WO2010137547A1 - 中枢神経細胞の増殖及び分化に係る中核因子を含む医薬組成物 - Google Patents
中枢神経細胞の増殖及び分化に係る中核因子を含む医薬組成物 Download PDFInfo
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- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5058—Neurological cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
- C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
Definitions
- the present invention relates to a pharmaceutical composition used for the treatment of cerebral cortical diseases and the like, and a pharmaceutical composition thereof administered to a non-human animal, the proliferation of neural stem cells or neural progenitor cells of the non-human animal
- the present invention relates to a method for inducing differentiation and a method for screening therapeutic agents for cerebral cortical diseases and the like.
- Recent advances in neuronal research have created the possibility of regenerating brains lost due to Alzheimer's disease or trauma. In such brain regeneration, it is important to elucidate factors that induce differentiation into nerve cells.
- Non-patent Document 1 a protein called Lhx2 induces neural stem cells or neural progenitor cells into the cortex and suppresses induction into the hippocampus.
- the nerve cell can be regenerated and used for treatment of cerebral cortical diseases such as Alzheimer's disease.
- the present invention has been made under the technical background as described above, and provides a new factor that induces differentiation into nerve cells.
- CRBN Cereblon
- Lhx2 a known cerebral cortical selector factor
- CRBN is a protein that forms a ubiquitin ligase complex, and its amino acid sequence is also known, but it has never been known that it has a function of inducing the proliferation and differentiation of central neural stem cells or neural progenitor cells. It was.
- the present invention has been completed based on the above findings. That is, the present invention provides the following [1] to [11].
- a pharmaceutical composition comprising 1) CRBN, 2) a nucleic acid encoding CRBN, or 3) a stem cell or neural progenitor cell in which CRBN is expressed.
- the pharmaceutical composition according to [1] wherein a nucleic acid encoding CRBN is inserted into a viral vector.
- the pharmaceutical composition according to [1] further comprising a protein that forms a ubiquitin ligase complex with CRBN in addition to CRBN.
- the pharmaceutical composition according to any one of [1] to [5] which is used for treatment of cerebral cortical diseases or surgical damage of cerebral cortex.
- the pharmaceutical composition according to any one of [1] to [5] which is used for regeneration of cerebral cortex.
- [8] A method of administering the pharmaceutical composition according to any one of [1] to [7] to a non-human animal, and proliferating neural stem cells or neural progenitor cells of the non-human animal.
- [9] A method of administering the pharmaceutical composition according to any one of [1] to [7] to a non-human animal and differentiating the neural stem cells or neural progenitor cells of the non-human animal into nerve cells.
- the method includes a step of bringing a test substance into contact with a ubiquitin ligase complex containing CRBN, and a step of measuring a ubiquitin ligase activity of the ubiquitin ligase complex and selecting a test substance having improved ubiquitin ligase activity.
- a screening method for a therapeutic agent for a cerebral cortical disease or a surgical damage of the cerebral cortex [11] The step of culturing neural stem cells or neural progenitor cells in the presence of the test substance, and the expression level of CRBN in the neural stem cell or neural progenitor cell are measured, and the test substance having an increased CRBN expression level is selected.
- a screening method for a therapeutic agent for a cerebral cortical disease or a surgical damage to the cerebral cortex which comprises a step.
- the CRBN contained in the pharmaceutical composition of the present invention induces proliferation and differentiation into central nerve stem cells or neural progenitor cells. Therefore, the pharmaceutical composition of the present invention is useful as a therapeutic agent for cerebral cortical diseases such as Alzheimer's disease. CRBN is also useful as a target substance for the development of new therapeutic agents for cerebral cortical diseases.
- Fluorescence micrograph of zebrafish embryo The upper left is a normal embryo, the upper right is an embryo in which the crbn gene is overexpressed, the lower left is an embryo in which the lhx2 gene is knocked down, and the lower right is an embryo in which the lhx2 gene is knocked down and the crbn gene is overexpressed.
- Micrograph of zebrafish embryo The right shows an embryo overexpressing the crbn gene, and the left shows an untreated embryo. The shooting magnification is the same for both. Photomicrograph of zebrafish transplanted with cells overexpressing the crbn gene (fluorescently labeled with rhodamine). The top row was taken at 200x and the bottom row was taken at 100x.
- the left column is a bright field image
- the right column is a fluorescent image.
- the left figure is a photograph of a normal individual, and the right figure is a photograph of a CRBN overexpressing individual.
