JP5531198B2 - Hearing loss model animal and its use - Google Patents
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Description
本発明は難聴モデル動物及びその用途に関する。詳しくは、加齢性難聴の表現型を示す齧歯類遺伝子改変動物、先天性難聴の表現型を示す齧歯類遺伝子改変動物、及びそれらを用いて難聴に有効な物質をスクリーニングする方法等に関する。 The present invention relates to a hearing loss model animal and its use. More specifically, the present invention relates to a rodent genetically modified animal exhibiting age-related deafness phenotype, a rodent genetically modified animal exhibiting congenital deafness phenotype, and a method for screening a substance effective for hearing loss using them. .
過去20年間、ヒト及びマウスでいくつかの難聴遺伝子が発見されてきた（非特許文献１）。従来、一部特定の遺伝子を欠損させた遺伝子改変ノックアウトマウスが難聴の表現型を示すことは知られている。しかしながら、ヒトの先天性難聴では、遺伝子欠損よりもむしろ遺伝子内の点変異が原因になる場合が多く、特定の遺伝子が欠損したノックアウトマウスを用いて難聴の発症機序を探るのは、ヒトの本来の病態を反映していない可能性がある。 In the past 20 years, several deafness genes have been discovered in humans and mice (Non-patent Document 1). Conventionally, it has been known that a genetically modified knockout mouse partially deficient in a specific gene exhibits a deafness phenotype. However, human congenital deafness is often caused by point mutations in the gene rather than gene deficiency, and it is not possible to investigate the pathogenesis of deafness using knockout mice lacking a specific gene. It may not reflect the original pathology.
ところで、ヒトは20歳を過ぎた頃より少しずつ聴力が低下し始め、70歳では約60%が加齢性難聴を発症することが報告されている。高齢化が進む中、加齢性難聴患者の数は今後さらに増加することが予想される。また、難聴は認知症を誘発する因子になるだけでなく、耳鳴りの直接的原因になる。このように、難聴は高齢者のQOL（Quality of Life：生活の質）に大きな影響を与える。来るべき超高齢化社会に向けて、加齢性難聴・耳鳴りを予防又は治療することはQOL向上につながるだけでなく、難聴の進行を予防することにより高齢者の認知症の発症や進行を遅らせる意味でも極めて重要な課題である。 By the way, it has been reported that human hearing begins to gradually decrease after the age of 20, and about 60% develop age-related hearing loss at the age of 70. As the population ages, the number of patients with age-related hearing loss is expected to increase further in the future. Hearing loss is not only a factor that induces dementia but also a direct cause of tinnitus. In this way, hearing loss has a great impact on the quality of life of the elderly. Preventing or treating age-related deafness and tinnitus not only leads to improved quality of life, but also prevents the progression of deafness by delaying the onset and progression of dementia in the elderly This is an extremely important issue.
以上の背景に鑑み本発明は、難聴や耳鳴りの予防又は治療手段の確立を目指し、難聴の表現型を示すモデル動物及びその用途を提供することを課題とする。 In view of the above background, an object of the present invention is to provide a model animal exhibiting a deafness phenotype and its use, aiming at establishment of a means for preventing or treating hearing loss and tinnitus.
上述の通り、特定の遺伝子の欠損と難聴の関係についての研究報告は存在するものの、特定の遺伝子に点変異を導入することによって先天性又は加齢性難聴を解析した例は未だない。本発明者らは神経成長因子の受容体型チロシンキナーゼであるRET/Ret遺伝子に注目し（慣例に倣い、ヒトの遺伝子を大文字で表記する）、RET/Retの活性化レベルが難聴に影響するとの仮定を立てた。尚、RET/Retは、ヒトのヒルシュスプラング病（巨大結腸症）、多発性内分泌腺腫症（MEN）、甲状腺癌の原因遺伝子であることが現在までに知られている。また、しかしながら、RET/retが聴力に関係していることを示す報告は、ヒトレベルでも動物レベルでも全くない。 As described above, although there are research reports on the relationship between a specific gene deficiency and hearing loss, there are no examples of analyzing congenital or age-related deafness by introducing a point mutation into a specific gene. The present inventors focused on the RET / Ret gene, which is a receptor-type tyrosine kinase of nerve growth factor (following the convention, the human gene is capitalized), and the activation level of RET / Ret affects hearing loss I made an assumption. It has been known that RET / Ret is a causative gene of human Hirschsprung's disease (macrocolosis), multiple endocrine adenomatosis (MEN), and thyroid cancer. However, there are no reports showing that RET / ret is related to hearing at the human or animal level.
上記仮定の下で本発明者らはまず、野生型マウスが正常聴力を獲得する生後18日までのRetタンパク質の発現レベル及び活性化レベルを解析した。その結果、Retタンパク質の活性化レベルが聴力の発達に重要な役割を果たすことが示唆された。次に、Retタンパク質の活性化に重要なアミノ酸が置換されるように点変異を導入したマウス（ノックインマウス）を作製した。具体的には、Retタンパク質の1063番目チロシンがフェニルアラニンに置換されるように遺伝子操作し、両方のアレルのRet遺伝子に点変異が導入されたホモ型のノックインマウス（ホモRet-Y1062F-ノックインマウス）と片方のアレルのRet遺伝子に点変異が導入されたヘテロ型のノックインマウス（ヘテロRet-Y1062F-ノックインマウス）を作製し、種々の検討を行った。その結果、ホモRet-Y1062F-ノックインマウスが先天性難聴の表現型を示し、当該マウスが先天性難聴モデルとして有用であることが判明した。また、ホモRet-Y1062F-ノックインマウスの内耳のらせん神経節は、生後の聴覚発達時期にアポトーシス陰性の重度の神経変性を示すことが明らかとなった。さらに、ヘテロRet-Y1062F-ノックインマウスでは、生後１月の時点において野生型マウスの聴力レベルと同等の聴力レベルを示す一方で、生後4月以降になると野生型マウスの聴力レベルと比較して高音域から聴力低下が加速し、生後10月ともなると高音域の聴力レベルが測定域外であった。即ち、加齢性難聴の表現型を示し、しかも非常に短期間で聴力レベルの低下が認められた。この事実は、加齢性難聴のモデルとして当該ヘテロRet-Y1062F-ノックインマウスが極めて有用であることを示す。尚、Retタンパク質の1063番目チロシンが腸神経系の形成及び腎形成に重要な役割を果たすことが報告されている（非特許文献２）。尚、ホモRet-Y1062F-ノックインマウスではヒルシュスプラング病が発症するが、ヘテロRet-Y1062F-ノックインマウスでは発症しない。 Under the above assumptions, the present inventors first analyzed the expression level and activation level of Ret protein up to 18 days after birth when wild-type mice acquire normal hearing. The results suggested that the level of Ret protein activation plays an important role in hearing development. Next, a mouse (knock-in mouse) into which a point mutation was introduced so that an amino acid important for activation of the Ret protein was substituted was prepared. Specifically, a homozygous knock-in mouse (homogeneous Ret-Y1062F-knock-in mouse) in which a point mutation was introduced into the Ret gene of both alleles was genetically manipulated so that the 1063rd tyrosine of the Ret protein was replaced with phenylalanine. Heterotype knock-in mice (hetero-Ret-Y1062F-knock-in mice) in which a point mutation was introduced into the Ret gene of one allele were prepared and various studies were performed. As a result, it was found that the homo-Ret-Y1062F-knock-in mouse showed a congenital deafness phenotype and was useful as a congenital deafness model. In addition, it was revealed that the spiral ganglia of the inner ear of homo-Ret-Y1062F-knock-in mice show severe neurodegeneration that is negative for apoptosis at the time of auditory development after birth. In addition, the hetero-Ret-Y1062F-knock-in mouse exhibits a hearing level equivalent to that of the wild-type mouse at 1 month of birth, but higher than that of the wild-type mouse at 4 months after birth. Hearing loss accelerated from the range, and in October, the hearing level in the high range was out of the measurement range. That is, it showed an age-related deafness phenotype, and a decrease in hearing level was observed in a very short period of time. This fact indicates that the hetero-Ret-Y1062F-knock-in mouse is extremely useful as a model for age-related hearing loss. It has been reported that the 1063rd tyrosine of the Ret protein plays an important role in the formation of enteric nervous system and nephrogenesis (Non-patent Document 2). In addition, Hirschsprung's disease develops in homo Ret-Y1062F-knock-in mice, but not in hetero Ret-Y1062F-knock-in mice.
一方、RET/Retと難聴との関係を更に検討するため、ヘテロRet-Y1062F-ノックインマウスと、恒常活性型RET遺伝子が導入された遺伝子改変マウス（242系：Kato M et al., Oncogene 18:837-42, 1999）とを交配した。交配によって得られたマウスの表現型を調べた結果、生後14月においても野生型と同等の聴力レベルを維持していた。つまり、恒常活性型RET遺伝子の導入によって、ヘテロRet-Y1062F-ノックインマウスに認められた加齢性難聴の表現型を改善する（加齢に伴う難聴を示さない）ことが示された。 On the other hand, in order to further examine the relationship between RET / Ret and hearing loss, hetero-Ret-Y1062F-knock-in mice and genetically modified mice introduced with a constitutively active RET gene (242 series: Kato M et al., Oncogene 18: 837-42, 1999). As a result of examining the phenotype of the mouse obtained by the mating, the hearing level equivalent to that of the wild type was maintained even in 14 months after birth. That is, it was shown that the introduction of the constitutively active RET gene improves the age-related deafness phenotype observed in hetero-Ret-Y1062F-knock-in mice (no deafness associated with aging).
