Amyloid β accumulation model animal

Abstract

[Problem] To provide amyloid β accumulation model cells. [Solution] A neurodegenerative disease model tissue and model animal, comprising brain cells derived from a non-human animal in which all or a part of a liver is replaced with human liver cells.

Description

Amyloid-β accumulation model animal

[0001] The present invention relates to a non-human animal with amyloid β (Aβ) accumulation, which can be used as an animal model for neurodegenerative diseases such as Alzheimer's disease.

[0002] The number of patients with Alzheimer's disease, which is considered the main cause of dementia, is increasing year by year worldwide and has become a major social concern. Therefore, the early establishment of therapeutic drugs, treatment methods, and prevention methods for Alzheimer's disease is desired. The detailed etiology of Alzheimer's disease is still unknown, but the commonly accepted theory regarding the cause, Aβ, has been that it originates from the brain (the theory that Aβ accumulation in the brain originates from the brain). However, in recent years, data has emerged that supports the hepatogenic theory (the theory that Aβ accumulation in the brain originates from the liver) (reference: Virginie Lam_doi.org / 10.1371 / journal.pbio.3001358).

[0003] To date, drug discovery based on the brain-origin theory of Aβ has not resulted in a complete cure for Alzheimer's disease. This suggests that an important etiology of Alzheimer's disease may be overlooked. Therefore, it is expected that recent drug discovery approaches based on the hepatic origin theory of Aβ may establish methods for preventing, treating, or treating Alzheimer's disease. On the other hand, when investigating methods for preventing, treating, or treating a specific disease, analysis is generally performed using model cells or model animals of that disease, but methods using conventional models do not necessarily produce useful results.

[0004] Manisha Prajapat_DOI 10.3389 / fnagi.2023.1296919Kaori Sato_doi.org / 10.1016 / j.jbc.2021.101004

[0005] In conventional Alzheimer's disease models, only a single or a few genes or proteins suspected to be involved in Alzheimer's disease have been focused on. Furthermore, because Alzheimer's disease is a brain disease, and the brain is isolated from the systemic blood circulation by a special barrier called the blood-brain barrier, attention has been limited to the interrelationships and intermediary factors between the brain and other organs. In order to develop better therapeutic or preventive drugs for Alzheimer's disease in the future, it is necessary to create a brain Aβ accumulation model that is based on the idea of ​​the hepatic origin of Aβ and also incorporates the interrelationships and intermediary factors between the brain and other organs.

[0006] As a result of intensive investigations to solve the above problems, the present inventors have found that a non-human animal in which all or part of the liver has been replaced with human hepatocytes can be used as the above-mentioned brain Aβ accumulation model, and have thus completed the present invention.

[0007] [1] A model tissue for a neurodegenerative disease, comprising brain cells derived from a non-human animal in which all or part of the liver has been replaced with human hepatocytes, the brain cells having amyloid beta accumulation. [2] The model tissue according to [1], in which the neurodegenerative disease is Alzheimer's disease. [3] The model tissue according to [1], in which the non-human animal is a mouse. [4] A model animal for a neurodegenerative disease, comprising a non-human animal in which all or part of the liver has been replaced with human hepatocytes. [5] The model animal according to [4], in which amyloid beta has accumulated in the brain. [6] The model animal according to [4], in which the neurodegenerative disease is Alzheimer's disease. [7] The model animal according to [4], in which the non-human animal is a mouse. [8] The model animal according to [4], in which the onset of a neurodegenerative disease has been accelerated by a high-fat diet and / or stress load. [9] The model animal according to [4], in which the animal has at least one finding selected from the group consisting of red and black stools, pale skin, and a decrease in an indicator reflecting red blood cell mass.

[10] The model animal according to [9], wherein the indicator reflecting the amount of red blood cells is hemoglobin and / or hemoglobin.

[11] A method for producing a model animal for a neurodegenerative disease, comprising the steps of replacing all or part of the liver of a non-human animal with human hepatocytes and obtaining an animal having accumulated amyloid beta in its brain.

[12] The method according to

[11] , comprising a step of raising the animal for at least 9 weeks after the replacement.

[13] The method according to

[11] , comprising a step of accelerating the onset of a neurodegenerative disease by a high-fat diet and / or stress loading.

[14] A method for screening for a therapeutic agent for a neurodegenerative disease, comprising contacting a candidate substance with the model tissue according to any one of [1] to [3], the model animal according to any one of [4] to

[10] , or the model animal produced by the method according to any one of

[11] to

[13] , and using the amount of accumulated amyloid beta as an indicator.

[15] A method for elucidating the cause of a neurodegenerative disease, comprising the steps of raising the model animal according to any one of [4] to

[10] or a model animal produced by the method according to any one of

[11] to

[13] under specified rearing conditions, evaluating the effect on the neurodegenerative disease, and identifying factors that have an effect on the neurodegenerative disease.

