dementia animal model (APPPS1)
본 실험에서 만 5개월령의 APP/PS1 생쥐를 실험동물로 사용하기 위해 준비하였다. 상기 APP/PS1 생쥐는 베타 아밀로이드(Aβ, β amyloid)를 과생성하도록 유전적으로 변이된 대표적인 알츠하이머 치매 동물모델이다. 이는 APPswe/PSEN1dE9(line 85)(The Jackson Lab; available through the JAX MMRRC Stock# 034829(formerly Jackson Lab Stock # 004462))(https://www.alzforum.org/research-models/appswepsen1de9-line-85) 라인으로, 이를 유지 및 판매하고 있는 KIST 연구동물 자원센터로부터 제공받아 사용하였다. 실험과정은 표준 설치류 식이와 물을 원하는 대로 섭취하게 하였고, 한국과학기술연구원(Korea Institute of Science and Technology)의 동물 보호 및 사용위원회의 엄격한 인가에 따라 처리하였다. 상기 실험동물은 1 주 동안 적응기간을 가지게 하였고, 일반 행동이상여부를 관찰하여 이상이 관찰되는 동물은 본 실험에 사용하지 않았다. 모든 실험동물은 실험기간동안 항온, 항습이 가능한 사육장에서 12 시간 채광, 12 시간 차광의 조건에서 식수와 사료를 자유롭게 섭취하도록 하였다.In this experiment, 5-month-old APP/PS1 mice were prepared for use as experimental animals. The APP/PS1 mouse is a representative Alzheimer's dementia animal model genetically mutated to overproduce beta amyloid (Aβ, β amyloid). This is APPswe/PSEN1dE9(line 85)(The Jackson Lab; available through the JAX MMRRC Stock# 034829(formerly Jackson Lab Stock #004462))(https://www.alzforum.org/research-models/appswepsen1de9-line-85) ) line, and was provided by the KIST Research Animal Resource Center, which maintains and sells it. In the experimental procedure, standard rodent diet and water were ingested as desired, and treatment was carried out in accordance with the strict approval of the Animal Protection and Use Committee of the Korea Institute of Science and Technology. The experimental animals were allowed to have an adaptation period for 1 week, and animals observed abnormalities by observing general behavioral abnormalities were not used in this experiment. All experimental animals were allowed to freely ingest drinking water and feed under conditions of 12 hours of light and 12 hours of shading in a kennel capable of constant temperature and humidity during the experiment period.
대조군(WT+GFP)은 베타 아밀로이드(Aβ, β amyloid)를 과생성하도록 유전적으로 변이가 이루어지지 않은 대조군을 사용하였다.As the control group (WT+GFP), a control group that was not genetically mutated to overproduce beta amyloid (Aβ, β amyloid) was used.
실험예 2. ZBTB16 단백질 녹다운 치매 동물모델 제조(APPPS1+shZtbt16)Experimental Example 2. ZBTB16 protein knockdown dementia animal model preparation (APPPS1 + shZtbt16)
ORIGENE(미국)로부터 Zbtb16의 유전자(서열번호 2)에 대한 shRNA(서열번호 7: 5'-TAGAGGTACGTCTTCTCTATCCTCCAGTC-3')를 발현하는 pGFP-C-shLenti-Zbtb16 shRNA 벡터(ORIGENE) 및 음성 대조군으로 특정부분에 무작위적인 서열로 shRNA를 발현하는 pGFP-C-shLenti-무표적 shRNA 대조군 벡터(TR30021)를 구입하였다.pGFP-C-shLenti-Zbtb16 shRNA vector (ORIGENE) expressing shRNA (SEQ ID NO: 7: 5'-TAGAGGTACGTCTTTCCTATCCTCCAGTC-3') against the gene (SEQ ID NO: 2) of Zbtb16 from ORIGENE (USA) and a specific portion as a negative control A pGFP-C-shLenti-non-targeting shRNA control vector (TR30021) expressing shRNA with a random sequence was purchased.
상기 벡터, third-generation packaging system(pMDLg/pRRE, pRSV-Rev, pMD2.G) 및 HEK293 T 세포주를 사용하여 제조사 매뉴얼에 따라 Zbtb16 shRNA 또는 대조군 shRNA를 발현하는 렌티바이러스를 제조하였다. 렌티바이러스를 함유하는 HEK293T 세포 배양액을 4℃에서 90 분 동안 50,000 g로 울트라원심분리(ultracentrifugation)시켜 농축시켰다. 이후, 상기 렌티바이러스 농축액을 Lenti-X™ p24 Rapid Titer Kit(clontech, 미국)을 사용하여 역가를 결정하였다.A lentivirus expressing Zbtb16 shRNA or control shRNA was prepared using the vector, third-generation packaging system (pMDLg/pRRE, pRSV-Rev, pMD2.G) and HEK293 T cell line according to the manufacturer's manual. HEK293T cell cultures containing lentivirus were concentrated by ultracentrifugation at 50,000 g at 4° C. for 90 minutes. Thereafter, the lentivirus concentrate was titrated using a Lenti-X™ p24 Rapid Titer Kit (clontech, USA).
2×106 형질도입 단위/ml의 재조합 렌티바이러스를 각각 실험예 1의 치매 동물모델(APPPS1)(5 개월령)과 정상 동물모델(WT)의 뇌량(Corpus callosum) 내에 도입하여, 대조군(WT+GFP), 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZtbt16)을 제조하였다. 이때 Nanofil 33G 평활 바늘과 Nanofil 시린지(World Precision Instrument) 그리고 미세 시린지 펌프(Eicom)를 이용하였다. 1개월간 사육하여 ZBTB16 단백질 녹다운 치매 동물모델(APPPS1+shZtbt16)을 제조하였고(6개월령), 비교 실험을 위한 대조군(WT+GFP), 치매 동물모델(APPPS1+GFP)도 동일한 조건으로 1개월간 사육하여 제조하였다(6개월령).2×10 6 transduction units/ml of recombinant lentivirus were introduced into the corpus callosum of the animal model of dementia (APPPS1) (5 months old) of Experimental Example 1 (5 months old) and the normal animal model (WT), respectively, and the control group (WT+) GFP), an animal model of dementia (APPPS1 + GFP), and an animal model of ZBTB16 knockdown dementia (APPPS1 + shZtbt16) were prepared. At this time, a Nanofil 33G smoothing needle, a Nanofil syringe (World Precision Instrument), and a micro syringe pump (Eicom) were used. ZBTB16 protein knockdown dementia animal model (APPPS1 + shZtbt16) was prepared by breeding for 1 month (6 months old), control group (WT + GFP) for comparative experiments, and dementia animal model (APPPS1 + GFP) were also bred for 1 month under the same conditions. prepared (6 months old).
실험기간동안 상기 동물모델은 항온, 항습이 가능한 사육장에서 12 시간 채광, 12 시간 차광의 조건에서 식수와 사료를 자유롭게 섭취하도록 하였다. During the experimental period, the animal model was allowed to freely ingest drinking water and feed under conditions of 12 hours of light and 12 hours of shade in a kennel capable of constant temperature and constant humidity.
실험예 3. 퇴행성 뇌질환 바이오마커 검증Experimental Example 3. Degenerative brain disease biomarker verification
본 발명에서는 실험예 1과 같이 대조군(WT+GFP)과 치매 동물모델(APPPS1+GFP)을 준비하고, ZBTB16 단백질 녹다운 치매 동물모델과 같이 1개월간 사육하여 6 개월령에 실험을 수행하였다. 이들은 각각 8개체씩 무작위로 할당하였다. 상기 동물모델에서의 ZBTB16 단백질 발현 여부를 평가하기 위하여 면역조직 염색 평가를 수행하였다. 상기 대조군(WT)과 치매 동물모델(APPPS1)을 각각 마취하고 관류한 후 뇌를 적출하여 4% PFA로 뇌조직을 고정하였다. 상기 뇌조직에서 선조체와 측좌핵 부분의 조직을 얻고, 상기 선조체와 측좌핵 부분의 조직에 각각 마우스 항-ZBTB16 항체(D-9; sc-28319, Santa Cruz Biotechnology, Santa Cruz, CA)를 붙였다(4 ℃, overnight). 그 다음 상기 조직에 바이오티닐화된 항-마우스 이차 항체를 처리하고, 그 뒤 발색을 위한 염색처리를 진행하였다. 이때 세포핵은 DAPI로 염색하였다.In the present invention, as in Experimental Example 1, a control group (WT+GFP) and an animal model of dementia (APPPS1+GFP) were prepared, and the experiment was performed at the age of 6 months by breeding for 1 month as in the ZBTB16 protein knockdown dementia animal model. They were randomly assigned to 8 subjects each. In order to evaluate the expression of ZBTB16 protein in the animal model, immunohistochemistry evaluation was performed. The control group (WT) and the dementia animal model (APPPS1) were anesthetized and perfused, respectively, and brains were removed and brain tissues were fixed with 4% PFA. The tissues of the striatum and the nucleus accumbens were obtained from the brain tissue, and a mouse anti-ZBTB16 antibody (D-9; sc-28319, Santa Cruz Biotechnology, Santa Cruz, CA) was attached to the tissues of the striatum and the nucleus accumbens, respectively ( 4°C, overnight). Then, the tissue was treated with a biotinylated anti-mouse secondary antibody, followed by staining for color development. At this time, the cell nucleus was stained with DAPI.
