Alzheimer′s disease animal model induced by amyloid-beta screening method of therapeutic agents for alzheimer's disease

Abstract

The present invention relates to an amyloid-beta-induced Alzheimer's disease animal model and a method for screening Alzheimer's disease therapeutic agents using the same. Unlike conventional animal models utilizing zebrafish in which cognitive impairment is induced by substances other than amyloid-beta, the amyloid-beta-induced Alzheimer's disease zebrafish animal model of the present invention induces Alzheimer's disease solely by amyloid-beta. Accordingly, the animal model of the present invention exhibits excellent reproducibility regarding the induction of Alzheimer's disease by amyloid-beta. Furthermore, the screening method for amyloid-beta-related disease therapeutic agents using the animal model of the present invention is useful for the development of new drugs related to diseases caused by amyloid-beta, as it allows for the accurate and convenient screening of therapeutic agents for such diseases.

Description

Technology Field

[0001] The present invention relates to an amyloid-beta-induced Alzheimer's disease animal model and a method for screening Alzheimer's disease treatments using the same. Background Technology

[0002] Alzheimer's disease is a degenerative brain disorder in which brain nerve cells die as abnormal proteins, such as amyloid-beta and tau, accumulate in the brain; it is known that more than 50% of all dementia patients exhibit symptoms caused by Alzheimer's disease. With the incidence rate rising annually—10% in those aged 65–74, 19% in those aged 75–84, and 47% in those aged 85 and older—Alzheimer's disease is emerging as a major social issue. However, to date, there are no known methods for prevention or established early diagnosis of Alzheimer's disease. Furthermore, no specific cure has been developed to date, and only medications that alleviate symptoms are currently in use.

[0003] Meanwhile, in order to develop therapeutic substances for Alzheimer's disease, it is essential to develop animal models that can actually be used for experiments before conducting clinical trials. Accordingly, research has been conducted on animal models created by transgenerating specific genes in mice to study the mechanisms of Alzheimer's dementia; however, these animal models had limitations in obtaining the desired results due to noise, such as the co-expression of the transgene.

[0004] Recently, as it has been revealed that zebrafish possess high genetic and functional similarity to the human genome, extensive research is being conducted worldwide for the study of human diseases. Furthermore, they are evaluated as an excellent model for screening candidate substances for disease treatments because the cost of purchasing and maintaining them per individual is very low and a large number of embryos can be secured. In addition, most organs, such as the nervous and circulatory systems, are formed within 24 hours of fertilization, and they can be used for large-scale screening experiments using 48-well or 98-well plates within a few days.

[0005] Recently, there has been an increasing number of cases where zebrafish are being used to replace conventional animal models for research on the pathogenesis of Alzheimer's disease and drug screening. A representative research example is a zebrafish animal model in which organ-specific biomarkers, such as fluorescent proteins, are expressed in microglia and macrophages. However, existing zebrafish animal models have shown limitations in terms of the reproducibility of Alzheimer's disease and the accuracy of drug screening. Prior art literature

[0006] (Patent Document 0001) KR 10-2584857 B1 The problem to be solved

[0007] In one aspect, the objective of the present invention is to provide an animal model of Alzheimer's disease with excellent reproducibility.

[0008] In one aspect, the objective of the present invention is to provide a screening method for Alzheimer's disease treatments that has excellent accuracy and reliability.

[0009] The problem to be solved by the present invention is not limited thereto, and may also include objectives or effects that can be identified from the means of solving the problem or embodiments described below. means of solving the problem

[0010] To achieve the above objectives, in one aspect, the present invention provides a zebrafish animal model of an amyloid-beta-induced amyloid-beta-related disease (e.g., Alzheimer's disease).

[0011] In addition, in one aspect, the present invention provides a method for screening amyloid-beta-related disease treatments using zebrafish.

[0012] In the above aspects, the present invention provides a method for screening an amyloid-beta-related disease treatment using zebrafish, comprising the steps of: treating a zebrafish with an amyloid-beta-related disease treatment candidate substance with an amyloid-beta-related disease (S1); and measuring one or more of i) the behavior of the zebrafish after treatment with the disease treatment candidate substance; and ii) the amount of amyloid-beta present in the ventricles of the zebrafish (S2).

[0013] In addition, the present invention provides a method for screening amyloid-beta-related disease treatments using zebrafish, wherein the method comprises measuring the behavior of zebrafish by measuring the avoidance response of zebrafish to a provided visual stimulus, and measuring the behavior of zebrafish by measuring one or more of the following: the proportion of zebrafish individuals that avoid the area where the visual stimulus is provided and are active in an area where the visual stimulus is not provided; and the time that zebrafish active in the area where the visual stimulus is not provided remain in said area.