[0008] The present invention provides a brain tissue model and a non-human animal model of Aβ accumulation, which enables screening of preventive and therapeutic drugs and elucidation of the causes of not only Alzheimer's disease but also other neurodegenerative diseases caused by interactions with the liver.

[0009] FIG. 1 is a diagram showing the results of immunostaining of amyloid β accumulated in the brain of a mouse. FIG. 2 is a diagram showing the results of immunostaining of amyloid β accumulated in the brain of a mouse. FIG. 3 is a diagram showing the results of immunostaining of amyloid β accumulated in the brain of a mouse. FIG. 4 is a diagram showing the results of immunostaining of amyloid β accumulated in the brain of a human. FIG. 5 is a diagram showing the correlation between human Aβ (1-42) and human Aβ (1-40). FIG. 6 is a diagram showing the scores of the Nesting Test.

[0010] 1. Overview The present invention relates to a model tissue of Aβ accumulation, which comprises brain cells of a non-human animal in which all or part (e.g., at least 70%) of the liver has been replaced with human hepatocytes, and to a model animal of a neurodegenerative disease comprising said non-human animal.

[0011] Based on the hepatogenic hypothesis of Aβ, PXB mice transplanted with human hepatocytes are expected to contain Aβ and related factors associated with Alzheimer's disease derived from the hepatocytes. ELISA measurements confirmed the presence of human Aβ in the blood of PXB mice. Furthermore, while most PXB mice remain healthy as experimental animals throughout the experimental period, we focused on PXB mice that exhibited behavioral and postural abnormalities in general. Their brains were formalin-fixed, paraffin-embedded, and immunostained with a human Aβ-specific antibody. Positive images of human Aβ were confirmed around the cerebral blood vessels and in the brain parenchyma of the mice.

[0012] The present invention aims to differentiate from a known mouse model in which the human amyloid precursor protein gene is forcibly expressed in the liver specifically (doi: 10.1371 / journal.pbio.3001358), and was completed based on the finding that human Aβ accumulation in the brain occurs in non-human animals that have undergone human hepatocyte transplantation, rather than by introducing and forcibly expressing a single gene. Specifically, the present invention has been developed to differentiate non-human animals in which some or all of the hepatocytes in the liver have been replaced with human hepatocytes (referred to as "human hepatocyte chimeric non-human animals"), which secrete human Aβ from human hepatocytes into the blood, and in individuals that meet the conditions described below, reproduce the characteristic of Alzheimer's disease in which Aβ crosses the blood-brain barrier and reaches the brain, where it accumulates. Furthermore, the present invention has been found to be useful as a neurodegenerative disease model, as PXB mice in which human Aβ has accumulated in the brain exhibit abnormal behavior and posture.

[0013] 2. Amyloid β Accumulation Model Animal In the present invention, the animal from which the human Aβ accumulation model tissue is derived is a human hepatocyte chimeric non-human animal, and the brain tissue of this animal is used as the model tissue. The "non-human animal" is preferably a mammal, more preferably a rodent. Examples of rodents include mice, rats, guinea pigs, squirrels, and hamsters, with mice or rats being preferred, as they are commonly used as experimental animals. A human hepatocyte chimeric non-human animal can be obtained by transplanting human hepatocytes into a liver-damaged immunodeficient non-human animal in accordance with known techniques (e.g., JP 2002-45087 A).

[0014] Typically, a liver-damaging immunodeficient non-human animal can be produced by subjecting a genetically immunodeficient non-human animal to a treatment for inducing liver damage, or by subjecting an animal with genetic liver damage to a treatment for inducing immunodeficiency. Examples of genetically immunodeficient animals include SCID mice, NUDE mice, RAG2 knockout mice, NOD mice, and NOG mice. Examples of genetically liver-damaged animals include transgenic animals into which a liver damage-inducing protein gene (e.g., uPA gene, tPA gene) has been introduced and which is linked under the control of an enhancer and / or promoter of a protein that is expressed specifically in hepatocytes.

[0015] In the present invention, the "human hepatocytes" to be transplanted into liver-damaged immunodeficient non-human animals may be hepatocytes of human origin. For example, human hepatocytes isolated from human liver tissue by standard methods such as collagenase perfusion can be used. The human liver tissue may be liver tissue from a healthy individual or from a patient suffering from diseases such as fatty liver or liver cancer, but is preferably liver tissue from a healthy individual. The collected human hepatocytes can be used as is, or they may be purified using a monoclonal antibody that specifically recognizes human hepatocytes or hepatocytes of a non-human animal. Furthermore, in the present invention, hepatocytes that have been reprogrammed by gene editing and then induced to differentiate, such as human iPS cells, can also be used.

