JP2018193309A - Agent for prevention or treatment of amyotrophic lateral sclerosis or frontotemporal dementia - Google Patents

Abstract

To provide a preventive or therapeutic agent showing excellent drug action on amyotrophic lateral sclerosis or frontotemporal dementia.SOLUTION: FUS protein, DNA molecule encoding FUS protein, and/or FUS protein production promoting material can be used as an agent for the prevention or treatment of amyotrophic lateral sclerosis or frontotemporal dementia.SELECTED DRAWING: None

Description

  The present invention relates to a preventive or therapeutic agent for amyotrophic lateral sclerosis (hereinafter ALS) or frontotemporal dementia (hereinafter FTD). More specifically, the disease has an effective preventive or therapeutic effect against ALS or FTD caused by abnormal elongation of a GGGGCC repeat sequence (hereinafter referred to as G4C2 repeat sequence) in the C9orf72 gene untranslated region. It is related with the preventive or therapeutic agent.

  Amyotrophic lateral sclerosis and frontotemporal dementia (hereinafter ALS / FTD) are refractory neurodegenerative diseases that share common genetic and neuropathological characteristics. Abnormal extension of the G4C2 repeat sequence in the untranslated region of the C9orf72 gene has been identified as the most frequent causative gene abnormality in familial and sporadic ALS / FTD (see Non-Patent Documents 1 and 2).

  In the brain of ALS / FTD patients caused by abnormal extension of G4C2 repeat sequences, abnormally extended repeat RNA transcribed from the C9orf72 mutant gene forms an RNA aggregate (hereinafter referred to as RNA foci) (see Non-Patent Documents 1 and 2). ). It has also been clarified that dipeptide repeat proteins (hereinafter referred to as DPRs) generated from the repeat RNAs form aggregates in the brain of the ALS / FTD patient by repeat-related non-ATG translation (hereinafter referred to as RAN translation). (Non-Patent Documents 3 to 5) These abnormally extended repeat RNAs and DPRs are considered to be the cause of neurodegeneration, but the molecular mechanism causing the pathogenesis of ALS / FTD has not been fully elucidated. .

  Conventionally, antisense oligonucleotides (hereinafter referred to as ASO) that inhibit translation of RNA derived from G4C2 repeat sequences in the untranslated region of the C9orf72 gene have been identified (Non-Patent Documents 6 to 8). Conventionally, a compound that binds to the G4C2 repeat sequence has also been identified (Non-patent Document 9). However, with these ASOs and compounds, the therapeutic effects of ALS / FTD have not been confirmed in animal models, and there are therapeutic drug candidates that show a positive effect on ALS / FTD caused by abnormal elongation of G4C2 repeat sequences. The current situation is not found.

DeJesus-Hernandez, M. et al. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245-56 (2011). Renton, A.E. et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257-68 (2011). Ash, P.E. et al. Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble molecules specific to c9FTD / ALS. Neuron 77, 639-46 (2013). Mori, K. et al. The C9orf72 GGGGCC repeat is translated into aggregating dipeptide-repeat proteins in FTLD / ALS. Science 339, 1335-8 (2013). Zu, T. et al. RAN proteins and RNA foci from antisense transcripts in C9ORF72 ALS and frontotemporal dementia.Proc Natl Acad Sci U S A 110, E4968-77 (2013). Donnelly, C. J. et al.RNA toxicity from the ALS / FTD C9ORF72 expansion is mitigated by antisense intervention.Neuron 80, 415-428 (2013). Lagier-Tourenne, C. et al.Targeted degenerateon of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degenerateon.Proc Natl Acad Sci U S A 110, E4530-9 (2013). Sareen, D. et al.Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion.Sci Transl Med 5, 208ra149 (2013). Su, Z. et al. Discovery of a biomarker and lead small molecules to target r (GGGGCC) -associated defects in c9FTD / ALS. Neuron 83, 1043-50 (2013).

  An object of the present invention is to provide a prophylactic or therapeutic agent exhibiting excellent drug efficacy against ALS or FTD.

  In order to elucidate the pathological mechanism of ALS / FTD caused by abnormal elongation of the G4C2 repeat sequence, the present inventors first created a fruit fly that expresses the G4C2 repeat sequence abnormally elongated in the compound eye and nervous system. , (I) exhibiting intense compound eye neurodegeneration, (ii) reduced survival to adults, shortening adult life, (iii) presenting progressive movement disorders, and (iv) G4C2 repeats It was confirmed that the sequence was transcribed into repeat RNA and further RAN-translated into DPRs, and found that it could be used as a model animal showing ALS / FTD pathology.

  Furthermore, in the Drosophila, expression of FUS protein, an RNA-binding protein, showed suppression of compound eye neurodegeneration, suppression of movement disorders, and suppression of RAN translation into DPRs, and improvement of ALS / FTD symptoms. confirmed. That is, the FUS protein has been found to have an excellent preventive or therapeutic effect on ALS / FTD, and the FUS protein, the DNA molecule encoding the FUS protein, and / or the FUS protein production promoter is ALS / It was found useful as a preventive or therapeutic agent for FTD. The present invention has been completed by further studies based on these findings.

