KR100517831B1 - Double Transgenic Alzheimer's mice expressing mutant PS2 gene and APPsw gene - Google Patents

Abstract

The present invention relates to a double transgenic dementia mouse expressing a PS2 mutant gene and an APPsw gene, and more particularly to a single expressing PS2 mutant dementia inducing gene derived from humans functionally linked to a promoter of brain-specific NSE genes. The present invention relates to a double transgenic dementia mouse produced by crossing a transgenic demented mouse and a single transgenic dementia mouse expressing a human-derived APPsw dementia causing gene functionally linked to a promoter of a brain-specific NSE gene. By expressing the two dementia genes at the same time, the dual transgenic demented mice exhibit characteristics such as brain dysfunction similar to dementia symptoms of human dementia patients, increased expression of toxic proteins such as amyloid beta-42, and increased activity of MAPK and ER. It can be used as an animal model which is very useful for the treatment of dementia disease or the early diagnosis of dementia disease.

Description

Double transgenic Alzheimer's mice expressing mutant PS2 gene and APPsw gene

The present invention relates to a double transgenic dementia mouse expressing a PS2 mutant gene and an APPsw gene, and more particularly to a single expressing PS2 mutant dementia inducing gene derived from humans functionally linked to a promoter of brain-specific NSE genes. The present invention relates to a double transgenic dementia mouse produced by crossing a transgenic demented mouse and a single transgenic dementia mouse expressing a human-derived APPsw dementia causing gene functionally linked to a promoter of a brain-specific NSE gene.

Dementia is a disorder accompanied by general disorders of systemic function, such as memory impairment and loss of judgment. There are many forms of dementia, about 50% of which are dementia of Alzheimer type, 20-30% of dementia of Vascular type, and alcoholic dementia and Parkinson's dementia. It is known that ˜20% develop both Alzheimer's and vascular dementia.

Alzheimer's disease (AD), the most important form of dementia, is an elderly dementia disease that occurs rarely before age 50 but gradually increases after age 60. With the increase of the elderly population due to the improvement of quality of life, the onset population is rapidly increasing not only in Korea but also in the world. The number of Alzheimer's disease patients registered in Korea over 65 years in 1995 was 241,000, accounting for 8.3% of the total population of the elderly. In 2020, it was predicted to be 619,000 [Health and Social Research Institute (1995), Ministry of Health and Welfare (1991). 4763) December 28, 1998]. Alzheimer's disease can only be diagnosed early and limited, so it is urgently needed to develop an accurate and early treatment method. However, the cause of the disease is not known exactly. .

The cause of Alzheimer's disease is not yet well understood, but the function of the following two proteins is attracting attention. The first is a neurotoxic protein called amyloid beta-39 / 43 (Amyloidβ-39 / 43, Aβ-39 / 43), which is concentrated at high concentrations to form plaques, which are responsible for memory and recognition of the brain. It deposits in the area, causing damage to the DNA of neurons, causing Alzheimer's. The other is a thin, thread-like protein called tau, which is twisted together to form a tangled tangle, resulting in the death of neuronal cells as the proteins are ideally phosphorylated.

Among them, amyloid beta-39 / 43, which is recently attracting attention as a key cause of AD disease, has 39 to 43 amino acids, and 28 amino acids of the extracellular domain of APP (Amyloid Precursor Protein) It consists of 11-15 amino acids of the transmembrane domain. Thus, Aβ-39 / 43 is induced and formed during the processing of APP genes. The formation of Aβ-39 / 43 is attributable to the function of the following key genes and protein enzymes. First, the production of Aβ-39 / 43 is known to be increased by the PS2 mutation (PS2mt, Volga German Families of N141I) among presenilins PS1 and PS2, which are early-induced AD genes. It is also known that the production of Aβ-39 / 43 is increased by APPsw expression.

In order to identify the cause of such dementia and develop a therapeutic substance, it is essential to develop an animal model for dementia that can be actually tested before the clinical trial. To this end, two types of dementia mice, such as 'Tg2575' and 'London mice', have been developed and used in the United States, but as described above, the PS2 mutant gene, the APPsw gene, or the two genes are simultaneously inserted to induce dementia. Mice have not been developed as a dementia model.

