JP6332723B2 - Method for screening compound for treating and / or preventing diseases in which aggregates of TDP-43 accumulate - Google Patents
Method for screening compound for treating and / or preventing diseases in which aggregates of TDP-43 accumulate Download PDFInfo
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- JP6332723B2 JP6332723B2 JP2013046451A JP2013046451A JP6332723B2 JP 6332723 B2 JP6332723 B2 JP 6332723B2 JP 2013046451 A JP2013046451 A JP 2013046451A JP 2013046451 A JP2013046451 A JP 2013046451A JP 6332723 B2 JP6332723 B2 JP 6332723B2
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Description
本発明は、凝集体形成能を有するTDP-43変異体及び該TDP-43変異体内のRRM1ドメイン、並びに該TDP-43変異体又はRRM1ドメインを利用したTDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニング方法に関する。また、本発明は、TDP-43の凝集体が蓄積する疾患のモデルとなるトランスジェニック非ヒト動物に関する。さらに、本発明は、TDP-43の凝集体に結合する抗体又は抗体断片、該抗体又は抗体断片を含む診断薬、診断用キット、並びに医薬組成物に関する。 The present invention relates to a TDP-43 mutant having an aggregate-forming ability, an RRM1 domain in the TDP-43 mutant, and a disease in which aggregates of TDP-43 using the TDP-43 mutant or the RRM1 domain accumulate. The present invention relates to a method for screening a compound for treatment and / or prevention. The present invention also relates to a transgenic non-human animal that serves as a model of a disease in which aggregates of TDP-43 accumulate. Furthermore, the present invention relates to an antibody or antibody fragment that binds to an aggregate of TDP-43, a diagnostic agent containing the antibody or antibody fragment, a diagnostic kit, and a pharmaceutical composition.
主要な認知症の1型である前頭側頭葉変性症(FTLD)と最難治性致死性神経難病である筋萎縮性側索硬化症(ALS)の原因タンパク質として、核タンパク質であるTDP-43 (TAR DNA-binding protein of 43kDa)が同定されている。TDP-43はFTLDやALSにおいて核の局在性が低下し、細胞質内で病的凝集体を形成するがその機序は不明である。 TDP-43, a nuclear protein, is the causative protein of frontotemporal lobar degeneration (FTLD), a major dementia type, and amyotrophic lateral sclerosis (ALS), the most refractory fatal neurological disease (TAR DNA-binding protein of 43 kDa) has been identified. TDP-43 has decreased nuclear localization in FTLD and ALS, and forms pathological aggregates in the cytoplasm, but the mechanism is unknown.
TDP-43は、FTLDとALSにおけるユビキチン化封入体において非常に高率に認められることが判明している。現在TDP-43の異常病理所見を呈する疾患は、TDP-43プロテイノパチーという疾患群と位置づけられ、各種の報告からTDP-43の機能異常がALS病態の本質である可能性が高まっている。 TDP-43 has been found to be found at a very high rate in ubiquitinated inclusion bodies in FTLD and ALS. Diseases presenting abnormal pathological findings of TDP-43 are currently positioned as a group of diseases called TDP-43 proteinopathy, and various reports indicate that TDP-43 dysfunction is likely to be the essence of ALS pathology.
このようにTDP-43の生理的・病理的機能を解明することはALSの克服に繋がる可能性があり、世界中で精力的な研究が進められている。TDP-43プロテイノパチーの最も明確且つ重要な病理所見は、TDP-43の核染色性の低下と細胞質での封入体形成である。この異所性局在の機能解明はALSの病態理解に不可欠である。 Thus, elucidating the physiological and pathological functions of TDP-43 may lead to overcoming ALS, and energetic research is underway all over the world. The most clear and important pathological findings of the TDP-43 proteinopathy are reduced nuclear staining of TDP-43 and inclusion body formation in the cytoplasm. Elucidation of the function of this ectopic localization is indispensable for understanding the pathology of ALS.
TDP-43の分子構造は、2ヶ所のRNA結合領域(RRM)とカルボキシル(C)末端側のグリシンリッチ領域、核移行シグナル(NLS)、及び核外輸送シグナル(NES)を有する。 The molecular structure of TDP-43 has two RNA binding regions (RRM), a glycine-rich region on the carboxyl (C) terminal side, a nuclear translocation signal (NLS), and a nuclear export signal (NES).
TDP-43は、本来核タンパク質であるが、FTLDやALSでは核外脱出と凝集体形成をし、それらがユビキチン化とリン酸化を受けることが特徴とされている。さらにその際、TDP-43が35kDa、25kDaの凝集性の高いC末端側の断片(C末断片)に切断されることが知られており、病巣における共通の所見である。しかしながら、これまでの研究の蓄積から、こうした現象は疾患の進行期のものであり、タンパク質構造異常を惹起する病初期の構造変化については未だ不明である。 TDP-43 is originally a nuclear protein, but FTLD and ALS are characterized by extranuclear escape and aggregate formation, which undergo ubiquitination and phosphorylation. Furthermore, it is known that TDP-43 is cleaved into 35 kDa and 25 kDa highly C-terminal fragments (C-terminal fragments), which is a common finding in lesions. However, from the accumulation of research so far, this phenomenon is in the advanced stage of the disease, and the structural change at the early stage of the disease causing protein structural abnormality is still unclear.
TDP-43のC末断片は、実際に患者組織に存在し、培養細胞や遺伝子改変マウスで細胞質内に封入体形成をすることが広く知られており(例えば、非特許文献１、２)、このシステムを用いた薬剤スクリーニングは存在する。しかしながら、これら断片化が疾患発症の初期に出現する証拠は皆無であり、動物実験でもこれら断片の過剰発現はALSモデルとしては非典型的且つ不完全である。
The C-terminal fragment of TDP-43 actually exists in patient tissues, and is widely known to form inclusion bodies in the cytoplasm in cultured cells and genetically modified mice (for example,
また、FTLD及びALS以外にもTDP-43の異所性局在を呈する疾患として、低悪性度グリオーマ、アルツハイマー病、ハンチントン病、ピック病、パーキンソン病、レビー小体病、大脳皮質基底核変性症、封入体筋炎、B細胞リンパ腫(M期)等が報告されている。 In addition to FTLD and ALS, ectopic localization of TDP-43 includes low-grade glioma, Alzheimer's disease, Huntington's disease, Pick's disease, Parkinson's disease, Lewy body disease, cortical basal ganglia degeneration. Inclusion body myositis, B cell lymphoma (M stage), etc. have been reported.
TDP-43は、頭部外傷や軸索の引き抜き損傷、挫滅損傷で一過性に細胞質に異所性局在することが報告されているが、この場合TDP-43の局在異常は一過性でFTLDやALSの病型を示さない(非特許文献３)。 TDP-43 has been reported to be transiently ectopically localized in the cytoplasm due to head trauma, axon pull-out damage, and crushing damage. Does not show FTLD or ALS disease type.
しかしながら、TDP-43 トランスジェニック(Tg)マウスにおいてC末断片の出現は、表現系出現の後期現象であり、C末断片の過剰発現のみでは細胞死は生じない。また、C末断片のTDP-43 TgマウスはALSの表現系を示さない。このように、インビボにおいてTDP-43のC末断片が疾患のトリガーとなることを示す証拠はない。また、全長TDP-43を用いたTDP-43プロテイノパチーのモデルは現在存在しておらず、全長TDP-43のミスフォールディングにいたる構造変化も不明である。 However, the appearance of the C-terminal fragment in TDP-43 transgenic (Tg) mice is a late phenomenon of the appearance of the phenotype, and cell death does not occur only by overexpression of the C-terminal fragment. The C-terminal fragment TDP-43 Tg mouse does not show an ALS expression system. Thus, there is no evidence that the C-terminal fragment of TDP-43 triggers disease in vivo. In addition, no TDP-43 proteinopathy model using full-length TDP-43 currently exists, and structural changes leading to misfolding of full-length TDP-43 are unclear.
そこで、本発明は、全長配列において凝集体形成能及び細胞毒性を有するTDP-43変異体、並びに該TDP-43変異体を利用したTDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニング方法を提供することを目的とする。また、本発明は、TDP-43の凝集体が蓄積する疾患のモデルとなるトランスジェニック非ヒト動物を提供することを目的とする。 Therefore, the present invention treats and / or prevents a TDP-43 mutant having aggregate-forming ability and cytotoxicity in the full-length sequence, and a disease in which aggregates of TDP-43 using the TDP-43 mutant accumulate. An object of the present invention is to provide a screening method for compounds. Another object of the present invention is to provide a transgenic non-human animal that serves as a model of a disease in which aggregates of TDP-43 accumulate.
本発明者らは、TDP-43の凝集体形成にRRM1の構造変化が関わること、更にRRM1の構造変化に重要な分子内配列を特定し、その突然変異体が培養細胞でFTLD、ALS患者と同様の病理変化を示す疾患モデルとなるという知見を得た。 The present inventors have identified the structural change of RRM1 in TDP-43 aggregate formation, and further identified an intramolecular sequence important for the structural change of RRM1, and the mutant was cultured with FTLD and ALS patients. The knowledge that it becomes the disease model which shows the same pathological change was acquired.
本発明は、これら知見に基づき、完成されたものであり、次のTDP-43変異体、RRM1ドメイン、化合物のスクリーニング方法、トランスジェニック非ヒト動物等を提供するものである。 The present invention has been completed based on these findings, and provides the following TDP-43 mutant, RRM1 domain, compound screening method, transgenic non-human animal, and the like.
(I) TDP-43変異体、RRM1ドメイン
(I-1) TDP-43のアミノ酸配列において、173位及び／又は175位のシステインが置換されたアミノ酸配列からなるTDP-43変異体。
(I-2) (I-1)に記載のTDP-43変異体内のRRM1ドメイン。
(I) TDP-43 mutant, RRM1 domain
(I-1) A TDP-43 variant comprising an amino acid sequence in which cysteine at position 173 and / or 175 is substituted in the amino acid sequence of TDP-43.
(I-2) The RRM1 domain in the TDP-43 mutant according to (I-1).
(II) 化合物のスクリーニング方法(A)
(II-1) 以下の工程を含む、TDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニング方法：
（１）(I-1)に記載のTDP-43変異体又は(I-2)に記載のRRM1ドメインに候補となる化合物を接触させる工程、
（２）該化合物の凝集体形成阻害活性を検出する工程、及び
（３）凝集体形成を阻害する化合物を選択する工程。
(II) Compound screening method (A)
(II-1) A method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) contacting the candidate compound with the TDP-43 mutant according to (I-1) or the RRM1 domain according to (I-2);
(2) a step of detecting the aggregate formation inhibitory activity of the compound, and (3) a step of selecting a compound that inhibits aggregate formation.
