WO2023171828A1 - Novel alzheimer model and construction method therefor - Google Patents

Abstract

Alzheimer's disease is the most common degenerative brain disease that causes dementia, and tau protein hyperphosphorylation is known to be the main cause. However, there are limitations in the screening and research of therapeutic candidate drugs due to the absence of cell models and animal models capable of completely implementing Alzheimer's disease. The present invention relates to a novel Alzheimer's model, and more particularly, to a neural cell model treated with a fusion protein in which a guide protein is coupled to tau protein. Since the cells can embody the characteristics of Alzheimer's disease as they are, it is expected that the cells can be used as a novel drug screening platform for the development of a therapeutic agent for Alzheimer's disease.

Description

Novel Alzheimer's model, and method of making the same

The present invention relates to a novel Alzheimer's model and method of producing the same.

Alzheimer's disease is the most common degenerative brain disease that causes dementia, and was first reported in 1907 by Dr. Alois Alzheimer, a German psychiatrist. Alzheimer's disease is characterized by a very slow onset and gradual progression. In the beginning, problems mainly occur with memory for recent events, but as the disease progresses, abnormalities in other cognitive functions such as language function and judgment occur, and eventually, all daily life functions are lost. Recently, with the rapid development of medicine, the average human lifespan has increased, and geriatric neurological diseases such as stroke, Alzheimer's disease (AD), and Parkinson's disease are increasing, but the most common among them is Alzheimer's disease.

The core mechanism of Alzheimer's disease appears to be the excessive production of a small protein called beta-amyloid, which is deposited in the brain and has a harmful effect on brain cells. In addition, studies have shown that hyperphosphorylation of tau protein, which plays an important role in maintaining the skeleton of brain cells, inflammatory response, and oxidative damage, also contribute to brain cell damage and affect the development of the disease. Neurogenic plaques (or senile plaques), a representative brain pathology, are associated with deposition of beta-amyloid protein, and neurofibrillary tangles are associated with tau protein hyperphosphorylation.

A fundamental treatment method for Alzheimer's disease has not yet been developed, and only drugs that can relieve symptoms and delay progression, such as acetylcholine enzyme inhibitors, are used in clinical settings. Accordingly, research is actively being attempted to find a fundamental treatment for Alzheimer's disease, but there are limitations in screening and research on candidate therapeutic drugs due to the absence of cell models and animal models that can fully represent Alzheimer's disease.

Accordingly, the inventors of the present invention completed the present invention after making continuous efforts to improve the above problems. The present invention relates to a novel Alzheimer's model, and more specifically, to a nerve cell model treated with a fusion protein in which a guide protein is bound to a tau protein. Since these cells can embody the characteristics of Alzheimer's disease, they are expected to be actively used as a new drug screening platform for developing treatments for Alzheimer's disease.

The present invention was conceived to solve the problems in the prior art as described above, and relates to a novel Alzheimer's model and a method of manufacturing the same.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

DETAILED DESCRIPTION OF THE INVENTION Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details, such as specific forms, compositions, and processes, are set forth in order to provide a thorough understanding of the invention. However, certain embodiments may be practiced without one or more of these specific details or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Accordingly, the phrases “in one embodiment” or “an embodiment” expressed in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable way in one or more embodiments.

Unless there is a special definition in the specification, all scientific and technical terms used in the specification have the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains.

In one embodiment of the present invention, neurodegenerative disease broadly includes all conditions in which nerve cells degenerate, but in general, it can be caused by factors other than distinct nervous system cells, such as cerebrovascular disorders, trauma, and metabolic abnormalities. It does not include what is there. Includes Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, polyglutamine disease including Huntington's disease, human prion disease, etc. In modern medicine, there is a lack of understanding of the clear cause of development or treatment methods.

