JP5876664B2 - Method for screening therapeutic agent for Alzheimer's disease and transgenic non-human animal - Google Patents

Description

The present invention is a screening method for Alzheimer's disease therapeutic agents, and to a transgenic non-human animal used in the screening methods.

  Dementia is a common disease that affects about 25% of the Japanese population over the age of 85, and Alzheimer's disease accounts for about half of all. Alzheimer's disease (AD) is a type of dementia whose main symptoms are cognitive decline and personality changes. Currently, there are approximately 1.6 to 1.8 million Alzheimer's disease patients in Japan, and the number of patients will continue to increase as the population ages.

  The amyloid cascade theory has been proposed as a mechanism of the onset of Alzheimer's disease. The amyloid cascade is triggered by amyloid accumulation associated with aging, and is a complex pathway that leads to inflammatory responses, accumulation of abnormal protein tau in neurons, and ultimately neuronal dysfunction and degeneration (cell death). It is a designation. That is, as described in Non-Patent Document 1 and Non-Patent Document 2, amyloid β precursor protein (APP) generates amyloid β protein (Aβ), and the amyloid β protein aggregates and deposits to cause brain neurons. It is believed that it may be causing destruction and cranial nerve loss.

  Three types of Aβ are known, Aβ40, Aβ42 and Aβ43, depending on the number of amino acids. Even though Aβ is responsible for the onset of Alzheimer's disease, it is not clear how neuronal cell death is induced. Toxicity studies using cultured cells have shown that monomers have low cytotoxicity and are highly toxic when aggregated into multimers.

  Currently, there is an active development of treatments for Alzheimer's disease. Inhibition of β-secretase and γ-secretase that produce amyloid β to reduce production from the recognition that neurodegeneration proceeds due to neurotoxicity of Aβ, particularly Aβ42, activation of neprilysin that degrades Aβ extracellularly Agents, Aβ polymerization inhibitors, antibody therapy against Aβ and the like have been developed.

  As a screening method for Alzheimer's disease therapeutic agents, Patent Document 1 describes a screening method for a brain function improving agent that administers indomethacin to Wistar rats, conducts a water maze test, and examines the time to reach the platform in the indomethacin administration group. ing. A screening method is also described in which healthy adults are given indomethacin and presented randomly selected numbers to examine how much they are remembered. However, with the former method, it is difficult to accurately determine the time to reach the platform because it depends on the muscle strength of each individual rat. Further, even in the latter method, it is difficult to make an accurate determination because the memory capacity differs from one to another.

  Patent Document 2 discloses that a test substance is brought into contact with a β-amyloid peptide having a turn structure between β-sheet structures based on amino acid sequences before and after the amino acid residues at positions 22 and 23 of Aβ42, and the aggregation activity thereof is measured. A method of measuring is described. In this method, a test substance and thioflavin T are added to this β-amyloid peptide and incubated, and a substance that lowers the fluorescence intensity is screened.

JP 2008-088122 A JP 2006-265189 A

Yankner BADuffy LK, Kirschner DA. 1990.Neurotrophic and neurotoxic effects of amyloid beta protein: Reversal by tachykinin neuropeptides.Science 250: p.279-p.282. Hyman BT. 1998. New neuropathological criteria for Alzheimer disease. Arch. Neural. 55: p.1174-p.1176.

  However, this method is a screening method at the enzyme level, and a screening method at the individual level is desired.

  The present invention has been made in view of such problems, and an object of the present invention is to provide a screening method for an accurate therapeutic agent for dementia at the individual level. Moreover, it aims at providing the transgenic non-human animal used for the screening method.

The method for screening a therapeutic agent for Alzheimer's disease according to the first aspect of the present invention comprises: a) Alzheimer's disease- related gene which is a gene encoding amyloid β protein (Aβ), amyloid β precursor protein (APP), or Tau protein An expression vector carrying the gene is introduced into the inner ear hair cells of the non-human animal to be tested to produce a transgenic non-human animal expressing the Alzheimer's disease- related gene, and b) the Alzheimer's disease- related gene is A candidate compound is administered to the expressed transgenic non-human animal, c) the hearing of the transgenic non-human animal administered the candidate compound is detected, and d) compared to a transgenic non-human animal not given the candidate compound , Waves derived from cochlear nerves in transgenic non-human animals administered with candidate compounds There when are emerging, and judging to be valid the candidate compound to Alzheimer's disease therapeutic agents.