도 2는 대조군(WT)과 치매 동물모델(APPPS1)에서 분리된 뇌조직(Brain)에서 배측선조체(dorsal striatum)과 측좌핵(nucleus accumbens, NAc)으로부터 ZBTB16 단백질 발현량을 면역조직염색법으로 측정한 결과이다. Figure 2 is a control (WT) and dementia animal model (APPPS1) from the brain tissue (Brain) from the dorsal striatum (dorsal striatum) and the nucleus accumbens (nucleus accumbens, NAc) from the ZBTB16 protein expression was measured by immunohistochemistry. is the result
도 2에 나타난 바와 같이, 치매 동물모델의 배측선조체(dorsal striatum)에서 ZBTB16 단백질의 발현이 유의미하게 증가하였음을 알 수 있다. 동물실험 후, 면역염색을 통해 각각에서의 ZBTB16 단백질발현을 살펴본 결과, 치매 동물모델에서 ZBTB16 단백질의 발현량이 유의미하게 증가하였으므로, ZBTB16 단백질을 통해 치매를 포함하는 퇴행성 뇌질환의 진단을 위한 정보를 제공할 수 있음을 알 수 있다.As shown in FIG. 2 , it can be seen that the expression of ZBTB16 protein was significantly increased in the dorsal striatum of the dementia animal model. After the animal experiment, as a result of examining the expression of ZBTB16 protein in each through immunostaining, the expression level of ZBTB16 protein in the dementia animal model was significantly increased, so the ZBTB16 protein provides information for the diagnosis of degenerative brain diseases including dementia know you can do it.
실험예 4. ZBTB16 녹다운이 치매 동물모델의 기억력 및 인지능력에 미치는 영향 조사Experimental Example 4. Investigation of the effect of ZBTB16 knockdown on memory and cognitive ability in an animal model of dementia
실험예 2의 대조군(WT+GFP)과 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에 대해 두 종류의 인지기능 검사(Y-maze test와 novel object recognition test)를 실시하였다.Two types of cognitive function tests (Y-maze test and novel object recognition test) were performed on the control (WT+GFP) and dementia animal model (APPPS1+GFP) and ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) of Experimental Example 2 carried out.
4-1. Y-maze 시험(YMT)4-1. Y-maze test (YMT)
실험동물들을 120° 각도로 세 갈래로 가지가 뻗은 Y-maze(42 × 3 × 12 cm)의 가지 끝에 두고 8분간 자유롭게 움직이도록 하고 그 움직임을 Ethovision system(Noldus IT b.v., Netherlands)을 이용하여 컴퓨터로 관찰하였다. 각 가지를 A, B, C로 정한 후 네 발이 모두 한 쪽 가지에 완전히 들어가야 1회 출입횟수로 인정하였다. 세 개의 서로 다른 가지에 차례로 들어간 경우를 자발적 변경 행동력(spontaneous alternation behavior)으로 규정하였다. Alternation score(%)는 다음 수식으로 계산하였다.Experimental animals were placed at the tip of the branch of the Y-maze (42 × 3 × 12 cm) branched into three branches at a 120° angle and allowed to move freely for 8 minutes, and the movement was monitored using a computerized Ethovision system (Noldus IT b.v., Netherlands). was observed with After each branch was designated as A, B, and C, all four feet must completely enter one branch to be counted as the number of entry and exit. The case of entering three different branches one after another was defined as spontaneous alternation behavior. Alternation score (%) was calculated by the following formula.
Alternation(%)=[(alternation 횟수)/(총 출입횟수-2)] × 100 Alternation(%)=[(number of alternations)/(total number of accesses-2)] × 100
4-2. 새로운 사물 탐색 시험(novel object recognition test, NOR)4-2. novel object recognition test (NOR)
신물질탐색 시험(Novel object recognition test)은 기존 방법을 변형한 것으로 적응기, 탐색기 및 신물질 인식기로 이루어져 있다. 이틀간 10분씩 생쥐를 개방장에 놓고 적응을 시킨 후, 두 개의 동일한 물체를 일정한 간격을 설치하였다. 실험동물을 3분간 자유롭게 탐색하도록 하면서 Ethovision으로 각 물체에서 머무르는 시간을 측정하였으며 이후 케이지에 30분간 돌려놓았다. 그리고 설치한 물체 중 하나를 신물질로 대체하고 실험동물의 움직임을 3분 동안 측정하였다. 물체의 선호도(recognition index(%))는 전체 탐색한 시간 중에서 각 물체에 머무르는 시간을 백분율로 환산하였다.The novel object recognition test is a modification of the existing method and consists of an adaptor, a searcher, and a new material recognizer. After acclimatization by placing the mice in an open field for 10 minutes for two days, two identical objects were installed at regular intervals. The time spent in each object was measured by Ethovision while the experimental animals were allowed to freely explore for 3 minutes, and then returned to the cage for 30 minutes. Then, one of the installed objects was replaced with a new material and the movement of the experimental animal was measured for 3 minutes. The object's recognition index (%) was converted into a percentage of the time spent in each object among the total search time.
실험의 통계분석을 위하여 대응표본 t-검정(paired t-test)을 사용하여 그룹 간 평균수치 유의차를 확인하였으며, 0.05보다 낮은 p 값을 가질 경우에는 *, 0.01보다 낮은 p 값을 가질 경우에는 **, 0.001보다 낮은 p 값을 가질 경우에는 ***로, 유의적 차이가 없는 경우 N.S로 유의적 차이를 표기하였다. 에러바는 S.E.M을 나타낸다.For statistical analysis of the experiment, the significant difference in mean values between groups was confirmed using the paired t-test. When the p value was lower than 0.05, *, and when the p value was lower than 0.01, **, a significant difference was indicated by *** when it had a p value lower than 0.001, and by N.S when there was no significant difference. Error bars indicate S.E.M.
도 3은 대조군(WT+GFP)과 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에 대한 Y-maze 시험(YMT) 결과를 나타낸 그래프이다. 3 is a graph showing the Y-maze test (YMT) results for the control group (WT+GFP), the dementia animal model (APPPS1+GFP), and the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16).
도 3을 통해 자발적 변경 행동력을 살펴본 결과, 대조군(WT+GFP)과 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)이 유사한 수준임을 확인하였다. 치매 동물모델(APPPS1+GFP)에서는 80% 이상으로 증가하였으나, 대조군과 비교하여 유의한 차이를 보이지 않았다.As a result of examining the spontaneous change behavior through FIG. 3 , it was confirmed that the control group (WT+GFP) and the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) had similar levels. In the dementia animal model (APPPS1+GFP), it increased to more than 80%, but there was no significant difference compared with the control group.
일반적으로 치매에 의한 인지기능 저하는 12 개월령부터 나타나므로, 대다수 치매 관련 실험은 12 개월령을 사용한다. 그러나 본 발명은 인지기능 증상이 없는 치매 위험군으로부터, 치매를 조기진단할 수 있는지, 치매 초기 발생하는 전조증상(정신적 증상)을 개선, 예방 또는 치료하는 효과를 살펴보는데 목적이 있으므로, 본 발명의 동물실험에서는, 12 개월령보다 6 개월이나 더 어린 6 개월령의 생쥐를 사용하였다. 따라서, 분명 치매가 유도되었음에도 불구하고 YMT 실험결과, 대조군과 치매 동물모델 및 ZBTB16 녹다운 치매 동물모델 간 유의한 차이가 나타나지 않음을 알 수 있다. 이는 본 발명자가 예상하던 바와 일치하는 결과이다.In general, cognitive decline due to dementia starts at the age of 12 months, so most dementia-related experiments use the age of 12 months. However, the present invention aims to examine whether dementia can be diagnosed early from a dementia risk group without cognitive function symptoms, and the effect of improving, preventing or treating the prognostic symptoms (mental symptoms) that occur in the early stages of dementia, so the animal of the present invention In the experiment, 6-month-old mice, which are 6 months younger than 12 months old, were used. Therefore, it can be seen that although dementia was clearly induced, there was no significant difference between the control group, the dementia animal model, and the ZBTB16 knockdown dementia animal model as a result of the YMT experiment. This result is consistent with what the inventors expected.
도 4는 대조군(WT+GFP)과 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에 대한 신물질탐색 시험(novel object recognition test, NOR) 결과를 나타낸 그래프이다. 도면 상에서 N은 신물질(Novel)이고, F는 기존 사물(familiar)을 의미한다.4 is a graph showing the results of a novel object recognition test (NOR) for a control group (WT+GFP), an animal model of dementia (APPPS1+GFP), and an animal model of ZBTB16 knockdown dementia (APPPS1+shZbtb16). In the drawing, N denotes a novel material, and F denotes an existing thing (familiar).
도 4를 통해 신물질탐색 평가를 실시한 결과, 대조군(WT+GFP), 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)이 유사한 수준임을 확인하였다.As a result of conducting the new material search evaluation through FIG. 4 , it was confirmed that the control group (WT+GFP), the dementia animal model (APPPS1+GFP), and the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) had similar levels.
