WO2016002854A1 - Préparation utilisée pour favoriser la prolifération de cellules souches neurales, préparation utilisée pour la prévention ou le traitement de maladies associées à une diminution des cellules souches neurales, préparation utilisée pour favoriser la formation de postsynapses, préparation utilisée pour la prévention ou le traitement de maladies associées à une diminution de la formation de postsynapses, et procédé de criblage - Google Patents

Préparation utilisée pour favoriser la prolifération de cellules souches neurales, préparation utilisée pour la prévention ou le traitement de maladies associées à une diminution des cellules souches neurales, préparation utilisée pour favoriser la formation de postsynapses, préparation utilisée pour la prévention ou le traitement de maladies associées à une diminution de la formation de postsynapses, et procédé de criblage Download PDF

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WO2016002854A1
WO2016002854A1 PCT/JP2015/069032 JP2015069032W WO2016002854A1 WO 2016002854 A1 WO2016002854 A1 WO 2016002854A1 JP 2015069032 W JP2015069032 W JP 2015069032W WO 2016002854 A1 WO2016002854 A1 WO 2016002854A1
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dna
pqbp
seq
neural stem
base sequence
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PCT/JP2015/069032
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Japanese (ja)
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岡澤　均
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国立大学法人東京医科歯科大学
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Publication of WO2016002854A1 publication Critical patent/WO2016002854A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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
    • G01N33/5073—Stem cells

Definitions

  • the present invention relates to a preparation used for promoting proliferation of neural stem cells, a preparation used for preventing or treating a disease associated with a decrease in neural stem cells, a preparation used for promoting post-synaptic formation, and a decrease in post-synaptic formation.
  • the present invention relates to a preparation used for prevention or treatment of a disease, and a screening method.
  • polyglutamine-tract binding protein 1 (hereinafter referred to as PQBP-1 in the present specification) is a protein responsible for an intermediate pathology in a degenerative disease polyglutamine disease (for example, see Non-patent Document 1). ).
  • Non-Patent Document 2 X chromosome linkage intellectual disability / mental retardation families
  • PQBP-1 has been reported to be a causative gene for intellectual disabilities and to be involved in RNA splicing. However, PQBP-1 is actually associated with any intellectual disability. It is still unknown.
  • the present invention relates to a preparation for use in promoting proliferation of neural stem cells containing a predetermined DNA encoding PQBP-1 or a polypeptide encoded by this DNA as an active ingredient, prevention of diseases associated with the reduction of neural stem cells Alternatively, it is an object of the present invention to provide a preparation used for treatment, a preparation used for promoting post-synapse formation, a preparation used for prevention or treatment of a disease associated with a decrease in post-synapse formation, and a screening method.
  • the present inventors have found that in mice knocked out with the PQBP-1 gene, the cell cycle of neural stem cells is increased and the formation of posterior synapses is reduced, and the present invention has been completed. More specifically, the present invention provides the following.
  • a disease associated with a decrease in neural stem cells comprising as an active ingredient the DNA according to any one of the following (a) to (d) encoding PQBP-1 or a polypeptide encoded by this DNA: A preparation used for prevention or treatment.
  • A DNA having the base sequence described in SEQ ID NO: 1
  • B DNA having a base sequence capable of hybridizing under stringent conditions with the base sequence described in SEQ ID NO: 1
  • C a DNA that encodes an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 2 and that has a base sequence that promotes proliferation of neural stem cells
  • D DNA comprising a nucleotide sequence having 90% or more homology with the nucleotide sequence of SEQ ID NO: 1 and promoting proliferation of neural stem cells
  • DNA of the following (a), (b), (e) or (f) that encodes PQBP-1, or a polypeptide encoded by this DNA as an active ingredient, Formulation used for promotion (A) DNA having the base sequence described in SEQ ID NO: 1 (B) DNA having a base sequence capable of hybridizing under stringent conditions with the base sequence described in SEQ ID NO: 1 (E) a DNA that encodes an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 2 and that has a base sequence that promotes post-synaptic formation (F) DNA comprising a base sequence having 90% or more homology with the base sequence described in SEQ ID NO: 1 and promoting post-synaptic formation
  • A DNA having the base sequence described in SEQ ID NO: 1
  • B DNA having a base sequence capable of hybridizing under stringent conditions with the base sequence described in SEQ ID NO: 1
  • E a DNA that encodes an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 2 and that has a base sequence that promotes post-synaptic formation
  • F DNA comprising a base sequence having 90% or more homology with the base sequence described in SEQ ID NO: 1 and promoting post-synaptic formation
  • a method for screening a candidate substance for a preparation used for the prevention or treatment of a disease associated with a decrease in neural stem cells Administering a test substance to an animal cell or an animal other than a human; Measuring the expression level of endogenous PQBP-1 or the degree of promotion of neural stem cell proliferation by PQBP-1 in the animal cells or animals other than humans to which the test substance has been administered; Selecting a test substance as a candidate substance of a preparation used for the prevention or treatment of a disease associated with a decrease in neural stem cells based on the measurement result.
  • a method for screening a candidate substance for a preparation used for the prevention or treatment of a disease associated with a decrease in post-synaptic formation Administering a test substance to an animal cell or an animal other than a human; Measuring the expression level of endogenous PQBP-1 or the degree of promotion of post-synaptic formation by PQBP-1 in the animal cells or animals other than humans to which the test substance has been administered; Selecting a test substance as a candidate substance for a preparation used for the prevention or treatment of a disease associated with a decrease in post-synaptic formation based on the measurement result.
  • the present invention can provide a preparation used for the prevention or treatment of the above, a preparation used for promoting post-synapse formation, a preparation used for prevention or treatment of a disease associated with a decrease in post-synapse formation, and a screening method.
  • FIG. 1 It is a figure which shows the image which analyzed the brain shape of the patient with which PQBP-1 gene was mutated by the magnetic resonance image.
  • FIG. 1 (a) is a horizontal plane image, (b) is a coronal plane image, and (c) is a sagittal plane image.
  • It is a photograph of the brains of a Nestin-Cre male and female adult mouse (2 months) knocked out of the PQBP-1 gene and a male and female adult mouse (2 months) not knocked out of the PQBP-1 gene.
  • FIG. 2 is a graph showing brain masses of Nestin-Cre male and female adult mice (2 months) knocked out with PQBP-1 gene and adult male and female mice (2 months) not knocked out with PQBP-1 gene.