以上の成果より、次の知見、即ち（１）RET/Retタンパク質のリン酸化レベル、即ち活性レベルが聴覚の形成、発達に重要な役割を担うこと、（２）先天性難聴の表現型を示すモデル動物を作製するためには、Retタンパク質の活性化を妨げるように両方のアレルのRet遺伝子に変異を導入することが有効であること、（３）加齢性難聴の表現型を示すモデル動物を作製するためには、Retタンパク質の活性化を妨げるように片方のアレルのRet遺伝子に変異を導入することが有効であること、及び（４）RET／Retが難聴の予防・治療の標的分子になること、が導き出された。
本発明は主として以上の知見に基づく。以下、本発明を列挙する。
［１］Retタンパク質の活性化に必須のアミノ酸の置換を伴う点遺伝子変異によって片方のアレルのRet遺伝子が機能障害されており、加齢性難聴の表現型を示す、齧歯類遺伝子改変動物。
［２］前記齧歯類がマウスであり、
前記アミノ酸が、Retタンパク質の1063番チロシン残基である、［１］に記載の齧歯類遺伝子改変動物。
［３］前記点遺伝子変異が、前記1063番チロシン残基がフェニルアラニンに置換される点遺伝子変異である、［２］に記載の齧歯類遺伝子改変動物。
［４］前記齧歯類がマウス以外の齧歯類であり、
前記アミノ酸が、該齧歯類のRetタンパク質においてマウスRetタンパク質の1063番チロシン残基に相当するチロシン残基である、［１］に記載の齧歯類遺伝子改変動物。
［５］以下のステップ（１）及び（２）を含む、加齢性難聴又は騒音性難聴の予防又は治療に有効な物質のスクリーニング法：
（１）［１］〜［４］のいずれか一項に記載の齧歯類遺伝子改変動物に被験物質を取り込ませるステップ；
（２）聴力の低下が抑制されたか否か評価し、評価結果に基づき被験物質の有効性を判定するステップ。
［６］ステップ（１）後の前記齧歯類遺伝子改変動物を用い、以下の指標、即ち、(a)内耳らせん神経節の細胞数、(b)内耳らせん神経節でのRet遺伝子の発現レベル及び(c)内耳らせん神経節でのRetタンパク質の活性化レベル、からなる群より選択される一以上の指標について検出し、検出結果に基づいてステップ（２）の評価を行う、［５］に記載のスクリーニング法。
［７］聴性脳幹反応によってステップ（２）の評価を行う、［５］に記載のスクリーニング法。
［８］Retタンパク質の活性化に必須のアミノ酸の置換を伴う点遺伝子変異によって片方のアレルのRet遺伝子が機能障害されており、先天性難聴の表現型を示す、齧歯類遺伝子改変動物。
［９］前記齧歯類がマウスであり、
前記アミノ酸が、Retタンパク質の1063番チロシン残基である、［８］に記載の齧歯類遺伝子改変動物。
［１０］前記点遺伝子変異が、前記1063番チロシン残基がフェニルアラニンに置換される点遺伝子変異である、［９］に記載の齧歯類遺伝子改変動物。
［１１］前記齧歯類がマウス以外の齧歯類であり、
前記アミノ酸が、該齧歯類のRetタンパク質においてマウスRetタンパク質の1063番チロシン残基に相当するチロシン残基である、［８］に記載の齧歯類遺伝子改変動物。
［１２］以下のステップ（１）及び（２）を含んでなる、先天性難聴の治療に有効な物質のスクリーニング法：
（１）［８］〜［１１］のいずれか一項に記載の齧歯類遺伝子改変動物に被験物質を取り込ませるステップ；
（２）聴力が回復したか否か評価し、評価結果に基づき被験物質の有効性を判定するステップ。
［１３］ステップ（１）後の前記齧歯類遺伝子改変動物を用い、(a)内耳らせん神経節の細胞数、(b)カルビンディン及び(c)シナプトフィジンからなる群より選択される一以上の指標について検出し、検出結果に基づいてステップ（２）の評価を行う、［１２］に記載のスクリーニング法。
［１４］聴性脳幹反応によってステップ（２）の評価を行う、［１２］に記載のスクリーニング法。
［１５］以下の（１）及び（２）を含むことを特徴とする、難聴の予防又は治療に有効な物質のスクリーニング法：
（１）RET遺伝子が発現している細胞であって、神経細胞及び神経芽細胞からなる群より選択される細胞を被験物質の存在下で培養するステップ；
（２）前記細胞における、RET遺伝子の発現レベル又はRETタンパク質の活性化レベルを検出し、検出結果に基づき被験物質の有効性を判定するステップ。
From the above results, the following findings are shown: (1) RET / Ret protein phosphorylation level, that is, the activity level plays an important role in auditory formation and development, (2) shows the phenotype of congenital deafness In order to produce a model animal, it is effective to introduce mutations into the Ret gene of both alleles so as to prevent the activation of Ret protein, and (3) a model animal showing a phenotype of age-related deafness In order to prevent the activation of Ret protein, it is effective to introduce a mutation into the Ret gene of one allele, and (4) RET / Ret is a target molecule for prevention / treatment of hearing loss It was derived.
The present invention is mainly based on the above findings. The present invention will be enumerated below.
[1] A rodent genetically modified animal in which the Ret gene of one allele is dysfunctional due to a point gene mutation accompanied by substitution of an amino acid essential for activation of the Ret protein, and exhibits a phenotype of age-related deafness.
[2] The rodent is a mouse,
The rodent genetically modified animal according to [1], wherein the amino acid is the 1063 tyrosine residue of the Ret protein.
[3] The rodent gene-modified animal according to [2], wherein the point gene mutation is a point gene mutation in which the 1063 tyrosine residue is substituted with phenylalanine.
[4] The rodent is a rodent other than a mouse,
The rodent genetically modified animal according to [1], wherein the amino acid is a tyrosine residue corresponding to the 1063 tyrosine residue of the mouse Ret protein in the rodent Ret protein.
[5] A screening method for a substance effective for the prevention or treatment of age-related hearing loss or noise-induced hearing loss, comprising the following steps (1) and (2):
(1) A step of incorporating a test substance into the rodent genetically modified animal according to any one of [1] to [4];
(2) A step of evaluating whether the decrease in hearing ability is suppressed and determining the effectiveness of the test substance based on the evaluation result.
[6] Using the rodent genetically modified animal after step (1), the following indicators are used: (a) number of cells in the inner ear spiral ganglion, (b) expression level of Ret gene in the inner ear spiral ganglion And (c) the activation level of the Ret protein in the inner ear spiral ganglion is detected for one or more indicators selected from the group consisting of, and the evaluation of step (2) is performed based on the detection results. The screening method described.
[7] The screening method according to [5], wherein step (2) is evaluated by an auditory brainstem reaction.
[8] A rodent genetically modified animal in which the Ret gene of one allele is dysfunctional due to a point gene mutation accompanied by substitution of an amino acid essential for activation of the Ret protein, and exhibits a congenital deafness phenotype.
[9] The rodent is a mouse,
The rodent genetically modified animal according to [8], wherein the amino acid is the 1063 tyrosine residue of the Ret protein.
[10] The rodent gene-modified animal according to [9], wherein the point gene mutation is a point gene mutation in which the 1063 tyrosine residue is substituted with phenylalanine.
[11] The rodent is a rodent other than a mouse,
The rodent genetically modified animal according to [8], wherein the amino acid is a tyrosine residue corresponding to the 1063 tyrosine residue of the mouse Ret protein in the rodent Ret protein.
[12] A screening method for a substance effective for the treatment of congenital hearing loss, comprising the following steps (1) and (2):
(1) A step of incorporating a test substance into the rodent genetically modified animal according to any one of [8] to [11];
(2) A step of evaluating whether or not hearing has been restored, and determining the effectiveness of the test substance based on the evaluation result.
[13] One or more selected from the group consisting of (a) the number of cells of the inner ear spiral ganglion, (b) calbindin and (c) synaptophysin using the rodent genetically modified animal after step (1) The screening method according to [12], wherein an index is detected and the evaluation of step (2) is performed based on the detection result.
[14] The screening method according to [12], wherein step (2) is evaluated by an auditory brainstem reaction.
[15] A screening method for a substance effective for the prevention or treatment of hearing loss, comprising the following (1) and (2):
(1) culturing a cell expressing a RET gene, which is selected from the group consisting of a neuron and a neuroblast, in the presence of a test substance;
(2) A step of detecting the expression level of the RET gene or the activation level of the RET protein in the cell, and determining the effectiveness of the test substance based on the detection result.
１．加齢性難聴モデル動物及びその用途
本発明の第１の局面は、加齢性難聴の表現型を示す齧歯類遺伝子改変動物及びその用途に関する。本発明の齧歯類遺伝子改変動物は加齢性難聴のモデルとして有用である。本明細書では、説明の便宜上、加齢性難聴の表現型を示す本発明の齧歯類遺伝子改変動物のことを「本発明の加齢性難聴モデル動物」と呼ぶ。
1. Age-related hearing loss model animal and use thereof The first aspect of the present invention relates to a rodent genetically modified animal exhibiting age-related hearing loss phenotype and use thereof. The rodent genetically modified animal of the present invention is useful as a model for age-related hearing loss. In the present specification, for convenience of explanation, the rodent genetically modified animal of the present invention showing the phenotype of age-related deafness is referred to as “age-related deafness model animal of the present invention”.
（１）加齢性難聴モデル動物
本発明の加齢性難聴モデル動物は、遺伝子変異によって片方のアレルのRet遺伝子が機能障害されている。即ち、本発明の加齢性難聴モデル動物はRet遺伝子に関してヘテロ変異体であり、この特徴によって加齢性難聴の表現型を示す。ここでの「遺伝子変異」は、Ret遺伝子がコードするタンパク質（即ちRetタンパク質）の活性化に必須のアミノ酸の置換を伴うものであり、障害されたRet遺伝子（以下、「変異型Ret遺伝子」と呼ぶ）は、活性化が抑制されたRetタンパク質をコードすることになる。
(1) Age-related hearing loss model animal In the age-related hearing loss model animal of the present invention, the Ret gene of one allele is dysfunctional due to gene mutation. That is, the age-related hearing loss model animal of the present invention is a heterozygous mutant with respect to the Ret gene, and exhibits a phenotype of age-related hearing loss by this feature. Here, the “gene mutation” refers to a substitution of amino acids essential for the activation of the protein encoded by the Ret gene (ie, the Ret protein), and an impaired Ret gene (hereinafter referred to as “mutant Ret gene”). Calls a Ret protein with suppressed activation.
齧歯類は特に限定されず、マウス、ラット、モルモット、ハムスター及びウサギを含む。好ましくはマウス（例えばC57BL/6J系統（日本チャールズリバーから入手可能））である。 Rodents are not particularly limited and include mice, rats, guinea pigs, hamsters and rabbits. Preferred is a mouse (eg, C57BL / 6J strain (available from Charles River Japan)).
齧歯類がマウスの場合、好ましくは、1063番チロシン残基が欠失又は他のアミノ酸に置換されたRetタンパク質を変異型Ret遺伝子がコードする。1063番チロシン残基は自己リン酸化を受けてRetタンパク質を活性化する重要なアミノ酸残基である。1063番チロシン残基の置換を伴う場合の置換後のアミノ酸の種類は特に限定されない。好ましくは、置換後のアミノ酸はフェニルアラニンとする。この態様の加齢性難聴モデル動物（モデルマウス）は、片方のアレルのRet遺伝子が、1063番チロシン残基がフェニルアラニンに置換された変異型Retタンパク質をコードしており、加齢性難聴の表現型を示すことになる。尚、マウスRet遺伝子には二つのアイソフォーム（Ret9とRet51）が存在することが知られている。Ret9遺伝子の配列を配列番号１に、Ret51遺伝子の配列を配列番号３にそれぞれ示す。また、Ret9タンパク質の配列を配列番号２に、Ret51タンパク質の配列を配列番号４にそれぞれ示す。 When the rodent is a mouse, the mutant Ret gene preferably encodes a Ret protein in which the 1063 tyrosine residue is deleted or substituted with another amino acid. The 1063 tyrosine residue is an important amino acid residue that undergoes autophosphorylation and activates the Ret protein. The type of amino acid after substitution when substitution of the 1063 tyrosine residue is involved is not particularly limited. Preferably, the amino acid after substitution is phenylalanine. In this embodiment of the model for age-related hearing loss (model mouse), the Ret gene of one allele encodes a mutant Ret protein in which the 1063 tyrosine residue is substituted with phenylalanine. Will indicate the type. The mouse Ret gene is known to have two isoforms (Ret9 and Ret51). The sequence of Ret9 gene is shown in SEQ ID NO: 1, and the sequence of Ret51 gene is shown in SEQ ID NO: 3, respectively. The sequence of Ret9 protein is shown in SEQ ID NO: 2, and the sequence of Ret51 protein is shown in SEQ ID NO: 4, respectively.