일반적으로 치매에 의한 인지기능 저하는 12 개월령부터 나타나므로, 대다수 치매 관련 실험은 12 개월령을 사용한다. 그러나 본 발명은 인지기능 증상이 없는 치매 위험군으로부터, 치매를 조기 진단할 수 있는지, 치매 초기 발생하는 전조증상(정신적 증상)을 개선, 예방 또는 치료하는 효과를 살펴보는데 목적이 있으므로, 본 발명의 동물실험에서는, 12 개월령보다 6 개월이나 더 어린 6 개월령의 생쥐를 사용하였다. 따라서 베타 아밀로이드의 축적에 의해 초기 치매가 유도되었음에도 불구하고 YMT 실험결과, 대조군과 치매 동물모델 및 ZBTB16 녹다운 치매 동물모델 간 유의한 차이가 나타나지 않음을 알 수 있으며, 이는 본 발명자가 예상하던 바와 일치하는 결과이다.In general, cognitive decline due to dementia starts at the age of 12 months, so most dementia-related experiments use the age of 12 months. However, the present invention aims to examine whether dementia can be diagnosed early from a dementia risk group without cognitive function symptoms, and the effect of improving, preventing or treating the prognostic symptoms (mental symptoms) that occur in the early stages of dementia, so the animal of the present invention In the experiment, 6-month-old mice, which are 6 months younger than 12 months old, were used. Therefore, it can be seen that although early dementia was induced by the accumulation of beta-amyloid, there was no significant difference between the control group, the dementia animal model, and the ZBTB16 knockdown dementia animal model as a result of the YMT experiment, which is consistent with what the present inventor expected. is the result
실험예 5. ZBTB16 녹다운이 치매 동물모델의 사회성에 미치는 영향 조사Experimental Example 5. Investigation of the effect of ZBTB16 knockdown on sociality in an animal model of dementia
5-1. 3CT(3-chamber test) 행동실험5-1. 3CT (3-chamber test) behavioral experiment
상기 실험예 2의 방법으로 제조한 치매 동물모델(APPPS1+GFP)과 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)이 초기 치매에 따른 음성증상을 나타내는지 확인하기 위하여 생쥐의 사회성을 확인하는 행동 실험 중 가장 널리 사용되는 3CT(3-chamber test) 행동실험을 수행하였다. In order to confirm whether the animal model of dementia (APPPS1 + GFP) and the animal model of ZBTB16 knockdown dementia (APPPS1 + shZbtb16) prepared by the method of Experimental Example 2 showed negative symptoms according to early dementia, the sociality of mice was checked during behavioral experiments The most widely used 3CT (3-chamber test) behavioral experiment was performed.
구체적으로, 투명한 벽을 통해 분리된 연속된 3개의 방 중 가운데 방에 측정 생쥐(6개월령, male)를 EthoVision XT 11.5(Noldus, Netherlands 제조)에 위치시키고, 양쪽 방 중 한쪽 방에 살아있는 생쥐(S1)를, 반대편 방에는 무생물인 물체(O)를 놓아두었으며, 또는 낯익은 생쥐(S1)와 새로운 생쥐(S2)를 놓아두었다. 양쪽 방 중에서 오래 머문 자리일수록 붉은색으로 표시하였다. 테스트는 10분간 수행하였고, 대조군으로는 정상 동물모델(WT+GFP)을 사용하였다.Specifically, a measuring mouse (6 months old, male) was placed in an EthoVision XT 11.5 (manufactured by Noldus, Netherlands) in the middle of three consecutive rooms separated by a transparent wall, and a live mouse (S1) was placed in one of the two rooms. ), an inanimate object (O) was placed in the opposite room, or a familiar mouse (S1) and a new mouse (S2) were placed. Among the two rooms, the longer you stayed, the more red. The test was performed for 10 minutes, and a normal animal model (WT+GFP) was used as a control.
실험의 통계분석을 위하여 대응표본 t-검정(paired t-test)을 사용하여 그룹 간 평균수치 유의차를 확인하였으며, 0.05보다 낮은 p 값을 가질 경우에는 *, 0.01보다 낮은 p 값을 가질 경우에는 **, 0.001보다 낮은 p 값을 가질 경우에는 ***로, 유의적 차이가 없는 경우 N.S로 유의적 차이를 표기하였다. 에러바는 S.E.M을 나타낸다.For statistical analysis of the experiment, the significant difference in mean values between groups was confirmed using the paired t-test. When the p value was lower than 0.05, *, and when the p value was lower than 0.01, **, a significant difference was indicated by *** when it had a p value lower than 0.001, and by N.S when there was no significant difference. Error bars indicate S.E.M.
도 5는 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 3-chamber test(3CT) 결과를 나타낸 도면으로, 도 5a는 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 무생물인 물체(0)와 살아있는 생쥐(S1)에 대한 관심 정도를 나타낸 도면이고, 도 2b는 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 낯익은 생쥐(S1)와 새로운 생쥐(S2)에 대한 관심 정도를 나타낸 도면이다.5 is a view showing the 3-chamber test (3CT) results of the animal model of dementia (APPPS1 + GFP), the animal model of ZBTB16 knockdown dementia (APPPS1 + shZbtb16) and the control (WT + GFP) prepared according to the present invention; FIG. 5a is an inanimate object (0) and a living mouse (S1) of the animal model of dementia (APPPS1+GFP), ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and control (WT+GFP) prepared according to the present invention. It is a view showing the degree, and Figure 2b is a familiar mouse (S1) and a new animal model (APPPS1 + GFP), ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and control (WT + GFP) prepared according to the present invention. It is a diagram showing the degree of interest in the mouse (S2).
도 5에 나타난 바와 같이, 무생물인 물체(0)보다 살아있는 생쥐(S1)에게 더 높은 관심을 보이는 현상은 치매 동물모델(APPPS1+GFP)에서도 정상적으로 나타나는 것을 확인되었다.As shown in FIG. 5 , it was confirmed that the phenomenon of showing a higher interest in a living mouse (S1) than in an inanimate object (0) is also normal in the dementia animal model (APPPS1+GFP).
그러나 도 5b에서, 대조군(WT+GFP)는 낯익은 생쥐(S1)보다 새로운 생쥐(S2)에 더 많은 시간과 상호작용을 나타내는 반면, 치매 동물모델(APPPS1+GFP)은 낯익은 생쥐(S1)와 새로운 생쥐(S2)에 대한 관심의 정도가 비슷함을 확인하였다(사회성 저하). 이에 반해 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)은 대조군과 유사하게 낯익은 생쥐(S1)보다 새로운 생쥐(S2)에 더 많은 시간과 상호작용을 나타냄을 확인하였다.However, in Figure 5b, the control group (WT+GFP) showed more time and interaction in the new mouse (S2) than the familiar mouse (S1), whereas the dementia animal model (APPPS1+GFP) showed the familiar mouse (S1). It was confirmed that the degree of interest in the new mouse (S2) was similar to that of the new mouse (S2) (decreased sociality). On the other hand, it was confirmed that the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) exhibited more time and interaction in the new mouse (S2) than the familiar mouse (S1), similar to the control group.
상술한 결과를 통해 베타 아밀로이드(Aβ, β amyloid)를 과생성하도록 유전적으로 변이된 치매 동물모델(APPPS1+GFP)은 새로운 생쥐에 대한 호기심과 친밀도가 감소하고 사회적 감성이 결여되는 증상을 나타냄을 알 수 있다.From the above results, it was found that the animal model of dementia (APPPS1+GFP) genetically mutated to overproduce beta amyloid (Aβ, β amyloid) exhibits symptoms of decreased curiosity and intimacy with new mice and lack of social sensitivity. can
상기 치매 동물모델의 뇌 내에 특이적으로 ZBTB16 단백질 발현을 억제할 경우, 새로운 생쥐에 대한 호기심과 친밀도가 증가하고 사회적 감성이 결여되는 증상이 정상수준을 회복됨 확인하였다.When the ZBTB16 protein expression was specifically suppressed in the brain of the dementia animal model, it was confirmed that curiosity and intimacy with new mice increased, and the symptoms of lack of social emotion returned to normal levels.
실험예 6. ZBTB16 녹다운이 치매 동물모델의 불안성에 미치는 영향 조사Experimental Example 6. Investigation of the effect of ZBTB16 knockdown on anxiety in an animal model of dementia
각각의 실험동물들이 갖는 불안 정도를 하기 실험을 통해 확인하였다.The degree of anxiety of each experimental animal was confirmed through the following experiment.
6-1. 고가식 십자 미로 실험(elevated plus maze test, EPM)6-1. Elevated plus maze test (EPM)
고가식 십자 미로 실험은 생쥐가 선천적으로 밝고 개방된 공간을 싫어하며, 새로운 환경에 자발적인 탐색 활동을 한다는 전제를 기반하여 실시한다. 고가식 십자 미로 장치는 공통의 중앙 플랫폼(5x5㎝)으로부터 확장된 두 개의 열린 가지(open arm)(30x5㎝) 및 두 개의 닫힌 가지(closed arm)(5x30㎝, 벽은 15㎝ 높이)로 구성되었다. 상기 구조는 대응되는 가지가 서로 마주보도록 배열된 더하기-기호(plus-sign)의 모양으로 형성되었다. 상기 장치는 지면(floor level) 위로 50㎝ 올라와 있다.The elevated cross maze experiment is conducted based on the premise that mice are innately dislike of bright and open spaces, and that they voluntarily explore new environments. The elevated cross maze apparatus consists of two open arms (30x5 cm) and two closed arms (5x30 cm, walls 15 cm high) extending from a common central platform (5x5 cm). became The structure was formed in the shape of a plus-sign with corresponding branches arranged to face each other. The device is raised 50 cm above floor level.
상기 고가식 십자 미로 장치에 상기 3개의 군에 속하는 생쥐를 넣고 각각 7분씩 관찰하였다. 각각의 실험동물은 개별적으로 개방 범위의 중앙에 위치하도록 하였으며, 각 열린 가지에 머무른 시간을 측정하였다. 행동관찰 영상은 소프트웨어(Smart Junior, Panlab SL, Barcelona, Spain)에 연결된 비디오카메라를 이용하여 촬영하였다. 이후 비디오 추적 소프트웨어(video tracking software)를 이용한 비디오 파일 분석을 통해 매초 단위로 생쥐가 머무른 시간을 측정하고, 그 결과는 도 6에 나타내었다.Mice belonging to the three groups were placed in the elevated cross maze device and observed for 7 minutes each. Each experimental animal was individually placed in the center of the open range, and the time spent on each open branch was measured. Behavioral observation images were taken using a video camera connected to the software (Smart Junior, Panlab SL, Barcelona, Spain). Thereafter, the time the mice stayed was measured in units of every second through video file analysis using video tracking software, and the results are shown in FIG. 6 .