  • FIG. 7 is a graph showing the brain masses of Synapsin1-Cre male and female adult mice (2 months) knocked out of the PQBP-1 gene and male and female adult mice not knocked out of the PQBP-1 gene, (a) Is a graph for male mice, and (b) is a graph for female mice.
  • FIG. 1 It is a photograph of the coronal plane of a Nestin-Cre male and female adult mouse (2 months) knocked out of the PQBP-1 gene and a male and female adult mouse (2 months) not knocked out of the PQBP-1 gene.
  • (A) is a photograph of a male adult mouse in which the PQBP-1 gene is not knocked out
  • (b) is a photograph of a Nestin-Cre male adult mouse in which the PQBP-1 gene is knocked out
  • (c) is a photograph.
  • D is a photograph of a Nestin-Cre female adult mouse in which the PQBP-1 gene was knocked out.
  • FIG. 6 is a graph of the relative percentage of neurogenesis, number of cells remaining in the stem cell pool, number of unlabeled neural stem cells 24 hours after intraperitoneal injection of BrdU into pregnant mice. It is a graph of the level of cerebral cortex cell death of Nestin-Cre-cKO mice knocked out of PQBP-1 gene and Nestin-Cre mice knocked out of PQBP-1 gene by TUNEL staining.
  • A shows a wild type mouse, a Nestin-Cre-cKO mouse knocked out of the PQBP-1 gene, a wild type mouse transformed with APC4, and a PQBP-1 gene transformed with APC4.
  • FIG. 1 It is a graph which shows the proliferation of the neural stem cell prepared from each E14 embryo of a Nestin-Cre-cKO mouse.
  • B is a photograph of the results of analyzing the expression levels of APC4, PQBP-1, Cyclin B, and GAPDH in each mouse embryo by Western blotting. According to FACS analysis, (a) a wild-type mouse, (b) a Nestin-Cre-cKO mouse knocked out of the PQBP-1 gene, and (c) APC4-transformed Nestin-Cre knocked out of the PQBP-1 gene -A graph showing the number of cells in neural stem cells extracted from E14 embryos of each mouse of cKO mice and (d) wild-type mice transformed with APC4.
  • Brain tissue of E18 after introducing APC4-GFP or GFP into each E13 embryo of a Nestin-Cre-cKO mouse knocked out of the PQBP-1 gene and a Nestin-Cre mouse knocked out of the PQBP-1 gene It is a photograph of. Brain tissue of E18 after introducing APC4-GFP or GFP into each E13 embryo of a Nestin-Cre-cKO mouse knocked out of the PQBP-1 gene and a Nestin-Cre mouse knocked out of the PQBP-1 gene It is a graph of the ratio of the buffy coat surface with respect to the apical surface in FIG.
  • Nestin-Cre male adult mice (2.5 months) knocked out with the PQBP-1 gene and male adult mice (2.5 months) knocked out with the PQBP-1 gene and introduced AAV (AAV-PQBP-1 vector) Month) is a graph of the ratio of the relative thickness of each layer to the total thickness of the cortex.
  • Nestin-Cre-cKO adult mouse knocked out of PQBP-1 gene and introduced with AAV (AAV-PQBP-1 vector) (3 months) Is a graph of the average time each mouse falls off the rod in a rotating rod test.
  • FIG. 6 is a graph of mouse rest time in a conditioned conditioning study for Cre-cKO adult mice (3 months). In a rotating rod test for wild type adult mice (3 months), Flox adult mice (3 months), Synapsin1-Cre-cKO adult mice knocked out of the PQBP-1 gene (3 months) It is a graph of the average time which falls from.
  • Fia for wild-type mouse (3 months), Flox mouse (3 months), Synapsin1-Cre adult mouse (3 months), Synapsin1-Cre-cKO mouse wild-type adult mouse (3 months) knocked out PQBP-1 gene It is a graph of the time when the mouse stopped in the conditioning test. It is a figure which shows the extent of the postsynaptic formation of Synapsin1-Cre mouse
  • A shows a spine image of a cerebral cortical neuron of a wild type mouse administered with an AAV-CMV-EGFP viral vector
  • B shows a 5 ⁇ FAD mouse administered with an AAV-CMV-EGFP viral vector
  • C shows an image of spine of cerebral cortex neurons of 5xFAD mice administered with AAV-CMV-EGFP virus vector and AAV-CMV-PQBP-1 virus vector. Show.
  • the preparation of the present invention contains a predetermined DNA encoding polyglutamine-tract binding protein 1 (PQBP-1) or a polypeptide encoded by this DNA as an active ingredient.
  • PQBP-1 polyglutamine-tract binding protein 1
  • a polypeptide encoded by this DNA as an active ingredient.
  • the preparation used for promoting the proliferation of neural stem cells of the present invention comprises the DNA according to any one of the following (a) to (d) encoding PQBP-1, or a polypeptide encoded by this DNA as an active ingredient: contains.
  • A DNA having the base sequence described in SEQ ID NO: 1
  • B DNA having a base sequence capable of hybridizing under stringent conditions with the base sequence described in SEQ ID NO: 1
  • C a DNA that encodes an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 2 and that has a base sequence that promotes proliferation of neural stem cells
  • D DNA comprising a nucleotide sequence having 90% or more homology with the nucleotide sequence of SEQ ID NO: 1 and promoting proliferation of neural stem cells
  • the present inventors have found that the cell cycle is prolonged by becoming PQBP-1 abnormal. By extending the cell cycle, the number of divisions of neural stem cells is reduced and neural stem cells are reduced. However, according to the preparation of the present invention, the cell cycle can be prevented from being prolonged, and thus the decrease in neural stem cells can be suppressed.
  • the preparation for use in the prevention or treatment of a disease associated with a decrease in neural stem cells of the present invention is a DNA according to any one of the following (a) to (d) encoding PQBP-1, or the DNA encoding this A polypeptide is contained as an active ingredient.
  • A DNA having the base sequence described in SEQ ID NO: 1
  • B DNA having a base sequence capable of hybridizing under stringent conditions with the base sequence described in SEQ ID NO: 1
  • C a DNA that encodes an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 2 and that has a base sequence that promotes proliferation of neural stem cells
  • D DNA comprising a nucleotide sequence having 90% or more homology with the nucleotide sequence of SEQ ID NO: 1 and promoting proliferation of neural stem cells
  • disease associated with a decrease in neural stem cells refers to a disease caused by a decrease in neural stem cells, such as microcephaly (for example, microcephaly with no abnormality in the shape of the brain).