The transgenic non-human animal according to the second aspect of the present invention has an expression carrying an amyloid β protein (Aβ), an amyloid β precursor protein (APP), or an Alzheimer's disease- related gene that is a gene encoding a Tau protein. A vector is introduced into inner ear hair cells to express its Alzheimer's disease- related gene.

  According to the present invention, a candidate compound is administered to a transgenic non-human animal expressed by introducing a dementia-related gene into an inner ear hair cell, and its hearing is detected. Therefore, accurate screening at the individual level is possible. Although senile disease takes time to develop, screening can be performed in a short time by using a transgenic non-human animal such as a mouse with a short generation change time. This will enable accurate development of therapeutic drugs for dementia, which not only improves intellectual dysfunction, which is a basic symptom of dementia, but also is associated with dementia such as decreased motivation, depression, and behavioral abnormalities. Secondary symptoms are also improved, improving the patient's QOL (Quality of Life).

It is a figure explaining the expression vector which carries a dementia related gene. It is a figure which shows the waveform data of the auditory brainstem reaction of a normal test animal.

  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.

(Transgenic non-human animals)
In the transgenic non-human animal according to the present embodiment, an expression vector carrying a dementia-related gene is introduced into an inner ear hair cell to express the dementia-related gene. Here, dementia-related gene is expressed not only when the gene is actually expressed in a non-human animal, but also in a state where the expression of the gene is temporarily suppressed.

  The transgenic non-human animal of this embodiment is a concept including not only an individual of the animal but also its tissue and cells. In addition, foreign genes other than dementia-related genes may be introduced into these individuals, tissues and cells.

  The dementia-related gene is, for example, a gene encoding one protein selected from the group consisting of amyloid β protein (Aβ), amyloid β precursor protein (APP) and Tau protein, and variants thereof, or a variant thereof. is there. Aβ includes Aβ40, Aβ42, Aβ43, and Aβ42-Arctic (Arc42 mutation introduced into Aβ42 which is one of the mutations of familial Alzheimer's disease).

  Inner ear hair cells are cells that convert physical stimuli into neural stimuli and transmit them to the cochlear ganglion cells. Inner ear hair cells include inner hair cells and outer hair cells, which are regularly arranged along the cochlear duct.

  The non-human animal used for the transgenic non-human animal is not particularly limited as long as it has inner ear hair cells. For example, mouse, rat, cow, pig, sheep, goat, rabbit, dog, cat , Guinea pigs, hamsters and the like. Preferred are mice, rats, rabbits, guinea pigs, or hamsters, among which rodents such as guinea pigs, hamsters, mice, rats, etc. are preferred, and mice are particularly preferred.

  Dementia includes Alzheimer's disease, Parkinson's disease, diffuse Lewy body disease and the like, and Alzheimer's disease is preferred. Alzheimer's disease is a dementia that presents symptoms such as progressive cognitive impairment (memory impairment, disorientation, learning impairment, attention impairment, spatial cognitive function, etc.), personality changes, and social inability to adjust. Is a disease. Alzheimer's disease includes both familial Alzheimer's disease and sporadic Alzheimer's disease. Here, familial Alzheimer's disease is hereditary Alzheimer's disease showing a complete autosomal dominant Mendelian inheritance pattern. In addition, sporadic Alzheimer's disease is Alzheimer's disease that occupies most of Alzheimer's disease, does not genetically develop, and develops in old age.

  Next, a method for producing a transgenic non-human animal according to this embodiment will be described.