6-2. 명암 이동 시험(light-dark box test, LDB)6-2. light-dark box test (LDB)
명암 이동 시험은 생쥐가 선천적으로 밝은 빛을 싫어하며, 새로운 환경에 자발적인 탐색 활동을 한다는 전제를 기반하여 실시한다. 명암 이동 시험은 명실(light box)과 암실(dark box)이 연결통로로 연결된 생쥐 케이지에서 암실에 상기 3개의 군에 속하는 실험동물을 넣고 10분 동안 관찰하면서, 명실로 처음 나갈 때까지의 지체시간, 명실에서 머문 시간, 각 암실과 명실을 이동한 빈도를 측정하였다. 그 빈도는 항불안 기능을 측정하는 지표(index)이다.The contrast shift test is conducted based on the premise that mice naturally dislike bright light and voluntarily explore new environments. In the light-dark movement test, the experimental animals belonging to the three groups are placed in a dark room in a mouse cage in which a light box and a dark box are connected by a connecting passage, and while observing for 10 minutes, the delay time until the first exit to the light room , the time spent in a dark room, and the frequency of moving between each dark room and light room were measured. Its frequency is an index measuring the anti-anxiety function.
명암 이동 시험에서도 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 결과를 비교하였고, 그 결과는 도 7에 나타내었다.In the light-dark movement test, the results of the dementia animal model (APPPS1+GFP), the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and the control group (WT+GFP) were compared, and the results are shown in FIG. 7 .
6-3. 통계6-3. statistics
실험의 통계분석을 위하여 일원 분산 분석(one-way ANOVA test)을 사용하여 그룹 간 평균수치 유의차를 확인하였으며, 0.05보다 낮은 p 값을 가질 경우에는 *, 0.01보다 낮은 p 값을 가질 경우에는 **, 0.001보다 낮은 p 값을 가질 경우에는 ***로, 유의적 차이가 없는 경우 N.S로 유의적 차이를 표기하였다. 에러바는 S.E.M을 나타낸다.For statistical analysis of the experiment, a one-way ANOVA test was used to confirm the significant difference in the mean value between groups, and when it had a p value lower than 0.05, *, and when it had a p value lower than 0.01, * *, Significant difference is indicated by *** when it has a p value lower than 0.001, and by N.S when there is no significant difference. Error bars indicate S.E.M.
6-4. 분석결과6-4. Analysis
도 6은 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 고가식 십자 미로 실험(EMP) 결과를 나타낸 도면이다.6 is a diagram showing the results of the elevated cross maze experiment (EMP) of the dementia animal model (APPPS1 + GFP), the ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and the control group (WT + GFP) prepared according to the present invention.
도 6에 나타낸 바와 같이, 치매 동물모델(APPPS1+GFP)은 열린 가지에서 머문 시간이 현저히 감소하였음을 확인하였다. ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)은 대조군(WT+GFP)과 유사한 수준으로 열린 가지에서 머문 시간이 증가한 것을 확인할 수 있다. 즉, 베타 아밀로이드 축적에 의해 초기 치매가 발발한 경우, 밝고 개방된 열린공간에서 머무르는 시간이 현저히 짧아졌음을 확인하였다. 즉, 베타 아밀로이드 축적에 의해 기억력 인지기능은 크게 저하되지 않은 초기 치매 상태임에도 불구하고 정신 및 행동 장애 중에서 불안 증상이 증가한다는 것을 알 수 있다. 이때, ZBTB16 단백질 발현을 억제하면, 초기 치매의 정신 및 행동 장애인 불안증상을 정상수준과 유사한 정도로 현저히 개선함을 알 수 있다.As shown in FIG. 6 , it was confirmed that the dementia animal model (APPPS1+GFP) significantly reduced the dwell time in the open branch. It can be seen that the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) increased the dwell time in the open branch to a level similar to that of the control group (WT+GFP). That is, it was confirmed that when early dementia occurred due to the accumulation of beta-amyloid, the time spent in a bright and open space was significantly shortened. In other words, it can be seen that anxiety symptoms increase among mental and behavioral disorders despite the early dementia state in which memory and cognitive function are not significantly reduced by the accumulation of beta-amyloid. At this time, it can be seen that when ZBTB16 protein expression is suppressed, anxiety symptoms, which are mental and behavioral disorders of early dementia, are remarkably improved to a level similar to the normal level.
도 7은 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 대조군(WT+GFP)의 명암 이동 시험(LDB) 결과를 나타낸 도면이다.7 is a diagram showing the results of a light-dark shift test (LDB) of an animal model of dementia (APPPS1+GFP), an animal model of ZBTB16 knockdown dementia (APPPS1+shZbtb16), and a control group (WT+GFP).
도 7에 나타난 바와 같이, 치매 동물모델(APPPS1+GFP)은 명실에 머무는 시간이 대조군(WT+GFP)에 비해 현저히 저하되었다. 이에 반해 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)은 대조군(WT+GFP)와 비슷한 수준으로 명실에 오래 머물며, 암실과 명실의 이동빈도가 유의하게 증가되었다.As shown in FIG. 7 , the dementia animal model (APPPS1+GFP) significantly decreased the time to stay in the light room compared to the control group (WT+GFP). In contrast, the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) stayed in the light room for a long time at a level similar to that of the control group (WT+GFP), and the frequency of movement between the dark room and the light room was significantly increased.
즉, 베타 아밀로이드 축적에 의해 기억력 인지기능은 크게 저하되지 않은 초기 치매 상태임에도 불구하고 정신 및 행동 장애 중에서 불안 증상이 증가한다는 것을 알 수 있다. 이때, ZBTB16 단백질 발현을 억제하면, 초기 치매의 정신 및 행동 장애인 불안증상이 정상수준과 유사한 정도로 현저히 개선됨을 알 수 있다. In other words, it can be seen that anxiety symptoms increase among mental and behavioral disorders despite the early dementia state in which memory and cognitive function are not significantly reduced by the accumulation of beta-amyloid. At this time, it can be seen that when ZBTB16 protein expression is suppressed, anxiety symptoms, which are mental and behavioral disorders of early dementia, are remarkably improved to a degree similar to the normal level.
실험예 7. ZBTB16 단백질 억제에 따른 자가포식 현상 분석Experimental Example 7. Analysis of autophagy according to ZBTB16 protein inhibition
7-1. 면역조직염색법7-1. immunohistochemistry
본 발명에 따른 ZBTB16 단백질 억제에 따른 p62 단백질 활성 및 자가포식 현상의 활성 정도를 파악하기 위하여 p62와 LC3를 마커로 사용해서 면역조직염색법을 수행하였다.Immunohistostaining was performed using p62 and LC3 as markers to determine the degree of p62 protein activity and autophagy activity according to the inhibition of the ZBTB16 protein according to the present invention.
실험예 2에 따라 제조된 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)을 준비하고, 이로부터 실험예 3에 따라 뇌조직 동결절편을 준비하였다.An animal model of dementia (APPPS1 + GFP) and a ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) prepared according to Experimental Example 2 were prepared, and from this, a frozen brain tissue section was prepared according to Experimental Example 3.
상기 절편에 1차 항체(anti-LC3;Novus Biologicals;NB100-2220, anti-p62 ;Abcam;ab91526)를 처리하고, 2차 항체(Goat anti-Rabbit IgG;Alexa Fluor 488, Goat anti-Mouse IgG;Alexa Fluor 594)를 처리하였다. 다시 PBS로 3회 세척하고 세포 내 핵 염색을 위해 DAPI 염색 후 공초점 현미경을 통해 p62 또한 LC3의 발현 정도를 관찰하였다. 결과는 도 8 및 도 9에 나타내었다. 면역조직염색법은 3회 이상의 독립적인 실험으로부터 대표적인 것을 도식화하였다.The section was treated with a primary antibody (anti-LC3; Novus Biologicals; NB100-2220, anti-p62; Abcam; ab91526), and a secondary antibody (Goat anti-Rabbit IgG; Alexa Fluor 488, Goat anti-Mouse IgG; Alexa Fluor 594). After washing again with PBS three times and DAPI staining for intracellular nuclear staining, the expression level of p62 and LC3 was observed through a confocal microscope. The results are shown in FIGS. 8 and 9 . The immunohistostaining method was schematically representative from three or more independent experiments.
도 8은 본 발명에 따라 제조된 치매 동물모델(APPPS1+GFP)에서 분리된 뇌조직에서 배측선조체(dorsal striatum)으로부터 LC3, p62 발현량을 면역조직염색법으로 측정한 결과이고, 도 9는 본 발명에 따라 제조된 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에서 분리된 뇌조직에서 배측선조체(dorsal striatum)으로부터 LC3, p62 발현량을 면역조직염색법으로 측정한 결과이다.8 is a result of measuring the expression levels of LC3 and p62 from dorsal striatum in brain tissue isolated from an animal model of dementia (APPPS1 + GFP) prepared according to the present invention by immunohistochemistry, and FIG. 9 is the present invention. LC3, p62 expression levels from the dorsal striatum in brain tissue isolated from the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) prepared according to the immunohistochemical staining method were measured.
도 8 및 도 9에 나타난 바와 같이, ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)은 치매 동물모델(APPPS1+GFP)에 비해 LC3는 증가하고 p62는 감소함을 확인하였다. 즉, 치매 동물모델에서 ZBTB16의 발현을 억제하였을 때 자가포식 현상이 증가하였음을 확인하였다.As shown in FIGS. 8 and 9 , it was confirmed that the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) increased LC3 and decreased p62 compared to the dementia animal model (APPPS1+GFP). That is, it was confirmed that autophagy increased when the expression of ZBTB16 was suppressed in an animal model of dementia.
