JP6850414B2 - Preparations used to promote neural stem cell proliferation, preparations used to prevent or treat diseases associated with neural stem cell depletion, preparations used to promote posterior synaptic formation, prevention or prevention of diseases associated with reduced posterior synaptic formation Formulations used for treatment and screening methods - Google Patents

Description

The present invention relates to preparations used to promote neural stem cell proliferation, preparations used to prevent or treat diseases associated with neural stem cell depletion, preparations used to promote postsynaptic formation, and reduction of postsynaptic formation. The present invention relates to a preparation used for prevention or treatment of a disease, and a screening method.

It is conventionally known that polyglutamine-tract binding protein1 (hereinafter referred to as PQBP-1 in the present specification) is a protein responsible for an intermediate pathological condition in the degenerative disease polyglutamine disease (see, for example, Non-Patent Document 1). ).

This mutation in the PQBP-1 gene was frequently found in the X-chromosome chain intellectual disability / mental retardation family (XLID / XLMR), and it has been reported that PQBP-1 may be the major causative gene of intellectual disability. (See, for example, Non-Patent Document 2). It has also been reported that PQBP-1 may be involved in RNA splicing (see, eg, Non-Patent Document 3).

Waragai et al. , "PQBP-1, a novel polyglutamine tract-binding protein, inhibits transcription activation by Brn-2 and impacts cell survival", HumM 977-987 Kalscheuer et al. , "Mutations in the polyglutamine binding protein 1 gene case X-linked mental retardation", Nature Genetics 2003; 35 (4): p. 313-315 Okazawa et al. , "Interaction beten Mutant Ataxin-1 and PQBP-1 Affects Transcription and Cell Death", Neuron, 2002; 34: p. 701-713

As mentioned above, it has been reported that PQBP-1 may be the causative gene of intellectual disability and that it is involved in RNA splicing, but that PQBP-1 is actually associated with any intellectual disability. Still unknown.

The present invention is a preparation used for promoting the proliferation of neural stem cells, which contains a predetermined DNA encoding PQBP-1 or a polypeptide encoded by this DNA as an active ingredient, and prevention of diseases related to the decrease 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 posterior synaptic formation, a preparation used for prevention or treatment of a disease associated with a decrease in posterior synaptic formation, and a screening method.

The present inventors have found that in mice in which the PQBP-1 gene has been knocked out, the cell cycle of neural stem cells is lengthened and the postsynaptic formation is reduced, and the present invention has been completed. More specifically, the present invention provides the following.

(1) Neural stem cell containing, as an active ingredient, the DNA according to any one of the following (a) to (d) encoding polyglutamine-tract binding protein 1 (PQBP-1), or a polypeptide encoded by this DNA. A formulation used to promote the growth of.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(C) It has 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, and can promote the proliferation of neural stem cells. DNA
(D) A DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and capable of promoting the proliferation of neural stem cells.

(2) The preparation according to (1), wherein the promotion of proliferation of the neural stem cells is due to suppression of cell cycle extension.

(3) A disease associated with a decrease in neural stem cells, which contains the DNA according to any one of the following (a) to (d) encoding PQBP-1 or the polypeptide encoded by this DNA as an active ingredient. A formulation used for prevention or treatment.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(C) A 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 and promoting the proliferation of neural stem cells.
(D) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting the proliferation of neural stem cells.

(4) Post-synaptic formation containing the DNA according to (a), (b), (e) or (f) below, which encodes PQBP-1, or the polypeptide encoded by this DNA as an active ingredient. A formulation used to promote.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(E) A 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 and promoting postsynaptic formation.
(F) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting postsynaptic formation.

(5) Post-synaptic formation containing the DNA according to (a), (b), (e) or (f) below, which encodes PQBP-1, or the polypeptide encoded by this DNA as an active ingredient. A formulation used to prevent or treat diseases associated with reduction.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(E) A 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 and promoting postsynaptic formation.
(F) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting postsynaptic formation.

(6) The preparation according to any one of (1) to (5), wherein the DNA is incorporated into a vector.

(7) A method for screening candidate substances for preparations used for the prevention or treatment of diseases related to the decrease of neural stem cells.
The step of administering the test substance to animal cells or animals other than humans, and
A step of 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 was administered.
A screening method comprising a step of selecting a test substance as a candidate substance for a preparation used for prevention or treatment of a disease related to a decrease in neural stem cells based on the measurement result.

(8) A method for screening candidate substances for preparations used for the prevention or treatment of diseases associated with a decrease in postsynaptic formation.
The step of administering the test substance to animal cells or animals other than humans, and
A step of measuring the expression level of endogenous PQBP-1 or the degree of promotion of postsynaptic formation by PQBP-1 in the animal cells or animals other than humans to which the test substance was administered.
A screening method comprising a step of selecting a test substance as a candidate substance for a preparation used for prevention or treatment of a disease associated with a decrease in postsynaptic formation based on the measurement result.

According to the present invention, a preparation used for promoting the proliferation of neural stem cells, which contains a predetermined DNA encoding PQBP-1 or a polypeptide encoded by this DNA as an active ingredient, a disease related to the decrease of neural stem cells. A formulation used for the prevention or treatment of, a formulation used for promoting posterior synaptic formation, a formulation used for the prevention or treatment of a disease associated with a decrease in posterior synaptic formation, and a screening method can be provided.