[0016] Human hepatocytes are transplanted into liver-damaged immunodeficient non-human animals by transplantation into the liver via the spleen of the non-human animal or directly via the portal vein. The non-human animals obtained by the above-mentioned method have all or part (e.g., at least 70%) of their livers replaced with human hepatocytes. A commercially available mouse ("PXB mouse," Phoenix Bio Inc.) with at least 70% of its liver replaced with human hepatocytes can also be used.

[0017] 3. Model Animals and Model Tissues The model animals of the present invention can be obtained by raising the human hepatocyte chimeric non-human animals prepared as described above. For example, the human hepatocyte chimeric non-human animals are raised for at least 9 weeks after transplantation (after replacement of mouse hepatocytes with human hepatocytes), preferably from 9 to 49 weeks (12 to 52 weeks of age).

[0018] From the animals bred as described above, animals with one or more of the following abnormal findings are selected: Animals with reduced cognitive functions, including learning functions, compared to wild-type animals (also referred to as control animals; the same applies below); Animals that exhibit abnormal posture or behavior compared to wild-type animals; Animals with pale skin; Animals that exhibit red or black stools, or brown or dark brown stools with blood, compared to brown or dark brown stools of wild-type animals.

[0019] These animals have reduced indices reflecting red blood cell mass, such as hemoglobin, hematocrit, and hemoglobin, compared to wild-type animals. For example, hemoglobin levels are 10.0 g / dL or less, hematocrit levels are 30% or less, and hemoglobin levels are 14.0 pg or less. These animals also have a reduced blood human Aβ(1-42) / human Aβ(1-40) ratio compared to wild-type animals. For example, the human Aβ(1-42) / human Aβ(1-40) ratio, measured in pmol, is 0.06 or less.

[0020] The model tissue of the present invention can be collected from the brain tissue of a human hepatocyte-chimeric non-human animal having the above findings.

[0021] The model tissue of the present invention can be cultured and maintained in a manner similar to the previously reported adult mouse brain slice culture (DOI: 10.21769 / BioProtoc.3869). For example, 300 μm-thick brain tissue slices cut with a vibratome or similar device can be maintained or passaged in a cell culture dish, tissue culture dish, multi-dish, or microplate equipped with a cell culture insert (e.g., made of hydrophilic PTFE with a pore size of 0.4 μm) using serum-free medium for brain tissue slices (e.g., Neuroblast A medium, etc.), optionally supplemented with supplements (e.g., B-27), amino acids, antibiotics, anti-inflammatory components, test substance solvents, etc.

[0022] The model tissue of the present invention can be obtained 0 to 35 days, preferably 0 to 21 days, after collection from the hepatocyte chimeric non-human animal. Furthermore, brain tissue collected from the hepatocyte chimeric non-human animal is characterized by excessive accumulation of human Aβ in brain cells and blood vessels, and is therefore considered to reproduce neurodegenerative diseases. "Excessive human Aβ accumulation" refers to a state in which extraneuronal accumulation of human Aβ and associated tissue changes are observed, such as intraneuronal phosphorylated tau accumulation, neuronal apoptosis and neuronal loss, and human Aβ accumulation in blood vessel walls, in addition to extraneuronal accumulation of human Aβ.

[0023] Examples of neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and polyglutamine diseases (Huntington's disease and spinocerebellar ataxia).

[0024] 4. Screening Method The screening method of the present invention is a method for screening therapeutic or preventive agents for Alzheimer's disease by contacting a candidate substance with a model tissue or model animal of the present invention, detecting human Aβ that accumulates in the brain tissue after contact, or human Aβ genes or proteins derived from the animal's human hepatocytes, and using the detection results obtained as an index. "Contacting" refers to exposing the model tissue or model animal of the present invention to a candidate substance, and examples of such contact include culturing the model tissue in the presence of the candidate substance or administering the candidate substance to the model animal.

[0025] Furthermore, the screening method of the present invention can include a method comprising the steps of (a) administering a test substance to a non-human animal of the present invention, and (b) evaluating the therapeutic and / or preventive effect against a neurodegenerative disease. The step (b) of evaluating the therapeutic effect against a neurodegenerative disease can include, for example, administering the test substance to a model non-human animal of the present invention exhibiting symptoms of various neurodegenerative diseases (e.g., Alzheimer's disease), and assessing the therapeutic effect against the disease using as indicators not only human Aβ accumulation in the brain but also phosphorylated tau accumulation in neurons, neuronal apoptosis and loss, human Aβ accumulation in vascular walls, the amount of the synaptic protein PSD-95, synaptic morphology and function, spatial memory and spatial working memory ability, coordinated movement, and motor learning.

[0026] Furthermore, the present invention involves adding a candidate substance to the culture medium of the model tissue of the present invention, and then measuring and evaluating not only human Aβ accumulated in the tissue, but also phosphorylated tau accumulation in neurons, neuronal apoptosis and neuronal loss, human Aβ accumulation in vascular walls, PSD-95, etc. The fixed period for adding the candidate substance is, for example, about 0 to 21 days after collection, but is not particularly limited thereto.