That is, this invention provides the invention of the aspect hung up below.
Item 1. A preventive or therapeutic agent for ALS or FTD, comprising as an active ingredient at least one selected from the group consisting of a FUS protein, a DNA molecule encoding the FUS protein, and a FUS protein production promoter.
Item 2. Item 2. The preventive or therapeutic agent according to Item 1, which is applied to familial ALS or familial FTD.
Item 3. Item 3. The preventive or therapeutic agent according to Item 1 or 2, which is used for improving neurodegeneration or movement disorder in ALS or FTD.
Item 4. Item 4. The preventive or therapeutic agent according to any one of Items 1 to 3, wherein the active ingredient is a FUS protein or a DNA molecule encoding the FUS protein.
Item 5. A protein translation inhibitor resulting from an abnormally elongated GGGGCC repeat sequence, comprising as an active ingredient at least one selected from the group consisting of a FUS protein, a DNA molecule encoding the FUS protein, and a FUS protein production promoter .

  According to the present invention, the pathology of ALS / FTD such as neurodegeneration and movement disorders can be improved by binding FUS protein to repeat RNA transcribed from an abnormally elongated G4C2 repeat sequence. Conventionally, ALS / FTD caused by abnormally elongated G4C2 repeat sequences has not been established as an effective treatment, and in recent years, patients with ALS / FTD who develop with age due to the arrival of an aging society The establishment of these treatments is an urgent issue, but the present invention shows the therapeutic effect of ALS / FTD for the first time in vivo and is useful as a novel treatment method for ALS / FTD. It is.

It is the result of having observed the compound eye of the transgenic Drosophila which expresses G4C2 repeat arrangement | sequence ((G4C2) ₅₀ , (G4C2) ₈₉ (W), and (G4C2) ₈₉ (S)) which extended abnormally with the optical microscope. Survival to adults (A) and life span for transgenic Drosophila expressing abnormally elongated G4C2 repeat sequences ((G4C2) ₅₀ , (G4C2) ₈₉ (W)) or normal G4C2 repeat sequences ((G4C2) ₉ ) It is the result of evaluating (B). It is the result of having performed the climbing assay about the transgenic Drosophila which expresses abnormally extended G4C2 repeat sequence ((G4C2) ₅₀ , (G4C2) ₈₉ (W)) or normal G4C2 repeat sequence ((G4C2) ₉ ). RNA transcribed from the repeat sequence and DPRs (poly (GR), poly (GA)) in an adult primordium of a transgenic Drosophila compound expressing an abnormally elongated G4C2 repeat sequence ((G4C2) ₈₉ (S)) , And poly (GP)) were analyzed by FISH and immunostaining. The result of having observed the compound eye of the transgenic Drosophila which expresses FUS, ILF2, or hnRNPK with the abnormally extended G4C2 repeat sequence ((G4C2) ₈₉ (S)) or EGFP with an optical microscope is shown. The result of having performed the climbing assay about the transgenic Drosophila which expresses FUS or EGFP with the abnormally extended G4C2 repeat sequence ((G4C2) ₅₀ ) is shown. Results of observation of compound eyes with an optical microscope and measurement of compound eye size for transgenic Drosophila expressing FUS, FUS-RRMmut, or EGFP together with abnormally elongated G4C2 repeat sequence ((G4C2) ₈₉ (S)) Indicates. DPRs (poly (GR), poly (GA)) in the adult eye of a transgenic Drosophila compound eye that expresses FUS, FUS-RRMmut, or EGFP with an abnormally elongated G4C2 repeat sequence ((G4C2) ₈₉ (S)) The results of immunostaining (A) and poly (GP)), the results of measuring the number of aggregates of each DRPs (B), and the results of measuring the amount of poly (GP) by the ELISA method (C) are shown. Abnormally elongated G4C2 repeat sequence ((G4C2) ₈₉ (S)) or EGFP-expressing transgenic Drosophila with caz ² mutations, compound eyes observed with light microscope and compound eye size measured The results are shown.

In this document, the notation “(G4C2) _n ” represents a G4C2 repeat sequence in which n GGGGCCs are linked repeatedly. “Poly (GR)”, “Poly (GA)”, and “Poly (GP)” indicate DPRs containing dipeptide units of glycine-arginine, glycine-alanine, and glycine-proline, respectively.

1. The preventive or therapeutic agent for ALS or FTD The preventive or therapeutic agent of the present invention is a drug used for the prevention or treatment of ALS or FTD, and comprises FUS protein, DNA molecule encoding FUS protein, and FUS protein It is characterized in that at least one selected from the group consisting of production promoting substances is an active ingredient. Hereinafter, the present invention will be described in detail.

Active ingredient In the present invention, at least one selected from the group consisting of a FUS protein, a DNA molecule encoding the FUS protein, and a FUS protein production promoting substance is used as an active ingredient.

(FUS protein)
The FUS protein itself exerts actions such as suppression of neurodegeneration, suppression of progressive movement disorders, and suppression of RAN translation to DPRs in ALS / FTD, and therefore, as an active ingredient of a preventive or therapeutic agent for ALS or FTD. Can be used.

  The FUS protein is also called ALS6, ETM4, HNRNPP2, POMP75, TLS, etc., and is a protein known as an RNA binding protein.

  Specific examples of the FUS protein used in the present invention include human FUS protein, orthologs thereof, and mutants thereof.

  As for the human FUS protein, for example, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is known.

  The ortholog of the FUS protein is not particularly limited. For example, mammals such as rats, hamsters, guinea pigs, mice, cows, sheep, pigs, goats, monkeys, and rabbits; birds such as chickens and ostriches; salmon and catfish The thing derived from fish etc. is mentioned. The origin of the FUS protein may be appropriately set according to the species to be administered.

