KR102078500B1 - Composition for diagnosing Alzheimer's disease - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing Alzheimer′s disease, including Dynamin 3 or adaptor related protein complex 3 subunit delta 1 (AP3D1) proteins, or genes encoding the same. When an expression level of the proteins of Dynamin 3 or AP3D1 or the genes encoding the same is low, it is possible to quickly and accurately diagnose that a person′s risk of developing Alzheimer′s disease is high, thus the composition can be advantageously used in a medical and pharmaceutical field.

Description

Composition for diagnosing Alzheimer's disease

The present invention provides a biomarker composition for diagnosing Alzheimer's disease, including Dynamin 3 or AP3D1 (adaptor related protein complex 3 subunit delta 1) protein or a gene encoding the same; A diagnostic composition for Alzheimer's disease comprising an agent measuring the protein or a gene encoding the protein; A method of providing information on the possibility of diagnosis or onset of Alzheimer's disease using the same; And it relates to a screening method of Alzheimer's disease treatment using the same.

Alzheimer's disease is a type of degenerative brain disease in which beta amyloid proteins and the like accumulate and cause cytotoxicity and neurons degenerate and lose cognitive ability. The cause of Alzheimer's disease is not yet fully understood, but associations such as beta amyloid protein, tau protein and ApoE protein have been reported. In the beta amyloid protein, amyloid beta precursor protein (APP) is cleaved by beta-secretase and gamma-secretase in the brain to form senile plaques. In addition, when the beta amyloid accumulates in the brain, neurofibrillary tangles are formed by inducing denaturation and accumulation of tau protein. Geriatric spots and nerve fiber bundles are characteristic lesions of Alzheimer's disease.

Accumulation of beta amyloid has been reported to cause the production of free radicals, destroy synapse formation of neurons and cause neuronal death. Therefore, many factors involved in the production and degradation of beta amyloid are expected to be involved in the induction and progression of Alzheimer's disease, but the detailed mechanisms that cause Alzheimer's disease are not well understood.

Heritability of Alzheimer's disease is more than 70%, and it will be possible to secure the effect of reducing prevalence when classifying high-risk groups through intensive risk prediction tests and managing them.

Also, a lipid-binding protein after which accounts for more than 95% of dementia patients ApoE, a major genetic risk factor for dementia that has stricken three homozygotes ApoEε2, ApoEε3, ApoEε4, human zygotes with the release of ApoEε4 type 2-3 Embryos and homozygotes are known to have a 5 times higher incidence of dementia (Reitz et al., 2014, Biochemical Pharmacology; Ba et al, 2016, Translational neurodegeneration).

Meanwhile, large-scale analysis of 74,046 people revealed CR1, BIN1, CD2AP, EPHA1, CLU, MS4A6A, PICALM, ABCA7, CD33, HLA-DRB5, PTK2B, SORL1, SLC24A4, DSG2, INPP5D, MEF2C, NME8, ZCWPW1, CELF The FERMT2 and CASS4 genes have been shown to be associated with late-onset Alzheimer's disease (Lambert et al., Nature Genetics, 2013). In addition, studies on the association between brain images and genes in normal and Alzheimer's patients (total 977) revealed that mutations in ABCA7, CLU, DSG2, EPHA1, FERMT2, PICALM, PTK2B, SORL1, and ZCWPW1 genes were associated with amyloid accumulation in the brain. Has been reported (Apostolova et al., JAMA Neurology, 2018).

However, the rate of explanation of known dementia-specific genes, such as APOE , is 25%, assuming 100% of heritability, and more than 75% of heritable missing heritability with no known genetic factors. The discovery of genetic factors that could explain this hidden heritability is expected to predict the risk of Alzheimer's disease.

Korean Patent Publication No. 10-2018-0130060

An object of the present invention is to provide a biomarker composition for diagnosing Alzheimer's disease comprising a protein of Dynamin 3 or AP3D1 (adaptor related protein complex 3 subunit delta 1) or a gene encoding the same.

Still another object of the present invention is to provide a composition for diagnosing Alzheimer's disease, which comprises an agent for measuring the expression level of a protein of Dynamin 3 or AP3D1 or a gene encoding the same.

Another object of the present invention is to measure the expression level of a protein or gene encoding the Dynamin 3 or AP3D1 from a biological sample isolated from the individual; And (b) comparing the measured expression level with a reference value obtained from a control sample, thereby providing an information providing method for diagnosing or developing Alzheimer's disease.

Still another object of the present invention is to provide a method for treating a cell in which a beta amyloid gene is expressed; (b) measuring the expression level of Dynamin 3 or AP3D1 protein or gene encoding the same in the cell of step (a); And (c) to provide a method for screening a therapeutic agent for Alzheimer's disease comprising the step of selecting a candidate substance of which the expression amount of step (b) is increased compared to the reference value.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing Alzheimer's disease comprising a protein of Dynamin 3 or AP3D1 (adaptor related protein complex 3 subunit delta 1) or a gene encoding the same.