- the left figure is a photograph of a normal individual, and the right figure is a photograph of a CRBN overexpressing individual.
- 1 indicates the pineal gland
- 2 indicates the ventral posterior node
- 3 indicates the raphe nucleus.
- a to D show cases where cells not expressing CRBN were transplanted
- E to H show cases where cells expressing CRBN were transplanted.
- a and E are bright-field images
- B and E are AlexaFluor (registered trademark) fluorescence images (showing tubulin distribution)
- C and G are rhodamine fluorescence images (donor cell distribution).
- D and H are fluorescence images of AlexaFluor (registered trademark) and rhodamine.
- the pharmaceutical composition of the present invention is characterized by comprising 1) CRBN, 2) a nucleic acid encoding CRBN, or 3) a stem cell or neural progenitor cell expressing CRBN.
- CRBN is a known protein, and the base sequence of the gene (crbn gene) that encodes it is also published on the database.
- the base sequence of the human-derived crbn gene, the base sequence of the mouse-derived crbn gene, the base sequence of the rat-derived crbn gene, and the base sequence of the zebrafish-derived crbn gene are GeneID: 51185, GeneID: 58799, It is registered with Entrez Gene with GeneID: 297498 and GeneID: 445491.
- the CRBN or crbn gene used may be natural, but consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of the natural CRBN, and is an active ubiquitin ligase complex
- a modified CRBN capable of forming a body or a gene encoding the same may be used.
- the pharmaceutical composition of the present invention may contain CRBN, a CRBN-encoding nucleic acid, a stem cell expressing CRBN, or a neural progenitor cell as an active ingredient.
- Preparation and use of pharmaceutical compositions containing these as active ingredients can be carried out in the same manner as known pharmaceuticals containing proteins, nucleic acids, stem cells, and progenitor cells as active ingredients.
- the nucleic acid encoding CRBN may be either DNA or RNA.
- the nucleic acid is preferably inserted into an appropriate vector so that it can act on neural stem cells in the brain.
- Such vectors can include viral vectors.
- virus vectors include adenovirus vectors, retrovirus vectors, and lentivirus vectors.
- the stem cells that express CRBN are preferably patient-derived iPS cells because rejection can be avoided, but other stem cells such as ES cells, adult stem cells, cord blood stem cells, and the like may be used.
- CRBN may be expressed in stem cells, but is preferably overexpressed.
- the method of expressing or overexpressing CRBN can be performed according to, for example, the method of Ando et al. (Andokaand Okamoto, Mar Biotechnol 8 (3): 295-303.
- the administration method of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately determined according to the type of active ingredient.
- CRBN or a nucleic acid encoding CRBN is used as an active ingredient, it can be administered by injection or infusion into the ventricle, intradermal, intraperitoneal, vein, artery, or spinal fluid.
- CRBN acts on neural stem cells or neural progenitor cells in the brain, it is preferable that treatment is performed so that it can pass through the cerebrovascular barrier, except when administered into the ventricle. Examples of such treatments include methods of binding to essential endogenous substances that are actively incorporated, structural modifications that avoid recognition of efflux transporters, and molecular weight reduction that includes only a minimal functional region.
- stem cells or neural progenitor cells are used as active ingredients, they are administered directly into the ventricle.
- the dose of the pharmaceutical composition of the present invention is not particularly limited, and can be appropriately determined according to the type of active ingredient.
- the daily dose for adults is preferably 0.5 mg to 100 mg when CRBN is administered, and preferably 1 mg to 200 mg when CRBN-encoding nucleic acid is administered, so that CRBN is expressed.
- stem cells or neural progenitor cells it is preferable to administer 500 to 5000 cells in an amount of about 50 ⁇ l to 500 ⁇ l.
- the pharmaceutical composition of the present invention When the pharmaceutical composition of the present invention is administered by injection or infusion, it may contain components usually contained in injection solutions or infusion solutions.
- Such components include liquid carriers (for example, potassium phosphate buffer, physiological saline, Ringer's solution, distilled water, polyethylene glycol, vegetable oils, ethanol, glycerin, dimethyl sulfoxide, propylene glycol, etc.), antibacterial agents And local anesthetics (eg, procaine hydrochloride, dibucaine hydrochloride, etc.), buffer solutions (eg, Tris-HCl buffer solution, Hepes buffer solution, etc.), osmotic pressure regulators (eg, glucose, sorbitol, sodium chloride, etc.) .