The FUS protein mutant is not particularly limited as long as it retains the ability to bind to a repeat RNA transcribed from the G4C2 repeat sequence. Specifically, the human FUS protein mutant includes the following: The mutants shown in (i) and (ii) are mentioned.
(i) a human FUS protein comprising the amino acid sequence shown in SEQ ID NO: 1 in which one or several amino acid residues are substituted, deleted, added and / or inserted in the amino acid sequence shown in SEQ ID NO: 1; A variant of the human FUS protein with equivalent RNA binding ability.
(ii) A human FUS protein having a sequence identity of 85% or more with respect to the amino acid sequence shown in SEQ ID NO: 1 and having an RNA binding ability equivalent to that of the human FUS protein consisting of the amino acid sequence shown in SEQ ID NO: 1. Mutant.

  In the mutant (i), the introduced amino acid modification may include only one kind of modification (for example, only substitution) among substitution, addition, insertion, and deletion, and two kinds The above modifications (for example, substitution and insertion) may be included. In the peptide of (i), the introduced substitution, addition, insertion or deletion may be one or several amino acids, for example, 1 to 30 or 1 to 20, preferably 1 to 15 1 to 10, 1 to 8, 1 to 5, or 1 to 4, more preferably 1 to 3, particularly preferably 1 or 2 or 1.

  In the mutant (ii), the sequence identity to the amino acid sequence shown in SEQ ID NO: 1 may be 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 99. % Or more.

  Here, in the mutant (ii), the sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 1 is the sequence identity calculated by comparison with the amino acid sequence shown in SEQ ID NO: 1. The term `` sequence identity '' refers to BLAST PACKAGE [sgi32 bit edition, Version 2.0.12; available from National Center for Biotechnology Information (NCBI)] bl2seq program (Tatiana A. Tatsusova, Thomas L. Madden, FEMS Microbiol. Lett., Vol. 174, p247-250, 1999) shows the identity value of amino acid sequences. The parameters may be set to Gap insertion Cost value: 11 and Gap extension Cost value: 1.

  Further, when amino acid substitution is introduced in the mutants (i) and (ii), conservative substitution can be mentioned as an aspect of the introduced amino acid substitution. That is, as the amino acid substitution in the mutants of (i) and (ii), for example, if the amino acid before substitution is a nonpolar amino acid, substitution with another nonpolar amino acid, If the amino acid before substitution is an acidic amino acid, it is substituted with another acidic amino acid, and if the amino acid before substitution is a basic amino acid, it is substituted with another basic amino acid. Substitution may be mentioned.

  In the present invention, “having RNA binding ability equivalent to the human FUS protein consisting of the amino acid sequence shown in SEQ ID NO: 1” means that the RNA binding ability is equivalent to the human FUS protein consisting of the amino acid sequence shown in SEQ ID NO: 1, It means that it has the same preventive or therapeutic effect against ALS or FTD as the human FUS protein.

  In the amino acid sequence shown in SEQ ID NO: 1 (human FUS protein), the amino acid residues at positions 286 to 371 form an RNA recognition motif region. Therefore, in the mutants (i) and (ii), It is desirable that the RNA recognition motif region is not mutated (substitution, deletion, and / or insertion).

  The FUS protein may be a natural protein or a recombinant produced by genetic engineering techniques.

(DNA molecule encoding FUS protein)
A DNA molecule encoding the FUS protein can be expressed in the ALS or FTD by expressing the FUS protein in an administered organism, such as suppression of neurodegeneration, suppression of progressive movement disorders, suppression of RAN translation into DPRs, etc. In order to exert the action, it can be used as an active ingredient of a preventive or therapeutic agent for ALS or FTD.

  The base sequence in the DNA molecule encoding the FUS protein is known. For example, the DNA molecule encoding the human FUS protein includes a DNA molecule containing the base sequence shown in SEQ ID NO: 2. Another example of a DNA molecule encoding the human FUS protein is an RNA binding ability equivalent to that of the human FUS protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and the nucleotide sequence shown in SEQ ID NO: 2. And a DNA molecule that hybridizes under stringent conditions with a DNA molecule comprising a base sequence complementary to the DNA molecule consisting of

  Here, “stringent conditions” means 0.5% SDS, 5 × Denhartz (Denhartz's, 0.1% bovine serum albumin (BSA), 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400) and 100 μg / ml salmon sperm DNA In 6 × saline sodium citrate (SSC) (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), and the temperature is kept at 50 ° C. to 65 ° C. for 4 hours to overnight.

Specifically, hybridization under stringent conditions is performed by the following method. In other words, a nylon membrane with a DNA library or cDNA library immobilized thereon was prepared, and nylon film was prepared at 65 ° C in a prehybridization solution containing 6 x SSC, 0.5% SDS, 5 x Denharz, 100 μg / ml salmon sperm DNA. Block the membrane. Then add each probe labeled with ³² P and incubate at 65 ° C. overnight. This nylon membrane was washed in 6 × SSC at room temperature for 10 minutes, in 2 × SSC containing 0.1% SDS for 10 minutes at room temperature, in 0.2 × SSC containing 0.1% SDS for 30 minutes at 45 ° C., then autoradio Take a graph and detect the DNA that specifically hybridizes with the probe.

  A DNA molecule encoding the FUS protein can be obtained by genetic engineering techniques based on its base sequence.

  Further, the DNA molecule encoding the FUS protein is preferably used by being incorporated into an expression vector so that the FUS protein can be expressed in vivo.

  Specific examples of the expression vector include plasmid vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, retrovirus vectors, lentivirus vectors, Sendai virus vectors, and the like. Among these, when a mammal such as a human is to be administered, a viral vector is preferable.

