WO2009136444A1 - Method of screening preventive or remedy for type i dipolar disorder - Google Patents

Method of screening preventive or remedy for type i dipolar disorder Download PDF

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WO2009136444A1
WO2009136444A1 PCT/JP2008/058665 JP2008058665W WO2009136444A1 WO 2009136444 A1 WO2009136444 A1 WO 2009136444A1 JP 2008058665 W JP2008058665 W JP 2008058665W WO 2009136444 A1 WO2009136444 A1 WO 2009136444A1
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mice
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dgkβ
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英彰 原
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カルナバイオサイエンス株式会社
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy

Definitions

  • the present invention relates to a method for screening a preventive or therapeutic agent for bipolar disorder type I.
  • Bipolar disorder is a type of mental illness also called bipolar mood disorder or bipolar manic depression. Mood disorders are divided into unipolar, where only one symptom of manic depression appears, and bipolar, where both symptoms appear, and in theory there are unipolar mania and unipolar depression. However, since many patients with unipolar mania later develop depressive symptoms, unipolar mania is now classified as part of bipolar disorder (DSM (Diagnostic and Statistical Manual of Mental Disorders)- IV-TR Classification and Diagnosis Guide for Psychiatric Disorders (New Revised Edition) Medical School March 1, 2005 New Edition 4th edition, text psychiatry Revised 3rd edition Nanzan-do April 10, 2007 3rd edition 1st edition etc. reference). Therefore, mood disorders are now classified into bipolar disorder and unipolar depression. Bipolar disorder is broadly divided into type I, which is severe in depression, and type II, which is severe in depression.
  • type I includes those with a long manic state and very mild depression, and those that have traditionally been unipolar (unipolar) manic, and that manic state affects social life. It has the feature that it is often intense enough to affect
  • Type II is not as violent as the type I addictive state, but has a longer depressive state compared to the addictive state, and the depressed state drives itself and sometimes attempts to commit suicide. It shows the feature that it is intense as there are cases.
  • bipolar disorder the current treatment methods for bipolar disorder are that type I may be used in combination with antipsychotic drugs and type II may be used in combination with antidepressants.
  • type I may be used in combination with antipsychotic drugs
  • type II may be used in combination with antidepressants.
  • the current situation is that there is only a common and symptomatic drug called “mood stabilizer” or “emotion modifier”, and there are almost no drugs exclusively for type I and type II.
  • Patent Document 1 suggests that human-derived DGK ⁇ (diacylglycerol kinase ⁇ ) may be related to bipolar disorder, and the authors of this document later published Patent Document 1 also describes the possibility.
  • DGK ⁇ diacylglycerol kinase ⁇
  • mice lacking the function of DGK ⁇ are commercially available (Taconic, TF0164 (catalog No.), etc.), but the relationship between the mice and bipolar disorder is not known.
  • mice deficient in the function of DGK ⁇ are not type II but clearly have type I symptoms of bipolar disorder.
  • the present invention has been completed and the object of the present invention is to provide a drug screening method using a disease model specialized in bipolar disorder type I. .
  • a prophylactic or therapeutic agent for bipolar disorder type I according to the first invention characterized in that a non-human animal in which a deficiency in DGK ⁇ function occurs in at least part of the forebrain to midbrain region is used. Screening method.
  • the disease model used in the method for screening a preventive or therapeutic agent for bipolar disorder type I of the present invention is characterized by comprising a non-human animal deficient in the function of DGK ⁇ .
  • Examples of the method for deleting the function of DGK ⁇ in a non-human animal include a method for deleting or mutating the DGK ⁇ gene by a known genetic engineering technique and inhibiting its expression or function.
  • a method using RNAi a method using antisense RNA, a method using a targeting gene with a modified target gene, a method for producing a mutant mouse using a transposon (International Publication No. WO 02/13602, Molecular and Cellular Biology (see vol.23, No.24, pp.9189-9207, 2003, etc.), etc., but among them, targeting genes with modified target genes can be more reliably suppressed in terms of expression.
  • the method used is preferred.
  • Such a mouse deficient in the function of DGK ⁇ can also be obtained as TF0164 (catalog No.) or the like from Taconic.
  • the non-human animal used as the disease model used in the screening method of the present invention may be any animal other than humans, and examples include rodents, dogs, cats, monkeys, horses, pigs, etc. Rodent animals are preferred. Examples of rodent animals include mice, rats, rabbits, hamsters, and guinea pigs. Among these, mice and rats are preferable, and mice are particularly preferable. For example, C57BL / 6N mice such as C57BL / 6N Cr / Cr / j (also referred to as B6 mice), Balb / c mice and the like are preferably used, and among these, B6 mice are preferable.
  • C57BL / 6N mice such as C57BL / 6N Cr / Cr / j (also referred to as B6 mice), Balb / c mice and the like are preferably used, and among these, B6 mice are preferable.
  • DSM-IV-TR classification and diagnosis of mental illness
  • diagnosis of mental illness Medical Shoin March 1, 2005
  • fourth edition of the new edition As described above, this includes not only those with a long manic state and very mild depression, but also those that have been conventionally regarded as unipolar (unipolar) mania.
  • screening in addition to so-called primary screening for selecting a desired preventive or therapeutic agent from a plurality of candidates, secondary screening for confirming the prophylactic or therapeutic effect of a test substance is possible. Screening (re-evaluation or confirmation test) is also included.
  • Tp is a method for obtaining various transgenic mice (hereinafter referred to as Tg mice) by utilizing transposition to various positions within the gene.
  • a mouse (DGK ⁇ -KO mouse) lacking the function of DGK ⁇ was selected from various Tg mice obtained by this method.
  • Tg mice having a Tp sequence and a Tg mouse (SB) having an enzyme (transposase) that causes translocation of this Tp sequence are crossed to create a variety of Tg mice.
  • SB Tg mouse
  • transposase enzyme that causes translocation of this Tp sequence
  • the transposase recognizes the repetitive sequence in the reverse direction present in the Tp sequence, excises the Tp sequence from the genomic DNA, and the excised Tp sequence is inserted again at an arbitrary position by the action of the transposase. This is because.
  • FIG. 12 (A) is a schematic diagram showing a donor site in which a plurality of Tp vector units are incorporated, and its cleavage site by Bgl II and Nco I in a Tg mouse having a Tp sequence.
  • sequence is Tp arrangement
  • this Tp gene incorporates a promoter sequence and LacZ and GFP sequences as markers.
  • DGK ⁇ -KO mice mice in which the Tp sequence was translocated between DGK ⁇ sequences
  • mice mice in which mutations occurred in the gene sequence.
  • the DGK ⁇ -KO mouse is selected by collecting Tg mouse genomic DNA, determining the genomic sequence of the Tp sequence insertion site by PCR, etc., and collating it with the mouse genome database, thereby translocating the Tp sequence into the DGK ⁇ gene. The method of identifying the mouse was performed.
  • FIG. 12 (B) is a schematic diagram showing a state in which the Tp sequence is incorporated into the DGK ⁇ sequence by Tp rearrangement and its cleavage site by BglII and NcoI.
  • the donor site does not exist in the selected mouse gene, the presence or absence of a neoplasm derived from a plasmid vector that exists only at the donor site was confirmed.
  • Southern blot analysis was performed by cutting the genomic genes of hetero (Tp / +) and wild type (WT (+ / +)) mice with SpeI and using the neo sequence as a probe. It was.
  • FIG. 14 is a schematic diagram showing a SpeI cleavage site in a donor site incorporating a plurality of Tp vectors.
  • Control indicates the analysis result of genomic DNA having one neo gene. From this genomic DNA, it is expected that a 4.2 kb band will be obtained by cleavage with SpeI, but a band of the expected size is detected. This confirmed that the probe complementary to the neo sequence used was indeed functioning.
  • KO DGK ⁇ -KO mouse
  • WT syngeneic wild-type mice
  • ⁇ no movement '' means that the mouse floats in the water without resistance and stays in the minimum movement necessary to bring the face out of the water surface (the movement of the forelimbs stops and one of the hind limbs is in posture). To maintain a slight movement).