즉, 베타 아밀로이드 축적에 의해 치매가 발병하면 뇌조직 내에서 자가포식 작용이 감소하여 질환 단백질이 제거되지 못하고 쌓여가므로, 질환의 증상이 점차 심각해짐을 유추할 수 있다. 여기에 shRNA를 처리하여 ZBTB16 단백질의 발현을 억제하면 뇌조직 내에서 자가포식 작용이 증가 및 회복되어 질환 단백질을 제거하므로 퇴행성 뇌질환의 정신 및 행동 장애가 현저히 개선, 예방 또는 치료되는 것으로 여겨진다.That is, when dementia occurs due to the accumulation of beta-amyloid, autophagy is reduced in brain tissue, and disease proteins cannot be removed but accumulate, so it can be inferred that the symptoms of the disease become progressively more serious. When shRNA is treated to suppress the expression of ZBTB16 protein, autophagy is increased and restored in brain tissue to remove disease proteins, so it is believed that mental and behavioral disorders of degenerative brain diseases are significantly improved, prevented or treated.
실험예 8. ZBTB16 단백질 억제에 따른 자가포식 현상 분석-12개월령Experimental Example 8. Analysis of autophagy according to ZBTB16 protein inhibition - 12 months of age
8-1. 12개월령(12mo) 동물실험8-1. 12-month-old (12mo) animal test
실험예 1로부터 제조된 대조군(WT+GFP)과 치매 동물모델(APPPS1+GFP) 및 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)을 제조하고, 1개월 대신에 7개월간 사육한 것을 제외하고는 실험예 2와 모두 동일하게 하여 12개월령(12mo) 대조군(WT+GFP)과 12개월령 치매 동물모델(APPPS1+GFP) 및 12개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)을 제조하였다.Experimental example except that the control (WT + GFP) and dementia animal model (APPPS1 + GFP) and ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) prepared from Experimental Example 1 were prepared and bred for 7 months instead of 1 month In the same manner as in 2, a 12-month-old (12mo) control group (WT+GFP), a 12-month-old dementia animal model (APPPS1+GFP), and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) were prepared.
8-2. 면역조직염색법8-2. immunohistochemistry
치매 발병 과정에 수반되는 자가포식(autophagy) 현상을 확인하기 위하여, 자가포식 마커 단백질인 LC3과 자가포식 기질 단백질은 p62를 확인하고자 하였다.In order to confirm the autophagy phenomenon involved in the onset of dementia, LC3, an autophagy marker protein, and p62, an autophagy matrix protein, were identified.
8-1에 따라 제조된 12 개월령(12mo) 대조군(WT+GFP), 치매 동물모델(APPPS1+GFP), ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)을 준비하고, 각각 마취하고 관류한 후 뇌를 적출하여 4% PFA로 뇌조직을 고정하였다. 상기 뇌조직에서 선조체와 측좌핵 부분의 조직을 얻어 동결건조하여, 뇌조직 동결절편을 준비하였다.Prepare a 12-month-old (12mo) control group (WT+GFP), an animal model of dementia (APPPS1+GFP), and an animal model of ZBTB16 knockdown dementia (APPPS1+shZbtb16) prepared according to 8-1, anesthetize and perfuse the brain, respectively After extraction, the brain tissue was fixed with 4% PFA. The tissues of the striatum and lateral nucleus pulposus were obtained from the brain tissue and freeze-dried to prepare a frozen section of the brain tissue.
상기 절편에 1차 항체(anti-LC3;Novus Biologicals;NB100-2220, anti-p62 ;Abcam;ab91526) 또는 마우스 항-ZBTB16 항체(D-9; sc-28319, Santa Cruz Biotechnology, Santa Cruz, CA)를 처리하고, 2차 항체(Goat anti-Rabbit IgG;Alexa Fluor 488, Goat anti-Mouse IgG;Alexa Fluor 594) 또는 바이오티닐화된 항-마우스 이차 항체를 처리하였다. 다시 PBS로 3회 세척하고 세포 내 핵 염색을 위해 DAPI 염색 후 공초점 현미경을 통해 p62의 발현 정도를 관찰하였다. 결과는 도 11 및 도 13에 나타내었다. 면역조직염색법은 3회 이상의 독립적인 실험으로부터 대표적인 것을 도식화하였다.Primary antibody (anti-LC3; Novus Biologicals; NB100-2220, anti-p62; Abcam; ab91526) or mouse anti-ZBTB16 antibody (D-9; sc-28319, Santa Cruz Biotechnology, Santa Cruz, CA) in the section was treated with secondary antibody (Goat anti-Rabbit IgG; Alexa Fluor 488, Goat anti-Mouse IgG; Alexa Fluor 594) or biotinylated anti-mouse secondary antibody. After washing again with PBS three times and DAPI staining for intracellular nuclear staining, the expression level of p62 was observed through a confocal microscope. The results are shown in FIGS. 11 and 13 . The immunohistostaining method was schematically representative from three or more independent experiments.
도 11은 12 개월령 대조군(WT+GFP(12mo))에서 분리된 뇌조직에서 배측선조체(dorsal striatum)로부터 ZBTB16, p62 발현량을 면역조직염색법으로 측정한 결과이고, 도 12는 12 개월령 치매 동물모델(APP/PS1+GFP(12mo))에서 분리된 뇌조직에서 배측선조체(dorsal striatum)로부터 ZBTB16, p62 발현량을 면역조직염색법으로 측정한 결과이며, 도 13은 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에서 분리된 뇌조직에서 배측선조체(dorsal striatum)으로부터 ZBTB16, p62 발현량을 면역조직염색법으로 측정한 결과이다.11 is a result of measuring the expression levels of ZBTB16 and p62 from the dorsal striatum in brain tissue isolated from a 12-month-old control group (WT+GFP (12mo)) by immunohistochemistry, and FIG. 12 is a 12-month-old dementia animal model. ZBTB16, p62 expression levels from the dorsal striatum in brain tissue isolated from (APP/PS1+GFP(12mo)) were measured by immunohistochemistry, and FIG. 13 is a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1). + shZbtb16), ZBTB16, p62 expression levels from the dorsal striatum in the brain tissue isolated from the immunohistochemical staining method.
도 11 및 도 12에 나타난 바와 같이, 12 개월령 대조군(WT+GFP(12mo))에 비해 12 개월령 치매 동물모델(APP/PS1+GFP(12mo))은 ZBTB16 발현이 증가하고, 자가포식 기질 단백질인 p62의 발현량도 증가하는 것을 확인하였다. 즉 치매가 발병하면 ZBTB16의 발현량이 증가하고 자가포식 현상은 감소하는 것을 알 수 있다.11 and 12, compared to the 12-month-old control group (WT+GFP (12mo)), the 12-month-old dementia animal model (APP/PS1+GFP (12mo)) increased ZBTB16 expression, and the autophagy matrix protein It was confirmed that the expression level of p62 also increased. That is, it can be seen that when dementia develops, the expression level of ZBTB16 increases and autophagy decreases.
다음, 도 13에 나타난 바와 같이, ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)은 치매 동물모델(APPPS1+GFP)에 비해 ZBTB16의 발현량이 감소하였고 p62가 정상수준으로 회복되는 것을 확인하였다. 즉, 치매 동물모델에서 ZBTB16의 발현을 억제하였을 때 자가포식 현상이 증가하였음을 확인하였다.Next, as shown in FIG. 13 , it was confirmed that the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) decreased the expression level of ZBTB16 compared to the dementia animal model (APPPS1+GFP), and the p62 was restored to a normal level. That is, it was confirmed that autophagy increased when the expression of ZBTB16 was suppressed in an animal model of dementia.
즉, 베타 아밀로이드 축적에 의해 치매가 발병하면 뇌조직 내에서 자가포식 작용이 감소하여 질환 단백질이 제거되지 못하고 쌓여가므로, 질환의 증상이 점차 심각해짐을 유추할 수 있다. 여기에 shRNA를 처리하여 ZBTB16 단백질의 발현을 억제하면 뇌조직 내에서 자가포식 작용이 증가 및 회복되어 질환 단백질을 제거하므로 퇴행성 뇌질환의 정신 및 행동 장애가 현저히 개선, 예방 또는 치료되는 것으로 여겨진다.That is, when dementia occurs due to the accumulation of beta-amyloid, autophagy is reduced in brain tissue, and disease proteins cannot be removed but accumulate, so it can be inferred that the symptoms of the disease become progressively more serious. When shRNA is treated to suppress the expression of ZBTB16 protein, autophagy is increased and restored in brain tissue to remove disease proteins, so it is believed that mental and behavioral disorders of degenerative brain diseases are significantly improved, prevented or treated.
실험예 9. ZBTB16 녹다운이 치매 동물모델의 사회성에 미치는 영향 조사-12개월령Experimental Example 9. Study on the effect of ZBTB16 knockdown on sociality in an animal model of dementia - 12 months of age
9-1. 3CT(3-chamber test) 행동실험9-1. 3CT (3-chamber test) behavioral experiment
상기 실험예 8-1의 방법으로 제조한 12 개월령 정상 동물모델(대조군(WT+GFP))과 12 개월령 치매 동물모델(APPPS1+GFP) 및 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)이 치매에 따른 음성증상을 나타내는지 확인하기 위하여 생쥐의 사회성을 확인하는 행동 실험 중 가장 널리 사용되는 3CT(3-chamber test) 행동실험을 수행하였다. 12-month-old normal animal model (control group (WT+GFP)) and 12-month-old dementia animal model (APPPS1+GFP) and 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) prepared by the method of Experimental Example 8-1 were dementia 3CT (3-chamber test), which is the most widely used behavioral experiment to check the sociality of mice, was performed to confirm whether they exhibit negative symptoms according to
구체적으로, 투명한 벽을 통해 분리된 연속된 3개의 방 중 가운데 방에 측정 동물모델(12 개월령, male)을 EthoVision XT 11.5(Noldus, Netherlands 제조)에 위치시키고, 양쪽 방 중 한쪽 방에 살아있는 생쥐(S1)를, 반대편 방에는 무생물인 물체(O)를 놓아두었으며, 또는 낯익은 생쥐(S1)와 새로운 생쥐(S2)를 놓아두었다. 양쪽 방 중에서 오래 머문 자리일수록 붉은색으로 표시하였다. 테스트는 10분간 수행하였고, 대조군으로는 실험예 8-1로부터 제조된 12 개월령 대조군(WT+GFP)을 사용하였다.Specifically, a measurement animal model (12 months old, male) was placed in the EthoVision XT 11.5 (manufactured by Noldus, Netherlands) in the middle of three consecutive rooms separated by a transparent wall, and live mice ( S1), an inanimate object (O) was placed in the opposite room, or a familiar mouse (S1) and a new mouse (S2) were placed. Among the two rooms, the longer you stayed, the more red. The test was performed for 10 minutes, and the 12-month-old control group (WT+GFP) prepared in Experimental Example 8-1 was used as a control.