  • congenital intellectual disabilities such as mental retardation, and developmental disorders such as autism and learning disabilities.
  • progenital intellectual disorders such as mental retardation, and developmental disorders such as autism and learning disabilities.
  • progenital intellectual disorders such as mental retardation, and developmental disorders such as autism and learning disabilities.
  • progenital intellectual disorders such as mental retardation, and developmental disorders such as autism and learning disabilities. It refers to an effect in which symptoms are improved or symptom deterioration is suppressed, and improvement or suppression is statistically significant.
  • microcephaly because it has a particularly high preventive or therapeutic effect.
  • the preventive or therapeutic effect of a disease associated with a decrease in neural stem cells is particularly effective for human males in the case of an X-linked disease.
  • the preparation used for promoting post-synaptic formation according to the present invention is a DNA according to the following (a), (b), (e) or (f) encoding PQBP-1, or a polypeptide encoded by this DNA Is contained as an active ingredient.
  • A DNA having the base sequence described in SEQ ID NO: 1
  • B DNA having a base sequence capable of hybridizing under stringent conditions with the base sequence described in SEQ ID NO: 1
  • E a DNA that encodes an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 2 and that has a base sequence that promotes post-synaptic formation
  • F DNA comprising a base sequence having 90% or more homology with the base sequence described in SEQ ID NO: 1 and promoting post-synaptic formation
  • the preparation for use in the prevention or treatment of a disease associated with a decrease in post-synaptic formation of the present invention is a DNA according to the following (a), (b), (e) or (f) encoding PQBP-1, Alternatively, it contains a polypeptide encoded by this DNA as an active ingredient.
  • A DNA having the base sequence described in SEQ ID NO: 1
  • B DNA having a base sequence capable of hybridizing under stringent conditions with the base sequence described in SEQ ID NO: 1
  • E a DNA that encodes an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 2 and that has a base sequence that promotes post-synaptic formation
  • F DNA comprising a base sequence having 90% or more homology with the base sequence described in SEQ ID NO: 1 and promoting post-synaptic formation
  • disease associated with decreased post-synaptic formation refers to a disease caused by decreased post-synaptic formation, such as dementia such as Alzheimer's disease, amyotrophic lateral sclerosis, mental Examples include developmental delay, frontotemporal dementia, Huntington's disease, Parkinson's disease, generalized Lewy body disease, and spinocerebellar ataxia.
  • the prevention or treatment effect of a disease associated with a decrease in post-synaptic formation means that at least one symptom of dementia such as Alzheimer's disease, amyotrophic lateral sclerosis, mental retardation is improved or worsened It refers to an effect that is suppressed and for which improvement or suppression is statistically significant. Among these, it is preferable to use it for Alzheimer's disease, amyotrophic lateral sclerosis, and mental retardation because it has a particularly high preventive or therapeutic effect.
  • the DNA of the present invention is any one of the above (a) to (f).
  • SEQ ID NO: 1 is a base sequence encoding human PQBP-1.
  • the base sequence described in SEQ ID NO: 1 promotes proliferation and synapse formation of neural stem cells.
  • a base sequence directly promotes proliferation or synapse formation of neural stem cells means that a polypeptide encoded by the base sequence promotes proliferation or synapse formation of neural stem cells.
  • Variants and homologues of DNA having the base sequence described in SEQ ID NO: 1 include, for example, DNA having a base sequence capable of hybridizing with the base sequence described in SEQ ID NO: 1 under stringent conditions.
  • stringent conditions for example, the reaction is carried out in a usual hybridization buffer at 40 to 70 ° C.
  • the conditions include washing in a washing solution having a concentration of 15 to 300 mM (preferably 15 to 150 mM, more preferably 15 to 60 mM, and still more preferably 30 to 50 mM).
  • SEQ ID NO: 2 is known to constitute human PQBP-1 protein.
  • the DNA of the present invention also includes DNA having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2.
  • “one or more” is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids, 1 amino acid).
  • it is desirable that the amino acid residue to be mutated is mutated to another amino acid in which the properties of the amino acid side chain are conserved.
  • hydrophobic amino acids A, I, L, M, F, P, W, Y, V
  • hydrophilic amino acids R, D, N, C, E, Q, G, H, K, S, T
  • amino acids having aliphatic side chains G, A, V, L, I, P
  • amino acids having hydroxyl group-containing side chains S, T, Y
  • sulfur atom-containing side chains Amino acids (C, M) having carboxylic acids and amide-containing side chains (D, N, E, Q), amino groups having base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses represent single letter amino acids).
  • amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence described in SEQ ID NO: 2 is preferably highly homologous to the amino acid sequence described in SEQ ID NO: 2.
  • the homology between the amino acid sequence described in SEQ ID NO: 2 (amino acid sequence of human PQBP-1) and the amino acid sequence of mouse PQBP-1 is 86.8% (Hitoshi Okagawa et al., “PQBP-1”).
  • Np / PQ A polyglutamine tract-binding and nuclear inclusion-forming protein, Brain Research Bulletin, 2001, Vol. 56, Nos. 3/4, pp. 273-280, human PQP mouse, protein PQP.
  • the homology between the amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence described in SEQ ID NO: 2 and the amino acid sequence described in SEQ ID NO: 2 is preferably 85% or more. Is more preferably 90% or more, still more preferably 95% or more (96% or more, 97% or more, 98% or more, 99% or more).
  • the most preferred embodiment of the above DNA is a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.
  • the DNA of the present invention further contains a prophylactic effect on the proliferation of neural stem cells or the prevention of diseases related to the reduction of neural stem cells or Examples include various mutants and homologs having therapeutic effects, and various mutants and homologs having preventive or therapeutic effects for diseases associated with promoting effects of post-synaptic formation or decreasing post-synaptic formation.
  • the N-terminal WW domain (SEQ ID NO: 3) and the C-terminal C terminal domain (SEQ ID NO: 4) of PQBP-1 are known.
  • the WW domain is a sequence that plays an important role in the action with RNA polymerase, and is known to bind specifically to proline-rich sequences.