  • KO mice had a significant decrease in immobility time compared to WT mice (**; p ⁇ 0.01 vs. WT).
  • This Pre test induces a kind of depression before starting the Post test, but as described above, it was found that depression was difficult to be induced in KO mice.
  • Test Example 2 Novel substance search test
  • a plastic open field (length 30 mm ⁇ width 30 mm ⁇ height 14 cm) was used for the test. The mice were habituated for 10 minutes each in an open fold for 3 days before the test.
  • mice The search time for each substance in mice for 10 minutes in acquisition and retention trials was measured using an EthoVision XT (Noldus, Wageningen, The Netherlands).
  • the mouse's search preference for novel substances in retention trials was calculated as a percentage of the total search time. The results are shown as an average of 8-10 mice in each group.
  • Test Example 3 Spontaneous exercise test
  • mice Using a group of mice different from those used in Test Examples 1 and 2, spontaneous movement in a novel environment was measured using a plastic open field (length 30 ⁇ width 30 ⁇ height 14 cm). Mice were placed in an open field and allowed to explore freely for 120 minutes.
  • mice The results are shown as an average of 4 mice in each group.
  • DGK ⁇ -KO mice showed an increase in locomotor activity at 120 minutes compared to WT mice.
  • Total movement distance (Fig. 4), time-dependent change of movement distance (Fig. 5), and movement speed (Fig. 6) are increased in KO mice compared to WT mice, respectively, Significant differences were seen.
  • FIG. 4 (*; p ⁇ 0.05 vs. WT)
  • FIG. 5 (*; p ⁇ 0.05 vs. WT) (**; p ⁇ 0.01 vs. WT)
  • Test Example 4 Open field test
  • the mouse used in Test Example 3 was placed in a plastic open field (length 30 ⁇ width 30 ⁇ height 14 cm) and allowed to search freely for 120 minutes.
  • the total movement distance of the mouse and the time spent in the center (15 ⁇ 15 cm) were automatically measured using a behavior tracking / analysis video tracking system (EthoVision XT TM, manufactured by NOLDUS).
  • the number of standing up and abdominal scratching actions during the first 10 minutes was measured manually.
  • DGK ⁇ -KO mice In DGK ⁇ -KO mice, an increase in exploratory behavior was observed in the open field test. DGK ⁇ -KO mice had increased time spent in the center of the open field and the number of intrusions into the center compared to WT mice (FIGS. 7 and 8).
  • KO mice were in an overactive state, that is, a kind of “snoring state” at the time of performing Test Example 4.
  • a large number of intrusions into the center indicates a low level of anxiety, that is, hyperactivity
  • a large number of standing up actions indicate overactivity
  • a large number of scratching actions indicate administration of excitatory drugs. It shows normal behaviors like psychiatric disorders similar to models.
  • Test Example 5 LacZ staining
  • a group of mice different from those used in Test Examples 1 to 4 was used, and LacZ staining was performed in order to confirm the “defect site of the DGK ⁇ gene in the mouse body”.
  • a 15-week-old male mouse from each of WT and KO mice was anesthetized with pentobarbital, and 0.1% M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde was injected into the left ventricle to perform perfusion fixation (perfusion pressure 130 cm H2O). After 20 minutes, the brain was removed and left overnight in the same solution. Subsequently, it was allowed to stand in a 0.1% M PB (pH 7.4) solution containing 25% sucrose for 24 hours. Thereafter, it was quickly frozen with powdered dry ice and stored at ⁇ 80 ° C. until sliced.
  • PB 0.1% M phosphate buffer
  • LacZ staining was performed using ⁇ -garactosidase staining kit (Actove motif, Carlsbad, CA). After infiltrating the slices with 0.2% glutaraldehyde and 1% formalin, the reaction solution containing 5-bromo-4-chloro-3-indoly- ⁇ -galactoside (X-Gal), a substrate of ⁇ -galactosidase, was kept for 2 hours. Colored by soaking.
  • X-Gal 5-bromo-4-chloro-3-indoly- ⁇ -galactoside
  • Tp translocation occurred in the forebrain-mesencephalon region of the mouse. That is, as a result of the translocation of the LacZ sequence promoter inserted as a marker in the Tp gene, LacZ is translated to express ⁇ -galactosidase, and the substrate X-gal (5-Bromo-4 -Chloro-3-Indolyl- ⁇ -D-Galactoside) was decomposed to produce an insoluble blue coloring substance. In other words, it can be seen that Tp translocation (DGK ⁇ deficiency) occurs in tissues exhibiting blue color development.
  • DGK ⁇ deficiency in the forebrain to midbrain region is one of the causes of bipolar disorder type I symptoms (such as hyperactivity and memory impairment).
  • Example 1 Screening method for preventing or treating bipolar disorder type I
  • a test substance is administered to the model of Reference Example 1, and a preventive or therapeutic drug for bipolar disorder type I is screened by comparing the results of Test Examples 1 to 4 before and after the administration.
  • the screening method of the present invention can be expected to develop an innovative preventive / therapeutic agent specialized in bipolar disorder type I.
  • * indicates that the standard deviation of the test result of the disease model mouse against wild type is p ⁇ 0.05
  • # indicates that the standard deviation of the second day relative to the first day is p ⁇ 0.05.
  • FIG. 1 It is a figure showing the result (time-dependent change of spontaneous exercise amount) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the spontaneous exercise test of the test example 3.
  • FIG. It is a figure showing the result (movement speed (cm / sec)) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the spontaneous movement test of the test example 3.
  • FIG. It is a figure showing the result (time (second) spent in the center of an open field) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the open field test of the test example 4.
  • FIG. A, C, E, G, and I represent staining results of olfactory bulb, striatum, hippocampus, substantia nigra, and cerebellum in KO mice.
  • B, D, F, H, and J represent staining results of the olfactory bulb, striatum, hippocampus, substantia nigra, and cerebellum in WT mice.
  • FIG. (A) is a schematic diagram showing a donor site into which a Tp sequence has been inserted, and its cleavage site by Bgl II and Nco I, used when preparing the disease model mouse (KO) of Reference Example 1.
  • FIG. (B) is a schematic diagram showing a DGK ⁇ sequence in which a Tp sequence is incorporated, and its cleavage site by BglII and NcoI.
  • B and N represent cleavage sites by Bgl II and Nco I, respectively.
  • the results of Southern blot analysis using the LacZ sequence as a probe for the genomic genes of wild type (WT (+ / +)), heterozygous (Tp / +), and homozygous (Tp / Tp) mice are shown.
  • FIG. 1 It is a schematic diagram which shows the donor site which inserted the Tp arrangement
  • S represents a site cleaved by SpeI.
  • the results of Southern blot analysis using a polynucleotide complementary to the neo sequence as a probe for each genomic gene of heterozygous (Tp / +) and wild type (WT (+ / +)) mice are shown.

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Abstract

[PROBLEMS] To provide a method of screening a preventive or remedy for type I dipolar disorder by using a type I dipolar disorder-specialized disease model. [MEANS FOR SOLVING PROBLEMS] A method of screening a preventive or remedy for type I dipolar disorder characterized in that a disease model of type I dipolar disorder, which comprises a nonhuman animal deficient in the DGKβ function, is employed.

Description

双極性障害Ｉ型の予防又は治療薬のスクリーニング方法Method for screening for preventive or therapeutic agent for bipolar disorder type I
本発明は、双極性障害のＩ型の予防又は治療薬のスクリーニング方法に関するものである。 The present invention relates to a method for screening a preventive or therapeutic agent for bipolar disorder type I.
双極性障害とは、双極性気分障害あるいは双極性躁鬱病とも呼ばれる精神疾患の一種である。
気分障害は、躁鬱のいずれかの症状のみが現れる単極性と、両方の症状が現れる双極性に二分され、単極性には、理論上は単極性躁病と単極性鬱病がある。しかし、単極性躁病患者の多くが、後に鬱症状を発症することから、現在では、単極性躁病は、双極性障害の一部として分類されている（DSM（Diagnostic and Statistical Manual of Mental Disorders）-IV-TR　精神疾患の分類と診断の手引（新訂版） 医学書院 2005年3月1日　新訂版第４刷，text精神医学　改訂３版　南山堂2007年4月10日３版１刷等参照）。
従って、現在では、気分障害は、双極性障害と、単極性鬱病に分類されるようになってきた。
そして、双極性障害は、躁状態の重いＩ型と、鬱状態が重いII型に、大別される。 Bipolar disorder is a type of mental illness also called bipolar mood disorder or bipolar manic depression.