It is a figure which shows the image which analyzed the brain shape of the patient which mutated the PQBP-1 gene by the magnetic resonance image. In FIG. 1, (a) is an image of a horizontal plane, (b) is an image of a coronal plane, and (c) is an image of a sagittal plane. It is a photograph of each brain of a Nestin-Cre male and female adult mouse (2 months) in which the PQBP-1 gene was knocked out, and a male and female adult mouse (2 months) in which the PQBP-1 gene was not knocked out. It is a graph which shows the brain mass of the Nestin-Cre male and female adult mouse (2 months) which knocked out the PQBP-1 gene, and the male and female adult mouse (2 months) which did not knock out the PQBP-1 gene. , (A) is a graph for male mice, and (b) is a graph for female mice. It is a photograph of the brain of Synapsin1-Cre male and female adult mice (2 months) in which the PQBP-1 gene was knocked out, and male and female adult mice (2 months) in which the PQBP-1 gene was not knocked out. It is a graph which shows the mass of the brain of Synapsin1-Cre male and female adult mice (2 months) in which the PQBP-1 gene was knocked out, and male and female adult mice in which the PQBP-1 gene was not knocked out, (a). Is a graph for male mice, and (b) is a graph for female mice. It is a photograph of the coronal surface of each of the Nestin-Cre male and female adult mice (2 months) in which the PQBP-1 gene was knocked out and the male and female adult mice (2 months) in which the PQBP-1 gene was not knocked out. , (A) is a photograph of a male adult mouse in which the PQBP-1 gene has not been knocked out, (b) is a photograph of a Nestin-Cre male adult mouse in which the PQBP-1 gene has been knocked out, and (c) is a photograph of a male adult mouse. It is a photograph of a female adult mouse in which the PQBP-1 gene has not been knocked out, and (d) is a photograph of a Nestin-Cre female adult mouse in which the PQBP-1 gene has been knocked out. 24 hours after intraperitoneal injection of BrdU into pregnant mice, it is a graph of the relative percentage of neurogenesis cells, the number of cells remaining in the stem cell pool, and the number of unlabeled neural stem cells. FIG. 5 is a graph of the level of cell death in the cerebral cortex of Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out and Nestin-Cre mice in which the PQBP-1 gene was not knocked out by TUNEL staining. In (a), a wild-type mouse, a Nestin-Cre-cKO mouse in which the PQBP-1 gene was knocked out, a wild-type mouse in which APC4 was transformed, and a PQBP-1 gene in which APC4 was transformed were knocked out. It is a graph which shows the proliferation of the neural stem cell prepared from the embryo of each E14 of a Nestin-Cre-cKO mouse. (B) is a photograph of the results of Western blotting analysis of the expression levels of APC4, PQBP-1, Cyclin B, and GAPDH in each mouse embryo. According to FACS analysis, (a) wild-type mice, (b) Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out, and (c) Nestin-Cre in which the PQBP-1 gene was transformed with APC4 were knocked out. It is a graph showing the number of each cell number in the neural stem cells extracted from the embryo of E14 of each mouse of -cKO mouse and (d) APC4 transformed wild type mouse. Brain tissue of E18 after introduction of APC4-GFP or GFP into the embryos of Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out and Nestin-Cre mice in which the PQBP-1 gene was not knocked out. It is a photograph of. Brain tissue of E18 after introduction of APC4-GFP or GFP into the embryos of Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out and Nestin-Cre mice in which the PQBP-1 gene was not knocked out. It is a graph of the ratio of the pia mater surface to the apical surface in. Wild-type mice (2 months), Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out (2 months), and Nestin in which the PQBP-1 gene was knocked out and AAV (AAV-PQBP-1 vector) was introduced. It is a photograph of the result of analysis of the protein in the brain of Cre-cKO mouse (2 months) by Western blotting. Wild-type mice (2 months), Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out (2 months), and Nestin in which the PQBP-1 gene was knocked out and AAV (AAV-PQBP-1 vector) was introduced. It is a graph which analyzed the ratio of the amount of protein in the brain of Cre-cKO mouse (2 months) by Western blotting. Nestin-Cre male and female mice in which the PQBP-1 gene was knocked out (2 months), and control male and female mice in which the PQBP-1 gene was not knocked out (2 months), and AAV (AAV-PQBP-1), respectively. It is a graph of the weight of the brain in the mouse (2 months) into which the vector) was introduced. (A) shows a graph of a male mouse, and (b) shows a graph of a female mouse. Brains of adult Nestin-Cre mice (2 months) in which the PQBP-1 gene was knocked out and male adult mice (2 months) in which the PQBP-1 gene was knocked out and AAV (AAV-PQBP-1 vector) was introduced. It is a photograph of the form of. Brains of Nestin-Cre male adult mice (2 months) in which the PQBP-1 gene was knocked out and male adult mice (2 months) in which the PQBP-1 gene was knocked out and AAV (AAV-PQBP-1 vector) was introduced. It is a photograph of the coronal plane of the mouse. (A) shows a photograph of the coronal plane of a mouse into which AAV (AAV-PQBP-1 vector) has not been introduced, and (b) is a photograph of the coronal plane of a mouse into which AAV (AAV-PQBP-1 vector) has been introduced. Shown in the photo. Nestin-Cre male adult mice in which the PQBP-1 gene was knocked out (2.5 months) and male adult mice in which the PQBP-1 gene was knocked out and AAV (AAV-PQBP-1 vector) was introduced (2.5 months). 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 in which the PQBP-1 gene was knocked out (3 months), Nestin-Cre-cKO adult mouse in which the PQBP-1 gene was knocked out and AAV (AAV-PQBP-1 vector) was introduced (3 months). Is a graph of the average time each mouse falls off the rod in a rotating rod test. Wild-type adult mouse (3 months), Nestin-Cre-cKO adult mouse in which the PQBP-1 gene was knocked out (3 months), Nestin- in which the PQBP-1 gene was knocked out and AAV (AAV-PQBP-1 vector) was introduced. FIG. 5 is a graph of mouse pause time in a fier conditioning test for adult Cre-cKO mice (3 months). In a rotary rod test, wild-type adult mice (3 months), Flox adult mice (3 months), and Synapsin1-Cre-cKO adult mice in which the PQBP-1 gene was knocked out (3 months), each mouse was a rod. It is a graph of the average time falling from. Fear of wild-type mice (3 months), Flox mice (3 months), Synapsin1-Cre adult mice (3 months), and Synapsin1-Cre-cKO mouse wild-type adult mice in which the PQBP-1 gene was knocked out (3 months). It is a graph of the mouse stop time in the conditioning test. It is a figure which shows the degree of synaptic posterior formation of Synapsin1-Cre mouse, Flox mouse, and Synapsin1-Cre-cKO mouse which knocked out the PQBP-1 gene. It is a graph of the diameter of a tree, the diameter of a protrusion head, and the number of dendrites in the posterior synapse of Synapsin1-Cre mice, Flox mice, and Synapsin1-Cre-cKO mice in which the PQBP-1 gene was knocked out. It is a figure which shows the time-dependent change of the degree of synaptic posterior formation of Synapsin1-Cre-cKO mouse which knocked out the PQBP-1 gene of Synapsin1-Cre mouse and Flox mouse. It is a graph of the dynamic morphological change of the dendrite of the synaptic posterior formation of Synapsin1-Cre mouse, Flox mouse, and Synapsin1-Cre-cKO mouse in which the PQBP-1 gene was knocked out. It is a photograph of the result of analyzing the expression of PQBP-1 in the mouse fed a high-fat diet by Western blotting. It is a graph of the result of analyzing the expression of PQBP-1 in the mouse fed a high-fat diet by Western blotting. It is a figure which shows the graph of the number of the postsynapse in the mouse which fed a high fat diet. It is a graph of the result of analyzing the expression of PQBP-1 by Western blotting. It is a graph of the dynamic morphological change of dendrites of postsynaptic formation in mice fed a high-fat diet. (A) shows an image of spines of cerebral cortical nerve cells of wild-type mice to which the AAV-CMV-EGFP viral vector was administered, and (b) is a 5xFAD mouse to which the AAV-CMV-EGFP viral vector was administered. (C) shows images of spines of cerebral cortical nerve cells of 5xFAD mice administered with AAV-CMV-EGFP viral vector and AAV-CMV-PQBP-1 viral vector. Shown. Wild-type mice (n = 8) to which the AAV-CMV-EGFP viral vector was administered, 5xFAD mice (n = 5) to which the AAV-CMV-EGFP viral vector was administered, and the AAV-CMV-EGFP viral vector and AAV. FIG. 5 is a graph for 5xFAD mice (n = 8) administered with the -CMV-PQBP-1 viral vector.

Hereinafter, specific embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments, and the present invention shall be carried out with appropriate modifications within the scope of the object of the present invention. Can be done. In addition, although the description may be omitted as appropriate for the parts where the description is duplicated, the gist of the invention is not limited.

<Formulation>
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. Hereinafter, the preparation of the present invention will be described.

(Preparation used to promote the proliferation of neural stem cells)
The preparation used for promoting the proliferation of neural stem cells of the present invention contains the DNA according to any one of the following (a) to (d) encoding PQBP-1 or the polypeptide encoded by this DNA as an active ingredient. contains.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(C) A 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 and promoting the proliferation of neural stem cells.
(D) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting the proliferation of neural stem cells.

The present inventors have found that the cell cycle is prolonged by causing PQBP-1 abnormality. This extension of the cell cycle reduces the number of neural stem cell divisions and reduces the number of neural stem cells. However, according to the preparation of the present invention, the extension of the cell cycle can be suppressed, and thus the decrease of neural stem cells can be suppressed.

(Preparation used for prevention or treatment of diseases related to neural stem cell depletion)
The preparation used for the prevention or treatment of a disease related to the reduction of neural stem cells of the present invention is the DNA according to any one of the following (a) to (d) encoding PQBP-1, or the DNA encoded by this DNA. Contains a polypeptide as an active ingredient.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(C) A 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 and promoting the proliferation of neural stem cells.
(D) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting the proliferation of neural stem cells.

In the present specification, "disease related to neural stem cell depletion" refers to a disease caused by neural stem cell depletion, for example, microcephaly (for example, microcephaly without abnormality in brain shape). , Congenital intellectual disability such as mental retardation, and developmental disability such as microcephaly and learning disability. Here, the "preventive or therapeutic effect of diseases related to the decrease of nerve stem cells" is at least one of congenital intellectual disabilities such as microcephaly and mental retardation, and developmental disorders such as autism and learning disabilities. An effect in which a symptom is improved or an exacerbation of a symptom is suppressed, and the improvement or suppression is statistically significant. Of these, it is preferably used for microcephaly because it has a particularly high preventive or therapeutic effect. Also, the prophylactic or therapeutic effect of diseases associated with the reduction of neural stem cells is particularly effective for human males in the case of X-linked diseases.

(Preparation used to promote postsynaptic formation)
The preparation used for promoting postsynaptic formation of the present invention is the DNA according to (a), (b), (e) or (f) below, which encodes PQBP-1, or the polypeptide encoded by this DNA. Is contained as an active ingredient.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(E) A 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 and promoting postsynaptic formation.
(F) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting postsynaptic formation.

(Preparations used to prevent or treat diseases associated with decreased postsynaptic formation)
The preparations used for the prevention or treatment of diseases associated with the reduction of postsynaptic formation of the present invention include the DNAs according to (a), (b), (e) or (f) below, which encode PQBP-1. Alternatively, the polypeptide encoded by this DNA is contained as an active ingredient.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(B) DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions.
(E) A 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 and promoting postsynaptic formation.
(F) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting postsynaptic formation.