  In the expression vector, the DNA molecule encoding the FUS protein only needs to be operably linked to a promoter capable of exhibiting promoter activity in cells in vivo to be administered. The promoter used in the expression vector is not particularly limited as long as it can function in the living body to which it is administered, for example, SV40-derived promoter, cytomegalovirus LTR, rous sarcoma virus LTR, MoMuLV-derived LTR, adenovirus Virus promoters such as promoters derived from mammals; β-actin gene promoters, PGK gene promoters, transferrin gene promoters, nestin gene promoters and other mammalian constituent protein gene promoters, etc .; Synapsin I, prion gene promoters and other neuron-specific gene promoters It is done.

  The expression vector preferably contains a transcription termination signal downstream of the DNA molecule encoding the FUS protein. Furthermore, the expression vector may further contain a selection marker gene for selecting transformed cells.

(FUS protein production promoter)
The FUS protein production promoting substance is a substance that promotes the production of FUS protein in vivo. The production-promoting substance improves the production ability of the FUS protein inherent in the living body in the administered living body, and the produced FUS protein suppresses neurodegeneration and prevents progressive movement disorder in ALS or FTD. Since it exerts effects such as suppression and suppression of RAN translation on DPRs, it can be used as an active ingredient in ALS or FTD preventive or therapeutic agents.

  The production-promoting substance acts at any stage of FUS transcription, post-transcriptional regulation, translation, post-translational modification, localization, and folding, as long as it promotes the expression of FUS protein in vivo. There may be.

Other Components The preventive or therapeutic agent of the present invention may contain other pharmacologically active components effective for the prevention or treatment of ALS or FTD in addition to the above active ingredients.

  In addition to the active ingredient, the prophylactic or therapeutic agent of the present invention may contain a pharmaceutically acceptable carrier as necessary in order to prepare a desired dosage form and pharmaceutical form. Specific examples of pharmaceutically acceptable carriers include suspending agents such as methyl cellulose, polysorbate 80, hydroxyethyl cellulose, gum arabic, tragacanth powder, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate; polyoxyethylene Hardened castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, Magrogol and other solubilizing agents; human serum albumin, dextran, methylcellulose, gelatin, sodium sulfite, sodium metasulfite and other stabilizers; human Serum albumin, lecithin, dextran, ethylene oxide / propylene oxide copolymer, hydroxypropylcellulose, methylcellulose, polyoxyethylene hydrogenated castor oil, polyethylene Adsorption inhibitor such as glycol; excipients such as sucrose, starch, mannitol, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, Binders such as gum arabic, polyethylene glycol, sucrose, starch; disintegrators such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, calcium citrate, lubricants such as magnesium stearate; water, physiological Diluents such as saline; lipids; buffering agents; emulsifiers; coloring agents; flavoring agents;

  In addition, in the preventive or therapeutic agent of the present invention, if a DNA molecule encoding FUS protein is used as an active ingredient, transfection reagents (liposomes, polyamines, etc.) used in gene therapeutic agents are included. It may be.

The preventive or therapeutic agent dosage form dosage forms the present invention is not particularly limited, may be appropriately set according to the active substance used type and dosage forms and the like. As the dosage form of the preventive or therapeutic agent of the present invention, specifically, liquid preparations such as injections, syrups, cell suspensions, liposome preparations; tablets, hard capsules, soft capsules, granules, powders, Examples include solid preparations such as pills. In addition, in the case of an injection, it may be in the form of a powder for preparation (for example, freeze-dried powder) that is dissolved in physiological saline before use.

Target for administration The preventive or therapeutic agent of the present invention suppresses neurodegeneration, movement disorders, and RAN translation into DPRs in ALS or FTD, and can improve the symptoms of ALS or FTD. used.

  The G4C2 repeat sequence in the C9orf72 gene untranslated region is repeated about 2 to 24 times in healthy individuals. On the other hand, in an ALS or FTD patient who develops due to abnormal extension of the G4C2 repeat sequence, it is repeated about 30 times or more, and in some cases, several thousand repeats or more. The preventive or therapeutic agent of the present invention is suitably used for ALS or FTD that develops due to such abnormal extension of the G4C2 repeat sequence. In particular, in familial and sporadic ALS or FTD, abnormal extension of the G4C2 repeat sequence occurs frequently, and can be said to be a suitable application target of the preventive or therapeutic agent of the present invention.

  In the prophylactic or therapeutic agent of the present invention, the organism to be administered may be an organism suffering from ALS or FTD, and in addition to humans, rats, hamsters, guinea pigs, mice, cows, sheep, pigs, goats, Mammals such as monkeys and rabbits; birds such as chickens and ostriches; fish such as salmon and catfish. What is necessary is just to set the origin of the active ingredient used by this invention according to the kind of organism used as administration object. For example, when preventing or treating human ALS or FTD, the active ingredient is selected from human FUS protein, a DNA molecule encoding human FUS protein, and a substance that promotes production of human FUS protein. Use it.

Administration method Examples of the administration form of the preventive or therapeutic agent of the present invention include oral administration, topical administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intravenous administration, transrectal, intradermal administration, and the like. What is necessary is just to set suitably according to the kind of active ingredient to be used. Among these dosage forms, the prevention or treatment of the present invention preferably includes local administration.

  Regarding the dose of the preventive or therapeutic agent of the present invention, it is effective for the prevention or treatment of ALS or FTD depending on the type of active ingredient to be used, the age, sex, weight, symptom level, dosage form, etc. of the administration subject. For example, when FUS protein is used as an active ingredient, an amount of about 0.1 to 100,000 μg per day may be administered separately in one or several times. In addition, if a DNA molecule encoding FUS protein / or a FUS protein production promoting substance is used as an active ingredient, the amount of FUS protein produced in vivo is appropriately set within the above range. do it.