- liquid carriers for example, potassium phosphate buffer, physiological saline, Ringer's solution, distilled water, polyethylene glycol, vegetable oils, ethanol, glycerin, dimethyl sulfoxide, propylene glycol, etc.
- the pharmaceutical composition of the present invention may contain 1) a protein that forms a ubiquitin ligase complex with CRBN in addition to CRBN, and 2) a ubiquitin ligase complex together with CRBN in addition to a nucleic acid encoding CRBN.
- a nucleic acid encoding a protein to be formed may be included, and 3) a stem cell or a neural progenitor cell in which a protein that forms a ubiquitin ligase complex with CRBN in addition to CRBN may be included.
- proteins that form a ubiquitin ligase complex with CRBN include DDB1 (Damaged DNA Binding protein), Cul4A (Cullin 4A), Cul4B (Cullin 4B), Roc1 (RBX1) and the like. These proteins are known proteins like CRBN, and the base sequences of the genes encoding them (ddb1 gene, cul4a gene, cul4b gene, roc1 gene) are also published on the database.
- the base sequence of human-derived ddb1 gene, the base sequence of ddb1 gene from mouse, the base sequence of ddb1 gene from rat, and the base sequence of ddb1 gene from zebrafish are GeneID: 1642, GeneID: 13194, GeneID: 64470, and GeneID: 393599, registered in Entrez Gene, human cul4a gene base sequence, mouse cul4a gene base sequence, rat cul4a gene base sequence, and zebrafish cul4a
- the base sequences of the genes are registered in Entrez Gene with GeneID: 8451, GeneID: 99375, GeneID: 361181, and GeneID: 394002, the base sequence of the cul4b gene derived from human, the base sequence of the cul4b gene derived from mouse,
- the nucleotide sequence of cul4b gene derived from rat and the nucleotide sequence of cul4b gene derived from zebrafish are GeneID: 8450, GeneID: 72584
- the ubiquitin ligase complex includes three types of proteins DDB1, CuL4A, Roc1, or DDB1, CuL4B, and Roc1.
- the pharmaceutical composition of the present invention preferably contains (or expresses) all of these three types of proteins (or nucleic acids encoding these proteins), but may contain only a part of these. .
- the pharmaceutical composition of the present invention can be used for treatment of cerebral cortex disease or surgical damage of the cerebral cortex or for regeneration of the cerebral cortex.
- cerebral cortical diseases include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Huntington's disease, progressive supranuclear palsy, cerebral cortex basal ganglia degeneration, and the like.
- the pharmaceutical composition of the present invention is used for humans, but may be administered to animals other than humans to induce proliferation of neural stem cells or neural progenitor cells or differentiation into neural cells of the animals.
- the target animals are mainly vertebrates, such as mice, rats, monkeys, dogs, ferrets, hamsters, chickens, Xenopus, zebrafish, and medaka.
- CRBN CRBN induces proliferation of neural stem cells or neural progenitor cells and differentiation into neural cells, as described in Examples.
- genome synteny is 70-80% conserved between humans and zebrafish (NatureNReviews Genetics 8, 353-367 (May 2007)). Therefore, CRBN is expected to show the same action as that confirmed in zebrafish in other vertebrates including humans.
- (A) a step of contacting a test substance with a ubiquitin ligase complex containing CRBN, and a step of measuring a ubiquitin ligase activity of the ubiquitin ligase complex and selecting a test substance having improved ubiquitin ligase activity. And a screening method for a therapeutic agent for a cerebral cortical disease or a surgical damage of the cerebral cortex.
- (B) The step of culturing neural stem cells or neural progenitor cells in the presence of the test substance, and the expression level of CRBN in the neural stem cell or neural progenitor cell are measured, and the test substance with an increased CRBN expression level is selected.
- the ubiquitin ligase activity can be measured, for example, according to the methods of Angers et al. (Nature 443, 590-593, 2006) and Groisman et al (Cell 113, 357-367, 2003).
- whether the test substance has improved the ubiquitin ligase activity is determined by measuring the ubiquitin ligase activity of the complex containing CRBN without contacting with the test substance and comparing it with the value. Can do.
- the substance selected by the method (A) has a function of improving the ubiquitin ligase activity of the complex containing CRBN present in neural stem cells or neural progenitor cells in vivo. Proliferation of neural stem cells or neural progenitor cells and differentiation into neural cells by CRBN are considered to be performed through the ubiquitin ligase activity of this complex. Therefore, the substance selected by the method (A) promotes proliferation and differentiation of neural stem cells or neural progenitor cells by CRBN and has therapeutic effects on cerebral cortical diseases and surgical damage of the cerebral cortex. Conceivable.