For the purpose of expressing the target gene in the inner ear hair cells, a minimal enhancer of Math1, which is a gene expressed specifically in sensory cells, is used. As shown in FIG. 1, Math1E-IRES the Math1 ^E and CMV promoter to express a variety of dementia associated factors group under Math1 ^E is coupled upstream of the MCS, as an expression vector having the BGHpA as polyadenylation site -Make EGFP :: L10a-BGHpA. The sequence of this expression vector is set forth in SEQ ID NO: 1.

  Since this expression vector expresses a fusion protein of EGFP and mouse ribosome L10a downstream of the IRES, it is easy to identify the expression cell of the dementia-related gene and analyze the gene expression profile. Then, the above-described dementia-related gene is introduced into the above-described expression vector to produce a transgenic non-human animal.

  The method for introducing the above-mentioned dementia gene into a non-human animal is not particularly limited. For example, the gene is introduced into a fertilized egg of a non-human animal, and the fertilized egg is implanted into a female of the animal. As a fertilized egg, it is preferable to use a male prosperous nucleus (about 12 hours after fertilization). A method for introducing a gene into a fertilized egg is not particularly limited. For example, a microinjection method, an electric pulse method, a calcium phosphate method, a lipofection method, a particle gun method, or the like can be used. A method for implanting a fertilized egg into a female is not particularly limited, and for example, a means for artificially transplanting and implanting into the oviduct of a pseudopregnant female animal can be used.

  Next, an individual that contains a dementia-related gene and expresses the gene is selected from offspring born from a female implanted with a fertilized egg, and the individual is passaged. Whether or not the introduced dementia-related gene is contained in a non-human animal can be confirmed by collecting DNA from the non-human animal and performing RT-PCR using the sequence of the introduced gene. .

  Whether or not a non-human animal containing a dementia-related gene is expressing a gene can be confirmed with a fluorescence monitor because the expression vector expresses an EGFP fusion protein via IRES. It is. As another method, it is possible to prepare a cell lysate from inner ear hair cells of a non-human animal and confirm by Western blotting using an antibody that recognizes a dementia-related gene. As another method, immunohistochemistry using an antibody that recognizes a dementia-related gene on a paraffin section prepared by perfusion-fixing inner ear hair cells of a transgenic non-human animal with paraformaldehyde according to a standard method. It is possible to confirm using

(Screening method for therapeutic drugs for dementia)
The transgenic non-human animal according to the present embodiment described above can be used for screening for a therapeutic agent for dementia. The therapeutic agent means a therapeutic agent for animals including humans, and the therapeutic agent includes any of prevention of disease, improvement of disease, and delay of progression of disease.

  The screening method for a therapeutic agent for dementia according to the present embodiment comprises: a) introducing an expression vector carrying a dementia-related gene into an inner ear hair cell of a non-human animal to be tested; Producing an expressed transgenic non-human animal; b) administering a candidate compound to the transgenic non-human animal expressing the dementia-related gene; and c) transgenic non-human administering the candidate compound. A step of detecting the hearing of a human animal, and d) the degree of hearing impairment in a transgenic non-human animal administered with the candidate compound is eliminated from the degree of hearing impairment in the transgenic non-human animal not receiving the candidate compound And determining that the candidate compound is effective as a therapeutic agent for dementia.

  First, based on the method for producing a transgenic non-human animal described above, a transgenic non-human animal in which a dementia-related gene is expressed is produced. Then, the candidate compound is administered to the transgenic non-human animal in which the dementia-related gene is expressed. Candidate compounds are compounds for which efficacy as therapeutic agents for dementia is to be judged. The administration method to a test animal is not specifically limited, For example, oral administration, intravascular administration by injection, etc. can be used. The dosage of the candidate compound administered to the transgenic non-human animal is not particularly limited as long as the candidate compound is an amount that acts on the transgenic non-human animal.

  Next, the hearing of the transgenic non-human animal to which the candidate compound is administered is detected. Auditory detection is preferably, for example, electrical signal detection based on an auditory brainstem response (ARB). The auditory brainstem reaction is a change in the potential of the brainstem portion when a sound stimulus is applied from the ear of the subject animal.