(III) トランスジェニック非ヒト動物
(III-1) (I-1)に記載のTDP-43変異体をコードするDNAが導入されたトランスジェニック非ヒト動物。
(III-2) 前記非ヒト動物がマウス、ラット、線虫又はショウジョウバエである、(III-1)に記載のトランスジェニック非ヒト動物。
(III-3) (III-1)又は(III-2)に記載のトランスジェニック非ヒト動物を用いることを特徴とする、TDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニング方法。
(III) Transgenic non-human animals
(III-1) A transgenic non-human animal into which a DNA encoding the TDP-43 mutant according to (I-1) is introduced.
(III-2) The transgenic non-human animal according to (III-1), wherein the non-human animal is a mouse, rat, nematode or Drosophila.
(III-3) To treat and / or prevent a disease in which an aggregate of TDP-43 accumulates, characterized by using the transgenic non-human animal described in (III-1) or (III-2) Screening method.
(IV) 抗体又は抗体断片
(IV-1) TDP-43の108〜116位に対応するアミノ酸配列からなるペプチドに結合する抗体又は抗体断片。
(IV-2) (IV-1)に記載の抗体又は抗体断片を含むTDP-43の凝集体が蓄積する疾患の診断薬。
(IV-3) (IV-1)に記載の抗体又は抗体断片を含むTDP-43の凝集体が蓄積する疾患の診断用キット。
(IV-4) (IV-1)に記載の抗体又は抗体断片を含む医薬組成物。
(IV-5) TDP-43の凝集体が蓄積する疾患の治療用及び／又は予防用である、(IV-4)に記載の組成物。
(IV) Antibody or antibody fragment
(IV-1) An antibody or antibody fragment that binds to a peptide consisting of an amino acid sequence corresponding to positions 108 to 116 of TDP-43.
(IV-2) A diagnostic agent for a disease in which an aggregate of TDP-43 containing the antibody or antibody fragment described in (IV-1) accumulates.
(IV-3) A diagnostic kit for a disease in which aggregates of TDP-43 containing the antibody or antibody fragment described in (IV-1) accumulate.
(IV-4) A pharmaceutical composition comprising the antibody or antibody fragment described in (IV-1).
(IV-5) The composition according to (IV-4), which is used for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates.
(V) 化合物のスクリーニング方法(B)
(V-1) 以下の工程を含む、TDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニング方法：
（１）(I-1)に記載のTDP-43変異体又は(I-2)に記載のRRM1ドメインに、(IV-1)に記載の抗体又は抗体断片と候補となる化合物を接触させる工程、
（２）該化合物の結合阻害活性を検出する工程、及び
（３）該抗体又は抗体断片の結合を阻害する化合物を選択する工程。
(V-2) 以下の工程を含む、TDP-43の凝集体が蓄積する疾患の診断用の化合物のスクリーニング方法：
（１）(I-1)に記載のTDP-43変異体又は(I-2)に記載のRRM1ドメインに、(IV-1)に記載の抗体又は抗体断片と候補となる化合物を接触させる工程、
（２）該化合物の結合阻害活性を検出する工程、及び
（３）該抗体又は抗体断片の結合を阻害する化合物を選択する工程。
(V) Compound screening method (B)
(V-1) A method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) A step of bringing the antibody or antibody fragment described in (IV-1) and the candidate compound into contact with the TDP-43 mutant described in (I-1) or the RRM1 domain described in (I-2) ,
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment.
(V-2) A method for screening a compound for diagnosis of a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) A step of bringing the antibody or antibody fragment described in (IV-1) and the candidate compound into contact with the TDP-43 mutant described in (I-1) or the RRM1 domain described in (I-2) ,
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment.
(VI) ペプチド、核酸
(VI-1) TDP-43の108〜116位に対応するアミノ酸配列を含むペプチド。
(VI-2) (VI-1)に記載のペプチドをコードする核酸。
(VI-3) (VI-1)に記載のペプチド又は(VI-2)に記載の核酸を含む、TDP-43の凝集体に対する抗体の産生を刺激又は増強するための医薬組成物。
(VI-4) ワクチンである(VI-3)に記載の組成物。
(VI) Peptides, nucleic acids
(VI-1) A peptide comprising an amino acid sequence corresponding to positions 108 to 116 of TDP-43.
(VI-2) A nucleic acid encoding the peptide according to (VI-1).
(VI-3) A pharmaceutical composition for stimulating or enhancing the production of an antibody against an aggregate of TDP-43, comprising the peptide according to (VI-1) or the nucleic acid according to (VI-2).
(VI-4) The composition according to (VI-3), which is a vaccine.
本発明により、TDP-43の凝集体形成に関わる機能ドメインとキー配列を初めて同定した。また、本発明のTDP-43変異体を発現する培養細胞は、患者の病理所見を再現した有効な初めての培養細胞モデルとなる。 According to the present invention, functional domains and key sequences involved in aggregate formation of TDP-43 were identified for the first time. Moreover, the cultured cells expressing the TDP-43 mutant of the present invention are the first effective cultured cell model that reproduces the pathological findings of patients.
本発明のTDP-43変異体及びRRM1ドメインは、インビトロ又は細胞内での凝集体形成阻害を指標とすることにより、TDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニングに活用可能である。 The TDP-43 mutant and RRM1 domain of the present invention are compounds for treating and / or preventing a disease in which aggregates of TDP-43 accumulate by using aggregate formation inhibition in vitro or in cells as an index. It can be used for screening.
また、本発明のTDP-43変異体をコードするDNAを動物に導入することで、TDP-43の凝集体が蓄積する疾患(例えば、ALS等)のモデル動物の作成が可能になり得る。 In addition, by introducing a DNA encoding the TDP-43 mutant of the present invention into an animal, it may be possible to create a model animal for a disease in which aggregates of TDP-43 accumulate (for example, ALS).
本発明の抗体及び抗体断片は、TDP-43細胞質封入体を特異的に認識できるので、TDP-43の凝集体が蓄積する疾患の早期診断や治療及び予防への適用が期待される。また、本発明の抗体又は抗体断片は、TDP-43の凝集体が蓄積する疾患の診断用及び治療用の化合物のスクリーニング方法への適用も期待される。 Since the antibodies and antibody fragments of the present invention can specifically recognize TDP-43 cytoplasmic inclusions, they are expected to be applied to early diagnosis, treatment and prevention of diseases in which aggregates of TDP-43 accumulate. Further, the antibody or antibody fragment of the present invention is expected to be applied to a method for screening a compound for diagnosis and treatment of a disease in which an aggregate of TDP-43 accumulates.
また、本発明のペプチド又は核酸は、TDP-43の凝集体形成前の病的状態に対する抗体の産生を刺激又は増強し得ることから、TDP-43の凝集体が蓄積する疾患の治療及び予防への適用が期待される。 In addition, since the peptide or nucleic acid of the present invention can stimulate or enhance the production of antibodies against a pathological condition before TDP-43 aggregate formation, it can be used for treatment and prevention of diseases in which TDP-43 aggregates accumulate. Is expected to be applied.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明者らはTDP-43における主なRNAの結合部位であるRRM1ドメインに着目した。RRM1の安定同位体タンパク質を用いた高圧NMRとRRM1凝集体の質量解析によって、RRM1が易凝集性を有し、その凝集に3つの特定アミノ酸配列(aa113-122、133-147、166-173)が関与することを突き止めた。特にaa166-173を含むβシート構造は、圧力や震盪刺激、酸化ストレスといった異常ストレス下でRRM1が異常会合する際の会合面となり、更に同部位に存在する2つのシステイン残基(C173とC175)がフリー状態でないと凝集体形成が促進されることを発見した。 The present inventors focused on the RRM1 domain, which is the main RNA binding site in TDP-43. By high-pressure NMR using stable isotope protein of RRM1 and mass analysis of RRM1 aggregate, RRM1 has easy aggregation property, and three specific amino acid sequences (aa113-122, 133-147, 166-173) Determined to be involved. In particular, the β-sheet structure containing aa166-173 is an association surface when RRM1 abnormally associates under abnormal stress such as pressure, shaking stimulation, or oxidative stress, and two cysteine residues (C173 and C175) present at the same site It was found that the formation of aggregates is promoted if is not free.
C173、C175をセリンに置換した変異体は、核内、細胞内で著明な封入体を形成し、細胞質においてALSやFTLD患者で見られるようにユビキチン化やリン酸化を認め、更に運動ニューロン培養細胞に神経毒性を示すことが明らかとなった。興味深いことに、C173/C175変異によるRRM1の構造変化は、遺伝性ALSで報告されているTDP-43の突然変異の凝集原性を著明に増悪し、これまで不明であった野生型TDP-43と変異型TDP-43の差異を明確に示したことから、ALSにおけるTDP-43の異常な構造変化に関与することが強く示唆された。 Mutants in which C173 and C175 are replaced with serine form marked inclusions in the nucleus and in the cell, and ubiquitination and phosphorylation are observed in the cytoplasm as seen in ALS and FTLD patients. It was revealed that the cells are neurotoxic. Interestingly, the structural changes in RRM1 due to the C173 / C175 mutation markedly exacerbated the aggregative nature of the TDP-43 mutation reported in hereditary ALS, and the previously unknown wild-type TDP- Clearly showing the difference between 43 and mutant TDP-43, it was strongly suggested to be involved in the abnormal structural change of TDP-43 in ALS.
この3つのアミノ酸配列に対するウサギポリクローナル抗体を作製し、培養細胞でaa113-122、166-173に対する抗体が正常の核内TDP-43を認識しないこと、C173、C175変異による細胞内凝集体を認識することを確認した。ALS患者脊髄を用いた組織染色を行い、疾患特異的なTDP-43の封入体がaa113-122に対する抗体で染色されることを明らかにし、C173、C175の封入体モデルが疾患の病態を反映することを確認した。 Rabbit polyclonal antibodies against these three amino acid sequences were prepared, and antibodies against aa113-122 and 166-173 did not recognize normal nuclear TDP-43 in cultured cells, and recognized intracellular aggregates due to C173 and C175 mutations It was confirmed. Tissue staining using the spinal cord of ALS patients revealed that disease-specific TDP-43 inclusions were stained with antibodies to aa113-122, and inclusion models of C173 and C175 reflect the disease pathology It was confirmed.
本発明におけるTDP-43の凝集体が蓄積する疾患としては、これらに限定されるものではないが、例えば、筋萎縮性側索硬化症、前頭側頭型認知症、低悪性度グリオーマ、アルツハイマー病、ハンチントン病、ピック病、パーキンソン病、レビー小体病、大脳皮質基底核変性症、封入体筋炎、B細胞リンパ腫(M期)等が挙げられるが、特に筋萎縮性側索硬化症及び前頭側頭型認知症である。 Diseases in which aggregates of TDP-43 in the present invention accumulate are not limited to these, for example, amyotrophic lateral sclerosis, frontotemporal dementia, low-grade glioma, Alzheimer's disease Huntington's disease, Pick's disease, Parkinson's disease, Lewy body disease, basal ganglia degeneration, inclusion body myositis, B-cell lymphoma (stage M), etc., especially amyotrophic lateral sclerosis and frontal side Head dementia.