In one embodiment of the present invention, Alzheimer's disease is a type of neurodegenerative disease and the most common degenerative brain disease that causes dementia, and was first reported in 1907 by Dr. Alois Alzheimer, a German psychiatrist. Alzheimer's disease is characterized by a very slow onset and gradual progression. In the beginning, problems mainly occur with memory for recent events, but as the disease progresses, abnormalities in other cognitive functions such as language function and judgment occur, and eventually, all daily life functions are lost. Recently, with the rapid development of medicine, the average human lifespan has increased, and geriatric neurological diseases such as stroke, Alzheimer's disease (AD), and Parkinson's disease are increasing, but the most common among them is Alzheimer's disease.

The core mechanism of Alzheimer's disease appears to be the excessive production of a small protein called beta-amyloid, which is deposited in the brain and has a harmful effect on brain cells. In addition, studies have shown that hyperphosphorylation of tau protein, which plays an important role in maintaining the skeleton of brain cells, inflammatory response, and oxidative damage, also contribute to brain cell damage and affect the development of the disease. Neurogenic plaques (or senile plaques), a representative brain pathology, are associated with deposition of beta-amyloid protein, and neurofibrillary tangles are associated with tau protein hyperphosphorylation.

A fundamental treatment method for Alzheimer's disease has not yet been developed, and only drugs that can relieve symptoms and delay progression, such as acetylcholine enzyme inhibitors, are used in clinical settings. Accordingly, research is actively being attempted to find a fundamental treatment for Alzheimer's disease, but there are limitations in screening and research on candidate therapeutic drugs due to the absence of cell models and animal models that can fully represent Alzheimer's disease.

In one embodiment of the present invention, Tau protein is a type of microtubule associated protein with a molecular weight of 50,000 to 70,000, and has the function of maintaining the stability of nerve cells by binding microtubules and preventing their collapse. It is a protein that performs. Abnormal aggregation of tau protein (tau) in brain nerve cells is a major feature of Alzheimer's disease (AD) and several other neurodegenerative diseases (collectively called tauopathies) (Biochimica et Biophysica Acta , 01 Jan 2005, 1739(2-3):331-354). In healthy nerves, tau stabilizes microtubules by promoting growth from axons and neuron polarization. When pathologically hyperphosphorylated, tau dissociates from microtubules and forms insoluble aggregates (Mol Neurodegeneration 4, 13 (2009). https://doi.org/10.1186/1750-1326-4- 13). Over the years, a structural framework for tau aggregation has been proposed, with evidence showing that insoluble filaments are formed from up to 10 soluble monomers and that these filaments are assembled into higher-order structures called neurofibrillary tangles (NFTs). It has been proposed. However, not much is still known about the pathophysiological significance of neurofibrillary tangles in tauopathies, the causes and molecular mechanisms that trigger this process, which suggests that reliable models exist to study tau aggregation under physiological conditions. It's because they don't do it. For this reason, the need for cell models or animal models that embody the characteristics of Alzheimer's disease caused by tau aggregation is increasing.

In one embodiment of the present invention, a guide peptide refers to a protein that binds to the N-terminal of a target protein and induces overexpression or inhibition of the function of the target protein. Therefore, in terms of a fusion protein in which a guide protein and a target protein are combined, the target protein is located at the C-terminal of the guide protein, and a linker, etc. may be located between the guide protein and the target protein. The orientation serves as an important factor in the function of the guide protein expected in the present invention. For example, as shown in Figure 1 of the present invention, HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), GST (glutathione Stransferase), and FD (T4 fibritin foldon) bound to the N-terminal of the target protein (Tau-4R) domain), DDT (D-dopachrome tautomerase), SCL (Streptococcus pyogenes collagen-like protein), CDA (human cytidine deaminase), NPM (nucleophosmin 1), HFQ (a host factor for bacteriophage Qβ RNA replication), HSP90α (heat- Shock protein 90 alpha isoform) can be used as a guide protein. The above target protein can be any protein involved in the progression of neurodegenerative disease, and is not limited thereto, but is preferably Tau, Aβ, or apoE, and more specifically, Tau, Aβ ₄₀ , Aβ ₄₂ , apoE2, and apoE3. , apoE4, or apoJ.