Mood disorders are divided into unipolar, where only one symptom of manic depression appears, and bipolar, where both symptoms appear, and in theory there are unipolar mania and unipolar depression. However, since many patients with unipolar mania later develop depressive symptoms, unipolar mania is now classified as part of bipolar disorder (DSM (Diagnostic and Statistical Manual of Mental Disorders)- IV-TR Classification and Diagnosis Guide for Psychiatric Disorders (New Revised Edition) Medical School March 1, 2005 New Edition 4th edition, text psychiatry Revised 3rd edition Nanzan-do April 10, 2007 3rd edition 1st edition etc. reference).
Therefore, mood disorders are now classified into bipolar disorder and unipolar depression.
Bipolar disorder is broadly divided into type I, which is severe in depression, and type II, which is severe in depression.
つまり、Ｉ型には、躁状態が長く、鬱状態がごく軽度であるものや、従来、単一性（単極性）躁病とされていたものが含まれ、その躁状態は、社会生活に影響を及ぼす程激しい場合が多いという特徴を有している。 In other words, type I includes those with a long manic state and very mild depression, and those that have traditionally been unipolar (unipolar) manic, and that manic state affects social life. It has the feature that it is often intense enough to affect
一方、II型は、躁状態がＩ型の躁状態ほど激しいものでは無い一方、躁状態に比べて鬱状態が長く、また、その鬱状態は、自分自身を追い込んで、時には自殺を図ってしまうケースもある程激しい，という特徴を示すものである。 Type II, on the other hand, is not as violent as the type I addictive state, but has a longer depressive state compared to the addictive state, and the depressed state drives itself and sometimes attempts to commit suicide. It shows the feature that it is intense as there are cases.
このように双極性障害と言っても、Ｉ型とII型とでは、症状がかなり異なることから、その原因も異なる可能性が高く、当然ながらその治療方法も違ったものであるべきである。 Thus, even if it is bipolar disorder, since the symptoms are quite different between type I and type II, the cause is likely to be different, and naturally the treatment method should be different.
それにも拘わらず、現在、双極性障害の治療方法としては、Ｉ型は抗精神病薬を，II型は抗鬱剤を、各々併用する場合があるものの、基本は、Ｉ型，II型ともに、「気分安定薬(mood stabilizer)」や「感情調整剤」という、共通の、しかも対症療法薬を用いるしか無く、Ｉ型とII型各々専用の薬は、殆ど無いと言うのが現状である。 Nevertheless, the current treatment methods for bipolar disorder are that type I may be used in combination with antipsychotic drugs and type II may be used in combination with antidepressants. The current situation is that there is only a common and symptomatic drug called “mood stabilizer” or “emotion modifier”, and there are almost no drugs exclusively for type I and type II.
そのため、双極性障害の、根本的な治療薬の開発のためには、Ｉ型とII型を区別した疾患モデル動物が必要とされていた。 Therefore, in order to develop a radical therapeutic agent for bipolar disorder, a disease model animal that distinguishes between type I and type II is required.
近年、特許文献１において、ヒト由来のDGKβ（ジアシルグリセロールキナーゼβ）が、双極性障害（bipolar disorder）に関連している可能性が示唆されており、当該文献の筆者等がその後に出した非特許文献１においても、その可能性が述べられている。 In recent years, Patent Document 1 suggests that human-derived DGKβ (diacylglycerol kinase β) may be related to bipolar disorder, and the authors of this document later published Patent Document 1 also describes the possibility.
しかし、この特許文献１においては、DGKβは、単極又は双極性の「鬱病」に関連している旨記載されており（［請求項１８］，［請求項２２］，［００４１］等）、これはむしろ、双極性障害のII型である。 However, in this Patent Document 1, it is described that DGKβ is related to monopolar or bipolar “depression” ([claim 18], [claim 22], [0041], etc.), This is rather a type II bipolar disorder.
しかも、この特許文献１での検証は、ヒトの遺伝子から、マウスで見つかったDGKβに相当するアミノ酸を発見し、それを、従来から示唆されていた、「マウスのDGKβと疾患との関わり」に結びつけたに過ぎず、発見した遺伝子と疾患との関わりを、直接確認したものではない。 In addition, the verification in this Patent Document 1 discovered an amino acid corresponding to DGKβ found in a mouse from a human gene, which was previously suggested as “relationship between mouse DGKβ and disease”. It is only a link, and it is not a direct confirmation of the relationship between the discovered gene and the disease.
一方、DGKβの機能を欠損させたマウスが市販されているが（タコニック（Taconic）社，TF0164（カタログNo.）等）、当該マウスと双極性障害との関連性については知られていない。 On the other hand, mice lacking the function of DGKβ are commercially available (Taconic, TF0164 (catalog No.), etc.), but the relationship between the mice and bipolar disorder is not known.
特表2002-540765号公報Special table 2002-540765 gazette
本発明者等は、DGKβの機能を欠損させたマウスについて、詳細な観察を行った結果、驚くべきことに、このマウスは、II型では無く、明らかに、双極性障害のＩ型の症状を示していることを見いだし、本発明を完成させたものであって、その目的とするところは、双極性障害のＩ型に特化した疾患モデルを用いた薬剤のスクリーニング方法を提供することにある。 As a result of detailed observations on mice deficient in the function of DGKβ, the present inventors have surprisingly found that these mice are not type II but clearly have type I symptoms of bipolar disorder. The present invention has been completed and the object of the present invention is to provide a drug screening method using a disease model specialized in bipolar disorder type I. .
上述の目的は、下記（第１の発明）乃至（第３の発明）等によって達成される。 The above object is achieved by the following (first invention) to (third invention) and the like.
（第１の発明）
DGKβの機能を欠損させた非ヒト動物を用いることを特徴とする、双極性障害Ｉ型の予防又は治療薬のスクリーニング方法。 (First invention)
A screening method for a preventive or therapeutic agent for bipolar disorder type I, wherein a non-human animal deficient in the function of DGKβ is used.
（第２の発明）
DGKβ機能の欠損が、前脳から中脳部位の少なくとも一部において発生している非ヒト動物を用いることを特徴とする、第１の発明記載の、双極性障害Ｉ型の予防又は治療薬のスクリーニング方法。 (Second invention)
A prophylactic or therapeutic agent for bipolar disorder type I according to the first invention, characterized in that a non-human animal in which a deficiency in DGKβ function occurs in at least part of the forebrain to midbrain region is used. Screening method.
（第３の発明）
非ヒト動物が、齧歯類であることを特徴とする、第１の発明又は第２の発明記載の、双極性障害Ｉ型の予防又は治療薬のスクリーニング方法。 (Third aspect of the present invention)
The method for screening a preventive or therapeutic agent for bipolar disorder type I according to the first or second invention, wherein the non-human animal is a rodent.
本発明のスクリーニング方法を用いることによって、双極性障害のＩ型に特化した、画期的な予防・治療薬の開発が期待できる。 By using the screening method of the present invention, the development of a breakthrough preventive / therapeutic agent specialized in bipolar disorder type I can be expected.
［本発明に用いる双極性障害Ｉ型の疾患モデル］
本発明の双極性障害Ｉ型の予防又は治療薬のスクリーニング方法に用いられる疾患モデルは、DGKβの機能を欠損させた非ヒト動物からなることを特徴とする。 [Disease model of bipolar disorder type I used in the present invention]
The disease model used in the method for screening a preventive or therapeutic agent for bipolar disorder type I of the present invention is characterized by comprising a non-human animal deficient in the function of DGKβ.