- neural stem cells to be used ES cells, iPS cells, adult stem cells obtained by inducing neural differentiation and the like are basically used.
- neural stem cells derived from adult mouse subventricular zone or adult rat hippocampus may be used.
- the expression level of CRBN can be measured by a method using an antibody against CRBN, an in situ hybridization method, an RT-PCR method, a northern blot method, or the like.
- whether or not the test substance increased the expression level of CRBN was determined by culturing neural stem cells or neural progenitor cells in the absence of the test substance, measuring the expression level of CRBN, and comparing it with the value. This can be determined.
- the substance selected by the method (B) has a function of increasing the expression level of CRBN in neural stem cells or neural progenitor cells in vivo. Accordingly, the substance selected by the method (B) promotes proliferation and differentiation of neural stem cells or neural progenitor cells by CRBN and has a therapeutic effect on cerebral cortical diseases and surgical damage of the cerebral cortex. Conceivable.
- AMO antisense morpholino oligonucleotide
- the basic principle is to cultivate embryos knocked down by one of the two types of genes (A), which are suggested to be functionally related (A), up to 6 hours after fertilization (the gastrulation stage). Expression of the other gene (B) is induced in the planned forebrain region by in vivo lipofection of the method (2) above. If the knockdown effect of A is remedied 24 hours after fertilization and the knockdown effect of B is not remedied by the reverse combination of A and B, it is determined that A is positioned at the functional upper level of B.
- Immunohistochemistry Antibody staining of zebrafish early neurons was performed by the following method. Embryos 24 to 28 hours after fertilization were fixed with 4% paraformaldehyde / phosphate buffer (pH 8.0) at 4 ° C. for 12 hours. After 4 washes for 15 minutes with phosphate buffer, 0.5% Blocking was performed at room temperature for 1 hour in 5% newborn goat serum dissolved in Triton X-100 / phosphate buffer.
- rhodamine dextran molecular weight 10,000
- a final concentration of 600 ng / ⁇ l of crbn RNA dissolved in zebrafish embryos at the 1-cell stage are injected under the conditions of the above method (1).
- a donor was used.
- 10-50 cells were aspirated with a glass microcapillary under fluorescent microscope observation and transplanted to the animal pole of the host (host) embryo at the same time.
- the host embryos were cultured as they were for 2 days, and then the localization of differentiation of donor-derived cells expressing CRBN was observed with a fluorescence microscope.
- As a negative control cells expressing green fluorescent protein (GFP), which has no effect on development, were also transplanted under the same conditions, and their differentiation localization was compared.
- GFP green fluorescent protein
- CRBN complex a binding protein
- SIGMA M2 FLAG agarose beads
- the purified CRBN complex is mixed with an aqueous solution containing Uba1 (E1), UbcH5b (E2), and GST-fused ubiquitin (Ub) recombinant protein, and after adding ATP, it is allowed to stand at 30-37 degrees for 2 hours. did.
- the reaction was stopped by SDS, and polyacrylamide gel electrophoresis and immunoblotting were performed to detect and measure ubiquitin ligase activity by visualizing self-ubiquitination and ubiquitination of the binding protein.
- This sample was reacted with an anti-glia monoclonal antibody (zrf-1 / zrf-2) or a rabbit anti-serotonin antibody at 4 ° C. overnight (12 to 18 hours).
- an anti-glia monoclonal antibody zrf-1 / zrf-2
- a rabbit anti-serotonin antibody at 4 ° C. overnight (12 to 18 hours).
- PBST + 5% goat serum for 1 hour at room temperature goat anti-mouse IgG antibody (Cy-2 conjugate. Glial cell staining) or goat anti-rabbit IgG antibody (Cy-5 conjugate. Serotonin producing cell staining)
- the secondary antibody reaction was performed. After reaction at 4 ° C. overnight (12 to 18 hours), the plate was washed with PBST for 1 hour at room temperature.
- CRBN in living cells self-ubiquitinates in cooperation with other factors to generate ubiquitinated proteins with various molecular weights.
- many of them are thought to be degraded by proteasomes in living cells.
- the proteasome was inhibited by MG132, and it was considered that a large number of bands were detected because the protein produced by self-ubiquitination remained.
- the right lane of FIG. 5 it is considered that almost no band was detected because most of the produced protein was degraded by the proteasome.