In order to understand the above technical reality and develop an animal model for diagnosing and treating Alzheimer's dementia disease, the present inventors have developed a single transgenic dementia mouse incorporating the dementia causing gene, the PS2 mutant gene or the APPsw gene. On the other hand, by crossing the two transgenic dementia mice it was possible to develop a double transgenic dementia mouse in which both genes are inserted into the chromosome. In other words, a PS2 mutant gene and an APPsw gene, which are expressed in an animal body and increase the production of a substance causing dementia, are introduced under NSE promoter control, respectively, to prepare a transgenic dementia mouse expressing the two genes and to cross them. The present invention was completed by discovering that a dual transgenic demented mouse that is capable of expressing two dementia genes early expresses the Alzheimer's phenotype and exhibits various pathological features observed in patients with dementia.

 Accordingly, an object of the present invention is to provide a double transgenic dementia mouse expressing the PS2 mutant gene and the APPsw gene simultaneously.

The above-described features of the present invention and other features can be well seen by those skilled in the art by the following detailed description of the present invention.

In order to achieve the above object, the present invention provides a single transgenic dementia mouse expressing a human PS2 mutant dementia causing gene (hPS2m) that is functionally linked to a promoter of a brain-specific Neuron-Specific Enolase (NSE) gene. And a double transgenic dementia mouse capable of expressing both genes by crossing a single transgenic dementia mouse expressing a human-derived APPsw dementia trigger gene functionally linked to a promoter of a brain specific NSE gene. It is characterized by.

That is, the present invention is a double transgenic dementia mouse in which the PS2 mutant gene and the APPsw gene are inserted into the chromosomes, and the two genes are functionally linked to the brain-specific NSE promoter, so that the two genes cause dementia. It is characterized by providing a dual transgenic dementia mouse as a dementia model animal that is expressed and consequently promotes expression of amyloid beta-42 protein and increases the activity of MAPK and ER.

Hereinafter, the present invention will be described in more detail.

Amyloid precursor protein (APP), which is present in normal brains, is usually metabolized by α-secretase and is present in most secreted APPs. However, in the brain of Alzheimer's patients, beta or gamma secretase (β- or γ-secretase) is activated and APP is processed to process the amyloid beta-39 / 43 consisting of 39 to 43 amino acids (Amyloidβ-39) / 43) will be produced in large quantities. Mutation of the PS2 gene also increases amyloid beta-42 production.

Amyloid beta-42, a toxic protein that damages the neurons responsible for memory and cognition in the brain, kills nerve cells by: First, it activates caspase-3, an enzyme that binds to the vesicle receptors of brain cells and kills nerve cells. That is, when amyloid beta-42 accumulates in the brain, nerve cells shrink due to its own toxicity, and signal transmission lines are cut off. It also increases free radicals that are harmful to cells and kills them with their toxins.

Amyloid beta-42, which is fatal to Alzheimer's, is closely related to mutations in the presenilin2 gene (PS2) and the amyloid precursor protein (APP) gene. have.

The PS2 mutant gene in the present invention is a mutant presenilin2 gene, which is one of the causal genes involved in causing human Alzheimer's disease. The PS2 gene is a gene located on human chromosome 1, and people whose mutants are inherited usually develop Alzheimer's disease before age 65.

APPsw gene in the present invention is human Alzheimer's is one of the causal genes involved in causing disease. The APP gene is a gene located on human chromosome 21. People whose mutants are inherited usually develop Alzheimer's disease before age 65. As a mutant form of APP, a mutant gene form in which amino acids at positions 670 and 671 are replaced with AL in KM is called an APP swedish gene.

Such PS2 variant genes and APPsw genes can be arbitrarily selected and used genes known in the art, for example, it is possible to easily obtain information about the sequence from a genetic information agency such as Genbank. These genes may be directly isolated from dementia patients or may be artificially synthesized.

The NSE promoter in the present invention is a promoter for controlling the human PS2 mutant gene and the APPsw gene to be well expressed in an animal model, and the genes are functionally linked to the NSE promoter. NSE promoter sequences are also readily available from genetic information databases known in the art.

In the present invention, the human PS2 mutation gene or the fusion gene NSE / hPS2m or NSE / APPsw in which the APPsw gene and the NSE promoter are coupled to each other can be introduced into various animals except humans. Such animals include, but are not limited to, mice or rats, and preferably, a mouse is used.