[0014] In addition, in one aspect, the present invention provides a method for screening an amyloid-beta-related disease treatment using zebrafish, wherein the method further comprises a step (S3) of determining the efficacy of a candidate substance for an amyloid-beta-related disease treatment, wherein S3 comprises one or more of the following: a ratio of zebrafish individuals that avoid a zone where visual stimulation is provided and are active in a zone where visual stimulation is not provided; determining whether one or more of the time spent by zebrafish active in the zone where visual stimulation is not provided in the zone increases compared to a group not treated with the disease treatment candidate substance; and determining whether the amount of amyloid-beta present in the ventricles decreases compared to a group not treated with the disease treatment candidate substance.

[0015] In addition, in one aspect, the present invention provides a method for screening an amyloid-beta-related disease treatment using zebrafish, wherein the method further comprises the step (preS1) of injecting amyloid-beta into the ventricles of a zebrafish to induce an amyloid-beta-related disease in the zebrafish.

[0016] In addition, in one aspect, the present invention provides a method for screening amyloid-beta-related disease treatments using zebrafish, wherein the amyloid-beta-related disease includes Alzheimer's disease. Effects of the invention

[0017] Unlike conventional animal models utilizing zebrafish in which cognitive impairment is induced by substances other than amyloid-beta, the amyloid-beta-induced Alzheimer's disease zebrafish animal model of the present invention induces Alzheimer's disease solely through amyloid-beta. Accordingly, the animal model of the present invention exhibits excellent reproducibility regarding the induction of Alzheimer's disease by amyloid-beta. Furthermore, the screening method for amyloid-beta-related disease treatments utilizing the animal model of the present invention is useful for the development of new drugs related to such diseases, as it allows for the accurate and convenient screening of treatments for diseases induced by amyloid-beta.

[0018] The various and beneficial advantages and effects of the present invention are not limited to those described above and may be more easily understood in the process of explaining specific embodiments of the present invention. Brief explanation of the drawing

[0019] Figure 1 is a stereomicroscope image (plan view (Fig. 1a), side view (Fig. 1b)) of a zebrafish Alzheimer's disease animal model, which is one aspect of the present invention, after injecting amyloid-beta. Figure 2 is a photograph observed with a fluorescence microscope after injecting amyloid-beta into a zebrafish Alzheimer's disease animal model, which is one aspect of the present invention. Figure 3 is a photograph showing the location of a zebrafish individual after injecting amyloid-beta into a zebrafish Alzheimer's disease animal model, which is one aspect of the present invention, and measuring the avoidance response to visual stimuli. Figure 4 is a fluorescence microscope image taken immediately after injecting amyloid-beta into a zebrafish Alzheimer's disease animal model, which is one aspect of the present invention (Figure 4a) and after a certain period of time (Figure 4b). Specific details for implementing the invention

[0020] The present invention is capable of various modifications and may have various embodiments, and specific embodiments are illustrated and described in the drawings. However, this is not intended to limit the invention to specific embodiments, and it should be understood that the invention includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the invention.

[0021] Furthermore, terms or words used in the specification and claims of the present invention are not limited to their ordinary or dictionary meanings, and must be interpreted in a meaning and concept consistent with the technical spirit of the present invention, based on the principle that the inventor can appropriately define the concept of the terms to best describe his invention.

[0022] Terms including ordinal numbers, such as second, first, etc., may be used to describe various components, but said components are not limited by said terms. Such terms are used solely for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the second component may be named the first component, and similarly, the first component may be named the second component. The term "and / or" includes a combination of a plurality of related described items or any of a plurality of related described items.

[0023] Furthermore, the terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as “comprising” or “having” are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0024] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this specification.

[0025] Hereinafter, embodiments will be described in detail with reference to the attached drawings, provided that identical or corresponding components are given the same reference number regardless of the drawing symbols, and redundant descriptions thereof will be omitted.

[0026] In one aspect, the present invention is a method for screening amyloid-beta-related disease treatments using zebrafish.

[0027] Amyloid-beta-related diseases refer to diseases that develop as a result of amyloid-beta directly or indirectly influencing their onset.

[0028] In one aspect, a method for screening an amyloid-beta-related disease treatment using a zebrafish according to the present invention may include the step (S1) of treating a zebrafish with an amyloid-beta-related disease treatment candidate substance; and the step (S2) of measuring one or more of i) the behavior of the zebrafish after treatment with the disease treatment candidate substance; and ii) the amount of amyloid-beta present in the ventricles of the zebrafish.

[0029] In one aspect, the candidate substance for treating amyloid-beta-related diseases may be treated by diluting it in an embryo culture medium. For example, the embryo culture medium may contain an E3 medium. The E3 medium may contain 3 to 7 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM MgSO4. In one aspect, the candidate substance diluted in the embryo culture medium may have a pH of about 6.8 to 7.4, or 7.0 to 7.2.