非ヒト動物のDGKβの機能を欠損させる方法としては、公知の遺伝子工学的手法によって、DGKβ遺伝子を欠損又は変異させ、その発現又は機能を阻害する方法が挙げられる。
具体的には、例えば、RNAiを用いる方法，アンチセンスRNAを用いる方法，標的遺伝子を改変したターゲッティング遺伝子を用いる方法，トランスポゾンを用いた変異マウス作成方法（国際公開番号WO　02/13602号，Molecular and Cellular Biology(vol.23, No.24, pp.9189-9207, 2003等参照）等が挙げられるが、中でも、より確実に発現を抑えることができるという点では、標的遺伝子を改変したターゲッティング遺伝子を用いる方法が好ましい。 Examples of the method for deleting the function of DGKβ in a non-human animal include a method for deleting or mutating the DGKβ gene by a known genetic engineering technique and inhibiting its expression or function.
Specifically, for example, a method using RNAi, a method using antisense RNA, a method using a targeting gene with a modified target gene, a method for producing a mutant mouse using a transposon (International Publication No. WO 02/13602, Molecular and Cellular Biology (see vol.23, No.24, pp.9189-9207, 2003, etc.), etc., but among them, targeting genes with modified target genes can be more reliably suppressed in terms of expression. The method used is preferred.
また、このようなDGKβの機能を欠損させたマウスは、タコニック（Taconic）社等から、TF0164（カタログNo.）等として入手することもできる。 Such a mouse deficient in the function of DGKβ can also be obtained as TF0164 (catalog No.) or the like from Taconic.
本発明のスクリーニング方法に用いられる疾患モデルとなる非ヒト動物としては、ヒト以外の動物であれば良く、例えば齧歯類動物，イヌ，ネコ，サル，ウマ，ブタ等が挙げられるが、中でも齧歯類動物が好ましい。
齧歯類動物としては、マウス，ラット，ウサギ，ハムスター，モルモットなどを挙げることができるが、中でも、マウスやラットが好ましく、特にマウスが好ましい。マウスは、例えば、C57BL/6N Cr/Cr/j等のC57BL/6Nマウス（B6マウスとも言う）、Balb/cマウス等が好ましく用いられ、中でも、B6マウスが好ましい。 The non-human animal used as the disease model used in the screening method of the present invention may be any animal other than humans, and examples include rodents, dogs, cats, monkeys, horses, pigs, etc. Rodent animals are preferred.
Examples of rodent animals include mice, rats, rabbits, hamsters, and guinea pigs. Among these, mice and rats are preferable, and mice are particularly preferable. For example, C57BL / 6N mice such as C57BL / 6N Cr / Cr / j (also referred to as B6 mice), Balb / c mice and the like are preferably used, and among these, B6 mice are preferable.
本発明における「双極性障害Ｉ型」とは、DSM-IV-TR　精神疾患の分類と診断の手引（新訂版） 医学書院 2005年3月1日　新訂版第４刷の判定基準によるものであり、上述した通り、躁状態が長く、鬱状態がごく軽度であるものの他、従来、単一性（単極性）躁病とされていたものをも含むものである。 “Dipolar disorder type I” in the present invention is based on the judgment criteria of DSM-IV-TR, classification and diagnosis of mental illness (new edition) Medical Shoin March 1, 2005, the fourth edition of the new edition As described above, this includes not only those with a long manic state and very mild depression, but also those that have been conventionally regarded as unipolar (unipolar) mania.
［本発明の双極性障害Ｉ型の予防又は治療薬のスクリーニング方法］
上記の疾患モデルを用いることによって、双極性障害Ｉ型の予防又は治療薬のスクリーニングを行うことができる。 [Method of screening for preventive or therapeutic agent for bipolar disorder type I of the present invention]
By using the above disease model, it is possible to screen for preventive or therapeutic agents for bipolar disorder type I.
尚、本発明で言うスクリーニングには、複数候補の中から、目的の予防又は治療薬等を選択するための、いわゆる一次スクリーニングの他、被験物の予防又は治療効果を確認するための、二次スクリーニング（再評価又は確認試験）も、含まれるものである。 In addition, in the screening referred to in the present invention, in addition to so-called primary screening for selecting a desired preventive or therapeutic agent from a plurality of candidates, secondary screening for confirming the prophylactic or therapeutic effect of a test substance is possible. Screening (re-evaluation or confirmation test) is also included.
以下、実施例によって、本発明を更に詳細に説明するが、本発明はこれら実施例に限定されるものでは無い。 EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.
［参考例１：双極性障害Ｉ型の疾患モデルマウス］
トランスポゾンを用いた変異マウス作成方法（国際公開番号WO 02/13602号, 及びMolecular and Cellular Biology(vol.23, No.24, pp.9189-9207, 2003等参照）は、転位性遺伝因子（トランスポゾン：以下、「Tp」と記載する。）が、遺伝子内で様々な位置に転位することを利用して、多様なトランスジェニックマウス（以下、Tgマウスと記載する。）を得る方法である。
本実施例では、この方法によって得られた各種のTgマウスから、DGKβの機能を欠損させたマウス（DGKβ－KOマウス）を選択した。 [Reference Example 1: Bipolar disorder type I disease model mouse]
Mutant mouse production methods using transposon (International Publication No. WO 02/13602, and Molecular and Cellular Biology (see vol.23, No.24, pp.9189-9207, 2003, etc.) include transposable genetic elements (transposon). : Hereinafter referred to as “Tp”) is a method for obtaining various transgenic mice (hereinafter referred to as Tg mice) by utilizing transposition to various positions within the gene.
In this example, a mouse (DGKβ-KO mouse) lacking the function of DGKβ was selected from various Tg mice obtained by this method.
具体的には、まず、Tp配列を有するTgマウスと、このTp配列の転位を起こさせる酵素（トランスポゼース）を有するTgマウス（SB）を掛け合わせ、多種多様なTgマウスを作成しうる「種マウス」を得た。得られた「種マウス」と「野生型マウス」を交配させ、種マウスの生殖細胞内でTpがゲノム上を移動することによって各種の変異を発現したTgマウスが得られた。 Specifically, first, a Tg mouse having a Tp sequence and a Tg mouse (SB) having an enzyme (transposase) that causes translocation of this Tp sequence are crossed to create a variety of Tg mice. " The obtained “seed mouse” and “wild type mouse” were mated, and Tg mice expressing various mutations were obtained by moving Tp on the genome in germ cells of the seed mice.
これは、トランスポゼースが、Tp配列内に存在する逆向きの反復配列を認識することによって、Tp配列をゲノムDNAから切り出し、切り出されたTp配列が、再びトランスポゼースの働きによって、任意の位置に挿入されたためである。 This is because the transposase recognizes the repetitive sequence in the reverse direction present in the Tp sequence, excises the Tp sequence from the genomic DNA, and the excised Tp sequence is inserted again at an arbitrary position by the action of the transposase. This is because.
図１２（Ａ）は、Tp配列を有するTgマウスの、Tpベクターユニットが複数組み込まれたドナーサイト，及びそのBgl II及びNco Iによる切断部位を示す模式図である。
配列中の、２つの太い矢印で表された逆向きの反復配列を含む部位が、Tp配列となっている。 FIG. 12 (A) is a schematic diagram showing a donor site in which a plurality of Tp vector units are incorporated, and its cleavage site by Bgl II and Nco I in a Tg mouse having a Tp sequence.
The site | part containing the repetitive sequence of the reverse direction represented by the two thick arrows in the arrangement | sequence is Tp arrangement | sequence.
図１２（Ａ）からも分かる通り、このTp遺伝子には、プロモーター配列や、マーカーとしてLacZ及びGFP配列が組み込まれている。 As can be seen from FIG. 12A, this Tp gene incorporates a promoter sequence and LacZ and GFP sequences as markers.
そしてこの遺伝子配列に変異が生じた各種のTgマウスの中から、DGKβ－KOマウス（DGKβ配列の間に、Tp配列が転位して来たマウス）を選択した。 Then, DGKβ-KO mice (mice in which the Tp sequence was translocated between DGKβ sequences) were selected from various Tg mice in which mutations occurred in the gene sequence.