その置換を伴う変異が加齢性難聴の表現型をもたらす限りにおいて、変異に係るアミノ酸残基は1063番チロシン残基に限定されるものではない。変異に係るアミノ酸の候補として、906番チロシン残基、1016番チロシン残基を挙げることができる。変異に係るアミノ酸の数は典型的には１であるが、加齢性難聴の表現型がもたらされる限りにおいて、変異に係るアミノ酸の数は２以上であることを妨げない。 As long as the mutation with the substitution results in an age-related hearing loss phenotype, the amino acid residue involved in the mutation is not limited to the 1063 tyrosine residue. Examples of amino acid candidates related to the mutation include the 906th tyrosine residue and the 1016th tyrosine residue. The number of amino acids involved in the mutation is typically 1, but it does not preclude that the number of amino acids involved in the mutation is 2 or more as long as an age-related hearing loss phenotype is provided.
齧歯類がマウス以外の場合には、好ましくは、当該齧歯類のRetタンパク質においてマウスRetタンパク質の1063番チロシン残基に相当するチロシン残基が他のアミノ酸に置換されたRetタンパク質を変異型Ret遺伝子がコードする。ここで、本明細書においてアミノ酸残基について使用する場合の用語「相当する」とは、比較されるタンパク質間においてその機能の発揮に同等の貢献をしていることを意味する。例えば、基準のアミノ酸配列に対して比較対象のアミノ酸配列を、一次構造（即ちアミノ酸配列）の部分的な相同性を考慮しつつ、最適な比較ができるように並べたときに（このときに必要に応じてギャップを導入し、アライメントを最適化してもよい）、基準のアミノ酸配列中の特定のアミノ酸に対応する位置のアミノ酸を「相当するアミノ酸」として特定することができる。BLASTやFASTAに代表される、容易に利用可能な配列アライメント用のソフトウエア又はアルゴリズムを利用することによって、「相当するアミノ酸」は容易に特定できる。 When the rodent is other than a mouse, preferably the Ret protein in which the tyrosine residue corresponding to the 1063 tyrosine residue of the mouse Ret protein is substituted with another amino acid in the Ret protein of the rodent Ret gene encodes. Here, the term “corresponding” when used with respect to amino acid residues in the present specification means that the compared proteins are equally contributing to the performance of their functions. For example, when the amino acid sequence to be compared with the reference amino acid sequence is arranged so that an optimal comparison can be made while considering partial homology of the primary structure (ie, amino acid sequence) (necessary at this time) And the alignment may be optimized), and the amino acid at the position corresponding to the specific amino acid in the reference amino acid sequence can be specified as “corresponding amino acid”. The “corresponding amino acid” can be easily identified by using a readily available software or algorithm for sequence alignment represented by BLAST and FASTA.
変異型Ret遺伝子は、典型的には、同種動物のRet遺伝子に変異が導入されたRet遺伝子である。一方で、異種動物のRet遺伝子に変異が導入された点変異型Ret遺伝子を変異型Ret遺伝子として採用することもできる。このような変異型Ret遺伝子の一例を挙げると、マウスRetタンパク質の1063番チロシン残基に相当する、1062番チロシン残基が他のアミノ酸（例えばフェニルアラニン）に置換されたRETタンパク質をコードする変異型RET遺伝子である。尚、RETには二つのアイソフォーム（RET9とRET51）が存在することが知られている。RET9遺伝子の配列を配列番号５に、RET51遺伝子の配列を配列番号７にそれぞれ示す。また、RET9タンパク質の配列を配列番号６に、RET51タンパク質の配列を配列番号８にそれぞれ示す。 The mutant Ret gene is typically a Ret gene in which a mutation is introduced into the Ret gene of the same animal species. On the other hand, a point mutation type Ret gene in which a mutation is introduced into the Ret gene of a heterologous animal can also be adopted as the mutant type Ret gene. An example of such a mutant Ret gene is a mutant that encodes a RET protein corresponding to the 1063 tyrosine residue of mouse Ret protein, in which the 1062 tyrosine residue is substituted with another amino acid (eg, phenylalanine). RET gene. RET is known to have two isoforms (RET9 and RET51). The sequence of the RET9 gene is shown in SEQ ID NO: 5, and the sequence of the RET51 gene is shown in SEQ ID NO: 7. The sequence of the RET9 protein is shown in SEQ ID NO: 6, and the sequence of the RET51 protein is shown in SEQ ID NO: 8, respectively.
本発明の加齢性難聴モデル動物は、例えば、ジーンターゲティングを利用してRet遺伝子に点変異を導入すること又は点変異を含む変異型Ret遺伝子を導入することによって作製できる。ジーンターゲティングは遺伝子の相同組換えを利用してゲノムに改変を加える技術である。ジーンターゲティングを利用すれば、特定の遺伝子が欠損した遺伝子改変動物（ノックアウト動物）、特定の遺伝子が導入された遺伝子改変動物（ノックイン動物）等を作製することができる。ジーンターゲティングを利用した遺伝子改変動物（ノックアウト動物、ノックイン動物）の作製法は、マウスの場合を例にとれば、大別して（ａ）ターゲティングベクターの構築、（ｂ）ES細胞へのターゲティングベクターの導入及び相同組換え体の同定、（ｃ）ブラストシストへの相同組み換え体の注入、（ｄ）仮親の子宮内への胚移植及び出産（キメラ仔マウス）、（ｅ）キメラ仔マウスと野生型マウスの交配（F1世代であるヘテロ接合体マウスの産出）、及び（ｆ）F1世代同士の交配（F2世代であるホモ接合体マウスの産出）からなる。ラットなど、他の齧歯類を用いた場合も同様の手順で遺伝子改変動物を得ることができる。尚、ジーンターゲティングによる遺伝子改変動物の作製法については例えばJoyner, A. L.:Gene Targeting (IRL press)、Capeccchi, M. R., Science, 244, 1288-1292 (1989)、Hua Gu, et al., Science, 265, 103-106 (1994)、McHugh, T. J. et al., Cell, 87, 1339-1349 (1996)、Shibata, H. et al., Science, 278, 120-123 (1997)等が参考になる。 The age-related hearing loss model animal of the present invention can be produced, for example, by introducing a point mutation into a Ret gene using gene targeting or by introducing a mutant Ret gene containing a point mutation. Gene targeting is a technique for modifying a genome using homologous recombination of genes. By using gene targeting, a genetically modified animal lacking a specific gene (knock-out animal), a genetically modified animal introduced with a specific gene (knock-in animal), and the like can be produced. Gene-modified animals (knockout animals, knockin animals) using gene targeting can be roughly classified into (a) construction of targeting vectors and (b) introduction of targeting vectors into ES cells. And identification of homologous recombinants, (c) injection of homologous recombinants into blast cysts, (d) embryo transfer and birth into uterus of surrogate parents (chimeric offspring mice), (e) chimeric offspring mice and wild type mice (F) generation of heterozygous mice that are F1 generations, and (f) crossing of F1 generations (production of homozygous mice that are F2 generations). When other rodents such as rats are used, genetically modified animals can be obtained by the same procedure. As for the method for producing a genetically modified animal by gene targeting, for example, Joyner, AL: Gene Targeting (IRL press), Capeccchi, MR, Science, 244, 1288-1292 (1989), Hua Gu, et al., Science, 265 103-106 (1994), McHugh, TJ et al., Cell, 87, 1339-1349 (1996), Shibata, H. et al., Science, 278, 120-123 (1997).
本発明の加齢性難聴モデル動物は変異型Ret遺伝子をノックインした動物（ノックイン動物）として得ることができる。本発明の加齢性難聴モデル動物は、変異型Ret遺伝子に関してヘテロ接合体であることから、変異型Ret遺伝子を挿入可能に構築したターゲティングベクターを用い、（ａ）〜（ｅ）の工程を行うことによって作製できる（マウスの場合）。 The age-related hearing loss model animal of the present invention can be obtained as an animal (knock-in animal) in which a mutant Ret gene is knocked in. Since the age-related hearing loss model animal of the present invention is heterozygous for the mutant Ret gene, the steps (a) to (e) are performed using a targeting vector constructed so that the mutant Ret gene can be inserted. (In the case of a mouse).
（２）加齢性難聴又は騒音性難聴を標的としたスクリーニング法
本発明の加齢性難聴モデル動物の用途の一つとして、加齢性難聴又は騒音性難聴の予防又は治療に有効な物質のスクリーニング法が提供される。本発明のスクリーニング法は以下のステップ（１）及び（２）を含む。
（１）本発明の加齢性難聴モデル動物に被験物質を取り込ませるステップ
（２）聴力の低下が抑制されたか否か評価し、評価結果に基づき被験物質の有効性を判定するステップ
(2) Screening method targeting age-related hearing loss or noise-related hearing loss As one of the uses of the age-related hearing loss model animal of the present invention, a substance effective for the prevention or treatment of age-related hearing loss or noise-related hearing loss A screening method is provided. The screening method of the present invention includes the following steps (1) and (2).
(1) A step of incorporating a test substance into the age-related hearing loss model animal of the present invention (2) A step of evaluating whether or not a decrease in hearing ability is suppressed and determining the effectiveness of the test substance based on the evaluation result
ステップ（１）では、本発明の加齢性難聴モデル動物を用意し、当該動物に被験物質を取り込ませる。被験物質の取り込ませ方は特に限定されない。例えば被験物質を含む餌又は飲料水を用意し、これを摂取させる。或いは、被験物質又は被験物質を含む溶液を用意し、これを投与する。投与方法として経口投与、経鼻投与、経気管投与、静脈内、動脈内、皮下、筋肉内又は腹腔内注射、標的組織又は器官への注入を例示することができる。被験物質を複数回投与することにしてもよい。その場合には各回の投与量は同一であっても異なっていても良い。 In step (1), the age-related hearing loss model animal of the present invention is prepared, and the test substance is taken into the animal. There is no particular limitation on the method of incorporating the test substance. For example, food or drinking water containing the test substance is prepared and ingested. Alternatively, a test substance or a solution containing the test substance is prepared and administered. Examples of the administration method include oral administration, nasal administration, tracheal administration, intravenous, intraarterial, subcutaneous, intramuscular or intraperitoneal injection, and injection into a target tissue or organ. The test substance may be administered multiple times. In that case, each dose may be the same or different.
被験物質としては様々な分子サイズの有機化合物又は無機化合物を用いることができる。有機化合物の例として、核酸、ペプチド（ダイペプチド、トリペプチド等のオリゴペプチドやポリペプチド）、タンパク質、脂質（単純脂質、複合脂質（ホスホグリセリド、スフィンゴ脂質、グリコシルグリセリド、セレブロシド等）、プロスタグランジン、イソプレノイド、テルペン、ステロイド、ポリフェノール、カテキン、ビタミン（B1、B2、B3、B5、B6、B7、B9、B12、C、A、D、E等）を例示できる。被験物質は天然物由来であっても、或いは合成によるものであってもよい。後者の場合には例えばコンビナトリアル合成の手法を利用して効率的なスクリーニング系を構築することができる。尚、細胞抽出液、培養上清などを試験物質として用いてもよい。また、既存の薬剤を被験物質としてもよい。 As a test substance, organic compounds or inorganic compounds having various molecular sizes can be used. Examples of organic compounds include nucleic acids, peptides (oligopeptides and polypeptides such as dipeptides and tripeptides), proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.), prostaglandins , Isoprenoids, terpenes, steroids, polyphenols, catechins, vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.) Test substances are derived from natural products In the latter case, an efficient screening system can be constructed using, for example, a combinatorial synthesis technique, and cell extracts, culture supernatants, etc. It may be used as a test substance, or an existing drug may be used as a test substance.