[0027] Furthermore, the step of evaluating the preventive effect against neurodegenerative disease in step (b) above can be exemplified by a method in which the test substance is administered to a model animal of the present invention before the animal exhibits symptoms of neurodegenerative disease, and the preventive effect against the onset of neurodegenerative disease is assessed using as indicators not only human Aβ accumulation in the brain but also phosphorylated tau accumulation in neurons, neuronal apoptosis and loss, human Aβ accumulation in blood vessel walls, the amount of PSD-95, synaptic morphology and function, spatial memory and spatial working memory ability, coordinated movement and motor learning.

[0028] Detection of human Aβ, mouse PSD-95, mouse phosphorylated tau, and apoptosis can be performed using immunohistochemistry (also known as immunostaining), RT-PCR, Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and other methods, either alone or in combination. Synaptic morphology and function are measured using morphological assessments using silver staining or Fluoro-Jade staining, and functional assessments based on synaptic plasticity. Spatial memory and spatial working memory abilities are measured using the Morris water maze test, Barnes circular maze test, and 8-arm radial maze test. Coordination and motor learning abilities are measured using the rotarod test, beam test, manual ladder walking test, and other methods.

[0029] The candidate substances to be evaluated are not particularly limited, and examples thereof include food-derived components, various natural or artificially synthesized peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotides (DNA, RNA), oligonucleotides (siRNA, etc.), peptide nucleic acids (PNA), etc.), low-molecular-weight or high-molecular-weight organic compounds, etc.

[0030] After contact with a candidate substance, in the model non-human animal of the present invention, 1) the blood human Aβ concentration is reduced compared to the control, 2) the blood human Aβ concentration is reduced or becomes below the detection limit, 3) the amount of human Aβ in brain tissue is reduced compared to the control, 4) the amount of human Aβ in brain tissue is reduced compared to the control or is undetectable, 5) the amount of mouse PSD-95 in brain tissue is increased compared to the control, 6) the amount of mouse PSD-95 in brain tissue is increased compared to the control or exceeds the upper limit of quantitation, 7) the amount of mouse phosphorylated tau in brain tissue is reduced compared to the control, 8) the amount of mouse phosphorylated tau in brain tissue is reduced compared to the control or is undetectable, 9) neuronal cell death, as indicated by TUNEL staining or the like, is reduced compared to the control. 10) neuronal cell death, as indicated by TUNEL staining or the like, is reduced or undetectable compared to controls; 11) the number of synapses that are positive for silver staining or Fluoro-Jade staining is increased compared to controls; 12) changes in parameters that indicate synaptic plasticity, such as long-term potentiation and long-term depression, are improved compared to controls; 13) results in the Morris water maze test, Barnes circular maze test, and 8-arm radial maze test are improved compared to controls; or 14) results in the rotarod test, beam test, and manual ladder walking test are improved compared to controls, the candidate substance is considered to have been able to suppress the accumulation of human Aβ, and can be evaluated as being usable as a therapeutic or preventive agent for neurodegenerative diseases (e.g., Alzheimer's disease).

[0031] Furthermore, in the model tissue of the present invention, if the results shown in 3) to 12) above are obtained, the candidate substance is considered to have an effect of improving human Aβ accumulation, and can be evaluated as being usable as a therapeutic or preventive agent for neurodegenerative diseases (e.g., Alzheimer's disease).

[0032] 5. Method for Elucidating the Etiology of the Present Invention The method for elucidating the etiology of the present invention comprises the steps of raising a model animal of the present invention under specified rearing conditions, assessing the effect on neurodegenerative disease, and identifying factors that affect the neurodegenerative disease. Specifically, the method for elucidating the etiology of the present invention involves setting various rearing conditions for the non-human animal of the present invention, detecting human Aβ accumulated in the brain tissue of the animal after the setting, or human Aβ genes or proteins derived from the animal's human hepatocytes, and using the detection results as an indicator to evaluate the effect on Alzheimer's dementia, followed by identifying factors involved in the effect. Rearing conditions include the number of animals housed in the same cage, the presence or absence of toys, exercise, sleep, food, drinking water, etc., and further include the daily regularity of activities such as exercise, sleep, food, and drinking water.

[0033] Specifically, the method for elucidating the etiology of the present invention can include the steps of (c) rearing the non-human animal of the present invention under specified rearing conditions, (d) evaluating the effect on Alzheimer's dementia, and (e) identifying factors that have influenced Alzheimer's dementia. The step (d) of evaluating the effect on Alzheimer's dementia can be carried out in the same manner as step (b) shown in the screening method of the present invention. The subsequent step (e) of identifying factors that have influenced Alzheimer's dementia can be exemplified by a method in which changes in genes, proteins, etc. are analyzed using the biological sample derived from the non-human animal obtained in step (d) to narrow down candidate causes, and then the effect of these candidates on Alzheimer's dementia is verified.