The term "disease associated with a decrease in posterior synaptic formation" as used herein refers to a disease caused by a decrease in posterior synaptic formation, for example, dementia such as Alzheimer's disease, muscular atrophic lateral sclerosis, and mental illness. Diseases such as developmental retardation, frontotemporal dementia, Huntington's disease, Parkinson's disease, generalized Levy's body disease, and spinal cerebral ataxia can be mentioned. Here, "preventive or therapeutic effect of diseases related to reduction of posterior synaptic formation" means that at least one symptom of dementia such as Alzheimer's disease, muscular atrophic lateral sclerosis, and mental retardation is improved or worsened. An effect that is suppressed and for which improvement or suppression is statistically significant. Of 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.

(DNA)
The DNA of the present invention is any of the above DNAs (a) to (f).

SEQ ID NO: 1 is a base sequence encoding human PQBP-1. The nucleotide sequence shown in SEQ ID NO: 1 promotes neural stem cell proliferation and synapse formation. Here, in the present specification, "the base sequence directly promotes the proliferation or synaptogenesis of neural stem cells" means that the polypeptide encoded by the base sequence promotes the proliferation or synaptogenesis of neural stem cells. Variants and homologues of DNA having the base sequence shown in SEQ ID NO: 1 include, for example, DNA having a base sequence capable of hybridizing with the base sequence shown in SEQ ID NO: 1 under stringent conditions. Here, as "stringent conditions", for example, the reaction is carried out in a normal hybridization buffer at 40 to 70 ° C. (preferably 50 to 67 ° C., more preferably 60 to 65 ° C.), and a salt is used. Conditions include conditions for washing in a washing solution having a concentration of 15 to 300 mM (preferably 15 to 150 mM, more preferably 15 to 60 mM, still more preferably 30 to 50 mM).

SEQ ID NO: 2 is known to constitute a human PQBP-1 protein. The DNA of the present invention also includes a 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. Here, "one or more" is usually 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (for example, 5 amino acids or less, 3 amino acids or less, 1 amino acid). In order to maintain the action of promoting the proliferation or synaptogenesis of neural stem cells, it is desirable that the mutated amino acid residue is mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, the properties of the amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids (C, M) with, carboxylic acid and amino acids with amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), aromatic-containing side chains Amino acids (H, F, Y, W) having (H, F, Y, W) can be mentioned (all in parentheses represent the one-letter notation of amino acids).

It is already known that a protein having an amino acid sequence modified by deletion, addition and / or substitution with another amino acid of one or more amino acid residues to an amino acid sequence maintains its biological activity. (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81,5662-5666, Zoller, MJ & Smith, M. Nuclear Acids Research (1982) 10,6487- 6500, Wang, A. et al., Science 224,1431-1433, Dalbadie-McFarland, G. et al., Protein. Natl. Acad. Sci. USA (1982) 79, 6409-6413). The 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 preferably has high homology with the amino acid sequence shown in SEQ ID NO: 2. Here, the homology between the amino acid sequence shown 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 Okazawa et al., "PQBP-1". (Np / PQ): A polyglutamine trac-binding and neural inclusion-forming protein ”, Brain Research Bulletin, 2001, Vol.56, Nos.3 / 4, pp.273-280), human P. Also, a high effect (promoting effect on neural stem cell proliferation, preventive or therapeutic effect on diseases related to neural stem cell depletion, promoting effect on posterior synaptic formation, or preventive or therapeutic effect on diseases related to decreased posterior synaptic formation). ) Is played. From this viewpoint, 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 shown in SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 2 is preferably 85% or more. Is more preferably 90% or more, and further preferably 95% or more (96% or more, 97% or more, 98% or more, 99% or more).

The most preferable embodiment of the above-mentioned DNA is a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1. However, the DNA of the present invention further has an effect of promoting the proliferation of neural stem cells or prevention of diseases related to the decrease of neural stem cells. It includes various mutants and homologs having a therapeutic effect, and various mutants and homologs having a preventive or therapeutic effect on diseases associated with a promoting effect of posterior synaptic formation or a decrease in posterior synaptic formation. Here, the WW domain on the N-terminal side of PQBP-1 (SEQ ID NO: 3) and the C-terminal domain on the C-terminal side (SEQ ID NO: 4) are known. The WW domain is a sequence that plays an important role in the action with RNA polymerase, and is known to bind proline-rich in a sequence-specific manner. The C terminal domain is also known as an intrinsically disordered protein that plays an important role in splicing. Therefore, in order to exert the function of PQBP-1, the DNA of the present invention preferably contains the sequence of any one or both of SEQ ID NOs: 3 and 4.

Further, the mutant or homolog of the DNA having the base sequence shown in SEQ ID NO: 1 includes DNA having a base sequence having high homology with the base sequence shown 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) homology with the base sequence shown in SEQ ID NO: 1. The homology of 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). When the base sequence is analyzed by BLASTN based on BLAST, the parameters are, for example, score = 100 and worldgth = 12. When the amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score = 50 and worldgth = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known (http://www.ncbi.nlm.nih.gov.).

In addition, the above-mentioned SEQ ID NOs: 1 to 4 are NCBI CCDS Database Report for CCDS14309.1 (current version) (http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgif It is derived from 1 & ORGANISM = 0 & BUILDS = CURRENTBUILDS).

The "DNA" in the present invention may be either a sense strand or an antisense strand (for example, it can be used as a probe), and its shape may be either single-strand or double-strand. 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 synthesizing by chemical synthesis, Known methods such as a method for isolating from a genomic DNA library and a cDNA library (see, for example, Japanese Patent Application Laid-Open No. 11-29599) can be mentioned.

(Polypeptide)
The polypeptide used in the prophylactic or therapeutic agent of the present invention is encoded by the above-mentioned 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 prepared, for example, by using a transformant into which an expression vector containing the above-mentioned DNA has been introduced. That is, first, this transformant is cultured under appropriate conditions to synthesize a protein (polypeptide) encoded by this DNA. Then, the polypeptide of the present invention can be obtained by recovering the synthesized protein from the transformant or the culture medium.

The transformant is appropriately selected from known nutrient media according to the type of transformant and the like so that a large amount of polypeptide can be easily obtained, and the temperature, pH of the nutrient medium, culture time, etc. are adjusted. It can be adjusted as appropriate (see, for example, Japanese Patent Application Laid-Open No. 11-29599).

The method for isolating and purifying the polypeptide is not particularly limited, and known methods such as a method using solubility, a method using a difference in molecular weight, and a method using electric charge (for example, JP-A-11-29599). (See Gazette). The vectors and transformants that can be used in the present invention will be described below.

(vector)
The expression vector can be prepared by inserting the above-mentioned DNA into an appropriate vector. The "appropriate vector" may be any vector that can replicate, retain or self-proliferate in various hosts of prokaryotes and / or eukaryotes, and can be appropriately selected depending on the purpose of use. For example, a high copy vector can be selected when a large amount of DNA is to be obtained, and an expression vector can be selected when a polypeptide is to be obtained. Specific examples thereof are not particularly limited, and examples thereof include known vectors described in Japanese Patent Application Laid-Open No. 11-29599.

In addition, the vector can be used not only for synthesizing a polypeptide but also for the preparation of the present invention. That is, the preparation of the present invention containing the above-mentioned DNA-incorporated vector can be directly introduced into humans to promote the proliferation of neural stem cells, prevent or treat diseases related to the decrease of neural stem cells, and to form postsynapses. It can be used for the prevention or treatment of diseases associated with promotion or reduction of postsynaptic formation. As the vector in this case, a vector that can be introduced into human cells is used. Examples of such a vector include an AAV vector.

(Transformant)
The transformant can be prepared by introducing a vector containing the above-mentioned DNA into a host. Such a host may be any host that is compatible with the vector of the present invention and can be transformed, and specific examples thereof are not particularly limited, but known natural cells such as bacteria, yeast, 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 method for introducing the vector can be appropriately selected depending on the type of vector, host, and the like. Specific examples thereof include, but are not limited to, known methods such as a protoplast method and a competent method (see, for example, Japanese Patent Application Laid-Open No. 11-29599) when a bacterium is used as a host.

In addition, when a human is used as a host, for example, when transforming a pregnant human using the above-mentioned AAV vector, the transformation can be performed by injecting into the abdomen.

As used herein, the term "active ingredient" refers to an effect that promotes neural stem cell proliferation, a preventive or therapeutic effect on a disease associated with neural stem cell depletion, an effect that promotes postsynaptic formation, or a disease associated with a decrease in postsynaptic formation. It refers to an ingredient contained in an amount necessary for obtaining a preventive or therapeutic effect, and other ingredients may be contained as long as the effect is not impaired to less than a desired level. In addition, the route of administration of the preparation may be either oral or parenteral, and is appropriately set.