試験群及び対照群に含まれる個体数は特に限定されない。一般に使用する個体数が多くなるほど信頼性の高い結果が得られるが、多数の個体を同時に取り扱うことは使用する個体の確保や操作（飼育を含む）の面で困難を伴う。そこで例えば各群に含まれる個体数を１〜５０、好ましくは２〜３０、さらに好ましくは３〜２０とする。 The number of individuals included in the test group and the control group is not particularly limited. In general, as the number of individuals used increases, a more reliable result can be obtained. However, handling a large number of individuals at the same time is difficult in terms of securing and operating (including breeding) the individuals to be used. Therefore, for example, the number of individuals included in each group is 1 to 50, preferably 2 to 30, and more preferably 3 to 20.
本発明のスクリーニング方法によって選択された物質が十分な薬効を有する場合には、当該物質をそのまま医薬の有効成分として使用することができる。一方で十分な薬効を有しない場合には化学的修飾などの改変を施してその薬効を高めた上で、医薬の有効成分として使用することができる。勿論、十分な薬効を有する場合であっても、更なる薬効の増大を目的として同様の改変を施してもよい。 When a substance selected by the screening method of the present invention has a sufficient medicinal effect, the substance can be used as it is as an active ingredient of a medicine. On the other hand, when it does not have a sufficient medicinal effect, it can be used as an active ingredient of a medicine after it has been modified by chemical modification to enhance its medicinal effect. Of course, even if it has a sufficient medicinal effect, the same modification may be applied for the purpose of further increasing the medicinal effect.
ステップ（２）では、被験物質の取り込みによって聴力の低下が抑制されたか否か評価し、評価結果に基づき被験物質の有効性を判定する。具体的には、被験物質の取り込みによって聴力の低下が抑制された場合、即ち、被験物質に聴力低下抑制作用ないし効果を認めた場合、被験物質が有効であると判定する。有効性が認められた複数の被験化合物を用いて再度ステップ（１）及び（２）を行い、有効性の高い物質の絞り込みを行うことにしてもよい。聴力の低下が抑制されたか否かの評価には、例えば聴性脳幹反応を利用できる。「聴性脳幹反応」とは、聴覚神経系を興奮させることによって得られる脳幹部での電位（脳幹の反応）である。所定の音刺激を与えた時の聴性脳幹反応（脳波）を解析することによって、聴力のレベルを再現性良く調べることができる。聴性脳幹反応を利用した検査法は難聴の判定に広く利用されている。聴性脳幹反応を利用した検査法の詳細については例えばABRハンドブック（加我君孝 編（金原出版））を参照することができる。 In step (2), it is evaluated whether or not the decrease in hearing ability is suppressed by taking in the test substance, and the effectiveness of the test substance is determined based on the evaluation result. Specifically, when the decrease in hearing is suppressed by the uptake of the test substance, that is, when the test substance exhibits an effect or an effect of suppressing the decrease in hearing, it is determined that the test substance is effective. Steps (1) and (2) may be performed again using a plurality of test compounds that have been confirmed to be effective to narrow down highly effective substances. For example, an auditory brainstem reaction can be used for evaluating whether or not the decrease in hearing ability is suppressed. The “auditory brainstem reaction” is a potential in the brainstem (brainstem reaction) obtained by exciting the auditory nervous system. By analyzing the auditory brainstem response (electroencephalogram) when a predetermined sound stimulus is applied, the level of hearing ability can be examined with good reproducibility. Test methods using the auditory brainstem reaction are widely used to determine hearing loss. For details of the test method using the auditory brainstem reaction, refer to the ABR Handbook (Edited by Kimitaka Kaga (Kanehara Publishing)).
(a)内耳らせん神経節の細胞数、(b)内耳らせん神経節でのRet遺伝子の発現レベル及び(c)内耳らせん神経節でのRetタンパク質の活性化レベルからなる群より選択される一以上の指標について検出し、検出結果に基づき、聴力の低下が抑制されたか否かを評価してもよい。これらの指標は互いに排他的なものではなく、二つ以上の指標を併用することもできる。また、ここで挙げた指標による評価に加えて、上記の聴性脳幹反応を利用した評価を行った上で最終的な判定をしてもよい。尚、(a)の指標の検出には組織染色が好適である。(b)の指標については、Ret遺伝子のmRNAの量をRT-PCRで定量することや、Retタンパク質の量を免疫学的に測定することによって検出可能である。(c)の指標の検出には、活性化Retに特異的な抗体を用いた免疫組織化学が好適である。 one or more selected from the group consisting of (a) the number of cells in the inner ear spiral ganglion, (b) the expression level of the Ret gene in the inner ear spiral ganglion, and (c) the activation level of the Ret protein in the inner ear spiral ganglion. May be detected, and based on the detection result, it may be evaluated whether or not the decrease in hearing is suppressed. These indicators are not mutually exclusive, and two or more indicators can be used in combination. Further, in addition to the evaluation based on the index mentioned here, the final determination may be made after performing the evaluation using the above-described auditory brainstem reaction. It should be noted that tissue staining is suitable for the detection of the index (a). The indicator (b) can be detected by quantifying the amount of Ret gene mRNA by RT-PCR or by measuring the amount of Ret protein immunologically. For the detection of the index (c), immunohistochemistry using an antibody specific for activated Ret is suitable.
２．先天性難聴モデル動物及びその用途
本発明の第２の局面は、先天性難聴の表現型を示す齧歯類遺伝子改変動物及びその用途に関する。この局面の齧歯類遺伝子改変動物は先天性難聴のモデルとして有用である。本明細書では、説明の便宜上、先天性難聴の表現型を示す本発明の齧歯類遺伝子改変動物のことを「本発明の先天性難聴モデル動物」と呼ぶ。
2. Congenital hearing loss model animal and use thereof The second aspect of the present invention relates to a rodent genetically modified animal exhibiting a congenital deafness phenotype and use thereof. The rodent genetically modified animal of this aspect is useful as a model for congenital deafness. In the present specification, for convenience of explanation, the rodent genetically modified animal of the present invention showing the phenotype of congenital deafness is referred to as “the congenital deafness model animal of the present invention”.
（１）先天性難聴モデル動物
本発明の先天性難聴モデル動物は、遺伝子変異によって両方のアレルのRet遺伝子が機能障害されている。即ち、本発明の先天性難聴モデル動物はRet遺伝子に関してホモ変異体であり（対立遺伝子の両方とも、活性化が抑制されたRetタンパク質をコードする変異型Ret遺伝子である）、この特徴によって先天性難聴の表現型を示す。
(1) Congenital hearing loss model animal In the congenital hearing loss model animal of the present invention, the Ret gene of both alleles is dysfunctional due to gene mutation. That is, the congenital hearing loss model animal of the present invention is a homo mutant with respect to the Ret gene (both alleles are mutant Ret genes encoding Ret proteins whose activation is suppressed). Indicates the deafness phenotype.
上記の第１の局面における齧歯類の説明及び変異型Ret遺伝子の説明は、この局面の先天性難聴モデル動物にも当てはまる。但し、本発明の先天性難聴モデル動物は、本発明の加齢性難聴モデル動物と異なり、変異型Ret遺伝子に関してホモ接合体であることから、ジーンターゲティングを利用して作製する場合、第１の局面で説明した「遺伝子改変動物（ノックアウト動物、ノックイン動物）の作製法」における（ａ）〜（ｆ）の工程を行うことになる（マウスの場合）。 The description of the rodent and the description of the mutant Ret gene in the first aspect also apply to the congenital deafness model animal of this aspect. However, unlike the age-related deafness model animal of the present invention, the congenital deafness model animal of the present invention is a homozygote with respect to the mutant Ret gene. The steps (a) to (f) in the “method for producing a genetically modified animal (knockout animal, knockin animal)” described in the aspect are performed (in the case of a mouse).
（２）先天性難聴を標的としたスクリーニング法
本発明の先天性難聴モデル動物の用途の一つとして、先天性難聴の治療に有効な物質のスクリーニング法が提供される。本発明のスクリーニング法は以下のステップ（１）及び（２）を含む。
（１）本発明の先天性難聴モデル動物に被験物質を取り込ませるステップ
（２）聴力が回復したか否か評価し、評価結果に基づき被験物質の有効性を判定するステップ
(2) Screening method targeting congenital deafness As one of the uses of the congenital deafness model animal of the present invention, a screening method for a substance effective for the treatment of congenital deafness is provided. The screening method of the present invention includes the following steps (1) and (2).
(1) A step of incorporating a test substance into the congenital deafness model animal of the present invention (2) A step of evaluating whether or not hearing ability has been restored and determining the effectiveness of the test substance based on the evaluation result
ステップ（１）は、第１の局面におけるスクリーニング方法のステップ（１）と同様の方法及び条件で実施することができる。ステップ（２）では、被験物質の取り込みによって聴力が回復したか否か評価し、評価結果に基づき被験物質の有効性を判定する。具体的には、被験物質の取り込みによって聴力が回復した場合、即ち、被験物質に聴力回復作用ないし効果を認めた場合、被験物質が有効であると判定する。有効性が認められた複数の被験化合物を用いて再度ステップ（１）及び（２）を行い、有効性の高い物質の絞り込みを行うことにしてもよい。ステップ（２）の評価は例えば、聴性脳幹反応を利用して行えばよい。また、加齢性難聴を発症したマウス内耳ラセン神経節で発現レベルが上昇するカルビンディン、シナプトフィジン等の指標についてその特異抗体を用いて検出し、検出結果に基づいて評価してもよい。あるいは、内耳らせん神経節の細胞数を指標として評価してもよい。二つ以上の指標を併用可能なこと、これらの指標による評価と聴性脳幹反応を利用した評価を組み合わせた総合評価によって最終的な判定を下すことにしてもよいことは、第１の局面の場合と同様である。その他、ここで特に言及しない事項については第１の局面の対応する説明を援用することにする。 Step (1) can be carried out under the same method and conditions as in step (1) of the screening method in the first aspect. In step (2), it is evaluated whether or not the hearing ability has been recovered by taking in the test substance, and the effectiveness of the test substance is determined based on the evaluation result. Specifically, when the hearing ability is recovered by the uptake of the test substance, that is, when the test substance exhibits a hearing recovery action or effect, it is determined that the test substance is effective. Steps (1) and (2) may be performed again using a plurality of test compounds that have been confirmed to be effective to narrow down highly effective substances. The evaluation in step (2) may be performed using an auditory brainstem reaction, for example. In addition, indicators such as calbindin and synaptophysin whose expression level is increased in mouse inner ear spiral ganglia that develop age-related deafness may be detected using the specific antibody and evaluated based on the detection result. Alternatively, the number of cells in the inner ear spiral ganglion may be evaluated as an index. In the case of the first aspect, two or more indicators can be used in combination, and the final judgment may be made by a comprehensive evaluation that combines an evaluation using these indicators and an evaluation using an auditory brainstem reaction. It is the same. For other matters not specifically mentioned here, the corresponding explanation of the first aspect will be incorporated.
３．RETを標的としたスクリーニング法
本発明の第３の局面は、難聴治療の新たな標的分子としてRETが有用であるとの知見に基づき、RETを標的分子としたスクリーニング法を提供する。この局面のスクリーニング法は以下のステップ（１）及び（２）を含む。このスクリーニング法によれば、難聴の予防又は治療に有効な物質をスクリーニングすることができる。
（１）RET遺伝子が発現している細胞であって、神経細胞及び神経芽細胞からなる群より選択される細胞を被験物質の存在下で培養するステップ
（２）前記細胞における、RET遺伝子の発現レベル又はRETタンパク質の活性化レベルを検出し、検出結果に基づき被験物質の有効性を判定するステップ
3. Screening Method Targeting RET The third aspect of the present invention provides a screening method using RET as a target molecule based on the finding that RET is useful as a new target molecule for treatment of hearing loss. The screening method of this aspect includes the following steps (1) and (2). According to this screening method, a substance effective for prevention or treatment of hearing loss can be screened.