In one embodiment of the present invention, guided oligomerization (GO) is a method of preparing a set of oligomeric conformers of any pathogenic protein or peptide with a soluble and biologically active structure, which is bound to the guide protein. Includes dimers, trimers, tetramers, pentamers and hexamers of the target protein. This is called an oligomeric conformer set. The set of oligomeric conformers can be prepared using any expression system utilizing bacterial, yeast, insect, and mammalian cells.

In one embodiment of the present invention, the drug screening platform refers to a system that performs screening of candidate drugs expected to have therapeutic effects on target diseases using the guide oligomerization conformer set of the present invention.

In one embodiment of the present invention, a fusion protein is provided in which a guide protein is bound to a tau protein, the tau protein is Tau-4R, and the guide protein is HRS (hepatocyte growth factor-regulated tyrosine). kinase substrate), GST (glutathione Stransferase), FD (T4 fibritin foldon domain), DDT (D-dopachrome tautomerase), SCL (Streptococcus pyogenes collagen-like protein), CDA (human cytidine deaminase), NPM (nucleophosmin 1), HFQ (a host factor for bacteriophage Qβ RNA replication), and HSP90α (heat-shock protein 90 alpha isoform). The fusion protein is a dimer, trimer, or tetramer. A fusion protein having a hexameric, pentameric, or hexameric three-dimensional structure is provided.

In another embodiment of the present invention, a gene encoding the above fusion protein is provided.

In another embodiment of the present invention, a vector containing the above gene is provided.

In another embodiment of the present invention, a transformant transformed with the above vector is provided, wherein the transformant is a cell isolated from an individual, and the transformant is a human Provides a transformant that is an excluded individual.

In another embodiment of the present invention, (a) preparing a fusion protein in which a guide protein is bound to a tau protein; and, (b) processing the fusion protein into nerve cells; providing a method for producing an Alzheimer's disease cell model, wherein the tau protein is Tau-4R; , The guide proteins include HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), GST (glutathione Stransferase), FD (T4 fibritin foldon domain), DDT (D-dopachrome tautomerase), SCL (Streptococcus pyogenes collagen-like protein), and CDA. Alzheimer's disease cells, which are at least one selected from the group consisting of (human cytidine deaminase), NPM (nucleophosmin 1), HFQ (a host factor for bacteriophage Qβ RNA replication), and HSP90α (heat-shock protein 90 alpha isoform). A method for manufacturing a model is provided, wherein the fusion protein has a dimer, trimer, tetramer, pentamer, or hexamer three-dimensional structure.

In another embodiment of the present invention, (a) preparing a fusion protein in which a guide protein is bound to a tau protein; (b) processing the fusion protein into nerve cells; and, (c) treating the nerve cells with a candidate substance for treating Alzheimer's disease; wherein the tau protein is Tau-4R. provides a screening method, and the guide proteins include HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), GST (glutathione Stransferase), FD (T4 fibritin foldon domain), DDT (D-dopachrome tautomerase), and SCL (Streptococcus pyogenes collagen). -like protein), CDA (human cytidine deaminase), NPM (nucleophosmin 1), HFQ (a host factor for bacteriophage Qβ RNA replication), and HSP90α (heat-shock protein 90 alpha isoform). Provided is a method for screening a substance for the treatment of Alzheimer's disease, wherein the fusion protein has a dimer, trimer, tetramer, pentamer, or hexameric three-dimensional structure.

The present invention relates to a nerve cell model treated with a fusion protein in which a guide protein is bound to tau protein. Since these cells can embody the characteristics of Alzheimer's disease, they are expected to be actively used as a new drug screening platform for developing treatments for Alzheimer's disease.