TDP-43変異体、RRM1ドメイン
本発明のTDP-43変異体は、TDP-43のアミノ酸配列において、173位及び／又は175位のシステインが置換されたアミノ酸配列からなることを特徴とする。
TDP-43 variant, RRM1 domain The TDP-43 variant of the present invention is characterized by comprising an amino acid sequence in which cysteine at positions 173 and / or 175 is substituted in the amino acid sequence of TDP-43.
ヒト由来のTDP-43は、414アミノ酸からなるタンパク質であり、一次構造はヘテロリボ核タンパク質(hnRNA)ファミリーと高い相同性を有している。TDP-43は2つの高く保存されたRNA認識モチーフ(RRM1、RRM2)を有し、C末端側にはグリシンリッチ領域を含んでおり、このグリシンリッチ領域はhnRNPファミリーのメンバーと結合する。 Human-derived TDP-43 is a protein consisting of 414 amino acids, and its primary structure has high homology with the heteroribonucleoprotein (hnRNA) family. TDP-43 has two highly conserved RNA recognition motifs (RRM1, RRM2) and contains a glycine-rich region on the C-terminal side, which binds to members of the hnRNP family.
ヒト由来のTDP-43タンパク質のアミノ酸配列は、GenBank Accession No.NP_031401として登録され、配列番号１に示されている。また、ヒト由来のTDP-43タンパク質をコードする遺伝子は、GenBank Accession No.NM_007375.3として登録され、cDNAは配列番号２に示されている。 The amino acid sequence of human-derived TDP-43 protein is registered as GenBank Accession No. NP_031401, and is shown in SEQ ID NO: 1. In addition, a gene encoding TDP-43 protein derived from human is registered as GenBank Accession No. NM_007375.3, and cDNA is shown in SEQ ID NO: 2.
本発明におけるTDP-43としては、上記配列番号１で表されるアミノ酸配列からなるタンパク質と同等の生物学的活性を有するものであれば、配列番号１で表されるアミノ酸配列からなるタンパク質の変異体であってもよい。 TDP-43 in the present invention is a mutation of a protein comprising the amino acid sequence represented by SEQ ID NO: 1 as long as it has a biological activity equivalent to that of the protein comprising the amino acid sequence represented by SEQ ID NO: 1. It may be a body.
本発明のTDP-43は、通常、動物由来であり、好ましくは哺乳動物由来、特に好ましくはヒト由来である。 The TDP-43 of the present invention is usually derived from an animal, preferably from a mammal, particularly preferably from a human.
本発明の「173位及び／又は175位のシステイン」とは、基本的には配列番号１で示されるアミノ酸配列の173位及び／又は175位のシステインであるが、他のアミノ酸配列を有するTDP-43については同様の位置が該当する。 The “cysteine at positions 173 and / or 175” of the present invention is basically a cysteine at positions 173 and / or 175 of the amino acid sequence represented by SEQ ID NO: 1, but has a different amino acid sequence. The same position applies to -43.
173位及び／又は175位のシステインを置換するアミノ酸の種類としては、本発明の効果が得られる限り特に限定されないが、好ましくはセリン又はアラニンである。 The type of amino acid that substitutes cysteine at positions 173 and / or 175 is not particularly limited as long as the effect of the present invention can be obtained, but is preferably serine or alanine.
本発明のTDP-43変異体は、更に核移行シグナル(NLS)が機能しない形で改変されていることが好ましい。 The TDP-43 mutant of the present invention is preferably further modified in such a way that the nuclear localization signal (NLS) does not function.
本発明のTDP-43変異体は、核内及び細胞内で封入体を形成し、運動ニューロン培養細胞に神経毒性を示す。また、上記TDP-43変異体内のRRM1ドメインだけであっても、容易に凝集体を形成する。そのため、本発明のTDP-43変異体及びRRM1ドメインは、以下に示すTDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニング方法等に使用可能である。 The TDP-43 mutant of the present invention forms inclusion bodies in the nucleus and cells, and exhibits neurotoxicity to motor neuron cultured cells. Moreover, even if only the RRM1 domain in the TDP-43 mutant is used, an aggregate is easily formed. Therefore, the TDP-43 mutant and the RRM1 domain of the present invention can be used in a method for screening a compound for treating and / or preventing the following diseases in which aggregates of TDP-43 accumulate.
RRM1ドメインは、配列番号１で示されるアミノ酸配列では103〜183位の部分である。
The RRM1 domain is a portion at
化合物のスクリーニング方法(A)
本発明の化合物のスクリーニング方法は、以下の工程を含むことを特徴とする：
（１）上記TDP-43変異体又は上記RRM1ドメインに候補となる化合物を接触させる工程、
（２）該化合物の凝集体形成阻害活性を検出する工程、及び
（３）凝集体形成を阻害する化合物を選択する工程。
Compound screening method (A)
The compound screening method of the present invention comprises the following steps:
(1) contacting a candidate compound with the TDP-43 mutant or the RRM1 domain;
(2) a step of detecting the aggregate formation inhibitory activity of the compound, and (3) a step of selecting a compound that inhibits aggregate formation.
上記方法により選択される、上記TDP-43変異体又は上記RRM1ドメインの凝集体形成を阻害する化合物は、TDP-43の凝集体が蓄積する疾患の治療及び／又は予防に有効であり得る。 The TDP-43 mutant or the compound that inhibits the RRM1 domain aggregate formation selected by the above method may be effective for the treatment and / or prevention of diseases in which the aggregate of TDP-43 accumulates.
候補となる化合物は、特に制限なく使用することができるが、例えば、低分子化合物、高分子化合物、生体高分子(タンパク質、核酸、多糖類等)等が挙げられ、このような化合物を含む種々の化合物ライブラリーを使用することができる。 Candidate compounds can be used without particular limitation, and examples include low molecular compounds, high molecular compounds, biopolymers (proteins, nucleic acids, polysaccharides, etc.), and various compounds including such compounds. The compound library can be used.
上記工程（１）における「接触させる」とは、上記TDP-43変異体又は上記RRM1ドメインにインビトロで候補となる化合物を接触させること、及び上記TDP-43変異体を発現する培養細胞に候補となる化合物を接触させることの両方を包含する。 “Contacting” in the above step (1) means that a candidate compound is brought into contact with the TDP-43 mutant or the RRM1 domain in vitro, and a cultured cell expressing the TDP-43 mutant is regarded as a candidate. Both contacting with the compound.
候補となる化合物の存在により、インビトロでの上記TDP-43変異体又は上記RRM1ドメインの凝集体の形成が、10%以上、好ましくは25%以上、より好ましくは50%以上、更に好ましくは75%以上低下した場合、当該化合物をTDP-43の凝集体が蓄積する疾患の治療及び／又は予防に有効なものとして選択できる。 Due to the presence of a candidate compound, in vitro formation of the TDP-43 mutant or the RRM1 domain aggregate is 10% or more, preferably 25% or more, more preferably 50% or more, and even more preferably 75%. In the case of a decrease, the compound can be selected as effective for the treatment and / or prevention of diseases in which aggregates of TDP-43 accumulate.
インビトロでの凝集体形成阻害活性の測定方法としては、特に制限なく使用できるが、例えば、吸光度計、ELISA法、SDS-PAGE又はウェスタンブロッティング法を用いることにより凝集体の存在を確認する方法が挙げられる。 The method for measuring the activity of inhibiting aggregate formation in vitro can be used without particular limitation, and examples thereof include a method for confirming the presence of aggregates by using an absorptiometer, ELISA method, SDS-PAGE or Western blotting method. It is done.
また、候補となる化合物の存在により、上記TDP-43変異体を発現する培養細胞中の(好ましくは細胞質中の)封入体の形成が、10%以上、好ましくは25%以上、より好ましくは50%以上、更に好ましくは75%以上低下した場合、当該化合物をTDP-43の凝集体が蓄積する疾患の治療及び／又は予防に有効なものとして選択できる。 Further, due to the presence of a candidate compound, the formation of inclusion bodies (preferably in the cytoplasm) in the cultured cells expressing the TDP-43 mutant is 10% or more, preferably 25% or more, more preferably 50 %, More preferably 75% or more, the compound can be selected as effective for the treatment and / or prevention of diseases in which aggregates of TDP-43 accumulate.
培養細胞を用いた凝集体形成阻害活性の測定方法としては、特に制限なく使用できるが、例えば、EGFPを結合させた上記TDP-43変異体を発現する培養細胞を蛍光顕微鏡を用いて観察することにより封入体の存在を確認する方法が挙げられる。 The method for measuring the aggregate formation inhibitory activity using cultured cells can be used without particular limitation.For example, observing cultured cells expressing the above TDP-43 mutant bound with EGFP using a fluorescence microscope Can be used to confirm the presence of inclusion bodies.
トランスジェニック非ヒト動物
本発明のトランスジェニック非ヒト動物は、上記TDP-43変異体をコードするDNAが導入されていることを特徴とする。
Transgenic non-human animal The transgenic non-human animal of the present invention is characterized in that a DNA encoding the TDP-43 mutant is introduced.
本発明における非ヒト動物とは、ヒトを含まない脊椎動物や無脊椎動物を意味し、トランスジェニック動物の作製が可能な非ヒト動物であれば特に限定されず、例えば、マウス、ハムスター、モルモット、ラット、ウサギ、ニワトリ、イヌ、ネコ、ヤギ、ヒツジ、ウシ、ブタ、サル、線虫、ショウジョウバエ等を挙げることができるが、マウス、ラット、線虫及びショウジョウバエが好ましく、マウスが特に好ましい。 The non-human animal in the present invention means a vertebrate or invertebrate that does not include humans, and is not particularly limited as long as it is a non-human animal capable of producing a transgenic animal. For example, a mouse, a hamster, a guinea pig, Examples include rats, rabbits, chickens, dogs, cats, goats, sheep, cows, pigs, monkeys, nematodes, and fruit flies, with mice, rats, nematodes and fruit flies being preferred, and mice being particularly preferred.
トランスジェニック非ヒト動物の作製方法は公知である。具体的には、上記TDP-43変異体をコードするDNAを動物の全能細胞に導入し、この細胞を個体へと発生させる。そして、得られた個体の中から、体細胞及び生殖細胞中に導入遺伝子が組み込まれた個体を選別することによって、目的とするトランスジェニック非ヒト動物を作製することができる。遺伝子を導入する全能細胞としては、受精卵や初期胚以外に、ES細胞のような培養細胞などが挙げられる。 Methods for producing transgenic non-human animals are known. Specifically, DNA encoding the above TDP-43 mutant is introduced into an totipotent cell of an animal, and this cell is generated into an individual. The target transgenic non-human animal can be produced by selecting individuals from which the transgene has been incorporated into somatic cells and germ cells from the individuals obtained. Examples of totipotent cells into which genes are introduced include cultured cells such as ES cells in addition to fertilized eggs and early embryos.
