JP2010043025A - Therapeutic composition for failure of central nervous system myelination, and method of promoting central nervous system myelination - Google Patents
Therapeutic composition for failure of central nervous system myelination, and method of promoting central nervous system myelination Download PDFInfo
- Publication number
- JP2010043025A JP2010043025A JP2008208155A JP2008208155A JP2010043025A JP 2010043025 A JP2010043025 A JP 2010043025A JP 2008208155 A JP2008208155 A JP 2008208155A JP 2008208155 A JP2008208155 A JP 2008208155A JP 2010043025 A JP2010043025 A JP 2010043025A
- Authority
- JP
- Japan
- Prior art keywords
- myelination
- nervous system
- central nervous
- plp1
- therapeutic composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
本発明は、ＰＬＰ１タンパク質の機能亢進によって生じる中枢神経髄鞘形成不全の治療用組成物および中枢神経髄鞘形成促進方法に関する。 The present invention relates to a composition for treating central nerve myelination dysfunction caused by enhancement of PLP1 protein function and a method for promoting formation of central myelination.
Pelizaeus-Merzbacher（ペリツェウス・メルツバッハ）病（ＰＭＤ）は、先天性の中枢神経髄鞘形成不全疾患である（例えば、非特許文献１参照）。ＰＭＤ患者は精神、言語、運動に強い精神障害を発症し、具体的病症として、頭部振戦、眼振、歩行時のふらつき、言語障害、視神経萎縮、進行性運動障害、精神運動発達退行等がみられる。 Pelizaeus-Merzbacher disease (PMD) is a congenital central nerve myelination dysfunction disease (see, for example, Non-Patent Document 1). PMD patients develop psychiatric disorders that are strong in mind, language, and movement. Specific illnesses include head tremor, nystagmus, wobbling during walking, language disorders, optic nerve atrophy, progressive movement disorders, psychomotor developmental regression, etc. Is seen.
この髄鞘形成不全の原因の一つとしてＸ染色体上に存在するProteolipid protein１（ＰＬＰ１）遺伝子が知られる。ＰＬＰ１遺伝子の重複、変異、欠失等の異常、および、その他原因不明の異常によって、中枢神経に特異的なグリア細胞であるオリゴデンドロサイトによる髄鞘形成不全が引き起こされ、ＰＭＤを発症する（例えば、非特許文献１、２参照）。 Proteolipid protein 1 (PLP1) gene present on the X chromosome is known as one of the causes of this myelination dysfunction. Abnormalities such as duplication, mutation, and deletion of the PLP1 gene and other abnormalities of unknown cause cause myelination dysfunction due to oligodendrocytes, which are glial cells specific to the central nerve, and develop PMD (for example, Non-patent documents 1 and 2).
しかしながら、現在までのところ、ＰＭＤの有効な治療薬および治療方法はなく、その開発が待ち望まれている。
本発明は、ＰＬＰ１タンパク質の機能亢進によって生じる中枢神経髄鞘形成不全の治療用組成物、その治療用組成物を含有する治療用薬剤、および中枢神経髄鞘形成促進方法を提供することを目的とする。 It is an object of the present invention to provide a therapeutic composition for central nerve myelination dysfunction caused by enhancement of PLP1 protein function, a therapeutic agent containing the therapeutic composition, and a method for promoting central myelination. To do.
本発明者らは、ＰＬＰ１タンパク質の過剰発現によって髄鞘形成不全を起こしたオリゴデンドロサイトにＭＡＰＫリン酸化酵素（ＭＡＰＫＫ）活性阻害物質であるＵ０１２６および／またはＰＤ９８０５９を作用させることによって、髄鞘形成不全症状が正常レベルまで改善されることを見出し、本発明の完成に至った。 The present inventors have made myelination hypoplasia by causing U0126 and / or PD98059, which are inhibitors of MAPK phosphorylase (MAPKK) activity, to oligodendrocytes that have become hypomyelinating due to overexpression of PLP1 protein. It was found that symptoms were improved to a normal level, and the present invention was completed.
なお、本明細書で、「ＭＡＰＫ」とは、マイトジェン活性化タンパク質リン酸化酵素（mitogen-activated protein kinase）ＥＲＫを意味する。また、「酵素の活性を阻害する」とは、「活性化酵素自体の活性を阻害する」こと、および「不活性の酵素の活性化を阻害する」ことの両方の意味を含む。例えば、「ＭＡＰＫＫ活性阻害物質」とは、活性化ＭＡＰＫＫの活性を阻害することによって、ＭＡＰＫ経路におけるＭＡＰＫＫの下流に位置するタンパク質の活性化を阻害する物質、および、不活性のＭＡＰＫＫに作用することによって、ＭＡＰＫＫ活性化を阻害することによって、ＭＡＰＫ経路におけるＭＡＰＫＫの下流に位置するタンパク質の活性化を阻害する物質、の両方を意味する。 In the present specification, “MAPK” means mitogen-activated protein kinase ERK. Moreover, “inhibiting the activity of an enzyme” includes both meanings of “inhibiting the activity of the activated enzyme itself” and “inhibiting the activation of an inactive enzyme”. For example, a “MAPKK activity inhibitor” refers to a substance that inhibits the activation of a protein located downstream of MAPKK in the MAPK pathway and an inactive MAPKK by inhibiting the activity of activated MAPKK. By means of inhibiting MAPKK activation, it means both substances that inhibit the activation of proteins located downstream of MAPKK in the MAPK pathway.