  • the C terminal domain is known as a naturally denatured protein that plays an important role in splicing.
  • the DNA of the present invention preferably contains one or both of SEQ ID NOs: 3 and 4 in order to exhibit the function of PQBP-1.
  • DNA variants and homologs having the base sequence described in SEQ ID NO: 1 include DNA comprising a base sequence having high homology with the base sequence described in SEQ ID NO: 1. Such DNA preferably has 90% or more, more preferably 95% or more (96% or more, 97% or more, 98% or more, 99% or more) of homology with the base sequence shown in SEQ ID NO: 1.
  • the homology between amino acid sequences and base sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) by Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990).
  • the “DNA” in the present invention may be either a sense strand or an antisense strand (for example, can be used as a probe), and the shape thereof may be either single-stranded or double-stranded. Further, it may be genomic DNA, cDNA, or synthesized DNA.
  • the method for obtaining the DNA of the present invention is not particularly limited, but a method for obtaining cDNA by reverse transcription from mRNA (for example, RT-PCR method), a method for preparing from genomic DNA, a method for synthesis by chemical synthesis, A known method such as a method of isolating from a genomic DNA library or a cDNA library (for example, see JP-A-11-29599) can be mentioned.
  • polypeptide used in the preventive or therapeutic agent of the present invention is encoded by the above-described DNA, and has, for example, the amino acid sequence set forth in SEQ ID NO: 2, or one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2 It has a substituted, deleted and / or added amino acid sequence.
  • the polypeptide encoded by the DNA of the present invention can be produced, for example, by using a transformant into which an expression vector containing the aforementioned DNA has been introduced. That is, first, the transformant is cultured under appropriate conditions to synthesize a protein (polypeptide) encoded by the DNA. Then, the polypeptide of the present invention can be obtained by recovering the synthesized protein from the transformant or the culture solution.
  • the culture of the transformant is appropriately selected from known nutrient media according to the type of transformant so that the polypeptide can be easily obtained in large quantities, and the temperature, pH of the nutrient medium, culture time, etc. It can be carried out with appropriate adjustment (for example, see JP-A-11-29599).
  • a method for isolating and purifying a polypeptide is not particularly limited, and a known method such as a method using solubility, a method using a difference in molecular weight, a method using charge, etc. (for example, JP-A-11-29599). Gazette).
  • a known method such as a method using solubility, a method using a difference in molecular weight, a method using charge, etc. (for example, JP-A-11-29599). Gazette).
  • the vectors and transformants that can be used in the present invention are described below.
  • An expression vector can be prepared by inserting the above-described DNA into an appropriate vector.
  • the “appropriate vector” is not particularly limited as long as it can be replicated and maintained in various prokaryotic and / or eukaryotic hosts, and can be appropriately selected depending on the purpose of use. For example, a high copy vector can be selected to obtain a large amount of DNA, and an expression vector can be selected to obtain a polypeptide. Specific examples thereof are not particularly limited, and examples thereof include known vectors described in JP-A-11-29599.
  • the vector can be used not only for synthesizing the polypeptide but also for the preparation of the present invention. That is, the preparation of the present invention containing the above-described vector incorporating the DNA is directly introduced into humans to promote proliferation of neural stem cells, prevention or treatment of diseases related to the decrease of neural stem cells, and formation of post-synaptic formation. It can be used for the prevention or treatment of diseases associated with promoting or reducing post-synaptic formation.
  • a vector that can be introduced into a human cell is used.
  • An example of such a vector is an AAV vector.
  • a transformant can be prepared by introducing a vector containing the aforementioned DNA into a host.
  • a host is not particularly limited as long as it is compatible with the vector of the present invention and can be transformed.
  • Specific examples thereof include known natural cells such as bacteria, yeasts, animal cells, and insect cells. Alternatively, artificially established cells (see JP-A-11-29599), or animals such as humans and mice can be mentioned.
  • the vector introduction method can be appropriately selected according to the type of the vector and the host. Specific examples thereof are not particularly limited. For example, when a bacterium is used as a host, known methods such as a protoplast method and a competent method (for example, see JP-A-11-29599) can be mentioned.
  • a human when used as a host, for example, when transforming into a pregnant human using the above-described AAV vector, it can be transformed by injection into the abdomen.
  • active ingredient refers to an effect of promoting proliferation of neural stem cells, an effect of preventing or treating a disease associated with a decrease in neural stem cells, an effect of promoting post-synaptic formation, or a disease associated with a decrease in post-synaptic formation It refers to a component contained in an amount necessary to obtain a prophylactic or therapeutic effect, and other components may be contained as long as the effect is not impaired to less than a desired level. Further, the administration route of the preparation may be either oral or parenteral, and is appropriately set.
  • the formulation may contain commonly used additives such as binders, inclusion agents, excipients, lubricants, disintegrants, wetting agents, tablets, granules, fine granules It is formulated into various forms such as powders and capsules.
  • the preparation may be in a liquid state such as an internal solution, suspension, emulsion syrup or the like, or may be in a dry state that is redissolved when used.
  • the formulation may also contain additives such as stabilizers, buffers, preservatives, isotonic agents and the like, usually contained in a unit dose ampoule or a multidose container or tube. It is distributed in the state.
  • the preparation may be formulated into a powder that can be re-dissolved with an appropriate carrier (such as sterile water) at the time of use.
  • the above-mentioned preparation of the present invention can be administered to humans in the manner described above to prevent or treat a disease associated with a decrease in neural stem cells or a disease associated with a decrease in post-synaptic formation. It can be used for prevention or treatment.
  • the screening method according to the present invention is a method for screening a candidate substance for a preparation used for the prevention or treatment of a disease associated with a decrease in neural stem cells, or a preparation used for the prevention or treatment of a disease associated with a decrease in posterior synapse formation. This is a screening method for candidate substances.
  • the method for screening a candidate substance of a preparation used for the prevention or treatment of a disease associated with a decrease in neural stem cells comprises a step of administering a test substance to animal cells or animals other than humans, and a test substance is administered.
  • Candidate substances for preparations used for the prevention or treatment of diseases related to the reduction of neural stem cells based on the step of measuring the degree of promotion of neural stem cell proliferation by PQBP-1 in animal cells or animals other than humans Selecting a test substance as
  • the method for screening a candidate substance for a preparation used for the prevention or treatment of a disease associated with a decrease in post-synaptic formation comprises a step of administering a test substance to animal cells or animals other than humans, and a test substance is administered. For measuring the degree of promotion of post-synaptic formation by PQBP-1 in animals other than human cells or animals other than humans, and formulations used for the prevention or treatment of diseases associated with decreased post-synaptic formation based on the measurement results Selecting a test substance as a candidate substance.