- glial cells Fig. 6 left diagram
- serotonin producing cells Fig. 7 left diagram
- the present invention is useful as a therapeutic agent for cerebral cortical diseases such as Alzheimer's disease. It is also useful for developing new therapeutic agents for cerebral cortical diseases.
Abstract
Description
即ち、本発明は、以下の〔1〕~〔11〕を提供するものである。
〔1〕1)CRBN、2)CRBNをコードする核酸、又は3)CRBNを発現させた幹細胞若しくは神経前駆細胞を含有することを特徴とする医薬組成物。
〔2〕CRBNをコードする核酸が、ウイルスベクター中に挿入されていることを特徴とする〔1〕に記載の医薬組成物。
〔3〕CRBNの他に、CRBNと共にユビキチンリガーゼ複合体を形成するタンパク質も含有することを特徴とする〔1〕に記載の医薬組成物。
〔4〕CRBNをコードする核酸の他に、CRBNと共にユビキチンリガーゼ複合体を形成するタンパク質をコードする核酸も含有することを特徴とする〔1〕又は〔2〕に記載の医薬組成物。
〔5〕CRBNの他に、CRBNと共にユビキチンリガーゼ複合体を形成するタンパク質も発現させた幹細胞若しくは神経前駆細胞を含有することを特徴とする〔1〕に記載の医薬組成物。
〔6〕大脳皮質疾患又は大脳皮質の外科的損傷の治療のために用いられることを特徴とする〔1〕乃至〔5〕のいずれかに記載の医薬組成物。
〔7〕大脳皮質の再生のために用いられることを特徴とする〔1〕乃至〔5〕のいずれかに記載の医薬組成物。
〔8〕〔1〕乃至〔7〕のいずれかに記載の医薬組成物を非ヒト動物に投与し、その非ヒト動物の神経幹細胞又は神経前駆細胞を増殖させる方法。
〔9〕〔1〕乃至〔7〕のいずれかに記載の医薬組成物を非ヒト動物に投与し、その非ヒト動物の神経幹細胞又は神経前駆細胞を神経細胞へ分化させる方法。
〔10〕被験物質をCRBNを含むユビキチンリガーゼ複合体に接触させる工程、及び前記ユビキチンリガーゼ複合体のユビキチンリガーゼ活性を測定し、ユビキチンリガーゼ活性を向上させた被験物質を選択する工程を含むことを特徴とする大脳皮質疾患又は大脳皮質の外科的損傷の治療薬のスクリーニング方法。
〔11〕被験物質の存在下で神経幹細胞又は神経前駆細胞を培養する工程、及び神経幹細胞又は神経前駆細胞中のCRBNの発現量を測定し、CRBNの発現量を増大させた被験物質を選択する工程を含むことを特徴とする大脳皮質疾患又は大脳皮質の外科的損傷の治療薬のスクリーニング方法。
(1)ゼブラフィッシュ胚の全身での遺伝子過剰発現
成魚は常時28.5℃で飼育し、照明オン14時間/オフ10時間の日周サイクルで継代、維持した。オスとメスの自然交配で受精卵を確保し、最初の卵割が始まる前(1細胞期)の胚に試験管内合成したCapped RNA水溶液を600 ng/μl の濃度で窒素ガス圧30 psi、30 msec(ミリ秒)間バルブ解放の条件で細胞質に注入した。lhx2遺伝子以外のすべての遺伝子(crbn遺伝子, six3.2遺伝子, crbnとE3ユビキチンリガーゼ複合体を構成する蛋白質をコードする遺伝子)の発現は全身での発現でも発生への影響が少ないのでこの方法で最初のデータを得た。
全身での発現で非特異的な胚の背側化を示すlhx2遺伝子および脳容積への影響を特異的に調べる精密な実験を行なう場合は、胚への予定頭部領域への生体内RNAリポフェクションを行なった。方法は、安藤らが開発、発表した技術(Ando and Okamoto, Efficient transfection strategy for the spatiotemporal control of gene expression in zebrafish. Mar Biotechnol 8(3):295-303. 2006)にすべて従った。
ノックダウンしたい遺伝子のcDNA配列の翻訳開始コドン周辺に対応した25-merのアンチセンスモルフォリノオリゴヌクレオチド (AMO,Gene Tools社)水溶液(700 ng/μl, 注入条件はRNAと同じ)を1細胞期の胚に注入した。crbn遺伝子のノックダウンに用いるAMOの配列は、5’AGAGCTGTAGCTGGTTCCCCATTTC 3’、lhx2遺伝子のノックダウンの場合は、5’TCTGCAACCCAAGATTTCCGTGAGA3’である。