すなわち、本発明に係るＰＬＰ１タンパク質の機能亢進によって生じる中枢神経髄鞘形成不全の治療用組成物は、ＭＡＰＫ経路活性化を阻害するための阻害物質を含有する。前記阻害物質はＭＡＰＫＫ活性阻害物質であってもよく、さらに、ＭＡＰＫＫ活性阻害物質は、Ｕ０１２６および／またはＰＤ９８０５９であってもよい。なお、上記中枢神経髄鞘形成不全はPelizaeus-Merzbacher（ペリツェウス・メルツバッハ）病（ＰＭＤ）であってもよい。 That is, the composition for the treatment of central myelination dysfunction caused by the enhancement of PLP1 protein function according to the present invention contains an inhibitor for inhibiting MAPK pathway activation. The inhibitor may be a MAPKK activity inhibitor, and the MAPKK activity inhibitor may be U0126 and / or PD98059. The central nerve myelination dysfunction may be Pelizaeus-Merzbacher disease (PMD).
さらに、本発明に係るＰＬＰ１タンパク質の機能亢進によって生じる中枢神経髄鞘形成不全の治療剤は、前記本発明に係る中枢神経髄鞘形成不全の治療組成物を含有することを特徴とする。なお、上記中枢神経髄鞘形成不全はPelizaeus-Merzbacher（ペリツェウス・メルツバッハ）病（ＰＭＤ）であってもよい。 Furthermore, the therapeutic agent for central nerve myelination dysplasia caused by the enhancement of the function of PLP1 protein according to the present invention comprises the therapeutic composition for central nerve myelination dysfunction according to the present invention. The central nerve myelination dysfunction may be Pelizaeus-Merzbacher disease (PMD).
本発明に係る中枢神経髄鞘形成促進方法は、in vitroで、及び、ヒトまたはヒト以外の脊椎動物において、ＰＬＰ１タンパク質の機能亢進によって中枢神経髄鞘形成不全を生じたオリゴデンドロサイトにおいて中枢神経髄鞘形成を促進する方法であって、ＭＡＰＫ経路活性化を阻害する工程を含有する。また、ＭＡＰＫ経路活性化の阻害は、ＭＡＰＫＫ活性を阻害することによって行ってもよい。さらに、上記ＭＡＰＫＫ活性の阻害は、Ｕ０１２６および／またはＰＤ９８０５９を投与することによって行ってもよく、Ｒａｆ活性を阻害することによって行ってもよい。 The method for promoting the formation of central nerve myelination according to the present invention is performed in vitro and in an oligodendrocyte in which dysplasia of central nerve myelination is caused by hyperfunction of PLP1 protein in humans or non-human vertebrates. A method of promoting sheath formation, comprising the step of inhibiting MAPK pathway activation. In addition, inhibition of MAPK pathway activation may be performed by inhibiting MAPKK activity. Furthermore, the inhibition of the MAPKK activity may be performed by administering U0126 and / or PD98059, or may be performed by inhibiting Raf activity.
本発明にかかるＰＬＰ１タンパク質の機能亢進によって生じる中枢神経髄鞘形成不全の治療方法は、ＭＡＰＫ経路活性化を阻害するための阻害物質を患者に投与する工程を含む。前記阻害物質はＭＡＰＫＫ活性阻害物質であってもよく、さらに、ＭＡＰＫＫ活性阻害物質は、Ｕ０１２６および／またはＰＤ９８０５９であってもよい。なお、上記中枢神経髄鞘形成不全はPelizaeus-Merzbacher（ペリツェウス・メルツバッハ）病（ＰＭＤ）であってもよい。 The method for treating central nerve myelination dysgenesis caused by the enhancement of PLP1 protein function according to the present invention includes the step of administering to a patient an inhibitor for inhibiting MAPK pathway activation. The inhibitor may be a MAPKK activity inhibitor, and the MAPKK activity inhibitor may be U0126 and / or PD98059. The central nerve myelination dysfunction may be Pelizaeus-Merzbacher disease (PMD).
本発明により、ＰＬＰ１タンパク質の機能亢進によって生じる中枢神経髄鞘形成不全の治療用組成物および治療用薬剤、および中枢神経髄鞘形成促進方法を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a therapeutic composition and therapeutic agent for central myelination dysplasia caused by enhancement of PLP1 protein function, and a method for promoting central myelination.