For oral administration, the formulation may contain additives such as commonly used binders, inclusions, excipients, lubricants, disintegrants, wetting agents, tablets, granules, fine granules. , Powders, capsules and the like. In addition, the preparation may be in a liquid state such as an internal liquid agent, a suspension agent, an emulsion syrup agent, or in a dry state in which it is redissolved at the time of use.

For parenteral administration, the formulation may also contain additives such as stabilizers, buffers, preservatives, isotonic agents, etc., usually contained in unit dose ampoules or multidose containers or tubes. It is distributed in the state. In addition, the formulation may be formulated into a powder that can be redissolved with an appropriate carrier (sterilized water or the like) at the time of use.

The above-mentioned preparation of the present invention is administered to humans by the method described above to prevent or treat a disease related to a decrease in neural stem cells, or a disease related to a decrease in postsynaptic formation. It can be used for the prevention or treatment of.

<Screening method>
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 related to a decrease in neural stem cells, or a preparation used for the prevention or treatment of a disease related to a decrease in posterior synaptic formation. This is a screening method for candidate substances.

(Screening method for candidate substances for preparations used for prevention or treatment of diseases related to neural stem cell depletion)
The method for screening a candidate substance for a preparation used for the prevention or treatment of a disease related to the reduction of neural stem cells of the present invention is a step of administering the test substance to an animal cell or an animal other than a human, and the test substance is administered. Candidate substances for preparations used for the prevention or treatment of diseases related to neural stem cell depletion 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 and the measurement results. It has a step of selecting a test substance as a subject.

(Screening method for candidate substances for preparations used for prevention or treatment of diseases associated with decreased postsynaptic formation)
The method for screening a candidate substance for a preparation used for the prevention or treatment of a disease associated with a decrease in posterior synaptic formation of the present invention is a step of administering a test substance to an animal other than an animal cell or a human, and the test substance is administered. A step of measuring the degree of promotion of posterior synaptic formation by PQBP-1 in animal cells or animals other than humans, and a preparation used for the prevention or treatment of diseases associated with a decrease in posterior synaptic formation based on the measurement results. It has a step of selecting a test substance as a candidate substance of the above.

According to each of the above screening methods, the test substance for which improvement in symptoms is detected can complement or replace the function of PQBP-1, and thus a disease related to a decrease in neural stem cells or a disease related to a decrease in postsynaptic formation. It can be identified as a candidate substance for a preparation used for the prevention or treatment of. Each step of both of the above screening methods will be described below.

(Administration process)
The administration step is a step of administering the test substance to animal cells or animals other than humans.

The test substance is not particularly limited, and may be any substance such as a natural or synthetic organic or inorganic low molecular weight or high molecular weight substance.

The animal cell is not particularly limited as long as it is a cell expressing PQBP-1 (nerve cell, neural stem cell, etc.), and conventionally known animal cells such as human and mouse can be used. In addition, the animal cell may be a wild type or one in which the expression of PQBP-1 is suppressed in advance.

Animals other than humans are not particularly limited, and for example, mammals such as mice, rats, dogs, cats, monkeys, pigs, cows, sheep, and rabbits can be used.

The administration method is not particularly limited, and for example, when administered to animal cells, a tissue sample containing animal cells and a test substance may be mixed, or animal cells may be cultured in the presence of the test substance. .. Alternatively, when it is administered to animals other than humans, it may be administered parenterally, for example. Parenteral administration may be, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or the like. It may also be administered orally.

(Measurement process)
The measurement step is the expression level of endogenous PQBP-1 or the degree of promotion of neural stem cell proliferation by PQBP-1 or promotion of posterior synaptic formation by PQBP-1 in animal cells or animals other than humans to which the test substance is administered. It is a process of measuring the degree of.

The measuring method is not particularly limited, and a conventionally known method can be used. For example, a test of animal cells after administration of the test substance or cells expressing PQBP-1 extracted from animals other than humans. By comparing the expression level of PQBP-1 before administration of the substance, or the number of neural stem cells or posterior synaptic formation, with the expression level of PQBP-1 after administration of the test substance, or the number of neural stem cells or posterior synaptic formation. , The degree of promotion of neural stem cell proliferation or the degree of promotion of posterior synaptic formation can be measured. Based on this measurement result, a candidate substance can be selected in the selection step described later.

(Selection process)
Based on the above measurement results, the selection step is a candidate substance for a preparation used for the prevention or treatment of a disease related to a decrease in neural stem cells, or a preparation used for the prevention or treatment of a disease related to a decrease in postsynaptic formation. It is a step of selecting a test substance as.

The selection method is not particularly limited, and the candidate substance may be selected by a conventionally known selection method. For example, candidate substances may be selected based on the expression level of PQBP-1, the degree of neural stem cell proliferation, or the degree of promotion of postsynaptic formation. These may be selected by comparing with the expression level of other PQBP-1, the degree of neural stem cell proliferation or the degree of promotion of postsynaptic formation. Specific examples of comparison targets include animal cells or animals other than humans in which the expression level of PQBP-1 is decreased, the degree of neural stem cell proliferation, and the degree of promotion of postsynaptic formation are decreased. It may be a negative control to which the test substance has not been administered. In addition, the test substance may be selected 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 postsynaptic formation is restored, and these are negative. You may select it by confirming that it is higher than the control.

(Preparation of PQBP-1 gene knockout mouse)
A gene essential for the development of an individual cannot be knocked out by a simple method, but it is possible to knock out the gene only under specific conditions by using a Cre-loxP system or the like. In this example, a knockout mouse (Nestin-Cre-cKO) targeting neural stem cells is utilized by utilizing the selective expression of Nestin in neural stem cells and the selective expression of Synapsin1 in mature neurons. Mice) and knockout mice (Synapsin1-Cre-cKO mice) targeting mature neural cells were created. Hereinafter, the Nestin-Cre-cKO mouse refers to a mouse in which the PQBP-1 gene has been knocked out in neural stem cells, and the Synapsin1-Cre-cKO mouse refers to a mouse in which the PQBP-1 gene has been knocked out in mature neural cells.

In order to prepare a targeting vector, first, three types of PQBP-1 genomic fragments were synthesized by PCR from a mouse BAC (bacterial artificial chromosome) library (ID: RP23-404N15). Among them, a 5'side fragment of about 3.6 kb containing exon 1 and exon 2 was incorporated upstream of the sequence of the neomycin resistance gene sandwiched between FLP recognition target sequences, and a fragment of about 3.9 kb containing exons 3 to 7 was incorporated. Is integrated between the two LoxP sequences, an intron fragment of about 4.1 kb is added to the 3'side, and a diphtheria toxin gene (DTA) is placed outside the 3'side sequence to prevent random insertion during recombination. Added. By this operation, a targeting vector was prepared.

The prepared targeting vector was introduced into ES cells (C57BL / 6) by electroporation, and after homologous recombination with G418 (manufactured by Sigma Co., Ltd., 200 mg / ml) was selected, the target gene was selected by PCR. 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 prepared by injecting the selected ES cells into C57BL / 6 mouse blastocysts. This chimeric mouse was crossed with a C57BL / 6 mouse to prepare a mouse having the allele of interest. The neomycin resistance gene was removed by mating with CAG-FLPe recombinant transgenic mice. The resulting PQBP-1-flox heterozygous female mouse was further mated with a Nestin-Cre male mouse (B6. Cg-Tg (Nes-Cre) 1 Kln / J; The Jackson Laboratory, Bar Harbor, ME). The mice were subjected to Nestin-Cre-knockout (cKO) mice. In addition, 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) to Synapsin1-Cre. -Knockout (cKO) mice were generated. Nestin-Cre-cKO mice and Synapsin1-Cre-cKO mice were produced for both male and female mice, respectively.
Since the PQBP-1 gene is a gene on the X chromosome, these cKO mice have the PQBP-1 gene 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.

(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) is a horizontal plane, (b) is a coronal plane, and (c) is a sagittal plane. As a result, it was confirmed that the patient had a mutated PQBP-1 and had a normal cortical structure.