  • a test substance in which symptom improvement is detected can complement or substitute the function of PQBP-1, and thus a disease associated with a decrease in neural stem cells or a decrease in post-synaptic formation It can be identified as a candidate substance for a preparation used for the prevention or treatment of the disease.
  • each process of both said screening methods is demonstrated.
  • the administration step is a step of administering the test substance to animal cells or animals other than humans.
  • test substance is not particularly limited, and may be any substance such as a natural or synthetic organic or inorganic low-molecular or high-molecular substance.
  • the animal cell is not particularly limited as long as it is a cell that expresses PQBP-1 (neural cell, neural stem cell, etc.), and conventionally known animal cells such as humans and mice can be used. Moreover, the animal cell may be a wild type or a cell in which the expression of PQBP-1 is previously suppressed.
  • Animals other than humans are not particularly limited, and for example, mammals such as mice, rats, dogs, cats, monkeys, pigs, cows, sheep, rabbits, and the like can be used.
  • the administration method is not particularly limited.
  • the test substance when administering to animal cells, the test substance may be mixed with a tissue sample containing animal cells, or the animal cells may be cultured in the presence of the test substance. .
  • parenteral administration when administering to the animal except a human, for example, parenteral administration may be sufficient.
  • Parenteral administration may be, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like. Oral administration may also be used.
  • the measurement method is not particularly limited, and a conventionally known method can be used. For example, test of an animal cell after administration of a test substance or a cell expressing PQBP-1 extracted from an animal other than a human is performed. By comparing the expression level of PQBP-1 before administration of the substance, or the number of neural stem cells or formation of the postsynapse with the expression level of PQBP-1 after administration of the test substance, or the number of neural stem cells or formation of the postsynapse The degree of neural stem cell proliferation promotion or the promotion of post-synaptic formation can be measured. Based on this measurement result, a candidate substance can be selected in a selection step described later.
  • the selection step is a candidate substance for a preparation used for the prevention or treatment of a disease associated with a decrease in neural stem cells, or a preparation used for the prevention or treatment of a disease associated with a decrease in post-synaptic formation. Is a step of selecting a test substance.
  • the selection method is not particularly limited, and a candidate substance may be selected by a conventionally known selection method.
  • the candidate substance may be selected based on the expression level of PQBP-1, the degree of neural stem cell proliferation, or the degree of promotion of post-synaptic formation. These may be selected by comparing with other PQBP-1 expression levels, the degree of neural stem cell proliferation, or the degree of promotion of post-synaptic formation. Specific examples for comparison include animal cells in which the expression level of PQBP-1 is reduced or animals other than humans, the degree of neural stem cell proliferation, and the degree of promotion of post-synaptic formation. Or a negative control in which no test substance is administered.
  • the selection may be made by selecting the test substance as a candidate substance by confirming that the expression level of PQBP-1, the degree of neural stem cell proliferation, or the degree of promotion of post-synaptic formation is restored. You may select by confirming that it is rising above the control.
  • the Nestin-Cre-cKO mouse refers to a mouse in which the PQBP-1 gene is knocked out by neural stem cells
  • the Synapsin1-Cre-cKO mouse refers to a mouse in which the PQBP-1 gene is knocked out by mature neurons.
  • a targeting vector In order to prepare a targeting vector, first, three types of PQBP-1 genomic fragments were synthesized by PCR from a mouse BAC (bacterial chromosomal chromosome) library (ID: RP23-404N15). Among them, an about 3.6 kb 5 ′ fragment containing exon 1 and exon 2 is incorporated upstream of the sequence of the neomycin resistance gene sandwiched between FLP recognition target sequences, and an about 3.9 kb fragment containing exons 3 to 7 Is inserted between the two LoxP sequences, a fragment of about 4.1 kb intron is added to the 3 ′ side, and a diphtheria toxin gene (DTA) is inserted outside the 3 ′ side sequence to prevent random insertion during recombination. Added. By this operation, a targeting vector was prepared.
  • a mouse BAC bacterial chromosomal chromosome
  • the prepared targeting vector is introduced into ES cells (C57BL / 6) by electroporation, ES cells subjected to homologous recombination with G418 (Sigma, 200 mg / ml) are selected, and then the target gene is obtained by PCR. Confirmed that was introduced. The selected ES cells were treated with restriction enzymes, and the subsequent genome was analyzed by Southern blotting to further confirm that the target gene was introduced.
  • Chimeric mice were produced by injecting the selected ES cells into C57BL / 6 mouse blastocysts. This chimeric mouse was crossed with a C57BL / 6 mouse to produce a mouse having the desired allele. The neomycin resistance gene was removed by mating with CAG-FLPe recombinant transgenic mice. The resulting PQBP-1-flox heterozygous female mice were further crossed with Nestin-Cre male mice (B6.Cg-Tg (Nes-Cre) 1 Kln / J; The Jackson Laboratory, Bar Harbor, ME). Nestin-Cre-knockout (cKO) mice were prepared.
  • PQBP-1-flox heterozygous female mice were mated with Synapsin1-Cre male mice (B6.Cg-Tg (Syn1-Cre) 671Jxm / J; The Jackson Laboratory, Bar Harbor, ME) and Synpsin1-Cre. -Knockout (cKO) mice were generated.
  • Nestin-Cre-cKO mice and Synapsin1-Cre-cKO mice were prepared for both male and female mice, respectively. Since the PQBP-1 gene is a gene on the X chromosome, in these cKO mice, the PQBP-1 gene is knocked out in males with one X chromosome, but PQBP-1 in females with two X chromosomes. The gene is heterozygous for normal and knockout.
  • FIG. 1 (Brain analysis) The structure of the brain of a patient with a mutated PQBP-1 gene was analyzed by magnetic resonance imaging. The result is shown in FIG. In FIG. 1, (a) shows a horizontal plane, (b) shows a coronal plane, and (c) shows a sagittal plane. As a result, it was confirmed that PQBP-1 had a normal cortical structure in patients mutated.