以下、上記知見に基づき完成した本発明の実施の形態を、実施例を挙げながら詳細に説明する。ただし、本発明は下記実施例に限定されない。 Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples. However, the present invention is not limited to the following examples.
実施の形態及び実施例に特に説明がない場合には、J. Sambrook, E. F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J.G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd.等の標準的なプロトコール集に記載の方法、あるいはそれを修飾したり、改変した方法を用いる。また、市販の試薬キットや測定装置を用いる場合には、特に説明が無い場合、それらに添付のプロトコールを用いる。 Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
なお、本発明の目的、特徴、利点、及びそのアイデアは、本明細書の記載により、当業者には明らかであり、本明細書の記載から、当業者であれば、容易に本発明を再現できる。以下に記載された発明の実施の形態及び具体的な実施例等は、本発明の好ましい実施態様を示すものであり、例示又は説明のために示されているのであって、本発明をそれらに限定するものではない。本明細書で開示されている本発明の意図ならびに範囲内で、本明細書の記載に基づき、様々に修飾ができることは、当業者にとって明らかである。 The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.
＝＝中枢神経髄鞘形成不全治療用組成物＝＝
本発明に係る、ＰＬＰ１タンパク質の機能亢進によって生じる中枢神経髄鞘形成不全治療用組成物は、ＭＡＰＫ経路活性化を阻害するための阻害物質のいずれか、もしくは、それらのうち２つ以上の組み合わせを含有することを特徴とする。この阻害物質は、ＭＡＰＫ経路の因子（例えば、K-Ras、H-Ras、N-Ras、A-Raf、B-Raf、c-Raf、ＭＡＰＫＫ、ＭＡＰＫ、など）の活性を阻害することにより、ＭＡＰＫの活性を阻害する物質であれば制限はなく、その物質は、低分子化合物やドミナントネガティブ型タンパク質変異体などであってもよいが、ＭＡＰＫＫ活性阻害物質であることが好ましく、Ｕ０１２６（1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene）またはＰＤ９８０５９（2′-Amino-3′-methoxyflavone）であることがさらに好ましい。
== Composition for treatment of central myelination dysplasia ==
The composition for treating central nerve myelination dysfunction caused by the enhancement of PLP1 protein function according to the present invention is any one of inhibitors for inhibiting MAPK pathway activation, or a combination of two or more thereof. It is characterized by containing. This inhibitor can inhibit the activity of factors of the MAPK pathway (eg, K-Ras, H-Ras, N-Ras, A-Raf, B-Raf, c-Raf, MAPKK, MAPK, etc.) there is no limitation as long as it is a substance that inhibits the activity of MAPK, the substance may be a low molecular compound or a dominant negative form protein variants but is preferably MAPKK active inhibitor, U0126 (1 4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene) or PD98059 (2′-Amino-3′-methoxyflavone).
＝＝中枢神経髄鞘形成不全治療剤＝＝
前記中枢神経髄鞘形成不全治療組成物のいずれか、もしくはそれらのうち２つ以上の組み合わせは、当業者に周知の薬学的に許容される担体、希釈剤、腑形剤等の製剤用添加物を用いて剤形化することができる。その形態は治療に適切な剤形であれば特に特定されず、例えば、経口剤として、錠剤、カプセル、顆粒、散剤、シロップ、腸溶剤、徐放性カプセル、カシュー、咀嚼錠、ドロップ、丸剤、内用液剤、菓子錠剤、徐放錠、徐放性顆粒等に剤形化してもよい。また、注射剤に剤形化してもよく、例えば、溶液性注射剤、乳濁性注射剤、または固形注射剤等が挙げられる。本治療剤には、上記製剤用添加物の他、異なる医薬組成物を配合することもできる。
== Therapeutic agent for central myelination dysplasia ==
Any of the above-mentioned compositions for treatment of central nerve myelination dysplasia, or a combination of two or more thereof, are pharmaceutical additives such as pharmaceutically acceptable carriers, diluents, and vaginal agents well known to those skilled in the art. To form a dosage form. The form is not particularly specified as long as it is a dosage form suitable for treatment. For example, as an oral preparation, tablet, capsule, granule, powder, syrup, intestinal solvent, sustained-release capsule, cashew, chewing tablet, drop, pill Alternatively, it may be formulated into liquid preparations for internal use, confectionery tablets, sustained release tablets, sustained release granules and the like. Moreover, it may be formulated into an injection, and examples thereof include a solution injection, an emulsion injection, and a solid injection. In addition to the above-mentioned pharmaceutical additives, a different pharmaceutical composition can be added to the therapeutic agent.