Next, the brain was removed from a 2-month-old adult mouse. Removal from the brain was performed according to a conventional method. Adult mice were Nestin-Cre male and female mice in which the PQBP-1 gene was knocked out (in neural stem cells), Synapsin1-Cre male and female mice in which the PQBP-1 gene was knocked out (in mature neurons), and PQBP-1. Control male and female mice in which the gene was not knocked out were used. 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, and FIG. 4 is a photograph of the PQBP-1 gene. Is a photograph of the brains of male and female Synapsin1-Cre males and female mice in which the PQBP-1 gene has not been knocked out, and male and female mice in the control in which the PQBP-1 gene has not been knocked out. In addition, the mass of each brain of each mouse was measured. The results are shown in FIGS. 3 and 5. 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 means PQBP-1-flox heterozygous male mice, "XFloxX" and ^{"X F} X" means PQBP-1-flox heterozygous female mice. The "+" symbol in the figure means a mouse produced by mating a mouse with the symbol with a Nestin-Cre mouse or a Synapsin1-Cre mouse, and the "-" symbol is the mouse. It means a mouse produced by mating a wild type C57BL / 6 without mating a mouse with a symbol with a Nestin-Cre mouse or a Synapsin1-Cre mouse. The numerical value on each bar graph of FIGS. 3 and 5 indicates the n number of each mouse.

From FIGS. 2 and 3, it was confirmed that in Nestin-Cre-cKO male mice in which the PQBP-1 gene was knocked out in embryonic neural stem cells, the brain became smaller and the mass of the brain was significantly reduced. This indicates that microcephaly due to a decrease in PQBP-1 is particularly likely to occur in human males. In addition, from FIGS. 4 and 5, it was shown that microcephaly did not develop in Synapsin1-Cre-cKO in which the PQBP-1 gene was knocked out in mature neurons. This indicates that microcephaly caused by a decrease in PQBP-1 is congenital.

In addition, a photograph of the coronal plane of a 2-month-old adult mouse was taken. As adult mice, Nestin-Cre male and female adult mice in which the PQBP-1 gene was knocked out, and male and female adult mice in which the PQBP-1 gene was not knocked out were used. In FIG. 6, (a) is a photograph of a male adult mouse in which the PQBP-1 gene has not been knocked out, and (b) is a photograph of a Nestin-Cre male adult mouse in which the PQBP-1 gene has been knocked out. c) is a photograph of a female adult mouse in which the PQBP-1 gene has not been knocked out, and (d) is a photograph of a Nestin-Cre female adult mouse in which the PQBP-1 gene has been knocked out.

FIG. 6 shows that no particular abnormality was observed in the structure of the brain in male Nestin-Cre mice in which the PQBP-1 gene was knocked out, as in humans in which the PQBP-1 gene was mutated (see FIG. 1). Was done. This indicates that microcephaly caused by a decrease in PQBP-1 is a type of microcephaly that is not accompanied by abnormalities in the structure of the brain.

(Analysis of cell cycle of Nestin-Cre-cKO mice)
The cell cycle of neural stem cells (NSPCs) in Nestin-Cre-cKO mice was analyzed. First, BrdU (manufactured by Sigma, 100 mg / kg body weight) was injected intraperitoneally into each pregnant mouse of E14 (14th day of fetal life) (the types of pregnant mice are shown in Table 1 below). BruU was repeatedly injected into pregnant mice (at 3-hour intervals), and cumulative labeling was performed. Each mouse removed its embryonic brain at 1, 1.5, 2, 3.5, 6.5, 15.5 or 24.5 hours after the first injection of BrdU. The removed embryo brain was fixed with 4% paraformaldehyde and incorporated into paraffin. Embryo brain fragments were made at 3 mm intervals, deparaffinized, rehydrated, and then microwaved for 15 minutes in 10 mM citrate buffer at pH 6.0. Then, antibody incubation was performed using mouse anti-BrdU antibody (1: 200, manufactured by BD Biosciences), rabbit anti-phospho-histone H3 (pH 3) antibody, and M-phase cell marker (1: 500, manufactured by Millipore). It was performed overnight at 4 ° C. The secondary antibody was incubated with Alexa Fluor 488 or Cy3 complex (1: 500, manufactured by Invitrogen). Percentage of BrdU / pH3 double-positive cells to pH3-positive cells in the ventricular zone calculated at 1, 1.5, and 2 hours after a single injection of BrdU to determine the length of the G2 / M phase. did. Y-axis intercepts (LI at 0 hours) were estimated from linear graphs of labeled index values (LIs) at 1, 1.5, 2, 3.5 and 6.5 hours and slopes were calculated. Since the growth rate (percentage of proliferating cells) in the ventricular zone of wild-type mice is close to 1.0, the LI and slope at 0 hours are the ratio of S phase to the total cell cycle (Ts / Tc). Represents the reciprocal of the entire cell cycle (1 / Tc). Ts and Tc represent the length of the S phase, the entire cell cycle. From these values (Ts / Tc and 1 / Tc), Ts and Tc were calculated. The length of each cell cycle is shown in Table 1.

From Table 1, it was confirmed that the cell cycle length was longer in Nestin-Cre-cKO mice as compared with the control. In particular, it was observed that the G2 / M phase was significantly extended by 67% compared to the control, and the G1 phase was slightly extended by 6%. Thus, it was shown that knocking out PQBP-1 prolongs the cell cycle, especially in the M phase.

(Analysis of the percentage of cells in Nestin-Cre-cKO mice)
Neurogenesis from the stem cell pool was analyzed by co-staining BrdU and Ki67. Twenty-four or 72 hours after intraperitoneal injection of BrdU into pregnant mice, the number of neurogenic cells (BrdU + / Ki67-), the number of cells remaining in the stem cell pool (BrdU + / Ki67 +), and unlabeled nerve stem cells (BrdU + / Ki67 +). Each embryonic brain of E15 was analyzed to calculate the number of BrdU− / Ki67 +). As a result, no difference was detected in the proportion of cells in each group after 24 hours and 72 hours. FIG. 7 is a graph showing the relative ratio of the number of neurogenesis cells, the number of cells remaining in the stem cell pool, and the number of unlabeled neural stem cells (BrdU- / Ki67 +) 24 hours after injection. Is.

(Analysis of cell death in Nestin-Cre-cKO mice)
The level of cell death in the cerebral cortex in Nestin-Cre-cKO and Nestin-Cre mice was assessed by TUNEL staining in E10, E15, E18, P0, and P60. The result is shown in FIG. From FIG. 8, it was confirmed that there was no difference in the number of cell-dead cells between the Nestin-Cre-cKO mouse and the Nestin-Cre mouse.

Conventionally, small head disease is caused by a mechanism such as an increase in the differentiation efficiency of neural stem cells (which depletes the stem cells), an increase in cell death of neural stem cells, and an impaired cell migration of differentiated neurons. Was thought to be. However, from the above results, the Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out developed microcephaly, but the above mechanism did not occur, and the cell cycle time was prolonged instead. It has been shown. This cell cycle prolongation is thought to reduce the number of neural stem cell divisions, resulting in reduced neuronal production.

(Analysis of genes strongly related to splicing by PQBP-1)
Exon levels were significantly altered by comparing the respective exon signals of wild-type mice and mice knocked out of the PQBP-1 gene in order to analyze genes that are strongly associated with PQBP-1 splicing. "Exon exon analysis" to find genes was performed. The difference was examined by Student's t-test based on the null hypothesis that the means of the two samples are the same. We also performed a "variance analysis" to detect changes in relative expression levels between exons. This was performed using an exon-level probe set in each gene of wild-type and PQBP-1 knockout mice. The difference was examined by F-test based on the null hypothesis that these two samples have the same pattern and variance. The exons of the mice in which the PQBP-1 gene was knocked out are the neural stem cells (Neural Stem Cell) of E15 Nestin-Cre-cKO mice, the cortex of 4-week-old Nestin-Cre-cKO mice, or the cortex of 4-week-old Nestin-Cre-cKO mice. The cortex of old Synapsin1-Cre-cKO mice was used and each was compared to wild-type mice. The results are shown in Tables 2-4. When the gene carries a plurality of exon probes, the lowest p-value was used. Table 2 shows the results of comparing the neural stem cells of E15 Nestin-Cre-cKO mice and wild-type mice. Table 3 shows the results of comparing the cortex of 4-week-old Nestin-Cre-cKO mice and wild-type mice. Table 4 shows the results of comparing the cortex of 4-week-old Synapsin1-Cre-cKO mice with wild-type mice.

The results (Table 2, Table 3, Table 4) show that APC1 and PQBP-1 in both analyzes were E15 Nestin-Cre-cKO mice, 4-week-old Nestin-Cre-cKO mice, and postnatal. It was confirmed that the expression level of each genotype of 4-week-old Synapsin1-Cre-cKO mice was significantly affected. On the other hand, APC4 was shown to be significant in neural stem cells, although the change in expression level was not significant in the cortex of Nestin-Cre-cKO mice. On the other hand, the two analyzes of exon array data (exon exon analysis and variance analysis) and the contribution of splicing / transcription are presumed to be correlated with the results. This speculation suggests that both transcription and splicing affect APC1 and PQBP-1, whereas transcription has a relatively large effect on APC4.