[0034] Narrowing down of etiological candidates based on changes in genes, proteins, etc. can be performed using serum, plasma, cerebrospinal fluid, as well as organ and tissue extracts, by using microarrays, RT-PCR, Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, etc., alone or in appropriate combinations. Methods for verifying the effect of the narrowed down etiological candidates on Alzheimer's dementia include: 1) exogenously administering the etiological candidate to the non-human animal of the present invention or forcibly expressing a gene; and 2) administering a factor that degrades or antagonizes the etiological candidate to the non-human animal of the present invention or suppressing gene expression. The verification method can be performed in the same manner as step (b) shown in the screening method of the present invention.

[0035] 6. Treatment for accelerating the onset of neurodegenerative disease Furthermore, in the present invention, when producing the model animal, the onset of disease can be accelerated by feeding the animal a high-fat diet and / or imposing various stresses. "Treatment for accelerating" the onset means hastening the onset and / or aggravating the neurodegenerative disease by feeding the animal a high-fat diet and / or imposing various stresses on the animal.

[0036] <Method 1> Feeding a high-fat diet (1) Animals used The animals used are preferably animals that have undergone transplantation of human hepatocytes, and are preferably animals that have been transplanted for 9 weeks (12 weeks of age) since transplantation.

[0037] (2) Method: Feed transplanted animals a high-fat diet. Feeding should begin at least 9 weeks after transplantation, preferably after 12 weeks of age. The diet should contain at least 30% fat of total calories, preferably 30-60%. The feeding period should be at least 4 weeks, preferably 4-40 weeks of age (16-52 weeks of age). The high-fat diet is placed in the animal cage and the animals are allowed to consume it ad libitum. <Reference Information> It is known that feeding Alzheimer's disease model mice into which human Aβ has been introduced by gene editing a high-fat diet for 3 months exacerbates Alzheimer's disease symptoms (DOI: 10.1038 / s41598-017-04412-2).

[0038] <Method 2> Stress loading: Stress is applied to the transplanted animal. Stress loading should begin at least after transplantation, preferably after 9 weeks (12 weeks of age). For example, the following stresses can be applied to the animal alone or in combination with two or more stresses. The following stress loading method will be explained using mice as an example, but it can also be applied to other animals.

[0039] (i) Mice are anesthetized using isoflurane. Anesthesia is stressful in mice because it increases the blood concentration of corticosterone, a type of stress hormone. The range of anesthesia conditions is a concentration of 1-4% (anesthetic flow rate of 150-600 mL / min) and anesthesia time of 1 minute to 1 hour. Preferred anesthesia conditions are a concentration of 2% (any flow rate within the above range) and anesthesia time of 2 minutes. The frequency of anesthesia is preferably at least once a day, but may be more frequent. The duration of anesthesia is at least one day per week, preferably five days per week.

[0040] (ii) Glass marbles are placed in the mouse cage. Glass marbles cause stress in mice by eliciting anxiety and / or aversion and by being perceived as potentially life-threatening objects. Examples of glass marbles include marbles. The number of glass marbles placed is not particularly limited, but is at least 1 per 200 cm2 of floor area of ​​the animal cage, preferably 1 per 9 cm2. The size of the glass marbles is at least 1.0 cm in diameter, preferably 1.6 cm in diameter. The period during which the glass marbles are placed in the mouse cage is at least 24 hours, preferably 120 hours twice (total of 240 hours).

[0041] (iii) Confining a mouse in a cylindrical device to restrain its body movements. Except when asleep, mice tend to move around for voluntary activities such as feeding and exploring. Therefore, restraining a mouse's body movements is stressful because it induces aversion, pain, and / or anxiety due to the restriction of voluntary activity. "Restraint" refers to a state in which a mouse's body is completely immobile while awake, a state in which the mouse's voluntary activity is restricted in space, or a state in which the movement of a portion of the mouse's body is restricted. Examples of methods for restraining a mouse include attaching its limbs to a restraining table (a plate-like device), placing the mouse in a conical tube (a cylindrical device), or having the mouse wear a restraining suit. The method of placing the mouse in a conical tube (a cylindrical device) is preferred. The restraint time is at least one minute, preferably five minutes. The frequency of restraint is preferably once a day, but may be more frequent. The duration of restraint is at least one day per week, preferably two days per week.

[0042] (iv) Removal of bedding from the mouse cage. Bedding not only provides a place for mice to hide and rest from the outside world, but also facilitates thermoregulation by utilizing its insulating effect and minimizes the effects of changes in environmental temperature. Removal of bedding causes psychological stress to mice due to the inability to hide, as well as physical stress due to changes in environmental temperature. The bedding removal time is at least one hour, preferably six hours. Bedding removal is preferably performed once a day, but may be performed more frequently. The duration of bedding removal is at least one day, preferably five days.