  • FIG. 2 is a photograph of the brains of a Nestin-Cre male and female mouse in which the PQBP-1 gene was knocked out, and a control male and female mouse in which the PQBP-1 gene was not knocked out.
  • FIG. 4 is a photograph of the PQBP-1 gene.
  • FIGS. 3 and 5 Is a photograph of the brains of Synapsin1-Cre male and female mice in which is knocked out and control male and female mice in which the PQBP-1 gene is not knocked out. In addition, the mass of each brain of each mouse was measured. The results are shown in FIGS. 3 and 5, (a) shows the results for male mice, and (b) shows the results for female mice. Incidentally, in FIG.
  • XY means a wild-type C57BL / 6 male mice
  • XX means a wild-type C57BL / 6 female mice
  • XFloxY and “X F Y” is PQBP-1-flox heterozygous male mice are meant
  • XFloxX and X F X mean PQBP-1-flox heterozygous female mice.
  • the symbol “+” in the figure means a mouse produced by mating a mouse with the symbol and a Nestin-Cre mouse or Synapsin1-Cre mouse
  • the symbol “ ⁇ ” It means a mouse produced by mating with wild-type C57BL / 6 without mating a mouse with a symbol and a Nestin-Cre mouse or Synapsin1-Cre mouse.
  • the numbers above each bar graph in FIGS. 3 and 5 indicate the n number of each mouse.
  • FIG. 6 shows that, as in humans with a mutated PQBP-1 gene (see FIG. 1), no abnormalities in brain structure are observed in males of Nestin-Cre mice knocked out of the PQBP-1 gene. It was done. This indicates that microcephaly caused by a decrease in PQBP-1 is a type of microcephaly that is not accompanied by abnormal brain structure.
  • Embryo brain fragments were made at 3 mm intervals, paraffin removed, rehydrated, and then heated in a microwave oven for 15 minutes in 10 mM citrate buffer at pH 6.0. Thereafter, antibody anti-BrdU antibody (1: 200, manufactured by BD Biosciences), rabbit anti-phospho-histone H3 (pH3) antibody, and M phase cell marker (1: 500, manufactured by Millipore) were used for antibody incubation. Performed at 4 ° C. overnight. Secondary antibody incubation was performed using Alexa Fluor 488 or Cy3 complex (1: 500, manufactured by Invitrogen).
  • the ratio of BrdU / pH3 double positive cells to pH3 positive cells in the ventricular zone was calculated at 1, 1.5, 2 hours after a single injection of BrdU to determine G2 / M phase length. did.
  • the Y-axis intercept (LI at 0 hour) was estimated by linear graphs of labeling index values (LIs) at 1, 1.5, 2, 3.5, and 6.5 hours, and the slope was calculated. Since the growth rate (ratio of proliferating cells) in the ventricular zone of wild-type mice is close to 1.0, the LI and slope at 0 hour are the ratio of the S phase to the total cell cycle (Ts / Tc), and Each represents the reciprocal of the total cell cycle (1 / Tc). Ts and Tc represent the length of the S phase and the whole cell cycle. Ts and Tc were calculated from these values (Ts / Tc and 1 / Tc). The length of each cell cycle is shown in Table 1.
  • FIG. 7 is a graph showing the relative proportion of neurogenesis, number of cells remaining in the stem cell pool, number of unlabeled neural stem cells (BrdU ⁇ / Ki67 +) 24 hours after injection. It is.
  • microcephaly is due to mechanisms such as increased differentiation efficiency of neural stem cells (and thus depletion of stem cells), increased cell death of neural stem cells, and impaired cell migration of differentiated neurons. It was thought.
  • the Nestin-Cre-cKO mouse knocked out from the PQBP-1 gene develops microcephaly, but the above mechanism does not occur, and instead the cell cycle time is prolonged. It has been shown. This prolongation of the cell cycle is thought to reduce the number of neural stem cell divisions, resulting in a decrease in neuron production.
  • the exon of the mouse in which the PQBP-1 gene was knocked out was a neural stem cell of an E15 Nestin-Cre-cKO mouse (Neural Stem Cell), the cortex of a 4-week-old Nestin-Cre-cKO mouse, or 4 weeks of age. Using cortex of aged Synapsin1-Cre-cKO mice, each was compared to wild type mice. The results are shown in Tables 2-4. In addition, when a gene has a plurality of exon probes, the lowest p-value was used. Table 2 shows the results of comparison of neural stem cells of E15 Nestin-Cre-cKO mice and wild type mice. Table 3 shows the results of comparing the cortex of 4 weeks old Nestin-Cre-cKO mice and wild type mice. Table 4 shows the results of comparing the cortex of a 4-week-old Synapsin1-Cre-cKO mouse and a wild-type mouse.
  • APC1 and PQBP-1 were found to be E15 Nestin-Cre-cKO mice, 4 weeks old Nestin-Cre-cKO mice, and postnatally in both analyses. It was confirmed that the expression level was significantly affected in all genotypes of 4-week-old Synapsin1-Cre-cKO mice.
  • APC4 was not significantly changed in the cortex of Nestin-Cre-cKO mice, but it was shown to be significant in neural stem cells.
  • two analyzes of exon array data exon exon analysis and variance analysis
  • splicing / transcription contributions are presumed to be correlated. From this assumption, it was suggested that both transcription and splicing affect APC1 and PQBP-1, whereas APC4 has a relatively large transcriptional effect.
  • GSEA Gene Set Enrichment Analysis
  • mice APC4 gene introduction test for Nestin-Cre-cKO mice
  • APC4 gene was introduced into mice in which PQBP-1 was knocked out.
  • mouse APC4 cDNA Genbank accession number NM — 024213
  • pIRES2-hrEGFPII manufactured by Stratagene
  • restriction enzymes with XhoI / BamHI and pApc4-IRES2-hrEGFPII was incorporated.
  • Produced Using this gene, it was introduced into the embryonic ventricles of wild-type mice and Nestin-Cre-cKO mice by electroporation.
  • FIG. 9 (a) shows the E14 of each of a wild type mouse, a Nestin-Cre-cKO mouse, a wild type mouse transformed with Apc4, and a Nestin-Cre-cKO mouse transformed with Apc4. Shows proliferation of neural stem cells prepared from embryos. As a result, it was confirmed that the proliferation of neural stem cells was significantly recovered by introducing APC4 into a mouse in which PQBP-1 was knocked out.