(1) A step of culturing a cell expressing a RET gene, which is selected from the group consisting of a neuron and a neuroblast, in the presence of a test substance (2) Expression of the RET gene in the cell Detecting the level or activation level of RET protein and determining the effectiveness of the test substance based on the detection result
ステップ（１）はではRET遺伝子が発現する細胞であって、神経細胞又は神経芽細胞に分類される細胞を用意する。使用可能な細胞の具体例を示すと、PC12細胞（理研細胞バンクから入手可能）、NB-1細胞（理研細胞バンクからから入手可能）である。 In step (1), cells that express the RET gene and are classified as nerve cells or neuroblasts are prepared. Specific examples of cells that can be used are PC12 cells (available from RIKEN Cell Bank) and NB-1 cells (available from RIKEN Cell Bank).
被験物質の添加のタイミングは特に限定されない。従って、被験物質を含まない培養液で培養を開始した後、ある時点で被験物質を添加することにしても、予め被験物質を含む培養液で培養を開始することにしてもよい。培養条件は使用する細胞に標準的なものを採用すればよい。尚、ステップ（１）はin vitroで行われる。 The timing of addition of the test substance is not particularly limited. Therefore, after culturing is started with a culture solution not containing the test substance, the test substance may be added at a certain point in time, or the culture may be started with a culture solution containing the test substance in advance. The culture conditions may be standard for the cells used. Step (1) is performed in vitro.
ステップ（２）では、被験物質存在下で培養した後の細胞におけるRET遺伝子の発現レベル又はRETタンパク質の活性化レベルを検出する。そして、検出結果に基づき被験物質の有効性を判定する。具体的には、被験物質の存在下で培養したことによってRET遺伝子の発現レベル又はRETタンパク質の活性化レベルが上昇した場合、即ち、被験物質にRET遺伝子の発現レベル上昇作用ないし効果、及び／又はRETタンパク質の活性化レベル上昇作用ないし効果を認めた場合、被験物質が有効であると判定する。有効性が認められた複数の被験化合物を用いて再度ステップ（１）及び（２）を行い、有効性の高い物質の絞り込みを行うことにしてもよい。 In step (2), the expression level of the RET gene or the activation level of the RET protein in the cells after culturing in the presence of the test substance is detected. Then, the effectiveness of the test substance is determined based on the detection result. Specifically, when the expression level of the RET gene or the activation level of the RET protein is increased by culturing in the presence of the test substance, that is, the action or effect of increasing the expression level of the RET gene on the test substance, and / or If the RET protein activation level elevating action or effect is observed, the test substance is determined to be effective. Steps (1) and (2) may be performed again using a plurality of test compounds that have been confirmed to be effective to narrow down highly effective substances.
RET遺伝子の発現レベルは、RET遺伝子のmRNA量をRT-PCRで定量することや、RETタンパク質の量を免疫学的に測定することによって検出可能である。一方、RETタンパク質の活性化レベルは、活性化RETに特異的な抗体を用いた免疫組織化学によって検出可能である。 The expression level of the RET gene can be detected by quantifying the amount of mRNA of the RET gene by RT-PCR or measuring the amount of RET protein immunologically. On the other hand, the activation level of the RET protein can be detected by immunohistochemistry using an antibody specific for activated RET.
この局面のスクリーニング法によって選抜された物質を上記スクリーニング法（第１の局面のスクリーニング法又は第２の局面のスクリーニング法）の被験物質とすることもできる。即ち、このスクリーニング法を一次スクリーニングとして用い、その後、上記スクリーニング法を実施することも可能である。このように２段階のスクリーニングを実施すれば、一層効率的に目的の物質を見出すことが可能となる。 A substance selected by the screening method of this aspect can be used as a test substance for the screening method (the screening method of the first aspect or the screening method of the second aspect). That is, this screening method can be used as a primary screening, and then the screening method can be performed. If the two-stage screening is performed in this manner, the target substance can be found more efficiently.
尚、特に言及しない事項（被験物質の種類、対照群の利用、選抜された物質の改変など）については、第１の局面におけるスクリーニング法に関する記述が援用される。 For matters not specifically mentioned (type of test substance, use of control group, modification of selected substance, etc.), the description relating to the screening method in the first aspect is incorporated.
＜聴力レベルを規定する分子の模索、及び難聴モデル動物の作製＞
１．方法
（１）難聴モデル動物の作製
点変異によってRetの1063番目のチロシンをフェニルアラニンに置換したc-Ret Y1062Fホモ及びヘテロノックインマウス作製の方法を以下に記す。ネオマイシン耐性遺伝子を含むターゲティングベクターを129svjマウス由来のES細胞に挿入し、薬物でターゲティングベクターを含むES細胞を選択した。その後、ES細胞クローンをC57BL/6Jマウスに胚盤胞移植を行い、キメラマウスを作製した（参考文献６）。
<Search for molecules that regulate hearing level and production of deafness model animals>
1. Method (1) Production of Hearing Loss Model Animal A method for producing c-Ret Y1062F homo and hetero knock-in mice in which the 1063 tyrosine of Ret is substituted with phenylalanine by point mutation is described below. A targeting vector containing a neomycin resistance gene was inserted into ES cells derived from 129svj mice, and ES cells containing the targeting vector were selected with drugs. Thereafter, ES cell clones were transplanted into C57BL / 6J mice to produce blastocysts to produce chimeric mice (Reference 6).
（２）聴力レベルを規定する分子の模索
得られたノックインマウスを聴性脳幹反応検査法（ABRハンドブック（加我君孝 編（金原出版））で聴力レベルを解析した。
(2) Searching for a molecule that regulates the hearing level The obtained knock-in mouse was analyzed for the hearing level by the auditory brainstem reaction test method (ABR Handbook (Edited by Kimitaka Kaga (Kanehara Publishing)).
（３）内耳の形態解析
マウス内耳はブアン固定液を用いて灌流固定した。得られた凍結切片、あるいはパラフィン切片について、抗体を用いた免疫染色とクリュー・バレラ染色を行った。電子顕微鏡解析の際は、マウス内耳はグルタールアルデヒドとパラホルムアルデヒドの混合固定液を用いて灌流固定した。
(3) Morphological analysis of the inner ear The mouse inner ear was fixed by perfusion using a Buan fixative. The obtained frozen section or paraffin section was subjected to immunostaining using an antibody and Clew-Barrera staining. During electron microscope analysis, the mouse inner ear was perfusion-fixed using a mixed fixative of glutaraldehyde and paraformaldehyde.
２．結果
（１）Retの1063番目のチロシンのリン酸化レベルと聴力の発達
神経成長因子の受容体型チロシンキナーゼであるRET/Retは、ヒトのヒルシュスプラング病（巨大結腸症）、多発性内分泌腺腫症（MEN）、甲状腺癌の原因遺伝子であることが現在までに知られている（参考文献２、３）。また、その1062番目（マウスRetでは1063番目）のチロシンは、RET/Retのキナーゼ活性及びシグナル伝達系に重要ないくつかの分子が結合する重要な部位であることが知られている（参考文献２〜４）。事実、点変異によってRetの1063番目のチロシンをフェニルアラニンに置換したc-Ret Y1062Fホモノックインマウス(c-Ret KIY1062F/ Y1062F-mice)は、腸神経の顕著な減少、及び腎臓の形成不全を示すことが報告されている（参考文献６）。
2. Results (1) Phosphorylation level and hearing development of the 1063 tyrosine of Ret RET / Ret, a receptor tyrosine kinase of nerve growth factor, is used in human Hirschsprung's disease (megacolon), multiple endocrine neoplasia ( MEN), it is known to date that it is a causative gene for thyroid cancer (reference documents 2, 3). In addition, the 1062nd (1063th in mouse Ret) tyrosine is known to be an important site where several molecules important for RET / Ret kinase activity and signal transduction system bind (References). 2-4). In fact, c-Ret Y1062F homoknock-in mice (c-Ret KIY1062F / Y1062F-mice) in which Ret 1063 tyrosine was substituted with phenylalanine by point mutations showed a marked decrease in enteric nerves and kidney dysplasia. Has been reported (Reference 6).
しかしながら、現時点において、RET/Retが聴力に関係していることを示す報告はない。そこで、まず野生型マウスが正常聴力を獲得する生後18日までのRetタンパク質の発現レベル及び活性レベル（1063番目のチロシンのリン酸化レベル）を免疫組織学的に解析した。その結果、生後14日のマウス内耳のらせん神経節、血管条、聴神経においてRetタンパク質およびRet1063番目のチロシンのリン酸化が検出された（結果を図示せず）。一方、Retは腸神経系の神経節の発達・維持に重要な役割を果たしていること（参考文献２、３）、さらに聴覚系の神経節は内耳から中枢神経系への音信号の伝達に重要な役割を果たしていること（参考文献５）が知られていることから、まず内耳聴神経節におけるRetの発現レベル及び活性レベル（1063番目のチロシンのリン酸化レベル）の経時的変化に焦点をおいて解析を行った。その結果、生後1-18日にかけて、Retタンパク質その自体はらせん神経節に常に発現しているのに対して（結果を図示せず）、Retの1063番目のチロシンのリン酸化レベルは、野生型マウスが聴力を獲得し始める二日前の生後6日以降に急激に上昇することが分かった（図１ａ）。このことから、らせん神経節におけるRetの1063番目のチロシンのリン酸化レベルが聴力の発達に重要な役割を果たしている可能性が出てきた。 However, there are currently no reports showing that RET / Ret is related to hearing. Thus, first, the expression level and activity level (phosphorylation level of the 1063rd tyrosine) of Ret protein until the 18th day after the wild-type mouse acquires normal hearing were analyzed immunohistologically. As a result, phosphorylation of Ret protein and Ret1063rd tyrosine was detected in the spiral ganglia, vascular streak, and auditory nerve of the 14-day-old mouse inner ear (results not shown). On the other hand, Ret plays an important role in the development and maintenance of the ganglia of the enteric nervous system (Refs. 2 and 3), and the auditory ganglia are important for the transmission of sound signals from the inner ear to the central nervous system. Since it is known that it plays an important role (Ref. 5), we first focused on the temporal changes in Ret expression and activity levels (1063 tyrosine phosphorylation level) in the inner ear auditory ganglia. Analysis was performed. As a result, from 1-18 days after birth, Ret protein itself is always expressed in the spiral ganglion (results not shown), whereas phosphorylation level of Ret 1063rd tyrosine is wild type It was found that the mouse increased rapidly after 6 days after birth two days before it began to acquire hearing (FIG. 1a). This suggests that the phosphorylation level of Ret 1063 tyrosine in the spiral ganglion may play an important role in hearing development.