실험의 통계분석을 위하여 대응표본 t-검정(paired t-test)을 사용하여 그룹 간 평균수치 유의차를 확인하였으며, 0.05보다 낮은 p 값을 가질 경우에는 *, 0.01보다 낮은 p 값을 가질 경우에는 **, 0.001보다 낮은 p 값을 가질 경우에는 ***로, 유의적 차이가 없는 경우 N.S로 유의적 차이를 표기하였다. 에러바는 S.E.M을 나타낸다.For statistical analysis of the experiment, the significant difference in mean values between groups was confirmed using the paired t-test. When the p value was lower than 0.05, *, and when the p value was lower than 0.01, **, a significant difference was indicated by *** when it had a p value lower than 0.001, and by N.S when there was no significant difference. Error bars indicate S.E.M.
도 14는 본 발명에 따라 제조된 12 개월령 치매 동물모델(APPPS1+GFP), 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 12 개월령 대조군(WT+GFP)의 3-chamber test(3CT) 결과를 나타낸 도면으로, 도 14a는 본 발명에 따라 제조된 12 개월령 치매 동물모델(APPPS1+GFP), 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 12 개월령 대조군(WT+GFP)의 무생물인 물체(0)와 살아있는 생쥐(S1)에 대한 관심 정도(3CT:sociability)를 나타낸 도면이고, 도 14b는 본 발명에 따라 제조된 12 개월령 치매 동물모델(APPPS1+GFP), 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16) 및 12 개월령 대조군(WT+GFP)의 낯익은 생쥐(S1)와 새로운 생쥐(S2)에 대한 관심 정도(3CT:novelty)를 나타낸 도면이다.14 is a 12-month-old dementia animal model (APPPS1 + GFP) prepared according to the present invention, a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1 + shZbtb16) and a 12-month-old control group (WT + GFP) 3-chamber test (3CT) results 14A is an inanimate object of a 12-month-old dementia animal model (APPPS1+GFP), a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and a 12-month-old control group (WT+GFP) prepared according to the present invention. (0) and a living mouse (S1) is a view showing the degree of interest (3CT:sociability), Figure 14b is a 12-month-old dementia animal model (APPPS1 + GFP) prepared according to the present invention, a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) and a 12-month-old control group (WT+GFP) show the degree of interest (3CT: novelty) in familiar mice (S1) and new mice (S2).
도 14a에 나타난 바와 같이, 무생물인 물체(0)보다 살아있는 생쥐(S1)에게 더 높은 관심을 보이는 현상은 12 개월령 치매 동물모델(APPPS1+GFP)에서도 정상적으로 나타나는 것을 확인하였다. As shown in Figure 14a, the phenomenon showing a higher interest in the living mouse (S1) than the inanimate object (0) was confirmed to appear normally in the 12-month-old dementia animal model (APPPS1 + GFP).
그러나 도 14b에서, 대조군(WT+GFP)는 낯익은 생쥐(S1)보다 새로운 생쥐(S2)에 더 많은 시간과 상호작용을 나타내는 반면, 치매 동물모델(APPPS1+GFP)은 낯익은 생쥐(S1)와 새로운 생쥐(S2)에 대한 관심의 정도가 비슷함을 확인하였다(사회성 저하;G③). 이에 반해 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)은 대조군과 유사하게 낯익은 생쥐(S1)보다 새로운 생쥐(S2)에 더 많은 시간과 상호작용을 나타냄을 확인하였다. 즉 ZBTB16 발현 억제를 통해 치매에 의한 사회성 저하 증상을 정상 수준으로 회복시킬 수 있다는 것을 확인하였다. However, in Figure 14b, the control group (WT+GFP) showed more time and interaction in the new mouse (S2) than the familiar mouse (S1), whereas the dementia animal model (APPPS1+GFP) showed the familiar mouse (S1). It was confirmed that the degree of interest in the new mouse (S2) was similar to that of the new mouse (S2) (decreased sociality; G③). On the other hand, it was confirmed that the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) exhibited more time and interaction in the new mouse (S2) than the familiar mouse (S1), similar to the control group. That is, it was confirmed that the symptoms of social deterioration caused by dementia could be restored to a normal level by suppressing the expression of ZBTB16.
상술한 결과를 통해 베타 아밀로이드(Aβ, β amyloid)를 과생성하도록 유전적으로 변이된 치매 동물모델(APPPS1+GFP)은 새로운 생쥐에 대한 호기심과 친밀도가 감소하고 사회적 감성이 결여되는 증상을 나타냄을 알 수 있다.From the above results, it was found that the animal model of dementia (APPPS1+GFP) genetically mutated to overproduce beta amyloid (Aβ, β amyloid) exhibits symptoms of decreased curiosity and intimacy with new mice and lack of social sensitivity. can
상기 치매 동물모델의 뇌 내에 특이적으로 ZBTB16 단백질 발현을 억제할 경우, 새로운 생쥐에 대한 호기심과 친밀도가 증가하고 사회적 감성이 결여되는 증상이 정상수준을 회복되는 것을 확인하였다. 즉 ZBTB16를 통해 인지기능 증상이 없는 치매 위험군(6개월령 생쥐)에서 뿐만 아니라 인지기능 증상이 나타난 치매군(12개월령 생쥐) 모두에서 치매 예방, 치료, 회복 효과를 나타내는 것을 알 수 있다.When specifically suppressing the expression of ZBTB16 protein in the brain of the animal model of dementia, it was confirmed that curiosity and intimacy with new mice increased, and symptoms of lack of social emotion were restored to normal levels. In other words, it can be seen that ZBTB16 exhibits dementia prevention, treatment, and recovery effects not only in the dementia risk group without cognitive symptoms (6-month-old mice) but also in the dementia group with cognitive symptoms (12-month-old mice).
실험예 10. ZBTB16 녹다운이 치매 동물모델의 기억력 및 인지능력에 미치는 영향 조사-12개월령Experimental Example 10. Study on the effect of ZBTB16 knockdown on memory and cognitive ability in an animal model of dementia - 12 months of age
실험예 8-1로부터 제조된 12 개월령 대조군(WT+GFP)과 12 개월령 치매 동물모델(APPPS1+GFP) 및 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)에 대해 두 종류의 인지기능 검사(Y-maze test와 novel object recognition test)를 실시하였다.Two types of cognitive function tests (Y -maze test and novel object recognition test) were performed.
10-1. Y-maze 시험(YMT)10-1. Y-maze test (YMT)
단기 기억 형태이며 순차적인 행동능력을 평가하기 위한 방법으로 Y 미로 실험을 실시하였다. 실험동물들을 120° 각도로 세 갈래로 가지가 뻗은 Y-maze(42 × 3 × 12 cm)의 가지 끝에 두고 8분간 자유롭게 움직이도록 하고 그 움직임을 Ethovision system(Noldus IT b.v., Netherlands)을 이용하여 컴퓨터로 관찰하였다. 각 가지를 A, B, C로 정한 후 네 발이 모두 한 쪽 가지에 완전히 들어가야 1회 출입횟수로 인정하였다. 세 개의 서로 다른 가지에 차례로 들어간 경우를 자발적 변경 행동력(spontaneous alternation behavior)으로 규정하였다. Alternation score(%)는 다음 수식으로 계산하였다. 실험동물로는 실험예 8-1의 방법으로 제조한 12 개월령 정상 동물모델(대조군(WT+GFP))과 12 개월령 치매 동물모델(APPPS1+GFP) 및 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)을 사용하였고, 이의 결과를 비교하였다.The Y-maze experiment was conducted as a method to evaluate the short-term memory type and sequential behavioral ability. Experimental animals were placed at the tip of the branch of the Y-maze (42 × 3 × 12 cm) branched into three branches at a 120° angle and allowed to move freely for 8 minutes, and the movement was monitored using a computerized Ethovision system (Noldus IT b.v., Netherlands). was observed with After each branch was designated as A, B, and C, all four feet must completely enter one branch to be counted as the number of entry and exit. The case of entering three different branches one after another was defined as spontaneous alternation behavior. Alternation score (%) was calculated by the following formula. As experimental animals, a 12-month-old normal animal model (control group (WT+GFP)) prepared by the method of Experimental Example 8-1, a 12-month-old dementia animal model (APPPS1+GFP), and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) ) was used, and the results were compared.
Alternation(%)=[(alternation 횟수)/(총 출입횟수-2)] × 100 Alternation(%)=[(number of alternations)/(total number of accesses-2)] × 100
10-2. 새로운 사물 탐색 시험(novel object recognition test, NOR)10-2. novel object recognition test (NOR)
신물질탐색 시험은 기존 방법을 변형한 것으로 적응기, 탐색기 및 신물질 인식기로 이루어져 있다. 이틀간 10분씩 생쥐를 개방장에 놓고 적응을 시킨 후, 두 개의 동일한 물체를 일정한 간격을 설치하였다. 실험동물을 3분간 자유롭게 탐색하도록 하면서 Ethovision으로 각 물체에서 머무르는 시간을 측정하였으며 이후 케이지에 30분간 돌려놓았다. 그리고 설치한 물체 중 하나를 신물질로 대체하고 실험동물의 움직임을 3분 동안 측정하였다. 물체의 선호도는 전체 탐색한 시간 중에서 각 물체에 머무르는 시간을 백분율로 환산하였다.The new substance discovery test is a modification of the existing method and consists of an adaptor, a searcher, and a new substance recognizer. After acclimatization by placing the mice in an open field for 10 minutes for two days, two identical objects were installed at regular intervals. The time spent in each object was measured by Ethovision while the experimental animals were allowed to freely explore for 3 minutes, and then returned to the cage for 30 minutes. Then, one of the installed objects was replaced with a new material and the movement of the experimental animal was measured for 3 minutes. The object preference was converted into a percentage of the time spent in each object among the total search time.