In the present invention, the fusion gene NSE / hPS2m or NSE / APPsw may be inserted into an animal chromosome according to various transformation methods well known in the art. Examples of such transformation methods include a method of microinjecting a fusion gene into an animal's fertilized egg or a method using a retroviral vector. Alternatively, the recombinant fusion gene may be injected into the sperm nucleus of a male mouse, and then combined with an egg nucleus of a female mouse to make a fertilized egg and implant it into another surrogate mother mouse. However, from the viewpoint of the safety of the operator performing the experiment, a method of microinjecting the fusion gene into the fertilized egg is more preferable.

In the following reference example of the present invention, the recombinant fusion genes NSE / hPS2m or NSE / APPsw, which are dementia-inducing genes as described above, are microinjected into fertilized eggs of BDF1 mice, and the fertilized eggs injected with such genes are transplanted into ICR mice, respectively. Converted dementia mouse pups were produced.

Then, a single transgenic dementia mouse produced as described above was crossed with each other to produce a dual transgenic dementia mouse in which the PS2 mutant gene and the APPsw gene were simultaneously inserted into the chromosome under NSE promoter control.

As described above, the double transformed demented mouse in which the PS2 mutant gene and the APPsw gene are inserted and expressed at the same time is a dementia-induced mouse model in which all of the PS2 mutation gene and the APPsw gene are expressed by the NSE promoter and thus exhibit various pathologies caused by Alzheimer's disease. In other words, the phenotype of Alzheimer's disease was measured and compared with a single transgenic dementia mouse expressing the prepared PS2 mutation gene, a single transgenic dementia mouse expressing the APPsw gene, or a normal untransformed mouse. As a characteristic pathology in patients, increased amyloid beta-42 production was confirmed, and water maze test showed abnormalities in brain function, and also increased MAPK activity and increased ER expression. Was confirmed. Through this, the double transgenic dementia mouse according to the present invention exhibits behavioral disorders and physiological characteristics very similar to those of dementia patients, and in particular, the single transgenic dementia mouse expressing and expressing only one gene of the two dementia causing genes. It can be seen that it is more clear than the comparison.

The double transgenic dementia mouse according to the present invention exhibits behavioral abnormalities similar to those exhibited by patients with dementia, and is characterized by almost perfect dementia symptoms such as not only amyloid beta-42 protein but also activation of MAPK and increased expression of ER. . Therefore, in the case of using the expression traits such as the increased expression of the dementia-related substances, it can be very useful for the study of the mechanism of dementia, and particularly useful for the development of a therapeutic or early diagnosis of dementia. Administration of dementia treatments developed or under development at home and abroad is currently used to treat various dementia effects under conditions similar to those of the human body, namely, inhibiting amyloid beta-42 accumulation, reducing behavioral abnormalities caused by dementia, inhibiting MAPK activity, and decreasing ER expression. By confirming that the dementia treatment effect of the dementia treatment agent can be assayed, and can also be used as an animal model to study the cause of dementia disease.

Hereinafter, the structure of the present invention will be described in more detail with reference to Production Examples and Test Examples, but the scope of the present invention is not limited thereto.

Reference Example 1 Preparation of Transgenic Dementia Mice Inserted with a PS2 Mutant Gene

① Gene acquisition and recombination

Among the genes used in this experiment, the NSE promoter was used by Dr. Sutcliffe (Research Institute of Scrippe Clinic) and the PS2 mutant gene hPS2m (N141I) was donated by Professor Kim Tae-wan of Columbia University.

Recombination of the gene was used in combination with the NSE promoter, PS2 gene, and the terminator. PS2 mutant genes (Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, Pettingell WH, Yu CE, Jondro PD, Schmidt SD, Wang K, et al. (1995) Candidate gene for the chromosome 1 familial Alzheimer's Disease locus.Science 269 (5226): 973-977; Gene Bank Accession Number: NM_012486) was first isolated from pUHD10-3 vector with pTet-Splice vector digested with restriction enzyme Eco RI / Xba I and digested with Eco RI / Spe I. After insertion, the Tet promoter was cleaved with Xho I / Eco RI and then removed, followed by insertion of the NSE promoter cleaved with Sal I / Eco RI to complete the fusion gene NSE / PS2m.

② Fine injection of fusion gene and injection into mouse fertilized egg

Recombinant fusion gene In order to purely separate NSE / PS2m, a gene region derived from bacteria was cut and removed, and only eukaryotic expression regions were harvested and dissolved in water. The purely isolated fusion gene was diluted to a concentration of 4 ng / μl and injected into the fertilized eggs of mice (BDF1). The fertilized egg implanted with the gene was implanted into a fertile pregnant ICR mouse to produce a transgenic dementia mouse expressing the PS2 mutant gene.