[0043] (v) Apply adhesive tape to the hind limbs of a mouse. Animal fur functions as a sensory organ. A foreign object attached to a mouse's fur can cause discomfort, but a foreign object that cannot be easily removed can be stressful for the mouse. Examples of foreign objects attached to the fur include adhesive tape, with a width of at least 1 mm, preferably 5 mm. The adhesive tape is applied to a site that cannot be easily removed, or in a manner that does not allow for easy removal. For example, the tape is applied to the area between the fibula and tibia and the tarsal bones of either the left or right hind limb, wrapping the tape around twice. The adhesive tape is applied for at least one hour, preferably six hours. The adhesive tape is preferably applied once a day, but may be applied more frequently. The adhesive tape is applied for at least one day per week, preferably two days per week.

[0044] (vi) Tilting the mouse cage. Normal animals have a sense of balance, so they feel uneasy when resting in a slanted position. Excessive tilting also affects voluntary activity, causing stress for the animal. The tilt angle of the mouse cage is 5 to 45 degrees, preferably 6 degrees. The tilt angle is maintained for at least 1 hour, preferably 120 hours.

[0045] (vii) A fasting period is established by removing food from the mouse cage, but water is provided. Mice consume approximately 1 to 5 g of food per day, and fasting from some or all of this food is stressful for the mice because it causes hunger and makes them feel hungry. The fasting period is 2 to 8 hours per day, preferably 6 hours. The fasting frequency is preferably once a day, but may be more frequent. The fasting period lasts at least one day per week, preferably five days per week.

[0046] The type of stress to be applied during the loading period is at least one type per week, preferably one to seven types per week, and the above stresses can be combined as appropriate.

[0047] <Reference information> It has been mentioned that stress may hasten the onset of Alzheimer's disease or worsen the condition (https: / / doi.org / 10.1016 / j.ynstr.2018.04.002).

[0048] <Method 3> Evaluation of the onset of neurodegenerative diseases Evaluation of the onset of neurodegenerative diseases can be performed, for example, by a nesting test. A standard paper material is placed in a mouse cage, and after a certain period of time, the degree of unraveling of the paper material and the quality of the resulting nest are quantified on a scale of 1 (not at all unraveled, no nest formed) to 5 (completely unraveled, a three-dimensional nest large enough to cover the mouse's body). The criteria for quantification are, for example, as follows (Deacon's evaluation method): Score 1: 90% or more of the nestlet shape remains Score 2: 50-90% of the nestlet shape remains Score 3: The nestlets are mostly unraveled, but no nest has formed Score 4: The nestlets are unraveled and gathered in one place, but the nest is flat Score 5: A nest has formed large enough to cover the mouse's body

[0049] For example, Nestlets (Ancare, Bellmore, NY, USA) can be used as the paper material. The paper material is placed in the mouse cage for at least 12 hours, preferably 12 to 168 hours.

[0050] <Reference Information> For details of the nesting test, please refer to the method by Deacon (doi: 10.3791 / 2607).

[0051] EXAMPLES The present invention will be explained in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0052] 1. Materials and Methods (1) Immunostaining of amyloid beta deposited in the brains of PXB mice. Reagents used: Amyloid beta antibody (Immuno-Biological Laboratories Co., Ltd. 11110), biotin-labeled anti-mouse Ig rabbit antibody (Nichirei 426032), peroxidase-labeled streptavidin (DAKO P0397).

[0053] Staining procedure i) Deparaffinization ii) Hydrogen peroxide treatment iii) Rinsing with running water iv) Buffer washing v) Bovine albumin block 30 minutes vi) Anti-amyloid β antibody reaction x 100 4℃, overnight vii) Buffer washing viii) Biotin-labeled anti-mouse Ig rabbit antibody x 500 37℃, 30 minutes ix) Buffer washing x) Peroxidase-labeled streptavidin x 500 37℃, 30 minutes xi) Buffer washing xii) DAB color reaction xiii) Rinsing with running water xiv) Hematoxylin nuclear staining 5 minutes xv) Dehydration, clearing, and mounting

[0054] (2) ELISA Measurement of Aβ (Types 42 and 40) Circulating Throughout the System of PXB Mice ELISA measurement of Aβ (Types 42 and 40) circulating through the system of PXB mice was performed using commercially available kits (Human β-Amyloid (1-40) ELISA Kit Wako II and Human β-Amyloid (1-42) ELISA Kit Wako, high sensitivity).

[0055] Preparation of Reagents: The antibody (BAN50)-immobilized microplate was used as is. A dilution series was prepared by diluting the standard solutions (human β-amyloid (1-40), 100 pmol / L, and human β-amyloid (1-42), 20 pmol / L) with standard diluent. The wash solution (20x) was diluted 20 times with deionized water. The HRP-labeled antibody (BA27 solution) (for human β-amyloid (1-40)) and the HRP-labeled antibody (BC05 solution) (for human β-amyloid (1-42)) were used as is. The TMB solution, stop solution, and plate seal were used as is.