（２）ホモ型Ret-Y1062F-ノックインマウスの表現型
上記可能性を個体レベルで検証する為に、Ret分子内の1063番目のチロシンに点変異を導入し、Ret分子内の1063番目のチロシンのリン酸化が殆ど検出されない（図１ｃ〜ｇ）ホモ型Ret-Y1062F-ノックインマウス（Ret機能高度障害マウス）を作製した（参考文献６を参照）。このマウスを用いて聴性脳幹反応（ABR）閾値（聴力レベル）を調べた。その結果、低音から高音域（4-40 kHz）まで、生後18日のRet機能高度障害マウスの聴力は78-85dBで重度の障害（聾）を受けていることが分かった（図１ｂ）。同じ日齢の野生型マウスの聴力は20-55dBで正常レベルであった（結果を図示せず）。このように、ホモ型Ret-Y1062F-ノックインマウスは先天性難聴の表現型を持つことが判明した。
(2) Phenotype of homozygous Ret-Y1062F-knock-in mouse In order to verify the above-mentioned possibility at the individual level, a point mutation was introduced into the 1063rd tyrosine in the Ret molecule, and the 1063rd tyrosine in the Ret molecule was introduced. Homo-type Ret-Y1062F-knock-in mice (highly impaired Ret function mice) were prepared (see Reference 6). These mice were used to examine the auditory brainstem response (ABR) threshold (hearing level). As a result, it was found that the hearing ability of Ret function highly impaired mice on the 18th day of life was 78-85 dB from the bass to the treble range (4-40 kHz) and suffered severe impairment (聾) (Fig. 1b). The hearing ability of wild-type mice of the same age was 20-55 dB, which was a normal level (results not shown). Thus, it was found that homozygous Ret-Y1062F-knock-in mice have a congenital deafness phenotype.
（３）1063番目のチロシンのリン酸化とらせん神経節の発達
次に、生後2.5、8、14日における、らせん神経節の細胞数を測定したところ、生後2.5日では有意な差はみられないものの（図１ｈ、ｉ、ｌ）、生後8、14日のホモ型Ret-Y1062F-ノックインマウス（Ret機能高度障害マウス）では、基底回転のらせん神経節が約20%あるいは35%と有意に減少していることが分かった（図１ｊ、ｋ、ｌ）。この結果と対応して、らせん神経節におけるc-Ret Y1062のリン酸化は生後1-5日まではほとんど検出されないが（図１ａ）、生後6日以降に顕著に上昇することから、生後6日以降のらせん神経節におけるc-Ret Y1062のリン酸化がらせん神経節の発達に重要であることが示唆された。
(3) Phosphorylation of the 1063rd tyrosine and development of the spiral ganglion Next, when the number of cells in the spiral ganglion was measured at 2.5, 8, and 14 days after birth, there was no significant difference at 2.5 days after birth. However, in the homozygous Ret-Y1062F-knock-in mice (Ret dysfunctional mice) at 8 and 14 days after birth (Fig. 1h, i, l), the spiral ganglion of basal rotation was significantly reduced to about 20% or 35%. (Fig. 1j, k, l). Corresponding to this result, phosphorylation of c-Ret Y1062 in the spiral ganglion is hardly detected until 1-5 days after birth (Fig. 1a), but it increases markedly after 6 days of birth, so that 6 days after birth. These results suggest that phosphorylation of c-Ret Y1062 in the subsequent spiral ganglion is important for the development of the spiral ganglion.
更に、らせん神経節減少が神経変性の原因となっているか否かについて検討する為に、らせん神経節に対する透過電子顕微鏡解析を行った。その結果、生後14日のホモ型Ret-Y1062F-ノックインマウス（Ret機能高度障害マウス）では、らせん神経節及びその核が明らかに縮んでいる形態変化を示した（図２ｂ）。さらに、その核膜が不連続になっており、その周辺に高度に凝集した異質染色質が観察された（図２ｄ）。一方、同じ日齢の野生型マウスのらせん神経節では、そのような形態変化は観察されず、核膜についてもはっきりした二重膜が観察された（図２ａ、ｃ）。尚、野生型、ホモ型Ret-Y1062F-ノックインマウスのいずれについても、らせん神経節にアポトーシスシグナルの表現型（タネル染色など）は検出されなかった。 Furthermore, in order to investigate whether spiral ganglion loss is the cause of neurodegeneration, transmission electron microscope analysis was performed on spiral ganglia. As a result, the 14-day-old homozygous Ret-Y1062F-knock-in mouse (Ret dysfunctional mouse) showed a morphological change in which the spiral ganglion and its nucleus were clearly contracted (FIG. 2b). Furthermore, the nuclear membrane was discontinuous, and a highly aggregated heterochromatin was observed around it (FIG. 2d). On the other hand, in the spiral ganglia of wild-type mice of the same age, such a morphological change was not observed, and a clear double membrane was also observed for the nuclear membrane (FIGS. 2a and c). In both wild-type and homozygous Ret-Y1062F-knock-in mice, no apoptotic signal phenotype (such as Tanel staining) was detected in the spiral ganglion.
以上の結果より、ホモ型Ret-Y1062F-ノックインマウスのらせん神経節は、生後の聴覚発達時期にアポトーシス陰性の重度の神経変性を示すことが明らかになった。c-Retが細胞死に伴うアポトーシスに関与するか否かについては、複数の研究グループから異なる結果が報告されており、興味ある課題である。 From the above results, it was revealed that the spiral ganglia of homozygous Ret-Y1062F-knock-in mice show severe neurodegeneration that is negative for apoptosis at the time of auditory development after birth. Whether c-Ret is involved in apoptosis associated with cell death or not has been reported by several research groups and is an interesting issue.
（４）ヘテロ型Ret-Y1062F-ノックインマウスの表現型
次に、同様の実験をヘテロ型Ret-Y1062F-ノックインマウス（Ret機能軽度障害マウス）を用いて行った。このマウスの寿命、体重、腸神経系、腎臓の発達については異常がないことが報告されている（参考文献６）。聴力測定（ABR）を行った結果、らせん神経節におけるRet-Y1062のリン酸化レベルが野生型よりも有意に減少しているヘテロ型Ret-Y1062F-ノックインマウスは、生後1ヶ月では野生型の聴力レベルと同等であるのに対して、生後4ヶ月以降、野生型と比較して高音域から聴力低下が加速し、10ヶ月に至っては、高音域の聴力レベルは測定域外（聾）であることが分かった（図３ａ〜ｃ）。更に、ヘテロ型Ret-ノックアウトマウスも同様に野生型マウスよりも加齢性難聴が進行（悪化）していることが分かった（結果を図示せず）。これらの結果と対応して、野生型マウスのらせん神経節の細胞数は加齢依存的に緩やかな減少を示したが、ヘテロ型Ret-Y1062F-ノックインマウスのらせん神経節の細胞数は、野生型と比較して急激な減少することが分かった（図３ｄ〜ｍ）。
(4) Phenotype of heterozygous Ret-Y1062F-knock-in mouse Next, a similar experiment was performed using heterozygous Ret-Y1062F-knock-in mouse (Ret function mildly impaired mouse). It has been reported that there are no abnormalities in the life span, body weight, enteric nervous system, and kidney development of this mouse (Reference 6). As a result of audiometry (ABR), heterozygous Ret-Y1062F-knock-in mice, in which the phosphorylation level of Ret-Y1062 in the spiral ganglion is significantly reduced compared to the wild type, Compared to the level, the hearing loss accelerates from the high range compared to the wild type after 4 months, and after 10 months, the high level hearing level is outside the measurement range (聾) Was found (FIGS. 3a-c). Further, it was found that heterogeneous Ret-knockout mice progress (aggravate) age-related hearing loss more than wild-type mice (results not shown). Corresponding to these results, the number of spiral ganglia in wild-type mice showed a gradual decrease in an age-dependent manner, whereas the number of spiral ganglia in heterozygous Ret-Y1062F-knock-in mice It was found that there was a sharp decrease compared to the mold (FIGS. 3d-m).
（５）加齢性難聴の回復実験
最後に、ヘテロ型Ret-Y1062F-ノックインマウスの加齢性難聴の回復実験を試みた。恒常活性型RETを持つRET遺伝子機能亢進マウス（RET-Tg-mice 242系：文献15 Kato M et a al. Oncogene 1999）とヘテロ型Ret-Y1062F-ノックインマウスを交配させ、恒常活性型RET遺伝子を導入したc-Ret-KIY1062F/+;RET-Tg-miceを作製した。14ヶ月齢において、c-Ret-KIY1062F/+の聴力レベルは野生型と比較して顕著に低下したのに対して、c-Ret-KIY1062F/+;RET-Tg-miceは野生型マウスと同等の聴力レベルを示した（図４ａ）。c-Ret-KIY1062F/+;RET-Tg-miceのらせん神経節におけるRet-Y1062のリン酸化レベルはc-Ret-KIY1062F/+よりも有意に増加していた（図４ｂ〜j）。この結果と対応して、ヘテロ型Ret-Y1062F-ノックインマウスと比較して、c-Ret-KIY1062F/+;RET-Tg-miceのらせん神経節の細胞数は有意に多いことがわかった（図４k〜o）。以上の結果より、ヘテロ型Ret-Y1062F-ノックインマウスの加齢性難聴の進行を恒常活性型RET遺伝子が部分的に阻止できる事が示唆された。
(5) Recovery experiment of age-related deafness Finally, the recovery experiment of age-related deafness of the hetero type Ret-Y1062F-knock-in mouse was tried. Mice with enhanced RET gene function with constitutively active RET (RET-Tg-mice 242 series: Reference 15 Kato M et a al. Oncogene 1999) and heterozygous Ret-Y1062F-knock-in mice were bred to produce a constitutively active RET gene. The introduced c-Ret-KIY1062F / +; RET-Tg-mice was prepared. At 14 months of age, the hearing level of c-Ret-KIY1062F / + was significantly reduced compared to wild-type, whereas c-Ret-KIY1062F / +; RET-Tg-mice was equivalent to wild-type mice The hearing level was shown (FIG. 4a). c-Ret-KIY1062F / +; The phosphorylation level of Ret-Y1062 in the spiral ganglia of RET-Tg-mice was significantly higher than that of c-Ret-KIY1062F / + (FIGS. 4b to j). Corresponding to this result, it was found that the number of spiral ganglion cells in c-Ret-KIY1062F / +; RET-Tg-mice was significantly higher than that in heterozygous Ret-Y1062F-knock-in mice (Fig. 4k-o). These results suggest that the homeostatic RET gene can partially block the progression of age-related deafness in heterozygous Ret-Y1062F-knock-in mice.