安藤ら(Ando et al., Lhx2 mediates the activity of Six3 in zebrafish forebrain growth. Dev Biol. 287(2):456-68. 2005)の手法に以下の過程以外はすべて従った。頭部特異的な遺伝子発現はRNAアンケージング(特開2002-315576、Ando et al., Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos. Nat. Genet. 28, 317-325, 2001)のかわりに上記(2)の方法の生体内リポフェクション法を用いた。基本的な原理は、機能的関連が示唆される2種の遺伝子の一方(A)を上記(3)の方法でノックダウンした胚を受精後6時間(原腸胚期)まで培養し、その予定前脳領域に上記(2)の方法の生体内リポフェクションで他方の遺伝子(B)の発現を誘導する。受精後24時間期でAのノックダウン効果が救済され、AとBの逆の組み合わせではBのノックダウン効果が救済されない場合はAはBの機能的上位に位置すると決定する。
ゼブラフィッシュの初期ニューロンの抗体染色は以下の手法で行なった。受精後24-28時間の胚を4%パラホルムアルデヒド/リン酸緩衝液(pH 8.0)で4℃、12時間固定した。リン酸緩衝液で15分間洗浄を4回行なった後、0.5%
Triton X-100/リン酸緩衝液に溶解した5%新生ヤギ血清中で常温1時間ブロッキングを行なった。同じ溶液中に1000倍希釈した抗アセチル化チューブリンモノクローナル抗体を含む溶液で4℃、12時間、一次抗体反応を行い、同様にリン酸緩衝液で洗浄後、一次抗体反応と同じ条件でAlexa Fluoro 488 (Molecular Probes社)をコンジュゲートした抗マウス抗体で二次抗体反応を行なった。リン酸緩衝液で洗浄した胚を30%/50%/70%グリセロール(リン酸緩衝液に溶解)中で透明化し、488 nmで励起し蛍光顕微鏡で観察した。
基本的にThisseらの手法(Nat. Protcol. 3 (1) 59-69, 2008)に従った。異なる点は、プローブのハイブリダイゼーション溶液のブロッカーに5 mg/mlのTorulaイーストRNAを使用した点と、抗体反応液中のブロッカーとして0.5%のBlocking reagent(Roche社)を用いた点である。プローブに用いた遺伝子のcDNA(six3.2遺伝子, emx1遺伝子, pax2.1遺伝子, foxg1遺伝子, otx2遺伝子)は、それぞれオリジナルな供与者から譲渡された。lhx2遺伝子のプローブは最初にクローン化した安藤のものを用いた。crbn遺伝子および関連遺伝子のプローブはゼブラフィッシュのESTデータベースからプライマーをデザインし、cDNAライブラリーからクローン化した。
1細胞期のゼブラフィッシュ胚に適宜濃度のローダミンデキストラン(分子量10,000)と最終濃度600 ng/μlのcrbn RNAを溶解したヌクレアーゼフリー水を上記(1)の方法の条件で注入しドナー(供与体)とした。ドナーが受精後3-4時間の時期に蛍光顕微鏡観察下で細胞をガラス製マイクロキャピラリーで10-50個吸引し、同時期のホスト(宿主)胚の動物極に移植した。そのままホスト胚を2日間培養した後、蛍光顕微鏡でCRBNを発現するドナー由来細胞の分化の局在性を観察した。またネガティブコントロールとして発生に影響がないとされる緑色蛍光蛋白質(GFP)を発現させた細胞も同じ条件で移植し、その分化の局在性を比較した。
基本的にGroisman らの方法に従った。まずFLAGエピトープタグを融合させたCRBNを発現する哺乳類細胞の破砕液よりM2 FLAGアガロースビーズ(SIGMA社)を用いてCRBNおよび結合タンパク質(CRBN複合体と呼ぶ)を精製した。次に精製したCRBN複合体をUba1 (E1), UbcH5b (E2),GST融合ユビキチン(Ub)組換えタンパク質の含まれた水溶液と混合し、ATPを加えた後30-37度で2時間静置した。その後SDSにより反応を停止させ、ポリアクリルアミドゲル電気泳動および免疫ブロッティングを行うことにより、自己ユビキチン化および結合タンパク質のユビキチン化を可視化させることでユビキチンリガーゼ活性を検出・計測した。