[0056] Measurement Procedure: (i) The microplate was allowed to return to room temperature before being removed from the aluminum pack. The wells to be used were removed. (ii) 100 μL of standard dilution was added to the sample blank wells. (iii) 100 μL each of the standard and sample (plasma) was added to the wells. (iv) The plate was sealed and incubated overnight in a refrigerator (4°C). (v) The plate seal was removed, and the liquid in the wells was aspirated using a plate washer or aspirator. 300 μL of washing solution was dispensed into each well and aspirated. This washing procedure was repeated four more times. (vi) 100 μL of labeled antibody was added to each well, and the incubation was continued for 2 hours (human β-amyloid (1-40)) or 1 hour (human β-amyloid (1-42)) in a refrigerator (4°C). (vii) The washing procedure was repeated five times in the same manner as in (v) above. (viii) 100 μL of TMB solution was added to each well, and the incubation was continued for 30 minutes in the dark at room temperature. (ix) 100 μL of stop solution was added to each well to stop the enzyme reaction. (x) The absorbance of the test sample, standard, and test sample blank was measured at 450 nm within 30 minutes. (xi) The concentration of human β-amyloid (1-40) or human β-amyloid (1-42) was calculated from the standard curve.

[0057] (3) Dissection and Clinical Observations The condition (posture, abnormal behavior) of the PXB mice was observed. After administering inhalation anesthesia, the mice were euthanized by cardiac exsanguination, and the following items were observed.

[0058] Macroscopic findings such as color and size of each organ

[0059] 2. Results (1) Immunostaining of Aβ Deposited in the Brain of PXB Mice The results of immunostaining of Aβ are shown in Figures 1 to 3. Figure 1 shows the results of immunostaining of Aβ in the brain of an individual with transplant number HSC0050-0151 shown in Table 1 below.

[0060] In Figure 1, the letters "A" and "B" represent the basal ganglia / perithalamic region and the hypothalamic region, respectively. "#1" to "#7" represent tissue sections numbered sequentially from the olfactory bulb side when the brain was cut into seven 2-mm-wide sections in the coronal plane. The upper panel shows hematoxylin and eosin staining, and the lower panel shows the results of Aβ immunostaining. Panel #3-A shows the stained image of the basal ganglia / perithalamic region in tissue section #3, panel #4-B shows the stained image of the hypothalamic region in tissue section #4, and panel #5-B shows the stained image of the hypothalamic region in tissue section #5. These results demonstrate the accumulation of Aβ in the brain tissue of PXB mice.

[0061] Figures 2 and 3 show the results of immunostaining for Aβ in the brain of an individual with transplant number HSC0033-0067, shown in Table 1 below. In Figures 2 and 3, the letters "A," "B," and "C" represent the cerebral cortex region, the periventricular region of the third ventricle, and the perilateral ventricle region, respectively. "#1" to "#7" represent tissue sections numbered sequentially from the olfactory bulb side when the brain was cut into seven 2-mm-wide slices in the coronal plane. Panel #3-A shows a stained image of the cerebral cortex region of tissue section #3, panel #4-B shows a stained image of the periventricular region of tissue section #4, and panel #4-C shows a stained image of the perilateral ventricle region of tissue section #4. The results in these figures demonstrate the accumulation of Aβ in the brain tissue of PXB mice.

[0062] Furthermore, Figure 4 shows the results of immunostaining human brain tissue with the Aβ antibody used in Figures 1, 2, and 3. The results in Figure 4 demonstrate that the Aβ antibody used in immunostaining also detects Aβ in human brain tissue. These results demonstrate that the Aβ detected in PXB mouse brain tissue is human Aβ.

[0063] (2) ELISA Measurement Results for Human Aβ (42 Types and 40 Types) The ELISA measurement results for Aβ (42 Types and 40 Types) are shown in Table 1.

[0064] Table 1 shows that human Aβ was not detected in the plasma of SCID231219-4 mice that had not been transplanted with human hepatocytes, but was detected in the plasma of PXB mice that had been transplanted with human hepatocytes. Furthermore, the correlation between human Aβ(1-42) and human Aβ(1-40) is shown in Figure 5. Figure 5 shows that the plasma concentrations of human Aβ(1-42) and human Aβ(1-40) have a positive correlation with an R value of 0.9 or greater.