Transgenic dementia mice expressing the PS2 mutant gene may be prepared according to the above method or from fertilized eggs deposited with the accession number KCTC 10420BP to the Korean Collection for Type Cultures.

REFERENCE EXAMPLE 2 Production of Transgenic Dementia Mice Inserted with the APPsw Gene

① Gene acquisition and recombination

Among the genes used in this experiment, the NSE promoter was used by Sutcliffe, Ph.D. (Research Institute of Scrippe Clinic), and the APPsw gene was donated by Professor Kim Tae-wan of Columbia University.

Recombination of the gene was used in combination with the NSE promoter, APPsw gene, the terminator. APPsw gene (Martin C, Tilman O, Christian H, Lisa M, Albert YH, Peter S, Carmen VP, Ivan L, Dennis JS (1992) Mutation of the β-amyloid precursor protein in familial Alzheimer's disease increases β-protein production. Nature 360: 672-674; Gen Bank Acession Number: Y00264) was used to identify primers specific for this gene (sense: 5'-TCTAG ATCGC GATGC TGC-3 ', antisense: 5'-GTCTA GAGTC TAGTT CTGCA-3'). After PCR amplification using the clone, the TA vector was cloned into Xba I / Spe I and inserted into the pNSE-splice vector digested with Xho I / Eco RI to complete the fusion gene NSE / APPsw.

② Fine injection of fusion gene and injection into mouse fertilized egg

Recombinant fusion gene In order to purely separate NSE / APPsw, a gene region derived from bacteria was cut and removed, and only eukaryotic expression regions were harvested and dissolved in water. The purified fusion gene NSE / APPsw was diluted to a concentration of 4 ng / μl and injected into the fertilized eggs of mice (BDF1). The fertilized egg implanted with the gene was implanted in a fertile pregnant ICR mouse to produce a transgenic dementia mouse expressing the APPsw gene.

Transgenic dementia mice expressing the APPsw gene may be prepared according to the above method or from fertilized eggs deposited with the accession number KCTC 10448BP to the Korean Collection for Type Cultures.

Preparation Example 1 Preparation of Double Transgenic Dementia Mice Following the Crossing of Transgenic Demented Mice Inserted with PS2 Mutant Gene and Transgenic Demented Mice Inserted with APPsw Gene

① PS2 mutant transgenic mice prepared in Reference Examples 1 and 2 and APPsw transgenic mice were crossed to produce litters.

② Identification of Transgenic Mouse

After cutting the tail of the offspring produced through the above to separate genomic DNA (genomic DNA), DNA-PCR was performed using primers specific to the respective recombinant genes (Gibco BRL Co. production request), Southern Southern blot hybridization was performed to confirm insertion of APPsw gene and PS2 mutant gene.

Each primer sequence specific for the recombinant gene is as follows.

APPsw gene specific primer

sense: 5'-TCGCG ATGCT GC-3 '

antisense: 5'-CTCTT CTCAC TGCAT GTCTC-3 '

PS2 Mutant Gene Specific Primer

sense: 5'-GAGGA AGAAG TGTGT GATGA G-3 '

antisense: 5'-CACGA TGACG CTGAT CATGA TG-3 '

In order to detect and confirm the inserted PS2 trans gene by Southern blot analysis, the tail genomic DNA of the PS2 litter was cut with Eco RI and transferred to the nitrocellulose membrane, followed by hybridization by labeling the PS2 specific DNA probe with ³² P. Bands were observed by photosensitizing the X-ray film.

In addition, in order to detect and confirm the APPsw trans gene by Southern blot analysis, the tail genomic DNA of the APPsw mountain was cut with BamHI, transferred to the nitrocellulose membrane, and then hybridized by labeling the APPsw specific DNA probe with ³² P. Was observed by sensitizing the X-ray film.

At this time, 5.2, 6.2 and 7.1 kb fragments of known sizes resulting from cleavage of lambda DNA with Hind III / Hae III were shown on the right side of the blot and the relative positions thereof were calculated to determine the size of the fragments. Irradiation at −70 ° C. for 3 days visualized specific hybridization signals. Both male and female are shown. The results are shown in FIG. The produced litters were produced in 25% of bitransgenic mice, 25% of APPsw transgenic mice, 25% of PS2 transgenic mice and 25% of normal mice.