本発明のトランスジェニック非ヒト動物としては、上記TDP-43変異体をコードするDNAを有するものであれば、トランスジェニック非ヒト動物のいずれの世代の動物であってもよい。また、本発明のトランスジェニック非ヒト動物としては、上記TDP-43変異体をコードするDNAをヘテロ又はホモのいずれで保持するものであってもよい。 The transgenic non-human animal of the present invention may be any generation of transgenic non-human animals as long as it has a DNA encoding the TDP-43 mutant. In addition, the transgenic non-human animal of the present invention may be one that retains the DNA encoding the TDP-43 mutant in either a heterozygous or homozygous manner.
本発明のトランスジェニック非ヒト動物は、導入されたTDP-43変異体が核内及び細胞内で封入体を形成し、運動ニューロン培養細胞に神経毒性を示すため、TDP-43の凝集体が蓄積する疾患のモデル動物として利用することができ、TDP-43の凝集体が蓄積する疾患の治療効果及び予防効果を評価することが可能である。そのため、本発明のトランスジェニック非ヒト動物を使用することにより、TDP-43の凝集体が蓄積する疾患を治療及び／又は予防するための化合物のスクリーニングを行うことができる。 In the transgenic non-human animal of the present invention, the introduced TDP-43 mutant forms inclusion bodies in the nucleus and in the cells, and exhibits neurotoxicity in cultured motor neuron cells. Therefore, aggregates of TDP-43 accumulate. It can be used as a model animal of the disease to be treated, and it is possible to evaluate the therapeutic effect and the preventive effect of the disease in which aggregates of TDP-43 accumulate. Therefore, by using the transgenic non-human animal of the present invention, a compound for treating and / or preventing a disease in which aggregates of TDP-43 accumulate can be screened.
例えば、本発明のトランスジェニック非ヒト動物に、候補となる化合物を投与し、該トランスジェニック非ヒト動物の運動能力などを評価することにより、該化合物のTDP-43の凝集体が蓄積する疾患に対する治療効果や予防効果を評価することができる。また、本発明のトランスジェニック非ヒト動物に候補となる化合物を投与し、投与後における脳内の細胞中のTDP-43封入体について分析することにより、該化合物のTDP-43の凝集体が蓄積する疾患に対する治療効果や予防効果を評価することもできる。 For example, by administering a candidate compound to the transgenic non-human animal of the present invention, and evaluating the ability of the transgenic non-human animal to exercise, etc., a disease in which aggregates of TDP-43 of the compound accumulate The therapeutic effect and the preventive effect can be evaluated. Further, by administering a candidate compound to the transgenic non-human animal of the present invention and analyzing TDP-43 inclusion bodies in cells in the brain after administration, aggregates of TDP-43 of the compound accumulate. It is also possible to evaluate the therapeutic effect and preventive effect on the disease.
抗体又は抗体断片
本発明の抗体又は抗体断片は、TDP-43の108〜116位に対応するアミノ酸配列からなるペプチドに結合することを特徴とする。
Antibody or Antibody Fragment The antibody or antibody fragment of the present invention is characterized by binding to a peptide consisting of an amino acid sequence corresponding to positions 108 to 116 of TDP-43.
ここで、結合の対象となるペプチドは、TDP-43の一部分の状態のもの、及び当該ペプチドのみからなるものの両方を含む。 Here, the peptides to be bound include both those in a partial state of TDP-43 and those consisting only of the peptide.
「108〜116位に対応するアミノ酸配列」とは、基本的には配列番号１で示されるアミノ酸配列におけるVLGLPWKTTであるが、他のアミノ酸配列を有するTDP-43については同様のアミノ酸配列が該当する。 The “amino acid sequence corresponding to positions 108 to 116” is basically VLGLPWKTT in the amino acid sequence represented by SEQ ID NO: 1, but the same amino acid sequence applies to TDP-43 having other amino acid sequences. .
TDP-43の108〜116位のアミノ酸残基は、TDP-43の凝集体において構造変化により外部露出しているため抗体と結合し易い構造になるが、野生型では抗体と結合し難い構造を取っていると考えられる。 The amino acid residues at positions 108 to 116 of TDP-43 are easily exposed to antibodies because they are externally exposed due to structural changes in the aggregates of TDP-43, but the wild type has a structure that is difficult to bind to antibodies. It is thought that it is taking.
上記抗体又は抗体断片は、公知の方法に従って取得することが可能であり、例えば、実施例に記載の方法により取得することができる。また、上記抗体はモノクローナル抗体又はポリクローナル抗体のいずれでもよく、またヒト化されたキメラ抗体であってもよい。抗体断片としてはFab、Fab'、F(ab')2、Fv、scFv等が挙げられ、抗体又は抗体断片は蛍光色素等により修飾されていてもよい。当該抗体又は抗体断片は、ELISA法、ウェスタンブロッティング法、プロテインチップによる解析法等に使用することで上記ペプチドを検出することができる。 The antibody or antibody fragment can be obtained according to a known method, for example, by the method described in the Examples. The antibody may be either a monoclonal antibody or a polyclonal antibody, or may be a humanized chimeric antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) 2 , Fv, scFv, and the like, and the antibody or antibody fragment may be modified with a fluorescent dye or the like. The peptide can be detected by using the antibody or antibody fragment in an ELISA method, Western blotting method, protein chip analysis method or the like.
本発明の診断薬及び診断用キットは、上記抗体又は抗体断片を含むことを特徴とし、TDP-43の凝集体を検出することが可能である。そのため、本発明の診断薬及び診断用キットにより、TDP-43の凝集体が蓄積する疾患の診断が可能となり得る。 The diagnostic agent and diagnostic kit of the present invention are characterized by containing the above antibody or antibody fragment, and are capable of detecting an aggregate of TDP-43. Therefore, the diagnostic agent and diagnostic kit of the present invention may enable diagnosis of diseases in which aggregates of TDP-43 accumulate.
本発明の医薬組成物は、ヒトを含む哺乳動物に投与されるものであって、上記抗体又は抗体断片を含むことを特徴とし、TDP-43の凝集体が蓄積する疾患の治療及び／又は予防の効果を有し得る。これは、TDP-43の108〜116位のアミノ酸残基は、疾患や変異によって構造変化が起こり、抗体と結合し易い構造になるためと考えられる。 The pharmaceutical composition of the present invention is administered to mammals including humans, and comprises the above antibody or antibody fragment, and is used for the treatment and / or prevention of diseases in which aggregates of TDP-43 accumulate. It can have the effect. This is presumably because the amino acid residues at positions 108 to 116 of TDP-43 undergo structural changes due to diseases and mutations, and become structures that easily bind to antibodies.
本発明の医薬組成物は、その使用形態に応じて、生物学的に許容される担体、賦形剤等を任意に含有できる。本発明の医薬組成物は、常套手段に従って製造することができる。例えば、必要に応じて糖衣や腸溶性被膜を施した錠剤、カプセル剤、エリキシル剤、マイクロカプセル剤等として経口的に、軟膏、硬膏等の外用剤、噴霧剤、吸入剤等として経皮的、経鼻的又は経気管的に、水又はそれ以外の薬学的に許容し得る液との無菌性溶液、懸濁液剤等の注射剤の形で非経口的に使用できる。 The pharmaceutical composition of the present invention can optionally contain biologically acceptable carriers, excipients and the like depending on the usage form. The pharmaceutical composition of the present invention can be produced according to conventional methods. For example, orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating or enteric coating as needed, or as an external agent such as an ointment or plaster, transdermally as a spray, inhalant, etc. It can be used nasally or tracheally, parenterally in the form of injections such as sterile solutions and suspensions with water or other pharmaceutically acceptable fluids.
本発明の医薬組成物の投与量は、剤型の種類、投与方法、患者の年齢や体重、患者の症状等を考慮して、最終的には医師の判断により適宜決定できる。 The dosage of the pharmaceutical composition of the present invention can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, and the like.
化合物のスクリーニング方法(B)
本発明の 化合物のスクリーニング方法は、以下の工程を含むことを特徴とする：
（１）上記TDP-43変異体又は上記RRM1ドメインに、上記抗体又は抗体断片と候補となる化合物を接触させる工程、
（２）該化合物の結合阻害活性を検出する工程、及び
（３）該抗体又は抗体断片の結合を阻害する化合物を選択する工程。
Compound screening method (B)
The compound screening method of the present invention is characterized by comprising the following steps:
(1) contacting the TDP-43 mutant or the RRM1 domain with the antibody or antibody fragment and a candidate compound;
(2) detecting the binding inhibitory activity of the compound, and (3) selecting a compound that inhibits binding of the antibody or antibody fragment.
上記方法により選択される、上記TDP-43変異体又は上記RRM1ドメインへの上記抗体又は抗体断片の結合を阻害する化合物は、TDP-43の凝集体が蓄積する疾患及び／又は予防、並びにTDP-43の凝集体が蓄積する疾患の診断に有効であり得る。 The compound that inhibits the binding of the antibody or antibody fragment to the TDP-43 variant or the RRM1 domain selected by the above method is a disease and / or prevention in which an aggregate of TDP-43 accumulates, and TDP- It may be useful for diagnosis of diseases in which 43 aggregates accumulate.
候補となる化合物は、特に制限なく使用することができるが、例えば、低分子化合物、高分子化合物、生体高分子(タンパク質、核酸、多糖類等)等が挙げられ、このような化合物を含む種々の化合物ライブラリーを使用することができる。 Candidate compounds can be used without particular limitation, and examples include low molecular compounds, high molecular compounds, biopolymers (proteins, nucleic acids, polysaccharides, etc.), and various compounds including such compounds. The compound library can be used.
候補となる化合物の存在により、上記の抗体又は抗体断片の結合が、10%以上、好ましくは25%以上、より好ましくは50%以上、更に好ましくは75%以上低下した場合、当該化合物をTDP-43の凝集体が蓄積する疾患の治療及び／又は予防、並びにTDP-43の凝集体が蓄積する疾患の診断に有効なものとして選択できる。結合阻害活性の測定方法としては、特に制限なく使用できるが、例えば、ELISA法が挙げられる。 When the binding of the antibody or antibody fragment described above is reduced by 10% or more, preferably 25% or more, more preferably 50% or more, still more preferably 75% or more due to the presence of a candidate compound, the compound is reduced to TDP- It can be selected as effective for the treatment and / or prevention of diseases in which 43 aggregates accumulate, and in the diagnosis of diseases in which aggregates of TDP-43 accumulate. The method for measuring the binding inhibitory activity can be used without particular limitation, and examples thereof include an ELISA method.