Gene Set Enrichment Analysis (GSEA) was performed to further analyze genes closely related to splicing by PQBP-1. Three species of Nestin-Cre-cKO mice and three wild-type mice were used for the analysis. PPI analysis was performed on genes related to the M phase selected by GSEA. As a result, it was confirmed that the genes strongly related to PQBP-1 in the M phase are U-515kd, APC2, and APC4.

(APC4 gene transfer test into Nestin-Cre-cKO mice)
A test was conducted in which the APC4 gene was introduced into mice in which PQBP-1 was knocked out. First, mouse APC4 cDNA (Genbank access number NM_024213) was prepared by RT-PCR, and this was incorporated into pIRES2-hrEGFPII (manufactured by Stratagene) treated with XhoI / BamHI restriction enzyme, and pApc4-IRES2-hrEGFP Made. This gene was used by electroporation into the ventricles of wild-type and Nestin-Cre-cKO mice embryos.

FIG. 9A shows the E14 of each of the wild-type mouse, the Nestin-Cre-cKO mouse, the wild-type mouse transformed with Apc4, and the Nestin-Cre-cKO mouse transformed with Apc4. It shows the proliferation of neural stem cells prepared from the embryo. From this, it was confirmed that the proliferation of neural stem cells was significantly restored by introducing APC4 into the mouse in which PQBP-1 was knocked out. In addition, FIG. 9B shows the expression levels of APC4, PQBP-1, Cyclin B, and GAPDH by Western blotting. As a result, it was confirmed that PQBP-1 was not expressed in the Nestin-Cre-cKO mice transformed with APC4, and that the expression level of APC4 was higher than that in the mice into which APC4 was not introduced. In addition, it was confirmed that the expression level of Cyclin B was increased in Nestin-Cre-cKO mice, whereas the expression level was suppressed in Nestin-Cre-cKO mice introduced with APC4.

In addition, the number of each cell was confirmed in the neural stem cells extracted from the E14 embryo of each mouse by FACS analysis. The result is shown in FIG. (A) is a wild-type mouse, (b) is a Nestin-Cre-cKO mouse, (c) is a Nestin-Cre-cKO mouse transformed with APC4, and (d) is a wild-type mouse transformed with APC4. The number of each cell in the type mouse is shown. As a result, in Nestin-Cre-cKO mice, the G2 / M phase ratio was higher than in wild-type mice, but it was confirmed that the introduction of APC4 suppressed the increase in the G2 / M phase ratio. It was.

It was also tested whether the ratio of the buffy coat surface to the apical surface affected the proliferation of EGFP-positive cells transformed by intrauterine electroporation. Specifically, first, the cDNA of mouse APC4 (Genbank access number NM_024213) was prepared by RT-PCR, and this was incorporated into pIRES2-EGFP (manufactured by Clontech) treated with XhoI / BamHI restriction enzyme, and pApc4. -IRES2-EGFP was prepared. PApc4-IRES2-EGFP or pIRES2-EG was introduced into the ventricles of E13 embryos of wild-type mice and Nestin-Cre-cKO mice, and the brain tissue of E18 embryos was analyzed. The results are shown in FIGS. 11 and 12. From this result, the ratio of the buffy coat surface to the apical surface was decreased in the mouse in which the PQBP-1 gene was knocked out, but it was recovered by adding APC4 from the outside. This showed that cell cycle prolongation was restored by the addition of APC4.

(PQBP-1 gene transfer test into Nestin-Cre-cKO mice)
First, the human PQBP-1 gene was introduced into a mouse (Nestin-Cre-cKO mouse) in which the PQBP-1 gene was knocked out. Specifically, an AAV vector plasmid was used as the vector. The AAV vector plasmid contains an expression cassette consisting of the human cytomegalovirus pre-early promoter (CMV promoter), behind which the cDNA encoding human PQBP1 or human PQBP1-EGFP, during the reverse terminal repetition of the AAV3 genome. Cytomegalovirus 40 polyadenylation signal sequence (SV40 poly (A)) was designed to follow. The recombinant AAV vector (AAV-PQBP-1 vector) is transient to HEK293 cells using a vector plasmid, AAV2 rep and AAV1 vp expression plasmids, and an adenovirus helper plasmid, pHelper (manufactured by Agent Technologies). Made by introduction. Recombinant virus was purified by separation and formation of two cesium chloride gradients over time and virus titers were determined by quantitative RT-PCR. In vivo administration of the AAV vector (AAV-PQBP-1 vector) is performed by intraperitoneally administering the AAV-PQBP-1 vector (AAV-PQBP-1 vector) to C57BL / 6J pregnant mice (pregnant E10 Nestin-Cre-cKO fetal mice). 2.0 × 10 ¹¹ genome copy) was 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.

Wild-type mice (2 months (10 weeks old)), Nestin-Cre-cKO mice (2 months (10 weeks old)), and Nestin-Cre-cKO mice introduced with AAV (AAV-PQBP-1 vector) ( Proteins in the brain at 2 months (10 weeks of age)) were analyzed by Western blotting. A photograph of Western blotting is shown in FIG. 13, and a graph showing the ratio of the amount of protein of PQBP-1 is shown in FIG. Since the amino acid sequence of the human PQBP-1 protein and the amino acid sequence of the mouse PQBP-1 protein have high homology as described above, they are detected as having substantially the same protein size even in Western blotting. From this, it was confirmed that the amount of protein of PQBP-1 was increased about 2.5 times by introducing the PQBP-1 gene.

In addition, the brain was taken out from a 2-month-old (10-week-old) adult mouse. As adult mice, 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) were used for each. The introduced mouse was used. The weight of the brain of each mouse is shown in FIG. (A) shows the results of a male mouse, and (b) shows the results of a female mouse. This significantly reduced brain weight, especially in male mice knocked out of PQBP-1 (white bar graph), by introducing AAV (AAV-PQBP-1 vector) (black bar graph). It was shown that it did.

In addition, a photograph of the brain morphology of a 2-month-old (10-week-old) adult mouse (FIG. 16) and a photograph of the coronal plane (FIG. 17) were taken. As adult mice, a Nestin-Cre male adult mouse in which the PQBP-1 gene was knocked out and a male adult mouse (Nestin-Cre-) in which the PQBP-1 gene was knocked out into which AAV (AAV-PQBP-1 vector) was introduced were introduced. cKO mouse) was used. In FIG. 17, (a) shows a photograph of the coronal plane of a mouse introduced with AAV (AAV-PQBP-1 vector), and FIG. 17B shows a photograph of a mouse introduced with AAV (AAV-PQBP-1 vector). A photograph of the coronal plane is shown. As a result, it was confirmed that in male mice in which PQBP-1 was knocked out, the weight of the brain was restored by introducing AAV (AAV-PQBP-1 vector) also in the morphology and coronal plane of the brain.

In addition, a quantitative analysis of the relative thickness of each layer with respect to the total thickness of the cortex of 2.5-month-old adult mice was performed. As adult mice, a Nestin-Cre male adult mouse (Nestin-Cre-cKO mouse) (white bar graph) in which the PQBP-1 gene was knocked out and a PQBP-1 into which AAV (AAV-PQBP-1 vector) was introduced were introduced. Adult male mice (Nestin-Cre-cKO mice) in which the gene was knocked out (black bar graph) were used. In addition, Cux, Tbr1, GAD67, and Foxp1 were used as specific markers for each layer. The result is shown in FIG. From this result, even if AAV (AAV-PQBP-1 vector) was introduced, there was no particular change in the layer thickness. Therefore, even if AAV (AAV-PQBP-1 vector) was introduced, the brain It was confirmed that there were no side effects such as affecting the thickness.

From the above results, it was shown that PQBP-1 can prevent or treat intellectual disorders such as microcephaly caused by a decrease in neural stem cells.

(Behavior analysis)
Rotating rods were tested on 3-month-old adult mice (Nestin-Cre-cKO mice, Nestin-Cre-cKO mice introduced with AAV (AAV-PQBP-1 vector)). First, the mouse was placed on a rotating rod (diameter 3 cm), the rotation speed was linearly increased from 3.5 rotations to 35 rpm in 300 seconds, and continued at a rotation speed of 35 rpm until 600 seconds had passed. There was a 10-minute break between each test, and 9 tests (3 tests per day for 3 consecutive days) were performed. The time of falling from the rod was recorded, and the average time of falling from the rod was recorded. The result is shown in FIG.