Figure 1 is a schematic diagram of an oligomer set prepared by applying guided oligomerization to Tau-4R, according to an embodiment of the present invention. In Figure 1, Tau-4R (gray) is represented as a recombinant protein linked to a guide peptide at the N terminus (white) and a spacer peptide containing a thrombin cleavage site (black). As the oligomeric structure of the guide peptide and the fusion structure with Tau-4R, DA / DB is a dimer, TA / TB / TC are a trimer, QA is a tetramer, PA is a pentamer, HA / HB is a hexamer, and Mw ( kDa) is the size of the oligomer as a fusion structure.

Figure 2 shows the results confirmed by SDS-PAGE without incubation (A) or with incubation (B) of the oligomer set prepared in the present invention, according to an embodiment of the present invention. In Figure 2, M is the molecular weight standard, lane 1 is M-4R (monomeric Tau-4R), lane 2 is DA-4R, lane 3 is DB-4R, lane 4 is TA-4R, and lane 5 is is TB-4R, lane 6 is DA-4R, lane 7 is PA-4R, and lane 8 is HA-4R.

Figure 3 shows the results of a toxicity test on SH-SY5Y cells of the oligomer set prepared in the present invention, according to an embodiment of the present invention.

Figure 4 shows the results of observing the cell morphology after treating hippocampal neurons derived from mouse embryos with the oligomer set prepared in the present invention, according to an embodiment of the present invention.

Figure 5 shows the results of a cytotoxicity test after treating hippocampal neurons derived from mouse embryos with the oligomer set prepared in the present invention, according to an embodiment of the present invention.

Figure 6 shows the morphology of cells after co-treating hippocampal neurons derived from mouse embryos with thrombin at various concentrations of oligomeric trimer TA-4R and TB-4R prepared in the present invention, according to an embodiment of the present invention. This is the result of observation.

Figure 7 shows cytotoxicity after co-treating oligomeric trimers TA-4R and TB-4R prepared in the present invention with thrombin at various concentrations to hippocampal neurons derived from mouse embryos, according to an embodiment of the present invention. This is the result of the test.

Figure 8 shows the results of confirming the LC50 values of the oligomer trimer (TB-4R), tetramer (QA-4R), or pentamer (PA-4R) prepared in the present invention according to an embodiment of the present invention.

One embodiment of the present invention relates to a fusion protein in which a guide protein is bound to a tau protein.

Another embodiment of the present invention relates to a gene encoding a fusion protein in which a guide protein is bound to a tau protein.

Another embodiment of the present invention relates to a vector containing a gene encoding the fusion gene.

Another embodiment of the present invention relates to a transformant transformed with the above vector.

Another embodiment of the present invention includes preparing a fusion protein in which a guide protein is bound to a tau protein; and processing the fusion protein into nerve cells. It relates to a method of producing an Alzheimer's disease cell model including.

Another embodiment of the present invention includes preparing a fusion protein in which a guide protein is bound to a tau protein; Processing the fusion protein into nerve cells; and treating the nerve cells with a candidate substance for treating Alzheimer's disease.

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

Example 1. Preparation of guided oligomerization (GO) set of tau protein

Full-length tau, represented by SEQ ID NO: 1, has an N-terminal projection domain (aa1-165), a proline-rich region (aa166-242), and an MT binding region (MTBR). , aa243-367) and C-terminal domain (aa368-441), and the MT binding region (MTBR), also named Tau-4R, consists of R1 (aa243-273), R2 (aa274-304) , contains four partially repeated sequences named R3 (aa305-335) and R4 (aa336-367). The above-mentioned Tau-4R tends to form β-strands, and is pointed out as the cause of tau aggregation (J Neurosci, 2008. 28(3): p. 737-48.).

The oligomer set shown in Figure 1 was prepared by applying guided oligomerization (GO) to the Tau-4R. More specifically, the target is expressed as a fusion structure whose N-terminal portion consists of a guide peptide and a short stretch of amino acids that are recognized and cleaved by the protease thrombin. Detailed sequence information and amino acid positions of the guide peptide are shown in Table 1.