中枢神経髄鞘形成不全治療剤を投与する対象の疾患は、ヒトまたはヒト以外の脊椎動物において、ＰＬＰ１タンパク質の機能亢進によって中枢神経において髄鞘が形成不全になる疾患であれば限定されないが、Pelizaeus-Merzbacher（ペリツェウス・メルツバッハ）病（ＰＭＤ）であってもよい。ここで、ＰＬＰ１タンパク質の機能亢進は、ＰＬＰ１タンパク質の過剰発現によるものでもよく、タンパク質自体の活性亢進によるものでもよい。
なお、中枢神経髄鞘形成不全治療剤の患者への投与方法および投与量は、投与目的、剤形、患者の状態などに応じ、当業者が適宜選択可能である。
The disease to which the therapeutic agent for central nerve myelination dysplasia is administered is not limited as long as it is a disease in which myelination in the central nerve becomes dysplastic in humans or non-human vertebrates due to hyperfunction of PLP1 protein, but Pelizaeus -Merzbacher disease (PMD) may also be used. Here, enhancement of the function of the PLP1 protein may be due to overexpression of the PLP1 protein, or may be due to enhancement of the activity of the protein itself.
The administration method and dose of the therapeutic agent for central nerve myelination dysplasia to a patient can be appropriately selected by those skilled in the art according to the purpose of administration, dosage form, patient condition and the like.
（１）ＰＬＰ１過剰発現によるオリゴデンドロサイトの髄鞘形成不全
本実施例では、まず、ＰＬＰ１タンパク質の機能亢進によって中枢神経において髄鞘が形成不全になる疾患のモデルとして、ＰＬＰ１を過剰発現させることで、髄鞘形成不全を起こすオリゴデンドロサイトを作製した。
(1) Myelination dysfunction of oligodendrocytes due to overexpression of PLP1 In this example, first, PLP1 is overexpressed as a model of a disease in which myelination is dysplastic in the central nerve due to hyperfunction of PLP1 protein. An oligodendrocyte causing dysmyelination was prepared.
妊娠１６日目のラット（Sprague-Dawley系）から胎仔の大脳を摘出した後、嗅球海馬、髄膜、血管を取り除いた。この大脳皮質を、２ｍｌのディスパーゼ（３Ｕ／ｍｌ、Roche社）溶液に入れ、３７℃のＣＯ２インキュベータ内で５分間処理した後、１ｍｌの０．０５％ＤＮａｓｅを加えた。再びインキュベータ内で１０分間静置後、ピペッティングによってオリゴデンドロサイトを分散した。 After removing the cerebrum of the fetus from a rat (Sprague-Dawley strain) on day 16 of gestation, the olfactory bulb hippocampus, meninges, and blood vessels were removed. This cerebral cortex was placed in 2 ml of dispase (3 U / ml, Roche) solution, treated for 5 minutes in a 37 ° C. CO 2 incubator, and then 1 ml of 0.05% DNase was added. After standing again in the incubator for 10 minutes, oligodendrocytes were dispersed by pipetting.
このように単離したオリゴデンドロサイトを、１０％ウシ胎児血清（ＦＣＳ、Hyclone社）添加ＭＥＭ培地に約１×１０７／１０ｍｌの細胞密度で播種し、１０ｃｍ径の培養皿で初代培養した。 Thus isolated oligodendrocytes, 10% fetal calf serum (FCS, Hyclone, Inc.) were seeded at a cell density of added MEM medium at about 1 × 10 7 / 10ml, and primary culture in a culture dish 10cm diameter.
６〜７日後、初代培養細胞を５ｍｌのダルベッコのリン酸緩衝生理食塩水で２回洗浄した。そこへ０．０５％トリプシン―ＥＤＴＡ（２ｍｌ／培養皿）を加え、３７℃のＣＯ２インキュベータ内で８分間静置した後、さらに培養皿を強く振盪して細胞を剥離させた。この細胞を、２ｍｌの１０％ＦＣＳ添加ＭＥＭ培地中でピペッティングにより分散させ、遠心（１００×ｇ、５分）によって３回洗浄した。次いで１０％ＦＣＳ添加ＭＥＭ培地を加えてピペッティングした後、約８×１０６／１０ｍｌの細胞密度で１０ｃｍの培養皿に播種し、培養した。 Six to seven days later, the primary cultured cells were washed twice with 5 ml Dulbecco's phosphate buffered saline. 0.05% trypsin-EDTA (2 ml / culture dish) was added thereto, and the mixture was allowed to stand in a CO 2 incubator at 37 ° C. for 8 minutes, and the culture dish was further shaken to detach the cells. The cells were dispersed by pipetting in 2 ml of MEM medium supplemented with 10% FCS and washed three times by centrifugation (100 × g, 5 minutes). The filtrate was pipetted by adding 10% FCS added MEM medium were seeded in culture dishes 10cm at a cell density of about 8 × 10 6 / 10ml, and cultured.
さらに６〜７日後、再度継代し、約３×１０６／１０ｍｌの細胞密度で播種し、培養した。 After another 6-7 days, and passaged again, they were seeded at a cell density of about 3 × 10 6 / 10ml, and cultured.