基本的にはOhtakeらの方法に従った。FLAGエピトープタグを融合させたCRBNを発現させた哺乳類細胞にプロテアソーム阻害剤であるMG132を投与し、静置した。その後、細胞を破砕し、その溶解液から上記と同様だがより厳格な条件でのFLAG精製を行い、CRBNを抽出し、免疫ブロッティングを行うことで、その自己ユビキチン化を検出・計測した。
ゼブラフィッシュは2日齢(グリア細胞染色は、受精後56時間のものを用い、セロトニン産生細胞染色は、受精後49時間のものを用いた。)のものを用いた。ゼブラフィッシュを4%パラフォルムアルデヒド(PFA)で固定後、リン酸緩衝液(PBS)で洗浄し、次いで、10μg/mlのプロテアーゼKで処理し、表皮を部分消化した。消化反応後PBST(PBS+0.5%Triton X-100)で20分間洗浄し、PFAで再固定した。これをPBSTで室温1時間洗浄し、その後PBST+5% ヤギ血清でブロッキングした。この標本を抗グリアモノクローナル抗体(zrf-1/zrf-2)またはウサギ抗セロトニン抗体で4℃、終夜(12~18時間)反応させた。その後、PBST+5% ヤギ血清で1時間、室温で洗浄し、ヤギ抗マウスIgG抗体(Cy-2コンジュゲート。グリア細胞染色)またはヤギ抗ウサギIgG抗体(Cy-5コンジュゲート。セロトニン産生細胞染色)に置換し二次抗体反応を行った。4℃、終夜(12~18時間)反応後、PBSTで室温1時間洗浄した。洗浄後30%, 50%, 70%グリセロール/PBSで置換し、全身をスライドグラス上にマウントし、プレパラートを作製した。Cy-2およびCy-5の励起波長で蛍光を観察し、グリア細胞またはセロトニン産生細胞の分布を記録した。なお、グリア細胞の観察はゼブラフィッシュの側面から、セロトニン産生細胞はそれらの正中線に沿った分布を観察するため背側から撮影した。
受精直後の1細胞期胚にCRBNをコードするRNA (700 ng/μl)と2%ローダミンデキストランの混合液を注入した。受精後4時間まで培養後、吸引キャピラリーを用いて細胞を10~20個ほど採取し、受精後30時間胚の間脳室へ注入し移植した。移植された魚をゼブラフィッシュ生理食塩水(E3リンガー)中で受精後3日まで飼育した後、4%パラフォルムアルデヒドで固定した。常法に従い抗アセチル化チューブリンモノクローナル抗体による一次抗体反応とAlexa Fluor(登録商標)抗マウスIgG (488 nm励起)抗体による二次抗体反応を行い、神経細胞軸索を蛍光標識して観察しながらローダミンデキストラン(543 nm励起)で標識された移植細胞の分布を調べた。
(1)Lhx2とCRBNの機能階層性の決定
lhx2遺伝子をノックダウンしたゼブラフィッシュの胚(図1左下)では、正常胚(図1左上)と比較し、脳の縮小がみられた。一方、lhx2遺伝子をノックダウンし、crbn遺伝子を過剰発現させた胚(図1右下)では、crbn遺伝子を過剰発現させた胚(図1右上)と同様に、脳の拡大がみられた。このことから、CRBNは、Lhx2の機能的下流に位置し、中枢神経幹細胞の増殖と神経細胞への分化を直接誘導していると考えられる。
CRBNが大脳で神経幹細胞への分化を誘導することを検証するため、ゼブラフィッシュ胚を用いた前脳および中脳でのCRBNの過剰発現実験を行なった。その結果、両脳は形態を保持したまま約1.5 倍の容積に拡大した(図2右)。なおかつ脳のニューロンネットワークは形態上正常であった(図2右)。
まず供与体(ドナー)胚にCRBNをコードするメッセンジャーRNAを蛍光物質(ローダミン)と共に注入し過剰発現させた。この胚胞を他個体に移植し、脳室内に分布したドナー由来細胞の分化を蛍光顕微鏡で観察した。その結果、移植したドナー由来細胞は、有意に魚類の終脳組織である嗅球に分化した(図3矢頭)。このことはCRBNを発現した細胞が脳室内に分配された場合、神経幹細胞に分化しRostral Migratory Stream (RMS)という細胞移動経路にのって哺乳動物では大脳皮質を発生する終脳背側部で脳組織に分化したことを示す。