  First, an electrode is attached to a predetermined part of the head of a test animal, sound stimulation is applied from the ear, and evoked potentials of cranial nerves are recorded over time to obtain an auditory brainstem reaction. In the measurement of the auditory brainstem reaction, it is preferable to perform the measurement in a state where the test animal is rested by pre-administering a sedative. Although the sedative is not particularly limited, for example, xylazine is suitable.

  An earphone-shaped speaker for sound stimulation is attached to the ear of the test animal. The shape and size of the loudspeaker are not particularly limited, but for example, those that are in close contact with the ear hole of the test animal are preferable. In order to prevent electromyogram-derived noise caused by moving the ear of the test animal, it is preferable to fix the ear of the test animal in advance when measuring the auditory brainstem response.

  There are no particular restrictions on the location where the electrode is attached to the head of the test animal as long as it can detect the evoked potential derived from the brainstem. For example, the plus electrode is at the top of the head, and the minus electrode is at the orbit and the ear root. It is possible to attach a ground to the middle of both eyes and the middle of the nose at the middle.

  The type, frequency, and magnitude of the sound stimulus are not particularly limited, and can be set as appropriate. For example, with a click sound, the stimulation sound pressure can be about 75 to 120 dBnHL, the stimulation frequency can be 10 to 40 times / second, and the stimulation duration can be 0.1 to 0.3 milliseconds.

  When a sound stimulus is applied from the ear, the stimulus is transmitted to the brain stem via the auditory nerve. The time from the application of the sound stimulus to the transmission of the stimulus to each part of the auditory nerve and brainstem is almost constant for each part. Therefore, the function of each part of the brain stem can be analyzed by recording the evoked potential transmitted from the auditory nerve to the brain stem over time.

  Next, the waveform data of the auditory brainstem response in the transgenic non-human animal administered with the candidate compound is compared with the waveform data of the auditory brainstem response in the transgenic non-human animal not given the candidate compound.

  FIG. 2 is waveform data of an auditory brainstem response of a normal subject animal. As shown in FIG. 2, seven positive waves are recognized in the waveform based on the auditory brainstem response. "I" is derived from the cochlear nerve, "II" is derived from the cochlear nucleus, "III" is derived from the upper olive complex nucleus, "IV" is derived from the outer hair band, "V" is derived from the lower hill, "VI" is from the inner knee Body-derived, “VII” is a positive wave derived from auditory radiation. The vertical axis represents the obtained evoked potential (Amplitude, added value), and the horizontal axis represents the time (Latency) after applying the sound stimulus.

  The waveform data of the auditory brainstem response in a transgenic non-human animal in which a dementia-related gene is expressed is unique waveform data in which a positive wave of “I” derived from the cochlear nerve is hardly detected. The hair cells and the cochlear nerve endings are connected at the synapse, and the hair cells convert physical stimuli into nerve stimuli and transmit them to the cochlear ganglion cells. However, if a dementia-related gene is expressed in the inner ear hair cells, the transmission of stimuli from the hair cells to the cochlear ganglion cells fails, and the positive wave of “I” is hardly detected.

  When the candidate compound is effective for dementia, the waveform data of the auditory brainstem response obtained from the transgenic non-human animal to which the candidate compound has been administered is close to the waveform data of the auditory brainstem response of a normal test animal. ”Is detected normally or almost normally. That is, the positive waveform “I” in the waveform data of the auditory brainstem response in the transgenic non-human animal administered with the candidate compound is compared with the waveform data of the auditory brainstem response in the transgenic non-human animal not given the candidate compound. If it appears, the candidate compound is determined to be effective as a therapeutic drug for dementia.

  On the other hand, if the candidate compound is not effective for dementia, the waveform data of the auditory brainstem reaction obtained from the transgenic non-human animal to which the candidate compound has been administered, the “I” positive wave remains hardly detected. That is, the positive wave of “I” in the waveform data of the auditory brainstem response in the transgenic non-human animal administered with the candidate compound appears in the same manner as the waveform data of the auditory brainstem response in the transgenic non-human animal not given the candidate compound. If not, the candidate compound is determined not to be effective as a therapeutic agent for dementia.