Targets & Guide Peptides	GenBank Accession No.	AA position
Tau-4R	NM_001123066.2 (SEQ ID NO: 2)	255-441
HRS (hepatocyte growth factor-regulated tyrosine kinase substrate)	NM_004712.3 (SEQ ID NO: 3)	1-221
Glutathione Stransferase (GST)	AAA72682.1 (SEQ ID NO: 4)	1-228
FD(T4 fibritin foldon domain)	NC_000866 (SEQ ID NO: 5)	457-483
DDT (D-dopachrome tautomerase)	NM_001084392.1 (SEQ ID NO: 6)	1-118
SCL (Streptococcus pyogenes collagen-like protein)	AF252861.1 (SEQ ID NO: 7)	106-256
CDA(human cytidine deaminase)	NM_001785.2 (SEQ ID NO: 8)	1-146
Nucleophosmin 1 (NPM)	NM_002520.6 (SEQ ID NO: 9)	1-122
HFQ (a host factor for bacteriophage Qβ RNA replication)	EU569043.1 (SEQ ID NO: 10)	1-102
HSP90α (heat-shock protein 90 alpha isoform)	NM_005348.2 (SEQ ID NO: 11)	293-732

4 μg of each set of oligomers prepared above was checked on a 4-20% gradient SDS-PAGE gel without incubation or with incubation at 95°C for 5 minutes, and the proteins were stained with Coomassie blue. The results are shown in Figure 2. As a result of the experiment, the stably maintained oligomeric structures of Tau-4R, namely trimer, tetramer, pentamer, and hexamer, migrated relatively more slowly in the SDS-PAGE gel when not heat-treated. However, not all oligomeric structures were maintained stably. DA-4R (lane 2), DB-4R (lane 3) as a dimer, and TB-4R (glutathione S-transferase) (lane 5) as a trimer maintain their identity as oligomers while running on an SDS-PAGE gel. It was found that it was not sufficiently stabilized to maintain. On the other hand, TA-4R (lane 4), QA-4R (lane 6), PA-4R (lane 7), and HA-4R (lane 8) maintained their oligomeric state well in SDS-PAGE.

Example 2. Confirmation of cytotoxicity of a set of tau protein oligomers

The cytotoxicity of the tau oligomer set prepared in Example 1 was confirmed in neural cell lines or primary cells.

First, to confirm toxicity to neural cell lines, the human neuroblastoma cell line SH-SY5Y was seeded in a ⁹⁶ -well culture dish at a density of 6 It was replaced with serum medium (3.5% FBS). Cell viability was measured daily for 3 days using PrestoBlue, and each measured value was converted to % based on the negative control and shown in Figure 3. The experimental results showed that among all oligomers, QA-4R was the most cytotoxic. TA-4R and PA-4R were also cytotoxic at low levels, but monomers, dimers, and hexamers had little cytotoxicity. These results imply that Tau-4R oligomeric conformations such as trimers, tetramers, and pentamers cause cytotoxicity. The cytotoxicity caused by Tau-4R oligomers increased significantly after one day of exposure to Tau-4R.

To confirm the toxicity of primary cultured cells, hippocampal neuronal cells were induced and cultured from mouse embryos. The primary cultured cells were seeded in a ²⁴ -well culture dish at a density of 1.2 It was replaced with . The procedure was divided into two groups: those with or without the addition of thrombin. Co-processing with thrombin can affect the function of target proteins by removing the guide peptide from the N-terminus, or can induce neuroprotective effects and neuroticism. Therefore, treatment with Tau-4R and any target protein in combination with thrombin was expected to better reflect the neuropathological state in vivo. Cells were treated with the Tau-4R oligomer set, and the morphology of the cells 6 days later is shown in Figure 4. From the above results, similar to the results using neuronal cell lines, it can be seen that unique neurodegeneration occurred in the test group administered oligomeric Tau-4R, including TA-4R, QA-4R, and PA-4R, in primary hippocampal neurons. I was able to. In particular, the neurodegenerative activity of TA-4R was analyzed to be worsened by the presence of thrombin. HA-4R, although weaker than TA-4R, also caused neurodegeneration due to the influence of thrombin. However, no neurotoxicity was observed when thrombin was treated alone or with M-4R. Non-neuronal cells, such as astrocytes, remained unaffected in their survival and proliferation under all conditions tested (see feeder cells in Figure 4).