一方、既知の情報として以下の論文が報告されている。即ち、(i)試験管内の実験によって、Retを介したシグナル伝達系は転写因子であるNF-κBの活性化に重要である事が報告されている（参考文献１７）。また、（ii）転写因子NF-κBの構成分子の一つであるp50タンパク質欠損マウスでは、calbindin D28Kタンパク質及びsynaptophysinタンパク質などの異常増加を伴うらせん神経節の変性が起こり、加齢性難聴の進行が早まることが報告されている（参考文献１６）。さらに、NF-κBの上流のシグナル伝達系が活性化すると、NF-κBは細胞質から核内に移行し、その転写因子としての機能を発揮することが知られている（参考文献１８）。以上の文献情報を踏まえ、NF-κB p50がヘテロ型Ret-Y1062F-ノックインマウスの加齢性難聴の進行に機能的に関わっているかどうかを解析する為、NF-κB p50のらせん神経節の核移行の程度を免疫組織染色（参考文献１９）により解析した。その結果、ヘテロ型Ret-Y1062F-ノックインマウスのNF-κB p50とDAPIで染色した核との共局在の程度（57％：図５c, d, i）と比較して、c-Ret-KIY1062F/+;RET-Tg-miceの共局在の程度は野生型、あるいはRET-Tg-miceのレベルまで顕著に増加した（75％：図５e, f, i）。以上の結果より、恒常型RETトランスジーンはヘテロ型Ret-Y1062F-ノックインマウスのNF-κB p50の核移行の低下（機能低下）を正常化することが示唆された。 On the other hand, the following papers have been reported as known information. That is, (i) In vitro experiments have reported that the Ret-mediated signal transduction system is important for the activation of the transcription factor NF-κB (Reference 17). In addition, (ii) in mice lacking the p50 protein, which is one of the components of the transcription factor NF-κB, degeneration of the spiral ganglion accompanied by abnormal increase in calbindin D28K protein and synaptophysin protein, and progression of age-related hearing loss Has been reported to be accelerated (Reference 16). Furthermore, it is known that when the signal transduction system upstream of NF-κB is activated, NF-κB moves from the cytoplasm into the nucleus and exerts its function as a transcription factor (Reference 18). Based on the above literature information, to analyze whether NF-κB p50 is functionally involved in the progression of age-related deafness in heterozygous Ret-Y1062F-knock-in mice, the nucleus of the NF-κB p50 spiral ganglion is analyzed. The degree of migration was analyzed by immunohistochemical staining (Ref. 19). As a result, c-Ret-KIY1062F was compared with the degree of co-localization of NF-κB p50 of hetero type Ret-Y1062F-knock-in mice with nuclei stained with DAPI (57%: Fig. 5c, d, i). / +; The degree of co-localization of RET-Tg-mice increased significantly to the level of wild-type or RET-Tg-mice (75%: FIG. 5e, f, i). These results suggest that the constitutive RET transgene normalizes the decrease in NF-κB p50 nuclear translocation (function decline) in heterozygous Ret-Y1062F-knock-in mice.
更に、転写因子NF-κBの構成分子の一つであるp50タンパク質欠損マウスで報告されている、calbindin D28K（図５k,n）やsynaptophysin（図５p,s）などのマーカータンパク質などの異常増加もヘテロ型Ret-Y1062F-ノックインマウスで観察された。興味深いことに、これらの異常増加はc-Ret-KI Y1062F/+;RET-Tg-miceでは野生型、あるいはRET-Tg-miceのレベルまで有意に減少しており（図５j-s）、恒常型RETトランスジーンはヘテロ型Ret-Y1062F-ノックインマウスのマーカータンパク質などの異常増加も正常化することが示唆された。以上の結果より、NF-κB p50はRet-Y1062を介したシグナル伝達系の下流に位置する転写因子として、ヘテロ型Ret-Y1062F-ノックインマウスの加齢性難聴の進行に機能的に関わっていることが示唆された。 Furthermore, abnormal increases in marker proteins such as calbindin D28K (Fig. 5k, n) and synaptophysin (Fig. 5p, s) have been reported in mice lacking the p50 protein, which is one of the components of the transcription factor NF-κB. Observed in heterozygous Ret-Y1062F-knock-in mice. Interestingly, these abnormal increases were significantly reduced to the level of wild-type or RET-Tg-mice in c-Ret-KI Y1062F / +; RET-Tg-mice (Fig. 5j-s) It was suggested that the type RET transgene also normalizes abnormal increases in marker proteins of hetero type Ret-Y1062F-knock-in mice. Based on the above results, NF-κB p50 is a transcription factor located downstream of the Ret-Y1062-mediated signal transduction system and is functionally involved in the progression of age-related hearing loss in heterozygous Ret-Y1062F-knock-in mice It has been suggested.
（６）まとめ
Ret遺伝子機能低下マウス（Ret-Y1062F-ノックインマウス）を用いた以上の解析結果により、Retのリン酸化レベルが生後の内耳らせん神経節の発達と生存に重要な役割を担っており、そのリン酸化レベルが低下すると、その程度によって先天性難聴と加齢性難聴の原因になることが示唆された。また、恒常活性型RETによる加齢性難聴の回復実験により、難聴治療の新たな標的分子としてRETが有用であることが示唆された。
(6) Summary
Based on the above analysis using Ret gene-reduced mice (Ret-Y1062F-knock-in mice), Ret phosphorylation level plays an important role in the development and survival of the inner ear spiral ganglion after birth. It was suggested that a decrease in the level may cause congenital deafness and age-related deafness. In addition, the recovery experiment of age-related hearing loss by constitutively active RET suggested that RET is useful as a new target molecule for the treatment of hearing loss.
３．考察
従来、先天性難聴と加齢性難聴は発症時期あるいは病態の明確な違いから、全く異なる疾患であるとみなされていた。本研究結果から、これら難聴も単一遺伝子により発症する可能性が示唆された。
3. Discussion Conventionally, congenital deafness and age-related deafness were considered to be completely different diseases because of the distinct difference in onset time or pathological condition. The results of this study suggested that these deafnesses may be caused by a single gene.
以上の実験結果より、以下の知見が得られた。
（１）RET/Retタンパク質のリン酸化レベル、即ち活性化レベルが聴覚の形成、発達に重要な役割を担う。
（２）先天性難聴の表現型を示すモデル動物を作製するためには、Retタンパク質の活性化を妨げるように両方のアレルのRet遺伝子に変異を導入することが有効である。
（３）加齢性難聴の表現型を示すモデル動物を作製するためには、Retタンパク質の活性化を妨げるように片方のアレルのRet遺伝子に変異を導入することが有効である。
（４）RET／Retが難聴の予防・治療の標的分子になる。
From the above experimental results, the following knowledge was obtained.
(1) The phosphorylation level of RET / Ret protein, that is, the activation level plays an important role in auditory formation and development.
(2) In order to produce a model animal exhibiting a congenital deafness phenotype, it is effective to introduce mutations in the Ret genes of both alleles so as to prevent the activation of the Ret protein.
(3) In order to produce a model animal exhibiting the age-related deafness phenotype, it is effective to introduce a mutation into the Ret gene of one allele so as to prevent the activation of the Ret protein.
(4) RET / Ret is a target molecule for the prevention and treatment of hearing loss.
＜臨床症例におけるRET遺伝子の変異＞
RETはヒルシュスプラング病（巨大結腸症）の原因遺伝子として知られているが、その一方でヒルシュスプラング病の患者は難聴のリスクが高まることが指摘されている。このことから、ヒルシュスプラング病の患者において、RETの点変異と難聴発症について関連する可能性がある。そこで、ヒルシュスプラング病とRET遺伝子の変異との関係を調べることにした。まず、ヒルシュスプルング病患児の内、全結腸型無神経節症（total colon aganglionosis）といわれる重症例（21例）についてRET遺伝子の全塩基配列を検索した。一方、聴覚検査を実施した結果、これら21例中、3例は「RET遺伝子変異に由来するヒルシュスプルング病における」聴覚障害であると確認された。これら3例には共通する特徴が認められた。即ち、全てが男児例であり、しかも重症例のなかでも特に重症とされる極型症例（全腸管型無神経節症）に集中していた。
<Mutation of RET gene in clinical cases>
RET is known as a causative gene for Hirschsprung's disease (macrocolonia), while patients with Hirschsprung's disease have been pointed to an increased risk of hearing loss. This may be related to RET point mutations and onset of hearing loss in patients with Hirschsprung disease. Therefore, we decided to investigate the relationship between Hirschsprung's disease and RET gene mutations. First, the complete nucleotide sequence of the RET gene was searched for severe cases (21 cases) called total colon aganglionosis among children with Hirschsprung disease. On the other hand, as a result of the hearing test, 3 of these 21 cases were confirmed to have hearing impairment “in Hirschsprung's disease derived from RET gene mutation”. These three cases showed common features. That is, all of them were boys, and among the severe cases, concentrated on extreme cases (all intestinal ganglia).
以上の検討より、ヒトにおいても、聴覚異常を規定する分子としてRETが重要であることが判明した。 From the above study, it was found that RET is important as a molecule that defines auditory abnormalities in humans.
（参考文献）
1. Brown SD, Hardisty-Hughes RE, Mburu P. Quiet as a mouse: dissecting the molecular and genetic basis of hearing. Nat Rev Genet. 2008 Apr;9(4):277-90. Epub 2008 Feb 19. Review.
2. Airaksinen MS, Saarma M. The GDNF family: signalling, biological functions and therapeutic value. Nat Rev Neurosci. 2002 May;3(5):383-94. Review.
3. Heanue TA, Pachnis V. Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies.Nat Rev Neurosci. 2007 Jun;8(6):466-79. Review. Erratum in: Nat Rev Neurosci. 2007 Jul;8(7):568.
4. Kato M, Takeda K, Kawamoto Y, Iwashita T, Akhand AA, Senga T, Yamamoto M, Sobue G, Hamaguchi M, Takahashi M, Nakashima I.Repair by Src kinase of function-impaired RET with multiple endocrine neoplasia type 2A mutation with substitutions of tyrosines in the COOH-terminal kinase domain for phenylalanine. Cancer Res. 2002 Apr 15;62(8):2414-22.
5. Rubel EW, Fritzsch B. Auditory system development: primary auditory neurons and their targets. Annu Rev Neurosci. 2002;25:51-101. Epub 2002 Feb 5. Review.
6. Jijiwa M, Fukuda T, Kawai K, Nakamura A, Kurokawa K, Murakumo Y, Ichihara M, Takahashi M.A targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric neurons and renal hypoplasia. Mol Cell Biol. 2004 Sep;24(18):8026-36.
7. Asai N, Fukuda T, Wu Z, Enomoto A, Pachnis V, Takahashi M, Costantini F. Targeted mutation of serine 697 in the Ret tyrosine kinase causes migration defect of enteric neural crest cells. Development. 2006 Nov;133(22):4507-16. Epub 2006 Oct 18.
8. Uesaka T, Nagashimada M, Yonemura S, Enomoto H. Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice. J Clin Invest. 2008 May;118(5):1890-8.
9. Bordeaux MC, Forcet C, Granger L, Corset V, Bidaud C, Billaud M, Bredesen DE, Edery P, Mehlen P. The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease. EMBO J. 2000 Aug 1;19(15):4056-63.
10. Cañibano C, Rodriguez NL, Saez C, Tovar S, Garcia-Lavandeira M, Borrello MG, Vidal A, Costantini F, Japon M, Dieguez C, Alvarez CV. The dependence receptor Ret induces apoptosis in somatotrophs through a Pit-1/p53 pathway, preventing tumor growth. EMBO J. 2007 Apr 18;26(8):2015-28. Epub 2007 Mar 22.
11. Spongr VP, Flood DG, Frisina RD, Salvi RJ. Quantitative measures of hair cell loss in CBA and C57BL/6 mice throughout their life spans. J Acoust Soc Am. 1997 Jun;101(6):3546-53.
12. White JA, Burgess BJ, Hall RD, Nadol JB. Pattern of degeneration of the spiral ganglion cell and its processes in the C57BL/6J mouse. Hear Res. 2000 Mar;141(1-2):12-8.
13. Hequembourg S, Liberman MC.Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL/6 mice. J Assoc Res Otolaryngol. 2001 Jun;2(2):118-29.
14. Ohlemiller KK. Reduction in sharpness of frequency tuning but not endocochlear potential in aging and noise-exposed BALB/cJ mice. J Assoc Res Otolaryngol. 2002 Dec;3(4):444-56. Epub 2002 Apr 9.
15. Kato M, Liu W, Akhand AA, Dai Y, Ohbayashi M, Tuzuki T, Suzuki H, Isobe K, Takahashi M, Nakashima I. Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines. Oncogene. 1999 Jan 21;18(3):837-42.