As can be seen from the results of FIG. 1, the hPS2m and APPsw genes are located under the control of the NSE gene promoter. In addition, a product of 442 bp derived from pNSE-hPS2m and a 509 bp product derived from pNSE-APPsw were confirmed (FIG. 1B). That is, 442 bp PS2 mutant gene product and 509 bp APPsw gene product can be identified from the produced tail DNA of the mouse.

As described above, the transgenic mouse expressing the PS2 mutant gene by inserting the NSE / PS2m fusion gene into the eukaryotic expression site of the mouse and transforming the transgenic mouse and the NSE / APPsw fusion gene into the eukaryotic cell expression site of the mouse are transformed. The fertilized eggs of double transgenic dementia mice produced through the crossing of transgenic mice expressing the APPsw gene, ie, the fertilized eggs of the double transgenic demented mice in which the PS2 mutant gene and APPsw were inserted into the chromosome, were transferred to the Korea Research Institute of Bioscience and Biotechnology. It was deposited with the Korean Collection for Type Cultures on July 4, 2003, and was granted accession number KCTC 10495BP.

Test Example 1 Expression of PS2m and APPsw Genes in Tissues of Double Transgenic Dementia Mice

Each tissue was isolated from the double transgenic demented mice to confirm whether PS2m and APPsw genes were expressed in a specific manner. To this end, tissue-specific expression of PS2m and APPsw genes under rat NSE promoter control was confirmed by RT-PCR using the brain, lung, heart, liver, kidney, small intestine, and muscle tissues of the transgenic demented mice. Β-actin signals and their transcripts (640 bp) as controls were shown to show the loading of RNA. Transcript and protein levels in various tissues were quantified by Kodak Electrophoresis Documentation and Analysis System 120 (FIG. 2C).

The results are shown in FIG. While transcript (Figure 2A) and protein (Figure 2B) levels of the PS2m transgene are highly expressed in lungs, kidneys and muscles, transcript and protein levels of the APPsw transgene are highly expressed in the brain resulting in a brain-specific manner. It can be seen that it is controlled by.

[Test Example 2] Measurement of brain dysfunction

Underwater maze tests to determine whether the double transgenic dementia mice have abnormal brain function compared to mice with a single dementia gene, ie, NSE / APPsw transgenic dementia mice and NSE / hPS2m transgenic dementia mice. Water Maze Test).

The results are shown in FIG. 3A, 3D, 3B, and 3C are results of measuring patterns of escape latency, escape distance, and swimming velocity across the front platform position in the underwater maze, respectively. [Water maze subjects Median values and SD of three experiments performed with n = 3, P <0.01]. NSE-PS2 / APPsw double transgenic dementia mice at 8 months of age showed a consistent trend with longer escape delays and escape distances than NSE / PS2 and NSE / APPsm single transgenic dementia mice.

On the other hand, in swimming speed, the swimming speed of the NSE-PS2 / APPsw double transgenic dementia mice of 8 months of age was observed to be slightly slower than that of demented mice of 5, 6, and 7 months of age.

As can be seen from the above results, it was confirmed that NSE-PS2 / APPsw double transgenic dementia mice exhibited behavioral abnormalities early due to abnormal brain function.

Test Example 3 Confirmation of Aβ-42 Deposition in the Brain of a Double Transgenic Dementia Mouse

It was confirmed by dot blot and immunostaining whether the deposition of amyloid β-42, a toxic protein that causes dementia, increased in the brain of a double transgenic dementia mouse according to the present invention.

The nitrocellulose filter transferred 40 μg of protein isolated from the brains of the double transgenic dementia mice, NSE / PS2 and NSE / APPsw single transgenic dementia mice and control mice were incubated with anti-human Aβ-42 antibody. The bands were quantified by densitometry to obtain relative levels of Aβ-42. The results are shown in Figure 4A. In addition, for immunochemical staining of Aβ-42, 20 μm-thick brain sections cut from each mouse were combined with anti-human Aβ-42 primary antibody and HRP-conjugated gut anti-rabbit IgG. Incubated. As a result, the tissue was observed under a microscope and shown in FIG. 4B.

Extensive distribution and accumulation of Aβ-42 accumulation in the cortex and hippocampus of 8-month-old transgenic demented mice was confirmed.

Test Example 4 Measurement of MAPK Activity of Double Transgenic Dementia Mice

The activation of three substances JNK, p38 and ERK belonging to the MARK group in double transgenic dementia mice, NSE / PS2 and NSE / APPsw single transgenic dementia mice and control mice according to the present invention was measured by Western blot. They are known to cause neuronal cell death.