Afterwards, sandwich ELISA was performed. Briefly, cells were treated with 0.5 ml of lysis buffer per well, and 100 ㎕ was dispensed and transferred to an ELISA plate pre-coated with anti-SNAP25 mouse monoclonal primary antibody. Since SNAP25 is expressed only in neuronal cells, it is used as a characteristic of neuronal cells. Plates were then washed, treated with affinity-purified rabbit polyclonal secondary antibodies, and the extent of captured SNAP25 was quantified using rabbit IgG and HRP-conjugated antibodies that recognize the TMB substrate. This was converted to % based on the negative control group and is shown in Figure 5. The experimental results showed that the level of SNAP25, a marker of healthy viable neurons, was consistent with the cytotoxicity results of the Tau-4R oligomer set (compare Figures 3 and 5). Neurotoxic and cytotoxic effects were analyzed in the following order: tetramer > trimer = pentamer > monomer = dimer = hexamer.

In addition, the cytotoxic effects of TA-4R and TB-4R as tau oligomer trimers on mouse hippocampal neurons (primary cultured cells) were tested when 0.5U, 2.0U, or 8.0U of thrombin was added. In the case of oligomeric dimers and trimers, the three-dimensional structures are quite unstable, so it is difficult to be sure which type of structure has more physiological effects unless experimentally determined. This can also be confirmed by the movement patterns of TA-4R and TB-4R in Figure 2. Therefore, the results of treating trimeric TA-4R and TB-4R with various thrombin concentrations are shown in Figures 6 and 7. As a result of the experiment, observation of cell morphology showed that both types of trimers had a neurotoxic effect by causing rapid neurodegeneration, but had no noticeable effect on non-neuronal cells. Therefore, under the same conditions, proliferation and survival of non-neuronal cells appeared to be unaffected (see feeder cells in Figure 6). Comparing the two trimers, the neurotoxicity of TA-4R was more pronounced than that of TB-4R. Also, considering TB-4R, neurotoxicity decreased as the concentration of thrombin increased.

Additionally, tests were conducted to determine the LC50 of trimer (TB-4R), tetramer (QA-4R), and pentamer (PA-4R) oligomeric fusion proteins. For this purpose, the human neuroblastoma cell line SH-SY5Y was seeded in 96-well culture dishes at a density of ⁶ -Replaced with serum medium (3.5% FBS). After 3 days of culture, cell viability was measured using PrestoBlue (n=3), and each average value was converted to % based on the negative control and shown in Figure 8. R-squared values were displayed on the trend line, and LC50 values were calculated using Prism software. As a result of the experiment, the LC50 values of TB-4R, QA-4R, and PA-4R were 36 nM, 16 nM, and 73 nM, respectively, showing that the toxicity was strong in the order of QA-4R > TB-4R > PA-4R. I was able to. In addition, while known fusion proteins that cause cytotoxicity form LC50 values at the μM level, the fusion protein of the present invention was found to form LC50 values at the nM level.

From the results of the above examples, it can be seen that the guide protein of the present invention has an excellent effect in regulating the function of the target protein, and includes dimers, trimers, tetramers, pentamers, and hexamers of the target protein in a state bound to the guide protein. It was found that the oligomer conformer set can be used as an excellent cell model or animal model for screening drugs for the treatment of the desired disease. In particular, guide proteins include HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), GST (glutathione Stransferase), FD (T4 fibritin foldon domain), DDT (D-dopachrome tautomerase), SCL (Streptococcus pyogenes collagen-like protein), and CDA. (human cytidine deaminase), NPM (nucleophosmin 1), HFQ (a host factor for bacteriophage Qβ RNA replication), or HSP90α (heat-shock protein 90 alpha isoform) is an oligomer bound to the N-terminus of Tau-4R as the target protein. When the conformer set is applied to nerve cells, the nerve cells embody the characteristics of Alzheimer's disease and can be used as a platform for screening drugs for the treatment of Alzheimer's disease.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, it will be apparent to those skilled in the art that various modifications and changes can be made to the practical scope of the present invention without departing from the technical spirit of the present invention as set forth in the claims.