From FIG. 19, the Nestin-Cre-cKO mouse introduced with AAV (AAV-PQBP-1 vector) stays on the rotating rod more than the Nestin-Cre-cKO mouse introduced with AAV (AAV-PQBP-1 vector). It was shown that the time to do was long.

Next, a Fear (fear) conditioning test was conducted on 3-month-old adult mice (wild-type mice, Nestin-Cre-cKO mice, Nestin-Cre-cKO mice introduced with AAV (AAV-PQBP-1 vector)). went. The test consisted of two parts, a conditioning trial and a test trial. Fear conditioning was performed in a clear plastic container equipped with a stainless steel grid floor (34 cm x 26 cm x 30 cm [H]). A CCD camera was mounted on the ceiling of the container and connected to a video monitor and a computer. The grid floor was connected to the shock generator. White noise (65db) was supplied by a loudspeaker (conditional stimulus, CS) as an auditory signal. A 0.4mA footshock (unconditional stimulation, US) for 2 consecutive seconds was performed for 30 seconds at the end of the CS period. The conditioning trial consisted of a two-minute exploration period with a combination of three CS-USs separated every 30 seconds. The test was performed 24 hours after the conditioning trial in the same conditioning chamber for 5 minutes without footshock. The rate at which the mouse stopped responding was measured as an index of fear memory. The result is shown in FIG. As a result, the Nestin-Cre-cKO mice in which the PQBP-1 gene was knocked out had less time to stop than the wild-type mice, but the introduction of AAV (AAV-PQBP-1 vector) caused the time to stop. It increased significantly.

(Behavioral analysis of Synapsin-1-Cre-cKO mice)
When wild-type mice, Flox mice, and Synapsin-1-Cre-cKO mice were tested for rotational speed with a rotating rod in the same manner as in the behavior analysis of the Nestin-Cre-cKO mice described above, Synapsin-1-Cre was tested. It was confirmed that the -cKO mice stayed on the rotating rod significantly shorter than the other control mice, similar to the Nestin-Cre-cKO mice (Fig. 21).

Furthermore, when a fear conditioning test was conducted on wild-type mice, Flox mice, Synapsin-1-Cre mice, and Synapsin-1-Cre-cKO mice by the same method as above, Synapsin-1-Cre-cKO mice were found. It was confirmed that, as with the Nestin-Cre-cKO mouse, the mouse stopped significantly less than the mouse of the other control (Fig. 22).

(Analysis of synapse formation in Synapsin-1-Cre-cKO)
Synaptic posterior formation of mature neurons in Synapsin1-Cre mice (n = 3, 4 weeks old), Flox mice (n = 3, 4 weeks old), Synapsin1-Cre-cKO mice (n = 3, 4 weeks old) The degree was measured. The measurement was performed by observing the mature nerve cells of each mouse using a two-photon microscope. More specifically, to each mouse in which AAV-GFP (5x1011 viral genome / ml) was previously injected into the retroplenial dysgranular (RSD) cortex (-2.0 mm in the anterior-posterior direction and 0.6 mm in the inward-external direction from bregma). , Inhalation anesthesia with isoflurane (0.1 ml / min) was performed, the skin of the observation site was incised, and the skull was thinly shaved using a drill to create a window for observation (thinned-skull method). Imaging of spines and dendritic protrusions using a two-photon laser microscope system FV1000MPE (manufactured by Olympus) equipped with a multi-photon laser MaiTai HP DeepSee-OL (manufactured by Spectra Physics) and a water-immersed objective lens XLPlanN25xW (manufactured by Olympus). I got the data. GFP was excited at a laser wavelength of 890 nm, RSD cortex was observed from a window by the thinned-skull method, and imaging data having a resolution of 1024 × 1024 pixels at 1 μm intervals was acquired. The observation result is shown in FIG. This confirmed that postsynaptic formation was reduced in Synapsin1-Cre-cKO mice in which the PQBP-1 gene was knocked out in mature neurons.

In addition, mature neurons of the above Synapsein1-Cre mice (n = 3, 4 weeks old), Flox mice (n = 3, 4 weeks old), and Synapsein1-Cre-cKO mice (n = 3, 4 weeks old). The diameter of the tree, the diameter of the protrusion head, and the number of dendrites (number / 10 μm) were measured at the posterior synapse of the mouse. For the measurement, the imaging data obtained from each of the above mice was read into IMARIS (Bitplane), and the filament tool (dendritic diameter: 0.297-4.950 μm, spine head diameter: 0.2-3. This was done by recognizing dendrites and spines using 0 μm) and constructing and analyzing a three-dimensional model. The result is shown in FIG. This confirmed that the diameter of the tree, the diameter of the process head, and the number of dendrites were significantly reduced in Synapsin1-Cre-cKO mice in which the PQBP-1 gene was knocked out in mature neurons. ..

In addition, the mature nerve cells of the above Synapsein1-Cre mice (n = 3, 4 weeks old), Flox mice (n = 3, 4 weeks old), and Synapsein1-Cre-cKO mice (n = 3, 4 weeks old). The time course of the degree of posterior synaptic formation was confirmed. For the measurement, observation of the imaging data of each of the above mice was performed at 0, 8, and 24 hours. The result is shown in FIG. This confirmed that the postsynaptic formation of Synapsin1-Cre-cKO mice in which the PQBP-1 gene was knocked out in mature neurons decreased with time.

In addition, the mature nerve cells of the above Synapsein1-Cre mice (n = 3, 4 weeks old), Flox mice (n = 3, 4 weeks old), and Synapsein1-Cre-cKO mice (n = 3, 4 weeks old). Changes in the dynamic morphology of dendrites of posterior synaptic formation were measured. For the measurement, the imaging data obtained from each of the above mice was read into IMARIS (Bitplane), and the filament tool (dendritic diameter: 0.297-4.950 μm, spine head diameter: 0.2-3. This was done by recognizing dendrites and spines using 0 μm) and constructing and analyzing a three-dimensional model. The result is shown in FIG. In FIG. 26, “Formation” indicates the proportion of newly formed postsynaptic posterior, “Elimination” indicates the proportion of formed posterior synaptic parts that have disappeared, and “Table” indicates no change. The percentage of the postsynaptic region is shown. From this result, it was confirmed that the proportion of newly formed postsynapses was reduced in Synapsin1-Cre-cKO mice in which the PQBP-1 gene was knocked out in mature neurons.

From the above, it was strongly suggested that the behavioral abnormalities (learning disability, cognitive impairment) of Synapsin1-Cre-cKO mice were correlated with the postsynaptic dysplasia.

(Analysis of genes related to PQBP-1 using Synapsin1-Cre-cKO)
Exon array data was acquired using GeneChip Mouse Exon 1.0 ST Array (Affymetrix) using the brains of 4-week-old Synapsin1-Cre-cKO (n = 3) and wild-type mouse (n = 3) as samples. Then, the exon array data was analyzed by the following procedure.

First, the signal value of the probe set corresponding to each exon was estimated according to the Affymetrix protocol. The signal values of each of the obtained probe sets were grouped for each transcript, and normalization was performed so that the total of the signal values of the probe sets corresponding to each transcript was constant. Next, for each transcript, a test of homoscedasticity regarding the variance of the signal values of the probe set group in each Synapsin1-Cre-cKO and wild-type group was performed, and the variance of the transcript changed significantly between the two groups. I made a list. Furthermore, for each probe set, a list of exons whose expression levels were significantly changed was prepared by testing the difference between the mean values of the signal values between the Synapsin1-Cre-cKO and the wild-type two groups.

Genes related to PQBP-1 were analyzed by finding a common set of the transcript list and the exon list obtained by the above operation. The results are shown in Table 5.

From Table 5, the PQBP-1 gene is a group of genes related to mental retardation (MR), Alzheimer's disease (ADD), amyotrophic lateral sclerosis (ALS), and synapse. , It was suggested that there is a close relationship.

From the above results, it was suggested that diseases such as Alzheimer's disease caused by decreased postsynaptic formation can be prevented or treated by PQBP-1.