ペプチド、核酸
本発明のペプチドは、 TDP-43の108〜116位に対応するアミノ酸配列を含むことを特徴とし、本発明の核酸は、該ペプチドをコードすることを特徴とする。
Peptide, Nucleic Acid The peptide of the present invention is characterized by including an amino acid sequence corresponding to positions 108 to 116 of TDP-43, and the nucleic acid of the present invention is characterized by encoding the peptide.
「108〜116位に対応するアミノ酸配列」とは、基本的には配列番号１で示されるアミノ酸配列におけるVLGLPWKTTであるが、他のアミノ酸配列を有するTDP-43については同様のアミノ酸配列が該当する。 The “amino acid sequence corresponding to positions 108 to 116” is basically VLGLPWKTT in the amino acid sequence represented by SEQ ID NO: 1, but the same amino acid sequence applies to TDP-43 having other amino acid sequences. .
本発明のペプチドは、 好ましくはTDP-43の108〜116位に対応するアミノ酸配列からなる。 The peptide of the present invention preferably consists of an amino acid sequence corresponding to positions 108 to 116 of TDP-43.
本発明の医薬組成物は、ヒトを含む哺乳動物に投与されるものであって、上記ペプチド又は上記DNAを含むことを特徴とし、TDP-43の凝集体に対する抗体の産生を刺激又は増強し得る。TDP-43の108〜116位に対応するアミノ酸残基は、疾患や変異によって構造変化が起こり、抗体と結合し易い構造になるが、野生型のTDP-43では、抗体と結合し難い構造をとっていると考えられる。そのため、本発明の医薬組成物は、TDP-43の凝集体が蓄積する疾患の予防の効果を有するワクチンとして機能し得る。また、本発明の医薬組成物は、TDP-43の凝集体が蓄積する疾患の治療及び／又は予防の効果を有し得る。 The pharmaceutical composition of the present invention is administered to mammals including humans, contains the peptide or the DNA, and can stimulate or enhance the production of an antibody against an aggregate of TDP-43. . The amino acid residues corresponding to positions 108 to 116 of TDP-43 undergo structural changes due to diseases and mutations, resulting in a structure that easily binds to antibodies, but wild-type TDP-43 has a structure that is difficult to bind to antibodies. It is thought that it has taken. Therefore, the pharmaceutical composition of the present invention can function as a vaccine having an effect of preventing diseases in which aggregates of TDP-43 accumulate. In addition, the pharmaceutical composition of the present invention may have an effect of treating and / or preventing a disease in which aggregates of TDP-43 accumulate.
本発明の医薬組成物は、その使用形態に応じて、生物学的に許容される担体、賦形剤等を任意に含有できる。本発明の医薬組成物は、常套手段に従って製造することができる。例えば、必要に応じて糖衣や腸溶性被膜を施した錠剤、カプセル剤、エリキシル剤、マイクロカプセル剤等として経口的に、軟膏、硬膏等の外用剤、噴霧剤、吸入剤等として経皮的、経鼻的又は経気管的に、水又はそれ以外の薬学的に許容し得る液との無菌性溶液、懸濁液剤等の注射剤の形で非経口的に使用できる。 The pharmaceutical composition of the present invention can optionally contain biologically acceptable carriers, excipients and the like depending on the usage form. The pharmaceutical composition of the present invention can be produced according to conventional methods. For example, orally as tablets, capsules, elixirs, microcapsules, etc. with sugar coating or enteric coating as needed, or as an external agent such as an ointment or plaster, transdermally as a spray, inhalant, etc. It can be used nasally or tracheally, parenterally in the form of injections such as sterile solutions and suspensions with water or other pharmaceutically acceptable fluids.
また、本発明の医薬組成物は、細胞又は組織内で上記ペプチドを発現させることができるDNAを使用した非ウイルスベクター又はウイルスベクターを含むものであってもよい。非ウイルスベクターによる投与方法としては、リポソームを用いてDNAを導入する方法、マイクロインジェクション法、遺伝子銃でキャリアとともにDNAを細胞に移入する方法等が挙げられる。ウイルスベクターを用いて投与する方法としては、組換えアデノウイルス、レトロウイルスなどのウイルスベクターを利用する方法が挙げられ、上記ペプチドを発現するベクターを無毒化したアデノウイルス、レトロウイルス等に導入し、細胞又は組織にこのウィルスを感染させることにより細胞又は組織内に遺伝子を導入することができる。 In addition, the pharmaceutical composition of the present invention may include a non-viral vector or a viral vector using DNA capable of expressing the peptide in cells or tissues. Examples of the administration method using a non-viral vector include a method of introducing DNA using a liposome, a microinjection method, a method of transferring DNA into a cell together with a carrier using a gene gun, and the like. Examples of the method of administration using a viral vector include a method using a viral vector such as a recombinant adenovirus and a retrovirus. The vector expressing the peptide is introduced into a detoxified adenovirus, a retrovirus, and the like. A gene can be introduced into a cell or tissue by infecting the cell or tissue with this virus.
本発明の医薬組成物の投与量は、剤型の種類、投与方法、患者の年齢や体重、患者の症状等を考慮して、最終的には医師の判断により適宜決定できる。 The dosage of the pharmaceutical composition of the present invention can be appropriately determined finally based on the judgment of a doctor in consideration of the type of dosage form, administration method, patient age and weight, patient symptom, and the like.
以下、本発明を更に詳しく説明するため実施例を挙げる。しかし、本発明はこれら実施例に何ら限定されるものではない。 Examples are given below to illustrate the present invention in more detail. However, the present invention is not limited to these examples.
＜プラスミド構築及びプロテイン精製＞
ヒトのRRM1 (aa 103-183)のcDNAは、テンプレートとして以前報告されたコンストラクト(pcDNA3-TDP-43-FLAG)を用いたPCRによってクローン化した(1)。システインをセリン(C/S)にする置換変異、家族性ALS関連変異体(A315T及びQ331K)、及びNLS変異体(mNLS)は以前報告されたように部位特異的突然変異導入を使用して生成した(1)。ヒトTDP-43のRRM1欠失変異体(ΔRRM1)は、除去される欠失部位へのプライマーを設計することでPCRによって生成した。これらのcDNAは、pcDNA3、培養細胞実験のためのpEGFP-N2 (Clontech, Palo Alto. CA)、及び組換えタンパク質を生成するためにpGEX-6p-1に、それぞれBamHI/XhoI、XhoI/BamHI及びBamHI/XhoIサイトでサブクローン化した。組換えタンパク質は、以前報告されているようにE. coliで産生させ精製した(1)。
(1) J. Neurosci. Res. 88, 784-797(2010)
<Plasmid construction and protein purification>
Human RRM1 (aa 103-183) cDNA was cloned by PCR using a previously reported construct (pcDNA3-TDP-43-FLAG) as a template (1). Cysteine to serine (C / S) substitution mutations, familial ALS-related mutants (A315T and Q331K), and NLS mutants (mNLS) are generated using site-directed mutagenesis as previously reported (1). An RRM1 deletion mutant of human TDP-43 (ΔRRM1) was generated by PCR by designing primers to the deleted site to be removed. These cDNAs are pcDNA3, pEGFP-N2 for cultured cell experiments (Clontech, Palo Alto. CA), and pGEX-6p-1 to produce recombinant proteins, respectively, BamHI / XhoI, XhoI / BamHI and Subcloned at the BamHI / XhoI site. Recombinant protein was produced and purified in E. coli as previously reported (1).
(1) J. Neurosci. Res. 88, 784-797 (2010)
＜抗体＞
ウサギポリクローナル抗TDP-43抗体(Sigma, St. Louis, MO)は、RRM1ドメインのウェスタンブロット分析のために使用した(1:1000希釈)。他のウサギポリクローナル抗TFP-43抗体(Proteintech, Chicago, IL)は、免疫蛍光のために使用した(1:1000)。マウスモノクローナル抗FLAG (M2) (Sigma)は、免疫蛍光染色、ウェスタンブロッティングとも1:500希釈で使用した。ウサギポリクローナル抗phospho-TDP-43抗体は、免疫蛍光、ウェスタンブロッティングともに1:500希釈で使用した。ウサギモノクローナル抗K48-specific ubiquitin (Millipore, Temecula, CA)は、免疫蛍光のために1:500希釈で使用した。RRM1ドメインのミスフォールド関連コアに対する抗血清を生成する方法は、以下に記載する。
<Antibody>
Rabbit polyclonal anti-TDP-43 antibody (Sigma, St. Louis, MO) was used for Western blot analysis of RRM1 domain (1: 1000 dilution). Another rabbit polyclonal anti-TFP-43 antibody (Proteintech, Chicago, IL) was used for immunofluorescence (1: 1000). Mouse monoclonal anti-FLAG (M2) (Sigma) was used at 1: 500 dilution for both immunofluorescence staining and Western blotting. Rabbit polyclonal anti-phospho-TDP-43 antibody was used at 1: 500 dilution for both immunofluorescence and Western blotting. Rabbit monoclonal anti-K48-specific ubiquitin (Millipore, Temecula, CA) was used at a 1: 500 dilution for immunofluorescence. Methods for generating antisera against misfold-related cores of the RRM1 domain are described below.
＜NMR測定＞
タンパク質のサンプルを、20 mM d-Tris-HClバッファー中に1.1 mM、pH 7.0で調製した。1H-NMRと15N/1H HSQC測定を、25℃で30 barから2000 barまで500 bar毎に行った。分析は、以前報告されているように高圧NMRセルと組み合わせたDRX600スペクトロメーター(Bruker biospin Co., Fallanden, Switzerland)を用いて行った(2)。溶液中のRRM1の3次元構造は、通常の三重共鳴NMR技術によって決定した。1H化学シフトはDSSのメチル共鳴を直接参照し、一方で15N化学シフトは、DSSの1H化学シフトを間接的に参照した。
(2) Chemical Reviews 106, 1814-1835(2006)
<NMR measurement>
Protein samples were prepared at 1.1 mM, pH 7.0 in 20 mM d-Tris-HCl buffer. 1 H-NMR and 15 N / 1 H HSQC measurements were performed at 25 ° C. from 30 bar to 2000 bar every 500 bar. Analysis was performed using a DRX600 spectrometer (Bruker biospin Co., Fallanden, Switzerland) combined with a high pressure NMR cell as previously reported (2). The three-dimensional structure of RRM1 in solution was determined by conventional triple resonance NMR techniques. The 1 H chemical shift directly referred to the DSS methyl resonance, while the 15 N chemical shift indirectly referred to the DSS 1 H chemical shift.