The present invention also provides a composition for diagnosing Alzheimer's disease, which comprises an agent for measuring the expression level of a protein of Dynamin 3 or AP3D1 or a gene encoding the same.

In one embodiment of the present invention, the agent for measuring the expression level of the protein is an antibody, interacting protein, ligand, nanoparticles or aptamer that specifically binds to the protein or peptide fragment. It may be.

In one embodiment of the present invention, the agent for measuring the expression amount of the gene encoding the protein may be a primer or probe that binds to the mRNA of the gene.

The present invention also provides a diagnostic kit for Alzheimer's disease comprising the composition.

In one embodiment of the present invention, the kit may be a reverse transcription polymerase chain reaction (RT-PCR) kit or a DNA chip kit.

In addition, the present invention comprises the steps of (a) measuring the expression level of the protein or gene encoding the Dynamin 3 or AP3D1 from a biological sample isolated from the individual; And (b) comparing the measured expression level with a reference value obtained from a control sample, thereby providing an information providing method for diagnosis or onset of Alzheimer's disease.

In one embodiment of the present invention, the biological sample may be tissue, cells, blood, plasma, serum or urine.

In one embodiment of the present invention, if the expression level of the protein of Dynamin 3 or AP3D1 or gene encoding it is lower than the reference value may further comprise the step of determining that the high likelihood of the development of Alzheimer's disease.

In one embodiment of the present invention, the measurement is reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time quantitative RT-PCR, RNase protection method (RNase protection method), Northern blot ), DNA chip technology assay (DNA chip technology assay), ELISA (Enzyme-linked immunosorbent assay), Western blot (western blot), may be performed by immunoblot (immunoblot) or immunocytochemistry (immunocytochemistry).

In addition, the present invention comprises the steps of (a) treating the candidate substance to the cell in which the beta amyloid gene is expressed; (b) measuring the expression level of Dynamin 3 or AP3D1 protein or gene encoding the same in the cell of step (a); And (c) provides a method for screening a therapeutic agent for Alzheimer's disease comprising the step of selecting a candidate substance of which the expression level of step (b) is increased compared to the reference value.

The composition according to the present invention can be quickly and accurately diagnosed as having a high possibility of developing Alzheimer's disease when the expression level of the Dynamin 3 or AP3D1 protein or gene encoding the same is low, and can be used to screen for treatment of Alzheimer's disease. It can be useful for the treatment of Alzheimer's disease.

1 shows that GMR-GAL4 and UAS-beta-amyloid are located on the balancer chromosomes CyO (chromosome 2) and TM6B (chromosome 3), respectively.
FIG. 2 shows a Drosophila strain in which Dynamin 3 RNAi is connected after UAS , inserted into the Drosophila genome, and crossed with Drosophila where beta amyloid gene is expressed, thereby suppressing Dynamin 3 gene expression and expressing beta amyloid gene.
FIG. 3 shows the Drosophila strain in which AP3D1 RNAi is connected after UAS , inserted into the Drosophila genome, and crossed with Drosophila where beta amyloid gene is expressed, thereby suppressing the expression of AP3D1 gene and expressing beta amyloid gene.
Figure 4 shows the results of comparing the phenotype after the beta amyloid gene expression or Dynamin 3 gene expression (RNAi) respectively or simultaneously in Drosophila eyes ( GMR-GAL4 : normal control; GMR-GAL4 UAS-β -amyloid: beta-amyloid gene is expressed and Drosophila group that is not the expression of genes inhibiting Dynamin 3; GMR-GAL4 UAS-Dynamin3RNAi: the no beta-amyloid gene is expressed, the expression of genes inhibiting Dynamin 3 Drosophila group; and GMR-GAL4 UAS-β-amyloid UAS-Dynamin3RNAi : Drosophila group in which beta amyloid gene is expressed and expression of Dynamin 3 gene is suppressed.
FIG. 5 shows the results of comparing beta amyloid gene expression or AP3D1 gene expression inhibition (RNAi), respectively or simultaneously, in the fruit fly's eye ( GMR-GAL4 : normal control; GMR-GAL4 UAS-β-). amyloid : Drosophila group expressing beta amyloid gene and suppressed AP3D1 gene expression; GMR-GAL4 UAS-AP3D1RNAi : Drosophila group expressing AP3D1 gene without beta amyloid gene expression; and GMR-GAL4 UAS-β amyloid UAS-AP3D1RNAi : Drosophila group in which the beta amyloid gene is expressed and the expression of the AP3D1 gene is suppressed).