(Analysis of PQBP1-1 expression in high-fat diet-fed mice)
The expression of PQBP-1 in the brains of mice fed a high-fat diet was analyzed by Western blotting. Mice (C57BL / 6) were divided into a high-fat diet group (n = 3, corresponding to "HFD6W" in FIG. 27-30) from 6 weeks of age to 6 weeks and a high-fat diet group (n = 3) from 11 weeks of age to 1 week. = 3, corresponding to "HFD1W" in FIG. 27-30) was used. For comparison, mice in the normal diet group (n = 3, corresponding to "HFD0W" in FIG. 27-30) not fed with a high-fat diet were used. The analysis was performed at 12 weeks of age in each mouse. As a high-fat diet, High Fat Diet 32 manufactured by Claire Japan Co., Ltd. was used, and the mice were allowed to freely ingest it. The high-fat diet group and the normal diet group during the period other than the high-fat diet were allowed to freely ingest the normal diet (CE-2 of Japan Claire Co., Ltd.). Water was also freely ingested, and the light and darkness of the breeding room was controlled every 12 hours, and the animals were bred at room temperature of 25 ± 2 ° C. and humidity of 50 ± 10%. A photograph of the analysis result is shown in FIG. 27, and a graph showing the ratio of the amount of protein is shown in FIG. 28. Traditionally, obesity has been considered to be a risk factor for Alzheimer's disease, but the results suggest that a decrease in PQBP-1 due to obesity causes Alzheimer's disease.

(Measurement of degree of synapse formation in mice fed a high-fat diet)
The number of posterior synapses was measured in the mice in the high-fat diet group fed with the above-mentioned high-fat diet and the mice in the normal diet group fed with the normal diet. In the measurement, first, at 10 weeks of age, AAV-GFP (5x1011 viral genome / ml) was injected into a retroplenial dysgranular (RSD) cortex (-2.0 mm in the anteroposterior direction and 0.6 mm in the inward and outward directions from bregma), and then 12 weeks. At age, inhalation anesthesia with isoflurane (0.1 ml / min) was performed, the skin of the observation site was incised, and the skull was thinly shaved using a drill to create a window for observation (thinned-skull method). Imaging of spines and dendritic protrusions using a two-photon laser microscope system FV1000MPE (manufactured by Olympus) equipped with a multi-photon laser MaiTai HP DeepSee-OL (manufactured by Spectra Physics) and a water-immersed objective lens XLPlanN25xW (manufactured by Olympus). I got the data. GFP was excited at a laser wavelength of 890 nm, RSD cortex was observed from a window by the thinned-skull method, and imaging data having a resolution of 1024 x 1024 pixels at 1 μm intervals was acquired. The obtained imaging data is read into IMARIS, and the dendrites and spines are recognized using a filament tool (dendritic protrusion diameter: 0.297-4.950 μm, spine head diameter: 0.2-3.0 μm). Measurements were made by constructing and analyzing a three-dimensional model. The result is shown in FIG. This confirmed that the number of posterior synapses was reduced by feeding a high-fat diet. This result suggests that a decrease in PQBP-1 due to obesity causes a decrease in postsynaptic formation, which causes Alzheimer's disease.

The dynamic morphological changes of the dendrites forming the posterior synapse of the mice fed the high-fat diet and the normal diet group were measured. For the measurement, the obtained imaging data was read into IMARIS, and the dendrite and spine were measured using a filament tool (dendritic diameter: 0.297-4.950 μm, spine head diameter: 0.2-3.0 μm). This was done by recognizing and constructing and analyzing a three-dimensional model. The result is shown in FIG. From this, it was confirmed that by feeding the mice with a high-fat diet, the proportion of the formed posterior synapses that disappeared increased.

From the above results, it was suggested that diseases such as Alzheimer's disease caused by a decrease in PQBP-1 due to obesity and, as a result, a decrease in postsynaptic formation, can be prevented or treated by PQBP-1. ..

(Observation of spine morphology of mouse cerebral cortical neurons by administration of AAV-PQBP-1 vector)
5.5 month-old 5xFAD mice (Alzheimer's disease model mice) or wild type mice, AAV-CMV-EGFP-PQBP -1 viral vector ^(1x10 9 vg / ml) or AAV-CMV-EGFP virus vector (1x10 ⁹ vg / Ml) 100 μl was filled in an osmotic pump, subcutaneously transplanted to the back of a mouse, and continuously administered to the subarachnoid space with a connected glass micropipette for 72 hours. 14 days after the start of virus administration, a spin-skull method (a method of thinly exfoliating the skull on the brain surface to a thickness of about 20-50 μm) is used to spin the nerve cells in the 1st and 2nd layers of the retrosplenial dysgranular (RSD) cortex. Was observed with a two-photon microscope. The number of spines per unit dendritic length was quantitatively evaluated using the image analysis software imaris 7.6.1 from the captured images. An image of spines of mouse cerebral cortical neurons is shown in FIG. FIG. 32 shows the number of dendrites (number / 10 μm) of mouse cerebral cortical neurons.

The 5xFAD mouse was obtained from The Jackson Laboratory. Further, the AAV-CMV-EGFP-PQBP-1 viral vector is used in the AAV vector plasmid containing the expression cassette consisting of the CMV promoter in the above-mentioned "Test for introducing the PQBP-1 gene into the Nestin-Cre-cKO mouse" described above. -A vector configured to express the cDNA encoding human PQBP-1. The AAV-CMV-EGFP viral vector is an AAV vector plasmid containing an expression cassette consisting of a CMV promoter, which is configured to express a cDNA encoding EGFP.

In FIG. 31, (a) is an image of spines of cerebral cortex nerve cells of wild-type mice to which the AAV-CMV-EGFP viral vector was administered, and (b) is an image of spines of wild-type mouse to which the AAV-CMV-EGFP viral vector was administered. It is an image of the spine of the cerebral cortex nerve cells of the 5xFAD mouse, and (c) shows the cerebral cortex nerve cells of the 5xFAD mouse to which the AAV-CMV-EGFP viral vector and the AAV-CMV-PQBP-1 viral vector were administered. This is an image of a spine. In FIG. 32, “WT + AAV-CMV-EGFP” is a graph of a wild-type mouse (n = 8) to which the AAV-CMV-EGFP viral vector was administered, and “5xFAD + AAV-CMV-EGFP” is AAV-. It is a graph about 5xFAD mouse (n = 5) to which CMV-EGFP viral vector was administered, and "5xFAD + AAV-CMV-PQBP-1 + AAV-CMV-EGFP" is AAV-CMV-EGFP viral vector and AAV-CMV-PQBP. It is a graph about the 5xFAD mouse (n = 8) to which -1 viral vector was administered.

As shown in FIG. 31, it was confirmed that the number of spines decreased in the 5xFAD mice as compared with the wild-type mice, whereas the number of spines increased in the 5xFAD mice administered with PQBP-1. Was done. Further, as shown in FIG. 32, the number of dendrites was significantly reduced in the 5xFAD mouse as compared with the wild-type mouse, whereas in the 5xFAD mouse to which PQBP-1 was administered, the number of dendrites was significantly reduced. It was confirmed that the numbers were significantly improved to the same extent as wild-type mice. From this result, it was found that PQBP-1 can treat or prevent Alzheimer's disease by promoting postsynaptic formation.

Claims (6)

Proliferation of neural stem cells containing the DNA according to any of the following (a) to (d) encoding polyglutamine-tract binding protein 1 (PQBP-1) or the polypeptide encoded by this DNA as an active ingredient. A formulation used to promote.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(C) It is possible to have a base sequence encoding an amino acid sequence in which one or several amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and promote the proliferation of neural stem cells. DNA that can be produced
(D) A DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and capable of promoting the proliferation of neural stem cells. The preparation according to claim 1, wherein the promotion of neural stem cell proliferation is due to suppression of cell cycle prolongation. Prevention or treatment of diseases related to neural stem cell depletion containing the DNA according to any of the following (a) to (d) encoding PQBP-1 or the polypeptide encoded by this DNA as an active ingredient. Formulation used for.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(C) A DNA having a base sequence encoding an amino acid sequence in which one or several amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and promoting the proliferation of neural stem cells.
(D) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting the proliferation of neural stem cells. A preparation used for promoting postsynaptic formation, which comprises the DNA according to the following (a), (e) or (f) encoding PQBP-1 or a polypeptide encoded by this DNA as an active ingredient.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(E) A DNA having a base sequence encoding an amino acid sequence in which one or several amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and promoting postsynaptic formation.
(F) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting postsynaptic formation. Prevention of diseases associated with decreased postsynaptic formation containing the DNA according to (a), (e) or (f) below, which encodes PQBP-1, or the polypeptide encoded by this DNA as an active ingredient. Or a formulation used for treatment.
(A) DNA having the nucleotide sequence shown in SEQ ID NO: 1
(E) A DNA having a base sequence encoding an amino acid sequence in which one or several amino acids are substituted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and promoting postsynaptic formation.
(F) DNA consisting of a base sequence having 90% or more homology with the base sequence shown in SEQ ID NO: 1 and promoting postsynaptic formation. The preparation according to any one of claims 1 to 5, wherein the DNA is incorporated into a vector.

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