  Thus, since the presence or absence of the positive wave of “I” in the waveform data of the auditory brainstem reaction is detected, screening for a therapeutic agent for dementia can be performed quantitatively. Furthermore, if the screening is performed for each aging day of the non-human animal to be tested, the drug effect of the candidate compound can be tested over time.

(Other embodiments)
In the above-described embodiment, the detection of the hearing of the transgenic non-human animal to which the candidate compound is administered was detection by an auditory brainstem reaction, but the scope of the present invention is not limited to such an embodiment. For example, the detection of hearing using operant behavior, which is a behavior pattern in which an animal learns to actively and voluntarily adapt to the environment, acquires a pleasant stimulus under a certain condition, or avoids an unpleasant stimulus It is also possible to do this. That is, the test animal's hearing is detected by having the test animal learn the avoidance of the condition of sound → electric shock in advance and performing the avoidance action only by listening to the sound. Transgenic non-human animals that express dementia-related genes have hearing impairments, so even if they learn to avoid the condition of sound → electric shock in advance, the avoidance behavior is insufficient even if they hear the sound . Then, a candidate compound is administered to a transgenic non-human animal in which a dementia-related gene is expressed, and a condition avoidance response to sound is observed. When the candidate compound is effective for dementia, the condition avoidance response of the transgenic non-human animal to which the candidate compound is administered is close to the condition avoidance response of the normal test animal. On the other hand, when the candidate compound is not effective for dementia, the transgenic non-human animal administered with the candidate compound remains insufficient in avoidance behavior.

  In the above-described embodiment, the presence or absence of the positive wave of “I” in the waveform data of the auditory brainstem reaction is detected, but the scope of the present invention is not limited to such an embodiment. For example, comparing the entire waveform data of an auditory brainstem response in a transgenic non-human animal to which a candidate compound is administered with the entire waveform data of an auditory brainstem response in a transgenic non-human animal not given the candidate compound, If the shape of the waveform data of the auditory brainstem response of a human animal is close, it is possible to determine that the candidate compound is effective as a therapeutic agent for dementia.

  It can be used to develop a treatment for dementia.

  SEQ ID NO: 1: vector

Claims (5)

a) Inner hair cell of non-human animal subject to test with expression vector carrying amyloid β protein (Aβ), amyloid β precursor protein (APP), or Alzheimer's disease related gene which is a gene encoding Tau protein To produce a transgenic non-human animal expressing the Alzheimer's disease- related gene,
b) administering a candidate compound to a transgenic non-human animal in which the Alzheimer's disease- related gene is expressed;
c) detecting the hearing of a transgenic non-human animal to which the candidate compound has been administered;
d) When a waveform derived from the cochlear nerve appears in a transgenic non-human animal to which a candidate compound is administered as compared to a transgenic non-human animal that does not receive the candidate compound, the candidate compound is treated as a therapeutic agent for Alzheimer's disease . A method of screening for a therapeutic drug for Alzheimer's disease , characterized in that it is determined to be effective. In c), the detection of the hearing screening method of Alzheimer's disease therapeutic agent according to claim 1, characterized in that the electric signals detected by the auditory brainstem response (ARB). The non-human animal, wherein a mouse, rat, cow, pig, sheep, goats, rabbits, dogs, cats, guinea pigs, or hamster, to claim 1 or 2 wherein characterized in that it is selected from the group consisting of Screening methods for Alzheimer's disease drugs. An expression vector carrying an Alzheimer's disease- related gene, which is a gene encoding amyloid β protein (Aβ), amyloid β precursor protein (APP), or Tau protein, is introduced into an inner ear hair cell, and the Alzheimer's disease- related gene A transgenic non-human animal characterized in that Transgenic claim 4 in which the non-human animal, mouse, rat, cow, pig, sheep, goats, rabbits, dogs, cats, guinea pigs, or, being selected hamsters, from the group consisting of Non-human animals.