一方、ヒトＰＬＰ１遺伝子FLJ45458（TOYOBO社）を鋳型に、下記の配列を持つオリゴヌクレオチドをプライマーとして、ExTaq polymerase（タカラバイオ社）でPCR増幅した。増幅したヒトＰＬＰ１遺伝子をpRetroX-IRES-ZsGreen１ベクター（Clontech-Takara Bio社、カタログ番号632520）のマルチクローニングサイトのBamHI切断（タカラバイオ社）部位に導入し、ＰＬＰ１組換えベクターを作製した。この組換えベクターを、Retro-X system (Clontech-Takara Bio社 カタログ番号631530)で産生されたレトロウイルスを介して、上述のように取得した培養オリゴデンドロサイトにトランスフェクトした。なお、pRetroX-IRES-ZsGreen１ベクターには、緑色蛍光タンパク質（ＧＦＰ）の遺伝子が組み込まれており、ＰＬＰ１を発現する細胞は緑色蛍光を発する。
プライマーＳ： ccgggatccatgggcttgttagagtgctgtgcaagatgtctg （配列番号１）
プライマーＡＳ： ccgggatcctcagaacttggtgcctcggcccatg （配列番号２）
On the other hand, PCR amplification was performed with ExTaq polymerase (Takara Bio) using the human PLP1 gene FLJ45458 (TOYOBO) as a template and an oligonucleotide having the following sequence as a primer. The amplified human PLP1 gene was introduced into the BamHI cleavage (Takara Bio) site of the multicloning site of the pRetroX-IRES-ZsGreen1 vector (Clontech-Takara Bio, catalog number 632520) to prepare a PLP1 recombinant vector. This recombinant vector was transfected into cultured oligodendrocytes obtained as described above via a retrovirus produced by the Retro-X system (Clontech-Takara Bio, catalog number 631530). The pRetroX-IRES-ZsGreen1 vector has a green fluorescent protein (GFP) gene incorporated therein, and cells expressing PLP1 emit green fluorescence.
Primer S: ccgggatccatgggcttgttagagtgctgtgcaagatgtctg (SEQ ID NO: 1)
Primer AS: ccgggatcctcagaacttggtgcctcggcccatg (SEQ ID NO: 2)
対照群として、ＰＬＰ１遺伝子を導入していない非組換えpRetroX-IRES-ZsGreen１ベクターを、上述と同様にして培養オリゴデンドロサイトにトランスフェクトした。 As a control group, non-recombinant pRetroX-IRES-ZsGreen1 vector into which no PLP1 gene was introduced was transfected into cultured oligodendrocytes in the same manner as described above.
各オリゴデンドロサイトを４％パラホルムアルデヒドで固定し、髄鞘形成のマーカーであるＭＢＰを認識する抗ＭＢＰ抗体（Millipore社、 カタログ番号AB15542、１００倍希釈）を用いて髄鞘を標識した。なお、二次抗体として、Alexa Fluor 594抗マウスＩｇＧ（Molecular Probes 社、カタログ番号A-11005、５００倍希釈）を用いた。その結果を図１に示す。 Each oligodendrocyte was fixed with 4% paraformaldehyde, and the myelin sheath was labeled with an anti-MBP antibody (Millipore, catalog number AB15542, diluted 100-fold) that recognizes MBP, a marker of myelination. As a secondary antibody, Alexa Fluor 594 anti-mouse IgG (Molecular Probes, catalog number A-11005, diluted 500 times) was used. The result is shown in FIG.
なお、図１Ａ、Ｂ、Ｃ、または図１Ｄ、Ｅ、Ｆは、それぞれ、異なる波長光の下で同一の領域を観察したものである。また、図１Ｃ及び図１Ｆは、それぞれ、図１Ａと図１Ｂ及び図１Ｄと図１Ｅの重ね合わせである。 1A, 1B, 1C, 1D, 1E, and 1F are obtained by observing the same region under different wavelength light. 1C and 1F are superpositions of FIGS. 1A, 1B, 1D, and 1E, respectively.
図Ａ〜Ｃに示すように、ＰＬＰ１を発現していない対照群において、ＧＦＰで標識されたオリゴデンドロサイト（図１Ｂ、矢印）は抗ＭＢＰ抗体によっても標識された（図１Ａ、矢印）。すなわち、ＰＬＰ１非発現オリゴデンドロサイトは髄鞘を正常に形成していた。 As shown in FIGS. AC, in the control group not expressing PLP1, oligodendrocytes labeled with GFP (FIG. 1B, arrow) were also labeled with anti-MBP antibody (FIG. 1A, arrow). That is, PLP1 non-expressing oligodendrocytes normally formed myelin sheaths.