[0065] (3) Clinical Observation and Necropsy In the case of HSC0050-0151 (24 weeks old at the time of necropsy), general observation at 24 weeks of age revealed recumbency, general paleness, decreased body temperature, decreased activity, and black stools, and the animal was deemed moribund and euthanized. At necropsy, the liver was pale-colored, and dark red contents were observed in the digestive tract. In the case of HSC0033-0067 (42 weeks old at the time of necropsy), general observation revealed a tilted posture from 39 weeks of age, and death was discovered at 42 weeks of age, so an immediate necropsy was performed. At necropsy, enlarged thymus and spleen were observed, as well as white spots / areas in the liver.

[0066] These results suggest that HSC0050-0151 had internal bleeding, as evidenced by black stool and dark red contents of the digestive tract, and that the spleen, a hematopoietic organ, may have enlarged to compensate for the internal bleeding in HSC0033-0067. The bleeding was caused by disruption of the vascular structure, and these two animals also had disruption of the cerebral vascular structure, resulting in the accumulation of human Aβ in the brain.

[0067] Ten male PXB mice (aged 20 weeks or older at the start of the experiment (Day 0)) were divided into two groups of five, one of which was an untreated control, and the other was fed methods 1 to 3 described above. Results are shown here. HFD60 (60% of total calories from fat) was used as the high-fat diet. The feeding period was 12 weeks, from Day 0 to Day 84. The following stress treatments were used: Stress loading was performed from Day 28 to Day 84, and the mice were housed in nestlets for one week.

[0068] Week of Day 28: Mice were anesthetized using isoflurane (5 days from Day 28-32, once a day for 2 minutes, anesthesia at a concentration of 2%) Week of Day 35: Glass marbles were placed in the mouse cage (120 hours from Day 35-39, capacity 1 marble / 9 cm2, glass marble diameter 1.6 cm) Week of Day 42: Mice were placed in a cylindrical device to restrain their body movement (2 days from Day 43 and 45, once a day for 5 minutes) Week of Day 49: Bedding was removed from the mouse cage (5 days from Day 49-53, once a day for 6 hours) Week of Day 56: Adhesive tape was applied to the mouse's hind legs (2 days from Day 57 and 59, once a day for 6 hours, applied to the right hind leg) Week of Day 63: The mouse cage was tilted (Day Week of Day 70: Food was removed from the mouse breeding cage to allow for a fasting period (Days 70-74, 5 days, once a day, 6 hours of fasting) Week of Day 77: Glass marbles were placed in the mouse breeding cage (Days 77-81, 120 hours, capacity 1 marble / 9 cm², glass marble diameter 1.6 cm)

[0069] Statistical analysis was performed using Tukey HSD multiple comparisons (IBM SPSS Statistics 30). The significance level was <0.05 (indicated by *). On Day 84 (evaluation the week after Day 77), the high-fat diet and stress group had significantly lower scores on the Nesting Test than the control group (Tables 2 and 3, Figure 6).

[0070]

Claims

1. A model tissue for neurodegenerative diseases, comprising brain cells derived from a non-human animal in which all or part of the liver has been replaced with human hepatocytes, the brain cells having accumulated amyloid beta.

2. The model tissue according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease.

3. The model tissue according to claim 1, wherein the non-human animal is a mouse.

4. A model animal for neurodegenerative diseases consisting of a non-human animal in which all or part of the liver has been replaced with human hepatocytes.

5. The model animal of claim 4, in which amyloid beta has accumulated in the brain.

6. The model animal according to claim 4, wherein the neurodegenerative disease is Alzheimer's disease.

7. The model animal according to claim 4, wherein the non-human animal is a mouse.

8. The model animal of claim 4, in which the onset of neurodegenerative disease is accelerated by a high-fat diet and / or stress loading.

9. The model animal according to claim 4, which has at least one finding selected from the group consisting of red and black stools, pale skin, and a decrease in an index reflecting red blood cell mass.

10. The model animal according to claim 9, wherein the indicator reflecting the amount of red blood cells is hemoglobin and / or hemoglobin.

11. A method for producing a model animal for a neurodegenerative disease, comprising the steps of replacing all or part of the liver of a non-human animal with human hepatocytes, and obtaining an animal in which amyloid beta has accumulated in its brain.

12. The method of claim 11, further comprising the step of rearing the animals for at least 9 weeks after replacement.

13. The method according to claim 11, comprising a step of accelerating the onset of a neurodegenerative disease by a high-fat diet and / or stress exposure.

14. A method for screening therapeutic agents for neurodegenerative diseases, comprising contacting a candidate substance with the model tissue according to any one of claims 1 to 3, the model animal according to any one of claims 4 to 10, or the model animal prepared by the method according to any one of claims 11 to 13, and using the amount of accumulated amyloid beta as an indicator.

15. A method for elucidating the cause of a neurodegenerative disease, comprising the steps of raising a model animal according to any one of claims 4 to 10, or a model animal produced by the method according to any one of claims 11 to 13, under specified rearing conditions, evaluating the effect on the neurodegenerative disease, and identifying factors that have an effect on the neurodegenerative disease.

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