The present invention provides a biomarker composition for diagnosing Alzheimer's disease, including Dynamin 3 or AP3D1 (adaptor related protein complex 3 subunit delta 1) protein or a gene encoding the same.

The present invention also provides a composition for diagnosing Alzheimer's disease, which comprises an agent for measuring the expression level of a Dynamin 3 or AP3D1 protein or a gene encoding the same.

In one embodiment of the present invention, the agent for measuring the expression level of the Dynamin 3 or AP3D1 protein is an antibody, interacting protein, ligand, nanoparticles or aptamer specifically binding to the protein or peptide fragment (aptamer) may be, but is not limited thereto.

In the present invention, the agent for measuring the expression level of the protein means an antibody that specifically binds to the protein, and includes all polyclonal antibodies, monoclonal antibodies, recombinant antibodies, and combinations thereof.

Such antibodies are polymorphic antibodies, monoclonal antibodies, recombinant antibodies and complete forms having two full length light chains and two full length heavy chains, as well as functional fragments of antibody molecules such as Fab, F (ab '). ), F (ab ') 2 and Fv. Antibody production can be readily prepared using techniques well known in the art to which this invention pertains, and antibodies made and commercially available can be used.

The composition of the present invention further includes not only an agent for measuring the expression level of the protein, but also a label for quantitatively or qualitatively measuring the formation of the antigen-antibody complex, conventional tools used for immunological analysis, reagents, and the like. can do.

Labels that enable qualitatively or quantitatively measuring the formation of the antigen-antibody complex include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but are not necessarily limited thereto. no. Enzymes that can be used as detection labels include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, and glucoseoxy Multidase, Hexokinase and GDPase, RNase, Glucose Oxidase War Luciferase, Phosphofructokinase, Phosphoenolpyruvate Carboxylase, Aspartate Aminotransferase, Phosphopyruvate Decarboxylase, β- Ratamases and the like. Fluorescent materials include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine and the like. Ligands include biotin derivatives. Luminescent materials include acridinium ester, luciferin, luciferase and the like. Microparticles include colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ⁸ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, but are not limited thereto. no.

Such tools or reagents include, but are not limited to, suitable carriers, solubilizers, detergents, buffers, stabilizers, and the like. If the label is an enzyme, it may include a substrate and a reaction terminator capable of measuring enzyme activity. Carriers include soluble carriers, insoluble carriers, and soluble carriers include physiologically acceptable buffers known in the art, such as PBS, and insoluble carriers include polystyrene, polyethylene, polypropylene, polyesters, polyacrylonitrile, Fluorine resins, crosslinked dextran, polysaccharides, other papers, glass, metals, agarose and combinations thereof.

In one embodiment of the present invention, the agent for measuring the expression level of the gene encoding the Dynamin 3 or AP3D1 protein may be a primer or probe that binds to the mRNA of the gene, but is not limited thereto.

As used herein, the term "primer" refers to a base sequence having a short free 3-terminal hydroxyl group, capable of forming base pairs with complementary templates, and as a starting point for template strand copying. Refers to a short sequence that acts. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. PCR conditions, sense and antisense primer lengths may be appropriately selected according to techniques known in the art.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA that can specifically bind to a gene, and is labeled to confirm the presence and expression of a specific gene. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, or the like. Selection of appropriate probes and hybridization conditions may be appropriately selected according to techniques known in the art.

Primers or probes of the present invention can be synthesized chemically using phosphoramidite solid support synthesis or other well known methods. Such sequences can also be modified through various methods known in the art. Examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides and modifications between nucleotides, such as uncharged linkages such as methyl phosphonate, phosphoester, phosphoramidate, carbamate, etc. ) Or a charged linker such as phosphorothioate, phosphorodithioate and the like.

In addition, the present invention comprises the steps of (a) measuring the expression level of the Dynamin 3 or AP3D1 protein or gene encoding the same from a biological sample isolated from the individual; And (b) comparing the measured expression level with a reference value obtained from a control sample, thereby providing an information providing method for diagnosis or onset of Alzheimer's disease.

The term "individual" in the present invention means an individual suspected of Alzheimer's disease, and may preferably be a human.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

Example 1 Preparation of Drosophila Lines Expressing Beta Amyloid Gene

The present inventors used the UAS-GAL4 system to produce a fruit fly expressing a beta amyloid gene showing human Alzheimer's disease-like trait. The UAS-GAL4 system allows the transfer of yeast transcriptional control systems into fruit flies to control gene expression (Duffy, 2002). GAL4 is a transcription factor that binds to an upstream activation sequence ( UAS ) to express a gene linked to the UAS . In this regard, the present inventors connect a GAL4 gene with a promoter that regulates gene expression in a specific tissue and connect the target gene to be expressed in a specific tissue behind the UAS , so that the GAL4 gene is expressed in a specific tissue, and the expressed GAL4 is UAS. To bind to express the target gene.