2. Abnormally extended G4C2 repeat sequence protein translation inhibitor As shown in the Examples section below, FUS protein, DNA molecules encoding FUS protein, and FUS protein production promoters were abnormally extended. It has the effect of suppressing protein translation caused by G4C2 repeat sequences. Therefore, the present invention further provides an abnormally elongated G4C2 repeat sequence comprising as an active ingredient at least one selected from the group consisting of a FUS protein, a DNA molecule encoding the FUS protein, and a FUS protein production promoter. A protein translation inhibitor resulting from The translation inhibitor can be used for the purpose of preventing or treating a disease associated with the formation of an aggregate of dipeptide repeat proteins resulting from an abnormally elongated G4C2 repeat sequence. In the translation inhibitor, the type, dosage form, administration method, and the like of the active ingredient are as described in the column “1. ALS or FTD preventive or therapeutic agent”.

  EXAMPLES The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.

1. Experimental materials and methods
1-1. Drosophila Stocks Drosophila stocks were all raised and bred at 23 ° C or 25 ° C with standard diet (containing glucose, corn meal, and dry yeast).

Transgenic Drosophila carrying the transgene of GMR-Gal4 is from Prof. Masamitsu Yamaguchi of Kyoto Institute of Technology, Transgenic Drosophila with UAS-EGFP (# 6874) and elav-Gal4 (# 8765) is Bloomington Drosophila Stock Obtained from the Center (Bloomington Drosophila Stock Center). Drosophila (caz ² mutant) having a mutation in caz, a homolog of Drosophila in FUS, was obtained from Dr. Erik Storkebaum at the Max Planck Institute, Germany.

  GMR is a tissue-specific promoter in the compound eye of Drosophila, and elav is a promoter site of a gene that is specifically expressed in the Drosophila nervous system. UAS is a sequence that activates its downstream transcription by Gal4 binding.

1-2. Generation of G4C2 repeat sequence constructs and transgenic Drosophila Transgenic Drosophila expressing abnormally extended G4C2 repeat sequences in compound eyes were established using the Gal4-UAS system. A specific manufacturing procedure is as follows.

Artificially synthesized (G4C2) _{50 in} which the 5 ′ terminal side was sandwiched by EagI recognition sequence and the 3 ′ terminal side was sandwiched by PspOMI recognition sequence was subcloned into T-vector pMD20. To generate a G4C2 repeat sequence with an increased number of repeats, pMD20- (G4C2) ₅₀ plasmid was treated with EagI and PspOMI to obtain a (G4C2) ₅₀ insert fragment and linearized with EagI pMD20- (G4C2) Ligation into ₅₀ plasmid vectors. The G4C2 repeat sequence in the ligated plasmid theoretically has a 50% probability that 100 GGGGCCs are linked repeatedly. The obtained plasmid was treated with EcoRI and HindIII, purified, and then subcloned into pcDNA ^™ 3.1 / myc-His (−) A vector (Invitrogen). The subcloned pcDNA ^™ 3.1 / myc-His (−) A- (G4C2) _n plasmid was amplified and sequenced. At this stage, a pcDNA ^™ 3.1 / myc-His (−) A- (G4C2) ₉ plasmid containing a G4C2 repeat sequence consisting of (G4C2) ₉ was accidentally obtained. Again, pcDNA ^™ 3.1 / myc-His (−) A- (G4C2) _n plasmid was treated with EcoRI and XbaI, purified, and then subcloned into the Drosophila pUAST vector. These constructs do not have an initiation codon (ATG) upstream of the G4C2 repeat sequence. These pUAST- (G4C2) _n plasmids were introduced into a recombinase-mutated SURE2 E. coli strain (Agilent Technologies) and amplified.

The pUAST-FUS plasmid, pUAST-ILF2 plasmid, and pUAST-hnRNPK plasmid were prepared using Geteway Vector Conversion System (Invitrogen). Human FUS cDNA (SEQ ID NO: 2), human ILF2 cDNA (SEQ ID NO: 3), and human hnRNPK cDNA (SEQ ID NO: 4) were subcloned into pENTR ^™ / D-TOPO plasmid (Invitrogen). In order to construct the Gateway destination vector pUAST-DEST, Gateway cassette A sequences (Invitrogen) was inserted into the pUAST vector. Using the plasmid, pUAST-DEST vector, restriction endonuclease, and ligase, pUAST-FUS plasmid, pUAST-ILF2 plasmid, and pUAST-hnRNPK plasmid were obtained. In addition, the FUS RRM mutation (FUS-RRMmut) construct (pUAST-FUS-RRMmut) was constructed by mutating the DNA sequence of the human FUS protein to mutate phenylalanine at amino acid positions 305, 341, 359, and 368. ) Was also produced. Each of the obtained plasmid DNAs was purified using PowerPrep HP Plasmid Maxiprep System (OriGene), and the number of GGGGCC repeats in the G4C2 repeat sequence was determined. For pUAST- (G4C2) ₈₉ plasmid, after induction of transcription factor insertion mutation using EZ-Tn5 ^TM <KAN-2> Insertion Kit (Epicentre), sequencing using the inserted sequence and repeated GGGGCC The number was determined.