(2) Chemical Reviews 106, 1814-1835 (2006)
＜組換えタンパク質と培養細胞溶解液の生化学的分析＞
組換えタンパク質は、22℃16時間800 rpmでマルチボルテックスミキサーにより震盪させ、次に4℃でインキュベートした。その後、タンパク質をLDSサンプリングバッファー(Invitrogen, Carlsbad, CA)と共に、70℃で20分間の反応よって変性させた後、SDS-PAGEを行った。パーフルオロオクタン酸(PFO, Fluorochem Ltd, Derbyshire, UK)は、細胞質と膜タンパク質の両方のタンパク質-タンパク質相互作用を保存する非解離性界面活性剤である。タンパク質サンプルは、同量のPFOサンプリングバッファー(100 mM Tris base, 2% NaPFO, 20% glycerol, 0.005% Bromophenol Blue, pH 8.0)と混合し、22℃で1時間インキュベートした。タンパク質サンプルは、予め冷却したランニングバッファー(0.5% NaPFO, 25 mM Tris-HCl, 192 mM Glycine, pH 8.5)を使用して4-12%勾配ポリアクリルアミドゲル(Novex, Tris-Bis gel, Invitrogen)で140Vで分離した。SDS-PAGE又はPFO-PAGEのために、サンプルは、ジチオスレイトール(DTT)を含む又は含まないプロテアーゼ阻害剤カクテル(Roche)及び／又はホスファターゼ阻害剤カクテル(Nacalai Tesque, Kyoto, Japan)を含む2%SDS-サンプルバッファーで変性した。サンプルは、SDS-PAGEによって分離し、ウェスタンブロッティングのためのPVDF膜に転移した。抗体検出は、増強した化学ルミネセンス(Nacalai Tesque)を用いて行った。PFO-PAGE後のゲルは、クマシーブリリアントブルー(CBB、ナカライテスク)によって染色した。
<Biochemical analysis of recombinant protein and cultured cell lysate>
The recombinant protein was shaken with a multi-vortex mixer at 800 rpm for 16 hours at 22 ° C. and then incubated at 4 ° C. Subsequently, the protein was denatured by a reaction at 70 ° C. for 20 minutes with LDS sampling buffer (Invitrogen, Carlsbad, Calif.), And then SDS-PAGE was performed. Perfluorooctanoic acid (PFO, Fluorochem Ltd, Derbyshire, UK) is a non-dissociating surfactant that preserves protein-protein interactions of both cytoplasmic and membrane proteins. The protein sample was mixed with the same amount of PFO sampling buffer (100 mM Tris base, 2% NaPFO, 20% glycerol, 0.005% Bromophenol Blue, pH 8.0) and incubated at 22 ° C. for 1 hour. Protein samples were run on 4-12% gradient polyacrylamide gels (Novex, Tris-Bis gel, Invitrogen) using pre-cooled running buffer (0.5% NaPFO, 25 mM Tris-HCl, 192 mM Glycine, pH 8.5). Separated at 140V. For SDS-PAGE or PFO-PAGE, samples contain protease inhibitor cocktail (Roche) and / or phosphatase inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) with or without dithiothreitol (DTT) 2. Denatured with% SDS-sample buffer. Samples were separated by SDS-PAGE and transferred to PVDF membrane for Western blotting. Antibody detection was performed using enhanced chemiluminescence (Nacalai Tesque). The gel after PFO-PAGE was stained with Coomassie Brilliant Blue (CBB, Nacalai Tesque).
＜チオフラビンTアッセイ＞
熱変性RRM1によるアミロイド形成は、チオフラビンTアッセイを使用して評価した。示した時間4℃での事後的なインキュベーション後に、タンパク質溶液は当モル量のチオフラビン誘導体BTA-1 (Sigma)と反応させた。蛍光は、マルチプレートリーダー(Tecan, Mannedorf, Switzerland)を使用して440 nm (励起)及び480 nm (発光)で測定した。
<Thioflavin T assay>
Amyloid formation by heat-denatured RRM1 was assessed using the thioflavin T assay. Following post-incubation at the indicated time of 4 ° C., the protein solution was reacted with an equimolar amount of the thioflavin derivative BTA-1 (Sigma). Fluorescence was measured at 440 nm (excitation) and 480 nm (emission) using a multiplate reader (Tecan, Mannedorf, Switzerland).
＜LC-MS/MS分析＞
プロナーゼ分解されたRRM1タンパク質凝集体のLC-MS/MS分析は、概ね以前に報告された方法に従って行った(3)。RRM1タンパク質凝集体(25μg)は、100μLの消化バッファー(50 mM Tris, 100 mM NaCl, 5 mM CaCl2, pH 8.0)に再懸濁し、5μgのプロナーゼ(Millipore)と混合し、37℃で1時間インキュベートした。反応混合液のタンパク質は100000 g、4℃で30分間超遠心によって沈降した。ペレットは20μLのバッファー(50 mM Tris, 500 mM NaCl, 6 M GdnHCl, 5 mM EDTA, 5 mM DTT, pH 8.0)中に回収し、NuTip C-18 (Glygen Co, Columbia, MD)を使用して脱塩した。バッファーは、蒸留水中の2%アセトニトリル及び0.1%トリフルオロ酢酸(Nacalai, Kyoto, Japan)で置換した。分解されたフラグメントは、LC-MS/MS (Paradigm MS4, AMR, Kyoto, Japan; Finigan LCQ Advantage MAX, Thermo Electron, Waltham, MA)で分析した。
(3) J. Biol. Chem. 286, 18664-18672(2011)
<LC-MS / MS analysis>
LC-MS / MS analysis of pronase-degraded RRM1 protein aggregates was generally performed according to previously reported methods (3). RRM1 protein aggregates (25 μg) were resuspended in 100 μL digestion buffer (50 mM Tris, 100 mM NaCl, 5 mM CaCl 2 , pH 8.0), mixed with 5 μg pronase (Millipore), and 1 hour at 37 ° C. Incubated. The protein in the reaction mixture was precipitated by ultracentrifugation at 100000 g at 4 ° C. for 30 minutes. The pellet is recovered in 20 μL buffer (50 mM Tris, 500 mM NaCl, 6 M GdnHCl, 5 mM EDTA, 5 mM DTT, pH 8.0) and using NuTip C-18 (Glygen Co, Columbia, MD). Desalted. The buffer was replaced with 2% acetonitrile and 0.1% trifluoroacetic acid (Nacalai, Kyoto, Japan) in distilled water. The degraded fragments were analyzed by LC-MS / MS (Paradigm MS4, AMR, Kyoto, Japan; Finigan LCQ Advantage MAX, Thermo Electron, Waltham, MA).
(3) J. Biol. Chem. 286, 18664-18672 (2011)
＜細胞培養、形質導入＞
全ての培養細胞は、5%CO2、湿度100%、37℃の状態にあった。HEK293A細胞(Invitrogen)は、10%ウシ胎児血清とペニシリン／ストレプトマイシン(PS)を含むダルベッコ改変イーグル培地で維持し、ヒトニューロブラストーマ細胞株SHSY-5Yは、15%ウマ血清、非必須アミノ酸(Invitrogen)を含むDMEM/F12-Ham培地で培養し、マウス運動ニューロン細胞株NSC34細胞(Cellutions biosystems, Vancouver, BC)は、10%FBSを含むDMEMで培養維持した。分化のために、培地を1%非必須アミノ酸(NEAA)を含むDMEM/F12-Ham中に3%FBSを含む分化培地に変更した。FuGene HD transfection kit (Roche, Basel, Switzerland)をプラスミド導入のために使用した。トランスフェクションの48時間後に、細胞に種々の分析を行った。
<Cell culture, transduction>
All cultured cells were in a state of 5% CO 2 , 100% humidity and 37 ° C. HEK293A cells (Invitrogen) are maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and penicillin / streptomycin (PS), and human neuroblastoma cell line SHSY-5Y contains 15% horse serum, non-essential amino acids (Invitrogen) The mouse motoneuron cell line NSC34 cells (Cellutions biosystems, Vancouver, BC) were cultured and maintained in DMEM containing 10% FBS. For differentiation, the medium was changed to a differentiation medium containing 3% FBS in DMEM / F12-Ham containing 1% non-essential amino acids (NEAA). FuGene HD transfection kit (Roche, Basel, Switzerland) was used for plasmid introduction. Cells were subjected to various analyzes 48 hours after transfection.
＜免疫細胞化学及び共焦点顕微鏡観察＞
4%PFAで固定化後、細胞は5%ヤギ血清及び0.2%Triton X100を含むPBS中で室温で1時間インキュベートした。細胞は0.1% Triton X100を含むPBS中で各種一次抗体と室温で1時間反応させ、その後、同じバッファー中で蛍光二次抗体(Alexa 488, Invitrogen; CF 568, Biotium, Hayward, CA)と反応させた。PBSで三回洗浄後に、2μg/mLを含むPBS中で細胞をインキュベートすることによって核を染色した。染色された細胞は、共焦点レーザー顕微鏡(Nikon, C1 SI, Tokyo, Japan)によって分析した。細胞カウント実験のために、7、8枚の写真をランダムに撮った(1画像当たり20〜50個のGFP陽性細胞)。全蛍光細胞と封入体陽性細胞の細胞カウントは、ImageJソフトウェアを使用して盲目様式で行った。
<Immunocytochemistry and confocal microscopy>
After fixation with 4% PFA, the cells were incubated for 1 hour at room temperature in PBS containing 5% goat serum and 0.2% Triton X100. Cells are reacted with various primary antibodies in PBS containing 0.1% Triton X100 for 1 hour at room temperature, and then reacted with fluorescent secondary antibodies (Alexa 488, Invitrogen; CF 568, Biotium, Hayward, CA) in the same buffer. It was. After three washes with PBS, nuclei were stained by incubating cells in PBS containing 2 μg / mL. Stained cells were analyzed with a confocal laser microscope (Nikon, C1 SI, Tokyo, Japan). For cell counting experiments, 7-8 pictures were taken randomly (20-50 GFP positive cells per image). Cell counts of total fluorescent cells and inclusion body positive cells were performed in a blinded fashion using ImageJ software.