  • FIG. 9B shows the expression levels of APC4, PQBP-1, Cyclin B, and GAPDH by Western blotting. This confirmed that PQBP-1 was not expressed in Nestin-Cre-cKO mice transformed with APC4, and that the expression level of APC4 was higher than that of mice not introduced with APC4. It was also confirmed that the expression level of Cyclin B was increased in the Nestin-Cre-cKO mice, whereas the expression level was suppressed in the Nestin-Cre-cKO mice introduced with APC4.
  • FIG. (A) is a wild-type mouse
  • (b) is a Nestin-Cre-cKO mouse
  • (c) is a Nestin-Cre-cKO mouse transformed with APC4
  • (d) is a wild-type transformed with APC4.
  • the number of cells in each type mouse is shown.
  • the ratio of G2 / M phase was higher than that of the wild type mouse, but it was confirmed that the increase in the ratio of G2 / M phase can be suppressed by introducing APC4. It was.
  • mouse APC4 cDNA (Genbank accession number NM_024213) was prepared by RT-PCR, and this was incorporated into pIRES2-EGFP (manufactured by Clontech) treated with restriction enzyme with XhoI / BamHI, and pApc4 -IRES2-EGFP was produced.
  • PApc4-IRES2-EGFP or pIRES2-EG was introduced into the cerebral ventricles of E13 embryos of wild-type mice and Nestin-Cre-cKO mice, and brain tissues of E18 embryos were analyzed.
  • the human PQBP-1 gene was introduced into a mouse (Nestin-Cre-cKO mouse) in which the PQBP-1 gene was knocked out.
  • an AAV vector plasmid was used as a vector.
  • the AAV vector plasmid contains an expression cassette consisting of a human cytomegalovirus immediate early promoter (CMV promoter), behind which is a cDNA encoding human PQBP1 or human PQBP1-EGFP, between the inverted terminal repeats of the AAV3 genome.
  • CMV promoter human cytomegalovirus immediate early promoter
  • SV40 poly (A) simian virus 40 polyadenylation signal sequence
  • Recombinant AAV vector (AAV-PQBP-1 vector) is transiently transferred to HEK293 cells using vector plasmids, AAV2 rep and AAV1 vp expression plasmids, and an adenovirus helper plasmid, pHelper (manufactured by Agilent Technologies). Made by introduction. Recombinant virus was purified by segregation of two cesium chloride gradients over time and virus titer was determined by quantitative RT-PCR.
  • AAV-PQBP-1 vector In vivo administration of an AAV vector (AAV-PQBP-1 vector) was carried out by intraperitoneally administering C57BL / 6J pregnant mice (E10 Nestin-Cre-cKO fetal mice pregnant) to AAV-PQBP-1 vector ( 2.0 ⁇ 10 11 genomic copies) were introduced into Nestin-Cre-cKO fetal mice. The following tests were performed using Nestin-Cre-cKO mice (2 months (10 weeks old)) into which this AAV-PQBP-1 vector was introduced.
  • mice include Nestin-Cre male and female mice in which the PQBP-1 gene was knocked out, control male and female mice in which the PQBP-1 gene was not knocked out, and AAV (AAV-PQBP-1 vector), respectively.
  • the introduced mouse was used.
  • the weight of the brain of each mouse is shown in FIG. (A) shows the results for male mice and (b) shows the results for female mice. This reduced the weight of the brain, particularly in male mice with PQBP-1 knocked out (white bar graph), with the introduction of AAV (AAV-PQBP-1 vector) (black bar graph).
  • AAV-PQBP-1 vector black bar graph
  • FIG. 16 a photograph of the brain morphology
  • FIG. 17 a coronal photograph of an adult mouse of 2 months (10 weeks old) were taken.
  • adult mice adult Nestin-Cre mice knocked out from the PQBP-1 gene and adult male mice knocked out from the PQBP-1 gene into which AAV (AAV-PQBP-1 vector) was introduced (Nestin-Cre- cKO mice).
  • FIG. 17 shows a photograph of the coronal plane of a mouse not introduced with AAV (AAV-PQBP-1 vector), and
  • (b) shows a mouse of AAV (AAV-PQBP-1 vector) introduced.
  • a photograph of the coronal plane is shown.
  • neural stem cells decrease and intellectual disorders such as microcephaly that can be prevented or treated by PQBP-1.
  • mice Three-month adult mice (Nestin-Cre-cKO mice, Nestin-Cre-cKO mice introduced with AAV (AAV-PQBP-1 vector)) were tested for rotating rods. First, the mouse was placed on a rotating rod (3 cm in diameter), and the rotation speed was increased linearly from 35 rotations to 35 rpm in 300 seconds, and continued at 35 rpm until 600 seconds passed. There was a 10 minute break between each test, and 9 tests (3 consecutive days, 3 tests per day) were performed. The time to drop from the rod was recorded and the average time to fall from the rod was recorded. The result is shown in FIG.
  • fia (fear) conditioning was tested. went. This test consisted of two parts: a conditioning trial and a test trial.
  • Fear conditioning was performed in a transparent plastic container equipped with a stainless steel lattice floor (34 cm ⁇ 26 cm ⁇ 30 cm [H]).
  • a CCD camera was installed on the ceiling of the container and connected to a video monitor and a computer.
  • the lattice floor was connected to a shock generator.
  • White noise (65 db) was supplied from a loudspeaker (conditional stimulus, CS) as an auditory signal.
  • a 0.4 milliamp foot shock (unconditional stimulus, US) for 2 seconds in a row was performed for 30 seconds at the end of the CS period.
  • the conditioning trial consisted of a two minute exploration period with three CS-US combinations separated every 30 seconds.
  • the test was performed in the same conditioning chamber for 5 minutes without a footshock 24 hours after the conditioning trial.
  • the rate at which the mouse stopped responding was measured as an index of fier memory.
  • the result is shown in FIG.
  • the stopping time was less than in the wild-type mice, but the stopping time was reduced by introducing AAV (AAV-PQBP-1 vector). Significantly increased.
  • GFP was excited at a laser wavelength of 890 nm
  • RSD cortex was observed from a window by a thinned-skull method, and imaging data having a resolution of 1024 ⁇ 1024 pixels at 1 ⁇ m intervals was obtained.