Transgenic Drosophila into which the respective pUAST vectors were introduced were prepared by a standard method. pUAST- (G4C2) _n of transgenic Drosophila and _{UAS- (G4C2) n, UAS- (} G4C2) 89 is what many expression amount when crossed with GMR-Gal4 line (G4C2) ₈₉ (S), less This was designated as (G4C2) ₈₉ (W). In addition, a transgenic Drosophila in which the transgene is expressed in a desired tissue was produced by crossing the Gal4 line and the UAS line.

1-3. Repeat-primed PCR analysis
Genomic DNA was isolated from transgenic Drosophila using FastDNA Green SPIN Kit (MP-Biomedicals). G4C2 repeat sequence in genomic DNA is described in the literature (Renton, AE et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 72, 257-68 (2011).) Repeat-primed PCR was performed according to In addition, fluorescence fragment length analysis of PCR fragments was performed with ABI 3130xl genetic analyzer (Applied Biosystems) equipped with Peak Scanner ^™ software v1.0 (Applied Biosystems). Thus, the number of GGGGCC repeats in the introduced G4C2 repeat sequence was determined, and it was confirmed that the G4C2 repeat sequence consisting of (G4C2) ₉ , (G4C2) ₅₀ , and (G4C2) ₈₉ was introduced.

1-4. Imaging of Drosophila compound eyes and quantification of pigment abnormalities and size of the compound eyes
Using a stereo microscope SZX10 (Olympus) equipped with a CCD camera PD21 (Olympus), an optical microscope image of a Drosophila compound eye was obtained. Drosophila compound eye pigment abnormalities and size quantification are described in the literature (Saitoh, Y. et al. P62 Plays a Protective Role in the Autophagic Degradation of Polyglutamine Protein Oligomers in Polyglutamine Disease Model Flies. Journal of Biological Chemistry 290, 1442-1453 ( 2015).). At least 10 compound eyes were analyzed for each genotype, and results were expressed as mean ± SEM.

1-5. Analysis of viability from egg to adult Crossing female Drosophila with heterozygous elav-Gal4 driver and male Drosophila with homozygous UAS- (G4C2) _n did. In F1, Drosophila that express G4C2 repeat sequences in neurons (ie, Drosophila with elav-Gal4 driver and UAS- (G4C2) _n ) were born. Drosophila expressing the G4C2 repeat sequence do not have a CyO balancer (ie, the phenotype is [Cy ⁻ ]), and Drosophila that do not express the G4C2 repeat sequence have a CyO balancer (ie, the phenotype is [Cy ⁺ ]). ).

Hatchability from egg until adult was calculated by the following method: phenotype [Cy ^-] a number of Drosophila phenotype of dividing the number of Drosophila [Cy ^+], further multiplied by 100 relative The value was calculated as the survival rate (%) from egg to adult.

1-6. Motor ability analysis using climbing assay 10-20 male Drosophila were placed in a glass vial with a height of 150 mm and an inner diameter of 22 mm so as not to stimulate the individual. After standing for 5 minutes, gently tap the bottom of the glass vial and record the height at which Drosophila reached the glass vial wall in 10 seconds with a digital video camera. Score 2 to 3.9 cm, 1 to 4 to 5.9 cm, 2 to 6 to 7.9 cm, 3 to 8 to 9.9 cm, 5 to 10 and above Turned into. This experiment was carried out 5 times at intervals of 20 seconds, and the results were expressed as mean values ± SEM.

1-7. Life analysis
About 30 Drosophila per vial were fed with standard food after emergence and reared for life analysis. Every 2 or 3 days, it was transferred to a vial containing fresh food and the number of individuals who died at that time was counted. Drosophila were raised until all individuals died. For each group, experiments were performed using 3 vials or more, and statistical analysis was performed.

1-8. Fluorescence in situ hybridization (FISH)
The 3rd instar larvae were dissected in ice-cold PBS, the compound eye adult primordium was removed, fixed with PBS (RNase-free) containing 4% paraformaldehyde (pH 7.0) for 30 minutes, and further dehydrated with 100% methanol. The fixed sample was rehydrated with PBS containing 75 v / v%, 50 v / v%, and 25 v / v% ethanol, and rinsed with PBS and distilled water (distilled water treated with diethyl pyrocarbonate). Subsequently, it was treated with distilled water containing 0.2N HCl for 20 minutes at room temperature and rinsed with distilled water. Further, the sample was treated with PBS containing 0.2% Triton X-100, rinsed with PBS for 5 minutes, and then fixed with PBS containing 4% paraformaldehyde (pH 7.0) for 20 minutes. Thereafter, the plate was washed twice with PBS for 5 minutes, and incubated for 15 minutes in PBS containing 2 mg / mL glycine twice. After acetylation treatment, the cells were incubated for 1 hour at 37 ° C. in hybridization buffer (containing 50% formamide, 2 × SSC, 0.2 mg / mL yeast tRNA, and 0.5 mg / mL heparin). Thereafter, the sample was incubated with a 5 ′ terminal Alexa594-labeled (GGCCCC) ₄ LNA (locked nucleic acid) probe or Alexa488-labeled (CCGGGG) ₄ LNA probe (5 nM) in a hybridization buffer at 80 ° C. overnight. In addition, those LNA probes synthesized by GeneDesign Inc were used. After hybridization, wash once for 5 minutes in 4 × SSC at 80 ° C., wash 3 times for 20 minutes in a solution containing 2 × SSC at 80 ° C. and 50% formamide, 0.1 × at 80 ° C. Washing was performed 3 times for 40 minutes in SSC and 1 time in PBT (PBS containing 0.5% Triton X-100) at room temperature. Thereafter, the sample was subjected to nuclear staining with DAPI, mounted on a slide glass using a SlowFade gold anti-fading agent (Thermo Fisher Scientific), and observed with a confocal laser scanning microscope (Zeiss LSM710).