当該DGKβ－KOマウスの選択は、TgマウスのゲノムDNAを採取し、PCR等によってTp配列の挿入部位のゲノム配列を決定し、マウスゲノムデータベースと照合することで、DGKβ遺伝子内部へTp配列が転位したマウスを同定する方法で行った。 The DGKβ-KO mouse is selected by collecting Tg mouse genomic DNA, determining the genomic sequence of the Tp sequence insertion site by PCR, etc., and collating it with the mouse genome database, thereby translocating the Tp sequence into the DGKβ gene. The method of identifying the mouse was performed.
図１２（Ｂ）は、Tp転位によって、DGKβ配列中にTp配列が組み込まれた状態，及びそのBgl II及びNco Iによる切断部位を示す模式図である。 FIG. 12 (B) is a schematic diagram showing a state in which the Tp sequence is incorporated into the DGKβ sequence by Tp rearrangement and its cleavage site by BglII and NcoI.
尚、Tp遺伝子に組み込んでおいたマーカー遺伝子（LacZ）をプローブに用いたサザンブロット解析により、DGKβ遺伝子以外のゲノム領域にTp配列が存在しないことを確認した（図１２（Ｂ），図１３参照）。 The Southern blot analysis using the marker gene (LacZ) incorporated into the Tp gene as a probe confirmed that no Tp sequence was present in the genomic region other than the DGKβ gene (see FIG. 12B and FIG. 13). ).
つまり、野生型（WT（＋／＋）），ヘテロ（Tp／＋），及びホモ（Tp／Tp）マウスの各々のゲノム遺伝子を、Bgl II及びNco Iで切断し、LacＺ配列をプローブとして用いた、サザンブロット分析を行った結果、ヘテロ，ホモのいずれにおいても、ドナーサイトにある筈の、8.9kbのバンドは、もはや検出されず、DGKβ内部に挿入された4.7kbのバンドが検出された。
つまり、選択したマウスにおいては、確かにTp転位が起こっており、もはやドナーサイトは存在しないこと、および、DGKβ遺伝子以外への転位も存在しないことが確認された。 In other words, wild-type (WT (+ / +)), heterozygous (Tp / +), and homozygous (Tp / Tp) mouse genomic genes were cleaved with BglII and NcoI, and the LacZ sequence was used as a probe. As a result of Southern blot analysis, the 8.9 kb band at the donor site was no longer detected in both heterozygous and homozygous, and the 4.7 kb band inserted inside DGKβ was detected. .
In other words, it was confirmed that Tp translocation certainly occurred in the selected mouse, there was no donor site anymore, and there was no translocation to other than DGKβ gene.
また、ドナーサイトが、この選択したマウス遺伝子中に存在していないことを裏付けるために、ドナーサイトにのみ存在する、プラズミドベクター由来のneo配列の有無を確認した。 In addition, in order to confirm that the donor site does not exist in the selected mouse gene, the presence or absence of a neoplasm derived from a plasmid vector that exists only at the donor site was confirmed.
具体的には、ヘテロ（Tp／＋），及び野生型（WT（＋／＋））マウスの、各々のゲノム遺伝子を、SpeIで切断し、neo配列をプローブとして用いた、サザンブロット分析を行った。 Specifically, Southern blot analysis was performed by cutting the genomic genes of hetero (Tp / +) and wild type (WT (+ / +)) mice with SpeI and using the neo sequence as a probe. It was.
図１４は、Tpベクターを複数組み込んだドナーサイト中の、SpeI切断部位を示す模式図である。 FIG. 14 is a schematic diagram showing a SpeI cleavage site in a donor site incorporating a plurality of Tp vectors.
分析の結果、ヘテロマウスにおいては、ドナーサイトにある筈の、1.2kbのバンドは、もはや検出されなかった（図１５）。 As a result of the analysis, in the hetero mouse, the 1.2 kb band at the donor site was no longer detected (FIG. 15).
尚、図１５において、Controlと記載したのは、neo遺伝子を１つ有する、ゲノムDNAの分析結果である。このゲノムDNAからは、SpeI による切断で、4.2kbのバンドが得られることが予想されるが、予想通りの大きさのバンドが検出されている。
これによって、使用したneo配列に相補的なプローブが、確かに機能していることを確認することができた。 In FIG. 15, “Control” indicates the analysis result of genomic DNA having one neo gene. From this genomic DNA, it is expected that a 4.2 kb band will be obtained by cleavage with SpeI, but a band of the expected size is detected.
This confirmed that the probe complementary to the neo sequence used was indeed functioning.
この結果、選択したマウスにおいては、確かにTp転位が起こっており、もはやドナーサイトは存在しないことが、更に裏付けられた。
これは、生殖細胞における減数分裂時の染色体間組換えとその後の染色体の分離によって、ドナーサイトとTp配列が別れたためと考えられる。 As a result, it was further confirmed that in the selected mice, Tp rearrangement had indeed occurred, and there was no longer any donor site.
This is probably because the donor site and the Tp sequence were separated by interchromosomal recombination at the time of meiosis in germ cells and subsequent chromosome segregation.
このDGKβ－KOマウス（以下、単に「KO（マウス）」と記載する。）を、以下の各種試験に供し、このＫＯマウスが、双極性障害Ｉ型の疾患モデルであることを確認した。
尚、比較のため、同系の、野生型マウス（以下、WT（マウス）と記載する。）を用いた。 This DGKβ-KO mouse (hereinafter simply referred to as “KO (mouse)”) was subjected to the following various tests to confirm that this KO mouse was a bipolar disorder type I disease model.
For comparison, syngeneic wild-type mice (hereinafter referred to as WT (mouse)) were used.
［試験例１：強制水泳試験］
DGKβ－KOマウスの抑鬱様症状（鬱状態）を検討するために強制水泳試験を行った。
強制水泳試験とは、以下の現象を利用するものである。
即ち、マウスを足の付かない深さの水に入れると、マウスは、溺れまいと手足を動かすが、時間経過に従って、絶望状態に陥り泳ぐのを諦め、無動状態となる。そして、しばらくすると、また泳ぎ始める。
この時の、無動状態の時間が長いほど鬱状態にあると判断することができる。 [Test Example 1: Forced swimming test]
A forced swimming test was performed to examine depression-like symptoms (depressed state) in DGKβ-KO mice.
The forced swimming test utilizes the following phenomenon.
That is, when the mouse is put into water of a depth that does not have a foot, the mouse moves limbs and limbs, but as time passes, it falls into despair and gives up to swim and becomes immobile. And after a while, I start swimming again.
At this time, it can be determined that the longer the non-moving state is, the more depressed it is.
具体的な試験は、Porsoltらの方法（Forced swimming in rats: hypothermia, immobility and the effects of imipramine. Eur J Pharmacol 57:431-436., 1979）に準じて行い、結果は、各群10匹のマウスの平均で示した。
初回の試行 (Pre-test)ではマウスを水 (25℃ ± 1)の入ったシリンダー (直径10 × 水深10 cm)に浮かべ5分間の無動時間を測定した。
Pre-testの24時間後の試行 (Post-test)において、同様にマウスをシリンダーに浮かべ再び無動時間を測定した。 The specific test was conducted according to the method of Porsolt et al. (Forced swimming in rats: hypothermia, immobility and the effects of imipramine. Eur J Pharmacol 57: 431-436., 1979). Shown as the average of mice.
In the first trial (Pre-test), a mouse was floated on a cylinder (diameter 10 × water depth 10 cm) containing water (25 ° C. ± 1), and the immobility time for 5 minutes was measured.
In a trial 24 hours after the pre-test (post-test), the mouse was also floated on the cylinder and the immobility time was measured again.
尚、「無動」とは、マウスが無抵抗に水に浮かび、水面から顔を出すのに必要な最小限の動きに留まっている状態(前肢の動きが停止し、片方の後肢が体勢を保つためにわずかに動いている状態)とした。 Note that `` no movement '' means that the mouse floats in the water without resistance and stays in the minimum movement necessary to bring the face out of the water surface (the movement of the forelimbs stops and one of the hind limbs is in posture). To maintain a slight movement).