실험동물로는 실험예 8-1의 방법으로 제조한 12 개월령 정상 동물모델(대조군(WT+GFP))과 12 개월령 치매 동물모델(APPPS1+GFP) 및 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)을 사용하였고, 이의 결과를 비교하였다.As experimental animals, a 12-month-old normal animal model (control group (WT+GFP)) prepared by the method of Experimental Example 8-1, a 12-month-old dementia animal model (APPPS1+GFP), and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) ) was used, and the results were compared.
실험의 통계분석을 위하여 대응표본 t-검정(paired t-test)을 사용하여 그룹 간 평균수치 유의차를 확인하였으며, 0.05보다 낮은 p 값을 가질 경우에는 *, 0.01보다 낮은 p 값을 가질 경우에는 **, 0.001보다 낮은 p 값을 가질 경우에는 ***로, 유의적 차이가 없는 경우 N.S로 유의적 차이를 표기하였다. 에러바는 S.E.M을 나타낸다.For statistical analysis of the experiment, the significant difference in mean values between groups was confirmed using the paired t-test. When the p value was lower than 0.05, *, and when the p value was lower than 0.01, **, a significant difference was indicated by *** when it had a p value lower than 0.001, and by N.S when there was no significant difference. Error bars indicate S.E.M.
도 15는 본 발명에 따라 제조된 12 개월령 정상 동물모델(대조군(WT+GFP))과 12 개월령 치매 동물모델(APPPS1+GFP) 및 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)의 Y-maze 시험(YMT) 결과를 나타낸 그래프(좌측)와 신물질탐색 시험(novel object recognition test, NOR) 결과를 나타낸 그래프(우측)이다.15 is a Y-maze of a 12-month-old normal animal model (control group (WT+GFP)) and a 12-month-old dementia animal model (APPPS1+GFP) and a 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) prepared according to the present invention. A graph showing the test (YMT) results (left) and a graph showing the novel object recognition test (NOR) results (right).
도 15에 나타낸 바와 같이, 정상 동물모델(대조군(WT+GFP))보다 12 개월령 치매 동물모델(APPPS1+GFP)의 변경 행동력이 유의하게 감소하는 것을 확인하였다. 즉 치매에 의해 기억력 및 주의 집중력이 현저히 감소하는 것을 알 수 있다.As shown in FIG. 15 , it was confirmed that the altered behavioral power of the 12-month-old dementia animal model (APPPS1+GFP) was significantly reduced than that of the normal animal model (control group (WT+GFP)). That is, it can be seen that the memory and attention and concentration are significantly reduced due to dementia.
반면, ZBTB16의 발현을 억제한 12 개월령 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)의 경우, 치매 동물모델(APPPS1+GFP)보다 변경 행동력이 유의적으로 증가하는 것을 확인할 수 있었다. 즉, ZBTB16 단백질 발현을 억제하면, 치매에 의해 저하된 기억력과 주의 집중력이 정상 수준으로 회복되는 것을 알 수 있다.On the other hand, in the case of the 12-month-old ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) in which the expression of ZBTB16 was suppressed, it was confirmed that the altered behavior was significantly increased compared to the dementia animal model (APPPS1+GFP). That is, when ZBTB16 protein expression is suppressed, it can be seen that the memory and attention and concentration deteriorated by dementia are restored to normal levels.
신물질탐색 시험은 인지능력과 기억력을 평가할 수 있다. 정상 동물모델(WT+GFP)은 신물질에 머무는 시간이 익숙한 물질에 머무는 시간에 비하여 유의성 있게 많은 반면, 치매 동물모델(APPPS1+GFP)은 두 물질에 머무는 시간에 유의적 차이가 없었다.The new substance discovery test can evaluate cognitive ability and memory. In the normal animal model (WT+GFP), the retention time for the new substance was significantly longer than that for the familiar substance, whereas there was no significant difference in the retention time for the two substances in the dementia animal model (APPPS1+GFP).
이에 반해 ZBTB16 녹다운 치매 동물모델(APPPS1+shZbtb16)은 대조군(WT+GFP)와 비슷한 수준으로, 신물질에 머무는 시간이 익숙한 물질에 머무는 시간에 비하여 유의성 있게 증가하는 것을 확인하였다.On the other hand, the ZBTB16 knockdown dementia animal model (APPPS1+shZbtb16) was similar to the control group (WT+GFP), and it was confirmed that the retention time in the new substance significantly increased compared to the retention time in the familiar substance.
즉, 베타 아밀로이드 축적에 의해 기억력 인지기능이 크게 저하된 치매 상태일 때, ZBTB16 단백질 발현을 억제하면, 치매로 인한 인지능력, 기억력 및 주의 집중력을 정상수준과 유사한 정도로 현저히 회복시킨다는 것을 알 수 있다.In other words, it can be seen that, in a dementia state in which memory and cognitive function are significantly reduced due to beta-amyloid accumulation, suppression of ZBTB16 protein expression significantly restores cognitive ability, memory, and attention concentration due to dementia to a level similar to normal levels.
Claims (21)
- ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 바이오마커 조성물.A biomarker composition for diagnosing degenerative brain disease, comprising the Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same as an active ingredient.
- 제1항에 있어서,According to claim 1,상기 ZBTB16 단백질은 서열번호 1의 아미노산 서열로 표시되는 것을 특징으로 하는 퇴행성 뇌질환 진단용 바이오마커 조성물.The ZBTB16 protein is a biomarker composition for diagnosing degenerative brain disease, characterized in that it is represented by the amino acid sequence of SEQ ID NO: 1.
- ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정할 수 있는 제제를 유효성분으로 포함하는 퇴행성 뇌질환 진단용 조성물.A composition for diagnosing degenerative brain disease, comprising, as an active ingredient, an agent capable of measuring the expression level of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same.
- 제3항에 있어서, 4. The method of claim 3,상기 유전자 발현수준을 측정하는 제제는 ZBTB16 단백질을 코딩하는 유전자에 특이적으로 결합하는 프로브, 프라이머 및 안티센스 올리고뉴클레오타이드로 이루어진 군으로부터 선택되는 어느 하나 이상이고,The agent for measuring the gene expression level is any one or more selected from the group consisting of probes, primers and antisense oligonucleotides that specifically bind to the gene encoding the ZBTB16 protein,상기 단백질 발현수준을 측정하는 제제는 상기 ZBTB16 단백질에 특이적으로 결합하는 항체, 펩타이드, 앱타머 및 화합물로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 퇴행성 뇌질환 진단용 조성물.The agent for measuring the protein expression level is a composition for diagnosing degenerative brain disease, characterized in that at least one selected from the group consisting of an antibody, peptide, aptamer, and compound that specifically binds to the ZBTB16 protein.
- 제3항에 있어서,4. The method of claim 3,상기 ZBTB16 단백질은 서열번호 1의 아미노산 서열로 표시되는 것을 특징으로 하는 퇴행성 뇌질환 진단용 조성물.The ZBTB16 protein is a composition for diagnosing degenerative brain disease, characterized in that it is represented by the amino acid sequence of SEQ ID NO: 1.
- 제3항에 따른 조성물을 포함하는 퇴행성 뇌질환 진단용 키트.A kit for diagnosing degenerative brain disease comprising the composition according to claim 3 .
- (1) 피검체로부터 분리된 생물학적 시료로부터 ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하는 단계;(1) measuring the expression level of a Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein or a gene encoding the same from a biological sample isolated from a subject;(2) 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 정상 대조군의 시료와 비교하는 단계; 및(2) comparing the expression level of the ZBTB16 protein or a gene encoding it with a sample of a normal control; and(3) 상기 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준이 정상 대조군의 시료보다 높을 경우 퇴행성 뇌질환이라고 판단하는 단계;를 포함하는 퇴행성 뇌질환 진단에 필요한 정보를 제공하는 방법.(3) determining that the expression level of the ZBTB16 protein or a gene encoding the ZBTB16 protein is higher than that of a normal control sample; determining that it is a degenerative brain disease;
- 제7항에 있어서,8. The method of claim 7,상기 피검체의 생물학적 시료로부터 LC3 및 p62 단백질 발현량을 측정하는 단계를 더 포함하는 것을 특징으로 하는 퇴행성 뇌질환 진단에 필요한 정보를 제공하는 방법.Method for providing information necessary for diagnosing degenerative brain disease, characterized in that it further comprises the step of measuring the expression levels of LC3 and p62 proteins from the biological sample of the subject.
- a) 퇴행성 뇌질환 동물 또는 환자에서 분리된 시료에 후보물질을 처리하는 단계;a) treating a candidate material in a sample isolated from an animal or patient with degenerative brain disease;b) 상기 후보물질 처리군에서 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현수준을 측정하는 단계; 및b) measuring the expression level of the ZBTB16 protein or a gene encoding the same in the candidate substance treatment group; andc) 상기 단계에서 측정된 ZBTB16 단백질 또는 이를 코딩하는 유전자의 발현 수준이 후보물질 비처리군(대조군)보다 낮아지면, 퇴행성 뇌질환 치료물질로 선정하는 단계;를 포함하는 퇴행성 뇌질환 치료물질의 스크리닝 방법.c) when the expression level of the ZBTB16 protein or the gene encoding it measured in the above step is lower than that of the non-treated group (control group) with the candidate substance, selecting a treatment material for degenerative brain disease; screening of a treatment material for degenerative brain disease Way.