16. Lang H, Schulte BA, Zhou D, Smythe N, Spicer SS, Schmiedt RA. Nuclear factor kappaB deficiency is associated with auditory nerve degeneration and increased noise-induced hearing loss. J Neurosci. 2006 Mar 29;26(13):3541-50.
17. Hayashi H, Ichihara M, Iwashita T, Murakami H, Shimono Y, Kawai K, Kurokawa K, Murakumo Y, Imai T, Funahashi H, Nakao A, Takahashi M. Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene. 2000 Sep 14;19(39):4469-75.
18. Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell. 2008 Feb 8;132(3):344-62. Review.
19. Talwalkar SS, Valbuena JR, Abruzzo LV, Admirand JH, Konoplev SN, Bueso-Ramos CE, Medeiros LJ. MALT1 gene rearrangements and NF-kappaB activation involving p65 and p50 are absent or rare in primary MALT lymphomas of the breast. Mod Pathol. 2006 Nov;19(11):1402-8. Epub 2006 Aug 18.
20. Song L, McGee JA, Walsh EJ. Consequences of combined maternal, fetal and persistent postnatal hypothyroidism on the development of auditory function in Tshrhyt mutant mice. Brain Res. 2006 Jul 26;1101(1):59-72. Epub 2006 Jun 15.
21. Yamamoto M, Li M, Mitsuma N, Ito S, Kato M, Takahashi M, Sobue G. Preserved phosphorylation of RET receptor protein in spinal motor neurons of patients with amyotrophic lateral sclerosis: an immunohistochemical study by a phosphorylation-specific antibody at tyrosine 1062. Brain Res. 2001 Aug 31;912(1):89-94.
22. Zheng QY, Johnson KR, Erway LC. Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear Res. 1999 Apr;130(1-2):94-107.
23. Tsujita K, Kaikita K, Hayasaki T, Honda T, Kobayashi H, Sakashita N, Suzuki H, Kodama T, Ogawa H, Takeya M. Targeted deletion of class A macrophage scavenger receptor increases the risk of cardiac rupture after experimental myocardial infarction. Circulation. 2007 Apr 10;115(14):1904-11. Epub 2007 Mar 26.
24. Ito M, Kizawa K, Toyoda M, Morohashi M. Label-retaining cells in the bulge region are directed to cell death after plucking, followed by healing from the surviving hair germ. J Invest Dermatol. 2002 Dec;119(6):1310-6.
(References)
1. Brown SD, Hardisty-Hughes RE, Mburu P. Quiet as a mouse: dissecting the molecular and genetic basis of hearing. Nat Rev Genet. 2008 Apr; 9 (4): 277-90. Epub 2008 Feb 19. Review.
2. Airaksinen MS, Saarma M. The GDNF family: signaling, biological functions and therapeutic value. Nat Rev Neurosci. 2002 May; 3 (5): 383-94. Review.
3. Heanue TA, Pachnis V. Enteric nervous system development and Hirschsprung's disease: advances in genetic and stem cell studies.Nat Rev Neurosci. 2007 Jun; 8 (6): 466-79. Review. Erratum in: Nat Rev Neurosci. 2007 Jul; 8 (7): 568.
4. Kato M, Takeda K, Kawamoto Y, Iwashita T, Akhand AA, Senga T, Yamamoto M, Sobue G, Hamaguchi M, Takahashi M, Nakashima I. Repair by Src kinase of function-impaired RET with multiple endocrine neoplasia type 2A mutation with substitutions of tyrosines in the COOH-terminal kinase domain for phenylalanine.Cancer Res.2002 Apr 15; 62 (8): 2414-22.
5. Rubel EW, Fritzsch B. Auditory system development: primary auditory neurons and their targets. Annu Rev Neurosci. 2002; 25: 51-101. Epub 2002 Feb 5. Review.
6. Jijiwa M, Fukuda T, Kawai K, Nakamura A, Kurokawa K, Murakumo Y, Ichihara M, Takahashi MA targeting mutation of tyrosine 1062 in Ret causes a marked decrease of enteric neurons and renal hypoplasia. Mol Cell Biol. 2004 Sep; 24 (18): 8026-36.
7. Asai N, Fukuda T, Wu Z, Enomoto A, Pachnis V, Takahashi M, Costantini F. Targeted mutation of serine 697 in the Ret tyrosine kinase causes migration defect of enteric neural crest cells.Development. 2006 Nov; 133 (22 ): 4507-16. Epub 2006 Oct 18.
8. Uesaka T, Nagashimada M, Yonemura S, Enomoto H. Diminished Ret expression compromises neuronal survival in the colon and causes intestinal aganglionosis in mice.J Clin Invest. 2008 May; 118 (5): 1890-8.
9. Bordeaux MC, Forcet C, Granger L, Corset V, Bidaud C, Billaud M, Bredesen DE, Edery P, Mehlen P. The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease.EMBO J. 2000 Aug 1 ; 19 (15): 4056-63.
10. Ca ñ ibano C, Rodriguez NL, Saez C, Tovar S, Garcia-Lavandeira M, Borrello MG, Vidal A, Costantini F, Japon M, Dieguez C, Alvarez CV.The dependence receptor Ret induces apoptosis in somatotrophs through a Pit -1 / p53 pathway, preventing tumor growth. EMBO J. 2007 Apr 18; 26 (8): 2015-28. Epub 2007 Mar 22.
11. Spongr VP, Flood DG, Frisina RD, Salvi RJ.Quantitative measures of hair cell loss in CBA and C57BL / 6 mice throughout their life spans.J Acoust Soc Am. 1997 Jun; 101 (6): 3546-53.
12.White JA, Burgess BJ, Hall RD, Nadol JB.Pattern of degeneration of the spiral ganglion cell and its processes in the C57BL / 6J mouse.Hear Res. 2000 Mar; 141 (1-2): 12-8.
13. Hequembourg S, Liberman MC.Spiral ligament pathology: a major aspect of age-related cochlear degeneration in C57BL / 6 mice.J Assoc Res Otolaryngol. 2001 Jun; 2 (2): 118-29.
14. Ohlemiller KK. Reduction in sharpness of frequency tuning but not endocochlear potential in aging and noise-exposed BALB / cJ mice. J Assoc Res Otolaryngol. 2002 Dec; 3 (4): 444-56. Epub 2002 Apr 9.
15. Kato M, Liu W, Akhand AA, Dai Y, Ohbayashi M, Tuzuki T, Suzuki H, Isobe K, Takahashi M, Nakashima I. Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein- I / ret transgenic mouse lines. Oncogene. 1999 Jan 21; 18 (3): 837-42.
16. Lang H, Schulte BA, Zhou D, Smythe N, Spicer SS, Schmiedt RA. Nuclear factor kappaB deficiency is associated with auditory nerve degeneration and increased noise-induced hearing loss.J Neurosci. 2006 Mar 29; 26 (13): 3541-50.
17. Hayashi H, Ichihara M, Iwashita T, Murakami H, Shimono Y, Kawai K, Kurokawa K, Murakumo Y, Imai T, Funahashi H, Nakao A, Takahashi M. Characterization of intracellular signals via tyrosine 1062 in RET activated by glial cell line-derived neurotrophic factor. Oncogene. 2000 Sep 14; 19 (39): 4469-75.
18. Hayden MS, Ghosh S. Shared principles in NF-kappaB signaling. Cell. 2008 Feb 8; 132 (3): 344-62. Review.
19. Talwalkar SS, Valbuena JR, Abruzzo LV, Admirand JH, Konoplev SN, Bueso-Ramos CE, Medeiros LJ.MALT1 gene rearrangements and NF-kappaB activation involving p65 and p50 are absent or rare in primary MALT lymphomas of the breast. Pathol. 2006 Nov; 19 (11): 1402-8. Epub 2006 Aug 18.
20. Song L, McGee JA, Walsh EJ. Consequences of combined maternal, fetal and persistent postnatal hypothyroidism on the development of auditory function in Tshrhyt mutant mice.Brain Res. 2006 Jul 26; 1101 (1): 59-72. Epub 2006 Jun 15.
21. Yamamoto M, Li M, Mitsuma N, Ito S, Kato M, Takahashi M, Sobue G. Preserved phosphorylation of RET receptor protein in spinal motor neurons of patients with amyotrophic lateral sclerosis: an immunohistochemical study by a phosphorylation-specific antibody at tyrosine 1062. Brain Res. 2001 Aug 31; 912 (1): 89-94.
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24. Ito M, Kizawa K, Toyoda M, Morohashi M. Label-retaining cells in the bulge region are directed to cell death after plucking, followed by healing from the surviving hair germ.J Invest Dermatol. 2002 Dec; 119 (6) : 1310-6.
本発明の齧歯類遺伝子改変動物は、難聴のメカニズムの研究、難聴に対する予防又は治療法の確立に有用である。 The rodent genetically modified animal of the present invention is useful for studying the mechanism of hearing loss and establishing prevention or treatment methods for hearing loss.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。
本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.
Claims (7)
（１）Retタンパク質の活性化に必須のアミノ酸である1063番チロシン残基がフェニルアラニンに置換される点遺伝子変異によって片方のアレルのみ、Ret遺伝子が機能障害されており、加齢性難聴の表現型を示す、遺伝子改変マウスに被験物質を取り込ませるステップ；
（２）聴力の低下が抑制されたか否か評価し、評価結果に基づき被験物質の有効性を判定するステップ。 A screening method for a substance effective for the prevention or treatment of age-related hearing loss or noise-induced hearing loss comprising the following steps (1) and (2):
(1) The phenotype of age-related deafness is that the Ret gene is dysfunctional only in one allele due to a point gene mutation in which the 1063 tyrosine residue, an amino acid essential for the activation of Ret protein, is substituted with phenylalanine. shown, steps of incorporating a test substance to genetically modified mice;
(2) A step of evaluating whether the decrease in hearing ability is suppressed and determining the effectiveness of the test substance based on the evaluation result.
（１）Retタンパク質の活性化に必須のアミノ酸である1063番チロシン残基がフェニルアラニンに置換される点遺伝子変異によって片方のアレルのみ、Ret遺伝子が機能障害されており、先天性難聴の表現型を示す、遺伝子改変マウスに被験物質を取り込ませるステップ；
（２）聴力が回復したか否か評価し、評価結果に基づき被験物質の有効性を判定するステップ。 A screening method for a substance effective for the treatment of congenital deafness comprising the following steps (1) and (2):
(1) The Ret gene is dysfunctional only in one allele due to a point gene mutation in which the 1063 tyrosine residue, which is an amino acid essential for the activation of Ret protein, is substituted with phenylalanine, resulting in a congenital deafness phenotype. Showing the step of incorporating the test substance into the genetically modified mouse ;
(2) A step of evaluating whether or not hearing has been restored, and determining the effectiveness of the test substance based on the evaluation result.
（１）RET遺伝子が発現している細胞であって、神経細胞及び神経芽細胞からなる群より選択される細胞を被験物質の存在下で培養するステップ；
（２）前記細胞における、RET遺伝子の発現レベル又はRETタンパク質の活性化レベルを検出し、検出結果に基づき被験物質の有効性を判定するステップ。 A screening method for a substance effective for the prevention or treatment of hearing loss, comprising the following (1) and (2):
(1) culturing a cell expressing a RET gene, which is selected from the group consisting of a neuron and a neuroblast, in the presence of a test substance;
(2) A step of detecting the expression level of the RET gene or the activation level of the RET protein in the cell, and determining the effectiveness of the test substance based on the detection result.
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