初回に行ったPre 試験（図１中の「一日目」）において、KOマウスではWTマウスと比べ、無動時間が有意に低下していた（＊＊; p＜ 0.01 vs. WT）。
このPre試験は、Post試験を始める前に、一種の鬱状態を誘発するものであるが、上記の通り、KOマウスでは、鬱状態が惹起され難かったことが判明した。 In the first pre-test ("Day 1" in FIG. 1), KO mice had a significant decrease in immobility time compared to WT mice (**; p <0.01 vs. WT).
This Pre test induces a kind of depression before starting the Post test, but as described above, it was found that depression was difficult to be induced in KO mice.
Pre試験の24時間後に行ったPost試験（「図１中の「二日目」）においてWTマウスは、Pre試験と比べ無動時間が有意に増加していた（＃; p＜ 0.05）。しかしながらKOマウスではその様な無動時間の増加は認められなかった。
また、Post試験においても、KOマウスにおける無動時間はWTマウスと比べ有意に低下していた （＊＊; p＜ 0.01 vs. WT）（図１）。 In the Post test ("Day 2" in Fig. 1) conducted 24 hours after the Pre test, the WT mice had a significantly increased immobility time compared to the Pre test (#; p <0.05). However, such an increase in immobility time was not observed in KO mice.
In the Post test, the immobility time in KO mice was also significantly lower than that in WT mice (**; p <0.01 vs. WT) (FIG. 1).
これらの結果は、KOマウスが、野生型に比べて、多動性が高いことを示し、この試験例１の実施時点において、DGKβ－KOマウスが、鬱症状への抵抗性がある、つまり一種の「躁状態」にあったことを示すものである。 These results indicate that KO mice have higher hyperactivity than wild type, and at the time of the implementation of Test Example 1, DGKβ-KO mice are resistant to depressive symptoms, This indicates that the vehicle was in the “drunken state”.
［試験例２：新奇物質探索試験］
試験例１で用いたマウスと同じマウス群（8-10匹）を用い、DGKβ 遺伝子が学習記憶に果たす役割を検討するために新奇物質探索試験を行った。 [Test Example 2: Novel substance search test]
Using the same group of mice (8-10 mice) as used in Test Example 1, a novel substance search test was conducted to examine the role of the DGKβ gene in learning memory.
この試験は物体認知記憶を測定する試験系である。
試験方法は、「Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, Liu G, Tsien JZ (1999) Genetic enhancement of learning and memory in mice. Nature 401:63-69.」を参考に、下記の通りにして行った。 This test is a test system for measuring object recognition memory.
See `` Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, Liu G, Tsien JZ (1999) Genetic enhancement of learning and memory in mice.Nature 401: 63-69. '' In addition, it went as follows.
試験にはプラスチック製のオープンフィールド(縦30 × 横30 ×高さ14 cm)を用いた。試験前の3日間はマウスをオープンフォールド内にそれぞれ10分間慣らした。 A plastic open field (length 30 mm × width 30 mm × height 14 cm) was used for the test. The mice were habituated for 10 minutes each in an open fold for 3 days before the test.
初回の獲得試行 (acquisition trial)では、オープンフィールドの中央に二つの同様の物質 (円柱: 直径2.5 cm × 高さ2.5 cm)を壁から5 cm離れた位置に対称に設置し、マウスを10分間自由に探索行動させた。 In the first acquisition trial (acquisition trial), two similar substances (cylinder: diameter 2.5 cm x height 2.5 cm) are placed symmetrically 5 cm away from the wall in the center of the open field and the mouse is placed for 10 minutes I was allowed to explore freely.
24時間後の保持試行 (retention trial)では、片方の物質を別の新奇の物質 (立方体: 縦2.5 × 横2.5 × 高さ2.5 cm)に交換し、同様に10分間マウスを自由に探索させた。 In a retention trial after 24 hours, one substance was replaced with another novel substance (cube: vertical 2.5 cm x horizontal 2.5 mm x height 2.5 cm), and mice were allowed to explore freely for 10 minutes as well. .
獲得試行及び保持試行における10分間のマウスのそれぞれの物質に対する探索時間をEthoVision XT (Noldus、Wageningen、The Netherlands)を用いて測定した。
保持試行における、マウスの新奇物質に対する探索嗜好性を、探索時間合計に対する割合として計算した。結果は、各群８－１０匹のマウスの平均で示した。 The search time for each substance in mice for 10 minutes in acquisition and retention trials was measured using an EthoVision XT (Noldus, Wageningen, The Netherlands).
The mouse's search preference for novel substances in retention trials was calculated as a percentage of the total search time. The results are shown as an average of 8-10 mice in each group.
初回に行った獲得試行においては、WTマウス及びKOマウス共に、左右の物質に対して同程度の探索時間を費やした。 In the first acquisition trial, both WT and KO mice spent the same amount of search time for the left and right substances.
獲得試行の24時間後に行った保持試行においては、WTマウスは新奇物質に対する探索時間が増加したが、KOマウスはその様な新奇物質に対する探索時間の増加は認められず、左右の物質に対し同程度の探索時間を示した（図２）。
（＊; p＜ 0.05 vs. WT）（＃; p＜0.05） In retention trials performed 24 hours after the acquisition trial, WT mice increased the search time for novel substances, while KO mice did not show an increase in the search time for such novel substances, and were similar for the left and right substances. A moderate search time was shown (FIG. 2).
(*; P <0.05 vs. WT) (#; p <0.05)
また、獲得試行及び保持試行ともにWTマウスとKOマウスの間に探索時間の合計に差は認められなかった（図３）。 In addition, there was no difference in the total search time between the WT mouse and the KO mouse in both the acquisition trial and the retention trial (FIG. 3).
これらの結果は、KOマウスの学習能力が低下していることを示しており、これは双極性障害の病態の一つである認知障害と関連している可能性が考えられる。 These results indicate that the learning ability of KO mice is reduced, which may be related to cognitive impairment, one of the pathologies of bipolar disorder.
［試験例３：自発運動試験］
試験例１，２で用いたマウスとは別のマウス群を用い、新奇環境における、自発運動をプラスチック製のオープンフィールド（縦30 × 横30 ×高さ14 cm）を用いて測定した。
マウスを、オープンフィールドに置き、120分間自由に探索行動させた。 [Test Example 3: Spontaneous exercise test]
Using a group of mice different from those used in Test Examples 1 and 2, spontaneous movement in a novel environment was measured using a plastic open field (length 30 × width 30 × height 14 cm).
Mice were placed in an open field and allowed to explore freely for 120 minutes.
結果は、各群４匹のマウスの平均で示した。 The results are shown as an average of 4 mice in each group.
DGKβ－KOマウスはWTマウスと比べ、120分における自発運動量の増加が認められた。総移動距離（図４），および移動距離の時間依存的変化（図５），移動速度（図６）が、KOマウスではWTマウスと比べて各々増加しており、図４，５については、有意差が見られた。 DGKβ-KO mice showed an increase in locomotor activity at 120 minutes compared to WT mice. Total movement distance (Fig. 4), time-dependent change of movement distance (Fig. 5), and movement speed (Fig. 6) are increased in KO mice compared to WT mice, respectively, Significant differences were seen.
図４（＊; p＜ 0.05 vs. WT）
図５（＊; p＜ 0.05 vs. WT）（＊＊; p＜ 0.01 vs. WT）
図６（p＝0.07） Figure 4 (*; p <0.05 vs. WT)
FIG. 5 (*; p <0.05 vs. WT) (**; p <0.01 vs. WT)
Figure 6 (p = 0.07)
これらの結果は、KOマウスが、試験例３の実施時点で、一種の「躁状態」にあったことを示すものである。 These results indicate that the KO mice were in a kind of “manic state” at the time of the test example 3.
［試験例４：オープンフィールド試験］
試験例３で用いたマウスを、プラスチック製のオープンフィールド(縦30 × 横30 ×高さ14 cm）に置き、120分間自由に探索行動させた。
マウスの総移動距離と中央部(15 × 15cm)で過ごした時間は、行動追跡・解析用ビデオ・トラッキングシステム〔EthoVision XT（商標）、NOLDUS社製〕を用いて自動的に測定した。
初めの10分間における立ち上がり行動及び腹部の引っかき行動の回数は、手動で測定した。 [Test Example 4: Open field test]
The mouse used in Test Example 3 was placed in a plastic open field (length 30 × width 30 × height 14 cm) and allowed to search freely for 120 minutes.