  • the observation results are shown in FIG. As a result, it was confirmed that the formation of the postsynaptic portion was decreased in Synapsin1-Cre-cKO mice in which the PQBP-1 gene was knocked out in mature neurons.
  • the signal value of the probe set corresponding to each exon was estimated according to the protocol of Affymetrix.
  • the signal values of the obtained probe sets were grouped in units of transcripts, and normalization was performed so that the sum of the signal values of the probe set group corresponding to each transcript was constant.
  • an equal variance test was performed on the variance of the signal values of the probe set group in each of the Synapsin1-Cre-cKO and wild type groups, and the transcripts whose variance was significantly changed between the two groups. Created a list.
  • a list of exons in which the expression level was significantly changed was prepared by testing the difference in the average signal value between the two groups of Synapsin1-Cre-cKO and wild type.
  • the gene related to PQBP-1 was analyzed by obtaining a common set of the transcript list and exon list obtained by the above operation. The results are shown in Table 5.
  • the PQBP-1 gene is a group of genes related to mental retardation (Mental Retardation (MR)), Alzheimer's disease (ADD), amyotrophic lateral sclerosis (ALS), and synapses. It was suggested that there is a close relationship.
  • MR mental retardation
  • ADD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • mice fed with high fat diet The expression of PQBP-1 in the brains of mice fed a high fat diet was analyzed by Western blotting.
  • mice were injected with AAV-GFP (5 ⁇ 1011 viral genome / ml) into retrograde dysgranular (RSD) cortex (front-rear direction -2.0 mm from bregma, 0.6 mm inward / outward direction), 12 weeks
  • RSD retrograde dysgranular cortex
  • inhalation anesthesia with isoflurane was performed (0.1 ml / min)
  • the skin of the observation area was incised
  • the skull was thinned with a drill to create an observation window (thinned-skull method).
  • GFP was excited at a laser wavelength of 890 nm
  • RSD cortex was observed from a window by a thinned-skull method, and imaging data having a resolution of 1024 ⁇ 1024 pixels at 1 ⁇ m intervals was acquired.
  • the obtained imaging data is read into IMARIS, and dendrites and spines are recognized using a filament tool (dendritic diameter: 0.297-4.950 ⁇ m, spine head diameter: 0.2-3.0 ⁇ m). Measurements were made by building and analyzing a three-dimensional model. The result is shown in FIG. Thereby, it was confirmed that the number of posterior synapses decreases by giving a high fat diet. This result suggests that a decrease in PQBP-1 due to obesity causes a decrease in post-synaptic formation, which causes Alzheimer's disease.
  • the change in the dynamic morphology of the ridges of the postsynaptic formation in the high-fat diet group given the above-mentioned high-fat diet and the normal diet group fed the normal diet was measured.
  • the measurement was performed by reading the obtained imaging data into IMARIS and using a filament tool (dendritic diameter: 0.297-4.950 ⁇ m, spine head diameter: 0.2-3.0 ⁇ m) and dendrites and spines. This was done by recognizing and analyzing a three-dimensional model. The result is shown in FIG. Thereby, it was confirmed that by giving a high-fat diet to mice, the proportion of the lost synapse formed was increased.
  • mice Spine morphology observation of mouse cerebral cortical neurons by administration of AAV-PQBP-1 vector
  • 5xFAD mice Alzheimer's disease model mice
  • wild type mice were treated with AAV-CMV-EGFP-PQBP-1 viral vector (1x10 9 vg / ml) or AAV-CMV-EGFP viral vector (1x10 9 vg).
  • 100 ⁇ l was filled into an osmotic pump, implanted subcutaneously on the back of the mouse, and continuously administered into the subarachnoid space for 72 hours with a connected glass micropipette.
  • FIG. 32 shows the number of dendrites (number / 10 ⁇ m) of mouse cerebral cortical neurons.
  • the 5xFAD mouse was obtained from The Jackson Laboratory.
  • the AAV-CMV-EGFP-PQBP-1 viral vector is an EGFP vector plasmid containing an expression cassette consisting of a CMV promoter in the above-mentioned “PQBP-1 gene introduction test for Nestin-Cre-cKO mice”.
  • the AAV-CMV-EGFP viral vector is a vector constructed so that cDNA encoding EGFP is expressed in an AAV vector plasmid containing an expression cassette consisting of a CMV promoter.
  • FIG. 31 (a) is an image of a spine of a cerebral cortical neuron of a wild type mouse administered with an AAV-CMV-EGFP viral vector, and (b) is an image administered with an AAV-CMV-EGFP viral vector.
  • (C) is an image of cerebral cortical neurons of 5xFAD mice administered with AAV-CMV-EGFP viral vector and AAV-CMV-PQBP-1 viral vector. It is an image of spine.
  • “5xFAD + AAV-CMV-EGFP” is AAV ⁇

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Abstract

L'invention concerne : une préparation qui peut être utilisée pour favoriser la prolifération de cellules souches neurales, une préparation qui peut être utilisée pour la prévention ou le traitement de maladies associées à la diminution des cellules souches neurales, une préparation qui peut être utilisée pour favoriser la formation de postsynapses, et une préparation qui peut être utilisée pour la prévention ou le traitement de maladies associées à une diminution de la formation de postsynapses, dont chacune contient un ADN spécifique codant pour PQBP-1 ou un polypeptide codé par l'ADN comme principe actif ; et un procédé de criblage. La préparation, qui peut être utilisée pour favoriser la prolifération de cellules souches neurales, contient un ADN de codage de PQBP-1 comprenant la séquence de nucléotides représentée par SEQ ID NO: 1 ou similaire, ou un polypeptide codé par l'ADN ou similaire, comme principe actif. La préparation, qui peut être utilisée pour favoriser la formation de postsynapses, contient un ADN de codage de PQBP-1 comprenant la séquence de nucléotides représentée par SEQ ID NO: 1 ou similaire, ou un polypeptide codé par l'ADN ou similaire, comme principe actif.
PCT/JP2015/069032 2014-07-02 2015-07-01 Préparation utilisée pour favoriser la prolifération de cellules souches neurales, préparation utilisée pour la prévention ou le traitement de maladies associées à une diminution des cellules souches neurales, préparation utilisée pour favoriser la formation de postsynapses, préparation utilisée pour la prévention ou le traitement de maladies associées à une diminution de la formation de postsynapses, et procédé de criblage WO2016002854A1 (fr)

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