In the present invention, in order to express the beta amyloid gene in the fruit and optic nerve of Drosophila, a Drosophila having GMR-GAL4 and UAS-beta-amyloid was produced by connecting a GMR4 gene with a glass multiple reporter (GMR) promoter that regulates expression in the eye. It was. In addition, in order to maintain the Drosophila line, GMR-GAL4 and UAS-beta-amyloid are located on the balancer chromosome (CyO, chromosome 2 and TM6B, chromosome 3) in which crossover between homologous chromosomes is suppressed, respectively. (FIG. 1).

Example 2. Dynamin 3  Production of Drosophila in which Gene Expression is Suppressed and Beta Amyloid Gene is Expressed

In order to determine whether the expression of the Dynamin 3 gene decreases the cytotoxicity of the beta amyloid, the inventors have produced a fruit fly in which the beta amyloid gene is expressed and at the same time the expression of the Dynamin 3 gene is suppressed. Inhibition of Dynamin 3 gene expression was used RNAi system that can suppress gene expression in Drosophila in vivo (Dietzl et al., 2007).

In the present invention, Dynamin 3 RNAi was connected to UAS followed by insertion into the Drosophila genome, and hybridized with Drosophila expressing the beta amyloid gene produced in Example 1, Dynamin 3 gene expression is suppressed, Drosophila that beta amyloid gene is expressed Was prepared (FIG. 2).

Example 3. AP3D1 (Adapter Related Protein Complex 3 Subunit Delta 1) Gene expression is suppressed, Drosophila making beta amyloid gene expression

In order to determine whether reduced expression of AP3D1 gene affects the cytotoxicity of beta amyloid, the present inventors have produced a fruit fly in which beta amyloid gene is expressed and at the same time expression of AP3D1 gene is suppressed. Inhibition of expression of the AP3D1 gene was performed using the RNAi system (Dietzl et al., 2007).

In the present invention, the AP3D1 RNAi was connected to UAS and inserted into the Drosophila genome, and hybridized with the Drosophila expressing the beta amyloid gene produced in Example 1, the expression of the AP3D1 gene was suppressed, and the Drosophila was expressed. (FIG. 3).

Example 4. Dynamin 3  or AP3D1  Cytotoxicity in Drosophila with Inhibitory Gene Expression and Beta-amyloid Gene Expression

The present inventors observed and photographed the fruit fly's eyes through an anatomical microscope to confirm whether beta amyloid-induced cytotoxicity when the expression of Dynamin 3 or AP3D1 gene is inhibited.

As a result, the beta amyloid gene is expressed When Dynamin 3 gene expression was suppressed in Drosophila ( GMR-GAL4 UAS-β-amyloid UAS-Dynamin3RNAi ), destruction of single -celled cells was significantly increased compared to the normal control group ( GMR-GAL4 ). Compared to Drosophila ( GMR-GAL4 UAS-β-amyloid ) without expression of Dynamin 3 gene or beta amyloid gene without expression of Dynamin 3 gene, DMR ( GMR-GAL4 UAS-Dynamin3RNAi ) It was confirmed that the destruction of the cells was further increased.

In addition, the beta amyloid gene is expressed When the expression of AP3D1 gene was suppressed in Drosophila ( GMR-GAL4 UAS-β-amyloid UAS-AP3D1RNAi ), the control group ( GMR-GAL4 ) also expressed the beta amyloid gene and suppressed the expression of AP3D1 gene ( GMR- GAL4 UAS-β-amyloid ) or beta amyloid gene is not expressed and the expression of AP3D1 gene was suppressed compared to the fruit flies ( GMR-GAL4 UAS-AP3D1RNAi ) it was confirmed that significantly increased the destruction of single cells.

Therefore, the present inventors have found that the cytotoxicity caused by beta amyloid increases when the expression of the Dynamin 3 or AP3D1 gene is reduced or inhibited by mutation, etc., and thus the possibility of developing Alzheimer's disease by the expression level of the gene or protein is increased. It was confirmed that can be predicted or diagnosed. In addition, screening for substances that can increase the expression level of the gene or protein, it was confirmed that the treatment of Alzheimer's disease may also be possible.

Claims (11)

delete delete delete delete delete delete delete delete delete delete (a) treating the candidate with an isolated cell in which the beta amyloid gene is expressed;
(b) measuring the expression level of Dynamin 3 or AP3D1 protein or gene encoding the same in the cell of step (a); And
(c) screening a candidate substance having an increased expression level in step (b) above a reference value.

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