The total movement distance of the mouse and the time spent in the center (15 × 15 cm) were automatically measured using a behavior tracking / analysis video tracking system (EthoVision XT ™, manufactured by NOLDUS).
The number of standing up and abdominal scratching actions during the first 10 minutes was measured manually.
DGKβ－KOマウスでは、オープンフィールド試験において探索行動の増加が認められた。
DGKβ－KOマウスは、WTマウスと比べオープンフィールドの中央部で過ごす時間および中央部への侵入回数が増加していた（図７，図８）。 In DGKβ-KO mice, an increase in exploratory behavior was observed in the open field test.
DGKβ-KO mice had increased time spent in the center of the open field and the number of intrusions into the center compared to WT mice (FIGS. 7 and 8).
図７（＊; p＜ 0.05 vs. WT）
図８（p＝0.059） Figure 7 (*; p <0.05 vs. WT)
FIG. 8 (p = 0.059)
またDGKβ－KOマウスは、立ち上がり(rearing) 回数の増加が認められた（図９）。 In DGKβ-KO mice, an increase in the number of standing spears was observed (FIG. 9).
興味深いことに、DGKβ－KOマウスは、腹部の引っかき(scratching) 回数の著しい増加が認められた（図１０）。（＊＊; p＜ 0.01 vs. WT） Interestingly, DGKβ-KO mice showed a marked increase in the number of abdominal scratching sputum (FIG. 10). (**; p <0.01 vs. WT)
これらの結果は、KOマウスが、試験例４の実施時点において、過活動状態，つまり一種の「躁状態」にあったことを示すものである。
すなわち、中央部への侵入の多さが、不安レベルの低さ，つまり過動性を示し、立ち上がり行動の回数の多さが、過活動を示し、引っかき行動の多さが、興奮性薬物投与モデル等と類似の、精神疾患様の常同行動を示している。 These results indicate that the KO mice were in an overactive state, that is, a kind of “snoring state” at the time of performing Test Example 4.
In other words, a large number of intrusions into the center indicates a low level of anxiety, that is, hyperactivity, a large number of standing up actions indicate overactivity, and a large number of scratching actions indicate administration of excitatory drugs. It shows normal behaviors like psychiatric disorders similar to models.
上記試験例１～４の結果、DGKβノックアウトマウスは、全て躁状態を示すことが判明し、明らかに、双極性障害のＩ型の症状を示す疾患モデルであることが確認できた。 As a result of the above Test Examples 1 to 4, it was found that all the DGKβ knockout mice showed a manic state, and it was clearly confirmed that this was a disease model showing type I symptoms of bipolar disorder.
［試験例５：LacZ染色］
試験例１～４で用いたマウスとは別のマウス群を用い、「マウス生体中でのDGKβ遺伝子の欠損部位」を確かめるために、LacZ染色を行った。 [Test Example 5: LacZ staining]
A group of mice different from those used in Test Examples 1 to 4 was used, and LacZ staining was performed in order to confirm the “defect site of the DGKβ gene in the mouse body”.
WT及びKOマウス各々の15週齢雄性マウスを、pentobarbital麻酔し、4% paraformaldehyde含有0.1 M phosphate buffer (PB; pH 7.4)を左心室内に注入して灌流固定 (灌流圧130 cm H2O)した。20分後に脳を取り出し、同液にて一晩放置した。ついで25% sucrose含有0.1 M PB (pH7.4)液に24時間放置した。その後、速やかに粉末状のドライアイスで凍結し、薄切するまで-80℃にて保存した。O.C.T. Compound (Sakura Finetechnical)にて凍結した脳を包埋し、cryostat (Leica)を用いて、-20℃で厚さ10 μmの切片を作製し、MASコーティングされたスライドグラス (S-9441, matsunami)にのせ、-80℃で保存した。 A 15-week-old male mouse from each of WT and KO mice was anesthetized with pentobarbital, and 0.1% M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde was injected into the left ventricle to perform perfusion fixation (perfusion pressure 130 cm H2O). After 20 minutes, the brain was removed and left overnight in the same solution. Subsequently, it was allowed to stand in a 0.1% M PB (pH 7.4) solution containing 25% sucrose for 24 hours. Thereafter, it was quickly frozen with powdered dry ice and stored at −80 ° C. until sliced. Embed the frozen brain with OCT Compound (Sakura Finetechnical), and use cryostat (Leica) to prepare a 10 μm thick slice at -20 ° C, and MAS-coated slide glass (S-9441, matsunami ) And stored at -80 ° C.
LacZ染色は、β-garactosidase staining kit (Actove motif、Carlsbad、CA)を用いて行った。切片を0.2% glutaraldehyde 及び 1% formalinに浸透させた後、β－ガラクトシダーゼの基質である5-bromo-4-chloro-3-indoly-β-galactoside (X-Gal)を含んだ反応液に2時間浸して発色させた。 LacZ staining was performed using β-garactosidase staining kit (Actove motif, Carlsbad, CA). After infiltrating the slices with 0.2% glutaraldehyde and 1% formalin, the reaction solution containing 5-bromo-4-chloro-3-indoly-β-galactoside (X-Gal), a substrate of β-galactosidase, was kept for 2 hours. Colored by soaking.
DGKβ－KOマウスでは、嗅球・線条体・海馬を初めとする前脳-中脳部位においてLacZ陽性細胞が認められたが（図１１Ａ，Ｃ，Ｅ）、WTマウスではLacZ陽性細胞は認められなかった（図１１Ｂ，Ｄ，Ｆ）。 In DGKβ-KO mice, LacZ positive cells were observed in the forebrain-mesencephalon region including the olfactory bulb, striatum, and hippocampus (FIGS. 11A, C, and E), but LacZ positive cells were observed in WT mice. None (FIGS. 11B, D, F).
このことは、マウスの前脳-中脳部位において、Tp転位が生じていたことを示している。
つまり、Tp遺伝子中にマーカーとして挿入されたLacZ配列のプロモーターが、転位によってONの状態となった結果、LacＺが翻訳されてβガラクトシダーゼを発現し、基質であるX-gal（5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside）を分解し、不溶性の青い発色物質を生成したことを示している。
つまり、青色の発色を呈した組織において、Tpの転位（DGKβの欠損）が起こっていることが分かる。 This indicates that Tp translocation occurred in the forebrain-mesencephalon region of the mouse.
That is, as a result of the translocation of the LacZ sequence promoter inserted as a marker in the Tp gene, LacZ is translated to express β-galactosidase, and the substrate X-gal (5-Bromo-4 -Chloro-3-Indolyl-β-D-Galactoside) was decomposed to produce an insoluble blue coloring substance.
In other words, it can be seen that Tp translocation (DGKβ deficiency) occurs in tissues exhibiting blue color development.
一方、黒質や小脳などの中脳-後脳においては、KOマウスWTマウスともに、LacZ陽性細胞は認められなかった（図１１Ｇ～Ｊ）。 On the other hand, in the midbrain-hindbrain of substantia nigra and cerebellum, LacZ positive cells were not observed in both KO mice and WT mice (FIGS. 11G to J).
これらの結果は、上記の試験で用いたモデルマウスにおいて、DGKβの欠損部位が、前脳から中脳部位において発生していることを示すものである。 These results indicate that, in the model mouse used in the above test, the DGKβ deficient site is generated from the forebrain to the midbrain.
線条体は、脳における運動を司る領域であり、海馬は記憶障害に関与していることが知られている。
従って、前脳から中脳部位におけるDGKβ欠損が、双極性障害I型の症状（過動状態や記憶障害等）を引き起こす原因の一つであることが裏付けられたと考えられる。 It is known that the striatum is an area that controls movement in the brain, and the hippocampus is involved in memory impairment.
Therefore, it is considered that DGKβ deficiency in the forebrain to midbrain region is one of the causes of bipolar disorder type I symptoms (such as hyperactivity and memory impairment).