＜細胞死アッセイ＞
生細胞に由来するプロテアーゼ活性と死細胞から放出される細胞外空間に由来するプロテアーゼ活性を測定することによって生細胞と死細胞を同時に測定することができる、市販のダブル蛍光アッセイ(Multitox-Fluor Multiplex cytotoxicity assay, Promega)を用いて、過剰発現した細胞の生存率と毒性を評価した。この方法は、ウェル間での異なるトランスフェクション効率を最小化することができる。NSC34細胞は、トランスフェクション1日前に、ウェル当たり1×104の濃度で96ウェル蛍光観察用黒色プラスチック培養容器(CN137101, Nunc)に播種した。WT又は各種変異体のFlagタグTDP-43のプラスミドをFuGene HD transfection kit (Roche)を使用して0.2μg/wellで導入し、次の日に分化培地に交換した。トランスフェクションの48時間後に、それぞれ生細胞と死細胞に対する蛍光指示薬である、細胞浸透性GF-AFCと細胞不透過性bis-AAF-R110を加えた。2時間のインキュベーション後に、生細胞については400/505 nm、死細胞については485/520 nmの励起/発光を各々使用し、マルチプレートリーダー(Infinite 200, TECAN, Mannedorf, Switzerland)でRFUを得た。Bis-AAF-R110蛍光は生存率を評価するためにGF-AFCの内部標準として使用した。細胞毒性比はベクターコントロールとの比として表した。
<Cell death assay>
A commercially available double-fluorescence assay (Multitox-Fluor Multiplex) that can measure live and dead cells simultaneously by measuring protease activity derived from living cells and extracellular space released from dead cells. The cytotoxicity assay (Promega) was used to assess the viability and toxicity of overexpressed cells. This method can minimize different transfection efficiencies between wells. NSC34 cells were seeded in a 96-well black plastic culture vessel for fluorescence observation (CN137101, Nunc) at a concentration of 1 × 10 4 per
＜凝集関連RRM1を標的化する抗体の生成＞
ウサギを、LC-MS/MSと高圧NMR分析で凝集関連配列として特定したキーホールリンペットヘモシアニン結合ペプチド(CVLGLPWKTT, CQVKKDLKTG, SQRHMIDGRWC; それぞれRRM1-a, RRM1-b, RRM1-cと称している)で免疫した。RRM1-a及びRRM1-cでの免疫化後に得られた抗血清は、組換えRRM1と全長TDP-43タンパク質に対して特異的且つ高い反応性を示した。抗体特異性は、ウェスタンブロッティングとELISAによって確認した。プロテインAを使用して精製したIgGは、免疫蛍光及び免疫組織化学(1:500)のために使用した。
<Generation of antibodies that target aggregation-related RRM1>
Keyhole limpet hemocyanin-binding peptide identified as an aggregation-related sequence by LC-MS / MS and high-pressure NMR analysis (CVLGLPWKTT, CQVKKDLKTG, SQRHMIDGRWC; called RRM1-a, RRM1-b, and RRM1-c, respectively) Immunized with. Antisera obtained after immunization with RRM1-a and RRM1-c showed specific and high reactivity against recombinant RRM1 and full-length TDP-43 protein. Antibody specificity was confirmed by Western blotting and ELISA. IgG purified using protein A was used for immunofluorescence and immunohistochemistry (1: 500).
＜ALS患者由来の検体の免疫組織化学＞
実験手順は、京都大学の倫理委員会のガイドラインによって承認され、当該ガイドラインの下で行われた。孤発性ALSの確定診断を有する3人の患者及び年齢が一致する3人の非ALS検体由来の腰髄を用いた。腰髄ブロックは切除後、直ぐに10%緩衝化ホルマリン中でインキュベートさせパラフィン包埋を行った。切片(6μm)は抗原賦活化のためにオートクレーブによって処理し(10 mMクエン酸ナトリウムバッファー中で121℃10分)、3%BSAを含むPBS中でRRM1-a又はRRM1-cに対する一次抗体で一晩4℃でインキュベートした。抗体反応は、色素原の3,3’-ジアミノベンジジン四塩酸塩と共にVectastain Elite ABC kit (Vector Laboratories, Burlingame, CA)を使用して可視化した。染色特異性は、一次抗体を3%BSAを含む適当な量のPBSと置き換えて、染色性を認めなかったことによって評価した。
<Immunohistochemistry of specimens from ALS patients>
The experimental procedure was approved by the guidelines of the Ethics Committee of Kyoto University and conducted under the guidelines. Lumbar spinal cords from 3 patients with a definitive diagnosis of sporadic ALS and 3 age-matched non-ALS specimens were used. After excision, the lumbar spinal block was immediately incubated in 10% buffered formalin and embedded in paraffin. Sections (6 μm) were processed by autoclaving for antigen activation (121 ° C. for 10 minutes in 10 mM sodium citrate buffer) and primed with primary antibody against RRM1-a or RRM1-c in PBS containing 3% BSA. Incubate overnight at 4 ° C. Antibody reactions were visualized using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, Calif.) With the
＜統計＞
多重比較は、Prism software (Graphpad, La Jolla, CA)を用いたNewman-Keuls多重比較テストでのone way ANOVAによって解析した。
<Statistics>
Multiple comparisons were analyzed by one way ANOVA in Newman-Keuls multiple comparison test using Prism software (Graphpad, La Jolla, CA).
試験例１
RRM1ドメインの部位特異的高次構造上の変動は、高圧NMR分光法を用いて調査した。圧力は、分子会合の潜在的リスクである部分的に乱れた高次構造を増加させるために使用した。通常多くのタンパク質で可逆的な構造変化を誘発する2000 barでの圧力処理前後の、30 barにおける該ドメインの15N/1H HSQCスペクトルの重ね合わせにより、化学シフトの不可逆的な変化が(特にaa 113-122, 133-147, 及び166-173における多くの残基で)明らかになった。
Test example 1
Variations in the site-specific conformation of the RRM1 domain were investigated using high pressure NMR spectroscopy. Pressure was used to increase partially disturbed conformation, which is a potential risk of molecular association. Overlapping the 15 N / 1 H HSQC spectrum of the domain at 30 bar before and after pressure treatment at 2000 bar, which usually induces reversible structural changes in many proteins, results in irreversible changes in chemical shift (especially aa 113-122, 133-147, and 166-173 with many residues).
NMRによって特定された3つのRRM1コア領域の役割を更に調査するために、非特異的プロテアーゼであるプロナーゼで分解された震盪誘導RRM1凝集体のマススペクトル分析を行った。多数の分解されなかったペプチドが特定されたが、これは物理的ストレスの間にこれらのコア領域が構造変化しプロテアーゼが接近できなくなったことを示している。面白いことに、プロテアーゼ抵抗性フラグメントの3つのクラスターは、NMRによって特定されたものにマッチしていた(core-a, core-b, core-c、図１)。特に4と5番目のβストランドに位置するcore-cは、最もプロテアーゼに抵抗性の非分解産物を豊富に含んでいた(図１)。 To further investigate the role of the three RRM1 core regions identified by NMR, mass spectral analysis of shake-induced RRM1 aggregates degraded with pronase, a non-specific protease, was performed. A number of undegraded peptides have been identified, indicating that during physical stress these core regions have undergone structural changes that render the protease inaccessible. Interestingly, the three clusters of protease resistant fragments matched those identified by NMR (core-a, core-b, core-c, FIG. 1). In particular, core-c located in the 4th and 5th β-strands was rich in non-degradation products most resistant to proteases (FIG. 1).
試験例２
RRM1凝集体は、5番目のβストランドのC173とC175により媒介されたジスルフィド結合によるオリゴマーの画分を含んでいた。C173とC175が酸化されてTDP-43のオリゴマーを形成することが以前報告されているので、C173とC175のシステインをセリンに置換すること(C/S変異体)でオリゴマー形成が抑制できるかどうかを調査した。しかしながら、1つのC/S変異体(C173SとC175S)は、静置した状態でも、むしろDTT感受性オリゴマー形成が促進された(図２A, B)。さらに、二重のC/S変異体(C173S/C175/S)はより不安定であり、震盪がない場合でさえ不規則なオリゴマーや凝集体を形成した。チオフラビンTアッセイにおいても、システイン置換がβシート形成を増加させ、二重のC/S変異体は1つのC/S変異体より高いチオフラビンT蛍光を示した(図３)。
Test example 2
RRM1 aggregates contained a fraction of oligomers by disulfide bonds mediated by C173 and C175 of the fifth β-strand. Since it has been previously reported that C173 and C175 are oxidized to form oligomers of TDP-43, is it possible to suppress oligomer formation by substituting cysteine of C173 and C175 with serine (C / S mutant)? investigated. However, one C / S mutant (C173S and C175S) promoted DTT-sensitive oligomer formation even when left standing (FIG. 2A, B). In addition, the double C / S mutant (C173S / C175 / S) was more unstable and formed irregular oligomers and aggregates even in the absence of shaking. Also in the thioflavin T assay, cysteine substitution increased β-sheet formation and the double C / S mutant showed higher thioflavin T fluorescence than one C / S mutant (FIG. 3).
試験例３
全長TDP-43の高次構造におけるC173とC175の役割を調査した。WT又はC/S変異体(C173S, C175S, C173S/C175S)RRM1を含むC末端EGFP融合TDP-43コンストラクトは、共焦点顕微鏡分析のためにHEK293A細胞に過剰発現させた。RRM1ドメイン分析から得られたデータと一致し、1つ又は二重のC/S変異は顕著な全長TDP-43の凝集体又は封入体の形成を生じた。それらは、主に核にあるが、一部で細胞質にも存在した(図４A)。RRM1はRNAとの結合能力を喪失すると、染色体のクロマチンに局在することで凝集体形成をすることが報告されている。RRM1 C/S変異を有するTDP-43の核封入体が単にDNA/RNAと結合できないためという可能性を除外するため、NLSを改変することによってC174S及び／又はC175Sを有する細胞質TDP-43変異体を生成した。その結果RRM1 C/S変異と変異体NLS (mNLS)を含むTDP-43は、クロマチンの存在しない細胞質においても丸い封入体を頻繁に形成した(図４B)。
Test example 3
The role of C173 and C175 in the higher-order structure of full-length TDP-43 was investigated. C-terminal EGFP fusion TDP-43 constructs containing WT or C / S mutants (C173S, C175S, C173S / C175S) RRM1 were overexpressed in HEK293A cells for confocal microscopy analysis. Consistent with the data obtained from RRM1 domain analysis, one or double C / S mutations resulted in the formation of significant full-length TDP-43 aggregates or inclusion bodies. They are mainly in the nucleus, but some were also present in the cytoplasm (FIG. 4A). It has been reported that when RRM1 loses its ability to bind RNA, it aggregates by localizing to chromosomal chromatin. Cytoplasmic TDP-43 variant with C174S and / or C175S by modifying NLS to exclude the possibility that TDP-43 nuclear inclusions with RRM1 C / S mutation simply cannot bind to DNA / RNA Was generated. As a result, TDP-43 containing RRM1 C / S mutation and mutant NLS (mNLS) frequently formed round inclusion bodies even in the cytoplasm where chromatin was not present (FIG. 4B).