一方、図１Ｄ〜Ｆに示すように、ＰＬＰ１を過剰発現しているオリゴデンドロサイトでは、ＧＦＰで標識されたオリゴデンドロサイト（図１Ｅ、矢印）は抗ＭＢＰ抗体によっては標識されなかった（図１Ｄ、破線丸）。このように、ＰＬＰ１を過剰発現するオリゴデンドロサイトでは髄鞘形成不全が生じていた。 On the other hand, as shown in FIGS. 1D to F, in oligodendrocytes overexpressing PLP1, oligodendrocytes labeled with GFP (FIG. 1E, arrows) were not labeled with an anti-MBP antibody (FIG. 1D). , Dashed circle). Thus, oligodendrocytes overexpressing PLP1 had myelination failure.
（２）ＭＡＰＫＫ活性阻害剤による髄鞘形成不全症状の改善
次に、ＭＡＰＫＫ活性阻害剤によって、この髄鞘形成不全症状が正常レベルまで改善されることを示す。
(2) Improvement of dysmyelination symptom by MAPKK activity inhibitor Next, it will be shown that this dysmyelination symptom is improved to a normal level by an inhibitor of MAPKK activity.
ＰＬＰ１組換えベクター、および非組換えベクターをレトロウイルスを介してオリゴデンドロサイトにトランスフェクトし、その２４時間後、ＭＡＰＫＫ活性阻害剤であるＵ０１２６（1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene、１０ μＭ、メルク社、カタログ番号662005）、またはＰＤ９８０５９（2'-Amino-3'-methoxyflavone、１０ μＭ、メルク社、カタログ番号513000）のＤＭＳＯ溶液を培地中に加え、３７℃のＣＯ２インキュベーターで７２時間インキュベートした。なお、対照群として、遺伝子導入培養オリゴデンドロサイトの培地中に同量のＤＭＳＯを加え、同様にしてインキュベートした。 PLP1 recombinant vector and non-recombinant vector were transfected into oligodendrocytes via retrovirus and 24 hours later, U0126 (1,4-Diamino-2,3-dicyano-1 which is an inhibitor of MAPKK activity) , 4-bis (2-aminophenylthio) butadiene, 10 μM, Merck, catalog number 662005) or PD98059 (2′-Amino-3′-methoxyflavone, 10 μM, Merck, catalog number 513000) in DMSO And incubated for 72 hours in a 37 ° C. CO 2 incubator. As a control group, the same amount of DMSO was added to the medium of the gene-introduced culture oligodendrocyte and incubated in the same manner.
インキュベート後のオリゴデンドロサイトを、上記のように抗ＭＢＰ抗体で染色し、染色後の細胞を蛍光顕微鏡下で観察し、標識細胞を計数した。その結果を図２に示す。 After incubation, the oligodendrocytes were stained with an anti-MBP antibody as described above, the stained cells were observed under a fluorescence microscope, and the labeled cells were counted. The result is shown in FIG.
ＰＬＰ１を強制発現させたオリゴデンドロサイト群を、ＭＡＰＫＫ活性阻害剤であるＵ１２０６またはＰＤ９８０５９で処理すると、図２に示すように、ＧＦＰ陽性オリゴデンドロサイトに対するＭＢＰ陽性細胞の割合がＰＬＰ１非発現対照群と同等のレベルまで改善された。 When the oligodendrocyte group in which PLP1 was forcibly expressed was treated with U1206 or PD98059, which is a MAPKK activity inhibitor, as shown in FIG. 2, the ratio of MBP positive cells to GFP positive oligodendrocytes was different from that in the PLP1 non-expression control group. Improved to the same level.
さらに、遺伝子導入培養オリゴデンドロサイト髄鞘形成不全症状が、上述のＭＡＰＫＫ活性阻害剤により改善されたことを確認するため、ウエスタンブロット法によりＭＢＰの発現を解析した。 Furthermore, in order to confirm that the symptom of dysmyelination of the gene-transferred cultured oligodendrocyte was improved by the above-described MAPKK activity inhibitor, the expression of MBP was analyzed by Western blotting.