Uba1(ユビキチン活性化酵素)、UbcH5b(ユビキチン転移酵素)、及びGST融合ユビキチン組換えタンパク質を含む水溶液(Ub/E1/E2)に、CRBN(FH-CRBN complex)を加えた場合には、CRBNを加えない場合には検出されないタンパク質が検出された(図4の左図、中央図、右図のレーン2)。タンパク質のユビキチン化には、ユビキチンのほか、ユビキチン活性化酵素、ユビキチン転移酵素、ユビキチンリガーゼの3種類の酵素が必要である。左図、中央図、右図のレーン1では、ユビキチンリガーゼが存在しないためユビキチン化したタンパク質が生じなかったのに対し、左図、中央図、右図のレーン2では、CRBNがユビキチンリガーゼとして働いたことにより、ユビキチン化したタンパク質が生じ、そのタンパク質が電気泳動によって検出されたと考えられる。
MG132を加えた場合には、CRBNを含むタンパク質のバンドが多数検出された(図5の右レーン)。一方、MG132を加えない場合には、CRBNを含むタンパク質のバンドはほとんど検出されなかった(図5の左レーン)。
CRBNを過剰発現させた個体では、正常な空間的分布を保持しつつ、グリア細胞(図6左図)やセロトニン産生細胞(図7左図)が増加していた。このことは、CRBNが脳の空間的なパターンを正常に認識しつつ細胞を増殖、分化させることを意味する。
CRBNを発現しない細胞を移植した場合、その細胞は脳組織に分化しないのに対し(図8A~D)、CRBNを発現する細胞を移植した場合、その細胞は脳組織に分化した(図8E~H)。
Claims (11)
- 1)CRBN、2)CRBNをコードする核酸、又は3)CRBNを発現させた幹細胞若しくは神経前駆細胞を含有することを特徴とする医薬組成物。
- CRBNをコードする核酸が、ウイルスベクター中に挿入されていることを特徴とする請求項1に記載の医薬組成物。
- CRBNの他に、CRBNと共にユビキチンリガーゼ複合体を形成するタンパク質も含有することを特徴とする請求項1に記載の医薬組成物。
- CRBNをコードする核酸の他に、CRBNと共にユビキチンリガーゼ複合体を形成するタンパク質をコードする核酸も含有することを特徴とする請求項1又は2に記載の医薬組成物。
- CRBNの他に、CRBNと共にユビキチンリガーゼ複合体を形成するタンパク質も発現させた幹細胞若しくは神経前駆細胞を含有することを特徴とする請求項1に記載の医薬組成物。
- 大脳皮質疾患又は大脳皮質の外科的損傷の治療のために用いられることを特徴とする請求項1乃至5のいずれか一項に記載の医薬組成物。
- 大脳皮質の再生のために用いられることを特徴とする請求項1乃至5のいずれか一項に記載の医薬組成物。
- 請求項1乃至7のいずれか一項に記載の医薬組成物を非ヒト動物に投与し、その非ヒト動物の神経幹細胞又は神経前駆細胞を増殖させる方法。
- 請求項1乃至7のいずれか一項に記載の医薬組成物を非ヒト動物に投与し、その非ヒト動物の神経幹細胞又は神経前駆細胞を神経細胞へ分化させる方法。
- 被験物質をCRBNを含むユビキチンリガーゼ複合体に接触させる工程、及び前記ユビキチンリガーゼ複合体のユビキチンリガーゼ活性を測定し、ユビキチンリガーゼ活性を向上させた被験物質を選択する工程を含むことを特徴とする大脳皮質疾患又は大脳皮質の外科的損傷の治療薬のスクリーニング方法。
- 被験物質の存在下で神経幹細胞又は神経前駆細胞を培養する工程、及び神経幹細胞又は神経前駆細胞中のCRBNの発現量を測定し、CRBNの発現量を増大させた被験物質を選択する工程を含むことを特徴とする大脳皮質疾患又は大脳皮質の外科的損傷の治療薬のスクリーニング方法。
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Also Published As
Publication number | Publication date |
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CN102448472A (zh) | 2012-05-09 |
US9611465B2 (en) | 2017-04-04 |
EP2436387A1 (en) | 2012-04-04 |
JP5645816B2 (ja) | 2014-12-24 |
EP2436387B1 (en) | 2018-07-25 |
US20120134969A1 (en) | 2012-05-31 |
US20150232826A1 (en) | 2015-08-20 |
JPWO2010137547A1 (ja) | 2012-11-15 |
EP2436387A4 (en) | 2013-07-17 |
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