試験例４
RRM1-C/S変異体で共有されるプロテイノパチーの病原性の特徴の程度を評価するために、ユビキチン化又はリン酸化に対する免疫細胞化学を行った。ヒト神経SHSY-5Y細胞において、RRM1-C/S変異TDP-43の核封入体のほとんどがリン酸化されない一方、細胞質封入体はphospho-TDP-43標的化抗体(図５A、d-i)又はK48結合ユビキチン抗体(図５A、m-o)に強い反応性を示した。さらに、改変NLSシグナルを有するRRM1-C/S変異によって引き起こされる細胞質TDP-43封入体は、S409とS410で頻繁にリン酸化(図６A、d-i)、及びユビキチン化(図６A、m-o)された。全ての細胞質封入体がユビキチン又はリン酸化によって修飾されていないことは注目すべき点であり、これは凝集が細胞質でのそのような修飾より先に起こることを示している。RRM1 C/S置換無しでは細胞質TDP-43は、抗phospho-TDP-43抗体(図６A, a-c)又は抗ユビキチン抗体(図６A、j-l)への明確な反応性を示さなかった。WT (図５B)又は変異NLS (図６B)を有するRRM1-C/S置換を含むFLAGタグTDP-43のウェスタンブロット分析は、RRM1-C/S TDP-43がプロテアソーム阻害剤の存在下で容易にオリゴマー化することを明らかにした。さらに、RRM1-C/S置換を含むTDP-43タンパク質は、特にプロテアソーム阻害剤ラクタシスチンの存在下で、WT TDP-43タンパク質より多くS409/S410においてリン酸化された(図５C)。さらに、RRM1-C/S変異体の異所性局在の形態は、ラクタシスチンが無い場合でさえ、S409とS410のリン酸化に対してより感受性があった(図６C)。
Test example 4
In order to assess the degree of pathogenic features of proteinopathy shared by RRM1-C / S mutants, immunocytochemistry against ubiquitination or phosphorylation was performed. In human neuronal SHSY-5Y cells, most of the nuclear inclusions of RRM1-C / S mutant TDP-43 are not phosphorylated, while the cytoplasmic inclusions are phospho-TDP-43 targeted antibodies (FIG. 5A, di) or K48 binding It showed strong reactivity with the ubiquitin antibody (FIG. 5A, mo). Furthermore, cytoplasmic TDP-43 inclusion bodies caused by RRM1-C / S mutations with modified NLS signals were frequently phosphorylated (FIG. 6A, di) and ubiquitinated (FIG. 6A, mo) at S409 and S410. . It is noteworthy that all cytoplasmic inclusion bodies are not modified by ubiquitin or phosphorylation, indicating that aggregation occurs prior to such modification in the cytoplasm. Without RRM1 C / S substitution, cytoplasmic TDP-43 did not show clear reactivity to anti-phospho-TDP-43 antibody (FIG. 6A, ac) or anti-ubiquitin antibody (FIG. 6A, jl). Western blot analysis of FLAG-tagged TDP-43 containing RRM1-C / S substitutions with WT (FIG. 5B) or mutant NLS (FIG. 6B) facilitates RRM1-C / S TDP-43 in the presence of proteasome inhibitors It was revealed that it oligomerizes. Furthermore, TDP-43 protein containing RRM1-C / S substitution was more phosphorylated at S409 / S410 than WT TDP-43 protein, especially in the presence of the proteasome inhibitor lactacystin (FIG. 5C). Furthermore, the ectopic localized form of the RRM1-C / S mutant was more sensitive to phosphorylation of S409 and S410, even in the absence of lactacystin (FIG. 6C).
試験例５
TDP-43プロテイノパチーの特徴に注目し、RRM1-C/S変異によって引き起こされるミスフォールドしたTDP-43の機能的な評価を行った。
Test Example 5
Focusing on the characteristics of TDP-43 proteinopathy, functional evaluation of misfolded TDP-43 caused by RRM1-C / S mutation was performed.
特にRRM1-C/Sを有するTDP-43の核内凝集体の形成が、核内TDP-43の細胞質への異所性局在を誘導することが、抗TDP-43抗体を使用した免疫細胞化学によって明らかとなった(図７)。さらに、RRM1-C/Sを有するTDP-43の核と細胞質の両方の封入体は、共発現したWT TDP-43-FLAGタンパク質と共局在した(図８、f及びi)。これはミスフォールドしたTDP-43が、核と細胞質の両方で野生型の核TDP-43を凝集体に吸着し得ることを示している。 In particular, the formation of intranuclear aggregates of TDP-43 with RRM1-C / S induces ectopic localization in the cytoplasm of nuclear TDP-43. Immune cells using anti-TDP-43 antibodies Revealed by chemistry (Figure 7). Furthermore, both nuclear and cytoplasmic inclusions of TDP-43 with RRM1-C / S colocalized with the co-expressed WT TDP-43-FLAG protein (FIGS. 8, f and i). This indicates that misfolded TDP-43 can adsorb wild-type nuclear TDP-43 to aggregates in both nucleus and cytoplasm.
試験例６
家族性ALS患者でこれまで同定されたTDP-43の突然変異体タンパク質は、優性形質を有するにも関わらず、WTと比べて変異TDP-43の著明な凝集傾向の違いを示さない(図９A a-c, B)。ところが、A315TとQ331Kの家族性ALS (FALS)関連変異TDP-43におけるRRM1-C/Sの導入は、明確にWTと比べてFALS変異体の凝集傾向を強めた(図９A d-f, B)。
Test Example 6
The mutant protein of TDP-43 identified so far in patients with familial ALS does not show a marked difference in aggregation tendency of mutant TDP-43 compared to WT, despite having a dominant trait (Fig. 9A ac, B). However, introduction of RRM1-C / S in the familial ALS (FALS) -related mutation TDP-43 of A315T and Q331K clearly strengthened the aggregation tendency of the FALS mutant as compared to WT (FIG. 9A df, B).
TDP-43プロテイノパチーにおいて神経毒性に直結するTDP-43の病原構造は不明である。特に、封入体の毒性の有無については一定の見解を見ない。よって、生存細胞と死細胞に関するマーカーの同時測定を行い、運動ニューロン細胞株NSC34細胞を使用してTDP-43の核又は細胞質凝集体の毒性を調査した。その結果、全ての改変されたNLSを有するRRM1-C/S変異体がベクターコントロールと比べて毒性を有意に増加させた一方、核に異所性局在するだけでは有意な毒性を示さなかった(図１０A、B)。さらに、核凝集形態(C173S, C175S)とFALS関連変異TDP-43は明らかな毒性を引き起こさなかった(図１０B)。 The pathogenic structure of TDP-43 that is directly linked to neurotoxicity in the TDP-43 proteinopathy is unknown. In particular, there is no certain opinion about the presence or absence of inclusion body toxicity. Therefore, simultaneous measurement of markers for live and dead cells was performed, and the toxicity of TDP-43 nuclei or cytoplasmic aggregates was investigated using the motor neuron cell line NSC34 cells. As a result, RRM1-C / S mutants with all modified NLS significantly increased toxicity compared to vector control, while ectopic localization in the nucleus did not show significant toxicity. (Figure 10A, B). Furthermore, the nuclear aggregate form (C173S, C175S) and the FALS-related mutation TDP-43 did not cause obvious toxicity (FIG. 10B).
試験例７
RRM1-C/S変異体によって引き起こされるTDP-43封入体がALSの病態に関連するか否かを調査するために、3つのRRM1コア領域(図１１)に対するウサギポリクローナル抗体(pAbs)を生成した。RRM1と全長TDP-43の両方に対して高い抗体価を示した2つの抗血清が、core-aとcore-cについて得られた(pAb-RRM1-aとpAb-RRM1-c)。培養細胞では、pHb-RRM1-aとpAb-RRM1-cは、核で高濃度に発現したTDP-43を除いて、内在性又は外部のWT TDP-43には殆ど反応しなかった(図１１)。一方、RRM1-C/S変異を有するTDP-43の凝集体は、両方のpAbによって明確に認識された(図１１)。これらの結果は、凝集に関連するコアはTDP-43 RRM1-C/S変異体による凝集体で外部露出されていることを示している。
Test Example 7
To investigate whether TDP-43 inclusion bodies caused by RRM1-C / S mutants are associated with the pathology of ALS, rabbit polyclonal antibodies (pAbs) against three RRM1 core regions (FIG. 11) were generated. . Two antisera with high antibody titers against both RRM1 and full-length TDP-43 were obtained for core-a and core-c (pAb-RRM1-a and pAb-RRM1-c). In cultured cells, pHb-RRM1-a and pAb-RRM1-c hardly reacted with endogenous or external WT TDP-43 except for TDP-43 expressed at high concentrations in the nucleus (FIG. 11). ). On the other hand, aggregates of TDP-43 carrying the RRM1-C / S mutation were clearly recognized by both pAbs (FIG. 11). These results indicate that the core associated with aggregation is externally exposed with aggregates from the TDP-43 RRM1-C / S mutant.
上記pAbsがALS患者におけるTDP-43封入体を認識するかを試験した。免疫組織化学的分析は、pAb-RRM1-aは孤発性ALS患者の脊髄運動ニューロン中のレビー小体様ヒアリン封入体を認識した(図１２)。核のTDP-43は、細胞質TDP-43凝集体の存在にも関わらず、運動ニューロンではpAb-RRM1-aによって染色されなかった。これは培養細胞の結果と一致している。これらの結果は、TDP-43のRRM1-C/S置換誘導封入体はALSのものと共通の高次構造変化を共有することを示唆している。 We tested whether the pAbs recognize TDP-43 inclusions in ALS patients. Immunohistochemical analysis revealed that pAb-RRM1-a recognized Lewy body-like hyaline inclusions in spinal motor neurons of sporadic ALS patients (FIG. 12). Nuclear TDP-43 was not stained by pAb-RRM1-a in motor neurons despite the presence of cytoplasmic TDP-43 aggregates. This is consistent with the results for cultured cells. These results suggest that RRM1-C / S substitution-induced inclusion bodies of TDP-43 share common conformational changes with those of ALS.
Claims (3)
（１）TDP-43のアミノ酸配列において、配列番号１で示されるアミノ酸配列の173位及び／又は175位に相当するシステインが置換されたアミノ酸配列からなるTDP-43変異体又は該TDP-43変異体内のRRM1ドメインに候補となる化合物を接触させる工程、
（２）該化合物の凝集体形成阻害活性を検出する工程、及び
（３）凝集体形成を阻害する化合物を選択する工程。 A method for screening a compound for treating and / or preventing a disease in which an aggregate of TDP-43 accumulates, comprising the following steps:
(1) A TDP-43 variant comprising the amino acid sequence in which the cysteine corresponding to positions 173 and / or 175 of the amino acid sequence shown in SEQ ID NO: 1 is substituted in the amino acid sequence of TDP-43, or the TDP-43 mutation Contacting the candidate compound with the RRM1 domain in the body,
(2) a step of detecting the aggregate formation inhibitory activity of the compound, and (3) a step of selecting a compound that inhibits aggregate formation.
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