上記のようにＭＡＰＫＫ活性阻害剤で処理した培養細胞を、細胞溶解バッファー（50 mM HEPES-NaOH pH 7.5、20 mM MgCl2、150 mM NaCl、1 mM dithiothreitol、1 mM phenylmethane sulfonylfluoride、1 μg/ml leupeptin、1 mM EDTA、1 mM Na3VO4、10 mM NaF、0.5% NP-40）に懸濁した後、遠心分離した（Miyamoto et al.、 Proceedings of the National Academy of Sciences of the USA、103、10444-10449、2006を参照）。ここで得られた上清に、あらかじめ抗ＭＢＰ抗体（５００倍希釈、Millipore社、カタログ番号05-675）と結合させたプロテインＧ樹脂ビーズを混合し、上清中のＭＢＰを沈降させ、回収した。この回収タンパク質を変性ＳＤＳポリアクリルアミドゲルで電気泳動し、ＰＶＤＦ膜に移行した後、膜上のタンパク質を抗ＭＢＰ抗体（カタログ番号：05-675、Millipore社、５００倍希釈）で標識し、ECL Advance Western Blotting Detection kit（ＧＥヘルスケア バイオサイエンス社、カタログ番号RPN2135）を用いて検出した。 Cultured cells treated with the MAPKK activity inhibitor as described above were treated with cell lysis buffer (50 mM HEPES-NaOH pH 7.5, 20 mM MgCl 2 , 150 mM NaCl, 1 mM dithiothreitol, 1 mM phenylmethane sulfonylfluoride, 1 μg / ml leupeptin). , 1 mM EDTA, 1 mM Na 3 VO 4 , 10 mM NaF, 0.5% NP-40) and then centrifuged (Miyamoto et al., Proceedings of the National Academy of Sciences of the USA, 103, 10444-10449, 2006). The supernatant obtained here was mixed with protein G resin beads previously bound with an anti-MBP antibody (500-fold diluted, Millipore, catalog number 05-675), and MBP in the supernatant was precipitated and collected. . The recovered protein was electrophoresed on a denaturing SDS polyacrylamide gel, transferred to a PVDF membrane, and then the protein on the membrane was labeled with an anti-MBP antibody (catalog number: 05-675, Millipore, 500-fold dilution), and ECL Advance Detection was performed using a Western Blotting Detection kit (GE Healthcare Bioscience, catalog number RPN2135).
図３に示すように、ＰＬＰ１を過剰発現したオリゴデンドロサイトではＭＢＰの発現が検出できず（レーンＢ）、この細胞群で髄鞘形成不全が生じていた。一方、ＰＬＰ１を過剰発現した細胞群を、Ｕ０１２６（レーンＤ）またはＰＤ９８０５９（レーンＦ）で処理した場合、ＰＬＰ１非発現群（レーンＡ、Ｃ、Ｅ；ＣとＥはそれぞれＵ０１２６、ＰＤ９８０５９処理群）と同等レベルのＭＢＰの発現が検出された。これは、これらのＭＡＰＫＫ活性阻害剤によって髄鞘形成不全症状が正常レベルまで改善されたことを示している。 As shown in FIG. 3, MBP expression could not be detected in oligodendrocytes overexpressing PLP1 (lane B), and myelination failure occurred in this cell group. On the other hand, when a group of cells overexpressing PLP1 was treated with U0126 (lane D) or PD98059 (lane F), a PLP1 non-expression group (lanes A, C, E; C and E are U0126 and PD98059 treated groups, respectively) Level of MBP expression was detected. This indicates that these MAPKK activity inhibitors have improved myelination dysplasia symptoms to normal levels.
Claims (4)
ＭＡＰＫ経路活性化を阻害するための阻害物質を含有する治療用組成物。 A composition for treating central myelination dysplasia caused by hyperfunction of PLP1 protein, comprising:
A therapeutic composition comprising an inhibitor to inhibit MAPK pathway activation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008208155A JP5391401B2 (en) | 2008-08-12 | 2008-08-12 | Composition for treating central myelination dysplasia and therapeutic agent for central myelination dysplasia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008208155A JP5391401B2 (en) | 2008-08-12 | 2008-08-12 | Composition for treating central myelination dysplasia and therapeutic agent for central myelination dysplasia |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2010043025A true JP2010043025A (en) | 2010-02-25 |
| JP5391401B2 JP5391401B2 (en) | 2014-01-15 |
Family
ID=42014718
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2008208155A Active JP5391401B2 (en) | 2008-08-12 | 2008-08-12 | Composition for treating central myelination dysplasia and therapeutic agent for central myelination dysplasia |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5391401B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018106782A1 (en) * | 2016-12-08 | 2018-06-14 | Case Western Reserve University | Methods and compositions for enhancing functional myelin production |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003011337A1 (en) * | 2001-07-30 | 2003-02-13 | Keio University | MEDICINAL COMPOSITIONS CONTAINING Fc RECEPTOR Ϝ-CHAIN ACTIVATOR |
-
2008
- 2008-08-12 JP JP2008208155A patent/JP5391401B2/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003011337A1 (en) * | 2001-07-30 | 2003-02-13 | Keio University | MEDICINAL COMPOSITIONS CONTAINING Fc RECEPTOR Ϝ-CHAIN ACTIVATOR |
Non-Patent Citations (3)
| Title |
|---|
| JPN6013014624; Ｊ Ｎｅｕｒｏｌ Ｓｃｉ．， Ｖｏｌ．２２８ Ｎｏ．２， ｐ．２０４-５． （２００５） * |
| JPN6013014626; Ｎｅｕｒｏｇｅｎｅｔｉｃｓ， Ｖｏｌ．６ ＮＯ．１， ｐ．１-１６． （２００５） * |
| JPN6013014628; Ｊ Ｃｅｌｌ Ｂｉｏｌ．， Ｖｏｌ．１８０ ＮＯ．５， ｐ．９４７-５５． （２００８ Ｍａｒ １０） * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018106782A1 (en) * | 2016-12-08 | 2018-06-14 | Case Western Reserve University | Methods and compositions for enhancing functional myelin production |