[0030] In one aspect, the above S2 can be performed after 2 to 10 days have elapsed since treating the zebrafish with the disease treatment candidate substance.

[0031] In one aspect, measuring the behavior of the above-mentioned zebrafish may include measuring the zebrafish's avoidance response to a provided visual stimulus.

[0032] In one aspect, measuring the behavior of the zebrafish may include measuring one or more of: the proportion of zebrafish individuals that avoid the area where visual stimulation is provided and are active in an area where visual stimulation is not provided; and the time that zebrafish active in the area where visual stimulation is not provided stay in that area.

[0033] In one embodiment, the avoidance response of the zebrafish to the provided visual stimulus can be implemented by the following experiment.

[0034] For example, regarding the avoidance response to visual stimuli, a limited area is divided into two equal areas, and one of the areas is set up to provide visual stimuli, and then multiple zebrafish are exposed to that environment. After that, the proportion of zebrafish that avoid the area providing visual stimuli for a certain period of time and move to the area where visual stimuli are not provided can be calculated.

[0035] Alternatively, measuring the behavior of the zebrafish may include measuring the time spent avoiding the visual stimulus and staying in an area where no visual stimulus is provided after exposure to the visual stimulus.

[0036] In one aspect, the color of the visual stimulus used in the above visual stimulus experiment is not limited. For example, red, yellow, blue, etc., may be used, but are not limited thereto.

[0037] In addition, the visual stimuli provided in the above visual stimulus experiment may include dynamic stimuli and static stimuli. For example, when dynamic stimuli are provided, light of a specific color moving in the area where the visual stimuli are provided may be provided, and when static stimuli are provided, stimuli of a specific color displayed on the floor surface of the area where the visual stimuli are provided may be provided.

[0038] In one aspect, measuring the amount of amyloid-beta present in the ventricles of the zebrafish can be performed by measuring the area of ​​the fluorescence-expressed zone due to fluorescently labeled amyloid-beta.

[0039] In one aspect, the amount of amyloid-beta and / or the change in the amount of amyloid-beta after treatment with a disease treatment candidate substance can be derived by the following formula.

[0041] [Mathematical Formula 1] Amyloid-beta removal rate :

[0042] Clearance Index (%) = (Area of ​​fluorescence zone on the day of amyloid-beta injection - Area of ​​fluorescence zone 2 to 10 days after amyloid-beta injection) / Area of ​​fluorescence zone on the day of amyloid-beta injection.

[0044] Additionally, the above method may further include a step (S3) of determining the efficacy of a candidate substance for treating amyloid-beta-related diseases.

[0045] In one aspect, the above S3 may include one or more of: the proportion of zebrafish individuals that avoid the area where visual stimulation is provided and are active in the area where visual stimulation is not provided; determining whether one or more of the time spent by zebrafish active in the area where visual stimulation is not provided in the said area increases compared to a group not treated with the disease treatment candidate substance; and determining whether the amount of amyloid-beta present in the ventricles decreases compared to a group not treated with the disease treatment candidate substance.

[0046] In addition, in one aspect, the method may further include the step of treating zebrafish with an amyloid-beta-related disease treatment.

[0047] In this case, the step of determining the efficacy of the amyloid-beta-related disease treatment candidate substance may further include comparing the experimental result of a positive control group treated with the amyloid-beta-related disease treatment substance with the group treated with the disease treatment candidate substance.

[0048] In one aspect, the amyloid-beta-related disease treatment may include, but is not limited to, donepezil, EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid).

[0049] In one aspect, if the experimental results of the group administered the above-mentioned disease treatment candidate substance show a statistically significant result (p-value ≤ 0.01) compared to the above-mentioned positive control group, the said candidate substance can be determined as a treatment for amyloid-beta-related diseases.

[0050] In one aspect, the method may further include a step (preS1) of inducing an amyloid-beta-related disease in the zebrafish by injecting amyloid-beta into the ventricles of the zebrafish.

[0051] In one aspect, the injected amyloid-beta may be labeled (or tagged) with a fluorescent material.

[0052] In addition, in one aspect, the injected amyloid-beta may be in monomer form or oligomer form, but preferably may comprise amyloid-beta in oligomer form.

[0053] In one aspect, the amyloid-beta may include amyloid-beta 1-42.

[0054] In one aspect, the amyloid-beta-related disease mentioned above may include, but is not limited to, Alzheimer's disease.

[0055] The present invention will be specifically described below through experimental examples. These examples are merely for illustrating the present invention, and therefore the scope of the present invention is not limited by these examples.

[0057] [Preparation Example] Preparation of an amyloid-beta-induced Alzheimer's disease animal model

[0058] Zebrafish embryos were incubated at approximately 30–45°C. An amyloid-beta-induced Alzheimer's animal model was completed by injecting approximately 1–10 nL of oligomeric amyloid-beta (Aβ 1-42) into the ventricles of zebrafish larvae.