1-9. Immunofluorescence staining
The 3rd instar larvae were dissected in ice-cold PBS to remove the compound eye adult primordium, fixed with PBS containing 4% paraformaldehyde (pH 7.0) for 30 minutes, and then washed 3 times with PBT. Next, after blocking the sample with PBT containing 5% goat serum, rat monoclonal anti-poly (GR) antibody (clone 5A2, Millipore MABN778), rabbit polyclonal anti-poly (GP) antibody (Cosmo Bio CAC-TIP) were used as primary antibodies. -C9-P01), or rabbit polyclonal anti-poly (GA) antibody (Novus Biologicals NBP2-25018) at 4 ° C. overnight. After washing 3 times with PBT, it was incubated with Alexa 633 labeled anti-rat IgG antibody (Invitrogen A-21094) or Alexa 488 labeled anti-rabbit IgG antibody (Invitrogen A-11008) as a secondary antibody. After washing with PBT three times, the cells were stained with DAPI, mounted on a slide glass using a SlowFade gold antifading agent, and observed with a confocal laser scanning microscope (Zeiss LSM710).

1-10. Poly (GP) protein measurement
The heads of 5-day-old Drosophila expressing EGFP, FUS, or FUS-RRMmut with G4C2 repeat sequence or (G4C2) ₈₉ were collected and stored at -80 ° C. Samples were prepared according to the method described in the literature (Tran, H. et al. Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD / ALS. Neuron 87, 1207-14 (2015).). Poly (GP) is described in the literature (Su, Z. et al. Discovery of a biomarker and lead small molecules to target r (GGGGCC) -associated defects in c9FTD / ALS. Neuron 83, 1043-50 (2014).) It was measured by sandwich ELISA method. Data were normalized by values when expressing EGFP and (G4C2) ₈₉ .

1-11. Quantification of aggregate number of DPRs
The number of aggregates of DPRs in the Drosophila compound eye disc was quantitatively measured using ZEN Imagen Software (Zeiss). The procedure is as follows. First, 13 eye photoreceptor neurons in the middle of 2 and 3 rows from the rear end of the compound eye disc were selected by DAPI staining. Next, the number of aggregates of DPRs having a diameter larger than 2 μm and localized in the cytoplasm was counted. Ten or more compound eye discs were analyzed for each genotype, and the results were expressed as mean ± SEM.

1-12. Statistical analysis All statistical analyzes were performed using public domain R program (http://www.r-project.org/) or GraphPad Prism 6 software.

2. Experimental result
2-1. Properties of transgenic Drosophila expressing an abnormally elongated G4C2 repeat sequence
2-1-1. Compound eye abnormal status elongated G4C2 repeat sequence diagram a result of observation by _{((G4C2) 50, (G4C2} ) 89 (W), and (G4C2) ₈₉ (S)) optical compound eye of transgenic Drosophila expressing microscope It is shown in 1. For comparison, FIG. 1 also shows the results of observation of compound eyes of transgenic Drosophila expressing EGFP or a normal G4C2 repeat sequence ((G4C2) ₉ ) with an optical microscope.

As can be seen from FIG. 1, transgenic Drosophila expressing an abnormally elongated G4C2 repeat sequence showed intense compound eye neurodegeneration, especially when it was expressed at (G4C2) ₅₀ and was killed at 25 ° C. . On the other hand, in Drosophila expressing EGFP or normal G4C2 repeat sequence, no abnormality was observed in the compound eye.

  That is, from this result, it was possible to confirm neurodegeneration, which is a typical symptom of ALS / FTD, in transgenic Drosophila expressing an abnormally elongated G4C2 repeat sequence.

2-1-2. For adult Drosophila expressing G4C2 repeat sequences ((G4C2) ₅₀ , (G4C2) ₈₉ (W)) or normal G4C2 repeat sequences ((G4C2) ₉ ) with an increased survival rate and adult lifespan abnormalities, The results of evaluating the survival rate up to are shown in FIG. 2A, and the results of evaluating the lifespan of adults are shown in FIG. 2B. From these results, transgenic Drosophila expressing an abnormally elongated G4C2 repeat sequence has a lower survival rate to adults than that of a normal G4C2 repeat sequence, and the adult life span is 1 / 4-1 It became clear that it was shortened to about / 5.

2-1-3. Results of climbing assay for transgenic Drosophila expressing G4C2 repeat sequence ((G4C2) ₅₀ , (G4C2) ₈₉ (W)) or normal G4C2 repeat sequence ((G4C2) ₉ ) with abnormal motor ability 3 shows. From this result, it was found that the transgenic Drosophila expressing the abnormally extended G4C2 repeat sequence had a markedly lower score in the climbing assay than the case of expressing the normal G4C2 repeat sequence. Especially in transgenic Drosophila expressing (G4C2) ₈₉ , the score decreased significantly with aging. That is, it was confirmed that the transgenic Drosophila expressing the abnormally elongated G4C2 repeat sequence exhibits progressive movement disorder, which is a typical symptom of ALS / FTD.

2-1-4. Transcription of abnormally elongated G4C2 repeat sequences and RAN translation Abnormally elongated G4C2 repeat sequences ((G4C2) ₈₉ (S)) in transgenic Drosophila compound eyes, RNA transcribed from the repeat sequences, and DPRs (poly (GR ), Poly (GA), and poly (GP)) are analyzed by FISH and immunostaining, and the results are shown in FIG.