- 제9항에 있어서, 10. The method of claim 9,상기 ZBTB16 단백질은 서열번호 1의 아미노산 서열로 표시되는 것을 특징으로 하는 퇴행성 뇌질환 치료물질의 스크리닝 방법.The ZBTB16 protein is a screening method of a degenerative brain disease treatment material, characterized in that represented by the amino acid sequence of SEQ ID NO: 1.
- 제9항에 있어서,10. The method of claim 9,상기 발현수준의 측정은 웨스턴 블랏, ELISA, 방사선면역분석, 방사선 면역 확산법, 오우크테로니 면역 확산법(Ouchterlony immunodiffusion), 로케트 면역전기영동(Rocket immunoelectrophoresis), 면역염색법, 면역침전 분석법, 보체 고정 분석법, FACS, 단백질칩, 중합효소반응(PCR), 정량적 중합효소반응(qPCR), 역전사 중합효소 반응(RT-PCR), 경쟁적 역전사 중합효소 반응(competitive RT-PCR), 정량적 실시간 중합효소반응(qRT-PCR), 실시간 역전사 중합효소반응(real time RT-PCR), RNase 보호 분석법, 노던 블롯 분석법(northern blot analysis) 및 DNA 칩 분석으로 이루어진 군으로부터 선택되는 어느 하나의 방법을 이용하여 측정하는 것을 특징으로 하는 퇴행성 뇌질환 치료물질의 스크리닝 방법.The expression level was measured by Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, polymerase reaction (PCR), quantitative polymerase reaction (qPCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (competitive RT-PCR), quantitative real-time polymerase reaction (qRT- PCR), real time reverse transcription polymerase reaction (real time RT-PCR), RNase protection assay, northern blot analysis, and measurement using any one method selected from the group consisting of DNA chip analysis, characterized in that A screening method for therapeutic substances for degenerative brain disease.
- 제9항에 있어서,10. The method of claim 9,상기 후보물질 처리군의 혈청에서 LC3 및 p62 단백질 발현량을 측정하는 단계를 더 포함하는 것을 특징으로 하는 퇴행성 뇌질환 치료물질의 스크리닝 방법.The screening method for a therapeutic substance for degenerative brain disease, characterized in that it further comprises the step of measuring the expression level of LC3 and p62 protein in the serum of the candidate substance-treated group.
- 제9항에 있어서,10. The method of claim 9,상기 LC3 단백질 발현량이 후보물질 비처리군(대조군)보다 증가하고, 상기 p62 단백질 발현량은 후보물질 비처리군(대조군)보다 감소한 경우, 상기 후보물질을 퇴행성 뇌질환 치료물질로 판단하는 단계를 더 포함하는 것을 특징으로 하는 퇴행성 뇌질환 치료물질의 스크리닝 방법.When the LC3 protein expression level increases than the candidate substance untreated group (control group), and the p62 protein expression level decreases than the candidate substance untreated group (control group), the step of determining the candidate substance as a degenerative brain disease treatment material is further A screening method for a therapeutic substance for degenerative brain disease, characterized in that it comprises.
- ZBTB16(Zinc finger and BTB domain-containing protein 16) 단백질의 발현 또는 활성을 억제할 수 있는 제제를 유효성분으로 포함하는 퇴행성 뇌질환 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating degenerative brain disease, comprising as an active ingredient a formulation capable of inhibiting the expression or activity of Zinc finger and BTB domain-containing protein 16 (ZBTB16) protein.
- 제14항에 있어서,15. The method of claim 14,상기 발현을 억제할 수 있는 제제는 ZBTB16 단백질을 코딩하는 유전자의 mRNA에 특이적으로 결합하는 siRNA(small interference RNA), shRNA(short hairpin RNA), miRNA(microRNA), 리보자임(ribozyme), DNAzyme, PNA(peptide nucleic acids) 및 안티센스 올리고뉴클레오타이드으로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것을 특징으로 하며, The agent capable of inhibiting the expression is siRNA (small interference RNA), shRNA (short hairpin RNA), miRNA (microRNA), ribozyme, DNAzyme, which specifically binds to the mRNA of the gene encoding the ZBTB16 protein. It is characterized in that it contains any one or more selected from the group consisting of peptide nucleic acids (PNA) and antisense oligonucleotides,상기 활성을 억제할 수 있는 제제는 ZBTB16 단백질에 특이적으로 결합할 수 있는 화합물, 펩티드, 앱타머, 단백질 및 항체로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 것을 특징으로 하는, 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물.The agent capable of inhibiting the activity is a compound capable of specifically binding to the ZBTB16 protein, a peptide, an aptamer, a protein and an antibody, characterized in that it comprises any one or more selected from the group consisting of, degenerative brain disease prevention or a therapeutic pharmaceutical composition.
- 제15항에 있어서,16. The method of claim 15,상기 shRNA는 재조합 아데노바이러스, 아데노-관련 바이러스(Adeno-associated viruses: AAV), 레트로바이러스, 렌티바이러스, 헤르페스 심플렉스 바이러스, 배시니아 바이러스, 리포좀 및 니오좀로 이루어진 군으로부터 선택되는 어느 하나의 전달체에 의해 전달되는 것을 특징으로 하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물.The shRNA is a recombinant adenovirus, adeno-associated viruses (AAV), retrovirus, lentivirus, herpes simplex virus, basinia virus, liposomes and niosomes in any one carrier selected from the group consisting of A pharmaceutical composition for preventing or treating degenerative brain disease, characterized in that it is delivered by
- 제15항에 있어서,16. The method of claim 15,상기 shRNA는 서열번호 2로 표시되는 염기서열을 표적서열로 하고, 서열번호 1로 표시되는 ZBTB16 단백질의 발현을 억제하는 것을 특징으로 하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물.The shRNA is a nucleotide sequence represented by SEQ ID NO: 2 as a target sequence, and a pharmaceutical composition for preventing or treating degenerative brain disease, characterized in that it suppresses the expression of the ZBTB16 protein represented by SEQ ID NO: 1.
- 제17항에 있어서,18. The method of claim 17,상기 shRNA는 서열번호 7 내지 10로 표시되는 염기서열 중에서 선택되는 어느 하나인 것을 특징으로 하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물.The shRNA is a pharmaceutical composition for preventing or treating degenerative brain disease, characterized in that any one selected from the nucleotide sequences represented by SEQ ID NOs: 7 to 10.서열번호 7 : TAGAGGTACGTCTTCTCTATCCTCCAGTCSEQ ID NO: 7: TAGAGGTACGTCTTCTCTATCCTCCAGTC서열번호 8 : TTGAGGCTGAACTTCTTGCCACAGCCATTSEQ ID NO: 8: TTGAGGCTGAACTTCTTGCCACAGCCATT서열번호 9 : GGTCATCCAGGTCCTCCGCCTTGGCTTGCSEQ ID NO: 9: GGTCATCCAGGTCCTCCGCCTTGGCTTGC서열번호 10 : CCACTCTCCTCGCTGGAATGCTTCGAGATSEQ ID NO: 10: CCACTCTCCTCGCTGGAATGCTTCGAGAT
- 제14항에 있어서,15. The method of claim 14,상기 퇴행성 뇌질환은 알츠하이머형 치매(Alzheimer-type dementia), 루이체 치매(Lewy body dementia), 전측두엽 치매(frontotemporal dementia), 뇌혈관 치매(cerebrovascular dementia), 파킨슨병(Parkinson's disease), 경도인지장애, 다운증후군(Down's syndrome) 및 헌팅톤병(Huntington's disease)로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물.The degenerative brain disease includes Alzheimer-type dementia, Lewy body dementia, frontotemporal dementia, cerebrovascular dementia, Parkinson's disease, mild cognitive impairment, Down's syndrome (Down's syndrome) and Huntington's disease (Huntington's disease), characterized in that any one or more selected from the group consisting of degenerative brain disease prevention or treatment pharmaceutical composition.
- 제14항에 있어서,15. The method of claim 14,상기 조성물은 퇴행성 뇌질환에서 유발되는 정신 및 행동장애(Behavioral and Psychological Symptoms of Dementia, BPSD)를 개선, 예방 또는 치료하는 것을 특징으로 하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물.The composition is a degenerative brain disease prevention or treatment pharmaceutical composition, characterized in that the improvement, prevention or treatment of mental and behavioral disorders (Behavioral and Psychological Symptoms of Dementia, BPSD) caused by degenerative brain disease.
- 제20항에 있어서,21. The method of claim 20,상기 정신 및 행동장애(Behavioral and Psychological Symptoms of Dementia, BPSD)는 망상(delusion), 환각(hallucination), 초조(agitation), 공격적 행동(aggression), 이상 행동(activity disturbances), 우울(depression), 불안(anxiety), 일중주기장애(diurnal rhythm disturbances), 기분의 들뜸(elation), 과민(irritability), 불안정(lability), 정서적 불안정성(affective lability), 자기조절 결손(defective self regulation), 비정상적인 반복행동(aberrant motor behavior), 불안과 공포(anxiety and phobias) 및 수면 장애로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 퇴행성 뇌질환 예방 또는 치료용 약학적 조성물.The Behavioral and Psychological Symptoms of Dementia (BPSD) include delusions, hallucinations, agitation, aggression, activity disturbances, depression, and anxiety. (anxiety), diurnal rhythm disturbances, elation, irritability, lability, affective lability, defective self regulation, abnormal repetitive behavior ( Aberrant motor behavior), anxiety and fear (anxiety and phobias) and a pharmaceutical composition for preventing or treating degenerative brain disease, characterized in that at least one selected from the group consisting of sleep disorders.
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