The present invention relates to a novel Alzheimer's model, and more specifically, to a nerve cell model treated with a fusion protein in which a guide protein is bound to a tau protein. Since these cells can embody the characteristics of Alzheimer's disease, they can be used as a new drug screening platform to develop a treatment for Alzheimer's disease.

Claims (17)

1. A fusion protein in which a guide protein is bound to the tau protein.
2. According to clause 1,

   The tau protein is Tau-4R, a fusion protein.
3. According to clause 1,

   The guide proteins include HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), GST (glutathione Stransferase), FD (T4 fibritin foldon domain), DDT (D-dopachrome tautomerase), SCL (Streptococcus pyogenes collagen-like protein), and CDA ( A fusion protein that is at least one selected from the group consisting of human cytidine deaminase (NPM), nucleophosmin 1 (NPM), a host factor for bacteriophage Qβ RNA replication (HFQ), and heat-shock protein 90 alpha isoform (HSP90α).
4. According to clause 1,

   The fusion protein is a fusion protein having a dimer, trimer, tetramer, pentamer, or hexamer three-dimensional structure.
5. A gene encoding the fusion protein of claim 1.
6. A vector containing the gene of claim 5.
7. A transformant transformed with the vector of claim 6.
8. According to clause 7,

   The transformant is a cell isolated from an individual.
9. According to clause 7,

   The transformant is an individual other than a human.
10. (a) preparing a fusion protein in which a guide protein is bound to a tau protein; and,

    (b) Processing the fusion protein into nerve cells; A method of producing an Alzheimer's disease cell model, comprising:
11. According to clause 10,

    A method for producing an Alzheimer's disease cell model, wherein the tau protein is Tau-4R.
12. According to clause 10,

    The guide proteins include HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), GST (glutathione Stransferase), FD (T4 fibritin foldon domain), DDT (D-dopachrome tautomerase), SCL (Streptococcus pyogenes collagen-like protein), and CDA ( An Alzheimer's disease cell model that is at least one selected from the group consisting of human cytidine deaminase (NPM), nucleophosmin 1 (NPM), a host factor for bacteriophage Qβ RNA replication (HFQ), and heat-shock protein 90 alpha isoform (HSP90α). Manufacturing method.
13. According to clause 10,

    A method of producing an Alzheimer's disease cell model, wherein the fusion protein has a dimer, trimer, tetramer, pentamer, or hexamer three-dimensional structure.
14. (a) preparing a fusion protein in which a guide protein is bound to a tau protein;

    (b) processing the fusion protein into nerve cells; and,

    (c) treating the nerve cells with a candidate material for treating Alzheimer's disease.
15. According to clause 14,

    A method for screening a substance for treating Alzheimer's disease, wherein the tau protein is Tau-4R.
16. According to clause 14,

    The guide proteins include HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), GST (glutathione Stransferase), FD (T4 fibritin foldon domain), DDT (D-dopachrome tautomerase), SCL (Streptococcus pyogenes collagen-like protein), and CDA ( For the treatment of Alzheimer's disease, which is at least one selected from the group consisting of human cytidine deaminase (NPM), nucleophosmin 1 (NPM), a host factor for bacteriophage Qβ RNA replication (HFQ), and heat-shock protein 90 alpha isoform (HSP90α). Methods for screening materials.
17. According to clause 14,

    A method for screening a substance for treating Alzheimer's disease, wherein the fusion protein has a dimer, trimer, tetramer, pentamer, or hexamer three-dimensional structure.

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