  As a result, in transgenic Drosophila expressing an abnormally extended G4C2 repeat sequence, RNA transcribed from the abnormally extended G4C2 repeat sequence is expressed, and DPRs (poly (GR), It was confirmed that poly (GA) and poly (GP)) were also formed with aggregates.

2-1-5. Summary In transgenic Drosophila that expresses abnormally elongated G4C2 repeat sequences, aggregates of RNA transcribed with G4C2 repeat sequences and DPRs that are RAN translation products of the RNA are observed, and neurodegeneration and movement disorders are also exhibited. The lifespan was shortened. These events are typically observed in ALS / FTD, indicating that the transgenic Drosophila can be used as a model animal for ALS / FTD.

2-2. Effects of proteins capable of binding to G4C2 repeat sequences on transgenic Drosophila expressing abnormally extended G4C2 repeat sequences FUS, ILF2 (Interleukin) with abnormally extended G4C2 repeat sequences ((G4C2) ₈₉ (S)) or EGFP FIG. 5 shows the result of observation of compound eyes of transgenic Drosophila expressing enhancer-binding factor 2) or hnRNPK (Heterogeneous nuclear ribonucleoprotein K) with an optical microscope.

  FUS, ILF2, and hnRNPK have been reported to be capable of binding to RNA transcribed from G4C2 repeat sequences. FUS inhibited compound eye neurodegeneration due to abnormally elongated G4C2 repeat sequences. On the other hand, ILF2 and hnRNPK did not inhibit compound eye neurodegeneration due to abnormally elongated G4C2 repeat sequences. That is, from this result, it became clear that the symptom improvement effect caused by the abnormally extended G4C2 repeat sequence is unique to FUS.

2-3. Effects of FUS on transgenic Drosophila expressing abnormally elongated G4C2 repeat sequences
2-3-1. FIG. 6 shows the results of a climbing assay performed on transgenic Drosophila expressing FUS or EGFP in the nervous system together with a G4C2 repeat sequence ((G4C2) ₅₀ ) with abnormally extended motor ability . For comparison, the results of transgenic Drosophila expressing FUS together with EGFP are also shown in FIG.

  As shown in FIG. 6, when FUS was expressed together with an abnormally elongated G4C2 repeat sequence, movement disorder could be effectively suppressed.

2-3-2. With compound eye abnormal status elongated G4C2 repeat sequence _{((G4C2) 89 (S)} ), FUS, FUS-RRMmut, or the transgenic Drosophila expressing EGFP in compound eye, results and compound eye observing the compound-eye with an optical microscope The result of measuring the size is shown in FIG. For comparison, FIG. 7 also shows the results of transgenic Drosophila expressing FUS, FUS-RRMmut, or EGFP together with EGFP.

  As a result, when FUS was expressed together with abnormally elongated G4C2 repeat sequences, compound eye neurodegeneration could be effectively suppressed. In addition, in the case of expressing FUS-RRMmut with a mutation in the RNA recognition motif region of FUS, compound eye neurodegeneration could not be suppressed. That is, these results revealed that FUS suppresses neurodegeneration by binding to RNA transcribed from the G4C2 repeat sequence.

2-3-3. Transcription of abnormally elongated G4C2 repeat sequences and RAN translation Abnormally extended G4C2 repeat sequences ((G4C2) ₈₉ (S)), as well as DPRs (poly (GR), poly (GA) in compound Drosophila expressing FUS or EGFP ) And poly (GP)) are shown in FIG. 8A. In addition, the results of measuring the number of aggregates of each DRPs in the transgenic Drosophila are also shown in FIG. 8B. Further, FIG. 8C shows the result of measuring the amount of poly (GP) in transgenic Drosophila expressing FUS, FUS-RRMmut, or EGFP together with the abnormally extended G4C2 repeat sequence ((G4C2) ₈₉ (S)) by ELISA. Shown in

  From this result, it was revealed that the number of DPRs aggregates and the amount of poly (GP) were reduced by expressing FUS in transgenic Drosophila expressing an abnormally elongated G4C2 repeat sequence.

2-3-4. Effect abnormality extended G4C2 repeat sequence of the Drosophila FUS homolog caz variant _{((G4C2) 89 (S)} ) or EGFP expressing and caz ² mutations introduced transgenic Drosophila (caz ² / +), a compound eye The results of observation with an optical microscope and the results of measurement of eye size are shown in FIG. For comparison, the results of transgenic Drosophila not introduced caz ² mutation (+ / +) also shown in FIG.

  As a result, it was confirmed that compound eye neurodegeneration was exacerbated when caz mutation, a Drosophila FUS homolog, was introduced.

Claims (5)

  Amyotrophic lateral sclerosis or frontotemporal dementia comprising, as an active ingredient, at least one selected from the group consisting of FUS protein, DNA molecule encoding FUS protein, and FUS protein production promoter Preventive or therapeutic agent.   The prophylactic or therapeutic agent according to claim 1, which is applied to familial amyotrophic lateral sclerosis or familial frontotemporal dementia.   The preventive or therapeutic agent according to claim 1 or 2, which is used for improving neurodegeneration or movement disorder in amyotrophic lateral sclerosis or frontotemporal dementia.   The preventive or therapeutic agent according to any one of claims 1 to 3, wherein the active ingredient is a FUS protein or a DNA molecule encoding the FUS protein.   A protein translation inhibitor resulting from an abnormally elongated GGGGCC repeat sequence, comprising as an active ingredient at least one selected from the group consisting of a FUS protein, a DNA molecule encoding the FUS protein, and a FUS protein production promoter .