[0060] [Experimental Example] Screening of Alzheimer's disease therapeutic substances using an amyloid-beta-induced Alzheimer's disease animal model

[0061] [Experimental Example 1] Evaluation of the efficacy of an Alzheimer's disease treatment substance utilizing visual stimulus avoidance response

[0062] After transferring the animal model prepared in the example to a container such as a well plate, avoidance response behavior caused by visual stimulation was confirmed.

[0063] Specifically, groups treated with the candidate substance and groups treated with a therapeutic agent such as donepezil were prepared in the ventricles of amyloid-beta-induced Alzheimer's zebrafish.

[0064] As shown in Fig. 4, a well plate was set up by dividing half into a zone where a visual stimulus (red ball) is provided and the other half into a zone where no visual stimulus is provided. For each of the group treated with the candidate substance prepared above and the group treated with the therapeutic agent, the number of individuals that avoided the provided visual stimulus and moved to the opposite zone was counted for a certain period of time (approximately 60 minutes), and a ratio value for that number was derived. Visual stimulus

[0065] In addition, the time spent in the area where no visual stimulus was provided was measured when exposed to visual stimuli.

[0066] Afterwards, the measured values ​​of the group treated with the therapeutic agent and the group treated with the candidate substance were compared, and when a statistically significant result (p-value ≤ 0.01) was obtained, the candidate substance was evaluated as having efficacy against amyloid-beta-related diseases.

[0068] [Experimental Example 2] Evaluation of the efficacy of an Alzheimer's disease therapeutic substance using changes in the area where amyloid-beta is measured

[0069] Specifically, for the group treated with the candidate substance and the group treated with a therapeutic agent such as donepezil in the ventricles of amyloid-beta-induced Alzheimer's zebrafish, the zebrafish fry were anesthetized and fixed and imaged with a fluorescence microscope on the day of amyloid-beta injection and 2 to 10 days after injection, respectively.

[0070] Specifically, the area of ​​the fluorescently expressed region in the brain region corresponding to the ventricles was measured using Image J, and efficacy was evaluated based on whether the area of ​​the fluorescently expressed region decreased after treatment with the candidate substance and therapeutic agent.

[0071] As a result, it was confirmed that the area of ​​the fluorescence expression region was significantly reduced in the group treated with the therapeutic agent, and through this, it was confirmed that the animal model of the preparation example can be used to screen for the therapeutic agent for amyloid-beta-induced Alzheimer's disease.

[0073] Although the invention has been described above with reference to embodiments, this is merely illustrative and does not limit the invention. Those skilled in the art will understand that various modifications and applications not exemplified above are possible within the scope of the essential characteristics of the embodiments. For example, each component specifically shown in the embodiments may be modified and implemented. Furthermore, differences related to such modifications and applications should be interpreted as being included within the scope of the invention as defined in the appended claims.

Claims

Claim 1 A method for screening amyloid-beta-related disease treatments using zebrafish, wherein the method comprises the step (S1) of treating a candidate substance for amyloid-beta-related disease treatments to a zebrafish with an induced amyloid-beta-related disease; and the step (S2) of i) the behavior of the zebrafish after treatment with the candidate substance for disease treatments; and ii) measuring the amount of amyloid-beta present in the ventricles of the zebrafish, wherein measuring the behavior of the zebrafish includes measuring the avoidance response of the zebrafish to a provided visual stimulus, and measuring the behavior of the zebrafish includes the ratio of zebrafish individuals that avoid the area where the visual stimulus is provided and are active in an area where the visual stimulus is not provided; and measuring the time that the zebrafish active in the area where the visual stimulus is not provided stays in the area. Claim 2 delete Claim 3 A method for screening an amyloid-beta-related disease treatment using zebrafish, wherein the method further comprises a step (S3) of determining the efficacy of a candidate substance for an amyloid-beta-related disease treatment, wherein S3 comprises one or more of the following: a ratio of zebrafish individuals that avoid a zone where visual stimulation is provided and are active in a zone where visual stimulation is not provided; determining whether one or more of the time spent by zebrafish active in the zone where visual stimulation is not provided in the zone increases compared to a group not treated with the candidate substance for the disease treatment; and determining whether the amount of amyloid-beta present in the ventricles decreases compared to a group not treated with the candidate substance for the disease treatment. Claim 4 A method for screening an amyloid-beta-related disease treatment using a zebrafish, wherein the method further comprises the step (preS1) of injecting amyloid-beta into the ventricles of a zebrafish to induce an amyloid-beta-related disease in the zebrafish. Claim 5 A method for screening amyloid-beta-related disease treatments using zebrafish, wherein, in any one of claims 1, 3 and 4, the amyloid-beta-related disease includes Alzheimer's disease.

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