| CN110249051A (en) * | 2016-12-08 | 2019-09-17 | 凯斯西储大学 | Enhance the method and composition that functional myelin generates |
| US10982216B2 (en) | 2016-12-08 | 2021-04-20 | Case Western Reserve University | Methods and compositions for enhancing functional myelin production |
| EP4276187A3 (en) * | 2016-12-08 | 2024-01-17 | Case Western Reserve University | Methods and compositions for enhancing functional myelin production |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5391401B2 (en) | 2014-01-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zheng et al. | Inhibition of EHMT1/2 rescues synaptic and cognitive functions for Alzheimer’s disease | |
| Rajbhandari et al. | Single cell analysis reveals immune cell–adipocyte crosstalk regulating the transcription of thermogenic adipocytes | |
| Gehrke et al. | PINK1 and Parkin control localized translation of respiratory chain component mRNAs on mitochondria outer membrane | |
| Tran et al. | Lipopolysaccharide and tumor necrosis factor regulate Parkin expression via nuclear factor-kappa B | |
| EP3102679B1 (en) | Micro-rnas and compositions comprising same for the treatment and diagnosis of serotonin-, adrenalin-, noradrenalin-, glutamate-, and corticotropin-releasing hormone- associated medical conditions | |
| US20180264140A1 (en) | Motor neuron-specific expression vectors | |
| CA2844012C (en) | Micro-rnas and compositions comprising same for the treatment and diagnosis of serotonin-, adrenalin-, noradrenalin-, glutamate-, and corticotropin-releasing hormone- associated medical conditions | |
| US20230057355A1 (en) | Gene networks that mediate remyelination of the human brain | |
| Huang et al. | mir-500-Mediated GAD67 downregulation contributes to neuropathic pain | |
| CN112654366A (en) | Methods of treating or inhibiting the onset of huntington's disease | |
| Leal et al. | The RNA-binding protein hnRNP K mediates the effect of BDNF on dendritic mRNA metabolism and regulates synaptic NMDA receptors in hippocampal neurons | |
| Park et al. | Kinetically stabilizing mutations in beta tubulins create isotype-specific brain malformations | |
| Rochat et al. | Astrocyte‐targeting RNA interference against mutated superoxide dismutase 1 induces motoneuron plasticity and protects fast‐fatigable motor units in a mouse model of amyotrophic lateral sclerosis | |
| JP5391401B2 (en) | Composition for treating central myelination dysplasia and therapeutic agent for central myelination dysplasia | |
| JP2011530484A (en) | Neuronal differentiation promoter | |
| Sagehashi et al. | Insulin enhances gene expression of midnolin, a novel genetic risk factor for Parkinson’s disease, via extracellular signal-regulated kinase, phosphoinositide 3-kinase and multiple transcription factors in SH-SY5Y cells | |
| Sun et al. | Inhibition of Galectin-3 in a Rat Model of Epilepsy and Kainate-Activated BV2 Cells Limits Microglial Activation Through the NLRP3/Pyroptosis Pathway | |
| Mezo‐González et al. | Adult rats sired by obese fathers present learning deficits associated with epigenetic and neurochemical alterations linked to impaired brain glutamatergic signaling | |
| JP5686284B2 (en) | Composition and therapeutic agent for treatment of central myelination dysfunction | |
| Wang et al. | The involvement of ADAR1 in chronic unpredictable stress-induced cognitive impairment by targeting DARPP-32 with miR-874-3p in BALB/c mice | |
| Herz et al. | Genome-wide transcriptome analysis of Echinococcus multilocularis larvae and germinative cell cultures reveals genes involved in parasite stem cell function | |
| Daud et al. | Study on Amyotrophic lateral Sclerosis: A Disease of Motor Neuron in Pakistan | |
| Huang et al. | Astragaloside IV enhances memory and modulates hippocampal synaptic plasticity by decreasing GABAergic inhibition through EGR-1 mediated BDNF/TrkB signaling pathway in mice | |
| Konttinen et al. | Microglial amyloid beta clearance is driven by PIEZO1 channels | |
| Bai et al. | Panax quinquefolium saponins attenuates microglia activation following acute cerebral ischemia‐reperfusion injury via Nrf2/miR‐103‐3p/TANK pathway |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20110801 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20130402 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20130502 |
|
| RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20130502 |
|
| A521 | Written amendment |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20130502 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20130903 |
|
| R150 | Certificate of patent or registration of utility model |
Free format text: JAPANESE INTERMEDIATE CODE: R150 |