［実施例１：双極性障害Ｉ型の予防又は治療薬のスクリーニング方法］
参考例１のモデルに、被験物を投与し、その投与前後における、試験例１～４の結果を比較すること等によって、双極性障害Ｉ型の予防又は治療薬のスクリーニングを行う。 [Example 1: Screening method for preventing or treating bipolar disorder type I]
A test substance is administered to the model of Reference Example 1, and a preventive or therapeutic drug for bipolar disorder type I is screened by comparing the results of Test Examples 1 to 4 before and after the administration.
本発明のスクリーニング方法によって、双極性障害のＩ型に特化した、画期的な予防・治療薬の開発が期待できる。 The screening method of the present invention can be expected to develop an innovative preventive / therapeutic agent specialized in bipolar disorder type I.
DGKβの機能を欠損させた参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例１の強制水泳試験に供した結果を表す図である。尚、図中の＊＊は、野生型に対する疾患モデルマウスの試験結果の、標準偏差がp＜ 0.01であることを表し、＃は、一日目に対する二日目の標準偏差が、p＜ 0.05であることを表す。It is a figure showing the result of having used the disease model mouse (KO) of the reference example 1 which made the function of DGKβ defective, and the wild type mouse (WT) for the forced swimming test of the test example 1. In the figure, ** indicates that the standard deviation of the test result of the disease model mouse against the wild type is p <0.01, and # indicates that the standard deviation on the second day relative to the first day is p <0.05. It represents that. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例２の新奇物質探索試験に供した結果（探索時間合計中の、新奇物質探索時間の割合（％））を表す図である。尚、図中の＊は、野生型に対する疾患モデルマウスの試験結果の、標準偏差がp＜ 0.05であることを表し、＃は、一日目に対する二日目の標準偏差が、p＜ 0.05であることを表す。Results of the disease model mouse (KO) of Reference Example 1 and the wild type mouse (WT) subjected to the novel substance search test of Test Example 2 (ratio of novel substance search time in the total search time (%)) FIG. In the figure, * indicates that the standard deviation of the test result of the disease model mouse against wild type is p <0.05, and # indicates that the standard deviation of the second day relative to the first day is p <0.05. Represents something. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例２の新奇物質探索試験に供した結果を（探索時間の総計（秒））表す図である。It is a figure showing the result of having used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the novel substance search test of the test example 2 (total of search time (second)). 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例３の自発運動試験に供した結果（総移動距離（cm））を表す図である。It is a figure showing the result (total movement distance (cm)) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the spontaneous movement test of the test example 3. FIG. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例３の自発運動試験に供した結果（自発運動量の時間依存的変化）を表す図である。It is a figure showing the result (time-dependent change of spontaneous exercise amount) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the spontaneous exercise test of the test example 3. FIG. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例３の自発運動試験に供した結果（移動速度（cm／秒））を表す図である。It is a figure showing the result (movement speed (cm / sec)) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the spontaneous movement test of the test example 3. FIG. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例４のオープンフィールド試験に供した結果（オープンフィールドの中央で過ごした時間（秒））を表す図である。It is a figure showing the result (time (second) spent in the center of an open field) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the open field test of the test example 4. FIG. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例４のオープンフィールド試験に供した結果（オープンフィールドの中央部へ侵入した回数（回））を表す図である。It is a figure showing the result (the frequency | count (times) which invaded the center part of the open field) which used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for the open field test of Test Example 4. . 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例４のオープンフィールド試験に供した結果（初めの10分間における立ち上がり回数（回））を表す図である。It is a figure showing the result (number of times of starting (times) in the first 10 minutes) of the disease model mouse (KO) of Reference Example 1 and the wild type mouse (WT) subjected to the open field test of Test Example 4. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例４のオープンフィールド試験に供した結果（初めの10分間における腹部の引っかき行動の回数（回））を表す図である。The figure showing the result (number of times of abdominal scratching behavior in the first 10 minutes (times)) of the disease model mouse (KO) of Reference Example 1 and the wild type mouse (WT) subjected to the open field test of Test Example 4 It is. 参考例１の疾患モデルマウス（KO）と、野生型マウス（WT）を、試験例５のLacZ染色に供した結果を表す図である。Ａ，Ｃ，Ｅ，Ｇ，Ｉが、KOマウスの、嗅球，線条体，海馬，黒質，小脳の染色結果を表す。Ｂ，Ｄ，Ｆ，Ｈ，Ｊが、WTマウスの、嗅球，線条体，海馬，黒質，小脳の染色結果を表す。It is a figure showing the result of having used the disease model mouse (KO) of the reference example 1, and the wild type mouse (WT) for LacZ staining of the test example 5. FIG. A, C, E, G, and I represent staining results of olfactory bulb, striatum, hippocampus, substantia nigra, and cerebellum in KO mice. B, D, F, H, and J represent staining results of the olfactory bulb, striatum, hippocampus, substantia nigra, and cerebellum in WT mice. （Ａ）は、参考例１の疾患モデルマウス（KO）を作製する際に用いた、Tp配列を挿入したドナーサイト，及びそのBgl II及びNco Iによる切断部位を示す模式図である。また、（Ｂ）は、Tp配列が組み込まれたDGKβ配列，及びそのBgl II及びNco Iによる切断部位を示す模式図である。尚、図中のB，Nは、それぞれ、Bgl II及びNco Iによる切断部位を表す。(A) is a schematic diagram showing a donor site into which a Tp sequence has been inserted, and its cleavage site by Bgl II and Nco I, used when preparing the disease model mouse (KO) of Reference Example 1. FIG. (B) is a schematic diagram showing a DGKβ sequence in which a Tp sequence is incorporated, and its cleavage site by BglII and NcoI. In the figure, B and N represent cleavage sites by Bgl II and Nco I, respectively. 野生型（WT（＋／＋）），ヘテロ（Tp／＋），及びホモ（Tp／Tp）マウスの各々のゲノム遺伝子について、LacＺ配列をプローブとして用いた、サザンブロット分析を行った結果を表す図である。The results of Southern blot analysis using the LacZ sequence as a probe for the genomic genes of wild type (WT (+ / +)), heterozygous (Tp / +), and homozygous (Tp / Tp) mice are shown. FIG. 参考例１の疾患モデルマウス（KO）を作製する際に用いた、Tp配列を挿入したドナーサイト，及びそのSpeI切断部位を示す模式図である。尚、図中のSは、SpeIによる切断部位を表す。It is a schematic diagram which shows the donor site which inserted the Tp arrangement | sequence used when producing the disease model mouse (KO) of the reference example 1, and its SpeI cutting site. In the figure, S represents a site cleaved by SpeI. ヘテロ（Tp／＋），及び野生型（WT（＋／＋））マウスの、各々のゲノム遺伝子について、neo配列に相補的なポリヌクレオチドをプローブとして用いた、サザンブロット分析を行った結果を表す図である。The results of Southern blot analysis using a polynucleotide complementary to the neo sequence as a probe for each genomic gene of heterozygous (Tp / +) and wild type (WT (+ / +)) mice are shown. FIG.

Claims (3)

  1. DGKβの機能を欠損させた非ヒト動物を用いることを特徴とする、双極性障害Ｉ型の予防又は治療薬のスクリーニング方法。 A screening method for a preventive or therapeutic agent for bipolar disorder type I, wherein a non-human animal deficient in the function of DGKβ is used.
  2. DGKβ機能の欠損が、前脳から中脳部位の少なくとも一部において発生している非ヒト動物を用いることを特徴とする、請求項１記載の、双極性障害Ｉ型の予防又は治療薬のスクリーニング方法。 The screening for a prophylactic or therapeutic agent for bipolar disorder type I according to claim 1, wherein a non-human animal in which a deficiency in DGKβ function occurs in at least a part of the forebrain to the midbrain region is used. Method.
  3. 非ヒト動物が、齧歯類であることを特徴とする、請求項１又は２記載の、双極性障害Ｉ型の予防又は治療薬のスクリーニング方法。 The method for screening a preventive or therapeutic agent for bipolar disorder type I according to claim 1 or 2, wherein the non-human animal is a rodent.
PCT/JP2008/058665 2008-05-09 2008-05-09 Method of screening preventive or remedy for type i dipolar disorder WO2009136444A1 (en)

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