The results are shown in FIG. 5A and B show measurement results of activation of JNK and c-Jun, respectively. The top result was the treatment of 10 μg of protein per sample against phopho-JNK and phosphoro-c-Jun. Total JNK proteins were measured using antibodies that recognize them regardless of their phosphorylation status. 5C shows the result of activation measurement of p38. The top results were treated with 40 μg of protein incubated with the antibody against phosphoro-p38. The results below represent a total of p38 with relatively constant levels. 5D is the result of ERK activation measurement. Quantification of the bands is indicative of the activity of each of the MAPK groups. Three samples per group were analyzed by Western blot (p <0.05).

[Test Example 5] Characteristics of ER in double transgenic dementia mice

Studies have shown that ER is increased in dementia patients, and the expression of ER in the brains of double demented mice has been shown to be expressed earlier than in NSE / PS2 and NSE / APPsw single transgenic demented mice. Significant increases in ERα and ERβ have been shown in converting dementia mice.

Proteins were extracted from the brain of each mouse and then incubated with anti-ERα and anti-ERβ antibodies. The increase in protein levels in NSE / PS2 and NSE / APPsw single transgenic dementia mice was similar to that of control mice, but the ERα and ERβ genes in the brain of the double transgenic dementia mice according to the present invention were expressed very high.

As described above, a single transgenic dementia mouse expressing a human-derived PS2 mutant dementia causing gene functionally linked to a promoter of brain specific NSE gene and a human functionally linked to promoter of brain specific NSE gene Dual transgenic dementia mice produced by crossing a single transgenic dementia mouse expressing the derived APPsw dementia-producing gene are inserted into the chromosome of both the PS2 mutant gene and the APPsw gene. It can be usefully used for developing dementia treatments or early diagnosis methods, as it has excellent effects showing almost perfect dementia symptoms such as increased expression of amyloid beta-42 protein, increased MAPK activity, and increased expression of ER. have.

1A shows the structure of pNSE-hPS2m inserted with the PS2 mutant gene and the structure of pNSE-hAPPsw inserted with the APPsw gene, and 1B is for confirming the insertion of the PS2 mutant gene and APPsw in the double transgenic dementia mouse according to the present invention. This is the result of Southern blot.

Figure 2 shows the results confirming the tissue-specific expression of the PS2m and APPsw gene in double transgenic dementia mice according to the present invention.

Figure 3 shows the results of the water maze test (Double Maze Tests) of the double transgenic dementia mice and NSE / PS2 and NSE / APPsw single transgenic dementia mice according to the present invention.

Figure 4 shows the results confirmed by dot blot and immunostaining the deposition of amyloid beta-42 (Amyloidβ-42) in the brain tissue of a double transgenic dementia mouse according to the present invention.

Figure 5 is the result of measuring the activity of the three substances belonging to the MAPK group in double transgenic dementia mice, NSE / PS2 and NSE / APPsw single transgenic dementia mice and control mice according to the present invention.

Figure 6 shows the results of measuring the expression of ERα and ERβ at the age of 8 months of the double transgenic dementia mice, NSE / PS2 and NSE / APPsw single transgenic dementia mice and control mice according to the present invention.

Claims (9)

delete delete delete delete A human-derived PS2 mutant gene is inserted into the chromosome, and the PS2 mutant gene is a single transgenic mouse (A) produced by implanting a fertilized egg of Accession No. KCTC 10420BP functionally linked to an NSE promoter, and a human-derived gene in the chromosome. Microinjection of the fusion gene isolated from the offspring produced by crossing a single transgenic mouse (B) produced by transplanting a fertilized egg of Accession No. KCTC 10448BP functionally linked to the NSE promoter with the APPsw gene inserted therein. Fertilization column of accession number KCTC 10495BP obtained by delete A double transgenic dementia mouse produced by transplanting a fertilized egg of Accession No. KCTC 10495BP according to claim 5. The method of claim 7, wherein the double transformed non-human animal is a dual transgenic dementia mouse characterized in that the PS2 mutant gene and APPsw gene is expressed causing dementia symptoms. 8. The method of claim 7, wherein the double transformed non-human animal expresses the PS2 mutant gene and the APPsw gene to induce dementia symptoms by increasing amyloid beta-42 expression, increasing MAPK activity or increasing ER expression. Characterized by the double transgenic dementia mouse.